REVIEW ARTICLE
The modular respiratory complexes involved in hydrogen and
sulfur metabolism by heterotrophic hyperthermophilic archaea
and their evolutionary implications
Gerrit J. Schut1, Eric S. Boyd2, John W. Peters2 & Michael W.W. Adams1
1
Department of Biochemistry & Molecular Biology, University of Georgia, Athens, GA, USA; and 2Department of Chemistry and Biochemistry and
the Astrobiology Biogeocatalysis Research Center, Montana State University, Bozeman, MT, USA
Correspondence: Michael W.W. Adams,
Department of Biochemistry and Molecular
Biology, Life Sciences Bldg., University of
Georgia, Athens, GA 30602-7229, USA.
Tel.: 706 542 2060; fax: 706 542 0229;
e-mail: [email protected]
Received 14 February 2012; revised 30 May
2012; accepted 8 June 2012. Final version
published online 12 July 2012.
DOI: 10.1111/j.1574-6976.2012.00346.x
MICROBIOLOGY REVIEWS
Editor: Diego de Mendoza
Keywords
hydrogenase; evolution; respiratory
complexes; archaea.
Abstract
Hydrogen production is a vital metabolic process for many anaerobic organisms, and the enzyme responsible, hydrogenase, has been studied since the
1930s. A novel subfamily with unique properties was recently recognized,
represented by the 14-subunit membrane-bound [NiFe] hydrogenase from the
archaeon Pyrococcus furiosus. This so-called energy-converting hydrogenase
links the thermodynamically favorable oxidation of ferredoxin with the formation of hydrogen and conserves energy in the form of an ion gradient. It is
therefore a simple respiratory system within a single complex. This hydrogenase shows a modular composition represented by a Na+/H+ antiporter
domain (Mrp) and a [NiFe] hydrogenase domain (Mbh). An analysis of the
large number of microbial genome sequences available shows that homologs of
Mbh and Mrp tend to be clustered within the genomes of a limited number of
archaeal and bacterial species. In several instances, additional genes are associated with the Mbh and Mrp gene clusters that encode proteins that catalyze
the oxidation of formate, CO or NAD(P)H. The Mbh complex also shows
extensive homology to a number of subunits within the NADH quinone oxidoreductase or complex I family. The respiratory-type membrane-bound hydrogenase complex appears to be closely related to the common ancestor of complex
I and [NiFe] hydrogenases in general.
Hydrogenase diversity
Hydrogenases are the key enzymes of microbial hydrogen
metabolism. They catalyze the reversible interconversion
of protons and electrons with H2 and can be found in all
three domains of life. The importance of H2 metabolism
for anaerobic organisms is illustrated by the fact that the
majority of microbial genomes encode one or more
hydrogenases. Hydrogenases can be divided into three
major groups based on the metal composition of their
catalytic center. Two of the classes typically contain one
or more iron–sulfur clusters and are differentiated by
their active site metalloclusters which contain either
nickel and iron ([NiFe] hydrogenases; (Volbeda et al.,
1995)) or only iron ([FeFe] hydrogenases; (Nicolet et al.,
2000)). The third group is comprised of [Fe]-only hydrogenases (5,10-methenyl tetrahydromethanopterin hydrogeª 2012 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
nase, Hmd) and is devoid of iron–sulfur clusters but
contain an iron–guanylylpyridinol cofactor in the active
site (Shima & Thauer, 2007; Hiromoto et al., 2009). They
have only been identified in hydrogenotrophic methanogens (McGlynn et al., 2010; Thauer et al., 2010). The
[NiFe] and [FeFe] hydrogenases contain the dinuclear
ligands CO and CN coordinated to an active site iron
atom while the Hmd hydrogenases only contain CO
ligands (van der Spek et al., 1996; Happe et al., 1997;
Lyon et al., 2004). In spite of this distinguishing characteristic of diatomic ligands (CN and/or CO) attached to
an iron site, these three enzyme classes are evolutionarily
unrelated, suggesting that their properties arose through
convergent evolution (Posewitz et al., 2008). All three
groups of hydrogenase require a suite of accessory proteins for maturation of their complex active sites. Intriguingly, maturation of the active site clusters/cofactors of
FEMS Microbiol Rev 37 (2013) 182–203
Modular Mbh-type complexes
these different hydrogenases requires a suite of distinct
accessory proteins for each class of enzyme and this
occurs by different mechanisms, consistent with their
independent evolutionary origins (Bock et al., 2006; Forzi
& Sawers, 2007; McGlynn et al., 2007; Thauer et al.,
2010).
[FeFe] hydrogenases have thus far only been identified
in the bacterial domain and in some unicellular eukaryotes but are absent from the archaeal domain (Vignais &
Billoud, 2007). [FeFe]-hydrogenase are generally involved
in the production of hydrogen by anaerobic heterotrophic
organisms such as Clostridium species, which use low
potential ferredoxins or flavodoxins as electron carriers
(Demuez et al., 2007). The diversity of [FeFe] hydrogenases can be catagorized on the basis of both primary
sequence and structural considerations, including the size
of the catalytic subunit and the presence of accessory
subunits that typically harbor additional FeS clusters
(Vignais et al., 2001). The catalytic center of [FeFe]
hydrogenases consist of a [4Fe-4S] cluster connected to a
binuclear iron site, forming the so-called H cluster (Nicolet et al., 2000). The conserved residues that coordinate
the H cluster are arranged in three conserved cysteinecontaining motifs termed L1, L2, and L3 (Vignais et al.,
2001). While these signature motifs are conserved in
[FeFe]-hydrogenase, significant variation exists in the
composition and abundance of additional FeS clusters
(so-called F clusters) as well as in the presence of accessory subunits (Meyer, 2007; Meuser et al., 2011).
In contrast to [FeFe]-hydrogenases, [NiFe] hydrogenases are widely distributed among both the bacterial and
archaeal domains but have yet to be identified in the eukarya. Although the subunit composition of [NiFe]hydrogenase vary, they consistently include a so-called
large subunit that contains the active [NiFe] site and a
small subunit that contains one or more FeS clusters. The
large and small subunits show homology with the subunits NuoD and NuoB of respiratory complex I, respectively, and the additional subunits in the multimeric
[NiFe] hydrogenases show sequence similarity to other
Nuo subunits (Friedrich & Scheide, 2000). The [NiFe]
active site is coordinated by four strictly conserved cysteine residues organized as two CXXC motifs located in the
N- and C-terminus of the large subunit, termed the L1
and L2 motifs (Volbeda et al., 1995, 2002). Conservation
in regions of the protein flanking the L1 and L2 motifs
has been used to categorize the [NiFe] hydrogenases into
four primary classes termed Groups 1–4. These show an
apparent relationship with the inferred function of the
enzyme and exhibit phylogenetic coherence (Fig. 1) (Vignais & Billoud, 2007).
The Group 4 hydrogenases are membrane bound and
are distinct in that their catalytic subunits show a surprisFEMS Microbiol Rev 37 (2013) 182–203
183
Fig. 1. Bayesian phylogram of [NiFe] hydrogenases based on
representative sequences of the large catalytic subunits that divide
these enzymes into four major groups. Group 4 also contains related
complexes that are not hydrogenases represented by Nuo (Complex I,
NADH ubiquinone oxidoreductases) and Mbx (the unknown
membrane-bound oxidoreductase involved in S0 reduction). Representative homologs (Table S1, Supporting information) were aligned
using CLUSTALX (version 2.0.8) with the Gonnet 250 protein matrix
and default gap extension and opening penalties (Larkin et al., 2007).
ProtTest (version 2.0) (Abascal et al., 2005) was used to select
WAG + I + G + F as the best-fit protein evolutionary model and the
phylogeny was evaluated using MrBayes (version 3.1.2) (Huelsenbeck
& Ronquist, 2001; Ronquist & Huelsenbeck, 2003), using the WAG
evolutionary model with fixed (F) amino acid frequencies and gammadistributed rate variation with a proportion of invariable sites (I+G).
Tree topologies were sampled every 500 generations over 5 9 105
generations (after a burnin of 4 9 105) at likelihood stationarity and
after convergence of two separate MCMC runs (average standard
deviation of split frequencies <0.03) by MrBayes. The composite
phylogram constructed from 1000 trees was projected with FigTree
(http://tree.bio.ed.ac.uk/software/figtree/). The depth of clades
proportionally reflects the extent of the diversity within those clades.
ingly low sequence similarity to hydrogenases that comprise the other three Groups, indicating a distinct
evolutionary history for these membrane-bound enzymes
(Vignais & Billoud, 2007). The Group 4 hydrogenases
also include a number of distinct enzymes such as the
formate hydrogen lyase or hydrogenase 3 (Hyc) of E. coli,
which oxidizes formate and evolves H2 (Sawers et al.,
1985; Sauter et al., 1992), and the CO-induced hydrogenases of Rhodospirillum rubrum and Carboxydothermus
hydrogenoformans, which are involved in generating
energy from the oxidation of CO to CO2 coupled with
the production of H2 (Soboh et al., 2002; Singer et al.,
2006). The Group 4 hydrogenases also include the sixsubunit energy-converting hydrogenase (EchA-F), which
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184
was originally characterized in Methanosarcina barkeri
where it was found to be required for growth on acetate
(Meuer et al., 1999). This hydrogenase can reversibly generate H2 from reduced ferredoxin with the concomitant
generation/utilization of an ion gradient; homologs of this
enzyme are found among anaerobic bacteria and some
methanogenic archaea. Group 4 [NiFe]-hydrogenase also
include the ferredoxin-reducing, energy-converting
hydrogenases of hydrogenotrophic methanogens (Eha and
Ehb) that contain 17–20 subunits. These hydrogenases are
thought to provide reduced ferredoxin for biosynthesis
and for the first step of methanogenesis: the ferredoxindependent reduction in CO2 to form formylmethanofuran
(Tersteegen & Hedderich, 1999; Major et al., 2010).
Finally, the Group 4 enzymes include the Mbh-type
energy-converting H2-producing enzymes that were first
described in hyperthermophile Pyrococcus furiosus within
the order Thermococcales (Sapra et al., 2000, 2003; Silva
et al., 2000). These hyperthermophilic enzymes are the
focus of this review.
Hydrogen and sulfur metabolism in the
Thermococcales
Species of Pyrococcus and Thermococcus constitute the
order of Thermococcales. These organisms are generally
heterotrophic thermophiles that reduce elemental sulfur
(S0) and ferment both simple and complex carbohydrates
as well as peptide-based substrates. Many species can also
grow on carbohydrates in the absence of S0 (Kengen
et al., 1996). The pathways of carbohydrate degradation
have been well studied in members of the Thermococcales, especially in Thermococcus kodakarensis and P. furiosus (Sakuraba et al., 2004; Verhees et al., 2004). During
growth on sugars, such as the disaccharide maltose, the
main fermentation products are H2, CO2 and acetate. The
lack of other fermentation products such as lactate or
ethanol indicates that all generated reducing equivalents
are disposed of as H2. Glycolysis in the Thermococcales proceeds through a modified version of the
Embden-Meyerhof pathway in which the classical glycolytic enzymes glyceraldehyde-3-phosphate dehydrogenase
(GAPDH) and phosphoglycerate kinase (PGK) are
replaced by one ferredoxin-linked enzyme, glyceraldehyde-3-phosphate ferredoxin oxidoreductase (GAPOR),
which does not generate ATP via substrate-level phosphorylation (Mukund & Adams, 1995; van der Oost
et al., 1998). Omitting the ATP generating step of PGK
therefore reduces the overall substrate-level ATP yield of
glycolysis in the Thermococcales to zero (Verhees et al.,
2004). Subsequently, pyruvate is oxidized to acetyl-CoA
by pyruvate ferredoxin oxidoreductase (POR) which also
uses the low potential electron carrier protein ferredoxin
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G.J. Schut et al.
as acceptor (Blamey & Adams, 1993). This means that
NADH is not formed during sugar catabolism and the
only ATP generated (2 per mol glucose) is as a result of
acetate production by acetyl-CoA synthetase (Mai &
Adams, 1996).
It has been reported that in T. kodakarensis the conversion of phosphoenolpyruvate (PEP) to pyruvate, normally
catalyzed by pyruvate kinase, can be performed by PEP
synthetase. PEP synthetase uses AMP and phosphate
instead of ADP for ATP generation and results in an
additional yield of up to 2 ATP per glucose (Sakuraba &
Ohshima, 2002; Sakuraba et al., 2004). A deletion strain
of pyruvate kinase still displayed growth on carbohydrates
but the deletion of PEP synthetase did not, indicating
that PEP synthetase is essential for growth on carbohydrates (Imanaka et al., 2006). However, kinetic data indicate that PEP synthetase in vitro functions in the
thermodynamically more favorable gluconeogenic direction (Hutchins et al., 2001). Currently, the amount of
substrate-level ATP formed during sugar degradation in
the Thermococcales is not clear. It is likely to be either
(1) 2 ATP produced per glucose molecule using only
pyruvate kinase or (2) 4 ATP produced using PEP synthetase. However, it is possible that both enzyme systems
are utilized in concert depending on the physiological
state of the cells, enabling metabolic flexibility and allowing for a greater energy yield under favorable conditions.
Ferredoxin is the electron acceptor for all oxidative
steps in the glycolytic pathway in Thermococcales.
Because of its low redox potential ( 480 mV), H2 production ( 420 mV) with ferredoxin as the electron donor
is thermodynamically favorable, and therefore, all reducing equivalents generated in sugar metabolism can be disposed of as H2 (Park et al., 1991; Smith et al., 1995;
Hagedoorn et al., 1998; Verhees et al., 2004). The enzyme
responsible for H2 formation in the Thermococcales is
a membrane-bound hydrogenase complex (Mbh). In
P. furiosus, Mbh is a complicated enzyme encoded by a
14-gene cluster (PF1423-PF1436, MbhA-N), in which the
MbhL subunit encodes the catalytic subunit (Fig. 2). Mbh
is unique among [NiFe]-hydrogenases in that it generates
both molecular H2 and can use the energy from this exergonic reaction to create an ion gradient (either H+ or
Na+) across the membrane (Sapra et al., 2003). For this
reason, Mbh has been postulated to represent the simplest
form of respiration in modern day metabolism. In effect,
ion pumping enables Mbh to regain some of the ATP
that is not synthesized owing to the absence of the phosphorylation step in the modified glycolytic pathway with
an estimated yield of 0.3 mol of ATP generated per mol
H2 formed (Sapra et al., 2003). Deletion mutagenesis
studies with T. kodakarensis show that Mbh is essential
for growth in the absence of S0 as no other pathway for
FEMS Microbiol Rev 37 (2013) 182–203
Modular Mbh-type complexes
185
(a)
(b)
(c)
Fig. 2. (a) Highly homologous multisubunit complexes and their catalytic subunits involved in evolving H2, reducing S0 and oxidizing electron
carriers such as ferredoxin and NADH. Subunits homologous to Mrp are indicated in blue, hydrogenase subunits are displayed in red, the MbhHtype subunits with both homology to Mrp and hydrogenase modules are colored purple, the MbhI subunit, unique to Mrp-Mbh, is colored white,
and active or potential active site containing subunit is boxed with red. The H subunit of Mrp-Mbh and Mrp-Mbx are most homologous to MrpD
and an MrpA homolog is absent. (b). Comparison of the [NiFe] binding motifs (termed L1 and L2) in Pyrococcus furiosus Mbh with the
comparable region in homologous proteins lacking hydrogenase activity. Cysteine residues are indicated in red and additional conserved residues
in blue, Rc: Rhodobacter capsulatus. (c). Schematic representation of the P. furiosus Mrp-Mbh complex. The coloring scheme is the same as in
Fig. 2a and subunits predicted to contain iron–sulfur clusters are indicated in blue.
cofactor recycling is available to sustain growth (Kanai
et al., 2011). As ferredoxin is used as the only electron
acceptor during sugar oxidation, there must be a mechanism of generating NADPH to supply reducing equivalents for biosynthesis. In P. furiosus, two cytosolic [NiFe]
hydrogenases (SHI and II) are present, and these can supply NADPH using H2 as electron donor (Ma & Adams,
2001). However, there must exist additional routes to
generate NADPH because the deletion of either or both
SHI and SHII did not display a growth defect (Lipscomb
et al., 2011). Moreover, not all Thermococcales species
contain both cytoplasmic hydrogenases and their true
physiological functions are unclear.
The genome of P. furiosus contains a 13-gene cluster
termed Mbx (PF1453-PF1441, MbxA-M) that is highly
homologous to Mbh (see Table 1). Interestingly, the ion
translocation modules as well as the [FeS] binding motifs
present in Mbh are conserved in Mbx, suggesting that this
complex could accept electrons from ferredoxin and create
an ion gradient analogous to Mbh (Silva et al., 2000; Sapra et al., 2003). Previous biochemical studies have shown
FEMS Microbiol Rev 37 (2013) 182–203
that Mbx has a distinct role in S0-dependent growth
of P. furiosus (Schut et al., 2007). The addition of S0
elucidated a primary response that included the downregulation of all hydrogenase-related genes (encoding
Mbh and two cytosolic hydrogenases SHI and SHII) and
the concomitant up-regulation of a number of genes presumably involved in the reduction of sulfur, including the
genes encoding the Mbx complex, a pyridine nucleotidedisulfide oxidoreductase domain containing protein
(PF1186), a glutaredoxin-like protein proposed to function as a protein disulfide oxidoreductase (Pdo, PF0094)
(Ladenstein & Ren, 2006), and an uncharacterized
transcriptional regulator cluster (PF2051-PF2052) (Schut
et al., 2007). The gene product of PF1186 was purified
from S0-grown cells and was characterized as a CoASHdependent NADPH elemental sulfur reductase [Nsr,
(Schut et al., 2007)]. This enzyme was implicated as the
key enzyme in the S0 reduction pathway. It was further
proposed that Mbx couples the oxidation of ferredoxin to
NADPH formation, which can donate electrons to the
soluble Nsr (Fig. 3). However, the exact physiological
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186
G.J. Schut et al.
Table 1. Properties and homologies of Mrp-Mbh and Mrp-Mbx genes*
Mbh locus
Gene
# Cys (mot)
Location (TMD)†
Mbx homolog
PF1423
PF1424
PF1425
PF1426
PF1427
PF1428
PF1429
PF1430
PF1431
PF1432
PF1433
PF1434
PF1435
PF1436
MbhA
MbhB
MbhC
MbhD
MbhE
MbhF
MbhG
MbhH
MbhI
MbhJ
MbhK
MbhL
MbhM
MbhN
1
0
0
0
0
0
1
2
0
5 [FeS]
0
5 [NiFe]
0
8 2x[FeS]
Mem
Mem
Mem
Mem
Mem
Mem
Mem
Mem
Mem
Cyto
Cyto
Cyto
Mem
Cyto
MbxA
MbxB
MbxC
MbxD
MbxF
MbxF
MbxG
MbxH, H’
MbxJ
MbxK
MbxL
MbxM
MbxN
(3)
(3)
(3)
(3)
(2)
(4)
(3)
(13)
(2)
(8)
Nuo homolog
Mrp homolog‡
EchA
NuoK§
NuoL, M, N¶
MrpE
MrpF
MrpG
MrpB
MrpB
MrpB
MrpC
MrpD
EchC
EchD
EchE
EchB
EchF
NuoB
NuoC**
NuoD
NuoH
NuoI
Ech homolog
*Homology searches were performed using NCBI blast and JCVI CMR (Altschul et al., 1997; Peterson et al., 2001).
†
The predicted cellular location is listed as Cyto, cytoplasm; Mem, membrane. The number of predicted transmembrane domains (TMD) is indicated [SOSUI, (Hirokawa et al., 1998)].
‡
No MrpA homologs are included in the Mrp-Mbh type complexes (see text for details).
§
MbhG has a low homology to NuoK (Mathiesen & Hagerhall, 2003).
¶
NuoL, M and N are homologous (Mathiesen & Hagerhall, 2002).
**Homology between EchD, MbhK, NuoC and MbxK is weak.
Mrp (multiple resistance and pH adaptation), Na+/H+ antiporter module; Mbh, membrane bound hydrogenase module; Mbx, membrane bound
oxidoreductase module (unknown substrate); Ech, energy converting hydrogenase; Nuo, NADH ubiquinone oxidoreductase.
Fig. 3. Schematic representation of the proposed physiological roles
of MBH and MBX in the production of H2 and hydrogen sulfide in
Pyrococcus furiosus and related organisms. Fdox and Fdred indicate
oxidized ferredoxin and reduced ferredoxin, respectively.
function of the Mbx complex has been hard to define, as
no ferredoxin-dependent NADP reduction or ferredoxindependent S0 reduction activities could be measured in
cell extracts or membrane preparations of P. furiosus
(Schut et al., 2007). Confirmation of Mbx involvement in
S0 reduction was recently provided by the disruption
mutant of Mbx (deletion of MbxL, potentially the active
subunit) whose growth in the presence of S0 is highly
impaired (Bridger et al., 2011). Surprisingly, the Nsr deleª 2012 Federation of European Microbiological Societies
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tion strain showed no obvious growth phenotype when
grown on S0, and the amount of sulfide produced via S0
reduction was not significantly affected. In contrast, the
MbxL deletion mutant produced no sulfide (above the
abiotic background) and exhibited a clear growth defect in
the presence of S0 with a reduction in cell yield of ~70%
(Bridger et al., 2011). These results demonstrate that Nsr
is not essential for growth with S0, but Mbx is an essential
component of the S0 reduction system. It is possible that
the NADPH produced by Mbx in Nsr deletion strain is
used by other enzymes in a promiscuous manner to
reduce S0, and such activity has been previously shown in
vitro for ferredoxin NADPH oxidoreductase (FNOR) and
both soluble hydrogenase I and hydrogenase II (Ma et al.,
1993; Ma & Adams, 1994). Nevertheless, these results
illustrate that Mbh and Mbx are highly analogous complexes that function to re-oxidize the reduced ferredoxin
produced in energy metabolism in the absence and
presence of S0, respectively.
Regulation of hydrogen and sulfur metabolism
The response to the availability of S0 in P. furiosus was
shown to be orchestrated by a transcriptional regulator
termed SurR that contains a CXXC motif redox switch
(Lipscomb et al., 2009). In its reduced state, SurR activates transcription of the genes encoding soluble hydrogenases I and II (SHI and SHII) and the membrane-bound
FEMS Microbiol Rev 37 (2013) 182–203
187
Modular Mbh-type complexes
hydrogenase (Mbh), as well as a number of other genes
involved in H2 metabolism (Lipscomb et al., 2009). At
the same time, reduced SurR represses the expression of
genes including those that are potentially involved in the
reduction in S0: Nsr, Mbx, Pdo, and the regulator cluster
PF2051-PF2052. By in vitro transcription and binding
studies it was shown that in the presence of S0 the CxxC
motif in SurR becomes oxidized and renders the protein
unable to bind to promoter DNA, resulting in the de-regulation of genes in the SurR regulon. As a result, the
three hydrogenases (Mbh, SHI and SHII) are no longer
expressed, while the genes involved in S0 metabolism are
no longer repressed and their transcript levels increase
(Lipscomb et al., 2009; Yang et al., 2010). The SurR regulator binds to a specific sequence (GTTn3AAC) in its target promoter (including its own promoter) and most of
the genes observed in the primary S0 response contain
this SurR binding motif. Bioinformatic analyses indicate
that this regulator is only found in Thermococcales, indicating that the SurR S0/hydrogen regulation uniquely
evolved within this order.
Based on biochemical and transcriptional data, we propose a core set of genes involved in the combined H2 and
sulfur metabolism of P. furiosus and of members of the
Thermococcales in general involving Mbh, SHI, SHII,
Mbx, Nsr, SurR, Pdo and PF2051/52 (Table 2). This
core set of genes is conserved within the Thermococcales,
with the exception of T. gammatolerans, which does not
encode any cytosolic [NiFe]-hydrogenases, and T. kodakaresis and P. horikoshii, which contain only a single
cytoplasmic hydrogenase. The genes encoding the glutaredoxin-like protein (Pdo) and SurR share the same
promoter region in all Thermococcales and both are subjected to SurR control. However, the precise role of Pdo
in sulfur metabolism is not clear and no biochemical
activity related to S0 reduction has been demonstrated
(Schut et al., 2007). The Pdo gene product is characterized as a protein disulfide oxidoreductase, capable of
reducing disulfide bonds in insulin using DTT as a reductant (Ren et al., 1998; Pedone et al., 2004). Homologs of
Pdo are widespread in both the archaeal and bacterial
domains and are also found in organisms that do not utilize sulfur to support their metabolism, indicating that
Pdo is not likely to be uniquely involved in S0 reduction.
In the bacterium Thermotoga maritima, a Pdo homolog
was characterized and it was shown that a thioredoxin
reductase could transfer electrons from NADPH to Pdo
(Yang & Ma, 2010). The genomes of the Thermococcales
also encode a homolog of the thioredoxin reductase and
the homolog in P. furiosus (PF1422) is also part of the
SurR regulon. This suggests that thiol-based electron flow
might also be involved in S0 reduction by the members
of the Thermococcales, although the enzymes and pathFEMS Microbiol Rev 37 (2013) 182–203
ways are unclear. Although the core set of H2 and sulfurrelated genes linked by SurR appear to be restricted to
the order of Thermococcales, homologs of both Mbh and
Mbx are found in other microbial systems in which they
also are related to H2 metabolism. These will be discussed
later, using the P. furiosus Mrp-Mbh as the point of reference.
Mrp-Mbh-type complexes and their
taxonomic distribution
The 14 subunits of the Mbh complex show modular composition, a mosaic of defined components that include
Mrp-type Na+/H+ antiporter homologs (MbhA-H) and
hydrogenase components (MbhH-N). MbhH-N exhibit
homology to all of the subunits of Ech-type hydrogenases
as well as to a number of components of complex I (see
Table 1) (Soboh et al., 2004; Hedderich & Forzi, 2005;
Swartz et al., 2005). The Mrp-like system was first genetically characterized in the alkaliphile Bacillus halodurans,
where the Mrp Na+/H+ antiporter was identified as indispensable for pH homeostasis under alkaline conditions
(Hamamoto et al., 1994). The Mrp system in this organism consists of seven genes (MrpA-G), all with transmembrane domains. Some degree of redundancy is
apparent: MrpA contains modular elements that are
homologous to MrpB and MrpD (Swartz et al., 2005),
suggesting that MrpA evolved from a gene fusion involving the B and D genes. Intriguingly, the Mrp module in
the P. furiosus Mbh cluster lacks MrpA and only encodes
homologs of MrpB-G (Table 1 and Fig. 2). It should be
noted that P. furiosus also contains another Mrp-like cluster (MrpB-MrpG: PF1153-PF1149) of unknown function
that is not connected to Mbh or Mbx domains. This type
of Mrp cluster is conserved throughout the Thermococcales as well as in many other archaea. Although the
function of this Mrp-like cluster is unknown, its presence
suggests that the Mrp-type systems in the Thermococcales
also evolved independently and under different selective
pressure from the hydrogenase components. It was demonstrated that the A1A0-type ATPase in P. furiosus forms
ATP using sodium rather than a proton gradient. This
suggests that the Mrp system could function in the
exchange of a proton gradient generated by the hydrogenase module for a sodium gradient, enabling the ATPase
to generate ATP (Pisa et al., 2007).
For the purposes of this review, we use the P. furiosus
nomenclature and its 14-subunit (MbhA-N) composition
as a template, defining Mbh by the modular composition
of Mrp-type genes (MrpB-G homologs) and Group 4
[NiFe] hydrogenase-type genes as annotated in Table 1.
Here we define the Mbh and Mbx clusters as Mrp-Mbh
and Mrp-Mbx to emphasize and distinguish modular
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188
GQS_02525-530
GQS_03675
GQS_03680
*Homology searches were performed using NCBI blast and JCVI CMR (Altschul et al., 1997; Peterson et al., 2001).
The genome of Thermococcus AM4 is unfinished and the ORF numbers are not consecutive.
Thermococcus sp. 4557
Thermococcus AM4†
Thermococcus barophilus
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†
GQS_02710-770
GQS_02460-75
GQS_04975-990
GQS_04305
TAM4_989-549
TERMP_00904-03
TAM4_1905
TERMP_00657
TAM4_2127
TERMP_00656
TAM4_474-490
TERMP_00865-53
TAM4_579-655
TERMP_00539-36
TAM4_307-399
TERMP_00067-70
TAM4_984
TERMP_02185
PF2051-52
PAB229-3022
PH0062-S001
PNA2_0599-98
PYCH_14930-940
TK1261-60
TGAM_0437-38
TON_0525-24
TSIB_1263-64
PF0094
PAB2245
PH0178
PNA2_0698
PYCH_16300
TK1085
TGAM_1302
TON_0319
TSIB_0991
PF0095
PAB0108
PH0180
PNA2_0699
PYCH_16310
TK1086
TGAM_1303
TON_0318
TSIB_0990
PF1186
PAB0936
PH0572
PNA2_1248
PYCH_07890
TK1299
TGAM_1050
TON_0305
TSIB_0263
PF1453-41
PAB0485-96
PH1456-46
PNA2_0044-32
PYCH_11530-410
TK1226-14
TGAM_0703-15
TON_0498-86
TSIB_1917-05
PF1329-32
PAB0638-41
PNA2_1555-51
PYCH_00020-50
TON_0055-52
TSIB_1520-23
PF1423-36
PAB1884-94
PH1427-40
PNA2_0015-28
PYCH_11230-360
TK2080-93
TGAM_0047-34
TON_1582-95
TSIB_1309-96
TSIB_1295-82
TAM4_1289-1255
TERMP_01498-85
TERMP_01484-71
GQS_07585-520
GQS_07515-445
Pyrococcus furiosus
Pyrococcus abyssi
Pyrococcus horikoshii
Pyrococcus NA2
Pyrococcus yayanosii
Thermococcus kodakarensis
Thermococcus gammatolerans
Thermococcus onnurineus
Thermococcus sibiricus
PF0891-94
PAB1784-87
PH1290-94
PNA2_1665-68
PYCH_08370-400
TK2072-69
TON_0537-34
TSIB_0282-85
Mrp-Mbx
SHII
SHI
Mrp-Mbh
Species
Table 2. Distribution of the core set of genes involved in hydrogen and sulfur metabolism in Thermococcales*
Nsr
SurR
Pdo
PF2051-2
G.J. Schut et al.
composition. MbhL contains the [NiFe] catatlyic site of
the hydrogenase and this is coordinated by two pairs of
cysteine residues termed L1 and L2 (Vignais & Billoud,
2007). The Group 4 [NiFe] hydrogenases can be readily
identified based on sequence conservation in the motif
flanking regions, as well as by phylogenetic clustering
(Fig. 1). As discussed later, based on their catalytic
subunits, the various Mbh-type enzymes can also be differentiated from other Group 4 [NiFe] hydrogenase in
genome sequences and also from each other. While the
subunit composition of the Mbx cluster is very similar to
the Mbh cluster and the genes comprising the Mbx cluster have homologs in Mbh (Table 1), these two complexes can be readily differentiated from each other. The
Mbx cluster is composed of 13 genes and the difference
in the number of genes when compared to the Mbh cluster (14 genes) is owing to the fact that MbxE resembles a
fusion of MbhE and MbhF, MbhH is represented by two
homologous subunits in Mbx (MbxH and MbxH’), and
no homolog of MbhI (which is of unknown function) is
present in the Mbx cluster. Mbx does not have significant
hydrogenase activity based on biochemical data (Adams
et al., 2001). Moreover, the L1 and L2 [NiFe] coordinating residues in Mbh are not conserved in Mbx as the second cysteine in each motif is absent (Fig. 2b) and
significant diversification is present in the L1 and L2
flanking regions relative to Group 4 hydrogenases (Schut
et al., 2007; Vignais & Billoud, 2007).
Mrp-Mbh-type complexes in Euryarchaeota
The Mrp-Mbh and Mrp-Mbx clusters are represented by
highly similar gene clusters in the available Thermococcales genome sequences, (Table 3). This conservation suggests that these gene clusters evolved early in the
evolution of the Thermococcales and that they perform a
central role in the metabolism of these organisms. Interestingly, Thermococcus sp. 4557, T. sibiricus, and T. barophilus contain a second copy of the Mrp-Mbh cluster
(Mrp-Mbh2) directly adjacent to the first copy (Table 3).
Mrp-Mbh2 differs from Mrp-Mbh1 in that the second
[NiFe] binding motif (L2) contains an additional cysteine
(CMCC). The function of this second Mrp-Mbh cluster is
not clear. Most methanogens also contain homologs of
Group 4 energy-converting membrane-bound hydrogenases of either the Ech- or Eha-/Ehb-type. These proteins
are similar to the Mrp-Mbh-type complex but have a different subunit composition (Vignais & Billoud, 2007;
Thauer et al., 2010). Interestingly, three methanogens of
the order Methanomicrobiales (Methanoplanus petrolearius, Methanospirillum hungatei and Methanocorpusculum
labreanum) do encode a bonafide Mrp-Mbh homolog
(Table 3); however, the presence of Mrp-Mbh in these
FEMS Microbiol Rev 37 (2013) 182–203
189
Modular Mbh-type complexes
organisms is puzzling considering that their genomes also
encode Eha- and Ech-type hydrogenases (Anderson et al.,
2009).
Aciduliprofundum boonei (Reysenbach et al., 2006; Reysenbach & Flores, 2008), which so far is the only cultivable representative of the Archaeal DHVE2 order, also
contains Mrp-Mbh and Mrp-Mbx homologs (Table 3). It
also contains a homolog of cytoplasmic hydrogenase SHI
but appears to lack SurR and Nsr. Aciduliprofundum
boonei is a strict anaerobe and is proposed to utilize
peptides as carbon and energy sources using a metabolic
pathway similar to that found in the Thermococcales with
sulfur or ferric iron as the electron acceptor (Reysenbach
& Flores, 2008). Representatives of the order of DHVE2
are thought to be very abundant thermoacidophiles in
hydrothermal vent systems and are most closely related to
the Thermoplasmales (Reysenbach et al., 2006).
Analysis of the available Thermococales genome
sequences has yielded additional gene clusters encoding
multi-domain Mrp-Mbh complexes (Fig. 4). For example,
T. onnurineus harbors four gene clusters containing MrpMbh modules (Lee et al., 2008). Two of these clusters
contain genes potentially coding for a formate dehydrogenase module. We will term these Mrp-Mbh-Fdh1 and
Mrp-Mbh-Fdh2 (see Table 3) (Lim et al., 2010). The conversion of formate to bicarbonate and H2 was until
recently not considered a means of energy generation but
rather a response to avoid accumulation of formate
(Bohm et al., 1990; Takacs et al., 2008). However, it was
shown that T. onnurineus could derive enough energy
from the conversion of formate to bicarbonate and H2 to
sustain growth (Kim et al., 2010). Through transcript and
mutational analysis it became clear that only the MrpMbh-Fdh2 cluster (TON_1563-TON_1580) is essential for
growth on formate. Mrp-Mbh-Fdh1 and Mrp-Mbh-Fdh2
are very similar in both sequence and gene synteny but
Mrp-Mbh-Fdh2 harbors an additional gene potentially
coding for a formate transporter. On the basis of the gene
organization of Mrp-Mbh-Fdh2 it was proposed that formate is transported into the cell where the Fdh module
converts formate to bicarbonate and electrons are shuttled
to the Mrp-Mbh module, which produces H2 with the
concomitant generation of an electrochemical gradient
ultimately leading to ATP formation (Kim et al., 2010).
Pyrococcus yayanosii and Thermococcus gammatolerans also
contain Mrp-Mbh-Fdh2 and may also have the potential
to utilize formate as an energy source in a manner similar
to T. onnurineus (Zivanovic et al., 2009; Jun et al., 2011).
The presence of an Mrp-Mbh-Fdh1 lacking a formate
transporter in P. abyssi and T onnurineus indicates that
formate potentially is formed intracellularly and that this
can be utilized to generate additional energy by conversion to hydrogen. The generation of formate intracelluFEMS Microbiol Rev 37 (2013) 182–203
larly could be accomplished through the activity of a
pyruvate-formate lyase; however, only the genomes of
T. kodakarensis and T. sibiricus encode such an enzyme
(TK0289, TSIB_0631) and neither harbors an Mrp-MbhFdh1-type gene cluster. The possible presence and source
of any intracellular formate therefore remains unknown
(Fukui et al., 2005; Mardanov et al., 2009). Interestingly,
T. kodakarensis contains a cluster of soluble formate
dehydrogenase-related genes just upstream of its MrpMbh locus (TK2076-TK2079) but their function is also
unknown.
Bioinformatic analyses also reveal an Mrp-Mbh-type
modular system with a carbon monoxide dehydrogenase
module in T. onnurineus, which we will term Mrp-MbhCodh, (Lee et al., 2008) (Table 3). Growth experiments
indeed demonstrated that T. onnurineus can grow in the
presence of CO and form H2 in an energy yielding fashion
(Yun et al., 2011). Carboxydotrophic growth has also been
shown for T. barophilus and Thermococcus AM4 and their
genomes also harbor clusters encoding an Mrp-Mbh-Codh
(Sokolova et al., 2004; Vannier et al., 2011), Table 3). The
cluster is composed of three functional domains, Mrp,
Mbh and a Codh domain (CooF and CooS), together with
a potential regulator and a Codh maturation factor (CooC)
encoded within the same cluster (Lim et al., 2010). This
cluster is analogous yet very distinct from the CO-oxidizing, H2-forming enzyme complex of the bacterium C. hydrogenoformans. This contains an Ech-type hydrogenase
and two CO dehydrogenase subunits [CooF and CooS;
(Soboh et al., 2002)] but lacks an Mrp module.
Mrp-Mbh-type complexes in Crenarchaeota
Homologs of Mrp-Mbh and Mrp-Mbx orthologs have a
limited distribution within the phylum Crenarchaeota.
Only one order, the Desulfurococcales, contain complete
homologs of Mrp-Mbh and Mrp-Mbx (Table 3). Its
members are characterized by a S0-dependent and heterotrophic life style analogous to that of the Thermococcales,
although a few strains are reported to not utilize S0 and
some can grow autotrophically (Vieille & Zeikus, 2001;
Boyd et al., 2007; Prokofeva et al., 2009). The genome
sequences of several members of the Desulfurococcales
are available as a number of type strains were sequenced
as part of the Genomic Encyclopedia of Bacteria and
Archaea project (http://www.jgi.doe.gov/programs/GEBA).
The homologs of Mrp-Mbh and Mrp-Mbx found in the
Desulfurococcales are present in gene clusters that are
rearranged compared with those found in the Thermococcales (Table 3). For example, both Mrp-Mbh and
Mrp-Mbx contain a fused homolog of the E and F subunit, the homolog to MbhI is poorly conserved, a fused
homolog of MbxJ and MbxK is present, there is only one
ª 2012 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
furiosus
Abyssi
horikoshii
NA2
yayanosii
ª 2012 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
Crenarchaeota
Desulfurococcus
kamchatkensis
Desulfurococcus
mucosus
Thermosphaera
aggregans
Ignisphaera aggregans
Thermococcus
kodakarensis
Thermococcus
gammatolerans
Thermococcus
onnurineus
Thermococcus sp.
AM4
Thermococcus
barophilus
Thermococcus
sibiricus
Thermococcus sp.
4557
Aciduliprofundum
boonei
Methanoplanus
petrolearius
Methanospirillum
hungatei
Methanocorpusculum
labreanum
Pyrococcus
Pyrococcus
Pyrococcus
Pyrococcus
Pyrococcus
Euryarchaeota
Species/Mbh-type
cluster
Igag_1902-14
Tagg_0624-36
Desmu_0818-06
DKAM_0419-07
DKAM_095645
Desmu_100213
Tagg_1025-36
GQS_02710770
Aboo_0386-74
TAM4_474690
TERMP_0086553
TSIB_1917-05
TGAM_070315
TON_0498-86
PF1453-41
PAB0485-96
PH1456-46
PNA2_0044-32
PYCH_11530410
TK1226-14
Mrp-Mbx
MbxNAD(P)H
Pep
Pep, CBH
Pep, CO
Pep, formate,
CO
Pep, CO
Pep, formate
Pep, CBH
Pep, CBH
Pep
Pep
Pep
Pep, CBH
Energy
source
Pep, CBH
Pep
Pep, CBH
Pep, CBH
H2
TAM4_10571048
TERMP_0115439
TON_1017-31
Mrp-MbhCodh
Mlab_0948-61
TGAM_006548
TON_1563-80
PYCH_11030200
Mrp-MbhFdh2
H2
GQS_02455380
TON_028166
PAB1389-02
Mrp-MbhFdh1
Mhun_2579-92
DKAM_101523
Desmu_006153
Tagg_005950
MbhNAD(P)H
H2
GQS_07515445
TERMP_014841471
TSIB_1295-82
Mrp-Mbh2
Mpet_1584-71
Aboo_0664-77
GQS_07585-520
TSIB_1309-96
TAM4_12891255
TERMP_01498-85
TON_1582-95
TGAM_0047-34
PF1423-36
PAB1884-94
PH1427-40
PNA2_0015-28
PYCH_11240360
TK2080-93
Mrp-Mbh
Table 3. Distribution of Mrp-Mbh type oxidoreductases in the domains archaea and bacteria*
H+
H+
H + , S0
H + , S0
CO2
CO2
H+, S0 or
Fe3+
CO2
H + , S0
H+ , S0
H+ , S0
H + , S0
H + , S0
H+ , S0
H+ , S0
H + , S0
H + , S0
H+, S0
Electron
acceptors
190
G.J. Schut et al.
FEMS Microbiol Rev 37 (2013) 182–203
FEMS Microbiol Rev 37 (2013) 182–203
COPR5265_092034
Amico_1275-62
Smar_0018-30
Mrp-Mbh
Mrp-Mbh2
Kole_057460‡
Smar_105571†
MbhNAD(P)H
Mrp-MbhFdh1
Mrp-MbhFdh2
Mrp-MbhCodh
*Homology searches were performed using NCBI blast and JCVI CMR (Altschul et al., 1997; Peterson et al., 2001)
†
S. marinus Mbh-NAD(P)H cluster contains homologs of all Mrp-Mbh subunits and three homologs to the MbhH subunit
‡
K. olearia Mbh-NADPH cluster contains homologs of Mrp-Mbh subunits except for MbhI.
Coprothermobacter
proteolyticus
Aminobacterium
colombiense
Thermoanaerobacter
wiegelii
Thermosipho
melanesiensis
Thermosipho
africanus
Fervidobacterium
nodosum
Kosmotoga olearia
Petrotoga mobilis
Bacteria
Thermotoga maritima
Thermotoga
neapolitana
Thermotoga sp. RQ2
Staphylothermus
marinus
Staphylothermus
hellenicus
Species/Mbh-type
cluster
Table 3. Continued
Thewi_003446
TM1205-17
CTN_136654
TRQ2_161301
Pmob_089785
Tmel_121022
THA_169682
Fnod_174835
Kole_206351
H+, S0, SS03
Pep, CBH
H+
Pep, amino
acids
CBH
H+
H+, S0, SS03
Pep, CBH
H+, S0, SS03
H+, S0, SS03
Pep, CBH
Pep, CBH
H+, S0, SS03
Pep, CBH
H+, S0, SS03
H+, S0, SS03
Pep, CBH
Pep, CBH
H+, S0, SS03
H+, S0, SS03
S0
H + , S0
Electron
acceptors
Pep, CBH
Pep, CBH
Pep, CBH
Shell_0146-34
Energy
source
Pep
MbxNAD(P)H
Smar_0645-57
Mrp-Mbx
Modular Mbh-type complexes
191
ª 2012 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
192
G.J. Schut et al.
Fig. 4. The utilization of the Mrp-Mbh-type complexes in enzymes involved in metabolism of formate, carbon monoxide, S0 and NAD(P)H by the
inclusion of additional functional modules. Based on the nomenclature of Pyrococcus furiosus Mrp-Mbh (Table 1) the modules are defined as
follow: Mrp, MbhA-H; Mbh, MbhH-N (MbhH is common to both Mrp and Mbh modules); Mbx, MbxH-N; Mrp-Mbh-Fdh, the formate
dehydrogenase module is represented by TON_1563 (FdhA) and TON_1564 (Lee et al., 2008); Mrp-Mbh-CodH, the carbon monoxide
dehydrogenase module is represented by TON_1017 (CooS) and TON_1018 (CooF) (Lee et al., 2008); Mrp-Mbh-NAD(P)H, the FAD/NAD(P)H
binding module is represented by Tagg_0059 [pyridine nucleotide-disulfide oxidoreductase domain, IPR013027 (Hunter et al., 2009)] and
Tagg_0058 ([FeS] containing (IPR001450, Hunter et al., 2009); Mrp-Mbx-NAD(P)H, the FAD/NAD(P)H binding module is represented by TM1217
[pyridine nucleotide-disulfide oxidoreductase domain (IPR013027, Hunter et al., 2009)]. The small light blue bar for the Mrp module in Mbh-NAD
(P)H represents two genes homologous to MbhH.
homolog to MbxH rather than two, and in the Mbx cluster an additional gene encoding an unknown membranebound subunit is present.
For other members of the Crenarchaeota, the presence
or absence of homologs of Mrp-Mbh and Mrp-Mbx is
indicative of their metabolic capability (Table 3). For
example, Ignisphaera aggregans does not contain an
ª 2012 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
Mrp-Mbx homolog and does not utilize elemental sulfur,
in fact, its growth is inhibited by S0 (Niederberger et al.,
2006). Ignisphaera aggregans was isolated from a terrestrial geothermal spring in New Zealand and grows by
fermenting carbohydrates. As a homolog of glyceraldehyde-3-phosphate ferredoxin oxidoreductase (GAPOR) is
encoded in the genome of I. aggregans (Igag_1482), it is
FEMS Microbiol Rev 37 (2013) 182–203
Modular Mbh-type complexes
presumed to utilize a modified EM-pathway analogous to
that in the Thermococcales where reducing equivalents in
the form of reduced ferredoxin are disposed of as H2
(Niederberger et al., 2006; Goker et al., 2011). Interestingly, I.aggregans does not encode any other hydrogenase
besides Mrp-Mbh, indicating a relatively simple H2 respiratory-type metabolism (Goker et al., 2011). In contrast,
Staphylothermus hellenicus is unusual in that its genome
does not seem to encode any recognizable hydrogenase,
although an Mrp-Mbx cluster is present. Perhaps not surprisingly, S. hellenicus is obligatory dependent on S0 for
growth and does not form H2 at all (Arab et al., 2000).
Staphylothermus hellenicus was isolated from a marine
environment and is reported to grow only on complex
media in the presence of S0 (Arab et al., 2000). In contrast, the growth of Thermosphaera (Ta) aggregans, which
has both Mrp-Mbx and Mrp-Mbh clusters, is inhibited
by the presence of S0 (Huber et al., 1998). These results
suggest that the Mrp-Mbh and Mrp-Mbx play important
roles in H2 and sulfur metabolism in several Desulfurococcales, although their exact role in sulfur reduction has
yet to be definitively demonstrated.
In addition to Mrp-Mbh and Mrp-Mbx homologs,
Desulfurococcus kamchatkensis, D. mucosus and Ta. aggregans contain another member of the Group 4 hydrogenases (Table 3). This complex consists of homologs of
subunits MbhH-MbhN of the Mbh hydrogenase module
and a potential NAD(P)H interaction module (Fig. 4).
The subunits for the Mrp antiporter module are absent
with the exception of two homologs of the MbhH subunit. The NAD(P)H module consists of two ORFs, one
with homology to a glutamate synthase subunit (pyridine
nucleotide-disulfide oxidoreductase domain) and the second with four potential iron–sulfur cluster-binding
motifs. Considering that this potential NAD(P)H-linked
hydrogenase, which we termed Mbh-NAD(P)H, contains
an energy-converting module, it may be the case that it
can use an electrochemical gradient to couple the oxidation of NAD(P)H to H2 production (Table 3). This is
utilized during the fermentation of peptides, where NAD
(P)H is formed by the deamination of amino acids but
the reoxidation of NAD(P)H coupled to H2 production is
thermodynamically unfavorable (Thauer et al., 1977).
This is precisely the reason that an external electron
acceptor such as S0 is needed during peptide-dependent
growth in the Thermococcales (Adams et al., 2001). In
the absence of external electron acceptors, Ta. aggregans
ferments peptides to H2, CO2 and various organic acids
presumably with the Mbh-NAD(P)H complex encoded in
its genome (Niederberger et al., 2006).
Interestingly, S. marinus contains a variant of an
Mbh-NADPH system, in which a complete Mrp module is encoded along with three copies of the MbhH
FEMS Microbiol Rev 37 (2013) 182–203
193
subunit (Table 3). This organism exhibits an obligatory
S0-dependent mode of growth on complex substrates
(yeast extract and peptone). When S0 is limiting, the
growth rate and final cell yield decrease and cells produce
H2 and hydrogen sulfide simultaneously (Hao & Ma,
2003). However, it should be noted that growth was
observed using a very low sulfur concentration
(0.01 g L 1) with minimal H2S production relative to H2
production [87 lM H2S vs. 1.78 mM H2 (Hao & Ma,
2003)]. This indicates that the Mbh-NAD(P)H complex
might function as an energy-converting NAD(P)H-linked
hydrogenase with H2 evolution from NAD(P)H using an
ion gradient to drive the reaction. This organism might
still need a minimal amount of S0 for other purposes. In
contrast, Hyperthermus butylicus and Ignicoccus hospitalis,
also within the order of Desulfurococcales, exhibit S0dependent growth; however, they do not contain homologs of Mrp-Mbx or Mrp-Mbh (Brugger et al., 2007;
Podar et al., 2008). These organisms most likely utilize a
membrane-bound
molybdopterin-containing
sulfur
reductase (Igni_0801-Igni_0803, Hbut_0373-Hbut_0371)
as described for Acidianus ambivalens (Laska et al., 2003).
Both organisms can generate energy by the oxidation of
H2 using a Group 1 membrane-bound uptake hydrogenase (Igni_1367-Igni_1369, Hbut_1368-Hbut_1371) coupled to S0 as the electron acceptor but only I. hospitalis
has been shown to grow autotrophically (Zillig et al.,
1990; Paper et al., 2007).
Of the Crenarchaeota, members of the order Desulfurococcales therefore make interesting examples of the
manner in which Mbh, Mrp and Mbx modules have been
utilized. Members of the order Acidilobales (Acidilobus
saccharavorans and A. sulfurireducens), a group of sulfurreducing organisms that characteristically inhabit acidic,
terrestrial geothermal springs, do not contain homologs
of Mbx or Mbh (E.S. Boyd, unpublished data), although
a close homolog [NADH dehydrogenase subunit D
(NuoD)] was incorrectly annotated as MbxL (Mardanov
et al., 2010). It is likely that these organisms utilize
a membrane-bound molybdopterin-containing sulfur
reductase (ASAC_1394-ASAC_1397) as suggested previously for H. butylicus and I. hospitalis. These results are
consistent with the physiological characterization of the
isolates as S0-dependent heterotrophs that are unable to
grow and produce H2 in the absence of an external electron acceptor (Boyd et al., 2007; Prokofeva et al., 2009).
Thermofilum pendens is so far the only organism within
the order Thermoproteales that contains representatives
of the Group 4 hydrogenases (Tpen_1070-Tpen_1077,
Tpen_0190-Tpen_0180) (Anderson et al., 2008). However, most of the genes encoding subunits of the Mrp
module are missing and the functions of these hydrogenases are not known.
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194
Mrp-Mbh-type complexes in Bacteria
The bacterial order Thermotogales is comprised of thermophiles and genome sequences of its members indicate
a relatively high percentage of genes obtained through
horizontal transfer (Nelson et al., 1999; Zhaxybayeva
et al., 2009). These also include Mrp-Mbh-type gene clusters (Table 3). This order is characterized by the ability of
its members to metabolize a wide variety of carbohydrates
(Huber et al., 1986) to produce acetate, CO2 and H2
from carbohydrate metabolism (Schroder et al., 1994).
Thermotoga maritima is the type species of this order and
oxidizes a wide variety of both simple and complex carbohydrates (Conners et al., 2006). However, the pathway
of electron flow is different from that found in the Thermococcales, which have a modified, ferredoxin-dependent
glycolytic pathway. In contrast, T. maritima uses the classical Embden-Meyerhof and Entner-Doudoroff pathways
to oxidize glucose (Selig et al., 1997). This means that
both ferredoxin and NAD function as physiological electron carriers and that both are recycled with the generation of H2. The generation of H2 is accomplished by a
novel so-called bifurcating [FeFe] hydrogenase that simultaneously oxidizes ferredoxin and NADH with concomitant production of H2 using the exergonic reaction of
ferredoxin oxidation to drive the endergonic oxidation of
NADH (Schut & Adams, 2009). One of the genes clusters
that was transferred from the Thermococcales to the
Thermotogales is an Mrp-Mbx-type cluster in which all
13 subunits found in P. furiosus Mrp-Mbx are conserved
both in sequence and synteny. However, the last subunit
in the operon (Tm1217, the N-terminus is homologous
to Mrp-MbxN) appears to be fused with a pyridine
nucleotide-disulfide oxidoreductase domain in the
Thermotogales (Table 3 and Fig. 4). Interestingly, the
NAD(P)H input module from T. maritima is homologous to the input module of the Mbh-NAD(P)H-type
hydrogenase found in some of the Desulfurococcales
mentioned previously, suggesting that they have a common origin. However, the function of this cluster, which
we have termed Mrp-Mbx-NAD(P)H, is not known.
Although several members of the Thermotogales can
use alternative electron acceptors for growth, such as
S0 and thiosulfate, very little is known about how these
compounds are metabolized (Ravot et al., 1995). Because
Mrp-Mbx is involved in S0 metabolism in the Thermococales (Schut et al., 2007; Bridger et al., 2011; Kanai et al.,
2011) it is tempting to speculate that the Mrp-Mbx
homolog plays a role in S0 or thiosulfate reduction in the
Thermotogales. However, this Mrp-Mbx-NAD(P)H
cluster is not universally conserved within the Thermotogales. It is not present in T. petrophila, T. lettingae, and
T. naphtophila; yet, these organisms are still capable of
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G.J. Schut et al.
reducing S0 and/or thiosulfate (Takahata et al., 2001; Balk
et al., 2002). This observation indicates that the mechanism of S0 reduction might not be dependent on these
Mrp-Mbx homologs and serves to emphasize our lack of
understanding of S0 metabolism in the Thermotogales.
Most of the Thermotogales species produce H2 as their
primary reduced product of fermentation and most of
them contain a homolog of the bifurcating [FeFe]
hydrogenase found in T. maritima (Schut & Adams,
2009). One exception is Kosmotoga olearia, which lacks
the trimeric [FeFe] hydrogenase and contains two potential monomeric [FeFe] hydrogenases (Kole_0172 and
Kole_1794), a Mrp-Mbx-NAD(P)H homolog (Kole_2063Kole_2051), and a Mrp-Mbh homolog with a NAD(P)H
input module (termed Mrp-Mbh-NAD(P)H, Kole_0574Kole_0560, Table 3). Interestingly, the NAD(P)H input
module consists of three subunits. One contains an iron–
sulfur motif and is homologous to that in Mbh-NADPH
system of the Desulfurococcales, while the other two
share homology with two of the subunits (gamma and
beta) of the cytosolic hydrogenases (such as P. furiosus
SHI) present in the Thermococcales. Although very little
information is available on K. olearia, it ferments carbohydrates to acetate, CO2 and H2 like other members of
the Thermotogales, (Dipippo et al., 2009). The absence of
a bifurcating hydrogenase indicates that the Mrp-MbhNAD(P)H hydrogenase complex, possibly in combination
with one or both monomeric [FeFe] hydrogenases, could
convert most reducing equivalents generated in sugar
degradation to H2 in which the Mrp-Mbh_NAD(P)H
complex functions as a NAD(P)H-linked hydrogenase
using the ion gradient to drive H2 production.
Two other bacteria, in this case non-thermophilic, contain homologs of Mrp-Mbh, Aminobacterium colombiense,
and Coprothermobacter proteolyticus [Amico_1275-Amico_1262, COPRO5265_0920- COPRO5265_0934, Table 3
(Chertkov et al., 2011)]. Both organisms were isolated
from waste water and ferment both complex substrates
and a variety of sugars mainly to acetate, H2, and CO2
(Ollivier et al., 1985; Baena et al., 1998; Etchebehere &
Muxi, 2000). Aminobacterium colombiense contains an
additional cytosolic [NiFe] hydrogenase cluster (Amico_1558-1553) of the Group 3d type (Vignais & Billoud,
2007). Interestingly, from sequence comparisons, the
[NiFe] active site subunit of the Mrp-Mbh in C. proteolyticus (COPRO5265_0933) does not appear to be synthesized as an inactive preprotein. It lacks the C-terminal
extension that is normally cleaved to activate the NiFesite, and accordingly, an associated protease is not
encoded in the adjacent cluster of accessory genes
(COPRO5265_0935- COPRO5265_0941). Coprothermobacter proteolyticus contains two additional [FeFe]
hydrogenases, one potentially of the bifurcating type
FEMS Microbiol Rev 37 (2013) 182–203
Modular Mbh-type complexes
(COPRO5265_0177- COPRO5265_0181) and a monomeric type (COPRO5256_0174). Strikingly, both bacteria
encode all homologs of the Thermococcales Mrp-Mbh
cluster. In A. colombiense, the synteny is conserved while
in C. proteolyticus the [NiFe] maturation genes are
co-localized with the Mrp-Mbh cluster. Given the limited
distribution of the Mbh type within the domain bacteria
and the organization in these two bacteria it is tempting
to speculate that the gene clusters encoding these MrpMbh homologs have been horizontally transferred from
an archaeal source relatively recently in evolutionary time.
Another striking case of potential horizontal transfer is
represented by the presence of an Mrp-Mbx homolog in
the moderate thermophilic anaerobe, Thermoanaerobacter
wiegelii (Table 3). It is not known whether this Mrp-Mbx
cluster is active or whether this organism is capable utilizing S0 as an electron acceptor.
Phylogeny of Mrp-Mbh-type complexes in
Archaea and Bacteria
Phylogenetic analysis based on the active site containing
L-subunit of the Mbh/Mbx module indicates that these
complexes likely originated in the archaeal domain, as
indicated by paraphyletic nesting of monophyletic
bacterial sequences within these lineages. Aquisition of
Mrp-Mbh/Mbx complexes in bacteria likely occurred via
horizontal gene transfer (HGT) early in the evolutionary
195
history of bacteria, with several additional HGT events
occurring more recently (Fig. 5). For example, the MrpMbh found in A. colombiense (Amico_1275-Amico_1262),
clearly clusters together with the Mrp-Mbh complexes of
the Thermococcales and the synteny of the genes in cluster is preserved, indicative of a relatively recent lateral
transfer event between these lineages (Fig. 5). This analysis also shows that the monophyletic Crenarchaeal and
Euryarchaeal Mrp-Mbh and Mrp-Mbx lineages nest
with bacterial lineages, suggesting that acquisition of
Mrp-Mbh and Mrp-Mbx in bacteria occurred after the
split between the Crenarchaea and the Euryarchaea
(Fig. 5). Intriguigingly, the Mrp-Mbh complexes that
contain additional catalytic domains (Mrp-Mbh-Fdh,
Mrp-Mbh-Codh and Mrp-Mbh-NADPH) appear to cluster together, suggesting that these complexes have a common ancestor despite the fact that Mrp-Mbh-NADPH is
found in Crenarchaeota while Mrp-Mbh-Fdh and MrpMbh-Codh are present in the Euryarchaeota and are so
far restricted to the Thermococales (Fig. 5). The most
parsimonious interpretation for this observation is that
the additional Fdh, Codh or NADPH module was initially
recruited to an ancestral Mrp-Mbh module, likely in a
member of the Euryarchaeota, and that this resulted in
the diversification of this lineage away from the MrpMbh module found in Euryarchaeota today. Importantly,
the conservation in Mrp-Mbh modular content observed
among the two primary lineages comprising Mrp-Mbh,
Fig. 5. Phylogenetic distribution of Mrp-Mbh/Mbx-type complexes and their relationship to Complex I. The terminal labels correspond to taxa
identified in Tables 2 and 3. The phylogram includes representative sequences from a number of Mrp-Mbh/Mbx functions (Table 3) with
additional examples for Ech (Thermoanaerobacter tengcongensis and Methanosarcina mazei), Nuo (Escherichia coli K12, Rhodobacter capsulatus,
Aeropyrum pernix, and Acidilobus saccharavorans), Fpo (M. mazei) and Eha and Ehb (Methanobacterium thermoautotrophicum). These were
aligned as described in Fig. 1. The composite radial Bayesian phylogram constructed from 1600 trees was projected with Dendroscope (Huson
et al., 2007). Terminal labels are colored to distinguish Bacteria (red) and Archaea (black), and branches are colored cyan, navy, and green to
indicate Mbh-, Mbx-, and Nuo-type complexes, respectively.
FEMS Microbiol Rev 37 (2013) 182–203
ª 2012 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
196
and the primary lineage that comprises Mrp-Mbh-Fdh/
Mrp-Mbh-Codh/Mrp-Mbh-NADPH, coupled with the
monophyletic nature of Mrp-Mbh in Crenarchaeota and
Euryarchaeota, suggests that Mrp-Mbh emerged very early
during the evolution of Archaea.
Mrp-Mbh-type complexes and the
evolution of respiratory chains
The protein modules discussed herein can be thought of
analogous to modern day circuit boards that have been
assembled together through evolutionary time to form
complexes with unique and selectable functions in
metabolism. In this regard, the Mrp-Mbh-type complex
is simply a circuit board with a H+/Na+ antiporter module (Mrp) and a Group 4 hydrogenase module (Mbh)
that can be supplemented with a formate dehydrogenase
module (Fdh), a CO dehydrogenase module (Codh) or a
NAD(P)H input module (FAD/NADP, see Fig. 4).
Through evolutionary time, these modules appear to
have been rearranged and assembled into multiple
enzyme complexes that yielded unique evolutionary
advantages to various organisms by encoding different
metabolic functions. Interestingly, this analogy can also
be extended to our understanding of the evolution of
NADH ubiquinone oxidoreductase (Nuo or Complex I),
where the large and small subunits of [NiFe]-hydrogenases show homology with the subunits NuoD and NuoB
of Complex I, respectively (Bohm et al., 1990; Friedrich
& Scheide, 2000).
Among the [NiFe]-hydrogenases, those that comprise
the Group 4 membrane-bound enyzmes (Ech, Mbh, etc.)
are clearly related to Complex I (Fig. 1) (Mathiesen &
Hagerhall, 2003; Vignais & Billoud, 2007). The energyconverting hydrogenase (Ech) from Methanosarcina sp.
contains six subunits (EchA-F) that all show extensive
homology to elements from Complex I. It was proposed
that these Ech homologs represent the catalytic core of
Complex I (Hedderich, 2004; Hedderich & Forzi, 2005).
Indeed, Ech might actually represent the ‘core’ complex
of Group 4 hydrogenases that contains the catalytic capability for the reversible generation of H2 from reduced
ferredoxin linked with the translocation of ions. The
EchA subunit connects the Mrp and the hydrogenase
modules with Complex I, as it shows homology to
MrpAD and NuoLMN, as well as to Mrp-MbhH
subunits. This indicates that these genes share a common
ancestor, and these proteins contain a conserved iontranslocating unit, as proposed for NuoLMN (Friedrich &
Scheide, 2000; Mathiesen & Hagerhall, 2002; Swartz et al.,
2005). While MrpA and MrpD are homologs, the larger
MrpA protein is composed of an MrpD-like domain as
well as an added MrpB domain, suggesting that MrpA
ª 2012 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
G.J. Schut et al.
evolved from a duplication and fusion of MrpD and
MrpB (Swartz et al., 2005). In all of the Mbh-type complexes, homologs of MbhH only contain an MrpD
domain, while both MrpD and MrpA domains are present in the Complex I. It has been proposed that an ancestral Mrp-type H+/Na+ antiporter (MrpA, C, B and D)
together with a soluble [NiFe] hydrogenase formed an
ancestral membrane-bound hydrogenase, which likely
contained all three proton-translocating transmembrane
subunits (NuoLMN-like) (Mathiesen & Hagerhall, 2003).
This ancestral membrane-bound hydrogenase is thought
to have been the progenitor to Complex I and various
types of membrane-bound hydrogenases. In addition, it
was proposed that NuoK shares homology with MrpC
and MbhG (Table 1), further strengthening the case that
Nuo and Mrp-Mbh complexes have a common evolutionary origin (Mathiesen & Hagerhall, 2003).
Although one cannot retrace how the membranebound hydrogenases, complex I, and Mrp-type
antiporters evolved, it is clear that these complexes use
homologous functional modules to catalyze similar
chemical reactions. Considering that the ancestral membrane-bound hydrogenase could have arisen by fusion of
an Mrp-type antiporter with an ancestral [NiFe] hydrogenase (e.g. the ancestor of Mbh and Ech), the MrpMbh-type hydrogenase resembles this more closely than
does Ech. In this scenario, the other Group 4 hydrogenases, such as Ech, could have evolved from an Mrp-Mbhtype hydrogenase by shedding the Mrp-like subunits to a
more minimal complex as illustrated in Fig. 6. This
would include loss of an MrpC homolog, which has been
retained in Complex I. On the other hand, the more
complex Eha and Ehb hydrogenases found in hydrogenotrophic methanogens, which consist of 17–20 subunits,
appear to have evolved from an Mrp-Mbh-type complex
by acquiring additional subunits or modules to function
in the methanogenic electron flow network (Tersteegen &
Hedderich, 1999; Thauer et al., 2010). The redox link
between CO oxidation and H2 production appears to
have evolved as two different but related complexes; a
CO dehydrogenase module fused with an Ech-type
hydrogenase, as found in C. hydrogenoformans, and the
fusion of an Mrp-Mbh-type hydrogenase with a CO
dehydrogenase, as found in T. onnurineus (Soboh et al.,
2002; Lim et al., 2010; Yun et al., 2011). The evolution of
complex I would have involved a more drastic rearrangement, such as modification of the [NiFe]-hydrogenase
active site, addition of a quinone-binding site, as well as
the acquisition of a NADH input module (NuoEFG) as
well as additional subunits (NuoAJ) (Friedrich & Scheide,
2000). The ancestral Mrp-Mbh might have contained
both MrpA and MrpD units, which in turn gave rise to
NuoLMN of Complex I. In support of such a scenario,
FEMS Microbiol Rev 37 (2013) 182–203
Modular Mbh-type complexes
197
Fig. 6. Hypothetical scheme of the evolutionary shuffling of Mrp-Mbh-type modules leading to: Mrp-Mbh-type hydrogenases, Mrp-Mbx-type
oxidoreductases, Ech (energy-converting) hydrogenases, and Nuo-type (Complex 1, NADH quinone oxidoreductase) complexes. Present day
complexes are shown with solid lines. An ancestral Group 4 [NiFe] hydrogenase might have evolved from the fusion of a [NiFe] hydrogenase with
an Mrp-type Na+/H+ antiporter. The present day Mrp-Mbh and Mrp-Mbx complexes might be closely related to the ancestral complex and likely
evolved to form the various subgroups by acquiring additional modules. This ancestral complex is likely to have contained both MrpA and MrpD
as these are homologous to NuoL and NuoM in Complex I. The Ech hydrogenase could have lost several of the Mrp subunits to represent a
minimal complex. The ancestral Nuo complex could have arisen from an ancestral Mrp-Mbh by shedding most Mrp subunits, retaining only
MrpA, MrpD, and MrpC (indicated by a small light blue bar), loss of the [NiFe] site, and acquisition of a quinone-binding site and of additional
subunits (NuoA and NuoJ). This ancestral Nuo complex could then have served to yield the archaeal 11 subunit Nuo complex, and NADH-linked
complex I which include the NADH input module NuoEFG as found in present day Bacteria and Eukarya (but not in the archaea). The NADH
input module shows an intriguing similarity to [FeFe] hydrogenases, which leads to the hypothesis that an ancestral NADH input module might
have evolved by fushion to a [FeFe]-type hydrogenase leading to an ancestral form of a bifurcating [FeFe] hydrogenase. This then served as the
precursor for the present day multimeric bifurcating [FeFe] hydrogenases as well as the complex I input module NuoEFG.
sequence comparisons indicates that NuoL is more similar to MrpA, and NuoM is more similar to MrpD, while
NuoN could have evolved by gene duplication of NuoM
(Mathiesen & Hagerhall, 2003). In the Mbh-type hydrogenases, it is not clear whether MrpA or MrpD is the precursor to MbhH, although MrpD is the more likely
candidate.
Interestingly, based on the homology of the active catalytic subunits, MbhL, MbxL, FpoD and NuoD, the
S0-related Mrp-Mbx-type clusters seem to be more closely
related to the Nuo (and Fpo-type) clusters than they are
to Mrp-Mbh (Fig. 5). In adddition, one of the two conserved cysteine residues comprising each of the L1 and L2
motifs in Mbh is conserved in MbxL and FpoD, but neither cysteine is conserved in NuoD (Fig. 1). This suggests
an evolutionary trajectory whereby Cys ligands were
FEMS Microbiol Rev 37 (2013) 182–203
sequentially lost from the ancestral Group 4 hydrogenase
subunit ultimately leading to the Complex I module in
which all conserved Cys residues in MbhL are absent.
On the other hand, the NADH input module (NuoEFG)
of Complex I is unrelated to the input module found
in Mrp-Mbh-NADPH or Mrp-Mbx-NADPH-type
complexes. Interestingly, the N-terminal iron–sulfur
cluster-binding domain (N1b, N4, and N5) of NuoG
shows distinct homology with [FeFe]-hydrogenases
(Albracht et al., 1997; Vignais et al., 2001; Sazanov &
Hinchliffe, 2006). In addition, two accessory subunits of
multimeric [FeFe] hydrogenase, such as the bifurcating
[FeFe] hydrogenases of T. maritima, are homologous to
NuoE and NuoF of Complex I (Verhagen et al., 1999;
Schut & Adams, 2009). Importantly, in contrast to aerobic bacteria, the genomes of aerobic archaea lack recogª 2012 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
198
nizable homologs to the NuoEFG module, indicating that
their Complex I might not accept electrons from NADH
but rather from another electron carrier such as
ferredoxin. In this way, the archaeal Complex I might be
similar to the Mrp-Mbh complexes and most likely
electron flow occurs through the interaction of ferredoxin
with the NuoI subunit. This is a homolog of MbhN and
is predicted to contain two iron–sulfur clusters (Silva
et al., 2000; Sapra et al., 2003; Moparthi & Hagerhall,
2011). In these aerobic archaea, both ferredoxin and
NADH are generated in the degradative pathways as well
as in the TCA cycle (Zaparty et al., 2009). While the
reduced ferredoxin that is generated can be reoxidized by
the archaeal-type Complex I, a single subunit NDH type
II has been described which can divert electrons from
NADH into the quinone pool without any ion translocation (Bandeiras et al., 2003; Kerscher et al., 2008). This
collection of fascinating observations suggests that
components of Complex I share a common ancestor with
elements of both the [NiFe]- and the [FeFe]-hydrogenases.
Moreover, bacteria encode homologs of the NuoEFG
module as well as [FeFe]-hydrogenase but archaea lack
homologs of NuoEFG and [FeFe]-hydrogenase, which
strongly suggests that NuoEFG and/or [FeFe]-hydrogenase evolved after the divergence of archaea and bacteria
from the last universal common ancestor.
As discussed previously, the evolution of Complex I is
likely to have involved the ancestral Group 4 [NiFe]hydrogenase, as well as an ancestral form of present
day [FeFe]-hydrogenases (Fig. 6). However, unlike the Nterminal domain of the catalytic subunit of [FeFe]hydrogenase (i.e. the F cluster), the C-terminus (i.e. the
H cluster) is not conserved in the G module (i.e. NuoG)
of Complex I. The simplest interpretation of these
observations is that an ancestral NADH input module
interacted with an ancestor of the present day [FeFe]
hydrogenase yielding both bifurcating [FeFe] hydrogenases and the NADH input module NuoEFG. Taken
together, all of these data suggest that the NADH input
module represented by NuoEFG (and domains therein)
and the Mrp-Mbh-type complexes represent prime and
ubiquitous examples of how electron flow pathways
evolve to connect a variety of redox-driven modules. In
addition, the incorporation of Mrp and [NiFe] hydrogenase modules in the form of an ancestral Mrp-Mbh complex can be seen as an evolutionary mechanism to link
electron flow to energy conservation in the form of an
ion gradient. The distinct electron flow pathways from a
variety of electron donors through conserved iron–sulfur
clusters to catalytic sites in bifurcating [FeFe] hydrogenases and Group 4 [NiFe] hydrogenases illustrates an amazing evolutionary path of electron flow mechanisms that
ultimately coalesce in present day Complex I.
ª 2012 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
G.J. Schut et al.
Acknowledgements
This research was supported by grants from the Chemical
Sciences, Geosciences and Biosciences Division (FG0595ER20175) and the Office of Biological and Environmental Research (FG02-08ER64690) of the Office of Basic
Energy Sciences, Office of Science, U.S. Department of
Energy, and by a grant from the Low Temperature Geobiology and Geochemsitry program of the National Science
Foundation (EAR-1123689). The Astrobiology Biogeocatalysis Research Center was supported by a grant from
the NASA Astrobiology Institute (NNA08C-N85A). We
thank Sanjeev Chandrayan, Gina Lipscomb, Michael
Thorgersen, Patrick McTernan, and Chris Hopkins for
invaluable discussions.
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Supporting Information
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Table S1. Sequences used in Fig. 1.
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