Bioenergetics in Mitochondria, Bacteria and Chloroplasts
Microbial hydrogen splitting in the presence
of oxygen
Matthias Stein*1 and Sandeep Kaur-Ghumaan*†
*Max-Planck-Institute for Dynamics of Complex Technical Systems, Molecular Simulations and Design Group, Sandtorstrasse 1, 39106 Magdeburg, Germany,
and †Department of Chemistry, University of Delhi, Delhi-110007, India
Abstract
The origin of the tolerance of a subclass of [NiFe]-hydrogenases to the presence of oxygen was unclear for
a long time. Recent spectroscopic studies showed a conserved active site between oxygen-sensitive and
oxygen-tolerant hydrogenases, and modifications in the vicinity of the active site in the large subunit could
be excluded as the origin of catalytic activity even in the presence of molecular oxygen. A combination
of bioinformatics and protein structural modelling revealed an unusual co-ordination motif in the vicinity of
the proximal Fe–S cluster in the small subunit. Mutational experiments confirmed the relevance of two
additional cysteine residues for the oxygen-tolerance. This new binding motif can be used to classify
sequences from [NiFe]-hydrogenases according to their potential oxygen-tolerance. The X-ray structural
analysis of the reduced form of the enzyme displayed a new type of [4Fe–3S] cluster co-ordinated by six
surrounding cysteine residues in a distorted cubanoid geometry. The unusual electronic structure of the
proximal Fe–S cluster can be analysed using the broken-symmetry approach and gave results in agreement
with experimental Mößbauer studies. An electronic effect of the proximal Fe–S cluster on the remote
active site can be detected and quantified. In the oxygen-tolerant hydrogenases, the hydride occupies an
asymmetric binding position in the Ni-C state. This may rationalize the more facile activation and catalytic
turnover in this subclass of enzymes.
Introduction
Hydrogen is one of the energy carriers of the future. The
investigation of hydrogen metabolism from bacteria and
archaea has a strong impact on possible scenarios for future
energy supply [1]. Hydrogenases are enzymes which catalyse
the reversible oxidation of molecular hydrogen into two
protons and two electrons:
H2 ˙ H+ + H− ˙ 2H+ + 2e−
(1)
They are classified according to the metal content of their
active sites as [NiFe]-, [FeFe]- and [Fe]-hydrogenases [2].
Most of the [NiFe]-hydrogenases are very sensitive to
the presence of molecular oxygen and some are reversibly
or irreversibly inactivated by it. A subclass of [NiFe]hydrogenase, however, appears to be tolerant to oxygen and
retains the catalytic activity in its presence. In particular,
[NiFe]-hydrogenases from hyperthermophilic (e.g. Aquifex
aeolicus [3]) and Knallgas (e.g. Ralstonia eutropha [4]) bacteria
have shown this increased oxygen-tolerance. The key feature
that defines oxygen-tolerance is the preservation of catalytic
Key words: bioenergetics, hydrogen conversion, iron–sulfur cluster (Fe–S cluster), oxygentolerant hydrogenase, protein structural modelling, structural biology.
Abbreviations used: BS, broken-symmetry; DFT, density functional theory; FTIR, Fouriertransform IR; HS, high-spin; MBH, membrane-bound hydrogenase(s); PH, periplasmic
hydrogenase(s); XAS, X-ray absorption spectroscopy.
1
To whom correspondence should be addressed (email matthias.stein@mpi-magdeburg.
mpg.de).
Biochem. Soc. Trans. (2013) 41, 1317–1324; doi:10.1042/BST20130033
hydrogen splitting under aerobic conditions, i.e. in the
presence of molecular oxygen [5,6].
Figure 1 gives a comparison of the redox states of the
active sites of ‘standard’ (periplasmic, PH) and ‘oxygentolerant’ (membrane-bound, MBH) [NiFe]-hydrogenases.
The active site consists of a heterobimetallic Ni–Fe cluster.
The nickel atom is co-ordinated by four cysteine residues,
two of which are bridging towards the iron atom. Three
small inorganic ligands (one carbon monoxide and two
cyanides) co-ordinate the iron atom. Common to both
types of hydrogenases is the paramagnetic oxidized ‘ready’
state Ni-B which can be reduced to give the EPR-silent
Ni-SIr and the catalytically active Ni-SIa states. Ni-C is a
catalytic intermediate in a paramagnetic Ni(III) S = 1/2 state
in which a hydride occupies the bridging position between
the nickel and iron atoms of the active site. Ni-C is lightsensitive and can be converted into a reduced Ni(I)–Fe(II)
species by photodissociation of the bridging hydride at low
temperature. The fully reduced Ni-R state corresponds to
the resting state of the enzyme (for an overview of enzymatic
redox states, see [7]). Only the oxygen-sensitive ‘standard’
hydrogenases exhibit an additional oxidized ‘unready’ NiA state and its reduced EPR-silent Ni-SU equivalence. This
state requires prolonged exposure to substrate H2 to become
catalytically active and has not been found for oxygentolerant [NiFe]-hydrogenases. The structural and molecular
differences between ‘standard’ and ‘oxygen-tolerant’ [NiFe]hydrogenases could not be elucidated for a long time and were
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Figure 1 Comparison of the redox states of ‘standard’ PH and ‘oxygen-tolerant’ MBH enzymes
The large subunit harbours the Ni–Fe active site, whereas the small subunit carries three Fe–S clusters which are responsible
for the electron transfer to and from the active sites. For PH, the electron acceptor is a cytochrome c, but for MBH, it is a
membrane-embedded cytochrome b. Paramagnetic states of the active sites are given in red.
the subject of intense spectroscopic, genetic and structural
investigations.
The large subunit and the active site
The redox states of the active site in [NiFe]-hydrogenase
give rise to characteristic signatures in EPR and FTIR
(Fourier-transform IR) spectra. The Ni-A, Ni-B, Ni-C and
Ni-L states are characterized by a distinct set of EPR gtensor values and their ligand environment can be probed
very accurately by double-resonance experiments such as
ENDOR or HYSCORE (for a review, see [7]). The three
small inorganic ligands at the iron atom of the active site are
very sensitive probes of the electron density at the metal.
EPR and FTIR were thus used to investigate the active-site
and ligand environment differences between PH and MBH.
For example, the membrane-bound chemolithotrophic
hydrogenase Hase I from A. aeolicus exhibits structural and
electronic features that are very similar to that of the standard
oxygen-sensitive hydrogenases [8]. FTIR and EPR studies
indicated that the active-site composition of the oxygentolerant enzyme is very similar to, if not identical with, that
of oxygen-sensitive ‘standard’ [NiFe]-hydrogenases [9,10].
The active site before catalytic turnover is called ‘Ni-B’
and was shown to carry a bridging hydroxide (OH − )
ligand between the Ni(III) and Fe(II) atoms [11,12]. A
major difference between ‘standard’ [NiFe]-hydrogenases
and oxygen-tolerant ones was the faster activation kinetics
from Ni-B in oxygen-tolerant [NiFe]-hydrogenases [13].
Analysis of the amino acid sequences of the ‘standard’
[NiFe]-hydrogenase from Desulfovibrio gigas and the
oxygen-tolerant [NiFe]-hydrogenase from R. eutropha
revealed a sequence identity of only 40 % for the large
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site, however, are identical with those in the oxygen-sensitive
periplasmic hydrogenase from D. gigas for which X-ray
structures were available in various redox states. The closest
non-identical amino acid residues were Tyr70 and Val71 in D.
gigas which correspond to Gly80 and Cys81 in R. eutropha
MBH. Single-point mutation at these positions as well as
at Val77 and Leu125 were introduced and investigated by
protein film voltammetry in terms of their kinetics and
oxygen-tolerance. Ludwig et al. [4] concluded that the
oxygen-tolerance of R. eutropha MBH cannot simply be
linked to those single point mutations in the vicinity of
the active site. The oxygen-sensitivity of MBH was not
significantly enhanced when the residues of the standard
hydrogenases were introduced. Thus restriction of diffusion
of inhibitory gases such as oxygen or carbon monoxide
due to sterically demanding amino acid residues in different
diffusion pathways could be also ruled out. Clearly, the
oxygen-tolerance of some [NiFe]-hydrogenases is a complex
factor and seemed to be determined by a well-adapted spatial
and electronic structure of the active site.
XAS (X-ray absorption spectroscopy) is an elementspecific technique to determine redox changes and the coordination environment of protein-bound metal atoms. The
spectroscopic features of the standard [NiFe]-hydrogenase
from D. gigas (for which crystal structures are available) were
compared with that of the oxygen-tolerant MBH from R.
eutropha [14]. In the nickel EXAFS, the spectra of reduced
MBH and PH were very similar. For the Ni–Fe active site,
the Ni-SIa state was indistinguishable between the PH and
MBH. Ni-A and Ni-C states were characterized for the
PH from D. gigas and gave structural parameters in good
agreement with those from X-ray structure analysis. For the
Bioenergetics in Mitochondria, Bacteria and Chloroplasts
Figure 2 The proximal Fe–S cluster-binding motif
Upper panel: multiple sequence alignment of the small subunits of PH and MBH enzymes: MBHS_AQUAE (A. aeolicus),
MBHS_CUPNH (Cupriavidus necator, R. eutropha), hupS_9BACT (symbiont bacterium from B. puteoserpentis, see the text
for details), PHNS_DESGI (D. gigas), PHNS_DESVM (Desulfovibrio vulgaris Miyazaki F). Amino acid residues co-ordinating or
in close distance to the proximal Fe–S cluster are highlighted: cysteine amino acid residues (yellow) and glycine residues
(magenta). Lower panel: details of the co-ordination of the proximal Fe–S cluster. Left: crystal structure of the proximal Fe–S
cluster from the PH from D. gigas (PDB code 1FRV). Right: protein structural model of the proximal Fe–S cluster from the
MBH from C. necator [17].
MBH, no Ni-A state was obtained, which is in agreement
with electrochemical studies. Structural parameters of the NiB state of the MBH were in good agreement with protein
structural data and those derived from EPR experiments.
There were no structural modifications detectable for the
active-site iron atom from iron EXAFS.
The small subunit and the Fe–S clusters
Comparing PH and MBH, stoichiometry and simulation of
the iron EXAFS gave iron–sulfur and iron–iron distances
that were in good agreement with crystal structures and
previous XAS data and the presence of two [4Fe–4S] clusters
and one [3Fe–4S] cluster in the small subunit of standard
hydrogenases [14]. For the MBH, a slightly diminished
content of iron atoms and a larger variability in iron–
iron distances of 2.6, 2.7 and ∼3.4 Å (1 Å = 0.1 nm) was
reported. This indicated some sort of modifications in the
small subunit (hoxK) of the MBH compared with the PH.
A definite rationalization of this heterogeneity in iron–
iron bond distances and its impact on oxygen tolerance,
however, was not possible from EXAFS and required further
bioinformatic and structural investigation.
Saggu et al. [15] were the first to report differences
in the Fe–S clusters between ‘standard’ and ‘oxygentolerant’ [NiFe]-hydrogenases. FTIR and EPR investigations
suggested an active-site composition and electronic structure
of the active site to be very similar between the different
subfamilies. EPR studies on the MBH from R. eutropha,
however, revealed an additional paramagnetic species at
high redox potential ( + 290 mV) which was found to
magnetically couple to both the medial [3Fe–4S] cluster of
the small and the Ni–Fe active site of the large subunit
[15]. Such a species was not found in ‘standard’ [NiFe]hydrogenases. Similar observations were later made for the
MBH Hase I from A. aeolicus. Redox titrations of the Fe–S
clusters of the small subunit revealed four one-electron redox
transitions. By combining the electrochemical titrations with
other spectroscopic techniques, the spatial resolution of the
clusters in the electron transfer chain could also be made
[16]. Apparently, one of the three Fe–S clusters is capable of
performing two electronic transitions. Closest to the [NiFe]
active site is an unusual Fe–S cluster which can take three
oxidation states in a relatively narrow potential window. In
‘standard’ hydrogenases, this is a cubane [4Fe–4S] cluster
which then would take oxidation states [4Fe–4S]3 + , [4Fe–
4S]2 + and [4Fe–4S] + known to exist in other redox enzymes.
A bioinformatics analysis of the small subunit of different
PH and MBH enzymes revealed modifications at or near
the proximal Fe–S cluster (Figure 2). In standard [NiFe]hydrogenases, such as that from D. gigas, the proximal Fe–S
cluster is a [4Fe–4S] cubane which is co-ordinated by four
cysteine residues (Cys17 , Cys20 , Cys112 and Cys148 ). Two
glycine residues are in the vicinity of the proximal Fe–S
cluster, but do not directly co-ordinate to it (Gly19 and Gly117 ;
Figure 2). In the oxygen-tolerant MBH from A. aeolicus and
R. eutropha, however, these glycine residues are replaced by
two additional cysteine residues (Figure 2). The role of these
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two additional cysteine residues and their potential structural
and/or redox modifications were investigated in detail. On
the basis of the sequence identity of 44 % of the small subunit
between MBH from R. eutropha and PH from D. gigas,
a protein structural model for the MBH from R. eutropha
was generated. The protein structural model suggested that
the two additional cysteine residues were in co-ordinative
distance of the proximal Fe–S cluster and may bind or interact
with it [17]. A cuboidal iron-sulfur cluster surrounded by six
cysteine residues within bonding distance is a new type of
Fe–S cluster and was not described previously. A [4Fe–4S]
cluster with five instead of four cysteine residues was found
in the CCG-domain [18] and also the tandem co-ordination
of two consecutive cysteine residues (Cys19 and Cys20 ) to two
different iron atoms of the cubane was described previously
[19,20].
Mutational experiments were performed and the two
additional cysteine residues, i.e. Cys19 and Cys20 , were
replaced by their glycine counterparts [17]. It was found
that the hydrogen-dependent growth at high oxygen
concentrations critically depended on these two extra cysteine
residues and that oxygen-sensitivity was reintroduced by the
glycine residues. In the absence of oxygen, the double mutant
behaves like the wild-type MBH. FTIR and EPR experiments
showed that the active-site formation in the mutants was
intact and its properties were indistinguishable from the wildtype MBH. Protein film voltammetry in combination with
EPR experiments showed that the proximal Fe–S cluster can
undergo an extra one-electron oxidation, a property that
suggests that the proximal Fe–S cluster efficiently reduces
attacking oxygen and thus provides a high-fidelity protection
of the active site against oxidative damage [17].
The key signature for oxygen-tolerance
This new Fe–S cluster-binding motif in the small subunit of C-X-C-C-X93/94 -C-X4 -C-X28 -C (additional cysteine
residues are in bold and underlined) can be used as a novel
signature to physiologically annotate [NiFe]-hydrogenases.
A motif search of this PROSITE pattern [21] in the SwissProt
database finds 12 matching sequences, all of which are
annotated as MBH from different species, e.g. Escherichia
coli, R. eutropha, Rubrivivax gelatinosus and Alcaligenes
hydrogenophilus. Thus the new binding motif is characteristic
of class I [NiFe]-hydrogenases and can be used to classify
known hydrogenases [17,22].
The new Fe–S cluster-binding motif can also be used as a
signature to functionally annotate the physiological oxygentolerance of unknown [NiFe]-hydrogenases. For example, a
previously uncharacterized symbiont in the deep-sea mussel
Bathymodiolus puteoserpentis from a hydrothermal vent was
shown to possess hydrogen-oxidizing properties [23]. The
sulfur-oxidizing symbiont of the deep-sea vent mussel uses
hydrogen as an energy source to drive its metabolic processes.
Amino acid sequence comparison of the large subunit
showed an identity of 74 % with the [NiFe]-hydrogenase
from R. eutropha (Figure 2). The small subunits of the
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from R. eutropha have a sequence identity of 75 %. Both
sequences show the characteristic additional two cysteine
residues signature (see Figure 2) which enables a functional
annotation of the symbiont to be a MBH with oxygentolerant properties. This is in good agreement with the living
conditions of the hydrothermal vent endosymbiont in sea
water and shows a perfect adaptation of the bacterium to its
environment in the gill of Bathymodiolus species.
The X-ray structure reveals a novel type of Fe–S
cluster
Recently, the protein crystal structure of the MBH from
R. eutropha in the reduced form was published [24]. It
revealed the similarity of co-ordination of the active-site Ni–
Fe cluster in the large subunit to ‘standard’ hydrogenases
which could explain the similarities in EPR and FTIR spectra.
The co-ordination and binding of the intermediate [3Fe–
4S] and the distal [4Fe–4S] cubane clusters in the small
subunit were also similar to the PH. For the proximal Fe–
S cluster, however, an even more complicated co-ordination
environment than expected from the protein structural model
was found (Figure 3).
The proximal cluster in R. eutropha MBH is ligated
by six cysteine residues, and the sulfur atom of one of
the extra cysteine residues (Cys19 ) substitutes for one
of the four inorganic sulfides of the cubane. One of
the iron atoms of the cluster is co-ordinated by two cysteine
residues (Cys120 and Cys149 ). Together this leads to an open
and distorted configuration of the proximal [4Fe–3S]–Cys6
cluster (Figure 3). Such a co-ordination pattern of a cubanoid
Fe–S cluster was not observed before and represented a novel
type of Fe–S cluster. This unusual architecture enables two
redox transitions at physiologically possible redox potentials
[16,17] which cannot be achieved by regular cubane [4Fe–4S]
clusters. Thus this new type of Fe–S cluster is a two-electron
storage facility and provides an electron-rich environment
for the active site. It must be assumed that the easy and fast
release of electrons is the crucial factor for the protection of
oxidative damage to the active site by reducing oxygen.
The electronic structure of the proximal
Fe–S cluster and its effect on the active site
The two redox transitions of the proximal [4Fe–3S] cluster
correspond to changes of the formal oxidation states of
the iron atoms leading to [4Fe–3S]3 + /4 + /5 + . The formal
individual oxidation states of the metals ions are 3Fe(II)–
1Fe(III)→2Fe(II)–2Fe(III)→1Fe(II)–3Fe(III). There are indications that these redox transitions are associated with
large structural changes of the proximal Fe–S cluster [25,26].
For the R. eutropha MBH, there is currently only a
protein structure of the reduced form available [24]. BS
(broken-symmetry) [27–29] calculations in the DFT (density
functional theory) framework were performed to elucidate
the electronic structure of the reduced [4Fe–3S] cluster using
Bioenergetics in Mitochondria, Bacteria and Chloroplasts
Figure 3 Structural and electronic properties of the proximal Fe–S cluster
Protein crystal structure of the MBH from R. eutropha in its reduced form (PDB code 3RGW) [24]. The large subunit is shown
in green, the small subunit is in blue. Details of the structure of the proximal Fe–S cluster are shown. The proximal Fe–S
cluster is a distorted cubane cluster and represents a new type of Fe–S cluster with an unusual co-ordinative motif. Cys20 ,
Cys17 and Cys115 bind to one iron atom each. Cys149 and Cys120 , although not consecutive in sequence, form a tandem
co-ordinating motif and bind two-fold to the same iron atom. There are only three inorganic sulfur atoms in the proximal
cluster and the fourth position is occupied by Cys19 .
ADF2013.01 [30,31]. Figure 3 shows the possible magnetic
exchange coupling pathways for the electronic spins of the
iron atoms.
From the HS (high-spin) optimized orbitals, there are
six possible combinations of spin flipping in the reduced
[4Fe–3S] cluster (Figure 3). The spin–spin exchange coupling
constants J for each pair of iron atoms were calculated
according to the formula by Yamaguchi et al. [32] (eqn 2)
which is valid over the entire range of possible exchange
coupling strengths:
J AB = −
<
S2
EHS − EBS
>HS − < S 2 >HS
(2)
where EHS and EBS are the energies of the HS and BS solutions
respectively, and <S2 > are the expectation values of the spin
operators. When J AB <0, then the anti-ferromagnetic coupling
of the BS solution over the HS solution is energetically
favoured.
Table 1 gives the calculated exchange coupling constants
J AB in the reduced [4Fe–3S]3 + –Cys6 cluster of the MBH from
R. eutropha.
The pure BP86 [33,34] and the hybrid functionals B3LYP
[33,35,36] and PBE0 [37] were used in conjunction with
the dispersive correction for van der Waals interactions by
Grimme and colleagues [38,39]. All calculations give a very
consistent picture of an anti-ferromagnetic coupling Fe–S
cluster leading to low-spin solutions. BP86 tends to favour
the low-spin configuration compared with hybrid functionals
Table 1 DFT calculated exchange coupling constants in
wavenumbers for BS DFT calculations
JAB refers to the iron atoms A and B for which the electron spin was
flipped. For the numbering of iron atoms and a schematic display of
exchange coupling interactions, see Figure 3.
JAB /cm − 1
BP86 + D3/TZP
B3LYP + D3/TZP
PBE0 + D3/TZP
J1,2
J1,3
− 147
− 133
− 94
− 91
− 80
− 77
J1,4
J2,3
J2,4
− 126
− 119
− 133
− 94
− 42
− 80
− 87
− 25
− 67
J3,4
− 28
− 84
− 70
such as B3LYP [40] and thus gives more negative J coupling
constants. In terms of structural parameters, BP86 seems to
outperform hybrid functionals for transition metal systems
[41], its accuracy in calculating J-coupling constants for Fe–S
clusters, however, is still a matter of discussion (see [42,43]).
The exchange-coupling constants obtained with the PBE0
functional are in between the BP86 and B3LYP results.
Table 2 gives the relative ordering of the BS solutions. The
relative ordering of BS solutions is identical for B3LYP and
PBE0 calculations; the pure GGA BP86 gives BS12 as the
lowest in energy.
The energy differences between different BS solutions are
generally small and only some can be excluded on an energetic
evaluation (BS23 , BS34 ). It is clear that energy cannot be the
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Table 2 Relative energies of the BS solutions of
exchange-coupled iron atoms in the reduced proximal Fe–S cluster
of the MBH from R. eutropha
Relative energy (kcal/mol)
BS solution
BP86 + D3/TZP
B3LYP + D3/TZP
PBE0 + D3/TZP
BS12
BS13
BS14
0.0
+ 3.0
+ 4.4
+ 0.1
+ 1.1
0.0
+ 1.1
+ 2.1
0.0
BS23
BS24
BS34
+ 5.9
+ 2.8
+ 24.9
+ 11.0
+ 3.2
+ 2.6
+ 10.9
+ 3.9
+ 3.3
only criterion to discriminate between different coupling
schemes.
Table 3 gives the multipole-derived atomic spin populations which are obtained from fitting the electron density
up to quadrupole level [44]. The spin population is given
as the difference between the fitted densities for α and β
electrons. BP86 results are very similar, but with slightly
smaller numbers due to higher covalency and are omitted
from the present paper.
BS12 , BS14 and BS24 all agree on assigning a + 5/2 spin [a
formal Fe(III) oxidation state] to the Fe(3) atom which is coordinated by two cysteine residues, Cys149 and Cys120 . BS13
can be excluded, since it yields an opposite spin population
for Fe(3). BS12 also appears unrealistic, since it involves the
formation of two anti-ferromagnetically coupled pairs Fe(1)–
Fe(3) and Fe(2)–Fe(4) and is not in agreement with Mößbauer
results (see below). The BS solutions BS14 and BS24 only
differ in the sign of spin population assigned to the spincoupled pairs of iron atoms 1 and 2 and we cannot positively
discriminate between them.
Mößbauer investigations of reduced MBH from A. aeolicus
supported the assignment of a + 3 oxidation state of the
reduced [3Fe–4S] cluster and yielded insight into the spincoupling scheme [45]. One iron atom giving rise to a doublet
with a large quadrupole splitting which we tentatively assign
to Fe(3) [bound to Cys149 and Cys120 ; formally a lowspin Fe(III)] and a more localized Fe(II) character with a
characteristic isomer shift [46] and less covalency which we
attribute to originate from Fe(4) which is co-ordinated by
Cys19 that substitutes for an inorganic sulfide in the cubane
cluster.
The effect of the proximal Fe–S cluster on the active site
at a distance of 11 Å can be quantified by a combination
of EPR, HYSCORE and ENDOR spectroscopy and DFT
calculations (Figure 4). The Ni-C g-tensor principal values
and the hyperfine coupling constants for a hydride and a
respective deuteride differ slightly between standard and
oxygen-tolerant hydrogenases. In the Ni-C state of standard
[NiFe]-hydrogenases, the μ-hydride occupies a symmetrical
binding position (distances of 1.6 Å for Ni–H − and 1.7 Å for
H − –Fe), whereas, in the oxygen-tolerant hydrogenase from
A. aeolicus, the binding is asymmetric and closer to the iron
atom (Ni–H − of 1.9 Å and H − –Fe of 1.7 Å). The hydride
binding is also weaker by 3–4 kcal·mol − 1 (1 kcal = 4.184
kJ) and this may explain the increased light-sensitivity of
Ni-C, the decreased stability and the more positive catalytic
potential of the hydride complex [47].
Conclusion and outlook
Oxygen-tolerance in some [NiFe]-hydrogenases is not due
to modifications or alterations at or near the active site in
the large subunit. Rather, the proximal [4Fe–3S] cluster is
an effective protectant against oxidative damage at the active
site which is approximately 11 Å from it. The possibility to
undergo two one-electron oxidation steps in a narrow range
of physiological redox potentials allows the storage of two
electrons in the proximal Fe–S cluster. The co-ordination of
the proximal [4Fe–3S] cluster is unusual and has not been
observed previously. In the reduced state, co-ordination by
six cysteine residues leads to a distorted cubanoid structure.
One cysteine residue occupies the position of one inorganic
sulfide atom in a cubane cluster and two residues not adjacent
in sequence bind to one iron atom. The structural plasticity
and its effect on the electronic properties of the proximal
Fe–S cluster will be investigated in more detail in future as
more crystal structures in different oxidation states become
available for the MBH from R. eutropha as has been obtained
for the MBH from E. coli [25] and Hydrogenovibrio marinus
[26]. These studies indicate a structural rearrangement and
redox-state-dependent co-ordination of the proximal Fe–S
cluster.
Table 3 Multipole-derived spin populations for BS solutions of the proximal Fe–S cluster
B3LYP + D3/TZP
PBE0 + D3/TZP
BS solution
Fe(1)
Fe(2)
Fe(3)
Fe(4)
Fe(1)
Fe(2)
Fe(3)
Fe(4)
Assigned spin state
BS12
− 2.53
− 2.59
+ 2.93
+ 2.57
− 2.63
− 2.69
+ 3.04
+ 2.66
− 4/2, − 4/2, + 5/2, + 4/2
BS13
BS14
BS24
− 2.52
− 2.63
+ 2.55
+ 2.72
+ 2.63
− 2.54
− 2.74
+ 2.97
+ 3.00
+ 2.68
− 2.59
− 2.57
− 2.63
− 2.73
+ 2.65
+ 2.83
+ 2.72
− 2.65
− 2.84
+ 3.06
+ 3.10
+ 2.77
− 2.68
− 2.67
− 4/2, + 4/2, − 5/2, + 4/2
− 4/2, + 4/2, + 5/2, − 4/2
+ 4/2, − 4/2, + 5/2, − 4/2
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Figure 4 Arrangement of the metallic cofactors of the reduced MBH from R. eutropha
Distances are given in Å. The unusual binding situation and electronic structure of the proximal [4Fe–3S]–Cys6 cluster (one
plausible BS solution is shown) has an effect on the active site at a distance of 11.7 Å. In the reduced Ni-C form, in PH the
hydride occupies a μ-binding position between the Ni(III) and Fe(II) sites. In the oxygen-tolerant [NiFe]-hydrogenases, the
bridging hydride shifts from a symmetry in standard hydrogenases to an asymmetric binding position in oxygen-tolerant
hydrogenase [Ni–H − distance increases from 1.6 Å (black) to 1.9 Å (red)].
This insight into Nature’s design principles to protect the
active site from oxidative inactivation will have an impact
on possible biotechnological applications of hydrogenases in
microbial fuel cells and photobiological hydrogen production
[48,49].
Acknowledgements
We acknowledge our collaborators for a truly interdisciplinary work.
Funding
This work was supported by the Max-Planck-Society for the
Advancement of Science and the Federal State of SaxonyAnhalt Excellence Programme ‘Research Center Dynamic Systems:
Biosystems Engineering (CDS)’. S.K.-G. is grateful to the Department
of Science & Technology (DST)/the Indo–German Science and
Technology Centre (IGSTC), India, and the Max-Planck-Society,
Germany, for a Max-Planck–India Visiting Fellowship.
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Received 8 August 2013
doi:10.1042/BST20130033