doi:10.1016/j.jmb.2010.07.041 J Mol Biol (2010) 402, 428–444 Available online at www.sciencedirect.com The Crystal Structure of the [NiFe] Hydrogenase from the Photosynthetic Bacterium Allochromatium vinosum: Characterization of the Oxidized Enzyme (Ni-A State) Hideaki Ogata⁎, Petra Kellers and Wolfgang Lubitz⁎ Max-Planck-Institut für Bioanorganische Chemie, Stiftstrasse 34–36, D-45470 Mülheim an der Ruhr, Germany Received 12 May 2010; received in revised form 19 July 2010; accepted 20 July 2010 Available online 29 July 2010 The crystal structure of the membrane-associated [NiFe] hydrogenase from Allochromatium vinosum has been determined to 2.1 Å resolution Electron paramagnetic resonance (EPR) and Fourier transform infrared spectroscopy on dissolved crystals showed that it is present in the Ni-A state (N90%) The structure of the A vinosum [NiFe] hydrogenase shows significant similarities with [NiFe] hydrogenase structures derived from Desulfovibrio species The amino acid sequence identity is ∼ 50% The bimetallic [NiFe] active site is located in the large subunit of the heterodimer and possesses three diatomic non-protein ligands coordinated to the Fe (two CN− , one CO) Ni is bound to the protein backbone via four cysteine thiolates; two of them also bridge the two metals One of the bridging cysteines (Cys64) exhibits a modified thiolate in part of the sample A mono-oxo bridging ligand was assigned between the metal ions of the catalytic center This is in contrast to a proposal for Desulfovibrio sp hydrogenases that show a di-oxo species in this position for the Ni-A state The additional metal site located in the large subunit appears to be a Mg2+ ion Three iron–sulfur clusters were found in the small subunit that forms the electron transfer chain connecting the catalytic site with the molecular surface The calculated anomalous Fourier map indicates a distorted proximal iron–sulfur cluster in part of the crystals This altered proximal cluster is supposed to be paramagnetic and is exchange coupled to the Ni3+ ion and the medial [Fe3S4]+ cluster that are both EPR active (S = 1/2 species) This finding of a modified proximal cluster in the [NiFe] hydrogenase might explain the observation of split EPR signals that are occasionally detected in the oxidized state of membrane-bound [NiFe] hydrogenases as from A vinosum © 2010 Elsevier Ltd All rights reserved Edited by R Huber Keywords: [NiFe] hydrogenase; Allochromatium vinosum; photosynthetic purple-sulfur bacterium; iron–sulfur cluster; Ni-A state *Corresponding authors E-mail addresses: ogata@mpi-muelheim.mpg.de; lubitz@mpi-muelheim.mpg.de Present address: P Kellers, Department of Photochemistry and Molecular Science, The Ångström Laboratories, Uppsala University, SE-751 20 Uppsala, Sweden Abbreviations used: EPR, electron paramagnetic resonance; FTIR, Fourier transform infrared; ICP-OES, inductively coupled plasma with optical atomic emission spectrometry; MAD, multiwavelength anomalous dispersion; D vulgaris H, Desulfovibrio vulgaris Hildenborough; D vulgaris MF, Desulfovibrio vulgaris Miyazaki F; PDB, Protein Data Bank 0022-2836/$ - see front matter © 2010 Elsevier Ltd All rights reserved The Structure of the A vinosum [NiFe] Hydrogenase Introduction Hydrogenases catalyze the reversible oxidation of molecular hydrogen They play a key role in the energy metabolism of various microorganisms Hydrogenases can be divided into three groups according to the metal content of their active site: [NiFe], [FeFe], and [Fe] hydrogenases.2–7 An alternative distinction of hydrogenases can be made based on their function: uptake (hydrogen splitting), production (hydrogen generation), bidirectional, and sensory/regulatory hydrogenases.8 They appear as either membrane-bound/associated or as soluble enzymes.1 Several organisms possess not only one but often more and even different hydrogenases participating in a variety of metabolic processes comprising variable catalytic activity levels, oxygen, and temperature tolerance.9,10 These differences determine the eligibility of particular hydrogenases for special biotechnological applications To date, four crystal structures of O2-sensitive hydrogen uptake [NiFe] hydrogenases from sulfatereducing Desulfovibrio species have been determined but not a single one from photosynthetic bacteria or other microorganisms.2,11–18 Their crystal structures revealed that [NiFe] hydrogenases are composed of two subunits forming a heterodimer by hydrophobic interaction These contain three iron–sulfur clusters and a bimetallic active site, located in the small and the large subunit, respectively The catalytic center is composed of a Ni and an Fe atom, which are bridged by two thiolates of cysteines from the protein backbone Three diatomic non-protein ligands are bound to the Fe They were assigned as two CN− and one CO based on Fourier transform infrared (FTIR) results.19,20 Furthermore, two other cysteine thiolates coordinate the Ni in a terminal fashion A third bridging ligand between these metals is temporarily present depending on the respective apparent oxidation state.21,22 Additionally, there is another metal site existent in the large subunit It was identified as Mg2+ and is assumed to participate in mediating the proton transfer through the protein matrix The substrate (H2) crosses the large subunit via adequate hydrophobic channels.14,21,23,24 It has also suggested that the Mg2+ ion functions as recognition site during the maturation steps.25 In addition, two crystal structures of [NiFeSe] hydrogenases are also available These hydrogenases were derived from proteins isolated from the sulfatereducing bacteria Desulfomicrobium baculatum26 and Desulfovibrio vulgaris Hildenborough (D vulgaris H).27 [NiFeSe] hydrogenases form a subdivision of [NiFe] hydrogenases that exhibit a selenocysteine instead of a cysteine involved in the coordination of the nickel atom All iron–sulfur clusters of [NiFeSe] hydrogenases are of the [Fe4S4] type, which is in contrast to [NiFe] hydrogenases that possess both [Fe3S4] and [Fe4S4] clusters Finally, the extra metal, a 429 Mg2+ in [NiFe] hydrogenases, is in this case most likely replaced by an Fe2+/3+ ion.26,27 The [NiFe] enzyme passes through various redox states during its catalytic cycle The “as-isolated” protein is in many cases a mixture of the two most oxidized but inactive states Ni-A (unready) and Ni-B (ready) Both are paramagnetic (Ni3+, S = 1/2) but exhibit different g-values (gx = 2.32, gy = 2.24, and gz = 2.01 for the Ni-A state and gx = 2.33, gy = 2.16, and gz = 2.01 for the Ni-B state) in electron paramagnetic resonance (EPR)28 and different activation rates.29,30 Crystallographic studies on these states of the hydrogenases from D vulgaris Miyazaki F (D vulgaris MF), Desulfovibrio gigas, and Desulfovibrio desulfuricans indicated that the third bridging ligand is probably not the same in Ni-A and Ni-B.16,17 In the case of the Ni-B state, a hydroxide (OH−) was identified,31 whereas in the Ni-A state, a di-oxo species in the form of a hydroperoxy (OOH−) ligand has been discussed.16,17 Furthermore, a modification of cysteine residues was observed in some hydrogenases in their oxidized states In the case of the [NiFe] hydrogenases from D vulgaris MF and Desulfovibrio fructosovorans, oxidation of the bridging cysteine residues Cys84 and Cys75, respectively, was found.16,17 The [NiFeSe] hydrogenase from D vulgaris H showed a terminal cysteine residue (Cys75) binding two oxygen atoms.27 The photosynthetic purple-sulfur bacterium Allochromatium vinosum (strain DSM 185) is phylogenetically distinct from sulfate-reducing organisms Its [NiFe] hydrogenase is a periplasmic, membrane-associated, heterodimeric protein of about 91 kDa†.32 As the other well-studied hydrogenases, such as the periplasmic soluble [NiFe] hydrogenase from D gigas, spectroscopic investigations of A vinosum hydrogenase suggest similar characteristics of the active site.28,32,33 In the small subunit, this protein contains two types of iron– sulfur clusters, namely, [Fe3S4]1+/0 and [Fe4S4]2+/1+, as derived from Mössbauer studies.34,35 Earlier EPR studies at low temperatures revealed a complex split EPR signal around g = due to an [Fe 3S ] 1+ cluster coupled to an Fe-containing moiety 29,34,36,37 This additional paramagnet is supposed to be only present in the oxidized form of the enzyme and probably mediates a magnetic coupling between the medial [Fe3S4]1+ cluster and the Ni3+ of the active site Either an extra Fe3+ species located close to the proximal iron–sulfur cluster or a modification of the proximal cluster itself has been suggested.29,32,36,38 The above-described † It is known that A vinosum has at least two hydrogenases One is a hydrogenase with two subunits that is studied in this article and another is a five-subunit hydrogenase that is known as a HoxEFUYH type.50 430 phenomenon was also reported for other hydrogenases such as the membrane-associated [NiFe] hydrogenases of Ralstonia eutropha10,37 and Aquifex aeolicus.39 So far, a consistent interpretation of the complex EPR split signal has not been achieved, the main reason being lack of structural information The electrons that are released during the heterolytic cleavage of hydrogen are transferred via the electron transfer chain, formed by the iron–sulfur clusters, to an external electron acceptor In the case of the membrane-associated uptake [NiFe] hydrogenase (Hup) from the closely related purple-sulfur bacte- The Structure of the A vinosum [NiFe] Hydrogenase rium Thiocapsa roseopersicina, an interaction partner, namely HupC, has been identified.40 This protein is suggested to be another unit of the hydrogenase complex catalyzing the H2-dependent reduction of quinones A similar mechanism for the A vinosum [NiFe] hydrogenase appears possible The electron acceptor may differ from the one identified in sulfate reducers (i.e., cytochrome c),13,41,42 since the physiological properties of the A vinosum hydrogenase are so far not established Recently, the complete genome of A vinosum (strain DSM 180) has been determined and cytochrome b was identified in the genome sequence—as found in other membrane-bound hydrogenases, where it acts as electron acceptor in the membrane No crystal structures of [NiFe] hydrogenases from species other than Desulfovibrio and Desulfomicrobium have been determined so far In the present work, the spectroscopically well-studied19,20,29,34,36,43–46 A vinosum hydrogenase has been crystallized and applied to X-ray crystallographic analysis The derived structure has a resolution at 2.1 Å and allows the direct comparison with other hydrogen-converting enzymes The crystallized enzyme is shown to be in the (oxygen-inhibited) oxidized Ni-A state by EPR and FTIR spectroscopy This opens the possibility to examine the structural details and elucidate the peculiar spectroscopic features of this important species that is considered to be the oxygen-inhibited state of the enzyme, since all oxygen-tolerant [NiFe] hydrogenases from aerobic bacteria investigated so far not show the Ni-A state.10,47 Results Preparation and spectroscopic characterization The protein preparation was conducted aerobically as described earlier.48 The cells were extensively washed with cold organic solvent and the protein was extracted from the obtained membranes with Fig (a) cw-EPR spectrum from [NiFe] hydrogenase from A vinosum at T= 100 K The sample was prepared using dissolved crystals combined from different crystallization batches in 50 mM Tris–HCl buffer, pH 8.0 Experimental details: X-band, microwave frequency=9.636 GHz, modulation amplitude =0.30 mT, mw power= 19.97 mW (b) cw-EPR spectrum recorded at T= 10 K The enlarged spectrum of the Ni region is shown in the inset Experimental details: microwave frequency = 9.635 GHz, modulation amplitude = 0.50 mT, mw power = 0.66 mW (c) FTIR spectrum recorded at T = 288 K The same sample was used to measure the spectra depicted in (a) and (b) The signals of 1945 cm− (CO) and 2082 cm− and 2093 cm− (both CN− ) belong to the Ni-A state An extra signal (1951 cm− ) is present marked with an asterisk (see text) 431 The Structure of the A vinosum [NiFe] Hydrogenase detergent-containing buffer The solubilized enzyme was further purified by five different column chromatographic steps.48 The last two polishing steps (ion exchange and gel filtration) were repeated and only the purest fractions were selected to achieve the highest degree of purity for crystallization These samples were characterized by cw-EPR and FTIR spectroscopy prior to the crystallographic experiments The EPR and FTIR spectra of asisolated samples are shown in Fig S1 The EPR spectrum recorded at 100 K demonstrated that the sample contained dominantly the Ni-A state (∼ 90%) and a small fraction of the Ni-B state (∼ 10%) At low temperature (10 K) in part of the sample, signals of the coupled [Fe3S4] cluster and the Ni center were observed (Fig S1b) as described earlier by Albracht et al.29,36,38 Furthermore, the FTIR spectrum presented in Fig S1c showed the typical Ni-A signals: CO stretching band at 1944 cm− and two CN− vibration bands at 2082 and 2091 cm− In addition, a shoulder at 1951 cm− was present and bands at 1909 (shoulder at 1919), 2056, and 2066 cm− were observed, which are probably related to that part of the sample showing the split signals in the EPR spectrum (see Fig S1b).49 Only few crystals were suitable for the highresolution X-ray diffraction experiments The other crystals were dissolved in 50 mM Tris–HCl, pH 8.0, buffer and characterized by EPR and FTIR spectroscopy (Fig 1) The sample obtained by dissolving the crystals (see Materials and Methods) demonstrated that the Ni-A state was dominant (∼ 90%) in the EPR spectra (Fig 1a and b) However, no extra splitting has been observed for Ni-A and for the [Fe3S4]+ cluster at low temperature In the FTIR spectra obtained from the same sample, only the Ni-A state and no other signals from the EPR-silent states were observed (Fig 1c) One additional signal was observed in the region of the CO stretching band (at 1951 cm− ) without a counterpart at the CN− vibrational region For this band, no clear assignment was achieved The overall structure The crystal structure of the A vinosum [NiFe] hydrogenase in its Ni-A state has been determined at 2.1 Å resolution After calculating the phases from the Fe-multiwavelength anomalous dispersion (MAD) data sets (5.0 Å resolution), the electron density peaks of the iron–sulfur clusters and the [NiFe] active site were clearly confirmed Four molecules (Molecules 1–4) were observed in the asymmetric unit Two of them formed a dimer with a 2-fold non-crystallographic symmetry (Fig 2a) The contact surface area between the small subunit and large subunit was determined to approximately 3800 Å2 The contact surface area between the molecules in the dimer was determined to approximately 2800 Å2 and the solvent-accessible area of each molecule to approximately 25,500 Å2 (Fig 2b) Rmsd's of the Cα atoms of the four molecules in the asymmetric unit showed values of 0.15–0.19 Å (Table S1 and S2) The comparison of the structures from the A vinosum and D vulgaris MF hydrogenases bears striking homology (Fig 2c) The electrostatic potential of the contact surface of the dimer with the 2-fold non-crystallographic symmetry showed a relatively neutral charge distribution (Fig 2d and e) Imidazole molecules that were part of the crystallization solution were also identified in the electron density map Some of them form a hydrogen-bond network including some amino acids, which most likely serves to stabilize the molecule Sequence alignments of selected [NiFe] hydrogenases The amino acid sequence alignment of the small subunit from selected [NiFe] hydrogenases is depicted in Fig S2 Cysteines that coordinate the active site and the iron–sulfur clusters are highlighted The amino acid identity values and rmsd values of the carbon atoms (Cα) between these structures are summarized in Table S3 Rmsd's of the small subunits between A vinosum and the other hydrogenases show values of 0.75– 0.84 Å The large subunits exhibit similar deviations (0.78–0.85 Å) The determination of the C-terminal amino acid sequence of the small subunit up to Leu271 was based on the electron density map of Molecule of the asymmetric unit The respective region of the three remaining molecules (Molecules 1, 3, and 4) could be defined up to the residue Pro270 and their temperature factors were refined to an average value of 52.0 A Due to the crystallographic packing, they show a slightly different conformation (Fig 3) This exposed C-terminal end of the small subunit, consisting of Val267-Ser268-Val269-Pro270 Fig (a) Overall crystal structure of the dimeric [NiFe] hydrogenase from A vinosum In one-half the iron–sulfur clusters (small subunit), the [NiFe] active site and the extra Mg metal (large subunit) are indicated (b) Molecular surface representation of the dimeric form The two molecules are represented in green and blue (the small subunit is shown in light color) (c) Superimposed ribbon representation of the structures of A vinosum (blue) and D vulgaris MF (red) Rmsd of the A vinosum and D vulgaris MF hydrogenases were 0.78 Å (small subunit) and 0.82 Å (large subunit), respectively (Table S3) (d) The electrostatic potential of one molecule depicting the dimerization contact region Positively charged surface is blue and negatively charged surface is red (e) The electrostatic potential after a 180° rotation of figure (d) 432 The Structure of the A vinosum [NiFe] Hydrogenase Fig (legend on previous page) 433 The Structure of the A vinosum [NiFe] Hydrogenase and Leu271, appears to have a neutral electrostatic potential (Fig 2d and e) At the N-terminus of the large subunit, Long et al recently identified five extra amino acids (Glu-GlnAla-Arg-Arg).50 Three of these amino acid residues (Ala-Arg-Arg) were confirmed in the electron density map of A vinosum hydrogenase The results of matrix-assisted laser desorption/ionization timeof-flight mass spectrometry showed that the small subunit of the purified enzyme has a molecular mass of 29.7 kDa (± 0.4%).48 This is in good agreement with the results obtained from the crystal structure presented here, which has a calculated molecular mass of 29.1 kDa The slightly smaller value is probably due to unidentified amino acid residues in the electron density map At the C-terminus of the large subunit, 15 amino acids after His561 exist in the precursor sequence of the A vinosum hydrogenase (Fig S3) His561 is the last amino acid of the conserved C-terminal [NiFe] cluster binding motif (DPCxxCxxH) These 15 amino acids are missing in the electron density map This can be explained by the final maturation step of the hydrogenase, where the tail after His561 is cleaved off by a specific endopeptidase, as has been shown for other hydrogenases.51 The [NiFe] active site in the Ni-A state In the electron density omit map, no diatomic ligand was observed at the third bridging position (indicated as X in Fig 4a and b) When a single oxygen atom was assigned as bridging ligand, the temperature factor of the oxygen atom converged to approximately 19.4 Å2 The distance between Ni and Fe exhibited a slightly larger value (3.0 Å) than that determined for the D vulgaris MF hydrogenase (2.70–2.80 Å in the oxidized states) The distances of Fe-O and Ni-O were very similar, that is, the bridging ligand was located almost symmetrically between the Ni and Fe atoms The Fe-O-Ni angle was refined to approximately 102° Earlier reports about the hydrogenase from Desulfovibrio sp suggested that the bridging ligand in the Ni-A state is assigned to a hydroperoxide ligand.16,17 When a bridging peroxide was modeled in A vinosum hydrogenase, a negative electron density at the second oxygen position of the hydroperoxide ligand was observed in the difference Fobs − Fcalc Fourier map Higher temperature factors (24.3 and 36.9 Å2, respectively) were also observed Furthermore, the possibility of a half-occupied sulfido ligand was examined This turned out to be not reasonable for a bridging ligand The sulfido ligand was refined with a bond length of rNi-S = 2.1 Å and rFe-S = 2.1 Å, an angle of Ni-S-Fe = 92°, and a temperature factor of 11.5 Å2 However, a residual electron density was observed in the difference Fobs − Fcalc Fourier map Therefore, the possibility of a diatomic ligand or a sulfido ligand at the bridging position between the metals was excluded for A vinosum Modifications of bridging cysteine residues by the coordination of oxygen atoms were observed in the Ni-A/Ni-SU state of Desulfovibrio hydrogenases.16,17 A small residual electron density near a sulfur atom of the bridging cysteine (Cys64) was also observed in A vinosum (Fig 4b, labeled Y) The distances between the sulfur of the bridging cysteine residues (Cys64 and Cys558) and the Ni atom were slightly longer than that of the sulfur of the terminal cysteine residues (Cys61 and Cys555) The glutamate residue (Glu14) near the terminal cysteine (Cys555) has been proposed to be the first residue accepting the proton in the proton transfer pathway.52 The carboxyl oxygen of Glu14 points towards the sulfur of Cys555 and lies within a distance of about 3.2 Å [S (Cys555)–Oɛ2 (Glu14)] Assignment of the additional metal site In order to clarify and assign the electron density originating from a possible metal site located at the Cterminus (Fig S4), we applied the Fe-MAD method After the calculation of the anomalous difference Fourier map using the phases from the model and a data set collected at the Fe K-edge (λ = 1.74014 Å), no electron density peak corresponding to iron was found in this position Furthermore, inductively coupled plasma with optical atomic emission spectrometry (ICP-OES) was carried out to determine the metal content of the enzyme The results confirmed that the A vinosum hydrogenase contains Mg, Ca, Ni, and Fe, respectively When the electron density peak was refined as a Mg2+ ion, the temperature factor converged to 16.0± 2.7 Å2 The averaged temperature factor of the surrounding amino acid residues (Glu42, Glu326, Ala506, and His561; see Fig S4) bound to Mg showed a value of 17.5 Å2 The possibility of Ca in the Fig (a) C-terminus of the small subunit: Superposition of four independently refined molecules observed in the asymmetric unit (Mol1, chains A and B; Mol2, chains C and D; Mol3, chains E and F; Mol4, chains G and H in PDB ID: 3MYR) Val267-Leu271 from molecule (Mol2 in green, chain C) is depicted in stick representation The C-terminus of the remaining molecules Mol1 (blue), Mol3 (yellow), and Mol4 (purple) is shown as ribbon diagram The C-terminus of Mol2 is exposed to the solvent region Due to the crystallographic packing, the C-terminus of Mol1, Mol3, and Mol4 are contacted to neighboring molecules (b) Ribbon and surface representations of four independently refined models in the asymmetric unit Mol4 is shown as surface representation The C-terminus region of Mol1 and Mol2 is indicated with arrows In Mol1, the C-terminus region contacts another molecule (Mol4) due to the crystallographic packing 434 The Structure of the A vinosum [NiFe] Hydrogenase Fig (legend on previous page) The Structure of the A vinosum [NiFe] Hydrogenase 435 Fig (a) Stereoview of the electron density omit map (Fobs − Fcalc, σ cutoff) including the refined model of the active site of the [NiFe] hydrogenase from A vinosum The third bridging ligand is labeled X (b) Stereoview of the electron density omit map (Fobs − Fcalc, σ cutoff) including the refined model without the bridging ligand and the diatomic ligand (CO) of the Fe atom The three meshed spheres show the electron density of the bridging ligand assigned as an oxygen, the diatomic ligand (CO), and the modification of the thiolate of Cys64 Only a small amount of density was observed near the thiolate of Cys64 (labeled Y) (c) Stick representation of the [NiFe] active site and possible hydrogen-bond network (black dotted lines) with neighboring amino acids The red broken line between Cys555 and Glu14 illustrates part of a possible proton pathway (see text).52 C-terminus was excluded since the crystallographic parameters converged with a relatively high temperature factor of 25.5 ± 3.5 Å2 and a negative residual electron density in the Fobs − Fcalc map was observed after the refinement Furthermore, no electron density corresponding to Ca was observed in the anomalous 436 The Structure of the A vinosum [NiFe] Hydrogenase Fig Stereoviews of models using the Fobs − Fcalc omit map (4 σ cutoff ) of the proximal iron–sulfur cluster in its two possible conformations (a) The distorted iron–sulfur cluster showing the Fe4 shifted towards Asp75 (b) The standard cubane [Fe4S4] cluster difference Fourier map from the Fe K-edge data set Therefore, the metal at the C-terminus was assigned to a Mg2+ ion It shows a distorted octahedral coordination and binds to Glu42, His561, Ala506, and three water molecules (Fig S4), which form additional hydrogen bonds to Gln505, Glu326, Glu42, and Lys377 No Ca ion could be identified in the electron density map The iron–sulfur clusters The anomalous difference Fourier map of the iron– sulfur clusters, calculated with the phase of the refined model and the data set collected at 1.0 Å of the X-ray wavelength, is presented in Fig S5 It clearly shows that the distal iron–sulfur cluster (Fig S5a) has four iron atoms and the medial cluster (Fig S5b) has three iron atoms The distal [Fe4S4] cluster in A vinosum is coordinated by three cysteines (Cys190, Cys215, and Cys221) and one histidine (His187), which are also highly conserved in Desulfovibrio species (Fig S2) The medial iron– sulfur cluster was confirmed to be an [Fe3S4] type coordinated by three cysteine residues (Cys230, Cys249, and Cys252) The four highly conserved cysteines, which are known to coordinate the proximal cluster in sulfate-reducing species (Fig S2), suggested that the proximal cluster of the A vinosum hydrogenase is of [Fe4S4] type, too The anomalous Fourier maps calculated using the final phases from the refined model and the anomalous differences from the remote data set (X-ray wavelength of 1.0 Å) showed an ambiguous moiety at the proximal iron–sulfur cluster (Fig S5c) These results indicate that the iron (Fe4) that is coordinated by sulfur atom (Fig S2) could exist in two different conformations The best interpretation of the electron density map shown in Fig was achieved by refining it as a superposition of two conformers One is the standard cubane [Fe4S4] cluster (Fig 5b) and the other is a distorted [Fe4S4] cluster (Fig 5a) with an estimated occupancy of approximately 40% Another data set collected at 2.35 Å resolution of the A vinosum hydrogenase showed similar results in the anomalous Fourier map The proximal iron–sulfur cluster was also refined as a distorted [Fe4S4] cluster with an estimated occupancy of approximately 90% and the rest of the electron density fitted as a standard cubane [Fe4S4] cluster (data not shown) This shows that there is a variation in the proportion of the two conformers Furthermore, Asp75 was found in close vicinity to Fe4 of the distorted cluster within a distance of The Structure of the A vinosum [NiFe] Hydrogenase 2.3 Å (Figs 5a and 6) This Fe4 atom was still bound to the sulfur atom of Cys19 within a distance of 2.3 Å and the coordinating sulfur (Fig S2) lies in a distance of 2.3 Å from Fe4 Asp75 is present in the A vinosum hydrogenase, while a glutamate residue was found in hydrogenases from sulfate reducers Additionally, a small amount of a residual electron density in the Fobs − Fcalc omit map was identified between the Fe4 and Fe2 atom of the distorted cluster An oxygen or sulfur atom inserted at this position gave the best fit to the electron density but had a low occupancy (∼ 0.1) Therefore, it was not modeled in the structure presented here Discussion Spectroscopic characterization It was reported in earlier spectroscopic studies that the activity and spectroscopic properties of the as-isolated samples of A vinosum hydrogenase vary from preparation to preparation.29,36,38 Activity measurements as well as EPR and FTIR spectra obtained here also showed similar problems with the sample after extensive aerobic purification of the oxidized enzyme before crystallization (Fig S1) The enzyme seems to be irreversibly inactivated, as a comparatively low hydrogen uptake activity was found in preparations of A vinosum, accounting for values as low as 10 μmol H − mg − hydrogenase An average H2 uptake specific activity of ∼70 μmol H2 min− mg− hydrogenase was 437 reported in earlier investigations,53 a several times higher value Most probably, the enzyme is quite sensitive to oxygen leading to structural alterations that causes differences in the spectra and in the enzyme activity Surprisingly, dissolved crystals (that were not exposed to X-rays) showed a typical Ni-A signal both in EPR and in FTIR At low temperature, the unusual spin coupling was not observed (see Fig 1b) The only irregular feature in the IR spectra was the band at 1951 cm− in a fraction of the sample This can therefore not be assigned to the spin-coupled Ni-A but may be related to another side product, for example, oxygenation of the cysteine sulfurs Obviously, a selective crystallization of only those enzyme molecules that had the intact (original) Ni-A state took place Since the crystallization required several weeks (sometimes even months) to form the crystals under aerobic conditions, the modified enzyme molecules with spin-coupled species, which probably involves irreversible inactivation of the enzyme, were obviously degraded An additional problem with respect to the comparison between spectroscopic and crystallographic data might arise from the fact that in the aerobically grown crystals, radiation damage might occur during the prolonged data collection in the high-brilliance X-ray beam of the synchrotron even at cryogenic temperatures This is expected to result in reactive oxygen species leading to (photo)reduction of some of the sensitive cofactors in the protein.54,55 Prominent targets for such reactions are the [NiFe] center and the [FeS] clusters, in particular the proximal [Fe4S4] cluster Fig Comparison of different proximal iron–sulfur clusters in hydrogenases including the possible interaction partners of the respective shifted Fe atom (Fe4): A vinosum, distorted conformation (orange); D vulgaris MF, standard cubane [Fe4S4] cluster (green); and D desulfuricans, superoxidized [Fe4S3O3] cluster (yellow) 438 The overall structure The overall structure of the A vinosum hydrogenase was found to be very similar to that of hydrogenases isolated from sulfate-reducing bacteria, in particular D vulgaris MF (Fig 2c), although the total amino acid sequences were less than 50% identical (Table S3) The relatively neutral charge distribution of the electrostatic potential explains most probably the apparent dimeric form of the A vinosum hydrogenase (Fig 2d) It is suggested that the enzyme interacts with a membrane or with an up to now unknown electron acceptor via an extended C-terminus (Val267-Ser268-Val269Pro270-Leu271) close to the distal iron–sulfur cluster, whereas these amino acids are not present in periplasmic Desulfovibrio hydrogenases In the case of other membrane-associated hydrogenases, it is also expected that this region interacts with a protein that is embedded in the membrane, such as a b-type cytochrome Membrane-bound hydrogenases, such as A vinosum and A aeolicus, are lacking an extra region at the N-terminus of the large subunit, which is considered to be the membrane integral anchor region in the case of the periplasmic [NiFe] hydrogenase from D vulgaris MF The active site The diatomic ligands coordinated to the Fe in the active site were assigned to two CN− molecules and one CO according to the results from FTIR spectroscopy (Fig 4a).20,49 CN− ligands are generally known as strong σ-donors They establish hydrogen bonds to nearby amino acids In the case of the A vinosum hydrogenase, these are Arg487 and Ser510, respectively (Fig 4c) This is in agreement with FTIR spectroscopic results, which reveal that the CO stretching frequency is more sensitive to changes of the environment as compared to the bands of the hydrogen-bonded CN− ligands.56 The amino acid residues Leu490 and Val508 in the vicinity of the CO ligand not build any hydrogen bonds (Fig 4c) Another residue, namely, His68, which is located near the bridging Cys558, might form a hydrogen bond to the thiolate of this cysteine as in the D vulgaris MF hydrogenase.57 In the active site of Desulfovibrio hydrogenases in the Ni-A state, a di-oxo ligand was found bridging the Ni and Fe atoms16,17 and was proposed to be a hydroperoxy (OOH−) species In the Ni-B state, however, a mono-oxo species is present between the two metals.16,17,31 This is in contrast to the present case of the A vinosum hydrogenase (N90% Ni-A) for which a mono-oxo bridging ligand was assigned Here, it is most probably a hydroxy (OH−) ligand.31 At present, the origin of this difference found for the bridging ligand between sulfate-reducing and photosynthetic bacteria cannot be explained However, The Structure of the A vinosum [NiFe] Hydrogenase it can not be completely excluded that the extensive X-ray dose used in the crystallographic experiments leads to modifications of the enzyme and the cofactors, by which the second oxygen could be lost On the other hand, ENDOR experiments on hydrogenase single crystals of D vulgaris MF in the Ni-A state have indicated that an OH− bridging ligand is more likely than an OOH− ligand in this state.22 The Ni-Fe distance in the crystal structure of the A vinosum hydrogenase showed values of around 3.0 Å, which is in approximate agreement with the distance of 2.8 Å found for D vulgaris MF hydrogenase In some of the Desulfovibrio sp hydrogenases, spectroscopic and crystallographic studies suggested that a sulfur species is bridging the two metals.11,13,58,59 However, in the refined structure of the A vinosum hydrogenase, the angle of the metals and the bridging ligand and its temperature factor indicate that an oxygen species is located at this position In the case of the D vulgaris MF hydrogenase (Ni-A, Ni-B, reduced states and CO-inhibited states), the thiolate of the terminal-bound Cys546 (numbering of D vulgaris MF) was modified, which was tentatively assigned to an oxygen species.16 The thiolate of the bridging Cys84 (numbering of D vulgaris MF) was also modified and tentatively assigned to an oxygen atom in the case of the D vulgaris MF hydrogenase in the Ni-A state and in the case of the D gigas hydrogenase in the Ni-SU state.16,17 These modifications of the bridging cysteine residue were assigned as a partially occupied atomic species A similar modification was observed in the A vinosum hydrogenase in the Ni-A state (Fig 4b) However, the observed electron density near the thiolate of Cys64 was small and thus difficult to assign unequivocally to an oxygen atom Therefore, no assignment was made for this electron density The terminal Cys555 appears to be unmodified in A vinosum hydrogenase (Fig 4b) Furthermore, very recent studies of the [NiFeSe] hydrogenase from D vulgaris H in its oxidized, “asisolated”, state showed that the thiolate of the terminal Cys75 (numbering of D vulgaris H) was modified by two oxygen atoms.27 In the A vinosum hydrogenase, no modification was observed at the respective cysteine, namely, Cys61 These results suggest that in photosynthetic bacteria, the inactivation process of the active site may be different from the sulfate-reducing Desulfovibrio sp hydrogenases Altered cysteine residues are considered to be a result of “oxidative damage” related to the aerobic purification and crystallization of the enzyme The additional metal site The C-terminus of the large subunit of the D vulgaris MF hydrogenase hosts a Mg atom The Structure of the A vinosum [NiFe] Hydrogenase coordinated by His552 (His561 in A vinosum).11 This histidine residue is highly conserved among various [NiFe] hydrogenases (Fig S3) The crystal structures of the [NiFeSe] hydrogenases from D baculatum and D vulgaris H assume an Fe atom instead of Mg in this site.26,27 For the A vinosum hydrogenase, results from the Fe-MAD method, ICP-OES, and the final refinement suggest that there is a Mg and no Fe atom present near the C-terminus The Mg site is located next to the [NiFe] active site within a distance of ∼ 13 Å The hydrogen-bond network between the [NiFe] active site and the Mg ion, involving Cys555, Glu14, His561, Glu42, and some water molecules, is probably part of the proton transfer pathway of the enzyme A similar pathway has been proposed for Desulfovibrio sp hydrogenases.60,61 Cys555 and Glu14, which are highly conserved in almost all [NiFe] hydrogenases, are considered to be the essential residues in these proton transfer pathways (Fig 4c).52 The iron–sulfur clusters Based on the sequence alignment of various hydrogenases and spectroscopic studies, two [Fe4S4] clusters and one [Fe3S4] cluster are expected to be located in the small subunit of the A vinosum hydrogenase The crystallographic parameters of the medial [Fe3S 4] and distal [Fe 4S4] clusters converged well EPR and Mössbauer studies suggested that in the A vinosum hydrogenase, an extra [Fe3S4] cluster or a low-spin Fe3+ may be located between the medial [Fe3S4] cluster and the [NiFe] active site that causes complex EPR split signals in the g = region.29,36,38 The signal varies according to the applied redox potential in electrochemical experiments and is pH dependent.62 In the crystal structure reported here, no additional metal species were observed based on the results of the anomalous difference map (Fig S5) However, it was found that the proximal [Fe4S4] cluster has two different conformations, a standard cubane and a distorted form (see Fig 5) Since the results of the EPR and FTIR spectroscopy (Fig 1) showed that the proximal [Fe4S4] cluster was present as the standard cubane at the beginning of the X-ray diffraction experiments, it must be assumed that during irradiation of the crystal, the enzyme was probably damaged by the X-ray beam during data collection‡ This led to the observation of a mixture of two conformers in the crystal The ‡ The oxidation state of the single crystal sample after the X-ray diffraction experiments could not be examined This is due to the difficulties of EPR spectroscopy on a single crystal, which requires a larger amount of sample volume not provided by the small single crystal size used here 439 distorted cluster is assumed to magnetically couple to the active site in the Ni-A state (Ni3+, S = 1/2) and to the [Fe3S4]+ cluster and might possibly explain the split signals observed earlier for the oxidized A vinosum enzyme.29,36,38 The midpoint potentials of the A vinosum hydrogenase were estimated by redox titrations following the changes in the EPR spectral region of g = 2.62 An increase of the g = 2.02 signal was observed during progressive reduction Further reduction resulted in a decrease of the respective signal These observations can probably be explained if the split signal involves the [NiFe] center interacting with the modified proximal cluster and if it is this cluster that is reduced in the first stage The medial [Fe3S4] cluster might be reduced in a subsequent second process Similar modifications of iron–sulfur clusters were observed in other enzymes The crystal structure of the D desulfuricans [NiFe] hydrogenase showed that its proximal iron–sulfur cluster was modified and appears to be an [Fe4S3O3] species.13 One of the iron atoms is shifted towards the carboxyl oxygen of Glu75 (numbering in D desulfuricans, Asp75 in A vinosum) and linked to it with a distance of 2.1 Å (Fig 6) This iron atom is connected by two oxobridges to neighboring Fe atoms and by an oxobridge to a cysteine residue (Cys20) In the case of A vinosum hydrogenase, the disordered iron (Fe4, Figs and 6) is linked to Asp75 and Cys19 Since it was also observed that the proximal iron–sulfur cluster in A vinosum exists in two superimposed conformations in the crystals, it is difficult to exclude the possibility of having [Fe4S3O] or [Fe4S3O2] cluster types present in the A vinosum enzyme (see Results) Figure shows the comparison of proximal iron– sulfur clusters from A vinosum (orange), D vulgaris MF (green), and D desulfuricans (yellow) hydrogenases The amino acid at position 75 (aspartate) in A vinosum hydrogenase was identified to be in the vicinity of the proximal iron–sulfur cluster within a distance of 2.3 Å In the D desulfuricans hydrogenase, a glutamate is linked to the distorted iron atom instead For other Desulfovibrio hydrogenase crystal structures, a glutamate was also located in the respective position but in a relatively long distance to the cluster.16 Asp75 is assumed to have an impact in terms of stabilizing the proximal cluster in its distorted [Fe4S4] form, particularly in the A vinosum enzyme Further experiments, for example, introduction of mutations, are necessary to verify this finding and investigate possible effects on the hydrogen conversion, enzymatic activity, or oxygen sensitivity Crystal structures of other hydrogenases depicting a similar complex EPR split signal would help to further investigate this issue In the case of the oxygen-tolerant MBH hydrogenase from R eutropha, three additional cysteine residues were found near the proximal iron–sulfur cluster (Fig S2).10,37 Moreover, the amino acid 440 sequence of the oxygen-tolerant MBH of A aeolicus63 reveals the same finding (Fig S2) In the majority of other [NiFe] hydrogenases, two of these residues are replaced by glycine, namely, Gly18 and Gly120 in the A vinosum hydrogenase These particular cysteines probably affect the redox potential of the proximal iron–sulfur cluster and the catalytic activity of the enzyme and might also be responsible for its oxygen tolerance.10,37,63 This implies that not only amino acids in the immediate vicinity of the active site but also residues close to the proximal iron–sulfur cluster should be considered as key players regarding oxygen sensitivity of hydrogenases The Structure of the A vinosum [NiFe] Hydrogenase localized in between the Ni3+ (S = 1/2) of the Ni-A state and the oxidized medial [Fe3S4]+ (S = 1/2) cluster of the enzyme Thus, magnetic coupling to both paramagnets could occur and lead to the observed spectra A similar situation is probably met in the split EPR spectra of the oxidized oxygen-tolerant [NiFe] hydrogenases from R eutropha10,37,65 and A aeolicus.47,63 In these species, it is, however, expected that the modified proximal [Fe4S4] cluster has a different electronic structure leading to the observed differences in these split EPR spectra Materials and Methods Conclusion In conclusion, the overall A vinosum hydrogenase structure presented here reveals significant similarity to the crystallized enzymes derived from sulfatereducing bacteria Despite the fact that the amino acid sequences are less than 50% identical and the metabolic and phylogenetic origin is different, general structural features such as the active [NiFe] center conformation including the non-protein ligand assignment, the extra metal binding site, and the medial and distal iron–sulfur cluster coordination were confirmed This implies that the hydrogenconverting ability of hydrogenases follows a general mechanism and appears to be species independent The hydrogenase has been crystallized in the Ni-A state as shown by EPR and FTIR measurements on dissolved crystals In this state, the extra bridging ligand between the Ni and the Fe atoms in the active site of the A vinosum enzyme is shown to be a monoatomic oxygen species, most likely a hydroxy ligand The same ligand has been found for the Ni-B state,31 but probably these have different conformations64 leading to the observed variations in spectroscopic properties The detection of a modified bridging cysteine (Cys64) in part of the crystallized sample is reminiscent of findings of other [NiFe] hydrogenases of sulfate-reducing bacteria.2,17 These modifications of Ni-A probably lead to diamagnetic states that are thus not detectable by EPR techniques An interesting observation made for the proximal [Fe4S4] cluster is that it can exist in two different conformations in the oxidized state One is identical with the normal cubane-type [Fe4S4]2+ cluster that is diamagnetic; the other one exhibits a disturbed open structure with one iron atom displaced ([Fe3S4 + Fe]) and probably changed (oxygenated) bridges to that iron This altered proximal cluster is believed to be paramagnetic in the oxidized state The existence of such a cluster in Ni-A samples of earlier investigations could very well explain the magnetically coupled EPR spectra observed in such preparations The oxidized paramagnetic proximal cluster is Protein preparation A vinosum was cultivated in a 1100-l glass fermenter and yielded about kg cells (wet weight) per batch Purification (∼50 mg protein per kg cells wet weight) and crystallization were carried out aerobically as described earlier.48 The samples were concentrated to 30 mg ml− and crystallized using the vapor-diffusion method containing the crystallization buffer (1.0 M sodium citrate and 0.1 M imidazole, pH 8.0) The enzyme was characterized biochemically before conducting the crystallization experiment using SDS-PAGE, a modified Clark-electrode setup for the H2 uptake activity assay, and by mass spectrometry using an Applied Biosystems matrix-assisted laser desorption/ionization time-of-flight mass spectrometry spectrometer Voyager-DE PRO Workstation.66 Furthermore, the preparation was characterized spectroscopically (see below) Subsequent analysis of crystals after performing the X-ray diffraction was ruled out due to their small size and layer thickness Therefore, possible changes in terms of structure or redox state caused by radiation damage cannot be excluded The metal content (3 mg protein in 50 mM Tris–HCl, pH 8.0) was determined by ICP-OES Spectroscopic analysis The oxidation state of the enzyme was confirmed by EPR and FTIR spectroscopy Two different samples were prepared, an as-isolated solution sample and a sample from dissolved crystals, respectively The EPR experiments were carried out with a Bruker ESP-300 (X-band) spectrometer equipped with an Oxford Instruments cryostat and an ITC 503 temperature controller The solution sample (as-isolated state) was measured at T = 10 K and T = 100 K For another data set, the sample was prepared by dissolving the crystals The crystals were recovered from 40 crystallization wells (each 10 μl) and then washed with the crystallization buffer in an Eppendorf tube After a short centrifugation at 6700g for min, the supernatant was removed and the crystals were dissolved in 50 mM Tris–HCl buffer, pH 8.0 Subsequently, the sample was transferred into an X-band EPR tube and frozen in liquid nitrogen FTIR spectroscopic experiments with the as-isolated dissolved crystal sample were carried out in a Bruker IFS 441 The Structure of the A vinosum [NiFe] Hydrogenase Table Data collection and refinement statistics of the native data set PDB ID Data collection Wavelength (Å) Space group Unit cell parameters (Å) a b c Resolution range (Å) (highest shell) Observed reflections Unique reflectionsa Completeness (%) Rmerge (%) 〈I/σ(I)〉 3MYR 1.00000 P21212 205.75 216.96 119.80 48.06–2.10 (2.19–2.10) 1,230,196 295,408 95.0 (90.2) 5.7 (29.6) 18.5 (4.9) Refinement Resolution range (Å) R/Rfree (%) Number of residues Number of solvent molecules Number of imidazoles Rmsd bond lengths (Å) Rmsd angles (°) Ramachandran plot (%) Favored region Allowed region Outlier region Average B-factors (Å2) Protein Ligand Water Imidazole od was applied using the program MOLREP.70 The [NiFe] hydrogenase from D vulgaris MF was used as a search model The model building was done with the program Coot.71 The anomalous Fourier map was obtained from the native data set (at the X-ray wavelength 1.0 Å) using the fast Fourier transform program with the phase calculation from the refined model The refinement was carried out with REFMAC5.72 During every refinement procedure, non-crystallographic symmetry restraints have been used The statistics of the refinement are summarized in Table The stereochemical properties were checked by RAMPAGE.73 The electrostatic potential was calculated by the program GRASP2.74 The molecular graphic has been generated by PyMOL.75 Protein Data Bank accession number 30–2.10 13.8/16.8 3313 1924 0.027 1.93 96.8 2.9 0.2 20.9 17.4 27.8 38.4 Numbers in parentheses represent the values for the highestresolution shell a The equivalent of Friedel pairs are scaled as unique reflections 66v/S FTIR spectrometer with a cm− spectral resolution at 288 K For the dissolved crystal sample, the sample used in the cw-EPR spectroscopy was recovered and directly loaded into the CaF2 window cell of the sample holder Data collection and baseline correction were carried out by using the OPUS software (Bruker) X-ray diffraction data collection A complete native data set (2.1 Å resolution) was recorded In addition, two-wavelength Fe-MAD data sets were collected from another single crystal to 5.0 Å resolution.48 All diffraction experiments were carried out at 100 K using the synchrotron beam lines PXIII at SLS (Villigen, Switzerland), BL41XU at SPring-8 (Hyogo, Japan), and BL14.2 at BESSY II (Berlin, Germany) Diffraction images were indexed using the program MOSFLM67 or XDS68 and processed employing the CCP4 program suite.69 The conditions used for data collection and the results obtained are summarized in Table Structure determination and refinement The initial phases were determined by the Fe-MAD method Subsequently, the molecular-replacement meth- Coordinates and structure factors have been deposited in the Protein Data Bank (PDB) with the accession number 3MYR Acknowledgements Siem P J Albracht and Winfried Roseboom (University of Amsterdam, Netherlands) are gratefully acknowledged for helpful discussions and their support in protein purification We thank Katrin Schwarzbach and Tanja Berndsen for technical assistance and Maria-Eirini Pandelia and Leslie J Currell (all MPI for Bioinorganic Chemistry, Mülheim, Germany) for their help with the spectroscopic measurements and helpful discussions Yoshiki Higuchi (University of Hyogo, Japan) is gratefully acknowledged for his advice and many discussions We thank the staff of BL14.2 at BESSYII (Berlin, Germany), BL41XU at SPring-8 (Hyogo, Japan), and PXIII at SLS (Villigen, Switzerland) and also Daniel Kurz (MPI for Iron Research, Düsseldorf, Germany) for performing the ICP-OES experiments The work was financially supported by the EU Framework Programme no 226716, the EU/Energy Network project SOLAR-H2 (FP7 contract 212508), the BMBF (03SF0318B, 03SF0355C), and the Max Planck Society Supplementary Data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/ j.jmb.2010.07.041 References Vignais, P M & Billoud, B (2007) Occurrence, classification, and biological function of hydrogenases: an overview Chem Rev 107, 4206–4272 442 The Structure of the A vinosum [NiFe] Hydrogenase Fontecilla-Camps, J C., Volbeda, A., Cavazza, C & Nicolet, Y (2007) Structure/function relationships of [NiFe]- and [FeFe]-hydrogenases Chem Rev 107, 4273–4303 Hiromoto, T., Warkentin, E., Moll, J., Ermler, U & Shima, S (2009) The crystal structure of an [Fe]hydrogenase–substrate complex reveals the framework for H2 activation Angew Chem., Int Ed 48, 6457–6460 Peters, J W., Lanzilotta, W N., Lemon, B J & Seefeldt, L C (1998) X-ray crystal structure of the Fe-only hydrogenase (Cpl) from Clostridium pasteurianum to 1.8 Å resolution Science, 282, 1853–1858 Shima, S., Pilak, O., Vogt, S., Schick, M., Stagni, M S., Meyer-Klaucke, W et al (2008) The crystal structure of [Fe]-hydrogenase reveals the geometry of the active site Science, 321, 572–575 Nicolet, Y., Piras, C., Legrand, P., Hatchikian, C E & Fontecilla-Camps, J C (1999) Desulfovibrio desulfuricans iron hydrogenase: the structure shows unusual coordination to an active site Fe binuclear center Struct Fold Des 7, 13–23 Pilak, O., Mamat, B., Vogt, S., Hagemeier, C H., Thauer, R K., Shima, S et al (2006) The crystal structure of the apoenzyme of the iron–sulphur cluster-free hydrogenase J Mol Biol 358, 798–809 Burgdorf, T., Lenz, O., Buhrke, T., van der Linden, E., Jones, A K., Albracht, S P J & Friedrich, B (2005) [NiFe]-hydrogenases of Ralstonia eutropha H16: modular enzymes for oxygen-tolerant biological hydrogen oxidation J Mol Microbiol Biotechnol 10, 181–196 Guiral, M., Tron, P., Aubert, C., Gloter, A., IobbiNivol, C & Giudici-Orticoni, M T (2005) A membrane-bound multienzyme, hydrogen-oxidizing, and sulfur-reducing complex from the hyperthermophilic bacterium Aquifex aeolicus J Biol Chem 280, 42004–42015 10 Lenz, O., Ludwig, M., Schubert, T., Bürstel, I., Ganskow, S., Goris, T et al (2010) H2 conversion in the presence of O2 as performed by the membranebound [NiFe]-hydrogenase of Ralstonia eutropha Chem Phys Chem 11, 1107–1119 11 Higuchi, Y., Yagi, T & Yasuoka, N (1997) Unusual ligand structure in Ni–Fe active center and an additional Mg site in hydrogenase revealed by high resolution X-ray structure analysis Structure, 5, 1671–1680 12 Higuchi, Y., Ogata, H., Miki, K., Yasuoka, N & Yagi, T (1999) Removal of the bridging ligand atom at the Ni–Fe active site of [NiFe] hydrogenase upon reduction with H2, as revealed by X-ray structure analysis at 1.4 Å resolution Structure, 7, 549–556 13 Matias, P M., Soares, C M., Saraiva, L M., Coelho, R., Morais, J., Le Gall, J & Carrondo, M A (2001) [NiFe] hydrogenase from Desulfovibrio desulfuricans ATCC 27774: gene sequencing, three-dimensional structure determination and refinement at 1.8 Å and modelling studies of its interaction with the tetrahaem cytochrome c3 J Biol Inorg Chem 6, 63–81 14 Montet, Y., Amara, P., Volbeda, A., Vernede, X., Hatchikian, E C., Field, M J et al (1997) Gas access to the active site of Ni–Fe hydrogenases probed by X-ray crystallography and molecular dynamics Nat Struct Biol 4, 523–526 Ogata, H., Mizoguchi, Y., Mizuno, N., Miki, K., Adachi, S., Yasuoka, N et al (2002) Structural studies of the carbon monoxide complex of [NiFe]hydrogenase from Desulfovibrio vulgaris Miyazaki F: suggestion for the initial activation site for dihydrogen J Am Chem Soc 124, 11628–11635 Ogata, H., Hirota, S., Nakahara, A., Komori, H., Shibata, N., Kato, T et al (2005) Activation process of [NiFe] hydrogenase elucidated by high-resolution X-ray analyses: conversion of the ready to the unready state Structure, 13, 1635–1642 Volbeda, A., Martin, L., Cavazza, C., Matho, M., Faber, B W., Roseboom, W et al (2005) Structural differences between the ready and unready oxidized states of [NiFe] hydrogenases J Biol Inorg Chem 10, 239–249 Volbeda, A., Charon, M H., Piras, C., Hatchikian, E C., Frey, M & Fontecilla-Camps, J C (1995) Crystal structure of the nickel-iron hydrogenase from Desulfovibrio gigas Nature, 373, 580–587 Bagley, K A., Duin, E C., Roseboom, W., Albracht, S P J & Woodruff, W H (1995) Infrared detectable groups sense changes in charge density on the nickel center in hydrogenase from Chromatium vinosum Biochemistry, 34, 5527–5535 Pierik, A J., Roseboom, W., Happe, R P., Bagley, K A & Albracht, S P J (1999) Carbon monoxide and cyanide as intrinsic ligands to iron in the active site of [NiFe]-hydrogenases—NiFe(CN)(2)CO, biology's way to activate H2 J Biol Chem 274, 3331–3337 Ogata, H., Lubitz, W & Higuchi, Y (2009) [NiFe] hydrogenases: structural and spectroscopic studies of the reaction mechanism Dalton Trans 7577–7587 Pandelia, M E., Ogata, H & Lubitz, W (2010) Intermediates in the catalytic cycle of [NiFe] hydrogenase: functional spectroscopy of the active site Chem Phys Chem 11, 1127–1140 Leroux, F., Dementin, S., Burlatt, B., Cournac, L., Volbeda, A., Champ, S et al (2008) Experimental approaches to kinetics of gas diffusion in hydrogenase Proc Natl Acad Sci USA, 105, 11188–11193 Dementin, S., Leroux, F., Cournac, L., De Lacey, A L., Volbeda, A., Léger, C et al (2009) Introduction of methionines in the gas channel makes [NiFe] hydrogenase aero-tolerant J Am Chem Soc 131, 10156–10164 Menon, N K., Robbins, J., DerVartanian, M., Patil, D., Peck, H D., Menon, A L et al (1993) Carboxyterminal processing of the large subunit of [NiFe] hydrogenases FEBS Lett 331, 91–95 Garcin, E., Vernede, X., Hatchikian, E C., Volbeda, A., Frey, M & Fontecilla-Camps, J C (1999) The crystal structure of a reduced [NiFeSe] hydrogenase provides an image of the activated catalytic center Structure, 7, 557–566 Marques, M C., Coelho, R., De Lacey, A L., Pereira, I A & Matias, P M (2010) The three-dimensional structure of [NiFeSe] hydrogenase from Desulfovibrio vulgaris Hildenborough: a hydrogenase without a bridging ligand in the active site in its oxidised, “asisolated” state J Mol Biol 396, 893–907 15 16 17 18 19 20 21 22 23 24 25 26 27 The Structure of the A vinosum [NiFe] Hydrogenase 28 Lubitz, W., Reijerse, E & van Gastel, M (2007) [NiFe] and [FeFe] hydrogenases studied by advanced magnetic resonance techniques Chem Rev 107, 4331–4365 29 Albracht, S P J., Kalkman, M L & Slater, E C (1983) Magnetic interaction of nickel(III) and the iron–sulfur cluster in hydrogenase from Chromatium vinosum Biochim Biophys Acta, 724, 309–316 30 Lamle, S E., Albracht, S P J & Armstrong, F A (2004) Electrochemical potential-step investigations of the aerobic interconversions of [NiFe]-hydrogenase from Allochromatium vinosum: insights into the puzzling difference between unready and ready oxidized inactive states J Am Chem Soc 126, 14899–14909 31 van Gastel, M., Stein, M., Brecht, M., Schröder, O., Lendzian, F., Bittl, R et al (2006) A single-crystal ENDOR and density functional theory study of the oxidized states of the [NiFe] hydrogenase from Desulfovibrio vulgaris Miyazaki F J Biol Inorg Chem 11, 41–51 32 Albracht, S P J (1994) Nickel hydrogenases—in search of the active site Biochim Biophys Acta, 1188, 167–204 33 Lubitz, W., van Gastel, M & Gärtner, W (2007) Nickel iron hydrogenases Met Ions Life Sci 2, 279–322 34 Surerus, K K., Chen, M., van der Zwaan, J W., Rusnak, F M., Kolk, M., Duin, E C et al (1994) Further characterization of the spin coupling observed in oxidized hydrogenase from Chromatium vinosum— a Mössbauer and multifrequency EPR study Biochemistry, 33, 4980–4993 35 Teixeira, M., Moura, I., Xavier, A V., Moura, J J G., LeGall, J., Dervartanian, D V et al (1989) Redox intermediates of Desulfovibrio gigas [NiFe] hydrogenase generated under hydrogen—Mössbauer and EPR characterization of the metal centers J Biol Chem 264, 16435–16450 36 Albracht, S P J., van der Zwaan, J W & Fontijn, R D (1984) EPR spectrum at 4, and 35 GHz of hydrogenase from Chromatium vinosum—direct evidence for spin-spin interaction between Ni(III) and the iron–sulfur cluster Biochim Biophys Acta, 766, 245–258 37 Saggu, M., Zebger, I., Ludwig, M., Lenz, O., Friedrich, B., Hildebrandt, P & Lendzian, F (2009) Spectroscopic insights into the oxygen-tolerant membraneassociated [NiFe] hydrogenase of Ralstonia eutropha H16 J Biol Chem 284, 16264–16276 38 Albracht, S P J., Fontijn, R D & van der Zwaan, J W (1985) Destruction and reconstitution of the activity of hydrogenase from Chromatium vinosum Biochim Biophys Acta, 832, 89–97 39 Brugna-Guiral, M., Tron, P., Nitschke, W., Stetter, K O., Burlat, B., Guigliarelli, B et al (2003) [NiFe] hydrogenases from the hyperthermophilic bacterium Aquifex aeolicus: properties, function, and phylogenetics Extremophiles, 7, 145–157 40 Palagyi-Meszaros, L S., Maroti, J., Latinovics, D., Balogh, T., Klement, E., Medzihradszky, K F et al (2009) Electron-transfer subunits of the NiFe hydrogenases in Thiocapsa roseopersicina BBS FEBS J 276, 164–174 41 Yagi, T (1984) Spectral and kinetic abnormality during the reduction of cytochrome c3 catalyzed by hydrogenase with hydrogen Biochim Biophys Acta, 767, 288–294 443 42 Yahata, N., Saitoh, T., Takayama, Y., Ozawa, K., Ogata, H., Higuchi, Y & Akutsu, H (2006) Redox interaction of cytochrome c3 with [NiFe] hydrogenase from Desulfovibrio vulgaris Miyazaki F Biochemistry, 45, 1653–1662 43 Bagley, K A., Van Garderen, C J., Chen, M., Duin, E C., Albracht, S P J & Woodruff, W H (1994) Infrared studies on the interaction of carbon monoxide with divalent nickel in hydrogenase from Chromatium vinosum Biochemistry, 33, 9229–9236 44 Gessner, C., Stein, M., Albracht, S P J & Lubitz, W (1999) Orientation-selected ENDOR of the active center in Chromatium vinosum [NiFe] hydrogenase in the oxidized “ready” state J Biol Inorg Chem 4, 379–389 45 Gu, Z J., Dong, J., Allan, C B., Choudhury, S B., Franco, R., Moura, J J G et al (1996) Structure of the Ni sites in hydrogenases by X-ray absorption spectroscopy Species variation and the effects of redox poise J Am Chem Soc 118, 11155–11165 46 van der Zwaan, J W., Coremans, J M C C., Bouwens, E C M & Albracht, S P J (1990) Effect of 17O2 and 13 CO on EPR spectra of nickel in hydrogenase from Chromatium vinosum Biochim Biophys Acta, 1041, 101–110 47 Pandelia, M E., Fourmond, V., Tron-Infossi, P., Lojou, E., Bertrand, P., Léger, C et al (2010) Membranebound hydrogenase I from the hyperthermophilic bacterium Aquifex aeolicus: enzyme activation, redox intermediates and oxygen tolerance J Am Chem Soc 132, 6991–7004 48 Kellers, P., Ogata, H & Lubitz, W (2008) Purification, crystallization and preliminary X-ray analysis of the membrane-bound [NiFe] hydrogenase from Allochromatium vinosum Acta Crystallogr., Sect F: Struct Biol Cryst Commun 64, 719–722 49 Bleijlevens, B., van Broekhuizen, F A., De Lacey, A L., Roseboom, W., Fernandez, V M & Albracht, S P J (2004) The activation of the [NiFe]-hydrogenase from Allochromatium vinosum An infrared spectro-electrochemical study J Biol Inorg Chem 9, 743–752 50 Long, M N., Liu, J J., Chen, Z F., Bleijlevens, B., Roseboom, W & Albracht, S P J (2007) Characterization of a HoxEFUYH type of [NiFe] hydrogenase from Allochromatium vinosum and some EPR and IR properties of the hydrogenase module J Biol Inorg Chem 12, 62–78 51 Devine, E., Holmqvist, M., Stensjö, K & Lindblad, P (2009) Diversity and transcription of proteases involved in the maturation of hydrogenases in Nostoc punctiforme ATCC 29133 and Nostoc sp strain PCC 7120 BMC Microbiol 9, 53 52 Dementin, S., Burlat, B., De Lacey, A L., Pardo, A., Adryanczyk-Perrier, G., Guigliarelli, B et al (2004) A glutamate is the essential proton transfer gate during the catalytic cycle of the [NiFe] hydrogenase J Biol Chem 279, 10508–10513 53 Coremans, J M C C., van der Zwaan, J W & Albracht, S P J (1992) Distinct redox behavior of prosthetic groups in ready and unready hydrogenase from Chromatium vinosum Biochim Biophys Acta, 1119, 157–168 54 Yano, J., Kern, J., Irrgang, K D., Latimer, M J., Bergmann, U., Glatzel, P et al (2005) X-ray damage to 444 55 56 57 58 59 60 61 62 63 64 the Mn4Ca complex in single crystals of photosystem II: a case study for metalloprotein crystallography Proc Natl Acad Sci USA, 102, 12047–12052 von Sonntag, C (2006) Free-Radical-Induced DNA Damage and Its Repair: A Chemical Perspective Springer-Verlag, Berlin, Germany Pandelia, M E., Ogata, H., Currell, L J., Flores, M & Lubitz, W (2009) Probing intermediates in the activation cycle of [NiFe] hydrogenase by infrared spectroscopy: the Ni-SIr state and its light sensitivity J Biol Inorg Chem 14, 1227–1241 Goenka Agrawal, A., van Gastel, M., Gärtner, W & Lubitz, W (2006) Hydrogen bonding affects the [NiFe] active site of Desulfovibrio vulgaris Miyazaki F hydrogenase: a hyperfine sublevel correlation spectroscopy and density functional theory study J Phys Chem B, 110, 8142–8150 Higuchi, Y & Yagi, T (1999) Liberation of hydrogen sulfide during the catalytic action of Desulfovibrio hydrogenase under the atmosphere of hydrogen Biochem Biophys Res Commun 255, 295–299 Vincent, K A., Belsey, N A., Lubitz, W & Armstrong, F A (2006) Rapid and reversible reactions of [NiFe]hydrogenases with sulfide J Am Chem Soc 128, 7448–7449 Galvan, I F., Volbeda, A., Fontecilla-Camps, J C & Field, M J (2008) A QM/MM study of proton transport pathways in a [NiFe] hydrogenase Proteins: Struct., Funct., Bioinform 73, 195–203 Teixeira, V H., Soares, C M & Baptista, A M (2008) Proton pathways in a [NiFe]-hydrogenase: a theoretical study Proteins: Struct., Funct., Bioinform 70, 1010–1022 Cammack, R., Rao, K K., Serra, J & Llama, M J (1986) The redox properties of the iron–sulfur cluster in hydrogenase from Chromatium vinosum, strain-D Biochimie, 68, 93–96 Pandelia, M E (2009) [NiFe] hydrogenases from Desulfovibrio vulgaris Miyazaki F and Aquifex aeolicus studied by FTIR, EPR and electrochemical techniques: redox intermediates, O2/CO sensitivity and lightinduced effects Dissertation, Technische Universität Berlin van Gastel, M., Fichtner, C., Neese, F & Lubitz, W (2005) EPR experiments to elucidate the structure of the ready and unready states of the [NiFe] hydroge- The Structure of the A vinosum [NiFe] Hydrogenase 65 66 67 68 69 70 71 72 73 74 75 nase of Desulfovibrio vulgaris Miyazaki F Biochem Soc Trans 33, 7–11 Saggu, M., Teutloff, C., Ludwig, M., Brecht, M., Pandelia, M E., Lenz, O et al (2010) Comparison of the membrane-bound [NiFe] hydrogenases from R eutropha H16 and D vulgaris Miyazaki F in the oxidized ready state by pulsed EPR Phys Chem Chem Phys 12, 2139–2148 Kellers, P (2008) Strukturelle und funktionelle Charakterisierung der [NiFe]-Hydrogenase aus Allochromatium vinosum Dissertation, Heinrich-HeineUniversität Düsseldorf Leslie, A G W (1992) Recent changes to the MOSFLM package for processing film and image plate data Joint CCP4 + ESF-EAMCB Newsletter on Protein Crystallography, 26 Kabsch, W (1993) Automatic processing of rotation diffraction data from crystals of initially unknown symmetry and cell constants J Appl Crystallogr 26, 795–800 Collaborative Computational Project No (1994) The CCP4 Suite: programs for protein crystallography Acta Crystallogr., Sect D: Biol Crystallogr 50, 760–763 Vagin, A & Teplyakov, A (1997) MOLREP: an automated program for molecular replacement J Appl Crystallogr 30, 1022–1025 Emsley, P & Cowtan, K (2004) Coot: model-building tools for molecular graphics Acta Crystallogr., Sect D: Biol Crystallogr 60, 2126–2132 Vagin, A A., Steiner, R A., Lebedev, A A., Potterton, L., McNicholas, S., Long, F & Murshudov, G N (2004) REFMAC5 dictionary: organization of prior chemical knowledge and guidelines for its use Acta Crystallogr., Sect D: Biol Crystallogr 60, 2184–2195 Lovell, S C., Davis, I W., Arendall, W B., de Bakker, P I W., Word, J M., Prisant, M G et al (2003) Structure validation by Calpha geometry: phi, psi and Cbeta deviation Proteins: Struct., Funct., Genet 50, 437–450 Petrey, D & Honig, B (2003) GRASP2: visualization, surface properties, and electrostatics of macromolecular structures and sequences Methods Enzymol 374, 492–509 Delano, W L (2002) The PyMOL Molecular Graphics System DeLano Scientific, Palo Alto, CA; http:// www.pymol.org  ... experiments to elucidate the structure of the ready and unready states of the [NiFe] hydroge- The Structure of the A vinosum [NiFe] Hydrogenase 65 66 67 68 69 70 71 72 73 74 75 nase of Desulfovibrio... to an oxygen atom in the case of the D vulgaris MF hydrogenase in the Ni-A state and in the case of the D gigas hydrogenase in the Ni-SU state.16,17 These modifications of the bridging cysteine... of the crystal, the enzyme was probably damaged by the X-ray beam during data collection‡ This led to the observation of a mixture of two conformers in the crystal The ‡ The oxidation state of