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:

 
formula
(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 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 ‘oxygen-tolerant’ (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 light-sensitive 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’ Ni-A 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 oxygen-tolerant [NiFe]-hydrogenases. The structural and molecular differences between ‘standard’ and ‘oxygen-tolerant’ [NiFe]-hydrogenases could not be elucidated for a long time and were the subject of intense spectroscopic, genetic and structural investigations.

Comparison of the redox states of ‘standard’ PH and ‘oxygen-tolerant’ MBH enzymes

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.

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 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 g-tensor 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 oxygen-tolerant 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 subunit. Amino acid residues in close vicinity to the active 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 element-specific technique to determine redox changes and the co-ordination 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 MBH, no Ni-A state was obtained, which is in agreement with electrochemical studies. Structural parameters of the Ni-B 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 ‘oxygen-tolerant’ [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 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].

The proximal Fe–S cluster-binding motif

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].

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].

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 wild-type 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 oxygen-tolerance 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 symbiont bacterium and the small subunit of the MBH 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 oxygen-tolerant 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).

Structural and electronic properties of the proximal Fe–S cluster

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.

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.

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) [2729] calculations in the DFT (density functional theory) framework were performed to elucidate the electronic structure of the reduced [4Fe–3S] cluster using 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:

 
formula
(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 JAB<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 JAB in the reduced [4Fe–3S]3+–Cys6 cluster of the MBH from R. eutropha.

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 −147 −94 −80 
J1,3 −133 −91 −77 
J1,4 −126 −94 −87 
J2,3 −119 −42 −25 
J2,4 −133 −80 −67 
J3,4 −28 −84 −70 
JAB/cm−1 BP86+D3/TZP B3LYP+D3/TZP PBE0+D3/TZP 
J1,2 −147 −94 −80 
J1,3 −133 −91 −77 
J1,4 −126 −94 −87 
J2,3 −119 −42 −25 
J2,4 −133 −80 −67 
J3,4 −28 −84 −70 

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 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.

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 0.0 +0.1 +1.1 
BS13 +3.0 +1.1 +2.1 
BS14 +4.4 0.0 0.0 
BS23 +5.9 +11.0 +10.9 
BS24 +2.8 +3.2 +3.9 
BS34 +24.9 +2.6 +3.3 
 Relative energy (kcal/mol) 
BS solution BP86+D3/TZP B3LYP+D3/TZP PBE0+D3/TZP 
BS12 0.0 +0.1 +1.1 
BS13 +3.0 +1.1 +2.1 
BS14 +4.4 0.0 0.0 
BS23 +5.9 +11.0 +10.9 
BS24 +2.8 +3.2 +3.9 
BS34 +24.9 +2.6 +3.3 

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 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.

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 −2.52 +2.72 −2.74 +2.68 −2.63 +2.83 −2.84 +2.77 −4/2, +4/2, −5/2, +4/2 
BS14 −2.63 +2.63 +2.97 −2.59 −2.73 +2.72 +3.06 −2.68 −4/2, +4/2, +5/2, −4/2 
BS24 +2.55 −2.54 +3.00 −2.57 +2.65 −2.65 +3.10 −2.67 +4/2, −4/2, +5/2, −4/2 
 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 −2.52 +2.72 −2.74 +2.68 −2.63 +2.83 −2.84 +2.77 −4/2, +4/2, −5/2, +4/2 
BS14 −2.63 +2.63 +2.97 −2.59 −2.73 +2.72 +3.06 −2.68 −4/2, +4/2, +5/2, −4/2 
BS24 +2.55 −2.54 +3.00 −2.57 +2.65 −2.65 +3.10 −2.67 +4/2, −4/2, +5/2, −4/2 

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 co-ordinated 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 spin-coupled 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 spin-coupling 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 low-spin 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].

Arrangement of the metallic cofactors of the reduced MBH from R. eutropha

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)].

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)].

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.

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].

Bioenergetics in Mitochondria, Bacteria and Chloroplasts: Third Joint German/UK Bioenergetics Conference, a Biochemical Society Focused Meeting held at Schloss Rauischholzhausen, Ebsdorfergrund, Germany, 10–13 April 2013. Organized and Edited by Fraser MacMillan (University of East Anglia, Norwich, U.K.) and Thomas Meier (Max Planck Institute of Biophysics, Frankfurt am Main, Germany).

Abbreviations

     
  • BS

    broken-symmetry

  •  
  • DFT

    density functional theory

  •  
  • FTIR

    Fourier-transform IR

  •  
  • HS

    high-spin

  •  
  • MBH

    membrane-bound hydrogenase(s)

  •  
  • PH

    periplasmic hydrogenase(s)

  •  
  • XAS

    X-ray absorption spectroscopy

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 Saxony-Anhalt 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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