Oxidation-Reduction. Part C - Dehydrogenases (II), Oxidases (II), Hydrogen Peroxide Cleavage. Third Edition (PDFDrive)

The Enzymes
VOLUME XI11
OXIDATION-REDUCTION
Part C
DEHYDROGENASES (II)
OXIDASES (II)
HYDROGEN PEROXIDE CLEAVAGE
Third Edition
CONTRIBUTORS
WINSLOW S. CAUGHEY GREGORY R. SCHONBAUM

BRITTON CHANCE DIANA L. STIGGALL
L. ERNSTER JOHN A. VOLPE
J. IEUAN HARRIS WILLIAM J. WALLACE
YOUSSEF HATEFI MICHAEL WATERS
J. B. HOEK CHARLES H. WILLIAMS, JR.
J. RYDSTROM TAKASHI YONETANI
SHINYA YOSHIKAWA
ADVISORY BOARD
BRITTON CHANCE BO MALMSTROM

LARS ERNSTER VINCENT MASSEY
THE ENZYMES
Edited by PAUL D. BOYER
Molecular Biology Institute and
Department of Chemistry
University of California
Los Angeles, California
Volume XI11
OXIDATIOWREDUCTION
Part C
DEHYDROGENASES (II)
OXIDASES (II)
HYDROGEN PEROXIDE CLEAVAGE
THIRD EDITION
ACADEMIC PRESS New York San Francisco London 1976

A Subeidiary of Harcourt Brace Jovanovich, Publishers
COPYRIGHT 6 1976, BY ACADEMIC PRESS, INC.
ALL RIGHTS RESERVED.
NO PART OF THIS PUBLICATION MAY BE REPRODUCED OR
TRANSMITTED IN A N Y FORM OR BY ANY MEANS, ELECTRONIC
OR MECHANICAL, INCLUDING PHOTOCOPY, RECORDING, OR A N Y
INFORMATION STORAGE AND RETRIEVAL SYSTEM, WITHOUT
PERMISSION IN WRITING FROM THE PUBLISHER.
ACADEMIC PRESS, INC.

111 Fifth Avenue, New York. New' York 10003
United Kingdom Edition published by

ACADEMIC PRESS, INC. (LONDON) LTD.
24/28 Oval Road, London N W l
Library of Congress Cataloging in Publication Data
Main entry under title:

The Enzymes.
Includes bibliographical references.

CONTENTS: v. 1. Structure and control.-v. 2. Ki-
netics and mechanism.-v. 3. Hydrolysis: peptide
bonds. (etc.]
1. Enzymes. I. Boyer,PaulD.,ed.
[DNLM: 1. Enzymes. QU135 B791eJ
QP601.E523 574.1 '925 75-117107
ISBN 0-12-122713-8
PRINTED IN THE UNITED STATES OF AMERICA

Contents
. . . . . . . . . . . . . . .
List of Contributors vii
Preface . . . . . . . . . . . . . . .. . . ix
Contents of Other Volumes . . . . . . . . . . . . . xi
1. Glyceraldehyde-3-phosphate Dehydrogenase
J. IEUANHARRISAND MICHAEL
WATERS
I. Introduction ..
11. Molecular Properties
. . . . . . . . . . . . . 1
. . . . . . . . . . . . , 3
111. Catalytic Properties . . . . . . . . . . . . . as
2. Nicotinamide Nucleotide Transhydrogenases
J. B. HOEK,AND L. ERNSTER
J. RYDSTRBM,
I. Definitions . . . . , . . . . . . . . . . . 51
11. BBSpecific Transhydrogenases . . . . . . . . . . . 52
111. ABSpecific Transhydrogenases . . . . . . , . . . . 62
IV. Physiological Roles of Nicotinamide Nucleotide Transhydrogenases . . 79
3. Flavin-Containing Dehydrogenases
CHARLES
H. WILLIAMS,
JR.
I. Introduction . . . . . . . . . . . . . . . 90
.
11. Pyridine Nucleotide-Disulfide Oxidoreductases . . . . . . 92
111. Lipoamide Dehydrogenase . . . . . . . . . . . . 106
IV. Glutathione Reductaae . . . . . .
. . . . . . . 129
V. Thioredoxin Reductase . . . . . . . . . . . . 142
VI. Microsomal Electron Transport . . . . . . . . . . 148
VII. NADH-Cytochrome b. Reductase . . . . . . . . . . 154
VIII. NADPH-Cytochrome P-450 Reductase . . . . . . . . . 185
4. Metal-Containing Flavoprotein Dehydrogenases
YOUSSEF L. STIGGALL
HATEFIAND DIANA
I. Introduction . . . . . . . . . . . . . . . 175
11. NADH Dehydrogenases . . . . . . . . . . . . 177
V
vi CONTENTS
. . . . . . . . . . . . . 222
I11 Succinate Dehydrogenases
. . . . . 256
IV . ~-Glycerol-3-phosphate Dehydrogenase (EC 1.1.995)
.
V Choline Dehydrogenase (EC 1.1.99.1) . . . . . . .
. . 280
. . . . . . . . . . . . 263
VI . Lactate Dehydrogenases
VII . Nitrite Reductases (EC 1.6.6.4) . . . . . . . . .
. . 273
VIII . Adenylyl Sulfate Reductases (EC 1.8.99.2) . . . . . .
. . 279
. . 286
IX . Sulfite Reductases (H&:NADPH Oxidoreductases) (EC 1.8.12)
X . Addendum . . . . . . . . . . . . . . . . 295
5 . Cytochrome c Oxidare
WINSLOW S. CAUGHEY.WILLIAMJ . WALLACE.
JOHNA . VOLPE.AND SHINYA
YOSHIKAWA
I . Introduction . . . . . . . . . . . . . . . 299
. . . . . . . . . . . .
I1 Isolation and Characterization 305
. . . . . . . . . .
I11. Chemical and Physical Properties 313
.
IV Mechanisms . . . . . . . . . . . . . . . 337
6 . Cytochrome c Peroxidare
TAKASHI
YONETANI
I. Introduction . . . . . . . . . . . . . . . 345
I1. Preparation and Molecular Properties . . . . . . . . . 347
I11. Structural Aspects . . . . . . . . . . . . . . 348
IV . Enzymic Activity . . . . . . . . . . . . . . 352
V. Reaction Mechanism . . . . . . . . . . . . . 353
. .
VI . Interaction with Cytochrome c . . . . . . . . . 356
VII . General Comments . . . . . . . . . . . . . 300
7 . Catalase
R . SCHONBAUM
GREGORY AND BRITTON
CHANCE
I . Introduction . . . . . .
. . . . . . . . . . 363
I1. General Enzyme Properties . .
. . . . . . . . . 366
. . . .
I11. The Nature of the Active Site . . . . . . 369
. . . .
IV . Catalase-Mediated Redox Reactions . . . . . 388
Author Index . . . . . . . . . . . . . . . . 409
Subject Index . . . . . . . . . . . . . . . . 435
Topical Subject Index for Volumes I-XIII . . . . . . . . . . 459
List of Contributors
Numbers in parentheses indicate the pages on which the authors’ contributions begin.
WINSLOW S. CAUGHEY (299), Department of Biochemistry, Colorado

State University, Fort Collins, Colorado
BRITTON CHANCE (3631, Johnson Research Foundation, University
of Pennsylvania School of Medicine, Philadelphia, Pennsylvania
L. ERNSTER (51),Department of Biochemistry, Arrhenius Laboratory,
University of Stockholm, Stockholm, Sweden
J. IEUAN HARRIS (1), Medical Research Council Laboratory of Molec-
ular Biology, University Postgraduate Medical School, Cambridge,
England
YOUSSEF HATEFI (175),Department of Biochemistry, Scripps Clinic
and Research Foundation, La Jolla, California
J. B. HOEK (51),Department of Biochemistry, University of Nairobi,
Nairobi, Kenya
J . RYDSTROM (51),Department of Biochemistry, Arrhenius Labora-
tory, University of Stockholm, Stockholm, Sweden
GREGORY R. SCHONBAUM (363), Department of Biochemistry,
St. Jude Children’s Research Hospital, and University of Tennessee
Center for the Health Sciences, Memphis, Tennessee
DIANA L. STIGGALL (175), Department of Biochemistry, Scripps
Clinic and Research Foundation, La Jolla, California
JOHN A. VOLPE (299), Department of Biochemistry, Colorado State
University, Fort Collins, Colorado
WILLIAM J. WALLACE (299), Department of Biochemistry, Colorado
MICHAEL WATERS (1),Department of Biochemistry, Monash Uni-
versity, Clayton, Victoria, Australia
vii
viii LIST OF CONTRIBUTORS
CHARLES H. WILLIAMS, JR. (89), Veterans Administration Hospital,

and Department of Biological Chemistry, University of Michigan, Ann
Arbor, Michigan
TAKASHI YONETANI (346), Department of Biochemistry and Bio-
physics, University of Pennsylvania School of Medicine, Philadelphia,
Pennsylvania
SHINYA YOSHIKAWA (299), Department of Biochemistry, Colorado
Preface
This is the final volume of the Third Edition of “The Enzymes.” It
completes the coverage of oxidation-reduction enzymes.
As with previous volumes, the quality and quantity of information a t
the molecular level in this volume are impressive. The first portion of the
volume includes the remaining chapters on the nicotinamide nucleotide-
linked dehydrogenases, namely, the transhydrogenases and the very im-
portant glyceraldehyde-3-phosphate dehydrogenase. The second portion
completes the treatment of the great family of flavin-containing enzymes,
with chapters on the flavoprotein dehydrogenases and the metalloflavo-
protein dehydrogenases. The last portion includes chapters on catalase
and peroxidase that use hydrogen peroxide, and on cytochrome oxidase,
the enzyme responsible for most of the oxygen consumption by animals.
The Third Edition has proved considerably longer and contains much
more information than was thought likely when the edition was launched.
The privilege of editing the treatise has given me a deep respect for
the collective accomplishments of the many scientists whose continued
efforts have made such a treatise possible. The quality and abundance of
information found in this edition are a tribute to the individual research
worker, often little recognized, and to the society that has made such a
work possible. I know of no finer recognition of man’s potentiality and
creativity than has been my fortune to experience in editing this multi-
volume treatise.
Again, it is a pleasure to acknowledge the indebtedness of the users of
these volumes to the Advisory Board that helped plan each volume, to
the contributors for their unusually high level of excellence, to the staff
of Academic Press for their high professional standards, and to Lyda
Boyer, whose editorial and other assistance made many tasks lighter.
PAULD. BOYER
ix
This Page Intentionally Left Blank
Contents of Other Volumes
Volume I: Structure and Control
X-Ray Crystallography and Enzyme Structure

David E isenberg
Chemical Modification by Active-Site-Directed Reagents

Elliott Shaw
Chemical Modification as a Probe of Structure and Function

Louis A. Cohen
Multienzyme Complexes
Lester J. Reed and David J . Cox
Genetic Probes of Enzyme Structure

Milton J . Scklesinger
Evolution o f Enzymes
Emil L. Smith
The Molecular Basis for Enzyme Regulation

D. E. Koshland, Jr.
Mechanisms of Enzyme Regulation in Metabolism

E. R. Stadtman
Enzymes as Control Elements in Metabolic Regulation
Daniel E. Atkinson
Author Index-Subject Index
xi
xii CONTENTS OF OTHER VOLUMES
Volume II: Kinetics and Mechanism
Steady State Kinetics

W . W . Cleland
Rapid Reactions and Transient States
Gordon B. Hammes and Paul R . Schimmel
Stereospecificity of Enzymic Reactions
G. Popjhk
Proximity Effects and Enzyme Catalysis
Thomas C . Bruice
Enzymology of Proton Abstraction and Transfer Reactions
Irwin A. Rose
Kinetic Isotope Effects in Enzymic Reactions
J . H . Richards
Schiff Base Intermediates in Enzyme Catalysis
Esmond E. Snell and Samuel J. DiMari
Some Physical Probes of Enzyme Structure in Solution
Serge N . Timasheff
Metals in Enzyme Catalysis
Albert S. Mildvan
Author Index-Subj ect Index
Volume 111: Hydrolysis: Peptide Bonds
Carboxypeptidase A
Jean A. Hartsuck and William N . Lipscomb
Carboxypeptidase B
J . E . Folk
Leucine Aminopeptidase and Other N-Terminal Exopeptidases
Robert J . DeLange and Emil L. Smith
Pepsin
Joseph S. Fruton
CONTENTS OF OTHER VOLUMES xiii
Chymotrypsinogen : X-Ray Structure

J . Kraut
The Structure of Chymotrypsin

I?.M . Blow
Chymotrypsin-Chemical Properties and Catalysis
George P . Hess
Trypsin
B. Keil
Thrombin and Prothrombin
Staflan Magnusson
Pancreatic Elastase
B. S. Hartley and D. M . Shotton
Protein Proteinase Inhibitors-Molecular Aspects

Michael Laskowski, Jr., and Robert W . Sealock
Cathepsins and Kinin-Forming and -Destroying Enzymes
Lowell M . Greenbaum
Papain, X-Ray Structure
J . Drenth, J. N . Jansonius, R. Koekoek, and B. G. Wolthers
Papain and Other Plant Sulfhydryl Proteolytic Enzymes
A . N . Glazer and Emil L. Smith
Subtilisin: X-Ray Structure
J . Kraut
Subtilisins : Primary Structure, Chemical and Physical Properties
Francis S. Markland, Jr., and Emil L. Smith
Streptococcal Proteinase
Teh-Yung Liu and S. D.Elliott
The Collagenases
Sam Seifter and Elvin Harper
Clostripain
William M . Mitchell and William F. Harrington
xiv CONTENTS OF OTHER VOLUMES
Other Bacterial, Mold, and Yeast Proteases

Hiroshi Matsubara and Joseph Feder
Volume IV: Hydrolysis: Other C N Bonds, Phosphate Esters
Ureases
F. J . Reithel
Penicillinase and Other p-Lactamases
Nathan Citri
Purine, Purine Nucleoside, Purine Nucleotide Aminohydrolases
C . L. Zielke and C. H . Suelter
Glutaminase and 7-Glutamyltransferases
Standish C . Hartman
L-Asparaginase
John C. Wriston, Jr.
Enzymology of Pyrrolidone Carboxylic Acid
Marian Orlowski and Alton Meister
Staphylococcal Nuclease X-Ray Structure
F. Albert Cotton and Edward E . Hazen, Jr.
Staphylococcal Nuclease, Chemical Properties and Catalysis
Christian B. Anfinsen, Pedro Cuatrecasas, and Hiroshi Taniuchi
Microbial Ribonucleases with Special Reference to
RNases TI, T,,N1, and Uz
Tsuneko Uchida and Fuji0 Egami
Bacterial Deoxyribonucleases
I. R. Lehman
Spleen Acid Deoxyribonuclease
Giorgio Bernardi
Deoxyribonuclease I
M . Laskowski, Sr.
CONTENTS OF OTHER VOLUMES xv
Venom Exonuclease
M . Laskowski, Sr.
Spleen Acid Exonuclease
Albert0 Bernardi and Giorgio Bernardi
Nucleotide Phosphomonoesterases
George I . Drummond and Masanobu Yamamoto
Nucleoside Cyclic Phosphate Diesterases
George I . Drummond and Masanobu Yamamoto
E. coli Alkaline Phosphatase
Ted W . Reid and Irwin B. Wilson
Mammalian Alkaline Phosphatases
H . N . Fernley
Acid Phosphatases
Vincent P. Hollander
Inorganic Pyrophosphatase of Escherichia coli
John Josse and Simon C. K. Wong
Yeast and Other Inorganic Pyrophosphatases
Larry G. Butler
Glucose-6-Phosphatase, Hydrolytic and Synthetic Activities
Robert C. Nordlie
Fructose-1,6-Diphosphatases
8.Pontremoli and B . L. Horecker
Bovine Pancreatic Ribonuclease
Frederic M . Richards and Harold W . Wyckoff
Volume V: Hydrolysis (Sulfate Esters, Carboxyl Esters, Glycosides) ,

Hydration
The Hydrolysis of Sulfate Esters

A. B. Roy
xvi CONTENTS OF OTHER VOLUMES
Arylsulf atases
R. G. Nicholls and A. B. Roy
Carboxylic Ester Hydrolases

Klaus Krkch
Phospholipases
Donald J . Hanahan
Acetylcholinesterase
Harry C. Froede and Irwin B . Wilson
Plant and Animal Amylases
John A. Thoma, Joseph E . Spradlin, and Stephen Dygert
Glycogen and Starch Debranching Enzymes
E. Y . C. Lee and W . J. Whelan
Bacterial and Mold Amylases
Toshio Takagi, Hirolco Toda, and Toshizo Isemura
Cellulases
D. R. Whitaker
Yeast and Neurospora Invertases
J . Oliver Lampen
Hy aluronidases
Karl Meyer
Neuraminidases
Alfred Gottschallc and A. S. Bhargava
Phage Lysozyme and Other Lytic Enzymes
Akira Tszlgita
Aconitase
Jenny Pickworth Glusker
p-Hydroxydecanoyl Thioester Dehydrase
Konrad Bloch
Dehydration in Nucleotide-Linked Deoxysugar Synthesis
L. Glaser and H.Zarkowslcy
CONTENTS OF OTHER VOLUMES xvii
Dehydrations Requiring Vitamin B,, Coenzyme

Robert H. Abeles
Enolase
Finn Wold
Fumarase and Crotonase
Robert L. Hill and John W . Teipel
6-Phosphogluconic and Related Dehydrases
W . A. Wood
Carbonic Anhydrase
S. Lindslcog, L. E. Henderson, K . K . Kannan, A. Liljas,
P. 0. Nyman, and B. Strandberg
Author IndexSubject Index
Volume VI: Carboxylation and Decarboxylation ( Nonoxidative),

lromerization
Pyruvate Carboxylase
Michael C.Scrutton and Murray R. Young
Acyl-CoA Carboxylases
Alfred W . Alberts and P. Roy Vagelos
Transcarboxylase
Harland G.Wood
Formation of Oxalacetate by CO, Fixation on Phosphoenolpyruvate
Merton F. Utter and Harold M . Kolenbrander
Ribulose-l,5-DiphosphateCarboxylase
Marvin I. Siegel, Marcia WGhnick, and M . Daniel Lane
Ferredoxin-Linked Carboxylation Reactions
Bob B. Buchanan
Amino Acid Decarboxylases
Elizabeth A. Boeker and Esmond E. Snell
Actoacetate Decarboxylase
Irwin Fridovich
xviii CONTENTS OF OTHER VOLUMES
Aldose-Ketose Isomerases
Ernst A . Noltmann
Epimerases
Luis Glaser
Cis-Trans Isomerization
Stanley Seltzer
Phosphomutases
W. J. Ray, Jr., and E. J . Peck, Jr.
Amino Acid Racemases and Epimerases
E lija h Adams
Coenzyme Bl,-Dependent Mutases Causing Carbon Chain
Rearrangements
H . A . Barker
Blz Coenzyme-Dependent Amino Group Migrations
Thressa C . Stadtman
Isopentenylpyrophosphate Isomerase
P . W . Holloway
Isomerization in the Visual Cycle
Joram Heller
A6-3-KetosteroidIsomerase
Paul Talalay and Ann M . Bemon
Volume VII: Elimination and Addition, Aldol Cleavage and condensation,

Other C C Cleavage, Phorphorolysir, Hydrolysis (Fats, Glycoriderl
Tryptophan Synthetase
Charles Yanojsky and Irving P . Crawjord
Pyridoxal-Linked Elimination and Replacement Reactions
Leodis Davis and David E. Metzler
The Enzymatic Elimination of Ammonia
Kenneth R . Hanson and Evelyn A . Havir
CONTENTS OF OTHW VOLUMES xix
Argininosuccinases and Adenylosuccinases

Sarah Ratner
Epoxidases
William B. Jakoby and Thorsten A. Fjellstedt
Aldolases
B. L. Horecker, Orestes Tsolas, and C. Y.Lai
Transaldolase
Orestes Tsolas and B. L. Horecker
2-Keto-3-deoxy-6-phosphogluconicand Related Aldolaseo
W. A. Wood
Other Deoxy Sugar Aldolases
David Sidney Feingold and Patricia Ann Hoflee
8-Aminolevulinic Acid Dehydratase
David Shemin
8-Aminolevulinic Acid Synthetase
Peter M . Jordan and David Shemin
Citrate Cleavage and Related Enzymes
Leonard B. Spector
Thiolase
Ulrich Gehring and Feodor Lynen
Acyl-CoA Ligases
Malcolm J. P. Higgim, Jack A. Kornblatt, and Harry Rudney
a-Glucan Phosphorylases-Chemical and Physical Basis of Catalysis
and Regulation
Donald J . Graves and Jerry H . Wang
Purine Nucleoside Phosphorylase
R. E. Parks, Jr., and R. P. Agarwal
Disaccharide Phosphorylases
John J . Mieyal and Robert H. Abeles
Polynucleotide Phosphorylase
T . Godejroy-Colburn and M . Grunberg-Manago
xx CONTENTS OF OTHER VOLUMES
The Lipases
P. Desnuelle
p-Galactosidase
Kurt Wallenfels and Rudolf Wed
Vertebrate Lysozymes
Taiji Imoto, L. N. Johnson, A . C. T. North, D. C. Phillips, and
J . A . Rupley
Volume VIII: Group Transfer, Part A: Nucleotidyl Transfer, Nucleoridyl

Transfer, Acyl Transfer, Phosphoryl Transfer
Adenylyl Transfer Reactions

E. R. Stadtman
Uridine Diphosphoryl Glucose Pyrophosphorylase
Richard L. T u r n p h t and R. Gaurth Hansen
Adenosine Diphosphoryl Glucose Pyrophosphorylase
Jack Preiss
The Adenosyltransferases
S. Harvey Mudd
Acyl Group Transfer (Acyl Carrier Protein)
P. Roy Vagelos
Chemical Basis of Biological Phosphoryl Transfer
S. J. Benkovic and K . J . Schray
Phosphofructokinase
David P. Bloxham and Henry A . L a d y
Adenylate Kinase
L. Noda
Nucleoside Diphosphokinases
R. E. Parks, Jr., and R. P. Agarwal
CONTENTS OF OTHER VOLUMES xxi
3-Phosphoglycerate Kinase
R. K. Scope
Pyruvate Kinase
F. J. Kayne
Creatine Kinase (Adenosine 5’-Triphosphate-Creatine
Phosphotransferase)
D.c. w a t t s
Arginine Kinase and Other Invertebrate Guanidino Kinases
J . F. Morrison
Glycerol and Glycerate Kinases
Jeremy W. Thorner and Henry Paulus
Microbial Aspartokinases
Paolo Truffa-Bachi
Protein Kinases
Donal A . Walsh and Edwin G. Krebs
Volume IX: Group Transfer, Part B: Phosphoryl Transfer, One-Carbon Group

Transfer, Glycosyl Transfer, Amino Group Transfer, Other Transferaser
The Hexokinases
Sidney P. Colowick
Nucleoside and Nucleotide Kinases
Elizabeth P. Anderson
Carbamate Kinase
L. Raijman and M . E . Jones
N5-Methyltetrahydrofolate-HomocysteineMethyltransferases
Robert T . Taylor and Herbert Weissbach
Enzymic Methylation of Natural Polynucleotides
Sylvia J. Kerr and Ernest Borelc
Folate Coenzyme-Mediated Transfer of One-Carbon Groups
Jeanne I. Rader and F. M . Huennekens
xxii CONTENTS OF OTHER VOLUMES
Aspartate Transcarbamylases
Gary R. Jacobson and George R. Stark
Glycogen Synthesis from UDPG
W . Stalmam and H , G. Hers
Lactose Synthetase
Kurt E. Ebner
Amino Group Transfer
Alexander E. Braumtein
Coenzyme A Transferases
W . P. Jencks
Amidinotransferages
James B. Walker
N-Acetylglutamate-5-Phosphotransferase
Giza De'nes
Author I n d e x a u b j e c t Index
Volume X: Protein Synthesis, DNA Synthesis and Repair,

RNA Synthesis, Energy-Linked ATPases, Synthetases
Polypeptide Chain Initiation

Severo Ochoa and Rajarshi Mazumder
Protein Synthesis-Peptide Chain Elongation
Jean Lucus-Lenard and Laszlo Beres
Polypeptide Chain Termination
W . P. Tate and C. T . Caskey
Bacterial DNA Polymerases
Thomas Kornberg and Arthur Kornberg
Terminal Deoxynucleatidyl Transferase
F . J . Bollum
Eucaryotic DNA Polymerases
Lawrence A. Loeb
RNA Tumor Virus DNA Polymerases
Howard M . Temin and Satoshi Mizutani
CONTENTS OF OTHER VOLUMES xxiii
DNA Joining Enzymes (Ligases)

I. R. Lehman
Eucaryotic RNA Polymerases
Pierre Chambon
Bacterial DNA-Dependent RNA Polymerase
Michael J . Chamberlin
Mitochondria1 and Chloroplast ATPases
Harvey S.Penefsky
Bacterial Membrane ATPase
Adolph Abrams and Jeffrey B. Smith
Sarcoplasmic Membrane ATPases
Wilhelm Hasselbach
Fatty Acyl-CoA Synthetases
John C. Londesborough and Leslie T . Webster, Jr.
Aminoacyl-tRNA Synthetases
Dieter Sol1 and Paul R. Schimmel
C T P Synthetase and Related Enzymes
D. E. Koshland, Jr., and A. Levitzki
Asparagine Synthesis
Alton Meister
Succinyl-CoA Synthetase
William A. Bridger
PhosphoribosylpyrophosphateSynthetase and Related
Pyrophosphokinases
Robert L. Switzer
Phosphoenolpyruvate Synthetase and Pyruvate, Phosphate Dikinase
R. A. Cooper and H . L.Komberg
Sulfation Linked to ATP Cleavage
Harr y D . Peck, J r .
Glutathione Synthesis
A1ton Meis ter
Glutamine Synthetase of Mammals
Alton Meister
xxiv CONTENTS OF OTHER VOLUMES
The Glutamine Synthetase of Escherichia coli:

Structure and Control
E . R. Stadtman and A. Ginsburg
Volume XI: Oxidation-Reduction, Part A: Dehydrogenases (II , Electron

Transfer (1)
Kinetics and Mechanism of Nicotinamide-Nucleotide-Linked

Dehydrogenases
Keith Dalziel
Evolutionary and Structural Relationships among Dehydrogenases
Michael G. Rossmann, Anders Liljas, Carl-Ivar Brandin, and
Leonard J . Banaszak
Alcohol Dehydrogenases
Carl-Ivar Brand&, Hans Jornvall, Hans Eklund, and Bo Furugren
Lactate Dehydrogenase
J. John Holbrook, Anders Liljas, Steven J. Steindel, and
Michael G. Rossmann
Glutamate Dehydrogenases
Emil L. Smith, Brian M . Awten, Kenneth M . Blumenthal, and
Joseph F. Nyc
Malate Dehydrogenases
Leonard J . Banaszak and Ralph A . Bradshaw
Cytochromes c
Richard E . Dickerson and Russell Timkovich
Type b Cytochromes
Bunji Hagihara, Nobuhiro Sato, and Tateo Yamanaka
Volume XII: Oxidatiorr-Reduction, Part B: Electron Transfer ( I l l ,

Oxygenases, Oxidares (I1
Iron-Sulfur Proteins
Graham Palmer
CONTENTS OF OTHER VOLUMES xxv
Flavodoxins and Electron-Transferring Flavoproteins

Stephen G. Mayhew and Martha L. Ludwig
Oxygenases : Dioxygenases
Osamu Hayaishi, Mitsuhiro Nozaki, and Mitchel T. Abbott
Flavin and Pteridine Monooxygenases
Vincent Massey and Peter Hemmerich
Iron- and Copper-Containing Monooxygenases
V . Ullrich and W . Duppel
Molybdenum Iron-Sulfur Hydroxylases and Related Enzymes
R. C. Bray
Flavoprotein Oxidases
Harold J . Bright and David J . T. Porter
Copper-Containing Oxidases and Superoxide Dismutase
B. G. Malmstrom, L.-E. Andrdasson, and B. Reinhammar

Dehydrogenase
J. IEUAN HARRIS MICHAEL WATERS
I. Introduction . . . . . . . . . . . . . . . . 1
11. Molecular Properties . . . . . . . . . . . . . . 3
A. Isolation . . . . . .. . . . . . . . . 3
B. Enryme Structure . . . . . . . . . . . . 5
111. Catalytic Properties . . . . . . . . . . . . . . 28
A. Studies of Pyridine Nucleotide Binding . . . . . . 28
B. Mechanism of Action of GAPDH . . , . . . . . 38
C. Metabolic Role of GAPDH . . . . . . . . . . 45
1. Introduction ( 1 )
Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) catalyzes re-

versibly the oxidation and phosphorylation of D-glyceraldehyde 3-phos-
phate (G-3P) to 1,3-diphosphoglycerate (DPGA) according to the
following reaction scheme:
CHO ocoPo~*-
HAOH + HPO,*- + NAD+ ~ HAOH + H+ + NADH
AHaOPOa2- LHaOPO2-
It is thus a key enzyme in the glycolytic conversion of glucose to pyruvic
1. Abbreviations used are as follows : GAPDH, GlyceraldehydeSphosphate
dehydrogenase ; G 3 P , glyceraldehgde 3-phosphate ; DPGA, 1,3-diphosphoglyceric
acid; LDH, lactate dehydrogenase; MDH, mdic dehydrogenase ; and ADH, alcohol
dehydrogenase .
1
2 J. IEUAN HARRIS AND MICHAEL WATERS
acid which represents an important pathway of carbohydrate metabolism

in most organisms. That the oxidation of G-3P was associated with the
coupled phosphorylation of adenine nucleotides was originally established
by Meyerhof ( l a ) and by Needham and Pillai ( 2 ) . Meanwhile the even-
tual isolation of the participating enzyme was prompted by the earlier
observations of Lundsgaard (3)and of Green et al. ( 4 ) on the inhibition
of glycolysis and of alcoholic fermentation by halogenacetic acids, and
by the subsequent work of Rapkine ( 6 ) ,associating this inhibition with
sulfhydryl groups of GAPDH. The precise nature of the enzymic reaction
was elucidated by Warburg and Christian (6) when they succeeded in
preparing GAPDH in pure crystalline form from yeast. Subsequently,
isolation of the crystalline enzyme from rabbit skeletal muscle was de-
scribed by Dixon and Caputto (7) and by Cori et al. (8),and in retro-
TABLE I
SOURCESOF PUREGAPDH’s
Source Ref.
Rabbit muscle 7, 8
Yeast 10
Cat, dog, pig muscle 14
Rabbit, ox, human, chicken, turkey, 16
pheasant, halibut, sturgeon, lobster muscle
E . coli 16
B . stearothermophilus 17,18
T . aquaticus 19
B . cereus 20
Coelacanth muscle dl
Cold-adapted Antarctic fish muscle 22
Insects 93
Rat muscle 24
Kangaroo muscle 25
Pea seed 26
Photosynthetic plants d7,28
la. 0. Meyerhof, Naturwissenschaften 25, 443 (1937).

2. D. M. Needham and P. Pillai, Nature (London) 140,65 (1937).
3. E. Lundsgaard, Bwchern. Z. 217, 162 (1930).
4. D. E. Green, D. M. Needham, and J. D. Dewan, BJ 31, 2327 (1937).
5. L. Rapkine, BJ 32, 1729 (1938).
6. 0. Warburg and W. Christian, Biochem. Z.303,40 (1939).
7. R. Caputto and M. Dixon, Nature (London) 156, 630 (1945).
8. G. T. Cori, M. W. Slein, and C. F. Cori, JBC 159,565 (1945).
1. GLYCERALDEHYDE-3-PHOSPHATE DEHYDROGENASE 3
spect there can be little doubt that K. Bailey’s “albumin” from rabbit
muscle (9)“exhibiting a pronounced sheen upon agitation’’ was in fact
GAPDH.
Glyceraldehyde-3-phosphate dehydrogenase occurs widely and abun-
dantly throughout nature. It comprises about 20% of the total soluble
protein in yeast (10) and up to 10% of the soluble protein from muscle
(8),and the relative ease of its preparation from a wide variety of differ-
ent species has contributed to its popularity among enzymologists, protein
chemists, and X-ray crystallographers (cf. 11). Moreover, study of the
active enzyme-NAD complex has been facilitated by the fact that
uniquely among NAD-linked enzymes crystalline muscle GAPDH con-
tains firm bound NAD. Detailed reviews of these early studies have been
given by Velick and Furfine ( l a ) and by Colowick et al. (IS).
II. Molecular Properties
A. ISOLATION
Pure crystalline GAPDH has been isolated from a number of different
sources (cf. Table I) (7,8,10,14-28). Methods of purification have relied
heavily upon its solubility as the enzyme-NAD complex in high concen-
9. K.Bailey, Nature (London) 145, 934 (1940).

10. E. G. Krebs, G. W. Rafter, and J. M. Junge, JBC 200, 479 (1953).
11. J. I. Harris, in “Structure and Function of Oxidation-Reduction Enzymes”
(A. Akeson and A. Ehrenberg, eds.), p. 639.Pergam n, Oxford, 1972.
12. S. F.Velick and C. Furfine, “The Enzymes,” pol. 7, p. 243,1963.
13. S.P.Colowick, J. Van Eys, and J. H. Park, Compr. Biochem. 14,l (1966).
14. P. Elodi and E. SzorGnyi, Acta Phgsiol. 9, 339 (1956).
15. W.S.Allison and N. 0. Kaplan, JBC 239,2140 (1964).
16. G. D’Alessio and J. Josse, JBC 246, 4319 (1971).
17. R. E. Amelunxen, BBA 122, 175 (1966).
18. K. Suzuki and J. I. Harris, FEBS (Fed. Eur. Biochem. Soc.) Lett. 13, 217
(1971).
19. J. D. Hocking and J. I. Harris, FEBS (Fed. Eur. Biochem. Soc.) Lett. 34,
280 (1973).
20. K. Suzuki and K. Imahori, J . Biochem. (Tokyo) 73,97 (1973).
21. E. Kolb and J. I. Harris, BJ 130, 26P (1971).
22. F. C.Greene and R. E. Feeney, BBA 220,430 (1970).
23. C. W.Carlson and R .W. Rrosemer, Biochemistry 10, 2113 (1971).
24. N. K.Nagradova and M. K. Guseva, Biokhimiya 36, 496 (1971).
25. R. J. Simpson and B. E. Davidson, Aust. J. Biol. Sci. 24, 263 (1971).
26. R. G.Duggleby and D. T. Dennis, JBC 249, 162 (1974).
27. W.Hood and N. G. Carr, BBA 146, 309 (1967).
28. B. A. Melandri, P. Pupillo, and A. Baccarini-Melandri, BBA 220, 178 (1970).
trations (up to 70% saturation) of ammonium sulfate so that the pure

muscle enzyme can be obtained from a low salt extract of blended muscle
by direct crystallization from 65 to 70% ammonium sulfate. Methods
for preparing enzyme from bacterial sources such as Escherichia coli (16)
and B. stearothermophilus (18) have been improved by the use of chro-
matography on ion exchangers, while more recently Hocking and Harris
(19) have prepared pure enzyme from the thermophiles B. stearotherrno-
philw and Thermus aquaticus by means of affinity chromatography on
immobilized NAD'. This method of preparation utilizes the strong affinity
of the enzyme for suitably immobilized NAD' (it remains bound to
NAD-Sephsrose in 0.7 M NaCl and is then eluted from the resin with
a pulse of 10 mM NAD') which allows it to be obtained pure and in
high yield from relatively crude bacterial extracts as shown in Fig. 1.
FIG.1. Purification of (A) T.aquaticua and (B) B. stearothermophilua GAPDH;

SDS-gel electrophoresis (a) before and (b) after NAD-Sepharose (cf. 37).
1. GLYCERALDEHYDE-%PHOSPHATE DEHYDROGENASE 5
B. ENZYME
STRUCTURE
1. Primary Structure
A study of the enzyme by chemical methods involving the specific
labeling of catalytically active cysteine residues ($9, 30) and the charac-
terization of peptide fragments produced by enzymic cleavage (31) led
Harris and Perham to conclude that GAPDH from a given source was
composed of subunits comprising approximately 330 amino acid residues
corresponding to a molecular weight of 36,000. These results, considered
in conjunction with the physicochemical data of Harrington and Karr
(32),showed that the active enzyme with a molecular weight of 146,000
was a tetramer and that it was in all probability composed of chemically
identical subunits (31). Proof that the subunits are of identical primary
structure was obtained by Harris and co-workers when complete amino
acid sequences were established for enzyme from lobster muscle (33),
pig muscle ( 3 4 ) , and yeast (36).Comparison of the three sequences
(Table 11) shows that they are strictly homologous. Moreover, 60%
of the residues occur in identical sequence in the three species showing
that the sequence of GAPDH has been conserved to a much greater ex-
tent than the sequence of other comparable enzymes such as, for example,
alcohol dehydrogenase ( 3 6 ) .Hocking and Harris (37) have subsequently
determined the sequence of GAPDH from the thermophilic bacterium
T . aquaticus, and comparison of this sequence with that of the lobster
muscle enzyme shows a sequence identity of 50% which is again signifi-
cantly higher than was found in comparison of bacterial and liver alcohol
dehydrogenase (38) or bacterial and muscle triosephosphate isomerase
(399)-
29. J. I. Harris, B. P. Meriwether, and J. H. Park, Nature (London) 198, 154
(1963).
30. R. N. Perham and J. I. Harris, JMB 7,316 (1963).
31. J. I. Harris and R. N. Perham, JMB 13,876 (1965).
32. W. F. Harrington and G . M. Karr, JMB 13, 885 (1965).
33. B. E. Davidson, M. Sajg6, H. F. Noller, and J. I. Harris, Nature (London)
218, 1181 (1967).
34. J. I. Harris and R. N. Perham, Nature (London) 219, 1025 (1988).
35. G. M. T. Jones and J. I. Harris, FEHS (Fed. Eur. Biochem. Soc.) Lett. 22,
185 (1972).
36. H. Jornvall, Proc. Nat. Acad. Sci. U . S. 70,2295 (1973).
37. J. D. Hocking and J. I. Harris, Ezperientia (1976) (in press); J. D. Hocking
Ph.D. Dissertation, University of Cambridge, 1974.
38. J. Bridgen, E. Kolb, and J. I. Harris, FEBS (Fed. Eur. Biochem. Soc.) Lett.
33, 1 (1973).
39. S. Artavanis, Ph.D. Dissertation, University of Cambridge, 1974.
TABLE I1
COMPARISON
OF THE AMINO OF GAPDH FROM PIOMUSCLE,
ACIDSEQUENCE LOBSTEBMUSCLE,AND YEAST^.^ Q,
10
Asn-Gly -Phe-Gly -Arg - Ile -Gly -Arg-Leu-Val
Yeast Val-Arg-Val-Ala- Ile Leu-Ser -&g-
40
Asn-Asp-Pro-Phe - Ile
Gly -Ala -Gln -Val
Pro-Asx-Val -Glx -Val (Ala &: Asx,Asx,Pro,Phe, Ile
50 60
Tyr-Asp-Ser -Thr-His -Gly
t
Val-Val-Glu Ser -Thr-Gly -Val -Phe
Ile -Val-Glu
Ala- Ile - Asp
120 130
Ala-Pro-Met-Phe-Val
Y
150 160
*C
Val-Ser -Asn-Ala-Ser-CYS-Thr-Thr-Asn-Cys-Leu-Ala-Pro
Ser - Lys-Asp-Met-Thr-Val
Leu
170 180
Glu -Gly -Leu-Met-Thr-Thr-Val -His A l a - Ile Thr-Ala -Thr-Gln-LYS-
Ala -Val
(Met-Thr, Thr, Val, His) Ser -Le
200
Thr-Val -Asp-Gly -Pro-Ger
210 220
Ser -Thr-Gly -Ah-Ala-Lys-Ala-Val-Gly -Lys-Val Gly -Lys-Leu-Thr-Gly - M e t - A h -
230 240 250

Phe-Arg-Val-Pro-Thr Val -Ser -Val-Val -Asp-Leu-Thr
Pro-Asp
Val - A s x Glu -Thr-Thr
260 270
Leu-Gly-Tyr-Thr-Glu-
GLx -
-a
TABLE I1 (Continued)
Asx Ala Ser Leu-Gly -Asp-Ser -His Ser
300 310
Ser-Trp-Tyr-Asp-Asn-Glu
Aax-Asx-Glx Tyr Thr
Val Asp-Leu Met-Val H i s Met-Ala-Ser-Lys-Glu

4
~~
a From (56).
b Sequences not experimentally determined for the yeast chain are given within brackets and in a pro-
visional order that maximizes sequence homology between the yeast and muscle enzymes.
c C;s-149 forms part of the active site.
1. GLYCEBALDEHYDE-%PHOSPHATE DEHYDROGENASE 9
The amino acid sequence results clearly imply a unique sequence for
each of the enzymes examined, and there is no decisive evidence for the
existence among GAPDH’s of tissue-specific isozymes that differ in pri-
mary sequence despite reports of the occurrence of multiple electro-
phoretic forms in several different organisms (40, 41). I n no case was
it demonstrated that these multiple forms are the products of different
genes, and it is entirely possible that electrophoretically different tetra-
mers may have arisen by amide loss [as in the case of muscle aldolases
(42)] or through differential binding of NAD (41).
2. X - R a y Structure of Hobenzyme
The elucidation of the subunit structure and of the amino acid se-
quences of the subunits of different GAPDH’s provided the necessary
framework for the interpretation of chemical modification studies as well
as of X-ray crystallographic studies of the tertiary and quaternary struc-
ture of the active enzymeINAD complex. The first X-ray diffraction data
for GAPDH were obtained by Watson and Banaszak (43) with crystals
of enzyme from lobster muscle. These crystals, which displayed the yel-
low color that is characteristic of the holoenzyme, were orthorhombic
(P2,2,2, space group) with the tetramer as the asymmetric unit. Essen-
tially similar results have also been obtained with enzyme crystals from
human muscle (4,46) and from B. stearothermophilus (cf. 18). A more
detailed study of the lobster muscle enzyme by Rossmann and co-workers
(46-48) led to the computation of the first interpretable high resolution
(3 A) structure for GAPDH. The first map (with the tetramer as asym-
metric unit) was interpreted by averaging the four chemically equivalent
but crystallographically different subunits and, with the aid of the amino
acid sequence (33),it then became possible to trace the polypeptide chain
within the individual subunits. A coordinate system of P , Q , and R axes
40. H.G. Lebherz and W. J. Rutter, Science 157, 1198 (1967).

41. S. F. Velick in “Pyridine Nucleotide-Dependent Dehydrogenases” (H. Sund,
ed.), p. 57. Springer-Verlag, Berlin and New York, 1970.
42. C.F. Midelfort and A. H. Mehler, Proc. Nut. Acad. Sci. U.S . 69, 1816 (1972).
43. H.C.Watson and L. J. Banaszak, Nature (London) 204,918 (1974).
44. A. I. Gorjunov, N. S. Andreeva, T. Baranowski, and M. Wohy, J M B 69, 421
(1972).
45. H.C.Watson, E. Due& and W. D. Mercer, Nature (London) 240, l$O (1972).
46. M. Buehner, G.C. Ford, D. Moras, K. W. Olsen, and M. G. Roasmann, Proc.
Nut. Acad. Sci. U.S . 70, 3052 (1973).
47. M. Buehner, G. C. Ford, D. Moras, K. W. Olsen, and M. G. Rossmann, J M B
82, 563 (1974).
48. M. Buehner, G. C. Ford, D. Moras, K. W. Olsen, and M. G. Rossmann, J M B
90, 25 (1974).
Q Q
P P
FIO.2. Diagrammatic comparison of the association of subunits in GAPDH (left)

and LDH (right) (48).
FIo. 3. Stereoviews of the Ca atom backbone in lobster muscle GAPDH: (a)

one subunit viewed to illustrate the NAD+-binding and catalytic domains; (b) the
NADtbinding domain viewed in the same orientation as in (a); (c) the catalytic
similar to that used previously for lactate dehydrogenase (LDH) (49)

and malate dehydrogenase (MDH) (60) has been used (Fig. 2) to define
the GAPDH structure in order to draw attention to the striking struc-
tural similarities that exist between the three dehydrogenases (46, 61).
The major feature of the structure is that, although exhibiting apparent
222 symmetry, the tetramer consists functionally of a dimer of dimers
related across the Q axis (cf. 46). The only true twofold axis is the Q
axis whereas the other axes exhibit pseudosymmetry within the limits
domain viewed in the same orientation as in (a) ; and (d) complete tetramer viewed
down the P axis demonstrating the dumbbell silhouette with the four active sites
close to the center of the molecule (48, 6.5).
49. M. J. Adams, A. McPherson, Jr., M. G. Rossmann, R. W. Schevitz, and A. J.
Wonacott, J M B 51, 31 (1970).
50. E. Hill, D. Tsernoglou, L. Webb, and L. J. Banaszak, J M B 72,577 (1972).
51. M. G. Rossmann, A. Liljas, C.-I. Branden, and J. J. Banaszak, Chapter 2,
Volume XI.
of resolution obtained. The region of major interaction between subunits

is across the P axis; Q-axis-related contacts are relatively few and not
highly conserved, while R-axis-related contacts are again more numerous
and highly conserved.
The conformation of C a backbone atoms in the GAPDH subunit is
shown in Fig. 3a. The subunit is envisaged as consisting of two domains
(Figs. 3b and 3c), each with a specific function. The first, comprising
residues 1-149, is mainly involved in NAD+ binding while the second
domain, comprising residues 149-334, provides residues for substrate
binding, specificity, and catalysis. The “catalytic” domain also contains
most of the residues that are involved in intersubunit contacts.
a. The NAD-Binding Domain. The fold of the NAD+-binding domain
in GAPDH is shown diagrammatically in Fig. 4. It consists of a six-
stranded parallel /3 sheet flanked by helices and is similar to analogous
nucleotide binding structures in LDH, MDH, and ADH (cf. 6 1 ) . The
NAD+ in each of the four subunits is bound close to the molecular waist
(cf. Figs. 2 and Fig. 3d) of the tetramer and close enough to interact
via a section of antiparallel sheet (comprising residues 179-200) that ex-
tends across the R axis into the adjacent subunit. This intersubunit inter-
action was thought to link Lys-183 in one subunit to the pyrophosphate
Fra. 4. Diagrammatic representation of the NAD+-binding domain showing the

six-stranded parallel p sheet flanked by helices (48,63).
moiety of NAD+ in the adjacent subunit (46, 48), compatible with earlier
chemical evidence implicating Lys-183 in coenzyme binding ( 5 2 ) . A re-
vised structure (@, 53) for this part of the molecule shows, however,
that Lys-183 does not interact directly with either NAD+ or substrate.
Nevertheless, it remains possible that interactions between other residues
in the S-shaped loop (such as, for example, Pro-188 and Trp-193 with
NAD+ in the adjacent subunit could be responsible for the cooperativity
of NAD+ binding (cf. Section III,A,l) and the NAD+-promoted tetramer-
ization of the dimeric moiety. I n this respect, and as shown in Fig. 2,
GAPDH differs from LDH where each molecule of NAD’ is bound
entirely within each subunit with little possibility for direct interaction
between binding sites within the tetramer. The conformation of the NAD+
in GAPDH is nevertheless similar to that found in LDH. Thus it is bound
in an open extended configuration in each of the four subunits but with
the important difference of a 180° rotation about the C-1 to N-1 glycosi-
dic bond linking the nicotinamide ring to the ribose. This change ensures
that the “B” face of the ring is exposed to the substrate for hydride ion
transfer giving GAPDH its B specificity. The B or syn configuration
is stabilized by hydrogen bonds formed between the carboxyamide group
and the invariant Asn-313 and with the nicotinamide phosphate. It should
be noted that the alternative “A” configuration of the ring that occurs
in MDH, LDH, and ADH is prohibited in GAPDH due to steric hin-
drance involving the main chain residues Ala-120 and Pro-121 and the
carboxyamide group of the nicotinamide ring. The main chain hydrogen
bonding scheme in the NAD+-binding domain is shown in Fig. 5 and the
topography of the NAD-binding site itself is shown diagrammatically
in Fig. 6. The adenine ring binds between Phe-34 and Phe-99; a t the
side of the adenine binding pocket there are hydrophobic residues Pro-33,
Met-77, and Pro-79, while the inside of the pocket is more hydrophilic
in character owing to the presence of Asn-6 and Asn-31. Aspartate-32
forms a hydrogen bond to the 02’ atom on the adenosine ribose while
Gly-7 approaches it closely from one side. The phosphates interact with
the part of the chain comprising Gly-9, Arg-10, and Leu-11; Gly-97 and
Ala-120 provide a hydrophobic environment for the nicotinamide ribose
while, as mentioned previously, the carbonyl group of the nicotinamide
forms a hydrogen bond to Asn-313. It should be noted that the residues
found to be interacting with NAD’ are highly conserved in different
52. J. H. Park, D. C. Shaw, E. Mathew, and B. P. Meriwether, JBC 245, 2946

(1970).
53. D. Moras, I<. W. Olsen, M. N. Sabeson, G . C. Ford, and M. G . Rossmann,
JBC 250, 9137 (1975).
FIG.5. The main chain hydrogen bonding scheme in the NAD+-binding domain
(residues 1-149)(48, 63).
FIG.0. Stereoview of the NAD'-binding site showing amino acid side chains inter-
acting with the coenzyme. Note Phe-34 and Phe-99 on either side of the adenine
ring. Aspartate-32 and Gly-7, which are close to the adenine ribose, preserve their
functions in other dehydrogenases (48,651.
FIG.7. Diagrammatic representation of the catalytic domain viewed to show the

large pleated sheet forming the subunit interface across the P axis (68).
GAPDH’s (Table 11).Moreover, residues corresponding to Asp-32 and

Gly-7 are also found in structurally equivalent positions in LDH and
ADH (61).
The C-4 atom of the nicotinamide ring is close to the SH group of the
active site cysteine (Cys-149), and this interaction (be it a covalent bond
to a tetrahedral carbon atom or a charge-transfer complex) appears to be
responsible for the “Racker band” in the holoenzyme (64,66) (cf. Section
111,A). Cysteine-149, to which the substrate G-3P is bound, occurs a t
the junction between the two domains in the very center of the subunit.
b. The CataEytic Domain. The second half of the subunit (residues
149-334) consists primarily of a nine-stranded antiparallel sheet (Fig.
3c and Fig. 7) which forms an intersubunit contact generated by the P
axis. On the other side of the sheet there are three approximately parallel
helices (comprising residues 147-166, 201-216, and 215-267, respectively)
and the polypeptide chain ends in a long helix (residues 313-334) that
is closely associated with the first domain so that the C-terminal residue
comes close to the N-terminus (Fig. 3a). The hydrogen bonding scheme
for the catalytic domain is shown in Fig. 8.
The essential thiol (Cys-149) initiates a short helical region in which
the nonreactive Cys-153 occurs after one turn so that it comes close to
Cys-149 (cf. Section II,B,4,a). The polypeptide chain then returns to the
vicinity of the active center as part of an antiparallel sheet that contains
His-176. There follows an S-shaped antiparallel sheet (residues 179-200)
that interacts with NAD+ in the R-axis-related subunit. This segment
of the chain also contains Lys-191 which has been implicated as a possi-
ble binding site for phosphate. Histidine-176, conserved in all species (36,
37), occurs within hydrogen bonding distance of Cys-149, and these two
residues are clearly implicated in the catalytic mechanism (see Section
II,B,4,d).
c. Conservation of Amino Acid Residues. The enzyme subunit com-
prises approximately 330 residues. Of these 36% are in helix and 40%
in p sheet, in fair agreement with the prediction of ORD (66, 67) and
infrared absorption (68) measurements. Table I11 lists diagrammatically
the residues that have been conserved between the three completely
54. E. Racker and I. Krimsky, Nature (London) 169, 1043 (1952).

55. I. Krimsky and E. Racker, Science 122,319 (1955).
56. P. Zavodsky, L. B. Abaturov, and Y. M. Varshavsky, Acta Biochim.Biophys.
1, 389 (1966).
57. M. E. Magar, JBC 242, 2517 (1967).
58. D. W. Darnall and T. D. Barela, BBA 236, 593 (1971).
FIG.8. The main chain hydrogen bonding scheme in the catalytic domain (residues
150-334) ( 5 5 ) .
TABLE I11
AMINOACIDFUNCTIONS I N GAPDHO
Symbols and abbreviations aa follows: (-*-) consewed completely among the

pig, yeast, and lobster sequences; (*) in active center region; P, in contact generated
by P axis; Q, in contact generated by Q axis; R, in contact generated by R axis; and
D, in contact region between first and second domains within a subunit (48).
analyzed GAPDH sequences (i.e., pig, lobster, and yeast, Table 11).
Noted also are the particular functions that have been recognized for
a given amino acid; that is, whether it is involved (a) in the active cen-
ter, (b) in one of three types of subunit contacts, or (c) in domain boun-
dary contacts. This information is summarized in Table IV.
Correlation of conserved and variable regions with the three-dimen-
sional structure (59) shows that residues involved in catalysis and in
intersubunit contacts are conserved to a much greater extent than others.
It follows that the sequence of the catalytic domain with its greater pro-
portion of active site and subunit contact residues is more highly con-
served than the sequence of the NAD-binding domain despite the fact
that the latter represents a highly conserved structure.
59. I(. W. Olsen, D. Moras, M. G. Rossmann, and J. I. Harris, JBC 250, 9313
(1975).
TABLE IV
OF AMINOACID RESIDUES
CONSERVATION I N GAPDHa
Domain * P Q R D AA
First
Number conserved 17 0 3 5 9 71
Total number contacts 19 0 3 5 13 148
Percent conserved 90 - 100 100 69 48
Second
Number contacts 11 27 2 10 2 124
Percent conserved 92 82 40 83 40 67
Both
Number contacts 28 27 5 15 11 195
Percent conserved 90 82 63 88 61 58
symbols and abbreviations as follows: (*) involved in active center; P, in a

P-axis-generated contact between subunits; Q, in a Q-axis-generated contact between
subunits; R, in a R-axis-generated contact between subunits; D, in a domain-domain
contact within a subunit; and AA, amino acids involved (48).
3. Structure of Apoenzyme
Apoenzyme prepared from muscle holoenzyme by treatment with char-
coal is unstable and difficult to crystallize (60, 61). Consequently, it
has not so far been possible to solve the three-dimensional structure of
apo-GAPDH by X-ray crystallographic methods. Suzuki and Harris (18)
were able to prepare stable crystals suitable for X-ray diffraction analy-
sis of both holo- and apoenzyme from the thermophile B. stearother-
mophilus. GAPDH from this source is considerably more stable than
enzyme from mesophiles (17,18), and this stability is retained even in
the absence of NAD+ (Fig. 9 ) . Wonacott and colleagues (62,cf. 18) have
shown that these holoenzyme crystals are orthorhombic with space group
P2,2,2; the unit cell, like that of the lobster muscle enzyme, consists
of four tetramers. Apoenzyme crystals were found to be monoclinic (space
group P 2 , ) , and the unit cell consists of two tetramers.
It is known that the binding sites for NAD’ are not equivalent and
that conformational changes occur when NAD+ interacts with apoenzyme
in solution (for references see Section 111,A). These changes have been
shown to involve a volume contraction of about 776, possibly because
60. C. S. Furfine and S. F. Velick, JRC 240,814 (1965).
61. P. M. Wassarman and H. C. Watson, in “Enzymes and Isoenzymes: Structure,
Properties and Function” (D. Shugar, ed.), p. 51. Academic Press, New York, 1970.
20 J. IEUAN HARRIS A N D MICHAEL WATERS
D
Temperature .C
FIG.9. Comparative thermal stabilities of GAPDH’s from (A)rabbit muscle,
( 0 )B. stearothermophilus, ( A ) T . aquaticus holoGAPDH; a n d y o ) T . aquaticus
apoGAPDH (ST).
of the expulsion of solvent molecules as would be expected to occur if

NAD+ binding gives rise to a more tightly packed tetramer. A separate
determination of the three-dimensional structure of apoenzyme and iden-
tification of the structural changes that occur when NAD+ binds to the
protein subunits will be necessary in order to understand fully the cooper-
ative phenomena associated with the mechanism of the enzymic reaction
(cf. Section 111,AJ).
4. Chemical Modification of Native Enzyme
a. Cysteine Residues. The cysteine content of GAPDH is variable,
ranging between five SH groups per subunit for the lobster muscle enzyme
(33) and one SH group per subunit for enzyme from T . aquaticus (19,
3 7 ) . The essential cysteine (Cys-149) is also the most reactive, and it
is the selective reaction of this conserved residue in GAPDH that is re-
sponsible for the inhibition of glycolysis and of fermentation by iodo-
acetic acid (6, 9.9).
Cysteine-149 also reacts selectively with a number of other reagents
such as p-fluorodinitrobenzene (63),tetrathionate (64-66), iodosobenzoate
62. A. J. Wonacott, unpublished results.
63. S. Shaltiel and S. Soria, Biochemistry 8,4411 (1969).
64. A. Pihl and R. Lange, JBC 237, 1356 (1962).
65. W. S. Allison and N. 0.Kaplan, Biochemistry 3, 1792 (1964).
66. D. J. Parker and W. 9. Allison, JBC 244,180 (1969).
(66, 671, and various organic mercurials (cf. 68, 69). It is also acylated
during the enzyme-catalyzed hydrolysis of p-nitrophenylacetate (29, SO)
and acetyl phosphate (70). Cysteine-153, conserved in all known
GAPDH's [with the one exception of T.a q w t i w (S7')], is not reactive
in the native structure although under certain conditions it is capable of
forming an intrachain disulfide bond with Cys-149 (66-68).The initially
reversible inactivation of GAPDH by iodosobenzoate or tetrathionate is
thought to result from the formation of either a sulfenic acid or a sulfenyl
thiosulfate derivative of Cys-149. Upon standing, and more especially upon
heating or the addition of urea, these derivatives are able to react with
Cys-153 (which occurs after one turn of a helix, and thus close to Cys-149
in the tertiary structure) to form the disulfide bond. Subsequent reduction
with thiol does not restore enzymic activity, perhaps because forma-
tion of the ring introduces an element of strain into the helix which may
then induce an irreversible conformational change, possibly associated
with the displacement of Tyr-311 from the active site. It is, therefore,
probably significant that Cys-153 has been replaced by serine in T.
aquaticus GAPDH (37) since disulfide ring formation could lead to oxi-
dative inactivation of the enzyme at the high growth temperature
(7Oo-75OC) of this organism. With lobster GAPDH formation of the
intrachain disulfide ring between Cys-149 and Cys-153 occurs following
reaction of Cys-149 with DTNB ( 7 1 ) . This leads to a gradual unfolding
of the structure so that the other three buried SH groups also become
exposed and reactive toward DTNB. It should also be noted that the
initial reaction of Cys-149 in the holoenzyme with DTNB causes NAD
to be released (69) prior to disulfide bond formation. Reaction of the
DTNB apoenzyme with Cys-153 and the irreversible changes accompany-
ing this process may be a contributory factor in the instability of the
apoenzyme derivative.
b. Lysine Residues. A lysine residue in GAPDH, identified as Lys-183
in the primary sequence (34, 72),is acetylated irreversibly when apoen-
zyme is allowed to react with either p-nitrophenylacetate or acetyl phos-
phate (7'0, 73, 7'4) at alkaline pH. N-Acetylation occurs not by direct
reaction with Lys-183 but as the result of an S to N migration of acetyl
67. J. I. Harris and R. N. Perham, Proc. Znt. Congr. Biochem., 6th, 1964 Vol.
32, Sect, IVS27, p. 293 (1964).
68. P. M. Wassarman, H. C. Watson, and J. P. Major, BBA 191, 1 (1969).
69. P. J. Harrigan and D. R. Trentham, BJ 124, 573 (1971).
70. E. Mathew, B. P. Meriwether, and J. H. Park, JBC 242, 5024 (1967).
71. P. M. Wassarman and J. P. Major, Biochemistry 8, 1076 (1969).
72. J. I. Harris and L. Polghr, JMB 14, 630 (1965).
73. L. Polg&r,A C ~Physiol.
Q 25, 1 (1964).
74. L. Polgdr, BBA 118, 276 (1966).
groups from Cys-149. This implies that Lys-183 is close to Cys-149 in

the quaternary structure of the apoenzyme, an observation that was con-
firmed by the use of cross-linking reagents ( 7 5 ) .The acyl migration reac-
tion does not occur in the presence of NAD', and it was not established
if the reaction occurs within or between monomers. Acetylation of Lys-
183 was found to inhibit NAD binding as well as dehydrogenase activity,
and for these reasons it was suggested ( 5 d ) that Lys-183 is itself involved
in coenzyme binding. With yeast enzyme (35) the acetylation reaction
appeared to be less specific; thus, at pH 8.5 some reaction was also noted
with lysine residues at positions 212, 216, and 266, which may reflect
differences in the mode of binding of NAD in the yeast enzyme.
Reaction of the rabbit muscle holoenzyme with pyridoxal phosphate,
which results in total inactivation, is specific for Lys-191 and Lys-212
( 7 6 ) . With apoenzyme, on the other hand, pyridoxal phosphate reacts
with Lys-212 only, suggesting that a conformational change involving
Lys-191 takes place when NAD' is removed from the holoenzyme ( 7 7 ) .
It is of interest that Lys-191 and Lys-212 are conserved in all the se-
quenced species of GAPDH (see Tables I1 and 111, also 35) ; Lys-183,
on the other hand, is not conserved and its replacement by arginine in
the B. stearotherrnophilus (78) and T . aquaticus (37) enzymes explains
why the S to N acetyl transfer reaction does not occur with these en-
zymes. The position of Lys-183 in the three-dimensional structure of the
lobster holoenzyme (53) indicates that it is about 10 A distant from the
phosphate groups of the NAD'; moreover, its C-NH, group is 20 A distant
from Cys-149 in the R-axis-related subunit. These distances are such as
to suggest that the quaternary structure of the apoenzyme must be sub-
stantially altered with respect to the holoenzyme to permit a much closer
approach of the two groups if direct S to N transfer (70, 73, 74) or cross-
linking with 1,5-difluoro-2,4-dinitrobenzene (75) is to occur. It follows
that there is no direct interaction of Lys-183 in the holoenzyme with
either NAD' or substrate and that this particular lysine has no obvious
role in the mechanism of the catalytic reaction normally performed by
GAPDH.
c. Tyrosine Residues. Iodination of lobster muscle GAPDH with
K [ lZ5I] led to the identification of tyrosine residues of differing reactiv-
75. S. Shaltiel and M. Tauber-Finkelstein, BBRC 44, 484 (1971).

76. B. G. Forcina, G . Ferri, M. C. Zapponi, and S. Ronchi, Eur. J . Biochem. 20,
535 (1971).
77. M. C. Zapponi, G . Ferri, B. G . Forcina, and S. Ronchi, FEBS (Fed. Eur.
Biochem. Soc.) Lett. 31,287 (1973.)
78. J. Bridgen rind J. I. Harris, Znt. Congr. Biochem, 9th, 1973 Abstract 2e.1, p.
59 (1973).
ity (79). Tyrosine-46 was found to be the most reactive residue in the
native holoensyme, but significant specificity was attained only in the
presence of limiting amounts of iodine. Two other residues, Tyr-39 and
Tyr-42, were moderately reactive and several of the other nine tyrosines
in the subunit were also found to react, albeit a t appreciably slower rates.
Iodination of Tyr-46 does not cause inactivation, and its special reactiv-
ity is entirely compatible with its exposed position on the outside of helix
aC in the. three-dimensional structure of the tetramer. Tyrosine-39 and
Tyr-42 are in the same helical segment but are partially shielded by in-
teractions with neighboring subunits. Tyrosine-46, followed by Tyr-39
and Tyr-42, were also found to be the most susceptible to iodination in
the pig holo- and yeast enzymes (79) again indicating that the three-
dimensional structure of GAPDH has been highly conserved. With pig
spoensyme, on the other hand, Libor and Elodi (80) found Tyr-137 and
Tyr-252 [which Thomas and Harris (79) found to be among the least
reactive in the holoenzyme] to be more reactive than Tyr-46, Tyr-42,
and Tyr-39, a result that is difficult to reconcile with the three-dimen-
sional structure. The extent to which the reactivities of tyrosines can
be correlated with their positions in the native three-dimensional struc-
ture is however difficult to assess with complete certainty owing to possi-
ble effects on the structure of side reactions such as oxidation of thiol
groups (cf. Section II,B,4,a) and iodination of histidine residues. Thus,
dependence of specificity on reaction time and reagent concentration
could be the consequence of slow but irreversible conformational changes
within the tetrameric structure, and in this connection it should be noted
that apoensyme is less stable than holoeneyme in the presence of iodine
a t alkaline pH (79).
d. Histidine Residues. Although histidine has been implicated in the
catalytic mechanism of GAPDH (see, for example, 81-83, and Section
III,B,l), there is, perhaps surprisingly, no convincing chemical evidence
for the direct involvement of a specific histidine in the active site. Thus,
for example, Moore and Fenselau (84) could not link Cys-149 to any
neighboring histidine residue in the rabbit enzyme with the bifunctional
dibromoacetone; Allen and Harris (86) were also unable to achieve spe-
cific labeling of an essential histidine in the B. stearothermophilus en-
79. J. 0. Thomas and J. I. Harris, BJ 119, 307 (1970).

80. S. Libor and P. Elodi, Em-. J. Biochem. 12, 336 (1970).
81. E. J. Olson and J. H. Park, JBC 239, 2316 (1964).
82. P. Friedrich, L. PolgLr, and G . Szabolcsi, Acta Ph,gsiol. 25, 217 (1964)
84. J. Moore. Jr. and A. Fenselau, Biochemistry 11, 3753 (1972).
85. G. A. Allen and J. I. Harris, unpublished results.
zyme with either 3-bromoacetypyridyine or bromopyruvate [reagents

known to react selectively with His-195 in LDH (86, 87)’J.Iodination
of His-50 had no effect on activity (86), and likewise Ovbdi and Keleti
(88) showed that four of the eleven histidines in the pig enzyme were
not essential for activity. The first positive evidence for histidine in the
active site came from the photooxidation studies of Park and colleagues
(89,90). These studies appeared to show that inactivation of the rabbit
muscle enzyme was associated with the specific destruction of His-38.
Significantly, however, this particular histidine does not occur in the
lobster and yeast enzymes, and its involvement in the active site was
therefore considered to be unlikely ( 3 6 ) .The three-dimensional structure
(47) confirms that an invariant histidine (His-176, Table 11) is present
in the active site within hydrogen bonding distance of Cys-149. Moreover,
residue 38 (glutamic acid in lobster GAPDH) was shown to be on the
surface of the tetramer and a considerable distance away from the active
site. A histidine in this position would be expected to be particularly sus-
ceptible to photooxidation, and it seems likely therefore that an alterna-
tive explanation must be sought for the observed effects of photooxidation
(89,90) on the activity of the rabbit enzyme.
5. Dissociation and Hybridization

Early discrepancies in determinations of the molecular weight of rabbit
muscle and yeast GAPDH’s (91-93) were resolved by the definitive sedi-
mentation equilibrium studies of Harrington and Karr (32) in conjunc-
tion with the chemical investigations of Harris and Perham ( 3 1 ) . These
established that the enzyme is a tetramer with a molecular weight of
146,000 and that the 7.5 S tetramer dissociates to an unfolded 1.85
monomer in 5 M guanidine. Loss of activity below pH 5 and above pH
11, and in 8 M urea, is also accompanied by dissociation and subsequent
unfolding of the subunits as evidenced by sedimentation velocity, light
86. C. Woenckhaus, J. Berghauser, and G. Pfleiderer, Hoppe-Seyler’s Z. Physwl.

Chem. 350,473 (1909).
87. M. G. Rossmann, M. J. Adams, M. Ruehner, G. C. Ford, M. L. Hackert,
P. J. Lentz, A. McPherson, R. Schevitz, and I. E. Smiley, Cold Spring Harbor
Symp. Quant.Biol. 36, 179 (1971).
88. J. Ov&diand T. Keleti, Acta Biochim. Biophys. 4, 365 (1909).
89. J. C. Bond, S. H. Francis, and J. H. Park, JBC 245, 1041 (1970).
90.S. H. Francis, B. P. Meriwether, and J. H. Park, Biochemistry 12, 346 (1973).
91. J. B. Fox, Jr. and W. B. Dandliker, JBC 218,53 (1950).
92. J. F. Taylor and C. Lowry, BBA 20, 109 (1950).
93. H-G. Elias, A. Garbe, and W. Lamprecht, Hoppe-Seyler‘s Z. Physbl. Chem.
319, 22 (1900).
scattering, difference spectroscopy, and ORD measurements (94-98).

High ionic strength also appears to promote dissociation (98-103), and
on the basis of these observations it may be concluded that electrostatic
interactions and hydrogen bonds are important in maintaining the tetra-
meric structure. There is evidence, too, that the rat muscle enzyme is
dissociated a t low temperature with release of hydrophobic residues (24);
that ATP promoted dissociation of yeast and rabbit muscle enzymes is
enhanced a t Oo (104); and that detergents can cause dissociation without
extensive unfolding of the subunits (67,106), showing that hydrophobic
interactions also play a part in stabilizing the tetrameric structure.
In all the above-mentioned studies, only the tetramer has catalytic
activity. The only claims for an active dimer (106,107)are not supported
by satisfactory experimental evidence. Deal and co-workers (104, 108)
have, on the other hand, presented extensive studies of the ATP-induced
dissociation of GPD to inactive dimers and monomers a t low tempera-
tures. Furthermore, these subunits display very little unfolding, which
has been taken to imply that dissociation is the major factor in the activ-
ity loss. These results were confirmed (24) with the rat skeletal muscle
enzyme, which dissociates a t Oo to inactive dimers in the absence of
ATP. In addition, the activity transport studies of Hoagland and Teller
(109) have given strong evidence that only the tetrameric form is active
and that the presence of all three substrates promotes tetramer formation.
It was also shown that rabbit muscle enzyme exists in a dimer-tetramer
equilibrium in dilute aqueous solution a t 5 O , with an association constant
94. P. Elodi, G. Jecsai, and A. Morolovsky, Acta Physiol. 17, 165 (1960).
95. P. Elodi and S. Libor, in “Pyridine Nucleotide-Dependent Dehydrogenases”
(H. Sund, ed.), p. 175. Springer-Verlag, Berlin and New York, 1970.
96. W. C. Deal and W. H. Holleman, Fed. Proc., Fed. Amer. SOC.Ezp. Biol. 23,
983 (abstr.) (1964).
97. Y. Shibata and M. J. Kronman, ABB 118, 410 (1967).
98. R. Jaenicke, D. Schmid, and S. Knof, Biochemistry 7,919 (1968).
99. E. A. Meighen and H. K. Schachman, Biochemistry 9, 1177 (1970).
100. R. Jaenicke, in “Pyridine Nucleotide-Dependent Dehydrogenases” (H. Sund,
101. K. Kirschner and I. Schuster, in “Pyridine Nucleotide-Dependent Dehydro-
genases” (H. Sund, ed.), p. 217. Springer-Verlag, Berlin and New York, 1970.
102. G. M. Spotorno and M. R. Hollaway, Nature (London)226,756 (1970).
103. K. Suzuki and J. I. Harris, J. Bwchem. (Tokyo) 77,587 (1975).
104. S. M. Constantinides and W. C. Deal, Jr., JBC 244, 5695 (1969).
105. I. A. Bolotina, D. S. Markovich, M. V. Volkstein, and P. Zavodsky, BBA
132, 280 (1967).
106. A. I. Agatova, Biokhimivn 32,915 (1967).
107. J. Ovadi, M. Telegdi, J. Batke, and T. Keleti, Eur. J. Biochem. 22, 430 (1971).
108. S. T. Yang and W. C. Deal, Jr., Biochemistry 8,2806 (1969).
109. V. D. Hoagland and D. C. Teller, Biochemistry 8, 594 (1969).
of 2 X lo6 liters/mole. Removal of bound NAD’ shifts this equilibrium

toward dissociation (KaS8apoenzyme = 0.4 X lo6 liters/mole) . Gener-
ally, similar conclusions were reached by others (1212, 99, 110-1112).
Other evidence that a dimer-tetramer equilibrium exists is provided
by hybridization studies with GAPDH’s from different sources. Thus,
Kirschner and Schuster (101), Spotorno and Hollaway (log),and Stall-
cup and Koshland (113)were able to obtain Y,R, hybrids of yeast and
rabbit muscle enzymes a t high enzyme concentrations and physiological
pH suggesting that interchange of dimeric subunits can occur. Moreover,
NAD’ was shown to prevent hybridizstion, which suggests that the coen-
zyme promotes tetramer formation. Lebherz et al (114) found that ATP-
induced hybridization of trout GPD’s was likewise blocked by NAD’ or
NADH. Using conditions of high ionic strength, Meighen and Schachman
(99) were able to obtain small amounts of SRs and SsR hybrids of
native and succinylated rabbit muscle enzyme, which implies that mono-
mer formation had also occurred. Suzuki and Harris (103)used 3 M NaCl
to hybridize a number of different GAPDH’s including the rabbit, pig,
lobster, yeast, and E . coli enzymes. As shown in Fig. 10 different pairs
of electrophoretically distinct enzymes gave rise to five-membered hybrid
sets indicating that dissociation to monomers had occurred. The relative
amounts of dimer and monomer formed by dissociation of tetramers com-
prising pairs of isologous associations will be determined by the tetramer-
dimer and dimer-monomer dissociation constants. In experiments in-
volving yeast enzyme the dimeric form appears to predominate while
with the rabbit, pig, lobster, and E. coli enzymes dimers readily dis-
sociate to monomers. The relative ease with which enzymes from such
phylogenetically different sources are able to form hybrid tetramers is
indicative of a high degree of conservation of tertiary and quaternary
structure among GAPDH’s. Moreover, the successful hybridization (103)
of pig enzyme with lobster enzyme, which had been inactivated by car-
boxymethylation of Cys-149 in all four subunits, to form four enzymically
active protein bands, including the tetramer that contained only one pig
subunit, showed that individual subunits are able to express their activity
independently even within hybrid tetramers formed with inactive chem-
ically modified subunits of another species.
110. L. A. Fahien, JBC 241, 4115 (1966).

111. S. Lakatos, P. Zavodaky, and P. Elodi, FEBS (Fed. Eur. Biochem. Soc. Lett.
20, 324 (1972).
112. G. D. Smith and H. K. Schachman, Biochemistry 12, 3789 (1973).
113. W. B. Stallcup and D. E. Koshland, Jr., I M B 80,41 (1973).
114. H. G. Lebhera, B. Savage, and E. Abacherli, Nature (London) New Biol.
245, 269 (1973).
1.
GLYCERALDEHYDE-%PHOSPHATE DEHYDROGENASE
FIG.10. Hybridization of GAPDH’s: (a) rabbit (R) and lobster (L) muscle, pig (P) and lobster (L)mus-
cle; (b) rabbit (R) and yeast (Y), pig (P) and yeast (Y). The hybrid bands were separated by electropho-
resis on cellulose acetate in 50 mM phosphate buffer, pH 7.0, and revealed in (a) by protein staining and in
(b) by activity staining (cf 103).
27
28 J . IEUAN HARRIS AND MICHAEL WATERS
111. Catalytic Properties
A. STUDIES
OF PYRIDINE
NUCLEDTIDE
BINDING
The binding of NAD+ and of coenzyme analogs to GAPDH has been
studied extensively, and a detailed discussion of earlier work has been
given by Colowick et al. (13). Racker and Krimsky (64) were the first
to show that binding of NAD’ to the apoenzyme produces a unique yellow
complex with ti broad absorption maximum around 365 nm. As normally
isolated, GAPDH’s contain from 3 to 4 moles of tightly bound NAD’
per mole of tetramer, although there are exceptions such as the yeast
and sturgeon muscle enzymes which are isolated with little or no bound
coenzyme (cf. 16).The “Racker band” is either diminished or abolished
by oxidation or alkylation of the active site thiol 149; and by acylation
with the substrate (DPGA) or substrate analogs such as acetyl phos-
phate (cf. 116-1173,Modification of the thiol group results in a decrease
in the binding constant for NAD’, although the cooperativity of binding
is maintained (118).Moreover, no major change in conformation of the
lobster muscle enzyme could be detected a t 6 A resolution (61) following
carboxymethylation of Cys-149. Crystals of carboxymethylated enzyme
were completely isomorphous with those of native enzyme and could be
distinguished only by the conspicuous lack of yellow color implying that
the loss of Racker band absorbance is an effect that is restricted to the
active site. In this connection it is of interest that NAD’ protects the
active site thiol against alkylation by most alkylating agents (119) with,
however, the notable exception of iodoacetic acid (cf. 117). Kosower
(120)suggested that the Racker band resulted from a charge-transfer
interaction between the nicotinamide ring and an electron donor in the
enzyme. The nucleophilic nature of Cys-149 pointed toward a thiolate
anion as the likely electron donor (117,121, 122), although it was also
suggested (123,124) on the basis of spectral studies with analogs that
115. E. Racker and I. Krimsky, JBC 198, 731 (1952).

116. D. R. Trentham, BJ 122,59 (1971a).
117. D. R. Trentham, BJ 109,603 (1968).
118. W. Boers and E. C. Slater, BBA 315, 272 (1973).
119. B. Eisele and K. Wallenfels, in “Pyridine Nucleotide-Dependent Dehydroge-
nases” (H. Sund, ed.), p. 91. Springer-Verlag, Berlin and New York, 1970.
120. E. M. Kosower, JACS 78,3497 (1956).
121. L. Boross and E. Cseke, Acta Biochim. Biophys. 2, 47 (1967).
122. E. Cseke and L. Boross, Acta Biochim. Biophys. 5,385 (1970).
123. G. Cilento and P. Tedeschi, JBC 238, 907 (1961).
124. S.Shifrin, BBA 81,205 (1964).
tryptophan might act as the donor in a charge-transfer complex with

the nicotinamide. However, there is no tryptophan in the vicinity of the
nicotinamide ring in the tertiary structure (47) whereas the thiol group
of Cys-149 is ideally placed to form either a covalent bond or a charge-
transfer complex with the NAD’. Strong interactions of this type between
the essential thiol and the nicotinamide could adequately account both
for the tight binding between NAD+ and the enzyme and for the loss
of the Racker band upon modification of the thiol group. Moreover, it
seems probable that this interaction would stabilize the thiolate anion.
The fact that 260 nm excitation of the enzyme-NADH complex led
to NADH emission a t 460 nm, as well as the apparent lack of spectral
shift on NADH binding, led Velick (126)and others (IS, 126) to suggest
that the nucleotide was bound to GAPDH in a folded conformation. The
basis for this suggestion was the finding that in aqueous solutions, NADH
emission a t 460 nm can be excited by light a t both 260 and 340 nm.
This led to the idea that adenine and nicotinamide rings were coupled
in parallel in a hairpin or folded conformation to allow for transfer of
excitation energy between the rings. Loss of 260 nm excitation maximum
and the accompanying spectral shift that occurs when NADH binds to
LDH (127) were therefore taken to indicate an unfolding of the nucleo-
tide into its open form. In addition, NMR studies have shown that the
pyridine nucleotides exist predominantly in the folded conformation in
solution (128) and that spectral shifts occur on binding to GAPDH (129)
that are opposite to those seen with other dehydrogenases in which the
coenzyme is known to bind in the open form. These interpretations are
in conflict with the X-ray crystallographic model which shows beyond
doubt that the coenzyme in GAPDH is bound, as in LDH, in the open
form. It is possible, however, that the 180° rotation about the glycosidic
bond linking the nicotamide ring to the ribose in NAD+ bound to GAPDH
may account for the observed differences in the NMR spectra, and in
this connection it is of interest that the spectral shifts in NADH absorp-
tion that occur on binding to B-specific dehydrogenases were also found
to occur with GAPDH (130). It seems clear therefore that NAD’ is
bound in the open form with the nicotinamide ring close to the reactive
125. S. F. Velick, in “Light and Life” (W. D. McElroy and B. Glass, eds.), p.
108. Johns Hopkins Press, Baltimore, Maryland, 1961.
1%. D. Eby and M. E . Kirtley, Biochemistry 10,2677 (1971).
127. S.F. Velick, JBC 233, 1455 (1958).
128. N. J. Oppenheimer, L. J. Arnold, and N. 0. Kaplan, Proc. Nut. Acud. Sn’.
U.S. 68, 3200 (1971).
129. C. Y. Lee, R. D. Eichner, and N. 0. Kaplan, Proc. Nut. Acud. Sn’. U. S.
70, 1593 (1973).
130. H. F. Fisher, D. L. Adija, and D. G. Cross, Biochemistry 8, 4424 (1969).
thiol as demanded by the observation (131) that a hydride ion is trans-

ferred directly from the substrate to the nicotinamide ring.
Substrate analog and kinetic studies gave some insight into the other
groups involved in nucleotide binding. Thus, of the many nicotinamide
ring analogs tested, only the acetyl pyridine, thionicotinamide, nicotinyl-
hydroxamic acid, and nicotinic acid hydrazide analogs of NAD' can act
as substrates, albeit with higher K , values (132).
The activity of the acetylpyridine analog shows the the amide group
of nicotinamide is not essential. The carbonyl group is important since,
as is shown in the crystallographic model, it stabilizes the B conformation
of the coenzyme by forming a hydrogen bond with Asn-313. It is of inter-
est that the pyridine 3-aldehyde analog which is inhibitory .does not show
Racker band absorption whereas the active acetyl pyridine analog does
(133).Both deamino NAD' and acetyl pyridine NAD' retain the negative
cooperativity of binding seen with NAD+ (126).
Of the adenine analogs tested, only the 6-deamino analog exhibits ac-
tivity, again with a high K , (126). From the model, it is likely that this
amino group would interact with the highly conserved residue gluta-
mate-76. Substitution of a phosphate on the 2 position of the ribose
(NADP') or removal of the hydroxyl group (deoxyadenosine) abolishes
activity and binding ability (108) in agreement with the binding of the
2-hydroxy group by Asp-32 seen in the model. Moreover, competitive
inhibition by hydrophobic probes for the NAD+-binding site (24) may
be the result of competition for the hydrophobic pocket bounded by
Phe-34 and Phe-99, which contains the adenine moiety.
The overall picture that emerges from these results is in general agree-
ment with the X-ray crystallographic evidence and shows that the bind-
ing interactions between the protein and the NAD' are well distributed
over the nicotinamide, pyrophosphate, ribose, and adenine parts.
Moreover, strong binding requires all these interactions to be effective
simultaneously. The fact that there is an adenine binding pocket that
is distinct from the catalytic site is in agreement with the binding model
proposed by Yang and Deal (108) and effectively disproves the closed
NAD+ model (126,126).
1. Cooperativity of NAD' Binding
The early studies of Velick et al. ( l S 4 ) first delineated the two aspects
of NAD+ binding that have provided the basis for subsequent work. These
131. W. S. Allison, M. J. Connors, and D. J. Parker, BBRC 34, 503 (1969).
132. B. M. Anderson and N. 0. Kaplan, JBC 234, 1226 (1959).
133. N. 0. Kaplan, M. M. Ciotti, and F. E. Stolzenbach, ABB 69, 441 (1957).
134. S. F. Velick, J. E. Hayes, and J. Harting, JBC 203, 527 (1953).
workers found that the dissociation constant for NAD’ changed as satu-
ration of the enzyme was approached, and that binding of NAD‘ to the
apoenzyme produced different solubility and stability characteristics,
which led them to suggest that “NAD’ binding is associated with changes
in the configurational state of the protein.’’ A number of subsequent stud-
ies showed that NAD’ binding produces changes in viscosity (136),and
in optical rotatory dispersion (106,136, 137) and electron paramagnetic
resonance spectra (138).NAD’ binding also alters the rate of exchange
of the peptide bond amide groups with deuterium oxide and produces
a small increase in sedimentation and diffusion parameters (66).
The large difference between kinetically and spectrally determined dis-
sociation constants (cf. la) and conflicting reports about the number
(110,139) and independence of NAD+-binding sites (140,1.41) led to
a period of confusion which was largely resolved by the finding of positive
cooperativity of NAD’ binding in the yeast enzyme (149)and negative
cooperativity of binding in the muscle enzyme (143,144).
a. The Yeast Enzyme-A Concerted Binding Mechanism. Using a
combination of equilibrium and rapid kinetic techniques, Kirschner and
co-workers (101,142, 146-147) have shown that the binding of NAD’
to the yeast enzyme can be described by the concerted model of Monod
et al. (148).According to this proposal, the enzyme exists in two sym-
metrical tetrameric forms, R and T, and a relatively slow T to R conver-
sion occurs on addition of NAD’ to the apoenzyme which exists in about
98% T form. The T form is enzymically inactive and possesses highly
reactive thiol groups. The R form, on the other hand, has a higher Racker
band absorbance than the T form and a greater affinity for NAD’ (Kd
lo-* M compared to 2 x M ) . The isomerization can therefore be
clearly distinguished both in terms of a time-dependent interconversion
process and by the separate characteristics of the two forms. The coopera-
135. P. Elodi and G. Szabolczi, Nature (London) 184,56 (1959).
136. B. H. Havsteen, Acta Chem. Scand. 23, 2193 (1969).
137. I. Listowsky, C. S. Furfine, J. J. Betheil, and S. Englard, JBC 240, 4253 (1965).
138. W. Balthasar, Eur. J . Biochem. 22, 158 (1971).
139. A. L. Murdock and 0. J. Koeppe, JBC 239, 1983 (1964).
140. A. Stockell, JBC 234, 1286 (1959).
141. B. Chance and J. H. Park, JBC 242, 5093 (1967).
142. K. Kirschner, M. Eigen, R. Bittmann, and B. Voigt, Proc. Nat. Acad. Sci.
U . S. 56, 1661 (1966).
143. A. Conway and D. E. Koshland, Jr., Biochemistry 7 , 4011 (1968).
144. J. J. M. De Vijlder and E. C. Slater, BBA 167, 23 (1968).
145. K. Kirschner, in “The Regulation of Enzyme Activity and Allosteric Interac-
tions” (E. Kvamme and A. Pihl, eds.), p. 39. Academic Press, New York, 1968.
146. K. Kirschner, J M B 58, 51 (1971).
147. K. Kirschner, E. Gallego, I. Schuster, and D. Goodall, J M B 58, 29 (1971).
148. J. Monod, J. Wyman, and J.-P. Changeaux, J M B 12, 88 (1965).
tivity is not readily detected a t 20° and pH 7.4, but increases as the
temperature or pH is raised to 40° and pH 8.5.Even a t the higher tem-
perature and pH, each form of the tetramer displays equivalent and inde-
pendent binding sites, as predicted by the concerted model; and there
is no evidence for significant quantities of the hybrid forms which would
be predicted by the sequential model of Koshland et al. (149).
Later studies (160) showed that the binding of NADH is hyperbolic
in contrast to that of NAD’. Thus, NADH binds equally well to both R
and T forms, with the lower affinity characteristic of NAD’ binding to
the T form (Kd NADH 2 x M).These observations have been
rationalized by postulating that strong interaction with the adenine and
pyridinium carboxamide sites is possible only in the enzymically active
R form, and since reduced NAD+ does not possess the quaternary pyridi-
nium ring, it cannot stabilize the R form on binding. Thus, the enzyme
would be active in the reverse reaction only in the presence of NAD’,
which would convert the enzyme to the R form, as has been observed
(161; but see Section III,B for an alternative hypothesis). In this connec-
tion it should be noted that bound NADH fluorescence is enhanced in
the yeast enzyme (160)and quenched in the muscle enzyme (197),which
would imply a difference in the nucleotide binding site, in keeping with
the difference in cooperativity characteristics.
Support for a concerted model for the yeast enzyme has come from
X-ray small angle scattering experiments (169) as well as from hydro-
dynamic and optical rotation studies (165, 164). A volume contraction
of about 5% occurs on binding of NAD’ to the apoenzyme, presumably
related to tightening of the tetramer and expulsion of water molecules.
The relation between NAD’ bound (R) and change of volume (Y) was
hyperbolic, in accord with the concerted model. It was later shown (166)
from buoyant density and preferential hydration studies that water is
indeed excluded from the yeast enzyme on binding to NAD’, such that
a volume contraction of about 6% occurs. Furthermore, fluorimetric and
calorimetric titrations over the range 5O-4Oo showed independence of
149. D. E. Koshland, G. Nkmkthy, and D. Filmer, Biochemistry 5, 365 (1966).

150. G. von Ellenrieder, K. Kirschner, and I. Schuster, Eur. J . Biochem. 26, 220
(1972).
151. J. J. M. De Vijlder and B . J . M. Harmsen, BBA 178, 434 (1969).
152. H. Durchschlag, G. Puchwein, 0. Kratky, I. Schuster, and K. Kirschner, Eur.
J . Biochem. 19,9 (1971).
153. R. Jaenicke and W. B. Gratzer, Eur. J . Biochem. 10,158 (1969).
154. R. Jaenicke, in “Pyridine Nucleotide-Dependent Dehydrogenases” (H. Sund,
ed.) p. 209. Springer-Verlag, Berlin and New York, 1970.
155. D. L. Sloan and S. F. Velick, JBC 248, 6419 (1973).
NAD' binding a t pH 7.4 (166) and positive cooperativity a t pH 8.5

(166), in agreement with earlier studies (101). Eisele and Wallenfels
(119) have also provided support for the concerted model with studies
on the rate of stereoselective inhibition of the yeast enzyme by the anti-
podes of u-iodopropionic acid and a-iodopropionamide a t varying NAD'
concentrations. I n similar experiments the muscle enzyme showed nega-
tive cooperativity.
b. The Muscle Enzyme-A Complex Sequential Binding Mechanism.
Conway and Koshland (143) showed, on the basis of equilibrium binding
studies, that each molecule of NAD+ binds with decreasing affinity (Kl
lo-", K 2 K s 3 X lo-', and K , 2.6 x M) to the tetrameric rabbit
muscle enzyme. These binding constants cannot be described by the sta-
tistical relationship predicted from the concerted model, where each mole-
cule binds to an equivalent site on the R state tetramer, and can only
be accounted for by introducing terms for negative interaction between
the subunits (149) implying the existence of hybrid states between the
R and T forms. Here, in contrast to the yeast enzyme, symmetry forces
are not as strong as ligand-induced conformational distortions; thus,
symmetry is not maintained. Slater and colleagues (14.4, 161, 167-159)
obtained independent evidence for negative cooperativity of NAD+ bind-
ing to both the rabbit and lobster muscle enzymes by means of equilib-
rium dialysis and ultracentrifugation as well as by stopped-flow, fluori-
metric, optical rotatory dispersion, and circular dichroism methods.
Quantitative estimation of the Racker band indicated that binding of
the fourth NAD' did not appear to contribute to the absorbance a t 365
nm. It has since been shown (160-162) that this effect is produced
either with enzyme in which the active site thiol is partly oxidized or
by an insufficient range of titration with NAD'.
Heterogeneity in NAD+-binding affinity, that was in addition indicative
of two pairs of equivalent and independent sites, was found for the
sturgeon muscle enzyme by Seydoux et al. (160). Subsequently, Allen
156. S. F. Velick, J. P. Baggott, and J. M. Sturtevant, Biochemistry 10,779 (1971).

157. J. J. M. De Vijlder, W. Boers, A. G. Hilvers, B . J . Harmsen, and E. C. Slater,
in "Pyridine Nucleotide Dependent Dehydrogenases" (H. Sund, ed.), p. 233.
Springer-Verlag, Berlin and New York, 1970.
158. W. Boers, C. Oosthuizen, and E. C. Slater, BBA 250, 35 (1971).
159. J. J. M. De Vijlder, A. G. Hilvers, J. M. J. Van Lis, and E. C I Slater, BBA
191, 221 (1969).
160. F. Seydoux, S. Bernhard, 0. Pfenninger, M. Payne, and 0. P. Malhotra, Bio-
chemistry 12,4290 (1973).
161. B. D. Peczon and H. 0. Spivey, Biochemistry 11,2209 (1972).
162. N. C. Price and G. K. Radda, BBA 235,27 (1971).
and Harris (163) found negative cooperativity of NAD' binding to apply

in the case of enzyme from a prokaryote B . stearothermophilus, showing
that the phenomenon is not confined to eukaryotic cells. Moreover, as in
the case of the sturgeon enzyme, the binding isotherm appeared to reflect
the presence of two distinct pairs of equivalent binding sites. Yeast
GAPDH is thus so far unique in possessing a concerted mechanism of
coenzyme binding.
I n accord with the complex sequential model, Koshland and colleagues
(143,164) have reported that binding of only the first molecule of NAD'
influences the reactivity of the active site Cys-149 in all four subunits.
The major structural changes in the tetramer, as evidenced by thiol reac-
tivity, appear therefore to have occurred in response to the binding of
the first molecule of NAD'. This phenomenon has been observed with
ORD (137) and other (165) optical techniques, as well as by difference
sedimentation ( I l a ) , calorimetric (156), stopped-flow (144), and small
angle X-ray scattering (166) studies. The intrinsic catalytic activity of
each of the four sites remains constant with addition of each mole of
NAD' when measured with substrate analogs (164), indicating that the
catalytic rate is directly proportional to the amount of bound NAD' and
that it is not affected by conformational changes accompanying NAD'
binding to neighboring subunits. This observation suggested a possible
physiological role for negative cooperativity in modulating the effects
of variations in cellular NAD+ levels on the activity of the enzyme
through changes in its affinity for NAD', although it has since (166)
been pointed out that the high and relatively invariant levels of NAD'
in the cell renders this possibility unlikely.
Schlessinger and Levitzki (167)have probed further into the mecha-
nism of negative cooperativity by using a variety of spectroscopic meth-
ods to study the interactions of rabbit muscle GAPDH with both NAD'
and a fluorescent derivative of the coenzyme. Their results give added
support to the concept that the structural transitions that occur when
NAD' binds to the enzyme are sequential and that the largest structural
change occurs in binding the first molecule of NAD'. I n addition, they
show that whereas each NAD' molecule induces the same conformational
change a t the nicotinamide subsite there are distinct and progressive
structural changes a t the adenine subsite which are transmitted to ad-
j acent vacant subunits thereby accounting for their decreased affinity for
163. G. A. Allen and J. I. Harris, Biochem. J. 151, 747 (1975).
164. J. Teipel and D. E. Koshland, Jr., BBA 198, 183 (1970).
165. W. Bloch, R. A. MacQuarrie, and S. A. Bernhard, JBC 246, 780 (1971).
166. I. Simon, Eur. J. Biochem. 30, 184 (1972).
167. J. Schleasinger and A. Levitzki, J M B 82,547 (1974).
NAD’. These results offer a molecular explanation for the negative coop-
erativity in coenzyme binding and a t the same time for the finding that
the intrinsic catalytic activity of each site is independent of NAD’ satu-
ration (164). It was further suggested that ligands interacting a t the
adenine subsite of the NAD’ binding site induce the “half of the sites
reactivity” effect that has been observed with a number of alkylating
reagents (see Section 1111A,2) and that both negative cooperativity in
coenzyme binding and half-site reactivity result from ligand-induced
conformational changes in an a priori symmetrical tetramer.
2. The Preexisting Asymmetry Model
Many subunit enzymes show the phenomenon of “half-site” reactivity ;
that is, the reaction with a substrate or substrate analog shows a stoichio-
metry equal to half the number of chemically identical subunits. A possi-
ble explanation which has been proposed for the half-site reactivity of
the enzyme cytidine triosephosphate synthetase with a substrate analog
(168) is that reaction with one substrate molecule induces a change in
an adjacent subunit such that a second substrate molecule is prevented
from reacting. Analogous studies with GAPDH showed that whereas the
four active site sulfhydryl groups per tetrameric molecule react equiva-
lently with alkylating reagents such as iodoacetic acid or iodoacetamide
only two of the sites react with the pseudosubstrate p- (2-furyl)acrylolyl
phosphate (FAP) unless forcing conditions are used to achieve higher
stoichiometry. These observations considered in conjunction with the
original crystallographic data of Watson and Banaszak (43) led Mal-
hotra and Bernhard (169) to postulate that the chemically identical
subunits in GAPDH are arranged asymmetrically and that the half-site
reactivity also applied to the normal physiological substrate, diphospho-
glyceric acid. Later work (69,160) has however shown that all four sites
are reactive toward DPGA. One must therefore conclude that half-site
reactivity is induced by certain ligands but not by others, and several
possible explanations have been put forward to account for the phenome-
non (cf. 168). In the case of GAPDH these may be summarized as
follows:
1. The tetramer contains subunits of different primary structure.
2. The active sites within the tetramer overlap so that binding of an
acyl group to one subunit prevents acylation of the adjacent subunit.
3. The four subunits in the tetramer are identical, but acylation of
168. A. Levitrki, W. B. Stallcup, and D. E. Koshland, Jr., Biochemistry 10, 3371

(1971).
169. 0. P.Malhotra and S. A. Bernhard, JBC 243, 1243 (1968).
one subunit induces conformational changes in the adjacent subunit

which lowers the reactivity of that subunit toward further acylation.
4. Identical polypeptide chains form an asymmetric dimer (sad in
which the conformation of the two subunits is not identical and the tetra-
mer is a dimer of asymmetric dimers (ma1) .
5. Half-site stoichiometry of acylation is induced by bound coenzyme
blocking acylation a t the R-axis-related subunit.
I n considering these possible explanations, model 1 is excluded by the
amino acid sequence results (cf. 36, 170) and model 5 is excluded by
the finding of half-site reactivity for the muscle apoenzyme (171)and
for the yeast apoensyme (113,172).Bernhard and co-workers (160, 173,
174) maintained that models 2 and 3 are excluded for several reasons.
First, a number of alkylating agents, including DTNB which is consider-
ably larger than FAP, give full-site reactivity. This tends to exclude the
overlapping active site model 2. Second, iodoacetate and iodoacetamide,
in addition to giving full-site reactivity, react independently a t each
site, which is not in accord with the prediction of model 3. Moreover,
the two unreacted sites in the diacyl enzyme can be alkylated with iodo-
acetate without affecting the NAD+-induced acceleration of deacylation
and NAD+-induced change in the spectrum of the di (2-furylacryloyl)
enzyme, implying that there is little interaction between adjacent sites
of the type expected in a cooperativity model.
There remains model 4, and MacQuarrie and Bernhard (176)have uti-
lized the full-site reactivity by iodoacetamide and half-site reactivity by
FAP to provide support for this model. Thus, di (2-furylacryloyl) ensyme
was prepared, and the two remaining sites were blocked with iodoacetate.
Acyl groups were then removed from this derivative by arsenolysis, and
the resulting dialkyl enzyme was tested for stoichiometry with FAP. Only
one acyl group could be incorporated into the dialkyl enzyme. This result
cannot be explained in terms of an induced asymmetry model, and indeed,
can only be explained by a preexisting asymmetry model if there is a
subunit rearrangement. I n addition, alkylation of the enzyme with vary-
ing quantities of iodoacetate, followed by acylation of these derivatives
with FAP, showed a 2: 1 ratio of alkylation to acylation, independent
170. J. I. Harris, in “Pyridine Nucleotide-Dependent Dehydrogenases” (H.Sund,

171. A. Levitzki, BBRC 54, 889 (1973).
172. W. B. Stallcup and D. E. Koshland, Jr., J M B 80,77 (1973).
173. R. A. MacQuarrie and S. A. Bernhard, Biochemistry 10, 2456 (1971).
174. 0. P. Malhotra and 5. A. Bernhard, Proc. N a t . Acad. Sci. U. S. 70, 2077
( 1973).
175. R. A. MacQuarrie and S. A. Bernhard, J M B 55,181 (1971).
of the degree of prior alkylation. This linear relationship is not predicted

by an induced asymmetry model or an overlapping active site niodel but
is consistent with the preexisting asymmetry model (174).
Similar studies with alkylating and acylating reagents carried out with
yeast GADPH by Stallcup and Koshland (113,172, 176) have shown
that, as in the case of NAD+ binding, the fungal enzyme again differs
from the muscle enzyme. Thus, for example, yeast enzyme showed only
half-site reactivity toward iodoacetic acid and iodoacetamide (confirming
earlier observations with the bifunctional reagent p-fluoro-rn,m'-dinitro-
phenylsulfone (177)) . In addition, and in marked contrast to the behav-
ior of muscle enzyme (117, 176), a nonlinear relationship was noted be-
tween extent of alkylation and loss of enzymic activity. Similar results
were obtained with a number of other alkylating and acylating agents
indicating that the structure of the modifying reagent has only a second-
ary influence. The half of the sites' effect was therefore considered to
result from ligand-induced negatively cooperative changes in the enzyme
tetramer. This view was strengthened by the finding that the slow rate
of alkylation of the third site in a dialkyl enzyme is the same for a re-
ordered enzyme prepared according to MacQuarrie and Bernhard (176)
as it is for a normal randomly alkylated enzyme. This showed, in accord
with the negative cooperativity hypothesis, that the reactivity of the
third site is a function of the number of alkyl groups bound rather than
the number of available reactive sites. Moreover, relative values of two
rapidly reacting groups, a third more slowly reacting, and a fourth very
slowly reacting group, would necessitate three different subunit conforma-
tions in a preexisting asymmetry model which is therefore considered to
be unlikely to apply in the case of the yeast enzyme.
In an extension of these studies to the natural substrate, Stallcup and
Koshland (172) showed further that acylation of the first two sites with
DPGA decreases the rate of acylation of the third site, and that
DPGA (or other acylating agents) attached to the first two sites increases
the rate of deacylation of the third site through subunit interactions.
These additional observations provide further support for the concept
of negative cooperativity as a cause for half-site reactivity. They could
also explain many of the results that have been obtained with muscle
enzyme where apparent half-site stoichiometry might have resulted from
rapid hydrolysis of the acyl enzyme (83).
In contrast to the reactive thiol (Cys-149) groups the Lys-183 residues
of yeast GAPDH were found to react independently with acylating and
alkylating reagents (17 2 ) . Significantly, however, modification of the
176. W. B. Stallcup and D. E. Koshland, Jr., J M B 80,63 (1973).
177. D. Givol, FEBS ( F e d . Eur. Biochem. Soc.) Lett. 5,163 (1969).
Lys-183 residues abolishes the half-site reactivity of Cys-149, so that

all four thiol groups now react independently with iodoacetate or iodo-
acetamide. The equivalent reaction rates of the four thiol groups in the
N-acetylated enzyme argues against preexisting asymmetry unless it were
to be envisaged that a ligand effect could override a preexisting
asymmetry.
In any event it seems clear that yeast GAPDH exhibits positive coop-
erativity in NAD+ binding and negative cooperativity in acylation,
whereas the muscle enzyme exhibits negative cooperativity in NAD+
binding and apparent preexisting asymmetry in acylation. These differ-
ences presumably reflect differences in binding sites and in subunit inter-
actions which cannot so far be identified from the X-ray crystallographic
model of the lobster muscle enzyme.
B. MECHANISM
OF ACTIONOF GAPDH
1. Physiological Activity
GAPDH catalyzes the reversible oxidative phosphorylation of G-3P
in a reaction that couples the oxidation of an aldehyde to the synthesis
of a high energy phosphate anhydride, l13-diphosph~oglycerate(cf. 178) ,
according to the equation
G-3P + NAD+ + Pi 1,3-DPGA + NADH
The use of arsenate instead of phosphate results in the formation of
l-arseno-3-phosphoglyceratewhich is rapidly and nonenzymically hydro-
lyzed to 3-phosphoglycerate rendering the overall reaction irreversible
and thus amenable to steady-state kinetic analysis. Other aldehydes can
also be oxidized giving rise to the corresponding acyl phosphates (179).
GAPDH also exhibits a number of other activities which, although
unphysiological, have greatly aided efforts to elucidate the mechanism
of the normal physiological reaction. The nature of these other activities,
such as acyl transfer and esterase activities, which have been studied
extensively [principally by Park and colleagues (cf. IS)] can be explained
in terms of the formation of acyl enzyme intermediates. The normal
catalytic mechanism involves the formation of a covalent phospho-
glyceroyl thioester, and proof for the existence of such a high energy inter-
mediate was first obtained by Krimsky and Racker (55) who succeeded
in preparing an acetyl enzyme with acetyl phosphate. This was shown
to be an enzymically active intermediate since the acetyl group could
178. F. Lipmann, Advan. Eneymol. 6,231 (1946).
179. A. P. Nygaard and J. B. Sumner, PBB 39, 119 (1952).
be reduced by NADH to give acetaldehyde and NAD'. Formation of

the acetyl enzyme was inhibited by iodoacetate, consistent with the pro-
posal of a thioester linkage between the acetyl group and the protein.
The reactive group involved in thioester formation was subsequently
identified as Cys-149 by Harris et al. (29).
The importance of thiol groups in the enzymic reaction had been re-
vealed by the early studies of Segal and Boyer (180), who postulated
that the primary step in the reaction was the formation of a thiohemi-
acetal intermediate. This proposal is consistent with the observation that
the rate of reaction of a number of aliphatic and aromatic aldehydes
with the enzyme is facilitated by electron withdrawing substituents on
the aldehyde group, which implies the attack of a nucleophilic group
(such as a thiol) on the carbonyl carbon (181). Moreover, the redox reac-
tion has been shown (182) to proceed by way of a direct hydride ion
transfer from the substrate to the p-4 position of the nicotinamide ring.
It does not involve a reduction of the enzyme itself (183), and the B
specificity of GAPDH is now known (see Section II,B,2,a) to be the re-
sult of a 180° rotation of the nicotinamide ring about the glycosidic link-
age which exposes the other face of the ring for attack at C-4 (47, @).
A second function of NAD+ in the reaction is unrelated to its redox
role since it is required as a cofactor for most of the nonoxidative reac-
tions involving the reactive thiol group, with the exception of the esterase
activity (13, 54, 184-188).
The mechanism originally postulated by Segal and Boyer (180) (and
shown schematically in Fig. 11) is in accord with the above-mentioned
observations, and has since received extensive support from the stopped-
flow kinetic studies of Trentham and colleagues (69, 83, 116, 117, 189,
190).
This type of ping-pong mechanism (cf. 191) is contrary to the random
or ordered sequential addition of reactants, with deacylation prior to dis-
180. H. L. Segal and P. D . Boyer, JBC 204, 265 (1953).
181. T. H. Fife, T. Rikihisa, and B. M. Benjamin, Biochemistry 10, 3875 (1971).
182. F. A . Loewus, H. R. Levy, and B. Vennesland, JBC 223, 589 (1956).
183. W. S. Allison, H. B. White, 111, and M. J. Connors, Biochemistry 10, 2290
(1971).
184. J. Harting and S. F. Velick, JBC 207, 867 (1954).
185. J. H. Park, B. P . Meriwether, P. Clodfelder, and L. W. Cunningham, JBC
236, 136 (1961).
186. E. L. Taylor, B. P. Meriwether, and J. H. Park, JBC 238, 734 (1963).
187. A . G. Hilvers and J. H. M.Weenen, BBA 58,380 (1962).
188. R. G. Duggleby and D. T . Dennis, JBC 249, 175 (1974).
189. D. R. Trentham, BJ 122, 71 (1971).
191. M. Lazdunski, Progr. Bioorg. Chem. 3, 82 (1974).
,S-CHOHR
E
NA1l' I-CHOHR
R Y E'ADt
/SH ,SCOR
NAD+
R +H+
C O O e k -7 O R , SCOR
ENAD+ E'
WAD+
where RCHO refers to GAP and
RCOOPOs to DPGA
FIQ.11. Reaction mechanism of GAPDH adapted from Segal and Boyer (180).
sociation of NADH, that was postulated on the basis of steady-state

studies (60, 192-194) with muscle enzyme. A recent steady-state analysis
(196) carried out with the pea seed enzyme (26)) under conditions that
were designed to eliminate product inhibition by DPGA is, however, en-
tirely consistent with the ping-pong mechanism, and it seems likely that
the earlier steady-state kinetic studies would have been influenced by
product inhibition with NADH or DPGA as well as by nonspecific
salt effects (85).
Dissociation of NADH prior to phosphorolysis is a feature common
to many mechanisms for GAPDH (cf. 13) since this is necessary to ex-
plain the NAD' requirement for phosphorolysis of the acyl enzyme (196).
Indeed, apart from the earlier steady-state kinetic studies, the only report
a t variance with this concept is that of Smith (19'7), who examined the
ternary acyl enzyme-NADH complex fluorimetrically and was unable
to displace the NADH even a t high concentrations of NAD'. It has, how-
ever, been suggested (117)that phosphate is required for this displace-
ment to occur.
192. T. Keleti and J. Batke, Acta Physiol. 28, 195 (1965).

193. B. A. Orsi and W. W. Cleland, Biochemistry 11, 102 (1972).
194. C. M. Smith and S. F. Velick, JBC 247, 273 (1972).
195. R. G. Duggleby and D. T. Dennis, JBC 249,167 (1974).
196. E. Racker and I. Krimsky, Fed. Proc., Fed. Amer. SOC.Eap. B i d . 17, 1135
( 1958).
197. T. E. Smith, Biochemistry 5,2919 (1966).
Trentham (116) has shown that NAD+ strongly accelerates the forma-
tion and breakdown of acyl enzyme from DPGA as well as from
G-3P. Moreover, the relationship is reciprocal, since acylation of the en-
zyme weakens NAD+ binding (69) and thus accounts, a t least in part,
for earlier discrepancies between the kinetically and fluorimetrically de-
termined NAD' dissociation constants (60), as well as for the strong
product inhibition by NADH (198). In fact, only if the holoenzyme
is acylated is the dissociation of NAD+ in the reverse reaction sufficiently
rapid to account for the catalytic rate.
The rate-limiting step of the oxidative phosphorylation is NADH re-
lease a t high pH (>7.5) and phosphorolysis of the acyl enzyme a t low
pH. This is because the rate of phosphorolysis is highly p H dependent,
possibly increasing more than 2 X 104-fold from pH 5.4 to 8.6, while the
rate of NADH release is independent of pH over this range, with the
result that the two converge around pH 7.5. The pH dependence of phos-
phorolysis (190) may reflect a requirement for the phosphate trianion
(PO,3-). At high enzyme concentrations (> 0.1 mg/ml) , the conversion
of the predominant gem-diol form of G-3p to its reactive aldehyde form
becomes rate limiting (146, 199). This in vitro interconversion does not
apply in vivo since the reactive aldehyde form of G-3P is the product
of both the aldolase and triosephosphate isomerase reactions, and the
hydration of the aldehyde is a slow process. I n the reverse reaction, the
rate determining step is a process associated with NADH binding, prob-
ably a conformational change, at high pH, and release of G-3P a t low
pH (189, 1 9 9 ~ )At
. high ionic strengths, acylation becomes rate limiting
(83)*
With the natural substrate it is now generally agreed that all four sites
of the muscle enzyme tetramer are simultaneously active both in the for-
ward and reverse reactions (160, 161, 189) despite earlier claims (169)
that only the fourth site turns over.
Smith and Velick (194) have undertaken an extensive steady-state
kinetic analysis of forward and reverse reactions catalyzed by the liver
and muscle enzymes, under pseudophysiological conditions, in a n effort
198. I. Krimsky and E. Racker, Biochemistry 2, 512 (1963).
199. D. R. Trentham, C. H. McMurray, and C. I. Pogson, BJ 114, 19 (1969).
199a. An alternative proposal now favored by Trentham which removes the neces-
sity to postulate an NADH-induced conformational change is that aldehyde release
is rate limiting under all conditions of low salt. The precursor for aldehyde release
is the NAD+-aldehyde enzyme. A t high pH this complex is in rapid equilibrium
with the NADH-acyl enzyme, which is the major species and therefore the predomi-
nant steady-atate intermediate. At low pH, however, the rapid equilibrium can favor
the aldehyde-apoenzyme complex suggesting that NAD' dissociation from the
NAD+-aldehyde enzyme is favored at low pH.
42 J. IEUAN HARRIS AND'MICHAEL WATERS
to understand the factors allowing gluconeogenesis through GAPDH in

liver. Although the conclusions with regard to the forward reaction are
complicated by product inhibition caused by DPGA, the results obtained
for the reverse reductive dephosphorylation reaction provide an explana-
tion for the possible metabolic significance of negative cooperativity in
muscle and in liver. At the low DPGA concentrations encountered in vivo,
NAD' acts cooperatively to convert the DPGA saturation curve from
a sigmoidal to a hyperbolic form, thus sensitizing the enzyme to the lower
concentrations of the acyl phosphate. At the same time, NAD+ acts to
abolish substrate inhibition by NADH toward nonacylated enzyme sites.
At higher concentrations of acyl phosphate, NAD' acts as a weak com-
petitive inhibitor toward NADH, in contrast to the strong competitive
inhibition by NADH toward NAD' seen in the forward reaction. The
latter observations can be explained by the decrease in binding affinity
for NAD' that occurs on acylation (GO),which Smith and Velick (194)
suggested is largely the result of an isomerization process (cf. 143).Pre-
sumably, because the enzyme is almost totally acylated and because of
the high NAD' concentrations, cooperative effectcl are not seen in the
forward reaction in vitro; i.e., the enzyme exists in a single conformation.
While there is no doubt that bound NAD+ enhances acylation (117)
as well as deacylation (7O),it is difficult to reconcile the above-mentioned
cooperative effects with the observation (165)that NAD' occupation of
one site in the rabbit tetramer did not affect the catalytic activity of
the other three sites toward aldehyde substrates. Evidence against a slow
isomerization of the T + R variety, which might be expected to affect
thiol reactivity in other subunits, is the fact that the sturgeon apoenzyme
is almost instantaneously active in acylation, following the addition of
NAD' (116). The nature of the NAD+ enhancement of acylation and
deacylation is of considerable interest since it has a bearing on the mech-
anism of enzyme catalysis in general, and also because it explains the
NAD' requirement of a number of the minor activities of GPD (see Sec-
tion III,B,2).
Studies with the simple alkylating agents iodoacetate and iodoaceta-
mide have shown that NAD' promotes alkylation of Cys-149 by nega-
tively charged iodoacetate, but inhibits alkylation by the uncharged
iodoacetamide molecule (117,196).Since NADH and the adenine nucleo-
tides do not facilitate alkylation by iodoacetate or arsenolysis of acetyl
phosphate (200-202), it has been suggested that the positively charged
pyridinium ring facilitates attack by negatively charged alkylating or
200. S. H. Francis, B. P. Meriwether, and J. H. Park, JBC 246, 5427 (1971).
201. S. H. Francis, B. P. Meriwether, and J. H. Park, JBC 248, 5433 (1971).
202. A. Feneslau, JBC 245, 1239 (1970).
deacylating agents through ion pair formation. This "ion pair" concept
is supported by the inhibitory effect of high ionic strength on both alkyla-
tion by iodoacetate and acylation by acetyl phosphate, which presumably
occurs because the positive charge is masked by interaction with anions
(83, 122). Cseke and Boross (122,203) have shown that the PKa of
Racker band absorbance and of thiol anion carboxymethylation is
lowered from about 8 in the apoenzyme to around 5.5 (204) in the pres-
ence of NAD'. This has been taken as evidence that NAD' has lowered
the pK, of Cys-149 (117,203), especially since the pK, of carboxy-
methylation and of Racker band absorbance vary together, depending
on the nature of the solvent anion. If the nucleophilicity of the thiol
anion is insensitive to its basicity, as the evidence suggests, then this
lowered thiol group dissociation induced by NAD' would explain the thiol
group reactivity a t lower pH (< 7.0). It cannot explain the fact that
the reactivity is only exhibited toward iodoacetate and not iodoaceta-
mide, since reactivity of the thiol group should be identical toward the
two alkylating agents. Consequently, it is still necessary to postulate ion
pair formation in the presence of NAD'. Whatever the nature of this
effect it is manifest a t low ratios of NAD+:enzyme (< 1 mole/mole)
which suggests migration of NAD' from alkylated sites to nonalkylated
sites induced by the lowered affinity of NAD' for alkylated subunits (117,
202).
While the scheme outlined above accounts for the NAD+-dependent
activation of thiol-149, it does not account for the fact that Cys-149 is
highly activated compared to simple aliphatic thiols even in the absence
of NAD'. It therefore becomes necessary to postulate the presence of
a basic group to activate the thiol group by hydrogen bonding to its
proton in a manner similar to that found with papain and thiol-subtilisin,
where histidine is the basic group (83, 205, 206'). The effect of NAD'
on the pK, of the thiol could then be explained by postulating that NAD'
binding alters the conformational alignment of the thiol and basic resi-
dues in order to draw a proton further away from the thiol group. The
inhibitory effect of anions and the anion-induced variation in the pK,
of the thiol base proton would then be the result of interference in both
base thiol and pyridinium thiol interactions (122).It has been suggested
that a strongly basic group adjacent to the thiol base pair is responsible
for anion binding and that the bound anion creates an electron-rich region
which in turn increases the pK, of the thiol base pair (122, 20od). The
203. E. Cseke and L. Boross, Acta Biochim. Biophys. 2, 39 (1967).
204. M.T.A. Behme and E. H. Cordes, JBC 242,5500 (1967).
205. G. Loae, Phil. Trans. Roy. Snc. London, Ser. B 257, 237 (1970).
206. L. Polghr, FEBS (Fed. Eur. Biochem. Soc.) L e t t . 38, 187 (1974).
+
H< ,SCOR- HB, /WOR
ENAD+
- NAD+
E
FIO.12. Reaction mechanism of GAPDH according to Harrigan and Trentham ( 8 3 ) .
crystallographic evidence shows that Lys-191 and Arg-231 contribute

toward the formation of this site which appears to be suitably located
to participate in binding inorganic phosphate during the phosphorolysis
reaction. These two residues are highly conserved (Table 11), and modifi-
cation of Lys-191 is known to cause inactivation ( 7 6 ) . Presumably the
powerfully inhibitory analog, threose 2,4-diphosphate, which forms a
stable acyl enzyme even in the presence of phosphate (207),also binds
a t this site. A second anion binding site created primarily by the nicotina-
mide ribose and located where the phosphate group of the substrate
(G-3P) would be expected to bind (46, 47) might also account for sub-
strate inhibition by phosphate (60) and for the greater affinity of the
enzyme for G-3P as compared to glyceraldehyde (179).
I n the crystallographic model the highly conserved His-176 occurs
within hydrogen bonding distance of Cys-149 (cf. Section II,B,4,d). It
is thus ideally placed to be the histidine residue that interacts with the
active site thiol group during catalysis. It may also be envisaged to be
the residue that extracts a proton from Cys-149 during the initial acyla-
tion step according to the reaction scheme postulated by Harrigan and
Trentham (83) and illustrated schematically in Fig. 12. The role of histi-
dine in deacylation but not in the redox reaction has also been discussed
by Francis et a2. (90).
2. Other Activities of GAPDH

I n addition to its dehydrogenase activity GAPDH possesses several
other activities. These have been reviewed extensively by Colowick et
a2. (IS) and will be referred to here only insofar as they help to clarify
the mechanism of the dehydrogenase reaction.
a. Acyltransferase Activity. GAPDH catalyzes the exchange of
into acyl phosphates, such as acetyl phosphate and DPGA, and arsenate
207. E. Racker, V. Klybas, and M. Schramm, JBC 234, 2610 (1969).

will induce an irreversible arsenolysis of acyl phosphate in the presence

of the enzyme (184,208).
CHaCOOPOsHz + Ha*'POc % CHSCOO''PO~HZ+ HaPo,
CH&OOPOaHz + HaAsOi % CH~COOASOIHZ+ Hap04
1Hz0
CHaCOOH + HshO4
It has been suggested (70)that NAD+ acts by facilitating deacylation
of the thiol ester, and the later kinetic studies (189) are in agreement with
this view. Only NAD+ analogs which are active in the oxidative phos-
phorylation reaction will substitute for NAD' in the enzyme-catalyzed
arsenolysis of DPGA (188) or acetyl phosphate (90). At higher pH (8.5)
an irreversible S to N transfer of acetyl groups occurs.
b. Esterase Activity. NAD+-free GAPDH catalyzes the hydrolysis of
aryl esters (e.g., p-nitrophenyl acetate) a t rates up to five times that
of chymotrypsin (186). This reaction also proceeds via a thiolester inter-
RCOOAr + ESH ES -+
ArOH
COR -
mediate with Cys-149 (29) and is, therefore, inhibited by iodoacetate.
Ha0
RCOOH
The rate of the reaction is related to the electron withdrawing ability

of the phenolic moiety (81) and may be compared to the reaction of
aromatic aldehydes with the enzyme (181). These reactions are in accord
with the concept of a nucleophilic attack of the thiol of the carbonyl
carbon.
Although NAD' inhibits the esterase reaction, adenine nucleotides
stimulate it, which suggests that NAD+ is blocking access of the aromatic
ester to the reactive thiol in a manner similar to its effect on iodoaceta-
mide alkylation (7'0). Acyl shifts involving Cys-149 and Lys-183 also
occur readily with p-nitrophenyl acetate a t higher pH and in the absence
of NAD' (70,73, 74).
C. METABOLIC
ROLEOF GAPDH
GAPDH is common to both the glycolytic and gluconeogenic pathways.
The muscle and liver enzymes are similar in structure and properties
(194, 209), and the different behavior of the enzyme in muscle and liver
must therefore be ascribed to differences in the cellular environment.
208. P. Oesper, JBC 201,421 (1954).

209. J. M. Lamhert and R. N. Perham, FEBS ( F e d . Eur. Biochem. Soc.) Lett
40, 305 (1974).
Evidence has been obtained (210, 211) that the rate of glycolysis in
rat liver is proportional to the cytosolic redox state (i.e., the ratio of
free XAD': NADH) . It was also inferred that the rate of gluconeogenesis
is inversely proportional to (but not necessarily controlled by) this ratio.
These conclusions were drawn from studies utilizing the changes in
cytosolic NAD': NADH ratios induced by different dietary and hormonal
states and presumably reflect the ability of GAPDH to influence the rate
of glycolysis by responding to changes in the redox state. Moreover, it
has been suggested (211) that the primary effect derives from an altera-
tion in the phosphorylation state of the adenine nucleotide pool since
the NAD+:NADH ratio is coupled to the ATP/ADP X Pi ratio through
the phosphoglycerate kinase reaction
"AD+] 1 [3-phosphoglycerate] [ATP]

=-x X-
[NADH] K [GAP1 [ADPI[Pil
Thus, a high NAD+:NADH ratio will be associated with a high

ATP:ADP X P, ratio, and with an increased glycolytic flux. Conversely,
a decrease in the NAD+:NADH ratio will be accompanied by a fall of
the ADP:ADP X Pi ratio and, paradoxically, a rise in the flux in the
direction of gluconeogenesis. That is, while the overall NAD': NADH
ratio is set by the redox potentials of the cytosolic dehydrogenases at
that pH, it is possible to vary this ratio and hence the direction of carbon
flow through GAPDH by changes in metabolite concentrations. The
major change in the ATP:ADP X Pi ratio results from raised ADP and
Pi levels, although an increase in the latter would to some extent counter
the increased gluconeogenic flow by a substrate effect on GAPDH (212).
The percentage fall in ATP level is only slight in starvation and alloxan
diabetes although the percentage rise in ADP is considerable (210,213).
The NAD+:NADH ratio in the cytoplasm can, under some conditions
(e.g., in starvation), be considerably lower in liver than in muscle (212,
214), giving rise to conditions more favorable for gluconeogenesis. I n
addition, oleate infusion was found (216)to promote gluconeogenesis in
rat liver, possibly as the result of a lowered NAD+:NADH ratio; and
sharply lowered ratios were also found in the livers of alloxan diabetic
210. R. L. Veech, L. Raijman, and H. A. Krebs, BJ 117,499 (1970).

211. K. A. Gumaa,. P. McLean, and A. L. Greenbaum, Essays Biochem. 7, 39
(1971).
212. D. H. Williamson, P. Lund, and H. A. Krebs, BJ 103, 514 (1967).
213. J. Elliott, E. Dade, D. M. W. Salmon, and D. A. Hems, BBA 343,307 (1974).
214. L. A. Jedeikin and S. Weinhouse, JBC 213,271 (1955).
215. J. R. Williamson, JBC 242,4476 (1967).
rats (211,216), in accord with the increased gluconeogenesis characteristic

of diabetes.
These considerations rest heavily on the assumption that GAPDH is
catalyzing an equilibrium reaction, and that it does not fulfill a regula-
tory function dependent upon inhibitory effectors. While this assumption
may hold true in liver under normal circumstances ( H I ) , there is evi-
dence to suggest that this is not always the case. Thus, for example, of
eight glycolytic enzymes studied in adipose tissue taken from alloxan
diabetic rats (217), the GAPDH-phosphoglycerate kinase system alone
did not move in parallel with the other enzymes studied, and was the
slowest to be restored with insulin. GAPDH was also found (218, 219)
to be considerably displaced from its equilibium value in mouse brain
and rat heart. Cross-over studies (216, 219) have demonstrated that
GAPDH becomes the rate-limiting (and possibly regulatory) gluconeo-
genic step during pyruvate and oleate infusion of rat livers, and there
is good evidence (220) that the enzyme can be rate-limiting in ascites
cells. It has also been suggested (221) that GAPDH in cerebral cortex
slices may form a regulatory system with pyruvate kinase, similar to
the hexokinase-phosphofructokinase system in that ATP resulting from
the action of GAPDH and phosphoglycerate kinase might be expected
to inhibit pyruvate kinase leading to an accumulation of DPGA and the
consequent product inhibition of GAPDH.
The activities of glycolytic and gluconeogenic enzymes have been de-
termined for most rat and human tissues (222). The values for GAPDH
(Table V) show that in liver it is 50 times higher than the activity of
the least active enzyme, glucokinase; while in muscle it is over 300 times
greater than the enzyme of lowest activity, hexokinase. For adipose tis-
sue, on the other hand, total GAPDH activity is only marginally higher
than the activity of the least active enzyme, phosphofructokinase, sug-
gesting that in this tissue (although presumably not in heart or brain
where GAPDH activity is about 20 times greater than the activity of
the rate-limiting enzyme) GAPDH might be expected to be a rate-limit-
ing or regulatory enzyme. In assessing these possibilities it should never-
theless be recalled that the effective GAPDH activity in the cell is con-
trolled by a variety of factors. Thus, in the first place, a t the normal
216. H. A . Krebs, Recent Ad7lan. Enzyme Regul. 5,409 (1967).
217. E. D. Saggerson and A. L. Greenbaum, BJ 115, 405 (1969).
218. 0. H. Lowry and J. V. Passoneau, JRC 239, 31 (1964).
219. J. R. Williamson, JBC 240, 2308 (1965).
220. D. P. Kosow and I. A . Rose, Fed. Proc., F e d . Amer. SOC.Exp. Biol. 31, 1219
(abstr.) (1972).
221. F. S. Rolleston and E. A. Newsholme, BJ 104,524 (1967).
222. C. E. Shonk and G. E. Boxer, Cancer Res. 24,709 (1964).
TABLE V
MAXIMAL
ACTIVITIES
OF GAPDH I N RAT AND HUMAN
TISSUES'
Heart Skeletal
Tissue Liver Kidney Brain muscle muscle Spleen Adipose
Rat 63 f 2.36 69 f 6.5 81 f 10.5 90 f 7.6 294 f 36 25 f 5.7 0.06 f 0.16

Human 65 63 9 61 284 2.1
a Micromoles per minute per gram wet tissue It S.E.M.
physiological pH the enzyme is operating at only about 20% of its maxi-

mum activity (92s). Moreover, this falls to as low as 5% a t p H 6.5,
a pH that might well be attained during peak production of lactate in
muscle (224, 225). GAPDH is also inhibited by various adenine nucleo-
tides (108, 900,224-926) although, in vivo, ATP is the only nucleotide
normally present at sufficiently high concentrations to cause significant
inactivation. Thus, a t normal physiological concentrations of ATP
GAPDH could be inhibited to the extent of 67% a t pH 7.4 and of 87%
a t pH 6.8 (225). I n muscle phosphocreatine could also be an effective
inhibitor of GAPDH (224). I n the erythrocyte GAPDH is operating a t
less than 1% of its maximum capacity (227) because of product inhibi-
tion by NADH and a high K , for phosphate. The enzyme is also suscepti-
ble to appreciable product inhibition of the forward reaction by
DPGA (96,60),which could account for its inhibition following pyruvate
infusion of rat hearts (819). It has also been suggested (228, 229) that
binding of GAPDH to erythrocyte membranes and to F-actin could be
a means of regulating the activity of the enzyme.
Consideration of these many factors illustrates the GAPDH could well
play an important regulatory role in glycolysis despite its apparent high
in vitro activity. Studies with the purified enzyme have, in addition, led
to some interesting suggestions concerning its possible in vivo behavior.
Thus, isolation of acyl enzyme from muscle led to the proposal (165)
that scyl enzyme itself is a glycolytic intermediate of considerable impor-
tance since the active site concentration of GAPDH in muscle is in excess
of 70 pill (60). Moreover, under conditions of sudden glycolytic flow (as
223. G. T. Cori, M. W. Slein, and C. F. Cori, JBC 173, 605 (1948).
224. M. Oguchi, E. Gerth, B. Fitzgerald, and J. H. Park, JBC 248,5562 (1973).
225. M. Oguchi, B. P. Meriwether, and J. H. Park, JBC 248, 5562 (1973).
226. N. K. Nagradova, M. K. Vorona, and R. A. Asriyants, Biokhimiya 34, 503
(1969).
227. G. C. Mills and F. L. Hill, ABB 146, 306 (1971).
228. H. Arnold and D. Pette, Eur. J . Biochem. 6,163 (1968).
229. B. C. Shin and K. L. Carraway, JBC 248, 1436 (1973).
in muscle contraction), acyl enzyme might act to “buffer” glycolytic flow

through the triose phosphates below GAPDH until phosphofructokinase
responded to the altered adenine nucleotide ratio. It has also been sug-
gested (166, 194) that deacylation of GPD under such conditions would
enhance the bind of NAD’ (since NAD’ does not bind as strongly to
the acyl enzyme) and thereby favor the forward reaction. Concomitantly,
product inhibition by DPGA would be relieved by ATP synthesis a t the
phosphoglycerate kinase step, in response to the lowered cytosolic
ATP:ADP X Pi ratio. In addition, the lowered intracellular p H result-
ing from lactate production would increase the NAD+:NADHratio, and
this would also favor glycolysis by its effect on the GAPDH reaction.
In the liver, no such glycolytic transients occur; thus, the enzyme would
be permanently acylated, thereby favoring NADH binding and the re-
verse reaction, especially in the more reduced hepatocyte (214). The pres-
ence of NAD’ would serve to activate the reverse reaction a t low DPGA
concentrations, while the higher level of NADH would induce positive
cooperativity of DPGA binding (194).
These considerations do not take into account the major glycolytic con-
trols exerted by the hexokinase-phosphofructokinase system (230) but
they do show that G3PDH could act as a regulatory enzyme in response
to the NAD+:NADHand ATP:ADP x Pi ratios in the cell. These ratios
in conjunction with appropriate substrate (i.e., G-3P, DPGA, and Pi)
concentrations prime the enzyme for glycolysis or gluconeogenesis in
accord with the particular environment and needs of the cell.
ACKNOWLEDGMENTS
The authors are grateful to Dr. M. G . Rossmann for providing original copies of
Figs. 2-8 and to Dn. D. R. Trentham and J. Armstrong for reading parts of the
manuscript and for helpful comments.
230. H. A. Krebs, Essays Biochem. 8, 1 (1972).

Nicotinamide Nucleotide
Transhydroenases
J . RYDSTROM J . B. HOEK L . ERNSTER
I . Definitions . . . . . . . . . . . . . . . . . 51
I1. BB-Specific Transhydrogenases . . . . . . . . . . . 52
A . Historical . . . . . . . . . . . . . . . 52
B . Occurrence . . . . . . . . . . . . . . . 53
C . Purification and Assay . . . . . . . . . . . 54
D . Molecular Properties . . . . . . . . . . . . 57
E . Reaction Mechanism and Regulation . . . . . . . 59
I11. AB-Specific Transhydrogenaaes . . . . . . . . . . . 62
A . Historical . . . . . . . . . . . . . . . 62
B . Occurrence . . . . . . . . . . . . . . . 64
C . Preparations and Assay . . . . . . . . . . . 66
D . Molecular Properties . . . . . . . . . . . . 69
E . Relationship to the Energy-Coupling System . . . . . 71
F. Kinetics and Reaction Mechanism . . . . . . . . 75
G . Reconstitution . . . . . . . . . . . . . 78
I V . Physiological Roles of Nicotinamide Nucleotide Transhydrogenaaes . 79
A . Redox State of Mitochondrial Nicotinamide Nucleotides . 81
B. Role of Transhydrogenme in Mitochondrial Monooxygena-
tion Reactions . . . . . . . . . . . . . 83
C. Role of Transhydrogenme in Mitochondrial Glutamate and
Isocitrate Metabolism . . . . . . . . . . . 85
D . Role of Transhydrogenase in Fatty Acid Synthesis . . . 88
.
1 Definitions
The term “nicotinamide nucleotide transhydrogenase” (EC 1.6.1.1) is

used in this chapter to denote those enzymes that catalyze the reversible
51
52 J. RYDSTR~M, J. B. HOEK, AND L. ERNSTER
transfer of hydrogen between the two naturally occurring nicotinamide

nucleotides, NAD ( H ) and NADP(H) ,i.e., the reaction
NADH + NADP+ NAD+ + NADPH
without the mediation of any further substrates. Thus, the term does not
include those enzymes that catalyze transhydrogenation between only
one naturally occurring nicotinamide nucleotide and an artificial nicotin-
amide nucleotide analog [most NAD (H)- or NADP (H)-specific flavo-
proteins belong in this category] ; nor does it include so-called dual-spe-
cific dehydrogenases, i.e., enzymes that react with both NAD(H) and
NADP(H) , but where a transhydrogenation between the two requires
the mediation of a third substrate (e.g., glutamate dehydrogenase) .
Finally, the present chapter does not deal with the enzyme DT-dia-
phorase (EC 1.6.99.2), a flavoprotein that oxidizes both NADH and
NADPH but does not seem to catalyze a transhydrogenation between
the two.
Nicotinamide nucleotide transhydrogenases may be divided into two
classes. One class is present in certain bacteria, and possibly in some
plants, is an easily extractable, water-soluble enzyme ; is not functionally
linked to the energy-transfer system of the bacterial membrane; is a
flavoprotein; and is specific for the 4B-hydrogen atom of both NADH
and NADPH. The other class is present in both certain bacteria and
in mitochondria ; is a firmly membrane-bound water-insoluble enzyme;
is functionally linked to the energy-transfer system of the bacterial or
mitochondria1 membrane; is not known to be a flavoprotein; and is spe-
cific for the 4A-hydrogen atom of NADH and the 4B-hydrogen atom
of NADPH. For the sake of convenience, the two classes of enzyme will
be referred to below as BB-specific and AB-specific transhydrogenases,
respectively.
II. BB-Specific Transhydrogenases
A. HISTORICAL
Investigations by Colowick et al. (1) on isocitrate dehydrogenase in
Pseudomoms fluorescens led to the discovery that in the presence of ex-
tracts of these bacteria NAD' could be reduced by isocitrate provided
a catalytic amount of NADP' was added. It was proposed that a specific
enzyme, called pyridine nucleotide transhydrogenase, catalyzed the for-
1. S. P. Colowick, N. 0. Kaplan, E. F. Neufeld, and M. M. Ciotti, JBC 195,
95 (1952).
2. NICOTINAMIDE NUCLEOTIDE TRANSHYDROGENASES 53
mation of NADP+ plus NADH from NAD+ plus NADPH. Determination

of the stoichiometry of oxidized and reduced nicotinamide nucleotides
( g ) ,respectively, indicated the net reaction
NAD+ + NADPH + NADH + NADP+
Indirect transfer of hydrogen ( I ) , transfer of phosphate (2, S) or nico-
tinamide moieties ( 4 ) could be eliminated, suggesting that reduction of
NAD’ by NADPH was direct and stereospecific (6). Regulation of
the reduction of NADP’ by NADH by various nucleotides (3)indicated
that the transhydrogenase was an allosteric enzyme. However, despite
extensive kinetic studies of the transhydrogenase carried out mainly by
Kaplan and co-workers ( 6 ) with a purified enzyme, its detailed reaction
mechanism and regulation are still unclear. The stereospecificity of the
Pseudomonas enzyme for the 4B hydrogen of both NADH and
NADPH (7), in contrast to those of the mammalian and certain bacterial
transhydrogenases which are AB specific, led Hoek et al. (8) to propose
that the so-called BB- and AB-specific transhydrogenases represent two
different classes of enzymes (see Section I). Several other laboratories
have been involved in elucidating the properties of BB-specific transhy-
drogenases from other sources, e.g., the laboratories of Veeger (9) and
Chung ( I O ) , who found that the transhydrogenase from Azotobacter
vinelandii is closely related to the Pseudomonaa enzyme.
B. OCCURRENCE
Nicotinamide nucleotide transhydrogenase was originally discovered
in Pseudomonas fluorescens. Part of the work done with these bacteria
by Kaplan and co-workers (see 7) appeared later to have involved
Pseudomonas aeruginosa. There is little doubt, however, that these two
strains contain transhydrogenases that are closely related. Kaplan and
co-workers (3) also demonstrated the presence of transhydrogenase in
2. N. 0. Kaplan, S. P. Colowick, and E. F. Neufeld, JBC 199, 107 (1952).

3. N . 0. Kaplan, S. P. Colowick, E. F. Neufeld, and M. M. Ciotti, JBC 205,
17 (1953).
4. N. 0. Kaplan, S. P. Colowick, L. J. Zatman, and M. M. Ciotti, JBC 205, 31
(1953).
5. A. San Pietro, N. 0. Kaplan, and S. P. Colowick, JBC 212, 941 (1955).
6. P. T. Cohen and N . 0. Kaplan, JBC 245, 4666 (1970).
7. D. D. Louie and N . 0. Kaplan, JBC 245,5691 (1970).
8. J. B. Hoek, J. Rydstrom, and B. Hojeberg, BBA 333,237 (1974).
9. H. W. J. van den Broek and C. Veeger, “Pyridine Nucleotide Dependent Dehy-
drogenases,” p. 335. Springer-Verlag, Berlin and New York, 1969.
10. A. E. Chung, J. Bucteriol. 102, 438 (1970).
Azotobacter vinelandii, Azotobacter chroococcurn, and Azotobacter agile.

The occurrence of transhydrogenases in plants, i.e., spinach, pea leaves,
turnip greens, parsley, watercress, and Euglena gracilis, was reported by
Keister et al. (11) ; among these the spinach enzyme appears to be identi-
cal with ferredoxin-NADP reductase (19, IS). The same holds for the
transhydrogenase obtained from the alga Burnilleriopsis filifomzis (14)
1 6 ) . Whether these plant and alga transhydrogenases may be classified
as BB- or AB-specific eneymes remains to be established, although their
kinetic properties partly resemble those of the Pseudomonas enzyme.
Keister and Hemmes (16) isolated a transhydrogenase from Chromatiurn,
with properties similar to those of the Azotobacter enzyme.
C. PURIFICATION
AND ASSAY
The first demonstration of transhydrogenase activity in Pseudornonas

jluorescem by Colowick et al. (1) was carried out with a crude extract
obtained from cells grown on citrate as the sole carbon source. This ex-
tract could be fractionated further by acetone precipitation followed by
calcium phosphate adsorption and subsequent elution with potassium
phosphate. A second acetone fractionation and calcium phosphate adsorp-
tion gave a total purification of about 200-fold. This preparation was
devoid of dehydrogenase activity using glutamate, isocitrate, lactate,
6-phosphogluconate, glucose 6-phosphate, and ethanol as substrates in
combination with either NAD+ or NADP+.A considerably more elaborate
and extensive purification was reported by Cohen and Kaplan ( l 7 ) ,using
a Pseudomonas aeruginosa strain grown on glucose medium. After sonic
disruption of the cells and centrifugation, the supernatant was fraction-
ated with ammonium sulfate. The sediment was redissolved and chroma-
tographed in two steps on DE-11 and hydroxylapatite columns. Finally,
a two-step differential centrifugation gave a total purification of about
800-fold, yielding a homogeneous preparation that could be crystallized
and that exhibited no dehydrogenase activity with a variety of substrates
tested.
Spinach transhydrogenase was isolated by Keister et al. (11) by acetone
extraction and precipitation with protamine sulfate followed by adsorp-
11. D. L. Keister, A. San Pietro, and F. E. Stolzenbach, JBC 235, 2989 (1960).
12. M. Shin, K. Tagawa, and D. Arnon, Biochem. Z.338,84(1963).
13. W. W. Fredericks rind J. M. Gehl, JBC 246, 1201 (1971).
14. P. Boger, Plantn 92, 105 (1970).
15. P. Boger, Proc. Znt. Congr. Photosyn. Res., 2nd, 1971 p. 449 (1972).
16. D. L. Keister and R. B. Hemmes, JBC 241,2820 (1966).
17. P. T. Cohen and N. 0. Kaplan, JBC 245,2825 (1970).
tion on bentonite. This preparation could be purified further on Dowex

50W,giving a total purification of 330-fold. The preparation was reported
to exhibit a number of other activities, e.g., NADPH-menadione reduc-
tase and NADPH-cytochrome c reductase. Shin et al. (12) extended this
purification by means of repeated DEAE and Sephadex chromatography
steps. A final ammonium sulfate precipitation yielded a crystalline prepa-
ration whose activity was not much different from that of the enzyme
prepared by Keister et al. (11).
The Chromatium transhydrogenase was isolated by Keister and
Hemmes (16) from a sonicated extract of Chromatium strain D. After
high-speed centrifugation and ammonium sulfate precipitation, two sub-
sequent steps of DEAE-cellulose chromatography were employed. The
purity of the preparation was tested for various reductase.and dehydroge-
nase activities.
The Azotobacter vinelandii transhydrogenase has been purified by two
different laboratories. Chung (10) described a method that employed heat
treatment and alternating DEAE-cellulose and calcium phosphate gel
chromatography. The purity of the preparation was not stated in terms
of contaminating activities. This procedure was later modified by Middle-
ditch et al. (18) to include gel filtration and sucrose gradient fractiona-
tion. A purification of about 900-fold was achieved. Information regard-
ing contaminating activities in this preparation was not given, but the
transhydrogenase sedimented as a single band in the ultracentrifuge.
Similarly to Chung (lo), van den Broek et al. (19) employed heat treat-
ment that precipitated a considerable amount of contaminating protein.
Fractionation with ammonium sulfate, calcium phosphate gel, and ammo-
nium sulfate, in that order, prior to two sequential differential centrifuga-
tion steps, gave about 650-800-fold purification. It appears that this prep-
aration was not completely devoid of contaminating activities such as
pyruvate dehydrogenase and transacetylase ; these activities could not be
separated from the transhydrogenase activity by ultracentrifugation.
The properties of the various preparations obtained from Pseudomoms
fluorescem, spinach, Pseudomonas aeruginosa, Azotobacter vinelandii,
and Chromatium, respectively, are summarized in Table I.
Reduction of NAD' by NADPH or reduction of NADP' by NADH
cannot be assayed directly since the spectral differences between NADH
and NADPH or between NAD' and NADP' are negligible. Either reac-
tion may be measured by removing aliquots from the reaction mixture
18. L. E. Middleditch, R. W. Atchison, and A. E. Chung, JBC 247, 6802 (1972).

19. H. W. J. van den Broek, J. S. Santema, J. H. Wassink, and C. Veeger, Eur.
J. Biochem. 24, 31 (1971).
56 J. RYDSTR~M, J . B. HOEK, AND L. ERNSTER
TABLE I
PREPARATIONS
OF BB-SPECIFIC
TRANSHYDROQENASE FROM VARIOUSSOURCES
Specific
activity
Purifi- (pmoles/ Re
cation min/mg covery
Source (-fold) protein) (%) Contaminations Ref.
Pseudomanas 200 11.5" 16 Devoid of various reductase 1

JEuoreacens and dehydrogenase activi-
ties tested
Spinach 300 1.35" 35 Several, e.g., NADPH-men- 11,18
adione reductase and
NADPH-cytochrome c
reductaseb
Chrmatium 275 24" 7 Devoid of various reductase 16
and dehydrogenase activi-
ties tested
Pseudomonas 800 217O 15 None (crystalline) 17
aeruginosa
Azotobacter 900 3000 14 Not stated lo
vinelandii
Azolobaeter 650-800 220-260" 15 Pyruvate dehydrogenase 19
vinelandii and transacetylase
Azotobacter 900 362" 4.5 Not stated 18
vinelandii
Substrates used were NADPH plus NAD+.

* These activities may be catalyzed by the purified enzyme since the ,preparation
was crystalline.
Substrates used were NADPH plus thio-NAD+.
and terminating the reaction by the addition of either acid or alkali (20).
I n the former case only oxidized nicotinamide nucleotides remain intact,
whereas in the latter case only reduced nicotinamide nucleotides remain
intact. After neutralization, the nicotinamide nucleotides can be deter-
mined enzymically. An alternative method is to keep the concentration
of one of the substrates constant by means of a suitable enzymic regen-
erating system and to measure the change in concentration of the other
substrate a t 340 nm. This type of assay was used originally by Colowick
et al. (1) and later by Chung (10) and van den Broek et al. (19). The
advantage with the regenerating system assay is that the reaction may
be followed directly in a spectrophotometer, provided that the specificity
as well as the capacity of the regenerating system is sufficiently high;
20. M. Klingenberg and W . Slenczka, Biochem. 2.331,486 (1959).

2. NICOTINAMIDE NUCLEQTIDE TRANSHYDROGENASES 57
i.e., suitably, the maximal rate of the regenerating system should exceed
that of the transhydrogenase reaction by a t least 10 times.
I n order to measure transhydrogenation spectrophotometrically with-
out a regenerating system, coenzyme analogs, e.g., thio-NAD (P) (absorp-
tion maximum for the reduced form a t 400 nm), have often been used
(6, 90). However, coenzyme analogs are not natural substrates and there-
fore care should be taken in interpreting kinetic data obtained with these
artificial substrates. On the other hand, the use of analogs appears to
be the method of choice in cases where rapid reactions are measured or
where low concentrations of products interfere (see 21).
D. MOLECULAR
PROPERTIES
Transhydrogenase from Pseudomonas aeruginma purified by Cohen
and Kaplan (17) was reported to have a molecular weight of several
million (21,92). Its structure, as revealed by electron microscopy, ap-
peared like unbranched filamentous aggregates of nonuniform length in
the range of 500-5000 A, and the widths of the majority of the filamen-
tous helixes had an apparent dimension of 80-100 A ( 1 7 ) . In the pres-
ence of 2’-AMP (or NADP’) these structures were found to dissociate
into smaller fragments of about 900,000 daltons, composed of some 20 sub-
units of 40,000-45,OOo daltons (81). The subunits were arranged in a
cylindrical fashion with six or eight larger units peripheral to the frag-
ment (91). Similarly, van den Broek et al. (19) found that, in the absence
of NADP’, the purified Azotobacter transhydrogenase forms long helical-
like structures approximately 120 A in width and 15,000-18,000 A in
length. Upon addition of NADP+ the aggregates were transformed into
shorter fragments with a minimum molecular weight of about 58,000.
These data were later confirmed by Middleditch et al. (18) who also
determined the amino acid composition of the Azotobacter transhydro-
genase. It is still not clear, however, whether nucleotide-dependent
transformations similar to those described by van den Broek et al. (19)
occur in vivo.
The kinetic properties (see Section II,E) and spectral characteristics
(7, 9, 10, 17, 19) of the BB-specific transhydrogenases strongly suggest
that these enzymes are flavoproteins. Consistent with this suggestion,
Louie and Kaplan ( 7 ) showed that, in the presence of 1 M urea, 3H was
taken up from the medium and was incorporated into the reduced nicotin-
amide nucleotide. Direct proof for the occurrence of FAD in both the
21. D. D. Louie, N. 0. Kaplan, and J. D. Lean, J M B 70,651 (1972).
22. D. D. Louie and N . 0. Kaplan, “Pyridine Nucleotide Dependent Dehydroge-
rimes," p. 351. Springer-Verlag, Berlin and New York, 1969.
58 J. RYDSTRBM, J . B. HOEK, AND L. ERNSTER
Pseudomonas and Azotobacter transhydrogenases was provided by Cohen

and Kaplan (17) and by van den Broek et al. (19), respectively, who
showed that inactivation by heat treatment could be reversed by addi-
tion of FAD. FAD could not be replaced by FMN. Reduction of the
enzyme with either NADH or NADPH largely increased the heat sensi-
tivity, whereas oxidized nicotinamide nucleotides or FAD had the oppo-
site effect (17, 19). The number of flavins per 50,000-dalton molecular
weight was calculated to be 0.58 to 1.1 ( 1 7 ) .
Sulfhydryl agents, e.g., p-hydroxymercuribenzoate, both inactivate and
activate the Pseudomoms enzyme, depending on the presence of oxidized
and reduced substrates, respectively ( 1 7 ) . Inactivated protein may be
reactivated by mercaptoethanol suggesting that p-hydroxymercuribenzo-
ate acts on sulfhydryl groups near or a t the active site. Reversible
effects of sulfhydryl agents were also observed with the Chromatium en-
zyme ( 1 6 ) . Proteolytic enzymes such as trypsin did not inactivate the
Pseudomonas transhydrogenase (8).
Transhydrogenase obtained from Pseudomonas (2, 17) and Azoto-
bacter (9, 10, 19) catalyzes a reversible reduction of NAD+ by NADPH,
which is specific for the 4B hydrogen of N A D ( P ) H (6, 7, 18); both
enzymes show a broad pH optimum with a maximum around 7 (1, 19).
The degree of reversibility appears to vary depending on the source of
the enzyme. The reaction catalyzed by the Pseudomonus enzyme is less
readily reversible than that catalyzed by the Azotobacter enzyme, al-
though this discrepancy may be diminished by specific activators. Thus,
Kaplan et al. (3,21, 22) found that 2'-AMP and other nucleotides were
specific and efficient allosteric activators of both the Pseudomoms and
Azotobacter transhydrogenases ; compounds lacking a ribose 2-phosphate
substituent did not activate (21,22). I n the presence of activators, high
concentrations of NADP+ or PI were inhibitory, suggesting a competitive
relationship between these inhibiting and activating agents.
Rydstrom et al. (23) demonstrated that reduction of NADP' by
NADH, catalyzed by a partially purified Pseudomonus transhydroge-
nase, was specifically activated by Ca2+even in the absence of 2'-AMP
and analogous compounds. They found, in addition, that the specificity
for the activating nucleotide was altered in the presence of low concen-
trations of Ca2+.A similar Caz+-dependentstimulation of the Azotobacter
transhydrogenase is indicated by data reported by van den Broek and
Veeger (9) and by van den Broek et al. (19). I n detailed investigations
by van den Broek et al. (9,19) and by Chung (lo), using highly purified
transhydrogenase preparations, the Azotobacter enzyme, in contrast to
23. J. Rydstrom, J. B. Hoek, and B. Hojeberg, BBRC 52,421 (1973).

the Pseudomoms enzyme, was found to be activated by 2'-AMP only

under certain conditions.
Other reactions catalyzed by the Pseudomonas enzyme are transfer
of hydrogen from NADPH to thio-NADP', thio-NAD+, pyridine-alde-
hyde-NAD+, deamino-NAD', acetyl-pyridine-NAD+, ethylpyridylketone,
NMN (17, 24) and 3'-NADP+ (25, 26a). These reactions are activated
by 2'-AMP provided that the concentration of NADPH is not saturating.
However, when NADH serves as hydrogen donor 2'-AMP has to be pres-
ent as activator. I n the case of the Azotobacter transhydrogenase thio-
NAD' and thio-NADP', but not acetyl-pyridine-NAD+ and pyridine-
NAD' (19, 26), may be used as hydrogen acceptors. Exchange of
NADPH for NADH as hydrogen donor does not alter substantially the
specificity for the hydrogen acceptor. Reactions catalyzed by transhy-
drogenases from Chromatium (16) and spinach (11) appear to be poorly
reversible and 2'-AMP has only inhibitory effects in these cases. The
stereospecificities and the sensitivities to Ca2+have not been tested with
the Chromatium and spinach transhydrogenases. Likewise, the substrate
specificities of these transhydrogenases were not investigated in detail,
but appear to resemble those of the Pseudomoms enzyme (11, 16). The
transhydrogenase activity of spinach ferredoxin-NADP reductase is sub-
ject to regulation by ferredoxin (13) which, depending on pH, affects
both maximal velocity and affinities for the substrates.
I n addition to transfer of hydrogen between various nicotinamide
nucleotides, the transhydrogenases from Pseudomoms (17), spinach (11,
IS), and Azotobacter (9, 19) also catalyze a diaphorase reaction, using
either NADH or NADPH plus an artificial acceptor, e.g., potassium fer-
ricyanide or dichlorophenolindophenol. As expected, 2'-AMP stimulates
the NADH-linked diaphorase reaction catalyzed by the Pseudomonas
enzyme ( 1 7 ) .
E. REACTION
MECHANISM
AND REGULATION
The reaction mechanism of the Pseudornonas and Azotobacter transhy-

drogenases has been extensively investigated. Studies of the steady-state
kinetics of Pseudomonas transhydrogenase by Cohen (97)and by Cohen
24. N. 0. Kaplan. Harvey Lect. 54, 105 (1972).

25. L. Shuster and N. 0. Kaplan, JBC 215, 183 (1955).
25a. Abbreviations as follows : thio-NAD', oxidized thionicotinamide adenine
dinucleotide; thio-NADP', oxidized thionicotinamide adenine dinucleotide phos-
phate ; and 3'-NADP', oxidized nicotinamide adenine dinucleotide 3'-phosphate.
26. H. W. J. van den Broek, Ph.D. Thesis. H. Veenman and Zonen N. V., Wagenin-
gen, 1971.
60 J. R Y D S T ~ M , J . B. HOEK, AND L. ERNSTER
and Kaplan ( 6 ) led to the proposal that the reaction proceeded by a

ping-pong bi-bi mechanism. This conclusion was based on experiments
with reduction of thio-NAD' by NADPH in the absence and in the pres-
ence of 2'-AMP, which gave parallel double reciprocal plots. The data
presented by Cohen (27) and by Cohen and Kaplan ( 6 ) did not allow,
however, an elimination of a ternary complex mechanism, with a dissocia-
tion constant for the first substrate-enzyme complex close to zero. I n fact,
a ternary complex, Theorell-Chance mechanism, was later reported as
an alternative mechanism (7). Similar results were obtained by van den
Broek and Veeger (9, 28) and van den Broek (26) for the reduction of
thio-NAD+ by NADH and NADPH, catalyzed by the Azotobacter trans-
hydrogenase. The latter authors found, however, that inhibition of the
above reactions by NADP', although complicated by substrate inhibition,
appeared to give different patterns. Inhibition of the NADPH-
thio-NAD' reaction by NADP' was dependent on both donor and accep-
tor concentrations. I n the presence of high concentrations of NADPH
and low concentrations of NADP+ and thio-NAD+, the inhibition was
linear and noncompetitive with respect to NADPH. At increasing accep-
tor concentrations there appeared to be a competitive relationship be-
tween NADPH and NADP', which, at high concentrations of NADP'
(60 a), was nonlinear and showed a second-order dependence on
NADPH. Inhibition of the NADH-thio-NAD' reaction by NADP' was
linear and showed a noncompetitive relationship between NAD' and
NADH and a competitive relationship between NAD+ and thio-NAD'.
Inhibition by NADP a t high acceptor concentrations was noncompetitive
with respect to NADH, whereas lower acceptor concentrations resulted
in nonlinear plots. At low NADP concentration there was an uncompeti-
tive inhibition with respect to thio-NAD+, which, a t higher NADP' con-
centrations, became noncompetitive.
Essentially the same inhibition patterns were obtained by Chung (10)
who proposed the existence of separate hydrogen donor and hydrogen
acceptor sites. On the basis of the primary plots and the inhibition pat-
terns, van 'den Broek and Veeger (9, 28) and van den Broek (26) con-
sidered the ping-pong bi-bi mechanism unlikely and suggested an alterna-
tive ternary complex mechanism involving two donors, two acceptors,
and a four-equivalent reduced state of the enzyme. Reduction of
NADP+ and thio-NAD (P) was proposed to proceed according to a rapid-
equilibrium random bi-bi mechanism.
Inhibition data on the Pseudomoms transhydrogenase reported by
27. P. T. Cohen, Ph.D. Thesis, TJniversity of Michigan, Ann Arbor (University
Microfilms, Ann Arbor, No. 67-16542), 1967.
28. H. W. J. van den Broek and C. Veeger, Eur. J . Biochem. 24, 72 (1971).
Cohen (27) and by Cohen and Kaplan (6) appear to be similar to those
obtained with the Azotobacter transhydrogenase. Thus, with NADH as
hydrogen donor and thio-NAD+ as acceptor, inhibition by NAD' was
competitive with respect to thio-NAD+ and noncompetitive with respect
to NADH. As seen with the Azotobacter transhydrogenase, inhibition by
NADP+ was more complicated than that by NAD'. At low concentra-
tions NADP' appeared to be competitive with NAPH, whereas higher
concentrations of NADP' gave a noncompetitive pattern. In the presence
of 2'-AMP and with NADPH as donor and thio-NAD' as acceptor, in-
hibition by NADP' gave parabolic curves. These various types of inhibi-
tion by NADP+ were interpreted to indicate that NADP' is a less effec-
tive activator than 2'-AMP or NADPH and that NADP+ displaces either
NADPH or 2'-AMP from a regulatory site. I n addition, the inhibitory
effect of NADP+ was proposed by Cohn (27) and by Cohn and Kaplan
( 6 ) to depend partly on the formation of a dead-end complex with the
oxidized form of the enzyme. In this connection i t should be pointed out
that the pronounced activating effect of Ca2+on the reduction of NADP+
by NADH catalyzed by the Pseudomoms transhydrogenase, reported by
Rydstrom et al. (B), occurred in the absence of 2'-AMP, and that satu-
ration with Ca2+abolished completely the inhibitory action of NADP'.
Moreover, in the presence of nonsaturating concentrations of Ca2+,acti-
vation by 2'-AMP or NADPH was considerably more efficient than in
the absence of Ca2+.It seems likely that Ca2+may prove to be a useful
tool in clarifying the mechanism of the reaction.
Additional evidence for the existence of multiple binding sites, includ-
ing a possible regulatory site, has been obtained from several independent
experiments. In the case of the Pseudomoms transhydrogenase the
parallel primary double reciprocal plots of reduction of thio-NAD' by
NADPH revealed a second-order dependence on NADPH but a first-
order dependence with respect to thio-NAD+ (6, 27). Accordingly, two
molecules of NADPH appeared to be bound to the enzyme in the course
of the reaction, possibly one to an active site and one to a regulatory
site. In the presence of 2'-AMP, reduction of thio-NAD' by NADPH
showed a first-order dependence on NADPH suggesting that the hypo-
thetical regulatory site now was occupied by 2'-AMP. The existence of
a single binding site for both hydrogen donor and hydrogen acceptor is
supported by experiments with 3'-NADP+ (see 8). With NADB as hydro-
gen donor and thio-NAD+ as acceptor, the enzyme was rather inactive
in the absence of activators. Addition of 2'-AMP increased the maximal
velocity, with a simultaneous pronounced increase of the affinity for
NADH; the change of the affinity for thio-NAD+ was less dramatic (6,
27). After addition of 2'-AMP maximal stimulation of the velocity was
reached with a half-time of about 50 msec ( d l ) . The existence of two

separate binding sites in Azotobacter transhydrogenase is strongly sup-
ported by data reported by van den Broek (26)and by van den Broek
et al. (299). Spectral titrations indicated that the two NADP+-enzyme
complexes, besides having different conformations (as evident from C D
and ORD measurements), also have markedly different dissociation
constants, the low affinity complex (Kd< 100 &) absorbing in the
300-400 nm region and the high affinity complex (& = 2-3 a)
ab-
sorbing in the visible region (29). The thio-NADP+-enzyme complex has
a much higher dissociation constant (&- 90 &) than the
NADP+-enzyme complex ; this difference may provide an explanation for
the weak inhibitory effect of thio-NADP+ as compared to that of NADP'.
Neither thio-NADP+ nor NADP+ could be bound to the apoprotein.
Boger (14) showed that the transhydrogenase activity catalyzed by
ferredoxin-NADP reductase obtained from Bumilleriopsis filiformis,
which is very similar to the spinach enzyme, is regulated by ferredoxin
and that one common nicotinamide nucleotide binding site is involved
in both the diaphorase and the transhydrogenase reactions.
111. AB-Speciflc Transhydrogenares
A. HISTORICAL
The discovery of nicotinamide nucleotide transhydrogenase in certain
bacterial extracts by Kaplan and co-workers (1) led to a search for the
enzyme in other bacteria as well as in various mammalian organs. I n
1953,Kaplan et al. (30) demonstrated the presence of transhydrogenase
in beef heart homogenate. I n contrast to the soluble enzyme from Pseudo-
m o m s aeruginosa ( 1 ) the beef heart enzyme appeared to be insoluble
and firmly bound to the mitochondria1 membrane (31-35), although a
partial solubilization was accomplished by treatment with digitonin, a
nonionic detergent ( 3 0 ) .Furthermore, differences between the soluble and
29. H. W. J. van den Broek, J. S. Santema, and C. Veeger, Eur. J. Biochem. 24,
55 (1971).
30. N. 0. Kaplan, S. P. Colowick, and E. F. Neufeld, JBC 205, 1 (1953).
31. N. 0. Kaplan, M. N. Swartz, M. E. Frech, and M. M. Ciotti, Proc. N u t .
Acad. Sci. U.S. 42, 481 (1956).
32. G. F. Humphrey, BJ 65, 546 (1957).
33. T. M. Devlin, JBC 234, 962 (1958).
34. W. W. Kielly and J. R. Bronk, JBC 230,521 (1958).
35. W. C. McMurray, G . F. Maley, and M. A. Lardy, JBC 230, 219 (1958).
insoluble enzymes with respect to kinetic and regulatory properties (30)

indicated that the enzymes were unrelated mechanistically.
The first indication of the existence of an energy-linked NADP’ reduc-
tion was brought forward by Krebs (36) who found that NADPH-
dependent dehydrogenase reactions in pigeon heart homogenate were.sen-
sitive to dinitrophenol. This finding formed the basis for the postulate
(36) that NADP+ was reduced by an ATP-controlled flavoprotein.
Klingenberg and Slenczka (20) measured the steady-state reduction
levels of NAD and NADP in intact mitochondria and found that,
indeed, NADP was always much more reduced than NAD. These obser-
vations led to the hypothesis (20) that mitochondria contain an asym-
metric energy-linked transhydrogenase which uses oxidative energy for
transfer of hydrogen from NADH to NADP’. Later findings by
Estabrook and Nissley (37) and others ( 3 8 4 5 ) provided further support
for this hypothesis.
36. H. A. Krebs, Bull. Johns Hopkins Hosp. 95, 34 (1954).
37. R. W. Estabrook and S. P. Nissley, in “Functionelle und morphologische
Organisation der Zelle” (P. Karlson, ed.), p. 119. Springer-Verlag, Berlin and New
York, 1963.
38. M. Klingenberg and P. Schollmeyer, Proc. Znt. Congr. Biochem., 6th, 1961
Vol. 5, p. 46 (1963).
39. E. C. Slater and J. M. Tager, in “Energy-Linked Functions of Mitochondria”
(B. Chance, ed.), p. 97. Academic Press, New York, 1963.
40. J. M. Tager, BBA 77,258 (1963).
41. R. W. Estabrook, R. W. Hommes, and J. Gonze, in “Energy-Linked Functions
of Mitochondria” (B. Chance, ed.), p. 143. Academic Press, New York, 1963.
42. J. M. Tager and E. C. Slater, BBA 77,227 (1963).
43. J. M. Tager, J. L. Howland, and E. C. Slater, BBA 77, 266 (1963).
44. M.Klingenberg, in “Energy-Linked Functions of Mitochondria” (B. Chance,
ed.), p. 129. Academic Press, New York, 1963.
45. M. Klingenberg, H. van Haefen, and G. Wenske, Biochem. 2.343, 452 (1965).
46. M. Klingenberg, Biochem. Z. 343,479 (1965).
47. K. van Dam and M. F. ter Welle, Regul. Metab. Processes Mitochondria,
Proc. Symp., 1966 BBA Libr., Vol. 7,p. 235 (1966).
48. K. van Dam, Ph.D. Thesis, University of Amsterdam, Jacob van Campen,
Amsterdam, 1966.
49. E. J. de Haan, J. M. Tager, and E. C. Slater, BBA 131, 1 (1967).
50. S. Papa and A. Francavilla, in “Mitochondria1 Structure and Compartmenta-
tion” (E. Quagliariello et al., eds.), p. 363. Adriatica Editrice, Bari, 1967.
51. S. Papa, J. M. Tager, A. Francavilla, E. J. de Haan, and E. Quagliariello,
BBA 131, 14 (1967).
52. D. G. Nicholls and P. B. Garland, BJ 114, 215 (1969).
53. J. M. Tager, S. Papa, E. J. de Haan, R. D’Aloya, and E. Quagliariello, BBA
172, 7 (1969).
54. S. Papa, J. M. Tager, A. Francavilla, and E. Quagliariello, BBA 172, 20 (1969).
55. S. Papa, in “Energy-Level and Metabolic Control in Mitochondria” (S.Papa
et al., eds.), p. 401.Adriatica Editrice, Bari, 1969.
More direct evidence for an energy-linked transhydrogenase reaction

was furnished by Danielson and Ernster (66-68)who showed that respi-
ratory energy or ATP markedly stimulated the rate of conversion of
NADH and NADP+ into NAD’ and NADPH as catalyzed by submito-
chondrial particles. Thermodynamically, energization led to a shift of
the apparent equilibrium toward an extensive reduction of NADPH (69).
Later, a similar energy-linked transhydrogenase reaction was demon-
strated with membrane fragments of both respiring (60-63) and photo-
synthetic bacteria (64, 66). The properties of these so-called AB-specific
transhydrogenases (see Section I), particularly those of the beef heart
enzyme, and their relationship to the energy-supplying systems, have at-
tracted considerable attention (see 66-72). Because of the direct and
striking kinetic and thermodynamic effects of energy on the transhydro-
genase reaction, this reaction has been widely used as a probe for low
energy levels in studies concerning energy coupling in both mammalian
and bacterial systems (see 7 3 ) .The properties and function of mitochon-
drial transhydrogenase have been reviewed previously by Ernster and
Lee (74).
B. OCCURRENCE
AB-Specific transhydrogenase was originally discovered in beef heart
by Kaplan et al. (SO) but was also found in kidney, liver, other muscle
56. L. Danielson and L. Ernster, BBRC 10, 91 (1963).
57. L. Danielson and L. Ernster, Biochem. Z . 338, 188 (1963).
58. L. Danielson and L. Ernster, in “Energy-Linked Functions of Mitochondria”
(B. Chance, ed.), p. 157. Academic Press, New York, 1963.
59. C. P. Lee and L. Ernster, BBA 81, 187 (1964).
60. P. S. Murthy and A. F. Brodie, JBC 239,4292 (1964).
61. R. J. Fisher and D. R. Sanadi, BBA 248, 34 (1971).
62. P. D. Bragg and C. Hou, Can. J . Biochem. 46,631 (1968).
63. A. J. Sweetman and D. E. Griffiths, BJ 121, 125 (1971).
64. D. L. Keister and N. J. Yike, BBRC 24,510 (1966).
65. D. L. Keister and N. J. Yike, Biochemistry 6, 3847 (1967).
66. L. Ernster and C. P. Lee, “Methods in Enzymology,” Vol. 10, p. 738, 1967.
67. J. Rydstrom, A. Teixeira da Cruz, and L. Ernster, Eur. J. Biochem. 17, 56
(1970).
68. A. Teixeira da Crua, J. Rydstrom, and L. Ernster, Eur. J. Biochem. 23, 203
(1971).
(1971).
70. J. Rydstrom, Eur. J. Biochem. 31, 496 (1972).
71. J. Rydstrom, Ph.D. Thesis, University of Stockholm (Chem. Commun. No.
VII), Stockholm, 1972.
72. J. Rydstrom, Eur. J. Biochem. 45,67 (1974).
73. F. Gibson and G. B. Cox, Essays Biochem. 9, 1 (1973).
74. L. Ernster and C. P. Lee, Annu. Rev. Biochem. 33,729 (1964).
tissues (30, 32, 7 5 ) , and arterial tissue ( 7 6 ) . Different animals showed

large discrepancies in distribution of transhydrogenase activity in various
organs (30, 3 2 ) , although heart muscle generally contained the highest
activity (30, 32, 7 5 ) . Tissues such as brain, prostate, seminal vesicle,
spleen, and testis showed low or negligible activities (30, 32, 7 5 ) . Cul-
tured cells from rat hepatoma contain a transhydrogenase that appears
to be similar to that in beef heart ( 7 7 ) .Subcellular distribution studies
have shown that transhydrogenase is localized in the mitochondria
(31-35) where it is tightly bound t o the inner membrane (31-35, 56-68,
78) with its nicotinamide nucleotide binding site(s) exposed to the
matrix. The latter is evident from the known impermeability of the mito-
chondrial inner membrane to nicotinamide nucleotides (79) and from the
fact that “inside out” sonic submitochondrial particles (56-58), but not
intact mitochondria ( 7 5 ) ,catalyze a rapid transhydrogenation with ex-
ternally added nicotinamide nucleotides. Further support for a superficial
location of the active site (s) of the transhydrogenase in submitochondrial
particles is provided by the high sensitivity of the enzyme to trypsin
(8 0) . As pointed out by Sweetman et al. (81) this does not exclude that
in intact mitochondria part of the transhydrogenase molecule, different
from the nicotinamide nucleotide binding site(s), may be exposed to the
intermembrane space.
Steroid-metabolizing mitochondria, e.g., those from beef adrenal cortex
(75, 82-84) and porcine corpus luteum (86, 86), were recently shown
to contain an active energy-linked transhydrogenase that appears to play
a role in steroid hydroxylation reactions (see Section I V ) . Under proper
assay conditions all mitochondria1 transhydrogenases hitherto described
may be coupled to respiratory energy or ATP.
Among respiring bacteria evidence for an energy-linked transhydroge-
nase was provided by Murthy and Brodie (60) and by Fisher and Sanadi
(61) with membrane fragments from Escherichia coli. A similar but ap-
parently non-energy-linked transhydrogenase was also found in Myco-
A. M. Stein, N. 0. Kaplan, and M. M. Ciotti, JBC 234, 979 (1959).
75.
V. K. Kalra and A. F. Brodie, BBRC 51, 414 (1973).
76.
C. DeLuca and R. P. Gioeli, Can. J. Biochem. 50,447 (1972).
77.
T. Kawasaki, K. Satoh, and N. 0. Kaplan, BBRC 17, 648 (1964).
78.
A. L. Lehninger, Harvey Lect. 49, 174 (1955).
79.
K. Juntti, U.-B. Torndal, and L. Ernster, i,n “Electron Transport and Energy
80.
Conservation” (J. M. Tager et al., eds.), p. 257. Adriatica Editrice, Bari, 1969.
81. A. d. Sweetman, A. P. Green, and M. Hooper, BBRC 58, 337 (1974).
82. B. JV. Harding and D. H. Nelson, Endocrinology 75,506 (1964).
83. W. Cammer and R. W. Estabrook, ARB 122,721 (1967).
84. S. B. Oldham, J. J. Bell, and B. W. Harding, ABB 123, 469 (1968).
85. J. Robinson and P. M. Stevenson, Eur. J . Biochem. 24, 18 (1971).
86. V. I. Uigiris, E. N. McIntosh, C. Alonso, and H. A. Salhanick, Biochemistry
10, 2916 (1971).
66 J. RYDSTR~M,J. B. HOEK, AND L. ERNSTER
bacterium phlei (60). Photosynthetic bacteria, e.g., Rhodospirillum

rubrum (64, 66), Rhodopseudomonas spheroides (87) , Rhodopseudo-
monas v i d i s (66))Rhodopseudomonas palustris (66),and Rhodo-
spirillum molischiunum (66) contain a transhydrogenase that may be
)
driven by light. Occurrence of an ATP-supported transhydrogenase was

also reported in phosphorylating membrane fragments from Micrococcus
denitrificans (88,89).
Little is known about transhydrogenase in plants and yeast. Chloro-
plasts from spinach do contain a transhydrogenase, but this activity is
most likely related to the ferredoxin-NADP’ reductase (11-13; see 66)
in these chloroplasts. Hasson and West (90) reported on the interesting
finding of a microsomal ATP-stimulated and 2,4-dinitrophenol-sensitive
transhydrogenase in the endosperm of seeds of the immature cucumber
Echinocystis macrocarpa. Sonic submitochondrial particles from Sac-
charomyces cerevisiae contain a very low transhydrogenase activity (91)
that does not appear to be energy-linked (98). Harlow et al. (93) demon-
strated the presence of energy-linked transhydrogenase in the protozoan
Entamoeba histolytica.
AND ASSAY
C. PREPARATIONS
Mitochondria1 nicotinamide nucleotide transhydrogenase can be esti-
mated in the intact organelle either by removing aliquots and determining
the individual oxidized and reduced nicotinamide nucleotides , (20) or by
measuring changes of the intrinsic absorption (80, 37) or fluorescence
(37) of endogenous reduced nicotinamide nucleotides. As a result of vari-
ous interfering NAD (P)-dependent reactions and lack of suitable sub-
strate-regenerating systems in intact mitochondria, these types of assays
are often inadequate for determinations of absolute rates (see Section
IV). With intact bacteria the assays are still more complicated since
the levels of total NADP generally are very low (94). In fact, no at-
tempt to estimate transhydrogenase activity in intact bacteria has so far
been reported.
87. J. A. Orlando, D . Sabo, and C. Curnyn, Plant Physiol. 41, 937 (1966).
88. A. Asano, K. Imai, and R. Sato, Seikagaku 37,647 (1965).
89. A. Asano, K. Imai, and R. Sato, BBA 143,477 (1967).
90. E. P. Hasson and C. A. West, ABB 155,258 (1973).
91. J. Rydstrom and E. Ross, unpublished observation (1974).
92. G. Schatr and E. Racker, BBRC 22,579 (1966).
93. D. R. Harlow, E. C. Weinbach, and L. S. Diamond, Comp. Biochem. Biophy8.
(in press).
94. A. F. Brodie and D. L. Gutnick, “Electron Transfer in Biological Systems,”
Vol. lB, p. 699. Dekker, New York, 1971.
In order to circumvent the permeability barrier in mitochondria and

bacteria to nicotinamide nucleotides, and make possible a reliable assay
with added substrates as well as purification, the inner membrane and
cell wall, respectively, must be broken. Thus, Kaplan e t al. (SO) em-
ployed mechanical disruption by means of a Waring blender to disrupt
beef heart tissue and supposedly also mitochondria. Detergents, e.g., digi-
tonin, and subsequently calcium gel adsorption were introduced by
Kaplan et al. (SO) for further fractionation of the beef heart submito-
chondrial particles, resulting in a preparation that was about 10 times
more active than the whole homogenate. More extensive purification
failed because of the high sensitivity of the enzyme to lipid-removing
agents such as acetone and bile salts (SO, 95). Extraction with digitonin
followed by ammonium sulfate fractionation was later pursued by Hum-
phrey (32) who did not achieve, however, significantly higher specific
activities. I n more elaborate attempts, Kaufman and Kaplan (96) and
Kaplan (97) reported on a 14-fold and a 25-fold purification, respec-
tively, relative to digitonin-treated mitochondria. The latter preparation
was obtained by repeated sucrose-density gradient centrifugation of the
digitonin extract. Again, further purification was hampered by the high
sensitivity of the enzyme to organic solvents, bile salts, and phospho-
lipases (95, 9’7). Similar findings were reported by Kramar and Salven-
moser (98) and by Salvenmoser and Kramar (99; see also 100).
Disruption of mitochondria by means of sonication (101) has proved
to be an excellent method for preparing submitochondrial particles that
carry out efficient phosphorylation (34, 35).As demonstrated by Daniel-
son and Ernster (SS-SS), these particles catalyze a rapid reduction of
NADP’ by NADH supported by either respiration or ATP. Depending
on the composition of the sonication medium either nonphosphorylating
or phosphorylating particles are obtained which drive respiration-sup-
ported transhydrogenase and respiration ; and ATP-supported trans-
hydrogenase, respectively (102-104). By the addition of low amounts
of oligomycin to the nonphosphorylating particles the capacity for phos-
95. N. 0. Kaplan, “Methods in Enzymology,” Vol. 2, p. 681, 1955.

96. B. Kaufman and N. 0. Kaplan, JBC 236,2133 (1961).
97. N. 0. Kaplan, “Methods in Enzymology,’’ Vol. 10, p. 317, 1967.
98. R. Kramer and F. Salvenmoser, Hoppe-Seyler’s Z. Physiol. Chem. 346, 310
(1966).
99. F. Salvenmoser and R. Kramar, Enzymologiu 40,322 (1971).
100. R. Kramar, M. Miiller, and F. Salvenmoser, BBA 162,289 (1968).
101. G. H. Hogeboom and W. C. Schneider, N u t w e (London) 166, 302 (1950).
102. C. P. Lee, G. F. Azzone, and L. Ernster, Nature (London) 201, 152 (1964).
103. C. P. Lee and L. Ernster. “Methods in Enzymology,” Vol. 10, p. 543, 1967,
104. R. J. Fisher, B. P. Sani, and D. R. Sanadi, BBRC 44, 1394 (1971).
phorylation as well as ATP-supported transhydrogenation is restored

(106,106).
Recently, Rydstrom et al. (107) demonstrated that transhydroge-
nase was solubilized efficiently and selectively from beef heart sonic sub-
mitochondria1 particles by lysolecithin, giving a preparation that was
about seven times as active as particles. In contrast to earlier prepara-
tions this lysolecithin extract was devoid of all cytochromes except cyto-
chrome c.
A number of preparations of various bacterial membrane fragments
exerting energy-linked transhydrogenase activities have been described.
The use of sonication (SO-SS),French press (64,66),Ribi cell fraction-
ator (108),and grinding with sand (109)give preparations that generally
are less active with respect to both non-energy-linked and energy-linked
transhydrogenase activities than submitochondrial particles. Of particu-
lar interest are chromatophores from Rhodospirillum rubrum from
which a protein can be isolated that is necessary for both non-energy-
linked and energy-linked transhydrogenase activities of the chromato-
phores; the isolated factor itself has no measurable activity (109-113).
Protein factors have also been isolated from both Rhodopseudomonas
spheroides (114, 116) and Escherichia coli (116).However, both of these
proteins appear to influence only the energy-linked transhydrogenase re-
action and may therefore function as a general energy-coupling factor
rather than a specific transhydrogenase factor (see 117).
Assay of AB-specific transhydrogenase is principally identical to that
of BB-specific transhydrogenase (see Section I1,C) ; i.e., with the natural
nicotinamide nucleotides, an enzymic regenerating system is used to keep
the concentration of one of the substrates constant (SO, 39, 6648,66)
105. C. P. Lee and L. Ernster, BBRC 18,523 (1965).
106. C. P. Lee and L. Ernster, Regul. Metab. Processes Mitochondria, Proc. Symp.,
2966 BBA Libr., Vol. 7, p. 218 (1966).
107. J. Rydstrom, J. B. Hoek, and T. Hundal, BBRC 60, 448 (1974).
108. G. B. Cox, F. Gibson, L. M. McCann, J. D. Butlin, and F. L. Crane, BJ
132, 689 (1973).
109. R. R. Fisher and R. J. Guillory, FEBS (Fed. Eur. Biochem. Soc.) Lett. 3,
27 (1969).
110. R. R. Fisher and R. J. Guillory, JBC 244, 1078 (1969).
112. R. R. Fisher and R. J. Guillory, JBC 246,4687 (1971).
113. A. W. T. Konings and R. J. Guillory, JBC 248, 1045 (1973).
114. J. A. Orlando, ABB 141, 111 (1970).
115. T. J. Berger and J. A. Orlando, ABB 159,25 (1973).
116. P. D. Bragg and C. Hou, FEBS (Fed. Eur. Biochem. Soc.) Lett. 28, 309
(1972).
117. A. W. T. Konings and R. J. Guillory, BBA 283, 334 (1972).
2. NICOTINAMIDE NUCLWTIDE TRANSHYDROGENASES 69
and a suitable energy source is added. Nicotinamide nucleotide analogs

of NADP', i.e., thio-NADP', and of NAD', i.e., acetyl-pyridine-NAD',
may be used in the absence of a regenerating system (78, 96,118).Inter-
actions between the transhydrogenase system and the energy pool of the
membrane fragments used can be follwed by 8-anilinonaphthalene l-sul-
fonate fluorescence (72, 119) or by active transport of lipid-soluble anions
(120).In Rhodospirillum rubrum chromatophores this type of interac-
tion can also be monitored by spectral changes of carotenoids (121).
D. MOLECULAR
PROPERTIES
The transfer of hydrogen between NAD(H) and NADP(H), catalyzed
by mitochondrial transhydrogenase, occurs without exchange with the
hydrogen atoms of the surrounding water phase (106, 122, 123). The en-
zyme is stereospecific for the 4A hydrogen of NADH and the 4B hydro-
gen of NADPH (78, 106,122, 123). The same stereospecificity has been
reported for the transhydrogenases of Escherichia coli ( 8 ) and Rhodo-
spirillum rubrum (112). It has been proposed (8, 68-71) that AB-specific
transhydrogenases have separate binding sites for NAD ( H ) and
NADP(H) (see also Section II1,F).
The natural nicotinamide nucleotides NADP+ and NAD' can be ex-
changed for various substrate analogs (118; see also 6 5 ) , e.g., thio-
NADP' and acetyl-pyridine-NAD+, respectively. The activities with
these analogs vary substantially and 3'-analogs of NADP are virtually
inactive (8, 7 0 ) .It should be pointed out that "transhydrogenation" (see
Section I) between NADH and acetyl-pyridine-NAD', catalyzed by im-
pure transhydrogenase preparations, is attributable to NADH dehydroge-
nase activity (124, 125) rather than to transhydrogenase activity (97,
100).
A variety of inhibitors of mitochondrial transhydrogenase have been
described, some of which are relatively unspecific, such as various SH
118. R. R. Fisher and N. 0. Kaplan, Biochemistry 12,1182 (1973).
119. R. J. van de Stadt, F. J. R. M. Nieuwenhuis, and K. van Dam, BBA 234,
173 (1971).
120. L. L. Grinius, A. A. Jasaitis, Y. P. Kadziauskas, E. A. Liberman, V. P.
Skulachev, V. P. Topali, L. M. Tsofina, and M. A. Vladimirova, BBA 216, 1 (1970). .
121. S. A. Ostroumov, V. D. Samuilov, and V. P. Skulachev, FEBS (Fed. Eur.
Biochem. Soc.) Lett. 31, 27 (1973).
122. C. P. Lee, N. Simard-Duquesne, L. Ernster, and H. D. Hoberman, BBA 105,
397 (1965).
123. D. E. Griffiths and A. M. Roberton, BBA 118,453 (1966).
124. T. Cremona and E. B. Kearney, JBC 240,3645 (1965).
125. M. Gutman, H. Mersmann, J. Luthy, and T. Singer, Biochemistry 9, 2678
(1970).
reagents (32, 96), triiodothyronine (41), Mg2+ (59,67, 186, 1271, Ca2+
(127), Mn2+(127),or D,O (72,128). Specific transhydrogenase inhibitors
include various adenine nucleotides which compete with the substrates
of the enzyme. I n the case of the AB-specific transhydrogenases those
inhibitors display a site specificity in the sense that 2’- or 3’-substituted
adenine nucleotides are competitive with respect to NADP(H) , and
adenine nucleotides without such a substituent are competitive with re-
spect to NAD(H) (70,129, 130). I n addition, it is found (70,129, 130)
that inhibitors competitive with NADP (H) show increasing potency with
increasing hydrophobicity. For example, palmityl-CoA, which is a com-
petitive inhibitor of AB-specific transhydrogenases with respect to
NADPH(H), is considerably more potent than CoA (70,129, 130). I n
contrast, palmityldephospho-CoA, which is a competitive inhibitor with
respect to NAD ( H ), is only slightly more potent than dephospho-CoA
(70).This pattern is suggestive of a hydrophobic environment of the
NADP ( H )-binding site, and a relatively hydrophilic environment of the
NAD (H)-binding site of the enzyme. A survey of different site-specific
inhibitors of mitochondrial transhydrogenase-which seems to be valid
for other Al3-specific transhydrogenases as well (8)-is shown in Table
11.
Further characterization of AB-specific transhydrogenase has so far
been hampered by the lack of a purified enzyme and most of the available
information on the properties of AB-specific transhydrogenase has there-
fore been obtained using various preparations of membrane fragments.
The partially purified transhydrogenase from beef heart was reported by
Kaufman and Kaplan (96) and by Kaplan (97)to be a complex lipo-
protein with a molecular weight of about 250,000; no flavin could
be detected (97).Being a membrane-bound protein and presumably a
lipid-dependent enzyme, it is not surprising that mitochondrial transhy-
drogenase is highly sensitive to lipid-removing agents such as detergents
(30, 96,98,107),organic solvents (30,96), and phospholipases (30, 131,
132). Direct evidence for a lipid dependence of transhydrogenase is lack-
ing as yet. However, lecithin has been shown by Pesch (13.9) to be stimu-
126. A. Hommes, in “Energy-Linked Functions of Mitochondria” (B. Chance, ed.),
p. 39. Academic Press, New York, 1903.
127. T. E. Andreoli, R. L. Pharo, and D. R. Sanadi, BBA 90, 16 (1964).
128. S. A. Margolis, H. Baum, and G. Lenaz, BBRC 25, 133 (1966).
129. J. Rydstrom, A. V. Panov, G. Paradies, and L. Ernster, BBRC 45, 1389 (1971).
130. J. Rydstrom, J. B. Hoek, R. Alm, and L. Ernster, in “Mechanisms in Bioener-
getics” (G. F. Azzone et al., eds.), p. 579. Academic Press, New York, 1973.
131. L. A. Pesch and J. Peterson, BBA 06, 390 (1965).
132. V. N. Luzikov, V. V. Kupriyanov, and T. A. Makhlis, Bioenergetics 4, 521
(1973).
133. L. A. Pesch, BBA 81, 229 (1964).
TABLE I1
SITE-SPECIFIC INHIBITORS OFMITOCHONDRIAL
TRANSHYDROOENASE'
Non-energy- Energy-
Inhibitor Specificity linked linked
Adenosine NAD(H) 500 500

5'-AMP NAD(H) 300 700
ADP NAD(H) 300 400
Dephospho-CoA NAD(H) 9 40
Acetyl-dephospho-CoA NAD(H) 11 40
2'-AMP NADP(H) 700 1200
3'-AMP NADP(H) 700 1200
CoA NADP(H) 200 700
Acetyl-CoA NADP(H) 200 700
Palmityl-CoA NADP(H) 0.15 0.15
3':5'-AMP NADP(H) 400 500
From Rydstrom (70).

latory with a transhydrogenase preparation obtained by extraction of
beef heart mitochondria with tert-amylalchol.
Obviously, the transhydrogenase protein is very labile and difficult to
isolate, and it appears that a new technique for isolation of this type
of hydrophobic protein is required. I n this respect the use of lysolecithin,
as recently reported by Rydstrom et al. (lor), may be promising.
An interesting 'development is the isolation of a transhydrogenase fac-
tor from Rhodospirillum rubrum by Fisher and Guillory (109-115).
Its molecular weight is about 70,000, it contains essential sulfhydryl
groups and at least two subunits, and it is heat-inactivated and trypsin-
sensitive (113). Later, Hoek et al. (8) showed that the membrane compo-
nent is also trypsin-sensitive. The factor apparently contains no flavin
and has no known enzymic activity (113). Both non-energy-linked (112)
and energy-linked (including light-driven) transhydrogenase activities
of the depleted chromatophores are absent unless transhydrogenase factor
is added (109-113). Binding of the factor to the membrane component
is strongly influenced by the substrates of the transhydrogenase reaction
(111). In contrast to the transhydrogenase factor isolated from Rhodo-
pseudomonas spheroides (114, 116), the Rhodospirillum rubrum factor
cannot be replaced by thiol reagents (117).
E. RELATIONSHIP
TO THE ENERGY-COUPLING
SYSTEM
AB-Specific transhydrogenases are functionally coupled to the energy-
transfer system of the membrane in which they are located. This coupling
is manifested by an energy-dependent increase in both the rate (57, 58,

60, 64, 89) and the extent (59, 67) of reduction of NADP’ by
NADH. I n mitochondria (56-58, 106) and respiring bacteria (6043),
the transhydrogenase reaction can be driven by energy generated either
by electron transport through any of the coupling sites of the respiratory
chain or by ATP hydrolysis. It can also be driven by a potassium ion
gradient across the mitochondria1 inner membrane in the presence of
valinomycin (134). In photosynthetic bacteria, energy can be generated
either by light-induced electron transport or by the hydrolysis of inor-
ganic pyrophosphate or ATP (64, 65,87),
Inhibitors of electron transport do not affect the ATP-driven transhy-
drogenase reaction (5648, 65) whereas energy-transfer inhibitors, e.g.,
oligomycin or N,N’-dicyclohexylcarbodiimide, do not inhibit the respira-
tion-, light-, or pyrophosphate-driven reactions, but inhibit the ATP-
driven reaction (56, 57, 61, 65, 78, 108). I n so-called nonphosphorylating
submitochondrial particles, oligomycin stimulates and may even be
obligatory for the energy-linked transhydrogenase reaction driven by
either respiration or ATP (103,106,135-137).
Uncouplers of oxidative and light-induced phosphorylation abolish the
energy-linked transhydrogenase reaction (47, 58, 60-65, 89, 106). Simi-
larly, the reaction is abolished by the combined effects of valinomycin
and nigericin in the presence of potassium iops (138). I n submitochon-
drial particles it has been found that both uncouplers and oligomycin
inhibit the ATP-driven transhydrogenase less efficiently than ATP-driven
reduction of NAD’ by succinate (106, 139). On the other hand, the two
reactions were equally sensitive (139, 140) to the mitochondrial
ATPase-inhibitor protein of Pullman and Monroy ( 1 4 1 ) , and it has been
suggested (139, 140) that the transhydrogenase and ATPase interact in
a direct molecular fashion. Interestingly, in bacterial membrane particles
the ATP-driven transhydrogenase reaction has been reported to be more
uncoupler-sensitive than the succinate-linked NAD’ reduction (62, 6 3 ) .
134. E. Conover, in “Energy Transduction in Respiration and Photosynthesis”

(E. Quagliariello, S. Papa, and C. S.Rossi, eds.), p. 999. Adriatica Editrice, Bari,
1971.
135. C. P. Lee and L. Ernster, BBRC 23, 176 (1966).
136. C. P. Lee and L. Ernster, “Round Table Discussion on Mitochondria1 Struc-
ture and Compartmentation” (E. Quagliariello et al., eds.), p. 353. Adriatica Editrice,
Bari, 1967.
137. C. P. Lee and L. Ernster, Eur. J . Biochem. 3, 385 (1968).
138. M. Montal, B. Chance, C. P. Lee, and A. Azzi, BBRC 34, 104 (1969).
139. L. Ernster, K. Juntti, m d K. Asami, Bioenergetics 4,351 (1972).
140. K. Asami, K. Juntti, and L. Ernster, BRA 205, 307 (1970).
141. M. E. Pullman and G. L. Monroy, JBC 238,3762 (1963).
Substrate Oxyqen
-K
ow
Oliqomycin
I -
NAN + NADP+
’
“F“
NADH+ NAD+ “ATP
FIQ.1. Relationship of the energy-linked transhydrogenase reaction to oxidative

phosphorylation. From Ernster et al. (166).
The available information thus suggests that the energy-linked trans-

hydrogenase reaction and electron transport-linked phosphorylation util-
ize a common energy pool. A consequence of this assumption would be
that the two processes are competing for the energy available in this pool.
Experiments carried out with the purpose of demonstrating such a compe-
tition in submitochondrial particles were not successful initially (47, 48).
However, it was later shown by Lee and Ernster (135-137) that under
favorable conditions, i.e., limited respiration and the presence of saturat-
ing concentrations of NADH and NADP’, a competition indeed occurred
between energy-linked transhydrogenation and oxidative phosphoryla-
tion. It was found that oxidative phosphorylation caused an increase in
K , of the energy-linked transhydrogenase for NADH and that the
energy-linked transhydrogenase reaction caused an increase in K , of the
oxidative phosphorylation system for Pi (135-137). A schematic rela-
tionship of the energy-treated transhydrogenase reaction to oxidative
phosphorylation in mitochondria is depicted in Fig. 1.
The energy expenditure of the mitochondria1 energy-linked transhydro-
genase reaction has been estimated to one high energy bond per NADP’
reduced (57, 58, 106, 137, 142?-1&). The same stoichiometry was reported
for the energy-linked transhydrogenases in Escherichia coli (63),
Rhodospirillum rubrum (66), and Micrococcus denitrifioans (89). The
overall ATP-driven transhydrogenase reaction may thus be written as
NADH + NADP+ + ATP 6NAD+ + NADPH + ADP + P,
Since the equilibrium constant of the non-energy-linked transhydroge-
nase reaction is 0.79 (30) and that of ATP hydrolysis is about lo5 M
142. 9. Papa, A. Alifano, J. M. Tager, and E. Quagliariello, BBA 153, 303 (1968).
143. D. W. Haas, BBA 82,200 (1984).
144. J. M. Tager, G . S. P. Groot, D. Roos, 8. Papa, and E. Quagliariello, in “The
Energy Level and Metabolic Control in.Mitochondria” (S. Papa et al., eds.), p.
453. Adriatica Editrice, Bari, 1969.
74 J. RYDSTRbf, J. B. HOEK, AND L. ERNSTER
(based on a AGO of 7.3 kcal/mole; see 146),the equilibrium constant of

the above overall reaction is also of the order of lo6 M . In spite of the
very unfavorable equilibrium, van de Stadt et al. (119) have succeeded
in demonstrating a reversal of the reaction, leading to net ATP synthesis
by using a very high nicotinamide nucleotide potential (defined as
[NADPH] [NAD+]/ [NADH] [NADP+]) and efficient regenerating sys-
tems for NADPH, NAD+, and ADP. Additional evidence for the reversi-
bility of the energy-linked transhydrogenase reaction was provided by
Skulachev and associates in both submitochondrial particles (120,
146-148) and chromatophores derived from Rhodospirillum rubrum
(IN), using transport of lipophilic anions as a probe for the high energy
state.
The mechanism by which energy is transferred between the various
energy-generating systems and the transhydrogenase is not known. The
problem is intimately related to the more general problem of the mecha-
nism of energy conservation in biological membranes (for reviews, see
149, 160). Attempts have been made to explain the energy-linked trans-
hydrogenase reaction in terms of one of the three main current hypotheses
of energy conservation, namely, the chemical (161, 168) conformational
(163), and chemiosmotic (164) hypotheses. Mechanisms related to the
chemical hypothesis involve energized forms of the substrates, i.e.,
NADH (or NADP -) as considered a t an early stage by Ernster and
H
associates (6648,106, 136, 137). Protonated species of the substrates

traversing the mitochondria1 inner membrane down a transmembrane pH
gradient according to the chemiosmotic hypothesis have been proposed
by Mitchell (166,166; see also 167).Later, this hypothesis provided the
basis for more elaborate mechanisms postulated by Skulachev and co-
J. Rosing and E. C. Slater, BBA 267,275 (1972).
145.
V. P. Skulachev, FEBS (Fed. Bur. Biochem. Soc.) Lett. 11, 301 (1970).
146.
V. P. Skulachev, Curr. Top. Bioenerg. 4, 127 (1971).
147.
A. E. Dontsov, L. L. Grinius, A. A. Jasaitis, I. I. Severina, and V. P.
148.
Skulachev, Bioenergetics 3,277 (1972).
149. L. Ernster, Fed. Eur. Biochem. SOC.Symp. 35,257.
150. H. Baltscheffsky and M. Baltacheffsky, Annu. Rev. Biochem. 43, 871 (1974).
161. F. Lipmann, “Currents in Biochemical Research,” p. 137. Wiley (Interscience),
New York, 1946.
152. E. C. Slater, Nature (London) 172, 975 (1953).
153. P. D. Boyer, in “Oxidases and Related Redox Systems” (T. E. King, H. 9.
Mason, and M. Morrison, eds.), Vol. 2, p. 994. Wiley, New York, 1965.
154. P. Mitchell, Nature (London) 191,144 (1961).
155. P. Mitchell, “Chemiosmotic Coupling in Oxidative and Photosynthetic Phos-
phorylation.” Glynn Res., Bodmin, Cornwall, England, 1966.
156. P. Mitchell, Bioenergetics 3, 5 (1972).
157. J. Moyle and P. Mitchell, BJ 132,571 (1973).
workers (1.20,146-148),derived from their demonstration of membrane

potential changes accompanying the transhydrogenase reaction. Based
on the conformation hypothesis of Boyer (153),a principally different
mechanism was put forward by Rydstrom e t al. (67,69, 71; see also
1681, who proposed that energization involves a conformational change
of the transhydrogenase molecule per se. Such a mechanism, in contrast
to the previous ones, provides a satisfactory explanation for both the
kinetic and the thermodynamic features of the energy-linked transhydro-
genase reaction. The experimental basis of this mechanism is provided
by comparative studies of the steady-state kinetics of the energy-linked
a,nd non-energy-linked transhydrogenase reactions (see Section II1,F).
A conformational change is also included in the so-called electromechano-
chemical model proposed recently by Green and J i (169).According
to this hypothesis, energy-linked transhydrogenation involves a respira-
tion- or ATP-dependent polarization of a “transhydrogenase supermole-
cule,” which is discharged in the course of the transhydrogenase reaction.
Probably, the final understanding of the mechanism of the energy-linked
transhydrogenase reaction will have to await the elucidation of the mech-
anism of energy conservation in biological membranes. An the other hand,
the energy-linked transhydrogenase reaction may provide a valuable tool
for reaching this goal.
F. KINETICSAND REACTIONMECHANISM
Teixeira da Cruz e t al. (68) and Rydstrom e t al. (69, 158) have
investigated the steady-state kinetics of the non-energy-linked and
energy-linked transhydrogenase reactions catalyzed by sonic submito-
chondrial particles from beef heart. The data obtained seem to establish
clearly that the reaction proceeds by way of ternary complex of very
short lifetime, i.e., a Theorell-Chance mechanism (Fig. 2 ) . This conclu-
sion was based on linear and convergent double reciprocal plots of initial
velocities vs. substrate concentrations, as well as on product inhibition
patterns that revealed competitive relationships between the oxidized and
reduced forms of the same nicotinamide nucleotide and noncompetitive
relationships between NAD+ and NADP+ and between NADH and
NADPH. This pattern of product inhibition indicated that the transhy-
drogenase has separate binding sites for NAD(H) and N A D P ( H ) .
Studies with site-specific inhibitors (70,71) suggested, furthermore, that
NAD(H) is the first substrate bound by the enzyme. As pointed out by
158. J. Rydstrom, A. Teixeira da Cruz,and L. Emster, in “Biochemistry and Bio-
physics of Mitochondria1 Membranes” ( G . F. Azzone e t al., eds.), p. 177. Academic
Press, New York, 1972.
159. D. E. Green and S. Ji, Proc. N u t . Acud. Sci. U. S . 69, 726 (1972).
1 )H P
h
1
I I
I
5 3 I 5 5.1 5
1.4
(5.3)
10. a II 0.3
(10.2)
3.0
(2.1)
I
I
20.6
(166.0)
(-0) I
I I
I I
I I
I/ \I
E+E-NADH+NADP+- E*NADHF=: NADPH-E*NAD++ NAD+- E e~

FIG.2. Reaction mechanism of mitochondrial transhydrogenase. Rate constants
within brackets refer to the energy-linked hydrogenase; kl, k,, k,, and ks are ex-
pressed as pM-' min-', where kl and ka are expressed as min-'. Fast reactions
are indicated by dashed lines. From Rydstrom et al. (69).
Cleland (160), steady-state kinetics of a Theorell-Chance mechanism can

generally apply also to a rapid-equilibrium random mechanism with two
dead-end complexes. However, in view of the data obtained with site-
specific inhibitors this latter mechanism is unlikely in the case of the
transhydrogenase (70,7 1 ). The proposed mechanism is also consistent
with the observation of Fisher and Kaplan (118)that the breakage of
the C-H bonds of the reduced nicotinamide nucleotides is not a rate-
limiting step in the mitochondrial transhydrogenase reaction.
At neutral pH, the maximal initial velocities of the two directions of
the non-energy-linked transhydrogenase reaction differ by a factor of
about five, the reduction of NADP+ being the slower reaction (SO, 69,
68, 71, 127). Reduction of NADP+ by NADH is maximally active a t
about pH 5.5., whereas reduction of NAD+ by NADPH shows a pH
optimum a t about 7.0 (30,67, 71, 7.2; see also 3.2). When the reduction
of NADP+ by NADH approaches equilibrium, the rate constant of the
reaction is increased (67), indicating an activation of the transhydroge-
nase that is related to the accumulation of the products NAD+ and
NADPH. It has been proposed (67, 69, 71) that this activation may in-
volve a conversion of the enzyme from an inactive to an active conforma-
tional state, similar to that proposed to occur upon energization (see
below).
Michaelis constants of the non-energy-linked beef heart transhydroge-
nase reaction are 9 rJM for NADH, 40 yN for NADP+, 28 p M for NAD',
and 20 for NADPH (68); these values are similar to those found
with other AB-specific transhydrogenases (66,89; see also 8).Dissocia-
tion constants for the E-NADH and E-NAD+ complexes, derived from
160. W. W. Cleland, "The Enzymes," 3rd ed., Vol. 2, p. 1, 1970.
the steady-state kinetic data (68),are 6.8 and 10.0 @ I , The

respectively.
individual rate constants for the partial reactions that were computed
from the experimentally determined Michaelis and dissociation constants
(68)are indicated in Fig. 2. The equilibrium constant of the transhydro-
genase reaction, calculated from the Haldane relationship and based on
these rate constants, showed good agreement with the directly determined
equilibrium constant (68), especially if the value was corrected for the
difference in maximal initial velocities of the two directions of the
reaction.
Addition of an energy source in the form of a respiratory substrate
or ATP leads to a 5- to 10-fold increase in maximal initial velocity of
the reduction of NADP' by NADH (66-68, 69, 71, 127) as well as to
alterations of both the Michaelis constants for NAD(H) and NADP(H)
and the dissociation constants of the E-NADH and E N A D + complexes
(69, 71). The relative increase in maximal initial velocity is dependent
on the energy level (72, 106,155-137), the substrate concentrations (69,
7 1 ) , pH (67, 72), and is also influenced by Mg2+ (69, 67, 126).Energy-
linked changes in the Michaelis constants are particularly apparent with
the oxidized nicotinamide nucleotides, giving values of 6.5 p M and 43.5
UJM for NADP' and NAD+, respectively (69). The dissociation constants
of the energy-linked reaction are approximately 0 for the E-NADH com-
plex and 172 pLM for the E-NAD+ complex, i.e., energization promotes
the binding of NADH and the release of NAD' (69). On the basis of the
above kinetic data the rate constants of the energy-linked reaction were
calculated (Fig. 2, values in parentheses). A comparison of these
rate constants with those derived for the non-energy-linked reaction re-
veals that k, and k, are the rate constants that are most influenced by
energization. Product inhibition patterns (69) and the order of substrate
binding (70, 71) for the energy-linked reaction are the same as for the
non-energy-linked reaction.
As pointed out previously (Section III,E), the mechanism by which
the transhydrogenase reaction is linked to energy is unknown. However,
since the substrates of the transhydrogenase reaction are highly water
soluble and therefore may be assumed to interact with the enzyme in
the aqueous phase, it appears likely that the observed energy-linked
changes in dissociation constants reflect a structural alteration of the
transhydrogenase molecule per se from an inactive to an active form,
rather than alterations of the nicotinamide nucleotides. Recently, evi-
dence was presented (72) indicating that an increased proton concentra-
tion mimics the effect of energy on the kinetics of the mitochondria1
transhydrogenase, and it was suggested (72; see also 118) that the
energy-linked conversion of the inactive into the active form of the en-
78 J. RYDSTR~M, J. B. HOEH, AND L. ERNSTER
zyme may involve a protonation of a specific group (or groups) of the

enzyme.
A functional relationship between Complex I (NADH-ubiquinone re-
ductase) and transhydrogenase was suggested by Hatefi (161) and by
Hatefi and Hanstein (168),on the basis of the oxidation of NADPH
by submitochondrial particles and Complex I. These workers postulated
the presence of a specific acceptor site for NADPH in NADH dehydro-
genase which is associated with transhydrogenase (see also 163).Subse-
quently, however, Rydstrom et al. (164) pointed out that NADPH
in the absence of NAD’ could be oxidized unspecifically by NADH dehy-
drogenase (see 166) in a palmityl-CoA insensitive manner (see 70) via
the NADH-binding site of the enzyme. This explanation is consistent
with the finding (97,100, 107) that transhydrogenase may be readily
separated from NADH dehydrogenase. An additional palmityl-CoA sen-
sitive pathway for the oxidation of NADPH by artificial acceptors in
submitochondrial particles, possibly constituting a partial reaction of
transhydrogenase, has been demonstrated by Ernster et al. (166).
G. RECONSTITUTION
Because of its functional relationship to the energy-conserving system
and its ready response to low energy levels (see Section III,E), the
energy-linked transhydrogenase reaction has been recognized as a valu-
able tool for studying reconstitution, particularly in bacterial systems.
The studies reported have as a rule concerned reconstitution of the
energy-coupling system, with the transhydrogenase being present in the
particles before the addition of coupling factors.
There are two exceptions from this generalization, one of which is the
transhydrogenase factor isolated from Rhodospirillum rubrum chroma-
tophores (109-118).This factor is obligatory for both energy-linked and
non-energy-linked transhydrogenation ; its properties and function have
already been reviewed (see Section 111,D). Butlin (167;see also 73) re-
161. Y. Hatefi, BBRC 50, 978 (1973); Ann. N . Y . Acad. Sci. 227, 504 (1974).
162. Y. Hatefi and W. G . Hanstein, Biochemistry 12,3575 (1973).
163. C. I. Ragan, W. R. Widger, and T. E. King, BBRC GO, 894 (1974).
164. J. Rydstiom, J. B. Hoek,and L. Ernster, BBA 303,694 (1973).
165. C. Rosai, T. Cremona, J. M. Machinist, and T. P. Singer, JBC 240, 2634
(1965).
166. L. Ernster, C. P. Lee, and U.-B. Torndal, in “The Energy Level and Metabolic
Control in Mitochondria” (S. Papa, et al., eds.), p. 439. Adriatica Editrice, Bari,
1969.
167. J. D. Butlin, unpublished observation.
ported on a mutant of Escherichia coli K12 (nut-) that was lacking both
non-energy-linked and energy-linked transhydrogenase.
Enhancement of both ATP-driven and respiration-driven transhydro-
genase in Escherichia coli by a protein factor was reported by Bragg
and Hou (116). This factor restored both ATPase activity as well as
energy-linked transhydrogenase activity in factor-stripped membrane
fragments. An energy-transfer factor from rat liver mitochondria was
found to exert a similar stimulation on respiration-driven transhydro-
genation in Escherichia coli (168).
A different factor was isolated from Rhodopseudomoms spheroides
by Orlando (114) which only stimulated the light-driven transhydroge-
nase reaction and could be replaced by thiols (116).
Various mutant strains of Escherichiu coli are lacking Mgz+-Ca2+-acti-
vated ATPase and ATP-driven transhydrogenase but retain respiration-
driven transhydrogenase (16‘9-172). Washing of membrane fragments
obtained from an ATPase-deficient Escherichiu coli strain and subse-
quent addition of purified Mg2+-Caz+-stimulatedATPase reconstituted
the ATP-driven transhydrogenase (171) . Butlin et al. (1‘73) concluded
that two proteins specified by the unc A and unc B genes in Escherichia
coli K12 were essential for ATP-driven transhydrogenase. A mutant
deficient in cytochromes was found to have an unimpaired ATP-driven
transhydrogenase reaction (17 4 ) .
IV. Physiological Roles of Nicotinamide Nucleotide Transhydrogenases
As pointed out by Krebs and Veech (1‘76), the relationship between

the redox states of NAD and NADP in mammalian cells would be gov-
erned to a large extent by the substrate levels of NAD- and NADP-
dependent dehydrogenases, interlinked by shared reactants. The coordi-
nation of these systems of interlinked dehydrogenases and, in particular,
energy-linked transhydrogenase has been a matter of controversy. Funda-
168. R. J. Fisher, K . W. Lam, and D. R. Sanadi, BBRC 39, 1021 (1970).

169. D. L. Gutnick, B. I. Kanner, and P. W. Postma, BBA 283, 217 (1972).
170. B. I. Kanner and D. L. Gutnick, FEBS (Fed. Eur. Biochem. Soc.) Lett. 22,
197 (1972).
171. G. B. Cox, F. Gibson, L. M. McCann, J. D. Butlin, and F. L. Crane, BJ
132, 689 (1973).
172. P. D. Bragg and C. Hou, BBRC 50, 729 (1973).
173. J. D. Butlin, G. B. Cox, and F. Gibson, BBA 292, 366 (1973).
174. A. P. Singh and P. D. Bragg, BBRC 5‘7,1200 (1974).
175. H. A. Krebs and R. L. Veech, in “The Energy Level and Metabolic Control
in Mitochondria” (S. Papa et el., eds.), p. 329. Adriatica Editice, Bari, 1969.
mental to this argument is the question of the redox states of mitochon-

drial NAD and NADP in vivo, and we shall devote some attention to
this problem in one of the following subsections.
The majority of synthetic reactions in mammalian cells takes place
in the cytosol. The intramitochondrial localization of transhydrogenase
excludes a direct participation in these anabolic processes. Substrate shut-
tle mechanisms (176, 177) are required to allow for the interaction be-
tween intra- and extramitochondrial nicotinamide nucleotide-dependent
reactions, In the first instance transhydrogenase can be regarded to be
functionally related to intramitochondrial NADP-linked reactions. A
number of studies on isolated mitochondria have elaborated these rela-
tionships in some detail, in particular with regard to mitochondria1 mono-
oxygenation reactions and to the metabolism of glutamate and isocitrate.
Whereas these studies primarily emphasize the role of energy-linked
transhydrogenase in the supply of reducing equivalents at the expense
of energy, other authors (I@, 166) regard the enzyme as a component
of a fourth coupling site of the respiratory chain, which, when catalyzing
the reduction of NAD+ by NADPH, may drive ion translocations (147,
167) and ATP synthesis (119).
Even less satisfactory than in the case of mammalian transhydrogenase
is the experimental evidence suggesting specific metabolic functions for
the enrymes occurring in bacterial systems. Keister and Hemmes (16)
pointed out that the inhibitory effect of NADP+ on transhydrogenase
from Chromatium would indicate a controlling function of the enzyme
in the transfer of reducing equivalents from NADPH (produced in the
glyoxylate cycle in this organism) to NAD+, thus providing a regulatory
site for carbohydrate synthesis. The transhydrogenase from Pseudomoms
and Azotobacter, being subject to a complex allosteric regulation (see
above), could well play a comparable role in controlling the pathway
of reducing equivalents from NADPH. The close physical association
of transhydrogenase with enzymes involved in pyruvate oxidation in
Azotobacter vinelandii led van de Broek et al. (19) to suggest that the
enzyme is involved in regulating the transfer of reducing equivalents from
pyruvate to either N, or 0,.Bragg et al. (178) observed that the transhy-
drogenase activity in Escherichia coli was suppressed by the presence
of amino acids in the growth medium, and they suggested the enzyme
to be involved in amino acid synthesis in this organism. The mechanism
178. A. J. Meijer and K. van Dam, BBA 346,213 (1974).

177. P. Borst, in “Functionelle und morphologische Organization der Zelle,” p.
137. Springer-Verlag, Berlin and New York, 1963.
178. P. D. Bragg, P. L. Davies, and C. Hou, BBRC 47, 1248 (1972).
was not elaborated in detail, but more information on this effect may
be expected with the recently announced isolation of a mutant of Escheri-
chia coli deficient in both energy-linked and non-energy-linked transhy-
drogenase [ i.e., presumably a mutant in the transhydrogenase enzyme
(73,167)1.
A. REWX STATE
OF MITOCHONDRIAL
NICOTINAMIDE
NUCLEOTIDES
By their careful analyses of oxidized and reduced nicotinamide nucleo-
tides in isolated mitochondria under a variety of metabolic conditions,
Klingenberg and co-workers (20, 38,179; see also 37, 40, 47) established
that under energized conditions mitochondrial NADP is highly reduced
compared to NAD. These findings form the main experimental support
for the existence of an energy-linked transhydrogenase functioning in
intact mitochondria. However, the ratio of oxidized and reduced NAD
and NADP as measured after extraction of the mitochondria is not
necessarily equal to their thermodynamic redox state (l79a); any factor
that influences the activity coefficient of the oxidized and reduced forms
of the nicotinamide nucleotides to a different extent would result in a
difference between these entities. The question arises to what extent the
relatively high reduction level of mitochondrial NADP reflects a true
difference in redox state between NAD and NADP. Moyle and Mitchell
(157)suggested that the reduction level of free NADP in isolated rat
liver mitochondria is much higher than that of total NADP to the extent
that the potential difference between NAD and NADP is sufficient to
provide the free energy for phosphorylation of ADP to ATP. A similar
conclusion is indicated by the tentative calculations of Williams (181)
and Greenbaum et al. (182),who estimated the redox state of mitochon-
drial NADP in the intact liver from the total tissue levels of metabolites
of isocitrate dehydrogenase by the metabolite indicator method (see 175,
180).
179. M. Klingenberg, “Zur Bedeutung der freien Nukleotide,” Vol. 11, p. 82.
Springer-Verlag, Berlin and New York, 1961.
179a. The following terms are used throughout this section: “total NAD and
NADP” refers to nicotinamide nucleotides as measured after appropriate extraction ;
“free NAD and NADP” refers to activities of nicotinamide nucleotides in a specific
cellular environment; “redox state of NAD and NADP” is used in its thermody-
namic sense and denotes the ratio of activities of oxidized and reduced NAD and
NADP, respectively (see 176, 180).
180. T. Biicher and M. Klingenberg, Angew. Chem. 70,552 (1958).
181. J. R. Williams, in “The Energy Level and Metabolic Control in Mitochondria”
(S. Papa et al., eds.), p. 385. Adriatica Editrice, Bari, 1969.
182. A. L. Greenbaum, K. A. Gumaa, and P. McLean, ABB 143, 617 (1971).
82 J. RYDSTR~M, J . B. HOEK, AND L. ERNSTER
Although valuable from a methodological point of view, these calcula-

tions are based on a large number of assumptions, some of which lack
sufficient experimental support (see 183) (this point is partially illus-
trated by the large variations in results found by Greenbaum et al. (182)
when applying different methods of calculation). In fact, preliminary re-
sults of Hoek and Ernster (184) indicate that there exist only minor
differences between the reduction level of free and total mitochondria1
NADP. I n contrast, Krebs and co-workers (176,186, 186) concluded
that, in vivo, the redox state of mitochondrial NADP is close to that
of NAD. These conclusions were based on the estimation of glutamate
and 8-hydroxybutyrate redox couples in freeze-clamped rat liver as indi-
cators of the redox state of mitochondrial NAD and NADP, respectively
(see also 187-189).Hoek and Tager (1YO) made similar calculations on
isolated rat liver mitochondria, equilibrated in the presence of metabolites
of glutamate and p-hydroxybutyrate dehydrogenases. They observed that
these metabolic couples were at the same redox potential under conditions
where total NADP was considerably more reduced than total NAD as
a result of the energy-linked transhydrogenase. Later work (191) indi-
cated that glutamate dehydrogenase (which was shown to be reactive with
both NAD and NADP in rat liver mitochondria) can maintain a near-
equilibrium condition with NAD, but not with NADP, suggesting
that the enzyme is unable to compete with the efficient reduction of
NADP by the energy-linked transhydrogenase under these conditions.
These findings indicate that caution is warranted in the interpretation
of calculations based on indicator metabolite levels. Thus, no definite
conclusion can be drawn as yet concerning the redox state of mitochon-
drial NADP in vivo and, in this situation, little can be said about the
possible role of transhydrogenase in maintaining a high reduction level
of this coenzyme. This point should be kept in mind when the results
of studies on isolated mitochondria that will be discussed in the following
sections are to be extrapolated to the in vivo situation.
183. J. B. Hoek, Ph.D. Thesis, University of Amsterdam, Mondeel Offsetdrukkery,

Amsterdam, 1972.
184. J. B. Hoek and L. Ernster, in “Alcohol and Aldehyde Metabolizing Systems”
(R. G . Thurman et al., eds.), p. 351. Academic Press, New York, 1974.
185. D. H. Williamson, P. Lund, and H. A. Krebs, B j 103, 514 (1967).
186. J. T. Brosnan, H. A. Krebs, and D. H. Williamson, BJ 117, 91 (1970).
187. K. 8. Henley and E. G . Laughrey, BBA 201,9 (1970).
188. R. A. F. M. Chamalaun and J. M. Tager, BBA 222, 119 (1970).
189. B. Willms, J. Kleineke, and H. D. Soling, BBA 215,438 (1970).
190. J. B. Hoek and J. M. Tager, BBA 325, 197 (1973).
191. J. B. Hoek, L. Ernster, E. J. de Haan, and J. M. Tager, BBA 333, 540 (1974).
B. ROLEOF TRANSHYDROGENME
IN MITOCHONDRIAL
MONOOXYGENATION
REACTIONS
Several monooxygenation reactions have now been demonstrated to
have an intramitochondrial localization in different tissues, including
llp- and 18-hydroxylation of steroids in adrenal cortex (192-194) ; side
chain cleavage of cholesterol or its sulfate ester in adrenal cortex (195,
196), ovary (197-199) , and other steroidogenic tissues (200-202) ; and
la-hydroxylation of 25-hydroxycholecalciferol in kidney (203, 204).
These reactions are all catalyzed by an electron transport chain con-
taining cytochrome P-450 as the terminal electron acceptor (see
205-207). A P-450 cytochrome has been suggested to be involved in the
26-hydroxylation of cholesterol in liver mitochondria (208).NADPH is
required as a specific hydrogen donor for these processes (for reviews,
see 205, 206, 209). Enzymes potentially involved in the supply of
NADPH to steroid hydroxylation in adrenocortical mitochondria include
transhydrogenase (84, 192, 210, a l l ) , NADP-linked “malic” enzyme
192. A. C. Brownie and J. K. Grant, BJ 57, 255 (1954).

193. A. C. Brownie, J. K. Grant, and D. W. Davidson, BJ 58, 218 (1954).
194. P. Greengard, S. Psychoyos, N. H. Tallan, D. Y . Cooper, 0. Rosenthal, and
R. W. Estabrook, ABB 121, 298 (1967).
195. I. D. K. Halkerston, J. Eichorn, and 0. Hechter, JBC 236, 374 (1961).
196. K. D. Roberts, L. Bandi, and S. Lieberman, BBRC 29, 741 (1967).
197. B. Tamaoki and G. Pincus, Endocrinology 69, 527 (1961).
198. P. F. Hall and S. B. Koritz, Biochemistry 3, 129 (1964).
199. S. Sulmovici and G. S. Boyd, Eur. J. Biochem. 3,332 (1968).
200. P. F. Hall and K . B. Eik-Nes, BBA 63, 411 (1962).
201. D. Toren, K. M. J. Menon, E. Forchielli, and R. I. Dorfman, Steroids 3,
381 (1964).
202. G. Morrison, R. A. Meigs, and K. J. Ryan, Steroids, Suppl. 2, 177 (1965).
203. D. R. Fraser and E. Kodicek, Nature (London) 228,764 (1970).
204. R. W. Gray, J. L. Omdahl, J. G. Ghazarian, and H. F. DeLuca, JBC 247,
7528 (1972).
205. C. J. Sih, Science 163, 1297 (1969).
206. M. Hamberg, B. Samuelsson, I. Bjorkhem, and H. Danielsson, in “Molecular
Mechanisms of Oxygen Activation” (0. Hayaishi, ed.), p. 29. Academic Press, New
York, 1974.
207. J. G. Ghaaarian, C. R. Jefcoate, J. C. Knutson, W. H. OrmeJohnson, and
H. F. DeLuca, JBC 249, 3026 (1974).
208. I. Bjorkhem and J. Gustafsson, JBC 249, 2528 (1974).
209. P. F. Hall, in “The Testis” (A. D. Johnson, W. R. Comes, and N. L. Van
Denmark, eds.), Vol. 2, p. 1. Academic Press, New York, 1970.
210. M. L. Sweat and M. D. Lipscomb, JACS 77,5185 (1955).
211. B. W. Harding, L. D. Wilson, S. H. Wong, and D. H. Nelson, Steroids, Suppl.
2, 51 (1965).
84 J. RYDSTR~M, J . B. HOEK, AND L. EBNSTER
($l8-214), and NADP-linked isocitrate dehydrogenase (212, 216).

Numerous studies have been carried out with isolated mitochondria to
identify the source of NADPH for llp-hydroxylation by studying the
effect of inhibitors and uncouplers of oxidative phosphorylation in the
presence of different hydrogen donors (83,192, 210, 212, 214-222). Part
of this work was inconclusive since several complicating features in the
metabolism of adrenocortical mitochondria were insufficiently taken into
account such as secondary inhibitory effects of the substrates, inhibitors,
or uncouplers used (83,214, 220, 222) ; the requirement of transport of
substrates across the mitochondrial membrane (223, 224) ; and the possi-
bility of intramitochondrial dismutation reactions (265).
A role for energy-linked transhydrogenase in the supply of NADPH
was advocated by several authors (83,84,211,216,218-222). I n contrast,
Simpson and Estabrook ($14) suggested that malate is the primary
hydrogen donor for llp-hydroxylation. These authors proposed that, in
vivo, intra- and extramitochondrial “malic” enzyme would cooperate,
ensuring a continuous shuttling of reducing equivalents to mitochondrial
NADP from the cytosol (see also 217, 226). However, their experimental
evidence supporting a predominant role of malic enzyme has been dis-
puted (219-223). At present, the bulk of the evidence indicates that either
of the NADP-linked enzyme systems can, under suitable conditions, pro-
vide the reducing equivalents required for llp-hydroxylation in isolated
adrenocortical mitochondria. To what extent this conclusion is valid in
vivo remains an open question: Little information is available yet on
the factors that regulate the contributions of the different pathways of
NADPH supply to 1lp-hydroxylation.
Reports on the effect of different hydrogen donors and respiratory
chain inhibitors on the side chain cleavage of cholesterol in adrenal cortex
212.J. K. Grant, BJ 64,559 (1956).
213.E. R. Simpson, W. Cammer, and R. W. Estabrook, BBRC 31, 113 (1968).
214.E. R. Simpson and R. W. Estabrook, ABB 125,384 (1969).
215.J. L. Purvia, G. R. Battue, and F. G. Peron, in “Functions of the Adrenal
Cortex” (K. McKerns, ed.), Vol. 11, p. 1007. Appleton, New York, 1968.
216. F. Guerra, F. G. Peron, and J. L. McCarthy, BBA 117, 433 (1966).
217. D. Foneo, B. W. Harding, and D. H. Nelson, Endocrinology $1, 605 (1967).
218. L. A. Sauer and P. J. Mulrow, ABB 134,486 (1969).
219. Y. Harano and J. Kowal, ABB 153, 68 (1972).
220. L. A. Sauer, ABB 139, 340 (1970).
221. K. 0. Klein and B. W. Harding, Biochemistry 9, 3653 (1970).
222. L. A. Sauer, ABB 149,42 (1972).
223. L. A. Sauer and R. Park, Biochemistry 12,643 (1973).
224. A. N. Launay, J. M. Michejda, and P. V. Vignais, BBA 347, 60 (1974).
226. F. G. Peron and B. V. Caldwell, BBA 164,396 (1968).
and ovary mitochondria are inconsistent. Uigiris et al. (86) observed

an inhibition of the reaction in beef corpus luteum mitochondria by
rotenone, antimycin, or cyanide with different added hydrogen donors.
They suggested that the major pathway for generation of NADPH in-
volves energy-linked transhydrogenase (see also 209), although a minor
contribution of NADP-linked isocitrate dehydrogenase could not be ruled
out when isocitrate was added as the hydrogen donor. [No indications
of malic enzymic activity in corpus luteum mitochondria have been found
(86, 2267.1 In contrast to these observations, a stimulation of the cleav-
age reaction by inhibitors of respiratory chain oxidation was noted by
Hall (227) in beef adrenocortical mitochondria and by Robinson and
Stevenson (86) in porcine corpus luteum mitochondria. These findings
were interpreted to indicate the involvement of non-energy-linked trans-
hydrogenase in the supply of reducing equivalents (85, 927).Hall ob-
served a correlation between the reduction level of mitochondria1 NAD
and the rate of side chain cleavage. However, the reduction level of
NADP was much higher than that of NAD and showed little variation
under different reaction conditions. When succinate or fatty acids were
used as the hydrogen donor, energy was found to stimulate the rate of
side chain cleavage (86, 86, 228-250) , suggesting the requirement for
reversed electron transport coupled to transhydrogenation (85, 86).
Robinson and Stevenson (230) proposed a direct, energy-dependent
reduction of NADP' by succinate not involving transhydrogenase, but
more experimental evidence is needed to substantiate this proposal. Hoch-
berg et al. (231) found that succinate addition did not stimulate choles-
terol side chain cleavage in adrenal mitochondria over the rate sustained
by endogenous substrates.
C. ROLEOF TRANSHYDROGENASE IN MITOCHONDRIAL GLUTAMATE

AND ISOCITRATE METABOLISM
Considerations on the role of transhydrogenase in glutamate metabo-
lism in rat liver mitochondria have been dominated largely by a postulate
226. P. M. Stevenson and P. 1,. Taylor, FEBS (Fed. Eur. Biochem. SOC.)Lett.
19, 251 (1971).
227. P. F. Hall, Biochemistry 11, 2891 (1972).
228. P. F. Hall, Biochemistry 6, 2791 (1967).
229. J. Robinson and P. M. Stevenson, FEBS (Fed. Eur. Biochem. SOC.)Lett.
17, 53 (1971).
230. J. Robinson and P. M. Stevenson, FEBS (Fed. Eur. Biochem. SOC.) Lett.
23, 327 (1972).
231. R. B. Hochberg, S. Ladany, M. Welch, and S. Lieberman, Biochemistry 13,
1938 (1974).
of Klingenberg and co-workers (20, 179) that glutamate dehydrogenase

in intact mitochondria reacts preferentially with NADP and not with
NAD, although the isolated enzyme is known to react almost equally
well with both coenzymes (932). An effect of the mitochondria1 energy
level on reactions involving glutamate dehydrogenase was demonstrated
by several authors (37-40, 45, 46, 49-50), and it was suggested that the
reduction level of NADP+, as set by the energy-linked transhydrogenase
reaction, is the main factor regulating the degree of domination of gluta-
mate in isolated mitochondria and in vivo (46, 4 9 ) . This conclusion was
challenged by Krebs and co-workers (175,185,186) who interpreted their
observations on metabolite levels of the glutamate and p-hydroxybuty-
rate redox couples in freeze-clamped liver as evidence that glutamate
dehydrogenase reacts with both NAD and NADP in the intact tissue
(see above).
A recent reinvestigation of the work on isolated mitochondria by Hoek
et al. (191) led to a reinterpretation of the experimental evidence on
the nicotinamide nucleotide specificity of glutamate dehydrogenase.
These studies indicated that glutamate dehydrogenase in intact isolated
rat liver mitochondria can react with both NAD and NADP, the degree
of reactivity with either coenzyme being determined by their relative con-
centrations. Therefore, in the presence of appropriate concentrations of
its substrates the enzyme can catalyze a transhydrogenation that coun-
teracts the energy-linked reduction of NADP+ by NADH via transhydro-
genase (see 175).
Two different isocitrate dehydrogenases are present in mammalian
mitochondria, one NAD-linked, the other NADP-linked (233, 234).
Thus, two pathways are available for the oxidation of isocitrate by oxy-
gen (cf. 235). I n one of these, the NADP-linked pathway, transhydroge-
Base is involved in the transfer of reducing equivalents from NADPH
to NAD+. Nicholls and Garland (52) compared the rate of isocitrate oxi-
dation in intact rat liver mitochondria with the maximal rate of the
reverse transhydrogenase reaction and concluded that in the absence
of other reactions for NADPH oxidation the NADP-linked pathway can
contribute only to a minor extent under energy-rich conditions (see 75,
236). This conclusion is in agreement with several other lines of evidence
indicating that the NAD-linked pathway for oxidation of isocitrate by
232. J. A. Olson and C. B. Anfinsen, JBC 202,841 (1953).
233. L. Ernster and F. Navazio, E z p . Cell Res. 11,483 (1956).
234. L. Ernster and F. Navazio, BBA 26, 408 (1957).
235. G. W. E. Plaut, “The Enzymes,”2nd ed., Vol. 7, p. 106, 1963.
236. A. M. Stein, J. H. Stein, and S. R. Kirkman, Biochemistry 6, 1370 (1967).
the respiratory chain is the prevalent one (for reviews, see 52, 74, 237,
238). Moyle and Mitchell (157) recently reemphasized the NADP-linked
pathway for isocitrate oxidation and argued that NAD-linked isocitrate
dehydrogenase is absent in rat liver mitochondria. Their experimental
evidence has, however, been criticized (239) and presently there are no
convincing arguments to doubt the predominant role of NAD-linked
isocitrate dehydrogenase in the mitochondrial oxidation of isocitrate
under physiological conditions. I n view of the high reduction level of
NADP in energized mitochondria, the possibility suggests itself that
NADP-linked isocitrate dehydrogenase may function primarily in isoci-
trate synthesis. Under appropriate conditions, the reductive carboxylation
of a-ketoglutarate was shown to be catalyzed by mitochondria from rat
liver and kidney in an NADPH-dependent reaction (54, 55, 184, 2 3 9 ~ ) .
The synthesis of isocitrate wiih NAD-linked substrates required a supply
of energy,’indicating the involvement of the energy-linked transhydrogen-
ase (53, 184). The NADPH-dependent intramitochondrial synthesis of
isocitrate is of particular interest in connection with a proposed a-keto-
glutarate-isocitrate shuttle, functioning in the transfer of reducing equiv-
alents from mitochondrial to cytosolic NADP (24, 5 5 ) . This substrate
shuttle would involve the intra- and extramitochondrial NADP-linked
isocitrate dehydrogenases and the translocators for a-ketoglutarate and
isocitrate in the mitochondrial inner membrane. Recently, the a-ketoglu-
tarate-isocitrate shuttle system was reconstituted with isolated rat liver
mitochondria plus added isocitrate dehydrogenase and NADP (184). Un-
der these experimental conditior,s, mitochondrial NADP-linked substrates
could be used as hydrogen donors for the reduction of extramitochondrial
NADP’ and, with appropriate enzyme systems added, for driving extra-
mitochondrial NADPH-linked reactions such as glutathione reduction and
microsomal hydroxylation reactions (240).Although these studies indicate
that, in principle, the a-ketoglutarate-isocitrate shuttle can operate, thus
providing a functional link between energy-linked transhydrogenase and
extramitochondrial NADPH-dependent reactions, the relevance of these
findings to the physiological role of transhydrogenase is questioncbble as
yet. In particular, it remains to be established why, under various condi-
237. G. W. E.Plaut, Cun.Top. Cell. Regul. 2, 1 (1970).

238. G. D. Greville, “Citric Acid Cycle,” p. 1. Dekker, New York, 1969.
239. J. B. Hoek, J. Rydstrom, and L. Ernster, BBA 305,669 (1973).
239a. C. R. Mackerer in “Energy Metabolism and the Regulation of Metabolic
Processes in Mitochondria” (M. Mehlman and R. W. Hanson, eds.), p. 271. Academic
240. J. B. Hoek and L. Ernster, unpublished observations (1974).
tions, the need for cytosolic NADPH cannot be met entirely by cytosolic
reactions (see 2.41-246).
D. ROLEOF TRANSHYDROGENASE
IN FATTY
ACID SYNTHESIS
The inhibitory effect of CoA-thioesters of long chain fatty acids, in
particular palmityl-CoA, on transhydrogenase activity in submitochon-
drial particles from beef heart (70, 71, 129,130) was suggested to indicate
a role of the enzyme in lipogenesis. Palmityl-CoA has earlier been shown
to inhibit several enzymes involved in fatty acids synthesis (246-261 ) ,
and it has been proposed (S@) that this compound may act as a regulator
of fatty acid metabolism in vivo. Intramitochondrial fatty acid elonga-
tion was reported to utilize preferentially NADH (269-964) or both
NADH and NADPH (266,266). Podack and Seubert (267)isolated an
NADPH-specific enoyl-CoA reductase from rat liver mitochondria. This
enayme was found to react preferentially with fatty acids of medium
chain length. Recently, the authors suggested (268) that the enzyme
is involved primarily in the mitochondria1 elongation of unsaturated fatty
acids. Transhydrogenase could well play a role in the supply of NADPH
for this process, but experimental evidence substantiating this proposal
is lacking as yet.
241. C. M. Nepokroeff, M. R. Lakshmanan, G. C. Ness, R. A. Muesing, D. A.

Kleinsek, and J. W. Porter, ABB 162, 340 (1974).
242. S. J. Wakil, in “Lipid Metabolism” (S. J. Wakil, ed.), p. 1. Academic Press,
New York, 1970.
243. R. G. Thurman and R. Scholz, Eur. J. Biochem. 10,459 (1969).
244,W. Cleland, Annu. Rev. Biochem. 36, 77 (1967).
246. P. E. Sluse, A. J. Meijer, and J. M. Tager, FEBS (Fed. Eur. Bwchem. Soc.)
Lett. 18, 149 (1971).
246. J. W. Porter and R. W. Long, JBC 233,20 (1958).
247. P. K. Tubbs, BBA 70,608 (1963).
248. 0.Wieland and L. Weiss, BBRC 13, 26 (1963).
249. P. K. Tubbs and P. B. Garland, BJ 93,550 (1964).
250. P. A. Srere, BBA 108,445 (1965).
251. J. A. Dorsey and J. W. Porter, JBC 243,3512 (1968).
252. J. V. Dahlen and J. W. Porter, ABB 127,207 (1968).
253. W. Colli, P. C. Hinkle, and M. E. Pullman, JBC 244, 6432 (1969).
254. E. M. Wit-Peeters, Ph.D. Thesis, University of Amsterdam, Mondell Offset-
drukkery, Amsterdam, 1971.
255. 9. J. Wakil, J . Lipid Res. 2,1 (1961).
256. E. Quagliariello, C. Landriscina, and P. Coratelli, BBA 164, 12 (1968).
257. E.R.Podack and W. Seubert, BBA 280,235 (1972).
258. W. Seubert and E. R. Podack, Mol. Cell. Biochem. 1,29 (1973).
Flavin-Containing Dehydrogenases
CHARLES H . WILLIAMS. JR .
I . Introduction . . . . . . . . . . . . . . . . 90
I1. Pyridine Nucleotide-Disulfide Oxidoreductasea . . . . . . 92
A . The Reactions Catalyzed-Chemical Similarities and
Cross-Reactivity . . . . . . . . . . . . 92
B. Similarities and Contrasts in Mechanism . . . . . 94
C . Similarities and Contrasts in Structure . . . . . . 99
I11. Lipoamide Dehydrogenase . . . . . . . . . . . . 106
A . Metabolic Functions . . . . . . . . . . . 107
B . Review of the Mechanism of Massey and Veeger . . . 111
C . Properties of the 2-Electron-Reduced Enzyme, EH1 . . 111
D . Kinetic Studies . . . . . . . . . . . . . 115
E . Role of NAD+ as a Modifier . . . . . . . . . 117
F. Structural Studies . . . . . . . . . . . . 120
G. Summary and Conclusions. . . . . . . . . . 126
IV . Glutathione Reductase . . . . . . . . . . . . . 129
B . Properties of the 2-Electron-Reduced Enzyme, EHp . . 133
C . Kinetic Studies . . . . . . . . . . . . . 138
D . Thiol Groups . . . . . . . . . . . . . 141
V . Thioredoxin Reductase . . . . . . . . . . . . . 142
B . Specificity of Thioredoxin Reductase . . . . . . . 144
C . General Properties of the E. coli Enzyme . . . . . 144
D . Reduced States of the Enzyme-Mechanism . . . . 145
E . Light-Activated Reduction-Neutral Semiquinone . . . 147
VI . Microsomal Electron Transport . . . . . . . . . . 148
A . The NADPH-Cytochrome P-450 Reductase-Containing
System . . . . . . . . . . . . . . . 149
B. The NADH-Cytochrome bs Reductase System . . . . 150
C . Possible Synergism between the Two Microsomal Systems . 151
1). NADPH-Dependent Mixed Function Amine Oxidase . . 153
89
90 CHARLES H. WILLIAMS, JR.
VII. NADH-Cytochrome bs Reductaae . . . . . . . . . . 154

A. Molecular Properties of the Amphipathic and Soluble
Forms of the Reductme, . . . . . . . . . 154
B. Review of the Mechanism of Strittmatter . . . . . 156
C. Mechanism of the Reductase Bound to the Microsome . 161
D. Structural Studies . . . . . . . . . . . . 162
E. The Functional Methemoglobin Reductase System of the
Mature Erythrocyte. . . . . . . . . . . 164
VIII. NADPH-Cytochrome P-450 Reductaae . . . . . . . . 165
A. General Properties . . . . . . . . . . . . 166
B. Catalytic Activities of the Reductme . . . . . . 167
C. Mechanism . . . . . . . . . . . . . . 169
I. Introduction
The five enzymes lipoamide dehydrogenase, glutathione reductase,

thioredoxin reductase, NADH-cytochrome b, reductase, and NADPH-
cytochrome P-450 reductase are all flavin-containing dehydrogenases
that do not contain metals. The terms “dehydrogenase” and “reductase,”
when applied to these nonmetalloflavoproteins, usually connote electron
transferases which, following reduction by their donor substrate, react
only slowly with oxygen. They are thus distinguished from the oxidases
which react rapidly with oxygen and from the hydroxylases which react
more rapidly with oxygen in the presence of their other substrates. This
rather qualitative distinction among simple flavoproteins on the basis of
oxygen reactivity correlates rather well with four other properties :
1. Most reductases and dehydrogenases form neutral (blue) semi-

quinones while most oxidases form anionic (red) semiquinones upon re-
duction by dithionite or anaerobic photoirradiation in the presence of
EDTA ( 1 ) .
2. Only oxidases react with sulfite to give an addition complex (W).
3. Reductases and dehydrogenases catalyze the rapid reduction of
1-electron acceptors such as ferricyanide or cytochrome c whereas oxi-
dases do not ( 8 ) .
4. Those reductases and dehydrogenases thus far tested form superox-
ide anion upon reoxidation of their reduced forms by oxygen; if oxidases
1. V. Massey and G.Palmer, Biochemistry 5,3181 (1966).

2. V. Maasey, F. Muller, R. Feldberg, M. Schuman, P. A. Sullivan, L. G . Howell,
9. G . Mayhew, R. G. Matthew, and G . P. Foust, JBC 244, 3999 (1969).
3. FLAVIN-CONTAINING DEHYDROGENASES 91
form superoxide anion upon reoxidation, it is so firmly bound as to be

undetectable (3).
It will be seen, as each enzyme is discussed in detail, that only one of
the five covered in this chapter possesses all of the properties considered
above as characteristic of reductases.
The pyridine nucleotide-disulfide oxidoreductases, lipoamide dehydro-
genase (4),glutathione reductase ( 5 ) , and thioredoxin reductase (6-8)
share so many properties in common that they will be compared and
contrasted before being considered separately. As their group name im-
plies, they catalyze the transfer of electrons between pyridine nucleotides
and disulfides. In spite of their similarities they function in widely diver-
gent metabolic roles.
NADH-cytochrome b, reductase (9) and NADPH-cytochrome P-450
reductase (10,11) are microsomal enzymes. The latter has been referred
to until very recently as NADPH-cytochrome c reductase, since that is
how it is assayed, but there is no cytochrome c in microsomes and its
physiological acceptor seems to be cytochrome P-450.It is thus distin-
guished from NADH-putidaredoxin reductase (la), NADPH-adrenodoxin
reductase (13),and NADH-rubredoxin reductase (14).The adrenodoxin
reductase and the rubredoxin reductase, together with their respective
iron-sulfur protein acceptors, each constitute a cytochrome P-450 re-
ductase system.
Lipoamide dehydrogenase (16,16) and NADH-cytochrome b, reduc-
tase (17)were covered in detail in the second edition of “The Enzymes.”
The material in these chapters has stood the test of time remarkably
well and will only need to be summarized here.
3. V. Mqssey, S. Strickland, S. G. Mayhew, L. G. Howell, P. C. Engel, R. G.

Matthews, M. Schuman, and P. A. Sullivan, BBRC 36,891 (1969).
4. V. Massey, BBA 37, 314 (1960) ; 30, 205 (1958).
5. R. E. Asnis, JBC 213, 77 (1955).
6. S. Black, E. M. Harte, B. Hudson, and L. Wartofsky, JBC 235,2910 (1960).
7. T. Asahi, R.. S. Bandurski, and L. G . Wilson, JBC 236, 1830 (1961).
8. E. C. Moore, P. Reichard, and L. Thelander, JBC 239, 3445 (1964).
9. P. Strittmatter and S. F. Velick, JBC 221,253 and 277 (1956).
10. C. H. Williams, Jr. and H. Kamin, JBC 237, 587 (1962).
11. A. H. Phillips and R. G. Langdon, JBC 237,2652 (1962).
12. I. C. Gunsalus, P. W. Trudgil, and R. DuBus, JBC 241, 1194 (1966).
13. T. Omura, E. Sanders, R . W. Estahrook, D. Y. Cooper, and 0. Rosenthal,
ABB 117,660 (1966).
14. T. Ueda and M. J. Coon, JBC 247, 5010 (1972).
15. V. Massey “The Enzymes,” 2nd ed., Vol. 7, p. 276, 1963.
16. D. R. Sanadi, “The Enzymes,” 2nd ed., Vol. 7, p. 307, 1963.
17. P. Strittmatter, “The Enzymes,” 2nd ed., Vol. 8, p. 113, 1963.
II. Pyridine Nucleotide-Disulfide Oxidoreductares
A. THE REACTIONS SIMILARITIES

CATALYZED-CHEMICAL
AND CROSS-REACTIVITY
Electron transfer between pyridine nucleotides and disulfide com-
pounds is catalyzed by several flavoproteins and three of these are well
characterized. Lipoamide dehydrogenase functions in the oxidative decar-
boxylation of a-keto acids catalyzing the reoxidation of reduced lipoate
by NAD' (18, 19). Glutathione reductase catalyzes electron transfer be-
tween NADPH and glutathione (20-22). Thioredoxin reductase catalyzes
the reduction of thioredoxin by NADPH (8) ; thioredoxin is a protein
of 12,000 molecular weight containing a single cystine residue which is
the electron acceptor (23).
It is not surprising that enzymes catalyzing such similar chemical reac-
tions should bear striking similarity to one another both structurally and
mechanistically. Lipoamide dehydrogenase (24-28), glutathione reduc-
tase (g9),and thioredoxin reductase (30,31) contain, in addition to
FAD, a reactive disulfide which is functional in catalysis. These flavopro-
teins consist of two identical or near identical polypeptide chains, each
with a reactive cystine residue, and two molecules of FAD ( 3 1 3 6 ) .
The specificity of these enzymes toward their disulfide substrates is
quite remarkable: There is virtually no cross-reactivity. Since it is quite
difficult to separate glutathione reductase from thioredoxin reductase it
18. I. C. Gunsalus, Fed. Proc., Fed. Amer. Soc. E x p . Biol. 13, 715 (1954).
19. L. J. Reed, Advan. Enzymol. 18,319 (1957).
20. N.U. Meldrum and H. L. A. Tarr, BJ 29, 108 (1935).
21. E.E.Conn and J. W. Vennesland, JBC 192,17 (1951).
22. T. W. Rall and A. L. Lehninger, JBC 194,119 (1952).
23. T. C. Laurent, E. C. Moore, and P. Reichard, JBC 239, 3436 (1964).
24. V. Maasey, Q.H. Gibson, and C. Veeger, BJ 77,341 (1960).
25. R. L.Searls and D. R. Sanadi,BBRC 2,189 (1960).
26. R.L. Searls and D. R. Sanadi, JBC 235, PC32 (1960).
27. V. Masaey and C. Veeger, BBA 48,33 (1961).
28. R. L. Searle, J. M. Peters, and D. R.. Sanadi, JBC 236, 2317 (1961).
29. V. Massey and C.H. Williams, Jr., JBC 240, 4470 (1965).
30. G. Zanetti and C. H. Williams, Jr., JBC 242,5232 (1967).
31. L. Thelander, Bur. J. Biochem. 4, 407 (1968).
32. V. Massey, T. Hofmann, and G. Palmer, JBC 237,3820 (1962).
33. B. D.Burleigh, Jr. and C. H. Williams, Jr., JBC 247,2077 (1972).
34. R.D.Mavis and E. Stellwagen, JBC 243,809 (1968).
35. E.T. Jones and C. H. Williams, Jr., JBC 250,3779 (1975).
36. C. H.Williams, Jr., G. Zanetti, L. D. Arscott, and J. K. McAllister, JBC 242,
5226 (1967).
is not possible, a t very low levels, to distinguish cross-reactivity from

cross-contamination (37). The glutathione reductase activity of thiore-
doxin reductase preparations is less than 0.01% (8, 37, 38),while the re-
verse measurement is about 0.4% ( 3 7 ) . These levels of activity are of
the same order of magnitude as the oxidase activity of thioredoxin reduc-
tase so that they must be considered upper limits (37, 38). The activity
of erythrocyte glutathione reductase toward lipoate is quite high being
about 3% that with glutathione as acceptor (38-40).The important point
is the very high degree of specificity of these enzymes. The specificity
of glutathione reductase toward mixed disulfides will be discussed later.
The three disulfide substrates are different in many respects. Dihydro-
lipoamide (6,8-dimercaptooctanoamide) forms a five-membered ring of
high stability upon oxidation. I n its physiological form, in the pyruvate
and a-ketoglutarate dehydrogenase complexes, the amide nitrogen is the
r-amino group of a lysine residue of dihydrolipoyl transacetylase (41-43)
or transsuccinylase; thus, it is a protein bound substrate. The three-en-
zyme complex composed of the transacetylase, the a-keto acid oxidative
decarboxylase, and lipoamide dehydrogenase has been the subject of ex-
tensive study and review ( 4 4 ) . Oxidized glutathione (GSSG), unlike lipo-
amide, forms two molecules of 7-glutamylcysteinylglycine (GSH) upon
reduction. Its reoxidation is therefore entropically less favorable. As
mentioned above, thioredoxin is a protein in which the disulfide is a cys-
tine residue. The two halves of the cystine are separated in the polypep-
tide chain by only two other residues. The sequence in the region of the
disulfide of the Escherichia coli protein is (46-47)
-Trp-Ala-Glu-Trp-ps -Gly-Pro-ps -Lys -Met -
S s
The fluorescence of the tryptophan residues changes markedly upon re-
duction and reoxidation of the disulfide ( 4 8 , 4 9 ) .
37. L. D. Arscott and C. H. Williams, Jr., unpublished observations.
38. L. Thelander, JBC 242, 852 (1967).
39. A. Icen, Scund. J. CZin. Lab. Invest. 20, 96 (1967) ; 27, Suppl. 116,5 (1971).
40. E. M. Scott, I. W. Duncan, and V. Ekstrand, JBC 238, 3928 (1963).
41. V. Massey, BBA 38,447 (1960).
42. H. Nawa, W. T. Brady, M. Koike, and L. J. Reed, JACS 82, 896 (1960).
43. K. Daigo and 1,. J. Reed, JACS 84,666 (1962).
44. L. J. Reed and D. J. Cox, “The Enzymes,” 3rd ed., Vol. 1, p. 213, 1970.
45. A. Holmgren, Eur. J. Biochem. 6,474 (1968).
46. D. E. Hall, A. Baldesten, A. Holmgren, and P. Reichard, Eur. J . Biochem.
23, 328 (1971).
47. B. M. Sjoberg and A. Holmgren, JBC 247,8063 (1972).
48. L. Stryer, A. Holmgren, and P. Reichard, Biochemistry, 6, 1016 (1967).
49. A. Holmgren, JBC 247, 1992 (1972).
The three enzymes are quite specific for their respective pyridine nucle-
otide substrates. Under conditions normally used for assay, lipoamide
dehydrogenase is less than 1% as active with NADPH as with
NADH (16) and thioredoxin reductase is less than 1% as active with
NADH as with NADPH (36, 36). Lipoamide dehydrogenase can transfer
electrons to a number of NAD' analogs (9'7).Yeast glutathione reductase
is quite specific for NADPH (60),but the erythrocyte enzyme is 20%
as active with NADH as with NADPH under the conditions of the stan-
dard assay (39,4O, 61).
B. SIMILARITIES
AND CONTRASTS
IN MECHANISM
I n this section on mechanism and in the section to follow on structure,

the comparisons will show that the relationship between lipoamide dehy-
drogenase and glutathione reductase is more marked than is the relation-
ship of either to thioredoxin reductase. Thus, in catalysis, lipoamide de-
hydrogenase and glutathione reductase cycle between the oxidized state
and a spectrally characteristic state in which the enzyme has accepted
two electrons and these are shared between the FAD and the active center
disulfide. This intermediate does not seem to be operative in thioredoxin
reductase, and in this enzyme the FAD and disulfide interact in a differ-
ent way. The oxidized forms of these enzymes can then be represented
as
The spectra of oxidized glutathione reductase and of the 2-electron-

reduced enzyme are shown in Fig. 1. This red intermediate, which has
been shown to be functional in catalysis (299))will be referred to as EH,
designating a half-reduced active center; it has also been referred to as
F (62, 65), but this can be confused with oxidized flavin in other nomen-
clatures. Its spectral characteristics are virtually identical with those of
the analogous species of lipoamide dehydrogenase (24, 27, 64). It has
50. V. Massey, C. H. Williams, Jr., C . Zanetti, and G. Foust, h o e . Znt. Congr.

Biochem., i'h, 1967 Abstracts, Vol. 1, p. 165 (1968).
51. G. E. J. Staal and C. Veeger, BBA 185,49 (1969).
52. J. E. Bulger and K. G. Brandt, JBC 246,5570 (1971).
53. J. E. Bulger and K. G. Brandt, JBC 246, 6578 (1971).
54. N. Savage, BJ 67, 146 (1957).
Wavelenqth (nm)
FIG.1. Yeast glutathione reductase. Spectra of the oxidized and 2-electron-reduced

forms of the enzyme recorded anaerobically (29): (-) oxidized enzyme, (---I
526 moles GSH, and (0) 1 mole T P N H in the presence of DPNase.
relatively high absorbance in the 450-nm region and a shoulder extending

to 650 nm. Two-electron reduction of free FAD results in almost complete
bleaching a t wavelengths greater than 440 nm; thus, the flavin of EH,
is not fully reduced and a second electron acceptor must be operating.
The second electron acceptor in these enzymes is the disulfide of a cys-
tine residue. The disulfide in EH, is also partially reduced. Several lines
of evidence support this, but one crucial experiment with pig heart lipo-
amide dehydrogenase should be described in some detail (27, 66). Arse-
nite complexes quite specifically with vicinal dithiols as shown in Eq. (1).
The spectrum of the oxidized enzyme is unaffected by arsenite. If one

equivalent of NADH is added anaerobically to enzyme in the presence
of arsenite, EH, is produced immediately. In the absence of arsenite this
species would be stable indefinitely. However, with arsenite present, the
flavin is seen to reoxidize almost completely; at the same time the pyri-
dine nucleotide is fully oxidized (Fig. 2). Thus two electrons have been
taken up by the enzyme, but the FAD is oxidized. A second electron
55. V. Massey and G. Palmer, JBC 237,2347 (1962).

0 500 600 700 800

Wavelength (nm)
Fro. 2. Anaerobic reduction of pig heart lipoamide dehydrogenase in the presence
of arsenite (97): 1, oxidized enzyme plus 1 mM arsenite; 2, after 1.0 equivalent
NADH; 3, after 2.2 equivalents NADH; 4, after 3.3 equivalents NADH; and 5,
after NADase.
acceptor is clearly indicated, and the known specificity of arsenite sug-

gests that i t is a disulfide-dithiol. Furthermore, the initial rate of cataly-
sis by lipoamide dehydrogenase is unchanged following preincubation
with arsenite alone, or NADH alone, but is strongly inhibited if NADH
is included in the preincubation with the arsenite. Addition of more than
one equivalent of NADH in the presence of arsenite leads initially to
EH, followed slowly by extensive reduction of the flavin as evidenced
by loss of absorbance at 455 nm; the enzyme takes on a green color with
a very broad absorption centered about 700 nm. (Free FADH, in dilute
solution is pale yellow with no absorption beyond 500 nm.) The extinction
of this species is enhanced by excess NAD' and by low temperature and
indeed is totally dependent on the presence of NAD+. This is demon-
strated in Fig. 2 by the loss of the long wavelength absorption upon addi-
tion of NADase (Neurospora crassa NAD' glycohydrolase) which hydro-
lyzes NAD' and NADP+ but is inactive with NADH and NAPH. These
properties have led to the suggestion that this form of the enzyme is
a charge transfer complex between FADH, and NAD’, and this is further
reinforced by the observation of a red shift in the extinction maximum
when an acceptor of more positive potential is used ( 5 5 ) . The complex
is not formed upon reduction with NADPH in the presence of arsenite
and excess NADP’. On the other hand, when glutathione reductase is
reduced in the presence of arsenite and excess NADP’ (its natural cofac-
tor) the charge transfer band is formed while it is not formed when
NADH and NAD’ are used ( 2 9 ) . Under these conditions the enzymes
have accepted four electrons per FAD as a result of the reaction of the na-
scent dithiol with arsenite. The charge transfer complex is formed too
slowly to be significant in catalysis. To reiterate: lipoamide dehydroge-
nase and glutathione reductase accept only two electrons upon reduction
with excess substrate to form EH,, but in the presence of arsenite this
intermediate is not stable and 4-electron reduction results.
The exact chemical nature of EH2,i.e., the status of the two electrons
shared between the FAD and the disulfide, has been much debated and
perhaps cannot be adequately described by any single nomenclature. It
has variously been referred t o as a biradical (11) (24, 26, 28), as a charge
transfer complex in which thiolate is the donor and FAD the acceptor
(111) (28, 56,56a),and as a covalent bond between FAD and sulfur (IV)
bm p p p?
(57). The roman numerals refer t o the structures below i n which only
Ey SH
(1) (n) (rn) (Iv) (V)

one of the two active centers of the dimeric enzyme is shown; (I) is the
oxidized enzyme and (V) is the 4-electron-reduced enzyme which is cata-
lytically inactive. None of these formulations reflects accurately the
properties of EH,. It is now clear that it is not a simple semiquinone.
Two types of simple semiquinones have been observed i n flavoproteins
(1). Spectra of these are shown i n Figs. 3a (58) and 3b. The neutral
semiquinone (FADH’) is blue and its spectrum has peaks at 580, 490,
390, and 340 nm with a broad shoulder extending t o 700 nm. The anion
semiquinone (FAD’) is red and its spectrum has peaks at 480,400, and
360 nm. Each type is associated with a distinct electron spin resonance
56. E. M. Kosower, in “Flavins and Flavoproteins” (E. C. Slater, ed.), Vol. 1,
p. 1. Elsevier, Amsterdam, 1966.
56a. V. Maeaey and S. Ghisla, Ann. N.Y. Acad. Sci. 227, 446 (1974).
57. G. Palmer and V. Massey, in “Biological Oxidations” (T.P. Singer, ed.), p.
263. Wiley (Interscience), New York, 1968.
58. G. Zanetti, C. H. Williams, Jr., and V. Massey, JBC 243, 4013 (1968).
Ii
10
09
as
P O0 67
X
I 0 1
04
03
02
01
00
m x.
3 0
W*Iqm Inml
1. Ibl
Fxo. 3. (8) Typical neutral (blue) flavin semiquinone produced upon anaerobic
irradiation of thioredoxin reductase in the presence of EDTA (68): (-) oxidized
thioredoxin reductase in 2 x lo-' M EDTA, (---) 1 hr light a t Oo, -
.) 2 hr light
( a
at O", (--) 2 hr 40 min light at 0", and ( 0 )4 hr light at 0". (b) Typical anionic
(red) flavin semiquinone produced upon anaerobic irradiation of oxynitrilase in the
presence of EDTA (I).
(ERS) signal ( 1 ) . The spectral characteristics of E H 2 are clearly different

from either of these, especially in the 450-600-nm region, and indeed
must arise from a n interaction between the FAD and another group,
namely, the redox active disulfide. No ESR signal is observed with E H 2
(1, 88, 69). Its long wavelength absorption is only slightly temperature
sensitive, and the response is in the opposite direction to that which
would be predicted for a charge transfer complex (55).The weak covalent
interaction of (IV) implies that the isoalloxazine ring is reduced, and
this is clearly inconsistent with the spectrum. The problem seems t o
rest with the molecular orbital theorist.
The importance of EH, in catalysis by lipoamide dehydrogenase and
glutathione reductase has been demonstrated by rapid reaction spectro-
photometry. It is produced upon reduction with NADH or NADPH, re-
spectively, in the dead time of the instrument (ca. 3 msec) and is rapidly
reoxidized by lipoamide or glutathione at rates commensurate with
catalysis (g4, 60, 5 4 ) .
A species similar to EH, has not been observed with thioredoxin re-
ductase (30,31). Its anaerobic titration with NADPH is shown in Fig.
4. Two equivalents are required to reach a stable spectrum which is char-
59. V. Massey, G. Palmer, C. H. Williams, Jr., B. E. P. Swoboda, and R. H. Sands,

in "Flavins and Flavoproteins" (E. C. Slater, ed.), Vol. 1, p. 133. Elsevier, Amster-
dam, 1968.
Wavelength (nm)
FIQ. 4. Anaerobic reduction of thioredoxin reductase with NADPH in 0.05 M

phosphate, pH 7.6, 5" (SO): (-1 oxidized thioredoxin reductase, (---) 0.5 mole
TPNH/mole FAD, -
.) 1.0 mole TPNH/mole FAD, (*--.)
( a 2.0 moles TPNH/
mole FAD, and ( .-. ' .) 4.0 moles TPNH/mole FAD.
0
acterized by virtually complete bleaching in the 450-nm region and ab-

sorption of very low extinction extending beyond 700 nm (SO, 31). Thus,
this enzyme can accept four electrons without apparent complex forma-
tion between its nascent dithiol and the FAD. Initial rates of catalysis
are not inhibited by preincubation with mercurial alone or with NADPH
alone, but preincubation with both results in complete loss of activity
(8, SO). If a second aliquot of enzyme (not preincubated) is added to
the inhibited mixture the inhibited enzyme is reactivated and double the
control activity is observed. The uninhibited enzyme in the time of mix-
ing has produced sufficient reduced thioredoxin (dithiol form) to reverse
the inhibition of the preincubated enzyme (SO). The enzyme is also sensi-
tive in the reduced state to arsenite and Cd*+ (8).
C. SIMILARITIES IN STRUCTURE
AND CONTRASTS
The gross structure of the pyridine nucleotide-disulfide oxidoreductases

is the same, i.e., two polypeptide chains each containing a redox active
disulfide (and no other disulfide) and two molecules of FAD (61).Gluta-

thione reductase (34, 39) and lipoamide dehydrogenase (32) have molec-
ular weights of about 100,000 while the molecular weight of thioredoxin
reductase is about 70,000 (31,36,37,62).
The FAD is very tightly bound and exhibits an extinction coefficient
(at the wavelengths of the visible maxima, Table I) (29, 31, 32,
36, 37) of 11.3 mM-l cm-1 which is equal to that of free FAD (at
its maximum, 448 nm) (69). The tightly bound flavin can be related to
the amino acid composition to give the minimum molecular weight. Table
I gives the amino acid composition of the three enzymes as isolated from
a prokaryote and from a eukaryote. The molecular weights calculated
from amino acid analysis do not agree well with some of the estimates
made by physical measurements, but, on the other hand, neither do mea-
surements made by different physical means agree with one another. In
one case an exact comparison can be made between molecular weight
of a flavoprotein from amino acid analysis data (70) and the molecular
weight subsequently obtained when the protein had been sequenced ( 7 1 ) .
The agreement is to less than 1%. The quality of such measurements
is subject to two types of error. The error resulting from losses in transfer
can be eliminated by making the spectral estimation of flavin directly
on the solution used for amino acid analysis (of course, the extinction
coefficient of the flavin must be known). The other error results from
the presence of apoenzyme; this is seldom serious with flavoproteins such
as these where the FAD is tightly bound.
Data from amino acid analysis of a number of simple flavoproteins
are now available: reductases (31, 33, 36, 60, 62, 63, 72-74) and oxidases
60. L. D. Arscott and C. H. Williams, Jr., unpublished observations.

61. C. H. Williams, Jr., B. D. Burleigh, Jr., S. Ronchi, L. D. Arscott, and E. T.
Jones, in “Flavins and Flavoproteins” (H. Kamin, ed.), Vol. 3, p. 295. Univ. Park
Press, Baltimore, Maryland, 1971.
62. S. Ronchi and C. H. Williams, Jr., JBC 247,2083 (1972).
63. R. G.Matthews, L. D. Arscott, and C. H. Williams, Jr., BBA 370, 26 (1974).
64. J. E.Wilson, ABB 144, 216 (1971).
65. M. L.Cohn and I. R. McManus, BBA 276,70 (1972).
66. M. L. Speranea, S. Ronchi, and L. Minchiotti, BBA 327, 274 (1973).
67. R.L.Spencer and F. Wold, Anal. Biochem. 32, 186 (1969).
68. P. M. Harrison and T. Hofmnnn, BJ 80, 38P (1961).
69. H. Beinert, “The Enzymes,” 2nd ed., Vol. 2,Part A, p. 339,1960.
70. S. G.Mayhew and V. Massey, JBC 244, 791 (1969).
71. M. Tanaka, M. Haniu, K. T. Yasunobu, S. Mayhew, and V. Massey, JBC 248,
4354 (1973).
72. G.Forti and E. Sturani, Eur. J. Biochem. 3,461 (1968).
73.P. C. Engel and V. Massey, BJ 125,879 (1971).
74. L.Spate and P. Strittmatter, JBC 248,793 (1973).
(76-80).I n comparing reductases with oxidases (and hydroxylases) only

one trend is apparent: The tryptophan content of dehydrogenases is low
(1-6 per flavin) and of oxidases is high (7-14 per flavin) . (Normalization
of the data to a constant molecular weight does not change the conclu-
sion.) This fact is reflected in the ratios of absorbance in the 280-nm
region to that in the 450-nm region with reductases showing a lower ratio
than oxidases. The relationship between this ratio and the tyrosine plus
tryptophan content has been used (Table I) to predict a tryptophan
value for yeast thioredoxin reductase.
The data in Table I do not show any other obvious trends. The fact
that the prokaryotes have markedly lower serine contents than do their
eukaryote pair-mates is very probably an anachronism.
Using average extinction coefficients a t 280 nm for tryptophan (5.6
mM-' cm-l) and tyrosine (1.3 mM-' cm-l) in proteins (81), the extinction
coefficient for enzyme-bound FAD a t that wavelength can be estimated
and is found to range from 34 to 40 mM-I ern-'. The extinction coefficient
for free FAD a t 280 nm is 21 (69),indicating a marked red shift upon
binding. This is important in the interpretation of modification studies
of flavoproteins which involve monitoring in the ultraviolet. It has been
pointed out, for example, that thiol determinations by the Boyer (82)
procedure are often inaccurate because of the dissociation of flavin during
the titration (8s).
The free carboxyl groups of pig heart lipoamide dehydrogenase have
been determined as 56 per FAD (6'4). The glutamine plus asparagine
content is, by difference, 35. It can then be calculated that the net charge
on the protein will depend on the effective pK values of the histidine
residues and will vary with pH from +5 to -6 per FAD. Amide data
for thioredoxin reductase (31) allow a similar calculation: The charge
can vary from +2 to -8 per FAD.
The special reactivity as well as the specificity of the active center
disulfides in these enzymes is determined by their environment in the
proteins. Crucial components in this environment are the near neighbors
of the half-cystines in the primary structure. Peptides have been isolated
75. J. H. Paeur, K . Kleppe, and A . Cepure, ABB 111,351 (1965).
76. A. DeKok and A. B. Rawitch, Biochemistry 8, 1405 (1969).
77. M. Schuman and V. Massey. BBA 227,500 (1971).
78. S. C. Tu, S. J. Edelstein, and D. B. McCormick, ABB 159, 889 (1973).
79. B. Curti, S.Ronchi, U. Branzoli, G . Ferri, and C. H. Williams, Jr., BBA 327,
266 (1973).
80. M. I. S. Flashner and V. Massey, JBC 249, 2579 and 2587 (1974).
81. H. Edelhoch, Biochemistry 6, 1918 (1967).
82. P. D. Boyer, JACS 76, 4331 (1954).
83. G. Palmer and V. Massey, BBA 58, 349 (1962).
AMINOACID ANALYSIS
TABLE I
OF PYRIDINE
NUCLEOTIDE-DISULFIDE
OXIDOREDFCTASES~ K
Lipoamide Lipoamide Glutathione Glutathione Thioredoxin Thioredoxin
dehydrogenase dehydrogenase reductase reductase reductase reductase
Amino acid (pig heart)* (E. eoli)" (yeast)d (E. coZi)* (yeast)' (E. wZi)~
Cysteic acid* 10 5 6 7 5 5
Aspartic acid 44 41 49 49 29 37
Threonine 26 27 23 30 28 25
Serine 24 15 27 16 20 15
Glutsmic acid 47 49 45 43 33 35
Proline 18 21 13 22 13 10
Glycine 53 52 43 45 29 37
Alanine 46 52 33 47 35 34
Valine 44 45 41 43 19 21
Methionine 9 9 7 11 7 8
Isoleucine 36 38 29 34 20 24
Leucine 31 36 31 30 26 31
Tyrosine 8 8 17 16 9 9
Phenylalanine 15 15 16 15 11 11
Lysine 36 42 39 26 24 14
d
Histidine 11 13 14 12 6 10
Arginine 14 16 16 14 11 16 F
Tryptophan' 2 4 5 3 4i 1 F
FAD 1 1 1 1 1 1
2!
Total amino acid residues 474 488 454 464 327 344 x
Minimum molecular weight/ 51,000 53 000 50,600 50 800 35 900 37,700
FAD (including FAD)
- 4 2 0 0 I A m s x via' 5.3
)
6.7 8.5
)
7.3
)
6.8 5.0
dE
r
Visible maximum (nm) 455 455 460 462 456 456
NHpterminal' Ala Ser GlY G~Y
"5
4
COOH-terminalm Ala-Lys-L ys
*r
0 Data are based on spectral measurement of FAD in the solutions used for amino acid analyses. Corrections have been made for
losses in serine and threonine upon hydrolysis by extrapolation of data a t various times of hydrolysis to zero time and for the slow
hydrolysis of some peptide bonds involving valine and isoleucine by using only those values obtained at later times of hydrolysis. +;.r
hIatthews el al. (63). Agreement with other analyses (32, 64,65) is excellent. The high value for arginine (32) has not been borne
out in subsequent analyses (63-65). 7
Williams et a[. (33, 36). 8
Arscott and Williams (60). Numerous errors are apparent in the previous analysis (29). 2k
" Arscott and Williams (60).
f Speransa el a/. (66).
5
z
0 Williams el al. (36, 62). Agreement with another analysis is excellent (31).The Trp value is taken from (31). 0
I, Determined on separate samples hydrolyzed in the presence of dimethylsulfoxide (67). U
' Determined on separate samples by a modification of the method of Spies and Chambers (68). x"
J Value assumed on the basis of ultraviolet absorption, see text. 3
Ratio of the absorbance a t 280 nm to that a t the visible maximum given in the next line. T10
I Thelander (31), Rlassey et al. (32),Burleigh and Williams (3.3, and Jones and Williams (35). PI
Burleigh and Williams (33). 2
01
H
m
Pig heart lipoamide dehydmgenase:

8
Glu-Lye -Asx-Glu-Thr- Lsu- Gly- Gly- Thr-
B
Cys- Lsu-Am-Val-
I
Cly -Cye -1le -Pro- Ser -Lys-Ala-Leu
Yeast glutathione reductase:

s s
I I
Lys-Ala-G4-Lys -Ala-Leu-Gly-Gly -Thr -Cys-Val- Asn-Val- Gly-Cye-Val-Pro-Lys -Val-Val-Met
E s c k e r i c k i a c o 1 f lipoamlde dehydrogenase:
s s
I I
Tyr- Asn -Thr --Leu - Gly -Gly-Val- Cys- Leu-Am-Val- Gly - Cys- Ile-Pro- Sar- Lys
E s c h e r fc h l a c o Ii thioredoxln reductase:
s s
A l a - 4 8 -Ala-Thr-
I
Cys -Asp-Gly- Phe
FIG.5. Sequences around the active center disulfide of the pyridine nucleotide-
disulfide oxidoreductases.
from E . coli (33, 61, 84) and pig heart (63, 86, 86) lipoamide dehydroge-
nase, from yeast glutathione reductase (561, and from E. coli thioredoxin
reductase (61, 62,87) containing both halves of the reactive cystine resi-
due. The sequences of these peptides are shown in Fig. 5 . A feature com-
mon to the disulfides is that the half-cystines are close to one another
in the primary sequence, being separated by four other residues in
lipoamide dehydrogenase and glutathione reductase and by two other
residues in thioredoxin reductase, thus conforming tight loops in the
polypeptide chain. This structural arrangement had been predicted for
lipoamide dehydrogenase (28).
The E. coli and pig heart lipoamide dehydrogenase sequences (a
prokaryote-eukaryote pair) are identical in 14 of 17 overlapping resi-
dues. It is suggested that these proteins have been derived by divergent
evolution from a common ancestor (86, 867, but a final judgment on this
point will have to await total sequencing. A very high degree of homology
also exists between the lipoamide dehydrogenases and glutathione reduc-
tase in this analogous region (36).The two substitutions in the immediate
disulfide area, valine for leucine and isoleucine, are conservative both
chemically and genetically. It may be of interest that the prokaryote
84. J. P. Brown and R. N. Perham, FEBS (Fed. Eur. Biochem. Soc.) Lett. 26,
221 (1972).
85. C. H. Williams, Jr. and L. D. Arscott, 2. Naturforsch. B 27, 1078 (1972).
86. J. P. Brown and R. N. Perham, BJ 137,505 (1974).
87. L. Thelander, JBC 245, 6026 (1970).
3. FL AVIN -CONTAI N I N G DEHYDROG E N ASES 105
E. coli lipoamide dehydrogenase has a valine residue preceding the first

half-cystine, whereas the eukaryote enzymes have a threonine residue.
Chemically this is a relatively conservative change since the side chains
of these amino acids are virtually identical in volume.
The lack of homology between lipoamide dehydrogenase and thiore-
doxin reductase, and the apolar nature of the residues around the active
center disulfide in lipoamide dehydrogenase, an enzyme with an apolar
substrate, led to the postulation that the disulfide region contained impor-
tant determinants for interaction with the respective substrates (33, 62,
85, 88).The very high degree of homology between lipoamide dehydroge-
nase and glutathione reductase suggests that some modification of that
postulation is in order; clearly, glutathione and lipoamide are very differ-
ent molecules. It is possible that the apolar region is important in the
binding of lipoamide while the determinants for binding the larger gluta-
thione molecule are elsewhere. One would predict that ionic interactions
play a role in the binding of anionic glutathione and the nearby lysine
residues may function in this way. Since the two enzymes share a common
intermediate in catalysis, it seems reasonable to suggest that the structure
of the disulfide region confers a special reactivity on that disulfide.
The resolved spectrum of FAD when bound to glutathione reductase
and lipoamide dehydrogenase indicates that the flavin is bound in a hy-
drophobic milieu. It has been suggested for lipoamide dehydrogenase (33)
that the tight loop in the polypeptide chain, imposed by the’proximity
of the half-cystines in the sequence, forms part of this milieu. The tight
loop would favor a flat conformation accommodating the planar isoal-
loxazine ring and allowing multiple van der Waals contacts. Electron
sharing between the flavin and the disulfide demands that they be close.
There is a growing list of enzyme families in which a high degree of
homology has been demonstrated around a common active site residue
(89). These include the serine proteases, the cysteine proteases, the car-
boxypeptidases, the ATP-guanidine phosphotransferases (creatine, argi-
nine, and lombricine kinases) , and the aldolases. The pyridine nucleotide-
disulfide oxidoreductases can now be added to this list. A parallel can
be drawn between this group and the aldolases where the closely related
aldolases A and B show extensive homology but do not show homology
with transaldolase. Such is the case with glutathione reductase and lipo-
amide dehydrogenase having a high degree of homology but lacking
homology with thioredoxin reductase around the active center cystine
residue.
88. A. L. Fluharty, G. I. Adelson, and B. P. Gaber, ABB 134, 346 (1969).
89. M. 0. Dayhoff, “Atlas of Protein Sequence and Structure,” Vol. 5 , p. 56. Nat.
Biomed. Res. Found., Washington, D. C., 1972.
111. Lipoamide Dehydrogenase
Lipoamide dehydrogenase has been isolated from many species and

these are listed in Table I1 (4, 56, 90-115). The mammalian enzymes
are of mitochondria1 origin. Where tested, the enzyme from eukaryotic
sources is isozymic, while that from prokaryotes is a single species (105).
There are marked differences in the sensitivity of the enzyme as isolated
from various sources to inhibition by excess NADH in the
NADH + lipS,NH, reaction and in the relief of that inhibition by NAD’
(27, 99, 105, 106, 108, 109, 116, 117). Attempts to make phylogenetic
90. F. B. Straub, BJ 33,787 (1939).
91. C. J. Lusty and T. P. Singer, JBC 239,3733 (1964).
92. T. C. Linn, J. W. Pelley, F. .H. Pettit, F. Hucho, D. D. Randall, and L. J.
Reed, ABB 148, 343 (1972).
93. C. J. Lusty, JBC 238,3443 (1963).
94. S. A. Millard, A. Kubose, and E. M. Gal, JBC 244, 2511 (1969).
95. J. K. Reed, JBC 248, 4834 (1973).
96. S. Ide, T. Hayakawa, K. Okabe, and M. Koike, JBC 242, 54 (1967).
97. C. A. Eberhard, A. H. Guindon, C. Kepler, V. Massey, and C. Veeger, Biol.
Bull. 123, 480 (1962).
98. D. K. Basu and D. P. Burma, JBC 235,509 (1960).
99. J. Matthews and L. J. Reed, JBC 238,1869 (1963).
100. L. L. Poulsen and R. T. Wedding, JBC 245,5709 (1970).
101. A. Wren and V. Massey, BBA 110,329 (1965).
102. E. Misaka, Y. Kawahara, and K. Nakanishi, J . Biochem. ( T o k y o ) 58, 436
(1965).
103. C. H. Williams, Jr., unpublished observations.
104. Y. Kawahara, E. Misaka, and K. Nakanishi, J . Biochem. ( T o k y o ) 83, 77
(1988).
105. W. H. Scouten and I. R. McManus, BBA 227, 248 (1971).
106. H. Fehrmann and C. Veeger, BBA 350, 292 (1974).
107. 0. Vogel and U. Henning, Eur. J. Bioehem. 35,307 (1973).
108. C. H. Williams, Jr., JBC 240,4793 (1965).
109. M. Koike, P. C. Shah, and L. J. Reed, JBC 235, 1939 (1960).
110. G. W. Notani and I. C. Gunsaius, Abstr. Pap., 134th Meet., Amer. Chem.
SOC.Div. Biol. Chem., p. 3C (1958).
111. D. S. Goldman, BBA 32, 80 (1959).
112. M. L. Baginsky and F. M. Huennekens, ABB 120, 703 (1967).
113. S. M. Klein and R. D. Sagers, JBC 242,297 and 301 (1967).
114. C. Veeger, J. Krul, T. W.Bresters, H. Haaker, J. H. Wassink, J. S. Santema,
and A. DeKok, in “Enzymes, Structure and Function” (J. Drenth, R. A. Osterbaan,
and C. Veeger, eds.), Proc. 8th FEBS Meet., p. 217. North-Holland Publ., Amster-
dam, 1972.
115. I. C. Gunsalus, in “The Mechanism of Enzyme Action” (W. D. McElroy
and B. Glass, eds.), p. 545. Johns Hopkins Univ. Press, Baltimore, Maryland, 1954.
116. R. L. Searls and D. R . Sanadi, JBC 238,680 (1961).
117. A. Wren and V. Massey, BBA 122,436 (1966).
TABLE I1
SOURCESFROM WHICHLIPOAMIDE HAS BEENISOL~~TSD
DEHYDROGENASE
Eukaryotes Ref. 1 Prokaryotes Ref.
Pig heart 4190 Escherichia coli B 36

Beef heart 91,98 Escherichia coli K12 107
Beef liver 93 Escherichia coli M191-6 108
Pig brain 94 Escherichia coli Crookes 109
Beef kidney 92 Leuconostoc mesenteroides 110
Rat liver 96 Mycobacterium tuberculosis 111
Human liver 96 Peptococcus glycinophilus 118,113
Dogfish liver 97 Azotobacter vinelandii 114
Spinach leaves 98,99 Proteus vulgaris 116
Cauliflower floral heads 100 Streptococcus faecalis 116
Saccharomyces eerevisiae 101 Serratia marcescens 106
Saccharomyces oviformis 102 Pseudomonas jluorescens 106
Torula 10s Bacillus subtilis 106
Candida krusei 104 Azotobacter agilis 106
Ncurospora crassa 106
Pythium ultimum 106
Phytophtora erythroseptica 106
distinctions based on these differences are perhaps somewhat premature

(105).The complications in such attempts lie in the fact that the sensi-
tivity and its relief are dependent on the pH and in the fact that lipo-
amide dehydrogenase from some species binds NAD' very tightly SO that
the NAD' present in most commercial NADH may be sufficient to acti-
vate the enzyme. There are also marked differences in the stability of
the 2-electron-reduced enzyme (EH,, the catalytically important inter-
mediate) in the presence of excess NADH, anaerobically in the absence
of an acceptor. Three categories can be distinguished: very stable, moder-
ately stable, and unstable. Enzyme isolated from mammals falls into the
first category (24, 26, 93) ; enzyme from lower eukaryotes (99, 101, 106)
and from an anaerobic bacterium (11%)falls into the second category;
while enzyme from other prokaryotes, facultative (108) and aerobic
(114) bacteria, falls into the third category. Differentiation between the
first and second categories has been made difficult by more recent findings
(106, 118), but spectral details will be discussed in a later section.
A. METABOLIC
FUNCTIONS
Lipoamide dehydrogenase, as its more proper name dihydrolipoamide
dehydrogenase implies, functions physiologically in the reoxidation of
118. C. Veeger and V. Massey, BBA 67,679 (1963).
dihydrolipoic acid, bound in amide linkage to the r-amino group of a

lysine residue in the transacetylase or transsuccinylase (41, 43) ; the elec-
tron acceptor is NAD+ (18, 19). The lipoic acid is reduced as a conse-
quence of the thiamine pyrophosphate-dependent oxidative decarboxyla-
tion of a-keto acids, pyruvate and a-ketoglutarate, yielding acetyl-CoA
CH,-CO-COO- + E,-TPP'- -
and succinyl-CoA, respectively, as shown below for pyruvate.
E,-TPPS--CO-CH,
+
+ CO~ (2)
E,-TPP'--CO-CH,+
t\-ip NH-E,
s-s
E,-TPPa-
d ip-NH-E,
8-co-CH,
(3)
lip-NH-E,
I \
+ CoA-SH lip-NH-E,
I \
+ CoA-S-CO-CH, (4)
HS S-CO-CH, HS SH
Lipoamide dehydrogenase can be isolated from the multienzyme com-
plexes that carry out these oxidative decarboxylations : from the a-keto-
glutarate dehydrogenase complex of heart (41,119-181) or E. coli ( l d b ) ,
or from the pyruvate dehydrogenase complex of heart (98, 183, 184) or
E. coli (107, 186). The complexes have recently been reviewed (186').
It is possible that lipoamide dehydrogenase also functions in the com-
plexes that oxidatively decarboxylate the a-keto acids resulting from the
transamination of valine, isoleucine, and leucine but these have proved
difficult to resolve (1a7). Lipoamide dehydrogenase also functions in the
pyridoxal phosphate and tetrahydrofolate-dependent oxidative decar-
boxylation of glycine in the anaerobic bacterium Peptococcus glyci-
nophilus. The reaction in which the protein-bound lipoic acid is reduced
is very complex and not yet fully understood; the ultimate electron ac-
ceptor is NAD+ (118, 113,128).
Lipoamide dehydrogenase is quite easily dissociated from the a-keto
119. R. L. Searls and D. R. Sanadi, JRC 235,2485 (1960).

120. N. Tanaka, K. Koike, M. Hamada, K. Otsuka, T. Sueniatsu, and M. Koike,
JBC 247, 4043 (1972).
121. F. H. Pettit, L. Hamilton, P. Munk, G. Namihira, M. H. Eley, C. R. Willms,
and L. J. Reed, JBC 248,5282 (1973).
122. B. B. Mukherjee, J. Matthews, D. L. Homey, and L. J. Reed, JBC 240,
PC2268 (1965).
123. T. Hayakawa and M. Koike, JBC 242, 1356 (1967).
124. T. Hayakawa, T. Kanzaki, T. Kitamura, Y. Fukuyoski;Y. Sakurai, K. Koike,
T. Suematsu, and M. Koike, JBC 244,3660 (1969).
125. M. Koike, L. J. Reed, and W. R. Carroll, JBC 238,30 (1963).
126. L. J. Reed, Accounts Chem. Res. 7,40 (1974).
127. Y. Namba, K. Yoshizawa, A. Ejima, T. Hayaski, and T. Kaneda, JBC 244,
4437 (1969).
128. J. R. Robinson, S. M. Klein, and R. D. Sagers, JBC 248, 5319 (1973).
acid dehydrogenase complexes (129) ; indeed, even when the complexes

are carefully prepared, free flavoprotein is present (91). It is felt that
this free enzyme arises chiefly from the pyruvate dehydrogenase complex
(199,130).The physiological significance, number, and origin of the elec-
trophoretically separable forms of heart and liver lipoamide dehydroge-
nase have been topics of considerable controversy since their original
observation (131). The most appealing (i.e., the simplest) theory is that
there are two major forms, one arising from each of the major a-keto
acid dehydrogenase complexes (132, 133), and that other forms come
from proteolytic degradation during isolation (64, 134) ; but this hypoth-
esis may be too simple to explain all the facts (65, 135) and has been
sharply criticized (130).While numbers of isozymes as high as thirteen
have been reported (136),the most commonly quoted number is six (65,
93, 131, 134, 135, 137). Chromatography on anion exchangers resolves
the mixture into two major bands (65, 93, 132, 133, 136) with further
resolution of the more anionic band if shallow gradients are employed
(93).The more anionic species seem to be associated with the a-ketoglu-
tarate dehydrogenase complex and the less anionic species with the pyru-
vate dehydrogenase complex (65,130,132,133,135).Attempts have been
made using a wide variety of physical and chemical methods, other than
electrophoretic mobility, to demonstrate differences between the iso-
zymes; with the possible exception of the 280:455-nm ratios of 5.5 for
the less anionic species and 4.9 for the more anionic species (661,the
differences fall within the limits of experimental error (130-136, 138,
139).
Despite the sizable differences in effective charge, there is a generally
129. V. Massey, “Methods in Enzymology,” Vol. 9,p. 272, 1966.
130. W.C.Kenney, D. Zakim, P. K. Hoguem, and T. P. Singer, Eur. J. Biochem.
28, 253 (1972).
131. M. R. Atkinson, M. Dixon, and J. M. Thornber, BJ 82, 29P (1962).
132. Y. Sakurai, Y. Fukuyoski, M. Hamada, T. Hayakawa, and M. Koike, JBC
245, 4453 (1970).
133. T. Hayakawa, Y. Sakurai, T. Aikawa, Y. Fukuyoski, and M. Koike, in
“Flavins and Flavoproteins” (K. Yagi, ed.), Vol. 2, p. 99. Univ. Park Press, Balti-
more, Maryland, 1908.
134. J. E. Wilson, “Flavins and Flavoproteins” (H. Kamin, ed.), Vol. 3, p. 313.
Univ. Park Press, Baltimore, Maryland, 1971.
135. M. L.Cohn, L. Wang, W. Scouten, and I. R. McManus, BBA 159, 185 (1968).
130. A. M.Stein and J. H. Stein, Biochemistry 4, 1491 (1965).
137. V. Massey and Q,. H. Gibson, Proc. Int. Congr. Biochem., 6th, 1961 Vol. 1,
p. 157 (1963).
138. Y. Sakurai, T.Hayakawa, Y. Fukuyoski, and M. Koike, J. Biochem. (Tokyo)
65, 313 (1909).
139. A. M. Stein, B. Wolf, and J. H. Stein, Biochemistry 4, 1500 (1965).
110 CHARLES -H.
WILLIAMS, JR.
held opinion that a single structural gene codes for lipoamide dehydroge-
nase. I n mammals this assumption is based on the fact that the enzyme
isolated from either complex can be used to reconstitute the other complex
and on the lack of differences referrred to above (130, 13%).It has been
suggested that the differences observed electrophoretically are induced
when lipoamide dehydrogenase binds to the transacetylase or transsuc-
cinylase (which serve structural as well as catalytic functions in the com-
plexes) since the optical rotary dispersion and circular dichroism of the
flavoprotein differ in the two complexes (132).These changes must be
reversible, however, since they cross-react in the reconstitution of the
complexes. In E . coli, lipoamide dehydrogenase isolated from one complex
can serve in the reconstitution of the other and a mixture of the enzyme
isolated from the two complexes is electrophoretically homogeneous ; on
this basis a single structural gene was hypothesized (140). This has now
been confirmed by chromosomal mapping in an extensive series of lipo-
amide dehydrogenase mutants of E . coli K12 (141-143).
Very recently another physiological function has been suggested for
lipoamide dehydrogenase in addition to the reoxidation of lipoic acid in
a-keto acid oxidation. The observations (14.4)are these: Metabolic con-
ditions which result in an increase in intramitochondrial GTP levels lead
to the almost complete inhibition of oxidation of NAD+-linked substrates,
while having no effect on FAD-linked substrates. This effect can be
mimicked by inhibitors of lipoamide dehydrogenase such as arsenite and
5-methoxyindole-2-carboxylic acid. The latter compound is a very poor
inhibitor, having a K, of about 3 mM (146).It must be emphasized
that no direct effect of GTP on isolated lipoamide dehydrogenase can
he demonstrated (14.4).It is hypothesized (144) that lipoamide dehydro-
genase serves as a transhydrogenase between two pools of pyridine nucle-
otide, i.e., NADH produced by NAD+-linked substrates and NAD' avail-
able to the electron transport chain. The transhydrogenase activity of
this enzyme has been well demonstrated (146, 147). This hypothesis has
been criticized on metabolic grounds, but the results have not been refuted
(148, 149). It should perhaps be treated as a very interesting but tenta-
tive hypothesis.
140. F. H. Pettit and L. J. Reed, Proc. Nut. Acad. Sci. U S . 58, 1126 (1967).
141. J. R. Guest and I. T. Creaghan, J . Gen. Microbiol. 75, 197 (1973).
142. J. R. Guest, J . Gen. Microbiol. 80,523 (1974).
143. J. R. Guest and I. T. Creaghan, J. Gen. Microbiol. 81, 237 (1974).
144. M. S. Olson and T. T. Allgyer, JBC 248,1582 and 1590 (1973).
145. J. Reed and H. A. Lardy, JBC 245, 5297 (1970).
146. M. M. Weber and N. 0. Kaplan, JBC 225,909 (1957).
147. A. M. Stein, B. R. Kaufman, and N. 0. Kaplan, BBRC 2, 354 (1960).
148. C. M. Smith, J . Bryla, and J . R. Williamson, JBC 249, 1497 (1974).
149. E. I. Walajtys, D. P. Gottesman, and J. R. Williamson, JBC 249, 1857 (1974).
B. REVIEWOF THE MECHANISM AND VEEGER

OF MASSEY
The mechanism proposed by Massey and Veeger (15,27), which was

based in large measure on the work of Massey et al. (24), has served
as the working hypothesis in the intervening 12 years. Chief feature of
the mechanism was the 2-electron-reduced intermediate, EH,, with the
electrons shared between the flavin and the reactive disulfide. I n catalysis
the enzyme was reduced to EH, and reoxidized once in each catalytic
cycle. An equilibrium of EH, (at a second site on EH, referred to as
the “Y” site) with NAD’ was proposed as a side reaction because of
the requirement for NAD’ in the oxidation of NADH by lipoamide a t
low p H ; however, in the discussion the authors emphasized that while
they had made this a side reaction in the scheme, it was likely that NAD’
was bound to the Y site in the catalytic cycle per se. Thus, the proposed
mechanism was of the ping-pong type but with provision for the role
of NAD+ (in NADH oxidation) in protecting the enzyme against over-
reduction when NAD’ was bound a t the Y site. I n addition to the cata-
lytic mechanism, the scheme also proposed side reactions to account for
the inhibited 4-electron-reduced state.
The five lines of evidence which led to this scheme can be summarized
as follows:
1. The two-electron stoichiometry of the reduction of the enzyme to
an intermediate in which the flavin had spectral properties intermediate
between those of FAD and FADH,.
2. The fact that the intermediate was formed and reoxidized in half-
reactions at rates commensurate with the kinetics observed in the overall
reaction.
3. The inhibition by arsenite preincubation in the presence of NADH
and the spectral effects in the presence of arsenite (Section 11,B).
4. The requirement for NAD’ in the oxidation of NADH at low pH
together with the four-electron reduction of the enzyme by NADH in
the presence of NADase to an inactive form.
5. The parallel line kinetics found when one substrate was varied a t
several levels of the second substrate.
The data will be discussed briefly by way of introduction to the next
three sections.
c. PROPERTIES OF THE 2-ELECTRON-REDUCED ENZYME,

EH,
The spectral characteristics of pig heart lipoamide dehydrogenase re-
duced anaerobically a t pH 7.6 and 2 5 O under the following conditions
are virtually identical: 1 equivalent (1 mole/mole of enzyme FAD) of
NADH with NADase present (66, Fig. 5) or excess dihydrolipoamide

(24, Fig. 1 ) . The extinction coefficient of EH, is 3.05 mM-l cm-l a t 530
nm and 8.7 - 9.2 mM-' cm-l a t 445 nm. A large excess of NAD+ (100
equivalents) with a small excess of NADH (2 equivalents) leads to the
formation of a distinct spectral species shown in Fig. 6 (118).Based on
its high absorbance in the 450-nm region, it is postulated that this is
a complex of NAD+ with EH, (118).Rapid reaction measurements show
that it is formed in less than 3 msec and thus represents a potential
catalytic intermediate (118).
Large excesses of NADH lead to the formation of a complex with EH,.
This complex was originally observed in glutathione reductase (62,63),
but retrospective examination of spectra of lipoamide dehydrogenase in-
dicates that it is present in this enzyme also (118, Fig. 2, control curve,
19 equivalents NADH and 160, Fig. 5, curve 2). The available data indi-
cate that a tight complex of this type is formed in lipoamide dehydroge-
nase of Pythium ultimum (106), Peptococcus glycinophilus (112), and
perhaps yeast (101) and spinach (99). The characteristics of this complex
will be discussed in connection with glutathione reductase, but the diag-
NADHI-NAD rotio(fin0l)-
320 700
Wavelength (nm)
FIQ.6. Complex formation between the 2-electron-reduced form of pig heart lipo-
smide dehydrogenase and NAD' (118).
nostic sign of its formation is a drop in extinction in the 450-nm region

without a proportional drop in the 530-nm region upon the addition of
large excesses of NADH to EH,. I n some cases there is an increase in
extinction a t 530 nm (150, Fig. 5 ) , and this is the case with glutathione
reductase (52, 53).
A number of conditions lead to the further reduction of EH,. I n the
presence of NADase, excess NADH reduces the enzyme completely (four
electrons) ( 9 7 ) . It has been argued that the protection of the enzyme
by NAD' against four-electron reduction is the result of its binding a t
the same site as would the second NADH molecule (27).An alternative
explanation is that the NAD+ acts to reverse an already unfavorable
equilibrium (151).The effective potential (reflecting true potential and
substrate binding) a t pH 7.6 of the 2-electron-reduced enzyme-4-elec-
tron-reduced enzyme couple must be more negative than that of dihydro-
lipoamide-lipoamide (-325 mV, p H 7.6) ( 4 ) since EH, is stable in the
presence of 20 equivalents of dihydrolipoamide (24). The potential of
the NADH-NAD' couple a t this p H is -340 mV (152), i.e., only about
15 mV more negative than that of lipoate; thus, the removal of the prod-
uct, NAD', is crucial if extensive reduction of EH, is to be observed
a t pH 7.6. This is illustrated in a direct comparison of the rate of reduc-
tion of EH, by excess NADH or NADPH in the presence of NADase
in lipoamide dehydrogenase and glutathione reductase, respectively.
NADP' is much more tightly bound to the 4-electron-reduced glutathione
reductase than is NAD+ to 4-electron-reduced lipoamide dehydrogenase ;
and the rate of reduction of EH, in the former is much slower since
NADP' or NAD' bound to their respective enzymes are not available
to the hydrolytic action of NADase ( 2 9 , 5 2 , 5 3 ) .At pH 6.3 the separation
of potential between dihydrolipoamide and NADH is much greater (ca.
55 mV). At this pH, about 20% reduction of EH, to the fully reduced
enzyme is observed (24, Fig. 2) in a slow reaction, and this overreduction
is prevented by NAD+ (137).This degree of overreduction implies that
the effective potential of the 2-electron-reduced enzyme-4-electron-re-
duced enzyme couple is about 7 mV more negative than the NADH-
NAD' couple at pH 6.3 (-298 mV). In the presence of lipoate, i.e., in
turnover, overreduction of EH, is rapid (137).
Formation of EH, upon reduction with 1 equivalent of dithionite is
only about 85% complete because of overreduction, and the enzyme is
fully reduced by 2 equivalents of dithionite (59).
Reduction of lipoamide dehydrogenase by NADH with arsenite present
150. L. Casola and V. Massey, JBC 241, 4985 (1966).
151. D. R. Sanadi, Proc. Int. Congr. Biochem., 6th, 1961 Vol. 5, p. 172 (1963).
152. F. L. Rodkey, JBC 213,777 (1955).
has been discussed in Section I1,B. It leads to the production of a charge

transfer complex between FADH, and NAD' that is characterized by
a broad absorption band centered a t 700 nm imparting a green color to
the enzyme (27). Similar results are observed if cadmium ion (26, 28,
163) or mercurials (160) are present or if the enzyme has been pretreated
with cupric ion (164); however, the mechanisms by which these three
reagents effect the conversion to the charge transfer complex are quite
different: Cadmium ion acts like arsenite bridging the nascent thiols (26,
28, 153) ; mercurials react with one or both of the nascent thiols of the
reactive disulfide (160) ; and cupric ion causes the oxidation of 2 thiols
to a disulfide, causing sufficient strain to weaken (but not totally prevent)
the interaction between the FAD and the sulfur in the 2-electron-reduced
enzyme (166, 156). Details of the modifications by cupric ion, by cad-
mium ion, and by mercurials will be discussed in Section II1,F. This same
species can be formed from EH,, which has been produced by the addition
of 1 equivalent of NADH, simply by cooling the enzyme to 4 O ; the dis-
mutation is slow, requiring more than 16 hr to form the equilibrium mix-
ture of oxidized enzyme and the charge transfer complex (65).
The stability of EH2 is very species dependent. All of the above re-
sults refer to the pig heart enzyme and, where tested, t o other mammalian
species. It was initially reported that no long wavelength absorption was
observed upon reduction of E. coli enzyme with NADH (109), but reduc-
tion by 1 equivalent of NADH or dihydrolipoamide leads to the forma-
tion of 25% of the maximal 2-electron-reduced species (108) and similar
results are obtained with the Azotobacter enzyme (114). That this species
is the catalytically important one in the E. coli enzyme as well as in
the mammalian enzyme has also been demonstrated (60).Reduction with
dihydrolipoamide in the rapid reaction spectrophotometer a t 2O results
in the full formation of EH, followed by the slow (k = 13 min-l, 1 mM
dihydrolipoamide) four-electron reduction. The spectrum of EH, gener-
ated in this way is shown in Fig. 7 and is identical with that of the pig
heart enzyme. The 2-electron-reduced form, EH2 of lipoamide dehydroge-
nase of spinach (99) may be somewhat unstable; however, spectrally
it is difficult to distinguish between instability and formation of the EH,-
NADH complex (see above) on the basis of available spectral data.
Either phenomenon could lead to inhibition by excess NADH. I n gluta-
thione reductase it is possible that the complex can be rapidly reoxidized
by glutathione (65),
153. A. M. Stein and J. H. Stein, JBC 248,670 (1971).
154. C. Veeger and V. Massey, BBA 64, 83 (1962) ; 37, 181 (1960).
155. L. Casola, P. E. Brumby, and V. Massey, JBC 241,4977 (1988).
156. R. G. Matthews and C. H. Williams, Jr., BBA 370, 39 (1974).
Wavelength (nm)
FIG.7. Escherichia coli lipoamide dehydrogenase. The spectrum of the oxidized
enzyme ( A ) . The spectrum of the 2-electron-reduced enzyme (0) was generated
from rapid reaction spectrophotometry as described in the text. The spectrum of
the 4-electron-reduced enzyme (0) was produced by anaerobic reduction by
12 moles dihydrolipoamide/mole FAD.
D. KINETICSTUDIES
The kinetics of the half-reactions for pig heart lipoamide dehydroge-
nase, i.e., the conversion of enzyme to EH, by NADH or dihydrolipo-
amide and the reoxidation of EH, by NAD' or lipoamide derivatives,
have been measured by rapid reaction spectrophotometry (24, 137). Re-
duction of the enzyme by NADH and reoxidation of EH, by NAD' are
complete in the dead time of the instrument which is 3 msec. The rate
of reduction of the enzyme by dihydrolipoamide is rate determining in
the overall reaction and is 33,000 min-I a t infinite reductant conceptra-
tion; the same rate is determined by conventional kinetics a t infinite
concentration of both substrates ( 2 4 ) .
Initial velocity patterns obtained for the reduction of NAD' by dihy-
droplipoamide give a series of parallel lines (reciprocal plots). The K,,,
for NAD- is 0.2 mM, and the K,, for dihydrolipoamide is 0.3. mM. On
the basis of these data the authors proposed what would now be referred
to as a bi-bi ping-pong mechanism (94). This can be represented as fol-
lows, where E is the oxidized enzyme, EH, is the 2-electron-reduced en-
zyme, lip (SH) ,NHz is dihydrolipoamide, and lipS,NH, is lipoamide:
ki
E + lip(SH)zNH2ekr [E-lip(SH)gNHZ EHz-~~~SZNHZ] (5)
kr
[E-lip(SH)nNHze EHrlipS~NH~]
e EHz
kr
+ lipS2NHz (6)
kr kr
EHz + NAD+ e [EHz-NAD+ E-NADH] e E + NADH (7,8)
ks kn
The pH optimum of the pig heart lipoamide dehydrogenase in the
direction of NAD+ reduction by dihydrolipoamide is 7.9 ( 4 ) .I n the direc-
tion of NADH oxidation by lipoamide the pH optimum is 6.5 ( 4 ) . I n
this latter direction there is an absolute requirement for NAD+ a t the
pH optimum (971, but this requirement disappears as the pH is raised
(116). It is therefore crucial to be aware of the pH of the measurements
in comparing kinetic data.
A more recent examination of the kinetics of this enzyme by initial rate
measurements has included product inhibition patterns and has led to
the conclusion that a t least under some conditions an ordered bi-bi mecha-
nism applies which involves a ternary complex of enzyme, NAD+, and
dihydrolipoamide (167).Clear spectral evidence is presented for the exis-
tence of a complex between NAD+ and the oxidized enzyme and this will
be discussed in Section II1,E. The product inhibition pattern for NAD+
tended toward that expected for this mechanism only at high NAD+
concentration.
A third study of the kinetics of lipoamide dehydrogenase has utilized
the enzyme isolated from rat liver (96). At 25O, the temperature of the
two previous studies, when dihydrolipoamide was varied a t fixed levels
of NAD+, the double reciprocal plots were concave down. At 3 7 O this
behavior was not observed. The detailed studies were carried out a t the
higher temperature. Rates were measured in both directions a t p H 8.0,
the pH optimum for the reduction of NAD+. Under these conditions,
initial velocity patterns for the forward and reverse reactions were a
series of parallel lines. The K, for NAD+ was 0.52 mM, for dihydrolipo-
amide was 0.49 mM, for NADH was 0.062 mM, and for lipoamide was
0.84 mM. The maximum rate for NAD+ reduction was 20,700 min-*/FAD
157. J. Viaser, H. Voetberg, and C. Veeger, in “Pyridine Nucleotide-Dependent
Dehydrogenases” (H.Sund, ed.), p. 359. Springer-Verlag, Berlin and New York,
1970.
and for NADH oxidation was 7,500 min-’/FAD. These data were consis-
tent with a bi-bi ping-pong mechanism. Further support for this mecha-
nism came from rates of exchange between 14C-NAD+and NADH; the
time course of this exchange was unaltered by the addition of a mixture
of lipoamide and dihydrolipoamide. The substrate inhibition patterns
were complex and a t variance with the previous study (157).Dependence
of pattern type on the concentration of the fixed substrate was observed.
The overall pattern was consistent with a “dead end” inhibition product of
EH,-NADH. This may prove to be a most interesting result in view of
the spectral indication of such a complex (see Section II1,C).
The kinetics of the yeast lipoamide dehydrogenase in the direction of
NAD’ reduction indicate a bi-bi ping-pong mechanism is operative in
this species also (117).If the enzyme from yeast indeed proves to have
a tighter EH,-NADH complex than does the mammalian enzyme, prod-
uct inhibitions studies should show impressive dependence on the fixed
substrate (96).
The kinetic studies cited above have assumed that in lipoamide dehy-
drogenase the two active centers are independent of one another. A recent
paper has indicated homotropic regulation is operative under some condi-
tions (157,158).
E. ROLEOF NADt AS A MODIFIER

The requirement for NAD+ in the oxidation of NADH by lipoic acid
derivatives a t low pH led to the proposal of a second binding site, referred
to as the ‘‘Y site,” for pyridine nucleotide in lipoamide dehydrogenase
(97).The argument that NADt acted in preventing overreduction simply
by reversing the unfavorable equilibrium between EH, and the 4-elec-
tron-reduced enzyme (161) rather than by combining a t a second site
has been discussed above (see Section 111,C). Several other lines of evi-
dence have been advances which are consistent with two pyridine nucleo-
tide sites, but none of these leads unequivocally to the conclusion that
both sites are involved in catalysis. The matter is complicated by the
fact that the binding constant of a second site might be high on EH2
but low on the oxidized enzyme. Some of the experiments to be outlined
have dealt only with the oxidized enzyme.
Binding of NAD+ to the oxidized enzyme causes small changes both
in the visible (157,159,160) and in the fluorescence spectra (161). These
158. C. S.Tsai, ABB 159,453 (1973).
159. A. M. Stein and G . Czerlinski, Fed. Proc., Fed. Amer. SOC.Exp. Biol. a,
842 (1967).
180. H. Muiswinkel-Voetberg and C. Veeger, Eur. J . Biochem. 33, 285 (1973).
161. G. Su and J. E. Wilson, ABB 143,253 (1971).
changes are highly temperature-dependent, and in this connection the

temperature dependence of the kinetics (96) (Section II1,D) should
be recalled. Although the fluorescence changes are small, the sensitivity
of the method allowed usable measurements down to NAD' concentra-
tions of 125 p M (161). At 5" and a t 25O plots of reciprocal fluorescence
change vs. reciprocal NAD+ concentration are clearly biphasic while a t
37O they are monophasic. At 25O K D values of 0.2 and 0.9 mM were esti-
mated; a t 37O the K D was 1.7 mM. The data were interpreted as indicat-
ing two sites for NAD+a t the lower temperatures.
Changes in the visible spectrum upon NAD+ binding are not sufficiently
large to allow measurements a t low NAD' concentrations (167, 160).
Maxima (positive) in the difference spectrum were observed a t 505 and
387 nm and minima (negative) at 477,450,430, and 370 nm. The relative
extinction changes varied with temperature. The largest change was a t
450 nm and was about 0.34 mM-' cm-l. The binding curves were not regu-
lar hyperbolas, and dissociation constants were evaluated from Stockell
plots. These gave dissociation constants of 50 and 200 pM a t 25O for
the changes a t 430 nm and indicated more than one site. The lowest con-
centration used in these experiments was 100 ~LM. The disparity between
the two sets of dissociation constants has been discussed by both groups
(160, 161).
Alkylation of native pig heart lipoamide dehydrogenase by concen-
trated iodoacetamide results in a 95% decrease in the NADH-lipoamide
reductase activity and in the loss of the ability to form the charge trans-
fer complex between NAD' and 4-electron-reduced enzyme (163). Since
the formation of this complex is absolutely dependent on the presence
of NAD', the authors interpreted their results as indicating that the
alkyation had destroyed the NAD' binding site by reaction with a thiol.
The groups alkylated were not characterized. It was indicated that the
modified enzyme could be reduced to EH, but that further reduction re-
sulted with excess NADH (153). Subsequently, enzyme modified by con-
centrated iodoacetamide has been characterized more fully (166). Two
thiols and one methionine were alkylated. The more slowly reacting of
the two thiols could be protected by high concentrations of NAD'. This
thiol was identified; of the 7-8 thiols in the pig heart enzyme, 7 have
been identified by association with unique sequences (63, 86) and these
will be discussed in the next section. It was shown that all of the modified
enzyme could be reduced to EH, by NADH thus indicating that the 5%
residual activity was not resulting from 5% unmodified enzyme but
rather from an inherent activity of the modified enzyme (166). Further,
this suggested that the substrate binding site was not the site affected
by alkylation. Since the modified enzyme could no longer form the charge
transfer complex with NAD' in the 4-electron-reduced state, the possibil-

ity was presented that a second pyridine nucleotide binding site was that
affected by alkylation (156).In view of the marked lowering of activity
these data do not preclude modification of the substrate binding site. If
indeed there is but one pyridine nucleotide binding site, the modification
may only prevent the exact alignment necessary for charge transfer.
Recent findings have clarified the role of NAD' in the activation of
NADH oxidation by lipoic acid (160). It was found that NAD' shifted
the pH optimum of this reaction from 6.2 to 5.0 and that NAD+ binding
was associated with the release of a proton. It was concluded that NAD'
binding lowered the pK of a group essential for activity in its deproto-
nated form, and the suggestion was made that the group might be a thiol
(160). If this is the case, i t is almost certainly the thiol which, when
alkylated, prevents charge transfer between NAD' and the 4-electron-
reduced enzyme. The apparent pK (6.6) of the group(s) giving rise to
the high pH arm of the pH vs. activity curve did not change upon "AD+
binding (160).The authors speculated on the identity of this latter group
suggesting that it might be the lysine residue near the active center disul-
fide (Fig. 5 ) , pointing out that the pK was low for a lysine residue but
not without precedent. This apparent pK should be ascribed to factors
having to do specifically with the reaction in the direction of NADH
oxidation when lipoic acid is the acceptor. If lipoamide is the acceptor,
this pK shifts to a higher value, ca. 7.5. Furthermore, in the direction
of NAD' reduction, a still higher but indeterminant pK is seen ( 9 5 ) .
A requirement for a lysine residue (or the terminal amino group) in
the charged configuration would be very interesting since electrophilic
catalysis can be an enhancement factor in thiol-disulfide interchange
(162) which is the first step in the reoxidation of the enzyme. I n this
role i t could, a t the same time, stabilize the increasing negative charge
on the substrate and the decreasing negative change on the enzyme. A
representation of a possible transition state is shown in Fig. 8A. Electro-
philic catalysis is not necessary in the direction of dihydrolipoamide
oxidation as shown in Fig. 8B, since the nascent thiolate anion is stabi-
lized in EH,. But the same residue, a t the higher pH optimal for the
reaction in this direction, might be expected to be largely uncharged and
in this state serve as an excellent general base in the formation of a
thiolate anion on the substrate (Fig. 8B). Lysine residues have been
found near the disulfide in E. coli (33) and pig heart (63, 85, 86)
lipoamide dehydrogenase, in yeast glutathione reductase (35), and in
thioredoxin ( 4 5 , 4 7 ) .
162. D. S. Garwood and D. C. Garwood, J. Org. Chem. 37, 3804 (1972).

(A1 (8)
-S
-FAD)0
pH 6.3 pH 8.0
FIG.8. (A) Hypothetical transition state in the electrophilic catalysis of thiol-di-
sulfide interchange. (B) General acid-base catalysis of thiol-disulfide interchange
by a hypothetical amino group.
Thus far a modifier role for NAD+has been discussed only in the direc-
tion of NADH oxidation. Recent studies suggest that NAD+ may also
have a double role in the direction of NAD+ reduction by dihydrolipo-
amide (163).
STUDIES
F. STRUCTURAL
The total half-cystine content of pig heart lipoamide dehydrogenase
is 10 per FAD (63). The basis for the protein quantitation has been dis-
cussed in Section I1,C. Older data suggested that there were 2 cystine
residues and 6 cysteine residues (83),but more recent data (61,63) give
strong evidence that the active center cystine residue is the only cystine
residue. Only 7 thiols react with DTNB under denaturing conditions
(63); however, recalculation of data of Brown and Perham (86),taking
51,000 as the molecular weight, indicates that under reducing and dena-
turing conditions 10 thiols are alkylated by iodoacetate. Thus it would
appear that the enzyme contains 8 thiols (one of which is very unreac-
tive) and the active center disulfide. Combining the data of two labora-
tories, Table I11 shows that unique compositions (and in some cases
sequences) are associated with 7 of the presumed 8 thiols. The thiol con-
163. C. Veeger, H. Voetberg, J. Pronk, and A. J. W. G. Visser, in “Structure and
Function of Oxidation Reduction Enzymes” (A. Akeson and A. Ehrenberg, eds.),
p. 476. Pergamon, Oxford, 1972.
TABLE I11
SEQUENCESAROUND THE RE.4CTIVE THIOLS"
Peptide
RI
Sequence 7
(3
51
18
DTC4a
0.00
0.00
0.00
1
-
Leu(Val,Cys(Cm),Ile,Gly)Arg
Val-Q@Cm)-IlyGly-Ar$
V+-Cy~(Cm)-IlfGly-Ar$
20 +0.14 Val(Cys(Cm),His,Ala,His,Pro,
Thr,Ser,Glx,Ala,Leu,Phe)Arg
DTC2n2 4-0.15 ~-C~(Cm)-HL-Al~-H~-Pro(Thr,Ser,Glx,Ala,Leu,Phe)Arg
cc
55 -0.23 Thr (Vtl,Cy!
(Cm),Ile, Glx )Lys,
0
DTC2b -0.27 Th-Val-Cys (Cm)-Ile-Glu-Lys D
m
10 Tyr(Ser,Glu,Ala,Leu,Gln,Gly,Asn,Gly,Ala,Ser,Cys(Cm),Glu,Asp,Ile,Ala)Arg 2
8 -0.51 (Gln,Gly,Asn,Gly,Ala,Ser,Cys(Cm),Glu,Asp,Iie,Ala)Arg
DTC2a2 -0.52 Gly,Ala-Ser,Cys(Crn)-Gl~-Aslj-Il~-Al~-Ar~
7777
52 -0.30 (Cys(Cm),Asp,Ser,Pro,VaL,Ile,Tyr)
No corresponding peptide
DTCla -0.87 Ala-Glx-Asx-Glx-Gly-Ile(Cys(Cm),Glx,Gly,Val,Met)
777777
DTClb3 -0.63 Ala-Gly-Val-Ile-Thr-Cys (Cm)-Asp-Val-Leu-Leu
7 7 7 7 7 - 1 7 7 1
Composition and sequence data (63,86).Simple numbers for peptides refer to Matthews et al. (63),while alpha-numeric designa-
0
tions refer to Brown and Perham (86). -,represents a residue placed by the dansyl-Edman or the subtractive-Edman procedures.
R, refers to peptide mobility on electrophoresis at pH 6.5 relative to the mobility of aspartic acid (-1.0). CL
r?
tained in peptides 18, 51, and DTC4a is the one thought to be protected
by NAD' from alkylation by concentrated iodoacetamide (156) (see Sec-
tion 111,E).
The thiols in the native enzyme are remarkable unreactive except with
mercurials (150, 164) and with cupric ion (154, 155, 165). Two thiols
react rapidly with phenyl mercuric acetate and 2 more slowly. Reduction
of mercurial treated enzyme (after removal of excess reagent) by NADH
results in the migration of phenyl mercury to the nascent thiols of the
active center (150).
Treatment of native pig heart lipoamide dehydrogenase with cupric
ion leads to loss of lipoate-linked activities and to a marked increase
(10- to 30-fold) in the NADH-DCI activity. Concomitantly there is a
drop of 2 in the number of titratable thiols. The action of cupric ion
is catalytic (154, 155). Amperometric titration in the presence of urea
before and after addition of Eulfite indicates that the cupric ion-treated
enzyme contains one disulfide in addition to the active center disulfide
(155).Sulfite reacts with disulfides as follows:
RS-S-R' + SOa*- F! RSSOa- + R'S-
Thus, an increase in the thiol titer of one upon treatment with sulfite
indicates the reaction of one disulfide. The thiols involved in the forma-
tion of this disulfide are contained in peptides designated 20 and 8/10
in Table 111 (156). It is of possible interest that peptide 20 contains
2 histidine residues, and it has been suggested that one or both of these
bind cupric ion prior to its catalysis of the formation of the disulfide
bond (156).Formation of the disulfide bond and the changes in the cata-
lytic activities can be reversed by dialysis against cysteine provided the
cupric ion treatment is not prolonged (155). If cupric ion is removed
after the rapid changes have occurred, and the enzyme stored a t Oo for
long periods (up to 7 months) , further oxidation of thiols appears to take
place. These changes are also reversed by treatment with cysteine and
are thus distinct from those observed upon prolonged reaction with cupric
ion which are not reversible by cysteine (155).This result may indicate
that a cluster of thiols exists and that these can oxidize sIowly in enzyme
already containing a disulfide between thiols 8/10 and 20.
The properties of the enzyme treated briefly with cupric ion, other than
the marked changes in catalytic activities, would indicate that the forma-
tion of the disulfide bond does not markedly alter the enzyme. Thus,
in the optical spectrum the peak in the visible is blue-shifted only 3 nm;
164. M. Nakamura and I. Yamazaki, BBA 267,249 (1972).
165. E. Misaka and K. Nakanishi, J. Biochem. (Tokyo) 80, 17 (1966);59, 545
(1966).
the fluorescence excitation spectrum of the enzyme-bound FAD is un-

affected; and the fluorescence emission spectrum of the aromatic amino
acid residues is unaffected (155). Moreover, the enzyme is rapidly re-
duced to EH, (the 2-electron-reduced form) by NADH and by dihydro-
lipoamide, though reduction with the latter agent is not as rapid as in
the native enzyme. However EH, is not stable in the presence of excess
reductant and slow further reduction ensues leading, when NAD' is pres-
ent, to the charge transfer interaction (154). Prolonged treatment with
cupric ion leads to secondary changes as evidenced by the loss of the
high NADH-DCI activity and large decreases in the fluorescence exita-
tion spectrum of enzyme-bound FAD and to large increases in the fluores-
cence emission spectrum of the aromatic amino acid residues (155).
The thiols in native lipoamide dehydrogenase are remarkably unreac-
tive with other reagents; only one thiol is a t all reactive with DTNB
or iodoacetate (61). Formation of the TNB-enzyme mixed disulfide is
greatly increased by low concentrations (0.7 M ) of guanidine hydrochlo-
ride (166). Its modification is associated with the destabilization of the
enzyme in 1 M guanidine hydrochloride which results in the slow reaction
of 6 additional thiols. If the denaturant and excess DTNB are removed
when the single thiol has reacted, the spectrum of enzyme-bound FAD
is unmodified and the enzyme retains almost full activity. It is concluded
that the thiol and the FAD are remote from one another in the protein
(166).
Another facet of the reversible denaturation of the pig heart enzyme
by concentrations of guanidine hydrochloride up to 1 M is the effect of
the perturbant on activity and on the FAD spectrum (166). The differ-
ence spectrum in the visible and near ultraviolet between enzyme in
guanidine hydrochloride and enzyme in an equal concentration of sodium
chloride arises from a generalized red shift and a drop in extinction a t
455 nm. The changes are complete within the 3-msec dead time of the
rapid reaction spectrophotometer. The difference spectra are reminiscent
of, but some 5-fold larger than, those effected by temperature (167). The
activity in the direction of lipoamide reduction falls to 20741,while in
the direction of dihydrolipoamide oxidation it falls to 10% a t 1.0 M
denaturant; the NADH-DCI activity rises slightly. All of the changes
are reversible (166). Slightly higher concentrations of guanidine hydro-
chloride lead to the dissociation of the FAD (168). The effect of sodium
dodecyl sulfate on the activity, fluorescence spectrum, fluorescence polar-
ization, circular dichromisms, and sedimentation coefficient have been
166. C. Thorpe and C. H. Williams, Jr., Biochemistry 13, 3263 (1974).
167. F. Muller, S. G . Mayhew, and V. Massey, Biochemktry 12, 4654 (1973).
168. A. H. Brady and S. Beychok, JBC 244,4634 (1969).
124 CHARLES H. WILLIAMS) JR.
reported (169).Binding of this agent to the protein takes place in two

phases, the first of which can be reversed by cooling to Oo. The effects
on activity in the NADH-lipoate reaction are dependent on a group with
pK of 6.6. At higher concentrations dissociation of the FAD and dissocia-
tion to the monomer are observed (169).
Lipoamide dehydrogenase is quite stable in 6.5 M urea provided it is
not frozen or reduced; full activity is regained upon dilution (32, 170).
However, if reduced with NADH in 6.5 M urea, EH,, which is initially
formed, is further reduced over a period of a few hours. The activity
remaining upon dilution during this period is directly proportional to the
amount of EH, remaining a t the time of dilution. NAD’ stabilizes EH,
in 6.5 M urea just as it does in the absence of urea. When reduction in
urea is allowed to go to completion, the spectrum upon reoxidation is that
of free FAD indicating dissociation as a result of reductive denaturation
(82, 170).
The apoenzyme of pig heart lipoamide dehydrogenase has been pre-
pared by two quite different procedures. Both forms of the apoenzyme
can be reactivated by FAD, but they differ from one another in several
respects. Apoenzyme prepared by precipitation once with ammonium sul-
fate at low pH in the presence of a high concentration of monovalent
anions (171)is a monomer, i.e., molecular weight 52,000 (172-174).This
preparation is moderately stable at Oo. It has less than 10% of the origi-
nal FAD, less than 5% of the original NADH-lipoate activity, and about
90% of the original NADH-DCI activity ; the latter activity probably
results from the residual FAD turning over a t a rapid rate. The yield
of apoenzyme in this preparation is inversely dependent on the protein
concentration. The fluorescence yield of the aromatic amino acids is en-
hanced in the apoenzyme (relative to that in the holoenzyme) , and excita-
tion maxima are exhibited a t 284 and 290 nm (173-176).In the second
procedure, apoenzyme is prepared by dialysis against 1.5 M guanidine
hydrochloride, pH 7.6 to which F M N is added a t a concentration equal
to that of the eneyme-bound FAD. The guanidine hydrochloride is re-
moved by dialysis against F M N and finally the F M N is removed by
169. H.van Muiswinkel-Voetberg and C. Veeger, Eur. J . Biochem. 33, 279 (1973).
170. V. Massey, JBC 235, PC47 (1960).
171. P. Strittmatter, JBC 236, 2329 (1961).
172. C. Veeger, D. V. Dervartanian, J. F. Kalse, A. DeKok, and J. F. Koster,
in “Flavins and Flavoproteins” (E. C. Slater, ed.), Vol. 1, p. 242. Elsevier, Amster-
dam, 1966.
173. J. F.Kalse and C. Veeger, BBA 159,244 (1968).
174. C.Veeger, in “Flavins and Flavoproteins” (K. Yagi, ed.), Vol. 2, p. 252. Univ.
Park Press, Baltimore, Maryland, 1908.
175. J. Visser and C. Veeger, BBA 206,224 (1970).
dialysis against buffer. The F M N stabilizes the aproprotein in the pres-

ence of the gunnidine hydrochloride but binds only very loosely, if a t
all (see below) (168).The apoenzyme prepared by this method seems to
be dimeric (17 6 ) .
The recombination of FAD with apoenzyme prepared by the acid am-
monium sulfate method (monomer) is a multistep process (172-175). It
can be divided into two phases though each is complex. I n the first phase
FAD combines rapidly (half-time less than 2 min, 5O) with the mono-
meric apoprotein ; the product, still monomeric, initially gains (half-time
also less than 2 min, 5 O ) a very high NADH-DCI activity and this de-
creases about 25% over a 5-min period to a level about 12 times that
of the native enzyme. This species has very low NADH-lipoate activity.
At 5O no second phase ensues; a t higher temperatures, however, dimeriza-
tion leads to a fully active holoenzyme. The half-time for this process
a t 2 5 O and a t apoprotein concentrations of 0.5-1.0 mg/ml is about 5 min.
The return of NADH-lipoate activity is roughly parallel with the de-
crease in NADH-DCI activity to levels near those of native enzyme.
The activation energy of the process is 15-20 kcal/mole. As would be
expected, the rate of the second phase is dependent on protein concentra-
tion; it is optimal a t pH 7.2 and a t 0.2 M phosphate concentration. Care-
ful examination of the properties of the reconstituted enzyme is possible
only after removal of denatured protein by chromatography on calcium
phosphate gel which also serves to remove excess FAD. Reconstitution
of holoenzyme is inhibited by FMN, ADP, ATP, NAD’, and pyrophos-
phate, indicating that multiple linkages are involved in the very tight
binding. Of the several flavin derivatives tested, only 3-methyl-FAD
binds to give enzyme with appreciable NADH-lipoate activity (173-175,
177). Detailed curve fitting indicates that binding of FAD in lipoamide
dehydrogenase introduces no new transitions, i.e., the same set of six
Gaussian bands can be accounted for in free and bound FAD (178).
A reversible dimer-monomer transition has been reported independent
of FAD dissociation (174, 179-182). This transition seems t o be potenti-
ated by the exhaustive removal of phosphate ion or by freezing of dilute
176.
V. Massey, personal communication.
J. Visser, D. B. McCormick, and C. Veeger, BBA 159, 257 (1968).
177.
A . H. Brady and S.Beychok, JBC 246,5498 (1971).
178.
J. Visser and C. Veeger, BBA 159, 265 (1968).
179.
180. C. Veeger, H. Voetberg, J. Visser, G. E. J. Staal, and J. F. Koster, in ‘‘Flavins
and Flavoproteins” (H. Kamin, ed.), Vol. 3, p. 261. Univ. Park Press, Baltimore,
Maryland, 1971.
181. H. van Muiswinkel-Voetberg, J. Visser, and C. Veeger, Eur. J . Biochem. 33,
265 (1973).
182. H. van Muiswinkel-Voetberg and C. Veeger, Eur. J . Biochem. 33, 271 (1973).
enzyme at low ionic strength; dimer is converted to monomer upon dilu-

tion following either treatment. The monomer is characterized by high
NADH-DCI activity and low NADH-lipoate activity. Immediately fol-
lowing thawing of dilute enzyme frozen a t low ionic strength, the NADH-
lipoate activity is 75% of normal, the NADH-DCI activity is raised,
and the peak in the visible is shifted from 455 to 452 nm. These three
parameters return to normal overnight. The presence of bovine serum
albumin, EDTA, or ammonium sulfate during freezing of dilute enzyme
protect against the changes seen upon thawing. Enzyme potentiated
toward dissociation to monomer can be stabilized by NAD+, by heating
to 70°, by alkaline pH, or by 0.2 M phosphate at pH 7.2 (174,179-182).
Lipoamide dehydrogenase from E . coli has been crystallized but there
appears to be more than one dimer per asymmetric unit making X-ray
structure determination difficult (183,184). The yeast enzyme has also
been crystallized as long yellow needles approximately 20 p in width (104,
186).
Measurements of fluorescence energy transfer have allowed estimation
of the intramolecular distance between one of the two tryptophan residues
of pig heart lipoamide dehydrogenase and the enzyme-bound FAD. This
distance is found to be 13-16 A (186).Another very interesting estimation
of distance by fluorescence energy transfer is that between the FAD of
lipoamide dehydrogenase and the thiamine pyrophosphate of pyruvate
dehydrogenase in the complex of E . coli (187).This distance is between
30 and 60 A. Lipoic acid, bound through amide linkage to the €-amino
group of a lysine residue in the transacetylase, must make contact with
three groups in the course of its reductive acetylation, acetyl transfer,
and reoxidation [see Eqs. (2), ( 3 ) , and ( 4 ) ] . The lipoic acid itself, to-
gether with the side chain of the lysine residue, is about 14 A long (126).
Thus, it would appear that small changes are required in the juxtaposi-
tion of the three enzymes within the complex as the dithiolane ring moves
from one site to another (187).
G. SUMMARY AND CONCLUSIONS
The chief feature of the mechanism of lipoamide dehydrogenase pro-

posed by Massey and Veeger (16,27) is the catalytic intermediate in
183. D. DeRosier, personal communication.
184. J. Hainfeld, Ph.D. Dissertation, University of Texas, Austin, 1974.
185. E. Misaka and K. Nakanishi, J. Biochem. (Tokyo) 53, 465 (1963).
186. A. J. W. G. Viwer, H. J. Grande, F. Muller, and C . Veeger, Eur. J . Biochem.
45, 99 (1974).
187. 0. A. Moe, Jr., D. A. Lerner, and G. G. Hammes, Biochemistry 13, 2552
(1974).
which the enzyme has accepted two electrons and these are shared be-
tween the FAD and the reactive disulfide. Furthermore, this intermediate,
BH,, turns over once in each catalytic c,ycle accepting two electrons from
dihydrolipoamide and donating them to NAD'. Figure 9 shows this cycle;
I is the oxidized enzyme and I11 is EH,, while I1 and IV are complexes
of EH, with the substrates. The enzyme catalyzes a readily reversible
reaction, but the physiological direction is clockwise. Very simple repre-
sentations such as this one do not adequately describe all that is known
about the enzyme, and they force a choice of one form over others which
may be equally good. For example, EH, is depicted as a charge transfer
complex between thiolate anion and FAD ; but, as was discussed in Sec-
tion II,B, this may be an inadequate description of the sharing of two
electrons between the sulfur and FAD. On the other hand, such represen-
tations act as working hypotheses and suggest features of the mechanism
to be tested. One such feature that has long eluded investigators is the
possibility of intermediates in the very rapid reduction of the enzyme
by NADH. It has been suggested that the FAD is reduced to FADH,
followed by intramolecular electron rearrangement (98).It can be argued
that without such a step the flavin does not play a true redox function
when EH, is a charge transfer complex. If FADH, is an intermediate
I II
NADp
f$ ~ 7;
SH
IE III
Fro. 9. Mechanism for lipoamide dehydrogenase.
128 CHARLES €WILLIAMS,
I. JR.
in catalysis, then lipoamide dehydrogenase and glutathione reductase

would share a common intermediate in catalysis with thioredoxin reduc-
tase (Sections IV,B and V,D) .
The scheme in Fig. 9 suggests that lipoamide dehydrogenase functions
by a simple binary complex mechanism, and this conforms to a vast body
of kinetic evidence (Section, II1,D) , Since the oxidized enzyme can form
a complex (or complexes) with NAD' and since EH, may form stable
complexes with both NAD+ and NADH, a classic binary complex mecha-
nism is too simple, Indeed, the substrate inhibition patterns are very com-
plex and do not conform to any classic pattern. Until spectral properties
and rates of formation and breakdown can be measured for each of these
complexes the simple binary complex mechanism must serve.
The reversibility of the reaction is also indicated by the scheme in
Fig. 9. At pH 8.0, optimal for the reaction in the direction of NAD' re-
duction, the extrapolated maximal rate of NADH oxidation is about one-
third that of NAD+ reduction, 7,500 and 20,000 min-' per enzyme-bound
FAD, respectively. Much higher rates of NADH oxidation are observed
a t lower pH provided NAD+ is present and the NADH concentration
is low; a t pH 6.5 and at infinite lipoamide concentration, rates in excess
of 80,000 min-l per FAD have been estimated. Many factors undoubtedly
contribute to the difference in pH optimum for the reaction in opposite
directions. The oxidation-reduction potentials of both substrate couples
change considerably between pH 8.0 and pH 6.5; a t the latter pH,
NADH is a much stronger reductant relative to dihydrolipoamide than
is the case a t pH 8.0. The oxidation-reduction potentials of the enzyme
must also change, and the combined changes result in the extreme sensi-
tivity to NADH and the protection against this inhibition by NAD'.
I n addition the predominant charge state of some groups on the enzyme
would be shifted over this pH range; a group serving as an acid-base
catalyst, so essential for thiol-disulfide interchange a t neutral pH (Fig.
81, would be effective in this role only a t pH values near its pK. Thus,
both enzyme and substrates are quite different at the pH optima of the
reaction in its respective directions.
The means by which NAD' affects the oxidation of NADH is still un-
certain. The evidence for two pyridine nucleotide binding sites is not com-
pelling. The alternative explanation that NAD+ functions by reversing
the equilibrium between EH, and 4-electron-reduced enzyme (EH,) is
shown in Eq. (9). There is some kinetic evidence for a dead end' complex
EH1+ NADH EHrNADH EHd-NAD+ EHI + NAD+ (9)
such as EH,-NADH. The kinetic evidence neither requires nor suggests
two sites. Binding studies suggesting two sites have been carried out with
the oxidized enzyme, whereas it is binding to EH, that is crucial t o the

problem. The chemical modification, resulting in the abolition of the abil-
ity to form the charge transfer complex between FADH, and NAD‘,
while not completely eliminating the ability of NADH t o reduce the
enzyme, may be explicable in terms of very specific and subtle changes
a t a single pyridine nucleotide site. Finally, it is of interest to consider
the structural consequences of two sites for pyridine nucleotide and one
for FAD on a monomer of 50,000 molecular weight. Between 90 and 140
amino acid residues are required to build up such a site (188). This would
require a large number of the 475 residues in the molecule. This alone
should make lipoamide dehydrogenase a prime candidate for crystallog-
raphy and sequencing.
IV. Glutathione Reductase
Oxidized glutathione will be referred to as GSSG, and reduced gluta-

thione will be referred to as GSH. Many of the properties of glutathione
reductase have been discussed in Section 11. The ubiquity of glutathione
reductase activity has been reviewed and some newer data given (39).
The enzyme from yeast and from human erythrocytes has been most
extensively studied. It has also been purified from r a t liver (189, IN),
germinated peas (191), E . coli (5, 192), Penicillium chrysogenum (193),
and sea urchin eggs (194). I n all cases the enzyme has been shown to
be a flavoprotein.
FUNCTIONS
A. METABOLIC
Glutathione reductase catalyzes the virtually irreversible reduction of
GSSG by NADPH. Its metabolic function is therefore synonymous with
that of the product GSH. Glutathione is the most abundant thiol-disulfide
pair in the cell by more than an order of magnitude; under most condi-
tions the GSH:GSSG ratio is about 20:l (195). Since the ratio of
188. M. G. R o m a n n , D. Moras, and K. W. Olsen, Nature (London) 250, 194
(1974).
189. C. E. Mize and R. G. Langdon, JBC 237, 1589 (1962).
190. J. A. Ruzard and F. Kopko, JBC 238, 464 (1963).
191. L. W. Mapson and F. A. Isherwood, BJ 86,173 (1963).
192. C . H. Williams, Jr. and L. D. Arscott, “Methods in Enzymology,” Vol. 17,
503, 1971.
193. T. S. Woodin and I. H. Segel, BBA 167,64 and 78 (1968).
194. I. Ii and H. Sakai, BBA 350, 141 and 151 (1974).
195. P. C. Jocelyn, ed., “Biochemistry of the SH Group,” p. 10. Academic Press,
New York, 1972.
GSH :GSSG a t equilibrium of the reductase reaction is very high (calcu-

lated from data in 191, 196), it can be concluded that the reduction of
GSSG is limited either by the enzyme level or by the availability of
NADPH.
GSH is utilized in two types of reactions: First, it is the substrate
for enzymes such as GSH peroxidase (EC 1.11.1.9) and for a group of
transhydrogenases, GSH-homocystine oxidoreductase (EC 1.8.4.1), GSH-
protein disulfide oxidoreductase (EC 1.8.4.2), GSH-CoASSG oxidoreduc-
tase (EC 1.8.4.3), and GSH-cystine oxidoreductase (EC 1.8.4.4). The
most thoroughly studied of these transhydrogenases is the GSH-protein
disulfide oxidoreductase, better known as glutathione-insulin transhydro-
genase (197,198).It catalyzes the first step in the sequential degradation
of insulin (199). One of the products of the reaction, the phenylalanyl
chain, is an inhibitor of glutathione reductase (200). It is not known
if this constitutes a physiologically important feedback mechanism. The
transhydrogenase contains either a reactive disulfide or a masked thiol
since it is sensitive to thiol inhibitors only in the presence of its substrate,
GSH (201). A nonspecific, GSH-protein disulfide transhydrogenase has
also been purified (202).
Second, GSH functions, presumably nonenzymically, in the reduction
of protein thiols which have become oxidized to mixed disulfides (%XI).
I n this latter function GSH in some cases converts inactive enzymes to
active ones, or vice versa, and may thus serve as a means of metabolic
control. Examples of this important possibility are glycogen synthetase
D (EC 2.4.1.11) and fructose-1,6-diphosphatase(EC 3.1.3.11). The D
form of glycogen synthetase is dependent for activity upon the presence
of glucose 6-phosphate. The enzyme is inactivated by GSSG and reacti-
vated by GSH (204). Mixed disulfide formation between thiols of the
enzyme and GSSG leads to a decrease in affinity of the enzyme for its
activator (205).
A recent symposium on glutathione has covered many emerging func-
tions of this peptide (206). Several potentially important effects have
196. W. M. Clark, “Oxidation-Reduction Potentials of Organic Systems,” p. 486.
Williams & Wilkins, Baltimore, Maryland, 1960.
197. H.H.Tomizawa and Y. D. Halsey, JBC 234,307 (1959).
198. H.H.Tomizawa, JBC 237,428 and 3393 (1962).
199. P.T.Varandani, BBA 320,249 (1973).
200. R. G.Langdon, JBC 235, PC15 (1960).
201. P.T.Varandani and H.Plumley, BBA 151,273 (1968).
202. F. Tietze, BBA 220, 449 (1970).
203. R. Nesbakken and L. Eldjarn, BJ 87,526 (1963).
204. M.J. Ernest and X. H. Kim, JBC 248,1550 (1973).
206. M. J. Ernest and K. H.Kim, JBC 249,5011 (1974).
206. L. Flohe, H.C. Benohr, H. Sies, H. D. Waller, and A. Wendel, eds., “Proceed-
been inferred by observation of the consequences of perturbing the GSH-

GSSG poise with the oxidizing agent, diamide [ (CH,) ,NCON=
NCON (CH,) ,I. These include nerve transmission (20?’), membrane
integrity (208), and protein synthesis (209). The relative specificity
of this reagent for GSH has been demonstrated (210). A possible
connection between a number of disease states, chiefly hemolytic
and glutathione reductase deficiency, has been proposed (211). The
conditions are characterized by a low GSH :GSSG ratio or by an inability
to restore normal GSH-GSSG poise following a challenge, e.g., by a drug.
In almost all cases the GSH deficiency has been traceable either to a
lack of NADPH secondary to low glucose-6-phosphate dehydrogenase
(211-215) or to riboflavin deficiency (212, 216-222). I n the latter
case, glutathione reductase activity of erythrocytes was restored by ad-
ministration of riboflavin (217-220). The enzyme has been purified and
shown to have a diminished FAD affinity ( 2 2 1 , 2 2 2 ~ ) .
GSH acts catalytically in the rearrangement of incorrectly formed
ings of the 16th Conference of the German Society of Biological Chemistry.’’ Thieme,
Stuttgart, 1974.
207. E. M. Kosower and N. S. Kosower, in “Proceedings of the 16th Conference
of the German Society of Biological Chemistry” (L. Flohe et al., eds.), pp. 287-302.
Thieme, Stuttgart, 1974.
208. N. S. Kosower and E. M. Kosower, in “Proceedings of the 16th Conference
of the German Society of Biological Chemistry” (L. Flohe et al., eds.), pp. 216227.
209. N. S. Kosower and E. M. Kosower, in “Proceedings of the 16th Conference
of the German Society of Biological Chemistry’’ (L. Flohe et al., eds.), pp. 276-287.
210. E. M. Kosower, W. Correa, B. J. Kinon, and N. S. Kosower, BBA 264, 39
(1972).
211. H. C. Benohr and H. D. Waller, in “Proceedings of the 16th Conference of
the German Society of Biological Chemistry’’ (L. Flohe et al., eds.), p. 184. Thieme,
Stuttgart, 1974.
212. E. Beutler, in “Proceedings of the 16th Conference of the German Society
of Biological Chemistry” (L. Flohe et at?, eds.), pp. 109-114. Thieme, Stuttgart,
1974.
213. E. Beutler and S. K. Srivastava, Noture (London) 226, 759 (1970).
214. E. Beutler, Pharmacol. Rev. 21, 73 (1969).
215. S. K. Srivastava and E. Beutler, RJ 114, 833 (1969).
216. D. Glatzle, F. Weber, and 0. Wiss, Ezperientia 24, 1122 (1968).
217. E. Beutler. Science 165, 613 (1969).
218. B. Mandula and E. Beutler, Blood 36,491 (1970).
219. E. Beutler, J . Clin. Invest. 48, 1957 (1969).
220. S. K. Srivastava and E. Beutler, Ezperientia 26,250 (1970).
221. G. E. J. Staal, P. W. Helleman, J. DeWael, and C. Veeger, BBA 185, 63,
(1969).
222. N. V. Paniker, S. K. Srivastava, and E. Beutler, BBA 215, 456 (1970).
222a. D. J. Worthington and M. A. Rosemeyer, Eur. J . Biochem. 48, 167 (1974).
TABLE IV
POSSIBLEALTERNATIVE
SUBSTRATESOF GLUTATHIONE
REDUCTASE
Rate relative
Substrate to GSSG ( % ) a Ref.
Yeast GR
G-SS-CO A 10 886,887
GS--SOs- 0.04 887
G-SS-Cy 0.2 8.97
G-SS-Pantothineb 0.1 887
GSS-~-93-Hemoglobin - 688
GS-SeS-G ca. 100 889
bis-N,N( 7-Glutamyl) cystine 21 830
Erythrocyte GR
DL-Lipoate 3.1 39
GCystine 0.1 39
DkHomocystine 0.1 39
DTNB 0.4 39
Cgstamine 0.2 39
D-Pantothine 0.5 39
0 As discussed in Section II,A, rates of less than 0.5% are of doubtful significance.
Progressive inhibitor in the presence of NADPH (831).
disulfides during protein folding. This reaction is catalyzed by a micro-

soma1 enzyme, protein disulfide isomerase (EC 5.3.4.1) (2.23,224). GSH
unmasks a thiol group in the enzyme; this thiol group catalyzes the
isomerization (22%).
The specificity of glutathione reductase toward its disulfide substrate
was emphasized in Section II,A, since there is virtually no reactivity with
the substrates of the other pyridine nucleotide-disulfide oxidoreductases.
Other authors have emphasized the lack of specificity of this enzyme
since it can catalyze the reduction of a variety of mixed disulfides pro-
vided that glutathione or y-glutamylcysteine comprises one-half (193,
212) ; Table IV summarizes these ($9, 226-231). It is important to distin-
223. S. Fuchs, F. DeLorenzo, and C. B. Anfinsen, JBC 242,398 (1967).
224. E. D. Corte and R. M. E. Parkhouse, BJ 136,697 (1973).
225. F. DeLorenzo, S. Fuchs, and C. €3. Anfinsen, Biochemistry 5, 3961 (1966).
226. R. N. Ondarza and J. Martinez, BBA 113,409 (1966).
227. B. Mannervik and 8.A. Eriksson, “Glutathione,” pp. 120-132. Thieme, Stutt-
gart, 1974.
228. S. K. Srivastava and E. Beutler, BJ 119,353 (1970).
229. H. E. Ganther, Biochemistry 10,8089 (1971).
230. J. E. Smith, BBA 242, 36 (1971).
231. B. Mannervik and G. Nise, ARB 134,90 (1969).
guish between this activity and that of the transhydrogenases (EC 1.8.4
group) (232-836). In rat liver supernatant, it is clear that glutathione
reductase catalyzes the NADHP-dependent reduction of G-S-S-CoA
(837) ; however, there is some evidence for a distinct enzyme catalyzing
this reaction in yeast (238, 239) though this has been questioned (8.40).
B. PROPERTIES EH,
OF THE 8-ELECTRON-REDUCED ENZYME,
The catalytic center of glutathione reductase can be represented as

in Fig. 10, Structure I. I n catalysis the enzyme accepts two electrons
from NADPH and donates two electrons to GSSG. The catalytic
intermediate, Structure 111, will, as before, be referred to as EH,.
The mechanism is formally identical to that given in Fig. 9 for lipoamide
dehydrogenase. The spectrum of oxidized glutathione reductase and of
EH, are shown in Fig. 1. The evidence for the similarity between gluta-
thione reductase and lipoamide dehydrogenase in mechanism and struc-
ture has been discussed in Sections II,B and I1,C. Early recognitions of
these similarities should be cited (241-243) as well as the suggestions
that the flavoprotein (now referred t o as thioredoxin reductase) involved
in the NADPH-linked reduction of methionine sulfoxide and “active sul-
fate” was remarkably like lipoamide dehydrogenase and glutathione re-
ductase (7, 841, 24s).
The principal features, in addition to EH,, common to lipoamide dehy-
drogenase and glutathione reductase deserve emphasis : the formation of
complexes between the oxidized enzymes and their respective oxidized
pyridine nucleotides; the formation of complexes between EH, and both
oxidized and reduced pyridine nucleotides ; the formation of charge trans-
fer complexes between 4-electron-reduced enzymes and oxidized pyridine
232. S. H. Chang and D. R. Wilken, JBC 241,4251 (1966).
233. A. Eriksson and B. Mannervik, FEBS (Fed. Eur. Bwchem. Sac.) Lett. 7 , 26
(1970).
234. M. Winell and B. Mannervik, BBA 184,374 (1969).
235. S. A. Eriksson and B. Mannervik, BBA 212,518 (1970).
236. P. L. Wendell, BBA 159, 179 (1968).
237. R. N. Ondarza, E. Escamilla, J. Gutierrez, and G . De La Chica, BBA 341,
162 (1974).
238. R. N. Ondarea, R. Abney, and M. Lopez-Colome, BBA 191, 239 (1969).
239. R. N. Ondarza and R. Abney, FEBS (Fed. Eur. Biochem. Soc.) Lett. 7 , 227
(1970).
240. S. Eriksson, C. Guthenberg, and B. Mannervik, FEBS (Fed. Eur. Bwchem.
Soc.) Lett. 39, 296 (1974).
241. S. Black and B. Hudson, BBRC 5, 135 (1961).
242. V. Massey and C. Veeger, Annu. Rev. Biochem. 32,579 (1963).
243. S. Black, Annu. Rev. Biochem. 32,399 (1963).
v /1
NADP+
Ip m
F I ~ 10.
. Mechanism for glutathione reductase.
nucleotide; and the high degree of homology in the sequences of amino

acid residues around the active site disulfide. In both enzymes, EH, is
formed and reoxidized a t rates commensurate with its function as an in-
termediate in catalysis.
The principal differences between the two enzymes should also be men-
tioned. Lipoamide dehydrogenase catalyzes a reaction which is freely
reversible over the neutral pH range while the glutathione reductase reac-
tion is essentially irreversible, except a t high pH together with efficient
removal of the product (GSH). Lipoamide dehydrogenase functions with-
in a three-enzyme complex, accepting electrons from an enzyme-bound
intramolecular dithiol, while glutathione reductase accepts electrons from
NADPH and is reoxidized by GSSG yielding two molecules of GSH. Each
enzyme is quite specific for its pyridine nucleotide, both in catalysis and
in the formation of various characteristic complexes.
Complex formation between EH, and pyridine nucleotide (NAD') was
first noted in lipoamide dehydrogenase (87,118). It was clear that the
spectra of EH, were different when the reductant was NADH or when
it was dihydrolipoamide; and in light of the NAD+ requirement in the
oxidation of NADH, a complex of EH, with NAD+ was hypothesized.
Thus, when reductant-dependent differences were observed in the spectra
of 2-electron-reduced glutathione reductase EH,, an EH,-NADP' com-

plex was proposed (23). However, i t has subsequently been shown quite
conclusively that the major complex is EH,-NADPH in glutathione re-
ductase (52, 5 3 ) . In order to demonstrate EH,-NADPH or EH,-NADP+
complexes, it was necessary to produce the uncomplexed EH,. On the
assumption that the very slow turnover of yeast glutathione reductase
with NADH was a reflection of the poor binding of this pyridine nucleo-
tide, the rates of reduction of the enzyme to EH, by various concentra-
tions of NADH were measured. A second-order rate constant was deter-
mined as 2 X lo4 M-' sec-*, and there was no indication of a Michaelis
complex ( 5 2 ) . Evidence was then obtained for both EH,-NADPH and
EH,-NADP+ ( 6 3 ) . The dissociation constant of the EH,-NADPH com-
plex was found to be 2 p M EH,-NADP+ proved to be un-
stable, and the product (formed too slowly to be of catalytic importance)
was not identified ( 5 3 ) . These results with the yeast enzyme (53) have
been confirmed with the E. coli enzyme where the dissociation constant
for the EH,-NADPH complex was found to be 12 pM (60).The spectrum
of EH,-NADPH is shown in Fig. 11. Compared to uncomplexed EH,
I I I I I I I
I I I I I
400 500 600 700

Wavelength (nm)
FIQ.11. Complex formation between the 2-electron-reduced ( E K ) form of E. coli
glutathione reductase and NADPH. EHz was produced by anaerobic reduction with
borohydride; time wm allowed for the slight excew of borohydride to react with
water before beginning the titration with NADPH. 1, Oxidized; 2, EHz; 3,
EK+0.45 equivalent NADPH; 4, EH,+0.90 equivalent NADPH; and 5,
EHz+ 2.75 equivalents NADPH.
1 1 I I I I I
12 i
Wavelength (nm)
FIO.12. Complex formation between the 2electron-reduced form of E. coli gluta-
+
thione reductase and NADP+.l, Oxidized ; 2, EH,; 3, EHn 1.72 equivalents NADP’ ;
+
and 4, EH2 1036 equivalents NADP’. The procedure was as in Fig. 11.
it has higher extinction a t long wavelengths and lower extinction a t 450

nm. The spectrum of EH,-NADP’ is shown in Fig. 12; it is similar in
all respects to the analogous spectrum of lipoamide dehydrogenase (118)
in that, compared with the free EH,, it has lower extinction a t 540 nm
and higher extinction a t 700 nm with the isosbestic point a t 590 nm. I n
both cases EH, was produced with borohydride and the stable spectrum
was recorded after the small excess of borohydride had reacted with water
(60). EH, from yeast produced in this way is relatively stable in air
(though these experiments were done anaerobically) and can be dialyzed
aerobically overnight with only minor reoxidation (W44). Only the half-
reduced NADPH-cytochrome P-450 reductase is comparably stable to
oxygen (946, 946).
The findings with glutathione reductase from yeast and E . coli are con-
244. V. Massey and C. H. Williams, Jr., in “Pyridine Nucleotide-Dependent Dehy-

drogenases” (H. Sund, ed.), p. 370. Springer-Verlag, Berlin and New York, 1970.
245. B. S. S. Masters, H. Kamin, Q. H. Gibson, and C. H. Williams, Jr., JBC
240, 921 (1965).
246. H. Kamin, B. S. S. Masters, Q. H. Gibson, and C. H. Williams, Jr., Fed.
Proc., Fed. Amer. SOC.E x p . Biol. 24, 1164 (1965).
sistent with the following equations based in large measure on the

schemes previously proposed ( 5 3 ) .
NADP+ NADPH NADP'
E-NADP+-L- E (E-NADPH)-EH,-NADP+-L-- EH, (10)
NADPH
EH,
EH,-NADP+ - EH,-NADPH
EH,-NADP (anion)
NADP'
(12)
EH,-NADPH - E H , - N A D P + - ~ - EH, (13)
The reactions in Eq. (lo), proceeding to the right from E, are part of
normal catalysis as shown in Fig. 10 (51, 6 3 ) . The association of E with
NADP+ leads to a dead end complex. I n the reaction of yeast glutathione
reductase with NADPH, EH,-NADPH appears to be formed in the dead
time of the rapid reaction spectrophotometer (ca. 3 msec) when observa-
tion is a t 540 nm (244) ; however, if 3.4 pM EH,(free) is mixed with
20 pkf NADPH, Eq. (11), a minimum rate of complex formation of
10,000 min-' can be estimated when observation is a t 590 nm (by assum-
ing a maximum half-time of 4 msec) ( 6 3 ) .Thus, EH,-NADPH is formed
a t a fixed concentration of NADPH (in the absence of GSSG, 2 5 O ) a t
a rate comparable with the overall maximal rate of catalysis, i.e., 15,000
min-' (89). Rates of reoxidation of EH, and EH,-NADPH by GSSG
have not been compared. Equation (12) represents the slow conversion
of EH,-NADP' to another species (53) which has been very tentatively
identified as the anion semiquinone-NADP' complex (60) on the basis
of its spectral properties which are shown in Fig. 13. Its formation has
never been observed to go to completion but it is favored by high NADP'
concentrations, high pH, and is inhibited by excess NADPH (53, 60).
An E P R signal forms as the conversion proceeds; the free radical concen-
tration was estimated to be 50% of the FAD concentration (60). The
extinction maxima a t 360, 410, and 480 nm are characteristic of the semi-
quinone anion ( l ) ,but the identification is made difficult by the presence
of large amounts of EH,-NADP+. Slow changes associated with the pro-
duction of an E P R signal (10% of the FAD) are observed with lipoamide
dehydrogenase in the presence of excess NAD', but the spectral changes
are insufficient to be interpreted (59).Equation (13) represents the four-
electron reduction of the enzyme and does not proceed unless NADP'
is removed from the equilibrium as with NADase. The rates of the com-
bined reactions [Eq. (13)] are a t least 10-fold slower in yeast glutathione
reductase than they are in lipoamide dehydrogenase in parallel experi-
Wavelength Inm)
FIG.13. Yeast glutathione reductase, semiquinone anion production from the 2-
electron-reduced form. Curve 1, oxidized enzyme, anaerobic conditions, pH 7.6 ; curve
2, 1 min after the addition of 1 equivalent of NADPH; curve 3, 22 hr later; curve
4, 1 hr after the addition of 10 equivalents of NADP'; curve 5, 23.5 hr later; curve
6, 18.5 hr after the addition of 5 equivalents of NADPH; and curve 7, 35 min
after opening to air.
ments with identical concentrations of NADase ( d 9 ) . Indeed, since EH2

forms a stable complex with NADPH in glutathione reductase, the
EH,/EH, couple must have an oxidation-reduction potential consider-
ably more negative than that of the NADPH-NADP+ couple.
C. KINETICSTUDIES
Equations (10) and (11) indicate three intermediates, EH,, EH,-
NADP', and EH,-NADPH, which are formed a t rates sufficient to re-
quire their consideration as reactants with GSSG. Figure 10 hypothesizes
a simple binary complex mechanism based on the early kinetic studies
with enzyme from erythrocytes (39, do), peas (191), yeast ( d 9 ) , and
P. chrysogenum (193). If this hypothesis is correct then NADP+ dissoci-
ates from EH2 prior to reaction with GSSG and only EH2 and EH,-
NADPH need be considered as possible reactants with GSSG. As was

mentioned, the rate of reoxidation of EH, by GSSG has been measured
(944), but it has not been compared with the rate of reoxidation of
EH,-NADPH. However, the tight binding of EH, to NADPH and the
rapid formation of this complex (see above) would seem to indicate that
it is a significant intermediate in catalysis; if it were not, NADPH should
be a more potent substrate inhibitor. Thus, the small deviations some-
times observed from the kinetics expected from a simple binary complex
mechanism might have their origin in different rates of reaction of GSSG
with forms such as EH, and EH,-NADPH.
The early kinetic studies on glutathione reductase did not include in-
vestigation of product inhibition, so vital to a proper interpretation of
kinetic data in the elucidation of the mechanism (947, 948). In the one
case where product inhibition patterns were observed, they were not inter-
preted by more recent kinetic theory (40). Subsequent kinetic analyses
(see below) , in which product inhibition patterns have been obtained,
were either completed prior to the discovery of the EH,-NADPH complex
(53) or have not considered it. Furthermore, the product inhibition pat-
terns have been carried out a t only a single level of the fixed substrate;
it is essential that the patterns be obtained a t more than one level of
fixed substrate, especially where dead end complexes are involved (949)
as has been so amply demonstrated with lipoamide dehydrogenase (95,
167). In spite of these deficiencies, the more recent kinetic studies have
yielded much useful information.
Reexamination of the kinetics of erythrocyte glutathione reductase using
a preparation of higher specific activity (51) confirmed the substrate and
product inhibition patterns and the salt effects of the earlier study (40).
Product inhibition by NADP+ was found to be competitive with NADPH
and noncompetitive toward GSSG. Since this was in conflict with the
predicted inhibition patterns for a binary complex mechanism, the au-
thors proposed that the mechanism was mixed, ordered sequential and
binary complex (51); these results have been confirmed (950).Further-
more, these studies (@, 51) showed that there was substrate inhibition:
a t high levels of GSSG; a t high levels of NADPH if the GSSG concentra-
tion was low; and that the inhibition was more pronounced a t low ionic
strength. Both the K , for GSSG and the V,,, were affected by ionic
strength: At 0.3 M phosphate, V,,, was 14,300 moles NADPH per min
247. K. Dalziel, BJ 84, 244 (1962).
248. W. W. Cleland, BBA 67, 104 (1969).
249. K. Dalziel, in “Pyridine Nucleoticl,e-Dependent Dehydrogenases” (H. Sund,
250. B. Mannervik, Acta Chem. Scand:W,2912 (1969).
per FAD, K,(NADPH) was 13 a,

and K,(GSSG) was 125 p M ; at
0.03 M phosphate, V,,, was 7,700, K,(NADPH) was 9.5 VJM, and
K,(GSSG) was 19 f l . Thus, high ionic strength stimulated only a t
GSSG concentrations higher than 100 pM (51).The kinetics with NADH
(40, 61) and with lipoate (40) were also investigated; they demonstrated
the less marked specificity of the erythrocyte enzyme as compared to
the highly specific yeast enzyme. Effects of a wide variety of cations
and anions have been reported ($9).The pH optimum is broad and cen-
tered a t 6.8 (.!40).
I$,
The binary complex mechanism can be formalized as follows:
ki kr
E + NADPH + E-NADPH S EHp + NADP+
kz k4
kh ki
EHe + GSSG Ske EHrGSSG ke E + 2 GSH
+
and V,,, is then k, * k,/(k3 k7). The rate of formation of EH,, k,,
is too rapid to be measured accurately at 25”, but it can be measured a t
5” and is found to be 5250 min-I. If k, could be measured, then
V,,, could be calculated and compared with that measured by
steady-state kinetics which a t 5O is 1960 min-I; correlation between
the calculated and measured rate would be evidence for the binary
complex hypothesis. The unusual stability of the 2-electron-reduced
form, EH,, of yeast glutathione reductase in the presence of oxy-
gen makes possible the preparation of EH, free of any reductant
by reduction with borohydride and dialysis. The rate of reoxidation
of EH, by GSSG is found to be 2900 min-I. The calculated V,,, is then
1880 min-I, in excellent agreement with that found by steady-state kinet-
ics (S44, 251). These data demonstrate that the presence of NADP’ or
NADPH does not alter the rate of reoxidation of EH, by GSSG. Neither
do these data preclude the reoxidation of EH,-NADP’ or EH,-NADPH
complexes a t rates similar to the rate of reoxidation of free EH,.
The yeast enzyme, like the erythrocyte enzyme, is inhibited by NADP’
and the inhibition is competitive with NADPH (&EL-254).The inhibition
is rather weak, the inhibition constant being a t least 10-fold higher than
the K,,,for NADPH. On the basis of these studies a mixed mechanism
251. C. H. Williams, Jr. and V. Massey, in “Flavins and Flavoproteins” (H. Kamin,
ed.), Vol. 3, p. 289. Univ. Park Press, Baltimore, Maryland, 1971.
252. B. Mannervik, in “Structure and Function of Oxidation Reductase Enzymes
(A. Akeson and A. Ehrenberg, eds.), p. 425. Pergamon, Oxford, 1972.
253. B. Mannervik, BBRC 53, 1151 (1973).
254. €3. Mannervik, in “Glutathione” (L. Flohe et al., eds.), p. 114. Thieme, Stutt-
gart, 1974.
3. -
FLAVI N CON TAI N I NG DEHY DROGEN ASES 141
has been proposed. It is claimed that the sequential mechanism applies

at high concentrations of GSSG and that the binary complex mechanism
applies a t low GSSG concentrations (253, 254). In this connection it
should be recalled that the physiological ratio, GSH:GSSG is always
high. The kinetics of the reverse reaction have been investigated and have
been taken as evidence for a sequential mechanism (255).The p H opti-
mum for the reverse reaction is about 8 (255) while that of the forward
reaction is about 7 (29).These studies have not considered the EH2-
NADPH complex or the possibility that this complex as well as the EH2-
NADP' complex could be reoxidized by GSSG. Since NADP' is not as
tightly bound to the oxidized enzyme, its rapid dissociation can be in-
ferred. A reasonable proposal has been made which potentially reconciles
all of the present data ( 5 3 ) . A hybrid binary complex mechanism has
been proposed for transcarboxylase from Propionibacterium shermanni in
which distinct sites exist for the two substrate pairs, analogous to
NADPH-NADP+ and GSH-GSSG with glutathione reductase (256).
From what is now known of pyridine nucleotide binding sites (188) and
of the disulfide loop of glutathione reductase (see Section I1,C) , indepen-
dent sites are an attractive hypothesis.
Kinetic constants for the yeast enzyme are remarkably like those for
the erythrocyte enzyme: V,, = 15,000 moles NADPH per min per mole
of FAD, K,(NADPH) = 3.8 p M and, K,(GSSG) = 55 pLM (89).
Specific anion effects on the yeast enzyme have been interpreted to indi-
cate two anion binding sites near the active site (257').
D. THIOL
GROUPS
Glutathione reductase from yeast and from E . coli contains 4 and 5
thiol groups per FAD, respectively (Table I), in addition to the active
center disulfide. Peptides containing 3 of the 4 thiols have been isolated
and sequenced : Cys-Asn Asp ; Lys-Ile-Ala-Cys-Pro-Gly-Asn-Val-Gln-
Lys ; Asp-Thr-Ile-Tyr- (His,Glx) -Val-Cys-Lys- (Thr,Gly,Ala,Leu,) ( 3 5 ) .
The thiols in the yeast enzyme, like those in lipoamide dehydroge-
nase, are relatively unreactive. Reactivity with phenyl mercuric acetate
(29) and with N-ethylmaleimide (258) have been reported; it is of inter-
est that the 2-electron-reduced enzyme is stable in the presence of
N-ethylmaleimide and excess NADPH (258), but four-electron reduc-
tion results in the presence of p-chloromercuriphenyl sulfonate ( 2 9 ) .
255. A. L. Icen, FEBS (Fed. Eur. Biochem.Soc.) Lett. 16,29 (1971).
256. D. B. Northrop, JBC 244, 5808 (1969).
257. G. Moroff and K. G. Brandt, ABB 159,468 (1973).
258. R. F. Colman and S. Black, JBC 240, 1796 (1965).
142 CHARLM H. WILLIAMS, JR.
Erythrocyte glutathione reductase is not sensitive to cupric ion (969).

An early study of the enzyme from rat liver showed that GSSG was
bound to the enzyme (1 GS-/44,000 g of enzyme) and that GSH was
released upon the addition of NADPH (960).This finding, made prior
to the knowledge that the enzyme was a flavoprotein, implies that a thiol
on the protein reacts with GSSG yielding 1 GSH and that the half of
the GSSG bound to the protein in mixed disulfide linkage was released
by reduction of that linkage by NADPH. A possible explanation of these
data is that the preparation was a mixture of glutathione reductase and
a glutathione transhydrogenase ; however, the very high specific activity
of the preparation strongly indicates the need for the reinvestigation of
this finding.
V. Thioredoxin Reductare
Some of the catalytic and structural properties of thioredoxin reduc-

tase as they relate to analogous properties of lipoamide dehydrogenase
and glutathione reductase have been covered in Section 11. The flavopro-
tein, thioredoxin reductase, catalyzes the electron transfer from NADPH
to thioredoxin, a protein of 12,000 molecular weight containing a single
disulfide. The reductase has a reactive disulfide in addition to FAD.
Thus, electron flow is from NADPH to the FAD-disulfide system of thio-
redoxin reductase, to the disulfide of thioredoxin, and finally to a variety
of acceptor systems.
A. METABOLIC FUNCTIONS
The metabolic function of the thioredoxin reductase-thioredoxin system
is to supply reducing equivalents to a wide variety of acceptors. B y far
the best characterized of these is the E . coli ribonucleotide reductase sys-
tem (93, 961); the reductase consists of two subunits, proteins B1 and
B2 (269, 963). The B1 protein contains three reactive dithiol-disulfide
pairs and appears to be the immediate acceptor of reducing equivalents
from thioredoxin. As isolated, the three pairs are in the reduced state
and, in the presence of the B2 protein, three molecules of ribonucleotide
can be reduced prior to any input of reducing equivalents from thiore-
259. N. S. Agar and J. E. Smith, Proc. Soc. Exp. Biol.' Med. 142, 562 U973).
260. C. E. Miae, T. E. Thompson, and R. G. Langdon, JBC 237, 1596 (1962).
261. P. Reichard, Eur. J . Bbchem. 3, 259 (1968).
262. N. C. Brown, Z. N. Canellakis, P. Reichard, and L. Thelander, Eur. J . Bio-
chem. €I 561
, (1969).
263. L. Thelander, JBC 248, 4591 (1973).
TABLE V
SYSTEMS Is THE POSSIBLE
FOR WHICHTHIOREDOXIN ELECTRON
DONOR
Ribonucleotide reductase, Escherichia coli (83,861-866)

Yeast (46, 267)
Lactobacillus leichmannii (868-870)
Mammalian liver supernatant (808)
Rat hepatoma (871)
T4-induced E. coli (47, 878)
Euglena gracilis (873)
Nonspecific protein disulfide reductase, pea seeds (874)
Methionine sulfoxide reductase, yeast (8, 876, 876)
“Active” sulfate reductase, yeast (7, 876-877)
&Proline reductase, Clostridium sticklandii (878, 979)
doxin (264).The B2 protein is an iron-sulfur protein (265). The ribo-

nucleotide reductase system of Lactobacillus leichmannii, in contrast to
the E. coli enzyme, is vitamin B,,-dependent (266). Table V (6, 7 , 23,
46, 47, 902, 261-265, 267-279) indicates the wide variety of systems for
which thioredoxin is the possible electron donor. While the term “thiore-
doxin” was coined in connection with the ribonucleotide reductase system
(23) this protein had been demonstrated in two systems previously (6, 7 ) .
)
Thioredoxin reductase (EC 1.6.4.5) has been purified from E . coli (8,
3 6 ) , yeast (6, 7, 66, 275), and rat liver supernatant (202, 280). Only
the enzyme from E . coli has been extensively characterized, and it will
be discussed exclusively below. The similarity of the transhydrogenase
from Azotobacter vinelandii to thioredoxin reductase has been noted
(281) .
264. L. Thelander, JBC 249, 4858 (1974).
265. C. L. Atkin, L. Thelander, P. Reichard, and G. Land, JBC 248, 7464 (1973).
266. R. L. Blakley and H. A. Barker, BBRC 16,391 (1964).
267. E. Vitols, V. A. Bauer, and E. C . Stanhrough, BBRC 41, 71 (1970).
268. E. Vitols and R. L. Blakley, BBRC 21,466 (1965).
269. M. D. Orr and E. Vitols, BBRC 25, 109 (1966).
270. W. S. Beck, M. Goulian, A. Larsson, and P. Reichard, JBC 241, 2177 (1966).
271. E. C. Moore and P. Reichard, JBC 239,3453 (1964).
272. 0. Berglund, JBC 244, 6306 (1969).
273. F. K. Gleason and H. P. C. Hogenkamp, JBC 245,4891 (1970).
274. M. D. Hatch and J. F. Turner, BJ 76,556 (1960).
275. P. G. Porque, A. Baldesten, and P. Reichard, JBC 245, 2363 (1970).
276. P. G. Porque, A. Baldesten, and P. Reichard, JBC 245, 2371 (1970).
277. L. G. Wilson, T. Asahi, and R. S. Bandurski, JBC 236, 1822 (1961).
278. T. C. Stadtman and P. Elliott, JBC 228, 983 (1957).
279. D. S. Hodgins and R. H. Abeles, ABB 130,274 (1969).
280. A. Lawon, Eur. J . Biochem. 35, 346 (1973).
281. H. W. J. van den Broek and C. Veeger, Eur. J . Biochem. 24, 63 (197111
144 CHARLES H. WILLIAMS, J R .
OF THIOREDOXIN
B. SPECIFICITY REDUCTASE
Thioredoxin reductase is specific for NADPH and moreover for the
hydrogen from the B side of the nicotinamide ring (282).
The specificity of the flavoprotein for various disulfides was discussed
in Section I1,A. The specificity of the E . coli reductase for thioredoxin
from several sources has been tested; thioredoxin from yeast is not re-
duced by NADPH in the presence of the E . coli reductase (275).The se-
quence of amino acids around the redox-active disulfide of yeast and E.
coli thioredoxin shows a high degree of homology (46). Thus, it would
seem unlikely that major determinants of substrate recognition lie in this
region as had been suggested (62).When E. coli are infected by T 4 phage,
a new thioredoxin is made simultaneously with the normal thioredoxin.
Both thioredoxins are reduced by NADPH plus thioredoxin reductase ;
K , values (apparent, measured a t 120 pM NADPH) for the normal and
T4-induced thioredoxins are 10 and 6 p M , respectively. The sequence of
amino acid residues around the reactive disulfide in the T4-induced thio-
redoxin is totally different from that of the normal thioredoxin; even
the 2 residues between the half-cystines are different: -Cys-Val-Tyr-
Cys- (47). It is possible that the determinants of substrate recognition
by the reductase will emerge from X-ray crystallography studies on the
thioredoxins now in progress (47).
c. GENERALPROPERTIES OF THE E.C O l i ENZYME

The reduction of thioredoxin by NADPH is virtually complete, an
equilibrium constant of 48 having been estimated a t pH 7 (8).The equi-
librium constant [T(SH)2][NADP+]/[T(S)2][NADPH], falls by a
factor of 10 for each unit rise in pH (8).The activity is stimulated about
2-fold in phosphate buffer as compared to Tris and the pH optimum is
about 7.7 (8).The molecular weight determined by sedimentation equili-
brium is 66,000 (38) and by amino acid analysis is 73,OOO-75,000 based on
2 moles of FAD per mole of enzyme (68).The partial specific volume is
0.724 ml/g (8). The spectrum of the oxidized enzyme is given in Figs. 3
and 4. The extinction coefficient of the FAD a t 456 nm is 11.3mM-' cm-l.
Spectral ratios are A(271 nm) :A(456 nm) = 5.8; A(380 nm) :A(456
nm) = 1.03 ( 3 6 ) .
Kinetic parameters are given in Table VI. Plots of l / v against 1/(S)
give parallel lines both a t 4O and 25O; on this basis the assumption has
been made that a binary complex mechanism is operative. As with lipo-
amide dehydrogenase and glutathione reductase, this assumption is com-
282. A. Larsson and L. Thelander, JBC 240,2691 (1966).
TABLE VI
THIOREDOXIN
REDUCTASE-KINETIC
CONSTANTS"
4 25OC
L(NADPH)(rM) 0.8 1.2

K,(thioredoxin) ( p M ) 1.7 2.8
Ki(NADP/) (&') - 15
Turnover number/FAD (min-I) 520 2 ,000
a Assays were carried out in 0.05 M phosphate buffer, pH 7.6, 1.5 mM EDTA.
* At 4", the disappearance of NADPH was followed at 340 nm in 5 cm pathlength
cells.
At 25", the assays contained 0.2 mM DTNB and the appearance of TNB anion
was followed at 412 nm; the assays also contained 10 mM glucose &phosphate and
glucose-&phosphate dehydrogenase to overcome inhibition by NADP/ except in the
experiment estimating NADP+ inhibition.
plicated by the product inhibition pattern in which inhibition by NADP'

is competitive with NADPH. The enthalpy of activation calculated from
the turnover numbers a t the two temperatures is 10,000 cal/mole
FAD (689).
D. REDUCED
STATESOF THE ENZYME-MECHANISM
The titration of thioredoxin reductase with NADPH is shown in Fig.
4. Four electrons per FAD seem to be required for reduction; this is con-
firmed in titrations with dithionite or NADPH in the presence of NADase
(to hydrolyze the product NADP'). It can be seen that the first incre-
ments of NADPH cause a relatively larger change than do later incre-
ments. It seems likely that this is indicative of an unequal sharing of
electrons between the FAD and the oxidation-reduction-active disulfide
which favors the FAD. The long wavelength absorption extending to 900
nm has been ascribed to charge transfer interaction between FADH, and
NADP'. It is absent when the titration is carried out in the presence
of NADase or in reduction by dithionite. I n these latter titrations, a dis-
tinct long wavelength absorption is observed which does not extend be-
yond 700 nm (see Section V,E) (SO). These results have been confirmed,
and in addition it has been shown that the enzyme can be partially re-
duced by excess reduced thioredoxin (31).
Early studies indicated that the enzyme contained a second electron
acceptor and that this acceptor was a disulfide (Section I1,B) (8, 30).
This conclusion is greatly strengthened by the observation that enzyme
283. S. Ronchi, G. Zanetti, and C. H. Williams, Jr., unpublished results.
146 CHARLES H . WILLIAMS, JB.
FIQ.14. Mechanism for thioredoxin reductase.
reduced by 1.5 equivalents of NADPH reoxidized partially upon the

addition of an excess of p-chloromercuriphenyl sulfonate and that in a
similar experiment using only 1.0 mole of NADPH reoxidation was com-
plete. Thus electrons can be trapped on the nascent thiols by mercurial.
The enzyme-bound FAD following such experiments is fully reduced by
1.0 mole of NADPH (30).
The reduction of thioredoxin reductase by NADPH observed in the
rapid reaction spectrophotometer a t 2O proceeds in three phases. The first
phase is complete in the dead time of the instrument (ca. 3 msec) and
results in a spectrum with enhanced absorption a t long wavelengths and
very little change in absorption in the 450-nm region. This species decays
to the 4-electron-reduced enzyme in two phases, the first having a rate
constant of 2600 min-I and the second of 300 min-l. From the rate of
the overall reaction a t low temperature (Table VI), it is concluded that
the first and second phases are catalytically significant, while the third
represents a side reaction ($84).
The scheme shown in Fig. 14 represents a working hypothesis for the
reaction mechanism of thioredoxin reductase. This constitutes a gross
oversimplification. Thioredoxin reductase may be more complex than are
284. V. Massey, R. G . Matthews, G. P. Foust, L. G . Howell, C. H. Williams,
Jr., G. Zanetti, and S. Ronchi, in “Pyridine Nucleotide-Dependent Dehydrogenases”
(H. Sund, ed.), p. 393. Springer-Verlag,Berlin and New York, 1970.
lipoamide dehydrogenase and glutathione reductase, where only one spec-

trally distinct species, EH,, and its complexes with oxidized and reduced
pyridine nucleotides is observed. I n thioredoxin reductase on the other
hand, intramolecular electron transfer between the FAD and the disulfide
does not involve a spectrally identifiable interaction between the two
redox groups. I n the scheme, catalysis proceeds in a clockwise direction
in the upper square when thioredoxin is in excess and I represents the
oxidized enzyme; I1 is considered the rapidly formed species. The spec-
trum of the species formed with a rate constant of 2600 min-' has not
been determined, but it is assumed to be 111, or more properly a mixture
of I11 and VI. If NADPH is in excess, catalysis might be pictured as
proceeding from I to I1 to I11 to IV to V to VI back to I11 and then
counterclockwise around the lower square. The slow reaction with rate
constant of 300 min-' may represent the dissociation of NADP' from
VI. Distinction of the subtle differences between this scheme and that
previously proposed (284) will require an extensive rapid reaction inves-
tigation. The electron distribution between FAD and the disulfide ob-
served to favor the flavin indicates that the species IV will have a very
transient existence.
E. LIGHT-ACTIVATED SEMIQUINONE
REDUCTION-NEUTRAL
The formation of a blue (neutral) semiquinone in high yield upon ir-
radiation of thioredoxin reductase in the presence of a large excess of
EDTA is shown in Fig. 3a. The semiquinone is further reduced to FADH,
a t an even slower rate with maximal semiquinone formation a t 4 hr. I n
contrast to this very slow semiquinone production, enzyme reduced by
NADPH in the dark and subsequently exposed to light is rapidly con-
verted to the semiquinone. The rate depends on the amount of NADPH
used in the reduction; with 0.5 mole NADPH per FAD the half-time is
less than 0.5 min, with 2.0 moles NADPH per FAD the half-time is about
2 min. The rate of free radical production (EPR) exactly parallels the
rate of increase in absorbance a t 580 nm. The exact spectral character-
istics of the semiquinone depend on the state of oxidation of the disulfide-
dithiol. In the dithiol form the maximum is a t 578 nm while in the disul-
fide form the maximum is at 588 nm. That the spectral properties are
determined by the redox state of the disulfide is indicated by three findings.
If semiquinone is produced by irradiation following reduction of the en-
zyme by 0.5 mole/FAD, the maximum is a t 588 nm, while if the semiqui-
none is formed following reduction by 2.0 moles/FAD, the maximum is a t
578 nm. Oxidation of enzyme irradiated in the presence of excess EDTA for
various lengths of time requires ferricyanide stoichiometric with the ob-
148 CHARLES H. WILLIAMS, J R .
served semiquinone for irradiation times less than 4 hr; but for longer
times the ferricyanide required is greater up to a maximum of 4 equiva-
lents. The semiquinone is not reduced by NADPH or reoxidized by thio-
redoxin; however, semiquinone exhibiting a maximum a t 578 nm, upon
addition of thioredoxin, shifts its maximum to 588 nm and NADPH
causes the opposite shift (58).
The lack of reactivity of the semiquinone per se with either thioredoxin
or NADPH shows that it cannot be involved in catalysis. The rapid pro-
duction of semiquinone by irradiation of partially reduced enzyme is a
light-activated disproportionation since it is totally dependent upon the
presence of some oxidized enzyme. Enzyme fully reduced by dithionite
forms no semiquinone, while enzyme partially reduced by dithionite
rapidly forms semiquinone upon irradiation. Furthermore, the light-acti-
vated disproportionation of enzyme first reduced with NADPH results
in the reduction of NADP'. Thus, FAD catalyzes the disproportionation
in keeping with the known photosensitizing nature of free flavins. This
reaction is reversed slowly (half-time a.150 min 25') in the dark. The
semiquinone is rapidly reoxidized by oxygen to yield an enzyme with un-
altered spectral and catalytic properties (58).Similar reactions have been
very briefly reported for lipoamide dehydrogenase ; the dark reverse reac-
tion is comparatively rapid, being complete in 30 min (163).
VI. Microsomal Electron Transport
It is generally accepted that liver microsomes contain two electron

transport systems each containing a flavoprotein reductase. The paths
of electron flow can be outlined as follows:
NADPH -+NADPH-cytochrome P-450 lipid- cytochrome P-450 -0,

reductase
NADH-NADH-cytochrome b , lipid_cytochrorne b , -"CN-sensitive

reductase factor"
t
0 2
The "CN-sensitive factor" is the only component which has not been
characterized as a molecular entity (284a). I n Section VI,A, the makeup
284a. P. Strittmatter, L. Spats, D. Corcoran, M. J. Rogers, B. Setlow, and R.
Redline, Proc. Nat. Acad. Sci. U. S. 71, 4566 (1974) ; added in proof: The desaturaae
is a single polypeptide chain of 63,000 daltona containing 62% nonpolar amino acids
and one atom of nonheme iron.
and metabolic functions of the NADPH-dependent system will be out-

lined; in Section VI,B, the NADH-dependent system will be outlined;
and in Section VI,C, the possibilities for interchain electron transfer will
be briefly surveyed.
A. THENADPH-CYTOCHROME P-450 REDUCTASE-CONTAINING SYSTEM

The mixed function oxidations (reactions requiring both oxygen and
reducing equivalents) of endogenous substrates such as steroids and fatty
acids, and of drugs, carcinogens, and other foreign compounds, are carried
out by the cytochrome P-450 system. Most of these reactions are either
hydroxylations or oxidative N-dealkylations in which half the oxygen
molecule is incorporated and half is reduced to water. The system is al-
ways present, but it can be induced to much higher levels by the chronic
administration of a potential substrate. There is some evidence that dis-
tinct systems are induced by different substrates containing spectrally
distinct cytochromes, cytochrome P-450 and cytochrome P-448. A very
good bibliography indicating documentation for these introductory com-
ments is contained in Lu et al. ( 2 8 5 ) .
This system has been solubiliaed from the liver microsomal membrane,
resolved into three components and reconstituted (285-29291). All three
components, flavoprotein, cytochrome P-450, and lipid, are required for
maximal substrate metabolism. The active component in the lipid frac-
tion has been found to be phosphatidylcholine (292). The system from
kidney microsomes has been partially resolved and reconstituted (293,
294). The system from yeast has been resolved into three similar fractions
and reconstituted; the yeast reductase and lipid fractions can be replaced
by analogous fractions from liver (295). Thus, the system is perhaps
ubiquitous in eukaryotic microsomes. As was mentioned in Section I, the
systems carrying out similar reactions in prokaryotes and adrenal mito-
285. A. Y. H. Lu, R. Kuntzman, S. West, M. Jacobson, and A. H. Conney, JBC
247, 1727 (1972).
286. A. Y. H. Lu, K. W. Junk, and M. J . Coon, JBC 244, 3714 (1969).
287. A. Y. H. Lu, H. W. Strobel, and M. J. Coon, BBRC 36, 545 (1969).
288. A. Y. H. Lu, H. W. Strobel, and M. J. Coon, J . M o l . Pharm. 6, 213 (1970).
289. K. Ichihara, E. Kusunose, and M. Kusunose, Eur. J . Bwchem. 38,463 (1973).
290. W. Levin, D. Ryan, S. West, and A . Y. H. Lu, JBC 249, 1747 (1974).
291. A. Y. H. Lu and W. Levin, BBA 334,205 (1974).
292. H. W. Strobel, A. Y. H. Lu, J. Heidema, and M. J. Coon, JBC 245, 4851
(1970).
293. K. Ichihara, E. Kusunose, and M. Kusunose, BBA 239, 178 (19’711..
294. K. Ichihara, E. Kusunose, and M. Kusunose, FEBS (Fed. Eur. Biochem.
SOC.)Lett. 20, 105 (1972).
295. W. Duppel, J. M. Lebeault, and M. J. Coon, Eur. J . Biochem. 36,583 (1973).
chondria contain an iron-sulfur protein and seem to have no lipid require-

ment. The yeast system shows optimal activity with lysophosphatidyl-
ethanolamine (896).
Although resolution and reconstitution represent the strongest evidence
for the makeup of the NADPH-cytochrome P-450system, other tech-
niques have contributed important indirect evidence. Immunochemical
techniques have been used to indicate functionality of individual compo-
nents, particularly the flavoprotein reductase ; antibodies have been pre-
pared against the purified reductase (296, 897) and shown to inhibit
such processes as aniline hydroxylation (296),o-hydroxylation of fatty
acids (898), ethylmorphine demethylase (8973, NADPH-cytochrome c
reductase and NADPH-cytochrome P-450 reductase activities (897),
21-hydroxylation of progesterone and 17-hydroxyprogesterone (899),
NADPH-peroxidase activity (300),heme oxygenase (301), and steroid
17,20-lyase (Sob).Other techniques indicating the integrity of the system
have been the increased turnover of the reductase in the microsomes of
animals induced with potential substrates for the system such as pheno-
barbital (303),competitive inhibition between potential substrates (Sod),
the influence of potential substrates on the activation energy of the reduc-
tase (SO6),and stimulation of NADPH-cytochrome P-450reductase ac-
tivity (actually of the rate of anaerobic formation of the CO difference
spectrum upon reduction with NADPH in microsomes) by potential sub-
strates such as N-ethylmorphine (S06).
B. THENADH-CYTOCHROME
b, REDUCTASE
SYSTEM
The history of the NADH-dependent microsomal electron transfer
system has been quite different from that of the NADPH-dependent sys-
296. T. Omura, in “Microsomes and Drug Oxidations” (J. R. Gillette et al., eds.),
p. 160. Academic Press, New York, 1969.
297. B. S. S. Masters, J. Baron, W. E. Taylor, E. L. Isaacson, and J. LoSpalluto,
JBC 246, 4143 (1971).
298. F. Wadam, H. Shibata, M. Goto, and Y. Sakamoto, BBA 162, 518 (1968).
299. B. S. Masters, E. B. Nelson, B. A. Schacter, J. Baron, and E. L. Isaacson,
Drug Metab. Disposition 1, 121 (1973).
300. E. G. Hrycay and P. J. O’Brien,ABB 157,7 (1973).
301. B. A. Schacter, E. B. Nelson, H. S. Marver, and B. S. S. Masters, JBC 247,
3601 (1972).
302. G. Betz, M. Roper, and P. Tsai, ABB 163,318 (1974).
303. H. Jick and L. Shuster, JBC 241,5366 (1966).
304. S. Orrenius and H. Thor, Eur. J . Biochem. 9, 415 (1969).
305. J. L. Holtzman and M. L. Carr, ABB 150,227 (1972).
306. J. L. Holtzman and B. H. Rumack, Biochemistry 12, 2309 (1973).
tem in that the flavoprotein, NADH-cytochrome b, reductase (9, 307)

and the cytochrome b, were purified and thoroughly characterized (17,
308, 309) long before data suggesting a function appeared. It was not
until 1967 that it was suggested that cytochrome b, was the electron
donor to a “cyanide sensitive factor” responsible for the desaturation of
long chain fatty acids (310, 311, 311a) though it had been shown earlier
that the CN-sensitive factor, and not cytochrome P-450,was involved in
this process (318,313).The CN-sensitive factor has still not been charac-
terized (284a). The system has been resolved and reconstituted, but the
CN-sensitive factor used in the reconstitution was very crude (314). A
requirement for lipid in the system had also been demonstrated (315,
316).An antibody to cytochrome b, has been shown to inhibit the desatu-
rase of plasmalogen biosynthesis (317).
C. POSSIBLE
SYNERGISM
BETWEEN THE SYSTEMS
Two MICROSOMAL
Interaction between components of the NADH-dependent system and
those of the NADPH-dependent system has been demonstrated, e.g., be-
tween NADPH-cytochrome P-450 reductase and cytochrome b, (318).
The physiological significance of these interactions has been a matter
of much controversy.
A series of studies have demonstrated that the addition of NADH stim-
ulates microsomal hydroxylation or oxidative N-demethylation utilizing
the normal donor NADPH (295, 318-321). This has led to the proposal
307. P. Strittmatter and S. F. Velick, JBC 228, 785 (1957).

308. P. Strittmatter, in “Flavins and Flavoproteins” (E. C. Slater, ed.), Vol. 1,
309. P. Strittmatter, Fed. Proc. Fed. Amer. SOC.Exp. Biol. 24, 1156 (1965).
310. N. Oshino, Y. Imai, and R. Sato, Proc. Znt. Congr. Biochem., Yth, 1967
Abstracts, p. 725 (1968).
311. N. Oshino, Y. Imai, and R. Sato, J . Biochem. (Tokyo) 69, 155 (1971).
311a. N. Oshino and R. Sato, J . Biochem. (Toleyo) 69,169 (1971).
312. N. Oshino and Y.Imai, and R. Sato, BBA 128, 13 (1966).
313. J. L. Gaylor, N. J. Moir, H. E. Seifried, and C. R. E. Jefcoate, JBC 245,
5511 (1970).
314. T. Shimakata, K. Mihara, and R. Sato, J . Biochem. (Tokyo) 72, 1163 (1972).
315. P. Jones and S. Wakil, JBC 242,5267 (1967).
316. P. W. Holloway and S. J. Wakil, JBC 245, 1862 (1970).
317. F. Paltauf, R. A. Prough, B. S. S. Masters, and J. M. Johnston, JBC 249,
2661 (1974).
318. B. S. Cohen and R. W. Estabrook, ABB 143,37 (1971).
321. A. Hildebrandt and R. W. Estabrook, ABB 143,66 (1971).
that one of the two electrons required by cytochrome P-450 for hydroxy-
lation or oxidative N-demethylation is supplied by NADH via NADH-
cytochrome b, reductase and cytochrome b, (320, 321). The role of
NADH has been shown not to be obligatory since the terminal reactions
proceed rapidly in reconstituted systems free of any cytochrome b, (322)
and in microsomes in the absence of NADH (318).The stimulatory role
of NADH seems to be twofold. The K,,, for NADPH in the reduction
of cytochrome b, is of the same order of magnitude as its K,. in oxidative
demethylation, 0.5 and 1 a, respectively (318). As will be seen in
Sections VII and VIII, the specificity of the two flavoproteins for their
respective reduced pyridine nucleotides is virtually absolute. Thus, reduc-
tion of cytochrome b, by NADPH in microsomes must be via NADPH-
cytochrome P-450 reductase. The presence of NADH then serves to pre-
vent the interchain transfer of electrons from NADPH, i.e., competition
between cytochrome b, and cytochrome P-450 for NADPH-cytochrome
P-450 reductase is prevented by maintaining cytochrome b, reduced
(321,322).The second function of NADH would then be that of electron
donor to cytochrome P-450 by way of cytochrome b, (320422).Indeed,
NADH can function as the sole electron donor in the hydroxylation of
3,4-benzpyrene1 and in this reaction cytochrome P-448 is more effective
than is cytochrome P-450 (323).NADH has also been shown to stimulate
NADPH-peroxidase (324) though there is some uncertainty whether the
interchain electron transfer involves cytochrome b, or if the transfer is
directly from NADH-cytochrome b, reductase to cytochrome P-450 (323,
324)
The predicted stoichiometry in microsomal mixed function oxidations
for NADPH :substrate: oxygen is 1:1:1 (326).In the absence of substrate
NADPH oxidase is measured and in the presence of substrate this back-
ground oxidase activity is present though oxygen consumption increases
(326,326).As the level of substrate increases the expected stoichiometry
is approached (326,326’). The addition of potential substrates which can-
not be hydroxylated, such as perfluoro-n-hexane, leads to increased oxy-
gen consumption and this has been termed “uncoupling” (327). It has
been demonstrated that some of the electrons lost from the system are
322. A. Y. H. Lu, S. B. West, M. Vore, D. Ryan, and W. Levin, JBC 249, 6701
(1974).
323. E. G. Hrycay and P. J. O’Brien,ABB 160,230 (1974).
324. S. B. West, W. Levin, D. Ryan, M. Vore, and A. Y. H. Lu, BBRC 58, 516
(1974).
325. D. Y. Cooper, R. W. Estabrook, and 0.Rosenthal, JBC 238, 1320 (1963).
328. S.Orrenius, J . Cell Biol. 26,712 (1965).
327. V. Ullrich and H. Diehl, Eur. J . Biochem. 20,509 (1971).
lost as superoxide anion (328). It has been suggested that a further spar-
ing effect of NADH in NADPH-dependent mixed function oxidations
is in reducing superoxide that has been lost from the system ; it was shown
that the sparing action depended upon cytochrome b, (329).
The physiological significance of interchain electron transfer is still
unknown. Efforts a t reconstitution of the two systems into a single lipid
matrix are just beginning (330). The possible interactions are indicated
below (331, 322) :
NADPH +NADPH-cytochrome P - 450 lipid: cytochrome P - 450 -+02
reductase
NADH+NADH-cytochrome b, cytochrome b, +CN-sensitive

reductase
+
factor
0 2
I n one respect such schemes are quite misleading since they indicate a
one-to-one relationship between components. It has been shown however
that the ratio of NADPH-cytochrome P-450 reductase to cytochrome
P-450is 1 to 50 (332).
MIXEDFUNCTION
D. NADPH-DEPENDENT AMINEOXIDASE
Microsomes contain, in addition to the two cytochrome reductases just
discussed, a flavoprotein which catalyzes the mixed function oxidation
of secondary and tertiary amines to the hydroxylamines and amine
oxides, respectively (333, 334). This flavoprotein, which contains about
2 moles of phospholipid and 1 mole of FAD per 70,000 g of protein, is
specific for NADPH (333, 334). The enzyme is also able t o catalyze the
further oxidation of the hydroxylamines to nitrones (336).The reactions
328. M. J. Coon, T. A. Van der Hoeven, R. M. Kaschnita, and H. W. Strobel,
Ann. N . Y . Acad. Sci. 212, 449 (1973).
329. H. Staudt, F. Lichtenberger, and V. Ullrich, Eur. J. Biochem. 46, 99 (1974).
330. A. I. Archakov, G. I. Bachmanova, V. M. Devichensky, I. Karuaina, N. S.
Zherebkova, G. A. Alimov, G. P. Kuznetsova, and A. V. Karyakin, BJ 144, 1 (1974).
331. H. A. Sasame, S. S. Thorgeirsson, J. R. Mitchell, and J. R. Gillette, Pharma-
cologist 15, 170 (1973).
332. R. W. Estabrook and B. Cohen, in “Microsomes and Drug Oxidat[ons” (J. R.
Gillette et al., eds.), p. 95. Academic Press, New York, 1969.
333. D. M. Ziegler, D. Jollow, and D. E. Cook, in “Flavins and Flavoproteins”
(H. Kamin, ed.), Vol. 3, p. 507. Univ. Park Press, Baltimore, Maryland, 1971.
334. D. M. Ziegler and C. H. Mitchell, ABB 150, 116 (1972).
335. L. L. Poulsen, F. F. Kadlubar, and D. M. Ziegler, ABB 164, 774 (1974).
154 CHARLES €WILLIAMS,
I. JR.
are insensitive to carbon monoxide, indicating that cytochrome P-450 is

not involved (336). It has been shown by immunochemical means that
this flavoprotein is distinct from microsomal NADPH-cytochrome P-450
reductase (337). N-Hydroxylamines can also be reduced to the amine
by a system consisting of NADH, NADH-cytochrome b, reductase, and
a third protein fraction (336,338).
VII. NADH-Cytochrome b, Reductare
Microsomal NADH-cytochrome b, reductase and its acceptor sub-

strate cytochrome bs are amphipathic proteins, that is, they are each
composed of a hydrophobic domain and a soluble domain (74, 339, 340).
The hydrophobic domains serve to anchor the proteins by strong nonco-
valent interaction with the lipid bilayer of the microsome. The soluble
domains, containing the active sites, FAD or heme, project into the sur-
rounding cytosol. The two domains, in each case, are connected by what
is presumed (because of their proteolytic lability) to be rather flexible
sections of polypeptide, imparting considerable mobility to the projecting
catalytic domains. The proteins have been shown also to have transla-
tional mobility. Interaction between the reductase and the cytochrome
is controlled by both types of mobility (34l-!&4).
A. MOLECULARPROPERTIES
OF THE AMPHIPATHIC
AND SOLUBLE
FORMS
OF THE REDUCTASE
NADH-cytochrome b, reductase was originally solubilized from the

microsomes by incubation with cobra venom, and in this form it was
thoroughly characterized and its mechanism worked out (17, 307, 308) ;
it has also been solubilized by the action of lysosomes normally contami-
nating the microsomal fraction (346-347). The two soluble forms were
336. F. F. Kadlubar, E. M. McKee, and D. M. Ziegler, ABB 158, 46 (1973).
337. B. S. S. Masters and D. M. Ziegler, ABB 145, 358 (1971).
338. F. F. Kadlubar and D. M. Ziegler, ABB 162,83 (1974).
339. A. Ito and R. Sato, JBC 243,4922 (1968).
340. L. Spate and P. Strittmatter, Proc. Nat. Acad. Sci. U.S. 68, 1042 (1971).
341. M. J. Rogers and P. Strittmatter, JBC 249, 895 (1974).
343. P. Strittmatter, M. J. Rogers, and L. Spate, JBC 247, 7188 (1972).
345. S. Takesue and T. Omura, J . Biochem. (Tokyo) 87,259 (1970).
346. S. Takesue and T. Omura, J. Biochem. ( T o k y o ) 67,267 (1970).
TABLE VII
AMINOACIDANALYSISOF VARIOUSFORMSOF NADH-
CYTOCHROME bs REDUCTASP
Detergent- Lysosomal- Chymotryptic

Amino acid extracted A extracted B A-B core
Cysteic acid 7 6 1 6
Aspartic acid 34 28 6 28
Threonine 16 14 2 11
Serine 19 13 6 12
Glutamic acid 38 29 9 26
Proline 33 29 4 28
Glycine 27 20 7 19
Alanine 21 15 6 14
Valine 28 16 12 19
Methionine 10 8 2 8
Isoleucine 24 19 5 19
Leucine 40 27 13 29
Tyrosine 13 8 5 9
Phenylalanine 18 13 5 13
Tryptophan 6 2 4 2
Lysine 25 20 5 20
Histidine 12 9 3 9
Arginine 20 16 4 16
Total residues 39 1 292 99 288
Molecular weight 44,185 32 ,840 11,340 32,526
Data from Spatz and Strittmatter ( 7 4 ) .
found to be essentially identical (347). The amphipathic form of the

flavoprotein was solubilized with detergents and tends to aggregate in
aqueous media (74, 348).
Amino acid analyses of the two forms are shown in Table VII. They
differ from one another by 100 residues, or about 11,000 in molecular
weight. The amino acid content of the hydrophobic domain has been cal-
culated by difference, and the composition is dominated by apolar amino
acids (74). Treatment of the detergent-extracted enzyme with chymo-
trypsin results in a soluble form of the protein; its amino acid content
is very similar to that of the lysosomal form (74). The spectra of the
lysosomal-extracted and the detergent-extracted reductase are identical
in the visible and near-ultraviolet, the extinction coefficient a t 461 nm
being 10.6 mM-' cm-'. Differences between these spectra in the 260-
280-nm region are accounted for by the additional tryptophan and
tyrosine residues ; the approximate extinction coefficients at the ultra-
348. E. s. Pnaiili and B. DeBernard, BBA 253,323 (1971).
156 CHARLES H . WILLIAMS, JR.
violet maxima are 62 and 90 mM-* cm-' for the lysosomal-extracted and
detergent-extracted enaymes, respectively ( 7 4 ) .
B. REVIEW
OF THE MECHANISM
OF STRITTMATTER
Studies leading to the proposal of a mechanism for NADH-cytochrome

b, reductase were carried out with the snake venom-extracted enzyme,
i.e., the catalytic domain. In Section VII,C, work will be described show-
ing that this mechanism obtains for the enzyme bound to microsomes
except that the rate limiting step changes because of a presumed restric-
tion in the diffusion rate in the lipid milieu as compared to an aqueous
medium (341, 342).
Table VIII (9, 74, 307, 349-362) compares the various catalytic activi-
ties of the enzyme. Turnover with ferricyanide as acceptor is as rapid
as with the natural acceptor. Oxygen and cytochrome c are very poor
acceptors. The influence of substitutions on the pyridine ring is large,
TABLE VIII
SUBSTRATE OF NADH-CYTOCHROME
SPECIFICITY bs REDUCTASE
Electron donor Electron acceptor Turnover numbera
NADH* Ferricyanide 29,000

NADH" Cytochrome bs 29,000
NADHd Cytochrome c 0
NADHa Oxygen 1.4
AcPyADHf Ferricyanide 3,500
PYA1ADHf Ferricyanide 400
NADPHe Ferricyanide 2.8
a Moles substrate oxidized per mole enzyme per minute at pH 8.1 and 25".
NADH, 125 p M ; ferricyanide, infinite (307).The apparent K , for NADH is 2.7
p M (ferricyanide, 5.5 p M ) (307).
"NADH, 500 p M ; cytochrome b6, infinite, i.e., varied from 12 to 50 p M ; cyto-
chrome c (25p M ) reduction monitored at 550 nm. The indicated K , for cytochrome
br is 20 p M (74, 349). Electron transfer between cytochrome b6 and cytochrome c
was demonstrated not to be limiting (9, 5007).
d Strittmatter and Velick (9).
* Strittmatter (360).
f Donor, 125 p M ; ferricyanide, 250 p M (561,368).
The apparent K , for ferricyanide
is 2.2 p M (NADH, 120 p M ) (307).AcPyADH, 3-acetylpyridine adenine dinucleotide;
PyAlADH, 3-pyridinealdehyde adenine dinucleotide.
349. A. Loverde and P. Strittmatter, JBC 243,5779 (1968).

350. P. Strittmatter, JBC 234,2665 (1959).
0.100-I
I I I I I I
300 3% 400 450
Wavelength (nm)
FIG. 15. Difference spectra of NADH-cytochrome b, reductase. Curve 1, enzyme
reduced by NADH in the sample compartment and enzyme in the reference com-
partment; curve 2, the same but with NADPH. Curve 3 was generated by subtract-
ing curve 2 from curve 1 (360).
a t least a t the concentrations of donor tested. NADPH is a very poor

donor. These differences have been exploited to great advantage in the
mechanistic studies.
Protection of the reductase by NADH from inhibition by thiol group
reagents suggested that the enzyme formed a stable complex with pyri-
dine nucleotide (353). Such a complex was readily demonstrated by
difference spectroscopy. When the enzyme was reduced by NADH or
AcPyADH a prominent positive band was observed a t 317 nm (Fig. 15) ;
this band was very small (and blue-shifted) when NADPH, a very poor
substrate, was the reductant. Furthermore, addition of NAD' following
NADPH resulted in a difference spectrum identical with that produced
by NADH. The dashed line in Fig. 15 represents the absorption resulting
from NAD' binding. Thus, this band was attributed to a reduced enzyme-
NAD' complex (350).
158 CHARLIB H . WILLIAMS, JR.
F
5
s
9
Wavelength Lnm)
FIG 10. NADH-cytochrome bs reductase. Curve 1. oxidized enzyme. anaerobic

conditions; curve 2, -after the addition of 1 equivalent'of PyAlADH ;o the oxidized
enzyme; curve 3, after the addition of 1 equivalent of NADH to the oxidized en-
zyme; and curve 4, after the addition of 1 equivalent of NADH to the complex
shown in curve 2 (564).
Reduction of the enzyme with NADH or PyAlADH give quite different

and informative results as shown in Fig. 16 (364). Anaerobic reduction
with 1 equivalent of NADH yields enzyme with spectral properties typi-
cal of FADH, but with very flat absorption extending into the near infra-
red. Reduction of 3-pyridinealdehyde adenine dinucleotide (PyAlADH)
on the other hand is only partial, and the resulting spectrum can be dupli-
cated by assuming a mixture composed of 0.35 equivalent of oxidized
enzyme and PyAlADH, and 0.65 equivalent of reduced enzyme-PyAlAD+
complex; again the long wavelength absorption is present and a t a level
higher than was the case with NADH. The fluorescence of the PyAlADH
is fully quenched, indicating that it is bound. Addition of NADH follow-
ing PyAlADH displaces this bound PyAlADH; curve 4 can be duplicated
by assuming a mixture of 1 equivalent each of PyAlADH and reduced
enzyme-NAD+ complex. Thus, the spectral evidence indicates complexes
both between oxidized enzyme and reduced pyridine nucleotide and be-
tween reduced enzyme and oxidized pyridine nucleotide as shown:
TABLE IX
COMPARISON
OF RATESFOR OVERALL TURNOVER
WITH RATES
OF ENZYMEFLAVINREDUCTION BY VARIOUS
PYRIDINE
NUCLEOTIDES(361, 366)"
kc for flavin H rate/D rate

Turnover reduction
Nucleotide number (sec-l) Turnover k
NADH 68.6 69
(a-D)NADH 18.7 19 3.66 3.6
(8-D)NAI)H 68.6 - 1.00 -
AcPyADH 8.33 8.5
(a-D)AcPyADH 0.8 0.97 10.4 8.8
(8-D)AcPyADH 8.33 8.5 1 .oo 1.0
PyAlADH 0.96 1.02 - -
(a-D)PyA1ADH 0.11 0.12 8.7 8.5
0 Data from Strittmatter (361, 3666).

* Moles substrate oxidized per mole enzyme
FAD per second at pH 8.1 and 0'.
Rate of absorbance change measured anaerobically in a rapid reaction spectro-
photometer at 480 nm, pH 8.1, and 0'.
Furthermore, the displacement of PyAlADH by NADH demonstrates

that the formation of these complexes is reversible (354).
The rate of reduction of the FAD by the enzyme-bound NADH is
the same as the rate of turnover with ferricyanide, indicating that the
electron transfer from reduced pyridine nucleotide to enzyme-bound
flavin is the rate limiting step. This was true with each pyridine nucleo-
tide tested (Table IX). Large deuterium isotope effects are observed but
only when the heavy atom is on the CY side of the pyridinium ring. There-
fore, direct hydrogen transfer takes place stereospecifically (351, 355).
As would be expected for complexes of this type, the stereospecificity
is retained in reversing the reaction which can be effected in 0.5 M borate,
pH 9.5. With (a-D)AcPyADH the deuterium is recovered totally on the
CY side of the ring following reduction of the enzyme and displacement
with borate (356). If flavin reduction is rate limiting, then during turn-
over the enzyme must be largely in the oxidized form. However, since
the enzyme is not affected by thiol group reagents during turnover, the
protection afforded by NADH must result from the reduced pyridine
nucleotide-oxidized enzyme complex (308). Regardless of the rates of
hydrogen transfer, which vary with different pyridine nucleotides, the

356. P . Strittmatter, JBC 239, 3043 (1964).
rates of complex formation are very fast and were complete in the dead
time of the rapid reaction spectrophotometer (354).
Reoxidation of the enzyme-pyridine nucleotide complex by ferricyanide
takes place in two one-electron steps. The rate of the first step is too
rapid to measure and the rate of the second step can be measured only
if NADPH is used to prereduce the enzyme. I n this case the complex
with NADP' is virtually nonexistent and the changes measured are those
of uncomplexed enzyme. These observations demonstrate that a species
is formed within the mixing time and that it decays rapidly (78 sec-',
Oo) . Repetition a t 10 nm intervals generates the spectrum of the interme-
diate which is that of the neutral semiquinone. The fact that both steps
in the reoxidation of the enzyme-pyridine nucleotide complexes (i.e.,
when NADH is the reductant) are complete in less than 2 msec demon-
strates that they meet the kinetic requirements of an intermediate, rapid
formation and reoxidation (356).
Reoxidation of the enzyme-pyridine nucleotide complexes by cyto-
chrome b, also takes place in two steps. The rate of the first step is
again too fast to measure. The rate of the second step is markedly depen-
dent upon the pyridine nucleotide involved being 190, 68, and 18 sec-l,
Oo for NADH, AcPyADH, and NADPH, respectively (356).
A mechanism has been proposed for NADH-cytochrome b, reductase
based on these elegant studies (Fig. 17) (308,366). The hydrogen which
is stereospecifically and directly transferred has been printed in boldface.
Catalysis proceeds in a clockwise direction, and after the first catalytic
cycle the dissociation of oxidized pyridine nucleotide and the association
of reduced pyridine nucleotide are shown as a single step since both are
very rapid processes and this emphasizes that the thiol is not (kinetically
speaking) exposed during catalysis. The interaction of the thiol and the
FIO.17. Mechanism for NADH-cytochrome bs reductase (308).

pyridine nucleotide is hypothetical, but it should be pointed out that in-

teractions between nucleophiles such as the thiol and pyridine nucleotides
typically exhibit an absorption near 320 nm as is the case with the com-
plexes formed by this enzyme (350). The interaction pictured between
the flavin and a tyrosine residue is also hypothetical and will be discussed
further in Section VI1,D.
OF THE REDUCTASE
C. MECHANISM BOUNDTO THE MICROSOME
The mechanism for NADH-cytochrome b, reductase described in Sec-
tion VI1,B was worked out with the soluble enzyme, and the question
of its applicability to the interaction of the amphipathic proteins can
now be considered.
The turnover numbers of the detergent-extracted reductase in the re-
duction of ferricyanide or detergent-extracted cytochrome b, by NADH
are 21 and 77% lower than those for the soluble proteins, respectively.
The apparent K , values for NADH and cytochrome b, are raised about
2-fold. The addition of detergent has no effect on the turnover with fer-
ricyanide but doubles the rate with cytochrome b,. These results indicate
that reduction of ferricyanide catalyzed by the amphipathic reductase
is nearly normal but that the reduction of cytochrome b, is inhibited
by polymerization ( 7 4 ) .
The detergent-extracted proteins can be rebound to microsomes ; in-
deed, vast excesses over endogenous levels can be bound (341444).All
of the excess bound protein is active (343, 344) and electron transfer
between different molecules of cytochrome b, does not occur a t an ap-
preciable rate (341).These facts have two important consequences. First,
it seems highly unlikely that a fixed array exists in which a reductase
molecule interacts with a dozen or so cytochromes (the endogenous ratio)
but rather that translational movement occurs in which the interacting
partners are constantly changing. Second, if this picture is correct, the
kinetic characteristics of the system bound to the microsome can be inves-
tigated by relatively conventional means, i.e., substrate concentrations
can be varied and rate limiting steps identified. The essential difference
is that diffusion may be restricted. The active sites on the other hand
should have the same conformations as in the soluble proteins since in
this picture they project into the aqueous phase.
Rebinding of the reductase and the cytochrome b, to liposomes in a
ratio of 1:13 completely restores the activity, inhibited in solution by
polymerization. Thus, the phospholipid is an essential component in the
interaction of the amphipathic proteins (342). The rate of reduction of
cytochrome b, is dependent on its concentration in the microsome and on
the enzyme concentration. The data indicate that no kinetically signifi-

cant complex forms between the reduced reductase and cytochrome b,
(3441).The relative rate effects on ferricyanide reduction of various ana-
logs of NADH are the same for the soluble and bound reductase. How-
ever, the overall rate in the microsomes is limited by the rate of electron
transfer to cytochrome b, rather than by the rate of intramolecular elec-
tron transfer between complexes as is the case in solution (S42). The
data for the reduction of cytochrome b, could be fitted to the steady-state
rate equation generated from the following sequence of reactions which
also conform to the other characteristics of the bound system just
outlined:
kl kr
+
EFAD NADH $ E:iEA
t 2
---t E:i&
ENAD+ + Cyt bs"" Rr

+ Cyt b P d
+ E:fEH.
ENAD+
FADH' + Cyt bs"" E:kg+ + Cyt bsred
+
k7
k$
Efi:,D++ + NAD+
EFAD
It can be seen that this sequence of reactions is simply an expression in

kinetic terms of the mechanism shown in Fig. 17 which was derived for
the soluble enzyme. Whereas in the soluble system k, was the rate limit-
ing step, k, (and perhaps k,) is rate limiting in the bound system. The
bound system is therefore diffusion-limited. The data indicate that in
the intact microsome, NADH-cytochrome b, reductase is turning over a t
about 60% of the maximum velocity (3.41).
D. STRUCTURALSTUDIES
The apoenzyme of NADH-cytochrome b, reductase is readily prepared
by precipitation at low pH in the presence of high concentrations of po-
tassium bromide (171). [Such inhibition of flavin rebinding by anions
was first observed with the Old Yellow Enzyme (567).]The apoenzyme
is stable a t low temperature and neutral pH. FAD recombination is fol-
lowed with great sensitivity by the quenching of fluorescence which is
total upon rebinding. When rebound, FAD is fully functional ; F M N and
riboflavin also bind and give activities of 66 and 2076,respectively. The
binding constants are FAD, less than 1 nM; FMN, 8 n M ; and riboflavin
approximately 20 tJM indicating that all portions of the FAD molecule
are important for tight binding. Reaction of the free thiols on the apo-
367. H. Theorell and A. Nygaard, Acta Chem. Scand. 8, 1649 (1954).

enzyme has no effect on F M N rebinding, but rebinding is totally inhibited

by reaction with 2 equivalents of iodine, provided the thiols have been
protected from the iodine by prior reaction with N-ethylmaleimide. Apo-
enzyme thus modified has ultraviolet spectral properties consistent with
the formation with a single diiodotyrosine residue. This residue may be
important in its interaction with enzyme-bound FAD (171). Relative
dissociation constants have been determined for the tight binding of
NADH and AcPyADH to the apoenzyme; binding results in the quench-
ing of pyridine nucleotide fluorescence. Their binding is strongly inhibited
by ADP-ribose. NAD+ is bound 25 times less tightly than is NADH,
but ADP-ribose is bound only 24 times less tightly, indicating that oxida-
tion of the pyridinium ring weakens binding as was noted in the consider-
ation of the mechanism (Section VI1,B). Binding of NADH to apo-
enzyme is inhibited by the reaction of a single thiol (358).
Reversible dissociation of FAD is effected by lowering the p H ; FAD
rebinds upon neutralization. The rebinding has been studied by optical
and fluorescence spectroscopy and by the return of the ability of NADH
to reduce the FAD. The kinetics are identical by the three criteria and
indicate a half-time of 5 min for the refolding of the FAD and NADH
binding sites ; simple first-order kinetics are approximated for only the
first half of the reactions. FAD is not essential for the refolding but does
accelerate it. The half-time for the reformation of the pyridine nucleotide
binding site in the absence of flavin is about 8 min. The acceleration
by FAD can also be demonstrated by observation of the changes in pro-
tein fluorescence during refolding (359, 360).
The reactivity of the thiols in the native and denatured reductase has
been investigated (352, 559, 360). In the native enzyme 3 thiols react
with p-mercuribenzoate or mersalyl. Addition of an anionic detergent
make an additional thiol available. At p H 2 where the apoenzyme is
in equilibrium with FAD, 5 thiols react with p-mercuribenzoate or mer-
salyl. If the pH is raised, 2 equivalents of reagent are displaced as the
protein refolds even if the FAD has been removed. This strongly indicates
that the reactivity of a particular thiol is not merely a matter of its
“availability” as is so often claimed, but is also a matter of the absolute
reactivity of that thiol in its unique environment; this can only be tested
with reversibly reacting reagents such as mercurials. Of the 2 thiols which
become unreactive upon raising the pH, the reactivity of 1 is altered
very quickly and the other with a half-time of about 5 min. ,As would
360. P. Strittmatter, in “Flavins and Flavoproteins” (K. Yagi, ed.), Vol. 2, p.
85. Univ. Park Press, Baltimore, Maryland, 1968.
be expected with equilibria, the half-time for FAD rebinding is dependent

on the mercurial concentration and upon its absolute reactivity; 1 mer-
salyl is displaced spontaneously in the rebinding and the second only
after addition of excess thiol (569,360).
N-Ethylmaleimide reacts with 3 of the 6 (Table VII) thiols in the
soluble reductase, with 2 rapidly and with another slowly. In the presence
of NADH, N-ethylmaleimide reacts with 1 thiol rapidly and with another
slowly. It is concluded therefore that one of the rapidly reacting thiols
is in the pyridine nucleotide binding site (36R).
The reactivity of the lysyl residues in NADH-cytochrome b, reductase
has been investigated and found to fall into three groups. Modification
of the first most reactive group (about half the total) results in loss of
the ability to interact with cytochrome bg. In the presence of NADH
all but one of the remaining lysyl residues react, resulting in destabiliza-
tion of the holoenzyme structure (349).
The soluble reductase exists in two conformational states in the pH
range 10.7 to 11.5, an active holoenzyme and an inactive flavoprotein
(347). The inactive species is less compact, the FAD while still bound
is in a more polar environment as are the 2 tryptophan residues, an addi-
tional thiol becomes reactive, and the protein is much more readily at-
tacked by trypsin. Prolonged digestion leads to a compact FAD-binding
peptide of 10,000 molecular weight (347).Tryptic digestion of the native
soluble reductase leads to the removal of 47 amino acid residues; the
remainder is composed of two polypeptide chains held together by non-
covalent forces and has 65% of the normal activity. The two polypeptides
can be separated and recombined into an active flavoprotein (361).
Elucidation of the total primary sequence of NADH-cytochrome b,
reductase is now well advanced (347).
E. THEFUNCTIONAL METHEMOGLOBIN SYSTEM

REDUCTASE
ERYTHROCYTE
OF THE MATURE
Methemoglobinemia has long been associated with the absence of an

NADH-linked diaphorase (362, 363). However, flavoproteins isolated
from the red cell were never sufficiently active to account for the methe-
moglobin reductase activity calculated to be necessary. It has now been
shown that a methemoglobin reductase system of high activity is com-
posed of soluble forms of cytochrome b, reductase and cytochrome b,
361. P. Strittmatter, R. E. Barry, and D. Corcoran, JBC 247, 2768 (1972).

362. E. M. Scott and D. D. Hoskins, Blood 13, 795 (1958).
363. E. M. Scott and J. C. McGraw, JBC 237,249 (1962).
(364-366). The reductase has been purified by two groups of investigators

(366, 3673, and its properties are very similar to those of NADH-cyto-
chrome b, reductase solubilized from liver microsomes by lysosomal di-
gestion (347). FAD is the prosthetic group (366, 367), and the spectral
properties are very similar to those of the microsomal enzyme (367).
Its molecular weight is 34,000 (367). The turnover number with cyto-
chrome b, coupled to cytochrome c is 1280 moles cytochrome b, reduced
per minute per FAD which is quite low compared to the microsomal en-
zyme (366). Other activities are also low; turnover with ferricyanide
is about 10,000 per minute. In the absence of cytochrome b,, it is vir-
tually inactive with cytochrome c, oxygen, and methemoglobin, which
is a pattern like that of the microsomal enzyme. The effects of ionic
strength, pH, and EDTA are all similar to those of the microsomal en-
zyme (366). This reductase has been established as the missing compo-
nent in methemoglobinemic erythrocytes (368). The mechanism of the
presumed transition from a bound enzyme in the blast cell to a soluble
enzyme in the mature erythrocyte will be of great interest.
VIII. NADPH-Cytochrome P-450 Reductase
The flavoprotein responsible for the reduction of cytochrome P-450 in

microsomes has only recently been solubilized in a form active in the
reconstitution of the many cytochrome P-450-linked systems (286,
369-371) (Section V1,A). In this form the reductase is not pure and has
been characterized only in very preliminary experiments (369, 371).
Studies on its mechanism have not been reported. Several modified forms
of this enzyme have been isolated which turn over a t high rates with
cytochrome c but are either inactive or only partially active with cyto-
chrome P-450 (337, 372-374). They have been referred to as NADPH-
364. D. E. Hultquist and P. G. Passon, Nature (London), New Biol. 229, 252
(1971).
365. P. G. Passon, D. W. Reed, and D. E. Hultquist, BBA 275,51 (1972).
366. P. G. Passon and D. E. Hultquist, BBA 275,62 (1972).
367. F. Kuma and H. Inomata, JBC 247,556 (1972).
368. F. Kuma, S. Ishizawa, K. Hirayama, and H. Nakajima, JBC 247,550 (1972).
369. A. F. Welton, T. C. Pederson, J. A. Buege, and S. D. Aust, BBRC 54, 161
(1973).
370. T. A. Van der Hoeven and M. J. Coon, JBC 249,6302 (1974).
371. J. L. Vermilion and M. J. Coon, BBRC! MI, 1315 (1974).
372. T. Omura and S. Takesue, J . Biochem. (Tokyo) 67,249 (1970).
373. T. Iyanagi and H. S. Mason, Biochemistry 12,2297 (1973).
374. T. C. Pederson, J. A. Buege, and S. D. Aust, JBC 248, 7134 (1973).
166 CHARLES H. WILLIAMS, JB.
cytochrome c reductase (10, 11, 375). The connection between this en-
zyme and the microsomal hydroxylation system was made (376, 377)
soon after the microsomal origin of the NADPH-cytochrome c reductase
was established (10,11).
A. GENERALPROPERTIES
NADPH-cytochrome P-450 reductase is composed of a single polypep-
tide chain of 70,000-80,000 molecular weight (369, 371, 373, 374) associ-
ated with one molecule of FAD and one molecule of F M N (370, 371,
373, 378, 379). These results apply to the enzyme whether solubilized
by proteolytic digestion or by detergent extraction. The minimum molec-
ular weight based on the flavin content is somewhat higher, 87,000 (3729,
possibly indicative of the flavin lability observed upon irradiation in am-
monium sulfate (380). The detergent-solubilized reductase has a lower
flavin content, 0.64 and 0.79 moles of FMN and FAD per 79,000 g of
enzyme (371). The absorbance ratio, 275 nm:455 nm of 6.5, indicates
a relatively low content of aromatic amino acids (372, 374). The extinc-
tion of the flavins a t 455 nm is 10.7 mM-l cm-l (373).
The demonstration of the amphipathic nature of NADH-cytochrome
b, reductase (74) suggests the question of whether NADPH-cytochrome
P-450 reductase, another microsomal enzyme, is also amphipathic. The
molecular weight of the detergent-solubilized reductase has been deter-
mined in two laboratories by sodium dodecyl sulfate (SDS) polyacryl-
amide gel electrophoresis, both finding values of 79,000 ($6’9,371). The
molecular weight of the bromelain-solubilized enzyme is 71,000 (374) and
that of the trypsin-solubilized enzyme is 68,000 (373), both determined
by SDS gel electrophoresis. This is suggestive of a molecular weight
difference of 10,000. However, if this is correct, the minimum molecular
weight derived from flavin analysis would indicate that the trypsin-solu-
bilized reductase is approximately 20% flavin depleted (873). Until en-
zyme with full flavin complement can be prepared, comparative amino
acid analysis such as that used to demonstrate the difference between
375. B. L. Horecker, JBC 183,593 (1950).
376. S.Orrenius, G. Dallner, and L. Ernster, BBRC 14, 329 (1964).
377. D. Y. Cooper, S. Levin, S. Narashimhulu, and S. Rosenthal, Science 147,
400 (1965).
378. H. Kamin, i n “Reactivity of Flavins-The Proceedings of a Symposium
Dedicated to the Late Professor Leonos Michaelis under the Auspices of. the
Japanese Biochemical Society” (K. Yagi, ed.), p. 137. Univ. Park Press, Baltimore,
1975.
379. B. S. S.Masters, R. A. Prough, and H. Kamin, personal communication.
380. J. P. Baggott and R. G. Langdon, JBC 245, 5888 (1970).
the forms of NADH-cytochrome b, reductase (74) will not settle this

question.
The NADPH-cytochrome c reductase of yeast is an FMN-containing
enzyme (381,382). It is also of microsomal origin (383).
B. CATALYTIC
ACTIVITIE~
OF THE REDUCTASE
The turnover of NADPH-cytochrome P-450reductase with its natural
acceptor can only be studied as a coupled reaction in which the cyto-
chrome P-450 is acting as a hydroxylase or N-demethylase. In the ab-
sence of a hydroxylatable substrate, cytochrome P-450acts as an oxidase
(370).The oxidase activity of the reductase is very low (10).
The various modified forms of NADPH-cytochrome P-450 reductase
have been routinely assayed as cytochrome c reductases. The kinetics
of this reaction have been studied both by steady-state and by rapid
reaction methods. The pH optimum is 7.6 to 8.2 (10, 11, 375)) and the
activity increases with ionic strength being optimal a t 0.2 M phosphate,
pH 7.6 (11, 384).Varying the NADPH concentration at a series of cyto-
chrome c concentrations results in a family of parallel lines in reciprocal
plots. V,,, is 1200 moles cytochrome c reduced per minute per mole of
flavin a t infinite concentrations of both substrates (245); K , for NADPH
is 4 p M and for cytochrome c is 5.5 pM (10, 245). The dissociation con-
stant for the reductase-cytochrome c complex is 4.6 pM (380).NADP'
and AMP are competitive inhibitors of the reaction (10, 385). The K ,
for both rises with ionic strength, an indication that one of the effects
of high salt concentrations is to displace NADP'. At 0.1 M phosphate,
the K i is 2 pM for both inhibitors (11). Specific activities are usually
reported on a protein basis and range in recent studies from 40 pmole
cytochrome c per minute per milligram for the lipase- and trypsin-solu-
bilized enzymes (337, 372, 373) to 56 units per milligram for the brome-
lain-solubiliaed reductase (374). Comparable figures for the detergent-
solubilized enzyme have not been measured a t the same temperature
(371).The turnover number at infinite concentration of both substrates
with dichlorophenolindophenol as acceptor is virtually identical with that
with cytochrome c a t the same pH, though the pH optimum for dichloro-
phenolindophenol may not be the same (386).The reductase is also very
381. E. Haas, B. L. Horecker, and T. R. Hogness, JBC 136, 757 (1940).
382. E. Haas, C. J. Harrer, and T. R. Hogness, JBC 143, 341 (1942).
383. G. Schatz and J. Klima, BBA 81, 448 (1964).
384. M. H. Bilimoria and H. Kamin, Ann. N . Y . Acud. Sci. 212, 428 (1973).
385. E. F. Neufeld, N. 0. Kaplan, and S. P. Colowick, BBA 17, 526 (1955).
386. B. S. S. Masters, M. H. Bilimora, H. Kamin, and Q. H. Gibson, JBC 240,
4081 (1965).
active with menadione (386), ferricyanide, and neotetrazolium (10) as

acceptors.
A very important conclusion was reached based on the effect of p-mer-
curibenzoate on the NADPH oxidase and the NADPH-cytochrome c
reductase activities of microsomes, namely, that the natural acceptor
might be a component reactive with oxygen and involved in hydroxyla-
tions or demethylations (11). It was found that in the absence of
cytochrome c, the oxidase activity was largely inhibited by p-mercuri-
benzoate. In the presence of cytochrome c, NADPH oxidation exceeded
cytochrome c reduction in the absence of p-mercuribenzoate and the two
rates equaled each other in the presence of p-mercuribenzoate. Thus, a
mercurial sensitive oxidase distinct from the reductase was indicated, and
this component was hypothesized to be connected with hydroxylation
and/or demethylation (11).
The influence of mercurials on the NADPH-cytochrome c reductase
activity is complex. The activity in microsomes is stimulated about 50%
by p-mercuribenzoate (11) . Mersalyl inhibits the NADPH-cytochrome
c reductase activity (587).
The lipase-solubilized reductase is inhibited by p-mercuribenzoate, is
protected from this inhibition by NADPH, and the inhibition is relieved
by thiols (10). Careful titration of this enzyme with p-mercuribenzoate
a t pH 6.5 results in an almost 3-fold stimulation upon addition of 2 moles
of mercurial per flavin; the control activity is again observed when 7
equivalents have been added. At pH 7.7, a stimulation of 70% is seen
with 1 equivalent and loss of activity is complete (extrapolated) with
6 equivalents (245).The protection of the enzyme by NADPH against
mercurial inhibition is reminiscent of the effects with NADH cytochrome
b, reductase (360).
NADPH-cytochrome P-450 reductase catalyzes the reduction of ferric
ion to ferrous ion in the presence of chelators (584,388). This reaction
is similar to the peroxidation of microsomal lipids which has been shown
to be dependent on the NADPH oxidase system of microsomes (389,390).
Lipid peroxidation is thought to be involved in prostaglandin biosynthesis
(590392).The microsomal system can be mimicked in the peroxidation
of extracted microsomal lipid by a combination of NADPH, NADPH-
cytochrome P-450 reductase, and ferric ion chelated with EDTA (374,
387. M. R. Franklin and R. W. Estabrook, ABB 14'3,318 (1971).

388. M. M. Weber, H. M. Lenhoff, and N. 0. Kaplan, JBC 220, 93 (1966).
389. P. Hockstein and L. Ernster, BBRC 12,388 (1963).
390. H. E. May and P. B.McCay, JBC 243, B98 (1968).
391. W. Lands, R. Lee, and W. Smith, Ann. N . Y . Acad. Sci. 180, 107 (1971).
392. B. Samuelsaon, Progr. Biochem. Pharmacol. 5,109 (1969).
393). I n this model system, as contrasted with the simple ferric ion reduc-
tase activity of the flavoprotein (388),the metal is not the ultimate elec-
tron acceptor but presumably serves the dual role of oxygen activation
and electron carrier. The reaction may involve superoxide anion since
it is inhibited by superoxide dismutase (erthrocuprein) (394). Xanthine
plus xanthine oxidase can also serve as electron donor, and this latter
model system is also inhibited by superoxide dismutase (396).Superoxide
dismutase also inhibits the menadione-mediated NADPH oxidase activity
of NADPH-cytochrome P-450 (396) as well as the reconstituted benz-
phetamine hydroxylation system (397). The involvement of NADPH-
cytochrome P-450 reductase in microsomal lipid peroxidation has been
confirmed by the demonstration that the reaction in microsomes is totally
inhibited by antibody to the purified reductase (374). It has been sug-
gested that lipid peroxidation by microsomes requires another component,
in addition to the reductase, which takes the place of the ferric ion chelate
in the model system (374).
C. MECHANISM
Studies on the mechanism of NADPH-cytochrome P-450 reductase
have been carried out thus far only with the trypsin- or lipase-solubilized
forms. Assuming that this enzyme is composed of several semi-auton-
omous domains, and assuming further that modification during solu-
bilization is restricted to the domain involved in the interaction with
cytochrome P-450,then, as was the case with NADH-cytochrome b,
reductase, mechanism studies on the soluble enzyme will contribute to the
ultimate understanding of the operation of the reconstituted system. The
fact that the soluble reductase is composed of a single polypeptide chain
gives hope that the modification is a subtle one.
Reduction of the lipase-solubilized enzyme by NADPH is more rapid
than either turnover with cytochrome c or the rates of reconstituted sys-
tems (646).In rapid reaction spectrophotometric studies, changes a t 550
nm are taken is indicative of flavin radical (FlH) ; the oxidized (Fl)
and reduced (FlH,) forms of the enzyme have negligible absorbance a t
this wavelength. Changes a t 500 nm indicate formation of FlH, (nega-
tive) or reoxidation of FlH, (positive) ; F1 and F l H are isosbestic a t
500 nm. Both FlH and FlH, are formed a t rates consistent with their
393. T. C.Pederson and S.D. Aust, BBRC 48,789 (1972).
394. R. A. Prough and B. S.S.Masters, Ann. N . Y . Acad. Sci. 212,89 (1973).
395. T. C. Pederson and S.D. Aust, BBRC 52,1071 (1973).
396. T. Lyanagi and I. Yamazaki, BB.4 172,370 (1969).
397. H. W. Strobe1 and M. J. Coon, JBC 246, 7826 (1971).
participation in catalysis. Although F1H appears to be formed more

rapidly than FlH,, there is no lag in FlH, production and it is suggested
that FlH2 is formed both directly (in the dead time of the apparatus,
ca. 3 msec) and from F1H. The rapid appearance of FIH2 is observed
even a t very low ratios of NADPH to flavin (0.2) (246, 898).
Anaerobic reduction with ratios of NADPH to flavin of less than 0.5
or aerobic reduction with excess NADPH lead to the production of a
spectrally characteristic species of the enzyme which is not oxidized by
oxygen or by cytochrome c (246). This species has long wavelength ab-
sorbance, characteristic of the neutral flavin semiquinone, but the absor-
bance in the 455-nm region is much higher than that of a typical neutral
semiquinone ( 1 ) . Masters et al. (,%?46) interpreted this species to be the
2-electron-reduced enzyme. Since the enzyme contains two flavins per
mole they maintained that each flavin was half-reduced (246). However,
it has been pointed out in several discussions that the spectral character-
istics of this species strongly suggested that it is a mixture of oxidized
flavin and semiquinone flavin (399). Anaerobic reaction with excess
NADPH leads to further reduction but only about half the long wave-
length absorbance disappears. The authors interpreted this species as
being fully reduced (four electron) enzyme with a small amount of semi-
quinone (246). The ratio of NADPH oxidized to cytochrome c reduced
is 1 : l in experiments in which stoichiometric enzyme is used, but this
ratio approaches the expected value of 2 in the presence of catalytic
quantities of enzyme. This, taken together with the fact that cytochrome
c does not fully reoxidize the enzyme, indicated to the authors that the
enzyme was cycling in catalysis between the 4-electron-reduced and the
2-electron-reduced species (246, 398). Furthermore, the kinetics of
enzyme reduction suggested that the flavins were not independent but
rather interacted with one another (386,398).
A reexamination of the mechanism of NADPH-cytochrome P-450
reductase has followed the crucial finding that all forms of this enzyme
(detergent-, lipase-, and trypsin-solubilized) contain equimolar amounts
of FAD and F M N suggesting that the flavins might have distinct roles
(373). Distinct roles have been found for the FAD and FMN in sulfite
oxidase (400, 401). The static spectral results with the lipase-solubilized
398. H. Kamin, B. S.S. Masters, and Q. H. Gibson, in “Flavins and Flavoproteins”
(E. C. Slater, ed.), Vol. 1, p. 306. Elsevier, Amsterdam, 1966.
399. P. Hemmerich, in “Flavins and Flavoproteins” (E. C. Slater, ed.), Vol. 1,
400. L. M. Siegel, H. Kamin, D. C. Rueger, R. P. Presswood, and Q. H. Gibson,
in “Flavins and Flavoproteins” (H. Kamin, ed.), Vol. 3, p. 523. Univ. Park Press,
Baltimore, Maryland, 1971.
401. E. J. Faeder, P. 8. Davis, and L. M. Siegel, JBC 249, 1599 (1974).
Wavelength (nm)
FIG.18. Anerobic titration of NADPH-cytochrome P-450 reductase with NADPH.
Curve 1, oxidized enzyme; curves 2-6 after the addition of 0.16, 0.24, 0.49, 0.98,
and 1.4 moles of NADPH per mole of total enzyme-bound flavin. The inset, B,
shows the changes at 455 and 585 nm as a function of the NADPH added (409).
enzyme cited above have been confirmed with the trypsin-solubilized en-
zyme but reinterpreted. On the basis of EPR quantitation, NADPH titra-
tions, and ferricyanide titrations, the air stable species has been shown
to be the 1-electron-reduced enzyme. The species formed by excess
NADPH has been shown to be a roughly equimolar mixture of 3-electron-
and 4-electron-reduced enzyme (373, 402). The anaerobic titration of the
enzyme with NADPH is shown in Fig. 18. It can be seen that curves
1, 2, and 3 are isosbestic a t 500 nm; in a separate experiment curve 3
is shown to be virtually identical with the spectrum of the air stable
species. Furthermore, the redox state of the enzyme in curve 3 is produced
by the addition of one electron per two flavins. Addition of increasing
amounts of NADPH results in further reduction of the enzyme-to the
final mixture of 3-electron-reduced and 4-electron-reduced enzyme. Titra-
tion of the enzyme with dithionite gives a clear end point upon the addi-
tion of 2 moles of reductant per mole of enzyme (two flavins) ; thus,
402. T. Iyanagi, N. Makino, and H. S. Mason, Biochemistry 13, 1701 (1974).
no electron accepting groups other than the two flavins are present (402).
It is reemphasized that these experiments were carried out with trypsin-
solubilized enzyme (373). Recalling the apparent molecular weight differ-
ence, it is possible that this modified form is catalytically different from
the lipase-solubilized form (337, 409). Titrations with NADPH and ferri-
cyanide have been repeated with the lipase-solubilized enzyme (404).
The results indicate that 2 moles of ferricyanide are required to reoxidase
the air stable form of the enzyme.
Redox potentials have been determined for each of the steps of reduc-
tion of the trypsin-solubilized reductase (402): step 1, one electron con-
sumed, Eo' = -109 mV; step 2, two electrons consumed, EO' = -276
mV; and step 3, one electron consumed, Eo' = -371 mV at p H 7.0, 25O.
As expected, the redox potential of step 3 is more negative than the poten-
tial of the NADPH-NADP+ couple and was determined from the dithio-
nite titration. The overall potentiometric-spectrophotometric titration
curves could be very closely fitted with a computer-generated curve based
on the assumptions of four one-electron reduction steps and extinction
coefficients of 4.9 and 4.5 mM-l cm-' for the semiquinones, F1,H and
F1,H; the 23', values assumed for steps 2 and 3 were -270 and -290
mV. The precise fit was very sensitive to all of the assumptions ( 4 M ) .
Three alternative mechanisms were proposed based only on the thermo-
dynamic data (402). All of these assumed distinct functions for each
flavin and interaction between the flavins. They also assumed that elec-
trons would be transferred to cytochrome P-450one a t a time; this has
been shown to be the case with cytochromes P-450that receive electrons
from iron-sulfur proteins rather than from the flavoprotein directly (or
through the indirect mediation of lipid) (406, 406). One of these mecha-
nisms ( 4 2 ) is shown below. It seems to fit best with the kinetic data
determined for the lipase-solubilized reductase (246,398).In this scheme,
SH is a hydroxylatable substrate and SOH its hydroxylated product, and
F1, and F1, are the high potential and low potential flavins, respectively.
NADPH + H+ + e NADP+ + FliHn
Fl1
FliHz + Flz FliH. + FlzH.
$
FlrH. + P-450"SH Flz + P-45O'+SH + H+
F!
P-45OS+--SH + 02 P-450*+--SH-01
NADPH + FIIH-/FII+ H+ 2 NADP+ + FliHn/FlzH*
FllHz + P-4501+--SH-Oz S FI1H. + P-450a+ + SOH + OH-
403. B. S. S. Masters, C. H. Williams, Jr., and H. Kamin, "Methods in Enzymology"
Vol. 10, p. 535, 1967.
404. B. S. S. Masters, R. A. Prough, and H. Kamin, Biochemistry 14,607 (1975).
405. J. J. Huang and T. Kimura, BBRC 44, 1065 (1971).
406. C. A. Tyson, J. D. Lipscomb, and I. C. Gunsalus, JBC 247, 5777 (1972).
The mechanism hypothesizes that electrons are donated to the cyto-

chrome P-450 by the two couples with potentials near -276 mV.
This mechanism together with the others that are consistent with the
thermodynamic data (408) now form the working hypotheses for kinetic
experiments which hopefully will distinguish between them and further
define the mechanism of this important system.
Metal-Containing Flavoprotein
Dehydrogenases
YOUSSEF HATEFI DIANA L. STIGGALL
I . Introduction . . . . . . . . . . . . . . . . 175
I1. NADH Dehydrogenases . . . . . . . . . . . . . 177
A. NADH Dehydrogenase of Mammalian Mitochondria . . 177
B. NADH Dehydrogenases of Yeast . . . . . . . . 216
C . NADH Dehydrogenase of Azotobader uinelandii . . . 22 1
I11. Succinate Dehydrogenases . . . . . . . . . . . . 222
A . Mammalian Succinate Dehydrogenase (EC 1.3.99.1) . . 222
B. Succinate Dehydrogenase in Microorganisms . . . . 254
I V . LGlycerol-%phosphate Dehydrogenase (EC 1.1.99.5) . . . . 256
V. Choline Dehydrogenase (EC 1.1.99.1) . . . . . . . . . 260
V I . Lactate Dehydrogenases. . . . . . . . . . . . . 263
A . L( +)-Lactate: Cytochrome c Oxidoreductase
(Cytochrome b z ) (EC 1.1.2.3) . . . . . . . . 263
B. D( -)-Lactate:Cytochrome c Oxidoreductase (EC 1.1.2.4). 269
C . DZHydroxyacid Dehydrogenase (EC 1.1.99.6). . . . 272
V I I . Nitrite Reductases (EC 1.6.6.4) . . . . . . . . . . 273
VIII . Adenylyl Sulfate Reductases (EC 1.8.99.2) . . . . . . . 279
I X . Sulfite Reductases ( H a : NADPH Oxidoreductases) (EC 1.8.1.2) . 286
A. NADPH-Sulfite Reductases . . . . . . . . . 287
B . Reduced Methyl Viologen-Sulfite Reductases . . . . 295
.
X Addendum . . . . . . . . . . . . . . . . . 295
.
1 Introduction
Flavoproteins are involved in a large variety of key metabolic reactions

in all forms of life. They catalvze over a potential span of several hundred
millivolts oxidation-reduction reactions involving alkanes. alkenes. alco-
175
176 YOUSSEF HATEFI AND DIANA L. STIGGALL
hols, aldehydes, ketones, inorganic and organic acids, amines,. thiols,

disulfides, quinones, nicotinamide-adenine dinucleotides, purines, pyrimi-
dines, pteridines, and transition metal complexes. They can also catalyze
one- and two-electron reduction of molecular oxygen. Many flavoproteins
contain metal such as iron, molybdenum, and zinc. The combination of
flavin and metal often serves to adjust electron transfer between single-
electron and double-electron donors and acceptors. Multiple-electron re-
duction of an acceptor without detectable loss of intermediates is
achieved, as will be seen below, by the device of having multiple flavins
and metals in the same enzyme molecule. Excellent sources of recent
publication on the chemistry of flavins and flavoproteins are available
(1-6).
The present chapter is concerned with metal-containing flavoprotein
dehydrogenases. The enzymes discussed are respiratory chain-linked
NADH dehydrogenases, succinate dehydrogenases, ~-glycerol-S-phos-
phate dehydrogenase, choline dehydrogenase, L (+)-lactate: cytochrome c
oxidoreductase, D (-) -lactate :cytochrome c oxidoreductase, D-2-hydroxy-
acid dehydrogenase, nitrite reductases, adenylyl sulfate reductases, and
sulfite reductases. Among these, NADH and succinate dehydrogenases
have been the subject of intensive study for the past 20 years, in part
because of their importance as the principal electron entry points into
the mitochondria1 respiratory chain. Progress of research on these two
enzymes has been reviewed by various workers. One laboratory alone
has produced more than twenty reviews of various sorts during as many
years (see, for example, 6-96).These are excellent reference sources, and
1. E. C. Slater, ed., “Flavins and Flavoproteins,” Symp. Adn Flavoproteins.
Elsevier, Amsterdam, 1986.
2. K. Yagi, ed., “Flavins and Flavoproteins,” 2nd Int. Symp. Univ. Park Press,
3. H. Kamin, ed., “Flavins and Flavoproteins,” 3rd Int. Symp. Univ. Park Press,
4. T. E. King, H. S. Mason, and M. Morrison, eds., “Oxidases and Related Redox
Systems.” Univ. Park Press, Baltimore, Maryland, 1973.
6. W. Lovenberg, ed., “Iron-Sulfur Proteins,” Vols. 1 and 2. Academic Press, New
York, 1973.
6. T. P. Singer and E. B. Kearney, in “The Proteins” (H. Neurath and K. Bailey,
eds.), 1st ed., Vol. 2, Part A, p. 123. Academic Press, New York, 1964.
7. T. P. Singer and E. B. Kearney, Proc. Znt. Congr. Biochem., 4th, 1968 Sympo-
sium 11,Vol. 11,209 (1960).
8. T. P. Singer, in “Biological Structure and Function” (T. W. Goodwin and
0. Lindberg, eds.), Vol. 2, p. 103. Academic Press, New York, 1961.
9. T. P. Singer, S. Minakami, and R. L. Ringler, Proc. Znt. Congr. Biochem., bth,
1961 Symposium V, p. 174 (1963).
10. T. P. Singer, “The Enzymes,” 2nd ed., Vol. 7, p. 345, 1963.
4. METAL-CONTAINING FLAVOPROTEIN DEHYDROGENASES 177
skillfully propound the position of that laboratory regarding NADH and

succinate dehydrogenases. I n recent years, new information has appeared,
however, which has clarified many of the basic controversial issues and
erroneous assumptions. In the following account of these two enzymes,
it will be attempted, therefore, to offer a critical analysis of the major
aspects rather than to present an exhaustive review of the chronological
development of the field.
II. NADH Dehydrogenarer
A. NADH DEHYDROGENASE
OF MAMMALIAN
MITOCHONDRIA
Preparations of NADH dehydrogenase from mammalian mitochondria
may be divided into three types: (1) NADH-ubiquinone reductase or
complex I of the electron transport system, (2) the high molecular weight
NADH dehydrogenases, and (3) the low molecular weight NADH dehy-
12. T. P. Singer and T. Cremona, in “Oxygen in the Animal Organism” (F. Dickens
and E. Neil, eds.), p. 179. Pergamon, Oxford, 1964.
13. T. P. Singer, in “Oxidases and Related Redox Systems” (T.E. King, H. S.
14. T. P. Singer, in “Non-Heme Iron Proteins” (A. San Pietro, ed.), p. 349. Antioch
Press, Yellow Springs, Ohio, 1965.
15. T. P. Singer, Compr. Biochem. 14, 127 (1966).
16. T. P. Singer, E. Rocca, and E. B. Kearney, in “Flavins and Flavoproteins,”
Symp. Adn Flavoproteins (E. C. Slater ed.), p. 391. Elsevier, Amsterdam, 1966.
17. T. P. Singer, in “Biological Oxidations” (T. P. Singer, ed.), p. 339. Wiley (Inter-
science), New York, 1968.
18. T. P. Singer and M. Gutman, in “Pyridine Nucleotide-Dependent Dehydroge-
nases” (H. Sund, ed.), p. 375. Springer-Verlag, Berlin and New York, 1970.
19. T. P. Singer and M. Gutman, Advan. Enzymol. 34,79 (1971).
20. T. P. Singer, M. Gutman, and E. B. Kearney, in “Biochemistry and Biophysics
of Mitochondria1 Membranes” (G. F. Azzone et al., eds.), p. 41. Academic Press,
New York, 1972.
21. T. P. Singer, in “Biochemical Evolution and the Origin of Life” (E. Schoffe-
niels, ed.), p. 203. North-Holland Publ., Amsterdam, 1971.
22. T. P. Singer, D. J. Horgan, and J. E. Casida, in “Flavins and Flavoproteins,”
2nd Int. Symp. (K. Yagi, ed.), p. 192. Univ. Park Press, Baltimore, Maryland, 1968.
23. T. P. Singer, M. Gutman, and V. Massey, in “Iron-Sulfur Proteins” (W. Loven-
berg, ed.), Vol. 1, p. 225. Academic Press, New York, 1973.
24. T. P. Singer, E. B. Kearney, and M. Gutman, in “Biochemical Regulatory
Mechanisms of Eukaryotic Cells” (E. Kun and S. Grisolia, eds.), p. 271. Wiley (Inter:
science), New York, 1972.
25. T. P. Singer, E. B. Kearney, and W. C. Kenney, Advan. Enzymol. 37, 189
( 1973).
26. T. P. Singer, E. B. Kearney, and B. A. C. Ackrell, in “Mechanisms in Bioener-
getics” (G. F. Azzone et at., eds.), p. 485. Academic Press, New York, 1973.
Mitochondria
FIa. 1. Scheme showing the fractionation of beef heart mitochondria into enzyme
complexes I, 11, 111, IV, and V with the use of deoxycholate (DOCA),cholate,
ammonium sulfate, and ammonium acetate. From Hatefi et al. (31).
drogenases. The latter two have also been referred to as type I and type
I1 NADH dehydrogenases, respectively. As will be seen, the two prepara-
tions of NADH dehydrogenase are related to complex I, except that one
appears to have irreversibly lost ubiquinone reductase activity and the
other has grossly modified enzymic properties.
1. NADH-Ubiquinone Reductase (Complex I )
NADH-ubiquinone reductase was isolated by Hatefi et al. in 1961
(27-29). A procedure was developed for the resolution of the mitochon-
drial electron transport system into four enzyme complexes. Recently, a
fifth fraction, which is capable of energy conservation and ATP-PI ex-
change, was also isolated (30,31). The overall scheme for the isolation
of the five component enzyme complexes of the mitochondria1 electron
transporhxidative phosphorylation system is given in Fig. 1. It is seen
27. Y. Hatefi, A. G. Haavik, and D. E. Griffiths, BBRC 4, 441 and 447 (1961).
28. Y. Hatefi, A. G. Haavik, and D. E. Griffiths, JBC 237, 1676 (1962).
29. Y. Hatefi, Compr. Biochem. 14, 199 (1966).
30. Y. Hatefi, D. L. Stiggall, Y. Galante, and W. G. Hanstein, BBRC 61, 313
( 1974).
31. Y. Hatefi, W. G. Hanstein, Y. Galante, and D. L. Stiggall, Fed. Proc., Fed.
Amer. Ism. Em. Biol. 34,1699 (1975).
TABLE I
COMPOSITION
OF COMPLEX
10
Concentration
Component (per mg protein)
FMN (acid-extractable) 1.4-1.5 nmoles

Nonheme iron 23-26 ng-atoms
Acid-labile sulfide 23-26 nmoles
Ubiquinone- 10 4.2-4.5 nmoles
Cytochromes <O. 1 nmole
Lipids 0 . 2 2 mg
a From Hatefi et al. (88, 38).
that the procedure, which yields complexes I to V in high yield, is rela-

tively simple. Deoxycholate and cholate are used in combination with
KC1, a neutral salt of low ionic strength, for differential solubilization of
mitochondria1 components ; and ammonium acetate and ammonium sul-
fate are employed for fractionation and isolation of the fragments. The
details of composition and enzymic properties of the enzyme complexes
shown in Fig. 1 have been described (99, 39). It was also shown in 1961
that complexes I, 11, 111, and IV interact stoichiometrically to reconsti-
tute a particulate unit with full activity and inhibitor-response proper-
ties for electron transfer from NADH and succinate to molecular oxygen
(97,29, 33). These and the subsequent studies of other laboratories (34-
36) have established that complex I represents in a highly purified form
the segment of the electron transport system from NADH to ubiquinone.
The complex carries site 1 of energy conservation and, under appropriate
conditions, it is capable of interaction with the isolated coupling factors
of mitochondria to couple the energy derived from oxidation of NADH
by ubiquinone to the synthesis of ATP (3 5 ,5 6 ).
a. Composition. The composition of complex I is shown in Table I.
Essentially all the flavin of complex I is acid-extractable, and according
to Rao et al. (37), more than 96% of the flavin is FMN. The ratio of
32. Y. Hatefi, W. G. Hanstein, K. A. Davis, and K. S. You, Ann. N. Y . Amd.

Sci. 227,504 (1974).
33. Y . Hatefi, A. G. Haavik, L. R. Fowler, and D. E. Griffiths, JBC 237, 2661
(1962).
34. N. R. Orme-Johnson, W. H. Orme-Johnson, R. E. Hansen, H. Beinert, and
Y. Hatefi, BBRC 44,446 (1971).
35. C. I. Ragan and E. Racker, JBC 248,2563 (1973).
36. C. I. Ragan and E. Racker, JBC 248,6876 (1073).
37. N. A. Rao, S. P. Felton, F. M. Huennekens, and B. Mackler, JBC 238, 449
(1963).
180 YOUSSEF HATEFI AND DIANA L. STIGQALL
I I I 1
' I
mM-' 0,
FIQ.2. Lineweaver-Burk plot of ubiquinone-6 reduction by complex ( I. Y. Hatefi,
unpublished).
flavin:iron:labile sulfide is 1:16-18: 16-18. Preparations of complex I

usually contain a total of about 0.1 nmole of cytochromes b c1 per +
mg protein, which represents a contamination of complex I by about 0.8%
complex 111. As in beef heart mitochondria, the ubiquinone of complex
I is ubiquinone-10 and the phospholipids, which comprise more than
90% of the total lipid of complex I, are phosphatidylcholine, phospha-
tidylethanolamine, and cardiolipin (38).The latter appears to be more
firmly bound to complex I proteins than phosphatidylcholine and phos-
phatidylethanolamine (36).The minimum molecular weight of complex
I is 6.5-7 X lo5 g protein (or 8-8.5 x lo5 g protein plus lipid) per mole
of flavin. However, acrylamide gel electrophoresis of complex I treated
with sodium dodecyl sulfate and mercaptoethanol reveals the presence
of at least ten polypeptide bands ranging in molecular weight from
10,000 to 70,000 (89, 4.0).
b. Activities. The physiological electron acceptor for complex I is
ubiquinone-10. However, because of its water insolubility, the lower
homologs, particularly ubiquinone-1 which is slightly water soluble, are
more efficiently reduced by preparations of complex I. Indeed, special
assay conditions are needed to demonstrate the in vitro reduction of
added ubiquinone-10 or ubiquinone-6. Figure 2 shows a double reciprocal
plot of data for the reduction of ubiquinone-6 by complex I and
NADH. The VmaXis 21.5 pmoles NADH oxidized by ubiquinone-6 per
min per mg protein, which is very close to the V,,, = 25 obtained with
ubiquinone-1 (41). However, the apparent K , for QB (41a) is 0.63 mM,
38. S. Fleischer, H. Klouwen, and G . Brierley, JBC 238, 2936 (1961).
39. R. A. Capaldi, ABB 183,99 (1974).
40. Y. Hatefi and K. E. Stempel, JBC 244, 2350 (1969).
41. A. J. Merola, R. Coleman, and R. Hansen, BBA 73,638 (1963).
41a. Abbreviations: Ql and Qd, uniquinone-1 and ubiquinoneb, respectively ;
APAD, acetylpyridine adenine dinucleotide; EPR, electron paramagnetic resonance;
TABLE I1
ACTIVITIESOF COMPLEX 1 WITH VARIOUS ELECTRON
DONORSAND ACCEPTORS5
Inhibition
Specific by Amytal,
Donor Acceptor activity* rotenone
NADH Ferricy anide 685"

NADH Ubiquinone-1 25e +
NADH
NADH
Ubiquinone-6
2,3-Dimethoxy-5,f5-dimethylbenzoquinone
21. 5c
<1.5
+
NADH 2,3-Dimethoxy-5-methylbenzoquinone <1.3
NADH 2-Methylnaphthoquinone (menadione) 1.9
NADH 2,6-Dichloroindophenol 1.5
NADH Vitamin K I 0.0
NADH a-Tocopherylquinone 0.0
NADH Lipoic acid 0.0
NADH Cytochrome c 3-4 +
NADPH
NADPH
Ubiquinone-1
Ferricy anide
+d
0.9e
+
NADH APAD 3.9'
NADPH APAD 0.3"
Succinate Ubiquinone-1, cytochrome c 0.0
From Hatefi el al. (88, 40, 80).

bSpecific activity is expressed as micromoles substrate oxidized per min per mg
protein a t 38".
5 Vmax with respect to acceptor concentration.
d The rate has not been reported.
c Measured at p H 6.5.
whereas the K , for Q1is of the order of 40-50 p M (36,40).This difference

probably reflects the water insolubility of Q6,thus resulting in an errone-
ously high K,. As in submitochondrial particles, the NADH-Q reductase
activity of complex I is inhibited by Amytal, rotenone, piericidin A, or
mercurials (28, 29, 41, 42). While Q1 is rapidly reduced by complex I,
the replacement of its isoprenyl side chain with a methyl group or a pro-
ton results in substantial loss of reactivity (28).The small degree
of reduction achieved with 2,3-dimethoxy-5,6-dimethylbenzoquinone
(aurantiogliocladin) or 2,3-dimethoxy-5-methyl-benzoquinoneis not in-
hibited by Amytal and rotenone (Table 11). a-Tocopherylquinone is not
NMR, nuclear magnetic resonance ; mV, millivolt ; pCMB, pchloromercuribenzoate ;
pCMS, p-chloromercuriphenyl sulfonate ; PMS, phenazine methosulfate ; EDTA,
ethylenediaminetetraacetate; SDS, sodium dodecyl sulfate ; TTFA, 2-thenoyltriflu-
oroacetone ; ETP, submitochondrial (electron transfer) particles; MVH, reduced
methyl viologen ; DEAE-cellulose, diethylaminoethyl cellulose ; succ, succinate ; APS,
adenosine 5'-phosphosulfate ; PAPS, 3'-phosphoadenosine 5'-phosphosulfate. Other
abbreviations are standard [see JBC 244,2 (1969)1.
42. Y. Hatefi, K. E. Stempel, and W. G. Hanstein, JBC 244, 2358 (1969).
TABLE I11
ACTIVITIEBOF THE NADH DEHYDROQENABEOF BAUQHAND KIN@
Acceptor K,(mM) Specific activityb
Ferricyanide 5.0 1,700

Juglone 0.3 64
2,3-Dicyano-5,6-dichloro-l14-benzoquinone 0.7 58
Cytoohrome c - 0.16
Duroquinone 1.5 3.8
Menadione 0.07 2.9
2,6-Dichloroindophenol 0.2 2.4
Q6
- 16
Q1
- 37
Q1 4.3 59
From Baugh and King (43).

a
Micromoles NADH oxidized x min-1 X mg-1 at 30' except for the Q homologs
which were assayed at 22'.
reduced by complex I, nor is the naphthoquinone vitamin K,.However,

2-methylnaphthoquinone (menadione, vitamin K,) is slowly reduced in
an Amytal-rotenone insensitive manner. As will be seen below, the low
molecular weight NADH dehydrogenase, which is derived from complex
I after destabilization of complex I structure, has a very high rotenone-
insensitive menadione reductase activity. Therefore, the small activity
seen in preparations of complex I might be due to the presence of small
amounts (<2%,) of partially destabilized complex I.
Among artificial electron acceptors only ferricyanide is rapidly reduced
by complex I ; dichloroindophenol and methylene blue are poorly effec-
tive. As seen in Table 11, preparations of complex I are also capable of
reducing cytochrome c a t a slow rate. This reaction is completely sensi-
tive to inhibition by rotenone or antimycin A and probably results from
the presence of traces of complex I11 in complex I preparations.
Baugh and King (4.3,4%) have recently reported the isolation of a prep-
aration from mitochondria with high NADH-ferricyanide and NADH-Q
reductase activities (Table 111). As compared to complex I, the prepara-
tion contains 20-25% less FMN, but more iron and labile sulfide, the
ratio of FMN :nonheme iron :labile sulfide being 1 :28 :28. The enzyme
is isolated from Keilin-Hartree particles (prepared from beef heart mito-
chondria) after treatment with Triton X-100 and subsequently with
cholate. It is claimed to be water soluble and free of phospholipids. HOW-
ever, satisfactory analytical data for the absence of lipid (e.g., phos-
43. R. F. Baugh and T. E. King, BBRC,49, 1165 (1972).
43a. C. I. Ragan, W. R. Widger, and T. E. King, BBRC 80, 894 (1974).
1 I 1 I I 1
5 -0.1
4
a
-0.2
400 450 500 550
nm
FIG.3. Difference spectrum of NADH-reduced minus oxidized complex I at 5 mg
protein per ml. Dotted line is the base line before addition of NADH to the sample
cuvette. From Hatefi e t al. (88).
phorus in ashed samples) and detergents (especially cholate which is

less easily removable than Triton X-100) have not been provided. As
seen in Table 111, the Km of the preparation of Baugh and King for
ubiquinone-1 is 4.3 mM. This is two orders of magnitude greater than
the K m of complex I. According to Ragan and Racker ( 3 6 ) ,it seems un-
likely that the reactions catalyzed by the preparation of Baugh and
King represent physiological events, because a t high ubiquinone-1 con-
centrations there is no phosphorylation a t site 1 linked to NADH oxida-
tion (44).
It has been shown by Ragan and Racker (36) that phospholipids are
necessary for the ubiquinone, but not the ferricyanide, reductase activity
of complex I. Thus, removal of about 50% of complex I lipids by extrac-
tion with cholate under special conditions resulted in a reversible loss
of ubiquinone reductase activity without affecting the ferricyanide reduc-
tase activity. More phosphatidylcholine and phosphatidylethanolamine
were removed by this procedure than cardiolipin. Readdition of either
phosphatidylcholine or phosphatidylethanolamine restored considerable
rotenone-sensitive ubiquinone-1 reductase activity, which was further
augmented when small amounts of cardiolipin were also added. Using
preparations depleted of ubiquinone-10 by pentane extraction, these
authors have also shown that enzyme-bound ubiquinone-10 is not neces-
sary for the reduction of added ubliquinone-1 by complex I or the inhibi-
tion of this reaction by rotenone.
c. Spectral Properties. The absorption spectrum of NADH-reduced
minus oxidized complex I is shown in Fig. 3. The bleaching afforded by
NADH is the result of the reduction of flavin and the iron-sulfur chromo-
phores, and the peak a t about 430 nm, which is superimposed on the
flavin plus iron-sulfur bleaching, is the result of the Soret absorption of
the reduced cytochromes of contaminating complex 111. Less than 50%
44. G . Schatz and E. Racker, JBC 241,1429 (1966).
of the bleaching a t 450 nm could be attributed to flavin reduction (assum-

ing full reduction by NADH). This discrepancy between the flavin con-
tent of complex I and the degree of bleaching afforded by NADH (or
by succinate in the case of complex 11, see below) a t 450 nm had sug-
gested that the reduction of nonheme iron is probably responsible for
the additional bleaching (29, 46). Subsequent isolation and spectral
studies of ferredoxins and demonstration of the presence of iron-sulfur
species in complexes I, 11, and 111 substantiated these early predictions
regarding the absorbancies of complexes I and I1 at about 450 nm not
accounted for by flavin.
The presence in complex I of NADH-induced, low-spin EPR signals
was shown in early studies on this enzyme complex (28). These measure-
ments were made a t near liquid nitrogen temperature, and the possible
relevance of these signals to the mechanism of electron transfer from
NADH to ubiquinone was recognized. Subsequently, the resolution of
complex I by chaotropic agents into three distinct fractions, each contain-
ing iron-sulfur components (see below), suggested that complex I might
contain more than one species of iron-sulfur moiety. Thus, E P R studies
of NADH-treated complex I a t near liquid helium temperature indicated
the presence of four iron-sulfur centers, which were designated centers
1, 2, 3, and 4 (34, 4 6 ) . These centers are identified in Fig. 4 with letters
q, r, s (center 1); 0, p (center 2) ; and 1, m, n (overlapping centers 3
and 4). The field positions of the prominent peaks on the g scale are
given in Table IV. As seen in A of Fig. 4, partial reduction of complex
I with NADH resulted only in the appearance of centers 2 and 3. Further
addition of NADH then produced centers 1 and 4 (B and C of Fig. 4).
This is an expression of the reduction potential of these iron-sulfur ten-
ters, which appears to be in the order 3 2 2 > > 4> 1 ( 4 6 ) . As seen in
C of Fig. 4, the overlapping signals resulting from centers 3 and 4 (1,
m, n) are emphasized a t high power and low temperature (7.7OK),
whereas under these conditions center 2 (0,p) appears to be considerably
saturated.
Kinetic experiments with NADH or reduced acetylpyridine adenine
dinucleotide (APAD), which reacts much more slowly than NADH,
showed that the sequence of appearance of signals resulting from reduc-
+
tion of the iron-sulfur centers was 2, 3 4, 1 (34, 3 6 ) . All centers were
reduced with NADH at 4 O within 6 msec ( 4 6 ) .Earlier studies of Beinert
and his colleagues (47) had shown that the half-time of the appearance of
45. Y. Hatefi, “The Enzymes,”2nd ed., Vol. 7, p. 495, 1963.
46. N. R. OrmeJohnson, R. E. Hansen, and H. Beinert, JEC 249,19!2!2 (1974).
47. H. Beinert, G. Palmer, T. Cremona, and T. P. Singer, EERC 12, 432 (1983) ;
JBC 240, 475 (1965).
I m n
0 P
q
A
FIG. 4. E P R spectra of NADH-reduced complex I showing iron-surfur centers

1 (4, r, s), 2 ( 0 , p), and 3 + 4 (1, m, n). A, reduced with 10.6 electron neq of
NADH; B and C, with 127 neq. Microwave frequency was approximately 9.2 GHz,
power 03 mW, modulation amplitude, 7.5 G ; temperature 13°K for A and B, 7.7"K
for C. From OrmeJohnson et al. (84).
TABLE I V
FIELDP O S I T I O N S A N D ABSIGNMENTS OF RESONANCES
OBSERVED I
IN COMPLEX AT 13'Ka
Iron-sulfur center Field positions* g averagec
1 2.022, 1.938, 1.923 1.96

2 2.054, 1.922 1.97
3 2.100, 1.886, 1.862 1.95
4 2 . 103,d 1 .864d
From Orme-Johnson et al. (46).
* The numbers are the measured field positions of prominent peaks given on the g
value scale.
The average g values were calculated by assuming tha t, the values measured at
the low and high field peaks correspond to g. and g., respectively, and by interpqlating
or extrapolating a probable value of gv from the position of the center line or peak.
d Since center 4 is only seen in the presence of center 3, i.e., the field position of
the combined resonances is measured, these values may only be approximate. It is
likely that the values differ somewhat more from those of center 3.
TABLE V
INTEGRATED
INTENSITIESOF EPR RESONANCES FROM IRONSULFUR
CENTERSOF COMPLEX I I N RELATION
TO THE SPECTRO-
PHOTOMETRICALLY DETERMINED FLAVIN CONTENT'
Ratio of concentration of
iron-sulfur centers to
Iron-sulfur center flavin concentration
1+2+3+4 4.0
1 0.81
2 0.89
1+2 2.2
3+4 by difference 1.8
, From Orme-Johnson et al. (46)
the g = 1.94 signal (at 77OK, the temperature used for the EPR experi-
ments, the signal mainly results from center 1) of NADH dehydrogenase
preparations treated with slowly reacting NADH analogs corresponded to
one catalytic cycle of the enzyme as measured by the reduction of ferricya-
nide. It is important to note that double integration of the signals of com-
plex I have indicated that on the basis of electron consumption the mo-
larity of each of the four iron-sulfur signals appears to be comparable to
that of the flavin (Table V).
Since the iron-sulfur centers appear to be of the ferredoxin type, this
means that each center might involve 2 or 4 iron atoms (depending on
whether they are plant type or clostridial type) and take up one electron.
This is rather interesting in view of the fact that in complex I the ratio
of flavin:iron:labile sulfide is very close to 1: 16: 16, which would agree
with the possibility of four clusters of 4 irons and 4 labile sulfides each.
Indeed, as will be seen below, the low molecular weight NADH dehydro-
genase isolated from complex I has a composition of 1 flavin:4 iron:4
labile sulfide. According to Beinert and his colleagues (467, complex I
takes up approximately 20 electron neq/mg protein, or 13-14 electrons
per mole of flavin. The four iron-sulfur centers (assuming clostridial fer-
redoxin-type clusters of 4 iron and 4 labile sulfide per center) plus FMN
would account for 6 electrons, and ubiquinone could account for another
5-6 (Table I).However, complex I and the high molecular weight NADH
dehydrogenase also contain a multiplicity of thiol groups (see below)
a t least one of which appears to become susceptible to inhibition by mer-
curials after treatment of the preparation with NADH (23,48). Assum-
48. D. D. Tyler, R. A. Butow, J. Gonze, and R. W. Estabrook, BBRC 10, 551
(1965).
ing utilization of a pair of electrons in this activation process, then the

titration data of Beinert and co-workers becomes remarkably accurate
for complex I.
DerVartanian et al. (49) have examined the E P R properties of the
dehydrogenase of Baugh and King ( 4 3 ) ,which has a flavin:iron:labile
sulfide ratio of 1:28:28. They have concluded that quantitation of the
four reduced iron-sulfur centers by double integration accounts for only
36% of the iron content of the preparation. They felt that in this prepara-
tion the behavior of the EPR resonances suggests the presence of uniden-
tified iron complexes in addition to iron-sulfur centers. Ohnishi et al. (50)
have examined a preparation of complex I made by Ragan and Racker
(36). According to the latter authors, their preparation has a ratio of
flavin :iron: labile sulfide of 1 :23 :22 (the flavin content is 1.26 nmoles/mg
protein). Ohnishi et a2. (50) found seven iron-sulfur centers in this prepa-
ration when examined a t different temperatures, high microwave powers,
and in the presence of redox mediators ranging from -445 to -189
mV. Data regarding quantitation of these signals and their kinetic com-
petence have not yet been presented. Such data are necessary in order
to weigh the significance of the additional signals reported by Ohnishi
et al. As stated above, iron-sulfur center 1 of complex I is relatively tem-
perature-insensitive and observable a t liquid nitrogen temperature. This
signal degenerates upon prolonged exposure of various preparations to
NADH and leads to the appearance of new signals (46,51).The interpre-
tation that these latter signals resulted from the presence of molybdenum
in complex I (56) has been abandoned ( 5 3 ) . Preparations of complex
I or mitochondria1 inner membrane fragments do not contain more than
trace amounts of molybdenum (0.03 atom/mole of acid-extractable
flavin) (34, 5 3 ) . These and other complications involved in the interpre-
tation of E P R data have been discussed by Beinert and his colleagues
(46,54) as well as by Albracht ( 5 5 ) .
2. High Molecular Weight N A D H Dehydrogenases
In 1962, Ringler et al. (see 56) reported the isolation of a NADH dehy-
drogenase from bovine heart mitochondria with the use of Naja naja venom
phospholipase A and Triton X-100. The preparation contained 0.9 nmole
49. D. DerVartanian, R. F. Baugh, and T. E. King, BBRC 50, 629 (1973).
50. T. Ohnishi, J. S. Leigh, C. I. Ragan, and E. Racker, BBRC 56, 775 (1974).
51. M. Kawakita and Y. Ogura, J . Biochem. (Tokyo) 66,203 (1969).
52. S. P. J. Albracht and E. C. Slater, BBA 223,457 (1970).
53. S. P. J. Albracht, H. VanHeerikhuiren, and E. C. Slater, BBA 2 5 6 , l (1972).
54. N. R. OrmeJohnson, R. E. Hansen, and H. Beinert, JBC 249, 1928 (1974).
55. S. P. J. Albracht, BBA 347, 183 (1974).
56. R. L. Ringler, S. Minakami, and T. P. Singer, JBC 238, 801 (1963).
of flavin per mg protein, 16 g-atoms of iron per mole of flavin, and 6.2%
lipid by dry weight. It catalyzed the reduction of ferricyanide by NADH
with a calculated turnover number of 6.6 x lo6 moles of NADH oxidized
per minute per mole of “flavoprotein” a t 38O. The flavin was a mixture
of 25-30% FAD and the rest F M N or FMN plus riboflavin. The enzyme
also contained 5’-AMP in amounts roughly equivalent to the sum of
FMN plus riboflavin. These results were somewhat reminiscent of the
split products of FAD in the NADH-cytochrome c reductase preparation
of Mahler et al. (57). Thus, the authors considered that the ffavin
moiety of the enzyme might be FAD or both FAD and FMN (56).The
identity of the flavin of mitochondria1 NADH dehydrogenase was settled
by Rao et al. (37)in 1963 by a careful analysis of the total and acid-
extractable flavin of several preparations from mitochondria, especially
complex I and its parent particle complex 1-111. They found that in these
preparations more than 96% of the flavin was acid-extractable FMN.
Subsequently, Cremona and Kearney (68) modified the preparation of
the dehydrogenase of Ringler et al. (66),deleted the use of Triton X-100,
and obtained a more purified preparation of the enzyme containing
1.232 0.02 nmoles of flavin per mg protein. The flavin was identified as
FMN. The sedimentation coefficient ( s ~ ~ of , ~the
) preparation a t pH 10
(to prevent aggregation) was estimated to be 14 2 0.5 in the concentra-
tion range of 6-10 mg/ml. However, a skewing of the sedimentation bound-
ary was observed, which was stated to result from 30-3576 colorless im-
purity (actual results not shown). On the basis of its flavin content, the
preparation would have a minimum molecular weight of 813,000per mole
of flavin. However, the authors corrected for the presumed 3 M 5 % im-
purity calculated from their sedimentation patterns a t pH 10, and con-
cluded that the molecular weight of the “pure flavoprotein” is of the order
of 550,000.Subsequent pubhations from Singer’s laboratory have used
this figure as the established molecular weight of the high molecular
weight NADH dehydrogenase (19, 23). Using a similar correction as
above, the turnover number of the dehydrogenase preparation has been
calculated to be 800,000 per minute at 30°.
This and the earlier preparation of Ringler et al. are claimed to be
water-soluble, even though both were isolated after phospholipase treat-
ment of particles, which results in the formation of detergent-like lyso-
lipids, and Triton X-100 was added to the preparation of Ringler et al.
to prevent aggregation. The NADH dehydrogenase preparation of Baugh
and King (43) is also stated to be water-soluble. Both Triton X-100 and
57. H. R. Mahler, N. K. Sarkar, L. P. Vernon, and R. A. Alberty, JBC 199, 685
(1052).
58. T.Cremona and E. B. Kearney, JBC 239,2328 (1964).
cholate were used during its isolation, but it is claimed that addition
of cholate resulted in the removal of Triton X-100 which was added
earlier, and that subsequent chromatography on agarose and sucrose
gradient centrifugation removed the added cholate. Huang and Pharo
(69) have isolated a NADH dehydrogenase with the use of Lubrol, which
is very similar in enzymic properties, absorption spectrum, and flavin
content (1.17 nmole/mg protein) to the preparation of Cremona and
Kearney. They stated, however, that detergent appears to be essential
for the solubility of the dehydrogenase since its removal resulted in an
insoluble, but active, preparation. Thus, it seems prudent to reserve judg-
ment on whether or not these preparations of high molecular weight
NADH dehydrogenase are truly water-soluble. By comparison to complex
I, they ought to contain a considerable amount of hydrophobic membrane
“proteins” or polypeptides, and the complete absence of lysolipids in the
preparation of Cremona and Kearney (58) and detergents in the prepara-
tions of Ringler et al. (56) and Baugh and King (43) has not been dem-
onstrated. On the other hand, while water solubility of a complex enzyme
is convenient for laboratory experiments, it may be of little physiological
significance when in the native state the complex is tightly membrane
bound. Table VI summarizes the composition and activities of various
preparations of high molecular weight NADH dehydrogenase and pro-
vides a comparison with complex I.
3. Low Molecular Weight NADH Dehydrogenases
The low molecular weight form of mitochondrial NADH dehydroge-
nase was first isolated from pig heart muscle by Edelhoch et al. (60)
and Mahler and his associates (57) in 1952. The mitochondrial origin
of the enzyme was demonstrated by de Bernard (61).These and similar
preparations reported subsequently by Mackler (62), Kumar et al. (651,
and Pharo et al. (64) were isolated by extracting the source material
(heart muscle or various submitochondrial preparations) with 9-1 1 %
ethanol at pH 4.8-5.3 and 430450, a procedure originally devised for
isolation of the Straub diaphorase (lipoyl dehydrogenase) ( 6 5 ) . TWO
other preparations of basically similar composition and catalytic proper-
P. C. Huang and R. L. Pharo, BBA 245, 240 (1971).
59.
H. Edelhoch, 0. Hayaishi, and L. J. Tepley, JBC 197, 97 (1952).
60.
B. de Bernard, BBA 23, 510 (1957).
61.
B. Mackler, BBA 50, 141 (1961).
62.
S. A. Kumar, N. A. Rao, S. P. Felton, F. M. Huennekens, and B. Mackler,
63.
ABB 125, 436 (1968).
64. R. L. Pharo, L. A. Sordahl, S. R. Vyas, and D. R. Sanadi, JBC 241, 4771
(1966).
65. F. B. Straub, BJ 33, 787 (1939).
TABLE VI
COMPOSITION
AND PROPERTIES
OF HIGHMOLECULAR
WEIGHT
NADH DEHYDROGENASES
Flavin
(nmole/mg Flavin :iron : KZADH K$ Refer-
Preparation protein) sulfide Reactions catalyzed (rM) (PM) ences
Complex I (1961) 1.4-1.5 1:16-18: 16-18 NADH + Q, K*Fe(CN),, APAD 4

7 44 27J28 0
Ringler et al. (1962) 0.9 1:16:?
NADPH + Q, KIFe(CN)6JNAD
NADH -+KIFe(CN), 66 s
u,
Cremons-Kearney (1964) 1.23 1: 17-18:27-28 NADH, NADPH -+ K*Fe(CN)*
108 68,83,84 4
NADH -+ APAD
HUmg-Phmo (1971) 1.17 1:26:? NADH + KIFe(CN), 69
xgI3
Baugh-King (1972) 1.13 1:28: 28 NADH + Q, &Fe(CN),, APAD M
- 4,300 43,43a
NADPH + K,Fe(CN),, NAD
9
Tottmar-Ragan (1971) 0.5-0.6 1:28:28 NADH, NADPH + KIFe(CN)s 83 133
(from C. utilis) Ei
3z
P
r
u,
Er
Temperature, *C
FIO.5. Kinetics of the resolution of complex I as a function of temperature of
the incubation medium. The release of menadione reductase activity was measured
as an index of resolution 2.5 min after incubation of complex I with 0.47 M NaClO,
at the temperatures indicated. From Davis and Hatefi (69).
ties are those of King and Howard (66) and Hatefi and Stempel (40,
67, 68). The former was extracted from Keilin-Hartree particles from
beef heart after incubation of the particles at 37O with boiled snake
venom (as a source of phospholipase) in the presence of CaC1,. The latter
was isolated after resolution of complex I with various chaotropic agents
(67, 69). The above dehydrogenase preparations are qualitatively similar
in composition and enzymic activity and have molecular weights (often
calculated from flavin content) between 7 and 12 X lo4. They contain
FMN, nonheme iron, and labile sulfide (wherever examined), and have
a wide electron acceptor specificity with respect to quinoid structures and
ferric compounds. They are all inhibited by mercurials. The quantitative
differences in composition and activity appear to be related to their isola-
tion and purification conditions. Among these dehydrogenases, the prepa-
ration of Hatefi and Stempel (40, 67, 68) has been more fully studied
and appears to have been obtained with the least damage since it exhibits
the highest enzymic activities and the highest content of flavin, iron, and
labile sulfide. Therefore, the properties of this preparation as an example
of the low molecular weight NADH dehydrogenases will be more thor-
oughly discussed.
The enzyme is isolated from complex I after resolution of the complex
with chaotropic agents. The resolution process is highly temperature-
dependent (Fig. 5 ; activation enthalpy from data of Fig. 5 AH' = $37
66. T.E. King and R. L. Howard, JBC 237, 1686 (1962).
67. Y. Hatefi and K. E. Stempel, BBRC 26,301 (1967).
68. Y. Hatefi, K. E. Stempel, and W. G . Hanstein, JBC 244, 2358 (1969).
69. K. A. Davis and Y. Hatefi, Biochemistry 8,3355 (1969).
0.8
0.6
k
0.4
0.2
Salt concentration (M)

FIQ.6. Effect of various chaotropes on the first-order rate constant (k) of the
resolution of complex 1. TBA, tribromoacetate; TCA, trichloroacetate; DCA, dichlo-
roacetate ; TFA, trifluoroacetate; MCA, monochloroacetate; and AcO; acetate.
From Hanstein et at. (71).
kcal/mole), irreversible, and retarded in the presence of NADH. Its rate

is finely controllable by the concentration and the potency of the chao-
tropic agent employed (Fig. 6), and strongly inhibited when the medium
H,O is replaced with the more structured solvent, D,O (Fig. 7 ) . The sig-
h C Q . [MI
FIG.7. Solvent isotope effect on the resolution of complex I by NaClO, at 20"
and 30"; k, first-order rate constant in min-'. From Hanstein et al. (73).
Fxa. 8. Absorption spectrum of the soluble iron-sulfur protein (4.3 mg/ml) isolated
from complex I. Dashed line, after treatment with dithionite; dotted line, after
treatment with sodium mersalyl to destroy the iron-sulfur chromophore. From Hatefi
et al. (3.9).
nificance of these results in relation to the effect of water structure on

the structural stability of complex I in aqueous media has been discussed
elsewhere (6‘9-73).
The resolved complex is composed of two fractions, a soluble part,
which comprises about 15% of complex I proteins, and a water-insoluble
part consisting of the rest of the protein and the bulk of complex I lipids.
The soluble fraction is easily separated from the inscluble material by
centrifugation. Upon fractionation with ammonium sulfate, it yields a
soluble flavoprotein containing iron and labile sulfide and a dark brown
protein, which contains large amounts of iron and labile sulfide but no
flavin. The latter appears t o be an iron-sulfur protein and exhibits an
EPR signal which is characteristic of iron-sulfur center 2 of intact com-
plex I (46‘). Its absorption spectrum is shown in Fig. 8. The insoluble
fraction also contains equimolar amounts of iron and labile sulfide and
little or 110 flavin.
The flavoprotein fraction represents the low molecular weight NADH
dehydrogenase and contains per mg protein 13.5-14.5 nmoles of F M N
(acid extractable), 60-65 ng-atoms of iron, and 58-60 nmoles of acid
70. Y. Hatefi and W. G. Hanstein, Proc. N a t . Acad. Sci. US. 62, 1129 (1969).
71. W. G. Hanstein, K . A. Davis, and Y. Hatefi, ABB 147, 534 (1971).
72. Y. Hatefi and W. G. Hanstein, “Methods in Enzymology,” Vol. 31, Part A,
p. 770, 1974.
73. W. G. Hanstein, K. A. Davis, and Y. Hatefi, ABB 163, 482 (1974).
labile sulfide. The ratio of flavin to iron to labile sulfide is, therefore,
close to 1:4:4, suggesting that iron and labile sulfide might be in a clos-
tridial ferredoxin-type cluster. The visible spectrum of the oxidized dehy-
drogenase analyzed for contributions of flavin and iron-sulfur chromo-
phore, plus the spectra of NADH- and dithionite-reduced enzyme are
shown in Fig. 9. Its enzymic properties and kinetic constants with respect
to various electron acceptors are given in Table VII. Comparative data
for complex I are provided in Table VIII. I n addition to ubiquinone-1,
the enzyme also reduces higher isoprenologs of ubiquinone a t appreciable
rates (40,67).
The dehydrogenase preparations obtained by acid-ethanol extraction
of particles a t elevated temperatures vary considerably in their content
of flavin, iron, and labile sulfide, and in their activities. These differences
appear to be largely a consequence of destruction of the iron-sulfur
chromophore a t acid pH. As seen in Fig. 10, incubation of the low molecu-
lar weight dehydrogenase preparation of Hatefi and Stempel a t pH 4.8
and 3 8 O resulted after 1 hr in nearly complete loss of labile sulfide (Fig.
1OC) and reductase activity with respect to menadione, cytochrome c,
I---
FIa. 9. Spectral characteristics of the soluble NADH dehydrogenaee (1.6 mg/ml)

isolated from complex I. Traces 1, spectrum of oxidized enzyme; 2, NADH-reduced
enzyme; 5, dithionite-reduced enzyme; 4, flavin contribution to 1 after destruction of
iron-sulfur chromophore with sodium mersalyl ; 3, iron-sulfur contribution to.1 ob-
tained by subtraction of 4 from 1; 6, 4 plus dithionite showing that after destruction
of the iron-sulfur chromophore with mersalyl and reduction of flavin with dithionite
the enzyme has no absorption in the visible region. From Hatefi and Stempel (40).
k , , , , , ,
100. - -- ------_- -
I
M 40-
20 - pHs8.0
I 1
, " 1 1
102030405060 102030405060
min at 38' min at 38'
(a) (b)
10 2 0 3 0 4 0 3 3 6 0
min at 38'
(C)
FIQ. 10. Effect of incubation at pH 4 8 and 38" on the activities [(a) pH 8.0,
(b) pH 4.81 and labile sulfide content (c) of NADH dehydrogenase.Ks, menadione. '
Key: (-) Ks, (---) cytochrome c, and (---) ferricyanide. From Hatefi (76).
and ferricyanide (Fig. 10B). By comparison, a sample of the enzyme

incubated a t pH 8.0 lost less than 20% labile sulfide (Fig. lOC), 40%
menadione and ferricyanide reductase activity, and very little cyto-
chrome c reductase activity (Fig. 10A).
Kumar et al. (63) have estimated the molecular weight of the acid-
ethanol-extracted dehydrogenase by Sephadex gel exclusion to be approx-
imately 70,000. Assuming one mole of FMN per mole of enzyme, the
molecular weight of 70,000 agrees with the flavin content of the prepara-
tions of Kumar et al. (6S),Pharo et al. ( 6 4 ) , and Hatefi and Stempel
(40).Kumar et al. (63) have also determined the amino acid composi-
tion of their preparation, except that the value for tryptophan is not
TABLE VII
ENZYMIC
PROPERTIES
OF SOLUBLE
NADH DEHYDROGENASE'
Inhibition by Inhibition by
Specific K:"" KFPtor 1 pM rotenone >0.25 mM
Reaction activitp VEDfeO cxps= (rM) (PM) (%I NADH
NADH + KIFe(CN)* 215 330 400" 65 1650' None -

NADH + cytochrome c 43 76 220 600 - 3
NADH + &I 150-160 250 175
64
133 167 10-20 + srn
NADH --+ menadione 160-170 330 190 133 160 10 + M I
%
NADH + 2,6-dichloroindophenol 100 125 133 62 3:
Reduced APAD + &I 3.8 715 + 2I
From Hatefi and Stempel (40). 3
*
b Expressed as micromoles of NADH oxidized per min per mg of protein at 38". All activities are completely inhibited with 0.1
mM pmercuriphenyl sulfonate. z
At 0.75 mM NADH.
E
9
r
rn
Br
F
tp
E
9
t:
0
0
TABLE VIII
PROPERTIES
NADH DEHYDROGENASE I A N D 1-111.
OF COMPLEXES
i5!
2
ci
Inhibition by I
0.5 mM 1 pM c
1PM 0
Enzyme Specific K:*”” KrPto. pCMS rotenone antimycin A w
Y
Reaction complex activity (pM) (pM) (%I (%I (%) NADH 9
z
NADH -+ K3Fe(CN)6 I lo@ 7 400W None None None >O.lmM tl
NADH 4 cytochrome c I 2d 14 12 100 100 100
NADH -+ cytochrome c 1-111 25-30 14 12 100 100 100 ii
NADH 4 &I 1-111 14 15-17 44 100 100 None >0.25mM ;s
NADH + menadione
NADH -+ 2,6-dichloroindophenol
I
I
1.9
1.5
None
None
None
None
None
None i%
0 From Hatefi and Stempel (40). e3
Per mole of flavin, this activity is considerably higher in complex I than in the soluble, low molecular weight dehydrogenase.
c A t 0.15 mM NADH; V ~ ~ ~ ( C= N685.
’6
d This activity results from the presence in complex I of 0 . 5 1 % complex I11 contamination.
given. According to these investigators, a considerable amount of flavin

can be removed from the enzyme by treatment with Florisil or Bio-Gel.
The depleted enzyme retains its ferricyanide reductase activity, but loses
considerable activity for reduction of dichloroindophenol and cytochrome
c. The latter is partially restored by addition of large amounts of
FMN. The authors concluded from these data that reducing equivalents
from NADH first go to the iron-sulfur moiety of the enzyme, then to
flavin. Ferricyanide accepts electrons from the iron-sulfur moiety, but
indophenol, quinones, and cytochrome c are reduced a t the flavin site.
I n agreement with this conclusion they have shown that chromatography
of the enzyme on DEAE-cellulose at pH 6.8 results in nearly complete
removal of flavin and labile sulfide, and about two-thirds of the iron.
This preparation had no reductase activity with any of the acceptors,
even when assayed in the presence of added FMN. The above mechanistic
conclusions are not generally accepted, however, because (a) the prepara-
tion of Kumar et al. has very low reductase activities (probably related
to its low content of iron and labile sulfide) , (b) the cytochrome c reduc-
tase activity restored by addition of FMN is only about 1% of the maxi-
mal cytochrome c reductase activity of the more active preparations of
the enzyme (do),and ( c ) no attempt was made to reconstitute the iron-
sulfur moiety of the DEAE-cellulose-treated enzyme by treatment with
sulfide and ferrous ions to see whether ferricyanide reductase activity
can be restored. It has been stated by Yang (74)that the partial loss
of ferricyanide reductase activity (and 450 nm absorption) of the prepa-
ration of Kumar et al. upon aging in air a t room temperature could be
restored to a considerable extent by treatment of the enzyme with 2-
mercaptoethanol, FeCl,, and Na,S followed by filtration through a col-
umn of Sephadex G-25. However, the validity of this type of reconstitution
experiment rests on several important controls which were not presented.
4. Relevance of the Low and High Molecular Weight Preparations
to Mitochondria1 NADH Dehydrogenase
The ubiquinone reductase activity of their low molecular weight dehy-
drogenase led Pharo et al. (64, 75) to conclude that the enzyme repre-
sented the mitochondria1 NADH-ubiquinone reductase. However, it has
been shown that the quinone reductase activity of the low molecular
weight dehydrogenase is different from that of intact respiratory particles
or complex I in many important respects, including kinetic constants, re-
74. C. S. Yang, in “Flavins and Flavoproteins,” 3rd Int. Symp. (H. Kamin, ed.),
p. 664. Univ. Park Press, Baltimore, Maryland, 1971.
75. D. R. Sanadi, R. L. Pharo, and L. A. Sordahl, in “Non-Heme Iron Proteins”
(A. San Pietro, ed.), p. 429. Antioch Press, Yellow Springs, Ohio, 1966.
mM DPNH
FIG. 11. Effect of NADH concentration on the ferricyanide reductase activities
of complex I and the soluble, low molecular weight dehydrogenase. From Hatefi
and Stempel (40).
sponse to inhibition by Amytal, rotenone, and piericidin A, and the ap-

parent involvement of several EPR-active iron-sulfur centers. As com-
pared to respiratory particles or complex I, the soluble enzyme exhibits
low ferricyanide reductase activity per mole of flavin and very high re-
ductase activities with respect to menadione, 2,6-dichloroindophenol, or
cytochrome c as electron acceptor. The latter activity, in contrast to that
found in submitochondrial particles or complex 1-111, is insensitive to
inhibition by rotenone, piericidin A, or antimycin A, and 4 s marked by
a very high K , for cytochrome c (600 p.M versus 12 p M in the case of
complex 1-111) (Tables VII and VIII; also see 40). In addition, the fer-
ricyanide reductase activity of complex I is sharply inhibited at NADH
concentrations above 0.1 mM whereas the activity of the soluble dehydro-
genase is not (Fig. 11). Conversely, the former activity is not inhibited
by mercurials, but the latter is. It has also been shown that the ubiqui-
none reductase activity of particles is 70% inhibited by 50 m M guanidine-
HCl, whereas the same activity catalyzed by the low molecular weight
dehydrogenase is 75% activated (68, 76). The cytochrome c reductase
activity of the soluble enzyme was first discovered by Mahler and
co-workers (5‘7); hence, the designation “Mahler’s DPNH-cytochrome
c reductase.” However, these investigators had recognized the unphysi-
ological nature of this activity, and Mahler and Glenn (7’7) pointed
76. Y. Hatefi, PTOC.Nat. Acad. Sci. U . S. 60,733 (1968).
77. H. R. Mahler and J. L. Glenn, in “Inorganic Nitrogen Metabolism” (W. D.
McElroy and B. Glass, eds.), p. 575. Johns Hopkins Press, Baltimore, Maryland,
1956.
out that the cytochrome c reductase activity of their dehydrogenase

might be because cytochrome c behaves as a single electron acceptor
similar to, but not identical with, the physiological electron acceptor
in the respiratory chain. This early prediction is interesting since a
single electron accepting iron-sulfur structure is very likely the natural
acceptor for the membrane-bound dehydrogenase. It should also be men-
tioned that interaction with cytochrome c is not a peculiar property of
the above enzyme. A number of flavoproteins containing FMN or FAD
as prosthetic group and utilizing NADH or NADPH as electron donor
are known which interact with cytochrome c (29, 46). Examples of this
unphysiological phenomenon are in the case of Old Yellow Enzyme (78)
and Straub’s diaphorase (lipoyl dehydrogenase) (79).
It is clear from the above section that the differences between the en-
zymic properties of the soluble, low molecular weight NADH dehydroge-
nase and the particulate system represented by complex I are very large,
even though the former is obviously a component of the latter enzyme
system. Singer and his colleagues believe that the low molecular weight
enzyme is a “peptide fragment” of the high molecular weight NADH
dehydrogenase (16, 19). While in essence the parent-progeny relationship
is obvious, the vigorously espoused views concerning peptide fragment
and the equivalence of the high molecular weight preparation to mito-
chondrial NADH dehydrogenase are no longer acceptable. It has been
pointed out that the small molecular weight dehydrogenase is not likely
to be a peptide fragment because the procedures leading to its isolation
are not likely to cleave peptide bonds (69, 80). Furthermore, as will be
seen below, complex I and the corresponding section of respiratory parti-
cles catalyze the dehydrogenation of NADPH without the intermediation
of NAD and the transhydrogenase reaction. Studies on complcx I with
NADH and NADPH as substrates have shown that flavin and iron-sulfur
center 1 are reduced by NADH, but apparently not by NADPH. There-
fore, there appears to exist in complex I a segment, containing flavin and
a portion of the total iron and labile sulfide, which is specific for
dehydrogenation of NADH. It is highly probable that the small molecular
weight NADH dehydrogenase represents this segment of complex I, except
that conversion from membrane-bound to soluble state has modified its
enzymic properties, some of which (e.g., wide acceptor specificity) might
be simply the result of better access of acceptors to the soluble enzyme.
Much has been written by Singer and his colleagucs in defense of thc
78. A. Akeaon, A. Ehrenberg, and H. Theorell, “The Enzymes,” 2nd ed., Voi. 7,
p. 477,1963.
79. V. Massey, BBA 30, 205 (1958).
80. Y. Hate6 and W. G. Hanstein, Biochemistry 12,3515 (1973).
thesis that their preparation is the true NADH dehydrogenase of the

respiratory chain (14, 15, 19, 2 3 ) . They have done extensive studies on
their preparation and have compared its composition, enzymic, and E P R
properties to complex I and the low molecular weight dehydrogenase
preparations. They have pointed out quite correctly that theirs is the
first preparation of a NADH dehydrogenase which displays a high
ferricyanide reductase activity comparable on the basis of NADH dehy-
drogenase flavin to the activity of complex I and intact respiratory chain
preparations. By comparison, the low molecular weight NADH dehy-
drogenases have very low ferricyanide reductase activity. However, as
compared to complex I, the dehydrogenase of Singer and co-workers ap-
pears to have several important differences. The comparison to complex I
as a reliable point of reference is valid since (a) the above dehydrogenase
resembles complex T in its size, ferricyanide reductase activity, and con-
tent of nonheme iron, and ( b ) complex I appears to be the smallest seg-
ment yet isolated which displays all the catalytic and inhibitor-response
properties of the NADH-ubiquinone reductase portion of intact respira-
tory chains, including the important ability of conserving oxidative
energy and reacting with mitochondria1 coupling factors to synthesize
ATP ( 3 5 ) .The differences are as follows:
a. The NADH dehydrogenase of Singer and co-workers is incapable
of reducing ubiquinone, which is the physiological electron acceptor for
complex I. This inability has been referred to the fact that the dehydro-
genase is essentially devoid of lipids. That phospholipids are necessary
for ubiquinone reduction by complex I has been demonstrated by Ragan
and Racker (36 ) as discussed above. However, the latter investigators
were able to restore ubiquinone reductase activity by adding phospho-
lipids to deficient complex I preparations. I n the absence of such activity
restoration experiments, the view that the preparation of Singer e t al.
has not sustained irreversible damage during isolation would remain an
assumption. According to Ragan and Racker ( 3 6 ) ,special reducing condi-
tions are needed during the removal of lipids from complex I in order
to preserve the ability of the complex to exhibit ubiquinone reductasc
activity upon readdition of lipids. It remains to be seen whether the dehy-
drogenase of Singer et al. can be isolated under similar reducing condi-
tions with preservation of ubiquinone reductase activity when assayed
in the presence of added lipids.
b. As stated above the ratio of flavin to iron to labile sulfide is
1:16-18:16-18 for complex I. This ratio is stated to be 1:17-18:27-28
for the NADH dehydrogenase. The molar excess of labile sulfide as com-
pared to iron is surprising and contrary to literature data for all species
of iron-sulfur proteins known. However, this high labile sulfide value
might have resulted from the low extinction coefficient (21,000 liters
mole-' cm-l) used by the authors (81) for labile sulfide determination by
the method of Fog0 and Popowsky (82). A more correct molar extinction
coefficient is between 27,500 and 30,000, which-when applied to the labile
sulfide value published for NADH dehydrogenase-would lower it to
about 20, a value in much better agreement with the iron content of the
preparation.
c. According to Watari et al. (83) and Singer and Cremona ( I d ) , the
K , for NADH of their preparation is 108 p M . This value is more than
15-fold greater than the K , of complex I for NADH (7 p i l l ) determined
similarly in the NADH-ferricyanide reductase assays (Table VIII) .
This difference is rather serious because the high K,,, value is character-
istic of the low molecular weight NADH dehydrogenase derived from
complex I. The K , for NADH of the low molecular weight enzyme,
also determined in the ferricyanide reductase assay, is about 65
(Table VII), and as detailed above it is generally agreed that the iso-
lated low molecular weight dehydrogenase shows major differences in
catalytic properties as compared to its membrane-bound counterpart.
Thus, with respect to its K,,, for substrate, the NADH dehydrogenase
of Singer and co-workers is similar to the modified, low molecular weight
enzyme, and differs from complex I and other submitochondrial particles.
This difference might be associated with structural modifications respon-
sible for the inability of the high molecular weight NADH dehydrogenase
to interact with ubiquinone under appropriate conditions.
Although it was not recognized as a reaction involving a separate mech-
anism, the published data of Singer's laboratory show clearly that their
preparation also has NADPH dehydrogenase activity (84). Conse-
quently, the high molecular weight NADH dehydrogenase preparations
appear to be segments of the respiratory chain related to complex I. These
preparations appear to have preserved the ferricyanide reductase activity
of the system but irreversibly lost the physiological ubiquinone reductase
activity. The argument as to which preparation-the high or the low
molecular weight enzyme-represents the respiratory chain NADH
dehydrogenase is perhaps irrelevant in view of our present knowledge.
Both are clearly derived from complex I. However, the low molecular
weight preparation has grossly modified enzymic properties (see also
Section II,B), and the high molecular weight preparation appears to
have retained the NADH and NADPH dehydrogenase activities of com-
81. C. J. Lusty, J. M. Machinist, and T. P. Singer, JBC 240, 1804 (1965).
82. J. K. Fog0 and M. Popowsky, Anal. Chem. 21,732 (1949).
83. H. Watari, E. B. Kearney, and T. P. Singer, JBC 238, 4063 (1963).
84. C. Rossi, T. Cremona, J. M. Machinist, and T. P. Singer, JBC 240, 2634 (1965).
plex I, but only with respect to ferricyanide as electron acceptor. These

considerations lead, therefore, to the conclusion that proper purification
of NADH dehydrogenase beyond the stage of complex I has yet to be
achieved.
5. Inhibitors of N A D H Dehydrogenase
Thiol reagents, barbiturates, rotenoids, and piericidin A inhibit NADH
dehydrogenation and ubiquinone reduction in appropriate preparations.
According to Singer and his colleagues (19,23, 85-87) there are five types
of --SH groups in various preparations of NADH dehydrogenase. Type
I is the -SH group found in small molecular weight NADH dehydroge-
nase, and is apparently involved in mercurial inhibition of its reductase
activities. According to Kumar et al. ( 6 3 ) , NADH treatment increases
the mercurial sensitivity of the enzyme with respect to ferricyanide and
cytochrome c, but not dichloroindophenol, reduction. Hatefi et al. (68)
have shown that (a) incubation of the enzymes with 1-2 pM pCMS results
in activation by as much as 20-300/0, while higher concentrations inhibit,
and (b) contrary to thc results of others (37) N-ethylmaleimide does
not cause inhibition even after partial inactivation by heat a t pH 4.8
or in the presence of 3 M urea. In contrast to the low molecular weight
enzyme, the ferricyanide reductase activity of the high molecular weight
dehydrogenase or complex I is not inhibited by mercurials, suggesting
that type I -SH groups are not accessible to mercurials in these prepara-
tions. The type I1 -SH group appears to be peculiar to the high molecu-
lar weight dehydrogenase of Singer et al. At Oo it reacts rapidly and
reversibly with -SH reagents without inhibiting ferricyanide reductase
activity, but incubation a t 15O-3Oo results in gradual and irreversible
inactivation. The temperature effect suggests structural destabilization
and recalls the temperature dependence of the resolution of complex I
by chaotropic agents. Davis and Hatefi (69) have shown that in the pres-
ence of moderate concentrations of NaC104 the resolution of complex I
does not occur at temperatures below 15O (Fig. 5 ) . Type I11 -SH groups
are found in particles and high molecular weight preparations. This type
of -SH group, which was discovered by Tyler et al. (48), reacts with
mercurials and results in inhibition of electron transport only after the
preparation is pretreated with NADH. The conditioning by NADH is
reversible inasmuch as addition of ferricyanide to the NADH-treated
enzyme reverts it to the mercurial insensitive state. This type of -SH
85. T. Cremona and E. B. Kearney, JBC 240,3645 (1965).
86. H. Mersmann, J. Luthy, and T. P. Singer, RBRC 25,43 (1966).
87. M. Gutman, H. Mersmnnn, J . Luthy, and T. P. Singer, Biochemistry 9, 2678
(1970).
204 YOUSSEF H A T E F I AND DIANA L. STIGGALL
group is considered to be located very near the substrate binding site,

but possibly not directly involved in electron transport activity, since
relative to the turnover number of the enzyme both the NADH condition-
ing and the ferricyanide reversal are slow processes (23).Type IV -SH
groups are also found in particles and high molecular weight prepara-
tions. According to Singer and co-workers (19, 23), they react readily
with low levels of mercurials, and mercaptide formation with this type
of thiol group is considered to affect the ferricyanide reductase reaction
by increasing both the K,,, and the V,,, for ferricyanide by severalfold.
Type V -SH groups are detectable only in complex I and parent parti-
cles because they affect ubiquinone reduction and piericidin binding.
Mercaptide formation with this type of -SH group requires relatively
high concentrations (30-80 p M ) of mercurials and results in inhibition
of electron transport from NADH to ubiquinone, but not to ferricyanide
and other acceptors reacting on the substrate side of ubiquinone (Table
VIII) . I n submitochondrial particles, mercaptide formation with type
V -SH groups also results in the loss of one of two specific binding sites
for rotenone and piericidin A, and a sigmoidal to hyperbolic change in
the piericidin inhibition titration curves (23, 88).
The ubiquinone reductase activity of complex I is inhibited by barbitu-
rates (e.g., Amytal and Seconal), Demerol, rotenone, or piericidin A.
These compounds appear to inhibit electron transfer from the iron-sulfur
centers of complex I to ubiquinone. Absorption and fluorescence spectro-
scopic studies on submitochondrial particles had suggested to Chance et
al. (89) the existence of two consecutive flavoproteins between NADH
and ubiquinone. These authors placed the site of rotenone and Amytal
inhibition between the two flavoproteins. Hatefi (76) showed that in com-
plex I there is only one type of flavoprotein, and that the additional
bleaching by substrate a t the wavelength pair 475 minus 510 nm used
by Chance et al. results from reduction of the iron-sulfur components
of complex I. Therefore, the site of Amytal and rotenone inhibition could
be between the flavoprotein and an iron-sulfur moiety of complex I. While
the conclusion regarding the absence of two consecutive flavoproteins in
the complex I region of the respiratory chain was correct and confirmed
(go), the interpretation regarding the site of inhibition of rotenone and
Amytal was not. It is now generally agreed that the flavin and all the
EPR-active iron-sulfur moieties of complex I are located on the sub-
strate side of the inhibition site of Amytal, rotenone, and piericidin A.
88. M. Gutman, T. P. Singer, and J. E. Casida, JBC 245, 1992 (1970).
89. B. Chance, L. Ernster, P. B. Garland, C.-P. Lee, P. A. Light, T. Ohnishi,
C. I. Ragan, and D. Wong, Proc. N u t . Acad. Sci. U.S. 57, 1498 (1967).
90. C. I. Ragan and P. B. Garland, BJ 10, 399 (1969).
Palmer et al. (91) have suggested that in addition to the above site,
rotenone and piericidin A also inhibit electron transport immediately on
the substrate side of cytochrome cl. This view has not been accepted by
others. Teeter et al. (9.2) have shown that secondary effects of rotenone
and piericidin can be observed a t other regions of the respiratory chain
when high concentrations of the inhibitors are used, as by necessity did
Palmer et al. in their EPR experiments.
The studies of Horgan, Singer, and their colleagues (19, 22, 23, 88,
93, 94) with radioactive piericidin A and rotenone have led these authors
to the following conclusions :
(a) There are specific and unspecific binding sites for both rotenone
and piericidin A, the latter being reversible by washing of the particles
with bovine serum albumin.
( b ) Rotenone (and several other rotenoids), piericidin A, and Amytal
bind noncovalently and inhibit a t the same specific binding site in phos-
phorylating and nonphosphorylating preparations.
(c) Piericidin binds more tightly than rotenone, and titration data in-
dicate that 2 moles of piericidin bind with comparable affinity per mole
of NADH dehydrogenase in submitochondrial particles.
(d) Titration curves relating the degree of NADH oxidase inhibition
to inhibitor concentration are sigmoidal, thus indicating that the two
binding sites are not equivalent in terms of their contribution to inhibi-
tion of electron transport.
(e) Unlike submitochondrial particles, the number of binding sites per
mole of NADH dehydrogenase in complex I and complex 1-111 is close
to unity.
Other aspects of rotenone and piericidin inhibition studied by Singer
and co-workers are related more to submitochondrial particles than to
complex I. These studies have been compiled in reviews (.22, 23) by these
investigators and will not be detailed here.
As stated above, the low molecular weight NADH dehydrogenase of
Pharo et al. (64) was considered incorrectly to be the NADH-ubiquinone
reductase of the respiratory chain. This was in part because the ubiqui-
none reductase activity of the preparation could be partially inhibited
by Amytal and by very low concentations of rotenone. It was demon-
strated by others that these effects were different from the inhibitions
91. G.Palmer, D.J. Horgan, H. Tisdale, T. P. Singer, and H. Beinert, JBC 243,
844 (1968).
92. M. E. Teeter, M. L. Baginsky, and Y. Hatefi, BBA 172, 331 (1969).
93. D. J. Horgan, T. P. Singer, and J. E. Casida, JBC 243, 834 (1968).
94. D.J. Horgan, H. Ohno, T. P. Singer, and J. E. Casida, JBC 243, 5967 (1968).
obtained with complex I or submitochondrial particles (19, 22, $2). The

results of Hatefi et al. (42) show the following differences:
(a) Substantial inhibition of NADH-ubiquinone-1 reductase activity
of the soluble, low molecular weight enzyme requires more than 100-fold
as much rotenone as is necessary for a comparable degree of inhibition
of complex I.
(b) Barbiturates inhibit the ubiquinone reductase activity of the
former enzyme only when higher ubiquinone isoprenologs are used as
electron acceptor. Unlike the reaction catalyzed by complex I, barbitu-
rates do not inhibit the ubiquinone-1 reductase activity .of the soluble
dehydrogenase.
(c) Piericidin A, which is the most potent inhibitor known for ubiqui-
none reduction by complex I and submitochondrial particles, is essentially
ineffective on ubiquinone reduction by the soluble enzyme.
Among several iron chelators used, only o-phenanthroline inhibited the
soluble dehydrogenase (42). It was shown by Hatefi et al. (42) that incu-
bation of the enzyme with o-phenanthroline results in the loss of labile
sulfide, while pretreatment with bathophenanthroline sulfonate, Tiron
(1,2-dihydroxybenzene 3,5-disulfonate) or ethylenediamine tetraacetate
protects the enzyme against the loss of labile sulfide and inhibition of
activity upon subsequent incubation with o-phenanthroline. The unique
destructive ability of o-phenanthroline has been demonstrated by these
investigators for several iron-sulfur proteins (95,96).
While in contrast t o complex I the ferricyanide reductase activity of
the low molecular weight dehydrogenase is not inhibited at high NADH
concentrations (>0.2 mM) , its quinone reductase activities are. Indeed,
there seems to be a correlation between NADH inhibition, the apparent
Km of the enzyme for NADH, and whether the acceptor is a single-elec-
tron (ferricyanide, cytochrome c) or a two-electron (ubiquinones, mena-
dione, and dichloroindophenol) recipient (40). With single-electron ac-
ceptors, the apparent K m for NADH is about 65 pM and the reaction
is relatively insensitive to the concentration of NADH. However, with
two electron acceptors, the apparent K , for NADH is twice as much
(133 UJM) and the reaction is sharply inhibited a t NADH concentrations
greater than 0.25 mM. Hatefi and Stempel (40) have suggested that these
phenomena might be a consequence of the dissimilar affinity for NADH
of the various oxidation-reduction states of the enzyme (i.e., oxidized,
half-reduced, fully reduced) transiently produced during electron transfer
to one-electron versus two-electron acceptors. Millimolar concentrations
95. Y. Hatefi and W. G. Hanstein, ABB 138,73 (1970).
96. R. M. Kaschnitz and Y. Hatefi, ABB 171,292 (1975).
of NAD partially inhibit NADH dehydrogenase ( 4 0 ) . A similar inhibi-

tion is caused by AMP, ADP, and ATP, but not by adenosine ( 4 2 ) .
Guanidinium hydrochloride and alkylguanidinium salts have been impli-
cated as inhibitors of electron transport and oxidative phosphorylation
a t site 1 (97, 98). Hatefi et al. (42) have shown that the ubiquinone
reductase activity of complex I is inhibited by guanidinium hydrochloride
(10-50 mM), but the ferricyanide reductase activity of complex I and
all the reductase activities of the low molecular weight dehydrogenase
are activated. Guanidinium ion was more potent than the alkylated
derivatives] and the activation effect analyzed for menadione reduction
indicated a decrease in K , for NADH and an increase in V,,,, both
of which were dependent on the concentration (10-100 m M range) of
guanidinium ion.
6. N A D P H Oxidation and N A D P H to N A D Tramhydrogenation b y
Complex I
The ability of submitochondrial particles to catalyze transhydrogena-
tion from NADPH to NAD has been known for many years. The reverse
reaction, i.e., from NADH to NADP, is slow but can be accelerated when
energy is supplied to the system (e.g., ATP). The energy requirement
of the reverse reaction and the different equilibria of the transhydroge-
nase reaction in the absence and presence of an energy supply are ~ U Z -
zling thermodynamic problems. The mitochondrial transhydrogenase
reaction has been under vigorous investigation (99-102), a recent review
is available (103), and the topic is covered in Chapter 2.
Until 1973, it was generally agreed that the mitochondrial respiratory
chain is incapable of oxidizing NADPH directly (103-105).NADPH oxi-
dation was considered to occur only through the transhydrogenase reac-
tion and with the obligatory intermediation of NAD (103-105). In 1973,
97. J. B. Chappell, JBC 238,410 (1963).
98. B. Chance and G. Hollunger, JBC 238,432 (1963).
99. J. Rydstrom, A. Teixeira da Cruz. and L. Ernster, Eur. J. Biochem. 17, 56
(1970).
100. A. Teixeira da Cruz, J. Rydstrom, and L. Ernster, Eur. J. Biochem. 23, 203
(1971).
(1971).
102. R.R.Fisher and N. 0. Kaplan, Biochemistry 12, 1182 (1973).
103. N.0.Kaplsn, Harvey Lect. 66, 105 (1972).
104. L. Ernster, C.-P. Lee, and U. B. Torndal, in “The Energy Level and Metabolic
Control in Mitochondria” (S. Papa et al., eds.), p. 439. Adriatrica Editrice, Bari,
1969.
105. F. A. Hommes, in “Energy-Linked Functions of Mitochondria” (B. Chance,
ed.), p. 39. Academic Press, New York, 1963.
(CI
FIQ.12. (A) First-derivative E P R spectra of complex I treated with NADH or

NADPH. Conditions: complex I, 45 mg/ml; temperature 14°K; microwave fre-
quency 9.225 GHz; power, 2 mW, modulation amplitude, 6.3 G ; gain, 50. g = 2
was at 3295 G. Where indicated 1.5 mM NADH or NADPH was added. Small letters
of the alphabet in this, (B), (C), and Fig. 13 denote the same signals as in Fig. 4.
(B) The EPR spectrum of NADH-treated complex I shown in (A) a t gain of 200
and 0.3 mW power. (C) The E P R spectrum of NADPH-treated complex I shown
in (A) at gain of 200 and 0.3 mW power. From Hatefi and Hanstein (80).
Hatefi (106-108) and Hatefi and Hanstein (80) demonstrated, however,

that NADPH is oxidized directly by the respiratory chain a t a site close
to, but apparently not identical with, the site of NADH oxidation. It was
found that NADPH oxidation by respiratory particles is very slow a t
neutral pH (about 50 nmoles/min/mg protein) , but quite appreciable a t
pH values between 5 and 6 ( 2 2 5 0 nmoles/min/mg protein). Indeed, the
rate difference between pH 9 and pH 6 was found to be about 35-40-fold,
106. Y.Hatefi, BBRC 50, 978 (1973).

107. Y. Hatefi, i n “Dynamics of Energy Transducing Membranes” (L. Ernster
R. W. Estabrook, and E. C. Slater, eds.), p. 125. Elsevier, Amsterdam, 1974.
108. Y.Hatefi, Fed. Proc., Fed. Amer. SOC.
Exp. Biol. 32,595 (1973).
I m n
0
t r s
-1 G=100 I- H- \/
FIQ. 13. Computer-derived difference of NADH-treated minus NADPH-treated
complex I shown in Fig. 12. From Hatefi and Hanstein (80).
an indication of the fact that others working a t neutral or more alkaline

pH values had missed (or dismissed as resulting from the presence of
traces of NAD) the direct oxidation of NADPH by the respiratory chain.
Both NADPH dehydrogenase (assayed with ferricyanide as acceptor)
and NADPH-to-NAD transhydrogenase activities of respiratory particles
were found to fractionate mainly into complex I. Electron paramagnetic
resonance studies on complex I a t neutral pH showed that NADPH re-
duced iron-sulfur center 2 and partially the overlapping iron-sulfur cen-
+
ters 3 4. Iron-sulfur center 1 was not detectably reduced by NADPH,
nor was the flavin of complex I as evidenced from the difference absorp-
tion spectra of NADPH-treated minus NADH-treated complex I. In
agreement with previous findings described above, all iron-sulfur centers
were reduced by NADH. These results are depicted in Figs. 12-14. Sub-
-0.0
400 500 600
Wavelength Inm)
FIG.14. Absorption spectrum of NADPH-treated minus NADH-treated complex

I. Conditions: complex I, 6 mg protein/ml of 0.66 M sucrose containing 50 mM
Tris-HC1 ( p H KO), 1 mM histidine, and 0.25% (v/v) Triton X-100. The sample
cuvette was treated with 200 pM NADPH, and the reference cuvette with 100 p M
NADH. Dashed line, untreated complex I in both cuvettes. From Hatefi and Han-
stein (80).
5 6 7 a 9
PH
FIQ.15. pH dependence of NADPH oxidase, NADPH to 3-acetylpyridine adenine
dinucleotide (AP-DPN) transhydrogenase, and NADH oxidase activities of submito-
chondrial particles (ETP), Conditions: oxidase activities were measured in the pres-
ence of 2 mM NADH or NADPH, 0.25 121 sucrose, 100 mM sodium phosphate
for pH values 6-9, and 100 mM sodium acetate for pH values 5.0 and 5.5. ETP
concentration was 2.16 mg/ml for the NADPH oxidase, and 0.216 mg/ml for the
NADH oxidase assays. The transhydrogenasc reaction was measured by the Aminco-
Chance spectrophotometer at 400 minus 450 nm. The extinction coefficient used for
reduced 3-acetylpyridine adenine dinucleotide a t 400 nm was 2300 liters mole-' cm-'.
Media were the same as in the oxidase assays. Dotted lines indicate uncertainty about
the pH 5 rates because of possible acidity damage to ETP. The ordinate refers
to nanomoles of NADPH or NADH oxidized min-' x mg-' of ETP protein at 30".
From Hatefi and Hanstein (80).
sequent studies of Hatefi and Bearden (109) indicated that as compared

to NADH reduced complex I, the low field signal due to overlapping cen-
+
ters 3 4 was not only smaller but also a t a position approximately 3 G
upfield (corresponding to Ag = 0.002) when it was generated with
NADPH as reductant. These results indicated, therefore, that the par-
+
tial center 3 4 reduction by NADPH might have resulted mainly or
entirely from center 3 (see Table IV). Thus, i t appears that a t neutral
pH NADPH can reduce those components of complex I (iron-sulfur
centers 2 and 3) whose reduction potentials appear to be close to zero,
but not those whose reduction potentials are between -300 and -200
mV (flavin and iron-sulfur centers 1 and 4, see Section II,A,7).
As stated above, submitochondrial particles and complex I exhibit both
NADPH dehydrogenase and NADPH-to-NAD transhydrogenase activi-
ties. These activities have similar pH dependencies (Fig. 15) and are
109. Y . Hatefi and A. J. Bearden, unpublished.
both specific with respect to abstraction of the 4-B hydrogen of

NADPH (10'7). The NADH dehydrogenase of the respiratory chain is
also 4-B specific, whereas transhydrogenation from NADH to NADP is
4-A specific in agreement with the 4-B specificity of the reverse reaction
with regard to NADPH. The similarities between NADPH dehydroge-
nase and NADPH-to-NAD transhydrogenase have suggested that both
reactions might be catalyzed by the same enzyme. They have also created
suspicion regarding the noninvolvement of NAD in NADPH dehydroge-
nation in spite of the differences detailed above in the reduction of respir-
atory components by NADH and NADPH and the demonstration of the
absence of detectable NAD in the experiments involving complex I
(submitochondrial particles contain about 0.2 nmole NAD per mg pro-
tein) (80). Two lines of evidence have furnished unambiguous evidence,
however, that NADPH oxidation by submitochondrial particles and com-
plex I can occur under conditions that the transhydrogenase reaction is
completely inhibited.
The differences in the reduction of respiratory components with NADH
or NADPH as substrate is reflected in the degree of bleaching afforded
by these substrates a t 475 minus 510 nm in complex I and rotenone-
or piericidin-treated submitochondrial particles. Thus, as seen in Fig. 16
(left-hand trace), addition of NADH doubles the bleaching a t 475 minus
510 nm obtained by addition of NADPH to piericidin-treated particles.
The middle trace shows a similar effect when NAD is added instead of
NADH. This results from the presence of transhydrogenase activity,
which yields NADH from NAD and excess NADPH. I n the right-hand
475.510 nm
i1 min+
#l
CI
0
>
FIG.16. Effect of palmitoyl-coenzyme A on reduction of chromophores a t 475
minus 510 nm in ETP via NADPH to NAD transhydrogenation. Conditions: ETP,
2.2 mg protein/ml; NADPH, 60 p M ; NADH, 60 p n l ; NAD, 140 pM; sodium suc-
cinate, 1.75 m M ; piericidin A , 5.3 pM ; antimycin A, 1 p M ; 2-thenoyltrifluoroacetone
(TTFA), 1 m M ; palmitoyl-CoA (P-CoA), 200 pM. From Hatefi and Hanstein (80).
trace the preparation has been treated with appropriate amounts of

palmitoyl coenzyme-A (110) to inhibit the transhydrogenase reaction.
The bleaching by NADPH is seen, the NAD effect resulting from the
transhydrogenase reaction is largely, abolished, but subsequent NADH
addition is still effective. These results clearly show that NADPH reduc-
tion of components as measured a t 475 minus 510 nm can occur under
conditions that transhydrogenation to NAD is inhibited. That the latter
reaction was substantially inhibited in these experiments is clear because
in the presence of a piericidin block even a slow production of NADH
would have resulted in bleaching just as NADPH did under conditions
of low electron flux (pH 7.5 and NADPH concentration equivalent to
0.1 K,,,). Similar results were reported for complex I (80).
I n addition, it has been shown by Djavadi-Ohaniance and Hatefi (111)
that trypsin treatment of submitochondrial particles can distinguish
among NADH oxidation, NADPH oxidation, and NADPH-to-NAD trans-
hydrogenation. The exceptional sensitivity of the latter reaction to tryp-
sin was demonstrated earlier by Ernster and his colleagues (119). Taking
advantage of this sensitivity, the former investigators have shown that
treatment of submitochondrial particles a t Oo with appropriate amounts
of trypsin can completely destroy the NADPH-to-NAD transhydro-
genase activity without affecting appreciably either the NADH or the
NADPH oxidase activity (Fig. 17). Incubation of the particles a t 30°
in the presence of trypsin then led to gradual loss of NADPH oxidase
activity without affecting NADH oxidase activity. These results clearly
demonstrate that the three reactions shown in Fig. 17 are independently
affected by trypsin: transhydrogenase activity is rapidly destroyed a t
Oo, NADPH oxidase is gradually destroyed a t 30°, while NADPH oxida-
tion is unaffected by trypsin under these conditions. An important ques-
tion that is raised by these experiments is the relationship between the
NADPH dehydrogenase and the NADPH-to-NAD transhydrogenase
activities of submitochondrial particles. On the one hand, they are clearly
distinguishable on the basis of their sensitivities to trypsin and palmitoyl
coenzyme-A. On the other hand, they exhibit common features with re-
gard to their stereospecificities for abstraction of the 4-B hydrogen of
NADPH, their extremely large response to pH change, and their copurifi-
cation into complex I. It is entirely possible that the two activities might
be catalyzed by the same enzyme because (a) many nicotinamide-
110. J. Rydstrorn, A. V. Panov, G. Paradies, and L. Ernster, BBRC 45, 1389 (1971).
111. L. Djavadi-Ohaniance and Y . Hatefi, JBC, in press.
112. K.Juntti, U. B. Torndal, and L. Ernster, in “Electron Transport and Energy
Conservation” (J. M. Tager et a l , eds.), p. 257. Adriatica Editrice, Bari, 1970.
m
5 15 25 40 55
(a) (b)
FIG. 17. Effect of trypsin on the NADH oxidase, NADPH oxidase and the
NADPH-to-NAD transhydrogenase activities of submitochondrial particles. The
particles suspended in 0.25 M sucrose and 100 mM sodium phosphate, pH 7.0, were
treated with 0.1 mg trypsin per mg particle protein and incubated a t (a) 0" or (b)
30". At the intervals shown samples were removed and assayed at pH 6.0 and 7.0
for the activities shown. Transhydrogenase activity was measured either directly
by reduction of 3-acetylpyridine adenine dinucleotide at 375 nm in the presence of
cyanide-treated particles or by the increase in the rate of NADPH oxidation by
submitochondrial particles after the addition of NAD. (A) NADH + O,, ( 0 )
NADPH + 02,and (0) NADPH + NAD. From Djavadi-Ohaniance and Hatefi
(111).
adenine dinucleotide dehydrogenases, including the mitochondrial NADH

dehydrogenase, exhibit transhydrogenase activity in the presence of a
suitable analog, and (b) the exceptional trypsin sensitivity of the trans-
hydrogenase reaction might be concerned mainly with the NAD ( H ) bind-
ing site. For example, it is possible that similar to Rhodospirillum rubrum
(113) the mitochondrial transhydrogenase system also involves a soluble
protein cofactor. This protein factor or its association with the mem-
branes might be susceptible to the action of trypsin. A more attractive
possibility is suggested by the recent work of Vallee and his colleagues
(114). They have found that in a number of enzyme-active sites arginyl
residues serve as the positively charged recognition sites for negatively
charged substrates, and have identified arginyl residues a t the NADH
binding site of a number of alcohol dehydrogenases from various sources.
A peptide bond involving the carboxyl group of arginine, if present a t
the substrate binding site of the mitochondrial transhydrogenase, could
be particularly susceptible to attack by trypsin.

114. L. G. Lange, 111, J. F. Riordan, and B. L. Vallee, Biochemistry 13, 4361
(1974) .
7. Energy Conservation and Coupling by Complex I

It has long been known that the first site of energy conservation in
the respiratory chain is located between NADH and ubiquinone-cyto-
chrome b. Schatz and Racker (44) demonstrated ATP synthesis by sub-
mitochondria1 particles a t the expense of NADH oxidation by externally
added ubiquinone-1, and more recently Ragan and Racker (36)reconsti-
tuted oxidative phosphorylation a t site 1 in a system composed of com-
plex I and appropriate membrane proteins, coupling factors, and phos-
pholipids. The important experiments of Ragan and Racker showed that
in principle the isolated and purified complexes are capable of energy
conservation and coupling. The studies of Hatefi, Galante, and You (106,
107, 115) have shown that comparable ATP yields (P/O = 2.62.8) are
obtained as a result of oxidation of NADH or NADPH by submitochon-
drial particles. Since the components of complex I reduced by both
NADH and NADPH are iron-sulfur centers 2 and 3 and ubiquinone,
the above experiments implicate these electron carriers. in site 1 energy
conservation. Gutman and his colleagues (116) have shown that in
piericidin-treated submitochondrial particles iron-sulfur center 2 remains
reduced after exhaustion of added NADH through the piericidin leak.
Iron-sulfur center 2 could be reoxidized by addition of ATP. This reaction
was sensitive to uncouplers, and appeared to result from energy-linked
reverse electron transfer to NAD. On the basis of these observations,
Gutman et al. have concluded that coupling site 1 is located on the oxygen
side of iron-sulfur center 1 and the substrate side of both iron-sulfur
center 2 and the site of roteaone-piericidin block. These conclusions are
in general agreement with the results of Hatefi et al. (80,106-108) regard-
ing the NADPH reducible iron-sulfur centers and P/O > 2 obtained
during NADPH oxidation.
It is also interesting to note that energy-linked transhydrogenation can
be induced by ATP or as a result of succinate oxidation in rotenone-
treated particles (117). Further, Van de Stadt et al. (118) and Skulachev
(119) have presented data regarding energy production in rotenone-
blocked particles as a result of transhydrogenation from NADPH to
NAD. Therefore, it appears that energy communication with the trans-
hydrogenase reaction also occurs a t or near site 1. This would indeed
115. Y. Hatefi, Y. Galante, and K. S. You, unpublished.

116. M. Gutman, T. P. Singer, and H. Beinert, Biochemistry 11, 556 (1972).
117. C.-P. Lee, G. F. Azzone, and L. Ernster, Nature (London) 201, 152 (1964).
118. R. J. Van de Stadt, F. J. R. M. Nieuwenhuis, and K. Van Dam, BBA 234,
173 (1971).
119. P. Skulachev, Curr. T o p . Bioenerg. 4, 127 (1971).
(SDH Fe/S)
+3omV
NADH t)(Center lo.lb)(Center3,4)-(Center

Rotenone
2) -(Center
1 5 ) +-+(R~eske’s Fe/S)- O2
Half- -305mV -245rnV -20rnV +4OrnV + 280rnV

reduction
potentiol
FIG. 18. Thermodynamic profile of iron-sulfur centers in pigeon heart mito-
chondria : SDH, succinate dehydrogenase ; Rieske’s Fe/S, iron-sulfur protein of com-
plex 111. From Ohnishi (121).
be plausible if as suggested by their similar enzymic features NADPH

oxidation and nicotinamide-adenine dinucleotide transhydrogenation
shared through a common enzyme the same linkage to the respiratory
chain.
Ohnishi et al. (120, 121) have suggested that coupling site 1 involves
not only iron-sulfur center 2 but also half of iron-sulfur center 1 (desig-
nated center l a ) . Their conclusion is based on an apparent change in
the reduction potential of these centers induced by ATP as estimated
from the measurement of E P R signals in the presence of redox mediators.
Similar criteria were used by Wilson and Dutton (122, 123) to identify
cytochromes b, and a3 as energy transducing components a t coupling
sites 2 and 3, respectively. However, these experiments have been criti-
cized by Caswell (124) and Lambowitz e t al. (125).They feel that com-
plications resulting from improper equilibration of the redox mediators
with the electron carriers under study and ATP-induced reverse electron
transfer to and from these components have been the underlying basis
of the measured redox potential changes brought about by A T P addition.
According t o Ohnishi (121) , the half-reduction potentials of the various
iron-sulfur centers of the respiratory chain a t pH 7.2 are as shown in
Fig. 18. The value for center 1 is essentially in agreement with the results
of Orme-Johnson et al. (46, 54) who found that reduced acetylpyridine
adenine dinucleotide (Eo’ = -248 mV) can only reduce this center by
50%, while its oxidized form can effectively oxidize iron-sulfur center
120. T. Ohnishi, D. F. Wilson, and B. Chance, BBRC 49, 1087 (1972).
121. T. Ohnishi, BBA 301, 105 (1973).
122. D. F. Wilson and P. L. Dutton, BBRC 39,59 (1970).
123. D. F. Wilson and P. L. Dutton, A B B 136, 583 (1970).
124. A. H. Caswell, A B B 144,445 (1971).
125. A. M. Lambowitz, W. D. Bonner, Jr., and M. K. F. Wikstrom, Proc. N u t .
Acud. Sci. U . S. 71, 1183 (1974).
NADPH-
NADP -.I FeS4
FIQ. 19. Proposed electron transfer pathways for oxidation and reduction
of NADH/NAD and NADPH/NADP, and energy coupling site 1 in com-
plex I. Where applicable broken arrows indicate energy-linked electron or hydride
ion transfer. FeS, iron-sulfur center.
1 in dehydrogenase preparations. These investigators feel, however, that

the reduction potential of center 3 is greater than or equal to that of
center 2, since upon titration of complex I with graded amounts of NADH
or dithionite reduced centers 2 and 3 appear long before centers 4 and
1 are reduced. Thus, it seems that complex I contains two iron-sulfur
centers (1 and 4) with reduction potentials close to that of NADH
(Eo’= -315 mV), and two centers (2 and 3) with potentials close to
that of ubiquinone (Eo’N +65 mV) . Accordingly, the largest single-step
energy drop in the NADH pathway is between iron-sulfur centers 4 and
2 + 3 (AE = -225 mV; AGO‘ for 2e = -10,400 cal), and in the
NADPH pathway is, so far as known, between NADPH and iron-sulfur
+
centers 2 3 (AE = -295 mV; AGO’ for 2e = -13,600 cal). I n theory,
therefore, the energy liberated a t each of these two steps appears to be
compatible with the amount required for ATP synthesis a t site 1 with
either NADH or NADPH as substrate. The above considerations are
summarized in Fig. 19.
B. NADH DEHYDROGENASES
OF YEAST
The NADH dehydrogenase of yeast is of considerable interest because

in Saccharomyces cerevisiae and Saccharomgces carlsbergensis coupling
site 1 is absent, whereas in Candida utilis its existence depends on the
growth phase of the cells and can be altered by adaptations to culture
conditions and by catabolite repression.
In 1961, Vitols and Linnane (126) reported that mitochondria isolated
from S. cerevisiae showed identical P/O values for oxidation of succinate
and NAD-linked substrates. These results were the first indication of
the absence of coupling site 1 in S. cerevisiae. Mackler et al. (127) and
126. E. Vitols and A. W. Linnane, J. Biophys. Biochem. Cytol. 9, 701 (1961).
127. B. Mackler, P. J. Collipp, H. M. Duncan, N. A. Rao, and F. M. Huennekens,
JBC 237, 2968 (1962).
Mahler and co-workers (128) isolated respiratory particles from bakers’

yeast, and showed that NADH oxidation by these particles was insensi-
tive to inhibition by Amytal, Seconal, and rotenone. The former authors
also demonstrated that, unlike mammalian respiratory particles, the
flavin of S. cerevisiae particles was exclusively FAD of which approxi-
mately 50% was acid-extractable. Extraction of the remainder required
prior digestion of the particles with trypsin. Duncan and Mackler (129)
isolated a low molecular weight NADH dehydrogenase from these parti-
cles by the acid-ethanol procedure (see above). The preparation con-
tained per mg protein 10.6 nmoles of FAD, 6.3 ng atoms of iron, and
no labile sulfide. The molecular weight was determined by equilibrium
sedimentation to be approximately 55,000. Thus, it appeared that during
isolation the enzyme had lost about 40% of its flavin, assuming one mole
of FAD per mole of enzyme. The possibility of iron and labile sulfide
loss is also suggested by the use of acid pH, prolonged dialysis, and
DEAE-cellulose chromatography for isolation and purification of the
enzyme. The dehydrogenase could react with ferricyanide, dichloroindo-
phenol, and cytochrome c as electron acceptors, and in all instances added
FAD increased the activity and p-mercuriphenyl sulfonate inhibited it.
Thus, in terms of size, acceptor specificity, and mercurial sensitivity, the
NADH dehydrogenase from S. cerevisiae respiratory particles appears
to be comparable to a similar preparation from mammalian mitochondria.
The major difference is that the flavin of the latter is FMN. According
to Kim and Beattie (130),the appearance of NADH dehydrogenase ac-
tivity in mitochondria of glucose derepressed S. cerevisiae is blocked by
cycloheximide, but not by chloramphenicol, suggesting that NADH dehy-
drogenase does not contain products of mitochondria1 protein synthesis.
The NADH dehydrogenase system of C . utilis is very similar to that
of mammalian mitochondria. Both systems are inhibited by rotenone and
piericidin A, conserve energy a t coupling site 1, contain multiple forms
of EPR-active iron-sulfur center, have F M N as their flavin prosthetic
group, react best with ferricyanide as electron acceptor, and are inhibited
a t high substrate concentrations (131-133).Biggs et al. (132) were unsuc-
cessful in dissociating a high molecular weight type enzyme from C. utilis
128. H. R. Mahler, B. Mackler, S. Grandchamp, and P. P. Slonimski, Biochemistry
3, 668 (1964).
129. H. M. Duncan and B. Mackler, Biochemistry 5, 45 (1966).
130. I. C.Kim and D. S. Beattie, Eur. J . Biochem. 36,509 (1973).
131. P.A. Light, C. I. Ragan, R. A. Clegg, and P. B. Garland, FEBS (Fed. Eur.
Biochem. Soc.) Lett. 1, 4 (1968).
132. D. R. Biggs, H. Nakamura, E. B. Kearney, E. Rocca, and T. P. Singer, ABB
137, 12 (1970).
133. S. 0. C. Tottmar and C. I. Ragan, BJ 124,853 (1971).
0.06 r
h
Wavelength (nm)
350 400 450 500 550 600 650 700

Wnvalongth (nm)
FIG.20. Absorption spectra of the purified NADH dehydrogenase of C. utilis at
0.8 mg/ml. Trace (a), oxidized enzyme; trace (b) after addition of 0.1 mM NADH;
inset, (b) minus (a). From Tottmar and Ragan (138).
particles by digestion with phospholipase A. However, Tottmar and

Ragan (133) have isolated such a preparation with the use of deoxy-
cholate and Triton X-100. The preparation contains per mg protein
0.5-0.6 nmole of flavin ( F M N ) , 15-17 ng-atoms of iron, and 15-17 nmoles
of labile sulfide. It reacts efficiently with ferricyanide as electron accep-
tor, but poorly with menadione, cytochrome c, dichloroindophenol, and
ubiquinone-1. The K , and K i of the enzyme with respect to NADH are,
respectively, 83 pM and 0.2 m M ; the K , for ferricyanide is 1.0 mM.
The preparation exhibits a g = 1.94 E P R signal, which is characteristic
of complex I and the high molecular weight dehydrogenases from heart
mitochondria. Its absorption spectrum (Fig. 20) is qualitatively compar-
able to that of the mammalian high molecular weight dehydrogenase,
and similar to complex I it can oxidize NADPH a t a slow rate (133)
(see Table V I ) .
The absence of rotenone-piericidin sensitivity and coupling site 1 in
S. cerevkiae and S. carlsbergensis, and their presence in C. utilis have
suggested a possible connection between the two phenomena. Light et
al. (131) demonstrated that in C. utilis iron-limited growth conditions
result in a decrease of mitochondria1 cytochromes and nonheme iron, loss
of site 1 phosphorylation, loss of sensitivity of NADH oxidation to in-

hibition by rotenone and piericidin A, and loss of piericidin A binding
capacity. These changes were not reflected, however, in the respiratory
activity of the mitochondria. Site 1 phosphorylation and rotenone-pieri-
cidin sensitivity were recovered when the iron-deficient cells were incu-
bated with FeS04 in the absence of an added carbon source. Others have
reported that under various growth conditions, energy conservation a t
site 1 reappears upon aeration of deficient cells concomitant with (134)
or without (135) the appearance of piericidin sensitivity. These develop-
ments have been reviewed by Ohnishi (f.21).
More recently, a careful study of the problem has been done by Gross-
man et al. (136) in which they have followed NADH dehydrogenase ac-
tivity, appearance and loss of piericidin sensitivity, and the nature of
various E P R signals and cytochromes during the exponential and sta-
tionary phases of C . utilis growth as well as after catabolite repression.
They found that the respiratory particles of cells harvested during the
exponential growth phase have very low piericidin sensitivity and NADH-
ferriayanide reductase activity, but high NADH-juglone (5-hydroxy-1,4-
naphthoquinone) reductase activity (Fig. 21). Successive washing of the
particles with low osmolarity buffer resulted in the loss of NADH dehy-
drogenase activit,y. The EPR spectra of antimycin-treated particles re-
duced with NADH or dithionite showed a n absence of signals resulting
+
from iron-sulfur centers 1 and 2, but centers 3 4 were present. Signals
were also present a t gll = 2.01 and gL = 1.92, which appeared t o result
from an iron-sulfur center. The temperature sensitivity of this new species
was similar t o that of center 1.
Particles of cells in the stationary phase showed an increase in P/O
ratio, suggesting the appearance of site 1 coupling, presence of piericidin
sensitivity, rise in ferricyanide reductase activity, loss of juglone reduc-
tase activity (Fig. 21), appearance of EPR signals resulting from centers
1 and 2, and loss of the new EPR signal seen in exponential phase parti-
cles. Further, the dehydrogenase of the stationary phase was stable to
washing of the particles. Catabolite repression of late stationary phase
cells by addition of ethanol resulted in the loss of the above characteris-
tics and reacquisition of exponential phase properties with regard to
enzymic activity, piericidin sensitivity, and the EPR signals asso-
ciated with NADH dehydrogenase. These changes were prevented by
cy cloheximide.
134. T. Ohnishi, P. Panebianco, and B. Chance, BBRC 49,99 (1972).
135. R. A. Clegg and P. B. Garland, BJ 124, 135 (1971).
136. S. Grossman, J. G. Cobley, T. P. Singer, and H. Beinert, JBC 249, 3819 (1974).
10 20 30 40 50
Hours
FIG.21. Characteristics of NADH oxidation by submitochondrial particles from
C. utilis during transformation from exponential to stationary phase. Candida utilis
was grown in 1.5% (v/v) ethanol in a fermentor at 30". Cells were harvested a t
the times shown for isolation of mitochondria and preparation of submitochondrial
particles. NADH oxidase activity is expressed as microatoms of oxygen per min
per mg protein at 30". NADH dehydrogenase activity is expressed as micromoles
of NADH oxidized per min per mg particle protein a t 25" at V,, with respect
to Fe(CN).'-; sensitivity to piericidin A (0.5 nmole/mg protein) is expressed as
percent inhibition of NADH oxidase; and turbidity is given as absorbance at 650
nm in 1 cm light path. The pH was maintained during growth by automatic addition
of 6 N KOH (pH-stat) at 5.0 until 25 hr, after which no further acid development
occurred but the pH rose to between 5.0 and 6.2. From Grossman et al. (136).
The authors feel that during transition from the exponential to the
stationary phase, a different type of NADH dehydrogenase is synthesized
(136). However, a very interesting possibility suggests itself when one
compares the characteristics of NADH dehydrogenase in the stationary
phase and exponential phase (or catabolite repressed) particles, respec-
tively, with the properties of complex I and chaotrope-destabilized com-
plex I (see above). Stationary phase particles and complex I are capable
of energy conservation a t site 1, are piericidin-sensitive, have high fer-
ricyanide reductase and low naphthoquinone reductase activities (juglone
was used with stationary phase particles and menadione with. complex
I ) , and exhibit EPR signals resulting from centers 1 (responsible for
g = 1.94 signal) and 2. In both cases, the membrane-bound dehydroge-
nase is stable. In contrast, destabilized complex I and particles from
exponential phase or catabolite-repressed C . utilis cells exhibit low ferri-

cyanide reductase and high naphthoquinone reductase activities,' loss of
piericidin sensitivity and g = 1.94 E P R signal, and appearance of new
EPR signals (46, 136). In the fractions isolated from destabilized com-
plex I, the modified EPR signal is seen in the soluble, low molecular
weight dehydrogenase ( 4 6 ) , which presumably originates from the tem-
perature-insensitive center 1 signal of the unperturbed complex. Further,
the dehydrogenase activities of both destabilized complex I and exponen-
tial phase or repressed particles are unstable. These analogies suggest,
therefore, that in C . utilis cells at exponential growth phase or subjected
to catabolite repression a t late stationary phase there exist species of
NADH dehydrogenase with features akin to the mammalian low molecu-
lar weight enzyme derived from destabilized particles. Assuming that the
low molecular weight dehydrogenase of mammalian mitochondria is a
bona fide component rather than a degradation product, then its different
properties when membrane bound might be a consequence of integration
into the membrane and interaction with other respiratory components.
By analogy, it is conceivable that during the exponential growth phase
a species comparable to the low molecular weight dehydrogenase is pres-
ent and in the process of being assembled into the respiratory chain, while
after catabolite repression the membrane becomes degraded and this
species of dehydrogenase exhibits once again the properties of the un-
bound enzyme.
C. NADH DEHYDROGENASE
OF Azotobacter winelandii
Low molecular weight NADH dehydrogenases have been isolated by

DerVartanian (137) from A. winelandii grown under normal and iron-
limited conditions. Both preparations are reported to have a molecular
weight of 56,500, and to contain FRIN. The enzyme from cells grown
under iron-limited conditions contained 1 g-atom of molybdenum, 2
g-atoms of iron, and 2 moles of labile sulfide per mole of FMN, while
in the enzyme from cells grown under normal conditions the iron and
labile sulfide content was twice as much. The two preparations exhibited
different EPR signals but comparable NADH-menadione and NADH-
ferricyanide reductase activities. It is stated that, unlike the mammalian
low molecular weight enzyme, the purification of the Azotobacter low
molecular weight NADH dehydrogenase is not accompanied by changes
in catalytic properties.
137. D. V. DerVartanian, Z. Naturforsch. 27, 1082 (1972).

111. Succinate Dehydrogenarer
A. MAMMALIAN DEHYDROGENASE
SUCCINATE (EC 1.3.99.1)
Succinate dehydrogenase is the only enzyme of the citric acid cycle
which is bound to the inner membrane of mitochondria. It is also one
of three flavoproteins known in which flavin is covalently linked to the
protein. The other two are monoamine oxidase of the outer membrane
of liver mitochondria (138) and Chromatiurn cytochrome c-552 (139).
Succinate dehydrogenase was solubilized from beef heart mitochondria
in 1954 (140) and purified in 1970 (141-143). In the intervening years
modified or new procedures for isolation and purification of the enzyme
were reported by Bernath and Singer (144), Basford et al. (145),Wang
et al. (l46‘),Keilin and King (147, 148),Veeger et al. (149), and Cerletti
et al. (160). The preparations of various laboratories differed in their
content of covalently bound flavin (hence in degree of purity), nonheme
iron, and labile sulfide, and exhibited different enzymic activities, the
most important of which was the ability of the enzyme to transfer elec-
trons to the respiratory chain. Consequently, unresolvable controversies
developed and strong positions were taken regarding the molecular
weight, composition, activities, and regulatory properties of succinate
138. E. B. Kearney, J. I. Salach, W. H. Walker, R. Seng, W. Kenney, E. Zeszotek,

and T. P. Singer, Eur. J. Biochem. 24, 321 (1971).
139. W. C. Kenney, D. Edmondson, R. Seng, and T. P. Singer, BBRC 52, 434
( 1973).
140. T. P. Singer and E. B. Kearney, BBA 15, 151 (1954).
141. Y. Hatefi, K. A. Davis, W. G. Hanstein, and M. A. Ghalambor, A B B 137,
286 (1970).
142. W. G. Hanstein, K. A. Davis, and Y. Hatefi, in “Energy Transduction in
Respiration and Photosynthesis” (E. Quagliariello, S. Papa, and C. S. Rossi, eds.),
p. 495. Adriatica Editrice, Bari, 1971.
143. K. A. Davis and Y. Hatefi, Biochemistry 10, 2509 (1971).
144. P. Bernath and T. P. Singer, “Methods in Enzymology,” Vol. 5, p. 597, 1962.
145. R. E. Basford, H. D. Tisdale, and D. E. Green, BBA 24, 290 (1957).
146. T. Y. Wang, C. L. Tsou, and Y. L. Wang, Sci. Sinicn 5, 73 (1956).
147. D. Keilin and T. E. King, Nature (London) 181, 1520 (1958).
148. T. E. King, JBC 238, 4037 (1963).
149. C. Veeger, D. V. DerVartanian, and W. P. Zeylemaker, “Methods in Enzymol-
ogy,” Vol. 13, p. 81, 1969.
150. P. Cerletti, G. Zanetti, G. Testolin, C. Rossi, F. Rossi, and G. Osenga, in
“Flavins and Flavoproteins,” 3rd Int. Symp. (H. Kamin, ed.), p. 629. Univ. Park
dehydrogenase. These issues have been discussed in a number of reviews

by Singer and his colleagues from their laboratory’s standpoint. Their
most recent are references 23 and 25.
1. Molecular Properties
Singer and co-workers extracted succinate dehydrogenase from mito-
chondrial acetone powder a t alkaline pH, and applied purification pro-
cedures involving adsorption on calcium phosphate gel and ammonium
sulfate fractionation (140, 144). Summarizing their results, Singer ( 1 5 )
concluded in 1966 that succinate dehydrogenase had the following molec-
ular properties: “The minimum molecular weight of homogeneous prepa-
rations from nonheme iron content is 49,000 and from flavin content
200,000. Physical measurements support a molecular weight of approxi-
mately 200,000. This value is also in accord with gel exclusion studies
on Sephadex G-200. Thus the enzyme contains 1 mole of flavin and 4
g-atoms of nonheme iron per mole. . . . The sedimentation velocity of
the beef heart enzyme at 10-15 mg protein/ml is 6.5 S . . . .” This prepa-
ration could oxidize succinate in the presence of ferricyanide or phenazine
methosulfate (PMS) as electron acceptor but was unable to transfer elec-
to be unable to interact with the respiratory chain.
Wang et al. (146) reported in 1956 the isolation of succinate dehydro-
genase from heart muscle preparations treated with succinate and cya-
nide. The enzyme was extracted with 20% aqueous ethanol a t p H 9.0.
The final product, after adsorption on calcium phosphate gel and ammo-
nium sulfate fractionation, was stated to be electrophoretically homoge-
neous and to contain 1 mole of flavin and 4 g-atoms of iron per 140,000-
160,000 g of protein. While apparently purer than the preparations of
Singer’s group, the cyanide-treated enzyme was also shown subsequently
to be unable to interact with the respiratory chain.
In 1958, Keilin and King (147) reported that succinate dehydrogenase
isolated essentially by the method of Wang et al. (146), but without the
use of cyanide, had an important property. Unlike other preparations,
the Keilin-King enzyme was capable of electron transfer to the respira-
tory chain (148). It was subsequently shown by King (148) that the
presence of succinate during extraction of the enzyme was essential for
its ability to interact with the respiratory chain. Preparations of the en-
zyme contained 2.4-3.6 nmoles of flavin per mg protein and high levels
of nonheme iron and labile sulfide. An average of several preparations
showed a ratio of iron to sulfide to flavin of 8.5:8.1: 1 (151). By compari-
151. T.E. King, BBRC 16,511 (1964)

son to the preparation of Singer and co-workers and Wang et al., the
Keilin-King enzyme contained twice as much iron per mole of flavin but
less flavin per unit weight of protein. These data suggested the presence
in the latter preparation of an additional iron-sulfur protein linking succi-
nate dehydrogenase to the respiratory chain. One difficulty with regard
to this possibility was that omission of succinate during enzyme isolation
resulted in a preparation with similar composition, absorption spectrum,
and dye reductase properties, but with complete lack of the ability to
interact with the respiratory chain. Further, Veeger et al. (149) showed
that modifications of the Keilin-King procedure yielded a preparation of
succinate dehydrogenase which retained the ability to interact with the
respiratory chain, but contained 1 mole of flavin, 8 g-atoms of iron, and
4-8 moles of labile sulfide per 200,00CL250,000 g of protein. Thus, it ap-
peared that the ability to reconstitute with the respiratory chain is an
important property of succinate dehydrogenase, which was lost in the
earlier low-iron preparations.
I n 1959, Ziegler and Doeg (152-164) reported the isolation of a particu-
late preparation from beef heart mitochondria which was capable of elec-
tron transfer from succinate to ubiquinone. This preparation was subse-
quently recognized by Hatefi et al. (27, 29) to be one of the four electron
transfer complexes of the respiratory chain and is now generally referred
to as complex 11. Preparations of complex I1 contain approximately 5
nmoles of covalently bound flavin per mg protein, and 7-8 g-atoms of
iron and 7-8 moles of labile sulfide per mole of flavin. I n addition, it
was shown by Ziegler and Doeg that complex I1 contained cytochrome
b at a molar concentration comparable to flavin. These data cast strong
doubt on the molecular weight of 200,000 for succinate dehydrogenase
since, on the basis of its flavin content, complex I1 had a similar molecu-
lar weight while containing in addition cytochrome b and possibly other
proteins. In agreement with this, Baginsky and Hatefi (155,156) showed
in 1969 that a preparation of succinate dehydrogenase with a flavin con-
tent of 6-7 nmoles/mg protein could be isolated from complex 11. I n spite
of these indications, it was generally assumed, however, that succinate
dehydrogenase had been purified and its molecular weight was 200,000.
The enzyme was finally purified in 1970 by selective resolution of com-
plex I1 with chaotropic agents (141-143). It was shown to have a flavin
152. D. M. Ziegler and K. A. Doeg, BBRC 1, 344 (1959).

153. D. M. Ziegler and K. A. Doeg, ABB 97,41 (1962).
154. D. M. Ziegler, in “Biological Structure and Function” (T. W. Goodwin and
0. Lindberg, eds.), Vol. 2, p. 253. Academic Press, New York, 1961.
155. M. L. Baginsky and Y. Hatefi, BBRC 32,945 (1968).
156. M. L. Baginsky and Y. Hatefi, JBC 244,5313 (1969).
content of 10.3 +- 4% nmoles per mg protein, a molecular weight of about

100,000, two unlike subunits, an iron:labile su1fide:flavin ratio of
7-8:7-8:1, very high dye reductase activity, and the ability to interact
with the respiratory chain. Certain features of the chaotrope-induced
resolution of complex I1 will be described because (a) the procedure is
novel and interesting and ( b ) insofar as the authors have been able to
ascertain this is the only published procedure which yields pure succinate
dehydrogenase (156~). The molecular and enzymic properties of the
above preparations are summarized in Table IX.
a. Resolution of Complex I I with Chotropic Ions. The purification
of succinate dehydrogenase is a simple process, which involves selective
extraction of the enzyme from complex I1 in the presence of a chaotropic
agent followed by precipitation with ammonium sulfate (72, 143). Before
the addition of ammonium sulfate to the resolved complex, it is necessary
to separate the soluble enzyme from the remainder of complex 11, which
is particulate, by centrifugation. Otherwise, the addition of ammonium
sulfate will reverse the resolution process, as will be seen below. Figure
22 shows the effect of several chaotropic agents on the resolution of com-
plex 11. The ordinate is percent succinate-ubiquinone (solid lines) or SUC-
cinate-PMS (dotted line) reductase activity, and the abscissa is time.
156a. Subsequent to the announcement of the above findings by Hatefi’s group

(141, I @ ) , two laboratories (167, 165) have claimed the purification of succinate
dehydrogenase by modifications of earlier procedures. Righetti and Cerletti (167)
have stated that using an earlier method (150) they have obtained pure succinate
dehydrogenase “containing 7-8 nmoles of peptide bound flavin.” Purification details
for this preparation, which according to its stated flavin content can be at best
80% pure, have, not been given. The earlier procedure (150), according to which
the above enzyme was made, showed in the best fraction a flavin content of 5.5-5.7
nmoles/mg protein. The other preparation claimed to be pure (23, 26) is that of
Coles et 01. (15,s).This preparation was made by the original procedure of Singer
and co-workers (144) with subsequent purification by chromatography on Sephadex
G-200 columns (details not given). However, Coles et al. (165) stated that “Although
the peak fractions from such columns appeared homogeneous in sucrose gradients,
their histidyl flavin content was no higher (4 to 5 nmoles/mg) than previously re-
ported ( 5 nmole/mg).” This agreed with the earlier results of Singer’s (16) quoted
above that their molecular weight of 200,000 was “in accord with gel exclusion studies
on Sephadex G-200.” Coles et al. further stated that “correction for impurities” ob-
served on polyacrylamide gels stained with Coomassie Blue “raised the histidyl flavin
content to 8 to 9 nmoles/mg. . . .” Such calculated values referred to elsewhere
should not be misunderstood, however, to represent the actual flavin content of the
preparation, which was not more than 5 nmoles/mg protein, thus indicating a t best
50% purity.
157. P. Righetti and P. Cerletti, FEBS (Fed. Eur. Biochem. Soc.) LetL.13, 181
(1971).
158. C. J. Coles, H . D. Tisdale, W. C. Kenney, and T. P. Singer, Physiol. Chem.
Phys. 4, 301 (1972).
TABLE IX
MOLECULAR PROPERTIES
AND ENZYMIC PREPARATIONS
OF REPRESENTATIVE OF SIJCCINATE
DEHYDROOENASE
Flavin Flavin :iron : Turnover

Molecular (nmole/mg labile sulfide number at KZa Reconstitution
Preparation weight protein) ratio v,PMS (mM) activity
Bernath-Singera 200,000 4.2-5.0 1:4:? -6,000 1.3 Absent

wang et al!
King"
140,OOO-160,000
329,000
6-7
3.04
1:4:?
1:8.5:8.1
2,00O-3,500
-6,000
0.58
0.3
Absent
Present
<
0
Veeger et a1.d 200,000-250,000 4-5 1:8:4-8 -3,900 - Present sm
Complex 118 200, OOO 5.0 1:7-8:7-8 10,000-11,000 0.3 Present
Davis-Hatefie 97,000 10.3 1:7-8 :7-8 10,000-11,000 0.3 Present X
From Singer (16)and Bernath and Singer (I&). The latest preparation of Coles el al. (168)haa similar properties.
2H
a
Turnover number calculated from data in Wang et al. (146). z

~Flavinand flavin:iron:labile sulfide are average values from (161).Turnover number and molecular weight calculated from &U
highest activity in King (171)and average flavin content, respectively.
d Turnover number calculated from V , activity at 25" in DerVertanian et al. ($18). E9
From Hate6 et d. (%), and references therein. z
P
r
m
2
P
E
FIG.22. Resolution of complex I1 with respect to succinate dehydrogenase by

various chaotropes. Complex I1 was suspended in 50 mM Tris-HC1, p H 8.0. After
addition of 0.6 M chaotrope the concentration of complex I1 was 8 mg/ml. After
addition of the salts, samples were taken a t the intervals indicated and assayed
for succinate-Q, and succinate-PMS reductase activities. Solid lines, succinate-
ubiquinone-2(&) reductase activity ; dotted line, succinate-PMS reductase activity.
Resolution temperature, 0" ; assay temperature, 38". The complex I1 preparations
used in the experiments of this and subsequent Figs. 24 and 25 had specific activities
between 40 and 45 pmoles Q2 reduced by succinate per min per mg protein. From
Davis and Hatefi (169).
It is seen t h a t before addition of chaotropes, both activities were stable.

When 0.6 M chaotrope was added ubiquinone reductase activity dimin-
ished, indicating resolution of the complex. However, PMS reductase ac-
tivity remained unchanged because complex 11-bound and soluble suc-
cinate dehydrogenases have the same PMS reductase activities. Both the
extent and the rate of resolution are functions of the concentration and
potency of the chaotrope used. Further addition of 0.9 M NaClO, re-
sulted in further resolution of complex I1 as evidenced by further loss of
ubiquinone reductase activity. Once again PMS reductase activity was
essentially unaltered. The small change is probably because the enzyme,
unprotected in these experiments with succinate and dithiothreitol, was
allowed to stand in the presence of 1.5 M NaC10, for many minutes.
When the above protective agents are added, the small activity loss is
prevented. I t has also been shown that the amount of succinate dehydro-
genase solubilizcd is a linear function of the concentration of complex I1
(the range tested was 2.5-10 mg complex I1 protein/ml).
The selectivity of the resolution of complex I1 with respect to succinate
dehydrogenase is depicted in Fig. 23 on SDS-polyacrylamide gels stained
FIQ.23. Profile of the purification procedure of succinate dehydrogenase depicted

on SDS-polyacrylamide gels stained with Coomassie Blue. 1, complex 11; 2, first
extraction of succinate dehydrogenase with 0.4 M NaClO,; 3, second extraction of
succinate dehydrogenase with 0.75 M NaClO,; 4, particulate fraction of complex
I1 remaining after twice removal of succinate dehydrogenase. From Davis and Hatefi
(143).
with Coomassie Blue. Tube 1 is the starting complex 11. Tube 2 shows
the two subunits of succinate dehydrogenase plus traces of impurity
(about 6%) extracted with 0.4 M NaC104, and tube 3 shows the result
of a second extraction of complex I1 with 0.75 M NaC104. The latter
is essentially pure succinate dehydrogenase. Tube 4 is the remainder of
complex I1 after the two successive extractions with 0.4 and 0.75 M
NaClO,. It is seen in tube 4 that the stain intensities of the various poly-
peptide bands of complex I1 are essentially unchanged, except for
the two bands corresponding to the extracted subunits of succinate
dehydrogenase.
As indicated by the results of Fig. 22, the resolution of complex I1
with respect to succinate dehydrogenase is an equilibrium process. At
I NaCIOe
-!/' ' ' ' ' 1
4 ' o '4 ' ' 8' ' 0 4 8

(a) Min (b)
FIG.24. Effect of temperature on the resolution of complex 11. Complex I1 was
suspended in a solution containing 30 mM Tricine, 100 mM boric acid, 20 mM
succinate and 5 mM dithiothreitol. pH of the buffer was adjusted to 8.1 with NaOH.
Complex I1 was resolved by addition of 1.0 M NaClO, at the temperatures indicated.
After addition of NaC10, the concentration of complex I1 was 8 mg/ml. (a) Resolu-
tion at O", lo", and 30". (b) Increased resolution and reconstitution as the tem-
perature was changed from (0) 0" to ( 0 ) 30" and from 30" to O", respectively.
Assays were performed a t 38". From Davis and Hatefi (169).
a given concentration of chaotrope, the extent of resolution increases with

temperature (Fig. 24a) and is reversible when the temperature is de-
creased (Fig. 24b). Enthalpy and unitary entropy changes calculated
from the data of Fig. 24 are, respectively, +4 kcal/mole and -15 eu.
Chaotrope-induced resolution, as discussed elsewhere (70-72) , results
from the destructuring effect of chaotropes on water and consequent
weakening of the hydrophobic attractions of the system under study.
Thus, it might be expected that increasing the structure of the aqueous
medium by water structure-forming ions might shift the equilibrium in
the direction of unresolved (or reconstituted) complex I1 (159).This is
shown in Figs. 25a and 25b. I n Fig. 25a, complex I1 is first resolved t o the
extent of about 50% by addition of 0.7M NaC104. Then various concen-
trations of ammonium sulfate are added. Since sulfate is a water struc-
ture-forming ion, the resolution is reversed and complete reconstitution
is achieved a t about 0.46 M sulfate. Figure 25b (upper panel) shows the
effect of other structure-forming (or antichaotropic) ions, and Fig. 25b
159. K.A. Davis and Y. Hatefi, ABB 149,505 (1972).

0.7M1 NaC104 [(NHdaSOI]
-: ; ;7f[: 'ol
.*
.r
x
80-
~1057~
8 -
0
t 60- ---a---DilUtion
,/- -
@8 -
* 40- t
(NH4)tSO4
Fm. 25(a). Resolution of complex I1 by NaC104, and reconstitution by increasing

concentrations of ammonium sulfate as indicated. Complex I1 was suspended in
a solution containing 50 mM Tris-HC1, pH 8.0, 20 mlll succinate, and 5 mM dithio-
threitol. After the addition of NaC104, the concentration of complex I1 was 8 mg/ml.
Other conditions were the same as in Fig. 22. From Davis and Hatefi (169).
(lower panel) shows that removal of C10,- by precipitation as KC10,

also results in reconstitution. That the degree of reconstitution is not a
function of ionic strength is shown in Fig. 25b. I n agreement with the
above, Davis and Hatefi (159) have also shown that chaotrope-induced
resolution of complex 11in D,O, which is believed to be a more structured
solvent than H,O, is considerably inhibited as compared to the same
reaction carried out in H,O. The solvent isotope effect of D,O on the
stability of membranous structures has been studied by Hanstein et al.
(73).
b. Subunits. It was shown in 1970 that succinate dehydrogenase is com-
posed of two unlike subunits (141-143). These subunits could be sepa-
rated by SDS-acrylamide gel electrophoresis or by resolution of succinate
dehydrogenase under special conditions. The molecular weights of the
subunits estimated from a number of separate experiments were
70,000 f 7% and 27,000 f 5% (143). Subsequent results of Righetti and
Cerletti (157) have indicated molecular weights of 68,500 and 30,000,
and those of Coles et at. (158) give molecular weights of 70,000 and
30,000, respectively, for the two subunits. The larger subunit carries the
covalently bound flavin (143).
Since treatment of succinate dehydrogenase with SDS for gel electro-
phoresis destroys the iron-sulfur chromophore and results in the loss of
labile sulfide, a technique was devised by Davis and Hatefi (141-143)
4. METAL-CONTAINING FLAVOPROTEIN DEHYDROGENASES 23 1
min
FIU.25(b). Resolution of complex I1 by NnC104 and reconstitution by (upper
panel) various nntichaotropic salts, and (lowrr panel) removal of C104- with K’.
Conditions were the same as in Fig. 25a. At the concentrations used here and in
Fig. 25~1,the antictiaotropic salts had no effect on the original activity of unresolved
complex 11. From Davis and Hatefi ( 1 5 0 ) .
to resolve the enzyme in such a manner that the distribution of iron and
labile sulfide in the two subunits could be determined (159a). The tech-
nique uses a combination of potent chaotropes and cold temperature,
taking advantage of the inverse relationship of temperature with the
strength of hydrophobic interactions. Thus, succinate dehydrogenase was
159a. I n spite of the known fact about the effect of SDS, Righetti and Cerletti
(167) made the following surprising statement in their note on the subunits of suc-
cinate dehydrogenase: “The polypeptides were eluted from the gel and SDS was
removed from the protein moiety. . . . Both subunits contain iron and labile
sulfide. . . .”
treated a t pH 7.0 with the chaotrope sodium trichloroacetate (71)and

subjected to repeated freeze-thawing a t 77OK (liquid N,) and room tem-
perature, respectively. The larger subunit formed an insoluble aggregate,
while the smaller subunit remained in solution. They were separated by
centrifugation. Figure 26 shows the effect of increasing concentrations
of the sodium salts of trichloroacetate (TCA) , SCN-, ClO,-, and of guani-
dinium hydrochloride (Gu-HCl) on the resolution of succinate dehydro-
genase as measured by the loss of PMS reductase activity. It is seen
that, as expected, the extent of resolution increases in a cooperative man-
ner with the increased concentration of chaotropes, and that addition of
high concentrations of chaotropes without freeze-thawing had no effect
on PMS reductase activity (Fig. 26, top right corner).
The subunits separated by the above technique indicated a distribution
of iron and labile sulfide as shown in Table X. Thus the larger subunit
contained flavin, iron, and labile sulfide in the approximate ratio of 1 :4 :4,
while the smaller subunit appeared to have the characteristics of a soluble
iron-sulfur protein. The absorption spectra of succinate dehydrogenase
and its two subunits analyzed to show the contributions of flavin and
the iron-sulfur chromophore in each preparation are given in Fig. 27.
c. Molecular Weight. Since flavin is covalently bound in succinate
0.1 0.2 0.3 0.4 0.5

Cawntration [MI
FIQ.26. Effect of chaotropic agents and freeze-thawing on the resolution of suc-

cinate dehydrogenase. The enzyme, at a protein concentration of 1.48 mg/ml in
50 mM sodium phosphate (pH 7.0) and 20 m M succinate, was treated with various
chaotropic agents as shown in the figure, and frozen in liquid nitrogen and thawed
at room temperature three times. Since in the presence of higher concentrations
of chaotropes the larger subunit precipitated after freeze-thawing, all samples were
centrifuged for 1 min at 35,000 rpm, and only the clear supernatant was assayed
for PMS reductase activity. F-T,freeze-thawing. From Hanstein et al. (166).
TABLE X
MOLECULAR PROPERTIES O F SUCCINATE DEHYDROGENASE'
Labile
Flavin Iron sulfide
Molecular
Molecule weight nmoles (ng-atoms)/mg protein
Succinate dehydrogenase 97,000 f 4% 10. 3b 70-80 70-80

Large subunit 70,000 k 7% 12-13 45-55 45-55
Small subunit 27,000 k 5% < 0.5" 95-110 90-100
From Davis and Hatefi (143).

a
* Average of
12 preparations.
c From the small subunit obtained by chaotrope-induced resolution of succinate
dehydrogenase ( 1 4 3 ) . The small subunit obtained by resolution and column chroma-

tography in presence of SDS is completely free of flavin.
Wavelength (nm)
(A) (6) (C)
FIG.27. Absorption spectra of succinate dehydrogenase (A), its larger subunit

(B), and its smaller subunit (C). ( A ) Trace 1, oxidized succinate dehydrogenase;
trace 2, flavin contribution to the spectrum of oxidized enzyme; trace 5, iron-sulfur
contribution to the spectrum of oxidized enzyme ; trace 4, oxidized enzyme treated
with dithionite ; trace 3, oxidized enzyme treated with sodium mersalyl and dithio-
nite. Protein = 1.48 mg/ml. (B) Trace 1, oxidized; trace 2, flavin contribution to
trace 1 ; trace 3, after treatment of 1 with sodium mersalyl and dithionite. Pro-
tein = 3.0 mg/ml. (C) Trace 1, oxidized; trace 2, after treatment with mersalyl;
trace 3, after treatment of 2 with dithionite. Protein = 0.8 mg,'ml. From Davis and
Hatefi ( 1 4 3 ) .
dehydrogenase, its concentration in a preparation would permit the calcu-

lation of a minimum molecular weight, The flavin content of succinate
dehydrogenase as reported by Davis and Hatefi (143) for a number of
preparations is 10.3 k 4% nmoles/mg protein. This value indicates a
minimum molecular weight of 97,000 f 4%, which agrees with the sum
of the subunit molecular weights (70,000 & 7% plus 27,000 f 5% =
*
97,000 6.6%). The relative mobility of succinate dehydrogenase on
Agarose columns corresponded to a molecular weight of 105,000 k 7.6%.
The molecular weight of about 100,000 for succinate dehydrogenase was
confirmed subsequently by others (25, 158) for the enzyme isolated from
complex I1 by the method of Davis and Hatefi. The above data also
suggested that the ratio of the two subunits in succinate dehydrogenase
must be close to unity. According to Coles et al. (158) and Righetti and
Cerletti (1573, their preparations contain more of the small subunit. This
has not been confirmed by others, but a 2 : 1 ratio in favor of the smaller
subunit (or an impurity which would coelectrophorese with the smaller
subunit) would account for the low flavin content (7-8 nmoles/mg pro-
tein) reported by the latter authors.
The sedimentation constant (s2,) of succinate dehydrogenase between
4 and 12 mg protein per ml was determined by Davis and Hatefi (143)
to be 9.22 S. This is considerably higher than the value of 6.5 S given
by Singer (15) and co-workers for their low iron, 50% pure preparation.
The high sedimentation constant was considered by Davis and Hatefi
to be either the result of the density and shape of the enzyme (159b)
or polymerization in solution. Ultracentrifugation of the enzyme in the
presence of potent depolymerizing agents (SDS, cetyldimethylethylam-
monium bromide, Triton X-100, or 0.5 M sodium trichloroacetate) a t
pH 7.0, 8.0, and 9.0 showed no change in the sedimentation constant.
These results and the relative mobility of succinate dehydrogenase on
Agarose columns did not agree with the polymerization possibility. HOW-
ever, Coles et al. (158) felt that succinate dehydrogenase purified accord-
ing to Davis and Hatefi exists in solution in a monomer-dimer equilib-
rium, and that a t protein concentrations of 1-10 mg/ml the molecular
weight obtained by sucrose gradient centrifugation and chromatography
on Sephadex G-200 is 175,000 & 10,000.
d. Prosthetic Groups. Succinate dehydrogenase contains one mole of
FAD per mole, and the flavin is covalently linked to the larger subunit
159b. D-Amino acid oxidase (Michaelis complex with benzoate) has been reported
( f / f ~ ) of 1.013
to have a molecular weight of 115,000 k 500, a frictional coefficient
(nearly spherical), and an S = 11.00 (160).
160. K . Yagi and T. Ozawa, BBA 62, 397 (1962).
FIG.28. Structure of histidyl-8-a-FAD (left) and the sequence of the flavin penta-
peptide (right) of succinate dehydrogenase. From Singer et al. ( 2 6 ) .
(143). As a result of collaborative work among the laboratories of Ehren-

berg, Hemmerich, arid Singer (161-163), the structure of the bound flavin
was elucidated to be 8 ~(N-3-histidyl)
- flavin adenine dinucleotide. The
structure of the histidyl flavin and the sequence of a flavin-bound penta-
peptide isolated by proteolytic digestion are shown in Fig. 28. Singer et
al. (23,25) have discussed the details of these studies.
I t has been shown recently by Ohnishi et al. (164) that a t temperatures
below 20°K, succinate dehydrogcnase or complex I1 exhibits E P R signals
indicative of two iron-sulfur centers, designated centers S-1 and S-2.
Center S-1 ( g = 2.03, 1.94, and 1.92 a t temperatures above 20°K) is the
same as that originally reported by Beinert and Sands (1666).Center S-2
exhibits signals a t the same field positions but with different line shapes.
These signals become prominent at temperatures below 20°K. The half-
reduction potentials (En&)given for these two centers a t pH 7.4 are
0 +- 10 and -260 +- 15 mV, respectively. The presence of two species of
EPR-active iron-sulfur centers in succinate dehydrogenase agrees with
the earlier finding of two iron-sulfur containing subunits. In the larger
subunit the iron : labile sulfide: flavin ratio suggests that this subunit
might contain a 4 iron-4 labile sulfide cluster as in clostridial ferredoxin.
In the smaller subunit, the data of Davis and Hatefi indicate the presence
of approximately 3 g-atoms of iron and 3 moles of labile sulfide per mole.
Since these values are clearly higher than 2 and the loss of iron and labile
sulfide as a result of resolution of the enzyme into subunits is quite prob-
161. P. Hemmerich, A. Ehrenbcrg, W. H . Walker, L. E . G . Eriksson, J. Salach,

P. Bader, and T. P. Singer, FEBS ( F e d . Eur. Biochem. Soc.) L e t t . 3, 37 (1969).
162. J. Salach, W. H. Walker, T. P. Singer, A. Ehrenberg, P. Hemmerich, S. Ghisla,
and U. Hartman, Eur. J. Biochem. 26, 267 (1972).
163. T. P. Singer, J. Salach, W. H. Walker, M. Gutman, P. Hemmerich, and A.
Ehrenherg, i n “Flavins and Flavoproteins,” 3rd Int. Symp. (H. Kamin, ed.), p. 607.
Univ. Park Press, Baltimore, Maryland, 1971.
164. T. Ohnishi, D. U. Winter, J. Lim, and T. E. King, BBRC 53, 231 (1973).
165. H. Beinert and R. H. Sands, BBRC 3, 41 (1960).
TABLE XI
DYE REDUCTASE
PROPERTIES
OF SUCCINATE
DEHYDROOENASE
Parameter Value
Succinate -+ PMSa activity 67-7gb

Succinate -+K3Fe(CN)aactivity 13. 5bsc
Turnover number at Vz:: 10,000-11,000d
::v 100-1 l o b
KEMs(mM) -0.48
K z c o(mM) ~ 0 . 3 ~
0 At 1.65 mM PMS.
* Micromoles of succinate oxidized X min-1X mg-I protein at 38".
At 3 mM ferricyanide.
d Moles of succinate oxidized X min-1 X mole-' enzyme at 38".
able, the smaller subunit might also have a clostridial-type iron-sulfur

center .
2. Enzymic Properties
a . Succinate Dehydrogenase. Soluble succinate dehydrogenase can oxi-
dize succinate in the presence of PMS or ferricyanide as electron acceptor.
The highest activities reported for various preparations of succinate
dehydrogenase are those of the preparation of Davis and Hatefi (143,
166). These values are shown in Table XI. The turnover number of the
enzyme a t V,,, with respect to PMS concentration is 10,000-11,000 moles
succinate oxidized per minute per mole of enzyme at 3 8 O . This value is
the same as the turnover number of complex I1 (per mole of flavin) with
either PMS or ubiquinone as electron acceptor (153, 154, 156). It is also
the same as the turnover number reported by Ziegler and Doeg (153)
and by King (167) for succinate oxidation by submitochondrial particles.
The values given by the former authors are 10,000 with ubiquinone, 9,700
with PMS, and 10,100 with oxygen as electron acceptors. According to
King, the turnover numbers of the succinoxidase system of intact heart
muscle preparations and reconstituted preparations are 7,500 and 10,000,
respectively. The latter value is the same as that given by Veeger et
al. (149) for reconstituted particles made with their preparation of succi-
nate dehydrogenase. In contrast to the consistent results of the above
authors, Singer (15,23,25) insisted that the turnover number of succinate
dehydrogenase in heart mitochondria is 17,000-18,000, and concluded that
166. W. G. Hanstein, K. A. Davis, M. A. Ghalambor, and Y . Hatefi, Biochemistry
10, 2517 (1971).
167. T. E. King, Advan. Enzymol. 28, 155 (1966).
complex I1 and soluble succinate dehydrogenases have, therefore, lost

activity as a result of preparative modification. Surprisingly, however,
the paper referred to by Singer for the high turnover number of 17,000-
18,000 is also by Singer et al. (168), and in the latter paper the turnover
number of succinate dehydrogenase in beef heart mitochondria a t V,,,
with respect to PMS and 3 8 O is 11,000-13,000 and in complex I1 it is
11,300-11,900 moles succinate oxidized per mole of bound flavin. The only
other high turnover number found in the literature is that of Cerletti
et al. (169) which does not give details. These stated differences might
also be based on the values used for the concentration of succinate dehy-
drogenase flavin in heart mitochondria and respiratory particles. Neither
Singer et al. (168) nor Cerletti et al. (169) gave the flavin values used
in these papers for the calculation of their turnover numbers. However,
literature values for submitochondrial particles range from 0.11-0.14 (15)
to 0.28 (170) nmole/mg protein.
The ferricyanide reductase activity given in Table XI was measured
in the presence of 3 mM ferricyanide and is far from V,,,. As shown
by King (17 1 ) ,higher ferricyanide concentrations are strongly inhibitory.
Others have stated, however, that the extrapolated turnover number of
the enzyme is the same with either PMS or ferricyanide as electron accep-
tor (172).The K , values given in Table XI for PMS and succinate are
essentially the same as the values reported by King (167) and King and
Takemori (173).The K, for succinate is also the same as has been found
for complex 11, submitochondrial particles (166),Keilin-Har,tree heart
muscle preparations, and King's reconstitutable enzyme ( 1 7 3 ) .The value
given by Singer (15) for the succinate-PMS reductase activity of heart
homogenates, determined under similar temperature ( 3 8 O ) and pH condi-
tions as in Table XI, is 1.3 mM. The I(, given by Singer et al. (25)
for succinate a t 2 2 O to 2 5 O is 0.5 mM.
According to Veeger et al. (149),succinate dehydrogenase can also oxi-
dize L-chlorosuccinate, L-methyl succinate, D-malate, and L-malate.
Brodie and Nicholls (174) have reported that monofluorosuccinate and
2,2-difluorosuccinate are also oxidized by Keilin-Hartree preparations
yielding oxaloacetate as a final product. D-Chlorosuccinate, D-methyl suc-
168. T. P. Singer, J. Hauber, and 0. Arrigoni, BBRC 9, 150 (1962).
169. P. Cerletti, R. Strom, M. Giordano, F. Balastero, and M. A. Giovenco, BBRC
14, 408 (1963).
170. P. V. Blair, T. Oda, and D. E. Green, Biochemistry 2, 756 (1963).
171. T. E. King, JBC 238, 4032 (1963).
172. W. P. Zeylemaker, A. D. M. Klaase, E. C. Slater, and C . Veeger, BBA 198,
415 (1970).
173. T. E. King and S. Takemori, JBC 239,3559 (19643.
174. J. D. Brodie and P. Nicholls. BBA 198,423 (1970).
TABLE XI1
DISSOCIATION
AND INHIBITION
CONSTANTS
OF COMPETITIVE
INHIBITORS
OF SUCCINATE
DEHYDROOENASE"
KD Ki
Inhibitor (mM) (mM)
Oxaloacetate 0.004 0.0015

Malonate 0.028 0.018
Fumarate 2.6 1.3
Methylene succinate 6.3 1.8
Maleate 7.0 6.2
Bicarbonate - 12
Glyoxylate - 21
Acetoacetate 80 40
Glycolate - 120
Formate - 120
0 From Zeylemaker et al. (I"$?) and DerVartanian and Veeger (176).
cinate, malonate, methylene succinate, maleate, acetoacetate, and oxalo-

acetate are competitive inhibitors (149). I n addition bicarbonate, for-
mate, glycolate, and glyoxylate are also competitive inhibitors, and
appear to bind a t two sites on the enzyme (172).The inhibitor constants
( K i ) and the dissociation constants ( K D ) of a number of these com-
pounds as reported by Veeger and his colleagues (172, 175) are shown
in Table XII. It is seen that among these oxaloacetate binds strongly
to the enzyme and is a very potent competitive inhibitor. The role of
oxaloacetate in controlling the activity of succinate dehydrogenase is dis-
cussed below. DerVartanian and Veeger ( 1 75,176) have studied the effect
of various substrates, inhibitors and analogs on the spectral properties
of their preparation of succinate dehydrogenase. Spectral changes in the
presence of competitive inhibitors are thought to arise from charge-trans-
fer complex formation between the electron-donating inhibitor and the
enzyme.
Both the membrane-bound and the soluble succinate dehydrogenases
are capable of catalyzing fumarate reduction in the presence of a suitable
electron donor such as FMNH,. I n the mammalian enzyme, this activity
is not more than a few percent of the succinate-PMS reductase activity
(23, 2 5 ) . Ringler and Singer (177) have also shown that in brain mito-
175. D. V. DerVartanian and C. Veeger, BBA 92,233 (1964).
176. C. Veeger, D. V. DerVartanian, J. F. Kalse, A. de Kok, and J. F. Koster,
in "Flavins and Flavoproteins," Symp Adn Flavoproteins (E.C. Slater, ed.), p. 242.
177. R. L. Ringler and T. P. Singer, JBC 234,2211 (1959).
chondria treated with antimycin A, the reduction of fumarate to succinate

could be linked to the oxidation of a-glycerophosphate to dihydroxyace-
tone phosphate. A seemingly interesting observation reported by Singer
is that alkali treatment of respiratory particles a t pH 9.3-9.4 results in
a much greater loss of succinate dehydrogenase activity than fumarate
reductase activity. However, i t should be remembered that the electron
donors and acceptors in these assays are not exactly the same. Therefore,
the difference might have resulted from preferential destruction by alkali
of the PMS reductase site.
b. Complex ZZ. Preparations of complex I1 catalyze the oxidation of
succinate by either PMS ( a t V,n,,) or ubiquinone a t a rate of about
45-55 pmoles/min/mg protein a t 38O. Thus the turnover number in either
reaction is about 10,000. It has been shown by Ziegler and Doeg (153)
that the PMS reductase activity of complex I1 is the same in the presence
or absence of added ubiquinone-2 (complex I1 preparations are essen-
tially devoid of bound ubiquinone). This is not in agreement with the
conclusions of others on ubiquinone effect in submitochondrial particles
(see, however, Section III,A,4).
It has been shown recently that the mitochondria1 electron transport
system contains a t least three different b-type cytochromes (178).Two
of these cytochromes are found in complex 111, and under appropriate
conditions are reducible with substrates. The third b-type cytochrome
was discovered by Davis et al. ( l 7 8 ) , and shown to fractionate exclu-
sively into complex 11. At 77OK, the cytochrome b of complex I1 exhibits
a double band a t 557.5 and 550 nm, a prominent p band a t 531 nm,
(Y
and a Soret band at 422 nm (Fig. 29). Cytochrome b,,,., appears to

have a low reduction potential. It is not detectably reduced by succinate
in either complex I1 or respiratory particles, but its dithionite reduced
form is rapidly oxidized by either fumarate or ubiquinone. The role of
this cytochrome in mammalian mitochondria is not known. Davis et al.
(178) have suggested that it might be an electron entry point for an
unknown ancillary tributary of the respiratory chain. Further, Bruni and
Racker (179) have shown that a preparation of cytochrome b is required
for reconstitution of succinate-ubiquinone reductase activity (see below).
c. Reconstitution. The reconstitution experiments involving succinate
dehydrogenase may be divided into three types. These are recombination
of succinate dehydrogenase with alkali-inactivated particles, reconstitu-
tion of succinate-ubiquinone reductase activity with partially purified
components, and reversible resolution of complex 11.
178. K. A. Davis, Y . Hatefi, K. L. Poff, and W. L. Butler, BBA 325,341 (1973).

179. A. Bruni and E. Racker, JBC 243,962 (1968).
I
400 420 440 460 400 500 520 540 560
Wavelength, nm
FIQ.29. Reduced minus oxidized spectrum of cytochrome bEnr.sin complex 11.
Complex I1 at 1.76 mg/ml of 40 mM potassium phosphate, pH 7.4, was treated
with a small amount of dithionite in order to reduce its succinate dehydrogenase
and minor (-10%) complex I11 contaminant, and its spectrum was recorded. This
spectrum was then subtracted from that of complex I1 fully reduced with dithionite.
From Davis et a.!. (f78).
It has been known since the early 1950’s that treatment of respiratory
particles a t alkaline pH ( >9.0) destroys succinoxidase activity. King
and his colleagues showed that addition of the Keilin-King type of succi-
nate dehydrogenase to these particles restored a substantial amount of
succinoxidase activity (147, 148, 167). The alkali inactivation of the par-
ticles was shown subsequently to result from inactivation of the particle-
bound enzyme (16, 166, 180) rather than loss of succinate dehydrogenase
as originally believed (148, 167). The work of Hanstein et a2. (166) with
pure succinate dehydrogenase clarified many aspects of this type of re-
constitution. Their results may be summarized as follows. Incubation of
submitochondrial particles a t pH 9.3 and 38O under an argon atmosphere
resulted in a rapid, exponential loss of succinoxidase activity. Presence
of succinate during incubation retarded inactivation considerably, and
concomitant presence of dithiothreitol offered additional protection (Fig.
30A). All the decay curves extrapolated to a zero time activity corre-
sponding to 90% ,of the activity of untreated particles (Fig. 30A). This
90% activity was totally recovered upon addition of sufficient amounts
of succinate dehydrogenase to the alkali-inactivated particles. Titration
180. T. Kimura, J. Hauber, and T. P. Singer, Nature (London) 198,362 (1968).

Min at pH 9.3 and 38' Min ot 0'

(A) (8)
FIG.30. (A) Kinetics of inactivation of succinate oxidase activity of submitochon-

drial particles (ETP) a t pH 9.3 and 38". E T P at 18.2 mg/ml of deoxygenated 035
M sucrose plus 10 mM Tris-HC1 (pH 8.0) was brought to pH 9.3 at 38" with a
predetermined amount of 1 N NaOH, incubated under an atmosphere of argon
and sampled for activity as indicated. Where present in the incubation mixture,
succinate was 20 mM and dithiothreitol (DTT) 5 mM. Succinoxidase assays were
conducted at 30". All samples were neutralized with one volume of 100 mM sodium
phosphate (pH 7.3), containing 20 mM succinate and incubated 3 min at 38" before
assay. To those not containing succinate in the original mixture 40 mM succinate
was added along with phosphate buffer at the time of neutralization, so that the
final succinate concentration in all samples was 20 mM. From Hanstein et al. (166).
(B) Loss of reconstitution activity of soluble succinate dehydrogenase at 0" as a
function of time as compared to the activity of the same preparation when bound
to alkali-treated submitochondrial particles (ETP). Succinoxidase activity was as-
sayed as in ( A ) . (U) SD/ETP = 0.10, ( A ) SD/ETP = 0.30,(0) No ETP.
of the particles with increasing amounts of succinate dehydrogenase (Fig.

31) indicated that full activity restoration required the addition of a t
least 5 times as much succinate dehydrogenase as was present in the par-
ticles. The same stoichiometry was found for reconstitution of activity
in alkali-treated complex 11. These results suggest that the equilibrium
of the system involving free and particle-bound succinate dehydrogenase
under these conditions (i.e., in the presence of alkali-treated particles)
is in favor of the free enzyme. Another possibility that cannot be ruled
out is that only 20% of the added succinate dehydrogenase was capable
of reconstitution. When succinate dehydrogenase was added to alkali-
inactivated complex I1 and the reconstituted complex isolated by centrif-
ugation, it was found that the active complex contained twice as much
flavin and the corresponding amount of additional protein (Table XIII).
These results showed, therefore, that in this type of reconstitution a
double-headed particle is formed containing one complement of active
SD/alk-ETP
Ql 0;2 , 0:3
1.6 t '
.
$ 1.2-
b
tI :
F
0.8
-
M S.A./mg alk-ETPprole
0-0
S.A./w SDpotsin aa
S.A./mq total protein
tP 0."4i 8
I .
16
alk- ETP/SD
24
' ,I
Fra. 31. Reconstitution of succinate oxidase activity. Fresh succinate dehydrogenase

was dissolved at a concentration of 7.8 mg/ml in 50 mM phosphate (pH 7.5), con-
taining 20 mM succinate and 5 mM dithiothreitol, then immediately divided in
50 pl quantities into small test tubes, frozen in liquid nitrogen, and stored at -70".
E T P preparations stored as centrifuged pellets were suspended a t a concentration
of 9.1 mg/ml in 50 mM Tris (pH 8.01, containing 0.66 M sucrose. They were inacti-
vated a t pH 9 8 essentially as in Fig. 30A (none), readjusted to pH 7.7,and supplied
with 20 mM succinate (protein concentration at this point was 8.35 mg/ml). Appro-
priate amounts of the alkali-treated E T P were then added to test tubes containing
frozen succinate dehydrogenase. The components were mixed a t room temperature
with the help of a stirring rod, incubated 2.5 min at 30°, and assayed at 30" for
succinate oxidase activity. alk-ETP, alkali-treated ETP. From Hanstein et al. (166).
(added) and one of inactive (alkali-treated) succinate dehydrogenase

(166).
As originally shown by King (148),the reconstitution, but not the PMS
reductase, activity of succinate dehydrogenase is extremely labile. Han-
stein et al. (166) showed that at Oo the reconstitution activity of the
soluble, but not the reconstituted, enzyme decays exponentially with a
half-life of 75 min (Fig. 30B). However, a t < - 6 5 O this activity was in-
definitely stable. King (148) showed that during extraction of the enzyme
from particles the presence of succinate was necessary for retention of
reconstitution activity. Davis and Hatefi (149) used both succinate and
dithiothreitol during the chaotrope-induced resolution of complex I1 and
salt precipitation of succinate dehydrogenase. They showed, however,
that dithiothreitol was much more effective than succinate for complete
preservation of reconstitution and PMS reductase activities (166). King
(148, 161) had shown that except for reconstitution activity succinate
dehydrogenases extracted in the presence and absence of succinate were
identical in composition, spectra, and dye reductase properties. However,
TABLE XI11
RECONsTlTUTION OF SUCCIN~4T~-UnIaUINONE
REDUCT.4SE ACTIVITY"
Succ' PMS Succ-t Q

Flavin (per mg (per mg
Protein (nmoles / total total
Preparation (mg) me) protein) protein)
Complex 11, p H 9.3,

0 rnin a t 38" 5.6 22.6 22.2
Complex 11, p H 9.3,
20 min a t 38" 5.6 4.92 <0.9 <0.8
+
Complex I1 succinate
dehydrogenase 5.6 + 12.6 8.Gb 43.0 7.7
+
Complex I1 succinate
dehydrogenase, spun
60 min
Supernatant 10.0 48.8 <1.0
Pellet 7.9 6.7 24.4 23.9
a Conditions: Complex I1 was inactivated by 20 min incubation a t pH 9.3 and re-

adjusted to p H 7.6 (protein 11.25 mg/ml). Succinate dehydrogenase was prepared
and stored as an ammonium sulfate-precipitated pellet for 1 day a t -70". I t was
dissolved in 50 mM Tris-HC1 (pH 8.0) containing 20 m M succinate and 3 m M di-
thiothreitol before using (protein 21.1 mg/ml). Alkali-treated complex I1 (0.5 ml)
and succinate dehydrogenase (0.6 ml) were mixed together, assayed, and centrifuged
for 60 min a t 49,000 rpm. The supernatant and the pellet were then separated, the
latter was suspended in Tris-succinate-dithiothreitol buffer and both fractions were
assayed as indicated. Activities shown are expressed as micromoles of succinate oxi-
dized X min-1 x mg-1 of total protein a t 38".
* Calculated. From Hanstein el al. (168).
Baginsky and Hatefi (155, 156) showed that loss of reconstitution activ-
ity appears to be related to a damage in the iron-sulfur system of the
enzyme which is not detectable by assay for iron and labile sulfide con-
tent. They obtained a preparation of succinate dehydrogenase from com-
plex I1 which exhibited no reconstitution activity but had an iron:labile
su1fide:flavin ratio close to 8:8:1. They were then able to reactivate this
enzyme for reconstitution by treating it with Na2S, ferrous ions, and
mercaptoethanol, essentially in the same manner as apoferredoxin had
been previously converted to ferredoxin (181, 182). The reactivated prep-
aration was able to reconstitute with alkali-treated submitochondrial
particles or complex 11. Analyses showed that the preparation had ac-
quired additional iron and labile sulfide, but control experiments indi-
cated that reconstitution activity was not a spurious effect. The reactiva-
181. J. Hong and J. C. Rabinowitz, BBRC 29,246 (1967).
182. K.McCarthy and W. Lovenberg, JBC 243,6439 (1968).
tion of succinate dehydrogenase by the above technique was confirmed

by others (182a).
As stated above, preparations of succinate dehydrogenase contain two
EPR-active iron-sulfur centers in agreement with the fact that the en-
zyme is composed of two iron-sulfur containing subunits. The tempera-
ture-insensitive E P R signal a t g = 1.94 (EmN 0 mV) is present in high
iron-labile sulfide preparations which are inactive (156) and active (143)
for reconstitution. I n addition, this signal was originally observed by
Beinert and Sands (165) in succinate dehydrogenase preparations of
Singer et al. and Basford et aZ., which according to Beinert and Sands
contained per mg protein 1.0 nmole flavin plus 4.7 ng-atoms iron and
4 nmoles flavin plus 8 ng-atoms iron, respectively. These results indicate
that (a) reconstitution activity may not be related to the g = 1.94signal,
and (b) that the signal is present in preparations with iron :flavin ratios
of 4:l and 2:l. The latter conclusion is only qualitatively correct, of
course, since titration and signal integration were not performed in these
early experiments, and possible heterogeneity in the enzyme preparations
tested cannot be ruled out. However, Ohnishi et al. (164) stated that
the stability of center S-1 mirrors the stability of the dye reductase activ-
ity of the enzyme, whereas the half-reduction potential of center S-2 in
soluble succinate dehydrogenase (King type) showed a strong tendency
to decrease from -260 mV to approximately -430 mV. They felt that
these results suggest that center 5-2 may be involved in linking the en-
zyme to the respiratory chain. This conclusion does not necessarily imply
that center 5-2 is an intermediate electron carrier since succinate does
not reduce this low potential iron-sulfur center.
The second type of reconstitution is demonstrated by the work of Bruni
and Racker (179).These investigators reconstituted a succinate-ubiquin-
one reductase system from a King-type preparation of succinate dehydro-
genase (2.6nmoles flavin per mg protein), a preparation of cytochrome
b (24-27nmoles heme per mg protein) solubilized and purified with the
use of bile salts and SDS, and mitochondria1 or soybean phospholipids.
The highest succinate-ubiquinone reductase activity achieved was 980
moles succinate oxidized per minute per mole of succinate dehydrogenase
flavin. While this activity is only 10% of the turnover number of complex
11, it is still quite appreciable for this type of reconstitution. Since the
preparations of succinate dehydrogenase and cytochrome b used were not
pure, it is not known what is the minimum number of components
needed for reconstitution of succinate-ubiquinone reductase activity. The
role and the exact nature of the b-type cytochrome used in these experi-
182a. T. E. King, D. Winter, and W. Steele, in “Structure and Function of Oxida-
tion Reduction Enzymes” (A. Akeson and A. Ehrenberg, eds.), p. 519. Pergamon,
Oxford, 1972.
4. METAL-CONTAINING FLAVOPROTEIN DEHYDROGJCNASES 245
ments are also not known (in complex I1 the ratio of cytochrome b to
flavin is approximately 1 :1; in the Bruni-Racker reconstituted prepara-
tion this ratio was 3 2 : l ) . On the other hand, there is no evidence for
the existence of a component in complex I1 which might act as an inter-
mediate electron carrier between succinate dehydrogenase and ubiquinone.
It is entirely possible that, as implied by Bruni and Racker, succinate
dehydrogenase bound to a membranous structure containing phos-
pholipids might be able to interact with ubiquinone. Recently, Beinert
and his colleagues (183, 184) have isolated an iron-sulfur protein from
mitochondria with a molecular weight of about 100,000. They stated that
preparations of complex I1 contain an iron-sulfur component with similar
EPR characteristics. However, they agreed with the previous work of
others (143) that the SDS-polyacrylamide gel pattern of complex I1 is
devoid of a species with the mobility characteristics of their 100,000 mo-
lecular weight iron-sulfur protein.
The third type of reconstitution has been discussed already in Section
III,A,l,a. It involves reversal of the chaotrope-induced resolution of com-
plex I1 with respect to succinate dehydrogenase either by removal (or
dilution) of the added chaotrope or by addition of an antichaotropic ion
to the resolved system. The basis of this resolution-reconstitution process
is the disordering and restructuring of the water in which complex I1
is suspended. The process in either direction is relatively rapid, and com-
plete structural and functional reconstitution is easily achieved (Figs.
25a and b). Earlier studies of Hatefi et al. ($7, 29) have also shown
that complex I1 interacts with complexes I, 111, and IV to reconstitute
the entire respiratory chain. However, contrary to claims in the literature
(167, 173) based on experiments in which highly impure preparations
of succinate dehydrogenase and complex I11 were used, pure succinate
dehydrogenase and complex I11 do not interact to reconstitute a succi-
nate-cytochrome c reductase system (185). These results indicate that
appropriate components of complex I1 are needed t o link succinate dehy-
drogenase to the mitochondria1 ubiquinone-cytochrome c reductaee
complex.
3. Inhibitors and Modifiers
It has been known since the work of Hopkins and his colleagues in
1938 (186) that succinate dehydrogenase contains -SH groups essential
for the catalytic activity of the enzyme, and that substrates and competi-
tive inhibitors protect the enzyme against inhibition by -SH reagents.
183. F. J. Rueicka and H. Beinert, BBRC 58,556 (1974).
184. H. Beinert, B. A. C. Ackrell, E. B. Kearney, and T. P. Singer, BBRC 58,
564 (1974).
185. K. A. Davis and Y. Hatefi, BBRC 44, 1338 (1971).
186. F. G. Hopkins, E. Morgan, and C. Lutwak-Mann, BJ 32, 1829 (1938).
However, it is not known whether the active thiol of succinate dehydroge-

nase is a t or near the enzyme active site. At low levels of mercurials,
the inhibition can be reversed, but a t high levels mercurials irreversibly
destroy the iron-sulfur chromophore of the enzyme (149, 187).The flavin
spectra of succinate dehydrogenase and its larger subunit in Fig. 27 were
obtained by treating the preparations with solid sodium mersalyl. I n
addition to mercurials, N-ethylmaleimide and certain alkyl bromides also
inhibit the enzyme. Similar to other iron-sulfur proteins, succinate dehy-
drogenase is slowly inactivated in the presence of o-phenanthroline, but
Tiron and bathophenanthroline are much less effective (166). 2-Thenoyl-
trifluoroacetone (TTFA), which is also considered to interact with iron
in the respiratory chain, is a strong and relatively specific inhibitor of
succinate-ubiquinone reductase activity, but does not inhibit PMS reduc-
tase activity in complex I1 and soluble succinate dehydrogenase. Since
in complex I1 and the reconstituted system of Bruni and Racker (179)
the only succinate-reducible species known are succinate dehydrogenase
and ubiquinone, Baginsky and Hatefi (156) have suggested that the site
of action of TTFA might be on succinate dehydrogenase itself. I n view
of our present knowledge regarding the two iron-sulfur-containing sub-
units of the enzyme, it is further possible that PMS and TTFA do not
interact with the same iron-sulfur species. According to Rossi et al. (188)
TTFA also inhibits the succinate dehydrogenase activity of submitochon-
drial particles. However, these data involve assay complications, which
will be discussed in Section 111,A14.
Since the isolation of succinate dehydrogenase from cyanide-treated
particles (146), a considerable amount of work has been done on the
effect of cyanide on succinate dehydrogenase. Treatment of particles with
cyanide diminishes succinate-PMS reductase activity by about 50% and
causes a large increase in the apparent I<,, of the dye. These effects are
prevented by succinate (189).The effect of cyanide is more drastic on
electron transfer from succinate to methylene blue or the respiratory
chain. These reactions are almost completely destroyed by cyanide. Ex-
periments with partially purified succinate dehydrogenase showed, how-
ever, that cyanide treatment did not alter the PMS reductase activity
(190). The reactive species has been shown to be CN-, which binds
strongly to succinate dehydrogenase (189-1 91). According to Zanetti et
187. V. Massey, BBA 30, 500 (1958).

188. E. Rossi, B. Norling, B. Persson, and L. Ernster, Eur. J. Biochem. 16, 508
(1970).
189. A. Giuditta and T. P. Singer, JBC 234, 666 (1959).
190. C.-P. Lee and T. E. King, BBA 59,716 (1962).
191. G. Zanetti, Y. M. Galante, P. Arosio, and P. Cerletti, BBA 321, 41 (1973).
al. (191) the stoichiometry is 2 moles of CN- per g-atom of iron. It has
been postulated that interaction of cyanide with succinate dehydrogenase
causes conformational changes which alter the dye reductase properties
of the enzyme and labilize its linkage to the respiratory chain (189, 191).
Other details of the cyanide effect are found in earlier reviews (15, 251,
and will be discussed below.
4. Regulatory Properties
It has been known since the early studies of Kearney (192) that SUC-
cinate dehydrogenase undergoes reversible activation by substrates,
competitive inhibitors, and phosphate. The activation of succinate dehy-
drogenase was shown to be a characteristic of both the soluble and parti-
cle-bound enzyme and a slow process requiring many minutes of incuba-
tion with the activator a t ambient or higher temperatures (activation
energy = 31-33 kcal/mole). It has been suggested that the enzyme exists
in a free equilibrium between the unactivated and the activated forms,
and that the activator interacts with the latter and establishes a new
equilibrium in favor of the activated state of the enzyme (23, 25, 193;
see also 194 for an expanded mechanism).
I n addition to succinate, malonate, fumarate, and phosphate, i t has
been reported in recent years that succinate dehydrogenase is activated
by ATP, ITP, IDP (195), reduced ubiquinone-10 (195-197), succinyl
coenzyme-A (198), formate, ClO,-, I-, Br-, C1-, NO,-, SO,2-, acid pH
( I % ) , FMNH,, light irradiation of enzyme in the presence of EDTA
(200), 2,4-dinitrophenol (201), and phospholipids (202-205; see also 20,
192. E. B. Kearney, JBC 229,363 (1957).

193. T. Kimura, J. Hauber, and T. P. Singer, JBC 242,4987 (1967).
194. R. G. McDonald-Gibson and M. B. Thorn, BJ 114,755 (1969).
195. M. Gutman, E. B. Kearney, and T. P. Singer, Biochemistry 10, 4763 (1971).
196. M. Gutman, E. B. Kearney, and T. P. Singer, BBRC 42, 1016 (1971).
197. M. Gutman, E. B. Kearney, and T. P. Singer, Biochemistry 10, 2726 (1971).
198. E. B. Kearney, M. Mayr, and T. P. Singer, BBRC 46, 531 (1972).
199. E. B. Kearney, B. A. C. Ackrell, M. Mayr, and T. P. Singer, JBC 249, 2016
(1974).
200. J. I. Salach and T. P. Singer, JBC 249,3765 (1974).
201. L. Susheela and T. Ramasarma, BBA 242, 532 (1971).
202. P. Cerletti, R. Strom, and M. G. Giordano, BBRC 18, 259 (1965).
203. P. Cerletti, M. A. Giovenco, M. G. Giordano, S. Giovenco, and R. Strom,
BBA 146, 380 (1967).
204. P. Cerletti, P. Caifa, M. G. Giordano, and G. Magni, in “Flavins and Flavo-
proteins,” 2nd Int. Symp. (K. Yagi, ed.), p. 178. Univ. Park Press, Baltimore,
Maryland, 1968.
205. P. Cerletti, P. Caifa, M. G. Giordano, and M. A. Giovenco, BBA 191, 502
(1969).
24, 26, 206-208). Among the nucleotides ATP does not activate complex
I1 and soluble preparations, while ITP and IDP do. IDP is much more
effective than ITP, but its effect relative to ATP could not be tested
because it does not readily penetrate the mitochondria1 membrane. The
activating effect of formate is probably related to its property as a com-
petitive inhibitor (see above). The order of effectiveness of the mono-
valent ions has been reported to be C10,-, I-, NO,- > Br- > C1-, which is
reminiscent of their order of potency as chaotropes (70-72). These ions are
effective a t much lower concentrations when used a t pH values a t or
below pH 6.
It was reported by Hanstein et al. (166) that pure succinate dehydro-
genase prepared by perchlorate extraction of complex I1 in the presence
of succinate and dithiothreitol was fully activated. However, when recon-
stituted with alkali-inactivated submitochondrial particles the enzyme
became deactivated and required preincubation a t 30° with succinate be-
fore maximal succinoxidase activity was attained. Ignoring the latter
observation, Singer and his colleagues chose to fault the pure enzyme
for being fully active as isolated on the assumed ground that it had lost
its regulatory properties (23, 26, 26, 168). Subsequent studies of Coles
et al. (209) showed, however, that the above preparation could be deacti-
vated by removal of succinate and perchlorate. When isolated from com-
plex I1 in the absence of succinate (as stated above succinate is not cru-
cial for preservation of reconstitution property of the Davis-Hatefi
enzyme) , then deactivation was achieved rapidly upon filtration of the en-
zyme through a Sephadex column. Such deactivated preparations could
be reactivated again by the usual procedures.
Most preparations of succinate dehydrogenase contain tightly bound
oxaloacetate apparently in a 1:1 molar ratio with respect to the deacti-
vated fraction (207,208). According to King and his colleagues (210,
S l l ) , oxaloacetate binds to a sulfhydryl group on the larger subunit of
the enzyme to abolish enzymic activity. Kearney and her colleagues have
shown that the tightly bound oxaloacetate can be dissociated by various
activators of the enzyme, such as succinate, malonate, IDP, ITP, and
high concentrations of anions a t elevated temperatures but not in the
206. P. Cerletti and A. Manzocchi, Acta Vitaminol. Enzymol. 27, 5 (1973).

207. T. P. Singer, G. Oestereicher, and P. Hogue, Plant Physiol. 52, 616 (1973).
208. G. Oestereicher, P. Hogue, and T. P. Singer, Plant Physiol. 52, 622
(1973).
209. C. J. Coles, H. D. Tisdale, W. C. Kenney, and T. P. Singer, JBC 249, 381
(1974).
210. A. Vinogradov, D. Winter, and T. E. King, BBRC 49, 441 (1972).
211. D. B. Winter and T. E. King, BBRC 58,290 (1974).
cold or by precipitation and gel exclusion of the enzyme (212, 213). The
activation energies for activation and release of oxaloacetate were found
to be the same. However, with some activators it was found that oxalo-
acetate release showed a considerable lag as compared t o recovery of
enzymic activity. Moreover, oxaloacetate-free preparations could be re-
versibly deactivated a t alkaline pH, and activation by anions or FMNH,
showed a lower activation energy than given above (199, 200). Thus,
Ackrell et al. (213) suggested that oxaloacetate binding is not the cause
of deactivation but a consequence of the deactivated conformation. That
activation-deactivation of succinate dehydrogenase might involve confor-
mational changes is suggested by ( a ) greater sensitivity of the activated
enzyme to inhibition by N-ethylmaleimide and bromopyruvate, which
has been interpreted as exposure of -SH groups ( S l d ) , and (b) the re-
port that activation involves a free-energy change of only about 1.8
kcal/mole but an entropy change of 51 eu ( 2 5 , 2 6 ) .An important consid-
eration with regard to the above studies is that activation-deactivation ex-
periments involving incubations of 30 min or longer a t elevated tempera-
<
tures, treatment a t p H 6, and filtration through Sephadex have been
carried out using the dye reductase activity of the enzyme as the sole
criterion. While the results are interesting and in some cases applicable to
the membrane-bound enzyme, it is very doubtful that the reconstitution
activity of soluble succinate dehydrogenase would survive the above
treatments.
Activation studies with ATP and reduced ubiquinone-10 are interesting
because these experiments involve membrane-bound succinate dehydro-
genase and the nature of the activators suggests physiological regulation.
.4TP activation does not appear to involve membrane energization and
reverse electron transfer, because it is insensitive to inhibition by oligo-
mycin. The possibility that ATP induces the formation of the activator
succinyl-CoA by reversal of the succinyl-CoA synthetase reaction is com-
plicated by the finding that in metabolic states of the mitochondrion (e.g.,
the active state 3) where succinyl-CoA is known to accumulate, succinate
dehydrogenase is in the deactivated state ( 2 3 ) .However, the deactivated
state of succinate dehydrogenase under these conditions is believed to
be governed mainly by the oxidized state of ubiquinone ( 1 9 7 ) .
The possible role of ubiquinone in controlling succinate dehydrogenase
was first suggested in 1970 by Rossi et al. (188), who made the following
observations on succinate-PMS reductase activity of lyophilized,
ubiquinone-depleted (by pentane extraction of the lyophilized particles),
212. E. B. Kearney, B. A. C. Ackrell, and M. Mayr, BBRC 49, 1115 (1972).
213. B. A. C. Ackrell, E. B. Kearney, and M. Mayr, JBC 249, 2021 (1974).
214. B. M. Sanborn, N. T. Feldberg, and T. C. Hollocher, BBA 227, 219 (1971).
and ubiquinone-replenished submitochondrial particles. I n lyophilized

and ubiquinone-replenished particles, TTFA and cyanide pretreatment
diminished VmaXwith respect to PMS by about 50%. In ubiquinone-
depleted particles, V,,, was already diminished by 50% and further
treatment with TTFA or cyanide had no additional effect. These results
were taken by the authors and by Singer and co-workers (23, 25) to
indicate (a) an effect of ubiquinone on succinate dehydrogenase activity;
(b) that as originally suggested by Singer’s laboratory there are two PMS
reduction sites in the particles, one of which is abolished by TTFA or
cyanide pretreatment of the particles; and (c) that ubiquinone is prob-
ably the second PMS reduction site in particles. Regarding point ( a ) , the
experiments of Rossi et al. appear to involve an assay complication, how-
ever. The final electron acceptor in the succinate-PMS reductase assay
was 2,6-dichloroindophenol, and both PMS and dichloroindophenol accept
electrons in submitochondrial particles from components on the oxygen
side of the TTFA block (214a). Thus, addition of TTFA, extraction of
ubiquinone, or pretreatment of the particles with cyanide would abolish
the latter route for electron transfer to PMS and dichloroindophenol,
leaving only the pathway of dye reduction through succinate dehy-
drogenase itself. Therefore, the loss of 50% activity as a result of extrac-
tion of ubiquinone is probably referable to interruption of electron
flow to the additional sites a t which PMS and dichloroindophenol are
reduced, rather than to an activating effect of ubiquinone on succinate
dehydrogenase.
Gutman e t al. (196, 197) have shown, however, that reduced ubiquin-
one might, indeed, be a regulator of succinate dehydrogenase activity.
Substrates (NADH and a-glycerophosphate) , which increased the level of
reduced ubiquinone in particles, activated succinate dehydrogenase as did
added reduced ubiquinone-10. Similarly, the resting state of mitochondria
achieved by respiratory control showed high succinate dehydrogenase
activity, which was rapidly diminished by the release of respiratory
control with ADP or uncouplers (195). The greater activity of succi-
nate dehydrogenase in the resting state has been interpreted as resulting
from the accumulation of reduced ubiquinone under these conditions. The
results of Gutman et al. are not subject to the complications of the work
of Rossi et al., even though the succinate dehydrogenase assay was the
same, i.e., with PMS and dichloroindophenol as electron acceptors. This
214a. Our preliminary results have shown that about 27% of the succinoxidase
+
or succinate + P M S 2.6-dichloroindophenol activity of subrnitochondrial particles
ran be measured in the absence of PMS with dichloroindophenol as electron acceptor.
This activity is enchanced in the presence of added cytochrome c, and inhibited
by antimycin A (80%) or TTFA.
4. METAL-CONTAINING FLAVOPHOTEIN DEHYDROGENASES 25 1
05
Succtnate activation -
I 1 -
5 10 I5 20 25
Time ( m i d
FIG.32. Activation of succinate dehydrogenase b y reduced ubiquinone-10. Phos-

phorylating submitochondrial particles ( ETPH) were resuspended in a sucrose-
Tris-Mg buffer, pH 7.4, a t 4 mg protein/ml. Antimycin A ( I nmole/mg) and cyanide
(1 m M ) were added and the sample placed under an atmosphere of NZ t o prevent
autoxidation of reduced ubiquinone-10. Ubiquinone-10 was reduced with borohydride,
neutralized with dilute acetic acid, and shaken till the first appearance of the yellow
color of oxidized uhiquinone-10 t o ensure removal of unreacted borohydride, all
a t 0". Activation of succinate dehydrogenase was started by adding 50 fi1 of either
reduced ubiquinone-10 (curve A) or of ubiquinone-I0 (curve B) in absolute ethanol
to 3 ml of enzymr giving 175 m M final concentration of the quinone. Curve C,
no addition. Samples were withdrawn a t intervals and assayed immediately at 17".
The horizontnl arrow indicates the maximal activation reached with succinate as
activator. From Gutman et al. ( 1 9 7 ) .
is because ( a ) all the preparations used by Gutman et al. contained

ubiquinone, and (b) in the experiments with NADH and added reduced
ubiquinone-10 thc activation of succinate dehydrogenase was time-depen-
dent (Figs. 32 and 33).
5. Mechaniswi
Study of the reaction mechanism of succinate dehydrogenase has been
complicated, partly because of the activation-deactivation properties Df
the enzyme, and partly because most of the carly preparations studied
had low iron content and low activities. Kinetic studies with activated,
soluble preparations have led Zeylemaker et al. (215) to propose the fol-
215. W. P . Zeglrmaker, D. V. DerVartanian, C. Veeger, and E. C. Slater, BBA
178, 213 (1969).
Time (min)
FIG.33. Activation of succinate dehydrogenase by NADH. A preparation of phos-
phorylating submitochondrial particles ( E T P d (succinoxidase activity = 1.18 pmoles
succinate per min per mg at 30") was washed by centrifugation in a sucrose-Tris-Mg
buffer (pH 7.4) and resuspended in the same buffer at 1 mg of protein/ml. Antimycin
A (1 nmole/mg protein) was added to slow the rate of aerobic oxidation of NADH,
followed by 0.25 mM NADH. Oxidation of the latter at 23" was monitored spectro-
photometrically at 340 nm (dashed line). Samples were removed periodically and
assayed immediately for succinate dehydrogenase activity in the presence of 0.33
mg of PMS/ml (solid line). A t 16 min a second aliquot of 0.25 mM NADH was
added. From Gutman et al. (197).
lowing sequence for the partial reactions involving the interaction of sub-
strate with the enzyme:
E +S=ESr
ESr 2 ESri
ESrr + A,, + E P f Aped
EP=E+P
where E, S, ESI, ESrr,A, and P are enzyme, substrate, ES complexes I and

11, electron acceptor, and product, respectively. Stereospecificity of the
enzyme for dehydrogenation of succinate is trans irrespective of which
hydrogen pair H,H, or H,H, is removed (216, 217). Monohalogen-substi-
tuted acids with the L-configuration have only one trans pair of hydro-
216. D. V. DerVartanian, W. P. Zeylemaker, and C. Veeger, in "Flavins and Flavo-
proteins," Symp. Adn Flavoproteins (E. C. Slater, ed.), p. 183. Elsevier, Amsterdam,
1966.
217. J. Rktey, J. Seibl, D. Arigoni, J. W. Cornforth,'G. Ryback, W. P. Zeylemaker,
and C. Veeger, Eur. J . Biochem. 14,232 (1970).
COOH
COOH
gens and serve as substrates. The D-stereoisomers do not serve as sub-

strates but bind as competitive inhibitors.
Electron paramagnetic resonance studies of DerVartanian et al. (218)
with the Veeger et al. succinate dehydrogenase (149) indicated that maxi-
mal radical concentration occurred a t a level corresponding to only about
20-300/0 of the bound flavin, and that the g = 1.94 signal accounted for
a t most 1 equivalent of unpaired electrons per mole of flavin. Recent
work of Beinert et al. (184) with the activated soluble and particulate
preparations indicates that succinate reduces not more than 60% of the
ferredoxin-type iron-sulfur center of succinate dehydrogenase within the
turnover time of the enzyme, and that flavin semiquinone formation
neither precedes nor significantly lags behind the reduction of the iron-
sulfur center. The larger subunit by virtue of carrying the bound flavin
is undoubtedly involved in substrate binding and electron transfer, and
the semiquinone form of the enzyme is considered to be a catalytically
significant intermediate. Iron-sulfur center S-1, which is substrate reduci-
ble and has an E m N 0 mV according to Ohnishi et al. (164), might be
the species that interacts with flavin. The role of iron-sulfur center 5-2
with Em N -260 mV is not known. Preparations of succinate dehydroge-
nase with as little iron as 2 or 4 g-atoms/mole of flavin exhibit PMS
reductase (albeit low) activity, g = 1.94 signal a t >20°K, and activa-
tion-deactivation effects. Therefore, it appears that these phenomena do
not require the intactness of both iron-sulfur centers. The presence of
PMS reductase activity in the 2-iron preparations further suggests the
possible presence in these preparations of a stable 2 iron-2 sulfur center
or of a fraction with higher iron-sulfur content which is responsible for
the g = 1.94 signal and PMS reduction.
The two subunits resolved by Davis and Hatefi (143, 166) by the use
of chaotropes and freeze-thawing were inactive, separately and in combi-
nation, for succinate oxidation or fumarate reduction. However, the possi-
bilities have not been fully explored. Nor has this sort of resolution been
performed on the cyanide-treated enzyme to see whether one or the other
subunit can be preferentially modified. Further work in these areas might
218. D. V. DerVartanian, C. Veeger, W. H. OrmeJohnson, and H. Beinert, BBA

191, 22 (1969).
provide important clues to the reaction mechanism of succinate dehydro-

genase and the role of the subunits.
B. SUCCINATE IN MICROORGANISMS
DEHYDROGENASE
All aerobic organisms, including yeast, appear to have a membrane-
bound succinate dehydrogenase containing iron and covalently bound
flavin (15, 16, 25). In contrast, the enzyme in anaerobic organisms is
found in the cytoplasm and appears to be more effective as a fumarate
reductase, a modification which is in accord with the physiological re-
quirements of the organism. I n facultative anaerobes such as E. coli and
S. cerevisiae, both the membrane-bound succinate dehydrogenase and the
cytoplasmic fumarate reductase are found, their synthesis and concentra-
tion depending on the growth conditions.
The succinate dehydrogenase of yeast mitochondria was isolated by
Singer et al. (15, 219) in 1957, and stated to have a molecular weight
of 200,000 and an iron:flavin ratio of 4:1, similar to the mammalian
enzyme. These studies antedated, however, the purification of mammalian
succinate dehydrogenase by Davis and Hatefi. Therefore, the exact mo-
lecular weight and composition of the yeast enzyme will have to be reex-
amined in light of present information.
Hatefi et al. (220) have isolated the succinate dehydrogenase of
Rhodospirillum rubrum by extraction of chromatophores with NaC10,.
The enzyme has two subnits of molecular weights of approximately
60,000 and 25,000 (Fig. 34) (22Oa). Both contain iron-sulfur chro-
mophores (221), and the larger subunit carries the covalently bound
flavin. I n the intact enzyme, the ratio of flavin:iron:labile sulfide is ap-
proximately 1:8 :8. The enzymic properties of this prokaryotic enzyme
are also very similar to the mammalian succinate dehydrogenase. Ferri-
cyanide and PMS are reduced a t comparable V,,,,, rates, and the former
inhibits above 1 mM. K , values with respect to succinate, PMS, and
ferricyanide are 0.23, 0.11, and 0.3 mM, respectively (220). At 77OK,
the succinate-reduced enzyme exhibits a free radical signal a t g = 2.00
and an iron-sulfur type of signal a t g = 1.93 (220).The absorption spec-
trum of the R . rubrum enzyme is very similar to that of mammalian
mitochondria.
A very interesting observation is that the R . rubrum succinate dehydro-
genase can cross-interact with alkali-inactivated mammalian respiratory
219. T. P. Singer, V. Massey, and E. B. Kearney, ABB 69, 405 (1957).
220. Y . Hatefi, K. A. Davis, H. Baltscheffsky, M. Baltscheffsky, and B. C. Johans-
son, ABB 152, 613 (1972).
220a. K. A. Davis, I. P. Crawford, and Y . Hatefi, in preparation.
FIG.34. Electrophoresis of Rhodospirillum rubrum succinate dehydrogenase on

SDS-polyacrylamide gel. The protein bands were visualized with Coomassie blue.
From Davis et al. ( 2 2 0 ~ ) .
particles to reconstitute succinoxidase activity (Fig. 35). This activity

is inhibited by TTFA. According to Hatefi et al. ( 2 2 0 ) , affinity of the
R. rubrum enzyme for reconstitution with the mammalian respiratory
chain is also similar to that of the mammalian succinate dehydrogenase.
Rhodospirillurn rubrum succinate dehydrogenase can also reconstitute
with alkali-inactivated R. rubrum chromatophores, but cross-interaction
of the latter with the mammalian enzyme, though it occurs, is not equally
efficient ( 2 2 1 ) .
Tisdale et al. (222) have shown that several isoeymes of fumarate re-
ductase occur in brewer’s yeast, ranging in molecular weight from 34,000
to 112,000. The predominant species had a molecular weight of 62,000-
221. Y. Hatefi, unpublished.
222. H. Tisdale, J. Hauber, G. Pragcr, P. Turini, and T. P. Singer, Eur. J. Biochem.
4, 472 (1968).
42rnin
FIQ.35. Reconstitution of succinoxidase activity of alkali-inactivated bovine heart

ETP with bovine and R . rubrum succinate dehydrogenases (SD). Left-hand trace :
114 pg alkali-treated ETP (alk-ETP) and 37 pg bovine SD per ml. Right-hand
trace: 172 pg alk-ETP and 46 pg R. rubrum SD per ml. I n both cases alk-ETP
and SD a t 10 times the concentrations indicated above were premixed with 10 m M
succinate and preincubated for 3 min a t 30" before addition t o the assay mixture.
Alk-ETP, bovine SD, or R . rubrum SD d o n e resulted in no oxygen uptake. Where
indicated, 5.9 m M TTFA and 0.15 mM PMS were used. Assay temperature 30".
S.A., specific activity. From Hatefi et nl. (220).
63,000. The enzyme contains noncovalently bound FAD, nonheme iron,

possibly copper, but no labile sulfide. Electron paramagnetic resonance
studies showed signals resulting from copper and high-spin ferric ions
a t g = 4.3.
IV. ~-Glycerol-3-phosphateDehydrogenase (EC 1.1.99.5 1
The oxidation of L-glycerol 3-phosphatc to dihydroxyacetone phos-

phate is catalyzed by two different enzymes. One is the cytoplasmic
NAD-linked a-glycerophosphate dehydrogenase, and the other is the
niitochondrial enzyme, which appears to contain flavin and iron. The
latter enzyme was first studied by Green in 1936 (223). It was shown
to be associated with respiratory particles, and widely distributed in ani-
mal tissues. The highest concentration of the enzyme was found in the
brain. Lardy and co-workers (264) studied the enzyme in deoxycholate-
solubilized particles obtained from skeletal muscle, confirmed the finding
223. D.E. Green, BJ 30, 629 (1936).

224. T. Tung, I,. Anderson, and H. A. Lardy, ABB 40,191 (1952).
I
400 500 600
Wavelength nm
FIG.36. Absorption spectrum of partially purified a-glycerophosphate dehydroge-

nase, protein concentration 13 mg/ml. (0) Spectrum of oxidized enzyme. The differ-
ence spectra (shown in the insert) were recorded after the addition of a few granules
of sodium hydrosulfite ( 0 )or 5 pmoles substrate (0) in a total volume of 0.2 ml.
I n the difference spectra, a decrease in optical density indicates bleaching. From
Ringler (226).
of Green with regard to the specificity of the enzyme for L-a-glycerophos-

phate as substrate, and demonstrated that the reaction product was dihy-
droxyacetone phosphate.
a-Glycerophosphate dehydrogenase has been solubilized by treatment
of pig brain mitochondria with phospholipase A (225). Only partial puri-
fication of the enzyme has been achieved. The best preparations of Ring-
ler and Singer (225-227) were shown to contain 1 mole of flavin per
2.1 X 10” g of protein and 1 g-atom of nonheme iron per 3.5 X lo5 g
of protein. I t has been claimed that the flavin is FAD (227). The absorp-
tion spectrum of the above preparation is shown in Fig. 36. Phenazine
methosulfate, ferricyanide, 2,6-dichloroindophenol, and methylene blue
have been used as electron acceptors. With PMS a s electron acceptor,
the best preparations of Ringler and Singer (226) from pig brain were
shown to oxidize a-glycerophosphate a t 38O and p H 7.6 a t a rate of 3.4
pmoles/min x mg protein. Under these conditions K, for a-glycerophos-
phate was shown to be 9.5 mM, the same as the particle-bound enzyme
(223). Dihydroxyacetone phosphate is a competitive inhibitor of the
mammalian enzyme; K , = 0.18 m M a t 3 8 O and p H 7.6. Attempts a t
reversing the action of a-glycerophosphate dehydrogenase in the presence
of dihydroxyacetone phosphate plus reduced FMN, leucobenzylviologen,
225. R. L. Ringler, JBC 236, 1192 (1961).

226. R . L. Ringler and T. P. Singer, “Methods in Enzymology,” Vol. 5, p. 432,
1963.
227. T. P. Singer, “The Enzymes,” 2nd ed., Vol. 7, p. 345,1963.
or leucomethylviologen as electron donor have not been successful (226).

a-Glycerophosphate dehydrogenase is believed to be located in the
outer phase of the mitochondria1 inner membrane (228). The enzyme
appears to donate electrons to the respiratory chain beyond the level a t
which Amytal inhibits NADH oxidation (229). It has been shown with
the use of pentane-extracted mitochondria that electron transfer from
the enzyme to the respiratory chain, but not to dyes, requires the presence
of ubiquinone, and is inhibited by antimycin A (230). Therefore, i t ap-
pears that a-glycerophosphate dehydrogenase interacts with the mito-
chondrial electron transport system a t the level of ubiquinone, which is
also the point of convergence of complexes I, 11, and I11 (29, 33, 231).
I n agreement with the above findings, Ringler and Singer (177) have
made the important observation that in antimycin-treated brain mito-
chondria the oxidation of a-glycerophosphate to dihydroxyacetone phos-
phate can be linked by way of succinate dehydrogenase to the reduction
of fumarate. Further, Szarkowska and Drabikowska (232) have demon-
strated the reduction of exogenous ubiquinone-6 by the particle-bound
and the phospholipase-solubilized a-glycerophosphate dehydrogenase
from pig brain. Both systems were inhibited by 3-phosphoglycerate. The
best rate reported by these investigators for the soluble enzyme is 0.24
pmole QG reduced/min x mg protein at 37O and pH 7.2, which is only
7% of the PMS reductase activity of similar preparations. This low
activity might in part have resulted from assay difficulties, which are
usually encountered when water-insoluble homologs of ubiquinone are
used as electron acceptor. It is also possible that (a) by analogy to the
early preparations of succinate dehydrogenase (see Section 111), the
preparation of a-glycerophosphate dehydrogenase used in these studies
was damaged with respect to ubiquinone reduction, and (b) the reduction
of ubiquinone by the dehydrogenase occurs by way of an unknown
electron carrier in which the enzyme preparation used was deficient.
Mitochondria from the flight muscle of house flies, Musca dornestica,
have been shown to oxidize a-glycerophosphate a t exceptionally high
rates (233, 234). This activity was shown to be inhibited by EDTA. It
is believed that in these and other mitochondria the combined action of
the soluble and the particle-bound a-glycerophosphate dehydrogenases
228. M. Klingenberg, Eur. J. Biochem. 13, 247 (1970).

229. B. Chance and B. Sacktor, ABB 76, 509 (1958).
230. J. I. Salach and A. J. Bednarz, ABR 157, 133 (1973).
231. Y. Hatefi, Clin. Chem. 11, 198 (1965).
232. L. Szarkowska and A. K. Drabikowska, LifeSci. 7,519 (1963).
233. R. W. Estabrook and B. Sacktor, JBC 233, 1014 (1958).
234. B. Sacktor and D. G. Cochran, ABB 74,266 (1958).
ouler membranes inner
Cytosol
Glycerol- 1 -P
Dihydroxy-
acetone-P
It
FIG.37. The a-glycerophosphate cycle for the oxidation of extramitochondrial
NADH by the mitochondrial respiratory chain. From Klingenberg (928).
provides the principal route for the transfer of reducing equivalents from
cxtramitochondrial NADH to the mitochondrial electron transport SYS-
tem (228, 229, 233-237). This process has been termed the “a-glycero-
phosphate cycle” (Fig. 37). Lee and Lardy (238) and others (239-242)
have found that in the rat the a-glycerophosphate dehydrogenase activity
of mitochondria from liver, kidney, adipose tissue, and heart were in-
creased severalfold upon feeding of desiccated thyroid glands. The activ-
ity increase appeared to be organ-specific and particularly marked in
liver, which showed a 20-fold increase after 10 days. The activity of the
enzyme in brain, lung, spleen, stomach, small intestine, and testis was
not appreciably increased. Thyroidectomy resulted in the decrease or dis-
appearance of particle-bound a-glycerophosphate dehydrogenase activity
in several organs, and a single injection of triiodothyronine restored the
activity within 48 hr. The increased activity of a-glycerophosphate dehy-
drogenase induced by the thyroid hormone appears to result from synthe-
sis of new enzyme (239).The possible role of this striking, organ-specific
effect of the thyroid hormone has been discussed in relation to increased
carbohydrate degradation in response to the increased oxidation of extra-
mitochondrial NADH by the a-glycerophosphate cycle (238), as well as
in relation to its effect on phospholipid synthesis ( 243) .
235. R. W. Estabrook and B. Sacktor, ABB 76, 532 (1958).
236. B. Sacktor, L. Packer, and R. W. Estabrook, .4BB 80, 68 (1959).
237. B. Sacktor and A. Dick, JBC 237,3259 (1962).
238. Y.-P. Lee and H. A. Lardy, JBC 240, 1427 (1965).
239. Y.-P. Lee, A. E. Takernori, and H. Lardy, JBC 234, 3051 (1959).
240. H. A. Lardy, Y.-P. Lee, and A. Takemori, Ann. N . Y. Acad. Sci. 86, 506
(1960).
241. 0. Z. Sellinger and K.-L. Lee, BBA 91, 183 (1964).
242. G. H. Isaacs, B. Sacktor, and T. A. Murphy, BBA 177, 196 (1969).
243. W. R. Frisell and J. R. Cronin, ir, “Electron and Coupled Energy Transfer
in Biological Systems” (T. E. King and M. Klingenberg, eds.), Vol. 1, Part A, p.
177. Dekker, New York, 1971.
Flavin-containing a-glycerophosphate dehydrogenases have been found

also in Streptococcus faecalis (244) and Propionibacterium arabinosum
(245). The enzyme from 8. faecalis is reported to contain FAD, have
a K , for a-glycerophosphate of 4 mM, and a pH optimum of 5.8. I n
addition t o dyes, this enzyme can interact directly with molecular oxygen
to form H,O,. The preparation from P . arabinosum is particle-bound,
has a K , for a-glycerophosphate of 26 p M , and is claimed to contain
flavin and nonheme iron.
V. Choline Dehydrogenase (EC 1.1.99.1 1
The oxidation of choline to betaine is catalyzed by two enzymes. First,

choline is oxidized to betaine aldehyde by a n enzyme which is found in
mitochondria in membrane-bound form. This enzyme is believed to be
a flavoprotein containing nonheme iron. Betaine aldehyde is then oxidized
to betaine by a soluble enzyme, which is NAD-linked. Betaine aldehyde
dehydrogenase appears to be present both in mitochondria and the soluble
fraction of liver (243,246‘).
The existence of choline dehydrogenase was first demonstrated by Mann
and Quastel in 1937 (247, 248) in extracts of rat liver and kidney. These
authors also obtained evidence that the first oxidation product of choline
was betaine aldehyde. Others showed subsequently that choline oxidase
activity resided in the mitochondria1 fraction of rat liver and is linked
to the respiratory chain (249, 250). Detergents (251, 252), solvent treat-
ment of fragmented mitochondria (253), and venom phospholipase
(254-256‘) have been used for extraction and solubilization of choline
dehydrogenase. Among these, the best method reported to date appears
to be the digestion of acetone-powdered mitochondria with venom phos-
pholipase. Choline dehydrogenase, partially purified from phospholipase
extracts of rat liver mitochondria, contains 1 mole of flavin and 4 g-atoms
of nonheme iron per 850,000 g protein. The flavin is claimed to be acid-
244. N. J. Jacobs and P. J. VanDemark, ABB 88,250 (1960).
245. N. Sone, J . Biochem. (Tokyo) 74,297 (1973).
246. J. L. Glenn and M. Vanko, ABB 82, 145 (1959).
247. P. J. G. Mann and J. H. Quastel, BJ 31, 869 (1937).
248. P. J. G. Mgnn, H. E. Woodward, and J. H. Quastel, BJ 32, 1025 (1938).
249. C. J. Kensler and H. Langemann, JBC 192, 551 (1951).
250. J. N. Williams, Jr., JBC 194, 139 (1952).
251. J. N. Williams, Jr. and A. Sreenivasan, JBC 203, 899 (1953).
252. M. Korgenovsky and B. V. Auda, BBA 29,463 (1958).
253. K. Ebisuzaki and J. N. Williams, Jr., BJ 60, 644 (1955).
254. G . Rendina and T. P. Singer, BBA 30,441 (1958).
255. G. Rendina and T. P. Singer, JBC 234, 1605 (1959).
256. T. Kimura and T. P. Singer, “Methods in Enzymology,” Vol. 5, p. 562, 1962.
extractable FAD, and the enzyme preparation is reported to contain trace

amounts of a b-type cytochrome.
For assay of the activity of membrane-bound enzyme, molecular oxy-
gen, cytochrome c, ferricyanide, PMS, and dichloroindophenol can be
used (254).The soluble enzyme reacts only with the latter two electron
acceptors (227, 255, 256), thus indicating that other acceptors interact
indirectly by way of the niitochondrial electron transport system. With
PMS as electron acceptor, the K , for choline a t 38O and p H 7.6 is about
7 mM (227, 255). The best preparations of Kimura and Singer (256)
oxidize choline a t a rate of 5.3 pmoles/min x mg protein a t 3 8 O . I n addi-
tion to choline, the particulate enzyme has been reported to oxidize
arsenocholine (248) and other choline analogs (25‘7).Choline dehydroge-
nase is very sensitive to thiol inhibitors, and choline has been reported
to protect against inhibition by p-mercuribenzoate (258).The oxidation
of choline is competitively inhibited by betaine aldehyde ( K i = 2 mM)
(256).Nitrogen mustard has also been reported to be a strong competi-
tive inhibitor ( 2 5 9 ) , but others have reported no inhibition of choline
dehydrogenase by nitrogen mustard ( 2 6 0 ) .
Information regarding the involvement of flavin and iron in enzyme
catalysis is not available. Rothschild et al. (258) have reported that
dialysis of rat liver particles resulted in the loss of choline-cytochrome
c reductase activity, which could be restored by addition of FAD but
not FMN. However, these results have not been substantiated by others
(255). Singer has stated that the difference spectrum of the enzyme
“shows bleaching by substrate in both the flavin and the iron regions”
(227).This spectrum has not been published.
Relation to the Electron Transport System
It was shown by Strength et al. (261) that the oxidation of choline
by a particulate preparation from rat liver was considerably enhanced
upon addition of NAD. Others showed that choline oxidation by isolated
rat liver mitochondria was completely inhibited by Amytal when oxygen,
cytochrome c, ferricyanide, or methylene blue was the electron acceptor
(262-664). Choline dehydrogenase activity of particles and soluble prepa-
257. I. C. Wells, JBC 207, 575 (1954).
258. H. A. Rothschild, 0. Cori, and E. S. G. Barrbn, JBC 208, 41 (1954).
259. E. S. G. Bnrron, G. R. Bartlett, and Z. B. Miller, J . E x p . M e d . 87, 489 (1948).
260. A . Sivak, A. J. Mahoney, Jr., and W. I. Rogers, Biochem. Pharmacol. 16,
1919 (1967).
261. D. R. Strength, J . R. Christensen, and L. J. Daniel, JBC 203, 63 (1953).
262. L. Packer, R. W. Estabrook, T. P. Singer, and T. Kimura, JBC 235,535 (1960).
263. L. Ernster, 0. Jalling, H. Low, and 0. Lindberg, Exp. Cell R e s . Suppl., 3,
124 (1955).
264. 0. Rendina and T. P. Singer, F e d . Proc., F e d . A m e r . SOC.Exp. Biol. 18,
308 (1959).
rations was reported to be insensitive to Amytal when assayed with P M S

as electron acceptor (227, 262). Bianchi and Azzone (265) confirmed the
findings of Strength et al. and showed that choline oxidation by intact
rat liver mitochondria, but not by swollen mitochondria, was partially
inhibited by rotenone. They further demonstrated that addition of choline
to liver mitochondria in the presence of A D P resulted in the reduction
of intramitochondrial nicotinamide nucleotides, and that under anaerobic
conditions choline oxidation could be linked to the reduction of oxaloace-
tate to malate in the absence of an energy supply. These and other results
led to the conclusion that reducing equivalents from choline dehydroge-
nase to the respiratory chain of intact mitochondria passed in part
through the rotenone- and Amytal-sensitive site 1 of phosphorylation in
the NADH oxidase pathway, and in part through another Amytal-sensi-
tive, but rotenone-insensitive . point to ubiquinone and cytochrome b
(243).
Kimura e t al. (266) showed that, unlike a-glycerophosphate, the oxida-
tion of choline to betaine aldehyde in anaerobic mitochondria could not
be linked to fumarate reduction. They also reported t h a t the choline oxi-
dase activity of rat liver mitochondria was partially resistant to inhibi-
tion by antimycin A and quinoline oxide. They concluded, therefore, t h a t
the mitochondria1 choline and succinate oxidase pathways were separate.
The two chains were interlinked only between cytochrome cI and oxygen,
and the choline chain involved an autoxidixable cytochrome b. These
complications and the NAD stimulation of choline oxidation were re-
solved to a considerable extent by the work of Feinberg et al. (267) and
Estabrook and his colleagues (268). The former group showed that the
NAD stimulation was abolished when semicarbazide was present during
choline oxidation. Under these conditions, semicarbazide interacted with,
and prevented the NAD-linked oxidation of, betaine aldehyde which was
formed as a result of choline oxidation. I n the absence of semicarbazide,
choline oxidation was stimulated by NAD. These and other workers con-
cluded that the rotenone inhibition of choline oxidation, which occurred
in intact mitochondria, even in the presence of semicarbazide, involved
an interference with the rate of entry of choline into intact mitochondria.
Estabrook and co-workers (268) showed very clearly ( a ) that the anti-
mycin-resistant oxygen uptake by mitochondria in the presence of choline
resulted from inhibitor-resistant oxidation of endogenous substrates ; (b)
that rotenone has little effect on choline oxidation by submitochondrial
265. G. Bianchi and G. F. Azzone, JBC 239, 3947 (1964).
266. T. Kimura, T. P. Singer, and C. J. Lusty, BBA 44, 284 (1960).
267. R. H. Feinberg, P. R. Turkki, and P. E. Witkowski, JBC 242, 4614 (1967).
268. D. D. Tyler, J. Gonze, and R . W. Estabrook, ABB 115, 373 (1966).
particles, whereas under the same conditions Amytal exerts a strong in-
hibitory effect; (c) that contrary to the previous report of others, the
oxidation of choline by submitochondrial particles in the presence of PMS
as electron acceptor was inhibited by Amytal; (d) that the concentration
of Amytal needed for 50% inhibition of NADH (or 3-hydroxybutyrate)
and choline oxidation by submitochondrial particles were 0.2 and 0.7 mM,
respectively; and (e) that on the basis of spectra recorded a t 77OK, the
nature and the degree of cytochromes reduced in r a t liver mitochondria
were the same when the system was allowed to reach anaerobiosis as
a result of succinate oxidation in the presence of rotenone or choline oxi-
dation in the presence of rotenone and malonate.
It has been shown that the oxidation of choline by isolated r a t liver
mitochondria is biphasic (269). The initial phase of choline oxidation
is slow and coupled to the uptake of inorganic phosphate. The ensuing
phase is 3-5 times faster and not coupled t o phosphorylation. The slow
phase can be extended in the presence of Mg2+and A D P or ATP. These
compounds are considered t o control the permeability of mitochondria
to choline ( 2 7 0 ) .Calcium ions and conditions which result in mitochon-
drial swelling and membrane disruption have been shown to increase
choline oxidation (266, 271).
VI. lactate Dehydrogenases
Three types of lactate dehydrogenase are found in yeast, which may

be considered as metal-containing flavoproteins. These are L-lactate :cy-
tochrome c reductase or cytochrome b,, D-lactatc dehydrogenase, which
is found in anaerobic yeast, and D-1actate:cytochrome c reductase, which
is associated with the mitochondria of aerobic cells.
A. CYTOCHROME
L (+)-LACTATE: c OXIDOREDUCTASE
(CYTOCHROME
b,) ( E C 1.1.2.3)
This enzyme [also known a t L (+)-lactate dehydrogenase] was first
extracted from bakers’ yeast by Bernheim in 1928 (272). Bach et al.
(273) showed in 1942 that lactate dehydrogenase copurified with a species
of cytochronie b, which contained protoheme as prosthetic group. The
269. T. Kagawa, D. R. Wilken, and H. A. Lardy, JBC 240, 1836 (1965).

270. D. R. Wilken, T. Kagawa, and H. A. Lardy, JBC 240, 1843 (1965).
271. G. R. Williams, JBC 235, 1192 (1960).
272. F. Bernlieini, BJ 22, 1179 (1928).
273. S. J. Bach, M. Dixon, and L. G. Zerfas, Nature (London) 149, 48 (1942).
cytochrome was designated cytochrome b, by Keilin and co-workers

(274). The enzyme was crystallized by Appleby and Morton in 1954
(275, 276) and shown to contain FhlN in amounts equimolar to heme.
This also marked the first crystallization of a cytochrome. These studies
were confirmed by others (277) and extended to show that lactate dehy-
drogenase was specific for L (+) -lactate, and inhibitable by p-mercuri-
benzoate and Atebrin. Appleby and Morton (278) showed subsequently
that the crystalline enzyme contained 5-6% DNA, but that the polynu-
cleotide was not essential for activity. The crystalline preparation con-
taining DNA is known as type I cytochrome b,, and the preparation
from which DNA has been removed is known as type I1 cytochrome b,.
These early studies have been reviewed (279-282).
1. Physical Properties
Cytochrome b, is found as a soluble protein in the autolysates of Sac-
charonayces cerevisiae. The crystalline preparations of Appleby and
Morton (278) were shown to sediment as a single peak in the ultracentri-
fuge. Minimum molecular weight based on amino acid analysis and a
heme extinction coefficient of 232 mM-l cm-’ was calculated to be 53,000
(283). The heme extinction coefficient was then corrected to 183 mM-l
cm-’, and the minimum molecular weight per mole of heme recalculated to
be 58,600 ( 2 8 4 ) . It was concluded that cytochrome b, is a tetrameric
structure. This conclusion agreed with the results of X-ray diffraction
studies on type I and type I1 crystals, which indicated molecular weights
of 235,000 2 10,000 and 234,000 & 8,000, respectively, for these two
preparations of cytochrome b, (285).The oxidized and reduced spectral
bands of cytochrome b, are given in Table XIV.
Subsequent studies showed that reduction and carboxymethylation of
crystalline cytochrome b, yielded two unlike subunits of approximately
274. S. J. Bach, M. Dixon, and D. Keilin, Nature (London) 149, 21 (1942).

275. C. A. Appleby and R. K. Morton, Nature (London) 173, 749 (1954).
276. C. A. Appleby and R. K. Morton, BJ 71, 492 (1954).
277. E. Boeri, E. Cutolo, M. Luzzati, and L. Tosi, ABB 56, 487 (1955).
278. C. A. Appleby and R. K. Morton, BJ 75,258 (1960).
280. A. P. Nygaard, “The Enzymes,” 2nd ed. Vol. 7, p. 557, 1963.
281. T. P. Singer, C. Gregolin, and T. Cremona, in “Control Mechanisms in Respi-
ration and Fermentation” (B. Wright, ed.) , p. 47. Ronald Press, New York, 1963.
282. A. P. Nygaard, in “Control Mechanisms in Respiration and Fermentation”
(B. Wright, ed.), p. 27. Ronald Press, New York, 1963.
283. C. Jacq and F. Lederer, E w . J . Biochem. 12, 154 (1970).
284. P. Pajot and 0. Groudinsky, Eur. J. Biochem. 12, 158 (1970).
285. C. Monteilhet and J. L. Risler, Eur. J. Biochem. 12, 165 (1970).
TABLE XIV
PROPERTIES
SPECTRAL b t (TYPE
OF CYTOCHROME II)O
Oxidized form Reduced form
x B x €
Band (nm) (mM-1 cm-1) (nm) (mM-' cm-l)
a 560 9.2 557 30.9

B 530 11.3 528 15.6
r 413 129.5 424 183
s 360-365 34.4 328 39
uv 275 83.5 269 88
From Pajot and Groudinsky (284).
21,000 and 36,000 daltons (286).These subunits had different amino acid
compositions, and other results suggested that the heme binding site is
on the heavy chain. At this time, Baudras (287) showed that L ( + ) -lac-
tate: cytochrome c reductase isolated from the yeast Hansenula anomala
was very similar to the Saccharamyces enzyme in molecular weight and
heme and flavin content, but was considerably more stable and six times
more active. Moreover, unlike the Saccharamyces enzyme, the activity
of Hansenula cytochrome b, was inhibited in the presence of excess sub-
strate. The Hansenula cytochrome b, appeared to be composed of four
subunits of approximately 61,000 ? 5,000 daltons each (288). Baudras
and Spyridakis (689) suggested, therefore, that the 21,000- and 36,000-
dalton subunits of the Saccharomyces enzyme were the result of artifac-
tual splitting during isolation and crystallization of the type I cyto-
chrome b,. The differences between the Hansenula and the Saccharomyces
preparations of cytochrome b, were resolved by Jacq and Lederer (290)
who showed that, when prepared in the presence of the protease inhibitor
phenylmethylsulfonyl fluoride, the Saccharomyces enzyme does not crys-
tallize as before, and shows a subunit size comparable to that of the
Hansenula cytochrome b,. The enzyme so prepared had considerably im-
proved stability and enzymic properties, and was inhibited at high lactate
concentrations. It was concluded that the uncleaved, physiological form
of Saccharomyces cytochrome b, has a molecular weight of 230,000, and
is composed of four identical subunits, each associated with one FMN
286. F. Lederer and A.-M. Simon, Eur. J . Biochem. 20, 469 (1971).
287. A. Baudras, Biochimie 53, 929 (1971).
288. F. Labeyrie and A. Baudras, Eur. J. Biochem. 25,33 (1972).
289. A. Baudras and A. Spyridakis, Biochimie 53, 943 (1971).
290. C. Jacq and F. Lederer, Eur. J. Biochem. 25, 41 (1972).
TABLE XV
PARAMETERS
MOLECULAR OF INTACT
A N D CLEAVED
CYTOCHROME
bp
Parameter Intact Cleaved
N-Terminal residues Glu

a-Subunit iLys
Val
C-Terminal residues Ala

a-Subunit
I
/%Subunit ( Ala
LYE
Ala
ASP
Minimum molecular weight 58,100+7% 53,000 i~ 3%

per heme (amino acids) (amino acids)
58,600 f 2%
(dry weight)
Molecular weight of peptide 57,500 =-Subunit 33,000-36,000
chains
8-Subunit 21,000
Molecular weight 220,000 f 10% 220,000 f 10%
(gel filtration) (gel filtration)
234,600 f 4%
(crystallography)
240,000 f 4%
(ultracentrifugation)
From Jacq and Lederer (291).
and one heme ($991).The amino acid composition of the enzyme prepared
in the presence of phenylmethylsulfonyl fluoride has been determined, and
it has been shown that alanine and glutamic acids are the C- and N-
terminal residues, respectively (Table XV) (2991).These results indicated
that, by comparison, the early crystalline preparations involved nearly
10% loss of peptide material, and circular dichroism spectra a t the Soret
region of cytochrome b, showed a modification of the heme environment
in the cleaved enzyme (2991).
2. Cytochrorne b, Core
Tryptic hydrolysis of cytochrome b, yields a polypeptide fragment
which carries the heme and has a molecular weight of approximately
291. C. Jacq and F. Lederer, Eur. J. Biochem. 41,311 (1974).
11,000 (292). This material, designated cytochrome b, core, resembles

the whole enzyme in its reduction potential, light absorption, and EPR
spectra. Moreover, cytochrome b, core was shown to resemble soluble
preparations of liver microsomal cytochrome b, in several respects, in-
cluding cytochrome absorption spectrum, extinction coefficient, reduction
potential, E P R signals at alkaline pH, and proton NMR spectra of the
oxidized and reduced preparations (293, 294). Recently, Guiard et al.
(295) have shown that the amino acid sequence of cytochrome b, core
is very similar to that of microsomal cytochrome b,. They have also
indicated that the amino acid sequence of cytochrome b, core is compati-
ble with the peptide chain folding recently determined by others for cyto-
chrome b,, and thus affords a similar heme environment as well.
Cytochrome b, is reduced in microsomes by the enzyme cytochrome b,
reductase, which is a flavoprotein. I n cytochrome b,, both the flavin and
the heme are found in association with the same polypeptide chain. Thus,
Guiard et al. (295) considered the possibility of a common ancestral
origin for cytochromes b, and b,. They suggested that a pair of genes
coding for cytochrome b, and cytochrome b, reductase might have fused
in the course of evolution leading to cytochrome b,.
Cytochrome b, is stereospecific for L ( + ) -lactate. It also oxidizes other
a-hydroxymonocarboxylic acids a t slow rates (280, 298). As electron ac-
ceptors ferricyanide, methylene blue, 2,6-dichloroindophenol, 1,a-naph-
thoquinone 4-sulfonate, and cytochrome c have been used. This wide
acceptor specificity is characteristic of a number of flavoproteins, which
are generally capable of reducing quinoid structures and ferric compounds
(297). However, as will be seen below, cytochrome c is considered to
be the physiological electron acceptor for the yeast L-lactate
dehydrogenase.
Much of the available enzymic work on cytochrome b, has been per-
formed on type I and type I1 enzymes which, as mentioned above, appear
to have suffered limited proteolysis and peptide cleavage of the subunits
292. F. Labeyrie, 0. Groudinsky, Y. Jacquot-Armand, and L. Naslin, BBA 128,

492 (1966).
293. H. Watari, 0. Groudinsky, and F. Labeyrie, BBA 131, 592 (1967).
294. R. Keller, 0. Groudinsky, and K. Wiithrich, BBA 328, 233 (1973).
295. B. Guiard, 0. Groudinsky, and F. Lederer, Proc. Nat. Acad. Sci. U . S., 71,
2539 (1974).
296. R. H. Symons and L. A . Burgoyne, “Methods in Enzymology,” Vol. 9, p.
314, 1966.
297. M. Dixon, BBA 226,269 (1971).
during purification. These preparations are very unstable and their en-
zymic properties as compared to crude yeast extracts reflect the structural
damage they have sustained during purification (290, 291 ) . Comparative
data regarding molar activities, K , values for substrate and cytochrome
c, and inhibition by high levels of substrate have been published for the
intact and cleaved Saccharomyces enzymes as well as for the intact cyto-
chrome b, isolated from Hansenula anomalu (287, 289-291).
It is generally agreed that the rate-limiting step is the transfer of re-
ducing equivalents from substrate to the enzyme, that the initial reaction
rate is first order with respect to substrate concentration, that flavin is
the first electron acceptor (298-300), and that the transfer of electrons
from flavin to the heme occurs intramolecularly (300). Anaerobic titra-
tion with L-lactate has indicated that the enzyme accepts three electrons
(301). It has also been shown by EPR studies that upon reduction of
the enzyme with L-lactate, a flavin semiquinone is formed to the extent
of about 20% of the flavin content of the enzyme (301). However, it
is not known whether the flavin semiquinone is a kinetic intermediate
during enzyme catalysis.
Ferricyanide appears to accept electrons from both the flavin and the
heme (299-302), and it is believed that heme is required for cytochrome
c reduction. Forestier and Baudras (30.2)have reported that, by treat-
ment with guanidinium chloride, preparations of cytochrome b, could be
rendered partially deficient in flavin and heme. Thus, enzyme prepara-
tions were obtained which contained 65-75% flavin and variable amounts
of heme from about 12 to 100%. The low heme preparations showed con-
siderably greater loss of cytochrome c reductase than ferricyanide reduc-
tase activity. When preparations with increasing content of heme relative
to flavin were tested, both the ferricyanide and the cytochrome c reduc-
tase activities increased as a linear function of heme to flavin ratio (up
to heme: flavin = 1) , but the increase in the heme content had a much
greater effect on the cytochrome c reductase activity of the enzyme. The
apoenzyme of cytochrome b , has been prepared. However, reconstitution
with FMN, heme, and F M N plus heme in all cases resulted in extremely
298. M. Iwatsubo, A. Baudras, A. di Franco, C. Capeillere, and F. Labeyrie, in
“Flavins and Flavoproteins,” 2nd Int. Symp. (K. Yagi, ed.), p. 41. Univ. Park Press,
299. A. Baudras, C. Capeillere-Blandin, M. Iwatsubo, and F. Labeyrie, in “Strbc-
ture and Function of Oxidation Reduction Enzymes” (A. Akeson and A. Ehrenberg,
eds.), p. 273. Pergamon, Oxford, 1972.
300. R. K. Morton and J. M. Sturtevant, JBC 239, 1614 (1964).
301. K. Hiromi and J. M. Sturtevant, JBC 240, 4662 (1965).
302. J.-P. Forestier and A . Baudras, in “Flavins and Flavoproteins,” 3rd Int.
Symp. (H. Kamin, ed.), p. 599. Univ. Park Press, Baltimore, Maryland, 1971.
low activities (SOZ-SO4). Now that intact preparations of cytochrome b,

are available, further efforts on these lines might yield clearer results re-
garding the roles of flavin and heme in the reduction of cytochrome c
and artificial acceptors.
The kinetics of cytochrome c reduction by L-lactate dehydrogenase are
somewhat complicated because the enzyme binds cytochrome c strongly
(K, = M ) (299, 300, 305). The cleaved enzyme binds one mole of
cytochrome c per mole, but the intact preparations of Haiisenula bind
4 moles of cytochrome c per mole of enzyme, i.e., one mole of cytochrome
c per subunit (287). The cytochrome b,-cytochrome c adduct of Sac-
charoinyces can be crystallized. Examination of the crystals have sug-
gested that the crystal lattice of cytochrome b, can accommodate cyto-
chrome c without an apparent change in the crystal structure (299, 305).
The association of cytochrome c with L-lactate dehydrogenase does not
depend on the oxidation-reduction state of either cytochrome, and similar
to L-lactate protects the enzyme against denaturation by 3 M urea (299,
305). The interaction of cytochrome c with L-lactate dehydrogenase is
considered to be specific. I n addition to the above results, i t has been
shown that lysozyme, which is similar to cytochrome c in size and charge,
does not compete for the binding of cytochrome c to the enzyme (299,
305).
The role of L-lactate dehydrogenase in the physiology of aerobic yeast
is not clear. It has been shown that its presence in yeast depends on
the availability of oxygen (306), and that in the presence of antimycin
A, which inhibits electron transfer to cytochrome c from NADH-linked
substrates, L-or D-lactate can partially support the growth of Saccharo-
myces cerevisiae (307). Under these conditions, cyanide inhibited the
growth. Therefore, i t has been concluded that L- and D-lactate-cyto-
chrome c reductases can feed electrons to the respiratory chain at the
level of cytochrome c and provide energy through the third site of oxida-
tive phosphorylation (30’7).
B. D(-)-LACTATE:CYTOCHROME (EC 1.1.2.4)

c OXIDOREDUCTASE
This enzyme is tightly associated with the mitochondria of aerobic
yeast. Similar to L-lactate: cytochrome c reductase, it is produced in yeast
303. R. K. Morton and K. Sheplcy, Biochem. Z . 338, 122 (1963).
304. M. Mevel-Ninio, P. Pajot, and F. Labeyrie, Biochimie 53, 35 (1971).
305. A. Baudras, M. Krupas, and F. Labeyrie, Eur. J. Biochem. 20, 58 (1971).
306. F. Labeyrie and M. Somlo, “Homologous Enzymes and Biochemical Evolu-
tion Colloquium” (Nguyen van Thoai*and J. Roche, eds.), p. 93. Gordon & Breach,
New York, 1968.
307. P. Pajot and M. Claisse, Proc. Znt. Congr. Biochem., 9,1973 p . 239 (1973).
during oxygen adaptation. It was suggested that both the D- .and the
L-lactate cytochrome c reductases arise during oxygen adaptation from
the D-2-hydroxyacid dehydrogenase of anaerobic yeast. However, this
hypothesis has not found experimental support (308-310).
1. Physical Properties
D-Lactate: cytochrome c reductasc has been extensively purified from
the respiratory particles of bakers’ yeast by two different methods (308,
311-313). One method involves the treatment of particles with acetone
and n-butanol, and the other involves treatment with Triton X-100, phos-
pholipase A, and bacterial protcase. The latter method appears to result
in greater purification, and higher yield, activity, and stability of the
enzyme (312, 313). According to Gregolin and Singer (312), purified
preparations of D-lactate: cytochrome c reductase contain 1 mole of FAD
*
per 50,000 5,000 g protein, and 1 g-atom of Zn2+ per 22,000-27,000 g
protein. They have concluded that the flavin content and the sedimenta-
tion constant of S = 6.8 suggest that the enzyme has a molecular weight
of about 100,000 and contains 2 moles of FAD and 6 6 g-atoms of Zn2+
per mole. These conclusions are subject to change, however, because the
diffusion constant and the partial specific volume of the enzyme are not
known, and partial loss of flavin during purification of the enzyme cannot
be ruled out.
D-Lactate :cytochrome c reductase can oxidize D-2-hydroxymonocar-
boxylic acids, but only D-lactate and D-2-hydroxybutyrate are oxidized
at appreciable rates. The enzyme exhibits a similar high specificity for
electron acceptors. It reacts with cytochrome c and phenazine methosul-
fate as electron acceptors, but not with ferricyanide, methylene blue, 2,6-
dichloroindophenol, and menadione (308, 312, 313). With Dlactate as
substrate and a t V,,, with respect to acceptor, phenazine methosulfate
is reduced a t 30° eight times as fast as cytochrome c (308). The K ,
values a t 30° and pH 7.5 are D-lactate, 0.29 mM; ~-2-hydroxybutyrate,
1.4 mM; phenazine methosulfate, 4.5 mM; and cytochrome c, 5.4 p M .
The turnover number of the enzyme, isolated with the use of Triton
308. C. Gregolin and T. P. Singer, BBRC 4, 189 (1961).
309. A. P.Nygaard, JBC 236, 1585 (1961).
310. T. P. Singer, E. B. Kearney, C. Gregolin, E. Boeri, and M. Rippa, BBA
54, 52 (1961).
311. A. P.Nygaard, JBC 236, 920 (1961).
312. C.Gregolin and T. P. Singer, BBA 67, 201 (1963).
313. T. P. Singer and T. Cremona, ‘‘Methods in Enzymology,” Vol. 9, p. 302,
1966.
4. METAL-CONTAINING FLAVOPROTEIN DEHYDROGENASES 27 1
TABLE XVI
I N H I B I T O R S O F D ( -)-LACTATk:: CYTOCHROME C REDUCTASEa
Concentration Inhibition
Inhibitor (M) (%)
p-Mercuriphenyl sulfonate 5 x 10-7 60

p-Mercuriphenyl sulfonate 5 x 10-3 60
H202 I x 10-3 0
Oxalate 6 x 10-8 22
Oxalate 1x 10-6 50
Oxalate 5 x 10-3 92
EDTA 4 x 10-3 25
EDTA 1x 10-2 51
o-Phenanthroline* 3.5 x 10-3 95
o-Phenan throline" 3.5 x 10-3 90
~
a From Gregolin and Singer (319).

* Overnight dialysis a t p H 6.5 against the indicated concentration of inhibitor.
Incubated for 15 min a t 30'.
X-100 and phospholipase A, is reported to be 90,000 moles lactate/

min x mole flavin at 30° and pH 7.5. The reaction of the enzyme does
not appear to be reversible (312).
D-Lactate :cytochrome c reductase is inhibited by p-mercuriphenyl sul-
fonate salts, metal chelators, and dicarboxylic acids such as oxalate and
oxaloacetate (Table XVI) (312, 314, 315). According to Nygaard (314),
salts (cations) inhibit a t the acceptor site, and dicarboxylic acids a t the
substrate site. Cremona and Singer (315) have studied the inhibitions
by metal chelators and by oxalate. They recognized two types of inhibi-
tion. One type of inhibition is that which is caused by E D T A or oxalate.
This kind of inhibition is reversed immediately upon dilution of the en-
zyme-inhibitor mixture. The second is that which results from addition
of o-phenanthrolinc. Enzyme preparations treated with o-phenanthroline
bind 2 moles of the chelator per mole of Zn2+.This complex is stable
and inactive, and does not result in the release of Zn2+.The inactive
o-phenanthroline-enzyme complex can be reactivated by dialysis, addi-
tion of divalent metal ions such as Zn2+, Co2+,Mn2+, and Fez+, or by
incubation at elevated temperaturcs ( 5 4 5 O ) (312, 3 1 5 5 1 7 ) . It has been
shown that heat treatment does not involve the release of o-phenanthro-
line. The authors suggested that thermal reactivation of the o-phenan-
314. A. P. Nygaard, JBC 236, 2128 (1961).
315. T. Cremona and T. P. Singer, JBC 239, 1466 (1964).
316. A. Ghiretti-Magaldi, T. Cremona, T. P. Singer, and P. Bernath, BBRC 5,
334 (1961).
317. T. Crernona and T. P. Singer, BBA 57, 412 (1962).
272 POUSSEF HATEFI AND DIANA L. STIGGALL
throline-enzyme complex is the result of a change in the conformation

of the enzyme molccule. Other studies have suggested to these authors
that Zn?+is involved in the binding of substrate to the enzyme (312).
It has been shown that by treatment with ammonium sulfate a t acid
pH, flavin can be partially removed from the enzyme (318). Addition
of FAD, but not FMN, reactivated the enzyme. Zinc is not removed under
these conditions, and its addition is not required for reactivation. The
metal appears to be very tightly bound to the enzyme (312) ; its removal
without protein denaturation has not been achieved.
c. D-2-HYDROXYACID DEHYDROGENASE
(EC 1.1.99.6)
It was discovered in 1958 that anaerohically grown yeast contains a
form of lactate dehydrogenase which is different from the D- and L-lac-
tate:cytochrome c reductases of aerobic yeast (306, 319). The enzyme
has been partially purified (320, 321), and shown to contain flavin
(320-322). Gel filtration studies have suggested a molecular weight of
about 100,000 (320, 321). Preparations of the enzyme oxidize several D-2-
hydroxyacids to the respective keto acids in a reversible manner (320).
For the forward reaction, ferricyanide, 2,6-dichloroindophenol, menadi-
one, and methylene blue have been used as electron acceptors, and for
the reverse reaction leucomethyl viologen and FMNH, are effective elec-
tron donors (320).A number of L-2-hydroxyacids and 2-keto acids have
been shown to be competitive inhibitors. Oxalate, cyanide, o-phenanthro-
line, and EDTA are also potent inhibitors (320, 321, 323, 324). The in-
hibition by metal chelators develops slowly and is reversed by addition
of Zn2+,Co2+,Mn2+,or Fez+ (320, 323-326). Substrates prevent the inhibi-
tion by chelators a t concentrations considerably lower than their respec-
tive K, values (327). It has been suggested that EDTA inactivation in-
volves the removal of a metal, most probably Zn2+,from the substrate
binding site of the enzyme (325, 326, 328, 329). However, others have
318. C. Gregolin and T. P. Singer, BBA 57,410 (1962).
319. P. P. Slonimski and W. Tysarowski, C . R. Acad. Sci. 246, 1111 (1958).
320. T. Cremona, JBC 239, 1457 (1964).
321. J. Rytka and W. Tysarowski, Acta Biochim. Pol. 12,229 (1965).
322. M. Iwatsubo, BBA 77, 568 (1963).
323. E. Boeri, T. Cremona, and T. P. Singer, BBRC 2,298 (1960).
324. A. Curdel, L. Naslin, and F. Labeyrie, C. R. Acad. Sci. 249, 1959 (1959).
325. A Curdel and F. Labeyrie, BBRC 4,175 (1961).
326. A. Curdel, C. R . Acad. Sci. 254, 4092 (1962).
327. F. Labeyrie and E. Stachiewicz, BBA 52, 136 (1961).
328. E. Stachiewicz, F. Labeyrie, A. Curdel, and P. P. Slonimski, BBA 50, 45
(1961).
329. M. Iwatsubo and A. Curdel, BBRC 6, 385 (1961).
shown that, similar to D-lactate :cytochrome c reductase, EDTA-treated

D-2-hydroxyacid dehydrogenase can be reactivated by dialysis or by in-
cubation a t elevated temperatures in the absence of added metals (320,
330, 331). The latter authors believe that chelator treatment results in the
formation of an inactive enzyme-chelator complex without the removal
of metal. This complex can be reactivated by addition of metal ions or
dialysis, which presumably will result in the removal of the chelator,
or by heat treatment, which converts the inactive complex to an active
form. A similar mechanism has been invoked for the inhibition of D-lac-
tate :cytochrome c reductase by o-phenanthroline.
Thus, the presence and possible role of Zn2+and the nature of the flavin
prosthetic group of D-Zhydroxyacid dehydrogenase have yet to be unam-
biguously demonstrated. Howeger, it might be added that the enzyme
can be inactivated by treatment with ammonium sulfate a t acid pH, and
reactivated by FAD, but not by F M N (329). Further, the flavin in
EDTA-inactivated preparations is not reduced by D-lactate, but addition
of Zn? results in rapid bleaching at 450 nm (330). These results have
been considered as evidence that the flavin prosthetic group is FAD, and
that the metal is necessary for the reduction of flavin by substrate.
Soluble D-lactate dehydrogenases with enzymic properties similar to
those of the D-2-hydroxyacid dehydrogenase of anaerobic yeast have been
isolated from rabbit kidney mitochondria (322-334) and from a species
of Mycobacterium ( 3 3 5 ) .It is not clear whether these enzymes are metal-
containing flavoproteins.
VII. Nitrite Reductases (EC 1.6.6.4)
Nitrate reduction and assimilation is a fundamental biological process

in plants and various microorganisms. In this process nitrate is reduced
ultimately to ammonia. Thus, as shown in Eq. ( 5 ) , the reduction of ni-
trate to ammonia requires eight electron or hydrogen equivalents.
HN03 +8H+ NH.7 + 3H20 (5)
The first reduction product of nitrate is nitrite. This reaction is catalyzed
330. A. Ghiretti-Magaldi, T. Cremona, T. P. Singer, and P. Bernath, BBRC 5,

334 (1961).
331. T. Cremona and T. P. Singer, Nature (London) 194,836 (1962).
332. P. K. Tubbs, BBRC 3, 513 (1960).
333. P. K. Tubbs and G. D. Greville, BJ 81, 104 (1961).
334. P. K. Tubbs, BJ 82,36 (1962).
335. T. Szumilo and M. Szymona, Physiol. Chem. Phys. 4, 407 (1972).
by the molybdenum- and FAD-containing enzyme, nitrate reductase,

which is discussed in Volume XII, Chapter 6, p. 402.
Enzyme systems which catalyze nitrite reduction have been observed
in bacteria (336-343), fungi (344-351), green algae (348, 352-355) , and
higher plants (344, 356-362). While the assimilatory nitrite reductases
convert nitrite to NH,, the denitrifying organisms reduce it to nitric oxide
(338, 340, 363) or nitrogen gas (336). Examples of denitrifying nitrite
reductases are the enzymes of Pseudomoms denitnficans ( S d O ) , and P .
aeruginosa (338, 364-366), which convert nitrite to nitric oxide, and of
P. stutzeri (336), which reduces nitrite to NO and N,. The nitrite reduc-
tase of P. denitrificuns has been partially purified. The enzyme reduces
336. C. W. Chung and V. A. Najjar, JBC 218,617 (1956).

337. D. Spencer, H. Takahashi, and A. Nason, J. Bacterial. 73, 553 (1957).
338. G. C. Walker and D. J. D. Nicholas, BBA 49, 350 (1961).
339. R. A. Lazzarini and D. E. Atkinson, JBC 236, 3330 (1961).
340. B. C. Radcliffe and D. J. D. Nicholas, BBA 153,545 (1968).
341. 0. Prakash and J. C. Sadana, ABB 148,614 (1972).
342. J. M. Vega, M. G. Guerrero, E. Leadbetter, and M. Losada, BJ 133, 701
(1973).
343. C. D. Cox, Jr. and W. J. Payne, Can. J. Microbial. 19, 861 (1973).
344. A. Nason, R. G. Abraham, and B. C. Averback, BBA 15, 159 (1954).
345. J. Rivas, M. G. Guerrero, A. Paneque, and M. Losada, Plant Sci. Lett. 1,
105 (1973).
346. D. J. D. Nicholas, A. Medina, and 0. T. G. Jones, BBA 37,468 (1968).
347. K. Yamafuji, Y. Osajima, H. Omura, and 8. Hatano, Enzymologia 21, 245
(1960).
348. E. Kessler, Annu. Rev. Plant. Physiol. 15, 57 (1964).
349. K. A. Cook and G. J. Sorger, BBA 177,412 (1969).
350. R. H. Garrett, BBA 264, 481 (1972).
351. M. A. Lafferty and R. H. Garrett, Abstr. 7Jrd. Annu. Meet Amer. Sac. Micro-
biol. p. 194 (1973).
352. E. Kessler and F. C. Czygan, Experientia 19, 89 (1963).
353. M. G. Guerrero, J. Rivas, A. Paneque, and M. Losada, BBRC 45, 82 (1971).
354. W. G. Zumft, BBA 276, 363 (1972).
355. A. Hattori and I. Uesugi, Plant Cell Physiol. 9, 689 (1968).
356. G. G. Roussos and A. Nason, JBC 235,2997 (1960).
357. R. H. Hageman, C. F. Cresswell, and E. J. Hewitt, Nature (London) 193,
247 (1962).
358. K. W. Joy and R. H. Hageman, BJ 100, 263 (1966).
359. K. Asada, G . Tamura, and R. S. Bandurski, JBC 244, 4904 (1969).
360. J. Cardenas, J. L. Barea, J. Rivas, and C. G. Moreno, FEBS (Fed. Eur.
Biochem. Sac.) Lett. 23, 131 (1972).
361. M. J . Dalling, N. E. Tolbert, and R. H. Hageman, BBA 283, 505 (1972).
362. M. J. Dalling, N. E. Tolbert, and R. H. Hageman, BBA 283, 513 (1972).
363. A. Nason, Bacterial. Rev. 26, 16 (1962).
364. T. Yamanaka, A. Ota, and K. Okunuki, BBA 44,397 (1960).
365. T. Yamanaka and K. Okunuki, BBA 67,379 (1963).
366. T. Yamanaka, Nature (London) 204, 253 (1964).
nitrite to nitric oxide in the presence of NADH or NADPH and FMN,

FAD, or riboflavin. It can also use artificial electron donors, such as re-
duced benzyl viologen or leucomethylene blue, in the absence of flavins.
Inhibitor studies have suggested the involvement of metals and active
thiol.
Walker and Nicholas (338)have reported the isolation and 600-fold
purification of an enzyme from P. aeruginosa, which reduces nitrite to
nitric oxide. The preparation contained 1.5 nmoles of FAD per mg pro-
tein, a c-type cytochrome and an absorption band a t 630-635 nm, sugges-
tive of copper. As electron donors, reduced FMN, FAD, riboflavin, pyo-
cyanine, and methylene blue were effective, but not NADH, NADPH,
or reduced cytochrome c. The preparation required phosphate or sulfate
for maximal activity. The cytochrome and the 630-635-nm band were
reduced under anaerobic conditions with a suitable electron donor and
readily oxidized by nitrite. The K , for NaNO, is reported to be 3.1 X lo-”
M . The presence of an active thiol in the enzyme is indicated by p-mercu-
ribenzoate inhibition and glutathione reactivation.
Yamanaka and co-workers (364-366) have crystallized a cytochrome
oxidase from P . aeruginosa which oxidizes Pseudomonus ferrocytochrome
0551. It is also capable of nitrite reduction with a turnover number of
4000 moles nitrite reduced under anaerobic conditions to nitric oxide per
minute a t 37O. It is an adaptive enzyme, nitrate being essential for its
biosynthesis. The enzyme has a molecular weight of 120,000, with two
subunits of equivalent molecular weight, 2 heme c and 2 heme d groups
per mole (Fig. 38) (36%). Nitrite reductase activity is 94% inhibited
by 8 X M KCN, but only by CO. The lack of CO inhibition appears
to be related to the fact that the enzyme has a greater affinity for nitrite
than for carbon monoxide.
Nitrite reduction in assimilatory nitrate-reducing Neurospora crassa,
Tomlopsis nitratophila, Azotobacter vinelandii, and Azotobacter chro-
ococcum appears to be catalyzed by enzyme systems which require flavin
and metals. The enzyme from N . crassa has been partially purified, and
its molecular weight has been estimated to be 300,000 (344, 346, 351,
367). The enzyme reduces both nitrite and hydroxylamine to ammonia
and utilizes NADH or NADPH as electron donor. It is reported to be
a FAD-dependent enzyme and to contain iron, copper, and active thiol
(346, 367). Three moles of NADH are oxidized per mole of nitrite re-
duced to ammonia. It has been suggested that the reduction of nitrite
occurs in three steps, each involving two electrons. Thus, hyponitrite and
hydroxylamine have been proposed as successive intermediates in the re-
366a. J. C. Gudat, J. Singh, and D. C. Wharton, BBA 292, 376 (1973); D. C.
Wharton, private communication.
367. A. Medina and D. J. D. Nicholas, BBA 25, 138 (1957).
Wavelength (nm)
FIG.38. The absorption spectra of crystalline Pseudomonas cytochrome oxidase.

The crystals were dissolved in 0,2 M phosphate buffer (pH 7.0). (---) Oxidized,
(-) reduced with sodium dithionite. From Yamanaka and Okunuki (366).
duction of nitrite to NH, (36'7). The nitrite reductase of N . crassa is

inducible by nitrate or nitrite and repressed by ammonia (350).
The nitrite reductase of Torulopsis nitratophila is specific for NADPH
and FAD, and can utilize reduced benzyl or methyl viologen as electron
donor, but not reduced flavins (345). With NADPH as electron donor,
nitrite reduction is inhibited by cyanide and mercurials. Michaelis con-
stants for FAD and nitrite have been reported to be 45 n M and 19 p M ,
respectively. Unlike the Neurospora enzyme, the nitrite reductase of T.
nitratophila could not reduce hydroxylamine in the presence of NADPH
and FAD.
That hydroxylamine might not be an obligatory intermediate, or occur
as a free intermediate, in the reduction of nitrite to ammonia is suggested
by the properties of nitrite reductases of Azotobacter chroococcum and
Escherichia coli. The former is an adaptive enzyme, the formation of
which requires nitrate or nitrite in the culture (342).It is FAD-depen-
dent and presumably contains metals and p-mercuribenaoate inhibitable
thiols. It reduces nitrite to ammonia in the presence of NADH as electron

donor and does not appear to produce hydroxylamine as an intermediate.
Cyanide competitively inhibits the reduction of nitrite with a K i= 32
nM. The K , values for nitrite and NADH are 5.5 and 15 p M , respec-
tively. The enzyme is inhibited upon preincubation with NADH. Nitrite
protects against NADH inhibition and reverses it. Sucrose density gra-
dient centrifugation has suggested a molecular weight of 67,000 for the
A . chroococcum enzyme.
The E. coli enzyme can reduce nitrite and hydroxylamine to ammonia
a t the expense of NADPH (339). However, with the use of 15N-nitrite
it was shown that hydroxylamine was not an intermediate in the reduc-
tion of nitrite. No cofactor requirements were shown for the E . coli en-
zyme, but similar to other flavin and metal requiring nitrite reductases
it was inhibited by cyanide and mercurials.
The nitrite reductase of Azotobacter vinelandii ( A . agile) was ex-
tracted in soluble form by Nason and his colleagues (337). The prepara-
tion reduced nitrite and hydroxylamine in the presence of reduced nico-
tinamide-adenine dinucleotides and required flavin for maximal activity.
FAD was shown to be specific for nitrite reduction, whereas both FAD
and FMN were active for hydroxylamine reduction. The hydroxylamine
reductase activity of the preparation was enhanced in the presence of
Mn2+.Ammonia was shown to be the product of nitrite reduction, but
the product of hydroxylamine reduction was not identified. Another
nitrite and hydroxylamine reductase, which had a MnZ+requirement, was
also isolated and partially purified in Nason’s laboratory from soybean
TABLE XVII
THEPHYSICAL
PROPERTIESOF PURIFIED NITRITEREDUCTASE
FROM Achromobacter fscherio
Sedimentation constant,, slo,w 5.2 s

Diffusion coefficient, D ~ o . ~ 5.56 X lo-’ om2 sec-l
Molecular weight (Archibald procedure)b 95,000 +_ 4,000
Molecular weight (calculated from diffusion and 84,000
sedimentation constants)b
Heme content (nmoles/mg protein) 19
Minimum molecular weight (from heme content) 52,500
Iron content 0.102-0.105%
Minimum molecular weight (from iron content) 54,000
Isoelectric point Around p H 4,. 5
From Prakash and Sadana (341).

In the calculation, a value for the partial specific volume of 0.73 ml/g for nitrite
reductase is assumed.
I
t
0.9
4 20
I!
i!
Wavelength (nm)
FIQ.39. The absorption spectra of Achromobacter fischeri nitrite reductase. Spectra
were recorded in 0.05 1cI phosphate buffer, p H 7.5, at 0.41 mg enzyme protcin/ml.
(-) Oxidized, (- * -) reduced with dithionite, (---I NO,- (or hydroxylamine)
added to the dithionite reduced enzyme. From Prakash and Sadana (341).
leaves (356).However, the enzyme preparation did not require flavin,

but had an absolute requirement for an unidentified, heat-stable factor,
which had an absorption peak a t 312 and 315 nm, respectively, in 0.1
N HC1 and 0.1 M pyrophosphate, pH 7.0. The peak shifted to 358 nm
in 0.1 N NaOH.
The nitrite reductase system of Achromobacter fischeri appears to be
composed of two separable enzymes (341). The first enzyme is a flavin
reductase and utilizes NADH or NADPH to reduce FMN or FAD. The
second interacts with the flavin reductase and converts nitrite and hy-
droxylamine to ammonia. The nitrite reductase enzyme has a molecular
weight of 95,000 f 4,000 (Table XVII), contains two heme c per mole,
and is inhibited by p-mercuribenzoate, cyanide, and carbon monoxide.
The latter inhibition is reversed by light. Urea inactivation-reactivation

studies showed parallel loss and recovery of nitrite and hyroxylamine
reductase activities, and nitrite was shown to inhibit hydroxylamine re-
duction. These results have suggested that the enzyme has a common
binding site for nitrite and hydroxylamine. The absorption spectra of
the A . fischeri enzyme (oxidized, reduced, and reduced plus nitrite or
hydroxylamine) are shown in Fig. 39.
VIII. Adenylyl Sulfate Reductases (EC 1.8.99.2)
Two major pathways are known for the reduction of sulfate. One is
the assimilatory pathway, which reduces sulfate to the extent necessary
for satisfying the nutritional requirements of the organism. I n this path-
way, which has been extensively studied in yeast by Robbins and Lip-
mann (368) and Bandurski and his colleagues (369, 370), sulfate is first
activated in the presence of ATP by the enzyme ATP-sulfurylase t o form
adenosine 5'-phosphosulfate (APS). Then in a second reaction, APS is
phosphorylated in the 3' position by ATP to form 3'-phosphoadenosine
5'-phosphosulfate (PAPS)
ATP + Solz--+ APS+ PP (6)
APS + ATP + +
PAPS ADP (7)
In the presence of appropriate enzymes, the sulfate group of PAPS can
be donated to various acceptors, such as carbohydrates, steroids and
phenols, or become reduced to sulfite for assimilatory purposes. Figure
40 shows a unified scheme for sulfate and sulfite assimilation by algae
as proposed by Abrams and Schiff (371).
The second pathway by which sulfate is reduced is the dissimilatory
pathway in which sulfate is the terminal electron acceptor and leads to
the formation of large quantities of H,S. During the dissimilatory reduc-
tion of sulfate, APS is formed as in Eq. ( 6 ) . Then APS is reduced directly
to sulfite and AMP by the enzyme APS-reductase. Table XVIII shows
the data of Peck (372) on the pathway of sulfate reduction in various
microorganisms.
Adenylyl sulfate (APS) reductase is a flavoprotein, which contains iron
and possibly acid-labile sulfide. It catalyzes the reduction of APS in the
368. P. W. Rohbins and F. Lipmann, JACS 78, 6409 (1956).

369. L. G. Wilson, T. Asahi, and R. S. Bandurski, JBC 236, 1822 (1961).
370. K. Torii and R. S. Bandurski, BBA 136, 286 (1967).
371. W. R. Abrams and J. A. Schiff, Arch. Mikrobiol. 94, 1 (1973).
372. H. D. Peck, Jr., J. Bacteriol. 82, 933 (1961).
TRANSFERASES h3
SULFATE m
ESTERS * 0
I
I
I
n
2
-o-s-o--
I
1 -0-2-0- [Cor-s-] .
; ; I 0 SULFURYLASE
SUFATE 1 SUFATE
OUTSIDE 1 INSIDE P-PI !
I
-
I FERREDOXIN
OXIDIZED
AMP
'R-S-
I X
I
t
--+--
I
E
-0-s - 0-
SULFITE
OUTSIDE
r
FIG.40. A proposed unified scheme of sulfate assimilation in algae. Adenylyl sulfate (APS) transfers the
sulfo group via APS-sulfotransferase to form Car-SSO; (Car = carrier), which is reduced further by thio-
sulfonate reductase to Car-SS- which yields the thiol group of cysteine. In addition, if sulfite is released from
Car-S-SOa- (i.e., by thiol or from mutated sites) or if it enters the cell from outside, i t can be reduced via a
separate sulfite reductase. From Abrams and Schiff (371).
TABLE XVIII
PATHWAY
OF SULFATE
REDUCTION TYPES
I N VARIOUS OF MICROORGANISMS~
APS reductaseb PAPS reductaseb
Met,hyl Methyl
Organism viologenc viologen NADPH
1. Assimilatory sulfate reducers

Escherichia coli 0.4 2.4 3.7
(grown aerobically)
E. coli 0.0 3.4 2.8
(grown anaerobically)
Yeast 0 0 1.1
Aerobacter aerogenes 0.6 5.6 7.3
Proteus mirabilis 0 0.1 8.9
P . vulgaris 0 3.3 -
Pseudomonas hydrophila 0 4.0 0.6
Aeromonas punctata 0 12.2 21.4
Clostridium kluyveri 0 0.1 0
C. pasteurianum 0 8.7 0
Rhodopseudomonas spheroides 0 0.3 0
R . palustris 0 0 0
2. Dissimilatory sulfate reducers
Desulfovibrio desulfuricans 1,750 0 0
Clostridium nigrificans 310 0.4 0.1
Vibrio cholinicus 907 0 0
3. Sulfur oxidizers
Thiobacillus thioparus 640
T . thiooxidans 162
T . denitri’cans 1,260
Chromatium sp. 0.3
a From Peck (372).

Specific activity is expressed aa nanomoles of acid-volatile sulfur formed per hr
per mg protein.
No activity was observed with NADPH.
presence of an appropriate electron donor [reduced methyl viologen, or

reduced cytochrome c3 in Desuljovibrio vulgaris (373, 37Sa) ] to sulfite
and AMP [Eq. ( S ) ] .
APS + 2e F! S0a2- + AMP (8)
It can also catalyze the reverse reaction when ferricyanide or cytochrome
c is used as electron acceptor (374, 375). The phosphosulfate bond of
373. H. D. Peck, Jr., Proc. N a t . Acad. Sci. U . S. 45,701 (1959).
373a. D. V. DerVartanian and J. LeGall, B B A 346,79 (1974).
374. H. D. Peck, Jr., B B A 49, 621 (1961).
375. R. M. Lyric and I. Suzuki, Can. J. Biochem. 48, 344 (1970).
APS is energy-rich (AGO = 18-19 kcal/mole). Therefore, the reversal of

reaction (8) is rather interesting, because it can capture oxidation energy
and convert it to a biologically utilizable form. For example, the enzyme
ADP-sulfurylase can catalyze the synthesis of ADP from APS and inor-
ganic phosphate as shown in Eq. (9) (376, 377).
AD P-sulfurylase
APS + Pi, ' ADP + SOa2- (9)
APS reductase is found in dissimilatory sulfate reducing bacteria, such
as Desulfovibrio and Desulfotomaculum, in certain Thiobacilli, in
Thiocapsa roseopersicina, and in the alga Chlorella pyrenoidosa. Table
XIX, compiled by Schiff (378), gives the properties of various APS re-
ductases from plants and microorganisms. I n Thiobacilli and Desulfovi-
brio, APS reductase constitutes as much as 1-5% of the cell protein,
which suggests the important role of this enzyme in the metabolism of
these organisms (375).
The APS reductase of Desulfovibrio vulgaris has been extensively
studied by Peck and his co-workers. The enzyme is reported to have a
molecular weight of 220,000, and to contain 1 mole of FAD and 6-8
g-atoms of nonheme iron per mole (379). The oxidized and reduced ab-
sorption spectra of the enzyme are shown in Fig. 41. Spectrophotometric
studies have shown that in the absence of AMP the enzyme is partially
bleached between 350 and 500 nm upon addition of sulfite. The rate of
bleaching achieved with sulfite was shown by stopped-flow kinetic mea-
surements to be comparable to the turnover number of the enzyme when
sulfite oxidation was assayed in the presence of ferricyanide as electron
acceptor. These findings, plus the increased absorption of the sulfite-
treated enzyme a t 320 nm, have suggested to Peck and co-workers that
sulfite oxidation involves the interaction of sulfite with the enzyme to
form a flavin-sulfite adduct in position N-5 of the isoalloxazine ring
(379). The authors pointed out that these results are analogous to the
data of Massey and co-workers (380, 381) on the effect of sulfite on
various flavoproteins. The latter authors found similar spectral changes
when sulfite was added to glucose oxidase, D- and L-amino acid oxidases,
oxynitrilase, lactate oxidase, and glycollate oxidase. They concluded that
the flavoproteins which are capable of interacting with oxygen (APS re-
376. H. D. Peck, Jr., JBC 237, 198 (1962).

377. H. D. Peck, Jr., T. E. Deacon, and J. T. Davidson, BBA 96, 429 (1965).
378. J. A. Schiff and R. C. Hodson, Annu. Rev. Plant Physiol. 24, 381 (1973).
379. G. B. Michaels, J. T. Davidson, and H. D. Peck, Jr., BBRC 39,321 (1970).
380. V. Massey, F. Miillcr, R. Feldberg, M . Schurnan, P. A . Sullivan, L. G. Howell,
S. G. Mayhew, R. G. Matthews, and G. P. Foust, JBC 244, 3999 (1969).
381. F. Miiller and V. Massey, JBC 244, 4007 (1969).
TABLE XIX
PROPERTIES
OF ADENYLYL FROM PLANTS
SULF.ATEREDUCTASES A N D MICROORGANISMS~
Electron
donor or PH
Organism Enzyme acceptor optimum K, MW Remarks
Bacteria
Desulfovibrio APS reductase Fe(CN)? 7.4 S032-,2 m M 220,000 Contains 1 mole FAD, 6-8
vulgaris g-atoms nonheme iron
T hiobacillus APS reductase Fe(CN)F 7.4 S032-, 2.5 m M 170,000
thioparus AMP, 0.1 m M
APS reductase Cytochrome c 9.5 S032-, 0.017 m M 170,000 Contains 1 mole FAD, 8-10
AMP, 0.0025 m M g-atoms nonheme iron
Thiobacillus APS reductase Fe(CN)2- 7.2 SO3$-, 1.5 m M - Contains 1 mole FAD, 6-11
denitri’cans AMP, 0.041 m M g-atoms nonheme iron
Thiocapsa APS reductase Fe(CN)63- 8.0 S032-, 1.5 m M 180,000 Contains 1 mole FAD, 4 g-
roseopersicina AMP, 0.073 m M atoms nonheme iron, 2 g-
APS reductase Cytochrome c 9.0 S032-, 0.093 m M 180,000 atoms heme iron. Purified
AMP, 0.059 m M 60-80-fold ; homogeneous
upon ultracentrifugation
Fungi
Saccharom yces PAPS reductase NADPH 7.5 Partially purified into 3 frac-
cerevisiae (tris) tions A, B, C. Some activ-
ity with APS. Fraction A
purified 60-fold, fraction
C to apparent homogene-
ity in ultracentrifugation
Algae
Chlorella APS reductase Thiol - - 330,000 Partially purified. PAPS is
p yrenaidosa active in the presence of
a 3’-nucleotidase
a From Schiff and Hodson (378).

o’6
0.5
1
-
0.4 -
w.
C
5 03-
n::
a
0.2-01
0.1 -
300 3K) 460 450 500 550 600

nrn
FIa. 41. Absorption spectrum of purified APS reductase from Desulfovibrio vul-
garis. The enzyme concentration was 2.5 mg/ml. Insert A : difference spectrum ob-
tained from tracing of “oxidized” and “reduced” enzyme. Insert B: spectrum ob-
tained after boiling APS reductase and removing protein by centrifugation. From
Peck et al. (377).
ductase reacts slowly with oxygen) can form a flavin-sulfite adduct, and
that the N-5 position of the isoalloxazine ring is very likely involved.
Addition of AMP to the sulfite-treated APS reductase resulted in fur-
ther bleaching between 350 and 500 nm. Peck et al. (382, 383) have
shown by EPR spectroscopy near liquid helium temperature that addition
of either sulfite or AMP alone does not result in the formation of an
iron signal a t g = 1.94. However, when AMP and sulfite are added to-
gether, a g = 1.94 signal is produced, which is approximately 80% of
that obtained when the enzyme is reduced with dithionite. Thus, the auth-
ors suggested that APS reductase catalyzes an intramolecular electron
transfer during sulfite oxidation as shown in Fig. 42 from Peck et al.
( 382).
Whereas Peck and his co-workers have not reported the presence of
acid-labile sulfide in the APS reductase of D.vulgaris, Lyric and Suzuki
(376) have shown that the enzyme from Thiobacillus thioparus contains
4-5 moles of labile sulfide per mole. The T . thioparus enzyme appears
to have a molecular weight of 170,000, and contains, in addition to labile
sulfide, 1 mole of FAD and 8-10 g-atoms of iron per mole. That the en-
382. H. D. Peck, Jr., R. Bramlett, and D. V. DerVartanian, 2. Nuturforsch. B
27, 1084 (1972).
383. R. N. Bramlett and H. D. Peck, Fed. Proc., Fed. Amer. SOC.E z p . Biol. 32,
668 (1973).
4. METAL-COKTAINING FLAVOPROTEIN DEHYDROGENASES 285
X = nonheme iron centers

FIG.42. A proposed mechanism for APS reductase. From Peck e t al. (388).
zyme of Peck et al. very likely contains labile sulfide is suggested both
by its absorption spectrum and by its characteristic iron-sulfur signal
centered a t g = 1.94.
Another APS reductase of interest is that which has been isolated by
Triiper and Roger (384) from Thiocapsa roseopersicina. The enzyme is
reported to have a molecular weight of 180,000 and to contain 1 mole
of flavin (presumably FAD), 4 g-atoms of nonheme iron, 6 moles of labile
sulfide, and 2 c-type hemes per mole. The spectral properties of the en-
zyme are shown in Fig. 43. It utilizes cytochrome c and ferricyanide as
0.7 1 iL17nm
Wavelength (nm)
FIG.43. Absorption spectra of the purified APS reductase from Thiocapsa roseoper-
sicina: ox, oxidized enzyme; red, enzyme reduced with 1 mg sodium dithionite per
ml. From Triiper and Rogers ( 3 8 4 ) .
384. H. G. Truper and L. A. Rogers, J. Bacterial. 108, 1112 (1971).
electron acceptors, and the reaction to cytochrome c is especially sensitive

to thiol inhibitors. The heme groups of the enzyme are suggested to be
involved in electron transfer from sulfite to added cytochrome c. How-
ever, it has not been shown that these heme groups can be reduced by
treatment of the enzyme with substrate ( 3 8 4 ~ ) .
IX. Sulfite Reductases (H,S:NADPH Oxidoreductases) (EC 1.8.1.2)
As pointed out in the preceding section, sulfate assimilation in yeast

has been shown to involve the activation of sulfate by ATP successively
to adenosine 5’-phosphosulfate and once again to 3’-phosphoadenosine
5’-phosphosulfate. The latter is then reduced in the presence of NADPH
to sulfite and 3’,5’-diphosphoadenosine (372). Enzymes catalyzing the
6-electron reduction of sulfite to sulfide have been observed in bacteria
(339,385-397), yeast (398-401), fungi (402-404), and higher plants (359,
405). These enzymes may be divided into two classes depending on
384a. Dr. H. G. Truper has informed us that the APS reductase of Chlorobium
limicola, recently purified in his laboratory, does not contain any heme groups, but
is otherwise similar to the APS reductases of sulfate reducing bacteria and
Thiobacilli.
385. M. Ishimoto, J. Koyama, and Y. Nagai, J . Biochem. (Tokyo) 42, 41 (1955).
386. J. Mager, BBA 41,553 (1960).
387. J. Dreyfuss and K. J. Monty, JBC 238, 3781 (1963).
388. J. M. Akagi, BBRC 21, 72 (1965).
389. J. LeGall and N. Dragoni, BBRC 23, 145 (1966).
390. L. M. Siegel and H. Kamin, in “Flavins and Flavoproteins,” 2nd Int. Symp.
(K. Yagi, ed.), p. 15.Univ. Park Press, Baltimore, Maryland. 1968.
391. N. Gilboa-Garber and J. Mager, BBA 220,602 (1970).
392. P. A. Trudinger, J . Bncteriol. 104, 158 (1970).
393. W.D. Hoeksema and D. E. Schoenhard. J . Bacteriol. 108, 154 (1971).
394. L. M.Siegel, H. Kamin, D. C. Rueger, R. P. Presswood, and Q. H. Gibson,
in “Flavins and Flavoproteins,” 3rd Int. Symp. (H. Kamin, ed.), p. 523. Univ. Park
395. K. Kobayashi, E.Takahashi, and M. Ishimoto, J . Biochem. (Tokyo) 72, 879
(1972).
396. J.-P. Lee, J. LeGall, and H. D. Peck, Jr., J . Bacteiiol. 115, 529 (1973).
397. L. M. Siegel, M. J. Murphy, and H. Kamin, JBC 248, 251 (1973).
398. T. Wainwright, BJ 83, 39P (1962).
399. N. Naiki, Plant Cell Physiol. 6,179 (1965).
400. A. Yoshimoto and R. Sato, BBA 153, 555 (1968).
401. K. Prabhakararao and D. J. D. Nicholas, BBA 180,253 (1969).
402. A. Yoshimoto, T. Nakamura, and R . Sato, J . Biochem. (Tokyo) 50,553 (1961).
403. A. Yoshimoto, T . Nakamura, and R. Sato, J . Biochem. (Tokyo) 62, 756 (1967).
404. L. M. Siegel, F. J. Leinweber, and K. J. Monty, JBC 240, 2705 (1965).
405. G. Tamura, J . Biochem. (Tokyo) 57,207 (1965).
COOH
I
FIQ.44. Postulated structural formula for the siroheme prosthetic group. From
Murphy et al. (418).
whether or not they can use reduced nicotinamide adenine dinucleotide

(specifically NADPH) for the reduction of sulfite. The NADPH-sulfite
reductases appear to contain flavin, nonheme iron, acid-labile sulfide, and
a novel heme (extractable by acid acetone) of the isobacteriochlorin type
with characteristic a-absorption peak a t 582-589 nm. This heme, in which
two adjacent pyrrole rings are reduced, has been named “siroheme” (Fig.
44). The sulfite reductases, which cannot utilize NADPH as reductant,
are generally of smaller molecular weight, do not require flavin, but ex-
hibit the cytochrome-like absorption peaks comparable to those of the
siroheme-containing enzymes. Sulfite reduction by this group of enzymes
is usually studied in the presence of appropriate dyes (e.g., reduced
methyl viologen) as electron donors. Enzymic and genetic studies have
suggested that NADPH-sulfite reductases are composed of a flavoprotein
(NADPH dehydrogenase) , and a hemoprotein (sulfite reductase) which
can utilize reduced methyl viologen as electron donor.
A. NADPH-SULFITEREDUCTASES
NADPH-sulfite reductases are found in E . coli (386, 390, 391, 397,
406-416)) Salmonella typhimurium (587, 394, 417-419)) yeast (598-401,
406. F. J. Leinweber and K. J. Monty, BBA 63, 171 (1961).

407. L. M. Siegel and H. Kamin, “Methods in Enzymology,” Vol. 17B, p. 539,
1971.
408. L. M. Siegel, E. J. Faeder, and H. Kamin, 2.Naturjorsch. B 27,1087 (1972).
420-424), and Neurospora crassa (404, 425, 426). The E. coli enzyme
has been purified and extensively studied by Kamin, Siegel, and their
colleagues (390, 397, 407-416). The enzyme has a molecular weight of
670,000, and contains 4 moles of FAD, 4 moles of FMN, 20-21 g-atoms
of iron, 14-15 moles of acid-labile sulfide, and 3 4 moles of heme per
670,000 g protein (390, 397). The absorption spectrum of E. c d i
NADPH-sulfite reductase is shown in Fig. 45. The oxidized enzyme
(trace A ) has absorption maxima a t 278, 386, 455, 587, and 714 nm. The
455-nm peak results largely from flavin and is bleached upon treat-
ment of the enzyme with NADPH (trace B) or dithionite (trace C ) .
Electron paramagnetic resonance studies have shown a signal centered
a t g = 6, which is characteristic of high-spin ferric heme, and only under
special conditions a signal a t g = 1.94, characteristic of an iron-sulfur
center, has been observed (413). The enzyme catalyzes electron transfer
from NADPH to sulfite, nitrite, hydroxylamine, cytochrome c, ferricya-
nide, dichloroindophenol, menadione, FMN, FAD, and molecular oxygen.
It is also capable of transhydrogenation from NADPH to acetylpyridine
adenine dinucleotide phosphate, and electron transfer from reduced
methyl viologen (MVH) to sulfite, nitrite, hydroxylamine, or NADP.
All the NADPH-dependent reductions, except the reduction of acetyl-
pyridine adenine dinucleotide phosphate, are inhibited by p-mercuri-
phenyl sulfonate, but not the reduction of sulfite, nitrite, and hydroxyl-
amine by MVH. The reduction of the latter compounds by NADPH or
~ ~~~
409. M. J. Murphy, L. M. Siegel, H. Kamin, D. V. DerVartanian, J.-P. Lee, J.

LeGall, and H. D. Peck, Jr., BBRC 54, 82 (1973).
410. M. J. Murphy and L. M. Siegel, JBC 248, 6911 (1973).
411. M. J. Murphy, L. M. Siegel, and H. Kamin, JBC 248, 2801 (1973).
412. M. J. Murphy, L. M. Siegel, S. Tove, and H. Kamin, Proc. Nut. Acad. Sci.
U. S. 71, 612 (1974).
413. L. M. Siegel, P. S. Davis, and H. Kamin, JBC 249, 1572 (1974).
414. L. M. Siegel and P. S. Davis, JBC 249, 1587 (1974).
415. E. J. Faeder, P. 9. Davis, and L. M. Siegel, JBC 249, 1599 (1974).
416. M. J. Murphy, L. M. Siegel, and H. Kamin, JBC 249, 1610 (1974).
417. J. Dreyfuss and K. J. Monty, JBC 238, 1019 (1963).
418. L. M. Siegel, E. M. Click, and K. J. Monty, BBRC 17, 125 (1964).
419. L. M. Siegel and K. J. Monty, BBRC 17,201 (1964).
420. T.Wainwright, BJ 103, 56p (1967).
421. A. Yoshimoto and R. Sato, BBA 153,576 (1968).
422. K.Prabhakararao and D. J. D. Nicholas, BBA 218, 122 (1970).
423. A. Yoshimoto and R. Sato, BBA 220, 190 (1970).
424. A. Yoshimoto, N. Naiki, and R. Sato, “Methods in Enzymology,” Vol. 17B,
p. 520, 1971.
425. F. J. Leinweber, L. M. Siegel, and K. J. Monty, JBC 240, 2699 (1965).
426. F.J. Leinweber and K. J. Monty, JBC 240, 782 (1965).
I I I I I I
so 400 450 MO 5 ~ )600 650 mo
Wavelength (nm)
FIG.45. Absorption spectra of E . coli sulfite reductase in the presence of reducing

agents. All experiments contained enzyme at a final concentration of 1.54 p M in
the sample cell. Spectra were recorded versus a buffer blank as soon as possible
after addition of components. A, enzyme in buffer; B, enzyme plus 0.3 mM NADPH
(0.1 ml of 23.1 fiM enzyme was added to 1.4 ml of a solution of NADPH which
had been bubbled with N, for 30 min); and C, enzyme plus sodium dithionite.
From Siege1 et al. (397).
MVH is inhibited by CO, cyanide, arsenite, and sulfide. Carbon monox-

ide, cyanide, and arsenite react only with the reduced enzyme. Spectral
modifications of the heme and other results have indicated that the heme
is the site of action of these inhibitors as well as the site a t which sulfite,
nitrite, and hydroxylamine are reduced. The Michaelis constants of the
enzyme for sulfite and NADPH are both about 4-5 ,AM.
Treatment of E . coli sulfite reductase with p-mercuriphenyl sulfonate
results in the specific release of F M N from the enzyme (390). FMN-
depleted sulfite reductase can be prepared also by photodestruction of
FMN. The enzyme-FMN dissociation constant is 10 n M a t 2 5 O , and light
irradiation can deplete the enzyme of F M N by destroying the released
flavin. These treatments do not lead to removal or destruction of other
components of the enzyme. The FMN-depleted enzyme is no longer capa-
ble of NADPH-dependent reduction of sulfite, nitrite, hydroxylamine,
and diaphorase-type acceptors such as ferricyanide, cytochrome c , and

menadione. However, it is fully capable of the reduction of acetylpyridine
adenine dinucleotide phosphate by NADPH, and the reduction of sulfite,
hydroxylamine, and nitrite by MVH. Further, the remaining flavin (es-
sentially FAD), but not the heme, is still reducible by NADPH as
rapidly as the most rapidly reduced flavin of the native enzyme (k = 190
sec-l) . These results and kinetic studies (416) have indicated that FAD
is probably the first acceptor of reducing equivalents from NADPH, that
FMN is the link between FAD and heme as well as the site of reduction
of diaphorase-type acceptors, and finally that the heme is the last compo-
nent of the enzyme t o be reduced. The FMN-depleted sulfite reductase
can be reactivated by added FMN, FAD, and a number of other flavins
(413, 415).
By treatment with 5 M urea and chromatography on DEAE-cellulose,
it has been possible to dissociate the E. coli NADPH-sulfite reductase
into a flavoprotein and a hemoprotein fraction. The flavoprotein fraction
has been shown to be an octamer of a single polypeptide of molecular
weight 58,000-60,000 and to contain F M N and FAD in equimolar
amounts, but no heme, nonheme iron, or labile sulfide. The hemoprotein
fraction is a tetramer of a polypeptide of molecular weight 54,000-57,000,
and contains heme, nonheme iron, and labile sulfide, but no flavin. Thus
NADPH-sulfite reductase is considered to be an enzyme of asp4 subunit
composition. The amino acid composition of the whole enzyme and the
flavoprotein and hemoprotein fractions have been determined (414).
The hemoprotein fraction has no NADPH-dependent activities, but
reduces sulfite in the presence of MVH. The flavoprotein fraction cata-
lyzes electron transfer from NADPH to diaphorase-type acceptors and
to acetylpyridine adenine dinucleotide phosphate. It does not reduce
sulfite, nitrite, or hydroxylarnine with either NADPH or MVH as elec-
tron donor. The molecular weight of the flavoprotein is estimated to be
470,000 (two-thirds of the whole enzyme). A similar flavoprotein with
a molecular weight of 460,000has been isolated from a S. typhimurium
mutant, which requires cysteine for growth. Other genetic data on the
S. typhimurium enzyme (994), which appears to be essentially identical
to the E. coli sulfite reductase, are in agreement with the above results.
Thus mutants lacking the flavoprotein or the hemoprotein component of
the enzyme and containing only the appropriate partial activities have
been obtained and the respective partial enzymes isolated. The absorption
spectra of sulfite reductase preparations from the wild type and from
these mutants are shown in Fig. 46, and the proposed structure for the
two components of the wild-type enzyme is shown in Fig. 47.Reconstitu-
tion of NADPH-sulfite reductase by recombination of the flavoprotein
FIG.46. Comparison of the absorption spectra of wild-type and mutant (cys G-439
and cys 1-68) sulfite reductases from Salmonella typhimurium. Spectra of S. typhi-
murium sulfite reductase, cys G-439 NADPH-cytochrome c reductase, and cys 1-68
NADPH-cytochrome c reductase, each dissolved in 0.05 M potassium phosphate
buffer, pH 7.7, containing 0.1 mM EDTA, were read against a blank containing
only buffer. The spectrum of each enzyme is presented in terms of its millimolar
extinction coefficients, assuming 8 moles of flavin per mole of enzyme. Light broken
line, calculated difference spectrum between those of wild-type and cys G enzymes
when both enzyme solutions contain equal concentrations of flavin. From Siegel
et al. (394).
and hemoprotein fractions separated by urea treatment has been achieved

(414). Similarly, appropriate partial enzymes isolated from S. typhimu-
Sum mutants have been recombined in vitro to reconstitute NADPH-
sulfite reductase activity.
cyt c
T
MV
FIG.47. Schematic diagram of the proposed structure and function of the S.

typhimurium NADPH-sulfite reductase and its component “subenzymes.” From
Siegel et al. (394).
co
CN
pCMPS MVH AsO;
NADPH.
AcPyADP’ ,
- - FAD FMN
/ \
*
,’
Heme - SO,’-.
NADP’ 1
Diaphorase
NOz-.
NH,OH
Acceptors.
0 2
FIG.48. Proposed minimum linear scheme of electron flow within the sulfite reduc-
tase molecule. The dotted arrow between FMN and heme indicates that the mecha-
nism of electron flow from flavin to heme is not clear. From Siege1 et al. (413).
The above results are summarized in the scheme shown in Fig. 48.
Thus, the NADPH-sulfite reductase of enterobacteria appears to be com-
posed of an octameric flavoprotein and a tetrameric hemoprotein, which
also contains iron and labile sulfide. The flavoprotein contains 4 moles
of FAD and 4 moles of F M N per mole, and appears to bind 1 mole of
NADP per mole of FAD. Electron transfer occurs from NADPH to FAD
to FMN, and the two flavin sequence is considered to be a device for
“stepping down” a two-electron donor, NADPH, to a one-electron accep-
tor, the heme (413). This is in agreement with the findings that flavin
free radical seems to appear after full reduction of the flavins, and that
the rate of FH. formation is too slow for the radical to serve as electron
donor in the diaphorase reactions (390). The flavoprotein segment cata-
lyzes electron transfer to the hemoprotein, to diaphorase-type acceptors,
and to acetylpyridine adenine dinucleotide phosphate. The latter reduc-
tion does not require the presence of FMN. The hemoprotein accepts elec-
trons from the flavoprotein or from appropriate dyes and in turn reduces
sulfite, nitrite, and hydroxylamine, apparently by direct electron transfer
through the heme. The role of iron and labile sulfide is not clear. They
might be involved in electronic communication between F M N and the
heme. It is also possible that electrons from MVH enter the system a t
the level of the iron and labile sulfide. The iron and labile sulfide are
likely associated in the form of clusters found in iron-sulfur proteins.
However, unlike most iron-sulfur proteins, these clusters appear to be
resistant to destruction by mercurials (397). Another interesting point
is that it has been suggested that both the heme and the iron-sulfur
moieties of NADPH-sulfite reductase have reduction potentials consider-
ably more negative than that of the electron donor, NADPH (415).
The NADPH-sulfite reductase of S. cerevisiae (398-401, 420-424) has
properties similar to the reductase from enterobacteria. The enzyme has
been purified to near homogeneity by Yoshimoto and Sato (400). It con-
tains 1 mole each of FAD and FMW and 5 g-atoms of iron per 350,000
g protein, and a hemelike chromophore with absorption peaks a t 386 and

587 nm. The oxidized enzyme is greenish yellow, and its spectrum (Fig.
49) is very similar to that of the E . coli sulfite reductase. The yeast
enzyme catalyzes the reduction of sulfite, nitrite, and hydroxylamine by
NADPH or MVH, and the reduction of diaphorase-type acceptors (Le.,
quinones and ferric compounds) by NADPH. The NADPH-dependent
activities are inhibited by NADP, 2’-AMP, and p-mercuribenzoate. The
Michaelis constants for NADPH, sulfite, nitrite, and hydroxylamine are
18-21 p M , 14 p M , 1 mM, and 4.5 mM, respectively. The NADPH-treated
enzyme is inhibited by carbon monoxide or cyanide (390,401). The latter
treatment results in the formation of a reddish violet color with peaks
a t 397 and 411 nm. Cyanide and carbon monoxide are considered to react
with the heme moiety and inhibit the enzyme a t the site of reduction
of sulfite, nitrite, and hydroxylamine.
Yoshimoto and Sat0 (421) have isolated sulfite reductases from four
mutants of S. cerevisiae incapable of sulfite assimilation. These enzymes
were inactive for sulfite reduction by NADPH, but could utilize MVH
as electron donor. All the mutant enzymes contained the chromophore
responsible for the 386- and 587-nm peaks, nonheme iron, and labile sul-
fide. Three of these mutant enzymes contained F M N ; no flavin was de-
tected in the fourth. The sedimentation coefficients of these preparations
1
2a
!i
e
::
9 1.0
1
300 4 00 500 600
Wavelength (nm)
FIG.49. Absorption spectra of purified NADPH-sulfite reductase from ’Saccharo-

myces cerevisiae. Curve A : 3.38 mg of enzyme protein in 2.0 ml of 0.3 M potassium
phosphate (pH 7.3) containing 1 mM EDTA. Curve B: a mixture containing 3.38
mg of enzyme protein, 0.2 pmole of NADP, 10 pmoles of glucose 6-phosphate, 8
units of glucose-6-phosphate dehydrogenase, 0.3 M potassium phosphate buffer (pH
7.3), and 1 mM EDTA in n final volume of 2.0 ml was incubated anaerobically
for 60 min. The reference cell contained all the components except sulfite reductase.
From Yoshimoto and Sat0 (400).
were 5.1 S for the enzyme lacking FMN, 6.6 S for the three enzymes
containing FMN, and 14.8 S for the wild-type enzyme. The authors have
concluded, therefore, that the yeast sulfite reductase is composed of a t
least three components, one each carrying FAD, FMN, and the heme.
The FAD-containing component is the site of NADPH oxidation, and
the heme-containing component the site of sulfite (also nitrite and hy-
droxylamine) reduction, Thus, the mutant enzymes lacking the former
component can reduce sulfite only in the presence of an artificial electron
donor such as MVH which could reduce both F M N and the heme (Fig.
50). These conclusions regarding the yeast sulfite reductase are essen-
tially in agreement with our current knowledge of the mechanism of
sulfite reduction by NADPH and MVH in the E. coli enzyme.
Since the reduction of sulfite to sulfide is a six-electron reaction, two-
electron reduction steps may be written as
so2- + (SO,2-) + (sol-) --f (15)
52-
However, in both the yeast and the E. coli systems, the stoichiometries
for NADPH:S2- and S03*-:S2- are 3 : l and 1:1, respectively. These re-
sults and the inability to detect 2-electron- and 4-electron-reduced inter-
mediates in these systems have suggested that such intermediates, if pres-
ent at all, must be firmly held on the surface of the enzyme. It has further
been suggested that the presence of multiple flavins and hemes in the
enzyme might be a device for achieving a rapid six-electron reduction
of sulfite without the release of intermediates (414). This situation is
analogous to the four-electron reduction of 0, to 2H,O by cytochrome
oxidase and the six-electron reduction of nitrite to ammonia by various
assimilatory nitrite reductases. However, unlike cytochrome oxidase,
,4,8
-
NADPH-reacting site
FAD FMN
M$=reacting site
j 587 Chromophore
(1) i (n) ; (rn) Wild-type
Strain 6 , l l and
20
5,,
I 587 Chromphore
cm, Strain 21
FIG.50. Schematic illustration of a tentative relationship between NADPH-sulfite

reductase from the wild-type strain of Saccharomyces cerevisiae and two categories
of MVH-sulfite reductases from various mutant strains. From Yoshimoto and Sat0
(421).
which does not seem to reduce H,O,, several nitrite and sulfite reductases
can reduce hydroxylamine to ammonia. This fact indicates that these
sulfite and nitrite reductases are capable of catalyzing a two-electron
reduction reaction. Indeed, sulfur compounds of oxidation states between
sulfite and sulfide have been observed during sulfite reduction by MVH-
sulfite reductases (395).
B. REDUCED
METHYLVIOLOGEN-SULFITE
REDUCTASES
The methyl viologen-sulfite reductases have been isolated from Asper-
gillus nidulans (4OZ, .4OoS), Desulfotomaculum nigrificans (392),Desulfo-
vibrio gigas (389, 427), Desulfovibrio vulgaris (396), and from higher
plants, such as spinach (359) and Allium odorum (405). These sulfite
reductases are incapable of utilizing NADPH or NADH as electron
donor. With the possible exception of the sulfite reductase of A . nidulans,
they also appear to lack flavin. They all exhibit, however, absorption
maxima characteristic of siroheme. Indeed, it has been shown by Murphy
and his colleagues (410, 412) that a number of sulfite and nitrite reduc-
tases appear to contain the siroheme-type tetrahydroporphyrin. In addi-
tion to heme, the sulfite reductase preparation of D.nigrificans also con-
tains nonheme iron, labile sulfide, and zinc. I n general, the methyl
viologen-sulfite reductases appear to have lower molecular weights than
the NADPH-sulfite reductases. For the enzymes from D . nigrificans,
spinach leaves, and D. vulgaris, the reported molecular weights are, re-
spectively, 145,000, 84,000, and 26,800. The physiological electron donor
for the MVH-sulfite reductases is not known. However, similar to the
ferredoxin-nitrite reductases, certain MVH-sulfite reductases have been
shown to use ferredoxin as electron donor (388, 389; see also 373a).
X. Addendum
This additional material is intended to bring to the readers’ attention

the recent major developments. For easy identification, the following
comments are marked by the same section designations to which they
pertain in the text of the chapter.
II,A,3 SDS-Acrylamide gel electrophoresis of the soluble NADH
dehydrogenase derived from complex I has shown that this enzyme is
composed of two subunits with molecular weights of approximately 28,000
and 56,000 (G. Dooijewaard and E. C. Slater, private communication).
427. H. D. Peck, BBRC 22, 112 (1966).

II,A,6 Hatefi et al. (428) have shown that incubation of submitochon-

drial particles or complex I with 2,3-butanedione, in the presence of
borate buffer a t pH 9.0, inhibits the NADPH to NAD transhydrogenase
activity with little or no effect on the NADH and NADPH dehydrogenase
activities. Presence in the incubation mixture of NAD, NADP, and more
+
effectively NAD NADP, prevented the inhibition of transhydrogenase
activity by butanedione. Since butanedione specifically reacts with pro-
tein arginyl residues, these findings agreed with the sensitivity of the
transhydrogenase activity to trypsin (see Fig. 17) and suggested that
the nucleotide-binding site of the transhydrogenase enzyme contains a
susceptible arginyl residue.
II,A,7 Ohnishi (429) has published revised Em values for the iron-
sulfur centers of complex I. These values for iron-sulfur centers 1, 2, 3
and 4 a t pH 7.2 are center 1, component a, -380 -+ 20 mV; center 1,
component b, -240 -C 20 mV; center 2, -20 *
20 mV; center 3, -240 *
20 mV; center 4, -410 f 20 mV. Additional centers ( 5 and 6 with Em
value of -260 f 20 mV) are also claimed by Ohnishi to exist in the
NADH-ubiquinone segment of the respiratory chain.
III,A According to Ohnishi and collaborators (430, 4 3 l ) , succinate
dehydrogenase preparations which are capable of electron transfer to
the respiratory chain contain 3 iron-sulfur centers, designated centers
S-1, S-2 and 5-3. The Em values of these centers a t pH 7.4 have been given
as follows: center S-1, O r + 10 mV; center 5-2, -4OOk 15 mV; center
S-3, +SO * 15 mV. Centers S-1 and S-2 are thought to contain one Fe,S2
cluster each. Center 5-3 is considered to contain one Fe,S, core, and to
be the oxygen-sensitive center necessary for electron transfer from
succinate dehydrogenase to the respiratory chain. The findings of Ohnishi
and co-workers reported here and above have not yet been confirmed by
other groups.
VI,A A communication from L. Lederer has pointed out that the
N-terminus of the intact cytochrome b, chain is Asn, not Glu, (cf. Table
XV). Also a note has appeared from the same laboratory (432) on addi-
tional similarities between cytochrome b, and liver microsomal cyto-
chrome b, (cf. Section VI,A,2).
428. Y. Hatefi, L. Djavadi-Ohaniance, and Y. Galante, in “Electron-Transfer

Chains and Oxidative Phosphorylation” (E. Quagliariello, et al., eds.) . North-
Holland Publ., Amsterdam (in press).
429. T. Ohnishi, BBA 387, 475 (1975).
430. T. Ohnishi, D. B. Winter, J. Lim: and T. E. King, BBRC 61, 1017 (1974).
431. T. Ohnishi, J. S. Leigh, D. B. Winter, J. Lim, and T. E. King, BBRC 61,
1026 (1974).
432. B. Guiard, F. Lederer, and C. Jacq, Nature (London) 255, 422 (1975).
VII According to Husain and Sadana (433), the earlier preparation

of Achromobacter fischeri nitrite reductase with a molecular weight of
95,000 2 40,000 (341) was found to be polydisperse. A monodisperse
preparation subsequently studied (433) had a molecular weight of 80,000,
and was shown to be composed of two subunits of approximate molecular
weights of 39,000 with methionine as the sole N-terminal residue. The
subunits are stated to be linked together by disulfide bridges.
I n a private communication, Henry Kamin has indicated to us that
(a) J. Vega, R. H. Garrett and L. Siege1 have demonstrated recently that
the nitrite reductase of Neurospora has siroheme as its prosthetic group,
and have given us permission to cite this new finding. Kamin has further
suggested that we emphasize the fact that enzymic properties and patterns
of repression and derepression clearly show that the E. coli nitrite reductase
is distinct from the sulfite reductase of this organism, which is also
capable of nitrite reduction (Section IX,A).
VIII Recent data of Bramlett and Peck (434) indicate that, as pre-
dicted in the above review, the adenylyl sulfate reductase of Desulfovibrio
vulgaris does contain acid-labile sulfide. The enzyme with a molecular
weight of 220,000 has been shown to contain 1 mole of FAD, 12 g-atoms
of iron and 12 moles of labile sulfide per mole. I n addition, SDS-gel
electrophoresis has revealed the presence of subunits with molecular
weights of 20,000 and 72,000.
X A new flavoprotein, containing iron and labile sulfide, has been
discovered in the mitochondria1 inner membrane independently by
Ruzicka and Beinert (435) and Hatefi et al. (436).The protein contains
acid-extractable FAD, and 4 g-atoms of iron and 4 moles of labile
sulfide per mole of flavin. The molecular properties and the enzymic
function of this iron-sulfur flavoprotein are not clear.
ACKNOWLEDGMENTS
The authors are grateful to the investigators whose work has been reviewed for
kindly providing them with reprints and preprints in advance of publication. They
also wish to thank Mrs. C. Schaeggl for typing the manuscript. The work of this
laboratory reported in Sections I1 and I11 was supported by USPHS grants AM08126
and CA13609 to Y. H.
433. M. Husain and J. Sadana, Rur. J. Biochem. 42, 283 (1974).

434. R. N. Bramlett and H. D. Peck, Jr., JBC 250, 2979 (1975).
435. F. J. Ruzirka and H. Beinert, BBRC 66, 622 (1975).
436. Y. Hatefi, Y. M. Galante, D. L. Stiggall, and L. Djavadi-Ohaniance, in “The
Structural Basis of Membrane Function” ( Y . Hatefi and L. Djavadi-Ohaniance,
edu.), p. 169. Academic Press, New York, 1976.
Cytochrome c Oxidase
WINSLOW S. CAUGHEY WILLIAM J . WALLACE
JOHN A . VOLPE SHINYA YOSHIKAWA
1. Introduction . . . . . . . . . . . . . . . . 299
A . The Role of Cytochrome c Oxidase in Biological Systems . 299
B . History . . . . . . . . . . . . . . . . 300
C . The Chemical and Physical Properties of Cytochrome c
Oxidase . . . . . . . . . . . . . . . 301
D . The Chemistry of Oxygen Reduction . . . . . . . 302
I1. Isolation and Characterization . . . . . . . . . . . 305
A . Preparation . . . . . . . . . . . . . . 305
B . Metal Components . . . . . . . . . . . . 307
C . Protein . . . . . . . . . . . . . . . . 309
D . Lipids . . . . . . . . . . . . . . . . 312
I11. Chemical and Physical Properties . . . . . . . . . . 313
A . Models . . . . . . . . . . . . . . . . 314
B. Electronic Spectroscopy . . . . . . . . . . . 315
C . Ligand Binding Studies . . . . . . . . . . . 319
D . Potentiometry . . . . . . . . . . . . . . 325
E . Electron Paramagnetic Resonance Studies . . . . . 329
F . Interaction of Cytochrome c Oxidase with Cytochrome c . 334
G . Kinetic Studies . . . . . . . . . . . . . 335
IV . Mechanisms . . . . . . . . . . . . . . . . . 337
I. Introduction
A. THEROLEOF CYTOCHROME
c OXIDASEIN BIOLOGICAL
SYSTEMS
Cytochrome c oxidase. the terminal oxidase in the respiratory metabo-
lism of all aerobic organisms. plants. animals. yeasts. algae. and some
bacteria. is responsible for catalyzing the reduction of dioxygen to water .
299
300 W. S. CAUGHEY, W. J. WALLACE, J. A. VOLPE, AND S. YOSHIKAWA
The electrons are provided by reduced cytochrome c in the following

overall reaction:
02 + 4 cyt c*+ + 4 H+ -+ 2 H20 + 4 cyt c3+ (1)
The free energy developed in oxygen reduction is used to promote oxida-
tive phosphorylation and, in consequence, becomes available, as ATP,
to satisfy the energy requirements of the cell. It is not surprising then
that this oxidase is found in high concentrations in tissues where the
energy requirements are high. Especially high levels have been observed
in heart muscle ( I ) , flight muscles of birds (2) and insects (S),red skele-
tal muscles ( 4 ) , liver mitochondria ( 5 ) , brain gray matter (6),corpora
lutea of sheep (Y), the parasitic worm Ascaris (8),and sugar cane roots
( 9 ) .It is estimated that 90% of the energy for heart muscle contraction
(1) and 96% of the energy for bird flight muscle contraction (2) is pro-
vided through aerobic metabolism via cytochrome c oxidase. Chronic
muscular disease gives rise to oxidase depletion (10). A further indi-
cation of its importance in the energetics of biological systems is the sug-
gcstion by Malmstrom (11) that 90% of biological oxygen consumption
is directed through the oxidase. The locus of this activity is in the inner
membrane of mitochondria in eukaryotes and in the plasma membrane
of prokaryotes.
B. HISTORY
In 1886, MacMunn ( 1 2 ) discovcred the respiratory pigment, myohema-
tin, which was widely distributed in plant and animal tissues. This impor-
tant observation attracted little attention a t the time of its publication
and became cffectively lost in the literature (IS). I n 1925, Keilin (14)
1. D. R. Challoner, N a l w e (London) 217, 78 (1968).

2. A. Tucker, Science 154, 150 (1966).
3. D. Keilin, Proc. R o y . SOC.,
Ser. B 98, 312 (1925).
4. M. S. Gordon, Science 159, 87 (1968).
5. D. L. Drabkin, Physiol. R e v . 31, 345 (1954).
6. S. Manocha and G. H. Bourne, E x p . Brain Res. 2, 230 (1966).
7. L. Arvy and P. Mauleon, C . R . SOC.Biol. 158, 453 (1964).
8. M. H. Smith, N a l w e (London) 223, 1129 (1969).
9. A. G. Alexander, J. Ag?. Unit!. P . R . 50, 131 (1966).
10. F. W. Booth and J. R. Kelso, Can. J. Physiol. Phurmucol. 51, 679 (1973);
V. P. Andrcev, Dokl. Akad. Nauk Beloniss. SSR 17, 470 (1973).
11. B. G. Malmstrom, Quart. Reu. Biophys. 6,389 (1973).
12. C. A. MacMunn, Phil. Trans. R o y . Soc. London 177, 267 (1886).
13. D. Keilin. in “The History of Cell Respiration and Cytochromes” (J. Keilin,
ed.), p. 95. Cambridge Univ, Press, London and New York, 1966.
14. D. Keilin, Proc. R o y . SOC.,Ser. B 98, 312 (1925).
5. CYTOCHROME C OXIDASE 301
rediscovered the MacMunn pigment, proved it to be a mixture‘ of three

spectroscopically identifiable components which he named cytochromes
a, b, and c, and showed them to be links in the respiratory chain that
connected activated substrates to activated dioxygen. Cytochromes a and
c showed a special relationship to each other; cytochrome a was the sole
physiological oxidizing agent for cytochrome c. Hence, the name cyto-
chrome c oxidase ( 1 5 ) .
The ligand binding and autoxidizability studies seemed compatible
with the presence of two components, cytochromes a and as, of which
only as was considered to be autoxidizable and able to combine with
carbon monoxide or cyanide. Keilin and Hartree (16) identified this a3
component spectroscopically with the Atinungsjerment which Warburg
had shown on the basis of the photochemical action spectrum of its CO
complex to be a heme protein ( 1 7 ) .Further advances in the understand-
ing of this important enzyme were not to come for another 25 years until
renewed interest and improved isolation techniques paved the way for
further progress. Lemberg (18) and Lemberg and Barrett (19) have sum-
marized the development of this understanding. Less extensive reviews
have been provided recently by Wharton (20) (emphasizing the role of
copper) , by Nicholls and Chance (21) (emphasizing kinetic measure-
ments) , and by Malmstrom (11) (emphasizing physicochemical
measurements).
C. THECHEMICAL
AND PHYSICAL
PROPERTIES
OF CYTOCHROME
c
OXIDASE
The focus of this article will be upon those aspects of the structure
and function of cytochrome c oxidase that contribute particularly to an
understanding of the chemical events that lead to the reduction of di-
oxygen to water. This important function is, however, only one aspect
of its physiological role. The functioning enzyme is provided with elec-
trons from the electron transport chain by cytochrome c, uses these elec-
trons to reduce dioxygen bound at the active site, communicates the
energy released in this reduction to the site of oxidative phosphorylation,
15. D. Keilin and E. F. Hartree, Proc. Roy. Soc., Ser. B 121, 173 (1936).
16. D. Keilin and E. F. Hartree, Nature (London) 141, 870 (1938).
17. 0. Warburg and €3. Negelein, Biochem. 2.214,64 (1929).
18. R. Lemberg, Physiol. Rev. 49, 48 (1969).
19. R. Lernberg and J. Barrett, “The Cytochromes.” Academic Press, New York,
1972.
D. C. Wharton, Metal Zons Biol. Syst. 3, 157 (1974).
20.
P. Nicholls and B. Chance, in “Molecular Mechanisms of Oxygen Activation”
21.
(0.
Hayaishi, ed.), p. 479. Academic Press, New York, 1974.
302 W. S. CAUGHEY, W. J . WALLACE, J . A. VOLPE, AND S. YOSHIKAWA
and is strictly controlled in these functions by respiratory control proces-

ses. The structure of the oxidase and its placement in the cell reflect
the multiplicity of roles it is required to play. The active site of the
enzyme, which contains iron, as heme A, and copper, is the locus for oxy-
gen binding and reduction; thus, it is necessary to provide pathways to
get electrons in and energy out of the active site. The multisubunit lipo-
protein in which the active site is embedded is itself embedded in the
inner mitochondria1 membrane in such a way that it, along with NADH
dehydrogenase and possibly cytochrome b, spans the membrane ( 2 2 ) .
Thus, a t each site of energy conservation there appears to be an electron
carrier which spans the entire membrane. Perhaps this transmembranous
configuration provides an extended surface for interaction with electron
carriers and may also provide for an important interaction with ATPase.
Such an interaction is supported by the observation of Wilson et al.
(23,24) that binding of ATP to ATPase influences the redox potentials
and ligand binding characteristics of oxidase.
Thus, cytochrome c oxidase appears very carefully tuned to a variety
of specific tasks. The components are so adjusted that a low energy path-
way is available to entering electrons, an efficient nonthermal energy
transport pathway is available for energy conservation, and a set of elec-
tron donors has been assembled into an array that will permit facile re-
duction of dioxygen via an efficient low energy pathway that is unprece-
dented in simple systems. Nonenzymic reduction of dioxygen to water
is often slow and usually involves a complex series of steps (25-27). The
enzymic reaction is fast and appears to be accomplished in either a single
step or in a series of concerted steps; no evidence for intermediate reduc-
tion products (i.e., superoxide or peroxide) has been found.
D. THECHEMISTRY REDUCTION
OF OXYGEN
The chemical inertness of dioxygen a t first seems surprising because

the transformation to water is so strongly thermodynamically favorable
( 4 3 0 kcal) (Fig. 1 ) (28, 2 9 ) . However, on the basis of the standard
redox potentials, the simplest reduction step, the one-electron step to
22. E. Racker, Hosp. Pract. p. 87 (1974).

23. J. G. Lindsay and D. F. Wilson, Biochemistry 11, 4613 (1972).
24. D. F. Wilson and K. Fairs, A B B 163, 491 (1974).
25. C. T. Mathews and R. G . Robins, Australas. Znst. Mining Met., Proc. C31
242, 47 (1972).
26. D. V. Stynes, H. C. Stynes, J. A. Ibers, and B. R. James, JACS 95, 1142
(1973).
27. I. A. Cohen and W. S. Caughey, Biochemistry 7,636 (1968).
28. M. S. Tsao and W. K. Wilmarth, Advan. Chem. Ser. 36, 113 (1962).
29. K. Sehested, 0. L. Rasmussen, and H. Fricke, J . Phys. Chem. 72,626 (1968).
m
Q-%O$B% d*%V
B A2OV
+0.82V
FIG.1. Standard oxidation-reduction potentials for the steps involved in the con-
version of dioxygen to water at 25” and pH 7.
superoxide, is thermodynamically highly disfavored (SO). Hence, reac-

tions involving dioxygen must either have enormous driving energies to
go through the superoxide or have access to a two-electron step to per-
oxide. Although this conclusion depends upon reasoning based upon stan-
dard potentials (SO) (all concentrations 1 M and pH 7), i t seems valid
since oxygen reduction by a low energy pathway is found to proceed via
the two-electron reduction to peroxide as the first recognizable product
(31) .
The other property of dioxygen that contributes to the slowness of its
reactions is its electronic structure (3.2). I n common with most stable
molecules dioxygen has an even number of electrons. Uncommonly,
though, the molecule is paramagnetic with two unpaired electrons in the
two highest occupied molecular orbitals. Since both peroxide and oxide
are completely spin paired, reactions involving dioxygen must involve
spin reversal and are therefore spin forbidden and slow. The forbiddenness
can be removed if dioxygen can interact with a paramagnetic center to
participate in exchange coupling. The transition metal ions frequently
have unpaired electrons and turn out to be excellent catalysts for dioxy-
gen reduction.
Despite the long history of transition metal ion induced reduction of
dioxygen surprisingly little is known about the mechanism of the reaction
( 2 5 ) .Perhaps the most studied of these metal ion oxygenation reactions
is that between the nitrogen ligand complexes of cobalt (11) and dioxygen.
The traditional method for preparing cobalt (111) ammine complexes was
to assemble the desired ligands on cobalt(I1) and oxygenate the solution.
Upon long oxygenation a cobalt (111) complex was formed ( 3 3 ) .It is now
known that the first step in this reaction is the formation of an unstable
Co”02 complex (34) and upon standing, a second cobalt(I1) ion is added
to produce the p-peroxo bridged complex [reactions (2) and (3)1. I n the
30. P. M. Wood, FEBS (Fed. Ew. Biochem. Soc.) Lett. 44, 22 (1974) ; P. George,
in “Oxidases and Related Redox Systems” (T. E. King, H. S. Mason, and M. Morri-
son, eds.), p. 3. Wiley, New York, 1965.
31. R. G. Wilkins, Adwnn. Chem. Ser. 100, 111 (1971).
32. H. Taube, J . Gen. Physiol. 49, 29 (1965).
33. A. Werner, 2. Anorg. Chem. 3, 267 (1893).
34. J. Simplicio and R. G. Wilkins, JACS 89,6092 (1967).
presence of a second bridging ligand, such as amido or hydroxo, the

p-peroxo complex is stabilized and can be isolated (36) [reaction ( 4 ) ] .
con-0, + con - com-0,

0-co"' (3)
L
corn- 0,0-corn coF0NL\
-o,Coul L = NR, or OR (4)
I n the absence of the second bridging ligand, further oxidation occurs

by a series of, as yet, not completely understood steps (36) and the co-
balt (111) product results [reaction ( 5 )1.
coin -0,
0-co'"
- 2 Com-OH
I n a similar way chromium(I1) (37) and copper(1) (38)react with di-

(5)
oxygen by way of a p-peroxo intermediate.

And, of special relevance to cytochrome c oxidase, the reactions of
hemes (27, 39, 40) as well as simple aquo ferrous iron (26, 41) with di-
oxygen seem to proceed through two-electron reduction of bridged inter-
mediates. Thus, dipyridine hemes react cleanly with dioxygen to form
py-FeII-py- py-Fen + py (6)
py-Fen + 0, -py-Fen-O, (7)
py-FeI1-O0, + py-Fen-py-Fem-O,-Fem-py (8)
py-FenI-O~Fem-pyt++Fdn-O-Fenl + 2 py (9)
Fem-O, -2 Fe"-O.
0 Fen'
FeIV-0. + py-Ferl-Feln-O-Fem + py (11)

35. M. Mori and J. A. Weil, JACS 89, 3732 (1967).
36. L. G. Stadtherr, R. Prados, and R. B. Martin, Znorg. Chem. 12,1814 (1973).
37. T. B. Joyner and W. K. Wilmarth, JACS 83, 516 (1961).
38. C. DeMarco, S. Dupre, C. Crifo, G. Rotilio, and D. Cavallini, ABB 144, 496
(1971).
39. I. A. Cohen and W. S. Caughey, in "Hemes and Hemoproteins" (B. Chance,
R. W. Estabrook, and T. Yonetani, eds.), p. 577. Academic Press, New York, 1966.
40. W. S. Caughey, J. L. Davies, W. H. Fuchsman, and S. McCoy, in "Structure
and Function of Cytochromes" (K. Okunuki, M. D. Kamen, and I. Sekuzu, eds.),
p. 20. Univ. of Tokyo Press, Tokyo, 1968.
41. P. George, JCS p. 4349 (1954).
p-oxobishemins (42) as shown by reactions (6) through ( 9 ) . Kinetic data

fully support reactions ( 6 ) through (8) but have not yet provided infor-
mation on the steps from the bridged oxygen species (presumably p-per-
oxobishemin) to p-0x0 dimer. When the solvent medium is able to provide
protons, solvolysis to produce H,O, would likely follow formation of the
p-peroxobishemins complex and sequence ( 6 ) , ( 7 ) , and (8). However,
where solvolysis cannot occur, as in aprotic solvents, formation of the
ferry1 (FexVO)intermediate as suggested by reactions (10) and (11) is
reasonable (40). But in no case does our certain knowledge about the
mechanism of the reduction reaction extend beyond the bridged dimer.
Apparently the peroxide formed in the initial reaction is a kinetically
inert (fully spin-paired) molecule that does not readily accept additional
electrons despite the favorable thermodynamics for reduction to water
(Fig. 1). The most commonly suggested mechanism (43) for the subse-
quent reduction steps in protic media is shown by reactions (12), (13),
and (14).
Mi--O\
0-MI
-H+
MI--,
0- H
+ M:
MI- 0,
0-H
+ M;+ - M,-o+ + M,OH+ (13)
MI--+ + H,O, -MIOH + 0, + H+ (14)

It is anticipated that despite the specially favored environment pro-
vided for oxygen reduction by the protein the fundamental principles of
chemistry in simple systems will apply to the enzyme. Thus, any proposed
mechanism for the enzymic reduction of dioxygen will have to accommo-
date two electron steps leading sequentially to peroxide and water and
provide a means to overcome the characteristic stability of the peroxide
intermediate.
II. Isolation and Characterization
A. PREPARATION
In general cytochrome c oxidase has been isolated from mitochondria
or mitochondria1 fragments by initial extraction of proteins with a sur-
42. N. Sadasivan, H. I. Eberspaecher, W. H. Fuchsman, and W. S. Caughey, Bio-
chemistry 8,534 (1969).
43. M. L. Kremer, Trans. Faraday Soc. 59, 2535 (1963); E. Zidoni and M. L.
Kremer, ABB 161, 658 (1974).
TABLE I
SPECIFIC ACTIVITIESO F CYTOCHROME C OXIDASE PREPAR.%TIONS FROM DIFFERENTISOL.4TION PROCEDURES
4
pl
Conditions of assay Specific activity
Initial
concn. cyto- pg s-l/mg
Preparation Buffer PH Temp. chrome c*+ protein/ml protein/3 ml Ref.
Yonetani 0.050 M phosphate 5.9 25 15 6.6 4.50 44

Griffiths and Wharton 0
7.0 38 18 0.55 2.70 45
Okunuki et al. 0.075 M phosphate 5.95 25 15 1.81 5.20 46
Horie and Morrison D
24 1.22 4.70 47
Sun and Jacobs 6.0 25 15 0.08 48
Wainio 0 . 1 0 M phosphate 6.0 25 (I
6.7 43
Fowler et al. 0.070 M phosphate 6.0 25 22 0.5 14.3 60
Kuboyama et al. 5.7 23 20 0.5 16.0 51
I-olpe and Caughey 0.10 M phosphate 5.9 22 15 0.33 15.4 5.9
a Data not provided.

face-active agent followed by removal of contaminating detergent and

protein (Table I ) , (44-52). Procedures differ in the nature and amount
of detergent used and retained. Recently, a preparation with high s o h -
bility in detergent-free media was reported ( 5 2 ) . The preparations ob-
tained vary widely in activity (Table I ) , and this suggests that the de-
tailed manipulative history is critical to the “nativeness” of the isolated
enzyme. Nevertheless, there is good reason to believe that the oxidases
isolated by many procedures are similar, but not identical, in response
to chemical and physical probes (18).
B. METALCOMPONENTS
It is now widely agreed that both copper and iron are essential compo-
nents (52-56). The metal content (11 nmoles/mg protein) and the iron
to copper ratio (1.0) are well established for the bovine enzyme, whereas
in yeast the reported metal contents are higher and more variable (5-15
nmoles of iron per milligram of protein) and the copper to iron ratio
is greater than unity (-1.5) (Table 11) (44-46, 48-52, 57-59). The iron
is present as the unusual heme, heme A, with an apparently unique struc-
ture (Fig. 2) (60). The coordination environment of copper is far less
clear, but the easy reducibility of copper seems to require a ligand envi-
44. T. Yonetani, JBC 236, 1680 (1961).

45. D. E. Griffiths and D. C. Wharton, JBC 236, 1850 (1961).
46. K. Okunuki, I. Sekuzu, T. Yonetani, and S. Takemori, J . Biochem. (Tokyo)
45, 847 (1958).
47. S. Horie and M. Morrison, JBC 238, 1855 (1963).
48. F. F. Sun and E. E. Jacobs, BBA 143,639 (1967).
49. W. W. Wainio, JBC 239, 1402 (1964).
50. L. R . Fowler, S. W. Richardson, and Y. Hatefi, BBA 64, 170 (1962).
51. M. Kuboyama, F. C. Yong, and T. E. King, JBC 247, 6375 (1972).
52. J. A. Volpe and W. S. Caughey, BBRC 61,502 (1974).
53. D. E. Griffiths and D. C. Wharton, JBC 236,1857 (1961).
54. H. Beinert, in “Biochemistry of Copper” (J. Peisach, P. Aisen, and W. E.
Blumberg, eds.), p. 213. Academic Press, New York, 1966.
55. E. C. Slater, B. F. Van Gelder, and K. Minnaert, in “Oxidases and Related
Redox Systems” (T. E. King, H. S. Mason, and M. Morrison, eds.), Vol. 2, p. 667.
Wiley, New York, 1965.
56. W. W. Wainio, in “Oxidasas and Related Redox Systems” (T. E. King, H. S.
57. M. S.Rubin and A. Tzagoloff, JBC 248, 4269 (1973.
58. T. L. Mason, R. 0. Poyton, D. C. Wharton, and G. Schatz, JBC 248, 1346
( 1973).
59. P. G. Shakespeare and H. R. Mahler, JBC 246,7649 (1971).
60. W. S. Caughey, G. A. Smythe, D. H. O’Keeffe, J. Maskasky, and M. L. Smith,
JBC 250, 7602 (1975).
TABLE I1
COMPOSITION
OF CYTOCHROMF: PREPARATIONS
c OXIDASE
Copper Iron
(nmole/mg (nmole/mg Phospholipid
Preparation Species protein) protein) (%) Ref.
a
Yonetani Bovine 7.2 10 44
Griffiths and Bovine 9.2-10.6 8.2-9.4 24 46
Wharton
a
Okunuki et al. Bovine 10.0 (I
46
Wainio Bovine (I
11.5 9 49
Fowler et al. Bovine 9.4 8.4-8.7 a 60
n
Sun,and Jacobs Bovine 8.2 22 48
Kuboyama et al. Bovine 11.8 11.1 20 61
Volpe and Bovine 11.0 10.9 20 68
Caughey
Rubin and Yeast 21.3 15.0 3.8 67
Tzagoloff
Mason et al. Yeast 15.8 9.4 2 68
Shakespeare and Yeast 6.2-11.6 5.5-7.2 a 69
Mahler
0 Data not provided.
ronment that stabilizes Cu(1) relative to Cu(I1). Thus, sulfur might

serve as a ligating atom (as in a disulfide) (61) as may an interaction
with the T system of the long side chain of the heme (6‘2). The latter
possibility is attractive because it provides a role for the uiiusual side
chain by affording a mechanism for electronic coupling between iron and
copper (Fig. 3 ) . Whatever the nature of the binding forces, the copper
must be well sequestered by the protein because it is not readily moved
by the usual complexing agents [EDTA, BCS (62a), and CN-] (53),and
there is no evidence that any ligand or any inhibitor binds directly to
copper a t the active site (53, 63-65). In addition, it is now clear that
there are two quite distinct kinds of copper. One kind is observed by
electron paramagnetic resonance (EPR) to be rapidly reduced. The other
61. P. Hemmerich, in ‘LBiocl~emistryof Copper” (J. Peisach, P. Aisen, and W. E.

Blumberg, eds.), p. 15.Academic Press, New York, 1966.
62. W. S. Caughey, Adunn. Chem. Ser. 100,248 (1971).
62a. BCS = bathocuproin sulfonate or 2,9-dimethy1-4,7-diphenyl-l,lO-phenanthro-
line sodium disulfonate.
63. Q.H.Gibson and C. Greenwood, JBC 240,2694 (1965).
64. K.J. H.Van Buuren, P. F. Zuurendonk, B. F. Van Gelder, and A. 0. Muijsers,
BBA 256, 243 (1972).
65. J. A. Volpe, M. C. O’Toole, and W. S. Caughey, BBRC 62, 48 (1975).
H2
F\
Y3
7H2
04\OH 04bi
FIG.2. The structure of heme A.
FIG.3. Schematic representation of a possible conformation of the 2-alkyl group

of heme A.
kind is not detected directly spectroscopically, but is reduced slowly

( 6 6 ) . Similar observations suggest that, despite the fact that the only
heme is heme A, two kinds of iron are present ( 6 6 ) .These observations
must mean that the minimal functional unit contains two iron atoms and
two copper atoms.
C. PROTEIN
The 11 nmoles of iron per milligram of protein of bovine preparations
corresponds t o an empirical molecular weight of about -90,000. When
20% lipid is added a total of 108,000 is obtained. If the functional unit
contains two iron and two copper atoms, the minimum molecular weight
then becomes -200,000 with the additional possibility that multiples of
66. C. R. Hartzell, R. E. Hansen, and H. Beinert, Proc. N u t . Acad. Sci. U S . 70,

2477 (1973).
310 W . S. CAUGHEY, W. J . WALLACE, J. A. VOLPE, AND 9. YOSHIKAWA
TABLE I11
AMINO ACID COMPOSITION c OXIDASE
OF CYTOCHROME
Number of residues
Kuboyama Matsubara
Amino acid el al." rt alSh
Lysine 28 39
Histidine 20 30
Arginine 21 31
Aspartic acid 52 60
Threonine 51 53
Serine 53 A4
Glutamic acid 52 60
Proline 48 46
Glycine 53 59
Alanine 55 62
Cysteine 7 7
Valine 45 51
Methionine 13 35
Isoleucine 40 43
Leucine 79 87
Tyrosine" 29 33
Phenylalanine 43 47
NH8 63 59
Tryptophan 27 30
Ethanolamine
Total residues 716 827
Molecular weightc 80,054 93,802
Molecular weightd 89,400
a From Kuboyama et al. (61).

* From Matsubara rt al. (YO).
c Based on amino acid composition; the prosthetic groups have not been included.
d Based on heme content.
this minimum unit could also be observed. Direct physical measure-

ments of the molecular weight have led to values of 72,000 for an inactive
preparation (67) and about 200,000 (68, 69) and 430,000 (51) for active
preparations. An empirical molecular weight for protein alone of about
90,000 (51,70) was suggested by amino acid analysis of two preparations
67. R S. Criddle and R. M. Bock, BBRC 1,138 (1959).

68. A. Tzagoloff, P. C. Yang, D. C. Wharton, and J. S. Reiske, BBA 96, 1 (1965);
W. W. Wainio, T. Laskawska-Klita, J. Rosmm, and D. Grebner, J. Bioenerg. 4, 455
(1973).
69. B. Love, S. H. P. Chan, and E. Stotz, JBC 245,6664 (1970).
70. H. Matsubara, Y. Orii, and K. Okunuki, BBA 97,61 (1965).
(Table 111).Extraneous protein and variable amounts of lipid can result

in differences in determined molecular weight values. However, a molecu-
lar weight of about 200,000 is widely accepted for functioning oxidase
with 20% lipid. Nevertheless, treatment with alkali (69) or with sodium
dodecyl sulfate (71) has been reported to result in a molecular weight
of about 100,000 for a monomer (72) which exhibited activity. Such a
catalytically active monomer has been difficult to reconcile with different
roles for each of two coppers and of two hemes. For this reason, the dimer
is still broadly considered the normal form of the native oxidase.
Subdivision below the monomer level occurs in the presence of sodium
dodecyl sulfate and thiols (mercaptoethanol). The oxidase is thus identi-
fied as a multisubunit protein. Both yeast (57, 73) and Neurosporu crussu
(74) oxidases were shown to be composed of seven subunits. Bovine heart
oxidase, on the other hand, has been reported to have between two (75,
76) and six (57, 76) subunits. The subunits from yeast have molecular
weights in the range of I, 40,000; 11, 33,000; 111, 22,000; IV, 14,000;
V, 12,700; VI, 12,700; and VII, 4,600 (57, 7 7 ) . The situation for bovine
heart is less clear but the six subunits are reported to have molecular
weights around I, 40,000; 11, 25,000; 111, 19,000; IV, 14,000; V, 10,000;
and VI, 8,000 ( 7 6 ) .When fewer than six subunits are found, their molecu-
lar weights invariably correspond to some of the six reported (75-78).
The subunit sizes differ for yeast and bovine heart ( 5 7 ) .That the protein
compositions differ is also reflected in the failure of antibodies against
subunits I1 and VI of yeast oxidase to cross-react with bovine heart oxi-
dase ( 7 9 ) .
In both yeast (80) and Neurosporu crassa (74) biosynthetic studies
have shown that the four small polypeptides are of cytoplasmic origin
while the three large polypeptides are of mitochondria1 origin. Similar
studies are unavailable for bovine heart. The polypeptide associations
of the copper and heme are still far from clear, but hints that the metals
71. Y. Orii, Y. Matsumura, and K. Okunuki, in “Oxidases and Related Redox
Systems” (T. E. King, H. S. Mason, and M. Morrison, eds.), p. 666. Univ. Park
72. S. H. P. Chan, B. Love, and E. Stotz, JBC 245,6669 (1970).
73. R. 0. Poyton and G. Schatz, JRC 250,752 (1975).
74. W. Sebald, W. Machleidt, and J. Otto, Eur. J. Biochem. 38, 311 (1973).
75. H. Komai and R. A. Capaldi, FEBS ( F e d . Eur. Biochem. SOC.) Lett. 30, 273
(1973).
76. T. Yamamoto and Y. Orii, J. Biochem. ( T o k y o ) 75, 1081 (1974).
77. R. A . Capaldi and H. Hayashi, FEBS (Fed. Eur. Biochem. SOC.) Lett. 26,
261 (1972).
78. J. J. Keirns, C. S. Yang, and M . V. Gilmour, BBRC 45,835 (1971).
79. R. 0. Poyton and G. Schatz, JBC 250, 762 (1975).
80. T. L. Mason and G. Schatz, JBC 248, 1355 (1973).
312 W. S. CAUGHEY, W. J. WALLACE, J. A. VOLPE, AND S . YOSHIKAWA
are coupled with the low molecular weight subunits have appeared (76,
78, 81). The picture that emerges, albeit imperfectly, from these studies
is of a multisubunit protein synthesized partly in the cytoplasm and
partly in the mitochondria (@). The subunits are then assembled in the
mitochondria1 membrane, where the functioning enzyme resides, with the
times of incorporation of copper, heme, and lipid quite uncertain.
D. LIPIDS
The role of the lipid component remains in doubt but is becoming some-
what clearer. The phospholipid content is reported for several prepara-
tions (Table IV) (83-86).Somewhat more diphosphatidylglycerol and
TABLE I V
PHOSPHOLIPID COMPOSITION OF CYTOCHROME C OXIDASK AND
MITOCHONDRIAFROM BOVINEHEART
Percent of total lipid
Phospha-
Phospha- Phospha- tidyl- Diphospha-
tidyl- tidyl- ethanol- tidyl-
Preparation investigators inositol choline amine glycerol Ref.
Mitochondria
Fleischer et al. 10 37 31 16 83
Awasthi et al. 5 38 30 18 84
Cytochrome c oxidase
Fleischer et al. 9 26 25 30 83
Brierley and Merola 11 27 21 31 86
Yu el al. 46 26 11 86
Awasthi et al. 32 30 30 84
Awasthi et al.@ 73 84
0 "Lipid-free" cytochrome c oxidase was obtained by treatment of mitochondria

with Triton X-100 and Triton X-114. The remaining lipids (27%) were not identified
but were possible breakdown products of diphosphatidylglycerol.
81. G.Schatz, G.S. P. Groot, T. Mason, W. Rouslin, D. C. Wharton, and J. Saltz-

gaber, Fed. Proc., Fed. Amer. SOC.Exp. Biol. 31,21 (1972).
82. W. L. Chen and F. C. Charalampous, BBA 294,329 (1973).
83. S. Fleischer, H.Klouwen, G. Brierley, E. Carpenter, and T. Moran, JBC 236,
2936 (1961).
84. Y . C. Awasthi, T. F. Chuang, T. W. Keenan, and F. L. Crane, BBA 226, 42
(1971).
85. G.P. Brierley and A. J. Merola, BBA 64,205 (1962)
86. C. Yu, L.Yu, and T. E. King, JBC 250,1383 (1975).
somewhat less phosphatidylethanolamine are found in the oxidase than

in whole mitochondria, but the amount and kind of phopholipid in an
enzyme preparation is dependent upon the treatment it has received ( 8 4 ) .
Conventional purification procedures preferentially remove phosphatidyli-
nositol, phosphatidylcholine, and phosphatidylethanolamine. These lipids
are extracted readily with acetone (84, 8 7 ) , methanol chloroform ( 8 8 ) ,
or nonionic detergents such as Triton X-100 or X-114 (89, 90) to leave
behind a small amount of lipid (-1.6-1.7 pg phosphorus per milligram
of protein) that is largely cardiolipin (Table IV) ( 8 4 ) . This residual
cardiolipin (about 2 moles/mole of oxidase) (87) is both difficult t o ex-
tract (only chloroform-methanol-ammonia proved efficacious) and insen-
sitive to digestion by phospholipase A under conditions where the phos-
pholipids of mitochondria are readily hydrolyzed ( 8 4 ) . As the other
phospholipids are removed, the enzyme becomes progressively less active
(87), but the activity is restored by adding back purified lipids, mitochon-
drial lipid, or detergent (such as Emasol) (84, 85, 8 7 ) . However, if the
residual cardiolipin is removed, restoration of activity is minimal ( 8 4 ) .
Such studies suggest a t least three ways in which phospholipid interacts
with and affects the activity of the enzyme: (1) in the incorporation of
the oxidase into membranes, with a concomitant increase in the acces-
sibility of the active site (91) ; (2) in the formation of a complex between
cytochrome c and the oxidase (84); and ( 3 ) in the stabilization of active
conformations ( 9 2 ) . It is evident that particular attention must be paid
to both lipid and detergent contents before many of the differences in
properties among preparations can be rationalized.
111. Chemical and Physical Properties
The chemical and physical properties of cytochrome c oxidase have

been widely studied in intact mitochondria, in mitochondria1 particles,
and as the isolated enzyme. Aside from variations in activity mentioned
above and the recently observed effect of detergent on intensities of elec-
tronic spectra (52), the properties have proved remarkably insensitive
87. W. L. Zahler and S. Fleischer, J . Bioenerg. 2, 209 (1971).
88. Y. C. Awasthi, T. F. Chuang, T. W. Keenan, and F. L. Crane, BBRC 39,
822 (1970).
89. E. E. Jacobs, F. H. Kirkpatrick, Jr., E. C. Andrews, W. Cunningham, and
F. L. Crane, BBRC 25, 96 (1966).
90. F. F. Sun, K. S. Preebindowski, F. L. Crane, and E. E. Jacobs, BBA 153,
804 (1968).
91. T . F. Chuang, Y . C. Awasthi, and F. L. Crane, J . Bioenerg. 5, 27 (1973).
92. T. F. Chuang and F. L. Crane, J. Bidenerg. 4, 563 (1973).
to its physical state. These studies have encompassed a wide range of

techniques, and in this section it is intended to review the information
available from them with particular emphasis upon the evaluation and
interpretation of those data which may be of importance in unraveling
the chemkal events that lead to the reduction of dioxygen to water.
A. MODELS
In the development of ideas about the fundamental mechanisms of di-
oxygen reduction one expects to be able to carry over into the protein
system the basic physical and inorganic chemistry of copper and heme
iron. Consequently, it is important to have a clear understanding of the
response of copper complexes, heme A, and, perhaps, copper-heme A com-
plexes to those chemical and physical probes that might be used with
the oxidase. Although copper (I) and copper (11) complexes have been
subjected to extensive investigation and their properties are quite well
understood, few probes are effective in following copper in the oxidase
and thus so little is known about the copper environment in the enzyme
that the transposition of knowledge from simple systems to the enzyme
system is difficult. The main probe is EPR spectroscopy where a portion,
and only a portion, of the copper(I1) is visible. There is a probable re-
quirement for copper to retain substantially the same coordination envi-
ronment in the oxidized and reduced forms under rapid turnover conditions
and for different environments for the two copper atoms. Fortunately,
there are more opportunities to probe the iron and its associated por-
phyrin. Several physical properties can be examined and the struc-
ture-property relationships have been quite extensively worked out with
some hemes, namely, heme B (protoheme) and other 2,4-disubstituted
deuterohemes (60, 62, 93). However, the heme studies to date are not
as relevant to the oxidase as future studies can be for two reasons. One
is that heme A, although its structure has recently been elucidated and
a few properties examined (60), has still not been as thoroughly studied
as heme B. This is important, since heme A, in those few properties that
have been studied differs significantly from heme B. A second factor is
the paucity of data on electron exchange interactions between any heme
system and another heme or a copper or another donor (or acceptor).
Several p-oxobishemins, including p-oxobishemin A, have been thor-
oughly characterized (60, 94) , p-hydrazine-bishemes have been preRared
93. W. S. Caughey, C. H. Barlow, D. H. O’Keeffe, and M. C. O’Toole, Ann. N . Y .
Acad. Sci. 206,296 (1973).
94. D. H. O’Keeffe, C. H. Barlow, G. A. Smythe, W. H. Fuchsman, T. H. Moss,
H. R. Lilienthal, and W. S. Caughey, Bioinorg. Chem. (in press).
(93),and copper-heme A interactions have been indicated (95); but,

nevertheless, in sum, relatively little is known about interactions be-
tween hemes, coppers, and other donor-acceptor systems of the types
likely to be present in cytoohrome c oxidase. Then, the application of
the ideas of inorganic chemistry to the oxidase must be largely inferential
but nevertheless can lead to useful generalizations about mechanistic
pathways that might be accessible to dioxygen en route to water. Further-
more, model studies cannot be expected to hold all the answers t o the
oxidase function because the protein is able to provide environments for
electron transfer and an active site geometry that may not be readily
duplicated in conventional chemical systems. Simple metal atom chemis-
try very rarely provides assemblies of metal ions in an ordered yet flexible
environment where cooperative interactions can facilitate, in a single
step, reactions that require a number of steps by the separate ions.
B. ELECTRONIC
SPECTROSCOPY
1. Absorption Spectra
Electronic spectra provide a simple and convenient way to monitor
changes induced in the oxidase by various chemical treatments. Indeed,
spectral observations were a t the core of the pioneering observations of
MacMunn ( l a ) , Keilin (96),and Warburg (97); and more recently
many investigators have examined the spectra of isolated oxidase, mito-
chondrial particles, and electron transport particles. The spectra of the
fully oxidized [oxidase ( I V ) ] (97a) and the fully reduced [oxidase ( 0 ) ]
oxidase have been well characterized (52) (Table V). In Table VI are
spectral parameters for ligand complexes of various oxidation states
(98-105). Although the spectra of most of these complexes have been
95. R. A. Bayne, G. A. Smythe, and W. S. Caughey, in “Probes of Structure and
Function of Macromolecules and Membranes” (B. Chance, T. Yonetani, and A. S.
Mildvan, eds.), Vol. 2, p. 613. Academic Press, New York, 1971.
96. D. Keilin, Proc. Roy. SOC.,Ser. B 98, 312 (1925).
97. 0. Warburg, Biochem. 2.152,479 (1924).
97a. Hereafter, the oxidation state (number of electrons removed) of cytochrome
oxidase will be represented by a roman numeral, 0 to IV, in parentheses.
98. W. H. Vanneste, Biochemistry 5, 838 (1966).
99. C. Greenwood, M. T. Wilson, and M. Brunori, BJ 137,205 (1974).
100. R. Lemberg and J. Stanbury, BBA 143,37 (1967).
101. A. 0. Muijsers, K. J. H. Van Buuren, and B. F. Van Gelder, B B A 333,
430 (1974).
102. R. Wever, A. 0. Muijsers, B. F. Van Gelder, E. P. Bakker, and K. J. H. Van
Buuren, BBA 325, 1 (1973).
103. Y. Orii and K. Okunuki, J. Bioehem. ( T o k y o ) 55,37 (1964).
316 W. S. CAUGHEY, W. J. WALLACE, J . A. VOLPE, AND S. YOSHIKAWA
TABLE V
ELECTRONIC
ABSORPTIONSPECTRAL DATAFOR CYTOCHROME
c
OXIDASEWITH A N D WITHOUT DETERGENT'
Fully oxidized: oxidase(1V)b
Xmax,nm 830 660 598 545 515 418
emx with detergente 1.2 2.4 8.7 8.2 8.3 79
e , ~without detergentd 1.1 2.0 6.6 6.3 6.3 59
Fully reduced: oxidase(0)~
Xmax,nm 603 560 517 443
C ~ M with detergent" 19.3 7.7 7.2 100
em^ without detergentd 14.5 6.3 5.3 78
a Volpe and Caughey (68).

As isolated.
"Detergent, Tween 20. Oxidase(1V) dissolved in 0.1 M phosphate buffer 0.75%
Tween 20, p H 7.4.
d Oxidase(1V) dissolved in 0.01 M phosphate p H 7.4.
Reduced with slight excess of sodium dithionite.
observed many times (18) and may, on this basis, be considered well
established, there remain some troublesome difficulties in their detailed
interpretation (11).
Assignment of frequency and intensity values to band maxima for
heme a, heme a,, and copper components (98) has been accepted rather
widely (18). However, the EPR evidence (66) for strong interaction
among the metal components in terms of facile electron exchange and
magnetic coupling indicates the likelihood that changes induced a t one
component (e.g., oxidation or ligand binding) will affect the electronic
spectra (and other properties) of the other components. Thus, it is risky
indeed to ascribe individuality to the hemes or coppers on the basis of
monotonic dependence of the spectra upon the states of the individual
TABLE VI
OF ABSORPTIONMAXIMAFOR VISIBLEAND SORET
WAVELENGTHS
SPECTRAOF COMPLEXESOF CYTOCHROMEc OXIDASE'
Oxidase-ligand
complexb Xrnnx.nrn Ref.
Oxidase(0) .CO 603 590(sh) 551 517 440(sh) 431. 98

Oxidase(II1). CO 590 547 429 99
Oxidase(0) . O p 603 550 428 100
Oxidase(II1) .02 605 550 428 99
Oxidase(1V) .F- 638 598 545 421 101
Oxidase(1V). N8- 660 598 545 421 108
Oxidase(1V) .CN- 598 545 425 10s
Preparative methods different but all contained detergent.

The precise nature of ligand binding has not been established in these complexes.
metal centers (11, 104, and conclusions drawn from experimental obser-
vations which depend upon this separation for quantitation and analysis
should be viewed with caution. These comments apply both to attempts
to synthesize spectra for cytochromes u and u3 on the assumption that
the properties of one heme are independent of the oxidation state of, and
ligands bound to, the other metals (98),and to the assignment of the
830-nm band to copper (105). Copper may be an active contributor to
the 830-nm band since changes in EPR signals resulting from copper (11)
have been noted to follow the intensity of the 830-nm band, but present
evidence does not show that copper is the unique contributor, or even
a contributor a t all, to the 830-nm band intensity (106-108).
A comparison of the spectral differences between cytochrome c oxidase
and heme A derivatives with those for hemoglobin or myoglobin and
heme B derivatives reveals similar effects of protein environment on the
heme moieties. The magnitude and direction of wavelength shifts upon
going from heme species to proteins are comparable for the three proteins
(Tables V, VI, and V I I ) . There is therefore little doubt that the 8-formyl
group of heme A remains intact upon incorporation into the apoenzyme.
The conversion from deoxy to carbon monoxy to oxy species results in
similar spectral shifts (Table VII) (52, 65, 109-112). An exception is
the blue shift in the 605-nm band upon reaction with CO, whereas hemo-
globin (Hb) and myoglobin (Mb) experience a red shift upon binding
CO. Nevertheless, the remaining spectral evidence supports similar ter-
minal CO to Fe binding for the three proteins as do infrared C-0 stretch
bands (113-116). Similar binding of 0, among the three proteins can
also be assumed; infrared 0-0 stretch band data have shown this to be
104. W. S. Caughey, Annu. Rev. Biochem. 36, 611 (1967).
105. D. C. Wharton and A. Tzagoloff, JBC 239,2036 (1964).
106. W. S. Caughey and S. McCoy, in “Biochemistry of Copper” (J. Peisach, P.
Aisen, and W. E. Blumberg, eds.), p. 271. Academic Press, New York, 1966.
107. L. N. Mackey, T. Kuwana, and C. R. Hartzell, FEBS (Fed. Eur. Biochem.
Soc.) Lett. 36, 326 (1973).
108. L. E. AndrCasson, B. G. Malmstrom, C. Stromberg, and T. Vanngard, FEBS
(Fed. Eur. Biochem. SOC.)Lett. 28,297 (1972).
109. R. Banerjee, Y. Alpert, A. F. Leterrier, and R. J. P. Williams, Biochemistry
8,2862 (1969).
110. Y. Sugita and Y. Yoneyama, JBC 246,389 (1971).
111. A. 0. Muijsers, R. H. Tiesjema, and B. F. Van Gelder, BBA 234, 481 (1971).
112. K. D. Hardrnan, E. H. Eylar, D. K. Ray, L. J. Banaszak, and F. R. N.
Curd, JBC 241, 432 (1966).
113. J. 0. Alben and W. S. Caughey, Biochemistry 7, 175 (1968).
114. S. McCoy and W. S. Caughey, in “Probes of Structure and Function of
Macromolecules and Membranes” (B. Chance, T. Yonetani, and A. S. Mildvan,
eds.), Vol. 2, p. 295. Academic Press, New York, 1971.
115. W. S. Caughey, R . A. Bayne, and S. McCoy, JCS,D p. 950 (1970).
116. W. S. Caughey, Ann. N . Y . Acnd. Sci. 174, 148 (1970).
TABLE VII
VISIBLEA N D SORETSPECTRAL
DATAFOR OXYAND CARBONYL COMPLEXES
OF HEMOGLOBIN, ?
MYOGLOBIN, c OXIDASE
A N D CYTOCHROME m
Complex Xmax.nm(ernM-') Ref.
Hba 53(13.3) 430(145) 109

HbCO 569( 15.0) 539(14.9) 420(208) 109 ?
HbOr 576(15.2) 542(14.3) 415(135) 109,110 4
Oxidase(0)b 603(14.5) 560(6.3) 517(5.3) 443(77) 51
Oxidase(0)COb 603( 14.0) s590(11.5) 551 (7.4) 517(6.3) 444 (50) 431(62) 66
Oxidase(0)OZc 603(12) 550(10) 428(84) 111
Mba 556(12) 434( 114) 111 F-1

MbCO 579(12.2) 540( 14.0) 424( 187) 111 ?
MbO, m(14.6) 543(13.7) 418(128) 111
a Here, Hb stands for hemoglobin and Mb for myoglobin.

determined in the absence of detergent.
e,,,~
determined in the presence of 1.0 % cholate.
srn~
m
true for HbO, (117)and MbOz (118) but not for oxidase(O).O, as
yet. The marked difference in the spectra of the oxidase(1V) and p-OXO-
bishemin A derivatives indicates a simple p-oxobishemin A structure is
not present. However, interactions of copper with a p-oxobishemin moiety
could result in the spectrum found for oxidase(1V) and explain also the
generally low sensitivities of the visible spectra of oxidase to changes
in ligation and oxidation state.
2. Circular Dichroic Spectra
The circular dichroic (CD) spectra of many cytochrome c oxidase de-
rivatives have been observed (119-123) with reproducible results. Here
also the insensitivity of the C D spectra to ligand substitution makes them
difficult to interpret. Thus, the general shapes of the C D spectra for cyto-
chromes a and a3 generated by the algebraic addition of spectra obtained
from a number of derivatives were sufficiently independent of the specific
ligands on the complexes used to generate the spectra that it was con-
cluded that the hemes were acting independently (121, 123). However,
detailed examination of the spectra revealed sufficient ligand-dependent
differences in band positions and intensities to suggest cooperative inter-
actions of the hemes (12U,122). Thus, directly opposed interpretations
of the same data were presented. On the one hand, the traditional concept
of identifiable cytochromes a and a3 is maintained, while on the other,
the increasingly supportable notion of cooperativity between the hemes
is suggested. Here also it has proved difficult to distinguish between the
hemes (cytochromes a and a,) in oxidase(0) and oxidase(1V). But these
hemes can be distinguished upon reaction with ligands or upon changing
the oxidation state. Current evidence favors heme-heme interaction which
results in changes induced a t one heme influencing the behavior and prop-
erties of the other.
C. LIGAND BINDING STUDIES

In addition to studies that have involved the determination of the ther-
modynamic states (potentiometric) and the observable valence states
117. C. H. Barlow, J. C. Maxwell, W. J. Wallace, and W. S. Caughey, BBRC 55,
91 (1973).
118. J. C. Maxwell, J. A. Volpe, C. H. Barlow, and W. S. Caughey, BBRC 58,
166 (1974).
119. D. W. Urry, W. W. Wainio, and D. Grebner, BBRC 27, 625 (1967).
120. D. W. Urry and B. F. Van Gelder, in “Structure and Function of Cyto-
chromes” (K. Okunuki, M. D. Kamen, and I. Sukuru, eds.), p. 210. Univ. of Tokyo
Press, Tokyo, 1968.
121. Y. P. Myer, JBC 248, 1241 (1971).
122. R. H. Tiesjema and B. F. Van Gelder, BBA 347,202 (1974).
123. F. C. Yong and T. E. King, BBRC 40, 1445 (1970).
(EPR)of the electron acceptors in cytochrome c oxidase, there have been

other chemical studies directed toward understanding the changes that
occur a t the catalytically active center as it goes from oxidized to reduced
and back to oxidized again. These studies, as with the potentiometric
and EPR studies, must be interpreted with some caution because they
must of necessity be conducted under nonturnover conditions. I n these
circumstances the steps that emerge from the analysis of the data may
not correspond to any transitions that occur in the functioning enzyme.
Nevertheless, such work has provided valuable insight into the interrela-
tionships between the components of the active site.
The binding of ligands and the consequent alteration of physical and
chemical properties have been used to probe for the identification and
differentiation of the cytochromes a and as (18).The observational tech-
niques employed have varied from UV-visible and CD spectroscopy (dis-
cussed above) through potentiometry (124, 125) and EPR spectroscopy
(126)( to be examined later), but there remains a measure of uncertainty
in the interpretation of the results. The stoichiometry and stereochemis-
try of complexes have both been inferred from the changes in electronic
spectra that accompany ligand binding and/or changes in the oxidation
state (98, 127). However, while such spectral changes are convenient
monitors of ligand interaction, they do not measure directly either the
nature or the extent of ligand binding. Consequently, the complexes under
study are frequently ill-defined despite the extensive examination that
they may have undergone.
1. Azide, Fluoride, and Cyanide
Fluoride (101, 128), azide (102, 128-150), and cyanide (103,128, 129,
151-156) have been observed spectrophotometrically to bind reversibly
to the oxidase. The absorbance changes are small in all cases and the
kinetics complex-at least two forms of complex are kinetically observ-
124. J. S. Leigh, Jr., D. F. Wilson, C. S. Owen, and T. E. King, ABB 160, 476
( 1974).
125. Y. Fujihara, T. Kuwana, and C. R. Hartzell, BBRC 61, 538 (1974).
126. C. R. Hartzell and H. Beinert, BBA (in press).
127. T. Yonetani, JBC 235, 845 (1960).
128. S. Yoshikawa and Y. Orii, J . Bioch.em. ( T o k y o ) 71, 859 (1972).
129. S. Yoshikawa and Y. Orii, J . Biochem. ( T o k y o ) 71,873 (1972).
130. R. Wever, A. 0. Muijsers, and B. F. Van Gelder, BBA 325,8 (1973).
131. P. W. Camerino and T. E. King, JBC 241,970 (1960).
132. K. J. H. Van Buuren, P. F. Zuurendonk, B. F. Van Gelder, and A. 0. Muijsers,
BBA 256, 243 (1972).
133. K. J. H. Van Buuren, P. Nicholls, and B. F. Van Gelder, BBA 256, 258 (1972).
134. S. Yoshikawa and Y. Orii, J . Biochem. ( T o k y o ) 73, 637 (1973).
135. S. Yoshikawa and Y. Orii, J . Biochem. ( T o k y o ) 76, 271 (1974).
able in each case. Where the stoichiometry of the ligand binding reaction
was tested (cyanide and azide), only a single (133) 1:l complex (102,
132) was formed. Fluoride was suggested, on the basis of its inhibitory
behavior, to bind 2 moles/mole of oxidase although EPR spectra suggest
little interaction with the heme iron (128). The site(s) of ligand binding
has, then, not been clear but the suggestion has been made (136, 137)
that azide inhibits a t a site common to both electron transport and energy
conservation. In support of this contention, Wilson (137)has shown that
5-13 (5-chloro-3-t-butyl-2'-chloro-4'-nitrosalicylamide) releases azide in-
hibition of ATPase and electron transfer and that 1.35 molecules of s-13
per respiratory chain are needed to release inhibition of respiration. I n
the presence of azide, the redox potential is altered, (138) and the EPR
visible iron undergoes a high- to low-spin transition (139).Infrared ob-
servations of oxidase(1V) in the presence of azide revealed that even
upon long standing the vNS- is not shifted from its free solution value
( V = 2047 em-', Av% = 28 cm-') (140). However, oxidase(I1) treated
with azide followed by reoxidation t o oxidase (IV) exhibited a frequency
(2038 cm-', h v , = 14 em-') consistent with iron-bound azide (140, 141).
Clearly, azide was bound to the iron in the latter case but not in the
former. It is important to discriminate among the possible ligand bonding
configurations, but such differences in binding could not be established
on the basis of electronic spectra alone. Infrared difference spectroscopy
has enjoyed considerable success in the elucidation of the nature of bind-
ing of a variety of ligands to hemes and heme proteins (116, 1411, and
since the method provides a direct measure of the character of the ligand
bonds it promises to be a powerful tool in probing oxidase ligands, even
within intact tissue (142), despite the large size of the enzyme.
2. Carbon Monoxide
Carbon monoxide binds readily to iron (11), but not iron (111), por-
phyrins to form complexes that are quite distinctive in terms of the spec-
tral properties both of the heme and of the bound CO. Thus, CO has
been widely used as a probe of the active site of heme proteins (113,
136. D. F. Wilson and B. Chance, BBRC 23,751 (1966).

137. D. F. Wilson, Biochemistry 8, 2475 (1969).
138. D. F. Wilson, J. G. Lindsay, and E. S. Brocklehurst, BBA 256, 277 (1972).
139. B. F. Van Gelder and H. Beinert, BBA 189, 1 (1969).
140. W. S. Caughey, C. H. Barlow, J. C. Maxwell, J. A. Volpe, and W. J. Wallace,
Ann. N . Y . Acad. Sci. 244, 1 (1975).
141. S. McCoy and W. S. Caughey, Biochemistry 9,2387 (1970).
142. J. C. Maxwell, C. H. Barlow, J. E. Spallholz, and W. S. Caughey, BBRC
61, 230 (1974).
114). In the past, such investigations with cytochrome c oxidase have

been limited to visible UV spectroscopy ; however, more recently, infrared
(66, 116), EPR (124), and potentiometric (138) techniques have been
employed to investigate the reaction of CO.
Carbon monoxide may bind a t iron(I1) (62, 115) or copper(1) (65,115)
0
II
either as a terminal (M-CO) or bridging (M-C-M) ligand. The pres-
ence of the C-0 stretch band a t 1963.5 cm-l for the CO complex with
oxidase(0) provided firm evidence for binding a t iron(II), and not a t
copper(I), in a terminal (end-on) fashion similar to HbACO (v-1951
cm-1) (115) and MbCO (v-1944 cm-1) (114). Furthermore, the extreme
narrowness of the band showed a well-ordered (nonrandom) environment
about the bound CO well isolated from external medium. A similar band
was found for CO bound to the oxidase in intact heart muscle (142).
These data are not consistent with suggestions that CO is bound coop-
eratively by both copper(1) and iron(I1) in CO (143).
The stoichiometry of CO binding to the oxidase has been considered
to be one CO per two hemes since Keilin's original interpretations of visi-
ble spectra (13). Results from many subsequent attempts (98, 144-147)
to establish such a stoichiometry though variable, were interpreted as
consistent with the Keilin suggestion. However, recent evidence from in-
frared intensities and exchange of CO from the oxidase to Hb gave clear
evidence for one CO for each heme A (66). The very narrow bandwidth
( A V ~ , ~6, cm-I) shows the vc0 values for each heme are either identical
or very nearly so since if more than one bond were present the frequencies
would necessarily be nearly the same if such a narrow width were to
result. Since vco is a very sensitive probe of cis, trans, and medium effects
in heme proteins (49, 6 2 ) , the essentially identical vc0 for each heme
A provides strong evidence that each heme A is of the same structure
including trans ligand (histidine?) and that the environment about each
bound CO ligand is the same. The CO ligands in HbACO are found at
1950 and 1952 cm-*, corresponding to the a! and p subunits, respectively,
to give a v l I Z of 8 cm-' (148). It is of interest that in the presence
of one iron(II1) heme, e.g., oxidase (11).CO, only one CO binds with
143. J. G. Lindsay and D. F. Wilson, FEBS (Fed. E w . Biochem. S o d Lett. 48,
45 (1974).
144. Q.H. Gibson and C. Greenwood, BJ 86,541 (1963).
145. M. Morrison and S. Horie, JBC 240, 1359 (1965).
146. Q. H. Gibson, G. Palmer, and D. C. Wharton, JBC 240, 915 (1965).
147. G. E. Mansley, J. T. Stanbury, and R. Lemberg, BBA 113, 33 (1966).
148. J. A. Volpe, J. C. Maxwell, W. J. Wallace, W. S. Caughey, and S. Charache,
Fed. Proc., Fed. Amer. SOC.Exp. Biol. 34, 687 (1975).
vc0 a t 1965 cm-’ slightly, but only slightly shifted from the value for
the fully reduced oxidase CO complex (149).
Little is known about the relative affinities of the two heme A sites
for CO. The infrared and H b exchange evidence noted above demon-
strates that two CO ligands can bind, a t least in certain enzyme prepara-
tions. However, it is reasonable to expect the first CO to be bound with
greater affinity than the second; therefore, in some preparations, only
one CO may bind. I n mono-CO complexes, the heme to which CO binds
can be called a3. But, there is no basis for knowing which of the
two liemes in the fully reduced oxidase represents the preferred binding
site or even whether there is a preferred binding site. Once CO becomes
bound to one heme ( a 3 ) the
, other heme may in consequence adopt differ-
ent properties and become heme a. A reasonable interpretation of infrared
(65) and other (99) data is one in which with either one or two electrons
added to the fully oxidized enzyme, one CO binds a t iron. And, as dis-
cussed below, potentiometric and EPR evidence that CO binding affects
the properties of other metal centers has been obtained (1.24, 137).
3. Dioxygen
The infrared spectra show that CO binds to the oxidase (65, 114) in
much the same way it binds to H b and Mb (113, 114). Hence, it.might
be expected that the enzyme would form oxidase.0, in the same way
that H b and M b form HbO, and MbO,. Formation of such an “oxygen-
ated” complex represents a quite logical initial step in the sequence of
reactions that lead to the reduction of oxygen to water by oxidase. Conse-
quently, there have been a number of attempts to identify and charac-
terize an “oxygenated” oxidase.
In 1958, Okunuki and Sekuzu discovered a new form of cytochrome
c oxidase, characterized by a Soret band a t 426-428 nm and designated
i t “oxygenated” oxidase (160).The presence of the band a t 428 nm has
been taken as a clear indication of the formation of the oxygenated com-
plex (100) and is, in fact, the only evidence for the formation of such
a complex. And, despite some uncertainty about its spectral characteris-
tics (151), all discussion of the oxygenated complex has been cast in
terms of the variation in both position and intensity of the band at 428
nm. I n the original concept of Okunuki and Sekuzu, dioxygen was thought
149. J. G. Lindsay, ABB 163, 705 (1974).
150. K. Okunrrki, B. Hagihara, I. Sekuzu, and T. Horio, in (‘Proceedings of the
International Symposium on Enzyme Chemistry, Tokyo and Kyoto, 1957” (K.
Ichihara, ed.), p. 264. Academic Press, New York, 1958.
151. H. Beinert, C. R. Hartaell, and W.. H. Orme-Johnson, in “Probes of Structure
and Function of Macromolecules and Membranes” (B. Chance, T. Yonetani, and
A. S.Mildvan, eds.), Vol. 2, p. 575. Academic Press, New York, 1971.
324 W. S. CAUGHEY, W. J . WALLACE, J. A. VOLPE, AND S. YOSHIKAWA
to bind reversibly to oxidase(O), but the only direct evidence for this
has been the observation (158) that upon evacuation the dioxygen ap-
pears t o be removed and replaced by carbon monoxide. Greenwood et al.
(99) have prepared the “oxygenated” oxidase by photolyzing ferricy-
anide oxidized oxidase(0) .CO in the presence of 0,. The complex is
formed readily and is fairly stable. Decay appears to occur through a
slow intermolecular transfer reaction (153-155). Clearly, the mixed
valence complex oxidase(II1) -0, is not readily formulated except on
the familiar HbO, bent end-on model since only one reduced heme iron
is available and there is no evidence to suggest that copper contributes
to ligand binding. With two hemes present, two different mono dioxygen
complexes are possible, but a t present there is no experimental basis upon
which to decide whether the two complexes have the same or different
properties or whether one is preferred over the other. However, the pre-
sumed observation of relatively stable mixed valence oxygenated oxidase
complexes suggests that it may be possible to subject intermediate steps
in the reduction of dioxygen to scrutiny by more discriminating tech-
niques (such as infrared) and that this may provide the clues that will
lead to a detailed understanding of the steps involved in the important
enzymic reduction.
The ability to observe oxidase(0) .O, a t low temperature and oxi-
dase(II.1) or (11)*02 a t room temperature is quite understandable in
chemical terms. Many of the other observations on “oxygenated” oxidase
are rather more difficult to rationalize. Both Gilmour et al. (156) and
Tiesjema et al. (157) observed that oxidase(0) reacts rapidly (<5 msec)
with 0, to produce what is judged from the spectrum to be a mixture
of oxidized [oxidase(IV)] and oxygenated oxidase. The proportions of
the oxidized and oxygenated components in the reaction mixture depend
on the concentration of dioxygen, more 0, gave more oxidase(O)-O,,
and the latter complex was only slowly transformed into oxidase(1V)
( t l I z= 1 hr) . Orii and King (158) found three sequentially formed com-
to oxidase (IV) 3 under not
to~ *IIIt~/~ahr
plexes [ I h J S n r i n to I I 5 / ~ ~ 6 m
152. A. J. Davison and W. W. Wainio, Fed. Proc., Fed. Amer. SOC.Ezp. Biol. 23,
323 (1964).
153. Q. H. Gibson, C. Greenwood, D. C. Wharton, and G. Palmer, JBC 240, 888
(1966).
154. D. C. Wharton and Q. H. Gibson, JBC 243, 702 (1968).
155. B. Chance, C. Saronio, and J. S. Leigh, Jr., Proc. N a t . Acad. Sci:U. S. 72,
1635 (1975).
156. M. V. Gilmour, R. Lemberg, and B. Chance, BBA 172, 37 (1969).
157. R. H. Tiesjema, A. 0. Muijsers, and B. F. Van Gelder, BBA 256,32 (1972).
158. Y. Orii and T. E. King, FEBS (Fed. Eur. Biochem. Soc.) L e t t . 21, 199
(1972).
very different conditions. These observations are difficult to rationalize

unless it makes a difference how oxygen binds to oxidase(0) in the initial
step. Perhaps it is possible (indeed, even likely) that oxygen can interact
with either of the two hemes [to form oxidase(0) -Or] or with both of
the hemes [to form oxidase(0) . (O,),]. This would be analogous to the
reaction of oxidase(0) with CO ( 6 5 ) . It is easy t o see the possibility
that those molecules that bound one dioxygen could go on to oxidase(1V)
but those that bound two dioxygens would be unable (not enough elec-
trons) to complete the cycle and would remain trapped a t the “oxygen-
ated” stage. The slow oxidation observed could then be accounted for
by slow loss of one dioxygen and subsequent oxidation. If this speculation
is correct, it would point out a requirement that only a single dioxygen
binding site be available in the operating enzyme, i.e., one reduced and
one oxidized heme iron.
D. POTENTIOMETRY
The redox properties of cytochrome c oxidase have been investigated
both by anaerobic reductive titrations (159) and by potentiometric titra-
tions (160).Since measurements of the latter kind are, a t least in princi-
ple, able to provide absolute potential values, they have been favored
in recent studies. The inconsistencies found in the early work (161-163)
may have resulted from the lack of equilibrium conditions in some cases,
from differences in the preparations, or simply from some incorrect inter-
pretations of data. The importance of establishing that equilibrium condi-
tions are attained has recently been recognized (107, lab, 125), but iden-
tical sets of measurements on the various types of preparations have yet
to be reported.
1. Electron Economy
There now seems little doubt that complete oxidation or reduction of
the isolated oxidase under anaerobic conditions requires four electron
equivalents ( 164, 165). When the potentiometric titration is followed
spectrophotometrically (at 605-630 nm) , high potential (at -350-375
mV) and low potential (210-230 mV) hemes (Table VIII) are indicated
D. C. Wharton and M. A. Cusanovich, BBRC 37, 111 (1969).
159.
D. F. Wilson and P. L. Dutton, ABB 136, 583 (1970).
160.
K. Minnaert, BBA 110, 42 (1965).
161.
A. 0. Muijsers, R. H. Tiesjema, R. W. Henderson, and B. F. Van Gelder,
162.
BBA 267, 216 (1972).
163. T. Tsudzuki and D. F. Wilson, ABB 145, 149 (1971).
164. B. F. Van Gelder, BBA 118,36 (1966).
165. W. R. Heineman, T. Kuwana, and C. R. Hartzell, BBRC 49, 1 (1972).
326 W. S. CAUGHEY, W. J. WALLACE, J . A. VOLPE, AND S. YOSHIKAWA
TABLE VIII
MIDPOINTPOTENTIALSFOR THE METALCENTERS c
OF CYTOCHROME
OXIDASEI N ISOLATEDPROTEINA N D I N MITOCHONDRIA
Heme a Copper
Investigators
Muijsers et al." 200-250 335-360

Tsudsuki and Wilsonb 225 375 225
Leigh et al." 210 360
Wilson et a1.d 220 380
Mackey et a1.C 215 f 10 340 k 10 215 k 10 350 f 10
Wilson et aZ.d*e 2.50
Wilson et a1.d.J 105 345
~~ ~
a In the absence of cytochrome c the oxidase titrated as a single component with

n = 1 and E = 280 mV.
Partially purified cytochrome c oxidase; purified oxidase titrated aa a single com-
ponent with n = 1 and E = 285 mV.
Isolated cytochrome c oxidase.
Mitochondria.
In the presence of a 50 % CO, 50 % argon gas mixture.
J In the presence of 20 mM aride.
(125,162, 166). Wilson (137)has interpreted these results in terms of

a, (high potential) and a (low potential) heme components. However,
as pointed out so clearly by Malmstrom (11), a highly cooperative sys-
tem, such as this one must surely be, does not lend itself to a unique
interpretation of data obtained in these potentiometric spectrophoto-
metric studies in terms of the assignment of low and high potential char-
acter specifically to a and a,. This does not mean that the hemes may
not reside in different environments rather it means that the experimental
observations do not require they do so.
2. Iron-Copper Coupling and Redox Potentials

One of the objectives of these studies has been to gather evidence about
the pathway of electrons through the oxidase to oxygen ; thus, there have
been a number of attempts to differentiate between the iron and the cop-
per in each of the redox steps. The earliest attempts a t the separation
were based upon the assignment of the 60S-nm band to heme iron and
the 830-nm band to copper. Although there is no doubt that the 830-nm
166. L. N. Mackey, T. Kuwana, and C . R. Hartzell, FEBS (Fed. Eur. Biochem.

Soc.) Lett. 36, 326 (1973).
band is sensitive to the oxidation state of the metals (53,167), the identi-
fication of this band as resulting from copper has been called into ques-
tion (107). Nevertheless, Gibson and Greenwood ( 1 6 7 ) )on the basis of
a kinetic study using the 830-band to monitor the copper oxidation state,
placed the copper ( E m 280 mV) between the two hemes (a and a,)
N
in redox potential. However, this value is the mean of the high and low
potential heme components and may represent contributions from both
components. In a potentiometric study, also based upon the 830-nm band,
Tsudzuki and Wilson (163) reported an Em, value of 225 mV and an
n value of 1.0 for copper, and when the decay of copper(I1) was fol-
lowed by EPR as a function of potential, the corresponding value of E m
was 250 mV (168). I n a more extensive study, Mackey et al. (107) gen-
erated oxidant and reductant coulometrically and followed the potential
as a function of the number of electron equivalents added. From a com-
puter analysis of the resultant potential-composition curves, they as-
signed midpoint potentials to each of the metal atoms in the enzyme as
shown in Table VIII. These results confirmed the previous observations
that reduction (or oxidation) proceeds through two iron-copper pairs,
one at high potential and one at low potential. Although it now seems
certain that the iron and copper components of the oxidase titrate as
two pairs, one a t high potential and one a t low potential, caution must
be applied in using these results to infer the relation between the heme
iron and the copper in the fully oxidized (or fully reduced) states. I n
such a highly cooperative system, the entry of a single electron may have
a considerable perturbing effect upon the relative electron affinities of
all the other electroactive centers in the molecule.
3. Effect of Ligand Binding o n R e d o x Potentials

The potentiometric ligand binding studies of Wilson et al.(l38) give
results that are consistent with the idea that in oxidase(I1) there is a
reduced iron-copper pair and an oxidized iron-copper pair and that the
hemes, at least, remain in interactive contact. In the presence of CO po-
tentiometric titration of the oxidase (0) in pigeon heart mitochondria by
ferricyanide indicates the presence of only a single electroactive compo-
nent (Em, = 250 mV, n = 1.0). This result was interpreted to mean that
CO has been bound to cytochrome LU? raising its Em to a value where
it is not accessible to reaction with ferricyanide leaving only the low
potential component cytochrome a as a reactive species in the titration.
Although it would have been more satisfying to have had the experiment
167. Q. H. Gibson and C. Greenwood, JBC 240, 2694 (1965).

168. D. F. Wilson and J. S. Leigh, Jr., ABB 150, 154 (1972).
performed on carefully characterized CO complex (es) [e.g., by infrared

spectroscopy (65)] , the observations are not out of line with what would
be expected for a system in which only a single CO is bound. Even though
it has been shown that two CO molecules will bind to oxidase(0) (65),
each CO is expected to bind to oxidase(0) with different affinities, and
there may well be circumstances under which one CO is bound per two
hemes in fully reduced oxidase. Then the rise in potential for the unli-
ganded heme A component would mean that the cytochromes are coupled
in such a way that the unliganded cytochrome is sensitive to the changes
in electron density produced a t the other cytochrome when it binds with
CO. Alternatively, Malmstrom has suggested (11) that the data could
be equally well interpreted in terms of the binding of CO to the low po-
tential component so that it became the high potential component (by
a Nernst law potential shift) and the cooperativity in the system then
causing the original high potential component to become less readily re-
ducible. However, even in these circumstances, the potentiometric mea-
surements would leave some unresolved problems. If the two heme-copper
couples are a t different potentials in the fully reduced oxidase, it would
be very surprising if the strongly electrophilic ligand CO bound prefer-
entially to the relatively electron deficient high potential component of
the oxidase. However, if CO does not bind to the high potential compo-
nent, it is necessary to suppose that in consequence of binding CO to
the low potential component the high potential component has become
less reducible (lower redox potential). A mechanism for this kind of
charge transfer is difficult to imagine. There are then difficulties in com-
plete rationalization of the experimental results when an initial difference
in electron affinity for the two hemes is assumed.
The potentiometric observations in the presence of azide are as ex-
pected, if azide binds only to the oxidized low potential component (137).
Here, too, the ligand appears to bind to the wrong component. If there
is an initial difference in reducibility between the two hemes, the high
potential (more readily reducible) component would be expected to have
the lowest electron density and hence represent the binding site of prefer-
ence for the cr-bonding ligand azide. If azide does bind to the high poten-
tial site, the potentiometric observat,ions are difficult to rationalize. Thus
here, too, the assumption of an initial or inherent difference in the elec-
tron affinities of the two heme sites leads to difficulty in the completely
rational interpretation of the experimental observations.
4. Interpretation and Summary

On balance there seem to be fewer problems in interpretation if the
two heme sites of oxidase(1V) or oxidase(0) are placed a t the same in-
herent electron affinities. The subsequent introduction (or removal) of

electrons or ligands will perturb the electron distribution among the elec-
troactive centers so that they are no longer equivalent (in the redox
sense). In a closely coupled set of electron acceptors entry of the first
electron should be most thermodynamically favored and subsequent elec-
trons, because of the increased electron density within the system, less
so. This suggestion of initially equivalent hemes is supported by the in-
frared studies of the binding of CO ( 6 6 ) .The vc0 values are found to
be very sensitive to differences in electron availability a t iron(I1) (93),
and these values for the two hemes of oxidase(0) * (CO), must be nearly
identical. Indeed, the visible spectra, which have been .widely used to
follow the changes in valence state of the hemes as a function of the
potential of the system, also fail to suggest any large differences in initial
state electron affinities between the hemes. The large difference in electron
density a t irons necessary to produce the observed potential differences
would be expected to lead to large splitting of the porphyrin spectral
bands (169).Attempts to resolve the visible band into components result-
ing from a and u3 have never been successful with the unperturbed en-
zyme system. It is, however, not surprising that spectral changes occur
upon partial oxidation or reduction of the active centers since porphyrin
spectra are sensitive to the electron densities at the metal centers (170).
There seems then no reason to propose differences in the inherent reduci-
bility of the hemes in oxidase(1V) on the basis of the evidence currently
available. In fact, the intuitively attractive notion of equivalent and
symmetrical heme-copper pairs seem adequate to explain the currently
available potentiometric data.
E. ELECTRON RESONANCE
PARAMAGNETIC STUDIES
1. Copper
The presence in cytochrome c oxidase of two copper atoms and

two iron atoms of variable oxidation states would lead one t o believe that
EPR studies would provide a rich fund of information about the oxidation
state changes that occur in the functioning enzyme. Accordingly, many
such investigations have been reported over the past decade. Much of
the early attention was focused upon copper because its strong sharp
signal in the vicinity of g = 2 was easily visible in both simple complexes
169. B. D. McLees and W. S. Caughey, Biochemistry 7,642 (1968).

170. W. S. Caughey, in “Inorganic Biochemistry” (G. L. Eichhorn, ed.), p. 797.
and proteins. Malmstrom and Vanngard (171) examined a number of

both simple and complex copper-containing compounds and found char-
acteristic spectra. Later Viinngard (172) tabulated spectra for many more
compounds and pointed out that the copper(I1) could be classified in
terms of All into Type I (All < 10 mK) and Type 2 (All > 14 mK). It is
possible that these two classes correspond to tetrahedrally distorted
coordination (Type I) and rhombohedrally distorted coordination
(Type 11). However, too little is known about the coordination environ-
ment of copper in proteins to take such assignments seriously and the
Type I and Type I1 classification should be considered only as a con-
venient empirical observation. Nevertheless, it is interesting that the
inherent copper of oxidase(1V) (911 = 2.17, gm = 2.03, All 5 3 mK) (173)
falls distinctly outside the range for Type I Cu2+. Thus, there appears
to be a clear distinction between copper that is integrally bound in the
oxidase and adventitious copper that appears as a variable contaminant
in most preparations. This difference was not clearly understood in the
early literature and led to some confusion about the role played by
copper (174). The copper(I1) atom&) that give rise to the character-
istic oxidase(1V) E P R signal are strongly bound within the enzyme and
are not readily removed by treatment with complexing agents (53, l o g ) ,
but upon denaturation of the protein a more normal copper(I1) E P R
signal is seen (175) and the copper then becomes susceptible to removal
by complexing agents (53). Griffiths and Wharton (53) were able to
show by a chemical method (176) that in oxidase(1V) both copper
atoms were in the +2 oxidation state and this was later confirmed by
the coulometric titrations of Heineman et al. (165).It was then surprising
to find that the intensity of the E P R signal corresponded to only about
40% of the total copper known to be present as copper(I1) in oxidase(1V)
(173) or, as now appears likely, 80% of one copper and none of the second
copper. The low signal intensity together with the lack of fine structure
in the E P R signal suggests that the copper(I1) interacts with other
metal centers in such a way as to quench the inherent paramagnetism
of divalent copper and produce the observed lowering of the E P R signal
intensity.
171. B. Malmstrom and T. Vanngard, J M B 2, 118 (1960).
172. T. Vanngard, in “Biological Applications of Electron Spin Resonance” (H. M.
Swartz, J. R. Bolton, and D. C. Borg, eds.), p. 411. Wiley (Interscience), New York,
1972.
173. H. Beinert, D. E. Griffiths, D. C. Wharton, and R. H. Sands, JBC 237, 2337
(1962).
174. A. Ehrenberg and T. Yonetani, Acta Chem. Scnnd. 15, 1071 (1961).
175. H. Beinert and G.Palmer, in “Oxidases and Related Redox Systems” (T.E.
King, H. S. Mason, and M. Morrison, eds.), Vol. 2, p. 567. Wiley, New York, 1965
176. G. Felsenfeld, ABB 87, 247 (1960).
2. Iron
The E P R signal resulting from iron(II1) because of its breadth and
partial submersion under the copper(I1) signal has proved much more
difficult to study quantitatively. Nevertheless, it now seems well estab-
lished that in oxidase(1V) iron(II1) is represented by signals a t g = 3,
2, and 1.5 with intensities that correspond to about 40% of the heme
iron present (126, 139). Although the oxidation state of the iron in oxi-
dase (IV) has never been directly determined, the electron capacity of
the fully oxidized enzyme suggests that all the iron must be iron(II1).
Then, the low E P R signal intensity is most conveniently interpreted in
terms of metal-metal interaction between iron(II1) (low spin) and some
other paramagnetic center which partially quenches the signal expected
from the S = .2 net spin on the iron. Both heme-heme and heme-cop-
per (11) interactions have been suggested (138). Further elucidation of
this problem might be forthcoming on the basis of careful magnetic su-
sceptibility and Mossbauer studies. Unfortunately, the single published
accounts of magnetic susceptibility (174) and Mossbauer (17'7) studies
are difficult t o interpret. Nevertheless, it is interesting to note on the most
naive basis that the magnetic susceptibility results of Ehrenberg and
Yonetani (17'4) correspond t o -80% of two unpaired electrons. This is
just as expected on the basis of the E P R observations on intrinsic copper
and iron.
3. The Effect of Valence State Changes on the EPR Spectrum
Behavior of the signals resulting from E P R visible iron and copper
during reduction provides clues that may be important to the understand-
ing of the chemistry involved in oxygen reduction. Experiments carried
out on a relatively long time scale show that the first electrons to enter
-
the system give rise to a diminution of the low-spin iron(II1) ( g 3) -
-
signal and a corresponding increase in the broad g 6 signal attributed
to high-spin iron(II1) (139). The g 6 signal reaches maximum inten-
sity (corresponding to about 40% of the heme A) when two electrons
per mole of oxidase have been supplied to the system and then declines
leaving a very small rhombohedra1 signal when four electrons have been
supplied. The copper (11) signal remains unchanged (perhaps increasing
slightly) during entry of the first pair of electrons and then diminishes
t o zero during entry of the second pair (139, 178). Leigh et al. .(124)
have shown that the high-spin iron (111) signal makes its appearance with
a half-reduction potential of 380 mV and disappears with a half-reduc-
177. G. Lang, S. L. Lippard, and S. RosBn, BBA 336,6 (1974).
178. C. R. Hartzell and H. Beinert, BBA (in press).
332 W. S. CAUGHEY, W. J. WALLACE, J. A. VOLPE, AND S . YOSHIKAWA
tion potential of 210 mV. The copper(I1) signal disappears with a half-
reduction potential of 250 mV. Similar observations have been made for
both isolated oxidase (124, 125) and pigeon heart mitochondria and phos-
phorylating submitochondrial particles (168). It is apparent then, that
the E P R results are in substantial agreement with the potentiometric
results in showing that the iron and copper are reduced as two iron-copper
pairs.
At low temperature (<15OK), the g 6 signal is resolvable into a
H
number of components. The dominant species are a broad rhombic signal

and a narrow axial signal but other small signals are seen as well (126),
depending upon the nature of the reductant used. I n ETP and mitochon-
dria the splitting of the rhombic component is quite marked (124,
168) but, as purification proceeds, the outer wings (g = 6.8 and 5.3)
broaden and the axial component becomes dominant. The two paramag-
netic components reduce with slightly different half-reduction potentials
and on this basis may be different species (168). However, Hartzell and
Beinert (126) pointed out that it is not clear, on the basis of the available
evidence, whether the two components of the g 6 signal are inherently
H
associated with the oxidase so that the distortion of the signal is asso-
ciated with a conformational change or heme-heme interaction induced
by partial oxidation or reduction or whether it is simply another heme
protein. Wilson and Leigh (168) point out that in purified cytochrome
oxidase only the symmetric component had been seen (159) but that the
two component signal appears in ETP and mitochondria1 particles and
by implication suggest that this may be due to alteration of the enzyme
during purification. However, it was shown by Wever et al. (179)and
by Leigh and Wilson (180) that this highly asymmetric signal appears
in small amount upon photodissociation of the CO complex of oxidase (11)
and may be a property of the oxidase although alteration of the pro-
tein cannot be completely discarded. The origin of the two species
represented by this signal is far from clear. But, if the system is as
highly coupled as many of the experimental observations seem to sug-
gest, it may be that the unpaired electron spin can go visiting on both
hemes and that, in fact, both hemes are partially magnetically visible.
4. The Effect of Ligand Binding
-
It is clear that the high-spin iron(II1) ( y 6) appears as the !ow-spin
H
iron(II1) (g 3) disappears. It is, however, not so clear whether the

two signals originate from the same or different hemes or, as suggested
179. R. Wever, J. H. Van Drooge, G . Van Ark, and B. F. Van Gelder, BBA 347,
215 (1974).
180. J. S. Leigh, Jr. and D. F. Wilson, BBRC 48, 1266 (1972).
Fe"-O-Fem A0 FeEOH, HO-Fe= -

N;
FeKOH, &-Fern
FIG.4. Reductive cleavage of a poxobisiron(II1) complex with subsequent binding

of aride to the unreduced iron atom.
above, a combination of contributions from both hemes. The results do

suggest though that it is an interaction between the two hemes in oxi-
dase (IV) that induces the low-spin electronic configuration in the heme
system. Upon partial reduction the stabilizing interaction is lost and the
iron (11)-iron (111) pair exhibits high-spin (weak ligand) characteristics
for the iron(II1) component. The presence of a bonding interaction
between the hemes in oxidase(1V) was also indicated by ligand binding
studies. Although some aspects of the binding of ligands like fluoride,
azide, and cyanide to oxidized oxidase remain unclear, there seems no
doubt that these ligands either do not react or react only very slowly
with iron under fully oxidized conditions (131). In most hemins and oxi-
dized hemeproteins, the reaction between iron (111) and anionic ligands
occurs a t a virtually diffusion-controlled rate (181). The slow reaction
with the iron (111)atoms of oxidase(1V) is then surprising. However upon
half-reduction of oxidase (IV) (i.e., addition of two electrons) the reac-
tion between the high-spin iron(II1) ahd anionic ligands such as azide
occurs at the expected rapid rate (139) to produce a stable low-spin
(g = 2.9, 2.2, and 1.67) (138, 168) iron(II1) azide complex. The binding
of azide to iron in these circumstances is also demonstrated by infrared
studies (182). The interpretation of these observations that is chemically
most appealing would implicate a bridging ligand as the protective inter-
action between the two oxidized heme A components. Such a bridging
ligand would need to have a number of rather sharply defined properties.
Effectively it would need to be (a) divalent (for charge saturation), (b)
less strongly bound to iron(II1) in the uncoupled than in the coupled
form (to allow for displacement by azide), and (c) pH sensitive [Wilson
observed pH dependence on the high potential component (137) 1. Al-
though there are many potential ligands to iron available in the protein,
it is difficult to find a protein constituent that fulfills all the requirements.
The one ligand that is available in aqueous solutions and which otherwise
meets all the requirements is oxide. If oxide is the bridging, protecting
ligand the uncoupling reaction can be represented as in Fig. 4.
5. p-Oxobishemin A as a Possible Component of Oxidase ( I V )
The p-0x0 complexes are the commonly observed products of the oxy-
gen-induced autoxidation of simple hemes, and their properties have been
181. Q. H. Gibson, L. F. Parkhurst, and G. Geraci, JBC 244, 4668 (1969).
182. J. A. Volpe and W. S. Caughey, BBRC (in press).
well described (60,94). Such Fe”’O-Fe”’ complexes are known, to be

strongly antiferromagnetically coupled (183) and not to exhibit an EPR
spectrum (94). Thus, the EPR results on oxidase(1V) are not consistent
with only a p-oxobishemin A system. However, EPR results for iron
could perhaps be accommodated by the introduction of a closely coupled
third paramagnetic center with spin one-half. This could be the “EPR
silent” copper. The close coupling of the spins of three spin one-half cen-
ters (two hemes and one copper) would give rise to resultant spin states
of S = Q and 4 of which the S = 4 is expected to be the lowest lying
(184). Such a coupled metal triplet could give rise to the set of ESR
signals attributed to low-spin iron(II1). The visible copper would then
be either independent of the coupled triad or only weakly coupled to
it. (Note that both visible copper and visible “iron” represent only ~ 4 0 %
of the signal expected if all were seen, but that if one copper were totally
silent then 80%, or nearly all, of the other copper is seen.) Scission of
a bridging p-0x0 linkage by partial reduction will lead to uncoupling of
one iron atom from the triad with replacement of the strong field oxide
ligand by a weak field hydroxo ligand as shown in Fig. 4.
F. INTERACTION
OF CYTOCHROME
c OXIDASEWITH CYTOCHROME
c
Cytochrome c is the sole physiological source of electrons for dioxygen
reduction a t cytochrome c oxidase (11). It is crucial to a discussion of
the mechanism of dioxygen reduction to know whether the binding of
cytochrome c to the oxidase is to a single site or to more than one site.
This will determine whether the reduction reaction can occur symmetri-
cally (e.g., two sites) or asymmetrically (one site).
On the basis of kinetic studies (186, 186) a 1:1 complex between cyto-
chrome c and the oxidase was suggested. This seems to correspond to
the ratio usually found in mitochondria (187). On the other hand, prepa-
rations have contained an isolated complex with one cytochrome c per
heme A (188, 189). There is no obvious basis upon which to reconcile
183. T. H. Moss, H. R. Lilienthal, C. Moleski, G. A. Smythe, M. C. McDaniel,

and W. S. Caughey, Chem. Commun. p. 263 (1972).
184. J. E Huheey, “Inorganic Chemistry : Principles of Structure and Reactivity,”
p. 35. Harper, New York, 1972.
185. T. Yonetani and G. S. Ray, JBC 240, 3392 (1965).
186. E. Mochan and P. Nicholls, BBA 267, 309 (1972).
187. J. N. Williams, Jr., BBA 162, 175 (1968).
188. Y. Orii, I. Sekuzu, and K. Okunuki, J . Biochem. ( T o k y o ) 51, 204 (1962).
189. T. E. King, M. Kuboyama, and S. Takemori, in “Oxidases and Related Redox
Systems” (T. E. King, H. S. Mason, and M. Morrison, eds.), Vol. 2, p. 707. Wiley,
New York. 1965.
these differences. Thus, there is no unequivocal stoichiometric basis for

ascribing either symmetry or asymmetry to the cytochrome c-oxidase com-
plex. However, the absence of evidence for a second binding site on the
oxidase ( 2 1 ) , the mitochondria1 ratio of cytochrome c to the oxidase
(1.0),and the EPR evidence for asymmetry in the reduction of the metal
components (126,138) are all consistent with a 1 : 1 complex. This suggests
that the two copper atoms are different and that it is always the same
copper that lies on the initial electron acceptor pathway.
G. KINETICSTUDIES
The study of transformations that can be observed kinetically in the
system cytochrome c: oxidase :0, has made important contributions to
the understanding of the oxidase function, and much of this work has
been reviewed previously (18, 21). Cytochrome c oxidase is reducible at
different rates by a variety of agents including dithionite (157, 190),
NADH ( 1 5 7 ) , ascorbate (191), and dichlorohydroquinone (153). How-
ever, the one that is of most interest is the natural substrate cytochrome
c which appears to be the sole reducing agent under physiological condi-
tions. Under both aerobic and anaerobic conditions the initial reaction
between reduced cytochrome c and oxidase (IV) exhibits first-order de-
pendence on cytochrome c (108, 185). However cytochrome c’+ acts as
a competitive inhibitor for the reaction, blocks the electron transport
binding site on the oxidase, and causes the kinetics to become complex
as i t accumulates in reaction mixtures containing the isolated oxidase
(185).
I n the absence of oxygen cytochrome c2+rapidly (t,,. = 10 msec) re-
duced oxidase (IV) to oxidase (11), but subsequent changes are slow even
in the presence of excess cytochrome c2+ and do not involve additional
electrons (66, 107, 153, 192-194). The initial rapid reduction which was
followed spectrophotometrically in the visible region by the disappearance
of cytochrome c?+ or in the EPR by the disappearance of the g 2 (cop-
- IV
per) and g 3 (low-spin iron) signals (66, 194) was found to be indepen-
dent of the presence of cyanide in the reaction mixture (192). These
observations have been interpreted in terms of the initial reduction of oxi-
190. R.Lemberg and M. V. Gilmour, BBA 143,500 (1967).

191. T.Ozawa, Y. Takahashi, A. N. Malviya, and K. Yagi, BBRC 61, 601 (1974).
192. E. Antonini, M. Brunori, C. Greenwood, and M. T. Wilson, Biochem. Sac.
Trans. 1, 34 (1973).
193. K.J. H.Van Buuren, B. F. Van Gelder, J. Wilting, and R. Braams, BBA
333, 421 (1974).
194. H. Beinert, R.E. Hansen, and C. R. Hartrell, BBA (in press).
dase (IV) being accommodated by the magnetically visible iron and cop-
per and the slow step corresponding to an electronic rearrangement in
which the electrons are passed to the other iron-copper pair. This interpre-
- -
tation receives its strongest support from the rapid flow EPR studies of
Beinert and co-workers (66, 194) in which the copper ( g 2) and low-
spin iron ( g 3) signals disappear upon introduction of the first elec-
trons into oxidase(1V). Upon standing many minutes the copper signal
-
reappeared qualitatively and quantitatively unchanged while the g 3
signal was replaced by a g 6 signal [high-spin iron (111)1. This would
-
suggest that the iron and copper of the magnetically visible pair are the
primary electron acceptors in oxidase (IV) , that a facile electron trans-
port pathway connects the iron and the copper, and that the initial elec-
tron receptor pair is isolated from the other pair (153,192). The oxi-
dase(I1) formed in this initial reaction should then be able to bind CO
or 0, a t the reduced iron site. Recent work on the partially reduced oxi-
dase indicates this to be the case (99,155). In fact, under these condi-
tions, as expected, the oxygen adduct appears quite stable and long-lived
(99),and the unreduced iron is able to bind cyanide (134).
For the reduction of oxidase(1V) by cytochrome cz+ in the presence
of oxygen, however, the situation changes dramatically. The initial, rapid
two-electron reduction is followed by a slower, but still rapid, further
reduction (108, 155, 192), which is sensitive to the concentrations of both
oxygen and cyanide in the reaction mixture (108). I n the presence of
excess cytochrome c*+ and high concentrations of oxygen the enzyme will
turn over rapidly (19.2).
The oxidation by oxygen of oxidase(0) formed by photolydng oxi-
(7cd -
dase(O)-CO was found by Greenwood and Gibson (195) to be rapid
8 X lo7 M-’ sec-l) , with the kinetics controlled by the rate of
diffusion of oxygen to the reaction site, but no evidence was adduced for
an “oxygenated” intermediate with a lifetime longer than a few microsec-
onds. The overall rate of oxygen reduction has since been confirmed by
a number of investigators (166,171, 185),but the nonformation of an
oxygenated adduct as the first step in reduction seems no longer tenable.
Chance et al. (155)on the basis of the spectrophotometric titration of
the oxidase (formed by photolysis of oxidase(O).CO a t low tempera-
ture) with oxygen, suggested that the reaction [reaction (15) ] is occurring
with K = 320 pM. The “oxygenated” oxidase is formed a t a rate of
Oxidase(0) + 02 % oxidase(O).O* (15)
-3 X lo4 M-’ sec-1 at - 7 8 O (E, = 9.9 kcal/mole) and decays by a first-
order step to a second complex at a rate of 0.45 sec-‘ (8, = 12.5 kcal/
195. C. Greenwood and Q. H. Gibson, JBC 242,1782 (1967).
mole). I n a parallel experiment oxidase(0) *CO was oxidized by ferri-

cyanide [to oxidase(I1) .CO] and photolyzed in the presence of 0, a t
low temperature. The spectral changes observed were interpreted in terms
of the initial, rapid, reversible formation of oxidase (11)* 0, which then
slowly decayed to an unidentified product spectrally distinct from the
decay product of oxidase(0) SO,.
Although not firmly established, a reasonable first product of the reac-
tion between oxidase(0) and 0, is one in which dioxygen interacts with
a single iron atom in the bent end-on fashion characteristic of isolated
heme sites as in HbO, (117)and MbO, (118). This is also supported
by the similarity in the frequencies of CO bound to oxidase(0) (65,115)
and to H b (113).
Despite the fact that very few of the kinetic studies have been carried
out under anything approaching turnover conditions for the oxidase, these
studies have provided important insights into the mechanism of its func-
tion. It seems quite clear that electrons enter the system through the
cytochrome c binding site with very low activation energy to reside on
the nearly independent iron and copper atoms referred to as EPR visible.
Oxygen is able to promote both the entry of a pair of additional electrons
into the system and the reoxidation of visible copper and iron, presumably
through the reduction of oxygen to water. However, the fully reduced oxi-
dase reacted faster with oxygen than did the half-reduced indicating that,
although the electron redistribution problem is improved by the inter-
action of oxygen with the half-reduced oxidase, the most favorable con-
figuration is obtained only upon full reduction. Electrons enter the oxidase
singly but must react with oxygen in pairs. This must be related to the
relative stabilities of the oxidation states of the reduced oxygen rather
than any problems associated with the binding of oxygen to l-electron-
reduced states.
IV. Mechanisms
The organization of cytochrome c oxidase to carry out the reduction

of dioxygen to water must reflect the mechanism by which electrons are
supplied, the thermodynamic requirements of the oxidation states acces-
sible to oxygen, and the chemical transformations of oxygen en route
to water. From the above discussions it can be seen that widely diverse
evidence relevant to each of these areas has been accumulating, and
this evidence is now sufficient to suggest new mechanistic insight into the
0, reduction process. Although any such mechanistic schemes must neces-
sarily be speculative, they are worthwhile not only because of their inher-
ent interest but also because they can stimulate and provide focus for
further experimentation to test their validity.
The kinetic and EPR evidence for the presence of two, a t most, weakly
interacting, copper-heme A pairs in cytochrome c oxidase is quite con-
vincing. Each pair appears to exchange electrons readily between the re-
spective copper and iron components but in one pair the magnetic cou-
pling between copper and iron, a t least in the fully oxidized species, is
greater than in the other pair. Diagrammatically this is represented in
Fig. 5. The Cu-Fe pair on the left is represented as having weak magnetic
coupling and the pair on the right as having strong magnetic coupling.
Little is known about the nature of the magnetic coupling in the fully,
or partially, reduced states except that the net effect is to reduce the
magnetism below that expected on the basis of the number of paramag-
netic centers present. The possibility for magnetic coupling between the
irons of oxidase(1V) was discussed above, and the idea of one strongly
and one weakly coupled heme copper pair seems entirely consistent with
the available data. Restriction of the ability to exchange electrons be-
tween the two pairs is represented by the dashed double arrow between
the pairs.
The evidence for O2 binding a t iron, and not a t copper, is strongly sup-
ported by the infrared evidence for CO binding a t iron only (14, 6 5 ) .
Furthermore, since each heme A iron can bind CO (65) and heme A de-
rivatives can form FeOFe bridges without undue steric restrictions (60,
6 2 ) , it is reasonable to suggest the possibility, in analogy to model heme
autoxidations (27, 40,9 4 ) , that a single oxygen molecule bridges between
the two heme irons.
With nonphysiological reducing agents small enough to gain access to
the active site, e.g., dithionite, it is reasonable to expect reduction to occur
through direct interaction between one or both irons and the reducing
agent. On the other hand, electron entry from cytochrome c appears
to be a t copper in a t least one Cu-Fe pair. This could occur symmetri-
cally (two cytochrome c binding sites) as in Fig. 6, or asymmetrically
(one cytochrome c binding site) as in Fig. 7. I n the asymmetric process
FIG.5. Schematic representation of the interrelationships between the metal centers

at the active site of cytochrome c oxidase showing an electronically coupled iron-cop-
per pair (left) and an electronically and magnetically coupled iron-copper pair
(right) which interact weakly with each other (---I.
[V]
Cytc2*----. , - _ _ _ CytcZ+
Fa,- = - = -.Fa
0 2 HlzO
FIG.6. Schematic representation of the symmetric reduction of cytochrome c oxi-
dase by cytochrome c . I n this case cytochrome c is shown introducing electrons
into both iron-copper pairs with dioxygen extracting electrons a t iron and being
converted to water.
(Fig. 7) the copper that receives electrons from cytochrome c is desig-

nated the proximal copper (Cup) and the iron with which it is paired
is the proximal iron (Fe,) . The metals of the other pair are termed the
distal copper (CU,) and distal iron (Fe,). There is, of course, no need
(or basis) to make a similar distinction between the coppers in the sym-
metrical process.
A symmetrical reduction process would involve separate electron entry
into each Cu-Fe pair from two cytochrome c binding sites. The symmetric
process is represented in Fig. 6. If this process is in fact followed, it would
be advantageous for both heme irons to go reduced virtually simultane-
ously and this introduces two possible mechanistic problems. Reduction
would be slowed by the requirement of two cytochrome c molecules bind-
ing t o one oxidase, and with both iron atoms reduced there is the possi-
bility of one dioxygen molecule binding a t each heme site. Both of these
circumstances would tend to retard the reaction. If, despite these obsta-
cles, the reaction does proceed with entry of electrons to both Cu-Fe
pairs, a reasonable mechanism for 0, reduction becomes that of 0, bind-
ing t o one iron(I1) as to oxyHb or oxyMb (117,118),then, to a p-peroxo
0, H120
Fro. 7. Schematic representation of the asymmetric reduction of cytochrome c
oxidase by cytochrome c. In this case cytochrome c is shown introducing electrons
into only one of the iron-copper couples (proximal) with reduction of dioxygen
to water induced by electrons that originate on only one of the iron atoms.
340 W. 5. CAUGHEY, W. J . WALLACE, J . A. VOLPE, AND 5. YOSHIKAWA
F%
Fe' -
O*
FeLOw0
Fe"
-
-ze
z n+
FeEOH
HO-Fr"
- F&O-FE*
FIG.
8. Representative steps in the symmetrical reduction of dioxygen to water.
intermediate, and, finally, to 2 FeIII-OH or Felll-O-Felll as repre-

sented in Fig. 8. In this case, the coppers would both serve as components
in the electron pathway to 0,. The cleavage of the 0-0 bond may pro-
ceed via a homolytic process to give two intermediate Fe1I1-0' species
followed by one-electron reduction of each. The driving force for this
cleavage would have to lie in the pressure exerted by the cytochrome c
electron transfer system since a similar cleavage has not been demon-
strated as a component in the nonenaymic decomposition of peroxides in
protic solvents.
However, present evidence for cytochrome c reductions suggest there
is only one site on the oxidase; therefore, the reduction has been termed
((asymmetric" and can be represented schematically as in Fig. 7. The
evidence that supports an asymmetric process to a greater degree than
the symmetric process includes that for only one cytochrome c binding
site and for one Cu-Fe pair reducing much more readily than the other
pair. Asymmetric electron entry imposes interesting requirements on
the 0 2 reduction processes. As shown in Fig. 9, 0 2 binding would occur as
in the symmetric case. With only one electron added to oxidase(IV),
i.e., to give oxidase(III), the 0 2 could bind to the Fe:! in the bent end-on
fashion characteristic of oxphemoglobin (116). This bonding results in
charge transfer from iron to dioxygen through interaction of the dr-pn*
systems of the metal and dioxygen, respectively. Nevertheless, on the
basis of reactivity studies of HbOz with nucleophiles (IN), the iron and
the oxygen are expected to retain appreciable Fe"-02 character and
not to attain the status of an Fe1"-02- ion pair. This is repreaented
diagrammatically in A of Fig. 9. In these circumstances, it is expected
to be difficult (i.e., high activation energy) to bring up a second electron
FIG.9. Dioxygen bridging between proximal and distal iron atoms as the initial
step in reduction.
196. W. J. Wallace, J. C. Maxwell and W. S. Caughey, BBRC 57, 1104 (1974).
through an already reduced iron because this would involve (in formal
valence terms a t least) transient formation of Fe1-02 or FeIT-02-.
Of course, the presence of reduced cytochrome c and Cug coupled to
the Feb1-02 would represent a forcing condition that could make the
transfer of electrons through iron to O2 more favorable. However, even
this extra "electron pressure" may not be enough to cause the reaction
to proceed without the system having some way t o redistribute the elec-
tron density on Feb' onto another center (other than 02).It is proposed
here that a suitably positioned distal iron(II1) atom might serve this
purpose by interacting with the partially negative terminal oxygen to
form a bridge between the irons across which an electron could pass to
form a transient Fe~l'-O-O-Fe~ intermediate (B of Fig. 9), which
with oxidation of Cug and Fe;' yields a p-peroxo complex with all metals
in their oxidized state (C of Fig. 9). It can be seen here th a t without the
driving force of the closely coupled electron source (Cui) there would be
little tendency for the 0 2 to bridge between iron(I1) and iron(II1) atoms.
This process permits ready formation of the thermodynamically stable
peroxide intermediate. It may also be noted that attack by the terminal
oxygen atom is likely to proceed by displacement of a ligand, presumably
hydroxyl or water, from Fe;'.
The next reasonable step involves cleavage of the 0-0 bond. This is
envisioned as proceeding in the manner shown in Fig. 10. Here cleavage
of the 0-0 bond of the p-peroxobisiron(II1) complex A must occur
without the entry of electrons into the distal Cu-Fe pair (no low energy
pathway across the p-peroxo bridge) , and consequently must be achieved
by an heterolytic process. This could occur uninfluenced by electron
pressure from cytochrome c (A + B -+ C) or under the influence of elec-
tron pressure (A + D + E + C). T o go from A to B can be thought of
cuf
1. FeFOH
HO-Fet
%"* C
D E
FIG.10. The alternate routes to asymmetric cleavage of the p-peroxobisiron(II1)

complex formed in the first stage of dioxygen reduction (see Fig. 9).
QO-FZ
cut
0
- QI0
0-Fe,
.CUD
FIG.11. Delocalization of the negative charge from oxygen over both iron and
copper atoms in close magnetic and electronic contact.
as a splitting t o give one positive oxygen (two-electron deficiency,

Fe;" 0+)and one negative oxygen (electron saturated, Fe': 0-). The
driving force for this cleavage is expected to be the stabilization of the
hydroxyl anion bound to iron(II1) promoted by the presence of distal
copper(I1) as illustrated in Fig. 11. The hydroxyl anion will be stabilized
by charge delocalization from oxygen over iron and copper atoms in a
way that is rather like that for the carbanion resulting from removal of a
proton from a carbon adjacent to a carbonyl group (Fig. 12).
The key to both the stereochemistry and energy of the heterolytic
cleavage of dioxygen is the polarization of the 0-0 bond induced by the
bridging interaction between the two iron centers. Thus, with a n addi-
tional electron in the proximal copper-iron pair, the 0-0 bond is more
polarized in D than in A. I n t,his way both the energy and polarity of the
0-0 bond cleavage can be modified to allow the reaction to follow a
low energy pathway of unique polarity. Then E will form from D more
readily than B from A. Coupling D to cytochrome c2+ will provide still
stronger electron pressure for facile cleavage of the 0-0 bond. The
mechanism can then be considered a sort of push-pull arrangement.
The build up (from cytochrome c2+) of electron density on the proximal
Cu-Fe pair results in an ability to place greater electron density on the
proximal oxygen atom to cause repulsion of the distal oxygen atom as
oxide thereby rupturing the 0-0 bond. The delocalization (i-e., sta-
bilization) of the negative charge on the oxide formed from the distal
oxygen atom serves to "pull" away the distal oxygen.
The product C of Fig. 10 could represent the normal form of the fully
oxidized oxidase, oxidase (IV). However, experience with hemins suggests
that the p-oxobishemin would form readily-unless the protein provides
special conditions that make its formation unfavorable. Clearly, if an
R
0
II H
-C-F;-
I I
V
-+-
0
FIG.12. Schematic representation of stabilization of a carbanion adjacent to IL

carbonyl group by electron delocalization.
FeOFe linkage forms, interactions with coppers (and with protein) are
required in explanation of the E P R and electronic spectral differences
between oxidase(1V) and p-oxobishemin A derivatives (60). Of course,
it is possible that resting oxidase(1V) could have an FeOFe linkage which
might never form under turnover conditions. It is also of interest that
the asymmetric mechanism presented does not require that the distal
Cu”-Fe“’ pair ever become formally reduced during enzyme function.
The mechanism also provides for the receipt of electrons readily one a t
a time from cytochrome c and delivers them to bound 0, under thermo-
dynamically acceptable conditions.
The nature of coupling betweeen copper and hemes in the proximal
and distal pairs has been the subject of interesting speculations (60, 69).
Although, as noted above, little firm evidence of the environments about
the coppers have been reported, possible ways in which copper can inter-
act with heme A have been presented (60). One intriguing possibility
has copper interacting with the terminal double bond of the farnesylethyl
side chain (Fig. 2) which can assume a conformation (Fig. 3) with n-elec-
tron overlap between its three double bonds and porphyrin. If cytochrome
c?+ can pass an electron to the copper, then, in this way, there could result
a fast, low activation energy pathway for the electron from copper to
heme iron and 0, (Fig. 13). This coupling could reasonably provide for
facile electron transfer without strong magnetic coupling, a necessary cri-
terion for the proximal Cu-Fe pair. Involvement of the farnesylethyl
group in this way also permits conformational control of the electron
transfer in that small changes in conformation could turn electron trans-
fer on or off by altering the effectiveness of w-orbital overlap. Such con-
formational changes could result from interactions with ATP and/or
ADP and thus provide a mechanism for respiratory control processes.
Other sites for copper interaction with heme A suggested include binding
of copper a t the 2-1’ hydroxyl and to porphyrin ring and side chain un-
saturation (60). These might serve in the tight coupling found for the
distal Cu-Fe pair. I n any case, the differences in copper-heme A interac-
f.-[ -cu-l&Jqe~o
Cyt c 0
FIG. 13. Schematic representation of a possible mechanism for electron transfer
from cytochrome c to the active site of cytochrome c oxidase. The pathway is repre-
sented as involving copper as a mediator between the heme edge of cytochrome
c and the “stacked” 2-alkyl chain of heme A followed by transmittal of the electron
through the T system of heme A to dioxygen bound a t iron.
344 W. 6. CAUGHEY, W. J . WALLACE, J . A. VOLPE, AND S. YOSHIKAWA
tions between the two hemes is not such as to have provided evidence
thus far that the two hemes are detectably different in fully reduced or
fully oxidized forms. Copper could also serve in electron transfer between
the two hemes, but present evidence appears compatible with bridging
oxygen serving this role, if needed, in the functioning enzyme.
ACKNOWLEDGMENT
Dr. Helmut Beinert and Dr.Britton Chance kindly provided information in ad-
vance of publication. Support from U.S.P.H.S. grant No. HL-19580 from the Na-
tional Heart and Lung Institute aided the preparation of this chapter.
Cvtochrome c Peroxidase
TAKASHI YONETANI
I. Introduction . . . . . . . . . . . . . . . . 345
11. Preparation and Molecular Properties . . . . . . . . . 347
111. Structural Aspects. . . . . . . . . . . . . . . 348
IV. Enzymic Activity . . . . . . . . . . . . . . . 352
V. Reaction Mechanism . . . . . . . . . . . . . . 353
VI. Interaction with Cytochrome c . . . . . . . . . . . 356
VII. General Comments . . . . . . . . . . . . . . 360
1. Introduction
Yeast cytochrome c peroxidase ( ferrocytochrome c :hydrogen-peroxide

oxidoreductase, E C 1.11.1.5) which catalyzes the oxidation of ferrocyto-
chrome c to ferricytochrome c in the presence of hydroperoxide, was dis-
H,Or + 2 ferrocytochrome c -+ 2 ferricytochrome c + 2 OH- (1)

covered in brewer’s yeast by Altschul et al. ( 1 ) in 1940. These investi-
gators preliminarily identified this enzyme as a soluble cytochrome oxi-
dase from yeast, because dithionite-reduced ferrocytochrome c was ap-
parently oxidized by this enzyme without addition of hydrogen peroxide.
Subsequently, these investigators (2) obtained a highly purified prepara-
tion of this enzyme and demonstrated that this enzyme contains pro-
toheme and reacts with a stoichiometric quantity of hydrogen peroxide
to form a red compound, the absorption spectrum of which is similar
1. A. M. Altschul, R. Abrams, and T. R. Hogness, JBC 136, 777 (1940).

2. R. Abrams, A. M. Altschul, and T. R. Hogness, JBC 142, 303 (1942).
345
346 TAKASHI YONETANI
to that of Compound I1 of horseradish peroxidase (3), and correctly iden-

tified this enzyme as cytochrome c peroxidase. The purification method
they devised for this enzyme (1, 2) was somewhat tedious and gave a
rather poor yield; thus, subsequent investigators failed to obtain this en-
zyme in such a high purity as the original investigators did. Conse-
quently, the majority of subsequent studies on cytochrome c peroxidase
(4-11) were performed with less pure preparations. It was considered
from these investigations that the functional properties of cytochrome
c peroxidase were essentially identical to those of other protoheme-con-
taining peroxidases, such as horseradish peroxidase (3-9, 11, 12) and tur-
nip peroxidase ( I S ) , except for differences in their substrate specificity.
In 1965, Yonetani and Ray (14) obtained a highly purified preparation
of cytochrome c peroxidase in an excellent yield using DEAE-cellulose
ion exchange chromatography. Shortly thereafter, Yonetani et al. (16)
crystallized this enzyme by isoelectric dialysis. Subsequently, Yonetani
and co-workers (16-40) carried out a series of extensive investigations
3. D. Keilin and T. Mann, Proc. Roy. SOC.,Ser. B 122, 119 (1937).

4. B. Chance, in “Enzymes and Enzyme Systems” (J. T. Edsall, ed.), p. 93. Har-
vard Univ. Press,Cambridge, Massachusetts, 1951.
5. B. Chance, Advan. Enzymol. 12, 153 (1951).
6. B. Chance, ABB 21, 416 (1949) ; 22, 224 (1949) ; 24, 389 (1949).
7. B. Chance, ABB 41, 416 (1952).
8. P. George, BJ 54, 267 (1953) ; 55,220 (1953).
9. P. George, JBC 201, 413 (1953).
10. J. Beetlestone, ABB 89, 35 (1960).
11. P. Nicholls, ABB 106, 25 (1964).
12. D. Keilin and E. F. Hartree, BJ 48, 88 (1951).
13. T. Hosoya, J. Biochem. ( T o k y o ) 47, 369 (1960).
14. T. Yonetani and G. S. Ray, JBC 240,4503 (1965).
15. T. Yonetani, B. Chance, and S. Kajiwara, JBC 241,2981 (1966).
16. T. Yonetani, JBC 240, 4509 (1965).
17. T. Yonetani and G. S. Ray, JBC 241, 700 (1966).
18. T. Yonetani, JBG 241, 2562 (1966).
19. T. Yonetani and T. Ohnishi, JBC 241, 2983 (1966).
20. T. Yonetani, H. Schleyer, and A. Ehrenberg, JBC 241, 3240 (1966).
21. T. Yonetani, D. F. Wilson, and B. Seamonds, JBC 241, 5347 (1966).
22. T. Yonetani, H. Schleyer, B. Chance, and A. Ehrenberg, in “Hemes and Hemo-
proteins” (B. Chance, R. W. Estabrook, and T. Yonetani, eds.), p. 293. Academic
23. T. Yonetani and H. Schleyer, JBC 242, 1974 (1967).
26. T. Yonetani, JBC 242, 5008 (1967).
27. T. Yonetani and T. Asakura, JBC 243, 3996 (1968).
28. T. Yonetani and T. Asakura, JBG 243, 4716 (1968).
29. T. Asakura and T. Yonetani, JBC 244 537 (1969).
6. CYTOCHROME c PEROXIDASE 347
on its functional properties. Meanwhile, Ellfolk (41) independently ob-

tained a crystalline preparation of cytochrome c peroxidase and inves-
tigated its molecular properties (42, 4s).
II. Preparation and Molecular Properties
Cytochrome c peroxidase is found exclusively in aerobically grown

yeast (1, 2, 19, 44). Chantrenne (43) demonstrated that the synthesis
of this enzyme in anaerobically grown yeast is induced by exposure to
oxygen. Sels and Cocriamont (44) showed that apocytochrome c peroxi-
dase is synthesized in anaerobically grown yeast and that oxygen is re-
quired for the in vivo conversion from the apoenzyme to the holoenzyme.
Commercially available yeast, such as baker’s and brewer’s yeasts,
are the most convenient sources for large-scale preparations of cyto-
chrome c peroxidase. However, the content of this enzyme in yeast varies
considerably depending on the type of strain and culture conditions ; thus,
preliminary checks to find the brand of commercial yeast, which is rich
in cytochrome c peroxidase, is advised before starting a large-scale prepa-
ration. The detailed method of chromatographic purification of cyto-
chrome c peroxidase has been described elsewhere (16, 15, 45). The en-
zyme can be readily extracted with aqueous solvents from yeast that
has been lysed in the presence of organic solvents such as toluene and
ethylacetate. The extracted enzyme is concentrated by salting out with
saturated ammonium sulfate and purified by two cycles of DEAE-cellu-
lose chromatography to yield a preparation with a purity index (the ratio
of absorbance at 408 nm to that a t 280 nm) of 0.7to 1.1. Repeated chro-
30. T. Asakura and T. Yonetani, JBC 244, 4570 (1969).

31. T. Yonetani and T. Asakura, JBC 244,4580 (1969).
32. T. Asakura, H. R. Drott, and T. Yonetani, JBC 244,6626 (1969).
33. T. Yonetani, Aduun. Enzymol. 33, 309 (1970).
34. A. F. W. Coulson, J. E. Erman, and T. Yonetani, JBC 246, 917 (1971).
35. T. Iizuka, M. Kotani, and T. Yonetani, JBC 246,4731 (1971).
36. A. F. W. Coulson and T. Yonetani, BBRC 48, 391 (1972).
37. T. Asakura and T. Yonetani, JBC 247,2278 (1972).
38. R. K. Gupta and T. Yonetani, BBA 29.2, 502 (1973).
39. J. Leonard and T. Yonetani, Biochemistry 13, 1465 (1974).
40. N. Ellfolk, Acta Chem. Scand. 21, 175 (1967).
41. N. Ellfolk, Actu Chem. Scand. 21, 1921 (1967).
42. N. Ellfolk, Actu Chem. Scand. 21, 2736 (1967).
43. H. Chantrenne, BBA 18, 68 (1955).
44. A. A. Sels and C. Cocriamont, BBRC 32, 192 (1968).
45. T. Yonetani, “Methods in Enzymology,” Vol 1 p. 336, 1967.
matography does not increase the purity. Cytochrome c peroxidase is

readily crystallized from the enzyme preparation have a purity index
of more than 0.7 by exhaustive dialysis against distilled water. Cyto-
chrome c peroxidase with a purity index of 1.25 to 1.30 can be prepared
by two or three cycles of this isoelectric crystallization.
The molecular weight of cytochrome c peroxidase has been determined
to be 34,100on the basis of a sedimentation constant of 3.55 S, a diffusion
constant of 9.44 F, and a partial specific volume of 0.733 ml/g (49).
The enzyme exists as a monodisperse monomer containing one ferric pro-
toporphyrin IX, which is noncovalently bound ( 1 , 2, 1 4 ) . No other transi-
tion metal is detected in crystalline preparations of the enzyme ( 2 2 ) .The
apoenzyme moiety is an acidic protein with an isoelectric point a t p H
5.0 to 5.2,which is made of 272 amino acid residues: Asp,,, Thr,,, Ser,,,
G~uz,,Prol5, Glyz3, A h , Vallz9 Mets, h,Leuz3, Tryl2, Phe16, Lys21,
His,, Arg,, Cys,, and Try, (37, 4 3 ) . The N- and C-terminal residues
have been identified as threonine and leucine, respectively. The sulfhy-
dry1 group of the cysteine residue reacts with sulfhydryl reagents more
rapidly in the apoenzyme than in the holoenzyme. This site can be
blocked by p-mercuribenzoate without affecting its enzymic activity. It
should be noted that cytochrome c peroxidase contains no carbohydrate
(41) , in contrast to other protoheme-containing peroxidases.
Crystalline cytochrome c peroxidase has unit cell dimensions of
108.0X 77.8 X 51.4A, a space group of P2,2,2,, and a 2 value of 4 (mol-
ecules per unit cell) (46).Several heavy atom derivatives, whose crystal-
line structure are isomorphous with that of the native enzyme, have been
prepared for the X-ray diffraction analysis of crystalline cytochrome c
peroxidase.
Cytochrome c peroxidase has been reversibly resolved into protoheme
and apoenzyme moieties (26) by a modification of Teale's acid butanone
technique (47).The apoenzyme may be combined with porphyrins and
metalloporphyrins to form synthetic holoenzymes containing unnatural
prosthetic groups (9793, 38-40). The apoenzyme (96') and some of the
synthetic holoenzymes have been crystallized.
111. Structural Aspects

Since the primary and three-dimensional structures of cytochrome c
peroxidase are yet to be established, any discussion on its molecular
46. L. 0. Larsson, L. 0. Hagman, P. Kierkegaard, and T. Yonetani, JBC 245,

902 (1970).
47. F. W. J. Teale, BBA 35, 543 (1959).
structure becomes somewhat speculative a t the present time. However,

the indirect experimental data available today are sufficiently numerous
to discuss a definitive molecular structure for this enzyme, particularly
a t the heme region. Unlike the globins from myoglobin and hemoglobin,
apocytochrome c peroxidase is resistant to freezing-induced denaturation
and thus can be stored frozen for an indefinite period in the heme-free
state. Apocytochrome c peroxidase has been crystallized with a habit of
crystallization apparently identical with that of its holoenzyme (26).The
crystal of the protoporphyrin IX-apoenzyme complex has been demon-
strated to be isomorphous with the crystal of the native holoenzyme ( 4 6 ) .
These observations suggest that the gross conformation of apocyto-
chrome c peroxidase is not significantly affected by the incorporation of
the prosthetic group.
An anionic aromatic dye, anilino-naphthalene sulfonic acid, interacts
with apocytochrome c peroxidase to form a stoichiometric complex in
competition with heme ( 4 8 ) . This interaction results in a significant en-
hancement of the fluorescence emission of the dye, indicating a hydro-
phobic nature of the dye- or heme-binding site of this enzyme in a similar
manner to those of myoglobin and hemoglobin ( 3 9 ) .The chemical modifi-
cation of heme side chains a t positions 2 and 4 has no appreciable effect
on either the enzymic activity or the affinity of the apoenzyme for the
prosthetic group (28).On the other hand, both the enzymic activity and
the affinity for the prosthetic group are greatly reduced by modification
of one or two propionic acid chains a t positions 6 and/or 7 (3U,32). On
the contrary, the modification of heme side chains a t positions 6 an
7 has no appreciable effect on the oxygen affinity of myoglobin and hemo-
globin as well as the subunit cooperativity in the latter, whereas the mod-
ifications a t positions 2 and 4 influence both the oxygen affinity and the
subunit cooperativity (49, 50). Since two propionic acid side chains of
protoheme are exposed to the exterior of the molecule in myoglobin and
hemoglobin (51,52),it is tempting to speculate that the heme group of
cytochrome c peroxidase may be embedded in a direction opposite t o that
of myoglobin and hemoglobin. I n other words, the heme side chains a t
positions 2 and 4 are exposed to the exterior, whereas propionic acid side
chains a t positions 6 and 7 face the interior of the molecule in cytochrome
c peroxidase (Fig. 1).
48. L. Stryer, J M B 13, 482 (1965).

49. A. Rossi-Fanelli, E. Antonini, and A. Caputo, Advan. Protein Chem. 19, 73
(1964).
50. Y. Sugita and Y. Yoneyama, JBC 246,389 (1971).
51. J. C. Kendrew, Brookhaven Symp. Biol. 15, 216 (1962).
52. M. F. Perutz, J M B 13, 646 (1965).
FIQ.1. A proposed orientation of the protoheme prosthetic group in cytochrome c

peroxid-.
The nitric oxide complex of ferrocytochrome c peroxidase exhibits an

electron paramagnetic resonance (EPR) spectrum of Thombic symmetry
(53). Its hyperfine structure has been interpreted as derived from the
interaction of the heme iron with two types of axially bound nitrogen
atoms, one obviously from nitric oxide and the other from a protein
group. The magnitude of the latter hyperfine coupling is consistent with
the proposed assignment of a histidyl nitrogen as the fifth ligand of the
prosthetic group (Fig. 1). Rose Bengal, an anionic aromatic dye, like
anilinonaphthalene sulfonic acid, stoichiometrically reacts with apocyto-
chrome c peroxidase in competition with protoheme ( 5 4 ) .The bound rose
bengal acts as an effective photooxidation sensitizer. Photooxidation of
the Rose Bengal-apoenzyme complex results in specific modifications
of one tryptophan and two histidine residues per mole of protein, only
one of the latter being essential to the enzymic activity. Apparently these
residues are located adjacent to the bound Rose Bengal in the heme crev-
ice. One of the destroyed histidine residues may be the fifth ligand.
The sixth coordination position of the heme iron in cytochrome c perox-
idase, which is normally occupied by H,O, is available for reactions with
extraneous ligands such as fluoride, cyanide, and azide, as well as sub-
strates and substrate analogs. The acidic-alkaline transition of a hemo-
protein, which is caused by the ionization of the bound water ligand,
is usually accompanied by significant spectral changes. However, the vis-
ible absorption spectrum of cytochrome c peroxidase is not appreciably
53. T. Yonetani, H. Yamamoto, J. E. Erman, J. S. Leigh, and G. Reed, JBC
247, 2447 (1972).
54. A. F. W. Coulson and T. Yonetani, Etrr. J. Biochem. 26, 125 (1972).
TABLE I
CHEMICAL AND ELECTRONIC EQUILIBRIA
OF CYTOCHROME C PEROXIDASE
Chemical compound
“Acidic” -pK 6.3

“Alkaline”
Electronic state CCP(FelI1) H 2 0 CCP(FelI1) OH-
“Thermally excited” Low- spin (S = 8) High- spin (S = 8)
I Activation
energy (.I
Transition
temperature (T,)
“Ground”
1
c = 1230 cm-*
T , = 274°K ( + l ” C )
1
High- spin (S = 9)
E
T,
=
=
1
1830cm-l
232°K (-41°C)
Low- spin (S
affected by changes in pH at room temperature. This anomaly has been

1
= 8)
resolved by spectrophotometric ( 2 1 ) , EPR ( 2 2 ) ,and magnetic suscepti-

bility (55) measurements of this enzyme a t cryogenic temperatures. The
above-mentioned pH-insensitive absorption spectrum of this enzyme can
be adequately interpreted by the assumption that cytochrome c peroxi-
dase is a mixture of two chemical compounds (“acidic” and “alkaline”
forms), each of which is an electronic mixture of thermally excited and
ground states a t acidic and neutral pH regions and room temperature,
as indicated in Table I ( 5 6 ) . Since both [‘acidic” and “alkaline” forms
are mixtures of respective high- and low-spin electronic states and thus
exhibit more or less similar absorption spectra a t room temperature, the
rracidic”-L‘alkaline”transition of this enzyme cannot be readily moni-
tored by spectrophotometry a t ambient temperatures. Only when both
“acidic” and “alkaline” forms are brought to their respective lowest
ground states by lowering the temperature below -lOO°C, do they become
pure “high-spin” and “low-spin” states with distinctive spectral charac-
teristics (5‘7). Thus, its “acidic”-“alkaline” transition may be readily
followed by cryogenic spectrophotometry (21). Cytochrome c peroxidase
becomes unstable and irreversibly modified on standing above pH 8.
Thus, it is difficult to follow the transition quantitatively especially a t
the alkaline end. Nevertheless, the pK value of its transition is estimated
to be about 6.3, which is significantly lower than those of myoglobin and
hemoglobin.
55. T. Iizuka, M. Kotani, and T. Yonetani, RBA 167, 257 (1968).

56. T . Iizuka and T. Yonetani, Advan. Biophys. 1, 155 (1970).
57. A. S. Brill and R. J. P. Williams, BJ 78,253 (1961).
IV. Enzymic Activity
The reaction of cytochr me c p roxid se follows a general form of com-

pulsory order mechanism for two-substrate enzymes as indicated by Eqs.
( 2 ) , ( 3 ) , and (4) where E is the enzyme, S, is hydroperoxide, Szis ferro-
ESi + Sz r ka
ESISZ (3)
kr
ESzSz E + P
+ (4)
cytochrome c, and P is ferricytochrome c. Since S, and S, are two-equiva-
lent oxidant and one-equivalent reductant, respectively, the overall reac-
tion requires a stoichiometry of Eq. (1). Furthermore, the reaction is
practically irreversible. I n other words, the reverse rates, if they exist,
are negligibly small in comparison with the corresponding forward rates.
Within these restrictions, some of these rate constants have been esti-
mated from initial steady-state rate measurements, as summarized in
Table I1 ( 1 7 ) .The association rates (k,) of the enzyme and hydroper-
oxides are an order of magnitude faster than the corresponding rates re-
ported for horseradish peroxidase ( 6 ) . The association rates of the ES1
intermediate and ferrocytochrome c (S,) of lo8 M-'sec-' are one of the
most rapid rates recorded for protein-protein interactions and closely ap-
proach the theoretical limit set of the collision rate of these two proteins
(92).
TABLE I1
KINETIC
CONSTANTS OXIDATIONOF FERROCYTOCHROME
OF PEROXIDATIC c
CATALYZED
B Y CYTOCHROMEc PEROXIDASE~
Kinetic constant
ki kz ka K,
Substrate (M-1 sec-1) (M-1 sec-1) (sec-1) (M)
Heart ferrocytochrome c
HzOz 1 . 4 X lo8 5 . 9 X lo8 2 . 6 X loa 4 . 5 X lo-'
C,HsOOH 2 . 5 X lo7 5 . 0 X lo8 2 . 0 X 10' 4 . 1 X lo-'
Yeast ferrocytochrome c
HzOz 1 . 2 X lo8 5 . 6 X lo8 1 . 4 X 10' 2.5 X
C zH 6 0 0 H 2 . 2 x 107 5 . 2 x 108 1 . 2 x 104 2 . 3 x 10-6
a Assays were made in sodium acetate buffer, pH 6.0, at 23°C.

Cytochrome c peroxidase is reversibly inhibited by ligands that can

combine with ferric heme-such as fluoride, azide, and cyanide-but it
is not inhibited by carbon monoxide, which indicates that the formation
of the ferrous form of the enzyme is not involved in its reaction cycle.
The activities of cytochrome c oxidase and cytochrome c peroxidase in
tissue may be readily differentiated by the use of carbon monoxide since
only the former enzyme is selectively inhibited by carbon monoxide (19).
Cytochrome c peroxidase can oxidize a number of reducing agents such
as ascorbate, pyrogallol, guaiacol, hydroquinone, and ferrocyanide as well
as mammalian and yeast ferrocytochrome c (1, 4, 1 4 ) . Ferrocytochromes
b, b,, and c, from mammalian sources and a majority of bacterial ferro-
cytochrome c are not oxidized. The molecular activity of cytochrome c
peroxidase for mammalian and yeast ferrocytochrome c (k,= lo4 sec-’)
is a t least two orders of magnitude larger than those for other reductants.
Thus, this enzyme has a high substrate specificity toward ferrocyto-
chrome c. Alkylhydroperoxides, such as methyl-, ethyl-, and propylhy-
droperoxides, can be effectively utilized as S, by this enzyme.
V. Reaction Mechanism
Altschul et al. (1, 2 ) originally discovered that cytochrome c peroxi-

dase reacts with a stoichiometric amount of hydroperoxide to form a
red “peroxide compound,” which will be referred t o hereafter as Com-
pound ES. It has a distinct absorption spectrum, as shown in Fig. 2. The
formation of Compound ES from the enzyme and hydroperoxides is very
rapid (k, > lo7 los M-’ sec-I). No intermediate, which precedes Com-
IU
pound ES, has been thus far detected. In the absence of reductants, or
S,, Compound ES is highly stable. The rate constant of its spontaneous
decay is of the order of sec-l ( 2 2 ) . The primary peroxide compound
(Compound I) of horseradish peroxidase decays much faster a t a rate
of sec-’ (6). This unusual stability of Compound ES allows one to
determine various physical and chemical parameters quantitatively and
reliably. Titrations of Compound ES with reductants such as ferrocyto-
chrome c (16, 20) and ferrocyanide (18, 34) have established that Com-
pound ES is two oxidizing equivalents above the original ferric .enzyme.
The absorption spectrum of Compound ES is essentially identical to
that of Compound I1 of horseradish peroxidase which contains one
oxidizing equivalent per mole in the form of Fe(1V). I n addition, EPR
examinations have revealed that Compound ES contains a stable free
radical, the spin concentration of which is approximately one equivalent
per mole (Fig. 3 ) . Therefore, it is reasonable to conclude that two oxidiz-
Wavelength (nm 1
FIQ.2. Absolute and difference light absorption spectra of cytochrome c peroxidaee
and Compound ES at pH 7 and 20": (-1 enzymes and (---I + CJLOOH.
ing equivalents in Compound ES are maintained in the form of Fe(1V)

and a free radical of a protein group (R"). Compound ES formed from
alkyl-hydroperoxide is indistinguishable from that derived from hydrogen
peroxide. Furthermore, the formation of Compound ES upon reaction
with ethyl hydroperoxide is accompanied by the release of 1 mole of ethyl
alcohol per mole. These observations indicates that the 0-0 bond in S,
has been broken upon the formation of Compound ES and that Com-
pound ES is not a so-called reversible enzyme-substrate complex as ES,
in Eq. (2) implies, but an enzyme intermediate carrying two oxidizing
equivalents per mole in a form other than the original substrate (S,).
Reduction of Compound ES with 2 moles of ferrocytochrome c gen-
erates the original enzyme rapidly. It has not been possible to detect
the formation of the one-equivalent, ferrocytochrome c-reduced interme-
diate of Compound ES and to determine the rate constants of reactions of
i
12.004
I 6.00
0 I000 2000
- T 1
H
3000 4000
._
( ocrstcmdr)
51 0
FIG.3. Electron paramagnetic resonance absorption spectra of cytochrome c per-

oxidase and Compound ES at pH 7 and at 77°K: (-) enzyme and (---)
Compound ES.
Compound ES with first, and second moles of ferrocytochrome c individ-

ually. However, using ferrocyanide as a reductant, it has been possible
to examine the mechanism of reduction of Compound ES to the original
enzyme in detail ( 3 4 ) . Comparison of optical and EPR titrations shows
that the reaction of Compound ES with ferrocyanide in a range from pH
5 to 8 is biphasic and strongly supports a mechanism in which two one-
equivalent intermediates are a t rapid equilibrium, as shown in Eq. (5)
Fe(1V) Fe(II1)-R* (5)
where Fe(1V) and Fe(II1) are ferry1 and ferric heme irons and R" is
a protein free radical. Optical and EPR parameters thus far available
are not sufficient to identify the chemical nature of the protein group
responsible for R". However, the spontaneous decay of Compound ES re-
sults in destruction of several amino acid residues (36).At pH 4 and
8, tyrosine and tryptophan are the residues principally affected. Thus,
it is possible that one of these residues may be responsible for the forma-
Horseradish peroxidase Cytochrorne c peroxidase
2-eq. Oxidation level Comp. I[Fe(IV)-P'] Comp. ES [ Fe(IV)-R*l
RoH4
(green) (red)
1-eq. Oxidation level

ROOH \--
/;mp. IIlFe(IV)J
(red) ROOH
Original ferrlc level HRP [Fe(UI)] CCP[ Fe(iII)l

(brown) (brown)
FIG.4. Reaction cycles of horseradish and cytochrome c peroxidases.
tion of the free radical, R", in Compound ES [Fe(IV)-R*] and its one-
equivalent reduced form [ Fe (111)-R"] . Low-temperature magnetic sus-
ceptibility (55) and Mossbauer spectroscopic (58) data are consistent
with assumption that Compound ES contains Fe(1V).
On reaction with a stoichiometric amount of hydroperoxide, catalase
and horseradish peroxidase are converted to a green colored intermediate,
Compound I ( 5 ) . The chemical nature of Compound I has been exten-
sively debated since its discovery by Theorell (59). Recently, Dolphin
et al. (60) have demonstrated that upon one-equivalent oxidation several
metalloporphyrins are converted to stable porphyrin *-cation radicals,
the absorption spectra of which possess the spectral characteristics of
Compound I, namely, a decreased Soret n-x" transition and an appear-
ance of the 620-670-nm absorption bands. Since Moss et al. (61) proposed
the presence of Fe(1V) in Compound I of horseradish peroxidase from
Mossbauer spectroscopic measurements, it is attractive to describe Com-
pound I as Fe (IV) -P", where P" is a porphyrin =-cation radical. Then,
Compound I and Compound ES become isoelectronic. Both contain
Fe(1V) and a radical: the former as a porphyrin radical (P") and the
latter as a protein radical (R"). Then the reaction cycles of horseradish
and cytochrome c peroxidases may be compared as shown in Fig. 4.
VI. Interaction with Cytochrome c
The elucidation of the mode of interaction between cytochrome c perox-

idase and cytochrome c is not only essential in our understanding of the
reaction mechanism of this enzyme but also provides important clues
for formulating a general mechanism of electron transfer in biological sys-
58. G. Lang and T. Yonetani, unpublished results.
59. H. Theorell, Enzymologia 10,250 (1941)t
60. D. Dolphin, R. H. Felton, D. C. Borg, and J. Fajer, JACS 92, 743 (1970).
61. T.H. Moss, A. Ehrenberg, and A. J. Bearhen, Biochemistry 8,4159 (1969).
tems such as mitochondria1 and microsomal electron transfer systems.

The question of whether or not the direct contact between two prosthetic
groups is a prerequisite for the electron transfer processes in biological
reactions is long-standing and yet to be answered. This problem becomes
experimentally approachable by the use of the cytochrome c peroxi-
dase-cytochrome c couple.
The formation of a reversible Michaelis-Menten-type complex of the
enzyme and ferrocytochrome c [ES,S, in Eq. (3) ] can be postulated from
initial steady-state kinetics of the cytochrome c peroxidase reaction ( 1 7 ) .
Since cytochrome c peroxidase and cytochrome c are acidic and basic
proteins, respectively, their interaction may be governed principally by
electrostatic attraction. This assumption is further supported by the fact
that several polycations which reversibly and irreversibly bind cyto-
chrome c peroxidase inhibit its enzymic activity in competition with fer-
rocytochrome c ( l 7 , 6 2 ) .
The 220-MHz proton-NMR spectrum of horse heart ferricytochrome
c is well characterized by the presence of two low-field methyl resonances
a t -35 and -32 ppm and two other relatively sharp high-field methyl
resonances a t +2.4 and +2.7 ppm relative to the standard 2,2-dimethyl-
2-silopentane-5-sulfonate reference at 25O ( 6 3 ) . The two low-field reso-
nances a t -35 and -32 ppm (Fig. 5 ) have been assigned to two methyl
side chains of the heme group a t positions 8 and 3, respectively (6'4)
(see Fig. 6 ) . I n the presence of equimolar amounts of cytochrome c per-
oxidase, the linewidths of these two low-field resonances broaden from
20 to -100 Hz. This is accompanied by a decrease in the separation
between these two resonances from 3.1 to 2.25 ppm, as the consequence
of mutual shifts of the -35 and -32 ppm resonance to up- and down-
field directions, respectively. Nuclear magnetic resonance (NMR) titra-
tions of ferricytochrome c with cytochrome c peroxidase as a function
of either the resonance linewidths or AV give a stoichiometry of 1 : l to
confirm the formation of a stoichiometric complex between these two
macromolecules (38).The observed approximately fourfold broadening
of the resonance linewidths is consistent with the expected change in the
tumbling correlation time of the whole molecule upon a 1 : 1 complexation.
When cytochrome c is present in an excess over cytochrome c peroxidase,
a time-averaged NMR spectrum of free and complexed ferricytochrome
c rather than a simple superposition of two distinction spectra is ob-
served. This indicates that the association and dissociation rates for the
62. E. Mochan and P. Nicholls, BBA 216,SO (1970).
63. K. Wiithrich, Proc. Nut. Acad. Sci. U . S . 63, 1071 (1961).
64. A. G. Redfield and R. K. Gupta, Cold Spring Harbor Symp. Quant. Biol. 36,
405 (1971).
1 I I
36 34 32
Field shifts in PPM
FIQ.5. ‘H-NMR spectra of ferricytochrome c in the presence (b) and absence
(a) of an equimolar cytochrome c peroxidase.
FIG.6.A proposed peroxidaae binding site in cytochrome c.

peroxidase-cytochrome c complex must be much greater than the inverse

of the frequency separation between complexed and free states. It is pos-
sible to set a lower limit of 200 sec-I on the dissociation rate, which is
approximately equivalent to k-, of Eq. (3).
The complexation induced shifts of the -35 and -32 ppm resonances
to -35.2 and -33 ppm, respectively (Fig. 5), indicate that the spin den-
sities on methyl groups a t positions 8 and 3 are decreased and increased
by approximately 276,respectively. This could be merely the electrostatic
effect of negatively charged cytochrome c peroxidase which repels the
unpaired electron cloud in the complex away from it implying that cyto-
chrome c peroxidase interacts with ferricytochrome c in the area adja-
cent to the methyl side chain a t position 8. I n other words, the enzyme
approaches cytochrome c in a general direction of 7 o'clock from the back
side, as indicated by the arrow in Fig. 6. I n this mutual orientation the
direct contact between the prosthetic groups of these two hemoproteins
will be prevented by the backbone peptide chain of cytochrome c. It
is also possible that the binding of the peroxidase produces a conforma-
tional change which alters the resonance positions. It is further observed
that the linewidths of the resonances in the cytochrome c peroxidase-cy-
tochrome c complex are not sensitive to changes in the electronic structure
(high-spin or low-spin) and consequently also the electronic relaxation
time of the heme iron of cytochrome c peroxidase upon complexing with
fluoride and cyanide. These observations indicate that the heme group
of cytochrome c is a considerable distance from the heme iron of the
enzyme in the complex (>25 A, assuming an electronic relaxation time
of the heme iron of cytochrome c peroxidase of approximately 10-losec).
The fluorescence studies of the interaction of cytochrome c with the
anilinonaphthalene sulfonate-apoenzyme and protoporphyrin-apoenzyme
complexes provide another line of evidence (39) in support of the above-
mentioned conclusion. Both fluorescence steady-state and lifetime
titrations of these fluorescence-labeled apoenzymes with ferro- and ferri-
cytochrome c indicates the formation of a 1:l complex, the affinity for
ferricytochrome c being less than that for ferrocytochrome c. From the
phosphorescence and fluorescence quenching, the distance between the
emitter (a fluorescence label) and the quencher (the heme of cytochrome
c ) can be calculated by assuming that no direct electronic interaction exists
between them, and that the quenching is derived from the Forster-type
energy transfer (65) to the heme. The distance from the apoenzyme-
bound emitter to the heme group of cytochrome c is estimated to be 19
A and 14 W for the anilinonaphthalene sulfonate and protoporphyrin
65. T.Forster, Discuss. Faraday SOC.

27, 7 (1959).
emitter, respectively. The latter should correspond to the heme-heme dis-

tance in the complex of the holoenayme and cytochrome c. These NMR
and fluorescence data preclude the electron transfer mechanism through
a direct contact of two prosthetic groups. The possibility of electron
transfer via the polypeptide chains or quantum mechanical tunneling
must be seriously considered.
VII. General Comments
The molecular structures of oxygen-carrying myoglobin and hemoglo-

bin as well as electron-transferring cytochromes, which are made of rela-
tively small apoproteins (a molecular weight of less than 20,000 per
heme-site), have been determined a t atomic levels. However, those of
heme enzymes such as peroxidases, catalases, and oxidases, which contain
larger apoproteins (a molecular weight of more than 30,000 per heme-
site), have not been established yet. Cytochrome c peroxidase provides
the opportunity not only to solve the molecular structure of a heme
enzyme but also to elucidate the mode of interaction of two hemoproteins
by X-ray crystallography, although cocrystallization of the peroxidase
with cytochrome c has not yet been accomplished. Various evidences thus
far available point to the substantial difference in the molecular struc-
ture, especially in the heme region, between these two categories of hemo-
proteins, as discussed in Section I11 and Fig. 1. Recent metal substitution
studies have shown that the chemical substitution of heme iron with
manganese produces enzymically active artificial peroxidases (27, 31)
but not oxygen-carrying artificial myoglobin and hemoglobin (31, 66, 67)
and that the substitution of heme iron with cobalt generates oxygen-car-
rying artificial myoglobin and hemoglobin (68, 69) but not enaymically
active artificial peroxidases (70). These observations indicate that the
chemical nature of apoprotein and the mode of its interaction strongly
control the physical and chemical characteristics of the prosthetic group
in holohemoproteins.
The detailed chemical mechanism of the interaction between cyto-
chrome c peroxidase and hydroperoxides to form Compound ES must be
further elucidated. The use of substrate analogs such as various oxidia-
66. T. Yonetani, H. R. Drott, J. S., Jr. Leigh, G . Reed, T. Asakura, and M. R.

Waterman, JBC 245, 2998 (1970).
67. M. R. Waterman and T. Yonetani, JBC 245,5847 (1970).
68. B. M. Hoffman and D. H. Petering, Proc. N a t . Acad. Sci. U . S . 67, 637 (1970).
69. T. Yonetani, H. Yamamoto, and G . V. Woodrow, 111, JBC 249, 691 (1974).
70. T. Yonetani, T. Iizuka, and H. Yamamoto, JBC 249,2168 (1974).
ing agents (8, 9 ) , aromatic peracids (71) as well as isotope-substituted

substrates may offer the opportunity to effectively shed a light on this
problem.
The true physiological role of cytochrome c peroxidase in yeast is yet
to be established. It may serve as a part of the systems which prevent
intracellular accumulation of harmful hydrogen peroxide. It would be
of interest to know if cytochrome c peroxidase is synthesized concurrently
with or in competition with the production of other peroxide-decomposing
systems such as catalase. Although cytochrome c peroxidase is present
in mitochondria of aerobically grown yeast in a concentration comparable
to that of cytochrome oxidase (19) and possesses an extremely high mo-
lecular activity (k, = lo4 sec-') toward yeast ferrocytochrome c ( 1 7 ) ,
it has not been unequivocally shown that ferrocytochrome c is a true
substrate of this enzyme.
ACKNOWLEDGMENTS
This investigation has been supported by NSF grant (BMS73-00970), NHLI grant
(HL-14508), and NIAAA grant (AA-00292).
71. G. R Schonbaum and S. Lo, JBC 247,3353 (1972).

Catalase
GREGORY R. SCHONBAUM BRITTON CHANCE
I. Introduction . . . . . . . . . . . . . . . . 363
11. General Enzyme Properties . . . . . . . . . . . . 366
111. The Nature of the Active Site . . . . . . . . . . . 369
A. Identity of Ligands at the Fifth and Sixth Coordination
Positions of the Prosthetic Group . . . . . . . 369
B. Identity of a Distal Ligand: Selective Modifications of the
Apoprotein . . . . . . . . . . . . . . 376
C. Ligand Exchange Reactions . . . . . . . . . . 385
IV. Catalase-Mediated Redox Reactions . . . . . . . . . 388
A. The Nature of Compound I . . . . . . . . . . 389
B. The Catalase Reaction Mechanism. . . . . . . . 390
1. Introduction
A quarter of a century has passed since the first contribution on cata-
lase to “The Enzymes” : lLEnzyme substrate compounds: Mechanism of
action of hydroperoxidases” ( 1 ) . I n this perspective, we can identify a se-
quence of steps in the development of ideas on the mechanism of enzymic
action and the nature of enzyme-substrate compounds. The identification
of these compounds and the approach to enzymic reactions a t concentra-
tions stoichiometric with the substrate caused a principal transition of
viewpoint on hemoprotein catalysis from free radical mechanisms ( 2 ) un-
related to an active center toward the acceptance of catalysis occurring
at the iron atom of the porphyrin (3-5). The latter concept followed natu-
1. B. Chance, “The Enzymes,” Vol. 2, Part 1, p. 428, 1951.
2. C. Oppenheimer and K. G. Stern, “Biological Oxidation.” Junk, The Hague,
1939.
3. 0.Warburg, “Heavy Metal Prosthetic Groups and Enzyme Action” (A. Lawson,
transl.). Oxford Univ. Press (Clarendon), London and New York, 1949.
363
364 GREGORY R. SCHONBAUM AND BRITTON CHANCE
rally from the impetus of Otto Warburg’s emphasis on iron itself (S),
together with enlargements of this idea to include the heme caused by
the broader views of David Keilin ( 4 ) . Their ideas on the iron atom
as the active center of the heme, which serves as a “vise” to hold the
iron in place, were focal points in the structural approach to the nature
of enzyme-substrate compounds; the protein was held to play a secon-
dary role. Thus, in the 1930’s and 1940’s, the experimental ap-
proaches-particularly those of Theorell (6) and Pauling (?)-were
focused upon the valence state of the iron, the magnetic properties of
the enzyme-substrate compounds, and the ionic or covalent nature of the
intermediates (6,7).It remained for X-ray structure studies of the heme
region of myoglobin (8) to bring appropriate attention to the active site
as a special environment necessary for enzymic action. The present re-
view emphasizes the nature of this site, the environment it affords for
the heme and for the oxidizing or the reducing substrate, and the nature
of the energy barriers through which the substrates must pass in order to
react a t the iron atom.
Other studies concerned the chemical nature of the enzyme-substrate
intermediate. It was early recognized that “the precise formula (for the
enzyme-substrate complex) may differ from that of a simple iron peroxidc
complex” ( 5 ). The development of ideas on electron delocalization and,
indeed, electron transfer in oxygen and peroxide compounds of hemopro-
teins has followed over this interval, slowly a t first, beginning with the
work of George on myoglobin and peroxidase compounds (9) and reach-
ing a much broader based generality with infrared studies of ferrous
iron-oxygen compounds (10). These approaches, together with belated
X-ray and nuclear magnetic resonance studies of the structure of the
protein (11) and of the active site of the hydroperoxidases ( l a ) , together
4. D. Keilin, in “The History of Cell Respiration and Cytochrome” (J. Keilin,

ed.). Cambridge Univ. Press, London and New York, 1966.
5. B. Chance, in “Biological Antioxidants” (C.G . MacKenzie, ed.), p. 54. Josiah
Macy, Jr. Found., New York, 1950.
6. H. Theorell, Advan. Enzymol. 7 , 265 (1947).
7. L. Pauling, “Nature of the Chemical Bond and the Structure of Molecules
and Crystals: An Introduction to Modern Structural Chemistry.” Cornell Univ.
Press, Ithaca, New York, 1960.
8. L. Stryer, J. C. Kendrew, and H. C. Watson, J M B 8,96 (1961).
9. P. George, in “Currents in Biochemical Research” (D. E: Green, ed.), Vol.
11, p. 358. Wiley (Interscience), New York, 1956.
10. W. Caughey, Chapter 5, this volume.
11. L. L. Larsson, L. 0. Hagman, P. Kierkegaard, and T. Yonetani, JBC 245,
902 (1970).
12. R. Hershberg and B. Chance. Biochemistry 14,3885 (1975).
7. CATALASE 365
with the multiple methods of electron paramagnetic resonance (13) and

infrared spectroscopy (14) of the enzyme-substrate analogues, ensure
that the present review will be only a milestone on the way toward a
deeper understanding of iron porphyrin proteins in biological catalysis.
Thus this review, which emphasizes the chemistry of the catalase reac-
tion, leads naturally to a second (15) on the “bifunctional” peroxidatic-
catalatic nature of catalase activity and its relationship to the physiologi-
cal function of catalase.
The chemistry of the colorful, perplexing, and challenging problem of
catalase mechanism has been set forth in numerous reviews. Those by
Brill (16), Nicholls and Schonbaum ( I $ ) , and most recently, Deisseroth
and Dounce (18) summarize the fundamental properties of catalase and
its reactions, and their physiological implications. Further, Feinstein (19)
and Aebi (20-22) have presented detailed evaluations of acatalasemia,
and de Duve (23) and others have discussed catalase biosynthesis
(23-26), its intracellular location (23-25) and its turnover (24, 26-28).
These facets of the catalase problem will not be reiterated. Instead, a
brief synopsis of the enzyme characteristics will be followed by a discus-
sion on the nature of the active site and the chemistry of the catalase
reaction mechanism.
13. J. S. Leigh, M. Maltempo, P. I. Ohlsson, and K. G. Paul, FEBS (Fed. Eur.

Biocnem. Soc.) Lett. 51, 304 (1975).
14. C. H. Barlow, P. I. Ohlsson, and K. G. Paul, Abstr., 170th ACS Meet. (1975).
15. B. Chance, N. Oshino, and H. Sies, in preparation.
16. A. S. Brill, Compr. Biochem. 14, 447 (1966).
17. P. Nicholls and G . R. Schonbaum, “The Enzymes,” 2nd ed., Vol. 8, p. 147,
1963.
18. A. Deisseroth and A. L. Dounce, Physiol. R e v . 50, 319 (1970).
19. R. N. Feinstein, Biochem. Genet. 4, 135 (1970).
20. H. Aebi and H. Suter, Advan. Hum. Genet. 2, 143 (1971).
21. H. Aebi, E. Bossi, M. Cants, S. Matsubara, and H. Suter, in “Hereditary
Disorders of Erythrocyte Metabolism” (E. Beutler, ed.), p. 41. Grune & Stratton,
New York, 1968.
22. H. Aebi, in “Isozymes” (C. L. Markert, ed.), Vol. I, p. 227. Academic Press,
New York, 1975,
23. C. de Duve, in “Alcohol and Aldehyde Metabolizing Systems” (R. G. Thurman
et al., eds.), p. 161. Academic Press, New York, 1974.
24. C. M. Redman, D. J. Grab, and R. Irukulla, ABB 152, 496 (1972).
25. R. S. Holmes and C. J. Masters, ABB 148, 217 (1972).
26. G. L. Jones and C. J. Masters, ABB 161,601 (1974).
27. M. Rechcigl, in “Enzyme Synthesis and Degradation in Mammalian Systems”
(M. Rechcigl, Jr., ed.), p. 273. Univ. Park Press, Baltimore, Maryland, 1971.
28. B. Poole, in “Enzyme Synthesis and Degradation in Mammalian Systems”
(M. Recheigl, Jr., ed.), p. 375. Univ. Park Press, Baltimore, Maryland, 1971.
II. General Enzyme Properties
Catalase (H,O,:H,O, oxidoreductase; EC 1.11.1.6) was one of the first

enzymes to be isolated in a high state of purity, and its crystallization
(29) from beef liver extracts ranked among the early triumphs of bio-
chemistry. Extensive physicochemical studies which followed (30-41) led
to an impressive elucidation of its properties, but as yet not to a definition
of the apoprotein function in the enzyme catalysis.
Typically, all carefully characterized catalases are oligomers (isoelec-
tric point -5.5) containing four (42-46) tetrahedrally arranged (47),
60,000-dalton subunits (48-60). Each subunit consists of a single poly-
peptide chain (41) that associates with a single prosthetic group, ferric
protoporphyrin IX (61). The subunits apparently function independently
of one another (66,6 3 ) .
I n view of the structural complexity of catalase, it is not surprising
29. J. B. Sumner and A. L. Dounce, JBC 121,417 (1937).
30. D. Keilin and E. F. Hartree, Proc. R o y . SOC.,Ser. B 121, 173 (1937).
31. J. B. Sumner and N. Gralen, JBC 125,33 (1938).
32. J. B. Sumner and A. L. Dounce, JBC 127,439 (1939).
33. J. B. Sumner, A. L. Dounce, and V. L. Frampton, JBC 136, 343 (1940).
34. H. Theorell and K. Agner, Ark. Kemi 16, 1 (1943).
35. B. Chance, Acta Chem. Scand. 1,236 (1947).
36. B. Chance, JBC 194, 471 (1952).
37. B. Chance, JBC 194, 483 (1952).
38. J. Yang and T. Samejima, JBC 238,3262 (1963).
39. M. J. Stansell and H. F. Deutsch, JBC 240, 4299 (1965).
40. A. Tasaki, J. Otsuka, and M. Kotani, BBA 140, 284 (1967).
41. W. A. Schroeder, J. R. Shelton, J. B. Shelton, B. Robberson, and G. Appell,
ABB 131, 653 (1969).
42. R. E. Greenfield and V. E. Price, JBC 220,607 (1956).
43. A. W. Galston, R. K. Bonnichsen, and D. I. Arnon, Acta Chem. Scand. 5,
781 (1951).
44. H. F. Deutsch, Acta Chem. Scand. 8, 1516 (1952).
45. D. Herbert and J. Pinsent, BJ 43, 193 (1948).
46. D. Herbert and J. Pinsent, BJ 43, 203 (1948).
47. B. K. Vainshtain, S. Ya. Karpukhina, N. I. Sosfenov, and L. A. Feigin, Dokl.
Akad. Nauk SSSR 207,1336 (1972).
48. H. Sund, K. Weber, and E. Molbert, Eur. J. Biochem. 1, 400 (1967).
49. K. Weber and M. Osborn, JBC 244, 4406 (1969).
50. H. Furuta, A. Hachimori, Y. Ohta, and T. Samejima, J. Biochem. ( T o k y o )
76, 481 (1974).
51. K. G. Stern, JBC 112, 661 (1936).
52. B. Chance, JBC 179, 1299 (1949).
53. M. L. Kremer, J. Theor. Biol. 29, 387 (1970).
7. CATALASE 367
that its biosynthesis occurs stepwise. The synthesis is governed by a

single autosomal determinant and proceeds, in the case of (rat) liver cat-
alase, in three distinct stages (23): (a) synthesis of approximately
60,000-dalton apocatalase subunits, (b) intercalation of heme, and (c)
formation of tetramers.
The resulting oligomer is relatively stable, a property recognized early
in the course of its isolation from liver (54, 55) blood erythrocytes, and
bacteria (45,46, 56, 5 7 ) . Indeed, dissociation of catalase generally entails
its irreversible denaturation [but, see Samejima and Yang ( 5 8 ) ] and
occurs most commonly under rather drastic conditions (48): below p H
3 (58, 5 9 ) , above p H 10 (48, 60, 6 1 ) , in the presence of detergents ( 4 8 ) ,
or following an extensive chemical modification of the apoprotein (50,
6 2 ) . Gentler modifications such as controlled acetylation with N-acetyl
imidazole (50) do not result in tetramer dissociation although the sub-
units do partly unfold. These changes are not accompanied by a pro-
nounced loss of enzymic activity, and the modified derivative, like the
native catalase ( S O ) , is nonreducible by dithionite.
Despite the size of the protein subunits, their integrity does not depend
on cross-linking via disulfide bonds (63) and no disulfide bridges have
been identified within the partially completed amino acid sequence (41,
6Sa). Nor is there any evidence that association of subunits depends
on covalent bonding; rather, it appears to involve mainly hydrophobic
interactions ( 5 0 ) .Of particular interest in this context is the observation
that some forms of acatalasemia are attributable to the formation of
a catalase variant, of approximately normal specific activity, but with
a tendency t o dissociate into subunits ( 6 4 ) .
These more recent studies (50, 58) as well as others involving photo-
oxidation (65) of the enzyme, its carboxymethylation ( 6 6 ) , and the de-
A. L. Dounce, JRC 143, 497 (1942).
54.
A. J,. Dounce and V. Frampton, Science 89, 300 (1930).
55.
P. Waentig and W. Gierisch, Fermentforschung 1, 165 (1914-1916).
56.
M. Tsuchihashi, Biochem. 2 140, 63 (1923).
57.
T. Samejima and J. Yang, JBC 238, 3256 (1963).
58.
C. Tanford and R. Lovrien, JACS 84, 1892 (1962).
59.
60. T. Samejima, J. Biochem. (Tokyo) 46, 1101 (1959).
61. M. Nagahisa, J. Biochem. (Tokyo) 51, 216 (1962).
62. K. Abe, M. Hiraga, and F. Anan, J. Biochem. (Tokyo) 74,889 (1973).
63. S. Morikofer-Zwea, M. Cantz, H. Kaufmann, J. P. von Wartburg, and H . Aebi,
Eur. J. Biochem. 11,49 (1969).
63a. W. A. Schroeder, personal communication.
64. H. Aebi, S. R. Wyss, B. Scherz, and F. Skvaril, Eur. J. Biochem. 48, 137 (1974).
65. M. Nakatani, J. Biochem. (Tokyo) 49,98 (1961).
66. M. Nakatani, J. Biochem. (Tokyo) 48, 476 (1961).
rivatization of its sulfhydryl group (67493, tentatively suggest that (a)

the prosthetic group is deeply intercalated but not necessarily strongly
bound within the protein matrix; (b) the functional groups a t the active
site are not readily accessible to modification; (c) sulfhydryl groups are
not essential to enzymic activity; (d) in peroxisomes which are charac-
terized by an extraordinarily high catalase content (69a), dissociation
of the enzyme (69b) cannot be central to the modulation of its activity
(69c) ; and (e) the perturbations of the protein mantle are not intimately
reflected in the catalytic properties of the active site. It seems, therefore,
rather unlikely that different conformations of catalase in the high-spin
ferric state-inferred from the EPR data (70)-play a key role in the
expression of enzymic activity, specifically in (1) the decomposition of
hydrogen peroxide into oxygen and water and (2) the peroxide-dependent
oxidation of various substrates.
The set of possible redox transformations previously observed in cata-
lase-mediated reactions is summarized in Eq. ( 1 )
Ferricatalase
111
pathways
where Compounds I, 11, and I11 are enzyme-peroxide derivatives in
formal oxidation states Fe(V), Fe(IV), and Fe(VI), respectively;
XHOH, two-electron equivalent donor (reductant) ; AH, one-electron
equivalent donor (reductant) ; and ROOH, a hydroperoxide (R = H,
alkyl or acyl) .
Interpretations of the reactions outlined in Eq. (1) cannot be divorced
67. A. Pihl, R. Lange, and A. Evang, Acta Chem. Scand. 15,1271 (1961).
68. H. R. Schutte and H. Nurnberger, Hoppe-Seyler’s Z. Physiol. Chem. 315, 13
(1959).
69. K. Abe, M. Hiraga, and F. Anan, Bull. Tokyo M e d . Dent. Univ. 14, 309 (1967).
69a. C. de Duve and P. Baudhuin, Physiol. R e v . 46,323 (1966).
69b. P. Jones and R. H. Pain, and A. Suggett, Nature (London) 217, 1050 (1968).
69c. G. H. Barlow and E. Margoliash, BBA 188, 159 (1969).
70. W. E. Blumberg and J. Peisach, in “Oxidases and Related Redox Systems”
(T.E. King, H. S. Mason, and M. Morrison, eds.), 2nd ed., Vol. 1, p. 299. Univ.
7. CATALASE 369
from the discussion of the prosthetic group-apoprotein interactions-spe-

cifically, those bearing on the nature and labilities of ligands in the inner
coordination sphere of the protoheme complex [L, and L,, Eq. ( 2 ) ] ,
the role of a distal (outer sphere) group YH, and ligand exchange
mechanisms.
4-Fe-h
-YH
111. The Nature of the Active Site
A. IDENTITY OF LIGANDS AT THE FIFTHA N D SIXTH COORDINATION

POSITIONS OF THE PROSTHETIC GROUP
Protoheinin is the major structural component of the active site, and,
indeed, is the principal determinant of activity and specificity in hemopro-
teins. While hemoproteins are generally categorized into the three classes
of oxidases, peroxidases, and electron transport components, the first two
groups may have a number of features in common in that the oxidase
activity may involve certain steps in which peroxide compounds are in-
volved, and the peroxidases may participate in oxidase activities under
appropriate conditions. Nevertheless, it is the purpose of this contribution
to explore how the apoenzyme determines the fine structure of the cata-
lytic activities of the hemoprotein ( 7 1 ) . In this context, two hypotheses
merit consideration: ( 1 ) that constraints imposed by the protein modu-
late hemin properties (1, 72-74) and (2) that the protein participates
intimately in the catalytic function (s) of the enzyme ( 1 7 , 7 5 ) .
Both notions are necessarily deductive since the configuration of the
apoprotein near the prosthetic group is unknown ; also, because the char-
acteristics of catalase or its derivatives do not lend themselves to singular
assignments (74, 76-80) of the proximal ligand (L5,L6; cf. Table I ) and
71. H. Theorell, Ark. Kemi 16A, 1 (1942).
72. P. Nicholls, BBA 60, 217 (1962).
73. P. George and R. J. L. Lyster, Proc. Nat. Acad. Sci. U . S . 44, 1013 (1958).
74. A. S. Brill and H. E. Sandberg, Biophys. J. 8, 669 (1968).
75. P. Jones and A. Suggett, BJ 110, 621 (1968).
76. D. W. Smith and R. J. P. Williams, Struct. Bonding (Berlin) 7, 2 (1970).
77. W. E. Blumberg and J. Peisach, Bioorg. Chem. 1, 271 (1971).
78. W. E. Blumberg and J. Peisach, in “Probes of Structure and Function of Mac-
romolecules and Membranes” (B. Chance, T. Yonetani, and A. S. Mildvan, eds.),
Vol. 2, p. 215. Academic Press, New York, 1971.
79. A. S. Brill and H. E. Sandberg, Proc. Nat. Acad. Sci. U . S . 57, 136 (1967).
80. T. Yonetani and H. Yamamoto, in “Oxidases and Related Redox Systems”
(T. E. King, H. S. Mason, and M. Morrison, eds.), 2nd ed., Vol. 1, p. 279. Univ.
TABLE I
SOME AXIAL LIGANDS
POSTULATED IN CATALASE
II
Ls LS Ref.
Carbox yl - (6)
Tyrosine - (7.2)
Nonnitrogenous -
(80)
Histidine (imidazole) - (74, 77, 7 8 , 7 9 )
Carbox yl Carboxyl (76, 81)
Carbox yl H,O (76, 81)
- OH (17)
(hydrogen bond to YH)
distal (YH) components of the active site (76, 81). There are, however,
three viewpoints which are consistent with the imidazole nature of one
of the two ligands.
Thus, Brill and Sandberg pointed out that whenever imidazole is coor-
dinated to the ferriprotoporphyrin (74, 79, 82) the difference spectra of
low-spin vs. high-spin complexes are characterized by an absorption band
~ ~ - ~ ~ ~ 6-12 x lo3 M-’ cm-l). Such
below 250 nm ( A c ~ approximately
a “diagnostic band,” attributable to charge transfer transitions from L,
to porphyrin orbitals, was noted in the difference spectrum of ferricata-
lase cyanide (low spin) vs. catalase (high spin) ( 7 4 ) ,thereby favoring
L, 5His.
The same thesis was advanced by Blumberg and Peisach (77, 7 8 ) but
for different reasons. From the EPR spectra they computed the rhombi-
city and tetragonality (8%) characterizing crystal fields of various low-
spin hemoproteins and found that for low-spin catalase derivatives such
parameters parallel those typical of complexes with a proximal (L,) his-
tidy1 group.
Finally, the primary structure of bovine liver catalase ( 4 1 ) , specifically
between Gly-215 and Lys-220 ( ~ J u )is, reminiscent of “proximal histidine”
sequences, particularly in some cytochromes and globins (Table 11) (41,
81. A. S. Brill and R. J. P. Williams, BJ 78, 246 (1961).
82. H. E. Sandberg and M. S. Balegh, BBA 295,37 (1973).
82a. Rhombicity is a function of the geometry of the complex, whereas tetragonal-
ity is governed by the charge density at the ferric ion. Hence, necessarily, it is
influenced by the charge characteristics of the apical ligands.
TABLE I1
AMINOACID SEQUENCES A N D PROPOSED
OF K N O W N PEPTIDES"
"PROXIMAL
Helical position in hemoglobino
Hemoprotein F1 F2 F3 F4 F5 F6 F7 F8 F9 FG1 Deleted FG2 Ref.
Cytochrome cz -
(Rhodospirillum rubrum)
R H P (chromatium) -
Cytochrome c (man) -
Catalase (bovine liver) - - - -
Peroxidase (horseradish) Leu - Ala Leu

Hemolobin (a)(man) Leu - Ala Leu
Myoglobin (sperm whale) Leu - - Leu
a (-) denotes nonidentical residues; (---) denotes deletions in the polypeptide chain.
6Sa, 83-87). This parallelism, although intriguing, could be entirely fortu-

itous. Indeed, other evidence, particularly the EPR signature of the fer-
rocatalase-nitric oxide complex (80, 88) and the optical spectrum of the
free enzyme (76) brings into question the histidyl identity of L,.
The EPR spectrum of the f e r r o ~ a t a l a s e - ~ ~ N
complex
O shows only a
single triplet hyperfine structure in the x-absorption due to 14N of NO
(80, 88). In contrast to the corresponding spectra of myoglobin, hemo-
globin, and some peroxidases, no superhyperfine structure is discernible,
suggesting a lack of coupling between the 14Nnucleus and the trans proxi-
mal ligand. Whether this rules out histidine a t L, is uncertain. Essen-
tially, the data indicate either that the unpaired electron of NO is not
significantly delocalized into L, or that its residence time is negligible
in the vicinity of the trans nucleus.
Equally debatable are the interpretations of the catalase optical spec-
trum (76, 81) (Table 111),which is distinguished by a band near 880
nm (74,89) and an exaltation of the transition a t 625 nm. These features,
although not typical of aquo hemoproteins when L, = His, are apparent
in their “anionic” complexes (Table 111) (74, 79, 80, 89-98) and should
also characterize high-spin hydroxo derivatives (99, 100). For these rea-
sons, it seems premature to accept the thesis of Williams et al. (76, 81)
that L, = carboxyl.
83. M. 0. Dayhoff, ‘[Atlas of Protein Sequence and Structure,” Vol. 4. Nat. Bio-
chem. Res. Found., Silver Spring, Maryland, 1969.
84. K. Dus, R. G. Bartsch, and M. D. Kamen, JBC 237, 3083 (1962).
85. R. E. Dickerson, T. Takano, D. Eisenberg, 0. B. Kallai, L. Samson, A. Cooper,
and E. Margoliash, JBC 246, 1511 (1971).
86. K. G. Welinder and L. B. Smillie, Can. J . Biochem. 50, 63 (1972).
87. K. G. Welinder, Abstr., Int. Congr. Biochem., Sth, 1973 p. 79 (1973).
88. T. Yonetani, H. Yamamoto, J. E. Erman, J. S. Leigh, Jr., and G. H. Reed.
JBC 247, 2447 (1972).
89. K. Torii and Y. Ogura, J. Biochem. (Tokyo) 64, 171 (1968).
90. F. L. Jajczay, Ph.D. Thesis, University of Alberta, 1970.
91. M. F. Peruts, P. D. Pulsinelli, and H. M. Ranney, Nature (London), New
Biol. 237, 259 (1972).
92. A. Hayashi, T. Suzuki, K. Imai, H. Morimoto, and H. Watari, BBA 194, 6
(1969).
93. J. C. Kendrew, Brookhaven Symp. Biol. 15, 216 (1962).
94. H. C‘. Watson and B. Chance, in “Hemes and Hemoproteins” (B. Chance,
R. W. Estabrook, and T. Yonetani, eds.), p. 149. Academic Press, New York, 1966.
95. P. Urnes, Ph.D. Thesis, Harvard University, Cambridge, Massachusetts, 1963.
96. G. I. H. Hanania, A. Yeghiayan, and B. F. Cameron, BJ 98, 189 (1966).
97. M. R. Mauk and A. W. Girotti, Biochemistry 13, 1757 (1974).
98. G. R. Schonbaum, JBC 248, 502 (1973).
99. P. George, J. Beetlestone, and J. S. Griffith, in “Haematin Enzymes” (J. E.
Falk, R. Lemberg, and R. K. Morton, eds.), p. 105. Pergamon, Oxford, 1961.
100. K. Yoshida, T. Iizuka, and Y. Ogura, J. Biochem. (Tokyo) 68, 849 (1970).
TABLE I11
SPECTROSCOPIC
CHARACTERISTICS
OF SOMEFERRIC
HEMOPROTEINS
Proximal ligands
Absorption bands
Hemoprotein LS LS Ref. Xrnax/€rnMa Ref.
Catalase (horse erythrocyte) Not Not - -880 625 505 406 (89, 90)
identified identified 1.1, 8.1 11.4 115
HemoglobinMil His COz- (911 -900 623 500 (91992)

a@z(67 Glu) 6.0 10.5
Metmyoglobin (sperm whale) His F- (94) 860 609 490 406 (95,96)
1.15 7.8 8.3 133
His Hz0 (93) 1040 634 502 409 (95,96)

0.89 3.6 9.9 168
Peroxidase (horseradish) His Not (74, 79,80,87,97) 1070 641 499 403 (98)
identified 0.61 3.2 11.3 102
0 Per hemin: at -pH 7, 25".
W
-a
W
1 I
350 400 450 500 550 600 650 700
X(nm)
FIQ. 1. Spectrum of horse erythrocyte catalase (---) and its formate derivative
(-1 ; pH 4.6, 25’ (101a).
Less controversial is L,. It is assumed to be a labile rraquo”or hydroxo

ligand (17, 37, 72), mainly because such a moiety should be both readily
replaceable by exogenous ligands and nonoxidizable. Further, water pro-
tons exchange at 2 5 O into the environment of the paramagnetic center
at a rate exceeding 2.4 X lo5 sec-’ ( 1 0 0 ~ )Even
. this minimum rate is
a t least tenfold greater than the rate of proton exchange into the inner
coordination sphere of metmyoglobin (101), where “water” is known to
occupy the L, position. Hence, a carboxyl group (76, 81) is not neces-
sarily a viable alternative to an aquo ligand. Moreover, catalase shows
a remarkable affinity toward exogenous carboxylate ligands such as for-
mate ( K a < M ) (37), and the spectra of the resulting derivatives
are distinct (cf. Fig. 1) ( 1 0 1 ~ from
) the spectrum of the free enzyme.
Neither should obtain if L, were a carboxyl since in a competition be-
tween intra- and intermolecular reactions the former would prevail.
The above argument assumes that formate invariably ligates to the
ferric ion. Hershberg (102) has recently pointed out that this is not neces-
sarily so, on the basis of NMR studies suggesting that formate interacts
100a. A. S.Mildvan and G . R. Schonbaum, unpublished observations.
101. A. S. Mildvan, N. M. Rumen, and B. Chance, in “Probes of Structure and
Function of Macromolecules and Membranes” (B. Chance, T. Yonetani, and A. S.
Mildvan, eds.), Vol. 2, p. 205. Academic Press, New York, 1971.
101a. G.R. Schonbaum, unpublished observations.
102. R. L. Hershberg, Ph.D. Thesis, University of Pennsylvania, Philadelphia, 1974.
7. CATALASE 375
principally with the apoenzyme, albeit in the vicinity (approximately

7 A) of the prosthetic group. Accordingly, he assigned the observed
spectroscopic changes to some unspecified perturbation of the hemin
environment.
The preceding discussion illustrates the continuing uncertainty as to
the nature of the L, and L, ligands. The properties of low-spin ferricata-
lase derivatives are consistent with the presence of histidine a t L, but,
seemingly, the characteristics of the free enzyme and of the low-spin
ferrocatalase-nitric oxide complex are not. It could be argued, of course,
that the orientation of L,, or even its identity, is not invariant in different
derivatives, but is mobile or interchangeable. Indeed, Samej ima and Kita
(103) studied the optical rotatory dispersion and circular dichroism spec-
tra of the free enzyme and its cyanide and azide derivatives and concluded
that these compounds had different amounts of helicity.
Still another and equally provocative alternative presents itself if (a)
the Ls ligand is hydroxo in character, and (b) the iron atom is displaced
toward the sixth coordination position, away from the proximal histidine
group. Such a configuration is outlined in formula ( I ) ,where YH remains
unspecified but, as shown later, might be another histidine residue.
(1)
Note that in such a configuration:
1. Hydroxyl being a weak field ligand (104), the optical absorption
spectrum of ferric catalase is similar to that expected for a high-spin
form of hydroxy- or fluorometmyoglobin (99) (Table 111).
2. The nonreducibility of catalase, although a pn’ori not predictable,
is a t least understandable ( 1 0 4 ~ )First,
. the hydroxy group, L,, should
stabilize the ferric state. Second, reduction to a high-spin ferrous ion,
with an attendant increase in ion radius and its repulsion from the por-
phyrin lattice, would not be compensated by proximal interactions, as it
103. T. Samejima and M. Kita, BBA 175, 24 (1969).
104. F. Basolo and R . G. Pearson, in “Mechanisms of Inorganic Reactions,” 2nd
ed., p. 67. Wiley, New York, 1967.
104a. It is generally assumed that nonreducibility of catalase by dithionite is of
thermodynamic origin. The pathways of dithionite-dependent reductions are, how-
ever, by no means simple (106, 106), and it would be unjustified to reject kinetic
factors in the catalase-dithionite system.
105. C. Creutz and N. Sutin, Proc. N n t . Acarl. Sci. U . S. 70, 1701 (1973).
106. D. 0. Lambeth and G. Palmer, JBC 248, 6095 (1973).
376 GREGORY R. SCHONBAUM AND BRITTON CHANCB
is in myoglobin or hemoglobin. However, the transition to a spin-paired

form, as in ferrocatalase-CO or -NO, should-and does-stabilize the low
redox state of the enzyme.
3. In low-spin ferric compounds, e.g., cyanide or azide (106~) com-
plexes, the reduction in the ion radius and incursion of r-bonding should
facilitate the entry of iron into the porphyrin plane (108,109) and
thereby lead to interactions which typify complexes having a proximal
histidine (77,79).
4. In contrast, the lack of superhyperfine splitting in the ferrocata-
lase-NO complex would not have been foreseen if L, = His. As pointed
out by Yonetani et al. (88),a fast relaxation of electron spin may explain
this behavior. Alternatively, if catalase-NO were to be a square pyra-
midal complex, the spin density may not extend to the nitrogen atom
of the proximal histidine, and hence no superhyperfine coupling need
occur.
The above rationalizations rest on the assumption that iron is displaced
toward L6, putting, perhaps, undue weight on the premise that enzyme
properties are solely governed by the relative positions of L6, L,, and iron.
At best, it is but one facet of the possible constraints imposed by the apo-
protein upon the environment of the prosthetic group.
B. IDENTITY
OF A DISTAL
LICAND:SELECTIVE
MODIFICATIONS
OF
THE APOPROTEIN
In spite of the apparent inaccessibility of the distal site to the typical
and traditional protein reagents, some progress has been made in delineat-
ing its properties. Pivotal to these developments was the observation by
Heim et al. (110)that 3-amino-lH-1,2,4- triazole (AT) irreversibly in-
,Fa
(AT)
H
(n)
hibits the catalatic function in liver. The reaction occurs only in vivo
and not with an isolated enzyme. This behavior was explained by Margo-
liash and Novogrodsky (lll), who noted that the inhibition depends
106a. The spin state of ferricatalase-aside is temperaturedependent, the contribu-
tion of a low-spin form increasing with the decreasing temperature (89,107).
107. K. Torii, T. Iizuka, and Y. Ogura, J . Biochem. (Tokyo) 68, 837 (1970).
108. J. L. Hoard, in “Hemes and Hemoproteins” (B. Chance, R. W. Estabrook,
and T. Yonetani eds.), p. 9. Academic Press, New York, 1966.
109. R. Countryman, D. M. Collins, and J. L. Hoard, JACS 91, 5166 (1969).
110. W. G. Heim, D. Appleman, and H. T. Pyfram, Science 122, 693 (1955).
111. E. Margoliash and A. Novogrodsky, BJ 68,468 (1958).
7. CATALASE 377
upon the presence of peroxides or compounds susceptible to autoxidation,

and proposed a reaction between catalase Compound I, an enzyme-per-
Catalase -
oxide derivative, and AT, the inhibitor:
H202 AT
Compound I -+ inhibited enzyme
Only the subunits with intact prosthetic groups are modified (112) and
(3)
the inhibited product contains approximately one equivalent of AT per

hematin bound at His-74 (112, 113) most probably as in formula (111)
(11Sa) :
R
(ID)
The derivative retains both the ferric, largely high-spin characteristics

and the oligomeric structure of the native enzyme (111, 114). It shows,
however, only marginal activity in mediating the decomposition of hydro-
gen peroxide (115)and is correspondingly inert toward the typical hemo-
protein ligands such as cyanide and fluoride. This set of properties is
best reconciled by assigning the locus of modification to the distal site
of the prosthetic group (116).
The mechanism of the inhibitory reaction is uncertain, although two
proposals have gained some currency (115-118).One of these invokes
“activation” of histidine, which is then subject to an unexpectedly selec-
tive nucleophilic attack by AT (or some related compounds) (115,116).
The other demands compulsory two-electron equivalent oxidation of A T
(117,118), giving an electrophilic intermediate which, prior to its solva-
tion, reacts with the imidazole group of histidine. Although AT is an
112. J. Y. Chang and W. A. Schroeder, ABB 148,505 (1972).
113. B. B. L. Agrawal, E. Margoliash, M. I. Levenberg, R. S. Egan, and M. H.
Studier, Fed. Proc., Fed. Amer. SOC.
E x p . Biol. 29, 732 (1970).
113a. B. B. Agrawal, E. Margolinsh, M. I. Levenberg, R. S. Egan, and M. H.
Studier, personal communication.
114. P. Nicholls, BBA 59, 414 (1962).
115. E. Margoliash, A. Novogrodsky, and A. Schejter, BJ 74, 339 (1960).
116. E. Margoliash, in “Probes of Structure and Function of Macromolecules and
Membranes” (B. Chance, T. Yonetani, and A. S. Mildvan, eds.), Vol. 2, p. 571.
Academic Press, New York, 1971.
117. G. R. Schonbaum, in “Probes of Structure and Function of Macromolecules
and Membranes” (B. Chance, T. Yonetani, and A. S. Mildvan, eds.), Vol. 2, p.
571. Academic Press, New York, 1971.
118. L. P. Hager, in “Molecular Basis of Electron Transport” (J. Schultz and
B. F. Cameron, eds.), p. 367. Academic Press, New York, 1972.
exceedingly poor reductant of Compound I, as are long-chain aliphatic

alcohols ( k , 0.6 M-l sec-’ a t pH 7, 2 5 O ) (118a),the latter interpreta-
N
tion seems preferable. This hypothesis does not attribute unique reactivi-
ties to AT or histidine but suggests that the efficiency ( 1 1 8 ~ of
) the inhib-
itory reaction is governed by the rate of diffusion of the oxidized AT from
the active site, by the rate of its solvation, and by the proximity of histi-
dine to the site of AT oxidation (118d).
Such a scheme lends itself to several alternative descriptions of the
oxidative reaction (11’7, 118). However, since the AT-Compound I
reaction is pH-invariant (116),the pK, of distal histidine could be “atypi-
cal” or, more likely, its modification is not rate determining in the reac-
tion sequence of Eq. (4).It is uncertain, however, whether k , or k, repre-
sents the slow step of the reaction. Kinetic or analytical demonstration
of a Compound I-AT complex is also lacking. Thus, under nonturnover
conditions using preformed Compound I, the redox reactions are first
order in Compound I and AT when [AT] 570 mM.
Compound I
Catalase- AT
(inhibited enzyme)
-
118a. k, = knbs - L,,/(AT) where k a b s is the observed first-order constant,
k., is the rate constant characterizing spontaneous decomposition of Compound I
(-3.5 & 0.5 x lo-* sec-’) and AT, the total concentration of 3-aminotriaeole such
that t,p 5 40 sec; k, was determined under nonturnover conditions using pre-
formed Compound I, thus minimizing possible artifacts resulting from competing or
side reactions (118b).
118b. R. P. White and G. R. Schonbaum, unpublished observations.
118c. For mammalian catalase, the efficiency of the modification-defined in terms
of molarities, as 100 x modified catalase/Compound I reduced-is of the order of
25 2 5% (118b).
118d. Put in those terms, the AT-dependent modifications are not uniquely “cata-
lase-specific” but should also occur with other proteins in oxidatively coupled reac-
tions. Indeed, this has been recently demonstrated with several peroxidases (119,
110).
119. J. Y. Chang and W. A. Schroeder, ABB 156,475 (1973).
120. H. Snyder and J. Schultz, Abstr. Znt. Congr. Biochem., 9th, 1973 p. 79 (1973).
7. CATALASE 379
Similar pathways are equally plausible in other reactions leading to

the inhibition of catalase ; for example, interaction between Compound
I and semicarbazide would give nitrogen and an iV-carbamyl derivative,
as in Eq. (5) :
Catalase o+c,NHa
Compound I H I
I Q semicarbazide
= LQ +N,
(5)
The proposal outlined in Eq. ( 5 ) has not been examined, but indepen-
dent arguments support it. First acylhydrazides (RCONHNH,) are
readily oxidized ( 1 2 2 ) and-presumably because of the intermediary for-
mation of acyl diimides or acyl diazonium ions, as in Eq. (6)-the reac-
tion products are powerful acylating agents. Thus, amines are effectively
converted to the corresponding acyl derivatives (122) [cf. Eq. (6) ] :
R’CONHNH, -[
oxidation
R‘CON=NH
RTON:
] RNH, ~
R’CONHR + N, (6)
Similarly, Compound I-mediated oxidation of semicarbazide should give

oxidation product (s) equally reactive toward a proximal nucleophile.
Second, under nonturnover conditions, with preformed compound I, the
oxidation of semicarbazide is accompanied by uptake of oxygen (118b),
suggesting the formation of an intermediate such as diimide (123, 124),
followed by its autoxidation [ cf. Eq. (6) 1 . I n a parallel reaction with
AT, no uptake of oxygen is to be expected, and none is obtained (118b).
The above interpretations are hypothetical, but they have led to other
inquiries showing that selective modifications of the active site of catalase
are not limited to reactions involving Compound I. Inhibition of catalases
with cyanogen bromide is an example of such a reaction (125).This mod-
ification occurs only with the free enzyme in a pH-invariant reaction
(pH 4.5-7.5) ; the inhibition resulting from the incorporation of one C moi-
ety of BrCN into the apoenzyme of each subunit (Table IV) (63, 90,
121. C. G. Overberger, J.-P. Anselme, and J . G. Lombardino, in “Organic Com-
pounds with Nitrogen-Nitrogen Bonds,” Chapter 6. Ronald Press, New York, 1966.
122. Y. Wolman, P. M. Gallop, A. Patchornik, and A. Berger, JACS 84, 1889 (1962).
123. P. C. Huang and E. M. Kosower, JACS 89, 3910 (1967).
124. P. C. Huang and E. M. Kosower, JACS 89,3911 (1967).
125. G. R. Schonbaum and F. L. Jajcray, in “Hemes and Hemoproteins” (B.
Chance, R. W. Estabrook, and T. Yonetani, eds.), p. 327. Academic Press, New York,
1966.
TABLE I V
TITRATION
O F CATALASE” WITH BrCN A N D THE CATALATIC
ACTIVITYOF THE RESULTING DERIVATIVES
% Ce
incorporation
BrCN*/ into the % Inhibited 10-8 x kl’
catalase apoprotein enzymed (M-1 sec-l)* % Activity
- 0 9.0 100
0.3 28 6.4 71
0.6 52 4.1 46
0.8 77 2.2 25
1.0 95 33 0.6 7
2 >99 0.05 0.6
12 >99 0.01 <0.1
aUsing enzyme in which approximately 14 sulfhydryl groups were modified by

controlled oxidation.
* Per hematin; in 5 mM phosphate, pH 7, 25”.
c Assayed, following extraction of hemin via Teale’s procedure (126).
d Estimated spectrophotometrically (independently of activity assays). Note:
(a) Catalase remains fully active when devoid of SH group [see also Morikofer-Zwez
et al. (SS)]and (b) there is a close correspondence between loss of activity and degree
of inhibition, suggesting the absence of any subunit cooperativities [Jajczay (go)].
166).The stoichiometry of the reaction is given in Eq. (7) :

YH + BrCN -+ “YCN” + Br- + H+ (7)
where YH represents an apoprotein functional group and YCN is the sim-
plest, but not necessarily the final, reaction product (166~).The identity
of Y remains unknown. Almost certainly it is not an -SH, a- or c-amino,
thioether, or an -OH group ( 9 0 ) .
There are striking similarities in the physical [Fig. 2 (166)and Fig. 3
(126b)l and chemical properties of the cyanylated enzyme (Table V)
and the aminotriazole-catalase derivative. Both retain the structural in-
tegrity and ferric oxidation state of the native enzyme. In addition, as
gauged by their optical spectra [Fig. 2, but see also Figs. 6A and 6B
of Margoliash and Novogrodsky (111)I, the modification of the apopro-
126. F. J. W. Teale, BBA 35, 543 (1959).
0
residue X H to Y-C-X
b
126a. Hydrolysis of YCN to Y- NH1 or an intramolecular cross-linking with a
would also accord with the analytical data.
II
NH
126b. G. R. Schonbaum, J. Peisach, and W. E. Blumberg, unpublished observations.
7. CATALASE 381
1
120.0
80.01
I A4 8 0-
\\
H
60-
4 0-
20.0Y 20-
FIQ.2. Spectrum of (A) horse erythrocyte catalase and (B)its cyanogen bromide
derivative in 0.01 M phosphate pH 7.15, 25". Extinction coefficients (M-' cm-') are
expressed in terms of heme-Fe (126).
tein elicits in both cases nearly identical heme-linked perturbations which

correlate with the loss of activity and ligand binding capacity. It is not
unlikely, therefore, that YH = His. With cyanogen bromide, histidine
Mognetic field
FIG.3. Electron paramagnetic spectra in the region of g = 6, 1.4"K for (A) horse
erythrocyte catalase and (B) its cyanogen bromide derivative. Note that the rhombi-
city of the catalase is greater than that of the inhibited enzyme (126b).
W
M
TABLE V h3
COMP.4RISON O F PHYSICAL A N DCHEMICAL PROPERTIES O F HORSEERYTHROCYTE
CATALASE AND
ITSCYANOGEN BROMIDE DERIVATIVES
Property Native Ref. Modified Ref.
Molecular weight 246,000 (*8,000) (39) -250 ,000 (90)

1013 szOnw 11.2 (50.05) (39) 11.2 ( & 0.05) (90)
Absorptivities X,, (nm) 277 406 505 538 625 -880 (89, 90) 277 402 506 533 633 -880 (125)
1 O - a ~ (M-1 cm-1) 90 115 11.4 9 . 8 8 . 1 1.1 90 102 12.1 11.2 7.0 0.9
0
Circular dichroism in UV A (nm)
10-3 (deg cm*/decimole)
190 200
81 4
210
-56
220
-47
(90) 190 200
72 2 . 5
210
-56
220
-47
(90) g
0
2
Magnetic susceptibility -
5.96 (107) (12.5) F
(Bohr magnetons) Kn
Oxidation state Ferric ; high spin Ferric; high spin 0
Reducibility by NazSz04 Nonreducible (30) Nonreducible (1265) 2

%
Affinity for HCN a t pH 7, K ( M ) -5 x 10-6 (90) No evidence of complex when (126) ‘1
[enzyme] 5 10 p M and [HCN] 5 9
1:
100 p M U
Catalatic activity’ 10-6kl’ 11 0.06 rf: 0.002b (90)

(M-1 sec-1) cj
(Kat.f.) (95,000) (-500 5 200)

-
a Per heme group.
Possibly resulting from some residual free enzyme.
7. CATALASE 383
TABLE VI
REGENERATION
OF ACTIVECATALASE FROM THE CYANYL.4TED ENZYME, 25'
Ligand 108 [A- + AH] los [AH] lo6 k

A- PH (MI (MI (M-1 sec-1)
Formate 7.0 100 N O . 06 G
Formate 4.5 100 15 15 f 3

Formate 4.5 200 30 15 f 3
Acetate 4.5 -5 -2.8
Acetate 4.5 200 110 -0.6
Fluoride 4.5 95 4 38 f 2
No significant regeneration within 100 hr [Jajczay (go)].
would give either an N-cyanoimidazole, as in formula (IV) (127) or its

solvolysis product, N-carbamyl imidazole as in formula (V) (128) :
N/-N/CN NCN/
'NH, H
LJ LJ
(N) (V)
Both (IV) and (V) are stable in nonpolar solvents but not in acidic or
basic solutions (127, 128). Similarly, the integrity of YCN is retained,
but only in the structurttlly intact protein, in the absence of potential
enzyme ligands (formic, acetic, or hydrofluoric acids). Such ligands pro-
mote a slow hydrolysis of YCN (Table VI) with a concomitant recovery
of full enzymic activity (90). Furthermore, acid denaturation of the
cyanylated enzyme at pH 2 is followed by complete hydrolysis of
YCN (half-time - 4 hr a t pH 2, 2 5 O ) ( g o ) , and the formation of car-
bon dioxide, possibly via Eqs. (8) and (9) :
YCN HCNO (+YH2+) (8)

n
HCNO CO, (+NH,+) (9)
The characteristics of the BrCN-catalase reaction indicate, therefore,
that activation of distal histidine-its conversion into a n electrophilic
reagent ( 1 16)-is not necessarily a prerequisite to inhibition. Neverthe-
less, the remarkable selrctivity of this reaction points to some syfiergic
facets of the BrCN-enzyme interaction which are unique to the distal
127. H. Giesenian, J. P m t k . Chem. r41 1, 345 (1955).

128. G. R. Stark, Biochemistry 4, 588 (1965).
384 GREGORY R. SCHONBAUM AND BRITTON CHANCM
region (168a). Such interactions may arise through mutual polarization

of the YH-BrCN system and its modulation by iron of the prosthetic
group (a Lewis acid) or by hydrogen bonding. These assumptions are
central to the hypotheses outlined in Eq. (9a), where the constellation
near the ferric ion (17)is represented as in (VI) .
Pr -Fe -
a:
1 k.
H
Pr-Fe--O(
I H
I Y C N
Scheme A brings into focus the well-known Lewis acid catalysis, of

which the immediately pertinent example is the reaction of cyanogen
chloride with benzene under Friedel-Crafts conditions (130-156):
ClCN + AlCls- [NC----ClAlClsl q5-CN + HC1 + AlCh
bensene
(10)
Alternatively, as in Scheme B, BrCN forms only an outer sphere complex,
with Fe(H,O) assuming the role of an acid catalyst. I n both schemes
the essential feature is a polarization of the YH-BrCN system in which
YH acts as a donor and BrCN as an acceptor, not unlike the situation
in molecular complexes of halogens with u and T donors (133, 134).
128a. A similar reaction occurs with sperm whale metmyoglobin (1291, most likely
at E 7 (distal) histidine.
129. F. L. Jajcsay and G. R. Schonbaum, Proc. Can. Biochem. SOC. 12, 73 (1969).
130. G. Olah, “Friedel-Crafts and Related Reactions,]’ Vol. I. Wiley (Intersciepce),
New York, 1963.
131. S. Nilsson, Actu Cheni. Scund. 27, 329 (1973).
132. A. A. Woolf, J . Chem. SOC.London p. 252 (1954).
133. R. Foster, “Organic Charge-Transfer Complexes.” Academic Press, New York,
1969.
134. L. J. Andrews and R. M. Keefer, Advan. Inorg. Chem. Radiochem. 3, 91
(1961).
7. CATALASE 385
The convergence of interactions, particularly as in Scheme B, may well

explain the specificity of the cyanogen bromide-dependent inhibition.
Furthermore, as both schemes imply, the reaction is pH-invariant; it
occurs only with the native enzyme; it is competitively inhibited, e.g.,
by cyanide or formate (126) ; and the resulting product (YCN) like cata-
lase itself, should-and does-exchange water or protons a t the sixth co-
ordination site (1OOa).This, as well as weak acid-dependent reactivations
(Table V ) , suggests that Y H is converted to YCN a t a functionally im-
portant residue. We propose, therefore, that YH acts as a general acid-
base catalyst in both redox and ligand exchange reactions.
C. LIGAND REACTIONS
EXCHANGE
Catalase reacts reversibly with some weak acids forming spectroscopi-

cally and magnetically distinct noncovalent derivatives. Of these, cata-
lase-cyanide, -azide, -fluoride, -formate, and -acetate complexes have
been extensively studied (37, 135, 136) and reviewed in some detail
(16-18). Briefly, there is a consensus that such reactions do not involve
heme-heme interaction ; and, with the possible exception of carboxylate
ligands ( l o g ) , all presumably result in replacement of the proximal
“aquo” ligand a t L, in a stoichiometric reaction shown in Eq. (11) :
Pr-Fe(H20) + AH + Pr-Fe(AH) + H2O (11)

where P r indicates protein and AH, a weak acid ligand. All reactions
are of the second order and are independent of the ionization state of
the enzyme in the p H range from 4.0 to 9.0. All entail, in the rate-limiting
step, an interaction of catalase with the undissociated form of the ligand
(Fig. 4) but occur without a concomitant net uptake or loss of protons.
The above parameters define the stoichiometric mechanism of the reac-
tion but have little bearing on its intimate character (137); thus, the
participation of the weak acid, AH, rather than its conjugate base, A-, in
the rate-limiting step of the reaction does not necessarily imply that AH
is ligated in the final product.
-
A couple of examples illustrate the issue. Consider first the catalase-
c,yanide system characterized by the affinity, K , 2 X lo5 M-*. There
is no doubt that H C N is the reacting entity ($7, 69) and that net proton
release or uptake does not occur (118b). Nevertheless, FeCN appears
135. K. Agner and H. Theorell, ABB 10, 321 (1946).

136. M. L. Kremer, Zsr. J. Chem. 8,799 (1970).
137. C. H. Langford and H. B. Gray, in “Ligand Substitution Processes,” p. 2.
Benjamin, New York, 1965.
6-
%3 I:
;\
Qp”i&
fluoride
I
Acetate
1 1 1 . 1 I I I
to be the only possible product. To emphasize this point, note that

CH,C = N, CH,N = C, or H C E CH do not form viable complexes-a
behavior not attributable to peculiar steric effects but reflecting their in-
trinsic reactivity, or lack of it. Similarly, the high affinity of “formic acid”
( K , 2 X lo5 M-l) contrasts with the virtual inertness of formamide
H
( K , 2 0.5 M-I), suggesting again that dissociation of the ligand

+
(AH 4A- H+) is a prerequisite of its effective ligation.
Such reactions may be expressed as in Eq. (12) :
It is therefore understandable that the aminotriazole- or BrCN-modified

enzyme no longer participates in ligand interchange reactions, particu-
larly if, as implied, YH acts as a general acid-base catalyst. That HA
rather than A- is the reactive species may therefore indicate that the
distal site, embedded in a hydrophobic environment, is largely inacces-
sible to anions and cations.
The reaction outlined in Eq. (12) is undoubtedly more complex than
indicated and may proceed via dissociative, associative, or interchange
mechanisms (137).In a t least one case, however, it was shown that direct
ligand replacements, via an associative pathway as in Eq. (13), do not
occur:
Fe(HA) + A’H + +Fe(A’H) + AH (13)

“A’H
7. CATALASE 387
where A’H is an entering ligand other than water. For example, the re-
placement of thiocyanate by cyanide does not proceed via the SN2 (lim)
associative mechanism (118b) since the rate of catalase-thiocyanate dis-
sociation (/cap* 2 x
H sec-’, pH 6.7, 2 5 O ) is essentially the same
in the presence and absence of cyanide. Accordingly, the reaction may
be governed by dissociation of thiocyanate, or alternatively, the ligand
interchange occurs in two steps: the first, involving replacement of thio-
cyanate by water, being rate-limiting [Eq. (14a) ] :
Pr-Fe- (frCS) + H,O w Pr-Fe-OH + (HSCN)

(14%)
1YA, L Y H ,
I n the second, fast step, the water molecule is replaced by the entering
cyanide as in Eq. (14b) :
Pr-Fe-OH + HCN +Pr-Fe-7N + H,O
L Y H , I Y H , ( 14b)
Whatever the mechanism, “YH” would facilitate the reaction by par-

ticipating in proton transfers between the entering and departing ligands,
perhaps as shown in Eq. (14c) :
Pr-Fe-0. /H + HCN +Pr-Fe-OH,
(144
+ Pr-Fe-CN (H,O) + P r - F e - C p + H,O

JH, &.Iz
Particularly intriguing observations by Ehrenberg and Estabrook

(158) point to the differences in catalase-ligand interactions in solution
and in the frozen state. In liquid phase, catalase does not effectively ligate
ammonia (158) although spectroscopic changes are discernible (SO).
However, in the frozen state, low-spin catalase-amine complexes are
greatly stabilized (1S9), possibly as a result of a heme-linked conforma-
tional change.
Analogous conclusions were drawn by Yoshida at al. (100) using frozen
glycine-catalase solutions, initially a t pH 2 8, in which the enzyme under-
138. A. Ehrenberg and R. W. Estabrook, Acta Chem. Scand. 20, 1667 (1966).
139. G. Heimberger and A. Ehrenberg, in “Probes of Structure and Function of
Macromolecules and Membranes” (B. Chance, T. Yonetani, and A. S. Mildvan,
eds.), Vol. 2, p. 561. Academic Press, New York, 1971.
goes a transition from a high-spin to a low-spin form with decreasing

temperatures. They attributed this to a thermal equilibrium of high- and
low-spin aquo and hydroxo states of the enzyme, rather than to glycine-
dependent effects. A reappraisal of these analyses seems warranted, how-
ever, since Ehrenberg failed to observe a low-spin form of catalase at
77OK (initially a t pH 10) and because the optical and EPR spectra
N
of the catalase amine complexes and of the low-spin aquo or hydroxo

components are nearly identical.
IV. Catalase-Mediated Redox Reactions
Cellular control of peroxides is one of the key biochemical reactions,

and a process in which catalase plays a significant role. Catalase is not
only an exceptional mediator of hydrogen peroxide decomposition (140)
as in Eq. (15)
but is also an effective catalyst (141-149) of peroxide-dependent oxida-

tions of hydrazoic, formic, and nitrous acids; of lower aliphatic alcohols;
and of hydroxylamine. Except for the oxidation of nitrous and hydrazoic
acids such reactions may be represented by a stoichiometric relationship
given in Eq. (16).
ROOH -
+ HXOH catalase XO + ROH + HoO
[R = H, alkyl, acyl; X = 0, NH, C = O , (CH,).-1,2,s].
As shown by Chance et a2. ( I @ ) , these reactions occur in a t least two
stages, outlined in Eqs. (17) and (18).
+ ROOH 4
Catalase
k
Compound I (17)
Compound I + HXOH 2 catalase + XO (18)
Both steps in the catalytic cycle are pH-invariant (36) when the sub-
strates are un-ionized. Except a t very high concentrations of hydrogen
peroxide (144, 146) both follow second-order kinetics (146).
140. B. Chance, D. S.Greenstein, and F. J. W. Roughton, ABB 37, 301 (1952).

141. D. Keilin and E. F. Hartree, BJ 39, 293 (1945).
142. L. A. Heppel and U. T. Porterfield, JBC 178,549 (1949).
143. H. Theorell and A. Ehrenberg, ABB 41,462 (1952).
144. Y . Ogura, ABB 57, 288 (1955).
145. P. Jones and A. Suggett, BJ 110, 617 (1968)
I
146. B. Chance, JBC 182,649 (1950).

7. CATALASE 389
These aspects of catalase-mediated reactions, comprehensively re-

viewed elsewhere (16-18, 147, 148), highlight two topics of immediate
interest: the nature of Compound I and its peroxidatic reactions.
I
A. THENATUREOF COMPOUND
The nature of the oxygen and peroxide compounds of catalases, peroxi-
dases, and, more recently, hemoglobin was questioned by Philip George,
who proposed (9) that electrons were transferred between the ligand and
the metal atom in the formation of such “compounds,” in contrast to
the more general term, “complex.” Evidence for the generality of this
designation has developed slowly over the years to the point where not
only can complete electron transfer, as considered by George, be demon-
strated but also more recently electron delocalization in a variety of
lesser degrees has been shown [ (149) cf. also Caughey et al., Chapter
51. This phenomenon has become especially prominent in the discussion
of the oxygen compounds of hemoglobin, where the designations range
from “covalently bound oxygen with an extensively delocalized electron”
to “loosely bound oxygen with only slight electron delocalization.” These
most interesting and significant approaches reiterate George’s query on
the point of nomenclature of “compounds” and “complexes”: What is
the proper term for an intermediate compound in which modest electron
delocalization has occurred as in oxyhemoglobin? This problem has been
raised most recently in the discovery of a series of cytochrome oxidase-
oxygen compounds (150) in which a plethora of electron transfer possi-
bilities are available, leading to what Greenwood et al. (151) have termed
“mixed valency states” of the respiratory chain, with varying degrees
of electron delocalization or electron transfer to oxygen (150). It seems
appropriate, therefore, to reserve the term “complex” for the first dissoci-
able products of the combination of enzyme and substrate-the “en-
zyme-substrate complex”-which, according to Michaelis and Menten
can be reversibly dissociated without alteration. Examples of this would
be afforded by the above-mentioned cytochrome oxidase-oxygen com-
pound and, indeed, oxyhemoglobin compounds which generally fulfill this
147. S. B. Brown, P. Jones, and A. Suggett, in “Inorganic Reaction Mechanisms”

(J. 0. Edwards, ed.), Vol. 13, p. 159. Wiley (Interscience), New York, 1970.
148. B. Chance, Advan. Enzymol. 12, 153 (1951).
149. W. S. Caughey, C. H. Barlow, J. C. Maxwell, J. A. Volpe, and W. J. Wallace,
Ann. N . Y . Acad. Sci. 244, 1 (1975).
150. B. Chance, C. Saronio, and J. S. Leigh, Jr., Proc. N a t . Acad. Sci. U. S . 72,
1635 (1975).
151. C. Greenwood, M. T. Wilson, and M. Brunori, BJ 137,205 (1974).
criterion. The chemical equation for an intermediate complex therefore

involves the reversible sequence:
S + E+ES+S + E (19)
This sequence is apparently applicable to the cytochrome oxidase-oxygen

enayme-substrate compound, where the apparent dissociation constant
is relatively large (0.5 mM a t -1200) (150). At higher temperatures,
however, compounds are formed in which the reversibility can no longer
be identified in spite of a number of efforts to do so (152, 169).
I n studies of catalase, much effort has been directed toward a deter-
mination of whether or not hydrogen peroxide could be dissociated from
the enayme-substrate intermediates of catalases and peroxidases. It
should be pointed out that catalase, as contrasted with cytochrome oxi-
dase, has been studied only a t room temperature, and if any lesson is
to be learned from the study of cytochrome oxidase (150), it is that the
“complexes” are most likely to be identified a t low temperatures, as pre-
cursors of the “compounds.” I n this sense, they are of first importance
and not to be ignored in our understanding of the mechanism of enzymic
reactions.
B. THE CATALASE
REACTION
MECHANISM
By analogy to other catalase-ligand interactions, Compound I was
initially taken to be an enayme-peroxide complex, even if a rather un-
usual one. However, the optical spectrum of Compound I (154, 155),
its magnetic susceptibility (156), and the lack of a discernible E P R sig-
nature (126b) are unlike those of other high- or low-spin catalase-ligand
derivatives.
In spite of these anomalies, the idea of a “complex” remained a working
hypothesis, particularly since kinetic studies failed to reveal any interme-
diate (9) preceding Compound I formation (144, 157) and since the inter-
action of bacterial catalase with methyl hydrogen peroxide could be ex-
pressed in terms of an equilibrium reaction (158).The latter proposition
152. F. A. Schindler, Ph.D. Thesis, University of Pennsylvania, Philadelphia, 1964.

153. S. Muraoka and E. C. Slater, BBA 180,227 (1969).
154. B. Chance, ABB 41, 404 (1952).
156. A. 5.Brill and R. J. P. Williams, BJ 78,253 (1961).
156. A. S. Brill, Free Radicals Biol. Syst., Proc. Sump., 1960 p. 53 (1961).
157. B. Chance, in “Currents in Biochemical Research” (D. E. Green, ed.), p.
308. Wiley (Interscience), New York, 1956.
158. B. Chance and G. R. Schonbaum, JBC 237,1962 (1962).
7. CATALASE
jibh
.14
152
.SO
100 200 3
391
Sacondr
(A1
Fra. 5. (A) Formation of Compound I using EtOOH. (B) Decomposition of EtOOH
in the presence of catalase (0); and in the absence of enzyme ( W). I n (A) and (B) :
M . lysodeikticus catalase (hematin) N 6.2 p M ; (EtOOH) 19.5 p M , pH 8, 25" ( 1 0 1 ~ ) .
AAW (maximum at 300 sec) Zl.8 p M Compound I. Concurrently, less than 0.15
r M Compound I1 was formed, as determined independently.
was based on the assumption that Compound I is adequately stable and

that it is the sole product of the enzyme-peroxide reaction-a point con-
sidered in detail in that publication. More recent data suggest that acetal-
dehyde is formed from ethyl hydrogen peroxide in the presence of M .
lysodeikticzls catalase (Fig. 5 ) . Similar results are obtained with bovine
liver and horse erythrocyte catalases (159, 159u), the major fraction of
ethyl hydrogen peroxide being converted into acetaldehyde ( 1 7 ) . It ap-
pears that the equilibrium of catalase and peroxide involves problems
similar to those of cytochrome oxidase (150) in that electron transfer
between enzyme and substrate occurs and that low temperature and rapid
reaction techniques may be required to study this equilibrium. The for-
mation of aldehyde appears to be an intrinsic property of the catalase-
ethyl hydrogen peroxide system, as noted in the early work of Stern
(160); it is not an artifact attributable to the presence of some adventi-
tious ethanol. Even in the presence of methanol or nitrite (both excellent
reductants of Compound I ) , approximately 60% 4 10% of EtOOH is
converted to acetaldehyde (Table V I I ) . Moreover, the extent of conver-
sion is nearly invariant of the donor concentrations. Both facets of the
reaction suggest that the enzyme mediates either dehydration of
EtOOH (Scheme 1) or a redox rearrangement (Scheme 11).
159. G. R. Schonbaum, Abstr., Wenner-Gren Symp. Struct. Funct. Oxidatiori-Re-

duclion Enzymes, 1970, p. 48 (1970).
159a. G . R. Schonbaum, Abstr., Int. Congr. Biochem., Sth, 1979 p. 49 (2b10)
(1973).
160. K. G. Stern. JBC 114,473 (1936).
TABLE VII
FORMATION OF ACETALDEHYDE FROM ETHYLHYDROQEN
PICROXIDE MEDIATED
BY M. lg8OdeikliCUS (MLC) AND HORSEERYTHROCYTE
(HEC) CATALASESO
Acetaldehyde from
Donor MLC HEC

concn. range
(mM) pM %of (EtO0H)i pM %of (EtO0H)i
Ethanolb 51 f 1 100 51 f 1 100

1.64.1
Methanol 36 It 1 70 32 f 1 64
2 .O-8.2
Nitrite 37 & 2 72 27 f 2 52
0.3-1 .3
(MLC)re 3.2 p M , (HEC) 0.76 p M , (EtOOH) 51 p M in 0.1 M phosphate, pH 7,

25".
Control experiments showing conservation of redox equivalents and not the origin
of aldehyde [Schonbaum (10Ia)l.
E + RCHO + H,O
f
(Catalase)
EO + RCH,OH
(Compound I)
I
SCHEME
E + RCH,OOH +(E .RCH,OOH) (EO .RCH,OH)
E + RCHO
Y k 2
+ H,O
\
EO +-RCKOH
SCHEME Ir
Scheme I1 is preferred because with methyl or butyl hydroperoxides

k, > k, (IOIa).Essentially, then, Compound I is not the primary en-
zyme-substrate complex (161). The formation of Compound I entails the
reduction of substrate (peroxide) a t the active site (compare Schemes
I and 11). The recent discovery that nearly one mole of Compound I
is formed in the 1 :1 reaction between catalase ferriheme and peracetic
161. P. George, ABB 45,21 (1953).
7. CATALASE 393
25pM CH,CO,H
1 $
4.25pMCH,COSH
I, 8.5pM CH,CO,H
,i
\ 4
- I
I
0 10 20 30
10' [CH,CO, H] (M)
FIG.6. Titration of -15 fiM horse erythrocyte catalase-ferriheme with peracetic

acid, 0.08 M phosphate, pH 7.23, 25" ( 1 0 1 ~ ) .
acid (Fig. 6) further supports this conclusion ( 1 6 1 ~also

; see 169, 166,
163). As in enzyme-hydrogen peroxide reactions, the unionized peracid
is the immediate substrate (162) and, according to Jones and Middlemiss
(163),one mole of acetic acid is released per mole of catalase ferriheme
converted to Compound I.
No information is available as to the individual steps of the reac-
tion, and none is likely to issue from kinetic studies with higher alkyl
peroxides (C 2 2) or peracids. The reasons are implicit in the data
of Table VIII (1,69, 101a, 162, 164), which show that the observed rate
constants for Compound I formation ( k ,),, with peracetic acid, hy-
droxymethyl hydroperoxide, and ethyl hydrogen peroxide are of the same
*
order of magnitude (2.5 0.5 X lo4 M-* sec-') in spite of differences
in the following:
0-0 dissociation energies for EtOOH (43 kcal/mole) and peracids
(-35 kcal/mole) ,
the basicities of the leaving groups (pK,:acetic acid, 4.76; ethanol,
18), and
the ionization properties of peroxides (pK, :peracetic acid, 8.2;
EtOOH, 11.8)
161a. The stoichiometry of the reaction is unaccountably variable, the average
of several titrations showing that approximately 0.87 & 0.08 mole of Compound I
is formed per mole of peracetic acid.
162. P. Jones and D. N. Middlemiss, BJ 130,411 (1972).
163. P. Jones and D. N. Middlemiss, BJ 143,473 (1974).
164. S. Marklund, BBA 289,269 (1972).
e:
7. CATALASE 395
We must assume, then, that the rate-limiting step is virtually indepen-

dent of the above parameters. Nor can it be involved in the redox-depen-
dent rearrangement of iron-protoporphyrin since the reactions with H,O,
and CH,OOH are faster than those with EtOOH or CH,CO,H.
A relationship between k, ap,, and van der Waals volumes of peroxides
is the only discernible pattern. This strongly suggests that enzyme-per-
oxide association is the rate-determining step and, as such, is rather irrel-
evant to the elucidation of the redox transformations.
The cocatalytic role of the apoprotein in facilitating Compound I gen-
eration is increasingly regarded as that of a general acid-general base
(17, 75,159, 159a), although different functional groups are seen as fulfill-
ing this role. Jones and Suggett ('?5),pointed to the possible involvement
of >C(NHr),+ group from an arginine residue (BH') and a carboxylate
(A-) as in Eq. (20).
H
, Hgoz + B H
,F l I ,
H
H,O-Fe O/O--Fe (20)
I ?I H,
H 9_ _ _ _ 0_ _ _ _ _ _ Fe
I ,
H
As already mentioned, a less detailed scheme may also be developed,
using only Y H 3 His [formulas (VII) and (VIII) ]
,'
3,
H,
' 7
Enzyme
p-".
Enz-yy 2 - R or I
H.,, 3
"0 Y'
H
(VE) (VIII)
The central theme that the apoprotein facilitates the scission of the
0-0 bond is based on the established mechanisms of peroxide heterolysis
(165).By invoking "concerted" proton transfer (s) in the transition state,
such schemes illustrate that oxygen-oxygen heterolysis need not be at-
tended by an electrostatically unfavorable charge separation. I n addition,
they offer some rationale for the observed high entropy of activation in
the primary H,O,-catalase reaction (-AS* 25 cal mole-' deg-I) (166).
N
This should be the case in a rigid lattice of interactions implied in Eq.

(20) and formulas (VII) and (VIII).
All such suggestions are clearly most tentative since the nature of the
oxidation product, Compound I, is still unresolved. The stoichiometry
of the enzyme-peroxide reaction merely demands that its formal oxida-
165. L. Bateman and K. R. Hargrave, Proc. Roy. Soc., Ser. A 224,339 (1954).
166. G. K. Strother and E. Ackerman, BBA 47,317 (1960).
tion state be Fe(V), i.e., two oxidation equivalents above the native en-
zyme, Fe (111).Such an assignment tallies well with the magnetic suscep-
tibility for a compound with three unpaired electrons (6000-6500 X lo-’’
emu) (166) but does not uniquely define their distribution. For example,
assuming that the oxidation is confined to the prosthetic group, other
structures compatible with the magnetic susceptibility data can be ex-
pressed as a radical combined with Fe(1V) or as a diradical in conjunc-
tion with low-spin Fe (111).None of these structures singularly reflects
the nature of Compound I but, as emphasized by Hamilton (167),all
in varying degrees could contribute to its resonance form, for the term
“oxidation state” has little chemical significance in compounds with a
substantial covalent character, as evident in coordination compounds
with delocalized ground states such as metal dithienes (168, 169) and
metal-nitric oxide complexes (170).
In Compound I, such polarization interactions should involve an exten-
sive delocalization of electrons from the porphyrin toward the metal ion;
an extreme case of which is a porphyrin-r-cation radical combined with
Fe(1V) as shown in Eq. (21),
where X.6+denotes a radical moiety.

Pertinent to this discussion are recent studies on porphyrin-r-cation
radicals derived from magnesium and cobaltic oct.aethy1 porphyrins (171,
172),and zinc and magnesium tetraphenyl porphyrins (171).In all cases,
the optical spectra of such radicals share features also found in Com-
pound I (Fig. 7). These are (a) a decrease of r-r” transitions associated
with the Soret band and (b) the appearance of bands between 600 and
700 nm. The chief objection to the proposal implicating “a free radical”
moiety stems from the absence of a distinct E P R signature for Compound
I. This is not an overriding restriction since, conceivably, an electron
localized on the porphyrin will couple through exchange interactions with
spin localized on the metal (173),resulting in broadening of the EPR
signal beyond the limit of detection.
167. G. A. Hamilton, Advnn. Enzymol. 32,55 (1969).

168. G. N. Schrauzer, Accounts Chem. Res. 2,72 (1969).
169. J. A. McCleverty, Progr. Znorg. Chem. 10, 49 (1968).
170. J. A. Lewis, Sci. Progr. (London) 46,506 (1959).
171. J. H. Fuhrhop and D. Mauzerall, JACS 90,3875 (1968).
172. R. H. Felton, D. Dolphin, D. C. Borg, and J. Fajer, JACS 91, 196 (1969).
173. D. Dolphin, A. Forman, D. C. Borg, J. Fajer, and R. H. Felton, Proc. Nut.
Acud. Sci. U.S. 68, 614 (1971).
397
120’
100-
7 80-
E -
-
‘I60-
5 40:
20-
OJ I ‘ 0
350 400 450 500 550 600 650 700
A (nm)
FIG.7. Spectrum of horse erythrocyte catalase (---) and its peracetic acid deriva-
tive (-) (Compound I) ; 0.08 M phosphate, pH 7.23, 25” ( 1 0 1 ~ ) .
Among other factors contributing to electron delocalizations, the influ-

ence of environment, particularly that of the trans ligands, must also
be taken into account. Ideally, such ligands should be polarizable but
not readily oxidizable. An imidazole group a t L, could fulfill such a func-
tion and, as L,, the oxygen anion would be appropriate because of its
ability to enter into u-type coordination and x-donor bonding.
The nature of Compound I being still unresolved, it is not surprising
that the mechanisms of its reduction have been variously expressed as:
transfer of oxygen from Compound I (EO) to the reductant (HXOH)
followed by the rearrangement of the “hydroxylated” intermediate (118)
[Eq.
hydride transfer (17,18,75,174,175) [Eq. (2311
EO + XHOH +
outer sphere electron exchange (17.9)

c .I -
EO HiCQ-,, E(H,O) + XO (23)
inner sphere electron transfer (17,159~4167) [Eq. (24)]
EO + HXOH + [ - E+-X
p---H
(/] E(H,O) + XO
(24)
174. L. L. Ingraham, “Biochemical Mechanisms,” p. 71. Wiley, New York, 1962.

175. P. Nicholls, BJ 00, 331 (1964).
17mM CH,OOH
421-480nm
I.3pM CH,OOH
FIG.8. Reduction of horse erythrocyte catalase Compound I by methyl hydrogen
-
peroxide; at pH 7, 4". Note also the ready oxidation of Compound I1 to Compound
I11 by H201where knpp 3 x 1oJ M-'sec-' (164).
The reactions outlined in Eqs.. (22)-(24) are widely recognized in

"model" redox systems. Their relevance to the catalase-mediated oxida-
tions is the central issue-the subject of a lively current debate.
I n this context, the following criteria and guidelines must be
considered :
1. The HXOH donors (hydrogen peroxide, hydroxylamine, formic acid,
and alcohols) and nitrous acid reduce Compound I to ferricatalase with-
out detectable participation of Compound I1 (176). Accordingly, the
reaction occurs either via two-electron equivalent reductions, or the rate
of Compound I1 formation is smaller than the rate of its reduction (17'7,
178) [Eq. (25)].
Compound I + HXOH +[Compound I1 XOH] +catalase + XO (25)
slow fast
If so, then Compound I1 should be reducible by radicals derived from

HXOH (e.g., HOz' and NO,'). Contrary to these expectations, Nicholls
(1'79) failed t o adduce evidence indicating reduction of Compound I1
by NO,'; and Compound I1 is the dominant derivative in the presence of
reagents which generate superoxide. Apparently, as in the corresponding
peroxidase reactions (180) catalase Compound I1 is not readily reducible
by an externally generated superoxide. These observations, although not
entirely conclusive, do bring into question the scheme of Eq. (25) but
pertain only to compound I-HXOH interactions. With other substrates,
for example, with hydroperoxides (16.4) (Fig. S), phenols (178), or oximes
(101a) one-electron equivalent reductions of Compound I do occur.
2. No pH dependence is apparent in EO--(HXOH) reactions, when
ionization state of the reductant is taken into account (36)(Fig. 9). All
176. B. Chance and D. Herbert, BJ 4,402 (1950).

177. D. Keilin and P. Nicholls, BBA 29, 302 (1958).
178. P. George, BJ 52, XIX (1952).
179. P. Nicholls, BBA 81, 479 (1963).
180. B. H. Bielski, D. A . Comstock, A. Haber, and P. C. Chan, BBA 350, 113
(1974).
7. CATALASE 399
4 6 tb 15' 0
PH
Fro. 9. Effect of pH upon activity of Compound I toward hydrogen donors (38).
reactions conform t o the rate law

+
d(EO)/dt = ~ ~ ' ( E O ) ( X T ) / [(Ka/H+)I
~ (26)
where XT is the total concentration of the reductant, Ka the ionization
constant [Ka = (H+)(HXO-)/HXOH], and k:/[l +
Ka/H+] = k4 app.
Thus, as in the reactions of the resting enzyme with ligands, only the
un-ionized donor interacts with Compound I. Yet, following the formation
of a n outer sphere complex, EO(HXOH), ionization of the substrate is a
likely prerequisite t o HXOH oxidation. The point a t issue is illustrated
an excellent donor (k:Tpp

inert ( 1 0 1 ~ )Similarly,
.
-
by the relative reactivities of formic acid and formamide: the former is
9 X 105 IT-' s-l ) (36) and the latter is
N-methyl hydroxylamine is a reductant compara-
ble t o ethanol but 0-methyl hydroxylamine is relatively inactive ( 1 0 1 ~ ) .
It is also a much weaker acid.
Donor CHsCHZOH CHSNHOH CH30NHz
k:',",,(M-' sec-1) 1000 350 < 10
3. The reactivity pattern ( k 4 a p p : CHaCH20H> CHaNHOH >
CH30NH2) would not be expected if either exchangeability of X--H
hydrogen or nitrogen nucleophilicity were of importance in governing
k d a p p , and the results are not peculiar to methoxy amine. I n general,
amines are not effective Compound I reductants (k!c;p <0.2 Ri1-l sec-' a t
25") and unlike in nonenzymic reactions (181-183), attempts to demon-
strate hydroxylations of R N H 2 (R = H, CHa) have so far proved unsuc-
cessful (118b). Nor is the failure to effect hydroxylations limited to
amines. Thus, reduction of Compound I in the presence of cyanide does
not result in the formation of cyanate (118b) and thiols, which are supe-
rior to alcohols as reductants of peroxides, are not the preferred donors
181. W. R. Dunstan and E. Goulding, J. Chem. Soc., London 75, 1005 (1899).
182. I(.M. Ibne-Rasa and J. 0. Edwards, JACS 84, 763 (1962).
183. S. N . Lewis, in "Oxidations" (R. L. Augustine, ed.), 1701. I, p. 213. Dekker,
New York, 1969.
in catalase-mediated oxidations (146):

Donor CHaCHnOH CHaCHnSH
kis&, (M-I 8ec-I) 1000 <70
Hence, barring the formation of EO (RSH) complex in a rate-determining
step, the nucleophilic attack by sulfur at EO oxygen, or transfer of hydro-
gen atom from S-H, does not appear to be the preferred reaction path-
way, Further, the reduction of Compound I by aaide does not conform
to the stoichiometric mechanism [Eq. (27) J (143, 176).
EO + HNa +I+E(N0-) + N, + H+ (27)
Nitrous oxide, and not nitrogen, is one of the major products (143). The
reaction sequence
HNa
EO
N=N=N--OH .--) Nn + NO- + H+
2 NO-
2 €I+
NzO + HzO
is thus precluded. Otherwise, Nz should be the dominant product.
Only in the coupled oxidation of nitrite using glucose oxidase-glucose-
l8OZ (as H1sOISOHgenerating system) and beef liver catalase, which
results in the formation of isotopically enriched nitrate (183u),are the
results compatible with an oxygen atom transfer mechanism [Eqs. (28)
and (29) 1. Even in this case, alternative interpretations are possible, such
as oxidation of nitrite via outer or inner sphere electron transfers [Eqs.
(30) and (31) ] followed by, or concurrent with, fast solvation:
~ 4 8 0 + HNO, -L 6+ 1
E"0 (NO,)
HBl H,O
-E(H,O) + '%NO,- + 'H
(30)
where B denotes a base.

183a. The degree of '*O incorporation is variable, but contrary to the preliminary
report (184) it is not marginal.
184. R. J. Olcott and G . R. Schonbaum, Fed. Proc., Fed. Amer. SOC.Ezp. Biol.
33, 1245 (No. 119) (1974).
7. CATALASE 401
TABLE IX
REDUCTION 10B Y SATURATED
O F COMPOUND PRIMARY
ALCOHOLS
Y-CHzOH
Y
~~ ~~
OH CH3 H FCHz ClCHa CF3 CHZOH
k, (M-l sec-1) 1200 1020 830 470 95 32 26

v Y ~ (La) 13.4 22.7 5.7 26.9 36.8 35.3 30.4
PK. - 15.9 15.1 - 14.3 12.4 14.8
Horse erythrocyte catalase; 5 m M phosphate, pH 7, 25".

bvan der Waals volumes of Y (186, 186) [Chance (56) and White and Schonbaum
(118b)l.
Little evidence is available, therefore, that reductants induce redistri-

bution of electronic charge within the prosthetic group in such a manner
that oxygen of EO becomes the locus of the nucleophilic attack. The best
reductants of Compound I are "hard" donors rather than polarizable sub-
strates (cyanide, thiols, carbon monoxide, and isonitriles)-observations
which cannot be readily reconciled with the "hydroxylation" mechanisms
(118) outlined in Eqs. (22), (32), and (33):
EO + "'" 0-
[ :Hy>m]- EO-N
E(H,O) + HN Y N * N
NJ
(32)
- 1- EO + H,O, E05O@
[H
E(H,O) + 0, (33)
4. The data on alcohol oxidation further underscore the difficulties of

mechanistic interpretation [Table IX (35, 118b, 185, 186) and Table X
(118b)1. Thus, although k,, values for the reduction of horse erythro-
cyte catalase Compound I by ethanol, n-propanol, and n-butanol (Table
X) support the classic view (35) that steric (volume) constraints influ-
ence the reaction rates, other effects became apparent on comparing the
rates of oxidation of methyl, n-propyl, allyl, and propargyl alcohols
(118b) (Tables IX and X ) . The latter is clearly a superior reductant,
not only relative to other three-carbon alcohols but also compared to
methanol. An inductive (electron withdrawing) effect of the acetylenic
group may be of importance here but hardly dominant since 2-fluoro-,
2-chloro-, and 2,2,2-trifluoromethyl ethanols are by no means the prefer-
185. J. T. Edwards, J. Chem. Educ. 47, 261 (1970).
186. A . Bondi, J. Phys. Chem. 68, 441 (1964).
TABLE X
OF COMPOUND Ia BY
REDUCTION TWO,THREE,A N D FOURC.4RBON ALCOHOLS
Alcohol
Ethyl Propyl Butyl Ally1 Propargyl
vRb 0.76 1.0 1.24 0.91 0.90

kc llpp (M-l sec-l) 1020 6.5 0.4 330 2500
PK. 15.9 16.1 16.1 15.5 13.6
0 Horse erythrocyte catalase; 5 mM phosphate, pH 7, 25' [White and Schonbaum
(118b)l.
* VR denotes van der Waals volumes relative to V R = 1 for propyl.
TABLE XI
STEREOSPECIFICITY
OF ETHANOLOXIDATION
I N BEEF LIVER
CATALASE-MEDIATED
REACTIONS(PRODUCTANALYSES)
Products"
CHaCHO CHsCDO
Substrate (%I (%)
S-( -)-Ethanol-1-d 0 100

Ethanol-1-d (racemic) 43k3 57f3
aFollowing approximately 65 f 10% of the reaction [Schonhaum (169)].
red donors (Table IX) . Accordingly, neither the steric nor electronic fac-
tors are, per se, adequate in accounting for the variation of k, app values,
but their interplay as well as the residence time of alcohol in an EO
(ROH) complex should be taken into account.
That orientation effects are important is particularly well illustrated
in the case of ethanol. Its oxidation occurs stereospecifically (Table XI)
(169,187,188) and involves loss of the pro-R hydrogen (188).
H,C *
- H,C-C9
iI'
0
*
(+2H+)
(34)
S-(-)-1-H*-ethanol
To account for other results in Tables IX, X, and XII, it couid be
argued that significant interactions in the EO (HXOH) complex would
187. H. Gang, A. I. Cederbaum, and E. Rubin, BBRC 54,264 (1973).
188. R. J. M. Corrall, H. M. Rodman, J. Margolis, and B. R. Landau, JBC 249,
3181 (1974).
7. CATALASE 403
I
'6 -i -i -j -i -5 -6 -+
TAS.(kcdmle-' 1
FIG.10. Enthalpy-entropy compensations in reductions of Compound I by hydrogen

donors: (1) 2-fluoroethanol, (2) ethanol, (3) ally1 alcohol, (4) methanol, (5) pro-
pargyl alcohol, (6) hydrogen peroxide, and (7) formic acid. Horse erythrocyte cata-
lase in 5 mM phosphate, pH 7 ( I I H J ] .
result in a more negative entropy of activation and that given a proper

orientation of the reactants the enthalpy barrier would be lower. Such
an entropy-enthalpy correlation is discernible in the oxidation of several
alcohols (Table XIII) but cannot be extended to oxidations of hydrogen
peroxide or formic acid (Fig. 10). This is not entirely unexpected since
the observed activation parameters reflect the sum of the standard quan-
tities pertaining to the equilibrium between Compound I and the
reductant
EO + HXOH+ EO(HX0H) AFo = -RT In K., (35)
and the activation quantities for the transformation of EO(HX0H) t o

the transition state. Moreover, different reaction steps appear t o be rate
limiting in the Compound I-donor interactions. Thus, significant iso-
TABLE XI1
REDUCTION OF COMPOUND 1 B Y SOME " T W O ELF:CTRON I)ONORS''''
Y-OH
OH N=O HC=O N Hz
k4 npp 1 . 8 x 107 1.2 x 107 9 x 106 24x 104

PKa 11.6 3.3 3.75 > 12
Data from Chance (36), Chance and Herbert (176), and Schonbaum (10Ia).
TABLE XI11
THERMODYNAMIC
APPARENT ACTIVATIONPARAMETERS FOR SOME
CATALASE COMPOUND I-MEDIATEDOXIDATIONS'
AHS ASS
Donor (kcal mole-') (cal mole-' deg-1)
Methanol 8.4 -17.4

Trideuteromethanol 9.3 -17.6
Ethanol 10.8 -8.9
1,1-Dideuteroethanol 11.1 -9.1
Ally1 alcohol 9.3 -10.2
2-Propy n-l-ol 0.4 -21.9
0 Data from White and Schonbaum (118b).
tope effects, which are principally attributable to AH* (Table XIII)

obtain in the oxidation of 1-deutero alcohols (CDaOH and CHaCD20H)
but not in the oxidation of deuteroformate (DC02H) (Tables XIV and
XV) or deuteroethanol (CHaCH20D)(Table XVI). In particular, the
large differences in k4 values and kH/kD ratios for formate and metha-
nol oxidations cannot be simply attributed to steric constraints. Rather
for formate, the formation of the Compound I-donor complex must be
assumed to be rate determining. This being the case no isotope effect is
expected; indeed, kFapp and k?,,, are nearly equal ( k H / k ~ 1.1 f 0.05) -
of formic acid with the resting enzyme (kl
with Compound I (k4
-
(Table XIV). Note also that rate constants characterizing the interaction
.,,- loe M-* sec-I) (37) and

0.9 X 106 M-1 sec-1) are nearly the same.
Apparently, the active site is equally accessible to substrates in different
TABLE XIV
ISOTOPE EFFECTSI N BEEF LNER CATALASE-MEDIATED
OXIDATIONS'
Formic acid Ethanol Methanol
Substrate HCOzH DCOpH CHaCHzOH CH,CDzOH CHsOH CDIOH
k4 sppo (M-1 sec-1) 486 400 890 455 720 130

10-6 k4'b 8.0s 7.64 - -
(M-1 sec-1)
kdkn 1.16 1.96 5.54
In 10 mM potassium phosphate, pH 7, 24.7'.

(I
For un-ionized form using pK.(HC02H) 3.75; pK.(DC02H) 3.78 [White and
Schonbaum (118b)l.
7. CATALASE 405
TABLE XV
ISOTOPE EFFECTS
I N CATALASE-MEDIATED
OXIDATIONS O F
A N D DEUTEROETHANOLS~
ETHANOL
Rate constant (M-l sec-l)
Horse
Substrate erythrocyte Beef liver &I. lysodeikticus
Ethanol 1020 f 20 890 f 20 21.5 f 0 . 5

S-(-)-1JDeuteroethanol 1020 f 20 20.0 f 0 . 2
1,l-Dideuteroethanol 460 f 10 455 f 10 *
14.2 0 . 3
-
Pentadeuteroethanol - 445 f 10
kcE,c~,on/kcHIcDtoH 2.22 1.98 1.51
a I n 10 m M potassium phosphate, p H 7, 24.7" [Schonbaum (lola)].
oxidation states of the enzyme. The simplest permissible reaction scheme

is therefore
EO + H X O H kk-il ' E O ( X H 0 H ) 2 E(H20) + XO (36)
and
Hence, if k ) > k-l, then k 4 k l . Seemingly, such conditions are met

in the Compound I-formic acid reaction.
I n contrast, since in the oxidation of methanol kH/kD > 5 , scission of
the C-H bond must be rate limiting, suggesting that k t < k-1, and
k l a p p = ( k l k z ) / k _ , where k l a p p 830 M-l sec-'l (Table IX).
N
TABLE XVI
SOLVENT ISOTOPE
EFFECTSI N HoRSle ERYTHROCYTE-MI.:DIATED
OXIDATION OF ETHANOL
Solvent kc npp (M-I sec-l)
H20n
90 % D20-10 % H i 0 (v/v)*
*
1020 20
960 k 30
knlo/kDno 1.06
I n 10 m M potassium phosphate, p H 7.0.

In 10 m M potassium phosphate, pD 7.1; 24.7' [White and Schonbaum ( l l 8 b ) l .
Therefore, k2/k-1 = 8 X 10-4, provided that as in the reactions of

catalase with methyl hydrogen peroxide (Table VIII) or with formic
acid (37), kl- 106 M-' sec-1. Further, since even a t 2 mM MeOH, the
kinetics of Compound I reduction by methanol obey the second-order
rate law ( I & ) , it follows that k-l/lcl 2 20 m M . Hence, k-1 2 2 X lo'sec-'
and kt 2 16 sec-'.
The above analysis, which merely indicates the possible lower limits
of k-1 Llpp and kz app, presupposes the formation of one EO(HX0H) inter-
mediate in the course of the redox reaction.
On this score definitive evidence is lacking. Even this minimum postu-
late may be inadequate to account for the observation that in reactions
of catalase with methyl or butyl hydroperoxides less than 15% of the
initial peroxide is converted into the corresponding aldehydes unlike the
catalase-ethyl hydrogen peroxide system where formation of acetal-
dehyde from EtOOH exceeds 50% (Table V I I ) . I n the reactmionwith
MeOOH, the low yield of formaldehyde could be attributed to a rapid
diffusion of methanol from the active site:
EO(HOCH*) %EO + CH,OH (38)

Conversely, high conversion of EtOOH to CH,CHO would result from
slower diffusion of ethanol from EO (HOC,H,) ; accordingly, the yield
of butyraldehyde from BuOOH should be a t least equally high. Since
this is not so, we can only conclude that, compared to ethanol, either
the residence time of butanol a t the active site is unaccountahly shorter
or that prior to oxidation of ROH, the outer sphere complex, TO (HOR b ,
rearranges to another intermediate ; an intermediate having a configura-
tion in which the slowly reacting alcohols (Tables IX and X) cannot
be readily accommodated. An outer sphere complex differing from
EO(HOR), the possible initial product of E ( R O 0 H ) reaction, or an inner
sphere complex [Eq. (24) ] could meet such criteria.
However, no distinctions can be even attempted a t IUP,S(i i r We shall
merely note the following:
(a) Lack of isotope effect in the oxidation of C H,OD (Table XV) is
inconsistent with the reaction pathway outlined in Ey. (23) ;
(b) Formation of an inner sphere complex would be subject to a strin-
gent steric control ;
(c) Significant displacement of metal ion frorn the porphyrin plane,. or
doming of the porphyrin macrocycle should be prerequisites to an inner
sphere ligation ;
(d) Addition a t iron, if any, cannot be random; otlierwise oxidations
would not proceed stereospecifically (Table XI) ;
7. CATALASE 407
(e) Oxidations proceeding by an inner sphere mechanism are formally

analogous to various metal-catalyzed reactions, among others by Cr (VI)
(189, 190), Pb(OAc), (191-193), or V ( V ) (194-196), and could be ex-
pressed as suggested by Taqui Khan and Martell (197) [Eqs. (38a)
and (38b)l:
/--
,.o -P0, H (384

OxE-F e y T__ E-Fe- + 0, + OH-
’“OH
where OxE is a two-electron oxidized form of the active site, or as out-

lined in Eqs. (39)-(41) and Fig. 11.
A reminder that the above suggestions are no more than working hy-
potheses should hardly be necessary.
189. K. B. Wiberg, in “Oxidations in Organic Chemistry” (K. B. Wiberg, ed.),
Part A, p. 69. Academic Press, New York, 1965.
190. J. K. Beattie and G. P. Haight, Jr., i n “Inorganic Reaction Mechanisms”
J. 0. Edwards, ed., Vol. 17, Part 11, p. 93. Wiley (Interscience), New York, 1972.
191. K. Heusler and J. Kolvoda, Angew. Chem., Int. Ed. Engl. 3, 525 (1964).
192. Y. Pocker and B. C. Davis, JACS 95,6216 (1973).
193. R. Criegee, in “Oxidations in Organic Chemistry” (X. B. Wiberg, ed.), Part
A, p. 277. Academic Press, New York, 1965.
194. K. Kustin and D. L. Toppen, Inorg. Chem. 12, 1404 (1973).
195. J. S. Littler and W. W. Waters, J . Chem. Soc., London p. 2767 (1960).
196. J. S. Littler, A. I. Mallet, and W. W. Waters, J. Chem. SOC.,London p . 2761
(1960).
197. M. M. Taqui Khan and A. E. Martell, “Homogeneous Catalysis by Metal
Complexes,” Vol. I, pp 139-142. Academic Press, New York, 1974.
ACKNOWLEDGMENTS
Research in the authors' laboratories and preparation of this review were supported
by USPHS GM-12202, AA-00292 and HL-16061 (to B.C.),by the Medical Research
Council of Canada (MT-12701, and by NSF-GB 41635 (to G.R.S.)
Author Index
Numbers in parentheses are reference numbers and indicate that an author’s work
is referred to, although his name is not cited in the text.
A Alm, R., 70, 88(130)

Alonso, C., 65
Abacherli, E., 26 Alpert, Y., 317, 318(109)
Abatwrov, L. B., 14, 31(56) Altschul, A. M., 345, 346(1, 2), 347(1, 21,
Abe, K., 367, 368, 393(69), 394(69) 348(1, 21, 353(1)
Abeles, R. H., 143 Amelunxen, R. E., 2(17), 3, 19(17)
Abney, R., 133 Anan, F., 367,368,393(69), 394(69)
Abraham, R. G., 274,275(344) Anderson, B. M., 30
Abrams, R., 345, 346(1, 21, 347(1, 21, 348 Anderson, L., 256
(1, 2), 353(1, 2) Andrkasson, L. E., 317, 336(108), 336(108)
Abrams, W. R., 279, 280 Andreev, V. P., 300
Ackerman, E., 395 Andreeva, N. S., 9
Ackrell, B. A. C., 177, 245, 247, 248(26), Andreoli, T. E., 70, 76(127), 77(127)
24906, 1991, 253(184) Andrews, E. C., 313
Adams, M. J., 10, 24 Andrews, L. J., 384
Adelson, G. I., 105 Anfinsen, C. B., 86, 132
Adija, D. L., 29 Anselme, J. P., 379
Aebi, H., 365, 367,379(63), 280(63) Antonini, E., 335, 336(192), 349
Agar, N. S., 142 Appell, G., 366, 367(41), 370(41), 371(41)
Agatova, A. I., 25 Appleby, C. A, 264
Agner, K., 366, 385 Appleman, D., 376
Agrawal, B. B. L., 377 Archakov, A. I., 153
Aikawa, T., 109 Arigoni, D., 252
Akagi, J. M., 286, 295(388) Arnold, H., 48
Akeson, A., 200 Arnold, L. J., 29
Alben, J. O., 317, 321(113), 322(113), 323 Arnon, D. I., 54, 55(12), 66(12), 66(12),
(1131, 337(113) 366
Alberty, R. A., 188, 189(57), 199(57) Arosio, P., 246, 247(191)
Albracht, S. P. J., 187 Arrigoni, O., 237
Alexander, A. G., 300 Arscott, L. D., 92, 93, 94(36), 100(36, 371,
Alifano, A., 73 103(36, 63), lOQ(61,6 3 , 105(85), 106
Alimov, G. A,, 153 (36), 107(36), 118(63), 119(63, 85),
Allen, G. A., 23,24(85), 34 120(61, 63), 121(63), 123(61), 129, 135
Allgyer, T. T., 110 (601, 136(60), 137(60), 143(36), 144
Allison, W. S., 2(15), 3, 20,21(66), 28(15), (36)
30, 39 Artavanis, S., 5
409
410 AUTHOR INDEX
Arvy, L., 300 Barlow, C. H., 314, 315(93), 319, 321, 322
Asada, K., 274,286(359), 295(359) (1421, 323(140), 329(93), 334(94), 337
Asahi, T., 91, 133(7), 143(7), 279 (117, 118), 338(94), 339(117,118), 365,
Asakura, T., 346,347,348(27, 28,29, 30, 31, 368, 389
32, 37), 349(28, 30, 321, 360(27, 31) Baron, J., 150
Asano, A., 66, 72(89), 73(89), 76(89) Barrett, J., 301
Asnis, R. E., 91 Barldn, E. S. G., 261
Asriyants, R. A., 48 Barry, R. E., 164
Asyis, R. Aat., 48 Bartlett, G. R., 261
Atchison, R. W., 54, 56(18), 57(18), 58 Bartsch, R. G., 371(84), 372
(18) Basford, R. E., 222
Atkin, C. L., 143 Basolo, F., 375
Atkinson, D. E., 274, 277(339), 286(339) Basu, D. K., 106, 107(98)
Atkinson, M. R., 109 Bateman, L., 395
Auda, B. V., 260 Batke, J., 25, 40
Auts, S. D., 165, 166(369), 167(374), 168 Bathe, G. R., 84
(374), 169(374) Baudhuin, P., 368
Averback, B. C., 274,275(344) Baudras, A., 265, 268(287, 289), 269(287,
Awasthi, Y. C., 312; 313(84) 299, 302)
Assi, A., 72 Bauer, V. A., 143
Azzone, G. F., 67, 72(102), 214, 282 Baugh, R. F., 182, 187, 188, 189, 190(43)
Baum, H., 70
B Bayne, R.. A., 315,317,337(115)
Baccarini-Melandri, A., 2(28), 3 Bearden, A. J., 210
Bach, S. J., 263.264 Beattie, D. S., 81(176), 217
Bachmanova, G. I., 153 Beattie, J. K., 407
Bader, P., 235 Beck, W. S., 143
Baggott, J. P., 33, 34(156), 166, 167(380) Bednars, A. J., 258
Baginsky, M. L., 106, 107(112), 108(112), Beetlestone, J., 346, 372, 375(99)
112(112), 205, 224, 236(156), 243, 244 Behme, M. T. A., 43
(156), 246(156) Beinert, H., 100, 101(69), 179, 184(34), 185
Bailey, K., 3 (34, 46), 186(46), 187(34,46), 193(46),
Bakker, E. P., 315, 316(102), 320(102), 205, 214, 215(46, 541, 216(116), 219,
321(102) 220(136), 221(46, 1361, 226(218), 235,
Balastero, F., 237 244, 245, 253, 297, 307, 309, 316(66),
Baldesten, A., 93, 143(46), 144(46, 275) 320, 321, 323, 330, 331(139), 332(126,
Balegh, M. S., 370 1391, 333(139), 335(66, 126), 336(66,
Balthasar, W., 31 194)
Baltscheffsky, H., 74, 254, 256(220) Bell, J. J., 65, 83(84), 84(84)
Baltscheffsky, M., 74, 254,256(220) Benjamin, B. M, 39, 45(181)
Banaszak, J. J., 10, 11(51), 12(51) Benohr, H. C., 130,131
Banaszak, L. J., 9, 10, 35, 317, 318(112) Berger, A., 379
Bandi, L., 83 Berger, T. J., 68,71(115), 79(115)
Bandurski, R. S., 91, 133(7), 143(7), 274, Berghauser, J., 24
279, 286(359), 295(359) Berglund, O., 143
Banerjee, R., 317, 318(109) Bernath, P., 222, 223(144), 225(144), 226,
Baranowski, T., 9 271, 273
Barea, J. L., 274 Bernhard, S., 33, 34, 35(160), 36(160), 37
Barela, T. D., 15 (174, 175), 41(160), 42(165), 48(165),
Barker, H. A., 143 49(165)
AUTHOR INDEX 411
Bernheim,. F., 263 (74,79,81),374(81), 376(79), 385(16),

Betheil, J. J., 31,34(137) 389(16), 390,396(156)
Betz, G., 150 Brocklehurst, E. S., 321,322(138), 326(138
Beutler, E.,131,132(212),132 327(138),331(138), 333(138), 335(138)
Beychok, S.,123, 125(168) Brodie, A. F., 64, 65, 66(60), 68(60), 72
Bianchi, G.,262 (60)
Bielski, B. H., 398 Brodie, J. D.,237
Biggs, D. R , 217 Bronk, J. R.,62,65(34), 67(34)
Bilimoria, M. H.,167, 168(384, 386), 170 Brosemer, R. W., 2(23), 3
(386) Brosnan, J. T., 82,86(186)
Bittmann, R.,31 Brown, J. P.,104,118(86),119(86), 120,121
Bjorkhem, I., 83 (86)
Black, S.,91,133, 141, 143(6) Brown, N. C., 142. 143(262)
Blair, P.V.,237 Brown, S.B., 389
Blakley, R. L.,143 Brownie, A. C., 83,84(192)
Bloch, W., 34,42(165),48(165),49(165) Brumby, P. E.,114, 122(155), 123(155)
Blumberg, W. E.,368, 369, 370(77, 78), Bruni, A., 239,244,246
376(77), 380,381(126b), 390(126b) Brunori, M.,315, 316(99), 323(99), 324
Bock, R. M.,310 (99),335, 336(99, 192), 389
Boger, P.,54, 62 Bryla, J., 110
Boeri, E.,264, 270, 272 Bucher, T., 81
Boers, W., 28,33 Buege, J. A., 165, 166(369, 3741, 167(374),
Bolotina, I. A., 25, 31(105) 168(374),169(374)
Bond, J. C, 24 Buehner, M., 9,10(46), ll(46, 48),24(47),
Bondi, A., 401 29(47), 39(47, 48), 44(46, 47)
Bonner, W.D., Jr., 215 Bulger, J. E.,94, 112(52, 53), 113(52, 53),
Bonnichsen, R. K.,366 114(53),135(52,531, 137(53), 139(53),
Booth, F.W.,300 141 (53)
Borg, D. C., 356,396,397(173) Burgoyne, L. A.,267
Boross, L., 28, 43 Burleigh, B. D., Jr., 92, 100(33), 103(33),
Borst, P.,80 104(33,61), 105(33), 119(33), 120(61),
Bossi, E., 365 123(61)
Bourne, G.H., 300 Burma, D. P., 106,107(98)
Boxer, G.E.,47 Butler, W. L.,239,240(178)
Boyd, G.S.,83 Butlin, J . D., 68,78,79
Boyer, P.D., 39,40,74, 75, 101 Butow, R.A.,186,203(48)
Braams, R.,335 Buzard, J. A.,129
Brady, A. H.,123,125(168)
Brady, W. T.,93 C
Bragg, P. D., 64,68(62), 72(62), 79, 80
Bramlett, R.,284,285(382),297 Caifa, P., 247
Branden, C. I., 10,11(51),12(51) Caldwell, B. V.,84,88(225)
Brandt, K.G.,94,112(52,53),113(52,53), Camerino, P. W.,320, 333(131)
114(53), 135(52,53),137(53), 139(53), Cameron, B. F.,372,373(96)
141(53) Cammer, W., 65,84(83)
Branzoli, U.,101 Canellakis, Z.N..142,143(262)
Bresters, T.W., 106, 107(114),114(114) Cantz, M.,365, 367,379(63), 380(63)
Bridgen, J., 5,22 Capaldi, R. A., 180, 311, 312(75)
Brierley, G.,180,312,313(85) Capeillere, C., 268, 269(299)
Brill, A. S.,351, 365,369, 370(74, 791,372. Caputo, A., 349
412 AUTHOR INDEX
Caputto, R., 2,3(7) Christian, W., 2

Cardenas, J., 274 Chuang, T.F.,312,313034)
Carlson, C. W.,2(23), 3 Chung, A. E., 53, 55, 56(10, 18), 57(10,
Carpenter, E.,312 181, 58(10, 181, 60
Cam, M.L.,150 Chung, C. W., 274
Carr, N. G.,2(27), 3 Cilento, G.,28
Carraway, K.L., 48 Ciotti, M.M.,30, 52, 53(1), 54(1), 56(l),
Carroll, W.R.,10s 58(1, 31, 62(1), 65(31), 86(75)
Casida, J. E.,177, 204, 205(22, 881, 206 Claisse, M., 269
(22) Clark, W.M.,130
Casola, L., 112(150), 113, 114(150), 122 Clegg, R.A.,217,219(131), 220(135)
(150, 155), 123(155) Cleland, W.W.,40, 76, 88, 139
Caswell, A. H.,215 Click, E. M.,288
Caughey, W. S., 302, 304, 305(40), 306 Clodfelder, P.,39,45(185)
(52), 307, 308(52), 313(52), 314(60, Cobley, J. G.,219,220(136), 221(136)
62), 315(52, 93), 316, 317(52, 651, 318 Cochran, D.G.,258,259034)
(52, 65), 319, 321(113, ll6), 322(62, Cocriamont, C., 347
65, 113, 114, 115, 1421, 323(65, 113, Cohen, B. S.,151, 152(318, 320), 153
114, l40), 325(65), 328(65), 329(65, Cohen, I. A., 302, 304, 338(27)
93), 330(104), 334(60), 337(15, 113, Cohen, P. T.,53, 54, 56(17), 57(6), 58, 59
115, 117, 118),338(27, 40, 80,62, 651, (17), 60, 61
339(117, 1181, 340(116), 343(60, 621, Cohn, M. L., 100, 103(65), lOQ(65)
364, 389 Coleman, R.,180, 181(41)
Cavallini, D., 304 Coles, C. J., 225, 226, 230(158), 234(15@,
Cederbaum, A. I., 81(176), 402 248(158)
Cepure, A., 101 Colli, W.,88
Cerletti, P.,222, 225(150), 230(157), 231, Collins, D. M., 376
234, 237, 246, 247(191), 248 Collipp, P.J., 216
Challoner, D. R., 300 Colman, R. F.,141
Chamalaun, R.A. F. M.,82 Colowick, S. P.,3, 28, 29(13), 38(13), 39
Chan, P. C., 398 (131,40(13), 44, 52, 53(1), 54, 56(1),
Chan, S. H.P.,310,311(69) 58(1, 2, 3, 5), 62(1 ), 65(30), 67(30),
Chance, B.,31, 72, 204, 207, 215,219,258, 69(30), 70(30), 73(30), 76(30), 167
259(229), 301, 321, 324, 335(21), 336 Comstock, D. A.,398
(1551, 346, 348(22), 351(22), 352(6, Conn, E. E.,92
22),353(4,6, 221, 356(5), 363(5), 364, Conney, A. H.,149
365, 366, 369(1), 372, 374(37), 385 Connors, M.J., 30, 39
(37), 388(37, 52), 388(36), 389, 390 Conover, E.,72
.(150), 391(150), 393(1), 394(1), 398 Constantinides, S. M.,25
(36,1541,399(36), 400(146), 401, 403, Conway, A., 31, 33, 34(143), 42(143)
404(37), 406(37, 146) Cook, D.E.,153
Chang, J. Y.,377,378 Cook, K.A.,274
Chang, 5. H.,133 Coon, M.J., 91, 149, 150(295), 151(295),
Changeaux, J. P.,31 153, 165(286), 166(370, 3711, 167(370,
Chantrenne, H.,347, 348(43) 371), 169
Chappell, J. B.,207 Cooper, A., 371(85), 372
Charache, S., 322 Cooper, D.Y.,83, 91, 152, 166
Charalampous, F. C., 312 Coratelli, P.,88
Chen, W.L.,312 Corcoran, D., 148, 151(284a), 164
Christensen, J. R.,261 Cordes, E. H.,43
AUTHOR INDEX 413
Cori, C. F., 2, 3(8), 48 Danielson, L., 64, 65(56, 57, 58), 67(56,
Cori, G. T., 2, 3(8), 48 57, 58), 68(56, 57, 581, 72(56, 57, 58),
Cori, O., 261 73(57, 581, 74(56, 57, 581, 77(56, 57,
Cornforth, J. W., 252 58)
Corrall, R. J. M., 402 Danielsson, H., 83
Correa, W., 131 Darnall, D. W., 15
Corte, E. D., 132 Davidson, B. E., 2(25), 3, 5,9(33), 20(33)
Coulson, A. F. W., 347, 350, 353(34), 355 Davidson, D. W., 83
(34, 36) Davidson, J. T., 282, 284(377)
Countryman, R., 376 Davies, J. L., 304, 305(40), 338(40)
Cox, C. D., Jr., 274 Davies, P. L., 80
Cox, D. J., 93 Davis, B. C., 407
Cox, G. B., 64,68, 78(73), 79, 81(73) Davis, K. A., 179, 191, 192(71, 73), 193
Crane, F. L., 68, 79, 312, 313(84) (32, 691, 200(69),. 203, 222, 224(141,
Crawford, I. P., 254, 256(220) 142, 143), 225(141, 142, 143), 226(32),
Creaghan, I. T., 110 227, 228, 229, 230(73, 141, 142, 143),
Cremona, T., 69, 78, 177, 184, 188, 189, 231, 232(71, 166), 233, 234, 235(143),
190(58, 841, 202, 203, 270, 271, 272, 236, 237(166), 239,240(166), 241(166),
273 (320) 242(166), 243(166), 244(143), 245
Cresswell, C. F., 274 (143), 253,254,246(143), 248(71,166),
Creutz, C., 375 254, 255, 256(220)
Criddle, R. S., 310 Davis, P. S., 170, 288, 290(413, 414, 4151,
Criegee, R., 407 291(414), 292(413, 415), 294(414)
Crifo, C., 304 Davison, A. J., 324
Cronin, J. R., 259, 260(243), 262(243) Dayhoff, M. O., 105, 371(83), 372
Cross, D. G., 29 Deacon, T. E., 282, 284(377)
Cseke, E., 28, 43 Deal, W. C., Jr., 25, 30(108), 48(108)
Cunningham, L. W., 39, 45(185) DeBernard, B., 155, 189
Cunningham, W., 313 de Duve, C., 365, 367(23), 368
Curdel, A., 272, 273(329) de Haan, E. J., 63, 82, 86(191), 87(53)
Curnyn, C., 66, 72(.87) Deisseroth, A., 365, 385(18), 389(18), 397
Curti, B., 101 (18)
Cusanovich, M. A., 325 De Kok, A., 101, 106, 107(114), 114(114),
Cutolo, E., 264 124, 125(172), 238
Czerlinski, G., 117 De La Chica, G., 133
Czygan, F. C., 274 De Lorenzo, F., 132
De Luca, C., 65
De Luca, H. F., 83
D De Marco, C., 304
Dennis, D. T., 2(26), 3, 39, 40(!26), 45
Dade, E., 46 (188), 48(26)
Dahlen, J. V., 88 De Rosier, D., 126
Daigo, K., 93, 108(43) DerVartanian, D. V., 124, 125(172), 187,
D’Allessio, G., 2(16), 3, 4(16) 221, 222, 224(149), 226, 236(149), 237
Dalling, M. J., 274 (149), 238(149), 251, 252, 253(149),
Dallner, G., 166 281, 284, 285(282), 288, 295(373a)
D’Aloya, R., 63,87(53) Deutsch, H. F., 366,382(39)
Dalziel, K., 139 Devichensky, V. M., 153
Dandliker, W. B., 24 De Vijlder, J. J. M., 31, 32, 33(144, 1511,
Daniel, L. J., 261 34(144), 41(159)
414 AUTHOR INDEX
Devlin, T. M., 62, 65(33) Eichner, R. D., 29

De Wael, J., 131 Eichorn, J., 83
Dewan, J. D., 2 Eigen, M., 31
Diamond, L. S., 66 Eik-Nes, K. B., 83
Dick, A., 259 Eisele, B., 28, 33
Dickerson, R. E., 371(85), 372 Eisenberg, D., 371(851, 372
Diehl, H., 152 Ejima, A., 108
di Franco, A,, 268 Ekstrand, V., 93, 94(40), 138(40), 139
Dixon, M., 2,3(7), 109, 263, 264, 267 (40), 140(40)
Djavadi-Ohaniance, L., 212, 213, 296, 297 Eldjarn, L., 130
Doeg, K. A., 224,236(153), 239 Eley, M. H., 108
Dolphin, D., 356, 396, 397(173) Elias, H. G., 24
Dontsov, A. E., 74,75(148) Ellfolk, N., 347, 348(40, 41, 42)
Dorfman, R. I., 83 Elliott, J., 46
Dorsey, J. A., 88 Elliott, P., 143
Dounce, A. L., 365, 366, 367, 385(18), 389 Elodi, P., 2(14), 3, 23, 25, 26, 31
(18), 397(18) Engel, P. C., 91,100
Drabikowska, A. K., 258 Englard, S., 31, 34(137)
Drabkin, D. L., 300 Eriksson, A., 133
Dragoni, N., 286, 295(389) Eriksson, L. E .G., 235
Dreyfuas, J., 286, 287(387), 288 Eriksson, S. A,, 132
.
Dro t t, H R., 347, 348(32), 349(32), 360 Erman, J. E., 347, 350, 353(34), 355(34),
DuBus, R., 91 372, 376(88)
Due& E., 9, lO(45) Ernest, M. J., 130
Duggleby, R. G., 2(26), 3, 39, 40(26), 45 Ernster, L., 64, 65(56, 57, 58), 67(56, 57,
(188), 4806) 58), 68(56, 57, 581, 68(66), 69(68, 69,
Duncan, H. M., 216 106), 70(59, 67), 72(56, 57, 58, 67),
Duncan, I. W., 93, 94(40), 138(40), 139 73(57, 58, 59, 102, 103, 106, 135, 136,
(40), 140(40) 137), 74(56, 57, 58, 106, 136, 1371, 75
Dunstan, W. R., 399 (67, 68, 69), 76(59, 67, 68,69), 77(56,
Duppel, W., 149, 150(295), 151(295) 57, 58, 59, 67, 68, 69, 106, 135, 136,
Dupre, S., 340 137), 78, 82, 86(191), 87(74, 1841, 88
Durchschlag, H., 32 (129, 130), 166, 168, 204, 207, 212, 214,
Dus, K., 371(84), 372 246, 249(188), 261
Dutton, P. L., 215, 325 Escamilla, E., 133
Estabrook, R. W., 63, 65, 66(37), 81(37),
E 83, 84(83), 86(37), 91, 151, 152(318,
Eberhard, C. A., 106, 107(97) 320, 321), 153, 168, 186, 203(48), 258,
Eberspaecher, H. I., 305 259(233), 261, 262(262), 387
Ebisuzaki, K., 260 Evang, A., 368
Eby, D., 29, 30(126) Eylar, E. H., 317, 318(112)
Edelhoch, H., 101, 189
Edelstein, S. J., 101 F
Edmondson, D., 222
Edwards, J. O., 399 Faeder, E. J., 170, 287, 288, 290/415), 292
Edwards, J. T., 401 (415)
Egan, R. S., 377 Fahien, L. A., 26, 31(110)
Ehrenberg, A., 200, 235, 330, 331(174), Fairs, K., 302
346, 348(22), 351(22), 352(22), 353 Fajer, J., 356, 396, 397(173)
(20,22), 356,387,388,400(143) Feeney, R. E., 2(22), 3, 26(22)
AUTHOR INDEX 415
Fehrniann, H., 106, 107(106), 112(106) Frisell, W. R., 259, 260(243), 262(243)
Feigin, L. A,, 366 Furhs, S.,132
Feinberg, R. H.,262 Fuchsman, W. H., 304, 305(40), 314, 334
Feinstein, R. X., 365 (941,338(40, 94)
Feldberg, N. T.,249 Fuhrhop, J. H., 396
Feldberg, R.,90,282 Fujihara, Y.,320, 325(125), 326(125), 331
Felsenfeld, G., 330 (125),332(125)
Felton, R. H.,356, 396,397(173) Fukuyoski, Y.,108, 109, llO(132)
Felton, S. P.,179, 188(37), 189, 195(63), Furfine, C.,3, 19,31(12), 34(137),40(60),
203(63) 41(60), 42(60), 44(60), 48(60)
Fenselau, A.,23,42,43(202) Furuta, H., 366,367(50)
Ferri, G., 22,44(76),101
Fife, T.H.,39,45(181) G
Filmer, D.,32,33(149)
Fisher, H.F., 29 Caber, B. P., 105
Fisher, R. J., 64, 65, 67, 68(61), 72(61), Gal, E. M.,106, 107(94)
79 Galante, Y.,178, 214, 246, 247(191), 296,
Fisher, R. R., 68, 69(112), 71(109, 110, 297
111, 112), 76, 77(118), 78(109, 110, Gallego, E., 31
111, 112), 207, 213 Gallop, P.M., 379
Fitzgerold, B., 48 Galston, A. W.,366
Flashner, M.I. S., 101 Gang, H., 402
Fleischer, S.,180,312,313 Ganther, H.E.,132
Flohe, L.,130 Garhe, A.,24
Fluharty, A. L.,105 Garland, P. B., 63, 86, 87(52), 88, 204,
Forster, T.,359 217,219(131), 220(135)
Fogo, J. K.,202 Garrett, R. H., 274,275(351),275(350)
Fonzo, D., 84 Garwood, D.C.,119
Forchielli, E.,83 Garwood, D.S.,119
Forcina, B. G., 22,44(76) Gaylor, J. L.,151
Ford, G. C.,9, 10(46), 11(46, 48), 24(47), Gehl, J. M.,54, 59(13), 66(13)
29(47), 39(47, 48), 44(46, 47) George, P.,304, 346, 361% 9), 364, 369,
Forestier, J. P., 268,269(302) 372, 375(99), 389(9), 392
Forman, A,, 396,397(173) Gerari, G., 333
Forti, G., 100 Gerth, E., 48
Foster, R., 384 Ghalambor, M. A., 222, 224(141), 225
Foust, G. P.,90,94,98(50), 146, 147(284). (141), 230(141), 232(166), 236, 237
282 (1661, 240(166), 241(166), 242(166),
Fowler, L. R., 179, 258(33), 306(50), 307, 243(166), 248(166), 253( 166)
308(50) Ghazarian, J. G., 83
Fox, J. B., Jr.. 24 Ghiretti-Magaldi, A.,271, 273
Frampton, V. L.,366,367 Ghisla, S.,97, 235
Francavilla, A,. 63,86(50), 87(54) Gibson, F., 64, 68, 78(73), 79, 81(73)
Francis, S. H., 24,42, 44, 45(90), 48(200) Gibson, Q . H., 92,94(24), 97(24),98(24),
Franklin, M.R.,168 107(24), 109, 111(24), 112(24), 113
Fraser, D.R.,83 (24, 1371, 115(24, 137), 116(24), 136,
Frech. M.E.,62,65(31) 167(245), 168(245, 386), 169(245),
Fredericks, W. W.,54, 59(13). 66(13) 170(245,3861, 172(245,398), 286, 287
Fricke, H.,302 (3941, 290(394), 291(394), 308, 322,
Friedrirh, P.,23 324, 327, 333, 335(153), 336(153)
416 AUTHOR INDEX
Gierisch, W., 367 258(33), 306(45), 307, 308(45, 53),

Gieseman, H., 383 327(53), 330(53)
Gilboa-Garber, N., 286, 287(391) Grinius, L. L., 69,74(120), 75(120, 148)
Gillette, J. R.,153 Groot, G. S.P., 73,312
Gilmour, M. V., 311, 312(78), 324, 335 Grossman, S., 219, 220, 221(136)
Gioeli, R. P.,65 Groudinsky, O.,264, 265, 267
Giordano, M.,237, 247 Gudat, J. C.,275
Giovenco, M.A.,237,247 Guerra, F.,84
Giovenco, S.,247 Guerrero, M. G., 274, 276 (342, 345)
Girotti, A. W.,372 Guest, J. R.,110
Giuditta, A.,246,247(189) Guiard, B.,267, 296
Givol, D.,37 Guillory, R. J., 68, 69(112), 71(109, 110,
Glatzle, D.,131 111, 112, 113, 117), 78(109, 110, 111,
Gleason, F.K.,143 112), 213
Glenn, J. L.,199,260 Guindon, A. H., 106, 107(97)
Goldman, D.S.,106, 107(111) Gumaa, K.A.,46, 47(211), 81
Gonze, J., 63, 186, 203(48), 262 Gunsalus, I. C.,91,92, 106, 107(110, 115),
Goodall, D.,31 lOS(lS), 172
Gordon, M.S.,300 Gupta, R. K.,347, 348(38), 357(38)
Gorjunov, A. I., 9 Gurd, F. R.N., 317, 318(112)
Goto, M.,150 Guseva, M.K.,2(24), 3,25(24), 30(24)
Gottesman, D.P.,110 Gustafsson, J., 83
Goulding, E.,399 Guthenberg, C.,133
GouLian, M.,143 Gutierrez, J., 133
Grab, D.J., 365 Gutman, M.,69, 177, 186(23), 188(19, 23),
200(19), 201(19, 23), 203(19, 231, 204
Gralen, N.,366
(19,23), 205(19, 23,881,206(19), 214,
Grandchamp, S.,217
216(116), 223(23), 224(23), 235(23),
Grande, H. J., 126 236(23), 238(23), 247(20, 231, 248(23,
Grant, J. K.,83,84(192) 24), 249(23, 197), 250(23, 195), 251
Grataer, W.B.,32 252
Gray, H.B.,385,386(137) Gutnick, D. L., 66,79
Gray, R. W.,83
Grebner, D.,310,319 H
Green, A. P.,65
Green, D.E.,2, 75,222,237,256,257(223) Haaker, H., 106, 107(114), 114(114)
Greenbaum, A. L.,46,47(211), 81,82 Haas, E.,167
Greene, J. C.,2(22), 3,26(22) Haavik, A. G., 178, 179(27, 28), 181(28),
Greenfield, R.E.,366 183(28), 184(28), 190(27,28), 224(27),
Greengard, P.,83 245(27), 258(33)
Greenstein, D.S.,388 Haber, A., 398
Greenwood, C., 315, 316(99), 322, 323 Hachimori, A.,366, 367(50)
(99), 324, 327, 335(153), 336(99, 153, Hackert, M.L.,24
192), 389 Hageman, R. H., 274
Gregolin, C., 270, 271, 272(312) Hager, L. P.,377, 378(118), 397(118), 401
Greville, G. D.,87, 273 (118)
Griffith, J. S.,372,375(99) Hagihara, B.,323
Griffiths, D. E.,64, 68(63), 69, 72(63), Hagman, L. O.,348, 364
73(63), 178, 179(27, 281, lSl(281, 183 Haight, G. P.,Jr., 407
(28), 184(28), 190(27, 281, 245(27), Hainfeld, J., 126
AUTHOR INDEX 417
Halkerston, I. D. K., 83 Hatch, M. D., 143

Hall, D.E.,93,143(46), 144(46) Hatefi, Y., 78, 178(30), 179(27, 291, 180,
Hall, P.F.,83, 85(209) 181(29), 183, 184(28, 291, 190(27, 28),
Halsey, Y.D.,130 191, 192(71, 73), 193(69), 194(40, 671,
Hamada, M.,108, 109, llO(132) 195, 196, 197, 198(40), 199(88), 200
Hamberg, M.,83 (29,45,69),203,204, 205,206(40,42),
Hamilton, G. A., 396, 397(167) 207(40, 421, 208, 209, 210, 211(80,
Hamilton, L.,108 107), 212(80), 213, 214, 216(40, 108),
Hammes, G. G.,126 222, 224(141, 142, 1431, 225(72, 141,
Hanania, G.I. H., 372, 373(96) 142, 143), 226, 227, 228, 229, 230(73,
Haniu, M.,100 141, 142, 143), 231, 232(71, l66), 233,
Hansen, R. E.,179, 180, 181(41), 184(34), 234, 235(143), 236(156), 237(166),
185(34, 461, 186(46), 187(34, 46), 193 239,240(166, 178),241(166), 242(166),
(461, 215(46, 54), 221(46), 309, 316 243(166), 244(143, 1561, 245(143), 246
(66),335(66), 336(66, 194) (143, 156), 248(70, 71, 72, 1661, 253,
Hanstein, W. G., 78, 178, 179, 181(80), 254, 255(220a), 256, 258(29, 331, 296,
191, 192, 193(32), 199(68), 200, 203 297, 306(50), 307 308(50)
(68),206(42), 207(42), 208, 209, 210, Hattori, A.,274
211(80), 212(80), 214(80), 222, 224 Hauber, J., 237,240,247,255
(141, 1421, 225(72, 141, 1421, 226 Havsteen, B. H., 31
(321,230(141, 1421, 232(71), 236, 237 Hayaishi, O., 189
(1661,240(166), 241, 242,243, 248(70, Hayakawa, T.,106, 107(96), 108, 109, 110
71, 721, 253(166) (132)
Harano, Y., 84 Hayashi, A.,372, 373(92)
Harding, B. W.,65, 83(84), 84(84, 211) Hayashi, H., 311
Hardman, K. D.,317, 318(112) Hayaski, T., 108
Hargrave, K. R.,395 Hayes, J. E.,30
Harlow, D.R.,66 Hechter, O.,83
Harmsen, B. J. M., 32, 33(151) Heidema, J., 149
Harrer, C.J., 167 Heim, W.G.,376
Harrigan, P. J., 21, 23, 35(69), 37(83), Heimberger, G.,387
39(69, 831, 40(83), 41(69, 1901, 43 Heineman, W.R.,325, 330
(831, 44 Helleman, P. W.,131
Harrington, W. F, 5, 24 Hemmerich, P.,170, 235, 308
Harris, J. I., 2(18, 19, 211, 3, 4(18, 19), Hemmes, R. B., 54, 55, 56(16), 58(16),
5, 9(18,33), 14(35, 371, 18, 19, 20(19, 59(16), 80
29, 33, 371, 21(29, 30, 34, 37), 22(35, Hems, D. A., 46
37), 23, 24(35, 85), 25, 26, 27(103), Henderson, R.W.,325,326(162)
34, 36(35), 39, 45(29) Henley, K. S., 82
Harrison, P. M., 100, 103(68) Henning, U.,106, 107(107), 108(107)
Harte, E.M.,91, 143(6) Heppel, L.A.,388
Harting, J., 30,39,45(184) Herbert, D.,366, 367(45, 46), 398, 403
Hartree, E.F.,301, 346, 366,367(30), 382 Hershberg, R. L.,364, 374, 385(102)
(30),387(30), 388 Heusler, K.,407
Hartzell, C. R.,309, 316(66), 317, 320, Hewitt, E.J., 274
323, 325(107, 125), 326(107, 125), 327 Hildebrandt, A., 151,152(321)
107), 330(165), 331(125), 332(125, Hill, E.,10
126), 335(66, 107, 1261, 336(66, 194) Hill, F. L.,48
Hasson, E. P.,66 Hilvers, A. G.,33,39,41(159)
Hatano, S., 274 Hinkle, P.C.,88
418 AUTHOR INDEX
Hiraga, M., 367, 368, 393(69), 394(69) Howard, R . L., 191

Hirayama, K.,165 Howell, L.G.,90,91,146, 147(284), 282
Hiromi, K.,268 Howland, J. L.,63
Hoagland, V. D., 25 Hou, C., 64, 68(62), 72(62), 79, 80
Hoard, J. L.,376 Hrycay, E.G., 150,152
Hoberman, H. D.,69 Huang, J. J., 172
Hochberg, R . B.,85 Huang, P. C.,189, 190(59), 379
Hocking, J. D.,2(19), 3, 4(19),5, 14(37), Hucho, F., 106, 107(92), 108(92)
20(19, 37), 21(37), 22(37) Hudson, B.,91, 133, 143(6)
Hockstein, P., 168 Huennekens, F. M.,106, 107(112), 108
Hodgins, D.S., 143 (112), 112(112), 179, 188(37), 189,
Hodson, R. C.,282, 283 195(63), 203(63), 216
Hojeberg, B., 53, 58(8), 61(8, 231, 69(8), Huheey, J. E., 334
70(8), 71(8), 7603) Hultquist, D.E.,165
Hoek, J. B., 53, 58(8), 61(8, 231, 68, Humphrey, G. F.,62, 65(32), 67, 69(32),
69(8), 70(8), 70(107), 71(107),76(8), 70(32), 76(32)
78(107), 82,86(191), 87(184), SS(130) Hundal, T.,68,70(107), 71(107), 78(107)
Hoeksema, W. D., 286 Husain, M.,297
Hoffman, B. M,360
Hofmann, T.,92, 100(32), 103(32, 68), I
124(32) Ibers, J. A.,302
Hogeboom, G. H., 67 Ibne-Rasa, K.M.,399
Hogenkamp, H. P.C., 143 Icen, A.,93, 94(39), 100(39), 129(39), 132
Hogness, T.R., 167,345,346(1, 21, 347(1, (39), 138(39), 140(39), 141
2),348(1,2),353(1,2) Ichihara, K., 149
Hogue, P.,248 Ide, S.,106, 107(96)
Hoguem, P.K.,109, llO(130) Ii, I, 129
Holleman, W.H., 25 Iizuka, T.,347,351, 356(55), 360,372,376,
Hollocker, T. C.,249 382(107), 387(100)
Holloway, M.R., 25,26; Imahori, K.,2(20), 3
Holloway, P.W., 151 Imai, K.,66, 72(89), 73(89), 76(89), 372,
Hollunger, G.,207 373(92)
Holmes, R. S.,365 Imai, Y., 151
Holmgren, A., 93, 119(45, 471, 143(46, Ingraham, L. L.,397
47), 144(46,47) Inomata, H., 165
Holtaman, J. L.,150 Irukulla, R., 365
Hommes, A.,70,77(126) Isaacs, G. H., 259
Hommes, F.A.,207 Isaacson, E. L.,150
Hommes, R. W., 63 Isherwood, F.A., 129, 130(191), 138(191)
Hong, J., 243 Ishimoto, M.,286,295(395)
Hood, W.,2(27), 3 Ishizawa, S.,165
Hooper, M.,65 Ito, A.,154
Hopkins, F.G.,245 Iwatsubo, M.,268, 269(299), 272, 273
Horecker, B. L.,166,167(375) (329)
Horgan, D.J., 177, 205(22), 206(22) Iyanagi, T.,165, 166(373), 167(373), 170
Horie, S.,306(47), 307, 322 (373), 171(373), 172(373, 402)
Horio, T.,323
Horney, D.L.,108 J
Hoskins, D.D.,164 Jacobs, E. E., 306(48), 307,308(48), 313
Hosoya, T.,346 Jacobs, N.J., 260
AUTHOR INDEX 419
Jacobson, M., 149 (390, 397), 289(390, 3971, 290(394,

Jacq, C., 264, 265, 266, 268(290, 291), 296 413), 291(394), 292(390, 397, 4131,
Jacquot-Armand, Y., 267 293(390), 295(412)
Jaenicke, R., 25, 32 Kaneda, T., 108
Jajczay, F. L., 372, 373(90), 379(90), 380 Kanner, B. I., 79
(1251, 381(125), 382(90, 125), 383, Kanzaki, T., 108
384, 385(125) Kaplan, N. O., 2(15), 3, 20, 28(15), 29,
Jalling, O., 261 30, 52, 53(1), 54(1), 56(1, 17), 57(6,
James, B. R., 302 71, 58(1, 2, 3, 5, 7, 21, 221, 59(17),
Jasaitis, A. A., 69, 74(120), 75(120, 148) 60(7), 61, 62(21), 65(30, 311, 67, 69
Jecsai, G., 25 (30, 78, 96, 971, 70(30, 961, 72(78),
Jedeikin, L. A., 46, 49(214) 73(30), 76(30), 77(118), 78(97), 86
Jefcoate, C. R., 83, 151 (75), 87(24), 110, 167, 168, 169(388),
Ji, S., 75 207
Jick, H., 150 Karpukhina, S. Ya., 366
Jocelyn, P. C., 129 Karr, G. M., 5, 24
Jornvall, H., 5 Karuzina, I., 153
Johansson, B. C., 254, 255(220), 256(220) Karyakin, A. V, 153
Johnston, J. M., 151 Kaschnitz, R. M., 153, 206
Jollow, D., 153 Kaufman, B. R., 67, 69(96), 70(96), 110
Jones, E. T., 92, 94(35), 100, 103(35), 104Kaufmann, H., 367, 379(63), 380(63)
(35), 119(35), 120(61), 123(61), 141 Kawahara, Y., 106, 107(102, 104), 126
(35) (104)
Jones, G. L., 365 Kawakita, M., 187
Jones, G. M. T., 5, 14(35), 22(35), 24(35),Kawasaki, T., 65, 69(78), 72(78)
36(35) Kearney, E. B., 69, 176, 177, 188, 189, 190
Jones, 0. T. G., 274, 275(346) (58, 83), 202, 203, 217, 222, 223(25,
Jones, P., 151, 368, 369, 388, 389, 393, 394 140), 224(25), 234(25), 235(25), 236
(1621, 395(75), 397(75) (25), 237(25), 238(25), 245, 24700,
Jose, J., 2(16), 3, 4(16) 25), 248(24, 25, 261, 249(25, 26, 197,
Joy, K. W., 274 199), 250(25, 195, 196, 1971, 251(197),
Joyner, T. B, 301 252(197), 253(184), 254(16, 251, 270
Junge, J. M., 2(10), 3 Keefer, R. M., 384
Junk, K. W., 149, 165(286) Keenan, T. W., 312, 313(84)
Juntti, K., 65, 72, 212 Keilin, D., 222, 223, 240(147), 264, 300,
301, 315, 338(14), 346, 363(4), 364,
K 366, 367(30), 382(30), 387(30), 388,
398
Kadlubar, F, F., 153, 154 Keirns, J. J., 311, 312(78)
Kadziauskas, Y.P., 69, 74(120), 75(120) Keister, D. L., 54, 55, 56(11, 16), 58(16),
Kagawa, T., 263 59(11, 16), 64, 66(11, 64, 65), 68(64,
Kajiwara, S., 346 65), 69(65), 72(64, 65), 73(65), 76
Kallai, 0. B., 371(85), 372 ( 6 9 , 80
Kalra, V. K., 65 Keleti, T., 24, 25, 40
Kalse, J. F., 124, 125(172, 173). 238 Keller, R., 267
Kamen, M. D., 371(84), 372 Kelso, J . R., 300
Kamin, H., 91, 136, 166(10), 167(10, 245), Kendrew, J. C., 349, 364, 372
168(10, 245, 384, 3861, 169(245), Kenney, W. C., 109, 110(130), 177, 222,
170(245, 3861, 172(245, 398), 176, 278 223(25), 224(25), 225, 226(158), 230
(3), 286, 287(390, 394, 397, 412), 288 (158), 234(25, 158), 235(25), 236(25),
420 AUTHOR INDEX
237(25), 238(25), 247(25), 24805, Konings, A. W. T., 68, 71(113, 117)

158), 249(25), 250(25), 254(25) Kopko, F., 129
Kensler, C. J., 260 Korgenovsky, M., 260
Kepler, C., 106, 107(97) Koritz, S. B., 83
Kessler, E., 274 Koshland, D. E., Jr., 26, 31, 32, 33(149),
Kielly, W. W., 62, g5(34), 67(34) 34(143), 35(164), 36(113), 37(113,
Kierkegaard, P., 348,364 172), 42(143)
Kim, I. C., 217 Kosow, D. P., 47
Kim, K. H., 130 Kosower, E. M., 28, 97, 131, 379
Kimura, T., 172, 240, 247, 260, 261(256), Kosower, N. S., 131
262(262), 263(266) Koster, J. F., 124, 125(172), 126(180), 238
King, T. E., 78, 176, 182, 187, 188, 189, Kotani, M., 347, 351, 356(55), 366
190(43, 43a), 191, 222, 223, 226(151), Kowal, J., 84
235, 236, 237, 240(147, 148, 1671, 242, Koyama, J., 286
244(164), 245(167, 1731, 246, 248, 253 Kramer, R., 67, 69(100), 70(98), 78(100)
(164), 296, 306(51), 307, 308(51), 310 Kratky, O., 32
(51), 312, 319, 320, 322(124), 323 Krebs, E. G., 2(10), 3
(124), 324, 325(124), 326(124), 331 Krebs, H. A., 46, 47, 49, 63, 79, 81(175),
(124), 332(124), 333(131), 334 82(175), 86
Kinon, B. J., 131 Kremer, M. L., 305,366,385
Kirkman, S. R., 86 Krimsky, I., 13,28,39(54), 40,41, 42(196)
Kirkpatrick, F. H., Jr., 313 Kronman, M. J., 25
Kirsohner, K., 25, 26, 31(101), 32, 33 Krul, J., 106, 107(114), 114(114)
(101), 41(146) Krupas, M., 269
Kirtley, M. E., 29, 30(126) Kubose, A., 106, 107(94)
Kita, M., 375 Kuboyama, M., 306(51), 307, 308(51),
Kitamura, T., 108 310, 334
Klaaae, A. D. M., 237, 238(172) Kuma, F., 165
Klein, K. O., 84 Kumar, S. A., 189, 195(63), 203
Klein, S. M., 106, 107(113), 108(113) Kupriyanov, V. V., 70
Kleineke, J., 82 Kustin, K., 407
Kleinsek, D. A., 88 Kusunose, E., 149
Kleppe, K., 101 Kusunose, M., 149
Klima, J., 167 Kuwana, T., 317, 320, 325(107, 125), 326
Klingenberg, M., 56, 57(20), 63, 66(20), (107, 125), 327(107), 330(165), 331
81(20, 38), 86(38, 45, 461, 258, 259 (125), 332(125), 335(107)
(228) Kuenetsova, G. P., 153
Klouwen, H., 180, 312
Klybas, V., 44 1
Knof, S., 25
Knutson, J. C., 83 Labeyrie, F., 265, 267, 268,269, 272(306)
Kobayashi, K., 286, .295(395) Ladany, S., 85
Kodicek, E., 83 Lafferty, M. A., 274, 275(351)
Koeppe, 0. J., 31 Lakatos, S., 26
Koike, K., 108 Lakshmanan, M. R., 88
Koike, M., 93, 106, 107(96, 109), 108, 109, Lam, K. W., 79
110(132), 114(109) Lambert, J. M., 45
Kolb, E., 2(21), 3,5 Lambeth, D. O., 375
Kolvoda, J., 407 Lambowitz, A. M., 215
Komai, H., 311, 312(75) Lamprecht, W., 24
AUTHOR INDEX 421
Land, G., 143 Lenaz, G., 70

Landau, B. R.,402 Lenhoff, H.M.,168,169(388)
Landriscina, C.,88 Lents, P.J., 24
Lands, W.,168 Leonard, J., 347, 348(39), 349(39), 359
Lang, G.,331,356 (39)
Langdon, R. G., 91,129,130, 142, 166(11), Lerner, D. A.,126
167(11, 380), 168(11) Leterrier, A. F.,317,318(109)
Lange, L. G., 111,213 Levenberg, M.I., 377
Lange, R.,20, 368 Levin, W.,149, 152
Langemann, H.,260 Levitzki, A.,34,35,36
Langford, C. H., 385,386(137) Levy, H.R.,39
Lardy, H.A., 110, 256, 259, 263 Lewis, J. A.,396
Lardy, M. A., 62, 65(35), 67(35) Lewis, S. N.,399
Larsson, A., 143, 144 Liberman, E. A., 69, 74(120), 75(120)
Larsson, L. L., 364 Libor, S., 23, 25
Larsson, L. O., 348 Lichtenberger, F.,153
Laskawska-Klita, T.,310 Lieber, C. S.,81(176)
Laughrey, E.G.,82 Lieberman, S.,83,85
Launay, A. N.,84 Light, P. A., 204, 217, 219
Laurent, T. C., 92, 142(23), 143(23) Lilienthal, H.R.,314,334
Lazdunski, M.,39 Liljas, A., 10,11(51), 12(51)
Lazzarini, R.A,, 274,277(339), 286(339) Lim, J., 235, 244(164), 253(164), 296
Leadbetter, E.,274, 276(342) Lindberg, O.,261
Lean, J. D., 57,58(21),62(21) Lindsay, J. G.,302, 321, 322(138), 323,
Lebeault, J. M., 149,150(295), 151(295) 326(138), 327(128), 331(138), 333
Lebherz, H.G., 9,26 ( 1381, 335(138)
Lederer, F., 264, 265, 266, 267, 268(290, Linn, T. C., 106, 107(92), 108(92)
2911, 296 Linnane, A. W.,216
Lee, C.P., 64,67,68(66), 69(106), 70(59), Lipmann, F.,38,74, 279
72(59, 102, 103, lW),73(106, 135, 136, Lippard, S. L., 331
1371, 74(106, 136, 137, 166), 76(59), Lipscomb, J. D.,172
77(59, 106, 135, 136, 1371, 78, 87(74), Lipscomb, M.D., 83, 84(210)
204,207,214,246 Listowsky, I., 31, 34037)
Lee, C.Y., 29 Littler, J. S.,407
Lee, J. P.,286,288,295(396) Lo, S., 361
Lee, K.L., 259 Low, H.,261
Lee, R., 168 Loewus, F. A., 39
Lee, Y.P.,259 Lombardino, J. G.,379
Lees, B.D,329 Long, R.W.,88
Le Gall, J., 281, 286, 288, 295(373a, 389, Lopez-Colome, M., 133
396) Losada, M.,274,276(342, 345)
Lehninger, A. L., 65, 92 LoSpalluto, J., 150
Leigh, J. S., 187, 296, 320, 322(124), 323 Louie, D.D.,53,57(7), 58(21, 22), 60(7),
(124), 324, 325(124), 326(124), 327, 62(21)
331, 332(124, 168), 333(168), 336 Love, B., 310, 311(69)
(155),350,360,365, 372,376(88), 389, Lovenberg, W.,176,243
390(150), 391(150) Loverde, A., 156,164(349)
Leinweber, F. J., 286,287, 288(404) Lovrien, R.,367
Lemberg, R.,301, 315, 316(18, 1001, 320 Lowe, G., 43
(18), 322, 323(100), 324, 335(18) Lowry, C., 24
AUTHOR INDEX
Lowry, 0. H., 47 Magni, G., 247

Lu, A. Y.,149,152,165(286) Mahler, H. R., 188, 189, 199, 217, 307,
Lund, P.,46,82,86(185) 308(59)
Lundsgaard, E.,2 Mahoney, A. J., Jr., 261
Lusty, C. J., 106, 107(91, 93), 109(91), Major, J. P.,21
202, 262, 263(266) Makhlis, T.A., 70
Luthy, J., 69, 203 Makino, N.,171, 172(402)
Lutwak-Mann, C.,245 Maley, G. F.,62,65(35),67(35)
Luzikov, V. N.,70 Malhotra, 0. P.,33, 35(160), 36(160),
Luzzati, M., 264 37(174), 41(160)
Lyanagi, T., 169 Mallet, A. I., 407
Lyric, R. M., 281, 282(375), 284 Malmstrom, B. G., 300, 301, 316(11), 317
Lyster, R.J. L., 369 (ll), 326, 328, 330,334(11), 335(108),
336(108, 171)
M Maltempo, M., 365
Malviya, A. N.,335
McAllister, J. K., 92, 94(36), 100(36), Mandula, B., 131
103(36), l06(36), 107(36), 143(36), Mann, P.J. G., 260,261(248)
144(36) Mann, T., 346
McCann, L. M., 68,79 Mannervik, B., 132, 133, 139, 140, 141
McCarthy, J. L., 84 (253, 254)
McCarthy, K., 243 Manocha, S.,300
McCay, P. B.,168 Mansley, G. E.,322
McCleverty, J. A., 396 Manzocchi, A., 248
McCormick, D. B.,101, 125 Mapson, L. W.,129, 130(191), 138(191)
McCoy, S.,304, 305(40), 317, 321, 322 Margoliash, E.,368,371(85), 372,376,377
(114),323(114), 337(115), 338(40) (lll), 378(115), 380(111), 383(116)
McDaniel, M. C.,334 Margolis, J., 402
McDonald-Gibson, R. G.,247 Margolis, S. A,, 70
McGraw, J. C., 164 Marklund, S.,393, 394(164)
Machinist, J. M., 78, 190(84), 202 Markovich, D.S.,25,31(105)
Machleidt, W.,311 Martell, A. E.,407
McIntosh, E.N.,65 Martin, R. B.,304
McKee, E. M., 154 Martinez, J., 132
Mackerer, C. R.,87 Marver, H.S., 150
Mackey, L. N.,317, 325(107), 326(107), Maskasky, J., 307, 314(60), 334(60), 338
327(107), 335(107) (60), 343(60)
Mackler, B., 179,188(37),189,195(63), 203 Mason, H. S., 165, 166(373), 167(373), 170
(63),216 (373), 171(373), 172(373, 4021, 176
McLean, P.,46, 47(211), 81 Mason, T.L.,307, 308(58), 311, 312
McManus, I. R.,100, 103(65), 106, 107 Massey, V., 90, 91, 92, 93, 94(15, 24, 27,
(1051,1O9(65) 29),95(27), 96(27), 97(1, 24, 29, 55),
MacMunn, C. A.,300, 315 98(1, 24,50, 55), 100(29, 32), 101, 103
McMurray, C. H.,41 (29,32),106(4,27),107(4,24,97,101),
McMurray, W. C., 62, 65(35), 67(35) io8(4i), 109,111, ii2(24,55,ioi, 118,
McPherson, A., 10,24 150), 113(4, 24, 27, 29, 59, 137), 114
MacQuarrie, R. A., 34, 36, 37(175), 42 (27, 55, 150), 115(24, 137), 116(4, 24
(165),48(165), 49(165) 27), 117(27, 1171, 120(83), 122(150,
Magar, M. E.,14,25(57) 154, 155), 123(154, 1551, 124(32), 125,
Mager, J., 286,287(386, 391) 126, 133, 134(27, 1181, 135(29), 136
AUTHOR INDEX 423
(118), 137(1, 29, 59, 244), 138(29), Meyerhof, O., 2, 28(la)

139(244), 140(244), 141(29), 146, 147 Michaels, G,B.,282
(284), 148(58), 170(1), 177, 153(23), Michejda, J. M.,84
188(23), 200, 201(23), 203(23), 204 Middleditch, L. E.,55, 56(18), 57(18),
(23), 205(23), 223(23), 224(23), 235 58(18)
(23), 236(23), 238(23), 246, 247(23), Middlemiss, D. N., 393,394(162)
248(23), 249(23), 250(23), 254, 282 Midelfort, C.F.,9
Masters, B. S. S., 136, 150, 151, 154, 165 Mihara, K., 151
(337), 166, 167(245, 3371, 168(245, Mildvan, A. S.,374, 385(100a)
386), 169(245), 170(245, 386), 172 Millard, S. A., 106, 107(94)
(245,337,398) Miller, Z. B.,261
Masters, C. J., 365 Mills, G. C.,48
Mathew, E.,11, 21, 22(52, 701, 42(70), Minakami, S.,176, 187, 188(56), 189(56),
45(70) 190(56)
Mathews, C.T., 302,303(25) Minchiotti, L.,100, 103(66), 143(66)
Matsubara, H.,310 Minnaert, K.,307, 325
Matsubara, S., 365 Misaka, E.,106, 107(102, 1041, 122
Matsurnura, Y.,311 Mitchell, C. H.,153
Matthews, J., 106, 107(99), 108, 112(99), Mitchell, J. R.,153
114(99) Mitchell, P.,74, SO(155, 157), 81(157), 87
Matthews, R. G., 90,91, 100, 103, 104(63), Mize, C.E.,129, 142
114, 118(63, 156), 119(63, 156), 120 Mochan, E.,334,357
(63),121(63), 122(156), 146, 147(284), Moe, 0.A,, Jr., 126
282 Moir, N. J., 151
Mauk, M. R., 372 Molbert, E.,366, 367(48)
Mauleon, P.,300 Moleski, C.,334
Mauzerall, D.,396 Monod, J., 31
Mavis, R.D., 92, lOO(34) Monroy, G.L.,72
Maxwell, J. C.,319, 321, 322(142), 323 Montal, M., 72
140), 337(117, 118), 339(117, 118), Monteilhet, C., 264
340, 389 Monty, K.J., 286,287(387), 288(404)
May, H.E.,168 Moore, E. C.,91, 92(8), 93(8), 99(8), 142
Mayhew, S. G.,90, 91, 100, 123, 282 (23), 143(8, 231, 144(8), 145(8). 156
Mayr, M, 247. 249(199) (9)
Medina, A.,274,275(346),276(367) Moore, J., Jr., 23
Mehler, A. H.,9 Moran, T.,312
Meighen, E. A., 25, 26(99) Moras, D.,9, 10(46), ll(46, 481, 18, 24
Meigs, R. A,, 83 (47),29(47), 39(47, 48), 44(46, 47),
Meijer, A. J., 80,88 129, 141(188)
Melandri. B. A..2(28), 3 Moreno, C. G., 274
Meldrum, N. U.,92 Morgan. E.,245
Menon. K.M. J., 83 Mori, M., 304
Mercer, W. D., 9, lO(45) Morikofer-Zwex, S.,367, 379(63), 380
Meriaether. B. P.,5. 11. 20(29), 21(29), Morimoto, H., 372,373(92)
22(52, 70), 24, 39(29), 42(70), 44(90), Moroff, G.,141
45(29, 70, 90, 185), 48(200) Morrison, G.,83
Merola, A. J., 180, 181(41), 312, 313(85) Morrison, M., 176, 306(47), 307, 322
Mersmann, H., 69, 203 Morton, R.K.,264,269(300)
Mevel-Ninio, M.,269 Moss, T.H., 314, 334(94), 338(94), 356
Meyer, A.J., 88 Moyle, J.,74, 75(157), 81(157), 87
424 AUTHOR INDEX
Mozolovsky, A., 25 Neas, G. C., 88

Muller, F., 282 Neufeld, E. F., 52, 53(1), 54(1), 56(1),
Muller, M., 67, 69(100), 78(100) 58(1, 2, 3), 62(1), 65(30), 67(30), 69
Muesing, R. A., 88 (30), 70(30), 73(30), 76(30), 167
Muijsers, A. O., 308, 315, 316(101, 1021, Newsholme, E. A., 47
317, 318(111), 320(101, 102), 321(102, Nicholas, D. J. D., 274, 275(346), 276
132), 324,325,326(162), 335(157) (367), 286, 287(401), 288, 292(401,
Muiswinkel-Voetberg, H., 117, 118(160), 4221, 293(401)
119(160), 124, 125, 128(181, 182) Nicholls, D. G., 63,86,87(52)
Mukherjee, B. B., 108 Nicholls, P., 237, 301, 320, 321(133), 334,
Muller, F., 90, 123, 126 335(21), 346, 357, 386, 369(17), 370
Mulrow, P. J., 84 (17, 721, 374(17, 721,377, 384(17), 385
Munk, P., 108 (171, 389(17), 391(17), 395(17), 397
Muraoka, S., 390 (17), 398, 400(175)
Murdock, A. L., 31 Nieuwenhuis, F. J. R. M., 69, 74(119), 80
Murphy, M. J., 286, 287(397), 288(397), (1191, 214
289(397), 292(397), 295(410, 412) Niluson, S., 384
Murphy, T. A., 259 Nise, G., 132
Murthy, P. S., 64, 65, 66(60), 68(60), 72 Nisley, S. P., 63, 66(37), 81(37), 86(37)
(60) Noeler, H. F., 5, 9(33), 20(33)
Myer, Y. P., 319 Norling, B., 246, 249(188)
Northrop, D. B., 141
N Notani, G. W., 106, 107(110)
Nagahisa, M., 367 Novogrodsky, A., 376, 377(111), 378(115),
Nagai, Y., 286 380(111)
Nagradova, N. K., 2(24), 3, 25(24), 30 Nurnberger, H., 368
(241, 48 Nygaard, A. P., 38, 44(179), 162, 264, 267
Naiki, N., 286, 287(399), 288,292(399,424) (280), 270, 271
Najjar, V. A., 274
Nakajima, H., 165 0
Nakamura, H., 217
Nakamura, M., 122 O’Brien, P. J., 150, 152
Nakamura, T., 286,295(402, 403) Oda, T., 237
Nakanishi, K., 106, 107(102, 104), 122, 126 Oesper, P., 45
(104), 128(104) Oestereicher, G., 248
Nakatani, M., 367 Oguchi, M., 48
Namba, Y., 108 Ogura, Y., 187, 372, 373(89), 376(89), 382
Narashimhulu, S., 166 (89, 1071, 387(100), 388, 390(144)
Naslin, L., 267, 272 Ohlsson, P. I., 365
Nason, A., 274, 275(344), 277(337), 278 Ohnishi, T., 187, 204, 215, 219, 235, 244,
(356) 253, 296, 346, 347(19), 353(19), 361
Navazio, F., 86 (19)
Nawa, H., 93 Ohno, H., 205
Needham, D. M., 2 Ohta, Y., 366, 367(50)
Negelein, E., 301 Okabe, K., 106, 107(96)
Nelson, E. B., 150 O’Keeffe, D. H., 307,314(60), 315(93), 329
Nelson, D. H., 65, 83, 84(211) (931, 334(60, 94), 338(60, 941, 343(60)
NkmBthy, G., 32, 33(149) Okunuki, K., 306(46), 307, 308(46), 274,
Nepokroeff, C. M., 88 275(364, 365), 276, 310, 311, 315, 316
Nesbakken, R., 130 (1031, 323, 334
AUTHOR INDEX 425
Olah, G., 384 Pajot, P., 264,265,269

Olcott, R.J., 400 Palmer, G., 90,92,95,97(l,55), 98(1, 55),
Oldham, S. B.,65, 83(84), 84(84) 100(32), 101, 103(32), 112(55), 113
Olsen, K.W.,9, 10(46), ll(46, 481, 18, 24 (591, 114(55), 120(83), 124(32), 137
(47), 29(47), 39(47, 481, 44(46, 471, (1, 591, 170(1), 205, 322,324, 330, 335
129, 141(188) (1531,336(153), 375
Olson, E.J., 23, 45(81) Paltauf, F., 151
Olson, J. A.,86 Panebianco, P., 219
Olson, M. S., 110 Paneque, A.,274,276(345)
Omdahl, J. L.,83 Paniker, N.V.,131
Omura, H., 274 Panov, A. V., 70, 88(129), 212
Omura, T.,91, 150, 154, 165, 166(372), Papa, S.,63, 73, 86(50), 87(53, 54, 55)
167(372) Paradies, G.,70,88(129), 212
Ondarza, R. N.,132, 133 Park, J. H.,3, 5, 11, &(29), 21(29), 22
Oosthuizen, C.,33 (52, 701, 23, 24, 28(13), 29(13), 31,
Oppenheimer, C.,363 38(13), 39(13, 291, 40(13), 42(70), 44
Oppenheimer, N.J., 29 (13, QO), 45(29, 70, 81, 90, 1851, 48
Orii, Y., 310, 311, 315,316(103), 320(103), (200)
321(128), 324, 334, 336(134) Park, R., 84
Orlando, J. A., 66, 68, 71(114, 115), 72 Parker, D.J., 20, 21(66), 30
(871, 79(115) Parkhouse, R. M. E., 132
Orme-Johnson, N. R., 179, 184(34), 185 Parkhurst, L. F.,333
(34), 186, 187(34, 461, 193(46), 215, Passon, P. G.,165
221 (46) Passoneau, J. V.,47
OrmeJohnson, W. H.,83, 179, 184(34), Patchornik, A., 379
185(34), 187(34), 226(218), 253, 323 Paul, K.G., 365
Orr, M.D.,143 Pauling, L.,364
Orrenius, S.,150, 152, 166 Payne, M.,33, 35(160), 36(160), 41(160)
Orsi, B.A.,40 Payne, W.J., 274
Osajima, Y.,274 Pazur, J. H., 101
Osborn, M.,366 Pearson, R.G.,375
Osenga, G., 222, 225(150) Peck, H.D.,Jr., 279, 281, 282, 284,285,
Oshino, N.,151, 365 286(372), 288,295(396), 297
Ostroumov, S. A.,69,74(121) Peczon, B. D., 33, 41(161)
Ota, A.,274,275(364) Pederson, T. C., 165, 166(369, 374), 167
O’Toole, M.C.,308,314,315(93), 317(65),
(374), 168(374), 169(374)
318(65), 322(65), 323(65), 325(65), Peisach, J., 368, 369, 370(77, 781, 376(77),
328(65), 329(65, 93), 337(65), 338(65)
380, 381(126b), 390(126b)
Otsuka, J.,366
Pelley, J. W.,106, 107(92), 108(92)
Otsuka, K.,108
Perham, R. N.,5, 24, 21(30, 34), 45, 104,
Otto, J., 311
118(86), 119(86),120, 121(86)
Ovadi, J.,24,25
Peron, F. G., 84,88(225)
Overberger, C.G.,379
Owen, C. S.,320, 322(124), 323(124), 325 Persson, B.,246,249(188)
(124),326(124), 331(124), 332(124) Perutz, M.F.,349,372,373(91)
Ozawa, T.,234, 335 Pesch, L. A.,70
Petering, D.H.,360
P Peters, J. M., 92,97(28),104(28), 114(28),
Packer, L., 259,261, 262(262) 127(28)
Pain, R.H., 368 Peterson, J., 70
426 AUTHOR INDEX
Pette, D., 48 0
Pettit, F. H.,106, 107(92), 108(92), 110
Pfennhger, O., 33, 35(160), 36(160), 41 Quagliaridllo, E., 63, 73, 87(53, 54), 88
( 160) Quastel, J. H.,260, 261(248)
Pfleiderer, G., 24
Pharo, R. L., 70, 76(127), 77(127), 189,
190(59), 195(64), 198(64), 205
Phillips, A. H.,91, 166(11), 167(11), 168 Rabinowitz, J. C., 243
(11) Racker, E., 13, 28, 38, 39(54), 40, 41, 42
Pihl, A,, 20, 368 (196), 44, 66, 179, 180(36), 181(36),
Pillai, P., 2 183, 184(36), 186, 187, 201(35), 214,
Pincus, G., 83 239,244,246,302
Pinsent, J., 366, 367(45, 46) Radcliffe, B. C.,274
Plaut, G.W.E., 86,87 Radda, G.K.,33
Plumley, H.,130 Rafter, G. W.,2(10), 3
Pnafili, E.S.,155 Ragan, C. I., 78, 179, 180(36), 181(36),
Pocker, Y.,407 182, 183, 184(36), 188, 187, 190(43a,
Podack, E.R., 88 133), 201(35), 204, 214, 217, 218(133),
Poff, K.L.,239,240(178) 219( 1311,
Pogson, C.I., 41 Raijman, L., 46
PolgBr, L., 21, 22(73, 741, 23, 43, 45(73, Rall, T. W., 92
74) Ramasarma, T., 247
Poole, B., 365 Randall, D.D.,106, 107(92), 108(92)
Popowsky, M.,202 Ranney, H.M.,372,373(91)
Porque, P. G.,143,144(275) Rao, N. A,, 179, 188, 189, 195(63), 203
Porter, J. W.,88 (631,216
Porterfield, U.T., 388 Rapkine, L., 2,20(5)
Postma, P. W.,79 Rasmussen, 0.L.,302
Poulsen, L. L., 106, 107(100), 153 Rawitch, A. B.,101
Poyton, R. O.,307,308(58), 311 Ray, D.K.,317, 318(112)
Prabhakararao, K., 286, 287(401), 288, Ray, G. S.,334, 335(185), 336(185), 346,
292(401, 422), 293(401) 348(14), 352(17), 353(14)
Prados, R., 304 Recheigl, M., 365
Prager, G., 255 Redfield, A. G.,357
Redline, R., 148, 151 (284a)
Prakash, O.,274,277,278,297(341)
Redman, C.M., 365
Presswood, R. P., 170, 286, 287(394), 290
Reed, D.W.,165
(3941,291(394)
Reed, G.,350, 360
Preabindowski, K.S.,313 Reed, G.H., 372, 376(88)
Price, N.C.,33 Reed, J. K.,106, 107(95), 110, 116(95),
Price, V. E., 366 117(95), 118(95), 119(95), 139(95)
Pronk, J., 120, 148(163) Reed, L. J., 92,93, 106, 107(92, 99, 1091,
Prough, R. A., 151,166,169,172 108(19, 43, 92),109(03), 110,112(99),
Psychoyos, S.,83 114(99,log), 126(126)
Puchwein, G.,32 Reichard, P., 91,92(8), 93031, 99(81, 142
Pullman, M.E.,72, 88 (23),143(8,23,261,2621, 144(8, 275),
Pulsinelli, P. D.,372,373(91) 145(8), 156(9)
Pupillo, P., 2(28), 3 Reiske, J S., 310
Purvis, J. L.,84 Rendina, G.,260,261(254, 255)
Pyfram, H.T., 376 RBtey, J.,252
AUTHOR INDEX 427
Richardson, S. W.,306(50), 307, 308(50) Rouslin, W.,312

Righetti, P.,225, 230(157), 231,234 Roussos, G.G.,274,278(356)
Rikihisa, T.,39,45(181) Rubin, E.,81(176), 402
Ringler, R. L.,176, 187, 188(56), 189, 190 Rubin, M.S.,307,308(57),311(57)
(56),238, 257, 258(226) Rueger, D. C., 170, 286, 287(394), 290
Riordan, J. F., 213 (3941,291(394)
Rippa, M.,270 Rumack, B.H.,150
Rider, J. L.,264 Rumen, N. M., 374
Rivas, J., 274, 276(345) Rutter, W.J., 9
Robberson, B., 366, 367(41), 370(41), 371 Ruzicka, F.J., 245,297
(41) Ryan, D.,149, 152
Robbins, P. W.,279 Ryan, K.J., 83
Roberts, K.D.,83 Ryback, G.,252
Robertson, A. M., 69 Rydstrom, J., 53, 58(8), 61(8), 64, 66, 68,
Robins, R. G.,302,303(25) 69(8, 68, 69, 70, 71, 721, 70(8, 67, 70,
Robinson, J., 65, 85(86) 72, 107), 71(8), 72(67), 75(67, 68, 69,
Robinson, J. R., 108 70,711,76(8, 67,68,70,71,721, 77(67,
Rocca, E., 177, 217,254(16) 68, 69, 70, 71, 72), 78(70, 1071, 87, 88
Rodkey, F. L.,113 (70,71,129, 1301,207,212
Rodman, H.M.,402 Rytka, J., 272
Rogers, M. J., 148, 151(284a), 154, 156
(341, 342), 161(341, 342, 344), 161 S
(341,,342)
Rogers, L.A., 285 Sabeson, M. N., 11
Rogers, W.I., 261 Sabo, D.,66, 72(87)
Rolleston, F. S.,47 Sacktor, B.,258, 259(229, 233, 234)
Ronchi, S.,2!2, 44(76), 100, 101, 103(62, Sadana, J. C.,274,277,278, !297(341)
661, 104(61,62), 105(62), 120(61), 123 Sadasivan, N.,305
(611, 143(66), 144(62), 145, 146, 147 Sagers, R. D.,106,107(113), 1W113)
(284) Saggerson, E. D.,47
Roos, D., 73 Sajg6, M.,5,9(33),20(33)
Roper, M.,150 Sakai, H., 129
Rose, I. A., 47 Sakamoto, Y.,150
Rosemeyer, M.A,, 131 Sakurai, Y.,108,109,llO(132)
Roskn, S.,331 Salach, J. I., 222, 235, 247, 249(200), 258
Rosenthal, O.,83, 91 Salhanick, H.A.,65
Rosenthal, S.,152,166 Salmon, D.M.W., 46
Rosing, J., 74 Saltzgaber, J., 312
Rosman, J., 310 Salvenmoser, F., 67, 69(100), 70(98), 78
Ross, E.,66 (100)
Rossi, C., 78, 190(84), 202, 222, 225(150) Samejima, T., 366,367(50),375
Rossi, E.,246, 249 Samson, L.,371 (85),372
Rossi, F., 222, 225(150) Samuelsson, B.,83, 168
Rossi-Fanelli, A.,349 Samuilov, V. D.,69, 74(121)
Rossmann, M. G.,9, 10(46), ll(46, 48, Sanadi, D. R., 64, 65, 67, 68(31), 70, 72
51), 12(51), 18,24(47), 29(47), 39(47, (61), 76(127), 77(127), 79, 91, 92, 97
48),44(46,471,129, 141(188) (26, 281, 104(28), 106, 107(25), 108,
Rothschild, H. A.,261 113, 114(26, 28), 116(116), 117(151),
Rotilio, G.,304 127(28), 189, 195(64), 198(64), 205
Roughton, F.J. W., 388 (64)
428 AUTHOR INDEX
Sandberg, H. E., 369, 370(74, 791, 372(74, Schramm, M.,44

791, 376(79) Schrauzer, G. N., 396
Sandborn, B. M.,249 Schroeder, W. A., 366, 367(41), 370(41,
Sanders, E., 91 63a), 371(41, 63a), 372(63a), 377, 378
Sands, R. H., 98, 113(59), 137(59), 235, Schultz, J., 378
244, 330 Schuman, M.,90,91, 101,282
Sani, B. P., 67 Schuster, I., 25,26,31(101), 32,33(101)
San Pietro, A., 53, 54, 55(11), 56(11), 58 Schutte, H. R., 368
(5), 59(11), W11) Scott, E. M.,93, 94(40), 138(40), 139(40),
Santema, J. S., 55, 56(19), 57(19), 58(19), 140(40), 164
59(19), 62, 80(19), 106, 107(114), 114 Scouten, W. H., 106, 107(105), 109
(114) Seamonds, B., 346, 351(21)
Sarkar, N. K., 188, 189(57), 199(57) Searls, R. L., 92, 97(26, 281, 104(28), 106,
Saronio, C., 324, 336(155), 389, 390(150), 107(25), 108, 114(26, 281, 116(116),
391(150) 127(28)
Sasame, H. A., 153 Sebald, W., 311
Ssto, R., 66, 72(89), 730391, 76(89), 151, Segal, H. L., 39, 40
154, 288, 287(400), 288, 292(400, 421, Segel, I. H., 129,132(193), 138(193)
423, 424), 293, 294, 295(402, 403) Sehested, K., 302
Satoh, K., 65,69(78), 72(78) Seibl, J., 252
Sauer, L. A., 84 Seifried, H. E., 151
Savage, B., 26 Sekuzu, I., 306(46), 307, 308(46), 323, 334
Savage, N., 94, 98(54) Sellinger, 0. Z., 259
Schachman, H. K., 25,26(99), 34(112) Sels, A. A., 347
Schacter, B. A., 150 Seng, R., 222
Schatz, G., 66, 167, 183, 214, 307, 308(58), Setlow, B., 148, 151(284a)
311, 312 Seubert, W., 88
Schejter, A., 377, 378(115) Severina, I. I., 74, 75(148)
Scherz, B., 367 Seydoux, F., 33, 35(160), 36(160), 41(160)
Schevitz, R. W., 10, 24 Shah, P. C., 106, 107(109), 114(109)
Schiff, J. A., 279, 280, 282, 283 Shakespeare, P. G., 307, 308(59)
Schindler, F. A., 390 ShaltiB1, S., 20, 22
Schlessinger, J., 34 Shaw, D. C., 11, ZZ(52)
Schleyer, 346s 348(ZZ)3 351(n)3 Shelton, J. B., 366, 367(41), 370(41), 371
(n), 353(20, '22) (41)
Schmid, D., 25 Shelton, J. R., 366, 367(41), 370(41), 371
Schneider, W. C., 67 (41)
Schoenhard, D. E., 286 Shepley, K., 269
Schollmeyer, P., 63,81(38), 86(38) Shibata, H., 150
Scholz, R., 88 Shibata, Y., 25
Schonbaum, G. R., 361, 365, 369(17), 370 Shifrin, s.,28
(17), 372, 373(98), 374(17), 377, 378
Shimakata, T.,151
(117), 379(118b), 2"25), 381(125,
Shin, B. c.,48
126b), 382(125), 384(17)9 385(17, 100at
Shin, M., 54, 55, 58(12), 66(12)
llsb, 125), 387(118b), 389(17), 390
(lola), 391(17, lola), 392, 393(101a, Shonkj c. E**47
159), 394(101a), 395(17, 159, 159a), Shusterf L.*59f 150
397(17, lola), 398(101a), 399(101a, Siege19 L. M.9 170, 286, %7(390, 394, 3979
118b), 400, 401, 402(118b, 159), 403 412), 288(390, 397, 404), 289(390), 290
(118b), 404, 405 (394,413,414,415), 291(414), 292(390,
AUTHOR INDEX 429
397, 415), 293(390), 294(414), 295(410, Smith, D. W., 369, 370(76), 372(76), 374
412) (76)
Sies, H., 130, 365 Smith, G. D., 26,34(112)
Sih, C. J., 83 Smith, J. E., 132, 142
Simard-Duquesne, N., 69 Smith, M. H., 300
Simon, A. M., 265 Smith, M. L., 307, 314(60), 334(60), 338
Simon, I., 34 (60), 343(60)
Simplicio, J., 303 Smith, T. E., 40
Simpson, E. R., 84 Smith, W., 168
Simpson, R. J., 2(25), 3 Smythe, G. A., 307, 314(60), 315, 334(60,
Singer, T. P., 69, 78, 106, 107(91), 109(91), 94),338(60,94), 343(60)
110(130), 176, 177, 184, 186(23), 187, Snyder, H., 378
188(19, 23, 561, 189(56), 190(56, 83, Soling, H. D.,82
841, 200(15, 191, 201(14, 15, 19, 231, Somlo, M., 269,272(306)
202, 203(19, 2 3 , 204(19,23), 205(19, Sone, N.,260
23), 205(19, 22, 23, 88), 206(19, 221, Sordahl, L. A., 189, 195(64), 198(64), 205
214, 216(116), 217, 219, 220(136), 221 (64)
(1%) 222, 22303, 25, 140, 144), 225 Sorger, G. J., 274
(23, 25, 1441, 226(158), 230(158), 234 So&, S., 20
(25, 158), 235(23), 236, 237(15), 238 Sosfenov, N. I., 366
( 2 3 , 240(15), 245, 246, 247(15, 20, Spallhols, J. E., 321, 322(142)
23, 25, 189), 248(23, 24, 25, 26, Spats, L., 100, 148, 151(284a), 154(74),
249(23, 25, 26, 197, 199, 200), 250(23, 155, 156(74), 161(74), 166(74), 167(74)
25, 195, 196, 1971, 251(197), 252(197), Spencer, D., 274,277(337)
253(1&1), 254(15, 16, 251, 255, 257, Spencer, R. L., 100, 103(67)
258(=), 260, 261(%7, 254, 2557 256)* Speranaa, M. L., 100, 103,143(66)
262(227, 2621, 263(266), 284, 2709 271, Spivey, H. O., 3, 41(161)
272(312), 273 Sportorno, G. M., 25,26
Singh, A. P., 79 Spyridakis, A., 265,268(289)
Singh, J., 275 Sreenivasan, A., 260
Sivak, A., 261 Srere, P. A., 88
Sjoberg, B. M., 93, 119(47), 143(47), 144 Srivastava, s. K., 131, 132
(47) Staal, G. E. J., 94, 125, 131, 137(51), 139
Skulachev, P., 214 (51), 140(51)
Skulachev, v. P., 69, 74(120, 121), 75(120, Stachiewicz, E., 272
146, 147, 1481, SO(146, 147) Stadtherr, L. G., 304
Skvaril, F., 367 Stadtman, T. C., 143
‘later’ E’ “’ 28’ 31’ 33(144)’ 34(144)9 41 Stallcup, W. B., 26, 35, 36(113), 37(113,
(159), 63, 74,86(39, 49), 176, 187, 237,
172)
238(172), 251, 307, 390
Slein, M. W., 2, 3(8), 48 Stanbrough, E. C., 143
Slencaka, W., 56, 57(20), 63, 66(20), 81 Stanbury, J’, 315, 316(100), 322, 323(100)
(20), 86(20) Stansell, M. J., 366,382(39)
Sloan, D. L., 32, 33(155) Stark, G. R., 383
Slonimski, P. P., 217, 272 Staudt, H., 153
Sluse, P. E., 88 Steele, W., 244
Smiley, I. E., 24 Stein, A. M., 65, 86(75), 109, 110, 114,
Smillie, L. B., 371(861, 372 117, 118(153)
Smith, C. M., 40, 41, 42, 45(194), 49(194), Stein, J-H., 86,109, 1 1 4 3 118(153)
110 Stellwagen, E., 92, 100(34)
430 AUTHOR INDEX
Stempel, K. E.,180, 181(40), 191, 194(40, Suzuki, T., 372, 373(92)

67), 195, 196, 197, 198(40), 199(68), Swartz, M.N.,62,65(31)
203(68), 206(40, 42), 207(40, 42), 216 Sweat, M.L.,83, 84(210)
(40) Sweetman, A. J., 64, 65, 68(63), 72(63),
Stern, K. G., 363,366,391,397(160) 73(63)
Stevenson, P. M.,65, 85(86) Swoboda, B. E. P., 98, 113(59), 137(59)
Stiggall, D.L.,178,297 Symons, R.H., 267
Stockell, A.,31 Szabolcsi, G.,23, 31
Stolzenbach, F. E.,30, 54, 55(11), 56(11), Szarkowska, L.,258
59(11), 66(11) Szorhyi, E.,2(14), 3
Stotz, E.,310,311(69) Szumilo, T., 273
Straub, F.B.,106, 107(90), 189 Szymona, M., 273
Strength, D.R.,261
Strickland, S., 91 T
Strittmatter, P.,91, 100, 124, 148, 151(9,
17, 284a), 15407, 74, 307, 308), 155 Tagawa, K., 54, 55(12), 56(12), 66(12)
(347), 156(74, 307, 341, 3421, 157 Tager, J. M.,63, 73, 81(40), 82, 86(39,
(3501, 158(354), 159(308, 351), 160 40,49,191),87(53,54),88
(308,354, 355), 161(74, 341, 342, 343, Takahashi, E.,286, 295(395)
344, 3501, 162(171, 341, 342), 163 Takahashi, H.,274, 277(337)
(171, 352), 164(347, 349, 352, 359, Takahashi, Y.,335
360), 165(347), 166(74), 167(74), 168 Takano, T., 371 (85),372
(360) Takemori, A.,259
Strobel, H. W.,149, 153, 169 Takemori, S.,237, 245(173), 306(46), 307,
Stromberg, C., 317,335(108), 336(108) 308(46),334
Strom, R.,237,247 Takesue, S., 154, 165, 166(372), 167(372)
Strother, G.K.,395 Tallan, N.H.,83
Stryer, L.,93,349,364 Tamaoki, B.,83
Studier, M.H., 377 Tamura, G., 274, 286(359), 295(359, 405)
Sturani, E.,100 Tanaka, M.,100
Sturtevant, J. M., 33, 34(156), 268, 269 Tanaka, N.,108
(300) Tanford, C., 367
Stynes, D.V.,302 Taqui Khan, M.M., 407
Stynes, H.C., 302 Tarr, H.L.A., 92
Su, G., 117, llS(l61) Tasaki, A., 366
Suematsu, T., 108 Taube, H., 303
Suggett, A., 368, 369, 388, 389, 395(75), Tauber-Finkelstein, M.,22
397(75) Taylor, E.L.,39
Sugita, Y.,317,318(110), 349 Taylor, J. F.,24
Sullivan, P.A.,90,91,282 Taylor, P. L.,85
Sulmovici, S.,83 Taylor, W.E.,150
Sumner, J. B.,38,440791,366 Teale, F.W.J., 348, 380
Sun, F.F.,306(48), 307, 308(48), 313 Tedeschi, P.,28
Sund, H.,366, 367(48) Teeter, M.E.,205
Susheela, L.,247 Teipel, J., 34, 35(164)
Suter, H., 365 Teixeira da Cruz, A., 64, 69(68, 691, 70
Sutin, N.,375 (67), 72(67), 75(67, 69), 76167, 68,
Suzuki, I.,281, 282(375), 284 691,77(67,68,69),207
Suzuki, K.,2(18, 20), 3, 4(18), 9(18), 19, Telegdi, M., 25
25, 26, 27(103) Teller, D.C.,25
AUTHOR INDEX 431
ter Welle, M. F., 63, 72(47), 73(47), 81 Tu, S. C., 101

(47) Tubbs, P. K., 88, 273
Tepley, L. J., 189 Tucker, A., 300
Testolin, G., 222, 225(150) Tung, T., 256
Thelander, L., 91, 92(8), 93(8), 98(31), 'Turini, P., 255
99(8, 311, 100(31), 101(31), 103(31), Turkki, P. R., 262
104, 142, 143(8, 262, 2631, 144(8, 38), Turner, J. F., 143
145(8, 31), 156(9) Tyler, D. D., 186,203, 262
Theorell, H., 162, 200, 356, 364, 366, 369, Tysarowski, W., 272
370(6), 385, 388, 400(143) Tyson, C. A., 172
Thomas, J. O., 23 Tzagoloff, A., 307, 308(57), 310, 311(57),
Thompson, T. E., 142 317
Thor, H., 150
Thorgeirsson, S. S., 153 U
Thorn, M. B., 247
Thornber, J. M., 109 Ueda, T., 91
Thorpe, C., 123 Uesugi, I., 274
Thurman, R. G., 88 Ullrich, V., 152, 153
Tiesjema, R. H., 317, 318(111), 319, 324, Urnes, P., 372, 373(95)
325, 326(162), 335(157) Urry, D. W., 319
Tietre, F., 130, 143(202) Uigiris, V. I., 65
Tisdale, H., 205, 222, 225, 226(158), 230
(158), 234(158), 248(158), 255 v
Tolbert, N. E., 274
Tomizawa, H. H., 130 Vanngard, T., 317, 330, 335(108), 336(108,
Topali, V. P., 69,74(120), 75(120) 171)
Topper, D. L., 407 Vainshtain, B. K., 366
Toren, D., 83 Vallee, B. L., 213
Torii, K., 279, 372, 373(89), 376(89), 382 Van Ark, G., 332
(89, 107) Van Buuren, K. J. H., 308, 315, 316(101,
Torndal, U. B., 65, 73(166), 78, 207, 212 102), 320(101, 102), 321(102, 132,133),
Tosi, L., 264 335
Tottmar, S. 0. C., 190(133), 217, 218 van Dam, K., 63, 69, 72(47), 73(47, 481,
(133) 74(119), 80(119), 81(47), 86(49), 214
Tove, S., 287(412), 288,295(412) Van Demark, P. J., 260
Trentham, D. R., 21, 23, 28, 35(69), 37 van den Brock, H. W. J., 53, 55, 56(19),
(83, 1171, 39, 40(83, 117), 41(69, 189, 57(9), 58(9), 59(9, 19), 60(9), 62, 80,
190), 42(116, 117), 43(83, 1171, 44, 45 143
Trudgil, P W., 91 Van der Hoeven, T. A., 153, 165, 166
Trudinger, P. A., 286, 295(392) (3701, 170(370)
Triiper, H. G., 285 van de Stadt, R. J., 69,74, 80(119), 214
Tsai, C. S., 117 Van Drooge, J. H., 332
Tsai, P., 150 Van Eys, J., 3, 28(13), 29(13),.38(13), 39
Tsao, M. S., 302 (13), 40(13), 44(13)
Tsernoglou, D., 10 Van Gelder, B. F., 307, 308, 315, 316(101,
Tsofina, L. M., 69,74(120), 75(120) 1021, 317, 318(1111, 319,320(101, 102),
Tsou, C. L., 222, 223(146), 226(146), 246 321(102, 132, 133), 324, 325, 326(162),
(146) 331(139), 332(139), 333(139), 335
Tsuchihashi, M., 367 (157)
Tsudzuki, T., 325, 326(163), 327 van Haefen, H., 63, 86(45)
432 AUTHOR INDEX
Van Heerikhuizen, H., 187 von Ellenrieder, G., 32

Vanko, M., 260 von Wartburg, J. P., 367,379(63), 380(63)
Van Lis, M. J., 33,41(159) Vore, M., 152
Vanneste, W. H., 315, 316(98), 317(98), Vorona, M. K., 48
320(98), 322(98) Vyas, S. R., 189, 195(64), 198(64), 205
Varandani, P. T., 130 (64)
Varshavsky, Y. M., 14,3166)
Veech, R. L., 46, 79, 81(175), 82(175), W
86(175)
Veeger, C., 63, 56, 58(19), 57(9, lg), W d a m , F., 150
58(9, 191, 59(9, 191, 60(9), 62, 80(19), Waentig, P., 367
92, 94(24, 27), 95(27), 96(27), 97(24), Wainio, W. W., 306(49), 307, 308(49),
98(24), 106(27), 107(24, 97, 106, 114), 310, 319, 322(49), 324
111(24), 112(24, 108, 1181, 113(24, Wainwright, T., 286, 287(398), 288, 292
27), 114(27, 114), 115(24), 116(24, (398, 420)
27), 117(27, 157), 118(157, 160), 119 Wakil, S. J., 88, 151
(1601, 120, 122(154), 123(154), 124, Walajtys, E. I., 110
m ( i 7 2 , 173, 174, 1751, i26(174, i79, Walker, G. C.,274, 275
180, 181, 1821, 131, 133, 134(27, 1181, Walker, W. E., 222, 235
136(118), 137(51), 139(51, 157), 140 Wallace, W. J., 319, 321, 323(140), 337
(51), 143, 148(163), 222, 224, 226 (1171, 339(117), 340, 389
(218), 236(149), 237, 238(149), 251, Wallenfels, K., 28, 33
252, 253 Waller, H. D., 130, 131
Vega, J. M., 274, 276(342) Wang, L., 109
Velick, S. F., 3, 9, 19, 29, 30(125), 31 Wang, T. Y., 222, 223, 226, 246(146)
(12), 32(127), 33(155), 34(156), 39, Wang, Y. L., 222, 223(146), 226(146),
40(60), 41(60), 42(60), 44(60), 45 246(146)
184, 194), 48(60), 49(194), 91, 92, Warburg, O., 2, 301, 315, 363, 364(3)
151(9) Wartofsky, L., 91, 14303
Vennesland, B., 39 Warnarman, P. M., 19,21, 28(61)
Vennesland, J. W., 92 Wassink, J. H., 55, 56(19), 57(19), 58(19),
Vermilion, J. L., 165, 166(371), 167(371) 59(19), 80(19), 108, 107(114), 114
Vernon, L. P., 188, 189(57), 199(57) (114)
Vignais, P. V., 84 Watari, H., 190(83), 202,267, 372, 373(922)
Vinogradov, A., 248 Waterman, M. R., 360
Visser, J., 116, 117(157), 118(157), 120, Waters, W. W., 407
124, 125,(175), 126(179, 180, 1811, 139 Watson, H. C., 9, 10(45), 19, 21, 28(61),
(1571, 148(163) 35, 364, 372
Vitols, E., 143, 216 Webb, L., 10
Vladimirova, M. A., 69, 74(120), 75(120) Weber, F., 131
Voetberg, H., 116, 117(157), 118(157), 120, Weber, K., 366, 367(48)
125, 126(180), 139(157), 148(163) Weber, M. M., 110, 168, 169(388)
Vogel, O., 106, 107(107), lOS(l0t) Wedding, R. T., 106, 107(100)
Voigt, B., 31 , Weenen, J. H. M., 39
Volkstein, M. V., 25,31(105) Weil, J. A., 304
Volpe, J. A., 306(52), 307, 308(52), 313 Weinbach, E. C., 66
(52), 315(52), 316, 317(52, 651, 318 Weinhouse, S., 46, 49(214)
(52, 65), 319, 321, 322(65), 323(65, Weiss, L., 88
1401, 325(65), 328(65), 329(65), 333, Welch, M., 85
337(65, 118), 338(65), 339(118), 389 Welinder, K. G., 371(86, 871, 3?2
AUTHOR INDEX
Wells, I. C., 261 137, 138, 1631, 327, 328(1379, 331(124,

Welton, A. F.,165, lsS(369) 138), 332(124, 168),333(137,138,168),
Wendel, A., 130 335(138), 346, 351(21)
Wendell, P. L.,133 Wilson, J. E., 100, 101(64), 103(64), 109
Wenske, G.,63, 86(45) (64), 117, 118(161)
Werner, A,, 303 Wilson, L. D.,83, 84(211)
West, C.A.,66 Wilson, L.G.,91,133(7), 143(7), 279
West, S., 149, 152 Wilson, M.T.,315, 316(99), 323(99), 324
Wever, R.,315, 316(102), 320(102), 321 (99),335,336(99,192),389
(1021, 332 Wilting, J., 335
Wharton, D. C., 275, 301, 306(45), 307, Winell, M.,133
308(45), 310, 312, 322, 324, 325, 327 Winter, D. B., 235, 244(164), 248, 253
(53), 330(53), 335(153), 336(153) (164), 296
White, H.B., 111, 39 Wiss, O., 131
White, R. P., 378, 379(118b), 385(118b), Witkowski, P.E.,262
387(118b), 399(118b), 401, 402(118b), Wit-Peeters, E. M.,88
403(118b), 404, 405 Woenckhaus, C., 24
Wiberg, K. B., 407 Wold, F.,100,103(67)
Widger, W. R.,78, 182, 190(43a) Wolf, B., 109
Wieland, O.,88 Wolman, Y., 379
Wikstrom, M. K. F., 215 Wolny, M., 9
Wilken, D.R., 133, 263 Wonacott, A. J., 10, 19, 20
Wilkins, R. G.,303 Wong, D.,204
Williams, C. H, Jr., 91,92,93, 94(29, 35, Wong, S.H., 83,84(211)
36), 97(29), 98(30, 501, 99(30), 100 Wood, P. M.,303
(29, 33, 36, 37), 101, 103(29, 631, Woodin, T. S., 129, 132(193), 138(193)
104(33, 35, 61, 62, 63), 105(33, 62, Woodrow, G.V.,111,360
85), 106(36), 107(36, 103, 108), 113 Woodward, H.E.,260,261(248)
(29, 59), 114(108), 118(63, 156), 119 Woolf, A. A.,384
(33,35, 63,85, 1561, 120(61, 631,121 Worthington, D. J., 131
(631, 122(156), 123(61), 129, 135(29, Wren, A., 106, 107(101), 112(101), 117
601, 136(60), 137(29, 59, 60, 244), 138 ( 117)
(291, 139(244), 140(244), 141(29, 351, Wiithrich, K.,267, 357
143(36), 144(36,62), 145(30), 146(30), Wyman, J., 31
147(284), 148(58), 166(10), 167(10, Wyss, S.R.,367
245), leS(l0, 245, 3861, 169(245), 170
(245), 172(245) Y
Williams, G.R., 263
Williams, J. N.,Jr., 260, 334 Yagi, K.,176,234,335
Williams, J. R.,81 Yamafuji, K.,274
Williams, R.J. P., 317,318(109), 351,369, Yamamoto, H.,350, 360, 369, 370(80),
370(76), 372(76, 811,374(76, 811, 390 372(80), 376(88)
Williamson, D. H.,46, 82, 86(185, 186) Yamamoto, T., 311
Williamson, J. R., 46, 47(215), 48(219), Yamanaka, T., 274,275,276
110 Yamasaki, I., 122, 169
Willms, B., 82 Yang, C.S.,198,311,312(78)
Willms, C. R., 108 Yang, J., 366,367
Wilmarth, W. K.,302, 304 Yang, P. C.,310
Wilson, D.F.,215,302, 320,321, 322024, Yang, S. T.,25, 30(108), 48(108)
138), 323(124, 137), 325(124), 326(124, Yasunobu, K.T.,100
AUTHOR INDEX
Yeghiayan, A,,372,373(96) Z
Yike, N. J., 64, 66(64, 651, 68(64, 65),
69(65), 72(64, 65), 73(65), 76(65) Zahler, W. L., 313
Yonetani, T.,306(44,46), 307,308(44,46), Zakim, D., 109, llO(130)
320, 330, 331(174), 334, 335(185), 336 Zanetti, G.,92, 94(36), 97, 98(30, 501, 99
(1851, 346, 347(19), 348(14, 26, 27, (30), 100(36), 103(36), 106(36), 107
28, 29, 30, 31, 32, 33, 37, 38, 39), 349 (36), 143(36), 144(36), 145(30), 146
(26, 28, 30, 32, 39, 461, 350, 351(21, (30), 147(284), 148(58), 222,225(150),
22), 352(17, 22), 353(14, 16, 18, 19, 216, 247(191)
20, 22, 34), 355(34, 361, 356(55), 357 Zapponi, M. C., 22,44(76)
(17,38), 359(39), 360(27, 31), 361(17, Zatman, L.J., 53
19), 364, 369, 370(80), 372(80), 376 Zawodsky, P.,14, 25, 26, 31(56, 105)
Yoneyama, Y., 317, 318(110), 349 Zerfas, L. G.,263
Yong, F. C.,306(51), 307, 308(51), 310 Zeszotek, E.,222
(511, 319 Zeylemaker, W. P., 222, 224(149), 236
Yoshida, K., 372,387 (149), 237(149), 238(149, 1721, 251,
Yoshikawa, S.,320, 321(128), 336(134) 252, 253(149)
Yoshimoto, A., 286, 287(400), 288, 292 Zherebkova, N. S., 153
(400, 421, 423, 424), 293, 294, 295 Zidoni, E.,305
(402, 403) Ziegler, D. M.,153, 154, 165(337), 167
Yoshiaawa, K.,108 (3371, 172(337), 224, 236(153, 1541,
You, K. S.,179, 193(32), 214, 226(32) 239
Yu, C.,312 Zumft, W. G., 274
Yu, L.,312 Zuurendonk, P. F.,308, 320, 321(132)
Subject Index
A glyceraldehyde-3-phosphate dehydro-
genase and, 30
Absorption spectra small NADH dehydrogenases and, 196
adenylyl sulfate reductase, 282, 284, 285 transhydrogenases and, 59,69
catalase, 372-374, 381, 382, 397 ubiquinone reductase and, 181, 184, 186,
cytochrome b?, 265, 267 215-216
cytochrome b, reductase, 155-156, 157- Acetylpyridine adenine dinucleotide
158 phosphate, sulfite reductase and,
cytochrome c oxidase, 315-319, 322,323, 288, 290, 292
327 Achromobacter fischeri, nitrite reductase,
cytochrome c peroxidase, 351,353,354 physical properties, 277-279
glutathione reductase, 95, 135-136, 137- Active site, lipoamide dehydrogenase, 105
138 Acyl hydrazides, catalase and, 379
a-glycerophosphate dehydrogenase, 257 Acyltransferase activity, glyceraldehyde-
lipoamide dehydrogenase, 118, 122, 123, 3-phosphate dehydrogenase, 44-45
126 Adenine nucleotides
nitrite reductase, 278 glyceraldehyde-3-phosphate dehydro-
small NADH dehydrogenase, 193, 194 genase and, 2, 45, 46, 48
succinate dehydrogenase, 232, 233 transhydrogenase and, 70
sulfite reductase, 288, 289,291, 293 Adenosine, transhydrogenase and, 71
thioredoxin reductase, 98 Adenosine diphosphate
ubiquinone reductase, 183-184 choline dehydrogenase and, 262,263
Acatalasemia, form of, 367 lipoamide dehydrogenase and, 125
Acetaldehyde, catalase and, 391-392, 406 NADH dehydrogenase and, 207
Acetate succinate dehydrogenase and, 250
catalase and, 383, 385 transhydrogenase and, 71
small NADH dehydrogenases and, 192 Adenosine diphosphate ribose, cyto-
Acetoacetate, succinate dehydrogenase chrome b, reductase and, 163
and, 238 Adenosine diphosphate sulfurylase,
Acetyl coenzyme A, transhydrogenase reaction catalyzed, 282
and, 71 Adenosine monophosphate
Acetyl dephospho coenzyme A, trans- adenylyl sulfate reductase and, 282,
hydrogenase and, 71 283, 284
N-Acetylimidazole, catalase and, 367 cytochrome P-450 reductase and, 167
Acetyl phosphate, glyceraldehyde-3-phos- NADH dehydrogenase and, 188, 207
phate dehydrogenase and, 21, 28, 38- transhydrogenase and, 71
39,43, 44-45 Adenosine 2’-monophosphate
Acetylpyridine adenine dinucleotide sulfite reductase and, 293
cytochrome bs reductase and, 156, 157, transhydrogenase and, 57,58,59,60,
159, 160, 163 61, 71
435
436 SUBJECT INDEX
Adenosine 3'-monophosphate, trans- Ally1 alcohol, catalase and, 401-402, 403,

hydrogenase and, 71 404
Adenosine 3',5'-monophosphate, trans- Amine oxidase, mixed function, 153-154
hydrogenase and, 71 Amino acid (s)
Adenosine 5'-phosphosulfate, formation conservation, glyceraldehyde-3-phos-
of, 279 phate dehydrogenase, 14-19
Adenosine triphosphatase cytochrome bs reductase composition,
cytochrome c oxidase and, 321 155
transhydrogenase and, 79 cytochrome c oxidase composition, 310
Adenosine triphosphate cytochrome c peroxidase composition,
choline dehydrogenase and, 263 348
cytocrome c oxidase and, 300, 302, 343 lactate dehydrogenase, 266
glyceraldehyde-3-phosphate dehydro- pyridine nucleotide-disulfide oxido-
genase and, 25,26, 48,49 reductases, composition, 100, 102
lipoamide dehydrogenase and, 125 sequences
NADH dehydrogenase and, 207 glyceraldehyde-%phosphate dehydro-
,redox potentials and, 215, 216 genases, 6-8
succinate dehydrogenase and, 247, 248, hemoproteins, 371
249 thioredoxins, 144
sulfate reduction and, 279 small NADH dehydrogenase composi-
transhydrogenation and, 63-64, 65, 67- tion, 195
68, 72, 73-74, 77, 80, 81, 207, 214 synthesis, transhydrogenase and, 80-81
Adenylyl sulfate reductase(s) 3-Arnino-1H-1,2,4-triazole, catalase and,
occurrence of, 282 376-378
properties, 279-286 Ammonia, catalase and, 387
Adipose tissue Ammonium sulfate
glyceraldehyde-3-phosphate dehydro- glyceraldehyde-3-phosphate dehydro-
genase in, 47, 48 genase and, 4
glycolytic enzymes, diabetes and, 47 lipoamide dehydrogenase and, 124,125,
Adrenal cortex 126
succinate dehydrogenase and, 229-231
transhydrogenase of, 65
Amytal
function, 83-85
choline dehydrogenase and, 261-262,
Aerobacter aerogenes, sulfate reduction
263
by, 281 respiratory particles and
Aeromonas punctata, sulfate reduction mitochondrial, 199
by, 281 yeast, 217
Alanine residues, glyceraldehyde-3-phos- ubiquinone reductase and, 181,182,
phate dehydrogenase, 11, 12 204, 205
Alcohols, catalase and, 388, 398, 401 Anaerobes, succinate dehydrogenase of,
Aldehydes, glyceraldehyde-3-phosphate 254
dehydrogenase and, 39 Aniline, hydroxylation of, 150
Algae, sulfate assimilation by, 279, 280 Anilino-naphthalene sulfonate
Alkali cytochrome c peroxidase and, 349,359
cytochrome c oxidase and, 311 transhydrogenase and, 69
respiratory particles and, 240-241, 248 Antimycin A
Alkyl bromides, succinate dehydrogenase cholesterol side chain cleavage and, 85
and, 246 choline dehydrogenase and, 262
Alloxan diabetes, cytosolic redox state a-glycerophosphate dehydrogenase and,
and, 46-47 268
SUBJECT INDEX 437
L-lactate dehydrogenase and, 269 nitrite reductase of, 275, 277

small NADH dehydrogenase and, 199 transhydrogenase of, 53,54
succinate dehydrogenase and, 239, 250 function, 80
ubiquinone reductase and, 182, 197 purification, 55, 56
yeast NADH dehydrogenase and, 219
Antiparallel sheet, glyceraldehyde-3- B
phosphate dehydrogenase, 13, 14 Bacillus stearothermophilus
Arginine residues glyceraldehyde-3-phosphate dehydro-
catalase, 395 genase of,2, 4, 9, 23
glyceraldehyde-3-phosphate dehydro- apoenzyme, 19
genase, 12, 22, 24 pyridine nucleotide binding, 34
transhydrogenases and, 213-214, 296 Bacteriophage T4, thioredoxin and, 144
Arsenate, glyceraldehyde-3-phosphate Barbiturates, NADH dehydrogenases
dehydrogenase and, 38, 44-45 and, 203, 204, 206
Arsenite Bathocuproin sulfonate, cytochrome c
lipoamide dehydrogenase and, 95-97, oxidase and, 308
110, 111, 113-114 Bathophenanthroline, succinate dehydro-
sulfite reductase and, 289 genase and, 246
thioredoxin reductase and, 99 Bathophenanthroline sulfonate, NADH
Arsenocholine, choline dehydrogenase dehydrogenase and, 206
and, 261 3,4-Benzpyrene
Arterial tissue, transhydrogenase in, 65 hydroxylation, pyridine nucleotides
Ascites cells, glyceraldehyde-3-phos- and, 152
phate dehydrogenase in, 47 Benzyl viologen, nitrite reductase and,
Ascorbate, cytochrome c peroxidase and, 275, 276
353 Betaine, formation of, 260
Asparagine residues Betaine aldehyde, choline dehydrogenase
glyceraldehyde-%phosphate dehydro- and, 261, 262
genase, 11, 12, 30 Betaine aldehyde dehydrogenase, occur-
lipoamide dehydrogenase, 101 rence of, 260
Aspartate residues, glyceraldehyde-3- Bicarbonate, succinate dehydrogenase
phosphate dehydrogenase, 11, 12, 30 and, 238
Atebrin, L-lactate dehydrogenase and, 264 Borohydride, glutathione reductase and,
Azide 136, 140
catalase and, 376,385,400 Brain
cytochrome c oxidase and, 320-321,328, glyceraldehyde-3-phosphate dehy-
333 drogenase in, 47, 48
cytochrome c peroxidase and, 350,353 a-glycerophosphate dehydrogenase of,
Azotobacter 256-258
lipoamide dehydrogenase of, 114 Bromelain, cytochrome P-450 reductase
transhydrogenase and, 166, 167
molecular properties, 57-59 Bromide
reaction mechanism and regulation, small NADH dehydrogenases and, 192
59-62 succinate dehydrogenase and, 247, 248
Azotobacter anile, transhydrogenase of, 54 3-Bromoacetylpyridine, glyceraldehyde-3-
Azotobacter chroococcum phosphate dehydrogenase and, 24
nitrite reductase of, 275,276-277 Bromopyruvate
transhydrogenase of, 54 glyceraldehyde-3-phosphatedehydro-
Azotobacter vinelandii genase and, 24
NADH dehydrogenase of, 221 succinate dehydrogenase and, 249
SUBJECT INDEX
Bumilleriopsis filiformis, transhydro- Cetyldimethylethylammonium bromide,

genase of, 54 succinate dehydrogenase and, 234
2,3-Butanedione, transhydrogenation Chemiosmotic hypothesis, transhydro-
and, 296 genase and, 74-75
n-Butanol, catalase and, 401402 Chloramphenicol, yeast NADH dehydro-
Butylhydroperoxide, catalase and, 392 genase and, 217
Butyraldehyde, catalase and, 406 Chlorella pyrenoidosa, adenylyl sulfate
reductase in, 282, 283
C Chloride, succinate dehydrogenase and,
247, 248
Cadmium, lipoamide dehydrogenase and, Chlorobium limicola, adenylyl sulfate
114 reductase of, 286
Calcium 5-Chloro-3-t-butyl-2-~11loro-4’-nitro-
choline dehydrogenase and, 263 salicylamide, cytochrome c oxidase
transhydrogenases and, 58, 61, 70 and, 321
Candida utilis, NADH dehydrogenase of, 2-Chloroethanol, catalase and, 401
216-218, 219, 221 p-Chloromercuriphenyl sulfonate
Carbohydrate glutathione reductase and, 141
degradation, a-glycerophosphate dehy- small NADH dehydrogenase and, 203
drogenase and, 259 thioredoxin reductase and, 146
synthesis, transhydrogenase and, 80 ubiquinone reductase and, 197
Carbon monoxide Chloroplasts, transhydrogenase of, 66
catalase and, 401 Chlorosuccinate, succinate dehydrogenase
cytochrome c oxidase and, 301, 317418, and, 237-238
322, 327-328,329, 333, 336-337, 338 Cholate
cytochrome c peroxidase and, 353 NADH dehydrogenase and, 189
heme iron and, 321-323 ubiquinone reductase and, 178, 182, 183
nitrite reductase and, 275, 278-279 Cholesterol, side chain cleavage, 83, 84-85
sulfite reductase and, 289, 293 Choline, oxidation to betaine, 260
Carboxylate groups, catalase, 395 Choline dehydrogenase
Cardiolipin electron transport system and, 261-263
cytochrome c oxidase and, 313 properties, 260-261
ubiquinone reductase and, 183 Chromatium
Carotenoids, transhydrogenase and, 69 sulfate reduction by, 281
Catalase transhydrogenase of, 54
active site function of, 80
distal ligand identity, 376-385 molecular properties, 58, 59
ligand exchange reactions, 385-388 purification, 55, 56
ligand identity at fifth and sixth Chromium
coordination positions, 369-376 complexes, oxygen and, 304
apoprotein, selective modifications, Chymotrypsin, cytochrome b5 reductase
376-385 and, 155
general properties, 366-369 Circular dichroism
historical background, 363-366 catalase, 382
hydroperoxide and, 356 cytochrome c oxidase, 319
redox reactions, 388-389 transhydrogenase and, 62
nature of Compound I, 389-390 Clostridium kluyveri, sulfate reduction
reaction mechanism, 390-408 by, 281
Catalytic domain, glyceraldehyde-3- Clostridium nigrijicans, sulfate reduction
phosphate dehydrogenase, 16 by, 281
SUBJECT INDEX 439
Clostridium pasteurianum, sulfate reduc- Cysteine residues

tion by, 281 glyceraldehyde-3-phosphate dehydro-
Cobalt genase, 5, 12-13, 14, 22, 28, 29, 34,
ammine complexes, oxygen and, 303- 38, 39, 44, 45,
304 modification of, 20-21
Coenzyme A, transhydrogenase and, 70, pK. of, 43
71 Cystine, glutathione reductase and, 132
Complex I, see under Nicotinamide Cystine residues
adenine dinucleotide lipoamide dehydrogenase, 120-122
Complex 1-111 properties of, 197 pyridine nucleotide-disulfide oxidore-
Complex 11, see also Succinate dehydro- ductases and, 95, 101, 104
genase thioredoxin, 92
properties of, 239 Cytochrome(s) ubiquinone reductase
Compound I, nature of, 389-390 and, 179, 180
Configuration, pyridine nucleotide bind- Cytochrome a, see Cytochrome c oxidase
ing and, 31 Cytochrome b
Conformation hypothesis, transhydro- choline dehydrogenase and, 261, 262
genase and, 75 succinate dehydrogenase and, 224, 239,
Copper 244-245
complexes, oxygen and, 304 Cytochrome b2, L-lactate dehydrogenase
cytochrome c oxidase and, 302,307- and, 263-264,266267,296
309, 314, 315, 317,319, 322, 329-330, Cytochrome bs, cytochrome b, and, 267,
338 296
lipoamide dehydrogenase and, 114, Cytochrome b5 reductase
122-123 cytochrome P-450 reductase and, 151-
nitrite reductase and, 275 153
Corpus luteum, transhydrogenase of, 65, function of, 150-151
85 mechanism, microsome bound, 161-162
Cyanide mechanism of Strittmatter, review,
catalase and, 376, 377, 382,385, 387, 156-161
399,401 methemoglobin reductase and, 164-165
cholesterol side chain cleavage and, 85 molecular properties, amphipathic and
cytochrome c oxidase and, 301,308, soluble forms, 154-156
320-321, 335, 336 structural studies, 162-164
cytochrome c peroxidase and, 350,353 Cytochrome c
2-hydroxyacid dehydrogenase and, 272 adenylyl sulfate reductase and, 281,
L-lactate dehydrogenase and, 269 285-286
microsomal electron transport and, 148, choline dehydrogenase and, 261
151 cytochrome b2 and, 267, 268-269
nitrite reductase and, 275, 276, 277,278 cytochrome b5 reductase and, 156
succinate dehydrogenase and, 223,246- cytochrome c oxidase interaction, 334-
247, 250 335
sulfite reductase and, 289, 293 cytochrome c peroxidase an!, 356-360
Cyanogen bromide, catalase and, 379-385 cytochrome P-450 reductase and, 167,
Cycloheximide, yeast NADH dehydro- 168, 169, 170
genase and, 217,220 dehydrogenases and, 90
Cystamine, glutathione reductase and, D-lactate dehydrogenase and, 270
132 nitrite reductase and, 275
Cysteine, lipoamide dehydrogenase and, small NADH dehydrogenases and, 194,
122 195, 196, 198, 199-200, 203,206
440 SUBJECT INDEX
succinate dehydrogenase and, 250 Cytoplasm, cytochrome c oxidase poly-

sulfite reductase and, 288, 290 peptides and, 311
transhydrogenase and, 68
ubiquinone reductase and, 181, 182, 197 D
yeast NADH dehydrogenase and, 217
Cytochrome c oxidase Deaminonicotinamide adenine dinucleo-
biological role, 299-300 tide
chemical and physical properties, 301- glyceraldehyde3phosphate dehydro-
302, 313-314 genase and, 30
electronic spectroscopy, 315-319 transhydrogenase and, 59
electron paramagnetic resonance, Dehydrogenase(s), characteristics of,
329-334 90-91
interaction with cytochrome c, 334- Demerol, ubiquinone reductase and, 204
335 Deoxycholate
kinetic studies, 335-337 ubiquinone reductase and, 178
ligand binding studies, 319-325 yeast NADH dehydrogenase and, 218
models, 314-315 Deoxyribonucleic acid, blactate dehydro-
potentiometry, 325-329 genase and, 264
historical background, 300-301 Dephospho coenzyme A, transhydro-
lipids of, 312313 genase and, 70, 71
mechanisms, 337-344 Desulfotomaculum, adenylyl sulfate re-
metal components, 307-309 ductase in, 282
occurrence of, 300 Desuljovibrio, adenylyl sulfate reductase
preparation, 305-307 in, 282
protein of, 309-312 Desuljovibrio desuljuricans, sulfate re-
Cytochrome c peroxidase duction by, 281
cytochrome c interaction, 356-360 Desuljovibrio vulgaris, adenylyl sulfate
enzymatic activity, 352353 reductase of, 281, 282484,285
general comments, 380361 Detergent, see also specific compounds
historical background, 345-347 cytochrome bs reductase and, 154-156,
preparation and molecular properties, 161, 163
347-348 cytochrome P-450 reductase and, 166
reaction mechanism, 353-356 glyceraldehyde-3-phosphate dehydro-
structural aspects, 348-351 genase and, 26
Cytochrome c reductase transhydrogenaae and, 70
succinate dehydrogenase and, 245 Deuterium, cytochrome b6 reductase
transhydrogenase and, 55 and, 159
Cytochrome cI Deuterium oxide
choline dehydrogenase and, 262 small NADH dehydrogenases and,
ubiquinone reductase and, 205 192-193
Cytochrome cs, adenylyl sulfate reductase succinate dehydrogenase and, 229-230
and, 281 transhydrogenase and, 70
Cytochrome P-450, monooxygenases and, Deuteroethanol, catalase and, 405
a3 Deuteroformate, catalase and, 404
Cytochrome P-450 reductase, 165-166 Diamide, glutathione and, 131
catalytic activities, 167-169 Diaphorase, transhydrogenase and, 59
cytochrome bs reductase and, 151-153 Dibromoacetone, glyceraldehyde-3-phos-
general properties, 166-167 phate dehydrogenase and, 23
mechanism, 169-173 Dichloroacetate, small NADH dehydro-
substrates and components, 149-150 genases and, 192
SUBJECT INDEX 441
Dichlorohydroquinone, cytochrome c oxi- Disulfide (s)

dase and, 325 lipoamide dehydrogenase and, 122
2,6-Dichlorophenolindophenol pyridine nucleotidedisulfide oxidore-
choline dehydrogenase and, 261 ductases and, 92, 98, 100, 105
cytochrome b, and, 267 Disulfide groups, thioredoxin reductase,
cytochrome P-450 reductase and, 167 145-146
a-glycerophosphate dehydrogenase and, 5,5'-Dithiobis(nitrobenzoate)
257 glutathione reductase and, 132
2-hydroxyacid dehydrogenase and, 272 glyceraldehyde-3-phosphate dehydro-
small NADH dehydrogenases and, 196, genase and, 21, 36
198, 199, 203, 206 lipoamide dehydrogenase and, 120,123
succinate dehydrogenase and, 250 Dithionite
sulfite reductase and, 288 adenylyl sulfate reductase and, 284
transhydrogenases and, 59 catalase and, 367,375, 382
ubiquinone reductase and, 181, 182, 197 cytochrome ba,., and, 239, 240
yeast NADH dehydrogenase and, 217 cytochrome c oxidase and, 335, 338
2,3-Dicyano-5,6-dichoro-l,4-benzo- cytochrome P-450 reductase and, 171-
quinone, ubiquinone reductase and, 172
182 iron-sulfur centers and, 216
N,N'-Dicyclohexylcarbodiimide, trans- lipoamide dehydrogenase and, 113
hydrogenase and, 72 small NADH dehydrogenase and, 193,
1,l-Dideuteroethanol, catalase and, 404, 194
405 semiquinone formation and, 90
1,5-Difluoro-2,4-dinitrobenzene,gly- sulfite reductase and, 288
ceraldehyde-3-phosphate dehydrogen- thioredoxin reductase and, 145
ase and, 22 yeast NADH dehydrogenase and, 219
2,2-Difluorosuccinate, succinate dehydro- Dithiothreitol, succinate dehydrogenase
genase and, 237 and, 227,240, 242,248
Digitonin, transhydrogenase preparation Dodecyl sulfate
and, 67 cytochrome c oxidase and, 311
Dihydrolipoamide, physiological form, 93 lipoamide dehydrogenase and, 123-124
Dihydroxyacetone phosphate, a-glycero- succinate dehydrogenase and, 227-228,
phosphate dehydrogenase and, 256- 230-231,234,244,245
257 ubiquinone reductase and, 180
1,2-Dihydroxybenzene 3,5-disulfonate, DT-diaphorase, transhydrogenation and,
see Tiron 52
2,3-Dime thoxy-5,6dimethylbenzo- Duroquinone, ubiquinone reductase and,
quinone, ubiquinone reductase and, 182
181
2,3-Dimethoxy-5-methylbenzoquinone,
ubiquinone reductase and, 181 E
2,4-Dinitrophenol
dehydrogenases and, 63 Echinocystis macrocarpa
succinate dehydrogenase and, 247 seeds, transhydrogenase of, 66
Dioxygen, see Oxygen Electromechanochemical model, brans-
Diphosphatidylglycerol, cytochrome c hydrogenase, 75
oxidase and, 312 Electron economy, cytochrome c oxidase,
Diphosphoglycerate, glyceraldehyde-3- 325-326
phosphate dehydrogenase and, 35,37 Electron paramagnetic resonance
38, 41, 42, 44, 47, 48, 49 adenylyl sulfate reductase and, 284
442 SUBJECT INDEX
catalase, 370, 372, 381, 396 glyceraldehyde-3-phosphate dehydro-

cytochrome c oxidase and, 308309, 314, genase in, 48
316, 317, 321, 322, 327, 335-336 methemoglobin reductase of, 164-165
copper and, 329-330 Escherichia coli
iron and, 331 glutathione reductase of, 102
ligand binding effects, 332-333 dissociation constants, 135
p-oxobishemin and, 333-334 glyceraldehyde-3-phosphate dehydro-
valence state changes and, 331332 genase, hybrids of, 26
cytochrome c peroxidase, 350,351,353, lipoamide dehydrogenase of, 102, 104,
355 105, 110, 114, 115, 126
fumarate reductase, 256 nitrite reductase of, 276, 277
succinate dehydrogenase, 235, 214, 253, ribonucleotide reductase of, 142-143
254 sulfate reduction in, 281
sulfite reductase, 288 sulfite reductase of, 287-290
ubiquinone reductase, 184-187 thioredoxin
yeast NADH dehydrogenase, 219, amino acid composition, 102
220 partial sequence, 93
catalase and, 389,396, 397 thioredoxin reductase
sulfite reductase and, 292, 294-295 general properties, 144-145
Electron transport specificity, 144
choline dehydrogenase and, 261-263 transhydrogenase of, 65, 66
cytochrome c oxidase and, 301-302 energy and, 73
a-glycerophosphate dehydrogenase and, function, 80-81
258 molecular properties, 69
microsomal, 148-149 reconstitution, 79
cytochrome b, reductase system, 150- Esterase activity, glyceraldehyde-3-phos-
151 phate dehydrogenase, 45
cytochrome P-450 reductase system, EthanoI, catalase and, 392,399,401402,
149-150 403, 404, 405
mixed function amine oxidase, 153- Ethylenediaminetetraacetate
154 cytochrome c oxidase and, 308
synergism between systems, 151-153 a-glycerophosphate dehydrogenase and,
mitochondrial, components of, 178-179 258
succinate dehydrogenase and, 223-224, Zhydroxyacid dehydrogenase and, 272-
245, 250 273
transhydrogenase and, 72-74 n-lactate dehydrogenase and, 271
Emasol, cytochrome c oxidase and, 313 lipoamide dehydrogenase and, 126
Energy, conservation and coupling by NADH dehydrogenase and, 206
complex I, 214-216, 296 succinate dehydrogenase and, 247
Energy-coupling, transhydrogenase and, thioredoxin reductase and, 147, 148
71-75 Ethyl hydrogen peroxide, catalase and,
Enoyl coenzyme A reductase, transhy- 391-392, 395
drogenase and, 88 N-Ethylmaleimide
Entnmoeba histolytica, transhydrogenase cytochrome b, reductase and, 163,
of, 66 164
Erythrocytes glutathione reductase and, 141
glutathione reductase of, 93,94, 138 small NADH dehydrogenase and, 203
kinetics, 139-140 succinate dehydrogenase and, 246, 249
substrates, 132 Ethylmorphine, demethylation of, 150
SUBJECT INDEX 443
Ethylpyridylketone, transhydrogenase Ferrocytochrome c, cytochrome c peroxi-

and, 59 daae and, 345, 352, 353,354-355, 357,
Euglena gracilis, transhydrogenase of, 54 361
Ferrous ions, succinate dehydrogenase
F and, 243
Ferrous iron, oxygen reduction and, 304-
Fatty acid(s) 305
cholesterol side chain cleavage and, 85 Flavin
desaturation of, 151 adenylyl sulfate reductase and, 279, 285
w-hydroxylation of, 150 choline dehydrogenase and, 260
synthesis, transhydrogenase and, 88 cytochrome bz and, 268-269
Ferredoxin a-glycerophosphate dehydrogenase and,
sulfite reductases and, 295 256, 257, 260
transhydrogenase and, 54, 59 high molecular weight NADH dehy-
Ferric compounds, sulfite reductase and, drogenase and, 188,190
293 Zhydroxyacid dehydrogenase and, 272,
Ferric ion, cytochrome P-450 reductase 273
and, 168-169 succinate dehydrogenase and, 223-225,
Ferricyanide 226,232234,237,241,243,244,253,
adenylyl sulfate reductase and, 281,282, 254
283, 285 sulfite reductases and, 287
bacterial NADH dehydrogenase and, Flavin adenine dinucleotide
221 adenylyl sulfate reductase and, 282
choline dehydrogenase and, 261 283, 284, 297
cytochrome bl and, 267, 268 choline dehydrogenase and, 261
cytochrome ba reductase and, 156, 159, fumarate reductase and, 256
160, 161, 162 a-glycerophosphate dehydrogenase and,
cytochrome c oxidase and, 321, 327,337 257, 260
cytochrome P-450 reductase and, 168, 2-hydroxyacid dehydrogenase and, 273
171, 172 n-lactate dehydrogenase and, 270,272
dehydrogenases and, 90 lipoamide dehydrogenase and, 123,124,
u-glycerophosphate dehydrogenase and, 125, 127
257 mitochondria1 protein and, 297
high molecular weight NADH dehy- NADH dehydrogenase and, 217
drogenase and, 188, 201,203-204 nitrite reductase and, 275,276, 277,278
2-hydroxyacid dehydrogenase and, 272 pyridine nucleotide-disulfide oxido-
methemoglobin reductase and, 165 reductases and, 92, 94, 95, 97,99,
small NADH dehydrogenase and, 194- 100, 101, 105
195, 196, 198, 199, 203, 206 succinate dehydrogenase and, 234-235
succinate dehydrogenase and, 223, 236, sulfite reductase and, 288, 290,292,294
237, 254 transhydrogenases and, 57-58
sulfite reductase and, 288, 290 Flavin rnononucleotide
thioredoxin reductase and, 148 bacterial NADH dehydrogenase and,
transhydrogenase and, 59 221
ubiquinone reductase and, 181, 182, cytochrome bs reductase and, 162
183, 186, 197, 199,203,207 2-hydroxyacid dehydrogenase and, 272
yeast NADH dehydrogenase and, 217, L-lactate dehydrogenase and, 264, 265
218,219, 220, 221 lipoamide dehydrogenase and, 124-125
Ferrocyanide, cytochrome c peroxidase low molecular weight NADH dehydro-
and, 353, 355 genases and, 191, 193-194, 198
444 SUBJECT INDEX
nitrite reductase and, 275, 277, 278 p- (2-Furylacrylolyl phosphath, glyceral-

succinate dehydrogenase and, 238, 247, dehyde3phosphate dehydrogenase
249 and, 35, 36
sulfite reductase and, 288, 289, 292,
293-294 G
ubiquinone reductase, 179-180, 182, 186
yeast NADH dehydrogenase and, 218 Glucokinase, activity, liver and, 47
Flavoprotein ( s ) Gluconeogenesis, glyceraldehyde-3-phos-
functions of, 175-177 phate dehydrogenase and, 45-49
mitochondrial, 297 Glucose-6-phosphate dehydrogenase,
sulfite and, 282 glutathione reductase and, 131
Fluorescence Glutamate, redox couple, 82
cytochrome bs reductase and, 162, 163 Glutamate dehydrogenase, transhydro-
glyceraldehyde-3-phosphate dehydrogenases and, 52, 82, 85-86
genase, 32 Glutamate residues, glyceraldehyde-3-
lipoamide dehydrogenase, 117-118, 123, phosphate dehydrogenase, 30
124, 126 Glutamine residues, lipoamide dehydro-
thioredoxin, 93 genase, 101
Fluoride bis-N,N(y-Glutamyl)cystine,gluta-
catalase and, 377, 383,385 thione reductase and, 132
cytochrome c oxidase and, 320421,333 Glutathione
cytochrome c peroxidase and, 350, 353 nitrite reductase and, 275
p-Fluorodinitrobenzene, glyceraldehyde- reduced : oxidized ratios, 129-130
3-phosphate dehydrogenase and, 20 reduction of, 87
p-Fluoro-m,m'-dinitrophenylsulfone, reoxidation of, 93
glyceraldehyde-3-phosphate dehydro- Glutathione-cystine oxidoreductase, 130
genase and, 37 Glutathione-homocystine oxidoreductase,
2-Fluoroethanol, catalase and, 401, 403 130
Glutathione-insulin transhydrogenase, 130
Formaldehyde, catalase and, 406
Glutathione peroxidase, 130
Formamide, catalase and, 386, 399
Glutathione protein disulfide oxidoreduc-
Formate
tase, 130
catalase and, 374-375,383, 385, 386,404 Glutathione reductase
succinate dehydrogenase and, 238, 247, amino acid composition, 102, 104, 105
248 cystine residues, 104
Formic acid, catalase and, 388,395,398, kinetic studies, 138-141
399, 403, 405-406 mechanism, 94,97-98,134
Freezing, lipoamide dehydrogenase, 125- metabolic functions, 129-133
126 reaction catalyzed, 92
Fructose-l,&diphosphatase, glutathione reduction of, 112, 113
reductase and, 130 specificity of, 92-93
Fumarate coenzymes and, 91
choline dehydrogenase and, 262 thiol groups, 141-142
a-glycerophosphate dehydrogenase and, two-electron-reduced enzyme, proper-
258 ties, 133-138
succinate dehydrogenase and, 238-239, Glyceraldehyde, glyceraldehyde-3-phos-
247, 253 phate dehydrogenase and, 44
Fumarate reductase Glyceraldehyde-3-phosphatedehydro-
anaerobes and, 254 genase
yeast, 255-256 catalytic properties,
SUBJECT INDEX
mechanism of action, 38-46 H

metabolic role, 45-49
pyridine nucleotide binding, 28-38 Halogenacetic acids, glyceraldehyde-3-
chemical modifications, 20-24 phosphate dehydrogenase and, 2
dissociation and hybridization, 24-27 Hansenula anomala, L-lactate dehydro-
distribution of, 3 genase of, 265, 268, 269
historical background, 2-3 Heart
maximal activities in rat and human cytochrome c oxidase of, 311
tissues, 47-48 glyceraldehyde-3-phosphatedehydro-
metabolic role, 4549 genase in, 47, 48
molecular properties, lipoamide dehydrogenase of, 102, 104
isolation, 3-4 111-114, 116
structure, 5-27 transhydrogenase of, 62-63, 64, 65
other activities of, 44-45 kinetic constants, 76-77
physiological activity, 38-44 molecular properties, 70-71
preexisting asymmetry model, 35-38 preparation, 67-68
pure, sources of, 2 Helix, glyceraldehyde-3-phosphate dehy-
reaction catalyzed, 1 drogenase, 13-14
~-Glycerol-3-phosphatedehydrogenase, Heme
properties, 256-260 cytochrome bf and, 268-269
a-Glycerophosphate, succinate dehydro- sulfite reductases and, 287, 288,289,
genase and, 239, 250 290, 292,293,294,295
a-Glycerophosphate cycle, function of, Heme az, nitrite reductase and, 275,297
259 Heme A, cytochrome c oxidase and, 307,
Glycine 309, 314-315
catalase and, 387-388 Heme G
oxidative decarboxylation of, 108 adenylyl sulfate reductase and, 285, 286
Glycine residues, glyceraldehyde-3-phos- nitrite reductase and, 278
phate dehydrogenase, 12 Heme oxygenase, cytochrome P-450 re-
Glycogen synthetase, glutathione reductase and, 150
ductase and, 130 Hemoglobin, absorption spectra, 317-319
Glycolate, succinate dehydrogenase and, Hemoprotein(s), amino acid sequences,
238 371
Glycolysis, glyceraldehyde-$phosphate Hepatoma, transhydrogenase in, 65
dehydrogenase and, 4549 Hexokinase, activity in muscle, 47
Glyoxylate, succinate dehydrogenase and, Histidine residues
238 catalase, 370-372,375,377-378,381,383,
Glyoxylate cycle, transhydrogenase and, 395
80 cytochrome c peroxidase, 350
Guanidine, glyceraldehyde-3-phosphate glyceraldehyde-3-phosphate dehydro-
dehydrogenase and, 24 genase, 14, 44
Guanidinium chloride modification of, 23-24
cytochrome bz and, 268 lipoamide dehydrogenase, 101
lipoamide dehydrogenase and, 123, 124 succinate dehydrogenase, 235
NADH dehydrogenases and, 199,207 Homocystine, glutathione redubtasq and,
succinate dehydrogenase and, 232 132
Guanosine triphosphate, oxidation of Horseradish peroxidase, reaction cycle,
NAD+-linked substrates and, 110 356
Guiacol, cytochrome c peroxidase and, Human, glyceraldehyde-3-phosphate
353 dehydrogenase of, 9
446 SUBJECT INDEX
Hydrazoic acid, catalase and, 388 phenylalanyl chain, glutathione reduc-

Hydrogen bonds, glyceraldehyde-3-phos- tase and, 130
phate dehydrogenase, 11, 14, 15,25, Iodide, succinate dehydrogenase and,
43 247, 248
Hydrogen peroxide Iodine
catalase and, 388,395,398,403 cytochrome ba reductase and, 163
cytochrome c peroxidase and, 345-346 glyceraldehyde-3-phosphate dehydro-
Hydrogen sulfide, formation of, 279 genase and, 22-23, 24
Hydroperoxides Iodoacetamide
catalase and, 398, 406 glyceraldehyde-3-phosphate dehydro-
cytochrome c peroxidase and, 352,353, genase and, 35, 36-37,38,42,43
354 lipoamide dehydrogenase and, 118-119,
Hydrophobic bonds, catalase and, 367 122
Hydroquinone, cytochrome c peroxidase Iodoacetate
and, 353 glyceraldehyde-3-phosphate dehydro-
D-2-Hydroxyacid dehydrogenase, proper- genase and, 20,28,35, 36-37,38,
ties, 272-273 39, 42-43, 45
p-Hydroxybutyrate, redox couple, 82 lipoamide dehydrogenase and, 120, 123
~-2-Hydroxybutyrate,D-laCtate dehydro- a-Iodopropionamide, glyceraldehyde-3-
genase and, 270 phosphate dehydrogenase and, 33
25-Hydroxycholecalciferol,hydroxylation a-Iodopropionic acid, glyceraldehyde-3-
of, 83 phosphate dehydrogenase and, 33
Hydroxylamine Iodosobenzoate, glyceraldehyde-3-phos-
catalase and, 388, 398 phate dehydrogenase and, 20, 21
formation of, 153 Ionic strength
nitrite reductase and, 275,276, 277,278, cytochrome P-450 reductase and, 167
279 glutathione reductase and, 139-140
sulfite reductase and, 288-289,290, 292, glyceraldehyde-3-phosphate dehydro-
293, 294, 295 genase and, 25, 26,43
p-Hydroxymercuribenzoate, transhydro- Iron, see also Nonheme iron
genase and, 58 adenylyl sulfate reductase and, 279, 297
cu-Hydroxymonocarboxylic acids, L-lactate carbon monoxide and, 321-323
dehydrogenase and, 267 catalysis and, 363-364
5-Hydroxy-1,4-naphthoquinone,see cytochrome c oxidase and, 302,307-
Juglone 309, 321
17-Hydroxyprogesterone, hydroxylation electron paramagnetic resonance, 331
of, 150 deficiency, NADH dehydrogenases
Hyponitrite, nitrite reductase and, 275 and, 219, 221
a-glycerophosphate dehydrogenase and,
I 256, 257, 260
high molecular weight NADH dehydro-
Imidazole, catalase and, 370 genases and, 188, 190,201-202
Infrared spectroscopy, cytochrome c low molecular weight NADH dehydro-
oxidase and, 321,322, 323 genases and, 191, 193-194,198
Inosine diphosphate, succinate dehydro- mitochondria1 flavoprotein and, 297
genase and, 247, 248 nitrite reductase and, 275
Inosine triphosphate, succinate dehydro- sulfite reductase and, 288, 290, 292,293,
genase and, 247, 248 295
Insulin ubiquinone reductase and, 179, 180, 182,
glycolytic enzymes and, 47 183-184, 186-187, 204, 209-210
SUBJECT INDEX 447
Iron-copper coupling, cytochrome c oxi- Lactobacillus leichmannii, ribonucleotide
dase, 326-327, 338-343 reductase of, 142
Iron sulfide, succinate dehydrogenase, Lecithin, transhydrogenase and, 70-71
230-232,235,243,244, 245,246,253, Leucine residues
254, 296 disulfide oxidoreductases, 104
Iron-sulfur centers glyceraldehyde-%phosphate dehydro-
bacterial NADH dehydrogenase and, genase, 11, 12
221 Leucomethylene blue, nitrite reductase
energy conservation and, 214-216, 296 and, 275
yeast NADH dehydrogenase and, 217, Leucomethyl viologen, 2-hydroxyacid
218, 219 dehydrogenase and, 272
Isocitrate dehydrogenase, transhydro- Lipase, cytochrome P-450 reductase and,
genase and, 52-53,81,84,86-88 167, 168,169,170-171, 172
Isoleucine residues, disulfide oxidoreduc- Lipid
tases, 104 cytochrome bs reductase and, 151, 158
Isonitriles, catalase and, 401 cytochrome c oxidase and, 309, 311,
Isozymes, lipoamide dehydrogenase,. 109 312-313
high molecular weight NADH dehy-
J drogenase and, 188
peroxidation, cytochrome P-450 reduc-
Juglone tase and, 168,169
ubiquinone reductase and, 182 transhydrogenase and, 70
yeast NADH dehydrogenase and, 219, Lipoamide dehydrogenase(s1
220 amino acid composition, 102, 104,105
apoenzymes, 124
K coenzyme specificity, 94
cystine residues, 104, 120-122
distribution, 106107
a-Keto acids
kinetic studies, 115-117
decarboxylation of, 108
mechanism, 94-97, 126-129
2-hydroxyacid dehydrogenase and, 272
a-Ketoglutarate, reductive carboxylation mechanism of Massey and Veeger,
review of, 111
of, 87
Kidney metabolic functions, 107-1 10
transhydrogenase in, 64 reaction catalyzed, 92
function, 83 role of NAD' as modifier, 117-120
Kinetic studies, cytochrome c oxidase, structural studies, 120-126
335-337 two-electron-reduced enzyme, proper-
ties, 111-115
1 Lipoate
glutathione reductase and, 93, 132, 140
Lactate dehydrogenase(s1, types of, 263 ubiquinone reductas? and, 181
n ( -)-Lactate dehydrogenase, 269-270 Lipoprotein, cytochrome c oxidase and,
enzymic properties, 270-272 302
physical properties, 270 Liver
L(+)-Lactate dehydrogenase glyceraldehyde-3-phosphate dehydro-
cytochrome b, core, 266-267 genase in, 47, 48
enzymic properties, 267-269 glycolysis, cytosolic redox state and, 46
historical background, 263-264 lipoamide dehydrogenase of, 116-117
physical properties, 264-266 transhydrogenase in, 64
SUBJECT INDEX
Lobster muscle cytochrome P-450 reductase and, 168,

glyceraldehyde-3-phosphate dehydro- 169
genase 2-hydroxyacid dehydrogenase and, 272
cysteine residues, 20,28 small NADH dehydrogenases and, 194-
hybrids of, 26-27 195, 196, 206
lysine residues, 22 sulfite reductase and, 288, 290
primary structure, 5, 6-8, 9, 18 ubiquinone reductaae and, 181,182, 197,
pyridine nucleotide binding, 33 207
tyrosine residues, 22-23 yeast NADH dehydrogenase and, 218,
Lubrol, NADH dehydrogenase and, 189 220
Lysine residues Menadione reductase, transhydrogenase
cytochrome bs reductase, 164 and, 55
dihydrolipoate and, 108 Mercaptoethanol
glutathione reductase, 105, 119 cytochrome c oxidase and, 311
glyceraldehyde-3-phosphatedehydro- small NADH dehydrogenase and, 198
genase, 11, 14, 44, 45 succinate dehydrogenase and, 243
modification of, 21-22,3748 Mercurials
lipoamide dehydrogenase, 119, 126 glyceraldehyde-3-phosphate dehydro-
thioredoxin, 119 genase and, 21
Lysolecithin, transhydrogenase and, 68,71 large NADH dehydrogenase and, 203-
Lysophosphatidylethaolamine, cyto- 204
chrome P-450 reductase and, 150 lipoamide dehydrogenase and, 114, 122
Lysosomes, cytochrome bs reductase and, low molecular weight NADH dehydro-
154-156 genases and, 191, 199, 203
nitrite reductase and, 276
M succinate dehydrogenase and, 246
sulfite reductase and, 292
Magnesium ubiquinone reductase and, 181, 186,
choline dehydrogenase and, 263 199, 203, 204
transhydrogenase and, 70,77 p-Mercuribenzoate
Magnetic susceptibility, catalase, 382,396 choline dehydrogenase and, 261
Malate cytochrome ba reductase and, 163
choline dehydrogenase and, 262 cytochrome c peroxidase and, 348
succinate dehydrogenase and, 237 cytochrome P-450 reductase and, 168
Maleate, succinate dehydrogenase and, L-lactate dehydrogenase and, 264
238 nitrite reductase and, 275, 276, 278
“Malic” enzyme, steroid hydroxylations sulfite reductase and, 293
and, 83-84 p-Mercuriphenyl sulfonate
Malonate n-lactate dehydrogenase and, 27l’
choline dehydrogenase and, 263 sulfite reductase and, 288, 289
succinate dehydrogenase and, 238, 247, yeast NADH dehydrogenase and, 217
248 Mersalyl
Manganese cytochrome bs reductase and, 163, 164
nitrite reductase and, 277 cytochrome P-450 reductase and, 168
transhydrogenase and, 70 small NADH dehydrogenase and, 193
Membrane(s) succinate dehydrogenase and, 246
integrity, glutathione and, 131 Metal(s)
Menadione transition, oxygen reduction and, 303
bacterial NADH dehydrogenase and, Methanol, catalase and, 391, 392, 401,
221 403,404,405,406
SUBJECT INDEX
Methemoglobin reductase, cytochrome ba Mitochondria

reductase and, 164-165 choline dehydrogenase and, 260-263
Methionine residues fatty acid synthesis in, 88
glyceraldehyde-3-phosphate dehydro- glutamate and isocitrate metabolism,
genase, 11 85-88
lipoamide dehydrogenase, 118 a-glycerophosphate dehydrogenase of,
5-Methoxyindole-2-carboxylate, lipo- 256-259
amide dehydrogenase and, 110 membranes, cytochrome c oxidase and,
Methylene blue 302
choline dehydrogenase and, 261 monooxygenase reactions, 83-85
cytochrome b, and, 267 nicotinamide adenine dinucleotide de-
a-glycerophosphate dehydrogenase and, hydrogenases, 177-178
257 energy conservation and, 214-216
2-hydroxyacid dehydrogenase and, 272 high molecular weight, 187-189
nitrite reductase and, 275 inhibitors of, 203-207
succinate dehydrogenase and, 246 low molecular weight, 189-198
ubiquinone reductase and, 182 relevance of low and high molecular
Methylene succinate, succinate dehydro- weight dehydrogenases, 198-203
genase and, 238 transhydrogenation and, 207-214
3-Me thylflavin adenine dinucleotide, ubiquinone reductase (Complex I),
lipoamide dehydrogenase and, 125 178-187
Methyl hydrogen peroxide, catalase and, nicotinamide nucleotides, redox state,
390-391, 398, 405 81-82
Methyl hydroperoxide, catalase and, 392 transhydrogenase of, 62, 65
N-Methyl hydroxylamine, catalase and, assay, 66-67
399 preparation, 67-68
o-Me thy1 hydroxylamine, catalase and, Models, cytochrome c oxidase, 314-315
399 Molecular weight, succinate dehydro-
2-Methylnaphthoquinone, see Menadione genase, 232-234
Methyl succinate, succinate dehydro- Molybdenum
genase and, 237-238
bacterial NADH dehydrogenase and,
Methyl viologen
221
adenylyl sulfate reductases and, 281
ubiquinone reductase and, 187
nitrite reductase and, 276
Monochloroacetate, small NADH dehy-
sulfite reductases and, 287, 288-289, 290,
drogenases and, 192
292, 293
Micrococcus denitrifians, transhydro- Monofluorosuccinate, succinate dehydro-
genase of, 73 genase and, 237
Microorganism(s) , succinate dehydro- Monooxygenase ( s ), transhydrogenases
genase of, 254-256 and, 83-85
Microsomes Musca domestica, a-glycerophosphate
electron transport, 148-149 dehydrogenases of, 258-259
cytochrome b, reductase system, 150- Muscle
151 glyceraldehyde-3-phosphate .dehydro-
cytochrome P-450 reductase system, genase in, 47,48
149-150 transhydrogenase in, 64
mixed function amine oxidase, 153- Mycobacterium, 2-hydroxyacid dehydro-
154 genase of, 273
synergism between systems, 151-153 Mycobacterium phlei, transhydrogenase
hydroxylation reactions in, 87 of, 65-66
450 SUBJECT INDEX
Myoglobin, absorption spectra, 317-319 glutathione reductase and, 134-137

Myohematin, 300 large NADH dehydrogenase and, 202
nitrite reductase and, 275,276, 278
N respiratory particles and, 200
l,2-Naphthoquinone 4-sulfonate1 cyto- sulfate reduction and, 281,283
chrome b, and, 267 sulfite reductases and, 287-295
Neotetrazolium, cytochrome P-450 re- transhydrogenation, complex I and,
ductase and, 168 207-214,296
Nerve transmission, glutathione and, 131 ubiquinone reductase and, 181
Neurospora crassa Nicotinamide adenine dinucleotide-ubi-
cytochrome c oxidase of, 311 quinone reductase, 178-179
nitrite reductase of, 275-276 activities, 180-183
Nicotinamide adenine deoxydinucleotide, composition, 179-180
glyceraldehyde-3-phosphate dehydro- spectral properties, 183-187
genase and, 30 Nicotinamide bdeaminoadenine di-
Nicotinamide adenine dinucleotide nucleotide, glyceraldehyde-3-phos-
choline dehydrogenase and, 261,262 phate dehydrogenase and, 30
cooperativity of binding, 30-35 Nicotinic acid hydrazide adenine dinucle-
cytochrome c oxidase and, 335 otide, glyceraldehyde-3-phosphate
glyceraldehyde-3-phosphate dehydro- dehydrogenase and, 30
genase and, 3, 4, 26,42,45, 48 Nicotinamide mononucleotide, trans-
amino acid modification and, 21, 22 hydrogenase and, 59
binding, 10-16, 28-38, 49 Nicotinamide nucleotide transhydro-
pK. and, 43 genase
inhibition by, 206-207 AB-specific
lipoamide dehydrogenase and, 117-120, historical, 62-64
125, 126, 128 kinetics and reaction mechanism, 75-
nitrite reductase and, 275, 277, 278 78
oxidized : reduced ratio, glycolysis and, molecular properties, 69-71
46, 49 occurrence, 64-66
succinate dehydrogenase and, 252 preparation and assay, 66-69
Nicotinamide adenine dinucleotide dehy- reconstitution, 78-79
drogenase relationship to energy-coupling sys-
Azotobacter vinelandii, 221 tem, 71-75
high molecular weight, 187-189, 190 BB-specific
relevance to mitochondrial enzyme, historical, 52-53
198-203 molecular properties, 57-59
inhibitors of, 203-207 occurrence, 63-54
low molecular weight, 189-198, 295 purification and away, 54-57
relevance to mitochondrial enzyme, reaction mechanism and regulation,
198-203 59-62
transhydrogenase and, 78 definition, 5162
yeast, 216-221 physiological roles, 79-81
Nicotinamide adenine dinucleotide 3'- fatty acid synthesis, 88
phosphate, transhydrogenase and, 59, mitochondrial glutamate and iso-
61, 69 citrate metabolism, 85-88
Nicotinamide adenine dinucleotide phos- mitochondrial monooxygenase reac-
phate tions, 83-85
cytochrome bs reductase and, 156, 157, redox state of mitochondrial nicotin-
160 amide nucleotides, 81-82
SUBJECT INDEX 45 1
Nicotinylhydroxamic acid adenine dinu- Oxalate

cleotide, glyceraldehyde-3-phosphate 2-hydroxyacid dehydrogenase and, 272
dehydrogenase and, 30 n-lactate dehydrogenase and, 271
Nigericin, transhydrogenase and, 72 Oxaloacetate
Nitrate choline dehydrogenase and, 262
reduction of, 273 D-lactate dehydrogenase and, 271
succinate dehydrogenase and, 227, 247, succinate dehydrogenase and, 237,238,
248 248-249
Nitric acid, ferrocytochrome c peroxi- Oxidase(s), characteristics of, 90-91
dase complex, 350 Oxidative phosphorylation, transhydro-
Nitric oxide genase and, 73
catalase and, 372,375,376 Oximes, catalase and, 398
nitrite reductases and, 274,275 p-Oxobishemin A, cytochrome c oxidase
Nitrite and, 333-334
catalase and, 391, 392,400 Oxygen
sulfite reductase and, 288-289,290,292, adenylyl sulfate reductase and, 282,284
293, 294, 297 choline dehydrogenase and, 261, 262
Nitrite reductase (s) cytochrome b, reductase and, 156
occurrence of, 274 cytochrome c oxidase and, 323-325,
properties of, 275-279, 297 336-337, 338-343
Nitrogen, nitrite reductases and, 274 cytochrome P-450 reductase and, 170
Nitrogen mustard, choline dehydrogenase electronic structure, 303
and, 261 a-glycerophosphate dehydrogenase and,
Nitrones, formation of, 153 260
p-Nitrophenylacetate, glyceraldehyde-3- heme absorption spectra and, 317419
phosphate dehydrogenase and, 21,45 lactate dehydrogenases and, 269,270
Nitrous acid, catalase and, 388,398 microsomal mixed function oxidations
Nitrous oxide, catalase and, 400 and, 149, 152
Nonheme iron, see also Iron reduction, chemistry of, 302-305
adenylyl sulfate reductase and, 282, succinate dehydrogenase and, 236
284, 285 sulfite reductase and, 288
choline dehydrogenase and, 260
succinate dehydrogenase and, 223-225, P
226
sulfite reductases and, 287 Palmitylcoenzyme A, transhydrogenase
Nuclear magnetic resonance, ferrocyto- and, 70,78, 88, 212
chrome c, 357-359 Palmityldephosphocoenzyme A, trans-
hydrogenase and, 70, 71
0 Pantothine, glutathione reductase and,
132
Oleate, gluconeogenesis and, 46, 47 Peas, glutathione reductase of, 138
Oligomycin Pea seed, glyceraldehyde-3-phosphate
succinate dehydrogenase and, 249 dehydrogenase of, 40
transhydrogenase and, 67-68, 72 Penicillium chrysogenum, glutathione
Optical rotatory dispersion reductase of, 138
glyceraldehyde-3-phosphate dehydro- Peptococcus glycinophilus
genase, 14 glycine decarboxylation by, 108
transhydrogenase, 62 lipoamide dehydrogenase of, 112
Ovary Peracetic acid, catalase and, 392-393,
transhydrogenase, function, 83,85 395, 397
452 SUBJECT INDEX
Perchlorate Phosphate
small NADH dehydrogenases and, 191, choline dehydrogenase and, 263
192, 203 glyceraldehyde-3-phosphate dehydro-
succinate dehydrogenase and, 227-228, genase and, 44,48
229,231,232, 247,248 nitrite reductase and, 275
Perfluoro-n-hexane, oxygen consumption succinate dehydrogenase and, 247
and, 152-153 Phosphatidylcholine
Peroxide, see also Hydrogen peroxide cytochrome c oxidase and, 312, 313
oxygen reduction and, 303,305 cytochrome P-450 reductase and, 149
PH Phosphatidylethanolamine, cytochrome c
cytochrome bs reductase and, 163,164 oxidase and, 312, 313
cytochrome c peroxidase and, 350-351 Phosphatidylinositol, cytochrome c oxi-
cytochrome P-450 reductase and, 167, dase and, 312, 313
168 3'-Phosphoadenosine 5'-phosphosulfate,
glutathione reductase and, 134, 140, 141 formation of, 279
glyceraldehyde-3-phosphate dehydro- Phosphocreatine, glyceraldehyde-3-phos-
genase and, 41,48,49 phate dehydrogenase and, 48
lipoamide dehydrogenase and, 116, 119, Phosphofructokinase
125, 128 activity in adipose tissue, 47
small NADH dehydrogenases and, 194- glycolysis and, 49
195, 203 3-Phosphoglycerate, a-glycerophosphate
succinate dehydrogenase and, 247, 248 dehydrogenase and, 258
thioredoxin reductase and, 144 Phosphoglycerate kinase
transhydrogenases and, 58, 76,208,210- glycolysis rate and, 46,49
211, 213
pyruvate kinase and, 47
o-Phenanthroline
Phospholipase (9)
2-hydroxyacid dehydrogenase and, 272 choline dehydrogenase and, 260
n-lactate dehydrogenase and, 271-272,
transhydrogenase and, 70
273
Phospholipase A
NADH dehydrogenases and, 206
cytochrome c oxidase and, 313
succinate dehydrogenase and, 246
Phenazine methosulfate
choline dehydrogenase and, 261, 262, 257
263 n-lactate dehydrogenase and, 270,271
a-glycerophosphate dehydrogenase and, NADH dehydrogenase and, 187
257, 258 Phospholipase C
n-lactate dehydrogenase and, 270 low molecular weight NADH dehydro-
succinate dehydrogenase and, 223,225, genase and, 191
227, 232,236,237, 238, 239,242, yeast NADH dehydrogenase and, 218
243, 246, 249-250, 253, 254 Phospholipid
Phenobarbital, cytochrome P-450 reduc- amine oxidase and, 153
tase and, 150 cytochrome bs reductase and, 161
Phenols, catalase and, 398 succinate dehydrogenase and, 244,245,
Phenylalanine residues, glyceraldehyde-3- 247
phosphate dehydrogenase, 11, 33 synthesis, a-glycerophosphate dehydro-
Phenyl mercuric acetate, glutathione genase and, 259
reductase and, 141 ubiquinone reductase and, 179, 180,
Phenylmethylsulfonyl fluoride, L-lactate 182-183, 201
dehydrogenase and, 265,266 Photoirradiation, semiquinones, 90
SUBJECT INDEX 453
Photooxidation, glyceraldehyde-3-phos- Protein disulfide isomerase, glutathione

phate dehydrogenase and, 24 and, 132
Photosynthesis, transhydrogenation and, Proteus mirabilis, sulfate reduction by,
64, 66 281
Piericidin A Proteus vulgaris, sulfate reduction by, 281
respiratory particles and, 199 Protoheme, cytochrome c peroxidase and,
small NADH dehydrogenase and, 199 345,346, 348,349
transhydrogenation and, 211-212 Protons, transhydrogenase and, 77-78
ubiquinone reductase and, 181,203, Protoporphyrin IX, catalase and, 366
204-206, 214 Pseudomonas
yeast NADH dehydrogenase and, 219, transhydrogenase
220 function, 80
Pig muscle, molecular properties, 58-59
glyceraldehyde-3-phosphate dehydro- reaction mechanism and regulation,
genase of, 6-8, 18, 24 59-61
hybrids of, 26-27 Pseudomonas aeruginosa
tyrosine residues, 23 nitrite reductase of, 274, 275
Plants, transhydrogenases of, 54 transhydrogenase of, 53
Plasmalogen, biosynthesis, 151 molecular properties, 57
Polyacrylamide gel electrophoresis, suc- purification, 54, 56
cinate dehydrogenase, 227-228,230, Pseudomonas denitrificans, nitrite reduc-
245 tase of, 274275
Potassium bromide, cytochrome bs re- Pseudomonas fluorescena
ductase and, 162 transhydrogenase of, 52-53
Potassium ions, transhydrogenase and, purification, 54, 56
72 Pseudomonas hydrophila, sulfate reduc-
Potentiometry tion by, 281
cytochrome c oxidase and, 322 Pseudomonas stutzeri, nitrite reductase
electron economy, 325-326 of, 274
interpretation and summary, 328-329 Pyocyanine, nitrite reductase and, 275
iron-copper coupling, 326-327 Pyridine adenine dinucleotide, transhy-
ligand binding, 327-328 drogenase and, 59
Progesterone, hydrdxylation of, 150 Pyridine aldehyde adenine dinucleotide
Proline residues, glyceraldehyde-3-phos- cytochrome bs reductase and, 156,158-
phate dehydrogenase, 11 159
n-Propanol, catalase and, 401-402 glyceraldehyde-3-phosphate dehydro-
Propargyl alcohol, catalase and, 401-402, genase and, 30
403 transhydrogenase and, 59
Propionibacterium arabinosum, a-gly- Pyridine nucleotidedisulfide oxidore-
cerophosphate dehydrogenase of, 260 ductases
2-Propyn-1-01, catalase and, 404 mechanism, similarities and contrasts,
Prostaglandin, biosynthesis, 168 94-99
Prosthetic groups, succinate dehydrogen- reaction catalyzed-chemical similarities
ase, 234-235 and cross-reactivity, 92-94
Protease, D-lactate dehydrogenase and, structure, similarities and contrasts,
270 99-105
Protein(s) Pyridoxal phosphate, glyceraldehyde-3-
catalase function and, 369-370 phosphate dehydrogenase and, 22
cytochrome c oxidase and, 309-312 Pyrogallol, cytochrome c peroxidase and,
synthesis, glutathione and, 131 353
SUBJECT INDEX
Pyrophosphate Rhodospiiillum rubrum

lipoamide dehydrogenase and, 125 succinate dehydrogenase of, 254-255
transhydrogenase and, 72 transhydrogenase of, 66,69, 213
Pyruvate energy and, 73,74
gluconeogenesis and, 47,48 molecular properties, 69,71
oxidation, transhydrogenase and, 80 preparation, 68
Pyruvate dehydrogenase reconstitution and, 78
lipoamide dehydrogenase and, 126 Riboflavin
transhydrogenase and, 55 cytochrome ba reductase and, 162
Pyruvate kinase, glyceraldehyde-3-phos- deficiency, glutathione reductase and,
phate dehydrogenase and, 47 131
Pythium ultimum, lipoamide dehydro- nitrite reductase and, 275
genase of, 112 Ribonucleotide reductase, thioredoxin
and, 142-143
0 Rose Bengal, cytochrome c peroxidase
and, 350
Quinoline oxide, choline dehydrogenase Rotenone
and, 262 cholesterol side cleavage and, 85
Quinones, sulfite reductase and, 293 choline dehydrogenase and, 262-263
small NADH dehydrogenases and, 196,
R 199
transhydrogenation and, 211
Rabbit muscle ubiquinone reductase and
glyceraldehydea-phosphate dehydro- mitochondrial, 181,182,183,197,204-
genase, 25 206,214-215
amino acid modification, 22,24 yeast, 217,219
hybrids of, 26-27
pyridine nucleotide binding, 33-35 S
Rat muscle, glyceraldehyde-3-phosphate
dehydrogenase of, 25 Saccharomyces carlsbergensis, NADH
Redox potentials dehydrogenase of, 216, 219-221
cytochrome c oxidase, ligand binding Saccharomyces cerevisiae, see also Yeast
and, 327-328 adenylyl sulfate reductase of, 283
cytochrome P-450 reductase and, 172 lactate dehydrogenase of, 269-270
Reductase(s), characteristics of, 90-91 L-lactate dehydrogenase of, 264-266,
Respiration, transhydrogenation and, 64 268, 269
65, 72 NADH dehydrogenase of, 216,217,
R hodopseudomonas palustris 219-221
sulfate reduction by, 281 succinate dehydrogenase of, 254
transhydrogenase of, 66 sulfite reductase of, 292-295
R hodopseudomonas spheroides transhydrogenase of, 66
sulfate reduction by, 281 Salmonella typhimurium, sulfite reduc-
transhydrogenase of, 66 tase of, 290-291
molecular properties, 71 Seconal
preparation, 68 ubiquinone reductase and
reconstitution, 79 mitochondrial, 204
Rhodopseudomonas viridis, transhydro- yeast, 217
genase of, 66 Semicarbazide
Rhodospirillum moliachianum, trans- catalase and, 379
hydrogenase of, 66 choline dehydrogenase and, 262
SUBJECT INDEX 455
Semiquinone molecular properties, 223-226, 296

cytochrome P-450 reductase, 170, 172 regulatory properties, 247-251
dehydrogenases and, 90, 97-98 microorganisms and, 254-256
glutathione reductase and, 137-138 Succinyl coenzyme A, succinate dehydro-
oxidases and, 90 genase and, 247
thioredoxin reductase, 147-148 Sulfate
Serine residues, pyridine nucleotide-disul- nitrite reductase and, 275
fide oxidoreductases, 101 reduction, pathways, 279,281
Serum albumin, lipoamide dehydrogenase succinate dehydrogenase and, 247
and, 126 Sulfhydryl groups, see also Thiol groups
j3-Sheet, glyceraldehyde-3-phosphate de-
catalase, 368
hydrogenase, ll, 14 glyceraldehyde-3-phosphate dehydro-
Soybean genase and, 2
leaves, nitrite reductase of, 277-278
Sulfhydryl reagents, transhydrogenase
Spinach and, 69-70
lipoamide dehydrogenase of, 112
Sulfide
transhydrogenase
acid-labile, ubiquinone reductase and,
molecular properties, 59 179, 180, 182, 184, 186-187, 204,
purification, 54-55, 56 209-210
Spleen, glyceraldehyde-3-phosphate de-
adenylyl sulfate reductase and, 284-
hydrogenase in, 48
285, 297
Starvation, cytosolic redox state and, 46
high molecular weight NADH dehy-
Steroid(s)
drogenases and, 90,201-202
metabolism, transhydrogenase ahd, 65,
83-84 low molecular weight NADH dehydro-
Steroid 17,20-lyase, cytochrome P450 re- genases and, 191, 193-194, 195,198,
ductase and, 150 206
Streptococcus jnecalis, a-glycerophos- mitochondria1 flavoprotein and, 297
phate dehydrogenase of, 260 succinate dehydrogenase and, 224, 226,
Sturgeon muscle, glyceraldehyde-3-phos- 243
phate dehydrogenase of, 28, 33, 42 sulfite reductase and, 287, 288, 289, 290,
Subunits 292, 293, 295
catalase, 366 Sulfite
cytochrome b?, 264-266 adenylyl sulfate reductase and, 282, 284
cytochrome c oxidase and, 311 lipoamide dehydrogenase and, 122
glyceraldehyde-3-phosphate dehydro- oxidases and, 90
genase, primary structure, 5 Sulfite reductaseb)
succinate dehydrogenase, 230-232 NADPH-dependent
Succinate occurrence, 286,287-288
cholesterol side chain cleavage and, 85 properties, 288-295
succinate dehydrogenase preparation reduced methyl viologen-dependent,
and, 223-224, 227,242-243, 248 295
Succinate dehydrogenase types of, 286-287
a-glycerophosphate dehydrogenase and, Superoxide
258 catalase and, 398
mammalian, 222-223 cytochrome P450 reductase and, 169
enzymic properties, 236-245 dehydrogenases and, 90
inhibitors and modifiers, 245-247 mixed function oxidations and, 153
mechanism, 251-254 oxygen reduction and, 303
456 SUBJECT INDEX
T Thionicotinamide adenine dmucleotide

glyceraldehyde-3-phosphate dehydro-
Temperature genase and, 30
glyceraldehyde-3-phosphate dehydro- transhydrogenase and, 57,59,60,6142,
genase and, 25 69
lipoamide dehydrogenase and, 116, 118, Thioredoxin
123, 125 Escherichia coli, general properties,
NADH dehydrogenases and, 203 144-145
succinate dehydrogenase and, 228-229, nature of, 92, 93
231-232 Thioredoxin reductase
Tetrathionate, glyceraldehyde-3-phos- amino acid composition, 102
phate dehydrogenase and, 20, 21 cystine residues, 104
2-Thenoyltrifluoroacetone, succinate de- light-activated reduction-neutral semi-
hydrogenase and, 246, 250, 255 quinone, 147-148
Thermua aquaticus, glyceraldehyde-3- mechanism, 94, 98-99
phosphate dehydrogenase of, 2, 4,6, metabolic functions, 142-144
20, 21, 22 reaction catalyzed, 92
Thiamine pyrophosphate, lipoamide de- reduced states, mechanism, 145-147
hydrogenase and, 126 specificity of, 92-93
Thiobacilli, adenylyl sulfate reductases coenzymes and, 94
in, 282 specificity of, 144
Thiobacillus denitrificans Threonine residues, lipoamide dehydro-
adenylyl sulfate reductase of, 283 genase, 105
sulfate reduction by, 281 Threose 2,4-diphosphate, glyceraldehyde-
Thiobacillus thiooxidans, sulfate reduc- 3-phosphate dehydrogenase and, 44
tion by, 281 Thyroid gland, a-glycerophosphate de-
Thiobacillus thioparus hydrogenase and, 259
adenylyl sulfate reductase of, 283, 284 Tiron
sulfate reduction by, 281 NADH dehydrogenase and, 206
Thiocapsa roseopersicina, adenylyl sulfate succinate dehydrogenase and, 246
reductase of, 282,283, 285-286 a-Tocopherylquinone, ubiquinone re-
Thiocyanate ductase and, 181-182
catalase and, 387 Torulopsis nitratophila, nitrite reductase
small NADH dehydrogenases and, 192 of, 275, 276
succinate dehydrogenase and, 232 Transacetylase
Thiol(s) dihydrolipoate and, 108
adenylyl sulfate reductase and, 283 transhydrogenase and, 55
catalase and, 399-400, 401 Transsuccinylase, dihydrolipoate and, 108
cytochrome bs reductase and, 160-161, Tribromoacetate, small NADH dehydro-
162-164 genases and, 192
lipoamide dehydrogenase, 118, 119, 120, Trichloroacetate
123 small NADH dehydrogenases and, 192
nitrite reductase and, 275, 277 succinate dehydrogenase and, 227,232,
Thiol groups, see also Sulfhydryl groups 234
cytochrome c peroxidase and, 348 Trideuteromethanol, catalase and, 404
glutathione reductase, 141-142 Trifluoroacetate, small NADH dehydro-
NADH dehydrogenases, 203-204 genases and, 192
succinate dehydrogenase, 245-246 2,2,2-Trifluoromethyl ethanol, catalase
ubiquinone reductase, 188, 203 and, 401
SUBJECT INDEX 457
Triiodothyronine solubility, ubiquinone reductase and,

a-glycerophosphate dehydrogenase and, 180-181, 182
259 succinate dehydrogenase and, 224,225,
transhydrogenase and, 70 236,239,243,245,246,247,244-251
Triton X-100 yeast NADH dehydrogenase and, 218
cytochrome c oxidase and, 313 Ubiquinone reductase, see under Nico-
o-lactate dehydrogenase and, 270-271 tinamide adenine dinucleotide
NADH dehydrogenase and, 187, 188- Urea
189 glyceraldehyde-3-phosphate dehydro-
succinate dehydrogenase and, 234 genase and, 21,24
ubiquinone reductase and, 182-183 L-lactate dehydrogenase and, 269
yeast NADH dehydrogenase and, 218 lipoamide dehydrogenase and, 122,124
Trout, glyceraldehyde-3-phosphate nitrite reductase and, 279
dehydrogenase of, 26 small NADH dehydrogenase and, 203
Trypsin succinate dehydrogenase and, 227
cytochrome b2 and, 266-267 sulfite reductase and, 290,291
cytochrome b5 reductase and, 164 transhydrogenase and, 57
cytochrome P-450 reductase and, 166,
167,169, 170-171, 172 v
transhydrogenases and, 58,65,71,212,
213, 296 Valence changes, cytochrome c oxidase,
yeast NADH dehydrogenase and, 217 331-332
Tryptophan residues Valine residues, disulfide oxidoreduc-
cytochrome b, reductase, 164 tases and, 104,105
cytochrome c peroxidase, 350,355 Valinomycin, transhydrogenase and, 72
glyceraldehyde-3-phosphate dehydro- Venom, cytochrome b5 reductase and, 154
genase, 29 Vibro cholinicus, sulfate reduction by,
lipoamide dehydrogenase, 126 281
reductases and oxidases, 101 Vitamin K,, ubiquinone reductase and,
thioredoxin, 93 181, 182
Turnover number, succinate dehydro-
genase, 236-237
Tyrosine residues
W
cytochrome b5 reductase, 163 Water, catalase and, 374,385, 387
cytochrome c peroxidase, 355
genase, 21 X
modification of, 22-23
reductases, 101 Xanthine oxidase, cytochrome P-450
reductase and, 169
U X-ray(s)
Ubiquinone(s1 genase
choline dehydrogenase and, 262 apoenzyme structure, 19-20
energy conservation and, 214 holoenzyme structure, 9-19
258 Y
large NADH dehydrogenase and, 201
small NADH dehydrogenases and, 194, Yeast, see also Saccharomyces
196, 206 cytochrome c oxidase of, 311
458 SUBJECT INDEX
cytochrome c peroxidase in, 347 lipoamide dehydrogenase of, 112,117,

glutathione reductase of, 94, 102, 138 126
diasociation constants, 135 sulfate reduction in, 281
kinetics, 140-141 thioredoxin reductase of, 102
substrates, 132
glyceraldehyde-3-phosphate dehydro- Z
genase of, 6-8,18,26 Zinc
amino acid modification, 22,23 2-hydroxyacid dehydrogenase and,
half-site reactivity, 37 272-273
hybrids of, 26-27 n-lactate dehydrogenase and, 270,
pyridine nucleotide and, 28,31-33 271-272
Topical Subject Index
VOLUMES I-XI11
A Acetylcholinesterase
acceleration, V, 111-114
Acetamidyllysine residues, proteinase esteratic site, V, 95-97
inhibitors, 111, 451452 historical background, V, 87-88
Acetate: coenzyme A lipase inhibitors
catalytic properties anionic site, V, 98-100
cation requirements, X, 479-480 esteratic site, V, 100-110
estimates of substrate affinity, X, 481 fluoride, V, 110-111
formation of enzyme-bound acetyl physical properties, V, 90-93
adenyla te, X, 478 purification, V, 89
selective modification of amino acid substrate binding, anionic site, V, 93-95
residues, X, 480-481 Acetyl coenzyme A-acyl carrier protein
steady state kinetics and reation transacylase
mechanism, X, 481-483 catalytic properties
substrates and inhibitors, X, 477-478 assays, VIII, 187
molecular properties, X, 474-475 mechanism, VIII, 187-188
Acetoacetate decarboxylase p H optimum and substrate speci-
historical background, VI, 255-256 ficity, VIII, 187
inhibition studies historical background, distribution and
borohydride, VI, 267-269 metabolic significance, VIII, 185-
p-chloromercuriphenyl sulfonate, VI, 186
269-270 molecular properties, VIII, 186-187
8-diketones, VI, 265-266 Acetyl coenzyme A carboxylase
hydrogen cyanide, VI, 267 distribution, VI, 54-56
monovalent anions, VI, 266-267 historical background and metabolic
2-oxopropane sulfonate, VI, 264 significance, VI, 53-54
kinetic properties, VI, 263-264 molecular characteristics, VI, 58-59
mechanism, VI, 261-263 reaction catalyzed, VI, 53
properties regulation of, VI, 79-82
assay, VI, 256-257 substrate specificity, VI, 56-58
latency, VI, 258-259 subunit structure and function
molecular weight, subunits and active subunits in Escherichia coli,
amino acid composition, VI, 260- VI, 60-64
261 biotin carboxylase, VI, 70-71
purification, VI, 257-258 biotin carboxyl carrier protein, VI,
stability, VI, 261 64-70
459
460 TOPICAL SUBJECT INDEX
reconstitution and, VI, 72-78 a-hydroxycarboxylic acid inhibition,

structure of liver and wheat enzymes, IV, 462465
VI, 78-79 ion effects, IV, 466
transcarboxylase, VI, 71-72 pH and substrate effects, IV, 457-458
N-Acetyl-n-glucosamine repimerase, surface inactivation, IV, 459
properties, VI, 377-378 liver
N-Acetyl-n-glucosamine 6-phosphate 2'- bovine, IV, 491493
epimerase, properties, VI, 377-378 mouse, IV, 489-491
N-Acetylglutamate-5-phosphotransferase rat, IV, 484-489
allosteric inhibition Neurospora crassa, IV, 497
kinetics, IX, 516-518 physical properties, IV, 476
temperature effect, IX, 519-520 plant, IV, 497
catalytic reaction preparation, IV, 466-468
assays, IX, 514-515 red cell
kinetics, IX, 515-516 general properties, IV, 477
pH optima and activating ions, IX, purification and separation of genetic
514 types, IV, 477-484
stoichiometry, IX, 513-514 Saccharomyces, IV, 497
substrate specificity, IX, 514-515 serum, IV, 495-496
historical background, IX, 511412 specificity, alkaline phosphatase and,
purification IV, 450-454
allosteric enzyme of Chlamydomonas, spleen, IV, 493495
IX, 513 staphylococcal, IV, 498
nonallosteric enzyme of Escherichia, transphosphorylation, IV, 472473
I X , 513 use as reagent, IV, 473-476
N-Acetylneuraminate aldolase, proper- Acid proteinase(s)
ties, VII, 298-299 pepsinlike
0-Acetylserine sulfhydrase, properties, chemical properties, 111, 728-730
VII, 54 distribution and isolation, 111, 724-
Acidic nuclear protein kinases, proper- 728
ties, VIII, 580 enzymic properties, 111, 734-740
Acid phosphatase(s) physical properties, 111, 731-733
amebic, IV, 498 renninlike, 111, 740-741
assay, IV, 457 distribution and isolation, 111, 741-
problems, IV, 454 742
bone, IV, 496-497 enzymic properties, 111, 743-744
distribution, IV, 450 physical and chemical properties,
Drosophila melanogaster, IV, 498 111, 742-743
electrophoretic behavior, IV, 454455, Aconitase
468-469 catalytic properties
Escherichia coli, IV, 498 assay, V, 423
functional groups and group reagents equilibrium concentrations, V, 424
iodination, IV, 469-471 mechanism of action, V, 433-439
sulfhydryl groups, IV, 469 pH optima, V, 423-424
tyrosine and tryptophan, IV, 471-472 single vs. dual catalytic site, V,
Gaucher, IV, 496 432-433
general, IV, 455457 specificity, V, 4-22
historical, IV, 450 cofactors
kine tics ferrous iron requirement, V, 422
fluoride inhibition, IV, 459-462 role of reducing agent, V, 423
TOPICAL SUBJECT INDEX 461
function, V, 413414 glutamine synthetase, X, 720-733

historical background, V, 414-415 j3-hydroxydecanoyl thioester dehy-
inhibitors drase, V, 453455
fluorocitrate, V, 4 W 3 0 invertase, V, 300301
iron-binding agents, V, 428 lipase, VII, 593-595
other carboxylic acids, V, 430-431 papain, 111, 496499
other inhibitors, V, 431432 activation, 111, 511414
intracellular distribution, V, 416-417 chemical modification, 111, 51.5-516
kinetics, half-cystine content, 111, 509-511
latent period, V, 424425 location of thiol group, 111, 514
Michaelis constants, V, 425426 staphylococcal nuclease, IV, 195-196
relative reaction rates, V, 425 streptococcal proteinase, 111, 626-627
scheme, V, 426-428 subtilisin, 111, 553-560, 575-584
mechanism of, 11, 302-304 thiolase, VII, 404-405
molecular properties triosephosphate isomerase, VI, 330-333
factors affecting stability, V, 419420 trypsin, 111, 260-262
physiochemical properties, V, 418-419 urease, IV, 20-21
purification, V, 417418 Active-site-directed reagents
occurrence, V, 415416 as adjuncts to physical methods
reactions catalyzed, stereospecificity, crystallography, I, 143
11, 164-168 other spectroscopic methods, I, 145
role of metals, 11, 516-518 spin labels, I, 143-145
Aconitate, cis-trans isomerization, VI, characterization of functional groups
394-395 and, I, 142
Active site(s) chymotrypsin and
alkaline phosphatase, IV, 404-406 chloromethyl ketone from tosyl-
amino acid decarboxylases, VI, 245-248 phenylalanine, I, 94-96
8-aminolevulinate dehydratase, VII, other alkylating agents, I, 97-99
331433 other neutral proteases, I, 102-103
aspartate transcarbamylase, IX, 262- other studies, I, 99-102
268 consecutive covalent modifications
carbonic anhydrase, V, 617422, 643- displacement, I, 115117
646 elimination, I, 115-116
carboxylesterases, V, 61434 intramolecular alkylation, I, 113-114
catalase, XIII, 3694388 photolysis, I, 114-115
chemical modification, I, 194-196 heme proteins and, I, 142
a-chymotrypsin, 111, 1!%-202 hydrolytic enzymes and,
y-chymotrypsin, 111, 202-204 amidases, I, 130-131
chymotrypsinogen, 111, 179-182 deaminases, 1, 127-128
creatine kinase, VIII, 439442 esterases, I, 124-127
deoxyribonuclease I, IV, 297-299 glycosidases, I, 128-129
enolase nucleases, I, 130
components, V, 532-534 proteolytic enzymes, I, 118-124
mapping with substrate analogs, V, lyases and
526-529 carbonic anhydrase, I, 139-140
number, V, 530-532 3-deoxy-o-arabino-heptulosonate-7-
fumarase, affinity labeling, V, 563-564 phosphate synthase, I, 139
p-galactosidase, VII, 6574358 fumarase, I, 140-141
glucose-6-phosphate isomerase, VI, 6-hydroxydecanoyl thioester dehy-
285-287 drase, I, 141
TOPICAL SUBJECT INDEX
2-ke to-3-deoxy-6-phosphogluconic ribonucleic acid biosynthesis, VIII,

aldolase, I, 139 20-26
oxidoreductases and Adenosine aminohydrolase
alcohol dehydrogenase, I, 136 catalytic properties
glutamate dehydrogenase, I, 137 mechanism, IV, 5943
guanosine5’-phosphate reductase, I, nature of active site, IV, 58-59
137 reaction parameters, IV, 5 6 5 7
inosinic acid dehydrogenase, I, 137- molecular properties
138 chemical and physical, IV, 55-56
lactate dehydrogenase, I, 136
purification, IV, 54-55
as selective enzyme inhibitors, I, 146
physiological function, IV, 63-64
transferases and
acyltransferases, I, 134-135 Adenosine aminohydrolase (nonspecific),
adenylyltransferase, I, 135 IV, 73-75
amidotransferase, I, 131-134 Adenosine diphosphate
triosephosphate isomerase and, I, 138 synthesis of derivatives
trypsin and related enzymes, I, 103- adenine-myonic acid dinucleotide
112 and adenylyl diphosphoglycerate,
Acyl carrier protein VIII, 33-35
distribution and intracellular Iocaliza- adenosine diphosphate glucose syn-
tion, VIII, 158-164 thesis, VIII, 3 2 3 3
function in fatty acid biosynthesis, general features, VIII, 3&32
VIII, 164-165 Adenosine diphosphate sulfurylase, X,
molecular properties 663-665
physical properties, VIII, 166 Adenosine diphosphoryl glucose pyro-
prosthetic group and primary se- phosphorylase
quence, VIII, 166-170 activator, general effects, VIII, 77-78
structure-activity relationships, VIII, Aeromonas formicam, VIII, 108-109
170-173 Chlorella pyrenoidosa, VIII, 90
historical background, VIII, 155-158 classification, VIII, 75-77
prosthetic group synthesis and turn- enterobacteriaceae
over, VIII, 173-176 activator effects on kinetic parame-
Acyl coenzyme A ligases ters, VIII, 97-99
assays, VII, 411
activator-inhibitor interaction, VIII,
general aspects of reaction, VII, 409-
99-100
410
energy charge and, VIII, 104-107
reaction catalyzed, VII, 407-409
Acyl transfer inhibitor effect on kinetic constants,
carboxyl anions and, 11, 226-235 VIII, 101-102
tertiary amino groups and, 11, 235-238 manganese effects, VIII, 102-104
Acyltransferases, modification of, I, 134- Entner-Duodoroff pathway and, VIII,
135 78-81
Adenine aminohydrolase, IV, 51-54 Escherichia coli, VIII, 102-107, 109-110
Adenine nucleotide aminohydrolase, IV, mutantb) SG5 and CL1138, VIII,
75-76 110-114
Adenosine mutantb) SG14, VIII, 115-117
phosphodiester derivative biosynthesis general background, VIII, 73-75
adenyl cyclase, VIII, 26-27 nonchlorophyllous plant t i m e , VIII,
adenylylation of glycoside antibi- 93-94
otics, VIII, 27-30 other leaves, VIII, 89-90
3-phosphoglycerate activation and Micrococcus lysodeikticua membrane

phosphate inhibition of, VIII, 91- electron microscopy, X, 422
92 FMC and, X, 424425
physical properties, VIII, 117-119 interaction with antibodies, X, 423
R hodospirillum rubrum, VIII, 81-83 localization, X, 422423
reaction mechanism, VIII, 86 release from, X, 421-422
temperature and, VIII, 83-86 size and catalytic properties, X, 422
Serratia marcescens, VIII, 107-108 subunits, X, 423-424
spinach leaf, VIII, -9 mitochondria1
Adenosine kinase assay, X, 377
assay, IX, 51-52 beef heart, X, 377-386
distribution and purification, IX, 51 rat liver, X, 387-389
kinetic and molecular properties, IX, yeast, X, 386-387
52-53 Rhodopseudoomonas spheroides mem-
substrate specificity, IX, 53-54 brane, X, 429
Adenosine monophosphate, fructose-l,& sarcoplasmic membrane
diphosphatase and, IV, 618-620,627- calcium-dependent, X, 445-450
628, 636-637 calcium-independent, X, 444-445
Adenosine triphosphatase(s) historical background, X, 432-434
Alcaligenes jaecalis membrane Streptococcus jaecalis membrane
catalytic properties, X, 429 active transport and, X, 414-416
molecular properties, X, 428 amino acid composition, X, 405-406
solubilization and purification, X, carbodiimide-resistant mutants, X,
428 413-414
Bacillus megaterium membrane dicyclohexylcarbodiimide inhibition,
catalytic properties, X, 427-428 X, 411-413
purification, X, 426 electron microscopy, X, 405
reassembly, X, 428 isolation of membranes, X, 400
release from membranes, X, 426 kinetics, X, 408-409
size, amino acid composition and molecular weight, X, 404-405
morphology, X, 427 nectin and, X, 410411
subunits, X, 427 purification, X, 402404
Bacillus stearothermophilus membrane reassembly, X, 409-410
properties, X, 425-426 release from membranes, X, 400-402
solubilization and purification, X, subunits, X, 406408
425 Adenosine triphosphate
bacterial membrane, X, 396-400 calcium-efflux dependent
other bacteria, X, 416 net formation, X, 457458
Streptococcus faecalis, X, 400, 416- phosphate exchange, X, 458-459
429 metal complexes, electronic structure,
chloroplast 11, 479-481
Euglena gracilis, X, 394 Adenosine triphosphate citrate lyase,
spinach, X, 389-394 VII, 368-369
Escherichia coli membrane assay and isolation, VII, 369-370
deficient mutants, X, 419421 catalytic properties
molecular weight and catalytic control, VII, 372
properties, X, 418 equilibrium and kinetics, VII, 371-
release from membranes, X, 416-418 372
subunit composition, X, 418-419 specificity, VII, 371
function of, X, 375-377 stereospecificity, VII, 372-373
molecular properties 5’-Adenylic acid aminohydrolase

cofactors, VII, 370 catalytic properties
inhibitors, VII, 370-371 kinetics, IV, 66-70
molecular weight, VII, 370 mechanism, IV, 70-71
stability, VII, 371 physiological function, IV, 71-73
sulfhydryl groups, VII, 370 specificity, IV, 66
reaction mechanism molecular characteristics
adenosine diphosphate-adenosine chemical and physical properties, IV,
triphosphate exchange, VII, 373 65-66
citryl coenzyme A aa intermediary, purification and homogeneity, IV,
VII, 376 6465
citryl-enzyme as intermediary, VII, Adenylosuccinase
375-376 5-amino-4-imidazole-N-succinocar-
citryl phosphate as intermediary, boxamide ribonucleotide and, VII,
VII, 374-375 182-183
oxygen transfer to orthophosphate, . mechanism of action, VII, 196-197
VII, 373 Neurospora, VII, 191-192
phosphoryl-enzyme as intermediary, assay procedures, VII, 192
VII, 373-374 catalytic properties, VII, 192
reaction scheme, VII, 377 purification, VII, 192
Adenosine triphosphate sulfurylase subunit structure, VII, 192-193
general properties, X, 656-658 sterospecificity of additional or elimi-
mechanism, X, 658-662 ination, VII, 195
occurrence and purification, X, 652- yeast, VII, 185-186
654 assay procedure, VII, 187
reactions and assay, X, 655-656 function and distribution, VII, 186-
Adenosyltransferase(s) , general back- 187
ground, VIII, 121-123, 152-154 pH optima and equilibrium, VII, 191
Adenylate energy charge, metabolic purification, VII, 187
regulation and, I, 470-476 substrate affinity and product inhi-
Adenylate kinase bition, VII, 189-190
biological aspects substrate specificity, VII, 187-188
distribution, VIII, 279-282 sulfhydryl reagents and, VII, 188-
function, VIII, 285-288 189
genetics and disease, VIII, 282-284 Adenylylsulfate kinase, X, 662-663
catalytic properties Adenylyl sulfate reductase(s), properties,
assay, VIII, 300-301 XIII, 279-286
equilibrium constants, VIII, 302 Adenylyltransferase, modification of, I,
mechanism, VIII, 302-305 135
metal requirement, VIII, 297-298 Adenylyl transfer reactions, general
nucleotide specificity, VIII, 298-300 background, VIII, 1-6
molecular properties Adipose tissue, hormone sensitive lipase,
composition, VIII, 291-293 VII, 609-610
physical properties, VIII, 295-297 Adrenal gland, glycogen synthetase of,
preparation and purity, VIII, 288- IX, 353
291 Aerobacter aerogenes, pullulanase of, V,
reactive groups, VIII, 293-296 195-201
role of metals in mechanism, 11,502 Aerobacter cloacae, phage polysaccharide
Adenyl cyclase, properties, VIII, 26-27 depolymerase, V, 398
Aeromonas formicans, adenosine diphos- inhibitor studies

phoryl glucose pyrophosphorylase coenzyme competitive inhibitors,
of, VIII, 108-109 XI, 181-182
Affinity labeling, chemical modification others, XI, 182-183
in general and, 91-94 kinetic aspects
Agaricaceae, y-glutamyltransferase of, coenzyme binding, XI, 160-163, 183-
184
IV, 95-96
dismutase reaction, XI, 166
Alanine dehydrogenase, regulation of,
half-site reactivity, XI, 166-167
I, 443444 ordered mechanism, XI, 165-166
Alanine racemase, spore germination and, reaction mechanism, XI, 185-186
VI, 506 substrate binding, XI, 163-165
Alcaligenes fueculis, membrane adenosine substrate specificity; XI, 184-185
triphosphatase of, X, 428-429 liver, XI, 5 6 5 7
Alcohol dehydrogenase(s1 kinetic studies, XI, 20-22
chemical modifications, XI, 141-145 mechanism for catalysis, XI, 168-171
arginine residues, XI, 179 metal content changes, XI, 145-147
cobalt enzyme, XI, 180 modification of, I, 136
cysteine residues, XI, 176177 other sources, XI, 186-187
denaturation, XI, 180-181 bacterial, XI, 187-188
histidine residues, XI, 177-179 insect, XI, 189-190
manganese enzyme, XI, 180 plant, XI, 188-189
properties, XI, 179-180 rat liver,
uncharacterized, XI, 179 multiple molecular forms, XI, 111-
112
coenzyme analogs and, XI, 150-152
primary structure, XI, 114-117
comparisons
tertiary structure
evolutionary aspects, XI, 140-141 crystallization and preliminary X-
structural and functional aspects, ray studies, XI, 117-118
XI, 136-140 electron density maps, XI, 118-119
denaturation studies, XI, 147-148 three-dimensional structure, XI, 1%
fluorescence and phosphorescence, XI, 136
148-150 yeast
functional aspects, chemical modifications, XI, 176181
activity in ethanol metabolism, XI, inhibitor studies, XI, 181-183
106 kinetic aspects, XI, 22-23, 183-186
physiological substrates, XI, 105-106 primary structure, XI, 173-176
gene duplication and, I, 309-311 purification and molecular proper-
horse liver ties, XI, 171-173
multiple molecular forms, XI, 107- Aldehyde oxidase, metal complexes and,
108 11, 533-534
primary structure, XI, 113-116 Aldolase(s)
historical review, VII, 213-215
human liver,
properties in bacteria and fungi, VII,
multiple molecular forms, XI, 109-
215-216
110
Schiff bases and, 11, 359-380
primary structure, XI, 116 yeast, role of metals, 11, 515-516
inhibitor binding, Aldolase(s) (Metallo)
binary complexes, XI, 152-157 distribution and general properties
ternary complex, XI, 158-160 catalytic properties, VII, 253-254
multiple forms in microorganisms, Aldose-ketose isomerases

VII, 255-256 general considerations, VI, 271-272
purification and metal content, VII, nonphosphorylated sugars and, VI,
254-255 340-354
reaction mechanism Algae
other functional groups, VII, 257- blue green, fructose-l,6diphospha-
258 tases of, IV, 640-642
possible molecular homology in Alkaline phosphatase
yeast and muscle enzymes, VII, active sites, number of, IV, 404-406
258 chelating agents and, IV,426-427
role of metal ion, VII, 256-257 chemical modification, IV, 391-392
Aldolase(s) (Schiff base-forming) arginine, IV, 390
developmental aspects histidine, IV, 390
embryonic tissues and, VII, 249-251 leucine, IV, 390
modification of structure, VII, 252- methionine and cystine, IV, 390391
253 phenylalanine, IV, 389-390
tumors and, VII, 251-252 tryptophan, IV, 390
mechanism of catalysis competitive inhibitors, IV, 394-396
isotope exchange reactions, VII, composition, IV, 423-425
216-217 analysis, IV, 378-380
Shiff base formation, VII, 217-219 sequence work, IV, 380
organ-specific crystal structure, IV, 389
molecular properties and subunit distribution, IV, 374-376
structure, VII, 221-223 histochemical and gel localization, IV,
nonidentical subunits and micro- 433-434
heterogeneity, VII, 223-224 historical background, IV, 373374
occurrence of isozymes in vertebrate in vitro assay, IV, 432-433
tissues, VII, 220-221 isozymes, IV, 384387
specificity, VII, 219-220 kinetic studies, IV, 409-416
phylogenetic studies factors affecting, IV, 434-436
comparison of active site peptides, inhibition and, IV, 442443
VII, 248-249 Kim and 'Vim.,,IV, 436-439
isolation from other species, VII, metal ions and, IV, 440-441
24k248 phosphoryl enzyme formation and,
rabbit liver and brain IV, 439
isolation and general properties, VII, transferase activity, IV, 439-440
241-243 mammalian
primary structure of active site, VII, assay techniques, IV, 432-434
243-244 chemical modification, IV, 427-428
distribution, IV, 420-421
rabbit muscle
function, IV, 421-422
active site sequence, VII, 236
general survey, IV, 417-420
amino acid composition, VII, 224
kinetic studies, IV, 434-443
functional groups, VII, 224-232
mechanism, IV; 443-447
isolation of crystalline enzyme, VII,
physical properties, IV, 423-427
224 purification procedures, IV, 422-423
primary structure, VII, 236-239 reaction catalyzed, IV, 430-432
reaction mechanism, VII, 232-236 substrate specificity, IV, 428-430
X-ray crystallography and electron mutations and, I, 251-254
microscopy, VII, 239-241 phosphoryl enzyme, IV, 396-401
physical properties, IV, 387-388 aromatic

purification, IV, 377-378 aspartate transcarbamylase, IX,
specificity, IV, 392-394 267-268
stability, IV, 425426 biosynthesis, I, 228-237
subunits, IV, 380-384 asparaginase composition, IV, 111-113
transphosphorylation and, IV, 406-409 aspartokinase(s) composition, VIII,
zinc and, IV, 401404 520, 542, 543
Alkaline proteinase(s) carboxylesterase composition, V, 52-53
diisopropylfluorophosphate-sensitive, creatine kinase composition, VIII,
111, 744-745 390-392
chemical properties, 111, 749-754 degradation, VI, 504-506
distribution and isolation, 111, 745- elastase sequence, 111, 341-343
749 fumarase and, V, 544-545
enzymic properties, 111, 758-763 a-glucan phosphorylase composition,
keratinase, 111, 763-765 VII, 446-447
physical properties, 111, 754-758 glucose-6-phosphate isomerase com-
8-Alkyl-L-cysteine sulfoxide lyase, position, VI, 27S279
properties, VII, 52 guanidino kinase
Alkylsulfatase(s), V, 15 composition, VIII, 469-470
Allylases, proton shifts and, 11, 299302 function, VIII, 477-482
D-Altronate dehydrase, properties, V, 579 hexokinase composition, IX, 41
Ameba, acid phosphatase of, IV, 498 inorganic pyrophosphatase and, IV,
Amidases, modification of, I, 130-131 512514
Amidinotransferase(s), reactions cata- microbial proteinases and, 111, 749-
lyzed, IX, 497-498 751, 770-772
Amidotransferases, modification of, I, pancreatic ribonuclease sequence, IV,
131-134 653-654
Amine oxidase (s) , papain composition and sequence, IV,
active site, substrate interaction, XII, 507-509
524-525 pepsin
catalytic mechanism, XII, 525-526 composition, 111, 128-130
definition and classification, XII, 511- sequence, 111, 1W133
513 phospholipase A,
inhibitor reactions, XII, 523-524 content, V, 80-81
metabolic function, XII, 513 sequence, V, 81-82
microsomal, properties of, XII, 228. prothrombin composition, 111, 313
230 pyridoxal reactions with, 11,339445
other copper-containing, XII, 526-527 sequences, carboxypeptidase B, 111,
prosthetic groups, XII, 519-523 64-66
purification, molecular weight and streptococcal proteinase
substrate specificity, XII, 513-518 active site, 111, 626-627
spectral properties, XII, 518-519 composition, 111, 624-625
Aminoacetone, formation of, VII, 355 N- and C-terminal, 11, 625-626
Amino acid(s) thrombin
activation, VIII, 6-11 composition, 111, 285-286
acyl carrier protein sequence, VIII, sequences, 111, 287-290
166-170 Amino acid decarboxylases
amylase composition, microbial, V, active site
239-244 absorption spectra of pyridoxal
analogs, mutations and, I, 262265 phosphatedependent, VI, 245-247
pyridoxal phosphate binding site, VI, Aminoacyl transfer ribonucleic acid,

247-248 enzymic deacylation, X, 509-510
distribution and general properties, VI, Aminoacyltransfer ribonucleic acid syn-
224-237 thetases
general considerations, VI, 217-219 amino acid activation, X, 505-506
mechanism of action assay, X, 506
glycine decarboxylase, VI, 240-241 binding parameters, X , 507-508
pyridoxal phosphate a-decarboxy- reaction product, X, 506
lation, VI, 237-240 substrate specificity, X, 506607
pyridoxal phosphate p-decarboxyla- amino acid biosynthesis and, X, 536
tion, VI, 241-243 chemical properties
pyruvate-containing, VI, 244 affinity labeling, X, 505
metabolic importance amino acid composition, X, 503
bacterial, VI, 219-221 chemical modification, X, 505
mammalian and plant, VI, 221-224 proteolytic modification, X, 504
subunit structure, VI, 248-253 terminal amino acids and sequence
n-Amino acid oxidase, molecular proper- analysis, X, 503-504
ties and kinetic mechanism, XII, general considerations, X, 489492
445-456 genetics of, X, 529-534
L-Amino acid oxidase, molecular proper- mechanism of reaction
ties and kinetic mechanism, XII, general, X, 510-511, 517-515
456-461 isoleucyltransfer ribonucleic acid
Amino acid racemases synthetase, X, 511-517
assay methods occurrence and distribution, X, 492-491
coupling to L- or n-specific enzymes, purification, X, 494-496
VI, 489 Crystallization, X, 496-497
polarimetric, VI, 489-490 regulation of biosynthesis, X, 534-535
cofactors size and subunit composition, X, 502-
flavin, VI, 496 503
metal ions, VI, 496-497 multichain with dissimilar subunits,
no pyridoxal phosphate, VI, 495 X, 502
pyridoxal phosphate, VI, 494-495 multichain with similar subunits, X,
status uncertain, VI, 495-496 499-502
equilibrium position, VI, 490-491 single chain, X, 497499
history and survey, VI, 481488 Amino group(s)
kinetic features, VI, 491494 aspartate transcarbamylase, IX, 265-
mechanism of action 267
aminoacyl complex and, VI, 501-502 chemical modification, I, 175
nonpyridoxal enzymes, VI, 498-500 elastase, 111, 386-373
pyridoxal enzymes, VI, 497-498 ribonuclease, IV, 677-682
physiological aspects tertiary, acyltransfer to, 11, 235-238
n-amino acids in animal tissues, VI, trypsin, 111, 269-270
508-507 w-Amino group migrations, VI, 547-548
biosynthesis and degradation of free reaction mechanisms
amino acids, VI, 504-506 cobamide coenzyme as hydrogen
cell wall biosynthesis, VI, 502-503 carrier, VI, 560-561
peptide antibiotic biosynthesis, VI, general considerations, VI, 559-560
503-504 hydrogen transfer reaction, VI, 561
spore germination, VI, 506 role in bacterial fermentations, VI,
substrate specificity, VI, 490 562-563
Amino group transfer inhibitors

basic chemical features amino acids and derivatives, VII,
congruent nonenzymic models, IX, 349-350
387-391 carboxylic acids, VII, 351
general characteristics of intermedi- sulfhydryl reagents, metals and
ate steps, IX, 391-392 porphyrins, VII, 350-351
formally similar processes, IX, 384- mechanism, VII, 351-355
387 metabolic significance, VII, 343-344
historical background, IX, 379-381 molecular properties
other substrates and, IX, 462463 assay, VII, 347
w-amino and 0-0x0 acids, IX, 473- high and low activity forms, VII,
474 344-315
glutamate-oxoglutarate or aspartate- isolation, VII, 346-347
oxalacetate, IX, 463473 stability, VII, 344
noncarboxylic acids, IX, 475-476 occurrence, VII, 341-342
as side reactions, IX, 47-80 Aminopeptidase A, 111, 111-112
two a-amino-a-oxomonocarboxylic Aminopeptidase B, 111, 112-113
acids, IX, 473 Aminopeptidase M, 111, 102-105
recent developments, IX, 381-384 Aminopeptidase P, 111, 115-116
5-Amino-4-imidazole carboxamide ribo- Aminotripeptidase, 111, 117-118
nucleotide transformylase, proper- Ammonia elimination
ties, IX, 204-205 enzymic
5-Amino-4-imidarole-N-succinocarbox- general considerations, VII, 79-88
amide ribonucleotide nomenclature, VII, 77-79
cleaving activity, VII, 183-184 free energy, VII, 88-90
assay, VII, 184-185 interpretation, VII, 92-94
enzymic properties, VII, 185 ionic species, VII, 90-91
function and distribution, VII, 184 standard states, VII, 91-92
purification, VII, 185 models for steric course,
8-Aminolevulinate dehydratase carbanion intermediate, VII, 114-116
active site, nature of, VII, 331-333 concerted elimination, VII, 110-114
stereochernistry, VII, 94-95
catalytic properties
configurations of amino acid chiral
assay, VII, 330
centers, VII, 95-98
kinetics, VII, 330-331
configurations and conformations of
pH optima, VII, 331
olefinic products, VII, 109-110
cation requirements, VII, 328-330 specificity toward amino acid pro-
mechanism of porphobilinogen syn- chiral centers, VII, 99-109
thesis, VII, 333-337 Amphibolic pathways
molecular weight, VII, 324326 input signals and, I, 476-479
aggregation and, VII, 326-327 interaction with biosynthetic pathways,
reaction catalyzed, VII, 323-324 I, 481482
Schiff base and, 11, 361-362 Amylase (8)
subunits, quaternary structure and, action pattern
VII, 327-328 effects of chain length, V, 173-182
6-Aminolevulinate synthetase hydrolysis and condensation spe-
analogous reactions, VII, 355356 cificity, V, 149-152
cofactors, substrate specificity and multiple attack, V, 165-173
kinetic constants, VII, 348-349 single chain or multichain attack,
historical background, VII, 339-340 V. 161-165
subsite model, V, 154-161 L-Arabinose isomerase, properties, VI,

substrate structural requirements, V, 348-349
152-154 Arabinose-bphosphate isomerase,
amino acid content, V, 127 properties, VI, 324-325
assay methods, V, 117-120 n-Arabonate dehydrases, properties, V,
biosynthesis, genetics and control, V, 582
182-189 L-Arabonate-n-fuconate dehydratases,
chemical modification, V, 129-132 properties, V, 581-582
classification, V, 115-117 Arene oxides, epoxidases and, VII, 211-
mechanism of action, 140-149 212
microbial Arginine kinase, see also Guanidino
amino acid composition, V, 239-243 kinases
assay, V, 265-266 role of metals in mechanism, 11, 501-
calcium and, V, 247-250 502
carbohydrate components, V, 245-257 Arginine monooxygenase, properties,
chemical modification, V, 261-263 XII, 203-204
denaturation and renaturation, V, Arginine residues
251-256 chemical modification, I, 174
fragmentation, V, 257-258 chymotrypsinogen, 111, 176-179
mechanism, V, 268-271 ribonuclease, IV, 689490
mode of action, V, 266% Argininosuccinase (8)
molecular weights, V, 250-251 assay procedures, VII, 171
purification, V, 236-239
bovine kidney
specificity, V, 263-265
catalytic properties, VII, 179
sulfhydryl and disulfide groups, V,
purification, VII, 178-179
244-245
terminal groups, V, 241-244 catalytic properties
thermostable and acid stable, V, inhibitors, VII, 172
258-261 pH optimum and equilibrium, VII,
origin, purification and molecular 173
variants, V, 121-127 substrate specificity, VII, 172
pH, temperature and salt effects, V, function and distribution, VII, 170-171
132-140 historical background, VII, 169-170
size and shape, V, 128-129 mechanism of action, VII, 196
subunits and multiple binding sites, molecular structure
V, 127-128 subunit constitution, VII, 176-177
Androstenolone sulfatase, V, 7-9 sulfhydryl groups and, VII, 177-178
Anhydrochymotrypsin, elimination re- Neurospora, catalytic and physical
actions and, I, 115-116 properties, VII, 180-181
Animals, nuclear ribonucleic acid poly- number of binding sites, VII, 174-175
merase, X, 262-300 pea seeds, catalytic and physical
Anions, carbonic anhydrase and, V, properties, VII, 181-182
646-652, 658-660 primary structure
Antibiotics amino acid composition, VII, 179
amino glycoside, adenylylation, VIII, antigenic properties, VII, 179
27-30 purification, VII, 171-172
Antithrombins, thrombin and, 111,305- regulation
306 cooperative substrate effects, VII,
n-Arabinose isomerase, properties, VI, 173-174
346348 nucleotide stimulation, VII, 174
reversible cold inactivation and sub- optical properties, IX, 407416

unit dissociation, physical parameters and macro-
control of, VII, 175 molecular structure, IX, 398-406
kinetics, VII, 175-176 primary structure and functionally
thermodynamic constants, VII, 176 important groups, IX, 416424
stereospecificity of addition or elimi- stereochemistry and active site
nation, VII, 194 topography, IX, 451455
Argininosuccinate, synthesis, VIII, 38-39 substrates, quasi substrates and
Arthropoda, glycogen synthetase of, IX, inhibitors, IX, 435-451
358-359 Aspartate residues, chymotrypsin, 111,
Arylsulfatase(s), V, 23 235-236, 243
type I, V, 3 4 , 23-26 Aspartate transcarbamylase
type 11, V, 4, 26-39 active site functional groups, IX,
Asparaginase (s) 262-263
amylase content, V, 127 amino groups, IX, 265-267
Escherichia coli aromatic amino acids, IX, 267-268
isolation, IV, 107-108 histidine residues, IX, 267
properties, IV, 109-116 sulfhydryl groups, IX, 263-265
guinea pig serum allosteric effectors
isolation, IV, 105-106 bindine site, IX, 270-273
properties, IV, 106-107 properties in presence of effectors,
occurrence, IV, 102-105 IX, 269-270
other, IV, 116-117 bacterial, IX, 297-302
physiological properties, IV, 117-121 biosynthesis and genetics
properties control of, IX, 295-297
amino acid composition, IV, 111-113 location of genes, IX, 292293
general, IV, 109-110 two chains-single operon?, IX,
structure, IV, 113-116 293-294
substrate specificity and inhibitor catalytic subunit
effects, IV, 110-111 primary structure, IX, 232-234
Asparagine synthetase, X, 578-580 size and substructure, IX, 231-232
bacterial sources, X, 568-572 comparison of native enzyme with
glutamine-dependent, X, 572578 subunits, IX, 277-278
historical background, X, 561-568 cooperative properties of modified
Aspartate enzyme
metabolism, regulation of, I, 457459 modification with partial specificity,
pyruvate carboxylase and, VI, 31-33 IX, 279-280
Aspartate ammonia-lyase other modified enzymes, IX, 284-285
catalytic process, VII, 135-137 specifically modified, IX, 280-284
distribution, purification and kinetic urea and p H effects, IX, 278
properties, VII, 116-118 cooperative substrate binding, IX,
size and constitution, VII, 119-121 268-269
Aspartate :oxoglutarate aminotrans- detailed subunit structure, IX, 239-243
ferase(s1 fungal, IX, 302-306
isoenzymes and multiple subforms, induced conformational changes
IX, 393-398 allosteric effectors, IX, 276-277
pig heart and animal tissues substrates and substrate analogs,
coenzyme analogs and, IX, 429435 IX, 275-276
dynamic spatial aspects, IX, 465-462 isolation and characterization
kinetics, IX, 424-429 away procedures, IX, 228-230
properties associated with regulation, Aspartokinase I, VIII, 515-516

IX, 227-228 chemical properties, VIII, 520-522
purification, IX, 238 conformational changes, VIII, 526-536
size and subunit composition, IX, distribution of two activities on, VIII,
230-231 536-540
kinetics of ligand binding, I X , 285-287 extinction coefficient, VIII, 517
mammalian, IX, 306-307 kinetic parameters, VIII, 519-520
mechanism, IX, 243-282 ligand binding, VIII, 523-525
mechanisms for cooperativity molecular weight, VIII, 517-518
structural models, IX, 290-292 purification and criteria of homo-
two-state major transition, IX, geneity, VIII, 516
287-290 stability, VIII, 517
plant, IX, 307-308 sulfhydryl titration effects, VIII,
reconstitution 525-526
importance of metals, IX, 237-238 tetrameric structure, VIII, 518-519
metals other than zinc, IX, 238-239 Aspartokinase I1
methods, I X , 237 amino acid composition, VIII, 541-542
regulatory subunit extinction coefficient, VIII, 541
metal binding site, IX, 236 kinetic parameters, VIII, 541
primary structure, IX, 235 molecular weight, VIII, 541
as regulatory protein, IX, 236-237 purification and criteria of homo-
size and substructure, IX, 234-235 geneity, VIII, 540
stoichiometry of ligand binding stability, VIII, 540
nucleotides, IX, 274-275 subunit structure, VIII, 541
substrates and substrate analogs, Aspartokinase I11
IX, 273-274 amino acid composition, VIII, $543
Aspartokinase (s) extinction coefficient, VIII, 542
assay of, VIII, 512-513 inhibition, VIII, 544
Escherichia coli, VIII, 513-544 kinetic parameters, VIII, 543
historical background, VIII, 509-51 1 molecular weight, VIII, 543
lysine-sensitive, adenylylation, VIII, purification and criteria of homo-
44-45 geneity, VIII, 542
other coliform bacteria, VIII, 544 Aspergillus, proteinases of, 111, 747, 769
reaction catalyzed, VIII, 511-512 Aspergillus nidulans
regulated by concerted feedback molybdenum hydroxylases of, XII,
inhibition 412414
Bacillus polymyxa, VIII, 546-548 Asymmetry
Bacillus stearothermophilus, VIII, molecular, notations of, 11, 129-134
550 Azotobacter
Bacillus subtilis, VIII, 548-550 phage polysaccharide depolymerase
other bacilli, YIII, 551 assay and purification, V, 397
other genera, VIII, 552 properties, V, 397-398
other nonsulfur photosynthetic Azotobacter agilis, glutaminase of, IV,
bacteria, VIII, 545446 97-98
pseudomonads, VIII, 551452
R hodopseudomonas capsulatus, VIII, B
544-545
Rhodopseudomonas spheroides, VIII, Bacillus
552-553 alkaline proteinases of, 111, 605-606
Saccharomyces cerevkiae, VIII, 553 proteinases of, 111, 767-768
Bacillus cereus, phospholipase C of, V, 5‘-nucleotidase of, IV, 338-340

83-84 photosynthesis in, XI, 509-516
Bacillus megalerium pro teases
membrane adenosine triphosphatase, acid, 111, 723-744
X, 426-428 diisopropylfluorophosphate sensitive,
phage G-induced lytic enzyme 111, 744-765
bound, V, 409-410 metal-chelator sensitive, 111, 765-786
soluble, V, 408-409 other, 111, 786-795
Bacillus polymyxa, aspartokinase of, respiratory chains
VIII, 546-548 denitrifiers, XI, 521426
Bacillus stearothermophilus inorganic reductants, XI, 519-521
aspartokinasc of, VIII, 550 less-mitochondrion-like, XI, 517-519
membrane adenosine triphosphatase mitochondrion-like,’XI, 516-517
of, X, 425-426 sulfate respiration and, XI, 526-534
phage lytic enzyme, V, 410411 ribonucleic acid polymerases
Bacillus subtilis background, X, 333-335
aspartokinase of, VIII, 548-550 catalytic properties, X, 344-374
deoxyribonucleic acid polymerases, molecular properties, X, 335-344
physiological role, X, 143-144 Bacterial cell wall
extracellular ribonuclease of, IV, lytic protease
239-240 Myxobacter, 111, 786-788
intracellular ribonuclease of, IV, 240 other, 111, 789-790
phage-induced exonuclease, IV, Sorangium, 111, 788-789
258-259
Bacteriophage A
Bacteria, see also specific organisms
endolysin
alcohol dehydrogenase of, XI, 187-188
catalytic properties, V, 391-392
aldolases of, VII, 215-216
chemical properties, V, 388-391
amino acid decarboxylases of, VI,
physiochemical properties, V, 387-388
219-221
amylases purification, V, 385-386
induced exonuclease and, IV, 253-254
molecular properties, V, 236-263 Bacteriophage F series
aspartate transcarbamylases of, IX, polysaccharide depolymerase
297-302 catalytic properties and biological
deoxyribonucleic acid methyltrans- significance, V, 393
ferases, IX, 190-192 enzyme assay, V, 392
deoxyribonucleic acid polymerases of, partial purification, V, 392-393
X, 119-144 stability, V, 393
elongation factors, X, 55-67 Bacteriophage G
endonuclease of, IV, 259-270 lytic enzyme
exonucleases of, IV, 252-259 bound, V, 409-410
ferredoxins of, XII, 37-46 soluble, V, 408-409
fructose-1,6-diphosphatasesof, IV, Bacteriophage N20F’
639-640 lytic enzyme
glutamate dehydrogenases catalytic properties, V, 384-385
bacilli, XI, 332 chemical properties, V, 383-384
Escherichia coli, XI, 332-333 physiochemical properties, V, 382-383
others, XI, 333-334 purification, V, 382
hyaluronidases of, V, 313-314 Bacteriophage P1,lytic enzyme, V,
neuraminidases of, V, 324-325 399-400
Bacteriophage P14, lytic enzyme V, creatine kinase of, VIII, 401-403

399400 glycogen synthetase of, IX, 353-354
Bacteriophage PAL,lytic enzyme, V, Bromelain, 111, 542545
4W-401 y-Butyrobetaine hydroxylase
Bacteriophage SP-3, induced exonuclease, catalytic properties, XII, 168-169
IV, 258-259 purification, XII, 167
Bacteriophage T2
induced exonuclease, IV, 255 C
lysozyme
catalytic properties, V, 381382 Calcium
chemical properties, V, 380-381 amylases and, V, 247-250
physicochemical properties, V, 380 binding, staphylococcal nuclease and,
purification, V, 379 IV, 163-171
Bacteriophage T 4 hydrolases and, 11, 524-525
induced endonucleases I1 and IV, IV, Calcium translocation and phosphoryl
266-269 transfer
induced exonuclease, IX, 255 backward reaction
lysozyme adenosine, phosphate and calcium
catalytic properties, V, 369-374 binding, X, 462463
chemical properties, V, 366-369 phosphoprotein formation, X,
enzyme assays, V, 361-364 463-465
physicochemical properties, V, 366 forward reaction
purification, V, 364-366 adenosine triphosphate-adenosine
role in life cycle, V, 376379 diphosphate exchange, X, 462
Bacteriophage T5, induced deoxyribo- adenosine triphosphate and calcium
nuclease, IV, 261 binding, X, 459-460
Bacteriophage T7, induced endonuclease, phosphoprotein formation and, X ,
IV, 266-2436 460-462
Bacteriophage TP-I, lytic enzyme of, Candida utilia
V, 410-411 fructose-1,6-diphosphatase
Biosynthetic pathways inhibition by AMP, IV, 636-637
input signals and, I, 474-481 purification and properties, IV,
interaction with amphibolic pathways, 635-636
I, 481482 regulation, IV, 640
Biotin relation to SDPase, IV, 638
acetyl coenzyme A carboxylase and, structure, IV, 637-638
VI, 64-71 Carbamate kinase
activation of, VIII, 18 assays, IX, 107-108
pyruvate carboxylase and, VI, 3-5 forward reaction, IX, 108-109
Blood reverse reaction, IX, 109-110
coagulation, proteolysis and, I, 416-417 distribution, IX, 101-102
Blood cells, glycogen synthetase of, IX, function and metabolite control, IX,
354-355 115-119
Blood platelets, thrombin and, 111, historical background, IX, 97-100
300-301 molecular properties
Bone, acid phosphatase, IV, 4-97 composition, size and subunit st’ruc-
Bovine pancreatic ribonuclease, see ture, IX, 103-104
Ribonuclease purification, IX, 102103
Brain stability, IX, 104-105
aldolase, VII, 241-244 sulfhydryl reagent effects, IX, 105
reaction catalyzed, IX, 105-107 thermodynamics and kinetics of zinc

specificity and cofactors, IX, 107 binding, V, 641-642
thermodynamics, kinetics and mecha- modification of, I, 139440
nism, IX, 110-115 molecular properties
Carbohydrate composition, V, 601-603
amylase composition, V, 245-247 immunological properties, V, 598-599
prothrombin composition, 111, 313 methods of isolation, V,593-595
thrombin composition, 111, 286-286 physical properties, V,599-601
Carbon-hydrogen fission polymorphism and nomenclature, V,
electron delocalization and, 11, 287-290 595-598
other catalyses, 11, 318-320 primary structure, V, 603-607
Carbonic anhydrase reactions catalyzed, V, 629-630
active-site-directed chemical role of metals, 11, 522-524
modifications structure
anionic reagents, V, 658-660 fluorescence spectroscopy, 11, 425-426
sulfonamides, V, 661 physical probes, 11, 401406
assay methods, V, 630-632 Carbon-nitrogen cleavage, general fea-
catalytic mechanism, V, 661-662 tures, VII, 168-169
catalytic reaction, V, 662885 Carboxyl anions, acyl transfer to, 11,
substrate binding, V, 662 2’26-235
conformation in solution Carboxylesterase (s)
hydrogen exchange, V, 628-829 active site, organophosphorus com-
spectroscopy of native enzymes, V, pounds and, V, 61-64
622-625 amino acid composition, V, 52-53
stability and denaturation, V, assay procedures, V, 64-85
626-628 equivalent weight, V, 50
titration and chemical modification, inhibitors, V, 64
V, 625-626 kinetics, V, 60-61
crystal structure investigations, V, molecular weight, V, 48-50
608-611 multiple molecular forms, V, 4546
active site region, V,617-622 pH optimum, V, 59-60
human enzyme structure, V, 611 physiological and pharmacological
secondary structures, V,611416 significance, V, 65-67
side chain locations, V, 616617 preparations and criteria of purity,
distribution and physiological function, V, 4748
v, 5 w 5 9 3 substrate specificity
historical outline, V, 588490 acyl group transfer, V, 59
inhibitors aromatic amides, V, 57-59
anions, V, 646-052 carboxyl esters, V, 53-57
other, V, 658 thioesters, V,57
sulfonamides, V, 652-658 subunit structure, V, 51-52
kinetic properties Carboxyl group
hydration of aldehydes, V, 639-640 activation of, VIII, 6-20
hydrolytic reactions, V, 636-639 chemical modification, I, 174
interconversion of CO, and HCOo-, ribonuclease, IV, 675-677
V, 632-636 Carboxypeptidase(s)
metal ion cofactor, V, 640-641 bovine, homologies, 111, 66-67
cobalt as probe of active site, V, fungal, 111, 790-791
643-646 gene duplication and, I, 308-309
specificity, V, 642-643 role of metals, 11, 519-522
Carboxypeptidase A Catalase
amino acid sequence, 111, 5 active site
crystallography, 111, 17-18 distal ligand identity, XIII, 376-385
description of structure, 111, 2146 ligand exchange reactions, XIII,
determination of structure, 111, 18-21 385-388
esterase activity, 111, 7 ligand identity at fifth and sixth
kinetics, 111, 7-10 coordination positions, XIII, 369-
mechanism of action, 111, 14-17 376
ester cleavage, 111, 50-51 apoprotein, selective modifications,
inhibition and activation, 111, 51-54 XIII, 376-385
metal studies, 111, 54-56 general properties, XIII, 366-369
peptide cleavage, 111, 46-50 historical background, XIII, 363-365
metals and, 111, 11-12 redox reactions, XIII, 388-389
side chain modification, 111, 1214 nature of Compound I, XIII, 389-390
structure reaction mechanism, XIII, 390-408
backbone conformation, 111, 29-31 Catalytic site, see Active site
correlation of sequence with, 111, Cathepsin C, see Dipeptidyltransferase
31-33 Catheptic endopeptidases
folding of chain, 111, 26-29 cathepsin B, 111, 478-479
general features, 111, 21-23 cathepsin D, 111, 476477
helical segments, 111, 23-26 cathepsin E, 111, 477478
interpretation of substrate specific- Catheptic exopeptidases
ity, 111, 43-44 cathepsin A, 111, 481482
side chain conformation and inter- cathepsin C, 111, 479-480
action, 111, 33-36 catheptic carboxypeptidases A and B,
pstructure, 111, 26 111, 480
substrate binding changes, 111, 40-42 Cellobiose-2’-epimerase, properties, VI,
substrate and inhibitor complexes, 377
111, 3 7 4 0 Cellulase (9)
success and failure of crystallog- action on cellulose and related sub-
raphy, 111, 44-46 strates, V, 287-289
substrate specificity, 111, 6-7 applications, V, 289-290
Carboxypeptidase B assay and detection, V, 275-277
activation and inhibition, 111, 75-76 C1 factors and, V, 280-282
amino acid sequences and end groups, induction and repression, V, 277-278
111, 64-86 physical and chemical properties, V,
chemical composition, 111, 62-64 282-285
distribution, 111, 59-60 production and isolation
historical background, 111, 57-59 cultural conditions, V, 278
kinetics and competitive inhibition, influence of cultural conditions on
111, 71-75 physical properties, V, 278-279
mechanism, comment on, 111, 77 isolation methods, V, 279-280
physical properties, 111, 61-62 significance and distribution, 8,274-275
purification and assay, 111,60431 substrate binding and catalytic proper-
specificity, 111, 69 ties, V, 285-287
esterase activity, 111, 71 Cellulose polysulfatase, V, 11-12
peptidase activity, 111, 70-71 Cell wall
use in protein structural analysis and structure and action of lytic enzymes
modification, 111, 77-79 chemical properties of cell walls, V,
Castor bean, lipase of, VII, 613-614 353455
mode of action of enzymes, V, 355 as primary structure probe

morphogenesis of cell wall and mem- chemical cleavage and, I, 181-182
brane, V, 352-353 location of modified residues, I, 182
Cerebroside sulfatase, V, 13-14 proteolysis and, I, 178-181
Chain extension, protein structure and, as probe of function,
I, 293-300 accessory sites, I, 196-197
Chain shortening, protein structure and, active site, I, 194-196
I, 292-293 multifunctional enzymes, I, 197-198
Charge-transfer forces, propinquity proteinase inhibitors, 111, 443447
effects and, 11, 254-264 reagent reactivity determinants,
Chemical modification catalytic factors, I, 158
alkaline phosphatase electrostatic interaction, I, 157
bacterial, IV, 389-392 local environment polarity, I, 158-159
mammalian, IV, 427-428 selective adsorption, I, 156
amylases, V, 129-130, 261-263 steric factors, I, 157-158
chymotrypsin, 111, 234, 238-239 reversible, I, 172-173
control of selectivity, amino groups, I, 175
reaction conditions and, I, 168-170 arginine, I, 174
reagent choice, I, 168 carboxyl groups, I, 174
determination of degrees and sites of histidine, I, 173
analytical methods, I, 170-171 methionine, I, 173
instability of modified residues, I, serine and threonine, I, 173
171-172 sulfhydryl groups, I, 174-175
in elucidation of noncovalent structure tryptophan, I, 173
intermolecular reactions, I, 191-194 tyrosine, I, 174
intramolecular reactions, I, 183-191 ribonuclease, IV, 675-697
functional group reactivity deter- subtilisin BPN’, I, 203-205
minants subtilisins, 111, 596-602
field or electrostatic effects, I, 154 transient, I, 176
hydrogen bonding, I, 152-154 trypsin, 111, 269-273
matrix effects, I, 155 unsuspected, I, 176-178
microenvironment polarity, I, X-ray crystallography and
151-152 comparisons of protein solutions
miscellaneous effects, I, 155-156 and crystals, I, 202-203
steric effects, I, 154-155 isomorphous heavy atom replace-
general principles, I, 166167 ments, I, 201-202
immunochemistry and, I, 199-200 Chemical reaction
inorganic pyrophosphatase, IV, 514-518 rate equations, derivation, 11, 61-63
insoluble enzymes and antigens, I, Chlamydomonas reinhardti, N-acetyl-
200-201 glutamate-5-phosphotransferase of,
lysozyme, I, 207-211 IX, 513
mechanism and reactivity Chlorella pyrenoidosa, adenosine diphos-
kinetic considerations, I, 159-162 phoryl glucose pyrophosphorylase of,
protein functional group nucleo- VIII, 90
philicities, I, 162-164 Chloroplast ( s )
superreactivity, I, 164-166 adenosine triphosphatase
papain, I, 205-207 Euglena gracilis, X, 394
active site, 111, 515-516 spinach, X, 389-394
other reactions, 111, 516518 ribonucleic acid polymerase of, X,
pepsin, 111, 133-137 329-330
Choline dehydrogenase arginine 145, 111, 176-179

electron transport system and, XIII, catalytic site, 111, 179-182
261-263 isoleucine 16, 111, 175-176
properties, XIII, 260-261 methionine 192, 111, 179
Choline sulfatase, V, 14 Chymotrypsinogen A, chemical struc-
Chondroitin, hyaluronidase and, V, 310 ture, 111, 187-189
Chondroitin sulfates, hyaluronidase and, Chymotrypsinogens A and B, activation
V, 310 products, 111, 185-187
Chondrosulfatase, V, 12 Circular dichroism
Chymopapains, 111, 537-538 protein structure and, 11, 381-382, 408
Chymotrypsin(s) active center amino secondary, 11, 382386
acids, interaction, 111, 245-248 tertiary, 11, 386-391
active-site-directed reagents and, I, 94- typical cases, 11, 381407
103 ribonuclease, IV, 719-723
individual amino acid function Cis-trans isomerization about double
aspartic acid 102, 111, 235-236 bonds
aspartic acid 194, 111, 243 enzymic
histidine 57, 111, 231-234 with bond migration, VI, 39@-395
isoleucine 16, 111, 236-243 without bond migration, VI, 381390
serine 195, 111, 217-231 nonenzymic
ribonuclease and, IV, 674 metals and metal ions, VI, 401406
structure nucleophilic catalysis, VI, 397-401
physical probes, 11, 391-396 photoisomerization, VI, 395-397
ultraviolet difference spectroscopy, reversible addition of radicals, VI,
11, 414-417 406
fluorescence spectroscopy, 11, 421-424 Cistron, types of mutations and, I, 243-
substrate specificity 245
acylamido interaction, 111, 208 Citramalate, cleavage and synthesis, VII,
amino acid side chain binding, 111, 431-432
205-208 Citrate lyase
locked substrates, 111, 209-212 assay and isolation, VII, 378-379
stereo specificity, 111, 207-209 catalytic properties
a-Chymotrypsin control, VII, 387
active center structure equilibrium and kinetics, VII, 380-
acyl enzyme, 111,20&202 381,386387
enzyme-inhibitor complex, 111, 199- specificity, VII, 380, 385-386
200 stereospecificity, VII, 381, 387
enzyme-product complex, 111, 198- molecular properties
199 cofactors, VII, 379
native enzyme, 111, 190198 inhibitors, VII, 380
X-ray diffraction, 111, 190-195 molecular weight and subunits, VII,
7-Chymotrypsin, active center structure, 379
111, 202-204 sulfhydryl groups, VII, 379380
8-Chymotrypsin, active center, 111,204 reaction mechanism, VII, 381382, 388-
1.-Chymotrypsin, actiye center, 111, 204 389
Chymotrypsinogen Citrate synthase
activation, 111, 167-169 assay and isolation, VII, 358-360
active site unblocking, 111, 244 catalytic properties
X-ray crystallography, 111, 169-176 control, VII, 362-383
activation refolding, 111, 182-183 equilibrium and kinetics, VII, 382
specificity, VII, 361-362 pH optimum and ion efficts, 111,

stereospecificity, VII, 363-364 706-707
molecular properties sulfhydryl requirement, 111, 707-708
activators, VII, 361 historical perspective, 111, 700-703
cofactors, VII, 360 inhibitors
inhibitors, VII, 361 affinity labeling, 111, 714-715
molecular weight and subunits, VII, competitive, 111, 712-714
360 origin of active site specificity, 111,
sulfhydryl groups, VII, 360-361 717-71 9
proton transfer and, 11, 316-317 purification and assay, 111, 704
reaction mechanism specificity
citric anhydride as intermediary, proteins and polypeptides, 111, 710-
VII, 367-368 712
citryl coenzyme A as intermediary, synthetic esters and amides, 111, 708-
VII, 367 710
enolization of coenzyme A, VII, 365- structural properties
366 amino acid composition, 111, 706
inversion of configuration of acetyl physical constants, 111, 704-705
coenzyme A, VII, 366-367 use in sequence analysis, 111, 716-717
keto or enol .form of oxalacetate, Cobalt carbonic anhydrase and, V, 643-
VII, 365 646
Citric acid cycle reactions Coenzyme A transferase (s)
stereospecificity catalytic properties
fumarase, 11, 171-176 assay, IX, 487
isocitrate dehydrogenase, 11, 168-171 mechanism and kinetics, IX, 488-
succinate dehydrogenase, 11, 176-179 496
synthesis of citrate, 11, 164-168 specificity, IX, 486-487
Clostridium histolyticum thermodynamics, IX, 487488
collagenases, 111, 662-663 properties, IX, 485436
catalytic properties, 111, 670-689 reactions catalyzed, IX, 483485
chemical and biosynthetic modifica- Coenzyme B12-dependent reactions, ste-
reochemistry, 11, 204-214
tion, 111, 669-670
Collagenase(s)
composition, 111, M.Wf37
culture of organism and enzyme
activation by transition metals, 111,
purification, 111, 663-665
683
molecular size, 111, 667-668
assay, 111, 684-685, 692
physical properties, 111, 669
cofactors, 111, 677-678
possible subunits, 111, 668-669
evidence for intrinsic metal compo-
Clostridium pasteurianum, phosphofruc-
nent, 111, 678-683
tokinase of, VIII, 256 mechanism, 111,688-689
Clostridium perfringem reaction catalyzed, 111, 670, 691
phospholipase C of, V, 84-85 specificity, 111, 670-677, 691
polynucleotide phosphorylase of, VII, substrate interaction, 111, 688
571-572 thermodynamics and kinetics, 111,
Clostripain 685-688
as collagenase contaminant, 111, 715- zinc containing peptides, 111, 683-684
716 Clostridium histolytlcum, 111, 662-689
definition, 111, 699-700 clostripain in. 111. 715-716
-aeneral features definition, 111, 652-653
human synovial fluid, 111, 693-696 reduction of oxygen, XII, 578-579

known enzymes and their functions, specificity, inhibition and steady-
111, 653-659 state kinetics, XII, 574475
nature of substrate, 111,650-652 historical background, XII, 507-511
properties, generalizations, 111,659-660 magnetic and spectroscopic properties
R a m catesbiana, 111, 689-693 definitions and distribution of copper
uses of, 111, 660-662 forms, XII, 563-566
Compartmentalization, metabolic regula- type 1 copper, XII, 566-567
tion and, I, 423426 type 2 copper, XII, 567-570
Complementation, mutations and, I, 249- type 3 copper, XII, 570-571
25 1 reducing dioxygen to hydrogen per-
Conformational states, frozen, I, 370-371 oxide
Control mechanisms amine oxidase, XII, 511-527
compensatory galactose oxidase, XII, 527-533
antagonism of endproduct inhibi- Cortisone sulfatase, V, 10
tion, I, 436437 Creatine kinase
metabolite activation, I, 434-436 active form of substrates
precursor substrate activation, I, guanidine, VIII, 414
438-439 nucleotide, VIII, 412413
Convergence, protein evolution and, I, adenosine polyphosphates as inhibitors,
328-329 VIII, 422
Cooperativity, molecular basis, I, 375- anion effects, VIII, 423427
379 catalytic site, formation and topog-
Cooperativity index, enzyme regulation raphy, VIII, 439442
and, I, 358 conformational changes, substrate-
Coordination schemes induced, VIII, 436-438
determination “essential” thiol group
confirmatory techniques, 11, 468-470 importance for catalytic activity,
provisional techniques, 11, 464-468 VIII, 443448
enzymes, metals and substrates, 11, structural involvement, VIII, 44%
463464 443
experimentally determined, 11, 470- substrate effects, VIII, 448-451
476 equilibrium, VIII, 428-431
techniques, 11, 464470 groups essential for activity
experimentally determined cysteine residues, VIII, 431432
higher metal complexes, 11, 475476 histidine, VIII, 434
higher substrate complexes, 11, 471- lysine, VIII, 43S434
475 tyrosine, VIII, 434-436
ternary complexes, 11, 470-471 historical background, VIII, 384485
Copper-containing oxidase(s) hybrid, VIII, 403
blue mechanism, VIII, 451455
biological distribution and function, metal ions and, VIII, 409412
XII, 558-559 other brain-type, VIII, 402403
history, XII, 557-558 other muscle enzymes
oxidation-reduction properties, XII, assay and specific activity, VIII, 4.p
571-574 purification, VIII, 400
purification and molecular proper- stability, VIII, 401
ties, XII, 560-563 ox brain
catalytic properties assay and specific activity, VIII, 401
reducing substrates, XII, 575-578 purification, VIII, 401
stability, VIII, 401-402 Cystathionine p-synthetase, properties,

rabbit muscle VII, 54-56
assay and specific activity, VIII, Cystathionine y-synthetase, properties,
395-398 VII, 60-61
purification, VIII, 395 Cysteamine oxygenase, properties, XII,
stability, VIII, 398-399 148-149
role of metals in mechanism, 11, 499- Cysteine lyase, properties, VII, 56-57
500 Cysteine oxygenase, properties, XII, 149-
structure 150
amino acid composition, VIII, 390- Cysteine residues
392 creatine kinase, VIII, 431432, 442-451
isoenzymes, interspecific hybrids, guanidino kinases, VIII, 477-480
conformers and genetic variants, Cysteine synthetase, properties, VII, 54
VIII, 386-390 Cystine-disulfide groups, ribonuclease,
molecular weight, VIII, 395 IV, 690-696
primary, VIII, 392-393 Cystine peptides, microbial proteinases,
secondary and tertiary, VIII, 393-394 111, 752-754
subunit shape and organization, Cytidine diphosphate-D-glucose oxidore-
VIII, 394-395 ductase, properties, V, 478479
substrate binding, VIII, 414-420 Cytidine kinas6 see Uridine kinase
substrate specificity Cytidine monophosphate kinase, see Uri-
guanidines and organization of cre- dine monophosphs te kinase
atine binding site, VIII, 403-407 Cytidine triphosphate synthetase
nucleotides and related inhibitors, allosteric control, X, 546-547
VIII, 407-409 cooperative effects, X, 547-548
temperature and, VIII, 420-422 6-diazo-5-oxonorleucine and half-the-
Crosslinks sites reactivity, X, 549-550
intramolecular, ribonuclease, IV, 696- historical, X, 540-541
697 purification, X, 541-542
Crolalus adamanleus reaction catalyzed, X, 539-540
venom, phospholipase AI of, V, 75-76 covalent chemistry, X, 543-546
Crotalus atroz related enzymes, X, 552-558
venom, phospholipase A2 of, V, 7.7-78 role of nucleoside triphosphates, X,
Crotonase, properties, V, 568-571 550-552
Cyanate, inorganic pyrophosphatase and, structure, X, 542-543
IV, 516-517 Cytochrome (s),
Cyanogen bromide, inorganic pyrophos- absorption spectra, XI, 398-400
phatase and, IV, 514 bacterial
Cyclic adenosine monophosphate common methodology in research,
protein kinase dependent XI, 506-508
mechanism, VIII, 568-572 evolution, XI, 540-547
nomenclature, VIII, 566 patterns in electron transport path-
properties, VIII, 572-578 ways, XI, 508-509
purification, VIII, 568 sequence and structure, XI, 534-540
tissue and subcellular distribution, type b, XI, 591-593
567-568 Cytochrome b,
Cyclodiene insecticides, epoxidases and, mammalian, purification of, XI, 563-
VII, 210-211 564
jj’-Cystathionase, properties, VII, 51-52 occurrence and function, XI, 550-551
y-Cystathionase, properties, VII, 57159 plant, XI, 587-591
reaction with substrates eukaryotic,

adenosine triphosphate-induced re- photosynthetic cytochromes f and
duction, XI, 561 CJSS,XI, 493497
antimycin effect, XI, 562 respiratory cytochrome c, XI, 400-
kinetics, XI, 560-561 489
redox change, XI, 562-563 respiratory cytochrome c,, XI, 489-
respiratory, XI, 551652 492
absorption spectra, XI, 554-558 metal complexes and, 11, 534-538
different types, XI, 552-554 principles of protein evolution and, I,
miscellaneous, XI, 563-564 274-285
oxidation-reduction potential, XI, respiratory
558-560 amino acid sequence, XI, 419429
Cytochrome bl, occurrence of, XI, 579- evolution, XI, 429-450
584 molecular folding and structural in-
Cytochrome bl, preparation and proper- tegrity, XI, 450463
ties, XI, 585-587 oxidation reduction mechanism, XI,
Cytochrome ba 463489
biological role, XI, 567 structure, XI, 405-419
distribution, XI, 566 Cytochrome cl, respiratory, XI, 489-492
isolation, XI, 567-568 Cytochrome cm, photosynthetic, XI, 493-
nomenclature, XI, 565-566 497
properties, Cytochrome c oxidase
chemical, XI, 668-569 biological role, XIII, 299-300
physical, XI, 569 chemical and physical properties,
spectral, XI, 589-571 XIII, 301-302, 313-314
structure, XI, 571-572 interaction with cytochrome c, XIII,
amino acid sequence, XI, 572-573 334-335
ternary, XI, 573-576 kinetic studies, XIII, 335-337
Cytochrome bs reductase models, XIII, 314-315
cytochrome P-450 reductase and, XIII, chemistry of oxygen reduction, XIII,
151-153 302-307
mechanism, microsome bound, XIII, electronic spectroscopy
161-162 absorption spectra, XIII, 315-319
mechanism of Strittmatter, review, circular dichroic spectra, XIII, 319
XIII, 156-161 electron paramagnetic resonance
methemoglobin reductase and, XIII, studies
164-165 copper, XIII, 329-330
molecular properties, amphipathic and iron, XIII, 331
soluble forms, XIII, 154-156 ligand binding effects, XIII, 332333
structural studies, XIII, 162-164 p-oxobishemin and, XIII, 333334
Cytochrome b-662, valence state changes and, XIII,
distribution and preparation, XI, 584 331-332
properties and structure, XI, 584-585 historical background, XIII, 300-301
Cytochrome(s) c, ligand binding studies, XIII, 319-320
bacterial, XI, 497-506 azide, fluoride and cyanide, XIII,
evolution, XI, 64&547 320-321
function, XI, 506-509 carbon monoxide, XIII, 321-323
photosynthesis, XI, 509316 dioxygen, XIII, 323-326
respiratory chain, XI, 518634 lipids of, XIII, 312-313
structure, and sequence, XI, 534-540 mechanisms, XIII, 337344
metal components, XIII, 307-309 arrangement of unit chains, V, 230-

potent iome try 233
electron economy, XIII, 325-326 average chain length, V, 229-230
iron-copper coupling, XIII, 326-327 enzymic action pattern, V, 233-234
ligand binding effects, XIII, 327-328 Debye forces, propinquity effects and, 11,
summary, XIII, 328329 254-264
protein of, XIII, 309-312 3-Decynoyl-N-acetylcysteamine,isomer-
Cytochrome c peroxidase ization of, V, 459-461
cytochrome c interaction, XIII, 356- Dehydratases, miscellaneous, VII, 53-54
360 Dehydration ( 8 )
enzymic activity, XIII, 352-353 metal ion-assisted
general comments, 360-361 L-arabonate-n-fuconate, v, 581-582
historical background, XIII, 345-347 n-arabonate and p-xylonate, V, 582
preparation and molecular properties, galactonate, V, 578479
XIII, 347-348 gluconate, V, 575-578
reaction mechanism, XIII, 353356 hexarate, V, 579-581
structural aspects, XIII, 348-351 n-mannonate and n-altronate, V, 579
Cytochrome f, photosynthetic, XI, 493- Schiff base-assisted
497 glucosaminate dehydrase, V, 586
Cytochrome 0,occurrence and proper- 2-keto-3-deoxy-~-arabonatedehydra-
ties, XI, 592-593 tase, V, 583-585
Cytochrome P-450 reductase, XIII, 165- 5-keto-4deoxyglucarate dehydratase,
166 v, 585-586
catalytic activities, XIII, 167-169 Dehydrogenases
general properties, XIII, 166-167 the bigger family
mechanism, XIII, 169-173 general XI, 94
Cytophaga, isoamylase of, V, 204-206 members, XI, 94-99
primordial mononucleotide binding
D proteins, XI, 101
structural relationships, XI, 99
Deaminases, modification of, I, 127-128 time scale, XI, 99-101
Debranching enzyme (s) characteristics of, XIII, 90-91
characterization of, V, 223-226 comparison of three-dimensional
classes of, V, 192-194 structure
direct, V, 194-208 known structures, XI, 64-65
indirect, V, 208-210 malate and lactate to alcohol and
assay of glucosidase-transferase ac- glyceraldehyde-3-phosphatede-
tivity, V, 211-212 hydrogenase, XI, 65-69
effect on glycogen structure, V, 217- mononucleotide binding unit, XI,
219 69-70
glucosidase-transferase in glycogen NAD binding structure, XI, 70-73
storage disease, V, 221-222 recognition of similar structural do-
other enzymes, V, 222-223 mains, XI, 73-74
pH dependence, V, 215-217 dissociation constants of enzyme-co-
purification and physical properties, enzyme compounds,
v, 210-211 cooperative effects, XI, 46-47
reversion reactions, V, 219-220 initial measurements and binding
specificity, V, 213-215 studies, XI, 38-44
in vivo roles, V, 226-228 pH effects and role of histidine, XI,
structure determination and, 228-229 44-46
domain and subunit assembly, yeast alcohol dehydrogenase, XI, 22-

conservation of contacts, XI, 91 23
gene fusion, XI, 89-90 nicotinamide adenine dinucleotide-
quaternary structure, XI, 91-93 linked, metal complexes and, 11,
quaternary structure-evolution, XI, 525-528
93 preliminary generalizations, XI, 3-4
equilibrium and kinetics of enzyme- quaternary structure, XI, 91-92
coenzyme reactions alcohol dehydrogenase subunit asso-
dissociation constants, XI, 34-42 ciation, XI, 93
kinetics, XI, 4247 evolution, XI, 93
flavodoxin and, XI, 94-96 P and R axis-cooperativity, XI, 91-
functional aspects of dinucleotide 92
binding domains, XI, 83-84 Q axis, XI, 91
adenosine monophosphate, XI, 87-88 rhodanese and, XI, 98
nicotinamide adenine dinucleotide, sequence comparisons based on struc-
XI, 84-87 tural alignments
nicotinamide mononucleotide, XI, glutamate dehydrogenase compari-
88-89 sons, XI, 79
some generalizations, XI, 89 lactate, alcohol and glyceraldehyde-
inhibition and activation 3-phosphate dehydrogenases, XI,
analogs, XI, 30-34 77-79
product, XI, 34-35 statistics of comparisons, XI, 79-83
substrate, XI, 25-30 sequence comparisons in the absence
initial rate equations of three-dimensional structural
ordered mechanism with isomeric information
enzyme-coenzyme compounds : significance, XI, 7677
conformation change, XI, 10-11 suggested homologies or analogies,
ordered and random mechanisms for XI, 74-76
three-substrate reactions, XI, 13-15 steady-state kinetics
preferred pathway mechanism, XI, cooperative rate effects, XI, 31-34
12-13 inhibition and activation by sub-
rapid equilibrium mechanism, XI, strates, substrate analogs and
11-12 products, XI, 22-31
simple ordered mechanism, XI, 7-10 initial rate equations for ordered
kinases and, XI, 96-98 and random mechanisms, XI, 6-14
kinetics of enzyme-coenzyme isotope exchange a t equilibrium, XI,
reactions, 14-16
conformational changes, XI, 50-52 kinetic studies with alternate sub-
velocity constants, XI, 47-50 strates, XI, 18-22
kinetics of transient phase, XI, 47-48 maximum rate and Haldane rela-
integrated rate equations, XI, 48-50 tions, XI, 16-18
lactate dehydrogenases, XI, 52-53 phenomenological initial rate equa-
liver alcohol dehydrogenase, XI, 50- tions, XI, 4-6
52 subtilism and, XI, 98-99
other enzymes, XI, 53-55 Dehydroluciferin, adenylylation of, VIII,
kinetic studies with alternative 19-20
substrates Deletions, protein structure and, I, 293-
liver alcohol dehydrogenase, XI, 20- 300
22 Deoxyadenosine kinase, properties, IX,
other enzymes, XI, 23-24 66-68
Deoxyadenosine monophosphate kinase, assay, X, 239-240

properties, IX, 86-87, 95-96 isolation of covalent intermediates
3-Deoxy-~-arabino-heptulosonate7-phos- deoxyribonucleic acid-adenylate, X,
phate synthase, modification of, I , 245-246
139 ligase-adenylate, X, 245
Deoxycytidine kinase, properties, IX, mechanism of phosphodiester bond
62-66 synthesis, X, 244-252
Deoxycytidine monophosphate kinase, physical homogeneity, X, 241
properties, IX, 88-89, 95-96 physical properties,
Deoxycytidylate hydroxymethyltransfer- amino acid analysis, X, 242
ase, properties, IX, 209-210 molecular weight, X, 241
Deoxyguanosine kinase, properties, IX, stoichiometry, X, 242-243
68-69 purification, X, 240-241
Deoxyguanosine monophosphate kinase, reversal of, 246-248
IX, 94-96, see Guanosine monophos- role in vivo
phate kinase bacteriophage-induced, X, 252-254
Deoxyribonuclease (s) Escherichia coli, X, 254-259
adenosine triphosphate-dependent, IV, steady state kinetics
259, 261-262 overall reaction, X, 248-249
catalytic properties partial reactions, X, 249-252
inhibitors, IV, 281-283 Deoxyribonucleic acid methyltransferases
phosphodiesterase activity, IV, 283 biological significance, IX, 194-195
substrate concentration, pH and occurrence, IX, 190
ions, IV, 280-281 properties
classification of, IV, 251-252 bacterial, IX, 190-192
spleen eukaryotic, IX, 192-193
components, IV, 275 regulation, IX, 193-194
dimeric structure, IV, 275-276 Deoxyribonucleic acid polymerase(s)
distribution, localization and role, catalytic reactions
IV, 285-287 basic features, X, 123-124
features of degradation, IV, 276-278 fidelity of replication, X, 127-129
general catalytic properties, IV, 280- implications of mechanism, X, 134-
283 135
isolation, IV, 272-273 multiple sites in active center, X,
mechanism, IV, 278-280 124-125
methods of investigation, IV, 278 polymerization step, X, 125-126
physical and chemical properties, IV, pyrophosphorolysis and pyrophos-
273-275 phate exchange, X, 126-127
specificity, IV, 283-285 ribonucleotides and, X, 133-134
Deoxyribonuclease I specialized functions, X, 135-137
active center, IV, 297-299 synthesis without template, X, 132-
chemical nature, IV, 292-297 133
historical background, IV, 289-291 synthetic product, X, 131-132
inhibitor, IV, 299-302 template-primer, X, 129131
ions and, IV, 302-303 classification
kinetics, IV, 303-308 native and denatured templates, X,
physiological role, I, 310 182-183
specificity, IV, 308-310 polyribonucleotide templates, X, 184
Deoxyribonucleic acid ligase(s) size, X, 183
adenylyl transfer functions, VIII, 45-48 6 comparison of properties, X, 144
definitions and measurements, X, 175- 2-Deoxyribose-5-phosphate aldolase

176 catalytic reaction
exonucleases associated, IV, 255-258 activators and inhibitors, VII, 320
fidelity of synthesis, X, 201-202 assay, VII, 319-320
historical, X, 174-175 equilibrium constant, VII, 320
inhibitors and activators, X, 199-201 pH optimum, VII, 320
initiators, X, 203-204 Schiff base formation, VII, 321
intracellular distribution substrate specificity, VII, 321
chloroplasts, X, 181 historical background, VII, 315
membranes and other structures, X, metabolic significance, VII, 316-317
mitochondria, X, 180-181 isolation, VII, 317-319
nuclei, X, 179-180 physical properties, VII, 319
isolation and physicochemical proper- occurrence, VII, 315-316
ties, X, 120-123 Deoxysugars
kinetics synthesis, general considerations, V,
extent of synthesis, X, 195-196 465467
temperature and, X, 194-195 Deoxy sugar aldolase (s) ,general, VII,
metal activators, X, 193-194 303-304
molecular properties Deoxythymidine diphosphate-D-glucose
antisera and, X, 192-193 oxido-reductase
kinetic properties, V, 469-470
homogeneity, X, 188-189
presence of nuclease, X, 189-190 molecular properties, V, 467469
pyridine nucleotide and
sulfhydryl groups, X, 190-191
substrate release, V, 474476
zinc, X, 191-192
subunit association, V, 476-478
occurrence, X, 176-179
reaction mechanism
pH and PI, X, 194
enzyme bound pyridine nucleotide,
properties of purified viral enzyme, X,
V, 472-473
218
intramolecular hydrogen transfer, V,
size, X, 219-220 470-472
storage and stability, X, 219 isotope effects, V, 473-474
proteolytic cleavage: two enzymes in Deoxythymidine kinase
one polypeptide, X, 138-139 distribution, purification and assay,
purification IX, 69-70
chromatography, X, 185-186 kinetic, molecular and allosteric prop-
extent, X, 186-188 erties, IX, 71-74
stability, X, 185 reaction mechanism; active site, IX,
subcellular fractionation, X, 184-185 74
substrates substrate specificity, IX, 70
requirements, X, 198-199 Deoxythymidine monophosphate kinase
specificity, X, 196-197 distribution and purification ; assay;
templates stability, IX, 91-92
deoxyribonucleic acid, X, 204-205 kinetic and molecular properties, IX,
ribonucleic acid, X, 206-207 93-96
variety of, X, 119-120 substrate specificity, IX, 92-93
viral, Deoxyuridylate hydroxymethyl transfer-
purification, X, 216-218 ase, properties, IX, 210
solubilization, X, 215 Dermatan sulfate, hyaluronidase and, V,
virus purification and, X, 214-215 310-311
2,5-Diaminohexanoate, lysine mutase Diphosphoglycerate mutase, properties,

and, VI, 554 VI, 476477
Diazonium-1H-tetrazole, inorganic pyro- Disaccharide phosphorylases
phosphatase and, IV, 517-518 general background, VII, 515-518
3,&Dideoxyhexoses, synthesis of, V, 479- substrate specificity, VII, 526-528
480 Disulfide bridges
7,&Dihydro-2-amino-4-hydroxy-6-hy- elastase, 111, 339341, 348-349
droxymethylpteridine pyrophospho- p-hydroxydecanoyl thioester dehy-
kinase, X, 627-628 drase, V, 452453
Diisopropylfluorophosphate, papain and, prothrombin, 111, 313
111, 516-517 thrombin, 111, 290-291
Dioxygenase(s) trypsin, 111, 271
biological function and general Disulfide groups, amylases, V, 244-245
properties Disulfide loop, proteinase inhibitor re-
double bond cleavage, XII, 123-125 active site and, 111,420-422
double hydroxylation, XII, 125 Divergence
miscellaneous, XII, 125-127 protein evolution, I, 314-316
sulfur-containing compounds, XII, factors influencing rate, I, 317-321
125 speciation of homologous proteins:
classification, XII, 121-123 genetic drift, I, 321-328
heme-containing Dopamine p-monooxygenase, properties
indoleamine 2,3-dioxygenase, XII, of, XII, 294-295
130-132 Double bonds
tryptophan dioxygenase, XII, 127- isolated, sterospecificity of reactions,
130 111, 179-186
Double reciprocal plots
history and definition, XI, 120-121
a-ketoglutarate, XII, 151-152 enzyme regulation and, I, 356-357
nonlinear, 11, 56-59
y-butyrobetaine hydroxylase, XII,
Drosophila melanogaster
167-169
acid phosphatase of, IV, 498
p-hydroxyphenylpyruvate hydroxyl-
molybdenum hydroxylase
ase, XII, 179-183
genetics of, XII, 406412
lysyl hydroxylase, XII, 165-167
mechanism, XII, 183-189
E
prolyl hydroxylase, XII, 152-165
pyrimidines and nucleosides, XII, Ehrlich ascites cells, 5'-nucleotidase of,
169-179
IV, 34-49
nonheme iron-containing, XII, 132-133 Elastase, see also Tosyl elastase
cysteamine oxygenase, XII, 148-149 activation, 111, 244-245
cysteine oxygenase, XII, 149-150
amino acid sequence determination
phenolic, XII, 133-148 complete sequence, 111,341-343
phenolic disulfide bridged peptides, 111,339-
extradiol, XII, 140-144 341
intradiol, XII, 133-140 assay methods
others, XII, 144-148 elastin and, 111, 325-326
Dipeptidases, 111, 116-117 synthetic substrates and, 111, 326-
Dipeptidyl aminopeptidase 1, see Dipep- 327
tidy 1-transferase criteria of purity, 111, 32-29
Dipeptidyltransferase, 111, 105-111, see crystals, activity of, 365-366
also Cathepsin C enzymic activity
inhibitors and activators, 111,337-338 delocaliration of ,!) charge, VII,

irreversible inhibitors, 111, 338-339 81-82
proteins and, 111, 332-333 modification of X , VII, 82-84
synthetic substrates and, 111, 333- solvent effects, VII, 85-86
337 stereoelectronic control, VII, 85
history and distribution, 111, 323-325 weak bases, VII, 86
physicochemical properties, 111, 329- fumarase, 11, 304-308
331 nonenrymic aspartate-fumarate inter-
proelastase and, 111, 331-332 conversion, VII, 86-88
purification methods, 111, 327-328 stereochemistry, 11, 309-312
reactivity and pK. of amino groups, ,!)-Elimination reactions
111, 367472 mechanism, VII, 66-72
competitive labeling technique, 111, dehydratases, VII, 39-48
366-367 y-Elimination and replacement
nitrous acid and, 111, 372 mechanism, VII, 72-73
valine and aspartate residues, 111, pyridoxal phosphate and, VII, 59-62
372-373 y-Elimination reactions, pyridoxal-
ribonuclease and, IV, 672-673 linked, VII, 57-59
sequence homologies in serine pro- Elongation
teinases stringent response and, X, 78-79,82-83
activation peptides, 111, 343-344 effect of ppGpp, X, 81-82
B chains, 111, 344-348 synthesis of MSI and MSII, X, 79-81
disulfide bridges, 111, 348-349 Elongation factor(s)
hypothetical models, 111, 349-352 bacterial, X, 55-57
stability, 111, 332 function of factor G, X, 64-67
X-ray crystallography, function of factor Tu, X, 58-63
crystals and, 111, 353-354 physical properties, X, 57-58
Fourier synthesis, 111, 355-356 role of factor Ts, X, 63-64
heavy atom derivatives, 111, 354-355 Elongation factor G
Electron delocaliration, enzymic-C-H interaction with ribosomes, X, 66-67
fission and, 11, 287-290 protein synthesis and, X, 65-66
Electron density maps, interpretation of, Elongation factor Tu
I, 46-52 complex interaction with ribosomes,
Electron microscopy, collagenase action X, 62-63
and, 111, 693 guanine nucleotide binding, X, 58-59
Electron paramagnetic reaonance, ternqry complex, X, 59-62
ribonuclease, IV, 723-725 Endolysin
Electron-transferring flavoprotein bacteriophage A
catalytic properties, XII, 116-118 catalytic properties, V, 391-392
function, XII, 109-110 chemical properties, V, 388391
molecular properties physicochemical properties, V, 387-
oxidation-reduction, XII, 116 388
properties of chromophore, XII, purification, V, 385-386
111-115 Endonuclease (8)
purification, molecular weight and bacterial
amino acid composition, XII, 111 nonspecific, IV, 259-262
a, ,!) Elimination reactions specific, IV, 262-270
aconitase, 11, 302-304 Enolase
factors influencing, VII, 79-81 active site
conjugation, VII, 81 components, V, 532-534
number, V, 530-532 loss of function, survival value, I,

chemical properties 332-334
amino acid composition, V, 503 metal linkage
end groups and terminal sequences, coordination geometry, 11, 492494
V, 503-506 nature of ligands, 11, 490-492
immunochemistry, V, 507 role of metals in mechanism, 11,498-
polypeptide chain identity, V, 506- 499
507 hydrolases, 11, 519-525
criteria of purity, V, 502-503 lyases, 11, 508-519
general conisderations, V,499-501 phosphoenolpyruvate carboxylation,
kinetic parameters, V,523-524 11, 507-508
magnesium and, V,524-526 phosphoryl and nucleotidyl transfer,
mechanism of dehydration reaction 11, 499-507
evidence for carbanion intermediate, specificity, historical background, 11,
v, 537 119-129
isotope effects, V, 535-536 structure in solution
molecular properties, summary, V, circular dichroism and optical ro-
518-519 tatory dichroism, 11, 381-408
monomer-dirner activity relationships, fluorescence spectroscopy, 11, 418-
v, 537-538 430
physical properties, V,507-508 geometry and quaternary structure,
electrophoretic mobility, V, 508-510 11, 440-442
subunit structure, V, 510-518 infrared spectroscopy, 11, 374-379
properties of reaction catalyzed ionizable groups, 11, 430440
assay, V, 519-523 secondary and tertiary structures,
equilibrium, V, 523 11, 373-374
substrates, V, 519 ultraviolet absorption of peptide
rabbit muscle, glycidol phosphate and, groups, 11, 379-380
v, 534 ultraviolet difference spectroscopy,
role of metals, 11, 508-509 11, 408-417
substrate specificity, active site map- regulation of concentration
ping with analogs, V, 526-529 balance between synthesis and
yeast degradation, I, 402-403
carboxymethylation, V, 533 catabolite repression, I, 400401
photooxidation, V, 533-534 feedback repression of synthesis, I,
Enterochrome-566, properties, XI, 592 401402
Enzyme (s) substrate induction of synthesis, I,
bridge complexes, 11, 477478 399-400
carbonyl containing Enzyme crystallography, state of the art,
other, 11, 356358 I, 86-87
pyridoxal phosphate, 11, 346-356 Enzyme crystals
covalent modification differences from other crystals, I, 5-7
glycogen phosphorylase, I, 413-415 Fourier description
glutamine synthetase, I, 409413 structure analysis and phase prob-
development of novel properties, I, lem, I, 29-32
334-335 as sum of waves, I, 26-29
intrachain repetitions, I, 335 growth of,
multichain enzymes, I, 335-337 , batch, I, 19
inactive, proteinase inhibitors and, 111, equilibrium dialysis, I, 19-22
454-457 vapor diffusion, I, 19
mounting and radiation damage, I, adenosine diphosphorylglucose pyro-

22-23 phosphorylase
structure and symmetry, I, 7-13 energy charge and, VIII, 104-107
Enzyme regulation manganese effect, VII, 102-104
diagnostic tests, I, 356358 mutants, VIII, 109-117
allosteric protein evaluation, I , 372- alkaline phosphatase, I, 251-254, IV,
375 373-415
cooperativity index, I, 358 asparaginase, IV, 107-116
double reciprocal plots, I , 356-357 aspartate metabolism, regulation of, I ,
equations of state, I, 386388 457-459
fitting saturation curves, I, 361365 aspartate transcarbamylase of, IX,
frozen conformational states, I, 225497
370-371 aspartokinases of, VIII, 513-544
Hill plot, I, 358-359 adenylylation of, VIII, 44-45
minimal substrate technique, I, 372 cytochrome b, of, XI, 579, 580, 581
Scatchard or Klotz plots, I, 359-361 cytochrome b-562, properties and
Leelocity curves, I, 368369 structure, X I , 584-585
Y X or N x versus log (X) plots, I, deoxyribonuclease, ATP-dependent,
357 IV, 259
gloasary, I, 395-396 deoxyribonucleic acid ligase
molecular models, qualitative features, isolation and physical properties, X,
I, 344-348 239-243
quantitative molecular parameters mechanism, X , 244-252
derivation of general equation, I, role in vivo, X, 254-259
34a353 deoxyribonucleic acid polymerase I,
simple models, I, 353-355 physiological role, X, 139-141
Epimerase(s) deoxyribonucleic acid polymerase 11,
definition and history, VI, 365-357 physiological role, X, 142
keto-enol rearrangement and, VI, deoxyribonucleic acid polymerase, 111,
373-374 physiological role, X, 142-143
noncarbohydrate, VI, 378 deoxyribonucleic acid polymerase,
oxidation-reduction and, VI, 369-371 exonucleases and, IV, 255-258
proton shifts and, 11, 295-298 endonuclease I, IV, 259-280
Epinephrine, glycogen synthetase and, endonuclease 11, IV, 204-285
IX, 338, 341 exonucleases I and 111, IV, 253
Epoxidase(s) exonuclease I V of, IV, 254-255
general considerations, VII, 199-200 3' + 5' exonuclease, IV, 256
metabolic roles, VII, 200-201 5' + 3' exonuclease, IV, 256-258
Equations of state, enzyme regulation fatty acid synthetase, 3decynoyl-N-
and, I, 365-368 acetyl-cysteamine and, V, 461-463
Equilibria fructose-l,6-diphosphatase of, IV, 63%
chemical, metabolic regulation and, I, 639
418-419 P-galactosidase, VII, 624-625
Erthrocytes, phosphofructokinase of, 257 8-galastosidase, mutations, I, 255-266
Eecherichia coli glutaminase, IV, 80-93
acetyl coenzyme A carboxylase sub- glutamine synthetase of, VIII, 40-44,
units, VI, 60-64 X, 755-807
N-acetylglutamate-5-phosphotrans- inorganic pyrophosphatase
ferase of, IX, 513 catalytic properties, IV, 518-526
acid phosphatase of, IV, 498 molecular properties, IV, 501418
membrane adenosine triphosphatase, Eukaryotes

X , 416-421 deoxyribonucleic acid polymerases of,
methyltransferase of, IX, 154-160 X , 173-209
phage lytic enzymes, V, 355-361 polypeptide chain initiation
F series polysaccharide depolymer- inhibitors, X, 43
ase, V, 392-393 initiation factors, X, 2943
X endolysin, V, 385-392 initiator aminoacyl-transfer ribo-
N20F’ lytic enzyme, V, 382-385 nucleic acid and, X, 28-29
T 2 lysozyme, V, 379-382 messenger ribonucleic acid transla-
T4 lysozyme, V, 361-379 tion, X,43-44
phosphofructokinase of, VIII, 256-257 ribonucleic acid polymerases, X,261-
polynucleotide phosphorylase of, VII, 331
548-570 Evolution
mutant, VII, 571-572 allosteric proteins, I, 390393
restriction endonucleases, IV, 263-264 principles, cytochrome c and, I, 274-
ribonucleases of, IV, 241-243 285
succinyl coenzyme A synthetase of, structure-function relationships in
X, 582591 proteins, I, 267-274
thioredoxin reductase, general proper- Evolutionary factors
ties, XIII, 144-145 expression in protein structure
tryptophan synthetase convergence, I, 328-329
catalytic properties, VII, 22-30 development of novel properties, I,
molecular properties, VII, 8-21 334-337
vitamin BIZmethyltransferase of, IX, divergence, I, 314-328
122-154 loss of function and survival value,
Ester(s), subtilisin and, 111, 592-593 I, 332-334
Esterase(s) parallelism, I, 329-332
classification and distribution, V, 43-45 Exonuclease(s), bacterial, IV, 252-259
modification of, I, 124-127
other, V, 67-89 F
Estrone sulfatase, V, 6-7
Fast reaction techniques
Ethanolamine deaminase
application of
activation by monovalent cations,
conformational changes, 11, 112-114
VI, 545
enzyme-substrate reactions, 11, 108-
assays, VI, 542
112
cobamide binding sites, VI, 543-544
Fatty acid(s)
inhibitors, cobamide, VI, 544-545
activation, VIII, 6-11
isolation, VI, 541-542 biosynthesis, acyl carrier protein and,
occurrence, VI, 540-541 VIII, 164-165
other properties, VI, 546-547 unsaturated, cis-trans isomerization,
physical properties, VI, 542-543 VI, 390-394
specificity of coenzyme requirement, Fatty acid: coenzyme A ligases
VI, 544 catalytic properties, general consid-
substrate specificity and binding, VI, erations, X, 475-477
545-546 Fatty acid synthetase(s), I, 226-228
a-Ethylmalate, synthesis, VII, 426 3-decynoyl-N-acetylcysteamine and,
Etiocholanolone sulfatase, V, 9-10 V, 461-463
Euglena gracilis, chloroplast adenosine Fatty acyl coenzyme A synthetases
triphosphatase, X, 394 distribution and isolation
acetate : coenzyme A ligase, X, 470 a-ketoisovalerate synthase, VI, 205

long chain fatty acid: coenzyme A phenylpyruvate synthase, VI, 205-207
ligase, X, 471-472 pyruvate synthase, VI, 197-201
medium chain fatty acid :I coenzyme reductive carboxylic acid cycle of
A ligase, X, 470-471 bacterial photosynthesis, VI, 207-
medium-long chain fatty acid: co- 213
enzyme A ligase, x, 472-473 reductive monocarboxylic acid cycle
other related acid: coenzyme A of fermentative metabolism, VI,
ligases, X, 473-474 213-214
scope of chapter, X, 469-470 Fibrinogen
Feedback inhibition derivatives, thrombin and, 111,298-299
metabolic regulation and, I, 403404 thrombin and, 111, 295-298
allosteric concept, I, 404406 Ficin, 111, 538-542
cooperative effect, I, 406408 Fish, glycogen synthetase of, IX, 358
terminology, I, 408 Flavin coenzyme(s), structure and
Feedback regulation chemistry, XII, 423-425
multifunctional pathways, I, 444445 Flavodoxins
aspartate metabolism, I, 457-459 composition, molecular weight and
concerted inhibition, I, 449-450 purification, XII, 59-60
cumulative inhibition, I, 452454 discovery, nomenclature and distri-
enzyme multiplicity, I, 445-447 bution, XII, 58-59
heterogeneous metabolic pool inhi- flavin mononucleotide in, XII, 65-66
bition, I, 454-455 flavin-protein interactions
multivalent repression, I, 455-457 apoprotein preparation and proper-
sequential controls, I, 447-449 ties, XII, 82-83
synergistic inhibition, I, 450452 modified flavins and, XII, 85-87
Fermentation, reductive monocarboxylic protein modification and, XII, 87-88
acid cycle and, VI, 213-214 thermodynamics and kinetics, XII,
Ferredoxin( s) 83-85
bacterial function, XII, 60-63
background, XII, 37-39 oxidation-reduction potentials, XII,
chemical properties, XII, 4 4 4 6 98-102
physical properties, XII, 39-44 reactivity
chemical properties comproportionation, XII, 102-103
apoproteins and reconstitution, XII, dithionite, XII, 103-104
25-28 oxygen and ferricyanide, XII, 105-
chemical modification, XII, 28-29 108
electron transfer reactions, XII, 29 redox proteins and, XII, 108-109
exopeptidases and, XII, 28-31 regulation by iron, XII, 63-65
two irons per center spectroscopic properties
background, XII, 15-20 circular dichroism, XII, 94-95
chemical properties, XII, 25-31 fluorescence, XII, 93-94
perturbants of EPR spectra, XII, magnetic resonance, XII, 95-98
23-25 optical absorption spectra, XII,
physical properties, XII, 20-23 88-91, 99
Ferredoxin-linked carboxylations single crystal absorbance, XII, 91-93
general considerations, VI, 193-196, structure, XII, 66-67
214-216 determination and comparison of
a-ketobutyrate synthase, VI, 203-204 chemical sequences, XII, 67-70
a-ketoglutarate synthase, VI, 201-203 three-dimensional, XII, 70-82
Flavoprotein monooxygenase(s) reductive half-reaction, XII, 432-

external, XII, 204-206 431
bacterial luciferase, XII, 226-229 Fluorescence, ribonuclease, IV, 718-719
m-hydroxybenzoate-4-hydroxylase, Fluorescence spectroscopy
XII, 225 protein structure, 11, 418421,429-430
m-hydroxybenzoate-6-hydroxylase, typical cases, 11, 421-429
XII, 224-225 Fluorocitrate, aconitase and, V, 428-430
p-hydroxybenzoate hydroxylase, XII, Formaldehyde (and congeners)
211-216 transfer
imidazoleacetate monooxygenase, deoxycytidylate hydroxymethyl-
XII, 225-226 transferase, IX, 209-210
kynurenine-3-hydroxylase,XII, deoxyuridylate hydroxymethyl-
230-231 transferase, IX, 210
melilotate hydroxylase, XII, 217- glycine decarboxylase, IX, 221-223
22I thymidylate synthetase, IX, 210-215
microsomal amine oxidase, XII, serine hydroxymethyltransferase,
229-230 IX, 215-221
orcinol hydroxylase, XII, 223-224 Formate (and congeners)
phenol hydroxylase, XII, 221-223 transfer
salicylate hydroxylase, XII, 206-211 5-amino-4-imidazole carboxamide
internal, XII, 193-194 ribonucleotide transformylase,
arginine, XII, 203-204 IX, 204-205
lactate, XII, 194-199 formiminoglutamate formimino-
lysine, XII, 199-203 transferase, IX, 206-207
Flavoprotein oxidase ( s ) formiminoglycine formiminotrans-
chemical mechanism ferase, IX, 206
flavin reduction, XII, 474-503 5-formiminotetrahydrofolate cyclo-
oxidation by oxygen, XII, 503-505 deaminase, IX, 202-203
definition of, XII, 421-423 N-formylglutamate transformylase,
kinetics IX, 207-208
computer simulation, XII, 442-443 5-formyltetrahydrofolate cyclode-
strategy, XII, 425-426 hydrase, IX, 207-208
summary, XII, 443-445 10-formyltetrahydrofolate deacylase,
mechanism IX, 200
confirmation, XII, 437442 10-formyltetrahydrofolate synthe-
correlation of steady-state and tase, IX, 198-200
transient kinetics, XII, 435-437 glycinamide ribonucleotide trans-
molecular properties and kinetic formylase, IX, 203-204
mechanism 5,lO-methenyltetrahydrofolate
n-amino acid oxidase, XII, 445-466 cyclohydrolase, IX, 201
L-amino acid oxidase, XII, 456461 methionyltransfer ribonucleic acid
glucose oxidase, XII, 461-466 transformylase, IX, 208-209
monoamine oxidase, XII, 466-471 Formiminoglutamate formiminotrans-
old yellow enzyme, XII, 471-473 ferase, properties, IX, 206-207
steady-state kinetics Formiminoglycine formiminotransferase,
rate equation, XII, 429-432 properties, IX, 206
velocity measurements, XII, 426- 5-Formiminotetrahydrofolate cyclode-
429 aminase, properties, IX, 202-203
transient-state kinetics N-Formylglutamate transformylase,
oxidative half-reaction, XII, 435 properties, IX, 207-208
5-Formyltetrahydrofolate cyclodehy- purification and properties, IV, 632-

drase, properties, IX, 201-202 633
10-Formyltetrahydrofolate deacylase, structure and relation to other
properties, IX, 200 enzymes, IV, 633-634
10-Formyltetrahydrofolate synthetase, physiological role, IV, 613-615, 644-
properties, IX, 198-200 645
Frog muscle, glycogen synthetase of, proteolysis, changes induced, IV, 618
IX, 357 purification and properties, IV, 629-
Fructose-1,Bdiphosphatase(8) 630, 632-633
activation by disulfide exchange optimum pH and cation effects, IV,
coenzyme A and acyl carrier pro- 617418
tein, IV, 623-624 purification procedures, IV, 616-617
cystamine and, IV, 622-623 substrate specificity, IV, 618
homocystine, IV, 624-625 regulation, I, 439-441,IV, 613-615,
as regulatory mechanism, IV, 625- 630431
626 modification of tyrosine residues
assay methods and mechanism of and, IV, 619-620
action, IV, 615-616 papain and, IV, 619
Candida utilis pH and, IV, 618419
inhibition by AMP, IV, 636-637 pyridoxal phosphate and, IV, 620
purification and properties, IV, Saccharomyces cerevisiae, IV, 640
635836 slime molds, IV, 640
regulation, IV, 640 sulfhydryl groups, activation and, IV,
relation to SDPase, IV, 638 621422
structure, IV, 637438 L-Fucose isomerase, properties, VI, 346-
comparative properties, IV, 645- 348
646 L-Fuculose 1-phosphate aldolase
Eschen'chia coli, IV, 638439 catalytic reaction
higher plants and blue-green algae assay, VII, 314
physiological role, IV, 642-643 equilibrium constant, VII, 314
purification and properties, IV, 640- substrate specificity, VII, 314
642 properties, VII, 313-314
regulation, IV, 643 Fumarase
historical review, IV, 612413 catalytic properties
kidney active site affinity labeling, P,563-
purification and properties, IV, 629- 564
630 catalytic site number, V, 562-563
regulation, IV, 630431 kinetics, V, 552-557
molecular structure substrate specificity, V, 557-562
binding sites for divalent cation, general considerations, V, 539-540
IV, 628-629 historical development, V, 540-541
binding sites for F D P and AMP, isotope exchange and, 11, 304-308
IV, 627-628 mechanism of action, V, 564-568
induced conformational changes, modification of, I, 140-141
IV, 629 molecular properties
molecular weight and subunit amino acid composition, end groups
structure, IV, 826-627 and peptide maps, V, 544-545
muscle dissociation and recombination of
evidence for presence. IV. 632 subunits, V, 546-549
physiological ;ole, IV; 634-835 physical properties, V, 542-544
subunit structure, V, 545-546 other sources, VII, 661-663

thiol groups, V, 549-552 pH dependence, VII, 644-645
preparation and assay, V, 541-542 specificity, VII, 641-644
stereospecificity, 11, 171-176 transfer reaction, VII, 658-660
Fungi, see also Molds general background, VII, 618
aldolase of, VII, 215-216 immunological properties, VII, 839-
aspartate transcarbamylases of, IX, 641
302-306 mutations and, I, 255-256
carboxypeptidases of, 111, 790-791 occurrence, VII, 619-620
glutamate dehydrogenases physiochemical properties
Neurospora crassa, XI, 323-329 associated forms, VII, 633-634
others, XI, 329-332 denaturation and renaturation, VII,
nuclear ribonucleic acid polymerase, 634-636
X, 310-311 size, shape and quaternary structure,
ribonucleases of, IV, 208-239 VII, 627-633
purification
animal tissues, VII, 626-627
G Escherichia coli, VII, 624-625
other microorganisms, VII, 625426
n-Galactarate dehydrase, properties, V, Galactosyl transferase
580-581 biological significance, IX, 377
Galactonate dehydrase, properties, V, catalytic properties
578-579 kinetics, IX, 371-376
Galactose oxidase reaction catalyzed, IX, 369
chemical and physical properties, XII, substrate specificity, IX, 369-371
528-529 purification and properties, IX, 367-
copper of, XII, 529-530 369
discovery and purification, XII, 527- Gastricin, gene duplication and, I, 308
528 Gastric lipase, properties, VII, 605-606
inhibitors, XII, 531 Gaucher’s disease, acid phosphatase and,
mechanism, XII, 532-533 IV, 496
molecular properties and kinetic Genes
mechanism, XII. 461-466 cistron and types of mutations, I,
optical properties, XII, 530-531 243-245
specificity, XII, 531 duplication, I, 3 W 3 0 3
p-Galac tosidase carboxypeptidases, I, 308-309
assay and standardization, VII, 620- ethanol dehydrogenaaes, I, 309-311
623 gastricin, I, 308
bacterial suspensions and animal a-lactalbumin, I, 311-314
material, VII, 624 lysozyme, I, 311-314
standard assays and units, VII, 623 pancreatic proteinases, I, 303-307
transferase assay, VII, 624 pepsin, I, 308
chemical properties, VII, 636-639 rennin, I, 308
enzymic properties sulfhydryl proteinases, I, 307-308
active site, VII, 657-658 Genetic drift, speciation of homologous
condensation reactions, VII, 660 proteins and, I, 321428
inhibition studies, VII, 660-661 Glucagon, glycogen synthetase and, IX,
kinetics, VII, 648-4351 341
mechanism, VII, 651-657 a-Glucan phosphorylases
metal activation, VII, 645-648 allosteric transitions, VII, 473-474
desensitization, VII, 480-482 historical

kinetic and structural basis, VII, phosphohydrolase activity, IV, 545-
474-480 546
catalytic reaction phosphotransferase activity, IV,
nucleotide activation, VII, 439-441 546-547
substrate specificity, VII, 437-438 kinetic studies, IV, 572-574
chemical properties activators and inhibitors, IV, 578-
amino acid analysis and end groups, 582
VII, 446-447 mechanism, IV, 582-592
primary structure, VII, 448-451 pH and, IV, 574-576
enzyme structure, physical properties, substrate concentration, IV, 576-577
VII, 441-446 temperature, IV, 577-578
general considerations, VII, 435-437 membranous nature, possible signifi-
mechanism of catalysis cance, IV, 562-564
kinetics, VII, 460-463 metabolic roles and regulation, IV,
protein functional groups, VII, 463- 596-597
466 control in vivo, IV, 597-599
vitamin Ba coenzyme, VII, 451-459 speculation, IV, 599
regulation phospholipids and, IV, 554-556
associationdissociation phenomena, reactions catalyzed
VII, 469-473 multifunctional nature, IV, 567-568
interconversion of a and b forms, substrate specificity, IV, 568-571
VII, 466469 relation to other enzymes, IV, 552
mechanism of allosteric transitions, solubilization and attempted purifica-
VII, 473-482 tion, IV, 553-554
o-Glucarate dehydrase, properties, V, 580 Glucose-6-phosphate
Gluconate dehydratase, properties, V, distribution
578 intracellular, IV, 548-551
Glucosamine-6-phosphate isomerase, phylogenetic and tissue, IV, 547-548
properties, VI, 314-318 glycogen synthetase and, IX, 341-344
Glucosaminate dehydrase, properties, V, Glucose-6-phosphate isomerase
586 catalytic properties
D-c%ICOSeisomerase active form of substrate, VI, 293-294
nature of, VI, 341-344 anomerase activity, VI, 294-296
properties, VI, 349-354 assay, VI, 278-279
Glucose8phosphatae.e kinetic parameters, VI, 289-293
distribution, IV, 600-610 mechanism of action, VI, 296-301
catalytic properties, IV, 565-588 molecular architecture
assay methods, IV, 566-567 active site amino acids, VI, 285-287
control of phosphotransferase ac- amino acid composition, VI, 278-279
tivity, IV, 592-595 conformational states, VI, 284-285
kinetics and reaction mechanism, molecular weight and related proper-
IV, 572-592 ties, VI, 276-278
reactions catalyzed, IV, 567-571 multiple forms, VI, 281-284
thermodynamic considerations, IV, subunit structure, VI, 279-28E
571-572 occurrence and function, VI, 272-276
detergents and, IV, 556-557 Glucosidase-transferase
detergent-like effects, IV, 559-560 assay of, V, 211-212
direct effects, IV, 557-559 glycogen storage disease and, V, 221-
modifying effects, IV, 560-562 222
Glucosulfatase, V, 11 purification of, XI, 307

Glutamate reaction mechanism, XI, 357-360
pyrrolidone carboxylate formation from, regulation of, I, 443-444, XI, 360-366
enzymic, IV, 133-139 stability, denaturation and dissocia-
nonenzymic, IV, 130-133 tion, XI, 319-320
D-Glutamate cyclo transferase, pyrroli- tertiary structure, XI, 318-319
done carboxylate formation and, IV, Glutamate mutase
133-136 assay, VI, 524-525
L-Glutamate cyclotransferase, pyrrolidone catalytic properties
carboxylate formation and, IV, 138 conditions affecting activity, VI,
Glutamate dehydrogenase(s), 531-532
cellular location, XI, 305-306 equilibrium, VI, 532
chemical modification, interaction of components and
coenzyme site and specificity, XI, coenzyme, VI, 528-530
352-354 mechanism, VI, 532-534
cysteine residues, XI, 347-348 substrate and coenzyme specificity,
histidine residues, XI, 346-347 VI, 530-531
lysine residues, XI, 343-346 purification and molecular properties
other modifications, XI, 348-349 component E, VI, 525-526
spectrophotometric studies, XI, 349 component S, VI, 526-528
substrate site, XI, 349-352 stereochemistry, 11, 206-210
composition, XI, 320-322 Glutaminase(s)
distribution and coenzyme specificity, Azotobacter agik's, IV, 97-98
XI, 296-297 Escheiichia coli, IV, 80-81
animals, XI, 300-305 acyl transferase reactions, IV, 84-85
bacteria, XI, 297-299 assay, IV, 81
fungi, XI, 297 deuterium oxide and, IV, 90
plants, XI, 299-300 6-diazo-5-oxonorleucine and, IV,
electrophoretic and spectrophotometric 85-87
properties, XI, 322-323 mechanism of action, IV, 90, 92-93
function and equilibrium, XI, 294-295 occurrence, IV, 81
genetics and regulation of enzyme other inhibitors, IV, 87
synthesis, pH effects on kinetic parameters,
bacteria, XI, 332-334 IV, 88,89
fungi, XI, 323-332 purification, IV, 82
plants, XI, 334-335 relationship to other acylases, IV,
historical background, XI, 295-296 90, 91
kinetic studies, XI, 354-357 specificity, IV, 82-84
metamorphosis and, XI, 306 temperature and, IV, 88
modification of, I, 137 survey, IV, 93-95
oligomer structure, Glutamine
hexameric model, XI, 315-317 occurrence and function of, X, 699-
immunochemistry, XI, 317-318 704, 755-757
molecular weight, XI, 314-315 pyrrolidone carboxylate formation
trimer formation, XI, 317 from
polymerization, enzymic, IV, 139-141
mechanism, XI, 308, 310-312 nonenzymic, IV, 130-133
nucleotide effects, XI, 312-314 L-Glutamine cyclotransferase, pyrroli-
significance, XI, 307-308 done carboxylate formation and,
primary structures, XI, 335-343 IV, 139-141
Glutamine synthetase, X, 757-759 methionine sulfoxime phosphate

active site mapping, X, 720-733 formation, X, 714
adenylylation and deadenylylation, boxoproline formation, X, 713-714
VIII, 4044 physical and chemical properties, X,
historical development of problem, 704-708
X, 784-786 physicochemical properties, X, 792-793
regulatory protein, X, 786-787 adenylylation site, X, 793-794
uridylylation and deuridylation of pyrrolidone carboxylate formation by,
regulatory protein, X, 787-789 IV, 136-137
adenylylation effect regulation of, X, 743-754, 775-792
biosynthetic activity, X, 794-796 repression of formation, X, 780-781
y-glutamyltransferase activity, X, structure
796-800 chemical composition, X, 769-771
adenylylation state determination physical characteristics, X, 763-769
enayme assays, X, 802-803 taut, relaxed and tightened forms, X,
spectral analysis, X, 802 771-775
cascade control, X, 789-792 Glutaminyl peptides, pyrrolidone car-
covalent modification of, I, 409-413 boxylate formation from, IV, 139-141
cumulative feedback inhibition, X, 776 y-Glutamyl amino acids, pyrrolidone
mechanism, X, 777-780 carboxylate formation from, IV,
divalent cation control 142-146
adenosine triphosphate: cation ratio, y-Glutamyl cyclotransferase, pyrrolidone
x,783-784 carboxylate formation and, IV,
relaxation phenomena, X, 781 141-146
specificity, X, 781-783 y-Glutamyl-cysteine synthetase
general catalytic properties, X, 708-709 general catalytic properties, X, 676-678
hybrid forms, X, 804-807 mechanism, X, 683-687
intermediate formation of y-glutamyl partial reactions
phosphate, X, 716-720 adenosine triphosphate hydrolysis,
mechanism, X, 733-743, 759-763 X, 678-679
multiple molecular forms, X, 800-801 enayme4activated glutamatelcom-
determination of state of adenylyla- plex formation, X, 683
tion, X, 802-803 exchange reactions, X, 680-681
heterologous interaction, X, 804-807 5-oxoproline formation, X, 679-680
preparation of partially adenylylated phosphorylation of methionine
form, X, 801-802 sulfoxime, X, 681-683
partially adenylylated pyrophosphate synthesis, X, 679
direct isolation, X, 801 physical properties, X, 675-676
in vitro preparation, X, 801 pyrrolidone carboxylate formation
mixing of forms, X, 802 and, IV, 136-137
subunit dissociation and reassocia- sources, purification and assay, X,
tion, X, 802 674-675
partial reactions y-Glutamyl lactamase, see y-L-Glutamyl
adenosine triphosphate formation, cyclotransf erase
X, 714-715 y-Glutamyltransferase
arsenolysis of glutamine, X, 713 agaricaceae, IV, 95-96
cycloglutamyl phosphate synthesis, survey, IV, 93-95
X, 715 y-Glutamyl transpeptidase
exchange reactions, X, 709-710 kidney, IV, 96-97
pyrrolidone carboxylate formation primary, XIII, 5-9

and, IV, 141 Glycerate kinases
Glutathione S-epoxidetransferase, VII, assay and distribution, VIII, 504-505
205-206 catalytic properties, VIII, 507-508
catalytic properties metabolic role, VIII, 505-506
factors influencing, VII, 209-210 molecular properties
kinetics and specificity, VII, 209 purification and state of purity,
measurement of reaction, VII, 208 VIII, 506-507
notes on mechanism, VII, 210 stability, VIII, 507
products of reaction, VII, 208 Glycerol kinases
molecular properties assay and distribution, VIII, 488492
molecular weight, VII, 207 catalytic properties
multiple forms, VII, 206-207 product inhibition, VIII, 501-502
purification, VII, 206 substrate specificity and kinetics,
stability, VII, 208 VIII, 497-501
Glutathione reductase thermodynamics, VIII, 502
kinetic studies, XIII, 138-141 metabolic role, VIII, 492-493
metabolic functions, XIII, 129-133 molecular properties
thiol groups, XIII, 141-142 chemical modification, VIII, 496-497
two-electron-reduced enzyme, proper- composition, VIII, 494
ties, XIII, 133-138 purification and state of purity,
Glutathione synthetase VIII, 493494
acyl phosphate intermediate, X,
size and subunit structure, VIII,
690-692
494-495
general catalytic properties, X, 689-690
stability, VIII, 495-496
historical background, X, 671-674
regulation
mechanism, X, 693-695
mammals, VIII, 504
partial reactions, X, 693
physical properties, X, 688-689 microorganisms, VIII, 502-503
Glyceraldehyde-&phosphate dehydro- L-Glycerol-3-phosphate dehydrogenase,
genase, properties, XIII, 256-260
historical background, XIII, 2-3 Glycidol phosphate, enolase and, V, 534
isolation, XIII, 3-4 Glycinamide ribonucleotide transformyl-
mechanism of action ase, properties, IX, 203-204
other activities, XIII, 44-49 Glycine, oxidative decarboxylation of,
physiological activity, XIII, 38-44 IX, 221-223
metabolic role, XIII, 45-49 Glycine amidotransferase
pyridine nucleotide binding, XIII, biological distribution, IX, 498499
28-30 catalytic properties
cooperativity of, XIII, 30-35 reaction mechanism, IX, 500-503
preexisting asymmetry model, XIII, substrate specificity, IX, 499-500
35-38 regulation, IX, 503305
reaction catalyzed, XIII, 1 Glycogen
structure structure determination, debranching
apoenzyme, XIII, 19-20 enzymes and, V, 228-234
chemical modification, XIII, 20-24 Glycogen phosphorylase,
dissociation and hybridization, covalent modification of, I, 413415
XIII, 24-27 regulation of, I, 441442
holoenzyme X-ray structure, XIII, Glycogen storage disease, glucosidase-
9-19 transferase and, V, 221-222
Glycogen synthetase physiological conditions and, IX,

adrenal, IX, 353 348347
assay, IX, 317418 proteolytic inactivation, IX, 331
association with glycogen, IX, 311-312 purification, IX, 312-313
blood reaction catalyzed, IX, 316-317
erythrocytes, IX, 355 reaction mechanism, IX, 321-322
leukocytes, IX, 354-355 regulation of, I, 441-442
platelets, IX, 355 spleen, IX, 354
brain, IX, 353-354 system of interconversion, IX, 325-326
control of activity tumors, IX, 356-357
epinephrine effect, IX, 338, 341, tissues sensitive to sex hormones, IX,
351-352 355-356
glucagon and, IX, 341, 351-352 Glycosidases, modification of, I, 128-129
glucose and, IX, 347-348 Glycosulfatase (s)
glycogen and, IX, 336, 340 ccllulose polysulfatase, V, 11-12
insulin activation, IX, 336-338, cerebroside sulfatase, V, 13-14
340-341, 348-351 chondrosulfatase, V, 12
muscle contraction events, IX, glucosulfatase, V, 11
338-340 Glyoxylate condensing enzymes, general
donor specificity, IX, 318 remarks, VII, 411412
general properties of a and b forms, Gout, molybdenum hydroxylase genetics
IX, 324-325 and, XII, 400-402
glucosyl acceptor Gram-negative bacteria
de novo synthesis, IX, 320-321 phage lytic enzymes
oligosaccharides, IX, 320 Aerobacter cloacae, V, 398
polysaccharides, IX, 319-320 Azotobacter, V, 397498
heart, IX, 340-341 Klebsiella pneumoniae, V, 394-395
historical background, IX, 310-311 Pseudomonas, V, 395-397
inactive form, IX, 330-331 Salmonella, V, 398
kinase, IX, 326-327 Gram-positive bacteria
liver, IX, 341-353 phage lytic enzymes
muscle, IX, 332340 Bacillus megaterium, V, 408410
nomenclature, IX, 322-324 Bacillus stearothermophilus, V,
nonmammalian organisms, IX, 357-361 410-411
phosphatase Micrococcus lysodeikticus, V, 401402
liver, IX, 328-330 staphylococci, V, 398401
muscle, IX, 327-328 streptococci, V, 402-408
physicochemical properties Guanidine hydrochloride, inorganic pyro-
muscle, IX, 313-315 phosphatase and, IV, 508-510
other tissues, IX, 316-316 Guanidino kinase(s)
properties of the two forms bivalent metal ions and, VIII, 471473
activity of, IX, 335-336 catalytic reaction, VIII, 459
glucose 6-phosphate and, IX, chemical stop assays
332-334, 341-344 forward reaction, VIII, 464
inorganic phosphate and, IX, 334, reverse reaction, VIII, 465
344-345 continuous recording assays
magnesium and other ligands, IX, potentiometric, VIII, 466
335, 345 spectrophotometric, VIII, 465406
nucleotides, IX, 334-335, 345 discovery and isolation, VIII, 459461
pH and, IX, 335, 346 distribution and function, VIII, 461-464
equilibrium, VIII, 485-486 nature of active site, X, 383

function of amino acid residues purification and stability, X, 377-378
cysteine, VIII, 477-480 5'-nucleotidase of, IV, 347-348
others, VIII, 480-482 succinyl coenzyme A synthetase of,
historical background, VIII, 457-459 X, 591-594
isotopic assays, VIII, 465 Heavy atom derivatives
mechanism, VIII, 482-485 globular macromolecules
molecular properties factors in derivative formation, I,
amino acid composition, VIII, 73-86
469470 preparation and classification, I,
immunological reactions, VIII, 70-73
470-471 useful heavy atom compounds, I, 73
molecular weight and subunit com- Helicorubin, properties, XI, 592
position, VIII, 466-468 Heme proteins, modification of, I, 142
stability, VIII, 468-469 Hemoglobin
pH optimum, VIII, 476-477 allosteric properties, I, 388-390
substrate specificity mutations and, I, 257-259
general comments, VIII, 473 Hemoprotein(s), cytochro,me M i k e ,
guanidines, VIII, 474-476
XI, 576-577
nucleotides, VIII, 476
Hexarate dehydrase(s), properties, V,
Guanine aminohydrolaw, IV, 76-77
579-581
Guanosine aminohydrolase, IV, 77-78
Guanosine diphosphate-n-mannose oxido- Hexokinase (s)
reductase properties, V, 479 aggregation phenomenon, IX, 40-41
Guanosine kinase, see Inosine-guanosine amino acid composition and essential
kinase groups, IX, 41
Guanosine monophosphate, synthesis, chemical studies
VIII, 39-40 comparison of native isoenzymes,
Guanosine monophosphate kinase IX, 10-12
assay, IX, 84 nature of proteolytic modification,
distribution and purification, IX, 82-84 IX, 12-13
kinetic and molecular properties, IX, comparative aspects, IX, 46-48
84-85 isoelectric point, IX, 41
substrate specificity; reaction mechan- isoenzymes and modification by endo-
ism, IX, 85-86 genous proteases, IX, 2-6
Guanosine 5'-phosphate reductase, mechanism
modification of, I, 137 adenosine triphosphatase reaction,
Guinea pig IX, 18-19
serum, asparaginase of, IV, 105-107 conformational changes, IX, 27-28
enzyme-substrate interaction and,
H IX, 43-44
equilibrium measurements, IX, 19-22
Heart kinetic studies, IX, 13-17, 41-43
aspartate transaminase of, IX, 398-462 phosphoenzyme intermediate, IX,
glycogen synthetase of, IX, 340-341 23-24, 44
mitochondria1 adenosine triphosphatase sulfhydryl groups and, IX, 25-27
catalytic properties, X, 382-383 modification by added proteases, IX,
cold inactivation, X, 380 6-7
inhibitors and activators, X, 385-386 molecular weight and subunit struc-
molecular properties, X, 378-380,381 ture, IX, 39-40
molecular weight in nondenaturing Hormones, phosphofructokinase and,

solvents, IX, 7-8 VIII, 277-278
multiple forms, IX, 31-33 Hyaluronate-endo-p-glucuronidase, V, 313
purification procedures, IX, 37-38 Hyaluronate lyase, V, 313-314
regulation, IX, 29-31, 4446 Hyaluronidase (s)
relation of soluble to insoluble forms, bacterial, V, 309
IX, 33-37 biological effects, V, 319-320
subunit size, IX, 8-10 historical background, V, 307-308
Hill plots, enzyme regulation and, I, kinetics, V, 318-319
358-359 leech, V, 313
Hirudin, thrombin and, 111, 304405 lysosomal, V, 312
Histidine ammonia-lyase microbial, V, 313-314
catalytic process preparation and assay, V, 315-317
function of prosthetic group, VII, properties, V, 318
159-162 submandibular, V, 312-313
metal ion activation, VII, 162-163 substrates
prosthetic group, VII, 154-159 additional, V, 311
rate limiting step, VII, 164-166 chondroitin, V, 310
reaction sequence, VII, 148-154 chondroitin 4- and B-sulfate, V, 310
distribution, purification and kinetic dermatan sulfate, V, 310-311
properties, VII, 137-142 hyaluronate, V, 309
mechanism of action, VII, 195-196 testicular, V, 311-312
testicular type, V, 308,311-313
size and constitution, VII, 146-148
Hydrogenase, proton transfer and, 11,
Histidine deaminase, role of metals, 11,
317-318
509-510
Hydrogen bonds, propinquity effects
Histidine decarboxylase, Schiff base and,
and, 11, 254-264
11, 356-357
Hydrolase(s), role of metals in
Histidine residues
mechanism, 11, 519-525
aspartate transcarbamylase, IX, 267
D-2-Hydroxy acid dehydrogenase, proper-
chemical modification, I, 173
ties, XIII, 272-273
chymotrypsin erythro-p-Hydroxyaspartate dehydratase,
catalytic reaction and, 111, 234 properties, VII, 47-48
chemical modification, 111, 234 m-Hydroxybenzoa te-4-hydroxylase,
pH and, 111, 231-233 properties, XII, 225
creatine kinase, VIII, 434 m-Hydroxybenroa te-6-hydroxylase,
phosphofructokinase, VIII, 272 properties, XII, 224-225
ribonuclease, IV, 685-689 p-Hydroxybenzoate hydroxylase, proper-
subtilisin, 111, 580-584 ties, XII, 211-216
Histone kinases, properties, VIII, 579-580 8-Hydroxydecanoyl thioester dehydrase
Homoarginine residues, proteinase in- distribution, V, 464
hibitors and, 111, 461-452 function of, V, 441443
Homocitrate synthase, properties, VII, general considerations, V, 441443
425-426 inhibition by substrate analogs, V,
Homocysteine synthetase(s1, properties, 455-456
VII, 61-62 acetylenic inhibitor reaction site, V,
Homoserine dehydratase, properties, 456-458
VII, 67-59 3-decynoyl-N-ace tylcysteamine
Homoserine dehydrogenase, see Asparto- and fatty acid synthetase, V,
kinase 461-463
inhibitor specificity, V, 458459 Immunochemistry, chemical modification

isomeriration of 3-decynoyl-N- and, I, 199-200
acetylcysteamine, V, 459-461 Inclusion compounds, propinquity effects
modification of, I, 141 and, 11, 274-279
mutants, V, 463 Indoleamine 2,3-dioxygenase
protein structure historical, XII, 130-131
active site, V, 453-455 molecular and catalytic properties,
disulfide bridges, V, 452-453 XII, 131-132
nitration, V, 455 Influenza virus
reaction mechanism neuraminidase
isotope effects and labeled sub- isolation and purification, V, 328
strates, V, 449-450 purification of virus particles, V,
kinetic studies, V, 446-449 327-328
trapping of intermediates, V, Infrared spectroscopy, enzyme structure
451-452 and, 11, 374-379
substrate specificity, V, 444-445 Inhibition studies
optical and geometric, V, 446 nomenclature and basics, 11, 18-21
thioester, V, 445-446 prediction of patterns, 11, 21-25
4-Hydroxy-2-ketoglutarate aldolase, types of experiments, 11, 25-43
properties, VII, 299-301 Initial velocity studies
5-Hydroxymethyl deoxycytidine mono- bireactant, 11, 7-10
phosphate kinase, properties, IX, prediction of patterns, 11, 10-13
94-95 terreactant, 11, 13-18
8-Hydroxy-8-methylglutarylcoenzyme A Initiation factors
cleavage discovery and function, X, 6-12
general, VII, 432-433 eukaryote
properties of beef liver enzyme, VII, miscellaneous, X, 42-43
433-434 ribosome associated, X, 29-33
,8-Hydroxy-8-methylglutaryl coenzyme A supernatant, X, 33-42
synthase initiation cycle and, X, 12-13
distribution, VII, 429 properties
general considerations, VII, 427428 factor-1, X, 26-28
properties of yeast enzyme, VII, factor-2, X, 13-17
429-431 factor-3, X, 7-26
stereochemistry of product, VII, 428 Inorganic ions
p-Hydroxyphenylpyruvate hydroxylase, metabolic regulation and
XII, 179-180 divalent cations, I, 420-423
catalytic properties, XII, 180-183 monovalent cations, I, 419-420
purification and molecular properties, Inorganic pyrophosphatase
XII, 180 catalytic properties
assay, IV, 534
I interaction with inhibitors, IV,
Imidazole rings, trypsin, 111, 269-270 525-526
Imidazolylacetate monooxygenase, ion and inhibitor effects, IV, 518-519
properties, XII, 225-226 kinetics, IV, 535-538
Imido adenylate derivatives mechanism, IV, 538-539
synthesis, VIII, 37 nature and binding of active sub-
argininosuccinate, VIII, 38-39 strate and role of magnesium,
guanosine monophosphate, VIII, IV, 522-525
39-40 pH effects, IV, 518
reversal of reaction, IV, 519-520 Insects, alcohol dehydrogenase of, XI,

substrate specificity and stoichio- 189-190
metry, IV, 520-522,534-535 Insulin, glycogen synthetase and, IX,
chemical composition 336338, 340-341
amino acid content, IV, 512 Intestine
cyanogen bromide cleavage prod- monoglyceride lipase of, VII, 603-605
ucts, IV, 514 5'-nucleotidase of, IV, 345
N- and C-terminal amino acids, IV, Intramolecular reactions, miscellaneous,
512-514 11, 238-250
tryptic digest maps, IV, 514 Invertase, V, 292-293
chemical modification, IV, 514-515 biosynthesis, V, 294-295
cyanate and diazonium-1H-tetrazole, catalytic properties, V, 305
IV, 516-518 active site, V, 300-301
2,4&trinitrobenzene sulfonate, IV, inhibitors, V, 301-302
515-516 kinetics, V, 302
molecular properties mechanism, V, 302-303
electron microscopy, IV, 506-508 determination, V, 292
homogeneity, IV, 502-503 localization and multiple forms, V,
metalloenzyme, IV, 532-534 293-294
physicochemical parameters, IV, properties, V, 298-300
530-531 purification, V, 295-298, 304-305
purification, IV, 501-502, 530 Invertebrates, cytochrome b-like pig-
reconstitution from subunits, IV, ments of, XI, 564
510-612 Ionizable groups, protein structure and,
reversible divalent cation binding, 11, 430-440
IV, 531-532 Iron-sulfur proteins,
size, IV, 504 categories
other, IV, 539-541 conjugated, XII, 3-4
physical properties ferredoxin, XII, 2
optical properties, IV, 505-506 high potential, XII, 2-3
sedimentation and diffusion coeffi- eight irons
cients, IV, 504 background, XII, 37-39
viscosity, IV, 505 chemical properties, XII, 4 4 4 6
subunits in quanidine hydrochloride physical properties, XII, 3944
optical properties, IV, 510 four irons per center
sedimentation and diffusion coeffi- background, XII, 31-33
cients and intrinsic viscosity, IV, high potential proteins, XII, 35-37
509 low potential proteins, XII, 34
size, IV, 509 Iron-sulfur enzymes, XII, 47-50
Inosamine phosphate amidinotransferase mitochondrial, XII, 56
biological distribution, IX, 505-506 model compounds, XII, 48-47
catalytic properties nitrogenase system, XII, 60-56
reaction mechanism, IX, 507-508 nomenclature, XII, 2
substrate specificity, IX, 506-507 xanthine oxidase, XII, 56
regulation, IX, 508-509 Isoamylase (8)
Inosine-guanosine kinase, properties, plant, V, 208
IX, 54-56 pseudomonad and Cytophaga, V,
Inosinic acid dehydrogenase, modification 204-206
of, I, 137-138 yeast, V, 206-208
Isocitrate dehydrogenase, stereospecific- proton transferase and, 11, 283-285

ity, 11, 168-171 rate equations, 11, 63-65
Isocitrate lyase, VII, 382-383
assay and isolation, VII, 383
molecular properties
cofactors, VII, 384 Jack bean
inhibitors, VII, 385 ureaae of, IV, 2-5
molecular weight and subunits, VII,
384 K
sulfhydryl groups, VII, 385
Isoleucine residues Kallikreins, 111, 482-483
chymotrypsin Keratinase, microbial, 111,763-765
chemical modification, 111, 238-239 p-Keto acid decarboxylase(s), Schiff
conformation and, 111, 239-243 bases and, 11, 359
pH and, 111, 236-237 p-Ketoacyl acyl carrier protein
chymotrypsinogen, 111, 175-176 synthetase
Isomerase(s) catalytic properties
aldo-keto, proton shifts and, 11,290-295 assays, VIII, 190
Isopentylpyrophosphate isomerase mechanism, VIII, 194-199
assays, evaluation, VI, 567-568 pH optimum, substrate specificity
function, VI, 566-567 and kinetics, VIII, 190-194
mechanism, VI, 570-572 historical background, distribution and
occurrence, VI, 565-566 metabolic significance, VIII,
properties 188-189
inhibitors, VI, 569 molecular properties, VIII, 189-190
metal ion and, 569 a-Ketobutyrate synthase, properties, VI,
Michaelis constant, VI, 569 203-204
pH optimum, VI, 568 2-Keto-3-deoxy-~-arabonate aldolase,
reversibility and position of equili- properties, VII, 296
brium, VI, 569-570 2-Keto3-deoxy-~-arabonate dehydratase,
synthesis of substrate and product, VI, properties, V, 583-585
567 2-Keto-3-deoxy-o-fuconate aldolase,
dsopropylmalate synthase properties, VII, 296
properties, VII, 423 2-Keto-3-deoxy-D-glucarate aldolase
effect of leucine, VII, 424-425 properties, VII, 297
Isotope effects 5-Keto-4deoxyglucarate dehydratase,
primary, 11, 284-285 properties, V, 585-586
2-Keto3-deoxy-manno-octosonate aldo-
examples, 11, 325-329
lase, properties, VII, 298
origin and magnitude, 11, 322-324
2-Keto-3-deoxy-6-phosphogalactonic
pepsin and, 111, 180
aldolase, properties, VII, 295-296
secondary, 11, 285-287 2-Keto3deoxy-6-phosphogluconic aldo-
examples, 11, 331-333 lase
origin and magnitude, 11, 329-331 general properties
Isotopic exchange Pseudomonas putida, VII, 283-285
kinetics Pseudomonas saccharophila, VII, 285
basic considerations, 11, 4 3 4 4 mechanism
ping pong mechanism, 11, 48-52 isotope exchanges, VII, 290-291
sequential mechanism, 11, 44-48 oxalacetate decarboxylation, VII,
lack of, 11, 287 291-292
role of an additional base, VII, fructose-1,6-diphosphatase

292-295 purification and properties, IV,
role of Schiff base formation, VII, 629-630
289-290 regulation, IV, 630-631
modification of, I, 139 7-glutamyl transpeptidase of, IV, 96-97
structure vitamin Ba methyltransferase of, IX,
other characteristics, VII, 287-288 164-165
subunit composition and arrange- Kineticist
ments, VII, 285-287 tools of, 11, 6-7
3-Keto-dihydrosphingosine, formation inhibition studies, 11, 18-43
of, VII, 355-356 initial velocity studies, 11, 7-18
a-Ketoglutarate dehydrogenase com- isotope exchange, 11, 43-52
plexes, I, 224-225 miscellaneous, 11, 52-61
a-Ketoglutarate synthase, properties, Kinetics,
VI, 201-203 basic theory, 11, 6
a-Ketoisovalerate synthase, properties, nomenclature and notation, 11, 2-5
VI, 205 Kinetic systems
A'3-Ketosteroid isomerase far from equilibrium
animal tissue computer solutions to rate equa-
catalytic mechanism, VI, 617-618 tions, 11, 74-83
distribution and properties, VI, integrated rate expressions, 11, 71-74
615-617 single substrate-single product
catalytic mechanism Michaelis-Menten mechanism,
hydrogen isotopes and, VI, 605-607 11, 74-79
proposal for, VI, 612-615 near equilibrium
ultraviolet and fluorescence studies, relaxation amplitudes, 11, 99-108
relaxation spectra, 11, 83-99
VI, 607-612
catalytic properties Kinin-destroying enzymes, 111, 483
Kinin-forming enzymes
active-sitedirected inhibitor, VI, 601
catheptic kininogenases, 111, 483
competitive inhibitors, VI, 6oo-gO1
kallikreins, 111, 482483
kinetic studies, VI, 604
Klebsiellu pneumoniae
metal ions, chelators and urea, VI,
phage polysaccharide depolymerase
800 enzyme properties and role, V,
pH effects, VI, 602 394-395
reversibility, VI, 603-604 preparation and assay, V, 394
substrate specificity, VI, 59WOo Klotz plots, enzyme regulation and, I,
thermodynamic parameters, VI, 359-361
602-603 Kynurenine3hydroxylase, properties,
historical, VI, 591-592 XII, 230-231
chemical modification, VI, 595 1
induction, purification and crystal-
lization, VI, 592-593 a-Lactalbumin
structure, VI, 598-599 bovine, VII, 681-682
ultracentrifugation, VI, 593-594 gene duplication and, I, 311-314
ultraviolet and fluorescence spectra, lactose synthetase and, IX, 366
VI, 594-595 8-Lactamase, see Penicillinase
Kidney Lactate dehydrogenase,
argininosuccinase of, VII, 178-179 away, XI, 199-200
TOPICAL SUWECT INDEX 507
coenzyme binding significance of abortive complex, XI,

oxidized, XI, 280-281 276-277
reduced, XI, 277-280, 281 D(-)-Lactate dehydrogenase, XIII,
fluorescence and spectroscopy 269-270
coenzyme, XI, 268 enzymic properties, XIII, 270-272
protein, XI, 264-268 physical properties, XIII, 270
histidine-195, state of protonation, L( +)-Lactate dehydrogenase
XI, 289 cytochrome b, core, XIII, 266-267
historical, XI, 192-195 enzymic properties, XIII, 267-269
isolation, XI, 200-202 historical background, XIII, 263-264
isozymes, physical properties, XIII, 264-266
evolution of genes, XI, 198-199 Lactate oxidase, properties, XII, 194-199
general, XI, 195 Lactic acid racemase, properties, VI, 380
known genes, XI, 195-198 Lactobacillus, histidine decarboxylase of,
D-lactate specific enzymes, XI, 199 11, 356-357
kinetic studies, Lactose synthetase
transient phase, XI, 57-59 historical background, IX, 364-365
mechanism, XI, 289-292 requirement for two proteins
modification, I, 136 identification of a-lactalbumin, IX,
crystals, XI, 261 366
mild proteolysis, XI, 257-258 relationship to lysozyme, IX, 366-367
side chains, XI, 258-261 resolution of, IX, 365466
physical properties, XI, 261-264 Leaves, adenosine diphosphoryl glucose
reactions catalyzed pyrophosphorylase of, VIII, 86-90
cyanide addition, XI, 270 Leech, hyaluronidase of, V, 313
glyoxylate and XI, 269-270 Leucine aminopeptidase
ketopyruvate reduction, XI, 268-269 assay, 111, 83-84
reviews, XI, 192 chemical properties, 111, 88-89
steady-state kinetics enzymic properties
noninhibiting substrate concentra- inhibitors, 111, 96-98
tions, XI, 270-273 mechanism of action, 111, 98-100
steady-state inhibitors, XI, 273-274 role of metal ion, 111, 89-91
structure, substrate specificity and kinetics,
amino acid sequence, XI, 202-205 111, 91-96
composition, XI, 202, 203 historical background, 111, 82-83
oligomer, XI, 250-257 physical properties, 111, 86-88
threedimensional structure, XI, purification, 111, 84-86
205-250 use in sequence studies, 111, 101-102
substrate binding, XI, 290-291 Lipase(s1
absence of enzyme-substrate com- activity, determination of, VII, 578-
pounds, XI, 281-282 680
active ternary complex, XI, 284-286 castor bean, VII, 613-614
enzyme-anion complexes, XI, 282 catalytic properties
enzyme :coenzyme :inhibitor com- active site, VII, 593-595
plex, XI, 282-284 chemical structure of substrate, VIZ,
ternary complex transient kinetics, 595-599
XI, 286-289 colipase and, VII, 595
substrate inhibition effectors and, VII, 594601
lactate. XI. 275-276 ester bond synthesis and, VII, 601-
pyruvate, XI, 274-275 602
phosphoglycerides and, VII, 602-603 catalytic properties, X, 388-389

positional specificity, VII, 591-593 molecular properties, X, 388
substrate physical state, VII, 586- purification, X, 387-388
591 5'-nucleotidase of, IV, 343-345
definition of, VII, 575-577 sulfatase A of, V, 27-36
distribution sulfatase B of, V, 37-38
animal, VII, 577 sulfite oxidase of, XII, 414-419
microorganisms, VII, 578 vitamin Bn methyltransferase of, IX,
plant, VII, 577-578 164-165
gastrointestinal London forces, propinquity effects and,
gastric, VII, 605-606 11, 254-264
intestinal, VII, 603-605 Long chain fatty acid:coenayme A ligase
microorganisms, VII, 614-616 catalytic properties
milk, VII, 611-613 partial and exchange reactions, X,
pancreatic, VII, 580-581 486-487
catalytic properties, VII, 586-603 substrates and inhibitors, X, 485-486
molecular properties, VII, 583-586 Luciferase, bacterial, properties, XII,
purification, VII, 581-583 226-229
tiasue Luciferin, adenylylation of, VIII, 19-20
adipose tissue hormone sensitive, Lyase(s1, role of metals in mechanism,
VII, 609-610 11, 508-519
lipoprotein lipase, VII, 606-609 Lysine, biosynthesis, VI, 504
liver lipases, VII, 611 Lysine monooxygenase, properties, XII,
Lipoamide dehydrogenase 199-203, 293-294
distribution, XIII, 106-107 n-cu-Lysine mutase, VI, 551-552
kinetic studies, VIII, 115-117 cofactor requirements, VI, 552
mechanism, XIII, 126-129 comparison to L-plysine mutase, VI,
mechanism of Maasey and Veeger, re- 554-555
view of, XIII, 111 cofactor requirements, VI, 555-559
metabolic functions, XIII, 107-110 2,5diaminohexanoate and, VI, 554
role of NAD' as modifier, XIII, 117- inhibitors, VI, 553
120 partial reactions, VI, 553-554
structural studies, XIII, 120-126 purification of complex, VI, 552
two-electron-reduced enzyme, proper- L-p-Lysine mutase
ties, XIII, 111-115 assay, VI, 548-549
Lipoate, activation of, 17-18 cofactor requirements, VI, 550
Lipoprotein lipase, properties, VII, 606- comparison to n-a-lysine mutase, VI,
609 554-555
Liver cofactor requirements, VI, 555-559
acetyl coenzyme A carboxylase of, VI, inhibitors, VI, 551
78-79 occurrence, VI, 548
acid phosphatases, IV, 484-493 purification and physical properties,
alcohol dehydrogenase of, XI, 20-22, VI, 549-550
56-57, 107-109, 111-117 reversibility, VI, 551
aldolase, VII, 241-244 Lysine residues
argininosuccinase of, VII, 171-178, 179 acylation
fructose-1,6-diphosphatase, IV, 618-633 biotin activating enzyme, VIII, 18
glycogen synthetase of, IX, 341-353 lipoate activating enzyme, VIII, 17-
lipases, VII, 611 18
mitochondria1 adenosine triphosphatase creatine kinase, VIII, 432-434
ribonuclease, IV, 801 tryptophan, VII, 792-796

subtilisin, modification of, 111, 596-598 tyrosine, VII, 796-797
Lysosomes, hyaluronidase of, V, 312 chromophore properties, VII, 799-802
Lysozyme, see abo Phage lysozyme absorbance, VII, 802-803
activity chromophore exposure, VII, 809
biological role, VII, 669-670 circular dichroism, VII, 807-809
muramidase and chitinase, VII, 667- fluorescence, VII, 803-806
669 solvent and saccharide perturbation,
analysis of rate enhancement, VII, VII, 809
864-865, 868 conformation of egg-white model
general acid catalysis, VII, 885 main polypeptide chain, VII, 692-
ion pairing and, VII, 867 699
substrate distortion, VII, 865-867 side chains, VII, 699-707
association with hydrogen ions, VII, water structure, VII, 707
745-746 crystallographic studiea of inhibitor
apparent ionization constants, VII, complexes, VII, 707-708
736-739 a- and p-N-acetylglucosamine, VII,
enthalpy and volume changes, VII, 708
735-736 enzyme-substrate complex structure,
environment of ionizable groups, VII, 712-714
VII, 739-745 supplementary binding studies, VII,
isionic and isoelectric pH, VII, 732 714-717
potentiometric titrations, VII, 732- tri-N-acetylchitotriose, VII, 708-711
735 denaturation, VII, 760-766
association with other ions and mole- guanidine hydrochloride, VII, 774-
cules, VII, 746-755 776
avian, VII, 677-4380 nonaqueous solvents, VII, 776-777
bacteriophage T2 nuclear magnetic resonance, VII,
catalytic properties, V, 381-382 777-780
chemical properties, V, 380-381 properties, VII, 770-772
physicochemical properties, V, 380 stability, VII, 766-770
purification, V, 379 thermal, VII, 772-774
bacteriophage T4 urea, VII, 776
catalytic properties, V, 369-374 early history, VII, 666-667
chemical properties, V, 366-369 fluorescence spectroscopy, 11, 426-429
enzyme assays, V, 361464 gene duplication and, I, 311-314
physicochemical properties, V, 366 hen egg-white
purification, V, 364-366 amino acid sequence, VII, 672-676
role in life cycle, V, 375-379 composition, VII, 672
bond rearrangement mechanism, VII, preparation and purification, VII,
855-856 670-672
chemical modification, I, 207-211 synthesis, VIT, 67M77
chemical modifications human, VII, 680-881
amino groups, VII, 780-785 hydrodynamic measurements, shape
arginine, VII, 785-786 and solvation, VII, 717-725
carboxyl groups, VII, 786-788 hydrogen exchange, VII, 730-732
cystine, VII, 789-790 large substrates
histidine, VII, 791 bacterial cells and cell walls, VII,
methionine, VIJ, 791-792 836-841
other reactions, VII, 797-798 chitin and derivatives, VII, 841-843
low molecular weight substrates, VII, saccharide binding, VII, 808-835

846-847 substrates, kinetics and mechanism,
bond cleaved, VII, 847 VII, 836-868
chain length and, VII, 847-848 X-ray studies, VII, 682-717
character of substrate, VII, 848-849 X-ray studies, I, 67-69
Hammett constant, VII, 851-852 analysis of structure, VII, 6824392
isotope effect, VII, 850 conformation of egg-white model,
kinetic constants, VII, 850-851 VII, 692-707
kinetics of saccharide binding, VII, crystallography of inhibitor com-
852-855 plexes, VII, 707-717
pH and, VII, 849-850 Lysyl hydroxylase
rate enhancement, VII, 850 catalytic properties, XII, 166-167
temperature and, VII, 849 purification and molecular properties,
mechanism, VII, 856-857 XII, 165
acetamido participation, VII, 863- o-Lyxose isomerase, properties, VI, 344-
864 345
environment, VII, 863
general acid catalysis, VII, 857-860 M
glycosyl carbonium ion and, VII,
862-863 Macromolecular complexes, metabolic
location of catalytic center, VII, 857 regulation and, I, 426-428
substrate distortion, VII, 860-862 Maanesium, enolase and, V, 524526
optical properties, VII, 725-730 Malate, cleavage of, VII, 431
relationship to a-lactalbumin, IX, 366- Malate dehydrogenase,
367 catalytic properties,
saccharide binding, VII, 809-810 active site structure, X I , 390-395
acceptor and substrate reactivity, kinetic analyses, XI, 385-390
VII, 822-825 distribution and preparation, X I , 370-
free energies of association, VII, 373
810-820 mitochondrial, environment of, XI,
multiple modes of binding, VII, 833- 395-396
835 molecular properties,
pH dependence, VII, 832-833 amino acid composition, XI, 375-
separation of group contributions, 376, 377
VII, 829-832 molecular weight, XI, 373-374
separation of site contributions, nature of subforms, XI, 376, 378
VII, 825-829 subunit structure, XI, 374375
thermodynamics of association, VII, structure of NAD'dependent cyto-
820-822 plasmic
self-association, VII, 756-757 interaction with coenzyme, X I , 382-
structure in crystal and solution, VII, 385
757-760 pig heart crystal structure, X I , 379-
transfer reactions, VII, 843-846 382
vertebrate types of, XI, 369-370
chemical modifications, VII, 780-809 Malate synthase
denaturation, VII, 760-780 condensation mechanism, VII, 420-422
general considerations, VII, 666-670 occurrence, VII, 412-414
physical properties, VII, 717-760 proton transfer and, 11, 316-317
preparation, composition and se- purification and properties, VII, 415
quence, VII, 670-682 reversibility, VII, 417
specificity, VII, 415-417 history, occurrence and function, VI,
stereochemistry, VII, 417419 302-304
Maleate isomerase, properties, VI, 38% molecular properties of yeast enzyme,
385 VI, 304-305
Maleic anhydride, pyruvate carboxylase Medium chain fatty acid:coenzyme A
and, VI, 22-23 ligases
Maleyl isomerase(s), properties, VI, 385- catalytic properties
390 formation of butyryl adenylate, X,
Malonyl coenzyme A-acyl carrier protein 484
transacy lase steady state kinetics and reaction
catalytic properties mechanism, X, 484-485
assays, VIII, 179-180 substrates and inhibitors, X , 483
mechanism, VIII, 180-185 Melilotate hydroxylase, properties, XII,
pH optimum, substrate specificity 217-221
and kinetics, VIII, 180 Metabolic functions
historical background, distribution and regulation of balance
metabolic significance, VIII, 176- allosteric determination of catalytic
178 function, I, 442444
molecular properties, VIII, 178-179 compensatory control mechanisms, I,
Mammals 434-439
amino acid decarboxylases of, VI, 221- metabolite interconversion systems.
224 I, 43@-434
glutamine synthetase of, X, 699-754 oppositely directed exergonic reac-
hexokinase tions, I, 439442
mechanism, IX, 4144 Metabolic regulation
occurrence of multiple forms, IX, adenylate energy charge and, I, 470-
31-33 476
purification and molecular proper- covalently bonded modifiers and, I,
ties, IX, 37-41
484
regulation, IX, 44-46
operational response curves, I, 482-483
relation of solubie to insoluble
principles
forms, IX, 33-37
chemical equilibria and, I, 418-419
neuraminidase of, V, 325-327
compartmentalization and, I, 423426
Mandelic acid racemase, properties, VI.
379-380
covalent enzyme modification, I,
D-Mannonate dehydrase, properties, V, 409-415
579 enzyme concentration, I, 399403
D-Mannose isomerase, properties, VI, feedback inhibition, I, 403-408
344-345 inorganic ions, I, 419-423
L-Mannose isomerase, properties, VI, macromolecular complexes, I, 426-
345-346 428
Mannosed-phosphate isomerase proteolysis, I, 416-417
catalytic properties unidirection of reversible reactions,
general kinetic parameters, VI, 307- I, 428-430
309 Metabolic systems, analogy with elec-
mechanism of action, VI, 310-314 tronic systems, I, 463-465
zinc effect on nonemymic isomeriza- Metal ( s )
tion, VI, 309-310 catalysis by, VI, 401406
characterization as zinc metalloen- kinetics and, 11, 59-61
zyme, VI, 305-307 microbial proteinases and, 111, 772-773
Metal bridge complexes Methionyl transfer ribonucleic acid

binary complex formation, 11, 494495 transformylase, properties, IX, 208-
development of concept, 11, 485-489 209
enzyme-metal linkage, 11, 490494 4-Methoxybenzoate O-demethyl-mono-
reactions within coordination sphere, oxygenase, properties, XII, 285-287
11, 497-498 3-Methylaspartate ammonia-lyase
ternary complex formation, 11, 495- catalytic procesa
497 evidence for carbanion mechanism,
Metal complexes VII, 127-135
reaction substrate and activator binding, VII,
chelation mechanisms, 11, 453-455 121-127
coordinated ligand reactions, 11, 455- distribution, purification and kinetic
463 properties, VII, 118-119
ligand substitution mechanisms, 11, size and constitution, VII, 119-121
450-453 p-Methylcrotonyl coenzyme A
Metal ions carboxylase
properties relevant to catalysis distribution, VI, 4 1
general, 11, 448-450 historical background and metabolic
reactions of metal complexes, 11, significance, VI, 39-40
450-463 mechanism of action, VI, 4 2 4 5
Metalloenzymes, enzymic properties, 111, molecular characteristics, VI, 42
76-77 reaction catalyzed, VI, 38-39
5,lO-Methenyltetrahydrofolate cyclohy- substrate specificity, VI, 4 1 4 2
drolase, properties, IX, 201 a-Methyleneglutarak mutase, VI, 534-
Methionine adenosyltransferase 535
catalytic properties assay, VI, 535
activators and pH effects, VIII, 133- catalytic properties
135 coenzymes, W,536
assay, VIII, 129-130 equilibrium, VI, 536-537
energetics, VIII, 139-141 mechanism, VI, 537
kinetics, VIII, 137-139 substrates and inhibitors, VI, 536
reversibility, partial reactions and purification and molecular properties,
mechanistic considerations, VIII, IV, 535-536
130-133
Methyl groups
substrate specificities and inhibition asymmetrical, stereospecificity of ma-
late synthesis, 11, 157-164
by substrate analogs, VIII, 135-137
7-Methyl-y-hydroxy-a-ketoglutaric al-
net reaction, VIII, 125-127
dolase, properties, VII, 301-302
purification and physical properties, Methylmalonyl coenzyme A epimerase,
VIII, 127-129 stereochemistry, 11, 206-210
regulation and genetics Methylmalonyl coenzyme A mutase, VI,
mammals, VIII, 142-143 511-512
microorganisms, VIII, 141-142 assays, VI, 51%513
significance and distribution, VIII, catalytic properties
123-125 coenzymes, VI, 517-519
Methionine residues equilibrium, VI, 519
chemical modification, I, 173 mechanism, VI, 519-524
chymotrypsinogen, 111, 179 pH and, VI, 519
ribonuclease, IV, 682483 substrates, VI, 517
subtilisin, modification of, 111, 598-599 distribution, VI, 513
purification and molecular properties conformation studies, 111, 755-758,

animal tissue, VI, 515-517 775-776
Propionibacterium shermanii, VI, molecular weight, physical constants
514-515 and isoelectric point, 111, 754-755,
Methylmalonyl coenzyme A racemase, 773
properties, VI, 378-379 stability, 111, 755, 773-775
N5-Methyltetrahydrofolate-homocysteine Micrococcus luteus
methyltransferases, see Methyltrans- endonuclease, ATPdependent, IV,
ferase, Vitamin Bn methyltrans- 261-262
ferase polynucleotide phosphorylase of, VII,
Methyltransferase 548-570
assay and purification, IX, 154-155 ultraviolet repair enzymes, IV, 269-270
catalytic properties and folate bind- Micrococcus lysodeikticus
ing, IX, 156-158 adenosine triphosphatase of, X, 421-425
folate substrates, IX, 161-162 phage-induced lytic enzyme
occurrence, IX, 160-161 catalytic properties, V, 401-402
physical properties, IX, 155-156 purification, V, 401
reactions catalyzed, IX, 121-122 stability, V, 402
repression of synthesis, IX, 158-160 Microorganisms, see also Bacteria elc.
Mevaldate reductase, stereospecificity, aldolases of, VII, 255-256
11, 186-189 amylase of, V, 239-271
Mevalonate lipases of, VII, 614-616
labeled, preparation and use, 11, 192- 3’-nucleotidases of, IV, 354
204 ribonucleoside 2’,3’-cyclic phosphate
Mevalonate kinase, stereospecificity, 11, diesterase of, IV, 356-363
186-189 succinate dehydrogenase of, XIII, 254-
Micelles, propinquity effects and, 11, 256
264-274 type b cytochromes in, XI, 577-584
Michaelis-Menten mechanism Microsomes
single substrate-single product, 11, 74- electron transport, XIII, 148-149
75 cytochrome b, reductase system,
early phase solutions, 11, 77 XIII, 150-151
general solutions to one intermedi- cytochrome P-450 reductase system,
ate mechanism, 11, 75-77 XIII, 149-150
steady state solutions, 11, 77-79 mixed function amine oxide, XIII,
Microbial proteinase(s) 153-154
chemical properties, 111, 728-730, 742- synergism between systems, XIII,
743, 749-754, 770-773 151-153
distribution and isolation, 111, 724-728, Milk, lipases, VII, 611-613
741-742, 745-749, 767-770 Milk xanthine oxidase
enzymic properties, 111, 734-740, 742- catalytic properties
743, 758-763,776-786 mechanism of action, XII, 365-388
inhibitors, 111, 762-763, 777-778 reactions catalyzed, XII, 344-365
kinetics and mechanism, 111, 763, historical background, XII, 303-304
optimum pH, 111, 758-759, 776-777 chemical modification, XII, 317-326
substrate specificity, 111, 759-762, composition and physical properties,
778-786 XII, 310-317
physical properties, 111, 731-733, 742- magnetic interactions of redox
743 groups, XII, 342-344
purification, XII, 304-310 nitrate reductase and, XII; 402406

redox group magnetic and optical xanthinuria and gout in man, XII,
properties, XII, 326-342 400-402
Mitochondria molecular properties, XII, 389-394
adenosine triphosphatase other, XII, 388-389
assay, X, 377 Monoamine oxidase, molecular proper-
beef heart, X, 377-386 ties, and kinetic mechanism, XII,
rat liver, X, 387-389 466-471
yeast, X, 386-387 Monooxygenase (s)
nicotinamide adenine dinucleotide de- copper-containing
hydrogenases, XIII, 177-178 dopamine, XII, 294-295
energy conservation and, XIII, 214- phenol, XII, 295-297
216 flavin and pterin-linked, background,
high molecular weight, XIII, 187-189 XII, 191-193
inhibitors of, XIII, 203-207 heme containing
low molecular weight, XIII, 189-198 flavoproteins and, XII, 269-280
relevance of low and high molecular general mechanisms, XII, 280-285
weight dehydrogenases, XIII, 198- iron-sulfur proteins as electron do-
203 nors, XII, 259-269
transhydrogenation and, XIII, 207- iron and copper-containing
214 functions, XII, 257-258
ubiquinone reductase (Complex I), historical aspects, XII, 253-255
XIII, 178-187 nomenclature, XII, 256-257
ribonucleic acid polymerase of, X, role in oxygen activation, XII, 256
318-328
iron-containing,
type b cytochromes in, XI, 564-565 heme, XII, 258-285
Molds, see abo Fungi nonheme, XII, 285-294
amylases
model studies and possible mecha-
nisms, XII, 241-262
molecular properties, V, 236-263
nonheme iron-containing
glycogen synthetase of, IX, 361
lysine, XII, 293-294
proteases
4methoxybenzoate 0-demethyl,
acid, 111, 723-744
XII, 285-287
diisopropylfluorophosphate sensitive,
111, 744-765 phenylalanine, XII, 287-289
metal-chelator-sensitive, 111, 765-786 proline, XII, 292-293
other, 111. 786-795 tryptophan, XII, 291-292
Mblybdenum iron-sulfurflavin hydroxy- tyrosine, XII, 290-291
lase pterin-linked, XII, 231
catalytic properties Multienzyme complexes
mechanism of reduction, XII, 394- biological significance, I, 237-240
397 biosynthesis of aromatic amino acids,
oxidizing substrates, XII, 397400 I, 228-237
distribution and biological importance, fatty acid synthetases, I, 226-228
XII, 301-303 a-ketoglutarate dehydrogenase, I, 224-
enzymes to be considered, XII, 300-301 225
genetic studies pyruvate dehydrogenase,
Aspergillus nidulans, XII, 412414 composition and organization, I, 215-
Drosophila melanogaster, XII, 406- 220
412 regulatory features, I, 220-224
"OhCAL SUBJECT INDEX
Multiple isomorphous replacement intracellular localization, IV, 364-365

how heavy an atom, I, 37-39 physiological role, IV, 365
location of heavy atoms, I, 32-35 properties and substrate specificity,
outline of method, I, 32 IV, 364
phase determination and error assess- Neuraminidase (s)
ment, I, 35-37 assay method, V, 339-341
Mung bean, 3'-nucleotidase of, IV, 353 biological significance, V, 341-342
Muscle historical background, V, 321-323
aldolase, VII, 224-241, 258 inhibitors, V, 339
creatine kinases of, VIII, 395-401 kinetic data, IV, 337-339
enolase, glycidol phosphate and, V, 534 occurrence, V, 323
fructose-l,6-diphosphatase bacterial, V, 324-325
evidence for presence, IV, 632 mammalian, V, 325-327
physiological role, IV, 634-635 viral, V, 324
purification and properties, IV, 632- properties, 329-331
633 purification
structure and relation to other en- influenza virus, V, 327-328
zymes, IV, 633-634 Vibrio cholerae, V, 328-329
glycogen synthetase of, IX, 332-340 substrate specificity
phosphofructokinase of, VIII, 254-256 configurational, V, 331-332
phosphorylase kinase of, VIII, 557-564 esterification of neuraminic acid, V,
Mutants 336-337
advantages and limitations of, I, 265- N-substitution of neuraminic acid,
266 v, 335
isolation of, I, 245-249 0-substitution of N-acetylneurami-
Mutations nic acid, V, 336
alkaline phosphatase and, I, 251-254 position of glycoside linkage, V, 334-
amino acid analogs and, I, 262-265 335
p-galactosidase and, I, 255-256 steric hindrance in natural sub-
hemoglobin and,, I, 257-259 strates, V, 332-333
other enzymes and proteins, I, 259-262 Neurospora
reversion, suppression and comple- adenylosuccinase of, VII, 191-193
mentation, I, 249-251 argininosuccinase of, VII, 180-181
tryptophan synthetase, I, 256-257 Neurospora crassa
types, cistron and, I, 243-245 acid phosphatase, IV, 497
Myelin, ribonucleoside 2',3'-cyclic phos- aspartate transcarbamylase, IX, 302-
phate diesterase in, IV, 364-365 306
Myokinase, see Adenylate kinase argininosuccinase of, VII, 180-181
Myrosulfatase, V, 15-17 invertase, V, 303-304
Myxobacter, protease of, 111, 786-788 catalytic properties, V, 305
purified preparations, V, 304-305
Neutral proteinase(s)
N metal-chelator-sensitive, 111, 765-766
chemical properties, 111, 770-773
Negative charge, proteinase inhibitors distribution and isolation, 111,767-
and, 111, 422-423 770
Nervous tissue enzymic properties, 111, 776-786
5'-nucleotidase of, IV, 346-347 physical properties, 111, 773-776
ribonucleoside 2',3'-cyclic phosphate Nicotinamide adenine dinucleotide, pyr-
diesterase of, IV, 363-364 uvate carboxylase and, VI, 33-34
Nicotinamide adenine dinucleotide de- mitochondrial monooxygenase reac-

hydrogenase tions, XIII, 83-85
Azotobacter vinelandii, XIII, 221 redox state of mitochondrial nico-
mammalian mitochondria, XIII, 177- tinamide nucleotides, XIII, 81-82
178 Nitrate reductase, molybdenum hydroxl-
energy conservation and, XIII, 214- ase “common cofactor,” XII, 402-
216 406
high molecular weight, XIII, 187-189 Nitrite reductase(s), properties of, XIII,
inhibitors of, XIII, 203-207 273-279
low molecular weight, XIII, 189-198 Nitrogenase, properties of, XII, 50-56
relevance of low and high molecular Nonheme iron proteinb), metal com-
weight, XIII, 198-203 plexes and, 11, 531-533
transhydrogenation and, XIII, 207- Nuclear magnetic resonance, ribonucle-
214 ase, IV, 723-725
ubiquinone reductase (Complex I), Nuclear preparations
178-187 rat liver, pyrrolidone carboxylate for-
yeast, XIII, 216-221 mation by, IV, 138-139
Nicotinamide adenine dinucleotide Nucleases, see abo Deoxyribo- and
kinase Ribonucleases
assay, IX, 77-78 modification of, I, 130
distribution, purification and stability, X-ray diffraction studies, I, 67-69
IX, 76-77 Nucleophilic catalysis, isomerization and,
kinetic and molecular properties, IX, VI, 397-401
78-79 Nucleoside 3’,5’-cyclic phosphate dies-
reaction mechanism, IX, 79-80 terase, IV, 365-366
substrate specificity, IX, 80-82 distribution, IV, 366
Nicotinamide nucleotide transhydrogen- inhibitors and activators, IV, 368-370
ase intracellular localBation, IV, 367-368
AB-specific metal ions, pH and substrate affinity,
historical, XIII, 62-64 IV, 368
kinetics and reaction mechanism, physiological function, IV, 370-371
XIII, 75-78 possibility of other diesterases, IV, 370
molecular properties, XIII, 69-71 substrate specificity, IV, 366-367
occurrence, XIII, 64-66 Nucleoside diphosphokinase(s)
preparation and assay, XIII, 66-69 assay methods,
reconstitution, XIII, 78-79 coupled, VIII, 321-325
relationship to energy-coupling sys- isotopic, VIII, 325
tem, XIII, 71-75 staining procedure, VIII, 325
BB-specific catalytic properties
historical, XIII, 52-53 conformational changes, VIII, 331
molecular properties, XIII, 57-59 metal requirements, VIII, 329-330
occurrence, XIII, 53-54 reaction catalyzed, VIII, 320
purification and assay, XIII, 54-57 specificity, VIII, 320-321
reaction mechanism and regulation, sulfhydryl groups, VIII, 330-331,
XIII, 59-62 distribution, VIII, 309-313
definition, XIII, 51-52 function in the cell, VIII, 331-333
physiological roles, XIII, 79-81 historical development, VIII, 307-309
fatty acid synthesis, XIII, 88 kinetics and catalytic mechanism
mitochondrial glutamate and iso- pH and temperature effects, VIII,
citrate metabolism, XIII, 85-88 328329
substrate concentration effect, VIII, Optical rotatory dispersion

326-328 protein structure and, 11, 381-382, 408
molecular properties secondary, 11, 382-386
occurrence of isozymes, VIII, 313- tertiary, 11, 386-391
314 typical cases, 11, 391-407
phosphorylated enzyme, VIII, 315- ribonuclease, IV, 719-723
320 Orcinol hydroxylase, properties, XII,
physical properties, VIII, 315 223-224
purification procedures, VIII, 314- Ornithine mutase, properties, VI, 559
315 Oxalacetate
Nucleotide kinases, reaction catalyzed, formation from phosphoenolpyruvate,
I X , 49-50 general considerations, VI, 117-
3’-Nucleotidase 119, 165-168
microorganisms, IV, 354 Oxidation-reduction reactions
mung bean, IV, 353 between metal complexes, 11,529-530
rye grass, IV, 353 cytochrome c, 11,534-538
wheat seedling, IV, 353-354 mechanistic principles, 11, 530-531
5’-Nucleotidase (s) nonheme iron proteins, 11, 531-533
bacterial, IV, 338-340 xanthine and aldehyde oxidases, 11,
bull seminal plasma, IV, 342-343 533-534
cardiac tissue, IV, 347-348 within metal complexes
comparison of, IV, 349-352 mechanistic principles, 11, 525
Ehrlich ascites cells, IV, 348-349 xficotinamide adenine dinucleotide-
intestinal, IV, 345 linked dehydrogenases, 11, 525-528
liver, IV, 343-345 vitamin BIZmechanisms, 11, 628-529
nervous tissue, IV, 346-347 Oxidoreductase (8)
other vertebrate tissues, IV, 348 stereospecificity
pituitary gland, IV, 346 absolute coenzyme configuration, 11,
potatoes, IV, 349 144-147
snake venom, IV, 342 historical background, 11, 134-137
yeast, IV, 341342 hydrogen transfer and, 11, 137-144
Nucleotide kinases, reaction catalyzed, significance of A- and B-side speci-
I X , 50 ficity, 11, 154-157
Nucleotidyl transferring enzymes, role of substrates and, 11, 147-151
metals in mechanism, 11, 502-504 without hydrogen transfer, 11, 151-
Nucleus 154
ribonucleic acid polymerases Oxygenases, see Dioxygenases, Mono-
animal, X, 262-300 oxygenases
higher plant, X, 311-318 Oxygen binding proteins, X-ray diffrac-
yeast and fungi, X, 300-311 tion studies, I, 53-63
0 P
Pancreas
Old yellow enzyme, molecular properties pig, phospholipase A, of, V, 76-77
and kinetic mechanism, XII, 471-473 Pancreatic elastase, see Elastase
Oleate Papain
formation from stearate, 11, 179-184 active site
hydration, stereospecificity, 11, 184-186 chemical modification, 111, 515-516
Oligonucleotides, polynucleotide phos- comparison with other proteinases,
phorylase and, XII, 549-552 111, 498-499
geometry of, 111, 496498 Pentacovalency, pseudorotation and,

location of thiol group, 111, 514 VIII, 214-219
amino acid composition and sequence, Penicillinase
111, 507-509 assay methods, IV, 35-39
assay methods, 111, 518-519 background, IV, 23-25
chemical modifications, I, 205-207 catalytic reaction, IV, 27
active site, 111, 515-516 conformation and function
other reactions, 111, 516-518 nonspecific transitions, IV, 44-15
crystallization of, 111, 486-488 specific transitions, IV, 45-46
fructose-1,6-diphosphatase and, IV, 619 definitions and specificity, IV, 25-26
heavy atom derivatives, 111, 488-490 factors affecting activity
kinetic studies activators and inhibitors, IV, 43-44
acyl enzyme intermediate, 111, 525- pH and temperature, IV, 42-43
532 immunological studies, IV, 46
deacylation, 111, 533-635 kinetics and substrate specificity, IV,
kinetic constants and pH, 111, 535- 39-40
537 molecular properties
pH, ionic strength and temperature composition and sequence analysis,
effects, 111, 525 IV, 31-35
partial reduction, 111, 617 purification and physical properties,
physical properties IV, 2731
hydrodynamic properties, 111, 503 occurrence, IV, 26
immunochemical studies, 111, 606 structural modification of, IV, 41-42
spectrophotometric and fluorescence substrate structural modifications, IV,
properties, 111, 503-505 40-41
stability, 111, 505-506
Pepsin
preparation and crystallization, 111,
action
502-503
esterase activity, 111, 151
specificity
organic sulfite cleavage, 111, 151-152
esterase and thiolesterase activity,
Specificity, 111, 142-151
111, 523-524
nucleophile binding, 111, 524-525 theories of, 111, 160-164
peptide and amide bond hydrolysis, amino acids
111, 519-523 composition, 111, 128-130
transamidation and transesterifica- sequence, 111, 130-133
tion, 111, 524 assay, 111, 124-125
three-dimensional structure autolysis of, 111, 139-140
description, 111, 491-496 chemical modification, 111, 133-137
electron density map, 111,490-491 condensation reactions, 111, 156
water-insoluble derivatives, 111, 117- denaturation of, 111, 137-138
518 electrophoretic mobility, 111, 127
Papaya latex, other proteolytic enzymes, formation from pepsinogen, 111, 138-
111, 537-538 139
Papaya peptidase A, 111,538 gene duplication and, I, 308
Parallelism, protein evolution and, I, historical background, 111, 120
329332 inhibition of, 111, 154-156
Pasteur effect, phosphofructakinase and, isotope effects, 111, 160
VIII, 274-276 molecular weight and shape, 111, 126-
Pea seeds, argininosuccinase of, VII, 181- 127
182 occurrence
classification and nomenclature, 111, distribution, purification and kinetic

121-123 properties, VII, 142-146
other pepsinlike enzymes, 111, 123 mechanism of action, VII, 195-196,
optical properties, 111, 127-128 size and constitution, VII, 146-148
pH dependence, 111, 152-154 Phenylalanine hydroxylase, properties,
protein cleavage by, 111, 140-142 XII, 232-238
purification of, 111, 124 Phenylalanine 4-monooxygenase,
ribonuclease and, IV, 673 properties, XII, 287-289
side chain specificity, 111, 145-147 Phenylpyruvate synthase, properties,
stereochemical specificity, 111, 147-151 VI, 205-207
transpeptidation reactions, 111, 157-160 Phosphagen kinase, see Guanidino
Pepsinogen, included with Pepsin kinase(s)
Peptide(s) Phosphate, adenosine diphosphoryl
synthesis, nucleic acid independent, glucose pyrophosphorylase and,
VIII, 11-17 VIII, 91-92
Peptide bonds Phosphate esters
cleavage, proteinase inhibitors and, hydrolysis of acyclic
111, 452-454 di- and triesters, VIII, 207-208
Peptide chain elongation, see also metaphosphate mechanism for
Elongation monoesters, VIII, 202-206
scheme of, X , 54 Phosphoenolpyruvate, enzymatic syn-
Peptide groups, ultraviolet absorption, thesis, X, 631-633
11, 379-380 Phosphoenolpyruvate carboxykinase
PH discovery, distribution and physiologi-
chymotrypsin and, 111, 231-233, 236- cal role, VI, 136-138
237 physical properties, VI, 143-148
p-galactosidase and, VII, 635, 644-645 reactions catalyzed and their properties
guanidino kinases and, VIII, 476-477 metal requirement, VI, 141-142
kinetic parameters and, 11, 52-56 nucleoside phosphate specificity, VI,
methylmalonyl coenzyme A mutase 140-141
and, VI, 519 oxalacetate or phosphoenolpyruvate
phosphoglucomutase and, VI, 455 formation, VI, 139
pyruvate carboxylase and, VI, 21 pH optimum, VI, 142-143
Phage, see also Bacteriophage pyruvate formation, VI, 139-140
Phage lysozyme, see also Lysozyme thiol reagents and, VI, 143
application to other biologically im- regulation
portant problems, V, 350-352 activity, VI, 151-154
early history, V, 344-345 concentration, VI, 150-151
recent developments, V, 345-349 intracellular location, VI, 148-150
Phenol o-monooxygenase, properties of, Phosphoenolpyruvate carboxylase
XII, 221-223, 296-297 discovery, distribution and physio-
Phenylalanine ammonia-lyase logical role, VI, 119-122
catalytic process kinetic and regulatory properties, VI,
function of prosthetic group, VII, 126-133
159-162 mechanism of action, VI, 133-136
metal ion activation, VII, 162-163 nature of reaction catalyzed, VI, 122-
prosthetic group, VII, 154-159 124
rate limiting step, VII, 164-166 role of metals in mechanism, 11, 507-
reaction sequence, VII, 148-154 508
structural studies, VI, 124-126 thiols, VIII, 269-272

Phosphoenolpyruvate carboxytrans- structural properties
phosphorylase Clostridium pasteurianum, VIII, 256
characteristics and mechanism of dilution effects, VIII, 259-260
catalyzed reaction, VI, 157-161 Escherichia coli, VIII, 255257
different forms, structure and catalytic isozymes, VIII, 257
activities, VI, 163-164 molecular weight, VIII, 253-254
discovery, distribution and physiologi- phosphorylation, VIII, 260-261
cal role, VI, 154-157 rabbit erythrocyte, VIII, 257-258
kinetic parameters, effectors and in- rabbit muscle, VIII, 254-256
hibitors, VI, 162 Phosphoglucomutase
Phosphoenolpyruvate synthetase activation, VI, 439442
catalytic properties assay, VI, 417-418
kinetic studies, X, 643-645 all-or-none, VI, 420421
mechanism, X, 638441 colorimetric, VI, 418419
metal ion requirements, X, 645 coupled, VI, 419
pH and equilibrium, X, 645-646 radiometric, VI, 419-420
regulation, X, 648-649 catalytic reaction
specificity, X, 646-647 central complex structural differ-
stoichiometry, X, 637-638 ences, VI, 432
molecular properties isomeric forms of phosphoenzyme,
bound divalent metal ion, X, 637 VI, 430-432
molecular interconversions, X, 635- isotope exchange reactions, VI,
636 429430
phosphoryl and pyrophosphoryl kinetics, VI, 426-429
enzyme, X, 636-637 reaction sequence, VI, 421-424
purification, X, 633-634 roles of glucose diphosphate, VI,
stability, X, 634-635 424426
sulfhydryl groups, X, 635 inhibition
Phosphofructokinase anions, VI, 442444
assay of, VIII, 243-244 cations, VI, 444-446
chemical modification, VI, 446-447
cation requirement, VIII, 247-248
miscellaneous, VI, 447
isotopic exchange, VIII, 252
kinetic studies, VIII, 248-252 poor substrates and analogs, VI, 444
phosphoryl acceptor specificity, VIII, metal ion effects
244-245 activation, VI, 448-449
phosphoryl donor specificity, VIII, addition and release of magnesium,
24&247 VI, 449-451
control of glycolysis addition and release of other metals,
hormones and, VIII, 277-278 VI, 451-452
Pasteur effect, VIII, 274-276 complexes in vivo, VI, 454
pyridine nucleotide oscillations, metal-binding site, VI, 453454
VIII, 276 structural changes, VI, 452-453
purification, VIII, 241-243 pH and temperature effecta, VI, 455
reaction catalyzed, VIII, 240-241 physical and chemical properties, VI,
regulation of, I, 439-441, VIII, 261-269 413-415
role of specific groups polymorphism, VI, 416-417
histidine, VIII, 272 purity, VI, 412-413
other functional groups, VIII, 272-274 stability, VI, 416
TOPICAL SUFLJECT INDEX 521
preparation phosphate transfer to water, VI, 472-

chromatography, VI, 409410 474
dephospho-enzyme, VI, 411412 physical and chemical properties, VI,
isolation, VI, 408-409 460-462
phosphate-labeled, VI, 410-411 preparation and purity, VI, 459-460
specificity, VI, 436-439 reaction sequence, VI, 464-468
structural studies Specificity, VI, 471
active site phosphate group, VI, stability and storage, VI, 462
455-456 thermodynamics, VI, 470-471
active site phosphopeptide, VI, Phosphoglycolate, triosephosphate iso-
456-457 merase and, VI, 335-336
conformational studies, VI, 457-458 Phospholipase(s), general, V, 71-73
thermodynamics Phospholipase A,, V, 73
hydrolysiv of phospho-enzyme, VI, isolation and purification, V, 74-75
434-436 Crotalus adamanteus venom, V,
overall reaction, VI, 433 75-76
phosphate transfer to glucose phos- Crotalus atrox venom, V, 77-78
phate, VI, 433-434 pig pancreas, V, 76-77
6-Phosphogluconate dehydrase physical and chemical characteristics
distribution, V, 573-574 amino acid content, V, 80-81
properties, V, 575-578 amino acid sequence, V, 81-82
3-Phosphoglycerate, adenosine diphos- molecular weight, V, 78-80
phoryl glucose pyrophosphorylase sources, V, 74
and, VIII, 91-92 substrates and mode of attack, V, 74
3-Phosphoglycerate kinase Phospholipase C
biological behavior isolation and purification
Bacillus cereus, V, 83-84
occurrence, VIII, 337-338
reaction catalyzed, VIII, 338-337 Clostridium perfringens, V, 84-85
species variation and genetics, VIII, sources, V, 82-83
Phospholipids, glucose-6-phosphatase
338-340
and, IV, 554-556
historical background, VIII, 335-336
Phosphomutase(s), other sugars, VI,
458-459
molecular weight, VIII, 342
Phosphoribosylpyrophosphate synthetase
primary structure, VIII, 342343
secondary structure, VIII, 344 conditions affecting activity, X, 617
tertiary structure, VIII, 344-346 equilibrium constant, X, 617
purification procedures, VIII, 340-341 mechanism, X, 618-621
reaction kinetics substrates and activators, X, 614-617
backward and forward reactions, inhibition by metabolites
VIII, 346-348 bacterial enzyme, X, 621-622
metal ion specificity, VIII, 349 mammalian enzyme, X, 622-623
nucleotide specificity, VIII, 348-349 physiological significance, A, 623
postulated mechanism, VIII, 349-351 occurrence and purification, X, 611-612
Phosphoglycerate mutase other regulatory aspects, X, 623-624
assay, VI, 462-464 physical and chemical properties, X,
inorganic and simple organic com- 612-614
pounds, effects, VI, 474-475 reaction catalyzed and assay methods,
kinetics, VI, 469-470 X, 608-611
pH and temperature effects, VI, 475 related enzymes, X, 607-608
Phosphorus alcohol dehydrogenase of, XI, 188-189

acyclic, nucleophilic reactions and, amino acid decarboxylases of, VI,
VIII, 208-214 221-224
Phosphorylase kinase aspartate transcarbamylase of, IX,
heart muscle, VIII, 564 307-308
liver and other mammalian tissues, cytochromes b,
VIII, 565 microsomes, XI, 591
nonmammalian sources, VIII, 565 mitochondria, XI, 589-591
skeletal muscle, VIII, 557-564 photosynthetic systems, XI, 587-589
0-Phosphorylethanolamine phospholy- fructose-l,6diphosphatase
ase, properties, VII, 5 2 5 3 physiological role, IV, 642-643
Phosphoryl transfer purification and properties, IV,
catalysis 640-642
intramolecular, VIII, 219-227 regulation, IV, 643
metal ion, VIII, 227-231 isoamylases of, V, 208
enzymic mechanisms, VIII, 232-233 nuclear ribonucleic acid polymerase,
bimolecular or aeaociative, VIII, X, 311-318
235-238 pullulanase of, V, 202-204
metaphosphate, VIII, 233-235 viral ribonucleic acids, terminal
Phosphotransferase, glucose-bphospha- sequences, X , 85-86
tase and, IV, 592-595 Pneumococci, endonucleases, IV, 260-261
Phosvitin kinase, properties, VIII, 5& Point mutations, protein structure and,
581 I, 286-292
Photoisomerization, related problems Polymerization
and, VI, 395-397 statistics of, X , 157-158
Photosynthesis oligomer synthesis, X, 159-180
bacterial, XI, 509-510 polymer synthesis, X, 158-159
green sulfur, XI, 514-516 Polynucleotide ( 8 )
purple nonsulfur, XI, 610-512 enzymic methylation, general con-
purple sulfur, XI, 512-514 siderations, IX, 167-168
reductive carboxylic acid cycle of,
polynucleotide phosphorylase and,
VI, 207-213 VII, 552-557
Physarum polycephalum, ribonuclease
staphylococcal nuclease and
PPI of, IV, 241
kinetic measurements, IV, 186-187
Physiological function
design requirements, I, 465-467, 484- specificity, IV, 185-186
488
Polynucleotide phosphorylase
catalytic reactions, VII, 545-546
adenylate energy charge, I, 470-476
interactions between input signals, Clostridium and mutant Escherichia
I, 476-484 enzymes, VII, 571-572
kinetic properties, I, 467470 enzyme-polynucleotide complex,
Pituitary gland, 5’-nncleotidase of, IV, 346 VII, 570
Plants exchange reactions, VII, 570-571
acid phosphatase of, IV, 497 other practical uses, VII, 572-574
adenosine diphosphoryl glucose pyrophosphorolysis, VII, 548-557
phosphorylase polymerization, VII, 557-570
algae, VIII, 90 specificity, VII, 546-548
leaves, VIII, 86-90 degradation of, VII, 540-542
nonchlorophyllous tissue, VIII, 93- general background, VII, 533-535
94 in vivo
control of synthesis and activity, Polysaccharide depolymerase

VII, 538-539 Aerobacter phages, V, 398
distribution and localization, VII, Azotobacter phage, V, 397-398
536-538 bacteriophage F series
physiological role, VII, 538 catalytic properties and biological
molecular weight and subunits, VII, significance, V, 393
543-545 enzyme assay, V, 392
polymerization by partial purification, V, 392-393
kinetics, VII, 557-566 stability, V, 393
mechanism, VII, 566-570 Klebsiella phage
purification, VII, 539-540 enzyme properties and role, V,
thermal stability, VII, 542-543 394-395
Polypeptide chain initiation preparation and assay, V, 394
eukaryotes Pseudomanas phages, V, 395-397
inhibitors, X, 43 Porphobilinogen
initiation factors, X, 29-43 synthesis, mechanism of, VII, 333-337
initiator aminoacyl-transfer ribo- Potatoes, 5’-nucleotidase of, IV, 349
nucleic acid and, X, 28-29 Procarboxypeptidase B, physical and
messenger ribonucleic acid transla: chemical properties, 111, 67-68
tion, X, 43-44 Proelastase, activation of, 111, 331-332
prokaryotes Prokaryotes
general, X, 2-5 polypeptide chain initiation
initiation factors, X,6-28 general, X, 2-5
initiator aminoacyl-transfer ribo- initiation factors, X, 6-28
initiator aminoacyl-transfer ribo-
nucleic acid and, X, 5-6
nucleic acid and, X, 5-6
regulation
Proline iminopeptidase, 111, 115
interference factors, X, 44-45
Proline 4-monooxygenase, properties,
messenger recognition, X, 46-51
XII, 292-293
Polypeptide chain termination
Proline reductase, Schiff base and, 11,
events of protein synthesis and, X,
358
114-117 Proline residues, proteinase inhibitors
mechanism and, 111, 423
interaction of release factors with Prolyl hydroxylase, XII, 152-154
ribosomes, X, 101-108 assays, XII, 161
in vitro assay, X, 100-101 catalytic properties, XII, 156-160
peptidyl-transfer ribonucleic acid nonvertebrate, XII, 163-165
hydrolysis, X, 108-114 purification and molecular properties,
role of guanine nucleotides, X, 106- XII, 154-156
108 regulation, XII, 161-163
requirements Propane-l,2-diol dehydrase, stereo-
soluble protein factors, X, 95-100 chemistry, 11, 210-214
terminator codons, X, 88-95 Propinquity effects
Polyprenyl biosynthesis activation parameters and kinetic
stereospecificity order, 11,250-254
3-hydroxy3-methylglutaryl coen- approximation through noncovalent
zyme A synthesis, 11, 186-189 forces
preparation and use of labeled charge-transfer, 11, 254-264
mevalonates, 11, 192-204 Debye, 11, 254-264
squalene biosynthesis, 11, 190-192 hydrogen bonds, 11, 254-264
inclusion compounds, 11, 274-279 prokaryotic, X, 95-99

London, 11, 254-264 cleavage by pepsin, 111, 140-142
micelles, 11, 264-274 functional group adenylylation, VIII,
evaluation of concepts, 11, 220-226 40-49
Propionyl coenzyme A carboxylase genetic phenomena in
distribution, VI, 48 chain shortening, I, 292-293
historical background and metabolic deletion, addition, chain extension,
significance, VI, 46-48 I, 293-300
mechanism of action, VI, 51-53 gene duplication, I, 3W314
molecular characteristics, biotin bind- point mutations, I, 286-292
ing site, VI, 50-51 homologous, speciation of, I, 321-328
reaction catalyzed, VI, 46 structure, common characteristics, I,
stereochemistry, 11, 208-210 87-89
substrate specificity, VI, 49 structure-function relationships, evolu-
a-(n-Propyl)malate, synthesis, VII, tion and, I, 267-274
426-427 Proteinase(s), see also Protease(s)
Prostate gland acid, see Acid proteinases
acid phosphatase alkaline, see also Alkaline proteinases
assay, IV, 457 bacterial, 111, 605-606
electrophoresis, IV, 468-469
inhibitor association with, 111, 428-433
functional groups, IV, 469-472
inhibitor complexes, dissociation of,
general, IV, 455-457
111, 434-436
kinetics, IV, 457466
physical properties, IV, 476 microbial, see Microbial proteinases
preparation, IV, 466-468 neutral, see Neutral proteinases
transphosphorylation, IV, 472-473 thiol, microorganisms and, 111, 791-795
use as a reagant, IV, 473-476 Proteinase inhibitors
Protease(s), see also .Proteinases assays
activation, proteolysis and, I, 416 active site titrants, 111,393-396
gene duplication and competitive enzyme assays, 111, 396
pancreatic, I, 303-307 errors in, 111, 396-400
dfhydryl, I, 307-308 association constants
hexokinases and, IX, 2-7 enzyme titration methods, 111,
modification of, I, 118-124 402403
serine, sequence homologies, 111, 343 physicochemical methods, 111,400-
352 402
X-ray diffraction studies, 1, 63-67 potentiometric method, 111, 403-406
Protein (8) competitive inhibition and, 111, 391-
allosteric 393
alternative approaches, I, 385-388 complex formation, changes in con-
evaluation of, I, 372-375 formation-sensitive parameters,
evolutionary considerations, I, 390- 111, 406-410
393 crystalline inhibitors and complexes,
hemoglobin, I, 388-390 111, 383-384
molecular basis of cooperativity, I, definition, 111, 378
375-379 disulfide loop, reactive site and, 111,
protein design, I, 379-381 422-423
status of simple models, I, 381-385 enzyme-susceptible bond in reactive
chain termination and site, 111, 419
eukaryotic, X, 99-100 historical background, 111, 376-378
identities and nomenclature, 111, 378- phosvitin kinases, VIII, 580-581

379 substrate-specific
kallikrein inhibitor and pancreatic phosphorylase kinase, VIII, 557-565
inhibitors, 111, 379-380 pyruvic dehydrogenase kinase, VIII,
serum inhibitors, 111, 382 565-566
soybean inhibitors, 111, 380-382 Pro teolysis
inhibition a t same reactive site, 111, metabolic regulation and
470-473 blood coagulation, I, 416417
molecular differences proteolytic enzyme activation, I, 416
analogies and homologies, 111,457- polynucleotide phosphorylase and,
463 VII, 557
specificity toward other enzymes, Prothrombin
111, 463473 activating enzyme, 111, 315-317
nonoverlapping reactive sites, 111, amino acid and carbohydrate compo-
466-468 sition, 111,313
overlapping independent reactive aasays of, 111, 308
sites, 111, 468470 disulfide bridges, 111, 313
physical properties, 111, 384-388 isolation and purity, 111, 308-311
reactive site model metabolism
chemical model of inhibitor, 111, biosynthesis, 111, 320-321
443-447 turnover rate, 111,320
control dissociation of complex, 111, number of polypeptide chains, 111,
437439 314-315
detection of reactive sites, 111, physical properties, 111, 311412
412-418 structural aspects
equilibria of hydrolysis, 111, 423428 models, 111, 318-319
general properties, 111, 418-423 proteolysis, 111, 317-319
kinetics of interaction, 111,428437 secondary proteolysis, 111, 319
nature of stable complex, 111, 450- N- and C-terminal analysis, 111,313-
451 314
objections to, 111, 451457 Proton dissociation-replacement reac-
overshoot of complex, 111,410-412 tions, 11, 312-313
residue replacement a t reactive site, citrate synthase, 11, 316-317
111, 439-441 hydrogenase, 11, 317-318
sites for other enzymes, 111, 441443 malate synthase, 11, 316-317
temporary inhibition, 111, 447-449 stereochemistry, 11, 313-315
special purification techniques, 111, 1,l-Proton shifts, epimerases and, 11,
389-391 295-298
N-terminal residue in reactive site, l,2-Proton shifts, aldo-keto isomerases,
111, 419-420 11, 290-295
virgin and modified, interconversion, l,3-Proton shifts, allylases and, 11,299-
111, 436437 302
Protein kinase(s) Protozoa, glycogen synthetase of, IX, 359
cyclic nucleotide-regulated, VIII, Pseudomonads
566-578 aspartokinases of, VIII, 551-552
historical background, VIII, 555-557 isoamylase of, V, 204-206
nonclassified, VIII, 578379 Pseudomonus, proteinases, of, 111, 769-
acidic nuclear protein kinases, VIII, 770
580 Pseudomonas aeruginosa, phage poly-
histone kinases, VIII, 579-580 saccharide depolymerase, V, 395-397
Pseudomonas putida, phage polysac- specificity

charide depolymerase, V, 397 carbohydrates, VII, 502-503
Pseudomonas testosteroni purines, VII, 500-502
As-3-ketosteroid isomerase subunit structure, VII, 514
catalytic properties, VI, 5 9 W sulfhydryl groups, VII, 513-514
mechanism, VI, 605-615 Pyridine nucleotide ( s )
molecular properties, VI, 592699 deoxythymidine diphosphate-D-glucose
Pseudouridine kinase, properties, IX, 62 oxidoreductase and, V, 474478
Pullulanase phosphofructokinase and, VIII, 276
Aerobacter aerogenes Pyridine nucleotide-disulfide oxidore-
preparation and physical properties, ductases
V, 195-197 mechanism, similarities and contrasts,
reversion reactions, V, 201 XIII, 94-99
substrate specificity and action reaction catalyzed-chemical similarities
pattern, V, 197-201 and crom-reactivity, XIII, 92-94
plant, V, 202204 structure, similarities and contrasts,
Purine aminohydrolase (s) XIII, 99-105
assay methods, IV, 51 Pyridoxal, reactions with amino acids,
distribution, IV, 49-51 11, 339-345
historical background, IV, 48 Pyridoxal-linked reactions
Purine nucleoside phosphorylase absorption spectra and, VII, 62-65
assays amino acids and, VII, 33-39
direct spectrophotometry, VII, 505 mechanisms, VII, 65-66
inorganic orthophosphate estimation, @-elimination and, VII, 66-72
VII, 504 7-elimination and replacement, VII,
isotopic assays, VII, 505 72-73
pentose estimation, VII, 503-504 stereochemistry, VII, 73-74
spectrophotometry coupled with Pyridoxal phosphate, fructose-1,6-
xanthine oxidase, VII, 504-505 diphosphatase and, IV, 620
catalytic mechanism Pyridoxal phosphate enzymes
equilibrium studies, VII, 511 apoenzymes and, 11, 346-348
reaction mechanism, VII, 512 classification, 11, 367-368
reaction sequence, VII, 511-512 comparative characteristics, 11, 363-364
distribution in nature, VII, 485-490 mechanism of reaction, 11, 349-356
historical development, VII, 483485 peptide sequences, 11, 366
kinetics structural and spectral properties, 11,
substrate concentration and, VII, 348-349
505-510 Pyrimidines, dioxygenase reactions of,
temperature and pH effects, VII, XII, 169-179
510-511 Pyrimidine deoxyribonucleoside 2'-hy-
metabolic functions droxylase, catalytic properties, XII,
bacterial metabolism, VII, 494 176178
chemotherapy, VII, 493-494 Pyrimidine nucleoside monophospho-
erythrocyte metabolism, VII, 4 9 2 kinases, IX, 87-88
493 Pyrocatechase
fish skin, VII, 494495 structure, physical probes, 11, 406-407
nucleoside metabolism, VII, 490492 Pyrophosphokinase(s), other, X, 624-628
properties, VII, 495-496 Pyrrolidone carboxylate
purification, VII, 495 derivatives, enzymic formation of,
reactions catalyzed, VII, 496-500 IV, 146147
detection and determination, IV, 125- temperature, VI, 19-20

127 urea and pH, VI, 20-21
enzymic formation from glutamate molecular parameters and quaternary
D-glUtaInate cyclotransferase, IV, structure, VI, 16-18
133-136 nicotinamide adenine dinucleotide
L-glutamate cyclotransferase, IV, 138 and, VI, 33-34
glutamine synthetase, IV, 136-137 phosphoenolpyruvate and, VI, 34
yglutamylcysteine synthetase, IV, product inhibition-two-site mechanism,
136-137 VI, 7-8
rat liver nuclear preparations, IV, regulation of synthesis, VI, 34
138-139 role of metals, 11, 511-515
enzymic formation from glutamine second partial reaction
and glutaminyl peptides nature of bound metal ion, VI, 10-11
L-glutamine cyclotransferase, IV, role of bound metal ion, VI, 11-15
139-141 Pyruvate dehydrogenase complexes
y-ghtamyl cyclotransferase, IV, 141 composition and organization, I, 215-
y-glutamyl transpeptidase, IV, 141 220
enzymic formation from y-glutamyl regulatory features, I, 220-224
amino acids Pyruvate kinase
y-L-glutamyl cyclotransferase and, assay, VIII, 371
IV, 142-146 catalytic mechanism, VIII, 379-382
historical background, IV, 124-125 control, VIII, 378-379
metabolism, IV, 149-151 historical background, VIII, 353-355
natural occurrence, IV, 127-130 kinetics
nonenzymic formation, IV, 130-133 inhibitors, VIII, 375-377
Pyrrolidone carboxylyl peptidase, IV, substrates and activators, VIII,
147-149 372-375
Pyrrolidonyl peptidase, 111, 113-114 molecular properties
Pyruvate carboxylase chemical modification, VIII, 360-361
acyl coenzyme A derivatives and composition, VIII, 358
general properties, VI, 24-27 ronformationnl change, VIII, 361-
parameters reflecting enzyme con- 364
formation, VI, 29-31 purification, VIII, 355-358
specificity of activation, VI, 27-29 structure, VIII, 358-359
aspartate and, VI, 31-33 muscle, role of metal in mechanism,
first partial reaction, VI, 9-10 11, 504-506
immunochemical studies, VI, 23 stoichiometry and specificity
general properties, VI, 2-3 cofactors, VIII, 366-370
generalized minimal mechanism, VI, number of active sites, VIII, 370-
6-7 371
monovalent cation effects, VI, 6 reaction catalyzed, VIII, 364
partial reactions, VI, 3-5 substrate specificity, VIII, 364-366
presence of bound biotin, VI, 3 thermodynamics, VIII, 371-372
requirements, VI, 5-6 yeast, role of metals in mechanism, 11,
historical background, VI, 1-2 506-507
mild denaturation and chemical Pyruvate, phosphate dikinase
modification catalytic properties
maleic anhydride, VI, 22-23 kinetic studies, X, 643, 644-645
other reagents, VI, 23 mechanism, X, 641-642
sulfhydryl reagents, VI, 21-22 metal ion requirements, X, 645
regulation, X, 648-649 alternative treatment of multistep

specificity, X,646 mechanisms, 11, 91-93
stoichiometry, X,638 analysis and interpretation, 11,95-99
molecular interconversions, X, 635- multistep mechanisms, 11, 89-91
636 one-step mechanisms, 11, 83-84
phosphoryl and pyrophosphoryl en- thermodynamically dependent reac-
zyme, X, 636-637 tions, 11, 93-95
purification, X, 633-634 transient and stationary solutions of
stability, X, 634 rate equations, 11, 84-87
sulfhydryl groups, X, 635 two-step mechanisms, 11, 87-89
Pyruvate synthase, properties, VI, 197- Rennin, gene duplication and, I, 308
201 R-enzyme, see Pullulanase
Pyruvic dehydrogenase kinase, proper- j3-Replacement reactions, pyridoxal-
ties, VIII, 565-566 linked, VII, 54-57
Retinal isomerase, pigment regeneration
Q and, VI, 587-589
Reverse transcriptase
Qj3 replicase, elongation factors and, X, biological role
83-85 noninfectious murine sarcoma virus,
X, 232
R other inhibitors, X, 233
rifamycins, X, 233
Rana cateebiana Rous sarcoma virus a, X, 231-232
collagenase, 111, 689-690 temperature sensitive Rous sarcoma
catalytic properties, 111, 691-693
virus, X, 232-233
preparation, 111, 69M91
comparison to other polymerases, X,
Rate equations
derivation 233-235
inhibitors
chemical reaction, 11, 61-63
rifamycins, X, 231
isotopic exchange, 11, 63-65
sulfhydryl reagents, X, 230-231
Reactions,
reversible, unidirection of, 428-430 nuclease activity
Red cell deoxyribonuclease, X, 220-221
acid phosphatase ribonuclease, X,221
general properties, IV, 477 ribonuclease H,X, 221-222
purification and separation of primer and direction of synthesis, X,
genetic types, IV, 477-484 225-226
Reduced nicotinamide adenine dinucleo- problems, X, 229-230
tide kinase, properties, IX, 82 properties, X, 218
Reduviin, thrombin and, 111, 304-305 sire, X, 219-220
Relaxation amplitudes storage and stability, X, 219
kinetic studies near equilibrium properties of catalytic reaction
calculation of amplitudes, 11, 105- deoxyribonucleoside triphosphate,
10s X,224
thermodynamic effects of chemical divalent cations, X, 224-225
reactions, 11, 101-105 other conditions, X, 225
transformation to normal concen- purification, X, 216-218
tration variables, 11, 99-101 serological relationships
Relaxation spectra avian leukosis viruses, X, 223
kinetic studies near equilibrium general considerations, X,222
mammalian C-type viruses, X, 223- thermodynamics, IV, 740-744

224 transitions in derivatives, IV, 738-
other viruses, X,224 740
solubilization, X, 215 urea and, IV, 731-733
template chemical modification of functional
fidelity of synthesis, X, 228-229 groups
preferences, X,226-228 amino groups, IV, 677-682
requirements, X, 226 arginine, IV, 689-690
size, X,229 carboxyl groups, IV, 675-677
virus purification and, X, 214-215 cystine-disulfide groups, IV, 690-696
Reversions, mutations and, I, 249-251 histidine, IV, 685-689
L-Rhamnose isomerase, properties, VI, intramolecular crosslinks, IV, 696-
345-346 697
L-Rhamnulose 1-phosphate aldolaae methionine, IV, 682-683
catalytic reaction other reagents, IV, 697
assay, VII, 308-309 chemical modification of functional
equilibrium constant, VII, 310-311 groups
metal ions and, VII, 309 serine and threonine, IV, 696
pH optimum, VII, 309 tyrosine, IV, 684-685
substrate binding and reaction chemical synthesis and S-peptide
sequence, VII, 311-313 summary, IV, 697-705
turnover number, VII, 309 classification of, IV, 205-207
historical background, VII, 304 discussion of mechanism and stability
metabolic significance, VII, 305 lysine 41 and, IV, 801
molecular properties opposite vs. adjacent attack, IV,
isolation, VII, 305 791-794
physical properties, VII, 306 pH and, IV, 801-806
structure, VII, 306-308 proton transfer and rate-limiting
occurrence, VII, 304305 step, IV, 795-796
Rhodopseudomonas capsulatus, asparto- role of oxygen, IV, 79-01
kinase of, VIII, 544-545 stabilization of intermediates, IV,
R hodopseudomonua spheroides 794-795
8-aminolevulinate synthetase of, VII, structure, IV, 785-788
344-345 transphosphorylation and hydroly-
aspartokinase of, VIII, 552-553 sis, IV, 788-791
membrane adenosine triphosphatase, enzymic cleavage of main chain
x,429 chymotrypsin, IV, 674
Rhodospirillum rubrum, adenosine di- elastase, IV, 672-673
phosphoryl glucose pyrophosphor- pepsin, IV, 673
ylase of, VIII, 81-86 subtilisin, IV, 669-672
Riboflavin kinase, properties, IX, 74-75 trypsin, IV, 673-674
Ribonuclease (s) fungal, general survey, IV, 208-211
aggregation of, IV, 744-746 historical background, IV, 647-649
assays, IV, 747-750 isolation and chromatography, IV,
rhain conformation and solvent-in- 649-653
duced changes, IV, 725-726 macromolecular inhibitors, IV, 758-759
added electrolytes, IV, 735-737 mechanism of catalysis
organic solvents, IV, 733-735 Mathias and Rabin et al., IV, 780-
thermal and acid transitions, IV, 781
726-731 Roberts et al., IV, 784
Usher, IV, 783-784 preparation, IV, 212-213

Wang, IV, 782-783 properties, IV, 213-214
Witzel, IV, 781-782 specificity and mode of action, IV,
microbial, 215-218
of interest, IV, 239-243 structure and function, IV, 218-222
list of, IV, 243-249 Ribonuclease Tz
physical parameters applications, IV, 229-230
diffusion coefficient, IV, 708-709 preparation, IV, 223-224
electrophoretic mobility, IV, 710-711 properties, IV, 224-225
fluorescence, IV, 718-719 specificity and mode of action, IV,
hydration and axial ratio, IV, 709- 225-229
710 Ribonuclease Uz
hydrogen ion equilibrium, IV, 711- applications, IV, 237-239
712 preparation, IV, 234-235
hydrogen exchange, IV, 712-714 properties, IV, 235
molecular weight, IV, 709 specificity, IV, 235-237
physical parameters Ribonucleic acid
nuclear magnetic resonance and synthesis, VIII, 20-21
electron paramagnetic resonance, polynucleotide adenylyltransferases,
IV, 723-725 VIII, 24-26
optical rotatory dispersion and polynucleotide phosphorylase, VIII,
circular dichroism, IV, 719-723 23-24
partial specific volume, IV, 705-707 ribonucleic acid polymerase, VIII,
radius of gyration, IV, 707-708 21-23
refractive index increment, IV, 707 Ribonucleic acid polymerase(s)
sedimentation behavior, IV, 709 animal
ultraviolet absorption spectra, IV, general properties, X, 280-283
714-717 historical, X, 262-264
viscosity, IV, 710 inhibitors, X, 295-299
reaction catalyzed, IV, 746-747 intracellular localization, X, 279-280
small molecule effectors, IV, 759-772 nomenclature, X, 264-266
specificity, IV, 750-751 physiological role, X, 299-300
base, IV, 754-758 purification, X, 266-269
phosphate, IV, 758 regulation in vivo, X, 300
sugar, IV, 752-754 stirnulatory factors, X, 293-294
steady state kinetic data structure, X, 269-279
ionic strength and, IV, 777-778 template specificity, X, 284-293
Michaelis constants and turnover bacterial
number, IV, 772-777 assay, X, 338-339
organic solvents and, IV, 779-780 chain elongation, X, 359-366
structure chain initiation, X, 353-359
amino acid sequence, IV, 653-654 chain termination, X, 366-370
physical probes, 11, 396-401 inhibitors, X, 370-374
three-dimensional, IV, 654-669 outline of reaction, X, 346-348
Ribonuclease N,, IV, 230-231 purification, X, 335-338
applications, IV, 232-234 template binding, X, 348-353
preparation, IV, 231 variety of reactions catalyzed, X,
properties, IV, 231-232 344-346
R.ibonuclease T, chain elongation
applications, IV, 222-223 kinetics, X, 364-366
nondissociable ternary complex, X, Ribonuceloside 2’,3’-cyclic phosphate

359-361 diesterase with 3’-nucleotidase
specificity, X, 361-363 activity
chloroplast, X, 329-330 cellular localization, IV, 361-362
covalent modification a metalloenzyme, IV, 362-363
diphosphopyridine nucleotide- physiological function, IV, 363
dependent, VIII, 48-49 properties
possible artifact, VIII, 49 kinetics and mechanism of action,
inhibitors, X, 370 IV, 358-361
agents affecting template, X, 373374 physical and chemical, IV, 358
agents interacting with enzyme, X, substrate specificity, IV, 357458
371-373 Ribonucleotides
fungal, X, 310-311 reduction, regulation of, I, 442-443
higher plant Ribose-5-phosphate isomerase
enzyme properties, X, 314-316 general, VI, 318-320
factors, X, 317-318 catalytic properties
function, X, 318 assay methods, VI, 321-322
molecular properties, X, 314 mechanism, VI, 323-324
solubilization and purification, X, Michaelis constants, VI, 322-323
311-314 molecular properties, VI, 320-321
mi tochondrial Ribosomal ribonucleic acid methyl-
enzyme properties, X, 325-328 transferase
molecular properties, X, 324-325 biological significance, IX, 189-190
solubilization and purification, X, isoIation and properties, IX, 187-189
318-324 occurrence, IX, 187
synthesis, X, 328 Ribosome(s)
nuclear elongation and, X, 67, 77-78
animal, X, 262300 sites involved, X, 09-77
higher plant, X, 311-318 structure and, X, 68-69
yeast and fungi, X , 300-311 elongation factors and, X, 62-03, 66-67
structure of bacterial enzyme release factor interaction
dissociation and reconstitution, X, bacterial, X , 101-103
342-343 mammalian, X, 103-106
missing subunit problem, X, 343-344 sites involved in elongation
molecular weight, X, 341-342 guanosine triphosphatase and factor
subunits and, X, 340-341 binding sites, X, 73-76
yeast, X, 300-301 peptidyltransferase center, X, 69-72
enzyme properties, X, 306-309 role of 30 S proteins, X, 76-77
function, X, 310 Ribulose-1,d-diphosphate carboxylase
molecular properties, X, 304-306 general considerations, VI, 169-173
solubilization and purification, X, kinetics and specificity, VI, 181-183
301-304 “carbon dioxide,” VI, 183-184
stimulatory factors, x, 309310 divalent metal ion, VI, 185-186
Ribonucleoside 2’,3’-cyclic phosphate inhibitors, VI, 186-187
diesterase ribulose diphosphate, VI, 184-185
nervous ‘tissue, IV, 363-364 light activating factors, VI, 191-192
intracellular localization, IV, 364-356 mechanistic considerations, VI, 187-191
physiological rale, IV, 365 molecular properties
properties and substrate specificity, composite quaternary structure, VI,
IV, 364 179-180
native enzyme, VI, 173-178 iso1ated

subunits, VI, 178-179 contaminants, X, 437
reaction, VI, 180-181 procedures, X, 435-437
o-Ribulose-5-phosphate 3’-epimerase, shape and size of vesicles, X, 437-439
properties, VI, 374375 lipid functions
~Ribulose-5-phosphate 4’-epimerase, enzyme properties, X, 465467
properties, VI, 372-373 membrane permeability, X, 465
Rubredoxin, lipoprotein structure, physical proper-
chemical properties, XII, 12-15 ties, X, 442-444
historical background, XII, 4-6 Saturation curves, fitting of, I, 361365
physical properties, XII, 6-12 Scatchard plots, enzyme regulation and,
Rye grass, 3’-nucleotidase of, IV, 353 I, 359-361
Schiff base(s)
s formation
Saccharomyces, see also Yeast general characteristics, 11, 336-339
acid phosphatase, IV, 497 nonenzymic catalytic effects, 11,
Saccharomyces cerevisiae 339-346
aspartate transcarbamylase of, IX, other than pyridoxal, TI, 345-346
302-306 Schiff base enzyme(s)
aspartokinase of, VIII, 553 noncarbonyl, 11, 358-359
fructose-l$-diphosphatase, regulation, aldolases and transaldolases, 11,
IV, 640 359-360
Salicylate hydroxylase, properties, XII, 8-aminolevulinate dehydratase, 11,
206211 361-362
Salmonella phage lytic enzyme, V, 398 comparative properties, 11, 369
Sarcoplasmic membrane(s) p-keto-acid decarboxylases, 11, 359
calcium binding by, X, 450451 Seminal plasma
calciumdependent adenosine triphos- bull, 5’-nucleotidase of, IV, 342-343
phatase Serine hydroxymethyltransferase, proper-
ion requirements, X, 446-447 ties, IX, 215-221
lipid depletion and, X, 449 Serine peptides, microbial proteinases
membrane permeability and, X, and, 111, 751-752
445446 Serine residues,
temperature and pH, X, 447 chemical modification, I, 173
thiol reagents and, X, 449-450 chymotrypsin
calcium-independent adenosine tri- amide hydrolysis and, 111, 224231
phosphate, X, 444445 ester hydrolysis and, 111, 218-224
calcium transport by ribonuclease, IV, 696
absence of precipitating anions, subtilisin, 111, 575-576
X, 451 conversion to cysteine, 111,577-580
adenosine triphosphate extra split- sequence in other proteinases, 111,
ting, X, 453-455 576-577
presence of precipitating anions, X, Serratia marcescens, adenosine diphos-
451453 phorylglucose pyrophosphorylase of,
release from preloaded vesicles, X, VIII, 107-108
455456 Serum, acid phosphatase in, IV, 495-496
composition Slime molds, fructose-l,6-diphosphatases
lipids, X, 442 of, IV, 640
proteins, X, 440-442 Solvation, physical organic models, 11,
in situ, X, 434-435 226-238
Sorangium, proteinases of, 111, 747, synthetic analogs and, IV, 199-204
752-754, 789-790 synthetic substrates and inhibitors
Spinach kinetic measurements, IV, 190-195
adenosine diphosphoryl glucose pyro- specificity, IV, 187-189
phosphorylase of, VIII, 86-89 thymidine-3',5'-diphosphate and cal-
chloroplast adenosine triphosphatase cium ion binding, IV, 163-171
assay, X , 389 ultraviolet difference spectroscopy, 11,
catalytic properties, X, 391393 413-414
cold inactivation, X, 390-391 a warning, IV, 174
molecular properties, X, 390, 391 Staphylococci, lytic phage enzymes, V,
nucleotide binding, X, 39-94 398-399
purification, X, 389-390 Staphylococcus, acid phosphatase of, IV,
Spleen 498
acid deoxyribonuclease Staphylococcus aurews
catalytic properties, IV, 276-285 phage lytic enzyme, V, 400-401
distribution, intracellular localiza- virolysin, V, 399-400
tion and biological role, IV, Starch
285-287 structure determination, debranching
physical and chemical properties, IV, enzymes and, V, 228-234
272276 Stearate, conversion to oleate, 11, 179-184
acid exonuclease, IV, 330-336 Stereochemistry
acid phosphatase, IV, 493-495 a,j3 elimination reactions, 11, 309-312
deoxyribonuclease of, IV, 272-287 proton transfer, 11, 313-315
glycogen synthetase of, IX, 354 Stereospecificity, nicotinamide adenine
Squalene, epoxidase and, VII, 211 dinucleotide-dependent oxidoreduc-
Staphylococcal nuclease tases, 11, 134-157
active site, stereochemical probes, Steroid sulfatase(s), V, 4-6
IV, 195-196 androstenolone sulfatase, V, 7-9
behavior in solution, IV, 183-184 cortisone sulfatase, V, 10
covalent structure, IV, 180-183 estrone sulfatase, V, 6-7
crystallographic studies, introduction, etiocholanolone sulfatase, V, 9-10
IV, 156-159 Strain, physical organic models, 11,
fluorescence spectroscopy, 11, 429 226-238
fragments, complementation of, IV, Streptococcal proteinase
196-199 activation, 111, 627-628
general, IV, 153-156 amino acids
historical background, IV, 177-178 active site, 111, 626-627
isolation, IV, 178-179 composition, 111, 624-625
mechanism, IV, 174-175 N- and C-terminal, 111, 625-626
peptide chain conformation, IV, assay methods, 111, 627
159-163 immunological properties, 111, 615-619
proposed future studies, IV, 175 kinetics of hydrolysis
studies in solution, IV, 17%174 dielectric constant and, 111, 636-637
substrate specificity and catalytic esterase vs. peptidase activity, 111,
mechanisms 638-639
polynucleotide substrates, IV, 185-187 p H and, 111, 633-636
size and specificity of active site, structural inhibitors, 111, 637-638
IV, 191-195 mechanism of action, 111, 639-647
synthetic substrates and inhibitors, physical properties
IV, 187-191 electrophoresis, 111, 614
gel filtration and chromatography, serine and, 111, 575-580

111, 613-614 chemical modification
molecular weight, 111, 614 lysine residues, 111, 596-598
stability, 111, 614-615 methionine residues, 111, 598-599
preparation and crystallization tyrosine residues, 111, 599-602
proteinase, 111, 611413 historical background and develop-
zymogen, 111, 610-611 ment, 111, 562-563
specificity inhibitors, dye binding and, 111,
esterase activity, 111, 631-632 602-605
peptide and amide bonds, 111, physical, chemical and stability
628-631 properties
transferase activity, 111, 632633 subtilisin Amylosacchariticus, 111,
sulfhydryl group 566-567
nature, 111, 622-623 subtilisin BPN’, 111, 565-566
reactivity, 111, 623-624 subtilisin Carlsberg, 111, 564-565
zymogen-to-enzyme transformation subtilisin Novo, 111, 566
autocatalytic, 111, 621 practical uses, 111, 606-607
bacterial cell walls, 111,621-622 detergents and, 111, 608
preformed streptococcal proteinase, protein sequencing, 111, 807
111, 621 primary structure
subtilisin, 111, 620-621 comparison of sequences, 111,
trypsin, 111, 619-620 571-575
Streptococci general comparison, 111, 567
endonucleases, IV, 260-261 subtilisin Amylosacchariticus, 111,
phage lytic enzyme, V, 402-403 569-571
enzyme assay, V,403-404 subtilisin BPN’, 111,567-569
group C phages and, V,404-406 subtilisin Carlsberg, 111, 569
other phages, V, 406-408 subtilisin Novo, 111, 509
Streptococcus faecalis, membrane adeno- ribonuclease and, IV, 669-672
sine triphosphatase, X,400-416 substrate specificity and enzymic
Streptomyces, proteinases of, 111, properties
746-747, 752-754, 768-769 mechanism of action, 111, 593-596
Structural information protein and peptide substrates, 111,
other methods of obtaining 584-586
Fourier difference maps and salt synthetic substrates, 111, 586-593
difference maps, I, 4145 transesterification and transpeptida-
noncrystallographic symmetry, I, tion, 111, 593
45-46 X-ray structure
single isomorphous replacement and background, 111, 547452
variations, I, 39-11 catalytic site, 111, 553-560
Submandibular gland, hyaluronidase of, comparison with subtilisin Carlsberg,
V, 312-313 111, 560
Substrate bridge complexes general description, 111, 552-553
electronic structure of, ATP, 11, Subtilisin BPN’, chemical modification,
478-481 I, 203-205
formation mechanism, 11,48143 Subunits
reaction mechanism, 11, 483-485 acetyl coenzyme A carboxylase, VI,
Subtilisin (s) 60-79
active site studies adenylosuccinase, VII, 192-193
histidine and, 111, 580-584 aldolases, VII, 221-224
!K)PICAL SUBJECT INDEX 535
amino acid decarboxylases, VI, 248-253 size, X, 584

argininosuccinase, VII, 176-177 sulfhydryl groups, X, 590-591
aspartate transcarbamylase, IX, other sources, X, 594
230-243 pig heart
aspartokinases, VIII, 518-519, 541 phosphoenzyme formation, X,
carbamate kinase, IX, 103-104 592-593
creatine kinase, VIII, 394-395 polymorphism, X, 593-594
fumarase, V, 545-549 quaternary structure, X, 591-592
glucose-6-phosphate isomerase, VI, size, X, 591
279-281 possible regulatory properties, X, 606
glycerol kinase, VIII, 494-495 reaction catalyzed, X, 581-582
guanidino kinases, VIII, 4-88 steady state kinetics, X, 603-605
hexokinase, IX, 8-10, 39-40 substrate specificity
2-keto-3-deoxy-6-phosphogluconic coenzyme A, X, 595
aldolase, VII, 285-287 nucleoside di- and triphosphates, X,
phosphofructokinase, VIII, 254-257 595-596
polynucleotide phosphorylase, VII, succinate, X, 594-595
543-546 Sucrose phosphorylase
purine nucleoside phosphorylase, VII, covalent glucose-enzyme
514 configuration of bond, VII, 524
thiolase, VII, 396-397 isolation and properties, VII, 521423
transcarboxylase, 95-101 kinetics, VII, 510-521
triosephosphate isomerase, VI, 327-329 mechanism of catalysis, VII, 524-526
uridine diphosphate-n-glucose 4’- purification and properties, VII,
epimerase and, VI, 366-368 518-519
Succinate dehydrogenase water and alcohols as acceptors, VII,
mammaliam, XIII, 222-223 528632
enzymic properties, XIII, 236-245 Sulfarnatases, V, 18-19
inhibitors and modifiers, XIII, Sulfatase A
245-247 other sources, V, 36-37
mechanism, XIII, 251-254 ox liver, V, 27-36
molecular properties, XIII, 223-236 Sulfatase B
regulatory properties, XIII, 247-251 other sources, V, 38-39
microorganisms and, XIII, 254-256 ox liver, V, 37-38
stereospecificity, 11, 176-179 Sulfatophosphate sulfohydrolases, V,
Succinyl coenzyme A mutase, stereo- 17-18
chemistry, 11, 206-210 Sulfate, activation of, VIII, 35-37
Succinyl coenzyme A synthetase Sulfate-activating enzymes, regulation
anomalous reactions, X, 605-606 and control, X, 665-669
catalytic intermediates Sulfhydryl groups, see also Cysteine,
others, X, 602603 Thiol groups
phosphoensyme, X, 596-600 amylases, V, 244-245
succinyl phosphate, X, 600-602 argininosuccinase and, VII, 177-178
Escherichia coli aspartate transcarbamylase, IX,
active site, X, 590 263-265
characterization of phosphoenzyme, streptococcal proteinase, 111,622-624
x, 583-584 Sulfhydryl reagents, pyruvate carboxyl-
purification, X, 582483 ase and, VI, 21-22
quaternary structure, X, 584-589 Sulfites
reactivity and stability, X, 689 organic, pepsin and, 151-152
Sulfite oxidases molecular properties

liver cysteine and amino acid composi-
catalytic properties, XII, 417-419 tion, VII, 202
molecular properties, XII, 414-417 molecular weight, VII, 202-203
Sullite reductase(s), XIII, 286-287 purification, VII, 202
NADPHdependent, properties, XIII, Temperature
287-295 creatine kinase and, VIII, 420-422
reduced methyl viologendependent, p-galactosidase and, VII, 634-635
XIII, 295 phosphoglucomutase and, VI, 455
Sulfonamide, carbonic anhydrase and, pyruvate carboxylase and, VI, 19-20
V, 652-658, 661 Terminal deoxynucleotidyl transferase
Superoxide dismutase biological directions, X, 169-171
away methods, XII, 539-540 historical background, X, 145-147
catalytic mechanism mechanism, X, 160-161
enzymic dismutation of 02-,XII, metal ligand inhibitors, X, 161-163
552-556 other inhibitors, X, 163-165
inhibition, XII, 556 unititiated synthesis, X, 165-168
model complexes, XII, 657 nature of the reaction
crystallization, XII, 542 enzyme, X, 154-157
determination of purity and concen- initiators, X, 149-152
tration, XII, 540 metal ions, X, 152-153
historical, XII, 533 nucleoside triphosphates, X, 147-149
molecular properties pyrophosphate, X, 153-154
apoenzyme, XII, 548-549 practical applications
native enzyme, XII, 542-548 oligomeric additions, X, 167-188
reconstitution, XII, 549-551 polymeric additions, X, 166-167
redox properties, XII, 551-552 random copolymers, X, 168-169
physiological role, XII, 533 N-Terminal exopeptidases, background,
prokaryotic and mitochondrial, XII, 111, 81-82
537-538 Terminator codons
purification biochemical identification, X, 93-95
methods, XII, 538-539 genetic aspects, X, 88-93
sources, XII, 538 Testicle, hyaluronidase of, V, 311-312
suppression, mutations and, I, 249- Tetranitromethane, p-hydroxydecanoyl
25 1 thioester dehydrase and, V, 455
Synovial fluid, collagenases, of, 111, Thiamine pyrophosphokinase, X, 624427
693-696 Thiolase
active site, amino acid sequence, VII,
T 404-405
biological function
Tabanin, thrombin and, 111, 304-305 control of enzymic activity, VII, 400
Tadpole liver, glycogen synthetase of, metabolic significance, VII, 399
IX, 357-358 regulation of synthesis, VII, 401
Tartrate epoxidase, VII, 201 catalytic properties
catalytic properties equilibrium, VII, 397
factors influencing, VII, 205 8-ketoacyl groups and, VII, 398-399
kinetics, VII, 204-205 thiol groups and, VII, 397
measurement of reaction, VII, inhibitors, VII, 401-402
203-204 historical background, VII, 391-392
specificity and products, VII, 204 isolation and stability, VII, 393-394
mechanism, VII, 402-404 homology and tertiary structure, 111,

molecular properties, VII, 394-395 291-292
occurrence, VII, 392-393 isolation and purity, 111, 283-284
subunit structure and reversible dis- occurrence, 111, 280-282
sociation, VII, 395-397 physical properties and molecular
Thiol groups, see also Cysteine, Sulf- weight, 111, 284-285
hydryl groups inhibitors
fumarase, V, 549-552 polypeptide, 111, 304-305
papain protein, 111, 305-306
location of, 111, 514 synthetic, 111, 302-304
modification of, 111, 515-516 sequence homology, 111, 290
phosphofructokinase, VIII, 269-272 N- and C-terminal 'amino acids, 111,
Thiolsubtilisin, displacement reactions, I, 287
116117 Thymidine 3',5'-diphosphate
Thioredoxin reductase binding, staphylococcal nuclease and,
general properties, XIII, 144-145 IV, 163-171
light-activated reduction-neutral Thymidine diphosphate-L-rhamnose
scmiquinone, XIII, 147-148 synthetase, properties, VI, 375-376
metabolic functions, XIII, 142-144 Thymidylate synthetase, properties,
reduced states, mechanism, XIII, IX, 210-215
145-147 Thymine 7-hydroxylase catalytic proper-
specificity of, XIII, 144 ties, XII, 174-176
Threonine residues Tosyl elastase
chemical modification, I, 173 tertiary structure
ribonuclease, IV, 696 comparison with hypothetical model,
Threonine, serine dehydratase(s) 111, 364-365
inhibition by serine, VII, 47 electron density map, 111, 357-358
mammalian liver, VII, 39-42 model of, 111, 358-363
microbial, VII, 4 2 4 5 Tosyllysine chloromethyl ketone, results
other sources, VII, 4647 obtained, I, 112-113
Threonine synthetase, properties, VII, Tosylphenylalanine chloromethyl ketone
59-60 chymotrypsin and, I, 94-96
Thrombin results obtained, I, 112-113
amino acid and carbohydrate com- Transaldolase
position, 111, 285-286 distribution and purification, VII,
assay of, 111, 282-283 262-265
catalytic properties enzymic properties, VII, 265-268
general, 111, 292-293 function of, VII, 259-260
inhibitors, 111, 302306 historical background, VII, 261-262
mechanism, 111, 306-307 mechanism of action, VII, 271-277
protein and polypeptide substrates, metabolic role, VII, 277-280
111, 295-300 molecular properties, VII, 269-271
purported substrates, 111, 300-302 Schiff bases and, 11, 359-360
synthetic substrates, 111, 293-295 Transamination, see Amino group
A and B chain amino acid sequences, transfer
111, 287-290 Transcarboxylase
disulfide bridges, 111, 290-291 catalytic properties
general, 111, 278 assay and general properties, VI,
historical background, 111, 278-280 ioaiog
equilibria and free energy, VI, tumor tissue, IX, 183-184

109-111 virus infection, IX. 182-183
kinetics and reaction mechanism, VI, 2,4,6Trinitrobensene sulfonate, inor-
111-1 15 ganic pyrophosphatase and, IV,
role of cobalt and zinc, VI, 111 515-516
electron microscopy, VI, 101-108 Triosephosphate isomerase
historical background, VI, 84-89 catalytic properties
molecular weight, metal content, and function in vivo, VI, 337-338
biotin content and linkage, VI, kinetic parameters, VI, 333335
92-95 mechanism, VI, 338-340
purification, VI, 91-92 phosphoglycolate and, VI, 335-336
role in propionic acid fermentation, substrate active states, VI, 336-337
VI, 89-91 history and general, VI, 326
subunits modification of, I, 138
dissociation, VI, 95-99 molecular properties, VI, 326-327
reconstitution, VI, 99-101 active site structure, VI, 330333
Transfer ribonucleic acid isoenzymes, VI, 329-330
aminoacylation molecular weight, VI, 327
assays, X, 509 subunit structure, VI, 327-329
reaction product; X, 508-509 Trypsin
enzyme complexes, formation and deactivation, 111, 244-245
tection, X, 521-522 active-site-directed reagents and, I,
“recogniiion” problem, X, 522-523 103-112
chemical modification and, X, chemical modification
524-525
activation and, 111, 271-272
conclusions, X, 528 amino groups and imidazole rings,
heterologous aminoacylation, X, 111, 269-270
525-526 disulfide bridges, 111, 271
tyrosine and tryptophan residues,
isoacceptor ribonucleic acid sequenc-
ing, X, 523-524 111, 270-271
water-insoluble derivatives, 111,
method of “dissected molecules,” X,
272-273
525
chemical structure, 111, 255-260
mutant ribonucleic acid analysis, X,
crystalline, heterogeneity of, 111, 254
524
fragment, inhibitor complex with, 111,
topology of synthetase complexes,
454
X, 526-528
inhibitors
structure, X, 518-521
low molecular weight, 111, 273-274
Transfer ribonucleic acid methyltrans- naturally occurring polypeptides, 111,
ferase 274-275
biological significance, IX, 184-186 inorganic pyrophosphatase and, IV,
occurrence, IX, 168-169 514
properties mechanism and active site, 111, 260-261
ionic stimulation, IX, 174-175 catalytic site, 111, 261-262
substrate specificity, IX, 172-174 specificity and binding sites, llI,
purification, IX, 169-172 262-263
regulation physicochemical properties and sta-
bacteriophage infection, IX, 182 bility, 111, 254-255
hormonal, IX, 179-182 ribonuclease and, IV, 673-674
inhibition, IX, 176-179 substrates and specificity
assays, 111, 267-269 Tyrosine 3-monooxygenase, properties,

modified substrates, 111, 267 XII, 290-291
role of side chain, 111, 263-265 Tyrosine residues,
role of substrate structure, 111, 266 chemical modification, I, 174
substitution of amino and carboxyl creatine kinase, VIII, 434-436
groups, 111, 265-266 fructose-l,6diphosphatase, IV, 619-620
Trypsin inhibitor ribonuclease, IV, 684-685
pancreatic, chemical modification of, subtilisin, modification of, 111, 599-602
111, 443-444 trypsin, 111, 270-271
Trypsinogen
activation of, 111, 261-254 U
preparation of, 111, 251
Tryptophanase, properties, VII, 4849
Ultraviolet absorption
L-Tryptophan 2,3-dioxygenase
peptide groups, 11, 379-380
catalytic properties, XII, 129-130
ribonuclease, IV, 714-717
historical, XII, 127-128
Ultraviolet difference spectroscopy
molecular properties, XII, 128-129
protein structure, 11, 408409
Tryptophan hydroxylase, properties,
solvent perturbation and, 11, 410-413
XII, 240-241
typical cases, 11, 413417
Tryptophan 5-monooxygenase, proper-
Urea
ties, XII, 291-292
a-galactosidase and, VII, 635
Tryptophan residues,
pyruvate carboxylase and, VI, 20-21
chemical modification, I, 173
Urease(s)
trypsin, 111, 270-271
Tryptophan synthetase
catalytic properties active site studies, IV, 20-21
kinetics, IV, 18-20
individual reactions, VII, 27-30
mechanism, IV, 15-16
reaction mechanism, VII, 24-26
substrate specificity, IV, 16-18
reactions catalyzed, VII, 22-24
historical background, VII, 1-4 jack bean
molecular properties enzymic activity measurement, IV,
a-p2 affinity and equilibrium, VII, 21 4-5
a-chain purification and properties, isolation and purification, IV, 2-4
VII, 8-12 molecular properties, IV, 5-8
p-component purification and prop- chemical composition and behavior,
erties, VII, 16-20 IV, 11-12
mutant a chain, VII, 13-16 derivatives, IV, 12-13
mutant p subunits, VII, 20-21 immunological behavior, IV, 13
mutations and, I, 256-257 molecular weight, IV, 8-10
occurrence, VII, 4-5 other, IV, 10-11
other organisms and, VII, 21-22, 30 other sources, IV, 13-15
tryptophanase and, VII, 6-8, 81 Uridine-cytidine kinase
use in studies on protein synthesis assay, IX, 57-58
and regulation, VII, 5-6 distribution and purification, IX, 56-
Tumor viruses, deoxyribonucleic acid 57
polymerases of, X , 211-235, see ako kinetic and molecular properties, IX,
Reverse transcriptase 59-60
p-Tyrosinase, properties, VII, 49-51 reaction mechanism, IX, 60-61
Tyrosine hydroxylase, properties, XII, regulatory properties, IX, 61-62
238-240 substrate specificity, IX, 58-59
Uridine diphosphate-N-acetyl-n-glucos- nature of bases, IV, 320-321

amine 2'-epimerase, properties, VI, nature of sugar, IV, 320
371-372 Vibrio cholerae, neuraminidase of, V,
Uridine diphosphate-&glucose 4'-epimer- 328-329
ase, VI, 357-358 Viruses
activators, VI, 359362 neuraminidases of, V, 324
bound pyridine nucleotide virion deoxyribonucleic acid polymer-
protein conformation and, VI, 368- ase
369 others, X, 214
subunit association and, VI, 365368 tumor viruses, X, 213-214
kinetics and specificity, VI, 358-359 Visual pigments(s)
mechanism of catalysis, VI, 362-366 light interaction
Uridine diphosphoryl glucose pyrophos- early intermediates, VI, 584-585
phorylase later intermediates, VI, 585-586
analytical and synthetic applications, overall reaction, VI, 583-584
VIII, 54-55 molecular properties
measurement of activity, VIII, 52-53 criteria of purity, VI, 576
metabolic function linkage between retinal and protein,
cytology, VIII, 55-56 VI, 580-582
metabolism, VIII, 5559 lipids, VI, 579-580
regulation, VIII, 59-62 preparation, VI, 575-576
properties protein, VI, 576-577
kinetics, VIII, 65-68 retinal chromophore, VI, 577-579
mechanism, VIII, 69-71 structure and color, VI, 582-583
optima, VIII, 62 nature of, VI, 573-574
specificity, VIII, 68-69 regeneration following illumination,
structure, VIII, 6 2 6 5 VI, 587489
purification, VIII, 53-54 sites in photoreceptor, VI, 575
Uridine monophosphate kinase, proper- Vitamin B,,
ties, IX, 90-91 mechanisms, metal complexes and, 11,
528-529
V Vitamin BIZcoenzyme
Velocity curves, enzyme regulation and, amino group migrations and, VI, 539-
I, 368-369 540
Venom mutases and, VI, 509-511
enzymes hydrolyzing phosphate Vitamin BIZcoenzyme-requiring dehy-
esters, IV, 328 drases
5'-nucleotidase of, IV, 342 apoenzyme properties, V, 496-497
Venom exonuclease coenzyme analogs and, V, 493-496
chemical nature, IV, 317-319 enzyme-coenzyme interaction, V, 492-
general, IV, 313-317 493
structural determination general considerations, V, 481-482
identification of (I and u terminals, nature of hydrogen transfer, V, 485-
IV, 326-328 492
ribooligonucleotide sequences, IV, substrate to product interconversion,
324-326 V, 482-485
substrate structural characteristics Vitamin BIZmethyltransferase
conformation, IV, 319-320 alkylation studies and light stability,
monophosphoryl group and, IV, 322- IX, 137-143
324 assay, Ix, 122-123
catalytic properties activation refolding, 111: 182-183

methyl transfers catalyzed, IX, 129- arginine 145,111, 176179
135 catalytic site, 111, 179-182
propyl iodide inhibition, IX, 127-129 isoleucine 16, 111, 175-176
radioactive folate binding, IX, 1 3 6 methionine 192, 111, 179
137 clastase, 111, 353-356
mechanism, IX, 151-154 glossary of symbols, I, 89
occurrence, IX, 162-164 molecular symmetry determination
physical properties and, I, 15-18
absorption spectrum, IX, 124-125 molecular weight determination and,
resolution-reconstitution and molec- I, 13-15
ular weight, IX, 125-127 power and limitations, I, 3-5
purification, IX, 123-124 sub tilisin
role of S-adenosyl methionine, IX, background, 111, 547-552
143-151 catalytic site, 111, 553-560
Vitamin B,. adenosyltransferase comparison with subtilisin Carlsberg,
catalytic properties 111, 560
activators and inhibitors, VIII, 151- general description, 111, 552-553
152 X-ray diffraction
assay, VIII, 148-149 globular macromolecules
kinetics and substrate specificity, heavy atom derivatives, I, 69-86
VIII, 150-151 molecules studied, I, 52-69
reversibility, partial reactions and lysozyme
mechanistic considerations, VIII, analysis of structure, VII, 682-692
149-150 conformation of egg-white model,
net reaction, VIII, 145-147 VII, 692-707
purification and physical properties, crystallography of inhibitor com-
VIII, 147-148 plexes, VII, 707-717
significance and distribution, VIII, origin of, I, 23-25
144-145 o-Xylonate dehydrase, properties, V, 582
D-Xylose isomerase
W properties, VI, 349-354
role of metals, 11, 511
Wheat
acetyl coenzyme A carboxylase of, VI, Y
78-79
3’-nucleotidase of, IV, 353-354 Yeast
adenylosuccinase of, VII, 185-191
X alcohol dehydrogenase of, XI, 2223,
171-186
Xanthine oxidase, see Milk xanthine oxi- aldolase of, 11, 515-516, VII, 258
dase enolase
metal complexes and, 11, 533-534 carboxymethylation, V, 533
properties of, XII, 56 photooxidation, V, 533-534
Xanthinuria, human, molybdenum hy- glycogen synthetase of, IX, 359-361
droxylase genetics and, XII, 400402 hex0kinases
X-ray crystallography chemical studies, IX, 10-13
carboxypeptidase A, 111, 1 7 4 6 mechanism, IX, 13-28
chemical modification and, I, 201-202 modification by added proteases, IX,
chymotrypsinogen, 111, 169-175 6-7
molecular weight and subunit struc- methyltransferase of, IX, 161

ture, IX, 7-10 mitochondria1 adenosine triphosphatase
regulation, IX, 29-31 properties, X, 386
two isozymes and endogenous pro- purification, X, 386-387
teases, IX, 2-6 nicotinamide adenine dinucleotide de-
p-hydrory-p-methylglutaryl coenzyme hydrogenase of, XIII, 216221
A synthase, VII, 429-431 nuclear ribonucleic acid polymerase,
inorganic! pyrophosphatase X, 301-310
catalytic properties, IV, 534-539 5'-nucleotidase of, IV, 341442
molecular properties, IV, 530-539 proteases
invertase, V, 292-293
acid, 111, 723-744
biosynthesis, V, 294-295
diisopropylfluorophosphate-sensitive,
catalytic properties, V, 300303
localization and multiple forms, V,
111, 744-765
293-294 metal-chelator sensitive, 111, 765-786
properties, V, 298-300 other, 111, 786-795
purification, V, 295-298
isoamylase of, V, 206-208 2
mannose-6-phosphate isomerase of, VI,
304-305 Zinc, carbonic anhydrase and, V, 641-642
A 6
8 7
C 0
D 9
E O
F 1
G 2
H 3
1 4
1 5

Oxidation-Reduction. Part C - Dehydrogenases (II), Oxidases (II), Hydrogen Peroxide Cleavage. Third Edition (PDFDrive)

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Oxidation-Reduction. Part C - Dehydrogenases (II), Oxidases (II), Hydrogen Peroxide Cleavage. Third Edition (PDFDrive)

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The Enzymes

WINSLOW S. CAUGHEY GREGORY R. SCHONBAUM

BRITTON CHANCE BO MALMSTROM

ACADEMIC PRESS New York San Francisco London 1976

ACADEMIC PRESS, INC.

United Kingdom Edition published by

Library of Congress Cataloging in Publication Data

Main entry under title:

Includes bibliographical references.

PRINTED IN THE UNITED STATES OF AMERICA

WINSLOW S. CAUGHEY (299), Department of Biochemistry, Colorado

CHARLES H. WILLIAMS, JR. (89), Veterans Administration Hospital,

X-Ray Crystallography and Enzyme Structure

Chemical Modification by Active-Site-Directed Reagents

Chemical Modification as a Probe of Structure and Function

Genetic Probes of Enzyme Structure

The Molecular Basis for Enzyme Regulation

Mechanisms of Enzyme Regulation in Metabolism

Volume II: Kinetics and Mechanism

Steady State Kinetics

Author Index-Subj ect Index

Volume 111: Hydrolysis: Peptide Bonds

Chymotrypsinogen : X-Ray Structure

The Structure of Chymotrypsin

Protein Proteinase Inhibitors-Molecular Aspects

Other Bacterial, Mold, and Yeast Proteases

Author Index-Subject Index

Volume IV: Hydrolysis: Other C N Bonds, Phosphate Esters

Author Index-Subj ect Index

Volume V: Hydrolysis (Sulfate Esters, Carboxyl Esters, Glycosides) ,

The Hydrolysis of Sulfate Esters

Carboxylic Ester Hydrolases

Dehydrations Requiring Vitamin B,, Coenzyme

Author IndexSubject Index

Volume VI: Carboxylation and Decarboxylation ( Nonoxidative),

Author Index-Subject Index

Volume VII: Elimination and Addition, Aldol Cleavage and condensation,

Argininosuccinases and Adenylosuccinases

Author Index-Subject Index

Volume VIII: Group Transfer, Part A: Nucleotidyl Transfer, Nucleoridyl

Adenylyl Transfer Reactions

Author Index-Subject Index

Volume IX: Group Transfer, Part B: Phosphoryl Transfer, One-Carbon Group

Volume X: Protein Synthesis, DNA Synthesis and Repair,

Polypeptide Chain Initiation

DNA Joining Enzymes (Ligases)

The Glutamine Synthetase of Escherichia coli:

Author Index-Subject Index

Volume XI: Oxidation-Reduction, Part A: Dehydrogenases (II , Electron

Kinetics and Mechanism of Nicotinamide-Nucleotide-Linked

Author Index-Subject Index

Volume XII: Oxidatiorr-Reduction, Part B: Electron Transfer ( I l l ,

Flavodoxins and Electron-Transferring Flavoproteins

Author Index-Subj ect Index

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) catalyzes re-

acid which represents an important pathway of carbohydrate metabolism

la. 0. Meyerhof, Naturwissenschaften 25, 443 (1937).

II. Molecular Properties