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 ABC Transporters
A Goffeau, Université Catholique de Louvain, Louvain-la-Neuve, Belgium
B De Hertogh, Facultés Universitaires Notre-Dame de la Paix, Namur, Belgium
ã 2013 Elsevier Inc. All rights reserved.
This article is a revision of the previous edition article by André Goffeau, Benoı̂t De Hertogh, and Philippe V. Baret, volume 1, pp. 1–5, ã 2004, Elsevier Inc.
Glossary
ABC1 Specific small amino acid sequence which is a
specific signature of ABC proteins.
C-, D-, H-, P-, and Q-loop Loops are located at the junction
of a-helices and are represented by conserved amino acid
residue in one letter code: C is cysteine, D is aspartate,
H is histidine, P is proline, and Q is asparagine.
NBD Nucleotide-binding domain of about 100 amino
acids which is characterized by a Walker A and Walker
B motif.
Pdr5p The major yeast ABC transporter involved in
pleiotropic drug resistance including the azole-antifungals.
P-gp The major ABC transporter (glycoprotein)
involved in mammalian multidrug resistance
The ABCs constitute the largest family of proteins. They are
present in all living species from Archaea to Homo sapiens.
They make up to 4% of the full genome complement of bacteria
such as Escherichia coli or Bacillus subtilis. Each eukaryote
genome contains several dozens of ABC proteins. They are
recognized by a consensus nucleotide-binding domain (NBD)
of approximately 100 amino acids which comprises a series
of structural and sequence motifs illustrated in Figure 1: the
Walker A motif, the ABC1 signature, the Walker B motif,
the Helical domain, and the P, Q-, C-, D-, and H-loops which
are structural junctions between a-helices marked by a con-
served proline, asparagine, cysteine, aspartate, or histidine resi-
due. The ABC proteins catalyze a wide variety of physiological
functions, many but not all of which are related to transport.
We wish to describe the major physiological and biochemical
functions as well as the structural properties of some of the best-
known ABC transporters using as examples the multidrug
exporters Sav1866 from Staphylococcus aureus, Pdr5p from
Saccharomyces cerevisiae, and P-gp from H. sapiens.
Topology
Each ABC transporter molecule contains, or is associated to,
one or two cytoplasmic adenosine triphosphate (ATP)-binding
domains named NBDs and one or two transmembrane
domains (TMDs). Each TMD comprises usually six transmem-
brane spans (TMSs) separated by the three extracytoplasmic
loops ECL1, ECL2, and ECL3 and the two intracytoplasmic
loops ICL2 and ICL3. Association of one TMD to one NBD
results in a half-size ABC transporter, which functions as
homo- or heterodimers so that the minimal functional organi-
zation of an ABC transporter is considered to be TMD–NBD/
TMD–NBD or NBD–TMD/NBD–TMD. In bacteria, two NBDs
and which is often activated during cancer
chemotherapy.
Transmembrane span (TMS) and transmembrane
domain (TMD) The existence of the transmembrane
span is predicted by frequency of hydrophobic residues in a
reading window of about 17 amino acids. In ABC
transporters, the transmembrane domains usually
comprise six contigous transmembrane spans.
Transporter classification (TC) Classification of
over 1000 transporter families, developed by Milton
Saier based on a combination of mechanistic and
phylogenetic criteria.
Walker A and B Small consensus of amino acid sequences
involved in ATP binding.
often associate with two TMDs either as four single subunits
encoded within the same operon or in various combinations of
fused subunits (Figure 2). Association of other proteins may
occur. The most prominent associated bacterial protein is the
periplasmic solute-binding receptor, which in Gram-negative
bacteria is found in the periplasm, and in Gram-positive bacte-
ria is often a lipoprotein bound to the external membrane
surface via electrostatic interactions. The three domains of the
bacterial ABC uptake transporters, namely the periplasmic-
binding receptor, the cytoplasmic NBD, and the membrane
TMD, are believed to have arisen from a common ancestral
ABC transporter in which these three proteins were already
present. However, during evolution the sequence of the peri-
plasmic solute-binding receptors diverged more rapidly than
that of the TMDs while that of NBDs remained the least diver-
gent. Thus, all NBDs are homologous, but this is not true for
the TMDs or the receptors.
In eukaryotes, two TMDs and two NBDs are often, but
not always, associated in one single molecule called ‘full-
sized ABC transporter’. The topological relation between
NBDs and TMDs is variable (Figure 3); additional TM spans
occur in some systems as well as extracytoplasmic domains
of presumed regulatory function.
Phylogeny
The different families of ABC proteins transport a wide variety
of substrates against their concentration gradient using the
energy of ATP hydrolysis carried out by NBD. In bacteria, the
transported substrates are either imported in or exported out
of the cell. In eukaryotes, only extracytoplasmic exporters
transporting substrates either out of the cell or into organelles
are known. Within the ABC transporters, a total of 173
7
8 Bioenergetics | ABC Transporters

Out
NBD

In C N Stalk
N C
NBD1 NBD2 NBD1 NBD2

TMD

N NBD1 NBD2 Figure 3 Reconstructed structure of the purified yeast Pdr5p

C reconstituted in lipid-containing particles and observed by cryo electron
microscopy.

This family is not detected in the animal kingdom. Con-

C N versely, the large human and mouse ABCA family identified
N C in 1994 by Giovanna Chimini is not represented in fungal
NBD NBD
genomes.
Figure 1 Example of topological relations between nucleotide-binding
domains (NBDs) and transmembrane domains (TMDs) in full-sized
and half-sized ABC transporters. Function and Diseases

The ABC transporters from Archaea, Gram-negative, and

Out
Periplasmic-binding
receptor
Substrate
TMD1 TMD2
Transmembrane domain
Cytoplasmic nucleotide-
In NBD1 NBD2 binding domain
Figure 2 Example of protein subunits constituting bacterial ABC
influx transporters.
phylogenetic families have been identified so far and classified
within the transporter classification (TC) system developed by
Milton Saier in San Diego. These families generally correlate
with substrate specificity and comprise both efflux and influx
transporters.
Since 1995, the in silico analysis of hundreds of full genome
sequences has allowed the identification of thousands of new
ABC proteins. For instance, within the 49 ABC proteins identi-
fied in the human genome, five major families of transporters
have been identified and named ABCA (or ABC1), ABCB (or
MDR including the famous P-gp described below), ABCC
(or MRP), ABCD (or ALD), and ABCG (or WHITE). This clas-
sification overlaps with the TC.
S. cerevisiae genome contains 32 ABC proteins among
which 22 are associated to TMDs. The largest yeast ABC family
is Pdrp identified in 1997 by Anabelle Decottignies and André
Goffeau and shown later to be present in all fungi and plants.
Gram-positive plasma membranes are widely different in
their organization and composition. The variety of substrates
transported: sugars, amino acids, lipids, ions, polysaccharides,
peptides, proteins, toxins, drugs, antibiotics, xenobiotics, and
other metabolites is reflected by the divergence of the periplas-
mic sensor and that of the TMD which control the specificity
and the direction (efflux or influx) of transport.
Even if all eukaryotic ABC transporters are effluxers that
comprise each a TMD fused to NBD, some of them are not
directly involved in moving substrates. For instance, in the
cystic fibrosis transmembrane regulator (CFTR) and in the sul-
fonylurea receptor (SUR), the hydrolysis of ATP appears to be
linked to the regulation of opening and closing of ion channels
carried by the ABC protein itself or other proteins .The presence
of NBD and TMD in all ABC transporters, however, suggests
that a basic coupling mechanism exists for efflux and influx
whatever the transported substrate. However, distantly related
proteins utilize an NBD to drive diverse nontransport processes
such as DNA repair or protein elongation or regulation of
RNAse activities.
One strong impetus for the study of mammalian ABC trans-
porters is their involvement in human diseases. Many mende-
lian diseases and complex genetic disorders are caused by
ABC transporters including cystic fibrosis, adrenoleukodystro-
phy, Stargardt disease, Tangier disease, immune deficiencies,
progressive familial intrahepatic cholestasis, Dublin–Johnson
syndrome, Pseudoxanthoma elasticum, persistent hyperinsuli-
nemic hypoglycemia of infancy due to focal adenomatous
hyperplasia, X-linked sideroblastosis and anemia, age-related
macular degeneration, familial hypoapoproteinemia, fundus
flavimaculatis, retinitis pigmentosum, and cone rod dystrophy.
Frequent cases of resistance to plant, fungal, or mammalian
pathogens and to chemotherapic treatments of cancers often
result from induction or activation mutations of pleiotropic
drug resistance (Pdrp), multiple drug resistance (MDR), or
multidrug resistance-related protein (MRP) transporters. Basic

studies of human, plants, or pathogen ABC transporters would
greatly benefit from heterologous expression of human ABC
transporter genes in yeast or other cells but this technology is
not fully satisfactory yet. Meanwhile, knockout technology in
the mouse allows physiological analysis of the mammalian
transporters.
Structure and Biochemical Mechanism
In 2010, over a dozen high-resolutive X-ray structures of func-
tional ABC transporters are available and have contributed to
the emergence of a consensus view of the catalytic mechanism
of transport. Recent analyses at the electron microscopy level of
purified bacterial (BmrA from B. subtilis) and a fungal (Pdr5p
from S. cerevisiae) drug efflux ABC transporter came to a
remarkably coherent set of conclusions. In both cases, the
basic structural unit seems to comprise four joining NBDs
that correspond to two full-size Pdr5p or four half-size BmrA.
The NBD are related to the TMDs through four distinct stalks.
Each NBD is oriented at a fixed 90º angle relative to its neigh-
bor NBDs. This raises the possibility of concerted rotation
movements of the NBDs implying a certain flexibility of the
stalks. No intramolecular or no intramembrane pores were
observed even though there is room (or chamber) between
the four stalks that join together at their NBD tips (Figure 4).
The nature of the stalks, TMD and NBD, was further identi-
fied by the analysis of the well-resolved structure of complete
dimeric ABC bacterial and eukaryote transporters such as
those obtained for the phospholipid flippase MsbA from
E. coli and Staphylococcus typhimurum, the vitamin B12 importer
BtuCD from E. coli, the importer of metal-chelate HI1470/1
from Haemophilus influenzae, the drug exporter Sav1866 from
S. aureus, and the mammalian multidrug exporter P-gp.
The folds of the membrane and stalk domains were found
to be dissimilar in the different structures analyzed so far.
This may not be surprising taking into account the different
species analyzed, the different cocrystallized ligands, the differ-
ent numbers of TMS, and the different functions of the proteins
analyzed. In particular, the communication between the NBD
and the TMD was found to be variable and, for instance, is
carried out either through a long and complex intracytoplas-
mic loop (ICL) in MsbA, through a short L-shaped ICL between
Consensus motifs in NBD
Helical
Walker A1
domain
P-loop Q-loop
90–110 aa
Figure 4 The consensus adenosine triphosphate (ATP)-binding region of a typical ABC protein is made of approximately 100 amino acids (aa),
including the Walker A motif, the ABC1 signature, the Walker B motif, the helical domain, and the P-, Q-, C-, D-, and H-loops (see section Glossary).
Bioenergetics | ABC Transporters 9
the transmembrane spans 6 and 7 in BtuCD, or through the ICL3
and ICL 6 in Sav1866.
A clear contribution of the X-ray structures is the observa-
tion that two molecules of ATP are bound at the interface of the
two NBDs. Each nucleotide-binding site comprises a Walker A
motif from one monomer and the ABC1 C motif from the
other monomer in a head-to-tail arrangement of the two inter-
acting NBD monomers.
As the folds of the various ABC transporters are highly
variable we focus on some basic structural properties of the
bacterial Sav1866 and the mammalian P-gp multidrug drug
exporters which have been reported, respectively, by Kaspar
Locher in 2006 and Goffrey Chang in 2009.
The homodimeric Sav1866 transporter has two identical
subunits, each comprising a NBD fused to a TMD made of six
transmembrane a-helices . It exports a wide series of unrelated
drugs such as the anticancer compounds doxorubicin and
vinblastin. Two coupling a-helixes, located in ICL3 and ICL6,
interact with grooves from the two NBDs. This structural motif
is conserved in all ABC transporters but its amino acid
sequence is not conserved. Individual TMS from the two
TMDs embrace each other and form two symmetric membrane
bundles each made of six mixed TMS comprising two mem-
brane a-helices from one TMD and four membrane a-helices
from the other TMD. A central cavity was identified between
the TMDs. As it faces outward, it was interpreted to represent
an open drug extrusion pocket.
P-gp is a full size NBD–TMD–NBD–TMD transporter which
is responsible for multiple drug resistance of mouse and
human cell and to which has been attributed failure of chemo-
treated cancer patients. The mouse P-gp was expressed in the
yeast Pichia pastoris, purified and crystallized. This 3.8 Å struc-
ture is a nucleotide-free, inward conformation arranged in two
symmetric halves each comprising an NBD and a TM bundle.
Each TM bundle comprises six mixed TMs in which spans from
the two TMDs embrace each other. A large drug-binding pocket
of about 6000 cubic A located at the intersection of the two TM
bundles is open to both the inner membrane leaflet and the
cytoplasm. A total of 73 amino acids’ aromatic and hydropho-
bic residues facing this cavity was accessible to solvent and
involved in the overlapping binding of three different ABC
inhibitors. Only two amino acid residues are common to the
three binding sites. Two portals are wide open to the inner
ABC 1 Walker B1
C-loop pro-loop D-loop H-loop
10 Bioenergetics | ABC Transporters
phospholipid bilayer and allow entry of hydrophobic drugs
which penetrate the membrane by passive diffusion.
Transport Mechanism
Recent structural observations have led to the disposal of the
early hypothetic mechanism of alternating ATP-binding sites
during the drug transport cycle. Instead, a consensus emerges
favoring a rather simple alternating outward–inward access
mechanism by which binding and hydrolysis of ATP by the
cytoplasmic NBD controls the binding and translocation of the
ligand through the TMD (Figure 5). Ligand binding to a high-
affinity pocket formed by the TMDs induces a conformational
change in the NBDs resulting in a higher affinity for ATP. Two
molecules of ATP each binding to both NBDs induce a close
dimeric conformation which promotes the extrusion of the
ligand. ATP hydrolysis triggers the opening of the NBD dimer.
Finally, phosphate and ADP are released and the ligand-free
NBD dimer conformation is restored.
This two-state alternating access mechanism is shared not
only by eukaryotic drug exporters but also by bacterial impor-
ters. This is shown by analyses of the type 1 bacterial importers
such as the maltose (MalFGK), histidine (HisPQM), and
methionine (MetNId/l) importers in which low-affinity sub-
strate-binding proteins mediate the delivery of substrates to the
ATP-bound form and also of the type 2 bacterial importers,
such as the vitamin B12 (BtuCD) and metal-chelate (Hi1470/1)
importers which involve a binding protein of high affinity
for the substrate even in the absence of ATP. The delivery
of substrates via a binding protein introduces an additional
conformational switch that is of appreciable structural and
mechanistic divergences between type 1 and type 2 importers.
The multiple conformation changes occurring during the
ATP-driven substrate transport from prokaryotic and eukary-
otic transporters are far from being unraveled; however, at
present, it is best to regard the importers and the exporters as
having partly distinct molecular mechanisms. Moreover, the
Drug
TMD TMD
NBD NBD
Figure 5 The flip-flop hypothesis for drug transport. Coupling between TMDs and NBDs is assured trough conserved – a-helices located in ICLs.
The inward conformation of the TM bundles binds drugs in its cavity which is open both to the cytoplasm and to the inner phospholipid leaflet of the
membrane. Upon binding of ATP, the NBDs dimerize and set the outward conformation in which the drug-binding site is exposed to the external
milieu. ATP hydrolysis releases drugs, disrupts NBD dimerization, and resets the inward conformation.
MDR family of mammalian ABC exporters may have mechan-
isms partially different from that of the Pdrp family of plant
and fungal multidrug transporters. Obviously, the resolution
of more complex mechanisms such as those involved in the
gated choride flux of CFTR, the regulation of the potassium
channel of SUR1, or the loading of antigenic peptide by TAP1
will have to wait until more structural and biochemical infor-
mation become available.
Substrate Specificity of the Multidrug Exporters and
Reversal Agents
One of the most intriguing contemporary biochemical pro-
blems is the characterization of the interactions of the TMDs
with their transported substrate. The most extraordinary fea-
ture in this context is the apparent lack of specificity of the yeast
Pdr5p and human Pgp that transports hundreds of different
chemicals, apparently contradicting the famous key/slot con-
cept described in all textbooks. It is now recognized that at least
three partly overlapping drug-binding sites operate in MDR,
MRP, and Pdrp multidrug transporters and that drug binding
depends on the hydrogen-bound acceptor properties, presence
of aromatic rings, and hydrophobicity properties as well as on
the surface volume of the substrates.
Since 1980, tremendous efforts have been made to identify
inhibitors that can reverse MDR in cancer cells or Pdrp in fungi.
Over 30 compounds with cabalistic names and structures have
been shown to inhibit the cancer-related efflux pumps ABCB1
(MDR), ABCC1 (MRP), or ABCG2 (BCRP), or the fungal Pdrp
transporters.
Such inhibitors may act directly by acting as pseudosub-
strate, as competitive inhibitor of ATP binding or as a noncom-
petitive inhibitor acting at remote sites. They may also act
indirectly on aspects of metabolism that affect efflux. The
variety of identified drug pump inhibitors indicates that the
development of efficient and specific inhibition of the antican-
cer and antifungal pumps should be possible. However, no
TMD TMD
NBD NBD
ATP

ABC pump inhibitor is yet used clinically. Obviously, many
pump inhibitors, such as the antimalarial quinine derivatives
or the immunodepressor FK506 are known to act on diverse
physiological functions. Moreover, ABC drug pumps seem
to be involved in important physiological functions not related
to drug efflux such as the development, differentiation, and
maturation of immune cells. This may explain the multiple
secondary effects of the inhibitors. One may expect that until
the molecular structure and mechanisms of drug efflux are fully
understood, the development of anti-MDR or Pdrp compounds
will remain inefficient and merely based on brute screening. The
ball is clearly in the hand of scientists studying basic mechan-
isms. However, some peculiar clinical treatments have been
considered. For instance, low concentrations of isobuprofen,
which is a potent anti-inflammatory drug, inhibits azole efflux
from Candida albicans, and has been proposed for combination
treatment of fungal infections with fluconazole.
In addition, alternative strategies such as regulating expres-
sion of drug transporters or uptake of anticancer drugs are also
being considered.
Conclusion
The next frontiers in the study of ABC transporters are chal-
lenging. Many aspects of the evolutionary history of this large
ubiquitous family are still to be unraveled. Better heterologous
overexpression systems have to be developed to allow further
biochemical and mechanistic studies. Additional atomic struc-
tures have to be produced to identify all conformation changes
during the ATP-driven transport cycle. The diverse mechanisms
Bioenergetics | ABC Transporters 11
of ABC transporters in bacteria, fungi, plants, and animals have
to be sorted out. Specific inhibitors of ABC drug importers and
exporters have to be screened for. The physiological mechan-
isms of ABC-linked diseases have to be further studied in
mouse knockouts. Systems prone to specific inhibition of
ABC transporters expression by interfering RNA have to be
explored. Genetic therapy of the ABC diseases has to be
developed.
See also: Lipids Carbohydrates Membranes and Membrane
Proteins: Multidrug Resistance Membrane Proteins.
Further Reading
Aller SG, Yu J, Ward A, et al. (2009) Structure of P-glycoprotein reveals a molecular
basis for poly-specific drug binding. Science 323: 1718–1722.
Cannon RD, Holmes AR, Mason AB, et al. (2009) Efflux-mediated antifungal drug
resistance. Clinical Microbiology Reviews 22: 291–321.
Dean M (2002) The Human ATP-Binding Cassette (ABC) Transporter Superfamily,
Monograph. Bethesda, MD: NCBI, National Library of Medicine (US).
Decottignies A and Goffeau A (1997) Complete inventory of the yeast ABC proteins.
Nature Genetics 15: 137–145.
Gottesman MM and Ambudkar SV (2001) Overview: ABC transporters and human
disease. Journal of Bioenergetics and Biomembranes 33: 453–458.
Gutmann DA, Ward A, Urbatsch IL, Chang G, and van Veen W (2010) Understanding
polyspecificity of multidrug ABC transporters: Closing in on the gaps in ABCB1.
Trends in Biochemical Sciences 35: 36–42.
Jones PM and George AM (2004) The ABC transporter structure and mechanism:
Perspective on recent research. Cellular and Molecular Life Sciences 61: 682–699.
Locher K (2009) Structure and mechanism of ATP-binding cassette transporters.
Philosophical Transactions of the Royal Society B Biological Sciences
364: 239–245.
 Algal Hydrogen Production
U M N Murthy and M L Ghirardi, Bioscience Center, National Renewable Energy Laboratory (NREL), Golden, CO, USA
ã 2013 Elsevier Inc. All rights reserved.
Glossary
Catalytic H-cluster The catalytic core of the
[FeFe]-hydrogenase (HydA), termed the ‘H-cluster’, exists
as a [4Fe–4S] subcluster linked by a cysteine thiolate to
a modified 2Fe subcluster with unique nonprotein
ligands.
Fuel cells An electrochemical cell that converts a
source fuel into an electrical current. It generates
electricity inside a cell through reactions between a
fuel and an oxidant, triggered in the presence of an
electrolyte.
Introduction
Fossil fuels have been utilized as a source of energy for many
centuries, resulting in congestion, pollution, and growing
environmental stress. Global oil supplies are rapidly being
depleted and production levels are increasingly being reported
to be close to their peak. More importantly, zero CO2 emission
fuels are becoming increasingly important due to the con-
straints of global climate change. Therefore, the development
of new systems to produce these fuels is one of the greatest
challenges facing our society. Solar energy is the most abun-
dant and accessible renewable energy source available for
future sustainable production of fuel and electricity. For effec-
tive use of solar energy, it is important to develop more cost-
effective systems with the improved ability to convert solar
energy into chemical energy. In this context, hydrogen (H2) is
considered to be a promising, clean energy carrier for future
technologies. The fact that the burning of H2 produces only
H2O increases its attractiveness, particularly in light of recent
advances in hydrogen fuel cell technology. The direct photo-
production of H2 by cyanobacteria and green algae is an alter-
native to utilizing the reductant generated from photosynthetic
water reduction for the fixation of CO2 into carbon-containing
molecules.
Hydrogen metabolism is primarily the domain of bacteria
and microalgae. Within these groups, it involves many taxo-
nomically diverse species, a variety of enzymes, and several
metabolic pathways and processes. Green algae and cyanobac-
teria are photoautotrophic organisms; they can grow under
only sunlight and CO2, without organic sources of carbon.
It has been known for more than 60 years that, under anaero-
bic conditions, unicellular green algae can metabolize H2,
either by uptaking it and using it as an electron donor in
CO2-fixation, or by evolving H2 in the light. Indeed, hydroge-
nase enzymes that either uptake or evolve H2 have been found
in many green algae. This article briefly examines green algal
hydrogen metabolism and how it can be adapted for commer-
cial H2-production processes.
66
Photobioreactor A transparent reactor that allows growth
of cells under illumination; this term mostly refers to closed
systems that have no direct exchange of gases and
contaminants with the environment, as opposed to open
ponds.
Radical SAM proteins A novel protein superfamily with
over 600 members that catalyze diverse reactions, including
unusual methylations, isomerization, sulfur insertion, ring
formation, anaerobic oxidation, and protein radical
formation.
Green Algal Pathways for H2 Production
The unicellular alga Chlamydomonas reinhardtii belongs to the
group of eukaryotic green algae and lives in freshwater or
in moist places. In addition to photosynthesis, unicellular
green algae are able to perform many biotechnologically inter-
esting metabolic reactions, such as fermentation and hydrogen
photoproduction. The starting point for all biological solar-
driven H2-production methods is the process of photosynthesis
(see Figure 1). Oxygenic photosynthesis involves a sequential
chain of reactions that include light absorption, charge separa-
tion, electron transport, and dark CO2 fixation. Oxygenic
organisms harness solar energy to extract electrons from H2O,
which are required for CO2 fixation. Electrons generated from
H2O molecules at photosystem II (PSII) are transferred to the
cytochrome b6/f complex (b6f) and subsequently delivered to
photosystem I (PSI). Through PSI, the electrons are transferred
to ferredoxin (Fd) (a 2Fe–2S protein). Normally, Fd shuttles
electrons to the enzyme ferredoxin–nicotinamide adenine
dinucleotide phosphate (NADP)-oxidoreductase (FNR) that
reduces NADPþ to NADPH, the direct source of reductant to
convert CO2 into carbohydrates in the Calvin–Benson cycle.
Concomitantly, protons (Hþ) are carried from the stromal
to the lumenal side of the thylakoids, creating a proton gradient
across the thylakoid membrane, which is utilized by the
enzyme adenosine triphosphate (ATP) synthase to generate
the ATP that is also required for CO2 fixation. Under anaero-
bic conditions, reduced Fd efficiently binds to an [FeFe]-
hydrogenase and donates electrons to its catalytic site, known
as the ‘H-cluster’ (HC). The function of the hydrogenase is to
combine protons (Hþ) and electrons (e) to form and release
molecular H2. Photosynthetic reducing power can thus be parti-
tioned between at least two pathways: CO2 reduction (under
aerobic conditions) and H2 production (under anaerobiosis). It
is important to point out that CO2 reduction requires ATP,
whereas H2 photoproduction does not. Instead, the latter is
downregulated in the absence of ATP production (see section
H2 Production: Issues and Challenges). As expected, removal

Oxidative
Starch
respiration
NA(P)DH
Low light saturation
Chl
antenna
H+
State transitions
e–
e– e–
PSII PQ pool
H+
H2O + gradient
O2 + H
Figure 1 Photosynthetic electron transport pathways in the green alga, Chlamydomonas reinhardtii. Photosynthesis extracts electron from H2O and
via the electron transport chain delivers them to the [FeFe]-hydrogenase where H2 is generated. The red circles denote the major barriers limiting
technological use of algae for photobiological H2 production. Adapted from Ghirardi ML and Mohanty P (2010) Oxygenic hydrogen
photoproduction – current status of the technology. Current Science 98: 499–507, with permission from Current Science.
of CO2 from the headspace enhances light-driven H2-evolution,
indicating competition for electrons between the CO2-fixation
and the H2-evolution processes.
Oxygenic H2 photoproduction results in the simultaneous
generation of H2 and O2 in an 2:1 ratio as long as O2 is con-
tinuously removed from the headspace. In the absence of active
removal of O2, this mechanism can operate for only 30–90s
since O2 is a powerful inhibitor of the [FeFe]-hydrogenase reac-
tion (and it is also a suppressor of [FeFe]-hydrogenase gene
transcription, which is discussed in detail in later sections).
An alternate source of electrons for H2 photoevolution is
endogenous substrate degradation. This pathway is dependent
on the reduction of the plastoquinone (PQ) pool by NADH
(released from the initial step of glycolysis), in a reaction
catalyzed by the PQ/NAD(P)H-oxidoreductase. Light absorp-
tion by PSI and ensuing electron transport elevates the redox
potential of these electrons to the redox equivalent of ferre-
doxin, thus permitting the generation of molecular H2.
Enzymes for Algal Photohydrogen Production
Hydrogenases are enzymes capable of producing or uptaking
molecular hydrogen. Hydrogenases are classified into three
main groups, according to the cofactor(s) they contain in
their catalytic site, as iron–iron [FeFe], nickel–iron [NiFe], or
Fe-only hydrogenase. Algal hydrogenases belong to the class
of [FeFe]-hydrogenases, they contain only Fe and S in their
catalytic site, and are typically involved in H2 production rather
than H2 oxidation. They catalyze the reversible reduction of
protons to H2:
2Hþ þ 2Fd ! H2 þ 2Fd
[FeFe]-hydrogenases are small, monomeric enzymes of
45–49-kDa size containing a novel type of [Fe–S] cluster com-
monly referred to as the ‘H-cluster’ at their catalytic center.
The HC comprises a conventional [4Fe–S] complex bridged by
Bioenergetics | Algal Hydrogen Production 67
CO2
H2
O2-sensitivity
Chl
antenna
NADPH
FeFe
FNR NADP+
H2ase
Competition for reductant
Fd ATP
ADP + Pi
PSI ATPase
Nondissipated proton
the sulfur atom from a cysteine residue to a unique binuclear
iron–sulfur subcluster. Three other cysteines coordinate the
[4Fe4S]-center to the protein in all known [FeFe]-hydrogenases.
Except for the bridging cysteine, the iron atoms of the 2Fe2S
center are coordinated to carbon monoxide (CO) and cyanide
(CN) ligands, which are responsible for stabilizing the reduced
form of the enzyme, and by a dithiomethylamine group linking
the two S atoms. One of the CO groups is found in a position
bridging both iron atoms when the enzyme is in an oxidized
state, but its orientation has been proposed to change toward
the distal Fe when the enzyme is in the reduced state. Highly
conserved residues comprising several hydrophobic amino acids
are thought to be involved in the formation of H2 channels, thus
connecting the catalytic site to the protein surface.
In C. reinhardtii, a monomeric [FeFe]-hydrogenase of
48 kDa (named HYDA1) with high specific activity was first
isolated and biochemically characterized. More recent research
revealed the presence and expression of a second [FeFe]-
hydrogenase (HYDA2) in C. reinhardtii that is 74% similar
and 68% identical to HYDA1. The transcripts for both hydro-
genases, HYDA1 and HYDA2, are selectively expressed upon
dark anaerobic incubation, suggesting regulation of gene
expression by O2. HYDA1, and most probably HYDA2, directly
interact with reduced ferredoxin. The nucleus-encoded algal
hydrogenases are synthesized in the cytosol as precursor pro-
teins but the mature proteins are localized in the chloroplast
stroma. A transit peptide domain that routes the [FeFe]-
hydrogenases from the cytoplasm across the chloroplast enve-
lope and into the chloroplast stroma has been identified at the
N-terminal region of the enzyme.
[FeFe]-Hydrogenases Are Sensitive to O2
The metallo-cluster of the [FeFe]-hydrogenases is very sensitive
to O2. This observation, together with the fact that hydrogenases
are located in the chloroplast, where PSII releases O2, causes H2
68 Bioenergetics | Algal Hydrogen Production
production rates to be usually low. In algal cells, O2 can
adversely affect enzymatic activity by deactivating previously
assembled [FeFe]-hydrogenases, and the isolated enzyme com-
petes with CO for the same binding site in the HC of [FeFe]-
hydrogenases. Oxygen inactivation is thought to occur by the
direct interaction of O2 with the [2Fe–2S] center on catalytic
HC. In contrast to O2 inactivation, inhibition by CO is largely
but not entirely reversible. The nature of the final oxygen
species bound to the inactivated enzyme is not known. How-
ever, it is known that diffusion of O2 gas is necessary for the
inactivation of the enzyme, since the catalytic site of the
enzyme is embedded in the protein structure.
Melis and his coworkers in 2000 demonstrated a novel
strategy to sustain green algal H2 production in the light.
The O2 sensitivity of H2 metabolism was bypassed in a two-
stage process, in which O2-evolution and H2-evolution reactions
were temporarily separated. In the first stage, normal photo-
synthesis, CO2-fixation, and release of O2 upon oxidation of
H2O enabled green algae to survive. In the second stage, the
authors selectively and partially inactivated the O2-evolving
activity of PSII by incubating algal cultures in a sulfur-depleted
medium. Since respiratory O2 consumption is not affected
by sulfur deprivation, inactivation of O2 evolution facilitated
the transition from the aerobic to an anaerobic state in a sealed
photobioreactor. The establishment of anaerobiosis in the
photobioreactor induced the expression of the two [FeFe]-
hydrogenases in algal cells and inhibited the expression of
ribulose-1,5-bisphosphate carboxylase oxygenase (Rubisco).
This resulted in redirection of the flow of electrons from the
photosynthetic electron transport chain in the chloroplast
away from carbon fixation and towards proton reduction. As
a consequence, sulfur-deprived algae were able to produce H2
for a total of 3–4 days.
[FeFe]-Hydrogenase Maturation
Biosynthesis of an active [FeFe]-hydrogenase is a complex
process. [FeFe]-hydrogenase maturation proteins were initially
discovered in the green alga C. reinhardtii. Posewitz et al. showed
that two novel radical S-adenosyl-methionine (radical SAM)
proteins, HYDEF and HYDG, are required for C. reinhardtii
[FeFe]-hydrogenase activity. Using a chemochromic sensor,
a hydef mutant that was unable to produce hydrogen was
isolated from a C. reinhardtii mutagenesis library. Moreover,
the HYDEF gene was found to be adjacent to the HYDG
gene, and both were shown to be present in all other [FeFe]-
hydrogenase-containing organisms, although their role was
unknown at that time. In the absence of a functional HYDEF,
both hydrogenase structural genes were induced but no
hydrogenase activity was detected in the mutant. Complemen-
tation of the mutant with genomic DNA containing a func-
tional copy of the HYDEF gene restored hydrogenase activity in
the algae. Later on, King et al. showed that the heterologous
expression of active algal [FeFe]-hydrogenase in the bacterium,
Escherichia coli, could be accomplished by co-transforming the
HYDA1 structural gene along with both the HYDEF and HYDG
genes, and that all these genes were required and sufficient for
the expression of a functional [FeFe]-hydrogenase in this bac-
terium. The algal HYDEF gene encodes for two unique protein
segments, HYDE and HYDF, that are exclusively found in other
organisms containing [FeFe]-hydrogenase but encoded as two
separate genes, HydE and HydF. The HydE proteins are highly
homologous to the biotin synthase radical SAM proteins, and
HYDF shares homology with the large family of nucleoside
triphosphate (NTP)-binding proteins.
The specific role of HydE, HydF, and HydG proteins in
[FeFe]-hydrogenase maturation is not clearly understood yet.
Peters et al. proposed that the maturation proteins use com-
mon amino acids or other metabolites to synthesize a pre-
cursor to the [FeFe]-hydrogenase active site, which is then
transferred to the structural enzyme. HydE and HydG act on
HydF to assemble an active-site precursor containing all the
nonprotein ligands of the binuclear Fe subcluster, which is
subsequently transferred to the hydrogenase. It seems that
HydE is probably responsible for the synthesis of CO and
CN ligands and HydG for the dithiomethylamine ligand.
The dithiomethylamine-bridged cluster is then transferred as
a whole to HydF to generate a complete, functional HC for
insertion to the [FeFe]-hydrogenase apoprotein.
The Interaction of [FeFe]-Hydrogenases with
Ferredoxin
In green algae, H2 production couples to the photosynthetic
electron transport chain via ferredoxin. Ferredoxins are small
soluble [Fe–S] proteins which can be found in every phyloge-
netic group. They are mainly low-potential electron carriers
involved in various metabolic pathways having diverse redox
partners. Ferredoxin distributes photosynthetic electrons not
only to hydrogenase but also to other electron-dependent
enzymes, the most important of which is ferredoxin-NAD(P)-
reductase (FNR) for CO2 fixation.
There are six ferredoxin genes in C. reinhardtii. The physio-
logical regulation and metabolic function of each of the six
ferredoxins has not yet been established, although it is assumed
that both hydrogenases may associate with the photosynthetic
electron transfer ferredoxin (PetF, also called FDX1) which
is involved in photosynthesis and it is a typical plant-type
[2Fe–2S] ferredoxin. Recently, Winkler et al. characterized the
kinetics of electron transfer processes between HydA1 and
PetF. Using an in vitro system by reconstituting part of the
photosynthetic electron transport chain consisting of plastocy-
anin, PSI, PetF, and [FeFe]-hydrogenase, they demonstrated
that residue K396 of HydA1 is crucial for a successful binding
and electron transfer between PetF and HydA1. However, other
studies by Terauchi et al. found that each of the six FDX genes is
differentially regulated in response to changes in nutrient sup-
ply and/or anoxia. In this regard, more detailed understanding
of the hydrogenase–ferredoxin association mechanism and
kinetics is necessary for engineering hydrogenase to divert a
greater electron flux to the H2 production pathway and thus to
optimize photobiological H2 production.
H2 Production: Issues and Challenges
Photobiological H2 production by C. reinhardtii has become an
important research field because of its potential to be applied

in renewable energy production. To realize the economic
potential of producing hydrogen photobiologically, several
challenges must be overcome. Foremost, cellular metabolism
and the basic biochemistry supporting this process must be
understood. Following are the major issues and challenges that
need to be addressed.
O2 Sensitivity
The simultaneous photoproduction of H2 and O2 from H2O
in green alga depends on O2 tolerance of the reversible
hydrogenase enzyme. When oxygenic photosynthesis is active,
and unless strict measures are taken to remove O2, green algal
H2-production activity is transient owing to the rapid inactiva-
tion of the reversible hydrogenase by photosynthetic O2. Sus-
tained hydrogen production will only be possible when the
hydrogenase enzyme remains active under O2-evolving condi-
tions. Different strategies for development of O2 tolerant
hydrogenases have included molecular engineering of the
hydrogenases by either random or site-directed mutagenesis
to remove O2 sensitivity. The latter has mostly focused on
physical restriction of O2 access catalytic site by reducing the
O2 diffusion rate or shielding the active catalytic site. Although
the results are encouraging from a scientific perspective in
understanding enzyme structure and function, none of these
approaches has yielded biotechnologically significant hydrogen-
producing strains yet.
An alternative approach to sustain H2 production in the
light is to use sulfur deprivation to partially inactive O2 evolu-
tion, as discussed above. The novel application of this process
has led to significant and sustainable light-dependent release
of H2 by algal cultures, although at low-conversion efficiencies
due to limited PSII activity. Several approaches have been
examined to increase the yield of hydrogen in C. reinhardtii
under sulfur-deprived condition, including optimization of the
light and pH conditions in the photobioreactors optimization
of the medium composition and synchronization of cell divi-
sion (see Figure 2). Kruse et al. reported a significant increase
in the rate and duration of H2 photoproduction in sulfur-
deprived mutants that are starch over-accumulators and
blocked in state transitions. Another advance related to the
sulfur-deprived process came with the recent discovery of a
mutant affected in sulfate permease activity, which is required
for transport of sulfate into the chloroplast. This mutant is a
potential candidate for H2 photoproduction without the need
to totally remove sulfate from the growth medium.
The reported rates of H2 production by sulfur-deprived
cultures is still far below the maximum potential rate of H2
photoproduction for an algal system, mainly due to partial
inactivation of PSII. Furthermore, the system is difficult to
scale up and maintain. These challenges, which are related
partly to the photobioreactor design, might be addressed by
immobilizing the green algae. The immobilization technique
effectively separates cells from the liquid phase, significantly
increases the cell density and, as a consequence, allows for
more efficient light utilization on a per area basis. Laurinavi-
chene, et al. in 2006 demonstrated that sulfur-deprived, immo-
bilized cells of a nonmotile mutant of C. reinhardtii on glass
fibers significantly increase the duration of H2 production.
More recently, Kosourov and Seibert, demonstrated that
Bioenergetics | Algal Hydrogen Production 69
Figure 2 Photograph of the photobioreactor used for H2 production
from Chlamydomonas reinhardtii culture showing probes used for
monitoring and the controlling the system. Hydrogen bubbles
emanate toward the surface of the culture and are collected in a
liquid-accumulating bottle (not shown).
immobilization of sulfur/phosphorous-deprived algae in thin
Ca2þ-alginate film matrices has extended H2 photoproduction
for more than 150 h and also increased the stability of H2
photoproduction activity in the presence of atmospheric O2.
This work shows potential for further significant improve-
ments in both the rate and efficiency of H2 production under
sulfur-deprived conditions.
Reduced Antenna Size
Photosynthesis and H2 production in unicellular green algae
can in principle operate with a nearly 100% photon utiliza-
tion efficiency, making them potentially efficient biocatalysts
for the generation of H2 from sunlight and water. However,
green algal cultures show poor light utilization efficiency under
direct sun light and can waste up to 80% of the absorbed
irradiance. The reason for this is that green algal photosynthe-
sis normally saturates at about one-fifth of full sunlight inten-
sity or less. This optical property of the cells would further
lower the productivity of a commercial hydrogen production.
Enhanced hydrogen production may be achieved by engineer-
ing the antenna size to suppress fluorescence and heat dissipa-
tion that causes a reduction on antenna efficiency.
Competition for Reductants
Competition from the CO2-fixation pathway for electrons
from H2O is a major concern for sustained H2 production,
as discussed earlier in the article. This concern is based on the
higher affinity of ferredoxin for FNR than hydrogenase, which
could result in substantial drainage of electrons away from H2
production. Therefore, improved H2 production depends on
interaction of [FeFe]-hydrogenases with the electron-carrying
protein ferredoxin. Agapakis et al. fused the hydrogenase
from C. acetobutylicum to ferredoxin from spinach or from
70 Bioenergetics | Algal Hydrogen Production
C. acetobutylicum using flexible protein linkers of various
lengths and showed a threefold increase in H2 production by
a nonphotosynthetic, recombinant E. coli system. Moreover,
chimeras between ferredoxin and various ferredoxin reductases
have been shown to be functional in vitro, with improved
electron transfer rates, presumably due to the increased local
concentration of reduced ferredoxin. This approach may pro-
vide new targets for metabolic engineering of H2 production in
the future.
Downregulation of Electron Transport by the Proton Gradient
Under sulfur-deprived conditions, very little CO2 is fixed into
glucose, mainly because of the decrease in Rubisco levels.
These sulfur-deprived, anaerobic cultures use very little ATP,
as a consequence, the proton gradient generated by photo-
synthetic electron transport is not dissipated. This causes a
decrease in the rate of electron transport as demonstrated by
the acceleration in the rate of H2 production observed upon
addition of uncouplers. Although, mutants defective in one or
other ATPase subunits have been generated, their uncoupling
properties are yet to be studied. These mutants may be able to
circumvent the cellular regulatory process that downregulate
PSII activity at the level of the plastoquinone pool and limit H2
production.
State Transition
Oxygenic photosynthetic organisms adapt to varying energetic
requirements by transitioning between linear and cyclic elec-
tron transport, through the so-called state transitions. If the
ATP requirement is higher than what is provided by linear
electron transport alone, the cultures transition to state 2,
where cyclic electron transport predominates. Anaerobiosis
leads to the establishment of state 2 and, as a result, additional
limitations are placed on the rates of H2 photoproduction.
The relevance of the state 2 limitation on H2 photoproduction
by sulfur-deprived culture was recently demonstrated by Kruse
et al., who used a chlorophyll fluorescence video-imaging
screening procedure to identify mutants of C. reinhardtii
blocked in state 1. They isolated one mutant, stm6, with rates
of H2 photoproduction 13 times higher than its wild-type
strain, and with extended H2 photoproduction activity under
sulfur deprivation. These results underscore the importance of
molecular engineering to address this particular limitation to
algal H2 production technologies.
Conclusion
Biological hydrogen production is not only a challenging but
also promising biotechnology area, with the potential to help
solve environmental and energy-related problems. The applica-
tion of biological H2 production as a mature technology, how-
ever, depends on research advances to address improvements in
H2-production rates and efficiency through a combination of
genetically engineering microorganisms and developing bior-
eactors. Significant progress is being made in understanding
the molecular regulation of the genes encoding hydrogenases
and accessory proteins needed for assembly of hydrogenases,
as well as in identifying and addressing the major barriers to a
future commercial process. With increasing interest from the
scientific community and the public in clean and renewable
energy sources, rapid progress could bring this technology
to fruition in the not-so-long future. Moreover, the possibilities
and challenges within synthetic biology should be further
explored for creating green algae with a high potential for H2
production.
See also: Bioenergetics: Ferredoxin; Hydrogenases, Structure and
Function; Renewable Hydrogen from Biomass.
Further Reading
Agapakis CM, Ducat DC, Boyle PM, et al. (2010) Insulation of a synthetic hydrogen
metabolism circuit in bacteria. Journal of Biological Engineering 4: 3.
Ghirardi ML (2006) Hydrogen production by photosynthetic green algae. Indian Journal
of Biochemistry and Biophysics 43: 201–210.
Ghirardi ML, Dubini A, Yu JP, and Maness PC (2009) Photobiological
hydrogen-producing systems. Chemical Society Reviews 38: 52–61.
Ghirardi ML and Mohanty P (2010) Oxygenic hydrogen photoproduction – current
status of the technology. Current Science 98: 499–507.
Ghirardi ML, Posewitz MC, Maness PC, et al. (2007) Hydrogenases and hydrogen
photoproduction in oxygenic photosynthetic organisms. Annual Review of Plant
Biology 58: 71–91.
Ghirardi ML, Zhang JP, Lee JW, et al. (2000) Microalgae: A green source of renewable
H-2. Trends in Biotechnology 18: 506–511.
King PW, Posewitz MC, Ghirardi ML, and Seibert M (2006) Functional studies of [FeFe]
hydrogenase maturation in an Escherichia coli biosynthetic system. Journal of
Bacteriology 188: 2163–2172.
Kruse O, Rupprecht J, Bader KP, et al. (2005) Improved photobiological H-2 production
in engineered green algal cells. Journal of Biological Chemistry 280: 34170–34177.
Laurinavichene TV, Fedorov AS, Ghirardi ML, Seibert M, and Tsygankov AA (2006)
Demonstration of sustained hydrogen photoproduction by immobilized,
sulfur-deprived Chlamydomonas reinhardtii cells. International Journal of
Hydrogen Energy 31: 659–667.
Melis A and Happe T (2001) Hydrogen production. Green algae as a source of energy.
Plant Physiology 127: 740–748.
Melis A, Seibert M, and Ghirardi ML (2007) Hydrogen fuel production by transgenic
microalgae. Transgenic Microalgae as Green Cell Factories 616: 108–121.
Melis A, Zhang LP, Forestier M, Ghirardi ML, and Seibert M (2000) Sustained
photobiological hydrogen gas production upon reversible inactivation of oxygen
evolution in the green alga Chlamydomonas reinhardtii. Plant Physiology
122: 127–135.
Mus F, Dubini A, Seibert M, Posewitz MC, and Grossman AR (2007) Anaerobic
acclimation in Chlamydomonas reinhardtii – anoxic gene expression, hydrogenase
induction, and metabolic pathways. Journal of Biological Chemistry
282: 25475–25486.
Peters JW, Szilagyi RK, Naumov A, and Douglas T (2006) A radical solution for the
biosynthesis of the H-cluster of hydrogenase. FEBS Letters 580: 363–367.
Posewitz MC, King PW, Smolinski SL, et al. (2004) Discovery of two novel radical
S-adenosylmethionine proteins required for the assembly of an active [Fe]
hydrogenase. Journal of Biological Chemistry 279: 25711–25720.
Prince RC and Kheshgi HS (2005) The photobiological production of hydrogen:
Potential efficiency and effectiveness as a renewable fuel. Critical Reviews in
Microbiology 31: 19–31.
Stripp ST and Happe T (2009) How algae produce hydrogen–news from the
photosynthetic hydrogenase. Dalton Transactions 45: 9960–9969.
Terauchi AM, Lu SF, Zaffagnini M, et al. (2009) Pattern of expression and substrate
specificity of chloroplast ferredoxins from Chlamydomonas reinhardtii. Journal of
Biological Chemistry 284: 25867–25878.
Winkler M, Kuhlgert S, Hippler M, and Happe T (2009) Characterization of the key
step for light-driven hydrogen evolution in green algae. Journal of Biological
Chemistry 284: 36620–36627.
 Amine Oxidases
G Floris, University of Cagliari, Monserrato (CA), Italy
A Finazzi Agrò, University of Rome, Rome, Italy
ã 2013 Elsevier Inc. All rights reserved.
Glossary
Amines Hydrocarbon compounds bearing an amine group.
They are called primary, secondary, or tertiary when
nitrogen binds two, one, or zero hydrogen atoms.
Oxidases Enzymes that oxidize substrates using
molecular oxygen.
Polyamines Hydrocarbon compounds bearing
both primary and secondary amino groups
Classification
The oxidative deamination of mono-, di-, and polyamines is
catalyzed by a number of enzymes called ‘amine oxidases’
(AOs) that exhibit different patterns of substrate specificity
and inhibitor sensitivity and also differ in their action mecha-
nism. AOs are divided into two main categories, depending on
the nature of the cofactor involved. Monoamine oxidases
(MAOs) and polyamine oxidases (PAOs) belong to the first
class and utilize flavin adenine dinucleotide (FAD) as the
redox cofactor (Figure 1(a)). The second class is represented
by enzymes having a tightly bound CuII ion and a carbonyl-type
moiety identified as 6-hydroxydopa quinone (2,4,5-trihydrox-
yphenethylamine quinone; TPQ or TOPA) at the active site
(Figure 1(b)).
Distribution, Reaction Mechanism, and
Physiological Roles
Monoamine Oxidases
Two forms of MAOs (MAOs, amine: oxygen oxidoreductase
(deaminating, flavin-containing); EC 1.4.3.4) have been de-
scribed, namely MAO A and MAO B, the former made by 527
amino acid residues and the latter by 520, with an overall 70%
identity. Both enzymes have a FAD covalently bound through
its C8a-position to a cysteine (Cys406 in MAO A and Cys397 in
MAO B) side chain (Figure 1(a)). Both forms of MAOs have
been crystallized from many different sources.
MAOs are ubiquitous in cells of most mammalian species,
the notable exception being erythrocytes, and are tightly asso-
ciated with the mitochondrial outer membrane. The distribu-
tion of MAOs has been mainly studied in brain: MAO A is
found in catecholaminergic neurons, whereas MAO B is abun-
dant in serotonergic and histaminergic neurons and glial cells.
Some tissues mainly contain MAO A (human placenta and
bovine tyroid), while other tissues contain predominantly
MAO B (human platelets and bovine liver and kidney).
MAOs oxidize primary amino groups of arylalkyl amines to
form an imine intermediate with the concomitant reduction
of flavin to FADH2. The imine is then hydrolyzed to the
(e.g., spermine and sperimidine) strongly interacting
with nucleic acids involved in many important cellular
functions.
Primary amines Metabolically derived from amino
acids by decarboxylation. Primary amines often
show potent pharmacological or hormonal activity
(e.g., histamine, serotonin, GABA, and
noradrenaline).
corresponding aldehyde and ammonia, and FADH2 is oxidized
back to FAD by oxygen with the formation of hydrogen perox-
ide. MAO A oxidizes dopamine, noradrenaline, and serotonin
whereas MAO B preferentially oxidizes benzylamine and phen-
ylethylamine. Adrenaline, kynuramine, tyramine, and trypt-
amine are substrates for both enzymes.
MAO A and B have been implicated in apoptosis, immuno-
suppression, cytotoxicity, cell growth, and proliferation. These
enzymes play a protective role in the body by preventing the
entry of amines at renal and intestinal level or by oxidizing
them in blood. Liver MAOs are important for the control of
blood pressure by regulating the amines level. For instance,
MAO A can oxidize circulating serotonin thus preventing its
effects on heart and vascular system. Both enzymes have
important roles in the metabolism of neurotransmitters and
other biogenic amines in the brain and are implicated in a large
number of neurological and psychiatric disorders. As a matter
of fact, MAO A inhibitors are used as antidepressants whereas
MAO B inhibitors have an important role in the treatment of
Parkinson’s disease.
A further important role of MAOs is recently emerging, that
is, their role in the demethylation of histone lysines and argi-
nines, an essential process in the regulation of gene expression
and genomic imprinting.
Polyamine Oxidases
PAOs (N1-acetylspermidine:oxidoreductase (deaminating); EC
1.5.3.11) have been classified as part of flavoprotein superfam-
ily but in this case FAD is noncovalently bound to the protein.
PAOs are monomeric enzymes with a molecular mass ranging
from 53 kDa to 63 kDa. PAOs are found in vertebrates
and plants. Enzymes with characteristics similar to those of
vertebrates’ enzymes are present in yeasts and amoebae,
whereas bacteria and protozoans contain enzymes resembling
those of plants. Plant PAOs have been isolated and character-
ized, particularly from the Gramineae oat, maize, barley,
wheat, and rye. The enzyme from maize seedlings has been
crystallized.
Much alike the catalytic mechanism of MAOs, the oxidation
of polyamines can be divided into two half-reactions: (1) flavin
87
88 Bioenergetics | Amine Oxidases

reduction upon polyamine oxidation followed by (2) flavin PAOs preferentially oxidize acetyl spermine and acetyl spermi-
reoxidation by molecular oxygen. PAOs catalyze the oxidation dine: spermidine and putrescine are the respective reaction pro-
of polyamines’ secondary amino groups yielding different pro- ducts, together with 3-aminopropanal and hydrogen peroxide.
ducts depending on the organism considered. Mammalian Oxidation of spermine by plant PAOs gives 1,3-diaminopro-
pane, hydrogen peroxide, and 1-(3-aminopropyl)-4-aminobu-
tanal. The latter spontaneously cyclizes to 1-(3-aminopropyl)
MAO O pyrrolinium that undergoes further spontaneous rearrange-
ments to 1,5-diazobicyclo[4.3.0.]nonane (Scheme 1). The oxi-
NH CH C
S R dation of spermidine by plant PAOs gives 1,3-diaminopropane,
CH2 hydrogen peroxide, and 4-aminobutanal that yields 1-pyrroline
H2C N N O
O by spontaneous cyclization. At variance with MAOs and copper/
topaquinone (TPQ) AOs, the oxidation of polyamines by PAOs
N
H3C N H does not release ammonia.
O PAOs play an important role in the regulation of intracellu-
O
(b) O lar polyamines level, and seem to be important for homeosta-
(a)
sis and cell survival.
O
NH CH C Copper Amine Oxidases
H CH2 Copper amine oxidases (amine: oxygen oxidoreductase (dea-
CH2 N minating; copper containing); EC 1.4.3.6) (CAOs) are homo-
CH2 dimers in which each subunit (Mw 70–90 kDa) contains a
tightly bound type II copper ion and a TPQ. TPQ is derived
CH2 O
from a posttranslational modification of a tyrosyl residue,
CH2 O found in a strictly conserved amino acid consensus sequence
NH CH asn-tyr-asp/glu (NYD/E). The pink color of CAOs stems from
an intense absorption band around 500 nm due to the oxi-
C O
dized TPQ. CAOs catalyze the oxidative deamination of pri-
(c)
mary amino groups of mono-, di-, and polyamines, abstracting
Figure 1 Structure of the (a) FAD covalently bound in MAOs; (b) the two electrons from amines and transferring them to molecular
oxidized form of TPQ; and (c) lysyl tyrosylquinone. oxygen, to form the corresponding aldehyde, ammonia, and

(P)
(1)
+ O2, H2O
H2N (CH2)3 NH (CH2)4 NH2 H2N (CH2)3 NH2 + HOC(CH2)3 NH2
− H2O2

+H2O −H2O

N
(P)
(a)
(2)
+ O2, H2O
H2N (CH2)3 NH (CH2)4 NH (CH2)3 NH2 H2N (CH2)3 NH2 + HOC (CH2)3 NH (CH2)3 NH2
−H2O2

+H2O −H2O

H +H+
N N NH2 (CH2)3 N
−H+
(c) (b)
(M)
(3)
+ O2, H2O
H2N (CH2)3 NH (CH2)4 NH2 H2N (CH2)2 CHO + H2N (CH2)4 NH2
−H2O2

(M)
(4)
+ O2, H2O
H2N (CH2)3 NH (CH2)4 NH (CH2)3 NH2 H2N (CH2)2 CHO + NH2 (CH2)4 NH (CH2)3 NH2
−H2O2

Scheme 1 Spermidine and spermine oxidation catalyzed by plant (P) and mammalian (M) PAOs. Spermidine (1, 3); Spermine (2, 4); (a) 1-pyrroline;
(b) 1-(3-aminopropyl)-pyrrolinium; and (c) 1,5-diazobicyclo[4.3.0.]nonane. Arrows indicate the cleavage of polyamines.

hydrogen peroxide. Again, the catalytic mechanism can be
divided into two half-reactions: (1) enzyme reduction at the
quinone moiety (TPQ ! TPQH2) by substrate followed by (2)
reoxidation by molecular oxygen
Eox þ R  CH2  NHþ þ
3 ! Ered  NH3 þ R  CHO [1]
Ered  NHþ þ
3 þ O2 þ H2 O ! Eox þ NH4 þ H2 O2 [2]
The group of CAOs includes the following:
1. Diamine oxidases (DAOs), ubiquitous enzymes occurring
in prokaryotes as well as eukaryotes (bacteria, fungi, plants,
and animals). Several DAOs have been crystallized from
bacteria, from the yeast Hansenula polymorpha, and from
pea seedlings. Plant DAOs from various species have been
purified to homogeneity and characterized, the best known
and studied being those from lentil (Lens esculenta) and pea
(Pisum sativum) and from latex of the shrub Euphorbia char-
acias. In mammals, the best-known enzymes are those from
pig kidney and intestine and human placenta. DAOs are
mainly active on short aliphatic diamines, such as putrescine
(1,4 diaminobutane) and cadaverine (1,5 diaminopentane).
The physiological role of DAOs is multifaceted, because
this class includes several enzymes with different localiza-
tions and substrates. Bacteria and yeasts can utilize amines
as nitrogen and carbon sources through the reaction with
AO. Plant DAOs are implicated in wound healing, in detox-
ification, and in cell growth by regulating the intracellular
di- and polyamine levels, and the aldehyde products might
have a key role in the biosynthesis of some alkaloids. The
function of DAOs in mammals is even more diverse and
elusive and a plethora of physiological functions, some-
times opposite, have been described. AO activity is found
in many tissues, the highest levels being in decidual cells of
placenta, in kidney tubular epithelial cells, and in intestinal
epithelial cells. These localizations may suggest a barrier
function for DAOs to prevent the entry of diamines and
polyamines in the body. Furthermore, these enzymes may
keep at bay the endogenous histamine, which otherwise
can cause several pathological conditions such as allergy,
peptic ulcer, and anaphylactic reactions. Several observa-
tions point to a relationship between AO activity and
growth, both in normal and tumor tissues, that is, in cell
proliferation and differentiation. DAOs have also been pro-
posed as immune response modulators. It has been demon-
strated that human placental DAO is identical to the
amiloride-binding protein and thus in some way involved
in the regulation of epithelial ion transport. In analogy with
MAOs, also DAOs have been proposed to be involved in
demethylation of histones, in particular of methylated argi-
nines. In this case, their importance might be not only the
modification of histones but also the production of hydro-
gen peroxide just at nuclear level.
2. The mammalian extracellular soluble and intracellular
tissue-bound AOs are able to metabolize mono-, di-, and
polyamines, even though in vitro they are preferentially active
toward nonphysiological amines such as benzylamine. Extra-
cellular AOs are generally called either plasma and serum
AOs or benzylamine oxidases (BzAOs). The better-known
BzAOs are those from bovine, swine, and equine plasma.
Bioenergetics | Amine Oxidases 89
The enzyme from bovine plasma has been crystallized.
Tissue-bound AOs are often indicated somewhat mislead-
ingly as semicarbazide-sensitive amine oxidases (SSAOs),
since all CAOs, at variance with FAD-dependent enzymes,
are inhibited by semicarbazide and other carbonyl reagents.
Intracellular SSAOs are widely distributed: smooth muscle
cells of vascular tissue of all mammalian species are good
sources of these enzymes, which have been also detected in
uterus, ureter and vas deferens, in ox dental pulp, in human
umbilical artery, in rat adipocytes, and chondrocytes.
Plasma AOs may be considered key enzymes in cell
growth and differentiation processes and, thus, in tumor
development and growth regulation. Moreover, the
increased BzAO activity in blood serum of pregnant
women supports the possibility that this enzyme has a
protective role against the polyamines released from the
fetoplacental unit. SSAOs seem to play an important role
in vascular wall. Moreover, these enzymes might be
involved in the regulation of glucose metabolism (perhaps
via the H2O2 produced) and in the regulation of leukocyte
trafficking in endothelial cells. SSAOs have also been shown
to be a new class of DNA-binding proteins: in the presence of
polyamines, they bind DNA and oxidize DNA-bound poly-
amines. A structural similarity between SSAOs and VAP-1, a
protein involved in cellular adhesion, has been observed and
a human SSAO/VAP-1 protein has been crystallized.
3. The mammalian extracellular matrix-bound lysyl oxidase
[EC 1.4.3.13] (LOX), a 30-kDa protein, differs from other
members of the group because a lysyl tyrosyl quinone
(LTQ) rather than TPQ is the redox cofactor (Figure 1(c)).
LTQ is formed by oxidation of a conserved tyrosine residue
and subsequent intramolecular cross-linking with the
e-amino group of a conserved lysine. LOX catalyzes the
oxidative deamination of lysyl residues in elastin and col-
lagen allowing the formation of cross-links essential for the
structure of these proteins. The genetic or acquired decrease
of LOX activity is accompanied by severe pathological con-
ditions such as tendon laxity and formation of aneurisms
in arteries.
A LOX from Pichia pastoris has been crystallized and
characterized. This enzyme, cloned and overexpressed,
shows TPQ as the redox cofactor. Pichia pastoris LOX dis-
plays catalytic activity toward tropoelastin (the un-cross-
linked form of elastin), and lysine and lysine-containing
peptides are the preferred substrates for this enzyme. In this
respect, it resembles the LTQ-containing mammalian AOs.
Concluding Remarks
In the past few years, evidence has accumulated about the
physiological relevance of hydrogen peroxide, which is gener-
ated in the catabolism of mono-, di-, and polyamines by all
AOs. Hydrogen peroxide, which is always formed in the reac-
tions catalyzed by AOs, is more and more considered either a
crucial reagent for important biochemical processes or a signal
and a defense molecule, rather than a noxious waste product.
This reactive oxygen species is toxic at high concentration.
However, at lower concentrations it regulates the cell number
90 Bioenergetics | Amine Oxidases
during embryonic development as well as the proliferation and
adhesive properties of endothelial and smooth muscle cells.
Hydrogen peroxide appears to be involved in the impairment
of cell growth and proliferation, in the regulation of many
genes and transcription factors, and in the transduction of
cellular signals in many living species. In plants, hydrogen
peroxide might be utilized by peroxidases in basic physiologi-
cal events, such as development, polymerization of lignin and
suberin precursors, and catabolism of indoleacetic acid, in
response to wounding or to pathogen invasion.
See also: Bioenergetics: Quinones.
Further Reading
Binda C, Coda A, Angelini R, et al. (1998) Crystallization and preliminary X-ray analysis
of polyamine oxidase from Zea mays L. Acta Crystallographica Section D
54: 1429–1431.
Ciccone DN, Su H, Hevi S, et al. (2009) KDM1B is a histone H3K4
demethylase required to establish maternal genomic imprints. Nature 461:
415–419.
Dandrifosse G (ed.) (2009) Biological Aspects of Biogenic Amines, Polyamines and
Conjugates. Trivandrum, India: Transworld Research Network.
De Colibus L, Li M, Binda C, et al. (2005) Three-dimensional structure of human
monoamine oxidase A (MAO A): Relation to the structures of rat MAO A and human
MAO B. Proceedings of the National Academy of Sciences of the United States
of America 102: 12684–12689.
Duff AP, Cohen AE, Ellis PJ, et al. (2003) The crystal structure of Pichia pastoris lysyl
oxidase. Biochemistry 42: 15148–15157.
Floris G and Mondovı̀ B (eds.) (2009) Copper Amine Oxidases. Boca Raton, London
NY: CRC Press, Taylor & Francis Group.
Jalkanen S and Salmi M (2001) Cell surface monoamine oxidases: Enzymes in
search of a function. New EMBO member’s review. The EMBO Journal 20:
3893–3901.
Janes SM, Mu D, Wemmer D, et al. (1990) A new redox cofactor in eukaryotic enzymes:
6-hydroxydopa at the active site of bovine serum amine oxidase. Science
248: 981–987.
Mure M, Mills SA, and Klinman JP (2002) Current topics. Catalytic mechanism
of the Topa quinone containing copper amine oxidases. Biochemistry
41: 9269–9278.
Shi Y, Lan F, Matson C, et al. (2004) Histone demethylation mediated by the nuclear
amine oxidase homolog LSD1. Cell 119: 941–953.
 Anaplerosis
R R Russell III, Yale University School of Medicine, New Haven, CT, USA
H Taegtmeyer, The University of Texas Medical School at Houston, Houston, TX, USA
ã 2013 Elsevier Inc. All rights reserved.
Glossary
Anaplerosis The entry of substrates into the citric acid cycle
as intermediates other than acetyl-CoA, thereby increasing
the citric acid cycle pool size.
Carboxylation The introduction of a carboxyl group into a
compound.
The word anaplerosis (from the Greek, meaning to fill up) was
coined in the 1960s by Sir Hans Kornberg to describe pathways
that replenish intermediates in metabolic cycles. A metabolic
cycle transforms a substrate with little or no change in the
concentration of the cycle’s constituents, the central process
in energy substrate metabolism. A classic example for a meta-
bolic cycle is the citric acid cycle. Here anaplerotic pathways
mediate the entry of metabolites into the cycle as compounds
other than acetyl-CoA and thereby replenish the pool size of
the citric acid cycle. Other anaplerotic pathways are present in
eukaryotic and prokaryotic organisms; however, this article
focuses on anaplerosis as it relates to the citric acid cycle in
mammalian tissues and the importance of anaplerosis in the
maintenance of organ function.
Cycles have evolved because in a competitive environment
the chances for survival are greatest if resources are used in an
optimal way. For example, energy transfer in heart muscle is
linked to a series of cycles, beginning with the circulation and
ending with actin–myosin cross-bridge formation (Figure 1).
Steady-state concentrations of metabolic intermediates in a
cycle depend on their rates of synthesis and degradation. In
other words, substrates entering the citric acid cycle as citrate
(via the condensation of acetyl-CoA with oxaloacetate) do not
cause a net change in the citric acid cycle pool size because the two
carbons of acetyl-CoA are ultimately lost as CO2 in the isocitrate
and the a-ketoglutarate dehydrogenase reactions, respectively. In
contrast, anaplerosis supplies compounds to the citric acid cycle
through reactions other than those catalyzed by citrate synthase.
These compounds replenish carbon intermediates lost primarily
as amino acids (glutamate or aspartate) or as oxaloacetate.
The role of anaplerosis in the citric acid cycle is threefold.
First, as intermediates are drained away during biosynthetic
processes such as gluconeogenesis, glyceroneogenesis, and
amino acid synthesis, anaplerotic pathways replace them,
assuring sufficient carbon flux for citrate synthesis and,
hence, allow citric acid cycle flux to proceed unimpaired.
These aspects of anaplerosis are especially important in the
liver and renal cortex. Second, anaplerosis facilitates energy
transfer in organs with high rates of energy turnover, such as
the heart and skeletal muscle, especially under situations such
as ischemia in which normal functioning of the citric acid cycle
is impaired. Third, anaplerosis also regulates whole-body
b-Oxidation The oxidative metabolism of fatty
acids through a cycle of reactions that removes
successive two-carbon units (acetyl-CoA) from the
fatty acid.
metabolic homeostasis through the regulation of insulin secre-
tion from the b-islet cells of the pancreas. We now focus on the
mitochondrial anaplerotic pathways.
Anaplerotic Pathways
Substrates for Anaplerosis
The known anaplerotic pathways for the citric acid cycle are
depicted in the schematic drawing of Figure 2. As a key com-
ponent of all living cells, the citric acid cycle is highly organized
and responds instantly to changes in its environment. The
citric acid cycle is tied into a distributive system of energy
transformation reactions (Figure 1) and flux through the ana-
plerotic pathways. The primary substrates for anaplerosis are
lactate and alanine as precursors of pyruvate, although gluta-
mate can be a physiologically significant anaplerotic substrate
as well. In addition, the branched chain amino acids valine and
isoleucine, as well as odd-chain fatty acids and their metabolic
end product, propionate, serve as anaplerotic substrates.
Entry at Malate and Oxaloacetate
In the heart the major anaplerotic substrate in the heart is
pyruvate. Although the majority of pyruvate entering the mito-
chondria is oxidatively decarboxylated (and most likely by CO2
arising from pyruvate decarboxylation) and enters the citric acid
cycle as acetyl-CoA, a portion is also carboxylated and enters the
four-carbon (C-4) pool of the citric acid cycle. This conversion is
catalyzed by either pyruvate carboxylase (PC, E.C. 6.4.1.1),
forming oxaloacetate or by nicotinamide adenine dinucleotide
phosphate (NADP)-dependent malic enzyme (E.C. 1.1.1.40),
forming malate. While the NADP-dependent malic enzyme
reaction is reversible, the reaction in the direction of malate
production is slow and unlikely to play a significant role in
anaplerosis. Another provider of C-4 intermediates in the citric
acid cycle is aspartate, which undergoes transamination with
a-ketoglutarate via aspartic aminotransferase to form oxaloace-
tate and glutamate. In contrast to the carboxylation of pyruvate,
the entry of aspartate as oxaloacetate is balanced by the loss of
a-ketoglutarate from the citric acid cycle, resulting in the homeo-
stasis of citric acid cycle intermediates.
105
106 Bioenergetics | Anaplerosis

Entry via a-Ketoglutarate is present in a variety of tissues, including the liver, kidney,
brain, heart, and skeletal muscle.
Mitochondrial a-ketoglutarate can be readily formed from
glutamate via transamination with pyuruvate (forming ala-
nine) or oxaloacetate (forming aspartate). The transamina- Entry via Succinyl-CoA
tion of glutatmate with oxaloacetate by glutamate
The citric acid cycle intermediate succinyl-CoA plays an impor-
oxaloacetate transaminase (E.C. 6.2.1.1) results in a gain of
tant role in fatty acid and amino acid metabolism because it is
a C-5 intermidate at the same time that a C-4 intermediate is
the entry point of odd-chain fatty acids, propionate, and the
lost from the citric acid cycle. Of importance is that the
branched chain amino acids valine and isoleucine into the
mitochondrial pools of a-ketoglutarate and glutamate are in
citric acid cycle. Substrate entry as a-ketoglutarate or succinyl-
equilibrium and therefore labeling patterns of glutamate are
CoA, in contrast to other anaplerotic pathways, is associated
identical to those for a-ketoglutarate. As discussed below, this
with the generation of energy-rich phosphates via a substrate-
relationship between a-ketoglutarate and glutamate can be
level phosphorylation. The reaction catalyzed by succinyl-CoA
exploited in measuring citric acid cycle flux, relative rates of
synthetase (E.C. 6.2.1.4) normally favors the conversion of
contribution of substrates to the citric acid cycle, and rates
succinyl-CoA to succinate and leads to substrate level phos-
of anaplerosis. Furthermore, glutamate can be converted to
phorylation of GDP to GTP. This energy-providing pathway
a-ketoglutarate and NHþ 4 by glutamate dehydrogenase, which becomes important under anaerobic conditions when ATP
generation by oxidative phosphorylation is inhibited, such as
in the setting of ischemia.
Ca2+
Circulation
Krebs NADH H+ Entry via Fumarate
cycle
H+ PCr
In addition to the amino acids phenylalanine and tyrosine
entering as fumarate, the purine nucleotide cycle enriches the
Ca2+
ATP citric acid cycle based on the net reaction
Ca2+
Aspartate þ GTP ! fumarate þ NH3 þGDP þ Pi
Cross Several workers have demonstrated that flux through the purine
bridges nucleotide cycle increases in skeletal muscle during intense exer-
cise. This increased flux has two effects: first, it can maintain the
Ca2+
pool of adenine nucleotides, and second, it can increase the citric
Figure 1 Cycles of energy transfer in heart muscle. The central role acid cycle pool size via anaplerosis. Both of these effects are
of the Krebs cycle is highlighted. See text for details. expected to improve energy metabolism in exercising muscle.

Lactate Fatty acid

Alanine Pyruvate Acetyl-CoA Acetoacetate

Citrate
Oxaloacetate

Aspartate Isocitrate
asparagine Glutamine
Malate
Phenylalanine
tyrosine a-KETOGLUTARATE Glutamate
Valine
isoleucine
Fumarate methionine
Histidine
AMP
Proline
Succinyl-CoA Arginine
Succinate

IMP Adenylosuccinate Propionate

Odd-chain fatty acid
Aspartate GDP
GTP
Figure 2 Summary of anaplerotic pathways leading to the citric acid cycle. See text for details.

Exit of Cycle Intermediates: Balancing Anaplerosis
Under steady-state conditions, substrates entering the citric
acid cycle via anaplerotic pathways are balanced by removing
an equivalent amount of citric acid cycle intermediates through
pathways that maintain a constant citric acid cycle pool size.
These pathways have been termed cataplerotic, although the
term is somewhat an oxymoron; instead, we prefer the more
appropriate term drainage. Drainage pathways generally involve
removing the citric acid cycle of cycle intermediates oxaloacetate
(as aspartate via transamination, or phosphoenolpyruvate via
decarboxylation by phosphoenolpyruvate carboxykinase (E.C.
4.1.1.32)), citrate via export from the mitochondrial matrix,
or a-ketoglutarate via transamination to glutamate. While
these drainage pathways are usually thought of as means to
balance anaplerotic pathways, they play critical roles in renal
gluconeogenesis and enterocyte energy production from gluta-
mine (see below).
Measuring Rates of Anaplerosis
Changes in Citric Acid Cycle Pool Size
There are two principal methods for assessing anaplerosis
based on changes in citric acid cycle pool size. The amount of
citric acid cycle pool intermediates can be measured enzymati-
cally or resolved with high-performance liquid chromatogra-
phy. While both methods can reliably assess changes in the
citric acid pool size, they do not provide insight into the path-
ways involved in anaplerosis nor do they determine relative
rates of citric acid cycle enrichment from analperotic sub-
strates. Furthermore, because anaplerosis is usually balanced
by the exit of intermediates, changes in citric acid pool size are
generally negligible.
Simply determining changes in citric acid cycle pool size
provides no information on rates of anaplerotic flux or on
specific anaplerotic reactions. Using substrates labeled with
tracer 14C and measuring incorporation into specific positions
in citric acid cycle intermediates is more revealing. This method
can determine rates of anaplerosis and is able to characterize
the pathways involved in the process. However, the method
is also laborious and prone to error. It requires sequential
enzymatic degradation of intermediates quantifying the release
of the 14C label. This method has generally been replaced
by 13C-NMR spectroscopy or mass spectroscopy (NMR, nuclear
magnetic resonance).
13
C-NMR/Mass Spectroscopy
13
C-NMR spectroscopy is used to assess quantitatively the rela-
tive contributions of various substrates to the citric acid cycle.
This method determines the anaplerotic enrichment of individ-
ual carbon positions in citric acid cycle intermediates, similar to
the radiotracer method discussed before, but without the need
for digesting the carbon skeleton. 13C-labeled compounds enter
the carbon skeleton of citric acid cycle intermediates at specific
positions through anaplerotic pathways or through dilution of
l3
C by entry of unlabeled anaplerotic substrates. The enrichment
of l3C at those positions is then assessed and rates of anaplerotic
flux are determined relative to rates of citric acid flux (a/c).
Bioenergetics | Anaplerosis 107
The method is illustrated in Figure 3 in which the enrichment
of the carbon skeleton of a-ketoglutarate (and therefore gluta-
mate, which is present in sufficient concentrations to be mea-
surable by NMR spectroscopy) at the C-3 and C-4 positions is
used to quantify anaplerosis. Label in these positions is gener-
ated from randomization of the C-4 carbon in the first turn of
the citric cycle. Therefore, the sum of the 13C enrichments of
the C-2 and C-3 carbons will be equal to the enrichment of the
C-4 carbon of glutamate. In contrast, with anaplerotic activity,
the labeled C-2 and C-3 carbons will be diluted by 12C carbons
arising from unlabeled anaplerotic substrates, and the sum of
the enrichments of the labeled C-2 and C-3 carbons will be less
than the enrichments of the C-4 carbon of glutamate. For
example, using [2-13C]acetyl-CoA as a substrate and conditions
where there is no anaplerotic flux, analysis of the enrichment
of C-2, C-3, and C-4 positions of glutamate reveals that a/c ¼ 0,
indicating no anaplerotic flux. Further refinements to the
NMR-based methods take into account the recycling of car-
bons in pyruvate (a major source of anaplerotic substrate)
derived from the citric acid cycle to improve the accuracy of
measurements of anaplerotic flux.
Expression and Activity of Proteins Regulating Anaplerosis
The enzyme pyruvate carboxylase was discovered half a century
ago, and over the last decade there has been much progress in
understanding its structure and function. Using methods out-
lined above, studies have focused on changes in enrichment of
citric acid cycle intermediates to assess the activity of anaplero-
tic pathways and explain changes in flux in terms of mass
action and allosteric regulation of key enzymes. While this is
most likely the case with acute changes in anaplerotic flux,
changes in the expression and activity of enzymes responsible
for anaplerosis must be taken into account when studying
chronic processes such as pressure-overload-induced hypertro-
phy, heart failure, or diabetes. In streptozocin-treated diabetic
rats, transcript levels of anaplerotic enzymes in heart and skel-
etal muscle are downregulated (Figure 4). Furthermore, mice
with germline deletion of peroxiosome proliferator-activated
receptor-a show an increase in pyruvate carboxylase mRNA
expression. Changes in transcript levels are paralleled by simi-
lar changes in protein expression. Although, the in vitro activ-
ities of the anaplerotic enzymes involved in the carboxylation
of substrates (e.g., pyruvate carboxylase) can be measured by
determining the incorporation of 14C from H14 CO1 3 , it is not
known whether changes in expression correlate with changes
in activity. However, in pressure overload-induced left ventric-
ular hypertrophy in the rat, compensatory increases in ana-
plerotic flux into the citric acid cycle are accompanied by
increases in transcript levels of malic enzyme.
Changes in Anaplerosis in Response to Environmental
Stress: Workload, Nutritional Status, and Disease
Ultimately, any metabolic process has functional conse-
quences, and anaplerotic pathways are no exception. Anaplero-
tic pathways play important roles in regulating a wide variety
of organ responses to conditions of metabolic stress ranging
from exercise to inborn errors of metabolism.
142 Bioenergetics | ATP in Plant Mitochondria: Substrates, Inhibitors, and Uncouplers
NAD(P)H NADH
Cyt b
Porin
NDE 1 NDE 2
NAD(P)H
H+ NADH
NADH O2 2H2O
NADH Alternative
NDI 1 oxidase
Complex I NAD(P)H
Succinate
NDI 2
Complex II
Cyt c
Ubiquinone
Figure 1 A schematic representation of the routes of NAD(P)H oxidation and the electron transport chain (ETC) of plant mitochondria. NDI refers
to internal matrix-facing nonphosphorylating NAD(P)H dehydrogenase, and NDE refers to external cytosolic nonphosphorylating NAD(P)H
dehydrogenase. UCP refers to the uncoupling protein. Reproduced from Rasmusson AR, Soole KL, and Elthon TE (2004) Alternative NAD(P)H
dehydrogenases in plant mitochondria. Annual Review of Plant Biology 54: 23–39.
mitochondria to oxidize externally supplied NAD(P)H,
which cannot pass through the inner membrane. The external
cytosolic-facing NAD(P)H dehydrogenase (NDE) feeds directly
into the ubiquinone pool, as external NAD(P)H oxidation has
an ADP/O ratio equivalent to succinate of less than 2.0. Matrix
NADH oxidation in plants has a component which is insensi-
tive to inhibition by the complex I inhibitor, rotenone. This
oxidation is catalyzed by internal inner mitochondrial mem-
brane transdehydrogenases (NDI). The rotenone-insensitive
alternative matrix NADH oxidation has an ADP/O ratio also
similar to succinate indicating that NDI also feeds electrons
into the ubiquinone pool, bypassing proton pumping at com-
plex I. The AOX is a cyanide and antimycin A-resistant ubiqui-
nol oxidase that catalyzes the reduction of oxygen to water with
electrons from ubiquinol and, thus, bypasses the proton
pumps at complexes III and IV. Sequence analysis of the
genes putatively encoding NADH(P)H dehydrogenases in var-
ious plant species has separated this gene family into three
subfamilies, NDA, NDB, and NDC, based on their homology
to the yeast and bacterial gene sequences. There is functional
evidence that links NDA to NDI activity and the NDB gene
family to NDE activities. It is as yet unknown whether NDC
encodes an NDI- or NDE-like activity.
Thus, it is possible to have electron transport/transfer with
no ATP synthesis in plant mitochondria. Therefore, due to the
bypasses that exist around key regulatory sites in glycolysis,
plant respiration can be totally independent of respiratory
control, which gives plants great metabolic flexibility. This
flexibility, which results in the loss of adenylate control of
respiration by the energy status of the cell, can theoretically
Cytoplasm
Outer membrane
Intermembrane space
H+
Complex V
H+ H+
H+ H+
O2
Complex III 2H2O UCP
α β α
Complex IV
ADP + Pi ATP
Matrix
allow the release of biosynthetic intermediates from the respi-
ratory pathway, independent of energy charge of the cell. This
role of the nonphosphorylating pathway is hypothesized and
has not been clearly demonstrated in vivo.
Substrates for ATP Synthesis
The plant mitochondrial ETC can oxidize matrix NADH and
NADPH to a much lesser extent. The source of this NADH is
from pyruvate dehydrogenase and the citric acid cycle, and
plants have the full complement of these enzymes in addition
to other unique enzymes. The presence of matrix pools of
NADPH is indicative of the mitochondria’s anabolic role.
NADP-dependent enzymes involved in folate and thymidylate
synthesis such as NADP-dihydrofolate reductase and methy-
lene tetrahydrofolate dehydrogenase have been found in plant
mitochondria and their continued operation requires the turn-
over of the NADPH generated via NDI2. Apart from NAD-
dependent isocitrate dehydrogenase, there is also another
NADP-dependent form in the matrix; however, its role in
plant metabolism is not yet clear. Additionally, via the action
of a soluble transhydrogenase detected recently in pea leaf
matrix, the mitochondrial oxidation of any strictly NAD-linked
substrates such as pyruvate would also be able to produce
NADPH.
Another unique enzyme found in plant mitochondria
is glycine decarboxylase, which generates NADH in the matrix,
is part of photorespiratory cycle and is important in photo-
synthetic metabolism. It is accepted that the mitochondrial
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 "Oh, don't mind us."

"Thank you. It's really for your benefit, so you'd better listen. Let me
see, where were we? Oh yes, 'One pound of beef, ninepence; three pounds
of potatoes, fourpence; one piece of emery paper for the blanc-mange,
tuppence; one pound of india-rubber——'"

"'Dahlia darling,'" interrupted Myra, in a fair imitation of Archie's

voice, "'how often have I told you that we can't afford india-rubber in the
cake? Just a few raisins and a cherry is really all you want. You mustn't be
so extravagant.'"

"'Dearest, I do try; and after all, love, it wasn't I who fell into the cocoa
last night.'"

"'I didn't fall in, I simply dropped my pipe in, and it was you insisted on
pouring it away afterwards. And then, look at this—One yard, of lace, 4s.
6d. That's for the cutlets, I suppose. For people in our circumstances paper
frillings are quite sufficient.'"

Archie and Dahlia listened to us with open mouths. Then they looked at
each other, and then at us again.

"Is there any more?" asked Archie.

"There's lots more, but we've forgotten it."

"You aren't ill or anything?"

"We are both perfectly well."

"How's Miss Dalton?"

"Dora," I said, "is also well. So is Miss Fortescue and so is Thomas. We

are all well."

"I thought, perhaps——"

"No, there you are wrong."

 "I expect it's just the heat and the excitement," said Dahlia, with a smile.
"It takes some people like that."

"I'm afraid you miss our little parable," said Myra.

"We do. Come on, Dahlia."

"You'll pardon me, Archibald, but Miss Blair is dancing this with me."

Archie objected strongly, but I left, him with Myra, and took Miss Blair
away. We sat on the stairs and thought.

"It has been a lovely week," said Dahlia.

"It has," I agreed.

"Perhaps more lovely for me than for you."

"That's just where I don't agree with you. You know, we think it's
greatly over-rated. Falling in love, I mean."

"Who's 'we'?"

"Myra and I. We've been talking it over. That's why we rather dwelt
upon the sordid side of it just now. I suppose we didn't move you at all?"

"No," said Dahlia, "we're settled."

"That's exactly it," I said. "I should hate to be settled. It's so much more
fun like this. Myra quite agrees with me."

Dahlia smiled to herself. "But perhaps some day," she began.

"I don't know. I never look more than a week ahead. 'It has been great
fun this week, and it will probably be great fun next week.' That's my
motto."

"Well, ye—es," said Miss Blair doubtfully.

 PART II

CHAPTER I

ONE OF THE PLAYERS

"Do I know everybody?" I asked Myra towards the end of the dinner,
looking round the table.

"I think so," said Myra. "If there's anybody you don't see in the window
ask for him."

"I can see most of them. Who's that tall handsome fellow grinning at me
now?"

"Me," said Archie, smiling across at us.

"Go away," said Myra. "Gentlemen shouldn't eavesdrop. This is a

perfectly private conversation."

"You've got a lady on each side of you," I said heatedly, "why don't you
talk to them? It's simply scandalous that Myra and I can't get a moment to
ourselves."

"They're both busy; they won't have anything to say to me."

"Then pull a cracker with yourself. Surely you can think of something,
my lad."

"He has a very jealous disposition," said Myra, "and whenever Dahlia
—— Bother, he's not listening."
 I looked round the table again to see if I could spy a stranger.

"There's a man over there—who's he? Where this orange is pointing."

"Oranges don't point. Waggle your knife round. Oh, him? Yes, he's a
friend of Archie's—Mr Derry."

"Who is he? Does he do anything exciting?"

"He does, rather. You know those little riddles in the Christmas
crackers?"

"Yes?"

"Yes. Well, he couldn't very well do those, because he's an electrical

engineer."

"But why——"

"No, I didn't. I simply asked you if you knew them. And he plays the
piano beautifully, and he's rather a good actor, and he never gets up till
about ten. Because his room is next to mine, and you can hear everything,
and I can hear him not getting up."

"That doesn't sound much like an electrical engineer. You ask him
suddenly what amperes are a penny, and see if he turns pale. I expect he
makes up the riddles, after all. Simpson only does the mottoes, I know. Now
talk to Thomas for a bit while I drink my orange."

Five minutes elapsed, or transpired (whichever it is), before I was ready

to talk again. Generally, after an orange, I want to have a bath and go
straight off to bed, but this particular one had not been so all-overish as
usual.

"Now then," I said, as I examined the crystallised fruit, "I'm with you in
one minute."

Myra turned round and looked absently at me.

 "I don't know how to begin," she said to herself.

"The beginning's easy enough," I explained, as I took a dish of green

sweets under my charge, "it's the knowing when to stop."

"Can you eat those and listen to something serious?"

"I'll try.... Yes, I can eat them all right. Now, let's see if I can listen....
Yes, I can listen all right."

"Then it's this. I've been putting it off as long as I can, but you've got to
be told to-night. It's—well—do you know why you're here?"

"Of course, I do. Haven't I just been showing you?"

"Well, why are you here?"

"Well, frankly, because I'm hungry, I suppose. Of course, I know that if

I hadn't been I should have come in to dinner, just the same, but—— Hang
it, I mean that's the root idea of a dining-room, isn't it? And I am hungry. At
least I was."

"Stave it off again with an almond," said Myra, pushing them along to
me. "What I really meant was why you're here in the house."

This was much more difficult. I began to consider possible reasons.

"Because you all love me," I started; "because you put the wrong
address on the envelope; because the regular boot-boy's ill; because you've
never heard me sing in church; because—stop me when I'm getting warm—
because Miss Fortescue refused to come unless I was invited; because——"

"Stop," said Myra. "That was it. And, of course, you know I didn't mean
that at all."

"What an awful lot of things you don't mean to-night. Be brave, and
have it right out this time."
 "All right, then, I will. One, two, three—we're going to act a play on
Saturday."

She leant forward, and regarded me with apprehension.

"But why not? I'll promise to clap."

"You can't, because, you see, you're going to act too. Isn't it jolly?" said
Myra breathlessly.

I gave what, if I hadn't just begun the last sweet, would have been a
scornful laugh.

"Me act? Why, I've never—I don't do it—it isn't done—I don't act—not
on Saturdays. How absurd!"

"Have you told him, Myra?" Dahlia called out suddenly.

"I'm telling him now. I think he's taking it all right."

"Don't talk about me as 'him'!" I said angrily. "And I'm not taking it all
right. I'm not taking it at all."

"It's only such a very small part—we're all doing something, you know.
And your costume's ordered and everything. But how awfully sporting of
you."

After that, what could I say?

"Er—what am I?" I asked modestly.

"You're a—a small rat-catcher," said Myra cheerfully.

"I beg your pardon?"

"A rat-catcher."

"You said a small one. Does that mean that I'm of diminutive size, or
that I'm in a small way of business, or that my special line is young ones?"
 "It means that you haven't much to say."

"I see. And would you call it a tragic or a pathetic part?"

"It's a comic part, rather. You're Hereditary Grand Rat-Catcher to the

Emperor Bong. Bong the Second. Not the first Bong, the Dinner Bong."

"Look here. I suppose you know that I've never acted in my life, and
never been or seen a rat-catcher in my life. It is therefore useless for you to
tell me to be perfectly natural."

"You have so little to do; it will be quite easy. Your great scene is where
you approach the Emperor very nervously——"

"I shall do the nervous part all right."

"And beg him to spare the life of his mother-in-law."

"Why? I mean, who is she?"

"Miss Fortescue."

"Yes, I doubt if I can make that bit seem quite so natural. Still, I'll try."

"Hooray. How splendid!"

"A rat-catcher," I murmured to myself. "Where is the rat? The rat is on

the mat. The cat is on the rat. The bat is on the cat. The——"

"Mr Derry will go through your part with you to-morrow. Some of it is
funnier than that."

"The electrical engineer? What do they know about rat-catching?"

"Nothing, only——"

"Aha! Now I see who your mysterious Mr Derry is. He is going to

coach us."
 "He is. You've found it out at last. How bright green sweets make you."

"They have to be really bright green sweets. Poor man! What a job he'll
have with us all."

"Yes," said Myra, as she prepared to leave me. "Now you know why he
doesn't get up till ten."

"In the rat-catching business," I said thoughtfully, as I opened the door,

"the real rush comes in the afternoon. Rat-catchers in consequence never
get up till ten-thirty. Do you know," I decided, "I am quite beginning to like
my little part."

CHAPTER II

ALARMS AND EXCURSIONS

I was, I confess, very late the next morning, even for a rat-catcher. Mr
Derry was in the middle of his breakfast; all the others had finished. We
saluted, and I settled down to work.

"There is going to be a rehearsal at eleven o'clock, I believe," said

Derry. "It must be nearly that now."

"I shall be there," I said, "if I have to bring the marmalade with me.
You're going to coach us?"

"Well, I believe I said I would."

"Though I have never assumed the buskin myself," I went on, "I have,
of course, heard of you as an amateur actor." (Liar.) "And if you could tell
me how to act, while I am finishing my bacon, I should be most awfully
obliged."
 "Haven't you really done any?"

"Only once, when I was very small. I was the heroine. I had an offer, but
I had to refuse it, I said, 'Alath, dear heart, I may not, I am married
already.'"

"Very right and proper," murmured Derry.

"Well, as it turned out, I had made a mistake. It was my first who had
been married already. The little play was full of surprises like."

Derry coughed, and took out his pipe. "Let me see," he began, "what's
your part?"

"I am—er—a rodent-collector."

"Oh yes—the Emperor's rat-catcher."

"Grand hereditary," I said stiffly. "It had been in the family for years."

"Quite so."

I was about to enlarge upon the advantages of the hereditary principle

when the door opened suddenly to admit Myra and Archie.

"You don't say you're down at last!" said Myra, in surprise.

"I hardly say anything at breakfast, as a rule," I pointed out.

"What an enormous one you're having. And only last night——"

"On the contrary, I'm eating practically nothing—a nut and one piece of
parsley off the butter. The fact is, I glanced at my part before I went to bed,
and there seemed such a lot of it I hardly slept at all."

"Why, you don't come on very much," said Archie. "Neither do I. I'm a
conjuror. Can any gentleman here oblige me with a rabbit? ... No, sir, I said
a rabbit. Oh, I beg your pardon, I thought you were coming up on to the
stage.... Any gentleman——"
 "Have some jam instead. What do you mean by saying I don't come on
very much?" I took the book out of my pocket, and began to turn the leaves.
"Here you are, nearly every page—'Enter R.,' 'Exit R.,' 'Enter L.'—I don't
know who he is—'Exeunt R.,'—why, the rat-catcher's always doing
something. Ah, here they're more explicit—'Enter R.C.' Hallo, that's funny,
because I'd just—— Oh, I see."

"One of our oldest and most experienced mimes," said Archie to Derry.
"You must get him to talk to you."

"No secret of the boards is hid from him," added Myra.

"Tell us again, sir, about your early struggles," begged Archie.

"He means your early performances on the stage," explained Myra.

"There's one very jolly story about Ellen Terry and the fireproof curtain.
Let me see, were you Macbeth then, or Noise of Trumpets? I always forget."

I drank my last cup of tea, and rose with dignity.

"It is a humorous family," I apologised to Derry. "Their grandfather was

just the same. He would have his little joke about the first steam-engine."

Outside, in the hall, there was a large crowd of unemployed, all talking
at once. I caught the words "ridiculous" and "rehearsal," and the connection
between the two seemed obvious and frequent. I singled out Thomas,
abstracted his pouch, and began to fill up.

"What is all this acting business?" I asked. "Some idea about a little
play, what? Let's toddle off, and have a game of billiards."

"They've let me in for a bally part," said Thomas, "and you needn't think
you're going to get out of it. They've got you down, all right."

"Thomas, I will be frank with you. I am no less a person than the

Emperor Bong's Hereditary (it had been in the family for years) Grand Rat-
catcher. The real rush, however, comes in the afternoon. My speciality is
young ones."
 "I'm his executioner."

"And he has a conjuror too. What a staff! Hallo, good morning,

Simpson. Are you anything lofty?"

"'Oh, I am the Emperor Bong,'" said Simpson gaily; "'I am beautiful,

clever and strong——'"

"Question," said Thomas.

"''Tis my daily delight to carouse and to fight, and at moments I burst

into song.'"

I looked at him in amazement.

"Well, just at present," I said, "all I want is a match.... A lucifer, Emp. A

pine vesta, Maj. Thanks.... Now tell me—does anybody beside yourself
burst into song during the play? Any bursting by Thomas or myself, for
instance?"

"Nobody sings at all. My little poem is recitative."

"If you mean it's very bad, I agree with you," said Thomas.

"I made it up myself. It was thought that my part should be livened up a

little."

"Well, why hasn't it been?"

"If you will give me two minutes, Simpson," I said, "I will liven up my
own part better than that. What rhymes with rat-catcher?"

"Cat-catcher."

"Wait a bit.... Yes, that's got it.

"'Oh, I'm on the Emperor's staff

I'm a rodent-collector (don't laugh)—
My record (in braces)
 Of rats and their races
Is a thousand and eight and a half.'"

"May we have that again?" said Myra, appearing suddenly.

"'Oh, I'm on——'"

"No," said Thomas.

"'Oh, I'm on——'"

"No," said Simpson.

"There is no real demand, I'm afraid."

"Well, I did just hear it before," said Myra. "I wish you'd make up one
for me. I think we might all announce ourselves like that, and then the
audience will have no difficulty in recognising us."

"They'll recognise Thomas if he comes on with an axe. They won't think

he's just trotted round with the milk. But what are you, Myra?"

"The Emperor's wife's maid."

"Another member of the highly trained staff. Well, go on, Simpson."

"'Oh, I am her Majesty's maid,'" declared Simpson. "We all begin with
'Oh,' to express surprise at finding ourselves on the stage at all. 'Oh, I am
her Majesty's maid, I'm a sad little flirt, I'm afraid.'"

"I'm respectable, steady and staid," corrected Myra.

"No," I said; "I have it—"

"'Oh, I am her Majesty's maid!

And her charms are beginning to fade,
I can sit in the sun
 And look just twenty-one,
While she's thirty-six in the shade.'"

Myra made a graceful curtsey.

"Thank you, sir. You'll have to pay me a lot more of those before the
play is over."

"Will I really?"

"Well, seeing as the Grand Hereditary One is supposed to be making up

to her Majesty's confidential attendant——"

Miss Fortescue came pushing up to us.

"It is too ridiculous," she complained; "none of us know our parts yet,
and if we have a rehearsal now—what do you think about it?"

I looked at Myra and smiled to myself. "I'm all for a rehearsal at once,"
I said.

CHAPTER III

A REHEARSAL

"Now this is a very simple trick," said Archie, from the centre of the
stage. "For this little trick all I want is, a hippopotamus and a couple of
rubies. I take the hippopotamus in one hand—so—and cover it with the
handkerchief. Then, having carefully peeled the rubies——"
 Thomas put the last strip of silver paper on to his axe, and surveyed the
result proudly.

"But how splendid!" said Myra, as she hurried past. "Only you want
some blood." And she jumped over the footlights and disappeared.

"Good idea. Archie, where do you keep the blood?"

"Hey, presto! it's gone. And now, sir, if you will feel in your waistcoat
pockets you will find the hippopotamus in the right-hand side and the red
ink in the left. No? Dear, dear, the hippopotamus must have been a bad
one."

"Be an artist, Thomas," I said, "and open a vein or two. Do the thing
properly, Beerbohm. But soft, a winsome maid, in sooth; I will approach
her. I always forget that sooth bit. But soft, a win——"

"Why don't we begin?" asked Simpson; "I can't remember my part

much longer. Oh, by the way, when you come up to me and say, 'Your
Majesty e'en forgets the story of the bull's-eye and the revolving bookcase
——"

"Go away; I don't say anything so silly."

"Oh, of course it's Blair. Blair, when you come up to me and say——"
They retired to the back of the stage to arrange a very effective piece of
business.

"Any card you like, madam, so long as it is in the pack. The Queen of
Hearts? Certainly. Now I take the others and tear them up—so. The card
remaining will be yours. Ah, as I thought—it is the Queen of Hearts."

"Archie, you're talking too much," said Dahlia, "and none of it comes
into your part really."

"I'm getting the atmosphere. Have you an old top hat on you, dear,
because if so we'll make a pudding. No top hat? Then pudding is horf."
 "But stay, who is this approaching? Can it be—I say, mind the
footlights. When are we going to begin?"

"There!" said Thomas proudly. "Anybody would know that was blood."

"But how perfectly lovely," said Myra. "Only you want some notches."

"What for?"

"To show where you executed the other men, of course. You always get
a bit off your axe when you execute anybody."

"Yes, I've noticed that too," I agreed. "Notches, Thomas, notches."

"Why don't you do something for a change? What about the trap or
whatever it is you catch your bally rats with? Why don't you make that?"

"It isn't done with a trap, Thomas dear. It's partly the power of the
human eye and partly kindness. I sit upon a sunny bank and sing to them."

"Which is that?"

"If we don't begin soon," began Simpson——

"Hallo, Emperor, what's that you're saying? Quite so, I agree with you. I
wonder if your High Fatness can lend me such a thing as a hard-boiled egg.
Simpkins, when this rehearsal is over—that is to say, to-morrow—I'll take
you on at juggling; I'm the best——"

Deny finished his conversation with Miss Fortescue and turned to the
stage.

"Now then, please, please," he said. "We'll just take the First Act.
Scene, The Emperor's Palace. Enter Rat-catcher. You come on from the
left."

I coughed and came on.

 My part was not a long one, but it was a very important one. I was the
connecting link between the different episodes of the play, and they wanted
some connecting. Whenever anybody came on to the stage, I said
(supposing I was there, and I generally was—the rat-catcher of those days
corresponding to the modern plumber)—I said, "But who is this?" or "Hush,
here comes somebody." In this way, the attention of the wakeful part of the
audience was switched on to the new character, and continuity of action was
preserved.

I coughed and came on.

"No," said Derry, "you must come on much more briskly."

"I can't. I've been bitten by a rat."

"It doesn't say so anywhere."

"Well, that's how I read the part. Hang it, I ought to know if I've been
bitten or not. But I won't show it if you like; I'll come on briskly."

I went out, and came on very briskly.

"That's better," said Derry.

"'His Majesty ordered me to be here at the stroke of noon,'" I said.

"'Belike he has some secret commands to lay upon me, or perchance it is
nought but a plague of rats. But who is this?'"

"'Oh,'" said Myra, coming in suddenly, "'I had thought to be alone.'"

"'Nay, do not flee from me, pretty one. It is thus that——' I say, Myra,
it's no good my saying do not flee if you don't flee."

"I was just going to. You didn't give me a chance. There, now I'm
fleeing."

"Oh, all right. 'It is thus that the rats flee when they see me approaching.
Am I so very fearsome?'"
 "'Orrid," said Archie to himself from the wings.

"One moment," said Derry, and he turned round to speak to somebody.

"Puffickly 'orrid," said Archie again.

"Nay, do not frown," Myra went on, "'tis only my little brother, who is
like unto a codfish himself, and jealous withal."

"Ay, ay, and I thought it was a codfish. So that I had e'en brought the
egg-sauce with me."

"Trouble not thyself for that," said Archie. "For verily the audience will
supply thee with all the eggs thou wantest. I say, we are being funny."

"I'm not, I'm quite serious, I really did think it was a co—— 'But tell
me, fair one,'" I said hurriedly, "'for what dost the Emperor want me?'"

"Yes, yes," said Derry, "I'm sorry I had to interrupt you. I think perhaps
we had better begin again. Yes, from the beginning."

The rehearsal rolled on.

* * * * * * *

"I think it went splendidly," said Myra. "If only we had known our parts
and come in at the right moments and been more serious over it."

"If there's any laughing to be done it will have to be done by us. The
audience won't laugh."

"'Mr Derry having explained that the author was not in the house, the
audience collected their cauliflowers and left quietly.' I think it's a rotten
play."

"Well, it isn't frightfully funny," said Myra, "but we can put that in
ourselves."
 "It's so jolly hard to say the lines properly—they're so unnatural,"
complained Thomas. "'Truly thou hast created a favourable impression with
the damsel'—well, I mean, it's absurd. Any ordinary person would say
'Truly thou art amongst them, old spot,' or something of that kind."

"Well, you say that, Thomas; you'll be all right,"

"We might put a few songs in," said Dahlia, "and a dance or two."

"I think you've forgotten that we've done only Act I.," remarked Archie.
"His Majesty's conjuror doesn't really let himself go till Act II. Still, I'm all
for a song and a dance. Simpson, come and Apache with me."

They dashed at each other fiercely.

"Oh, we'll make it go all right," said Myra.

CHAPTER IV

LAST MOMENTS

"Has anybody here seen Kelly?" asked Dahlia, putting her head in at the
billiard-room door. "I mean Archie."

"I'm waiting here for Kate," I said. "I mean Myra."

"Oughtn't you to be dressing? It doesn't matter about me—I'm not on for

a long time."

"A rat-catcher's best suit is not an elaborate one; I can put it on in about
five minutes. It is now seven-thirty, we begin at eight-thirty—hence the
billiard cue. More chalk."
 "Oh, why aren't you nervous? How you can stand calmly there——"

"I am nervous. Look." I aimed carefully and put the red into a pocket
some miles away. "There you are. Have you ever seen me do that in real
life? Of course not. If my hand had been steady I should have been a foot to
the right. Still more chalk."

"Well, I want Archie, and I shall cry if I don't find him. That's how I
feel." She sat down and got up again.

"My dear Dahlia," I said solemnly, "now you can understand a father's
feelings—I mean, now, you see what you women have brought on
yourselves. Who suggested a play? The women. Who dragged me into it?
The women. Who said rat-catchers always wore whiskers? The women.
Who is designing me a pair of whiskers at this moment? The wom——
Simpson. Who but for whom (this is going to be a very difficult sentence)
who but for whom, would be just thinking of dressing leisurely for dinner,
instead of which we had a hasty snack, and have now got to put on heaven
knows what? The women. Well, it serves you right."

"Don't be horrid. I want Archie." She got up for the third time and
drifted out of the room.

I chalked my cue and went into a pocket without touching anything.

When I say I went in I mean that the ball I was playing with went in. You
do see that? Very well, then. I took it out and began to squint along my cue
again, when two hands came suddenly over my eyes and a voice said;
"Guess who is is."

"The Queen of Sheba," I tried.

"Right," said Myra.

I turned and looked at her.

"Golly, you do, you really do!" I said at last. "Did they always dress like
that in the Bong era? Short skirts, long pigtail, bare arms—lovely!"
 "'I can sit in the sun and look just twenty-one,'" sang Myra as she
dropped into the sofa.

"Well, just at present you're sitting in the billiard-room and looking

about fifteen.... How are you getting on with your French this term? I had a
very bad report in the holidays from your governess. The extra ninepence a
week seems to have been simply thrown away."

"Aren't you excited?" said Myra, looking at me with sparkling eyes.

"As for calisthenics, well, what I say is, 'My daughter is Church of
England, and if you don't like it, she can come away. I'm not going to have
her stuffed up with all that nonsense.'"

Myra jumped up. "Aren't you excited?" she insisted.

"Feel my tongue—I mean my pulse, it's quite normal. And why?

Because I've forgotten my part, and I'm going to bed."

"It's a great responsibility our beginning the play."

"It is. Have you ever thought that, if we refused to begin, the play
couldn't continue, and then the audience would be able to go home? My
idea was to tackle the people as they arrive, and come to terms with them.
I'm sure there's money in it."

"You aren't bothering, are you?"

"Of course, I am. I'd give a hundred pounds to be out of it. No, I
wouldn't—I'd give a hundred pounds if you'd always wear that frock and do
your hair like that. Will you? And you shall go on with your French, child."

Myra curtsied prettily.

"And I'll go on with my whiskers. You haven't seen me in those yet,

have you?" There was a loud noise without. "Here they are, coming in."

It was not the whiskers, however, but Archie and Thomas in full
costume; Archie in green and Thomas in black.