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Iron-Sulfur Clusters
in Chemistry and
Biology
Characterization, Properties, and Applications
Edited by
Tracey Rouault
Volume 1
DE GRUYTER
Editor
Tracey Rouault M.D.
Eunice Kennedy Shriver National Institute of Child Health and Human Development
National Institutes of Health
traceyrouault@icloud.com
Bethesda, MD. 20892
ISBN 978-3-11-047850-1
e-ISBN (E-BOOK) 978-3-11-048043-6
e-ISBN (EPUB) 978-3-11-047855-6
Library of Congress Cataloging-in-Publication Data

A CIP catalog record for this book has been applied for at the Library of Congress.
Bibliographic information published by the Deutsche Nationalbibliothek

The Deutsche Nationalbibliothek lists this publication in the Deutsche Nationalbibliografie;
detailed bibliographic data are available on the Internet at http://dnb.dnb.de.
© 2017 Walter de Gruyter GmbH, Berlin/Boston
Cover image: Rouault, Maio, 2017

Typesetting: Compuscript Ltd. Shannon, Ireland
Printing and binding: CPI books GmbH, Leck
∞ Printed on acid-free paper
Printed in Germany
www.degruyter.com
Preface
Iron-sulfur (Fe-S) clusters are versatile prosthetic groups that enable their associated
proteins to perform numerous functions, ranging from electron transport to substrate
ligation, structural support and DNA repair. Fe-S proteins did not become a focus of
research until the late 1950’s, when spectroscopy techniques evolved sufficiently to
identify features that were specific for Fe-S clusters. Initially identified in mamma-
lian succinate dehydrogenase, Fe-S clusters were subsequently found in numerous
bacterial proteins that performed complex functions, including nitrogenase, which
transforms atmospheric nitrogen into ammonia, generating an accessible source of
nitrogen for synthesis of proteins and nucleic acids. Understanding how Fe-S clusters
and proteins work has occupied many scientists for decades, and important break-
throughs regarding the mechanisms of nitrogenase and hydrogenase have occurred
in just the last few years.
Not only is it a challenge to understand how Fe-S proteins work, but it is also
a challenge to understand how Fe-S clusters are synthesized and inserted into Fe-S
proteins in living organisms. Studies originally performed in bacterial model systems
have revealed basic mechanisms of biogenesis that are conserved in all the kingdoms
of life. Moreover, it has become apparent that flaws in the Fe-S assembly process
cause several human diseases. As a result, biomedical researchers working on the
pathophysiology of rare diseases such as Friedreich’s ataxia have begun attending
conferences at which chemists and physicists discuss Fe-S research based on complex
spectroscopic studies and computational analyses. Researchers from different ends
of the spectrum have struggled to bridge the large gap between the physics and chem-
istry of Fe-S clusters and the important biological questions associated with their
functions.
Despite a growing need for cross-disciplinary communication, there was no
single book devoted to Fe-S proteins that provided a basic and broad overview of the
subject as it evolved over the last several decades until the first edition of this book
was published in 2014. This book represents the second edition of “Iron-sulfur clus-
ters in chemistry and biology”, which was written to make the subject of Fe-S proteins
more widely accessible to students and researchers by including a short history of
Fe-S research, chapters that highlight the unique chemistry of Fe-S clusters and tech-
niques important in analysis, and reviews from leading researchers on well-known
Fe-S proteins such as nitrogenase and hydrogenase. In addition, numerous chapters
focus on Fe-S synthesis and regulation in model organisms, and in mammalian bio-
genesis, DNA metabolism and human disease. Concluding with a discussion on the
potential role of Fe-S clusters in capturing reducing power and contributing to the
origin of life on earth, the final chapter touches on questions about how metabolic
pathways initially developed. Because of the rapid growth of the field, this book is now
divided into two volumes. The first volume focuses more on fundamental chemistry
vi Preface
and important enzymatic mechanisms. The second focuses more on Fe-S proteins
in biological systems, the mechanisms by which Fe-S clusters are synthesized and
correctly targeted to recipient proteins, and important regulatory functions of Fe-S
proteins. Multiple chapters were updated to reflect rapid progress, and new chapters
were added to expand coverage of methodologies used for characterization of Fe-S
proteins, chemical principles that render Fe-S clusters unique, including their sensi-
tivity to nitric oxide, and roles in DNA signaling and repair. Other new chapters cover
the Fe-S biogenesis process in E. coli, new insights into how Fe-S recipient proteins
acquire their clusters, and expansion of the chapters on human diseases that result
from failures of Fe-S protein biogenesis and function.
I am indebted to my many outstanding and generous colleagues, who spent con-
siderable time and effort in writing the chapters in this book. I hope that this book will
be useful to those interested in the subject of Fe-S from many different perspectives,
and that researchers from related disciplines will gain a greater sense for the context
of their own work.
I want to thank Stephanie Dawson, who perceived that there was an unmet intel-
lectual need and initiated this project in 2014 while she was an editor at De Gruyter.
I also gratefully thank Julia Lauterbach, Ria Fritz, Anne Hirschelmann, and Vivien
Schubert of De Gruyter for their tireless support and guidance in turning this book
into a reality. My family and friends have graciously supported me when I needed
time to work on the project long known to them as “the book”, and I’m thankful for
their help.
Tracey Rouault, July, 2017

Tracey A. Rouault biography
Tracey A. Rouault is a leading researcher in

the area of mammalian iron-sulfur proteins,
an area she began to pursue after discovering
an important role for an iron-sulfur protein in
the regulation of mammalian iron metabo-
lism. She received a degree in Biology from
Yale College and an MD degree from Duke Uni-
versity Medical School, where she completed
her training in internal medicine. She com-
pleted a medical fellowship at the National
Institutes of Health in Bethesda, Maryland,
and has since focused on the regulation of
mammalian iron metabolism and its relati-
onship to human diseases. Her main inte-
rests include elucidating mechanisms of
mammalian iron-sulfur cluster biogenesis and exploring the pathophysiology of
diseases related to ineffective iron-sulfur cluster biogenesis, several hematologic dis-
orders, genetic cancer syndromes, and neurodegenerative diseases. Her early research
in the role of iron-sulfur proteins in regulation led to a productive collaboration with
Helmut Beinert, a researcher responsible for numerous ground-breaking advances
related to iron-sulfur proteins. She has also collaborated with Richard Holm, whose
pioneering work led to the inorganic synthesis of numerous iron-sulfur clusters and
revealed that many properties of iron-sulfur proteins derive from intrinsic features of
their iron-sulfur clusters. She is an active member of the rapidly growing iron-sulfur
protein research community.
Contents
Preface v
Tracey A. Rouault biography vii
List of contributing authors xvii
Francesco Bonomi and Tracey A. Rouault

1 Iron-sulfur proteins: a historical perspective 1
1.1 Framing the scene 1
1.2 The early days of “nonheme iron” 1
1.3 Of proteins and analogues 2
1.4 Beyond electron shuttles 6
1.5 How are FeS clusters synthesized in cells? 7
Acknowledgments 8
References 8
Toshiko Ichiye
2 Chemistry of iron-sulfur clusters 11
2.1 Introduction 11
2.2 Electronic structure of Fe-S complexes 12
2.2.1 Spin-polarization and strong metal-ligand bonds 12
2.2.2 Spin-coupling and metal-metal bonds 14
2.2.3 Spin resonance delocalization in mixed-valence iron pairs 14
2.3 Unique properties of Fe-S clusters 15
2.3.1 Stable rigid clusters mean low reorganization energy 15
2.3.2 Polynuclear clusters mean multiple valency 16
2.3.3 Resonance delocalization and [Fe4S4(Cys)4] cluster conversion 16
2.4 Summary 18
Acknowledgments 18
References 18
Louis Noodleman
3 From the quantum chemistry of iron sulfur clusters to redox energetics and
reaction pathways in metalloenzymes 21
3.1 Introduction 21
3.2 Iron sulfur cluster geometric coordination and electronic structure 22
3.3 Spin polarized DFT – fundamentals 24
3.4 Exchange correlation energies and potentials 27
3.5 Electron densities, unitary transformations, and invariants for energies
and properties 28
3.6 Spin polarization and the inverted level scheme 29
x Contents
3.7 Spin coupling and BS 31

3.8 Spin barycenter energy 34
3.9 Electron localization and delocalization 35
3.10 Electron trapping – inner and environmental effects 37
3.11 Protein and solvent interactions with the quantum cluster 38
3.11.1 Poisson-Boltzmann PC: Model 1 39
3.11.2 te, Poisson-Boltzmann self-consistent reaction field (PB-SCRF):
Model 2 39
3.12 Redox potential and pKa fundamentals 40
3.13 Rieske cluster and electron-proton coupling 41
3.14 Hyperfine coupling 42
3.15 Polynuclear systems – redox potentials and spin dependent terms 44
3.16 Iron-sulfur nitrosyl complexes 49
3.17 Iron-sulfur cluster enzymes in pathogens 58
3.17.1 Adenosine 5ʹ-phosphosulfate reductase (APSR) 58
3.17.2 Isoprenoid synthesis enzyme H 64
3.18 Concluding remarks 68
Acknowledgments 69
References 69
Yisong Guo and Jikun Li

4 Bioinorganic spectroscopy of iron sulfur proteins—an overview 77
4.1 Introduction 77
4.2 Optical techniques 79
4.2.1 Electronic absorption spectroscopy 79
4.2.2 CD Spectroscopy 81
4.2.3 Raman and IR spectroscopic techniques 85
4.3 Magnetic techniques 95
4.3.1 Electron paramagnetic resonance 95
4.3.2 Pulsed EPR spectroscopy 108
4.3.3 Mössbauer spectroscopy 121
4.4 Concluding remarks 129
Acknowledgments 130
References 130
Doros T. Petasis and Michael P. Hendrich

5 Quantitative interpretation of EPR spectroscopy with applications for
iron-sulfur proteins 135
5.1 Introduction 135
5.2 Basic EPR theory 136
5.3 g Factor anisotropy 138
Contents xi
5.4 Hyperfine structure 138

5.5 Ligand interactions 140
5.6 Spin Hamiltonian 141
5.7 Basic EPR instrumentation 142
5.8 Simulation of powder spectra 143
5.9 Quantitative aspects 145
5.10 Examples 147
5.10.1 S = 1/2 systems 147
5.10.2 Spin systems with S = 3/2 , 5/2, 7/2, etc. 151
5.10.3 Spin systems with S = 1, 2, 3, etc. 156
5.11 Conclusion 160
References 160
Mrinmoy Chakrabarti and Paul A. Lindahl

6 The utility of Mössbauer spectroscopy in eukaryotic cell biology and
animal physiology 163
6.2 Transitions associated with MBS 163
6.3 Coordination chemistry of iron 165
6.4 Electron spin angular momentum and EPR spectroscopy 167
6.5 High-spin vs low-spin FeII and FeIII complexes 167
6.6 Isomer shift (δ) and quadrupole splitting (ΔEQ) 167
6.7 Effects of a magnetic field 168
6.8 Slow vs fast relaxation limit 169
6.9 MB properties of individual Fe centers found in biological
systems 170
6.10 Magnetically interacting Fe aggregates 172
6.11 Insensitivity of MBS and a requirement for 57Fe enrichment 173
6.12 Invariance of spectral intensity among Fe centers 174
6.12.1 Mitochondria 174
6.12.2 Vacuoles 177
6.12.3 Whole yeast cells 178
6.12.4 Human mitochondria and cells 179
6.12.5 Blood 179
6.12.6 Heart 181
6.12.7 Liver 181
6.12.8 Spleen 182
6.12.9 Brain 182
6.13 Limitations of MBS and future directions 184
Acknowledgments 185
References 186
xii Contents
Nathaniel S. Sickerman, Markus Ribbe, and Yilin Hu

7 The interstitial carbide of the nitrogenase M-cluster: insertion pathway
and possible function 191
7.2 Proposed role of NifB in carbide insertion 192
7.3 Accumulation of a cluster intermediate on NifB 194
7.4 Investigation of the insertion of carbide into the M-cluster 196
7.5 Refining methyltransfer and hydrogen atom abstraction steps in
NifB 199
7.6 Tracing the fate of carbide during substrate turnover 201
References 202
Thomas Spatzal, Susana L. A. Andrade and Oliver Einsle

8 The iron-molybdenum cofactor of nitrogenase 205
8.2 The metal clusters of nitrogenase 206
8.3 Structure of FeMoco 207
8.4 Redox properties of FeMoco 209
8.5 An overlooked detail: the central light atom 210
8.6 The nature of X 212
8.7 Insights into the electronic structure of FeMoco 216
8.8 A central carbon – consequences and perspectives 217
Acknowledgments 219
References 219
Joseph T. Jarrett
9 Biotin synthase: a role for iron-sulfur clusters in the radical-mediated
generation of carbon-sulfur bonds 223
9.2 Sulfur atoms in biomolecules 224
9.3 Biotin chemistry and biosynthesis 225
9.4 The biotin synthase reaction 227
9.5 The structure of biotin synthase and the radical
SAM superfamily 229
9.6 The [4Fe-4S] cluster and the radical SAM superfamily
2+ 233
9.7 The [2Fe-2S]2+ cluster and the sulfur insertion reaction 236
9.8 Characterization of an intermediate containing 9-MDTB
and a [2Fe-2S]+ cluster 237
9.9 Other important aspects of the biotin synthase reaction 238
9.10 A role for iron-sulfur cluster assembly in the biotin synthase
reaction 240
Contents xiii
9.11 Possible mechanistic similarities with other sulfur insertion radical SAM
enzymes 241
Acknowledgments 243
References 243
Russ Hille
10 Molybdenum-containing iron-sulfur enzymes 249
10.2 The xanthine oxidase family 250
10.2.1 D. gigas aldehyde:ferredoxin oxidoreductase 251
10.2.2 Bovine xanthine oxidoreductase 253
10.2.3 Aldehyde oxidases 261
10.2.4 CO dehydrogenase 264
10.2.5 4-Hydroxybenzoyl-CoA reductase 268
10.3 The DMSO reductase family 269
10.3.1 DMSO reductase and DMS dehydrogenase 271
10.3.2 Polysulfide reductase 281
10.3.3 Ethylbenzene dehydrogenase 285
10.3.4 Formate dehydrogenases 286
10.3.5 Bacterial nitrate reductases 296
10.3.6 Arsenite oxidase and arsenate reductase 304
10.3.7 Pyrogallol:phloroglucinol transhydroxylase 308
10.4 Prospectus 310
References 311
Nicholas D. Lanz and Squire J. Booker

11 The role of iron-sulfur clusters in the biosynthesis of the lipoyl
cofactor 327
11.2 Discovery of LA 327
11.3 Functions of the lipoyl cofactor 328
11.3.1 LA in primary metabolism 328
11.3.2 LA as an antioxidant 330
11.4 Pathways for lipoyl cofactor biosynthesis 331
11.4.1 The exogenous pathway 331
11.4.2 The endogenous pathway 332
11.5 Characterization of LipA 333
11.5.1 The discovery of LipA 333
11.5.2 In vivo characterization of LipA 333
11.5.3 LipA is an iron-sulfur enzyme 335
11.5.4 LipA is an RS enzyme 336
xiv Contents
11.5.5 Product inhibition of LipA 340

11.5.6 LipA contains two [4Fe-4S] clusters 341
11.5.7 Two distinct roles for the two iron-sulfur clusters in LipA 343
11.5.8 A unique intermediate in LipA catalysis 344
11.5.9 Evidence for substrate-based radicals during LipA catalysis 346
11.5.10 Structural insights into LipA catalysis 347
11.5.11 A proposed mechanism for the biosynthesis of the lipoyl cofactor 350
11.6 Conclusions 350
Acknowledgments 351
References 351
Yvain Nicolet and Juan C. Fontecilla-Camps

12 Iron-sulfur clusters and molecular oxygen: function, adaptation,
degradation, and repair 359
12.2 Fe-S clusters – reasons for their abundance 360
12.2.1 Origin of Fe-S clusters 360
12.2.2 Functions of Fe-S clusters 361
12.3 Oxygen and Fe-S clusters 363
12.3.1 Properties of molecular oxygen and its partially reduced species 363
12.3.2 Oxidative damage to Fe-S clusters 365
12.3.3 Molecular mechanisms of oxidative damage to Fe4S4 clusters 366
12.3.4 Fe3S4 to Fe2S2 cluster conversion in FNR 367
12.3.5 X-ray crystallographic studies 367
12.3.6 Alternative reactions can occur and compete 369
12.3.7 Structural changes 370
12.4 Adaptation to oxygen 370
12.4.1 Switch between metabolisms or restriction to niches 372
12.4.2 O2-tolerant NiFe hydrogenases 373
12.4.3 Protective systems against ROS 376
12.4.4 Evolutionary replacement of Fe-S clusters to keep essential functions
in aerobic organisms 377
12.5 Conclusions 378
References 379
Erin L. Dodd, Jason C. Crack, Andrew J. Thomson, and Nick E. Le Brun

13 Reactivity of iron-sulfur clusters with nitric oxide 387
13.1.1 Structure and chemistry of iron-sulfur (Fe-S) clusters that function in
biological sensing 387
13.1.2 Chemistry and biology of NO 389
Contents xv
13.1.3 The biology of NO sensing via Fe-S cluster proteins 391

13.2 Biophysical techniques useful for studying Fe-S cluster reactions
with NO 392
13.2.1 EPR spectroscopy 393
13.2.2 Mössbauer spectroscopy 394
13.2.3 Infrared spectroscopy 394
13.2.4 RR spectroscopy 395
13.2.5 Nuclear resonance vibrational spectroscopy 395
13.2.6 Stopped-flow absorbance spectroscopy 396
13.2.7 Electrospray ionization mass spectrometry 397
13.3 Metal nitrosyls 397
13.3.1 The metal-NO bond 397
13.3.2 Iron-nitrosyl complexes 399
13.3.3 Formation and interchange of Fe-S cluster nitrosyl species 401
13.3.4 Nitrosylated species can be converted back to [2Fe-2S] and [4Fe-4S] Fe-S
clusters 403
13.3.5 Effect of cluster protein environment on nitrosylation 403
13.4 Physiologically relevant reactions of NO with Fe-S cluster proteins 404
13.4.1 “Secondary” vs. “dedicated” biological NO sensors 404
13.4.2 NsrR 405
13.4.3 FNR regulator 412
13.4.4 FnrP 415
13.4.5 Wbl family proteins 415
13.4.6 SoxRS 419
13.4.7 IRP1 and other aconitases 422
13.4.8 HcpR2 and the Hcp 424
13.5 Perspectives and future directions 425
Acknowledgments 425
References 426
List of contributing authors
Susana L. A. Andrade Oliver Einsle

Institut für organische Chemie und Biochemie Institut für organische Chemie und Biochemie
Universität Freiburg Universität Freiburg
Freiburg, Germany Freiburg, Germany
chapter 8 e-mail: einsle@bio.chemie.uni-freiburg.de
chapter 8
Francesco Bonomi
DeFENS Juan Fontecilla-Camps
University of Milan Institute de Biologie Structurale J.P. Ebel
Milan, Italy Grenoble, France
e-mail: francesco.bonomi@unimi.it e-mail: juan.fontecilla@ibs.fr
chapter 1 chapter 12
Squire Booker Yisong Guo

Department of Chemistry Department of Chemistry
Pennsylvania State University Carnegie Mellon University
University Park, PA, USA Pittsburgh, PA, USA
e-mail: sjb14@psu.edu e-mail: ysguo@andrew.cmu.edu
Mrinmoy Chakrabarti Michael Hendrich

Department of Chemistry Department of Chemistry
Texas A & M University Carnegie Mellon University
College Station, TX, USA Pittsburgh, PA, USA
e-mail: mrinmoy.chak@chem.tamu.edu e-mail: hendrich@cmu.edu
chapter 6 chapter 5
Jason C. Crack Russ Hille

Centre for Molecular and Structural Department of Biochemistry
Biochemistry University of California
University of East Anglia Riverside, CA, USA
Norwich, England e-mail: russ.hille@ucr.edu
Erin L. Dodd Yilin Hu

Centre for Molecular and Structural Molecular Biology & Biochemistry
Biochemistry University of California, Irvine
University of East Anglia Irvine, CA, USA
Norwich, England e-mail: yilinh@uci.edu
xviii List of contributing authors
Toshiko Ichiye Louis Noodleman

Department of Chemistry Department of Integrative Structural and
Georgetown University Computational Biology
Washington, DC, USA The Scripps Research Institute
e-mail: ti9@georgetown.edu La Jolla, CA, USA
chapter 2 e-mail: lou@scripps.edu
chapter 3
Joe Jarrett
Department of Chemistry Doros T. Petasis
University of Hawai’i at Manoa Department of Physics
Honolulu, HI, USA Allegheny College
e-mail: jtj@hawaii.edu Meadville, PA, USA
chapter 9 e-mail: dpetasis@allegheny.edu
chapter 5
Nicholas D. Lanz
Departments of Biochemistry and Molecular Markus Ribbe
Biology and Chemistry Molecular Biology & Biochemistry
The Pennsylvania State University University of California, Irvine
University Park, PA, USA Irvine, CA, USA
e-mail: ndl111@psu.edu e-mail: mribbe@uci.edu
Nick E. Le Brun Nathaniel S. Sickerman

Centre for Molecular and Structural Department of Molecular Biology and
Biochemistry Biochemistry
University of East Anglia University of California
Norwich, England Irvine, CA, USA
e-mail: n.le-brun@uea.ac.uk chapter 7
chapter 13
Thomas Spatzal
Jikun Li Institut für organische Chemie und Biochemie
Department of Chemistry Universität Freiburg
Carnegie Mellon University Freiburg, Germany
Pittsburgh, PA, USA chapter 8
chapter 4
Andrew J. Thomson
Paul Lindahl Centre for Molecular and Structural
Department of Chemistry Biochemistry
Texas A & M University University of East Anglia
College Station, TX, USA Norwich, England
e-mail: lindahl@chem.tamu.edu chapter 13
chapter 6
Yvain Nicolet
Institute de Biologie Structurale J.P. Ebel
Grenoble, France
e-mail: yvain.nicolet@ibs.fr
chapter 12
1 Iron-sulfur proteins: a historical perspective
Francesco Bonomi and Tracey A. Rouault
1.1 Framing the scene
Although iron-sulfur proteins (Fe-S) are now recognized as being pervasive throughout
all three kingdoms of life, they were not among the prosthetic groups that were recog-
nized or studied during the first half of the twentieth century [1]. One reason for their
relatively late appearance on the research scene was that they often lacked a distinc-
tive visible color that commanded attention, unlike proteins that incorporate a heme
cofactor or other metallic cofactors. Furthermore, Fe-S centers are often destabilized
by exposure to oxygen, and working with Fe-S proteins requires special techniques for
measuring iron and sulfur and equipment, such as anaerobic hoods, electron para-
magnetic resonance (EPR) machinery and Mössbauer spectroscopy, in addition to the
more commonly used ultraviolet and visible spectrophometric methods. Upon consi-
deration of the importance of new instrumentation and techniques for the discovery
and characterization of Fe-S centers, Helmut Beinert [2] concluded in a retrospective
about Fe-S research that, “there was scarcely a way that these discoveries could have
been made earlier.”
1.2 The early days of “nonheme iron”
In 1951, researchers observed that a dark brown fraction from ammonium sulfate frac-
tionation of leaf extracts was able to catalyze reduction of met-hemoglobin [3]. This
report likely represents one of the earliest mentions of Fe-S activity, but no further
insight into the nature of these reducing proteins was reported. In the ensuing years,
from 1956 to 1958, tightly bound nonheme iron was reproducibly detected in animal
tissues, particularly in lysates from their mitochondria [4].
A burst of knowledge was unleashed by the use of EPR imaging techniques,
which were developed to assess materials that contained unpaired electrons during
the 1940s and 1950s and became commercially available in 1956 [2]. EPR signals ema-
nating from this nonheme iron were first detected in succinate dehydrogenase (SDH)
[5, 6]. The development of sensitive microassays for iron and sulfide contents indica-
ted that SDH also contained labile sulfide [7, 8]. Mitochondria and chloroplasts were
the subject of intensive investigations by many gifted scientists, providing an ideal
“testing ground” for the application of these novel techniques. This combination led
to the first EPR spectra of FeS components in the respiratory chain and to a first coarse
outline of the essential participation of these redox carriers to the electron flow within
the system (Fig. 1.1) [9, 10].
DOI 10.1515/9783110480436-001
2 1 Iron-sulfur proteins: a historical perspective
Fig. 1.1: The first spectra of Fe-S proteins obtained by EPR and published in 1960 and 1961.
(Modified from Beinert H, Sands RH, Biochem Biophys Res Commun, 3, 41–46, 1960, and Beinert H,
Lee W, Biochem Biophys Res Commun, 5, 40–45, 1961.)
Shortly after the pioneering work on mitochondrial nonheme iron proteins was
performed, some of the most stable and abundant FeS proteins, namely clostridial
(4Fe-4S) ferredoxins, were isolated and named by a group led by Len Mortenson, then
at the DuPont Co. [11]. Len Mortenson was later instrumental in building the Chemistry
Department at the University of Georgia (USA), laying the groundwork for the develop-
ment of the Center for Metalloenzyme Studies, which grew during the ensuing years.
Multiple other types of FeS proteins were discovered, including proteins from the
anaerobic photosynthetic purple sulfur bacterium Chromatium vinosum in which a
nonstandard redox form of a [4Fe-4S] cluster was identified [12]. Others were found in
which two histidine residues replaced half of the standard cysteines as iron ligands [13]
and others in which a single iron atom was tetrahedrally coordinated by four cystei-
nes in the absence of additional “inorganic sulfide,” known as rubredoxin [14]. The
relevance of all these contributions (and of many more that cannot be mentioned here
for lack of space) found expression in the milestone book Non Heme Iron Proteins,
which was edited by Anthony San Pietro and appeared in 1965.
1.3 Of proteins and analogues
No computers were involved when a structural model for a [2Fe-2S] cluster was propo-
sed as early as 1966 based on the interpretation of the g = 1.94 EPR signal of plant-type
ferredoxin [15]. Isotopic substitution and analysis of the hyperfine splitting pattern
in EPR spectra later confirmed that the proposed structure was correct and that the
two sulfur (or selenium) atoms were indeed indistinguishable [16]. In this regard,
Fig. 1.2: A picture of Helmut Beinert at work (left) with two colleagues. (Courtesy of the University
of Wisconsin and Dr. Elizabeth Craig.)
it is again worth remembering that the FeS proteins field has represented a very signi-
ficant environment in which methodologies that work at the interface among physics,
chemistry, and biochemistry have been deployed (Fig. 1.2). These methodologies cover
the whole gamut of the electromagnetic spectrum, from microwaves to X-rays and beyond.
Confirming these hypothetical structures by X-ray crystallography required
several years. Crystals of clostridial-type 2[4Fe-4S] ferredoxins had been obtained
as early as 1966 [17], but it was not until the 1970s that crystallographic structures
became available. The Lovenberg group presented a structure of rubredoxin [18], and
this was quickly followed by reports on the structure of HiPIP [19] and of clostridial-
type ferredoxin [20]. The structure of a plant-type ferredoxin [21] was solved and later
supported by data from nuclear magnetic resonance (NMR) spectroscopy [22]. Appli-
cations of NMR to this particular field have been important because of the intrinsic
difficulties associated with characterizing paramagnetic centers [23].
After those pioneering efforts, numerous structures were solved at high resolu-
tion. The complexity of the investigated systems also grew progressively from the
1960s to the present. Studies progressed from single-iron rubredoxins and two-iron
ferredoxins to incredibly complex flavo-molybdo-iron proteins, often made up of
several subunits, and included cases where metal clusters shared ligands from sepa-
rate polypeptides or included non-amino acid ligands. The first proposed structure
of nitrogenase [24] was an exciting milestone, and the intricacy of the chemistry
and structural biochemistry of these complicated systems is still a subject of intense
research interest.
In the 1970s, chemists were able to synthesize and characterize a number of
structural analogues of FeS clusters at the atomic level [25]. The relative stability of
these clusters as a function of their nuclearity and of the nature, size, and reactivity
of terminal ligands was investigated by the Holm (Fig. 1.3) group and by many others
(Fig. 1.3). These collective efforts led to the elucidation of the sequence of individual
reactions resulting in the self-assembly of the clusters (for a comprehensive review of
Fig. 1.3: Richard Holm, with valued colleagues, synthesized most types of FeS clusters in vitro
and thereby proved that FeS clusters could interconvert and assemble independently of protein
structure. (Courtesy of Dr. Richard Holm.)
30 years of progress, see [26]). The original work was carried out in nonaqueous systems,
but shortly afterward, it was shown that essentially the same chemistry worked in
micellar systems and aqueous buffers as well as with other metals using enzymes to
catalyze some individual steps of the overall chain of events (for example, [27]). The
ability to reconstruct a replicate of various FeS centers found in proteins proved that
the protein structure was unnecessary for the sites’ existence [1] and supported the
concept that FeS centers are modular centers that have an unusual ability to inter-
convert between species, allowing 2[2Fe-2S] clusters to readily form a single [4Fe-4S]
complex [26] (Fig. 1.4). Moreover, FeS clusters proved to be more robust and cofactor-like
than had been originally thought [1], and the chemical characteristics of sulfur were
recognized for their unique contribution to the chemistry of FeS clusters [28].
In short, roughly one decade after the San Pietro book mentioned in Section 1.2,
the knowledge in the field required had grown to the point that a two-volume book
(properly titled Iron-Sulfur Proteins and edited by Walt Lovenberg) was not sufficient
and was followed by a third volume several years later. The wealth of information
within the book was great, and a wide variety of approaches and techniques origina-
ting from chemistry, physics, and biochemistry were put to synergistic use to clarify
many puzzling issues. An important breakthrough occurred when researchers recog-
nized that particular EPR signals emanated from two interacting iron ions, a ferrous
ion and a ferric ion (reviewed in [2]), rather than from a single metal site (Fig. 1.3).
By the mid-1970s, there was enough information on the structural features of FeS
proteins and on their distribution throughout the kingdoms of life to begin to consider
when FeS proteins first appeared in life. Studies in molecular evolution led to incre-
asing awareness that FeS structures and the proteins around them likely had been
around since the earliest days of anaerobic life on this planet, and these structures may
have been merged, reshuffled, and repurposed through fusion and duplication [29].
2
RS SR
Fe
RS
SR
9 1
S RSSR 3R
S , .4 S
, 5/2
RS RS
2 * O2 SR
2 3
RS S SR RS S S SR
HS
Fe Fe [Fe(SR)4] 1 Fe Fe Fe
RS S SR RS S S SR
10a 11
* e
e * 4RS
O2 2 ,
[Fe2S2(SR)4] 3
* Fe 2
S S S
FeS
RS, RSSR Fe Fe Fe
2 RS SR
L
[Fe4S4(SR)4] 2 S Fe 12 S SR
RS
Fe S
13a
e Fe S Fe 2, * e
e e
RS S Fe
* SR Fe 2
[Fe4S4(SR)4] 3 13b [Fe3S4(SR)3] 3
,
S
S L RS * M1, 2, e
5R OH
3. 4RSSR 4S
L 1, 2, 3
Fe 3
3.5 2 Fe 3 M
RS SR R S
S S S M0, 1
R Fe R 2.5
S S Fe Fe Fe
R S RS SR
R
RS Fe S
Fe SR S SR
Fe
S S M1 Cu, Ag, Tl
R RS R
35 M2 Mn, Co, Ni, Zn, Cd
Fig. 1.4: Synthetic routes to assembly of FeS analogues. The structures most often encountered in
proteins are highlighted. FeS clusters are highly interchangeable, and the integrity of FeS clusters does
not depend on protein scaffolds. (Redrawn from Rao VP, Holm RH, Chem Rev, 104, 527–559, 2004.)
Thus, proteins might have evolved in the primordial environment around submarine
volcanic vents and incorporated FeS centers into fundamental biochemical processes.
These concepts, along with the apparent ease of self-assembly of FeS s tructures
and of their relative tolerance toward various types of ligands resulted in hypo-
theses based on the supposition that life arose in an “iron-sulfur” world. Bioche-
mistry, as we know it, was hypothesized to have taken place first on the positively
charged surface of pyrite crystals [30], and genuine FeS structures (not dissimilar
from those “captured” by protein thiolates in a later stage of evolution) could have
been responsible of providing the earliest catalysts in a nonprotein world, perhaps
in separate compartments, which might be regarded as the earliest protocells [31].
Although still much debated [32, 33], these hypotheses continue to fascinate because
they address critical questions about how various life forms may capture and store
energy from the environment.
1.4 Beyond electron shuttles
The ability of FeS centers to accept and donate single electrons had led to the focus on
their roles as electron shuttles. Helmut Beinert was once again among the first to reco-
gnize that a non-redox enzyme – namely, mammalian mitochondrial aconitase – was
an iron-sulfur protein and to understand that transition from the non-active to the
active form of the enzyme required conversion of a [3Fe-4S] into a [4Fe-4S] cluster [34].
Assessing this unequivocally took a rather unique combination of spectroscopic skills
and analytical accuracy. Helmut Beinert had both, as testified by the back-to-back
reports that appeared in 1983 [35, 36]. On a more personal note, it is worth remembe-
ring that, during the celebration of Helmut’s ninety-second birthday in Madison in
2005, the distinguished spectroscopist Eckard Munck spoke about having introduced
the concept of “millibeinerts” to score the reliability and accuracy of measurements
that were performed in his own laboratory (FB, personal recollection). The conver-
sion of a [3Fe-4S] cluster into a [4Fe-4S] cluster in mitochondrial aconitase apparently
required only the addition of iron and a reducing agent, and the reverse conversion
appeared to occur spontaneously when iron was not present. In fact, the fourth labile
iron was involved in the direct ligation of the substrate, citrate or isocitrate [37]. Thus,
a new role for FeS in ligating enzyme substrates was discovered.
In the early 1990s, yet another potential role of FeS proteins as sensors emerged
when investigators were studying the regulation of intracellular iron metabolism.
The mammalian protein responsible for regulating the translation of ferritin and
stabilizing the transcript that encodes the transferrin receptor, known as the iron-
responsive element binding protein (IRE-BP), was identified, and it unexpectedly
had a high sequence similarity to mitochondrial aconitase [38], which had been
crystallized and further characterized [39]. Mammalian cells had been known to
possess a second aconitase, which was in the cytosol, and purification of the aco-
nitase activity and peptide sequencing revealed that the IRE-BP, which was an apo-
protein [40], and cytosolic aconitase with its [4Fe-4S] cluster were identical proteins
[41]. To encompass the two activities of the proteins, it was renamed iron regulatory
protein 1, and multiple studies revealed that the key to the transition from func-
tioning as an active aconitase to an iron regulatory protein involved the loss of the
[4Fe-4S] cluster (reviewed in [42, 43]).
These new concepts and the underlying evidence were discussed in a memorable
meeting held in Konstanz in 1994, to celebrate Helmut’s eightieth birthday and to
present all these “novel” breakthroughs. Not long after, another example in which the
FeS cluster served as a sensor was uncovered in bacteria in studies of fumarate nitrate
1.5 How are FeS clusters synthesized in cells? 7
reductase, where a labile Fe-S cluster was recognized as the key to sensing oxygen
and remodeling transcription to direct a switch from aerobic to anaerobic metabolism
(reviewed in [44]).
Scores of non-redox functions for Fe-S proteins accumulated over the years. In a
review that appeared in 1997, the accumulated evidence was summarized by stating
that, “Iron-sulfur clusters now rank with such biological prosthetic groups as hemes
and flavins in pervasive occurrence and multiplicity of function” [1]. New roles conti-
nue to emerge, and Fe-S proteins are now recognized to play an important role in DNA
metabolism and maintenance of DNA integrity [45, 46] and in human diseases [47].
1.5 How are FeS clusters synthesized in cells?
Despite the “self-assembling” nature of Fe-S clusters discussed earlier, it was not
clear how cells could synthesize Fe-S clusters without encountering problems with
the cytotoxicity of sulfide and with the insolubility of iron(III) sulfides. Protein-bound
zero-valence sulfur had been found as a cysteine-bound persulfide at the active site of
sulfurtransferases, and this form of “elemental” sulfur was demonstrated to undergo
easy reduction to sulfide by addition of suitable thiols [48]. Bovine liver rhodanese
was recognized as the epitome of this class of enzymes, and in the mid-1970s, it was
found that liver rhodanese could rescue damaged Fe-S clusters in mitochondrial SDH
by replenishing some of the missing cluster sulfide [49] or serve as a source of cluster
sulfide in Fe-S proteins [27] and in their chemical analogues [27]. Nevertheless, rhoda-
nese was known to be absent from scores of FeS-rich organisms, making it difficult to
consider that rhodanese activity was of general relevance [50]. Indeed, evidence was
accumulating that cysteine was a likely source of sulfide for the biogenesis of Fe-S
structures in chloroplasts [51].
Advances in genetics, sequencing, biochemistry, and biophysics led to the dis-
covery of the bacterial genes involved in nitrogen fixation (the nif gene cluster) in
Azotobacter vinelandii [52] and to the identification of a cysteine desulfurase as
the essential sulfide-generating component of the system (reviewed by [53]). Later,
the isc (iron-sulfur cluster assembly) operon used for the general synthesis of Fe-S
proteins was discovered in A. vinelandii [54], in other bacteria [55], in yeast model
systems (see the review by [56]), and in mammals [57]. The role of scaffold prote-
ins as intermediates in the assembly process was discovered [58], along with the
importance of a chaperone-co-chaperone pair for cluster delivery [59, 60] and pro-
posed roles for intermediate scaffolds [61]. Studies of Fe-S proteins and chemistry
are ongoing, the field is vibrant, and unexpectedly, mutations in FeS assembly pro-
teins have proven to be the cause of several important human diseases, including
Friedreich ataxia, ISCU myopathy, a rare type of sideroblastic anemia, and lactic
acidosis in infants (reviewed in [47]).
Indeed, how these FeS proteins work and are generated is the subject of many
excellent ongoing research, which will be described in chapters that follow in this
book. Topics range from nitrogen fixation, to hydrogenase function, to plant growth,
and to the origin of life itself, with numerous implications for industrial processes,
food production, and for human disease.
Acknowledgment
We thank Jacques Meyer for generously providing his overview of the history of iron-
sulfur research in his tribute to Helmut Beinert.
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Another random document with
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in South Africa, and to produce what, thank God! he had failed
in producing—a racial war." Mr. Chamberlain retorted that Sir
William Harcourt's attitude was unpatriotic and injurious to
the cause of peace. He denied aggressiveness in the policy of
the government, asserting that the South African Republic had
been spending millions on armaments imported from abroad, in
view of which the strengthening of the British garrison at the
Cape by an additional regiment and three batteries was no
unreasonable measure. Mr. Balfour, also, begged the House and
the country to believe that the troops were sent only as a
measure of precaution, to maintain admitted rights.
{478}
SOUTH AFRICA: The Transvaal: A. D. 1897 (May-October).

The British assertion of suzerainty and declination of
proposal to arbitrate disagreements.
On the 7th of May, 1897, the Acting State Secretary of the

South African Republic addressed to the British Agent at
Pretoria a communication of great length, reviewing the
positions taken by Mr. Chamberlain in his several arraignments
of the government of the Republic for alleged violation of the
London Convention of 1884, and proposing an arbitration of the
questions involved. "The complaint," he wrote, "which Her
British Majesty's Government has advanced in an unmistakably
pronounced manner over an actual or possible breach of the
Convention has deeply grieved this Government, as it thinks
that it has fulfilled its obligations. It sees in the
fulfilment of the mutual obligations under the Convention one
of the best guarantees for the maintenance of a mutual good
understanding and for the promotion of reciprocal confidence.
To this good understanding and that confidence, however,
severe shocks have been given by events which cannot be
lightly forgotten. And if it were not that this Government
wishes to guard itself against adopting a recriminating tone,
it might put the question whether, for example, the incursion
of Dr. Jameson, whether considered as a breach of the
Convention or a grievance, is not of immeasurably greater
importance than the various matters adduced by Her British
Majesty's Government would be, even if the contention that
they constitute breaches of the Convention could be accepted.
There should, in the view of this Government, be a strong
mutual endeavour to restore the shocked confidence and to calm
the excited spirit which this Government with sincere regret
sees reigning throughout almost the whole of South Africa.
This Government is anxious to co-operate for this end, for the
desire of the Republic, with the maintenance of its
independence and rights, is for peace, and where for the
reasons given it has been unable to entertain the proposal of
Her British Majesty's Government in the matter of the Aliens
Law,—and it appears very difficult to arrive at a solution of
the question by means of correspondence,—it wishes to come to
a permanent good understanding along a peaceful course, not
only with respect to its undisturbed right to make an alien
law, but also with regard to all points touching the
Convention which are referred to in the two Despatches under
reply by Her British Majesty's Government. While it respects
the opinion of Her British Majesty's Government, it takes the
liberty, with full confidence in the correctness of its own
views, to propose to Her British Majesty's Government the
principle of arbitration with which the honourable the First
Volksraad agrees, in the hope that it will be taken in the
conciliatory spirit in which it is made. …
"Although this Government is firmly convinced that a just and

impartial decision might be obtained even better in South
Africa than anywhere else, it wishes, in view of the
conflicting elements, interests, and aspirations, which are
now apparent in South Africa, and in order to avoid even the
appearance that it would be able or desire to exercise
influence in order to obtain a decision favourable to it, to
propose that the President of the Swiss Bondstate, who may be
reckoned upon as standing altogether outside the question, and
to feel sympathy or antipathy neither for the one party nor
for the other, be requested to point out a competent jurist,
as has already oftener been done in respect of international
disputes. The Government would have no objection that the
arbitrator be subject to a limitation of time, and gives the
assurance now already that it will willingly subject itself to
any decision if such should, contrary to its expectation, be
given against it. The Government repeats the well-meant wish
that this proposal may find favour with Her British Majesty's
Government, and inasmuch as the allegations of breaches of the
Convention find entrance now even in South Africa, and bring
and keep the feelings more and more in a state of suspense,
this Government will be pleased if it can learn the decision
of Her Majesty's Government as soon as possible."
Mr. Chamberlain's reply to this proposal was not written until

the 16th of the following October, when he, in turn, reviewed,
point by point, the matters dealt with, in the despatch of Mr.
Van Boeschoten. With reference to the Jameson raid he said: "Her
Majesty's Government note with satisfaction that the
Government of the South African Republic see in the fulfilment
of the mutual obligations under the London Convention one of
the best guarantees for the maintenance of a mutual
understanding and for the promotion of reciprocal confidence.
Her Majesty's Government have uniformly fulfilled these
obligations on their part, and they must strongly protest
against what appears to be an implication in the Note under
consideration that the incursion of Dr. Jameson can be
considered as either a breach of the Convention by Her
Majesty's Government or a grievance against them. That
incursion was the act of private individuals unauthorised by
Her Majesty's Government, and was repudiated by them
immediately it became known. The immense importance to the
Government of the South African Republic of that repudiation,
and of the proclamation issued by the High Commissioner under
instructions from Her Majesty's Government, is recognised
throughout South Africa. Her Majesty's Government maintain
strongly that since the Convention of 1881 there has never
been any breach or even any allegation of a breach on their
part of that or the subsequent Convention, and, as the subject
has been raised by the implied accusation contained in the
Note under consideration, Her Majesty's Government feel
constrained to contrast their loyal action in the case of the
Jameson raid with the cases in which they have had cause to
complain that the Government of the South African Republic
failed to interfere with, if they did not countenance,
invasions of the adjacent territories by its burghers in
violation of the Convention, and they feel bound to remind the
Government of the Republic that in one of these cases Her
Majesty's Government were compelled to maintain their rights
by an armed expedition at the cost of about one million
sterling, for which no compensation has ever been received by
them."
Concerning the proposal of arbitration, the reply of the

British colonial secretary was as follows: "In making this
proposal the Government of the South African Republic appears
to have overlooked the distinction between the Conventions of
1881 and 1884 and an ordinary treaty between two independent
Powers, questions arising upon which may properly be the
subject of arbitration.
{479}
By the Pretoria Convention of 1881 Her Majesty, as Sovereign
of the Transvaal Territory, accorded to the inhabitants of
that territory complete self-government subject to the
suzerainty of Her Majesty, her heirs and successors, upon
certain terms and conditions and subject to certain
reservations and limitations set forth in 33 articles, and by
the London Convention of 1884 Her Majesty, while maintaining
the preamble of the earlier instrument, directed and declared
that certain other articles embodied therein should be
substituted for the articles embodied in the Convention of
1881. The articles of the Convention of 1881 were accepted by
the Volksraad of the Transvaal State, and those of the
Convention of 1884 by the Volksraad of the South African
Republic. Under these Conventions, therefore, Her Majesty
holds towards the South African Republic the relation of a
suzerain who has accorded to the people of that Republic
self-government upon certain conditions, and it would be
incompatible with that position to submit to arbitration the
construction of the conditions on which she accorded
self-government to the Republic. One of the main objects which
Her Majesty's Government had in view was the prevention of the
interference of any foreign Power between Her Majesty and the
South African Republic, a matter which they then held, and
which Her Majesty's present Government hold, to be essential
to British interests, and this object would be defeated by the
course now proposed. The clear intention of Her Majesty's
Government at the time of the London Convention, that
questions in relation to it should not be submitted to
arbitration, is shown by the fact that when the delegates of
the South African Republic, in the negotiations which preceded
that Convention, submitted to Her Majesty's Government in the
first instance (in a letter of the 26th of November, 1883,
which will be found on page 9 of the Parliamentary Paper C.
3947 of 1884) the draft of a treaty or convention containing
an arbitration clause, they were informed by the Earl of Derby
that it was neither in form nor in substance such as Her
Majesty's Government could adopt."
Great Britain, Papers by Command.

C.—8721, 1898.
SOUTH AFRICA: Natal: A. D. 1897 (December).

Annexation of Zululand.
See (in this volume)

AFRICA: A. D. 1897 (ZULULAND).
SOUTH AFRICA: Cape Colony: A. D. 1898.

The position of political parties.
The Progressives and the Afrikander Bund.
"The present position of parties at the Cape is as unfortunate

and as unwarranted as any that the severest critic of
Parliamentary institutions could have conjured up. … The Cape
has always had the curse of race prejudice to contend with.
Time might have done much to soften, if not to expunge it, if
home-made stupidities had not always been forthcoming to goad
to fresh rancour. The facts are too well known to need
repetition. It is true not only of the Transvaalers that 'the
trek has eaten into their souls,' and up to the time of
emancipation and since, every conceivable mistake has been
committed by those in authority. Thus, when the breach was, to
all appearances, partly healed, the fatal winter of 1895 put
back the hands of the clock to the old point of departure. As
Englishmen, our sympathies are naturally with the party that
is prevalently English, and against the party that is
prevalently Dutch; but to find a real line of political
difference between them other than national sentiment requires
fine drawing. … According to our lines of cleavage both
Bondsmen [Afrikander Bund] and Progressives are Conservatives
of a decided type. Practically they are agreed in advocating
protective duties on sea-borne trade, although in degree they
differ, for whilst the Bond would have imposts as they are,
the Progressives wish to reduce the duties on food stuffs to
meet the grievance of the urban constituencies, and might be
induced to accord preferential treatment to British goods. On
the native question neither party adopts what would in England
be considered an 'advanced' programme, for education is not
made a cardinal point, and they would equally like, if
possible, to extend the application of the Glen Grey Act,
which, by levying a tax on the young Kaffirs who have not a
labour certificate, forces them to do some service to the
community before exercising their right of 'putting the
spoon,' as the phrase is, 'into the family pot.' Neither party
wishes to interfere with the rights of property or the
absolute tenure of land under the Roman-Dutch law. A tax on
the output of diamonds at Kimberley has been advocated by some
members of the Bond as a financial expedient, but it is
understood to have been put forward rather as a threat against
Mr. Rhodes personally than as a measure of practical politics.
Questions of franchise are tacitly left as they are, for no
responsible politicians wish to go back upon the enactment
which restricted the Kaffir vote to safe and inconsiderable
limits. The redistribution of seats was the subject of a Bill
upon which the last House was dissolved, after the rebuff that
the Ministry received upon a crucial division, but it has been
dealt with rather for practical than theoretical reasons. Two
schemes of redistribution have been formulated, and each has
been proposed and opposed with arguments directed to show the
party advantage to be derived. For political reform, in the
abstract, with or without an extension of the suffrage, there
is no sort of enthusiasm in any quarter. Railway
administration furnishes, no doubt, an occasional battle-field
for the two sides of the House. Roughly, the Progressives
favour the northern extension, and are willing to make
concessions in rates and charges to help on the new trade with
Rhodesia; whilst the Bond declare themselves against special
treatment of the new interests, and would spend all the money
that could be devoted to railway construction in the farming
districts of the colony itself. Mr. Rhodes, however, has
warned the Cape that any hostile action will be counteracted
by a diversion of traffic to the East, and it is unlikely that
any line of policy will be pursued that is likely to injure
the carrying trade of the southern ports. Between the
followers of Mr. Rhodes and the followers of Mr. Hofmeyr there
is no wide divergence of principle on public affairs of the
near future, so far as they have been or are to be the subject
of legislation; where the difference comes in is in the
attitude they severally assume towards the two republics and
the territories of the north, but when talk has to yield to
action it is improbable that there will be much in their
disagreement."
N. L. W. Lawson,
Cape Politics and Colonial Policy
(Fortnightly Review, November 1898).
{480}
SOUTH AFRICA: The Transvaal: A. D. 1898 (January-February).
Re-election of President Kruger.

Renewed conflict of the Executive with the Judiciary.
Dismissal of Chief-Justice Kotze.
The Presidential election in the South African Republic was

held in January and February, the polls being open from the 3d
of the former month until the 4th of the latter. President
Kruger was re-elected for a fourth term of five years, by
nearly 13,000 votes against less than 6,000 divided between
Mr. Schalk Burger and General Joubert, who were opposing
candidates. Soon afterwards, the conflict of 1897 between the
Judiciary and the Executive (see above: A. D. 1897,
JANUARY-MARCH), was reopened by a communication in which
Chief-Justice Kotze, of the High Court, called the attention
of the President to the fact that nothing had been done in
fulfilment of the agreement that the independence of the Court
and the stability of the Grondwet should both be protected by
law against arbitrary interference, and giving notice that he
considered the compromise then arranged to be ended. Thereupon
(February 16) President Kruger removed the judge from his
office and placed the State Attorney in his seat. Justice
Kotze denied the legality of the removal, and adjourned his
court sine die. In a speech at Johannesburg, some weeks
afterwards, he denounced the action of President Kruger with
great severity, saying: "I charge the President, as head of
the State, with having violated both the constitution and the
ordinary laws of the land; with having interfered with the
independence of the High Court; and invaded and imperilled the
rights and liberties of everyone in the country. The
guarantees provided by the constitution for the protection of
real and personal rights have disappeared, and these are now
dependent on the 'arbitrium' of President Kruger."
SOUTH AFRICA: Rhodesia and the British South Africa Company:

A. D. 1898 (February).
Reorganization.
In February, the British government announced the adoption of

plans for a reorganization of the British South Africa Company
and of the administration of its territories. The Company,
already deprived of military powers, was to give up, in great
part, but not wholly, its political functions. It was still to
appoint an Administrator for Rhodesia south of the Zambesi,
and to name the majority of members in a council assisting
him, so long as it remained responsible for the expenses of
administration; but, by the side of the Administrator was to
be placed a Resident Commissioner, appointed by the Crown, and
over both was the authority of the High Commissioner for South
Africa, to whom the Resident Commissioner made reports. At
home the status of the Board of Directors was to be
considerably altered. The life directorships were to be
abolished, and the whole Board of Directors in future to be
elected by the shareholders,—any official or director removed
by the Secretary of State not being eligible without his
consent. The Board of Directors was to communicate all
minutes, etc., to the Secretary of State, and he to have the
power of veto or suspension. Finally, the Secretary of State
was to have full powers to inspect and examine all documents;
Colonial Office officials named by him were, in effect, to
exercise powers like those of the old Indian Board of Control.
SOUTH AFRICA: Cape Colony: A. D. 1898 (March-October).
Election in favor of the Afrikander Bund.

Change in the government.
Elections to the Upper House of the Cape Parliament, in March,
gave the party called the Progressives, headed by Mr. Rhodes, a
small majority over the Afrikander Bund—more commonly called
the Bond. The Parliament opened in May, and the Progressive
Ministry, under Sir Gordon Sprigg, was defeated in the Lower
House in the following month, on a bill to create new
electoral divisions. The Ministry dissolved Parliament and
appealed to the constituencies, with the result of a defeat on
that appeal. The Bond party won in the elections by a majority
of two, which barely enabled it to carry a resolution of want
of confidence in the government when Parliament was
reassembled, in October. The Ministry of Sir Gordon Sprigg
resigned, and a new one was formed with Mr. Schreiner at its
head.
SOUTH AFRICA: The Transvaal: A. D. 1898-1899.

Continued dispute with the British Government
concerning Suzerainty.
During 1898 and half of 1899, a new dispute, raised by Mr.

Chamberlain's emphatic assertion of the suzerainty of Great
Britain over the South African Republic, went on between the
British Colonial Office and the government at Pretoria.
Essentially, the question at issue seemed to lie between a
word and a fact and the difference between the disputants was
the difference between the meanings they had severally drawn
from the omission of the word "suzerainty" from the London
Convention of 1884. On one side could be quoted the report
which the Transvaal deputation to London, in 1884, had made to
their Volksraad, when they brought the treaty back, and
recommended that it be approved. The treaty, they reported,
"is entirely bilateral [meaning that there were two sides in
the making of it] whereby your representatives were not placed
in the humiliating position of merely having to accept from a
Suzerain Government a one-sided document as rule and
regulation, but whereby they were recognized as a free
contracting party. It makes, then, also an end of the British
suzerainty, and, with the official recognition of her name,
also restores her full self-government to the South African
Republic, excepting one single limitation regarding the
conclusion of treaties with foreign powers (Article 4). With
the suzerainty the various provisions and limitations of the
Pretoria Convention which Her Majesty's Government as suzerain
had retained have also, of course, lapsed."
On the other side, Mr. Chamberlain could quote with effect

from a speech which Lord Derby, then the British Colonial
Secretary, who negotiated the Convention of 1884 with the Boer
envoys, made on the 17th of March, that year, in the House of
Lords. As reported in Hansard, Lord Derby had then dealt with
the very question of suzerainty, as involved in the new
convention, and had set forth his own understanding of the
effect of the latter in the following words: "Then the noble
Earl (Earl Cadogan) said that the object of the Convention had
been to abolish the suzerainty of the British Crown. The word
'suzerainty' is a very vague word, and I do not think it is
capable of any precise legal definition.
{481}
Whatever we may understand by it, I think it is not very easy
to define. But I apprehend, whether you call it a
protectorate, or a suzerainty, or the recognition of England
as a paramount Power, the fact is that a certain controlling
power is retained when the State which exercises this
suzerainty has a right to veto any negotiations into which the
dependent State may enter with foreign Powers. Whatever
suzerainty meant in the Convention of Pretoria, the condition
of things which it implied still remains; although the word is
not actually employed, we have kept the substance. We have
abstained from using the word because it was not capable of
legal definition, and because it seemed to be a word which was
likely to lead to misconception and misunderstanding."
Great Britain,
Papers by Command: C. 9507, 1899, pages 24 and 34.
SOUTH AFRICA: The Transvaal: A. D. 1899 (March).
Petition of British subjects to the Queen.
A fresh excitement of discontent in the Rand, due especially

to the shooting of an Englishman by a Boer policeman, whom the
Boer authorities seemed disposed to punish lightly or not at
all, led to the preparation of a petition to the British
Queen, from her subjects in the South African Republic,
purporting to be signed in the first instance by 21,684, and
finally by 23,000. The genuineness of many of the signatures
was disputed by the Boers, but strenuously affirmed by those
who conducted the circulation of the petition. It set forth
the grievances of the memorialists at length, and prayed Her
Majesty to cause them to be investigated, and to direct her
representative in South Africa to take measures for securing
from the South African Republic a recognition of their rights.
The petition was forwarded to the Colonial Office on the 28th
of March.
Great Britain, Papers by Command: 1899, C. 9345.
SOUTH AFRICA: The Transvaal: A. D. 1899 (May-June).

The Bloemfontein Conference between President Kruger and
the British High Commissioner, Sir Alfred Milner.
There seems to be no mode in which the questions at issue

between the British and the Boers, and the attitude of the two
parties, respectively, in their contention with each other,
can be represented more accurately than by quoting essential
parts of the official report of a formal conference between
President Kruger and the British High Commissioner in South
Africa, Sir Alfred Milner, which was held at Bloemfontein, the
capital of the Orange Free State, during five days, May
31-June 5, 1899. The meeting was arranged by President Steyn,
of the Orange Free State, with a view to bringing about an
adjustment of differences by a free and full discussion of
them, face to face. In the official report of the
conversations that occurred, from which we shall quote, the
remarks of President Kruger are given as being made by the
"President," and those of the High Commissioner as by "His
Excellency." The latter, invited by the President to speak
first, said:
"There are a considerable number of open questions between Her

Majesty's Government and the Government of the South African
Republic on which there is at present no sign of agreement. On
the contrary, disagreements seem to increase as time goes on.
… In my personal opinion the cause of many of the points of
difference, and the most serious ones, arises out of the
policy pursued by the Government of the South African Republic
towards the Uitlander population of that Republic among whom
many thousands are British subjects. This policy, the bitter
feeling it engenders between the Government and a section of
Uitlanders, and the effect of the resulting tension in South
Africa, and the feeling of sympathy in Great Britain, and even
throughout the British Empire generally, with the Uitlander
population, creates an irritated state of public opinion on
both sides, which renders it much more difficult for the two
Governments to settle their differences amicably. It is my
strong conviction that if the Government of the South African
Republic could now, before things get worse, of its own motion
change its policy towards the Uitlanders, and take measures
calculated to content the reasonable people among them, who,
after all, are a great majority, such a course would not only
strengthen the independence of the Republic but it would make
such a better state of feeling all round that it would become
far easier to settle outstanding questions between the two
Governments. … The President, in coming here, has made a
reservation as to the independence of the Republic. I cannot
see that it is in any way impairing the independence of the
Republic for Her Majesty's Government to support the cause of
the Uitlanders as far as it is reasonable. A vast number of
them are British subjects. If we had an equal number of
British subjects and equally large interests in any part of
the world, even in a country which was not under any
conventional obligations to Her Majesty's Government we should
be bound to make representations to the Government in the
interests of Her Majesty's subjects, and to point out that the
intense discontent of those subjects stood in the way of the
cordial relations which we desire to exist between us. I know
that the citizens of the South African Republic are intensely
jealous of British interference in their internal affairs.
What I want to impress upon the President is that if the
Government of the South African Republic of its own accord,
from its own sense of policy and justice, would afford a more
liberal treatment to the Uitlander population, this would not
increase British interference, but enormously diminish it. If
the Uitlanders were in a position to help themselves they
would not always be appealing to us under the Convention. …
"President.—I shall be brief. I have come with my commission,

in the trust that Your Excellency is a man capable of
conviction, to go into all points of difference. … I should
like His Excellency to go point by point in this discussion,
so that we can discuss each point that he thinks requires
attention, not with a view to at once coming to a decision,
but to hear each side, and we can go back on any point if
necessary, and see if we can arrive at an understanding. I
would like to give concessions as far as is possible and
practicable, but I want to speak openly, so that His
Excellency may be able to understand. I should like to say
that the memorials placed before Her Majesty's Government came
from those who do not speak the truth. I mean to convey that
we do give concessions wherever we think it practicable to do
so, and after we have discussed it in a friendly way Your
Excellency will be able to judge whether I or the memorialists
are right. I have said that if there are any mistakes on our
side, we are willing to discuss them. Even in any matter
concerning internal affairs I would be willing to listen to
his advice if he said it could be removed in this way or that
way. But when I show him that by the point we may be
discussing our independence may be touched, I trust he will be
open to conviction on that subject. …
{482}
"His Excellency.—I think the point which it would be best to

take first, if the President agrees, … would be the Franchise.
… There are a number of questions more or less resting upon
that. … I should like to know a little more about the
President's views. I want to know more because if I were to
begin and say I want this, that, and the other, I know I
should be told this was dictation. I do not want to formulate
a scheme of my own, but I can, if necessary.
"President.—As long as I understand that it is meant in a

friendly manner, and you mean to give hints, I won't take it
that they are commands. It has already been arranged that you
give me friendly hints and advice, and I will not take it as
dictation, even though it should be on points on which I
should consider you have no right to interfere. … I would like
you to bear one point in view, namely, that all kinds of
nations and languages, of nearly all powers, have rushed in at
the point where the gold is to be found. In other countries …
there are millions of old burghers, and the few that come in
cannot out-vote the old burghers, but with us, those who
rushed in to the gold fields are in large numbers and of all
kinds, and the number of old burghers is still insignificant;
therefore we are compelled to make the franchise so that they
cannot all rush into it at once, and as soon as we can assure
ourselves by a gradual increase of our burghers that we can
safely do it, our plan was to reduce the time for anyone there
to take up the franchise, and that is also my plan. … As His
Excellency doubtless knows, I have proposed to the Volksraad
that the time should be reduced by five years, and gradually
as more trusted burghers join our numbers, we can, perhaps, go
further. There are a number who really do not want the
franchise, but they use it as pretext to egg on people with
Her Majesty. … You must remember, also, on this subject, that
the burghers in our Republic are our soldiers, who must
protect the land, and that we have told these men to come and
fight when we have had difficulties with the Kaffirs. They
wanted the vote, but they would not come and fight. Those who
were willing to help obtained the franchise, but it appears
that many do not want to have it.
"His Excellency.—They did not want to take the obligations

without the rights of citizenship, and in that I sympathize
with them. If they should obtain that right, then naturally
they would have to take those burdens upon them.
"President.—Those who want the franchise should bear the

burdens.
"His Excellency.—Yes. Immediately they get the franchise they

take upon themselves the obligations connected therewith."
[From this the talk wandered to the subject of commandeering,

until the High Commissioner brought it back to the franchise
question.]
"His Excellency.—If I made a proposal to admit strangers under

such conditions as to swamp the old burghers it would be
unreasonable. But the newcomers have, at present, no influence
on the legislation of the Republic, which makes an enormous
difference. They haven't got a single representative. The
First Volksraad consists of 28 members, and not one member
represents the feelings of the large Uitlander population.
"President.—Men from any country could after two years vote

for the Second Volksraad, and after two years more sit in the
Second Raad. There are Englishmen who have obtained the full
franchise in that way, and are eligible for the Volksraad. And
now I have proposed to shorten the last ten years of the
period required for the full franchise and make it five years.
"His Excellency.—There are a great many objections of the

gravest kind to the process by which men may now obtain
burgher rights. First of all, before he can begin the process
of gradually securing burgher rights—which will be completed
in 14 years at present, and in 9 years according to the
President—he has to forswear his own allegiance. Take the case
of a British subject, which interests me most. He takes the
oath, and ceases to be a British subject by the mere fact of
taking that oath; he loses all the rights of a British
subject, and he would still have to wait for 12 years, and
under the new plan 7 years, before he can become a full
citizen of the Republic. British subjects are discouraged by
such a law from attempting to get the franchise. Even if they
wanted to become citizens, they would not give up their
British citizenship on the chance of becoming in 12 years
citizens of the Republic.
"President.—The people are the cause of that themselves. In

1870 anyone being in the land for one year had the full
franchise.
"His Excellency.—That was very liberal.
"President.—In 1881, after the war of independence, some of

our officials and even members of our Raad then said that they
were still British subjects, although they had taken the oath
of allegiance, and I had to pay back, out of the £250,000,
what I had commandeered from them. That was the reason the
oath had to be altered. …
"His Excellency.—In 1882, after all this had happened, there

was a franchise law in the Transvaal, which demanded five
years' residence, but it did not require the oath that is now
taken. It required a simple declaration of allegiance to the
State, though all this that the President refers to happened
before. Why was not it necessary to introduce this alteration
then?
"President.—The people who, before the annexation, had taken

that oath, but had not forsworn their nationality, 1887, sent
a lying memorial, as they are sending lying memorials now, to
say that everybody was satisfied, as they now say that
everybody is dissatisfied.
"His Excellency.—I think I must just explain a little more

clearly my views on the point we are now discussing. … I think
it is unreasonable to ask a man to forswear one citizenship
unless in the very act of giving up one he gets another, and I
think it is also unnecessary to ask him to do more than take
an oath of fealty to the new State, of willingness to obey its
laws and to defend its independence, when it is known and
certain that the taking of that oath deprives him of his
existing citizenship. I think the oath should be a simple oath
of allegiance, and that it should not be required of a man
until the moment he can get full rights in a new State. Now
that was the position under the law of 1882, and all these
reasons which the President has been giving are based on what
happened before that.
{483}
Why were they not considered and acted upon when the law of
1882 was made? … As for the period required to qualify for the
full franchise, I do not see why the length of time should be
longer in the South African Republic than in any other South
African State. They are all new countries. In the new country
which is springing up in the north, and which is getting a new
Constitution this year, the period is one year. The people who
have conquered that country for the white race may find that
the newcomers are more numerous than they are. But I do not
expect that anything like that will be done in the South
African Republic; something far short of that would be
reasonable. What I do think and desire, and that is the object
of my suggestion, is this: that the numerous foreign population
engaged in commerce and industry—to which the country, after
all, owes its present great position in wealth and influence—
should have a real share in the government of the Republic,
not to over-rule the old burghers—not at all—but to share the
work of Government with them, to give them the benefit of
their knowledge and experience, which is in many cases greater
than that of the old burghers, so that through their gradual
co-operation a time may come when, instead of being divided
into two separate communities they will all be burghers of the
same State. It is not enough that a few people should be let
in. It is obvious, however, that you could not let in the
whole crowd, without character or anything—I do not ask
it—but you want such a substantial measure that in elections
of members of the Volksraad the desires of the new industrial
population should have reasonable consideration. They have not
got it now, and when the questions that interest them come
before the Volksraad it is too evident that they are discussed
from an outside point of view. The industrial population are
regarded as strangers. … I do not want to swamp the old
population, but it is perfectly possible to give the new
population an immediate voice in the legislation, and yet to
leave the old burghers in such a position that they cannot
possibly be swamped.
"President.—I hope you will be open to conviction on that

point. I would like to convince you on the subject, and to
show you that it would be virtually to give up the
independence of my burghers. In the Republic the majority of
the enfranchised burghers consider they are the masters. Our
enfranchised burghers are probably about 30,000, and the
newcomers may be from 60,000 to 70,000, and if we give them
the franchise to-morrow we may as well give up the Republic. I
hope you will clearly see that I shall not get it through with
my people. We can still consult about the form of oath, but we
cannot make the time too short, because we would never get it
through with the people—they have had bitter experience. I
hope His Excellency will think about what I have said, and
weigh it well.
"His Excellency.—I see your point, and want to meet it.
"President.—I will think over what has been said, and will try
and meet every difficulty.
At the opening of the Conference on the second day the

President spoke of reports of an increase of British forces in
South Africa, which the High Commissioner assured him were
untrue. The latter in turn referred to accounts that had
appeared of an extensive purchase of arms in the Transvaal;
and was assured by the President that the armament of the
burghers was only for their proper preparation to deal with
the surrounding natives. The President then produced a
memorial purporting to be signed by 21,000 Uitlanders,
contradictory of the representations contained in the memorial
sent to the Queen in March (see above). After discussion upon
this, the conversation returned to the question of the
franchise.
"His Excellency.—What makes this whole discussion so difficult

is the intense prejudice on the side of the present burghers,
and their intense suspicion of us. They think Her Majesty's
Government wants to get their country back in one way or
another. Her Majesty's Government does not; but what it does
desire is that it should have such a state of rest in the
country as will remove causes of friction and difficulty
between the Republic and Her Majesty's possessions in South
Africa, and the whole of the British Empire, and my
suggestions here are directed to that end. I do not want to
say it over and over again, I say it once for all. …
"President.—I should like to make a slight explanation to His

Excellency. His Excellency yesterday mentioned that in some
States those going in from outside speedily got burgher
rights, but he must not forget, as I said before, they are

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Iron Sulfur Clusters in Chemistry and

Biology Volume 1 Characterization

Iron Sulfur Clusters in Chemistry and Biology Volume 2

Advances in Semiconductor Nanostructures. Growth,

Advances in Mathematical Chemistry and Applications

Nanomaterials and Nanocomposites: Synthesis,

Low Dimensional and Nanostructured Materials and

Luminescence in Electrochemistry Applications in

The Alkaloids Chemistry and Biology Volume 77 Hans-

Quantum Systems in Physics Chemistry and Biology

New-Generation Bioinorganic Complexes

Methods in Protein Biochemistry

Protein Tyrosine Phosphatases

Library of Congress Cataloging-in-Publication Data

Bibliographic information published by the Deutsche Nationalbibliothek

© 2017 Walter de Gruyter GmbH, Berlin/Boston

Cover image: Rouault, Maio, 2017

Tracey Rouault, July, 2017

Tracey A. Rouault is a leading researcher in

Francesco Bonomi and Tracey A. Rouault

3.7 Spin coupling and BS 31

Yisong Guo and Jikun Li

Doros T. Petasis and Michael P. Hendrich

5.4 Hyperfine structure 138

Mrinmoy Chakrabarti and Paul A. Lindahl

Nathaniel S. Sickerman, Markus Ribbe, and Yilin Hu

Thomas Spatzal, Susana L. A. Andrade and Oliver Einsle

Nicholas D. Lanz and Squire J. Booker

11.5.5 Product inhibition of LipA 340

Yvain Nicolet and Juan C. Fontecilla-Camps

Erin L. Dodd, Jason C. Crack, Andrew J. Thomson, and Nick E. Le Brun

13.1.3 The biology of NO sensing via Fe-S cluster proteins 391

Susana L. A. Andrade Oliver Einsle

Squire Booker Yisong Guo

Mrinmoy Chakrabarti Michael Hendrich

Jason C. Crack Russ Hille

Erin L. Dodd Yilin Hu

Toshiko Ichiye Louis Noodleman

Nick E. Le Brun Nathaniel S. Sickerman

1.1 Framing the scene

1.2 The early days of “nonheme iron”

1.3 Of proteins and analogues

1.4 Beyond electron shuttles

1.5 How are FeS clusters synthesized in cells?

SOUTH AFRICA: The Transvaal: A. D. 1897 (May-October).

On the 7th of May, 1897, the Acting State Secretary of the

"Although this Government is firmly convinced that a just and

Mr. Chamberlain's reply to this proposal was not written until

Concerning the proposal of arbitration, the reply of the

Great Britain, Papers by Command.

SOUTH AFRICA: Natal: A. D. 1897 (December).

See (in this volume)

SOUTH AFRICA: Cape Colony: A. D. 1898.

"The present position of parties at the Cape is as unfortunate

SOUTH AFRICA: The Transvaal: A. D. 1898 (January-February).

Re-election of President Kruger.

The Presidential election in the South African Republic was

SOUTH AFRICA: Rhodesia and the British South Africa Company:

In February, the British government announced the adoption of

SOUTH AFRICA: Cape Colony: A. D. 1898 (March-October).

Election in favor of the Afrikander Bund.

SOUTH AFRICA: The Transvaal: A. D. 1898-1899.

During 1898 and half of 1899, a new dispute, raised by Mr.