O2 reactions at the six-iron active site (H-cluster) in [FeFe]-hydrogenase

Irreversible inhibition by molecular oxygen (O(2)) complicates the use of [FeFe]-hydrogenases (HydA) for biotechnological hydrogen (H(2)) production. Modification by O(2) of the active site six-iron complex denoted as the H-cluster ([4Fe4S]-2Fe(H)) of HydA1 from the green alga Chlamydomonas reinhardtii was characterized by x-ray absorption spectroscopy at the iron K-edge. In a time-resolved approach, HydA1 protein samples were prepared after increasing O(2) exposure periods at 0 °C. A kinetic analysis of changes in their x-ray absorption near edge structure and extended X-ray absorption fine structure spectra revealed three phases of O(2) reactions. The first phase (τ(1) ≤ 4 s) is characterized by the formation of an increased number of Fe-O,C bonds, elongation of the Fe-Fe distance in the binuclear unit (2Fe(H)), and oxidation of one iron ion. The second phase (τ(2) ≈ 15 s) causes a ～50% decrease of the number of ～2.7-? Fe-Fe distances in the [4Fe4S] subcluster and the oxidation of one more iron ion. The final phase (τ(3) ≤ 1000 s) leads to the disappearance of most Fe-Fe and Fe-S interactions and further iron oxidation. These results favor a reaction sequence, which involves 1) oxygenation at 2Fe(H(+)) leading to the formation of a reactive oxygen species-like superoxide (O(2)(-)), followed by 2) H-cluster inactivation and destabilization due to ROS attack on the [4Fe4S] cluster to convert it into an apparent [3Fe4S](+) unit, leading to 3) complete O(2)-induced degradation of the remainders of the H-cluster. This mechanism suggests that blocking of ROS diffusion paths and/or altering the redox potential of the [4Fe4S] cubane by genetic engineering may yield improved O(2) tolerance in [FeFe]-hydrogenase.     

On understanding proton transfer to the biocatalytic [Fe-Fe](H) sub-cluster in [Fe-Fe]H(2)ases: QM/MM MD simulations

Proton transfer to the [Fe-Fe](H) sub-cluster in the Desulfovibrio desulfuricans (DdH) and Clostridium pasteurianum (CpI) [Fe-Fe] hydrogenases was investigated by a combination of first principles and empirical molecular dynamics simulations. Pathways that can be inferred from the X-ray crystal structures of DdH and CpI, i.e., (Glu159→Ser198→Glu156→water460→Cys178→DTMA([Fe-Fe](H)) and (Glu282→Ser319→Glu279→water612→Cys299), respectively, were considered. Proton transfer from Cys178 to DTMA in the [Fe-Fe](H) sub-cluster in DdH was readily observed in our results, specifically when [Fe-Fe](H) was in the reduced state ([Fe(I)-Fe(I)]) or in the mixed valence state for the protonated distal iron Fe(d) ([Fe(I)-Fe(II)-H(-)](H)). A concerted mechanism is proposed, where proton transfer in DdH from Glu159 to Glu156 via Ser198 and Glu156 to Cys178 via water460 readily occurred, as well as from Glu282 to Glu279 via Ser319 and Glu279 to Cys299 via water612 in CpI. The theoretical prediction of the proton transfer characteristics is consistent with the assumed biocatalytic mechanism of the [Fe-Fe] hydrogenases in which the proton binds at Fe(d), providing confirmation that has not been explored so far. The computational results were qualitatively validated by the agreement with experimental hydrogen production activity data for mutated CpI enzymes, relative to the wild-type protein. Finally, the insight provided by the simulations, combined, in part, with experimental validation, are important for establishing an approach in future exploration of proton transfer to the active site in this class of enzymes, and possibly also for biomimetic analogs.     

On the novel H2-activating iron-sulfur center of the "Fe-only" hydrogenases

The two hydrogenases (I and II) of the anaerobic N2-fixing bacterium Clostridium pasteurianum (Cp) and the hydrogenases of the anaerobes Megasphaera elsdenii (Me) and Desulfovibrio vulgaris (strain Hildenborough, Dv), contain iron-sulfur clusters but not nickel. They are the most active hydrogenases known. All four enzymes in their reduced states give rise to EPR signals typical of [4Fe-4S]1+ clusters but exhibit novel EPR signals in their oxidized states. For example, Cp hydrogenase I exhibits a sharp rhombic EPR signal when oxidized under mild conditions but the enzyme is inactivated by over-oxidation and then exhibits an axial EPR signal. A similar axial signal is observed from mildly oxidized hydrogenase I after treatment with CO. EPR, M?ssbauer and ENDOR spectroscopy indicate that the EPR signals from the oxidized enzyme and its CO derivative arise from a novel spin-coupled Fe center. Low temperature magnetic circular dichroism (MCD) studies reveal that an EPR-silent Fe-S cluster with S greater than 1/2 is also present in oxidized hydrogenase I. From a study of all spectroscopic properties of Cp, Dv, and Me hydrogenases, it is concluded that the H2-activating site of all four is a novel Fe-S cluster with S greater than 0 and integer, which in the oxidized state is exchange-coupled to a S = 1/2 species. The data are most consistent with the S = 1/2 species being a low spin Fe(III) center. The H2-activating site is susceptible to oxidative rearrangements to yield both active and inactive states of the enzyme. We discuss the possible implications of these finding to methods of enzyme oxidation and purification procedures currently used for hydrogenases.     

Oxygen tolerance of the H2-sensing [NiFe] hydrogenase from Ralstonia eutropha H16 is based on limited access of oxygen to the active site

Hydrogenases, abundant proteins in the microbial world, catalyze cleavage of H2 into protons and electrons or the evolution of H2 by proton reduction. Hydrogen metabolism predominantly occurs in anoxic environments mediated by hydrogenases, which are sensitive to inhibition by oxygen. Those microorganisms, which thrive in oxic habitats, contain hydrogenases that operate in the presence of oxygen. We have selected the H2-sensing regulatory [NiFe] hydrogenase of Ralstonia eutropha H16 to investigate the molecular background of its oxygen tolerance. Evidence is presented that the shape and size of the intramolecular hydrophobic cavities leading to the [NiFe] active site of the regulatory hydrogenase are crucial for oxygen insensitivity. Expansion of the putative gas channel by site-directed mutagenesis yielded mutant derivatives that are sensitive to inhibition by oxygen, presumably because the active site has become accessible for oxygen. The mutant proteins revealed characteristics typical of standard [NiFe] hydrogenases as described for Desulfovibrio gigas and Allochromatium vinosum. The data offer a new strategy how to engineer oxygen-tolerant hydrogenases for biotechnological application.     

The [FeFe]-hydrogenase maturase HydF from Clostridium acetobutylicum contains a CO and CN ligated iron cofactor

Biosynthesis of the [FeFe] hydrogenases active site (H-cluster) requires three maturation factors whose respective roles are not understood yet. The clostridial maturation enzymes (CaHydE, CaHydF and CaHydG) were homologously overexpressed in their native host Clostridium acetobutylicum. CaHydF was able to activate Chlamydomonas reinhardtii [FeFe] hydrogenase apoprotein (CrHydA1_apo) to almost 100% compared to the native specific hydrogen evolution activity. Based on electron paramagnetic resonance spectroscopy and Fourier-transform infrared spectroscopy data the existence of a [4Fe4S] cluster and a CO and CN⁻ ligand coordinated di-iron cluster is suggested. This study contains the first experimental evidence that the bi-nuclear part of the H-cluster is assembled in HydF.     

The structure and mechanism of iron-hydrogenases

Hydrogenases devoid of nickel and containing only Fe-S clusters have been found so far only in some strictly anaerobic bacteria. Four Fe-hydrogenases have been characterized: from Megasphaera elsdenii, Desulfovibrio vulgaris (strain Hildenborough), and two from Clostridium pasteurianum. All contain two or more [4Fe-4S]1+,2+ or F clusters and a unique type of Fe-S center termed the H cluster. The H cluster appears to be remarkably similar in all the hydrogenases, and is proposed as the site of H2 oxidation and H2 production. The F clusters serve to transfer electrons between the H cluster and the external electron carrier. In all of the hydrogenases the H cluster is comprised of at least three Fe atoms, and possibly six. In the oxidized state it contains two types of magnetically distinct Fe atoms, has an S = 1/2 spin state, and exhibits a novel rhombic EPR signal. The reduced cluster is diamagnetic (S = 0). The oxidized H cluster appears to undergo a conformation change upon reduction with H2 with an increase in Fe-Fe distances of about 0.5 A. Studies using resonance Raman, magnetic circular dichroism and electron spin echo spectroscopies suggest that the H cluster has significant non-sulfur coordination. The H cluster has two binding sites for CO, at least one of which can also bind O2. Binding to one site changes the EPR properties of the cluster and gives a photosensitive adduct, but does not affect catalytic activity. Binding to the other site, which only becomes exposed during the catalytic cycle, leads to loss of catalytic activity. Mechanisms of H2 activation and electron transfer are proposed to explain the effects of CO binding and the ability of one of the hydrogenases to preferentially catalyze H2 oxidation.     

氢化酶结构研究进展

氢化酶是微生物代谢产氢过程中的关键酶,也是目前生物制氢领域的研究热点。本文综述了厌氧发酵产氢微生物中氢化酶的分类及特点,以及[Ni—Fe]、[Fe—Fe]和[Fe—Scluster—free]三种氢化酶晶体结构和活性中心结构;阐述了多种微生物来源的氢化酶结构的研究进展,对几种典型氢化酶的结构及活性中心进行了对比分析,并根据当前研究热点,对氢化酶的研究方向进行了展望。本文阐述的内容信息量丰富且具有一定的实用性,对于氢化酶相关领域研究具有重要的意义。     

Interactions of metal ions with two quinolone antimicrobial agents (cinoxacin and ciprofloxacin). Spectroscopic and X-ray structural characterization. Antibacterial studies

Several novel metal-quinolone compounds have been synthesized and characterized by analytical, spectroscopic and X-ray diffraction methods. The crystal structure of the four compounds, Na(2)[(Cd(Cx)3)(Cd(Cx)3(H2O))].12H2O, [Co(Cp)2(H2O)2].9H2O, [Zn(Cp)2(H2O)2].8H2O and [Cd(HCp)2(Cl)2].4H2O, is presented and discussed: HCx=1-ethyl-1,4-dihydro-4-oxo(1,3)-dioxolo(4,5-g)cinnoline-3-carboxylic acid and HCp=1-cyclopropyl-6-fluoro-1,4-dihydro-4-oxo-7-(1-piperazinyl)-3-quinoline carboxylic acid. In all these compounds the quinolone acts as a bidentate chelate ligand that binds through one carboxylate oxygen atom and the exocyclic carbonyl oxygen atom. Complexes of ciprofloxacin were screened for their activity against several bacteria, showing activity similar to that of the ligand. In addition, the number of bacteria killed after 3 h of incubation with the ligand, [Co(Cp)2(H2O)2].9H2O, Ni(Cp)2.10H2O and Cu(Cp)2.6H2O, was determined against S. aureus ATCC25923. There is a direct relationship between the growth rate and the lethal rate. Against growing bacteria, the ligand is the most bactericidal and Cu(Cp)2.6H2O is the less bactericidal. On the contrary, against non-dividing bacteria, the complexes were more bactericidal than the ligand, with Cu(Cp)(2).6H(2)O the most bactericidal compound.     

Cell-free H-cluster synthesis and [FeFe] hydrogenase activation: all five CO and CN⁻ ligands derive from tyrosine

[FeFe] hydrogenases are promising catalysts for producing hydrogen as a sustainable fuel and chemical feedstock, and they also serve as paradigms for biomimetic hydrogen-evolving compounds. Hydrogen formation is catalyzed by the H-cluster, a unique iron-based cofactor requiring three carbon monoxide (CO) and two cyanide (CN⁻) ligands as well as a dithiolate bridge. Three accessory proteins (HydE, HydF, and HydG) are presumably responsible for assembling and installing the H-cluster, yet their precise roles and the biosynthetic pathway have yet to be fully defined. In this report, we describe effective cell-free methods for investigating H-cluster synthesis and [FeFe] hydrogenase activation. Combining isotopic labeling with FTIR spectroscopy, we conclusively show that each of the CO and CN⁻ ligands derive respectively from the carboxylate and amino substituents of tyrosine. Such in vitro systems with reconstituted pathways comprise a versatile approach for studying biosynthetic mechanisms, and this work marks a significant step towards an understanding of both the protein-protein interactions and complex reactions required for H-cluster assembly and hydrogenase maturation.     

Light induced H2 evolution in a hydrogenase-TiO2 particle system by direct electron transfer or via rhodium complexes

Three different hydrogenases (isolated from Clostridium pasteurianum, Desulfovibrio desulfuricans strain Norway 4 and D. baculatus 9974) added to a suspension of TiO2 (anatase) powder are able to catalyze H2 evolution under band gap illumination of the semiconducting particles, and in the presence of EDTA or methanol as electron donor. This H2 production can be obtained by the direct electron transfer from the conduction band of the TiO2 particles to the active site of the enzyme at pHs higher than 7. This mediator-independent charge transfer is more efficient with C. pasteurianum and D. baculatus 9974 hydrogenases, and in the presence of methanol. Rhodium tris- and bis-bipyridyl complexes can act efficiently as electron carriers from the supporting particles to the adsorbed enzyme molecules in cases where the direct transfer is inefficient.     

New Insights into [FeFe] Hydrogenase Activation and Maturase Function

[FeFe] hydrogenases catalyze H₂ production using the H-cluster, an iron-sulfur cofactor that contains carbon monoxide (CO), cyanide (CN^–), and a dithiolate bridging ligand. The HydE, HydF, and HydG maturases assist in assembling the H-cluster and maturing hydrogenases into their catalytically active form. Characterization of these maturases and in vitro hydrogenase activation methods have helped elucidate steps in the H-cluster biosynthetic pathway such as the HydG-catalyzed generation of the CO and CN^– ligands from free tyrosine. We have refined our cell-free approach for H-cluster synthesis and hydrogenase maturation by using separately expressed and purified HydE, HydF, and HydG. In this report, we illustrate how substrates and protein constituents influence hydrogenase activation, and for the first time, we show that each maturase can function catalytically during the maturation process. With precise control over the biomolecular components, we also provide evidence for H-cluster synthesis in the absence of either HydE or HydF, and we further show that hydrogenase activation can occur without exogenous tyrosine. Given these findings, we suggest a new reaction sequence for the [FeFe] hydrogenase maturation pathway. In our model, HydG independently synthesizes an iron-based compound with CO and CN^– ligands that is a precursor to the H-cluster [2Fe]_H subunit, and which we have termed HydG-co. We further propose that HydF is a transferase that stabilizes HydG-co and also shuttles the complete [2Fe]_H subcluster to the hydrogenase, a translocation process that may be catalyzed by HydE. In summary, this report describes the first example of reconstructing the [FeFe] hydrogenase maturation pathway using purified maturases and subsequently utilizing this in vitro system to better understand the roles of HydE, HydF, and HydG.     

Oxygen-tolerant H2 oxidation by membrane-bound [NiFe] hydrogenases of ralstonia species. Coping with low level H2 in air

Knallgas bacteria such as certain Ralstonia spp. are able to obtain metabolic energy by oxidizing trace levels of H2 using O2 as the terminal electron acceptor. The [NiFe] hydrogenases produced by these organisms are unusual in their ability to oxidize H2 in the presence of O2, which is a potent inactivator of most hydrogenases through attack at the active site. To probe the origin of this unusual O2 tolerance, we conducted a study on the membrane-bound hydrogenase from Ralstonia eutropha H16 and that of the closely related organism Ralstonia metallidurans CH34, which was purified using a new heterologous overproduction system. Direct electrochemical methods were used to determine apparent inhibition constants for O2 inhibition of H2 oxidation (K I(app)O2) for each enzyme. These values were at least 2 orders of magnitude higher than those of "standard" [NiFe] hydrogenases. Amino acids close to the active site were exchanged in the membrane-bound hydrogenase of R. eutropha H16 for those from standard hydrogenases to probe the role of individual residues in conferring O2 sensitivity. Michaelis constants for H2 (K M H2) were determined, and for some mutants these were increased more than 20-fold relative to the wild type. Mutations resulting in membrane-bound hydrogenase enzymes with increased K M H2 or decreased K I(app)O2 values were associated with impaired lithoautotrophic growth in the presence of high O2 concentrations.     

Magnetic circular dichroism and electron paramagnetic resonance studies of hydrogenases I and II from Clostridium pasteurianum

The two iron-only hydrogenases (I and II) from Clostridium pasteurianum have been investigated by variable temperature magnetic circular dichroism (MCD) and electron paramagnetic resonance (EPR) spectroscopies. Samples were studied both reduced with dithionite under an atmosphere of H2 and after oxidation with thionine. The results are consistent with four and two [4Fe-4S]1+,2+ (F)-clusters in hydrogenases I and II, respectively. All four F-clusters are reduced and paramagnetic in reduced hydrogenase I, with up to one exhibiting an S = 3/2 ground state and the remainder having conventional S = 1/2 ground states. Both F-clusters have S = 1/2 ground states in reduced hydrogenase II; however, one appears to be only partially reduced under the conditions used for reduction. MCD studies of the oxidized enzymes show no temperature-dependent features in the visible region which can be attributed to the EPR-active S = 1/2 hydrogen-activating cluster, suggesting predominantly oxygen and nitrogen coordination for the iron atoms of this center. However, temperature-dependent MCD transitions arising from a hitherto undetected S greater than 1/2 Fe-S clusters are apparent in both oxidized hydrogenases. Detailed EPR studies of oxidized hydrogenase I revealed resonances from an S = 3/2 species, however, spin quantitation reveals this to be a trace component that is unlikely to be responsible for the observed low temperature MCD spectrum. The nature and origin of these S greater than 1/2 Fe-S clusters are discussed in light of the available spectroscopic data for these and other iron-only hydrogenases.     

Purification of hydrogenases by affinity chromatography on Procion Red-agarose.

The agarose-coupled triazine dye Procion Red HE-3B has been demonstrated to be applicable as an affinity gel for the purification of five diverse hydrogenases, namely the soluble, NAD-specific and the membrane-bound hydrogenase of Alcaligenes eutrophus, the membrane-bound hydrogenase of the N2-fixing Alcaligenes latus, the reversible H2-evolving and the unidirectional H2-oxidizing hydrogenase of Clostridium pasteurianum. In the case of the soluble hydrogenase of A. eutrophus, chromatography on Procion Red-agarose even permitted the separation of inactive from active enzyme, thus yielding a 2-3-fold increase in specific activity. For the homogeneous enzyme preparation obtained after two column steps (Procion Red-agarose, DEAE-Sephacel), a specific activity of 121 mumol of H2 oxidized/min per mg of protein was determined. Kinetic studies with free Procion Red provided evidence that the diverse hydrogenases are competitively inhibited by the dye, each with respect to the electron carrier (NAD, Methylene Blue, Methyl Viologen), indicating a specific interaction between Procion Red and the catalytic centres of the enzymes. For the highly purified preparations of the soluble and the membrane-bound hydrogenase of A. eutrophus, in 50 mM-potassium phosphate, pH 7.0, Ki values for Procion Red of 103 and 19 microM have been determined.     

Importance of the protein framework for catalytic activity of [FeFe]-hydrogenases

The active center (H-cluster) of [FeFe]-hydrogenases is embedded into a hydrophobic pocket within the protein. We analyzed several amino acids, located in the vicinity of this niche, by site-directed mutagenesis of the [FeFe]-hydrogenases from Clostridium pasteurianum (CpI) and Chlamydomonas reinhardtii (CrHydA1). These amino acids are highly conserved and predicted to be involved in H-cluster coordination. Characterization of two hydrogenase variants confirmed this hypothesis. The exchange of residues CrHydA1Met(415) and CrHydA1Lys(228) resulted in inactive proteins, which, according to EPR and FTIR analyses, contain no intact H-cluster. However, [FeFe]-hydrogenases in which CpIMet(353) (CrHydA1Met(223)) and CpICys(299) (CrHydA1Cys(169)) were exchanged to leucine and serine, respectively, showed a structurally intact H-cluster with catalytic activity either absent (CpIC299S) or strongly diminished (CpIM353L). In the case of CrHydA1C169S, the H-cluster was trapped in an inactive state exhibiting g values and vibrational frequencies that resembled the H(trans) state of DdH from Desulfovibrio desulfuricans. This cysteine residue, interacting with the bridge head nitrogen of the di(methyl)amine ligand, seems therefore to represent an essential contribution of the immediate protein environment to the reaction mechanism. Exchanging methionine CpIM(353) (CrHydA1M(223)) to leucine led to a strong decrease in turnover without affecting the K(m) value of the electron donor. We suggest that this methionine constitutes a "fine-tuning" element of hydrogenase activity.     

A radical solution for the biosynthesis of the H-cluster of hydrogenase

Fe-only or FeFe hydrogenases, as they have more recently been termed, possess a uniquely organometallic enzyme active site, termed the H-cluster, where the electronic properties of an iron-sulfur cluster are tuned with distinctly non-biological ligands, carbon monoxide and cyanide. Recently, it was discovered that radical S-adenosylmethionine enzymes were involved in active hydrogenase expression. In the current work, we present a mechanistic scheme for hydrogenase H-cluster biosynthesis in which both carbon monoxide and cyanide ligands can be derived from the decomposition of a glycine radical. The ideas presented have broader implications in the context of the prebiotic origin of amino acids.     

The interaction of polymeric viologens with hydrogenases from Desulfovibrio desulfuricans and Clostridium pasteurianum.

The interaction between hydrogenases from either Desulfovibrio desulfuricans or Clostridium pasteurianum and electron donors methyl viologen or polymeric viologens was examined. Extracts from each organism contained a single gel electophoretic band of active hydrogenase. The hydrogenase of D. desulfuricans was much more stable than that of Cl. pasteurianum. With methyl viologen apparent Km and Vm values were 0.5 mM and 0.62 mumole H2/min per milligram protein for the Cl. pasteurianum and 0.7 and 6.2 mumole H2/min per milligram protein, respectively, for the D. desulfuricans enzyme. The hydrogenases bound the polymeric viologens more tightly than methyl viologen, more so for the enzyme of D. desulfuricans than for Cl. pasteurianum. Maximal rate of hydrogen production was less with the polymeric than with methyl viologen. The results suggest that the D. desulfuricans enzyme in conjunction wiion than that from Cl. pasteurianum.     

Energy-Converting [NiFe] Hydrogenases from Archaea and Extremophiles: Ancestors of Complex I

[NiFe] hydrogenases are well-characterized enzymes that have a key function in the H2 metabolism of various microorganisms. In the recent years a subfamily of [NiFe] hydrogenases with unique properties has been identified. The members of this family form multisubunit membrane-bound enzyme complexes composed of at least four hydrophilic and two integral membrane proteins. These six conserved subunits, which built the core of these hydrogenases, have closely related counterparts in energy-conserving NADH:quinone oxidoreductases (complex I). However, the reaction catalyzed by these hydrogenases differs significantly from the reaction catalyzed by complex I. For some of these hydrogenases the physiological role is to catalyze the reduction of H+ with electrons derived from reduced ferredoxins or poly-ferredoxins. This exergonic reaction is coupled to energy conservation by means of electron-transport phosphorylation. Other members of this hydrogenase family mainly function to provide the cell with reduced ferredoxin with H2 as electron donor in a reaction driven by reverse electron transport. As complex I these hydrogenases function as ion pumps and have therefore been designated as energy-converting [NiFe] hydrogenases.     

Homologous and heterologous overexpression in Clostridium acetobutylicum and characterization of purified clostridial and algal Fe-only hydrogenases with high specific activities

Clostridium acetobutylicum ATCC 824 was selected for the homologous overexpression of its Fe-only hydrogenase and for the heterologous expressions of the Chlamydomonas reinhardtii and Scenedesmus obliquus HydA1 Fe-only hydrogenases. The three Strep tag II-tagged Fe-only hydrogenases were isolated with high specific activities by two-step column chromatography. The purified algal hydrogenases evolve hydrogen with rates of around 700 micromol H(2) min(-1) mg(-1), while HydA from C. acetobutylicum (HydA(Ca)) shows the highest activity (5,522 micromol H(2) min(-1) mg(-1)) in the direction of hydrogen uptake. Further, kinetic parameters and substrate specificity were reported. An electron paramagnetic resonance (EPR) analysis of the thionin-oxidized HydA(Ca) protein indicates a characteristic rhombic EPR signal that is typical for the oxidized H cluster of Fe-only hydrogenases.     

The crystal structure of a reduced [NiFeSe] hydrogenase provides an image of the activated catalytic center.

BACKGROUND: [NiFeSe] hydrogenases are metalloenzymes that catalyze the reaction H2<-->2H+ + 2e-. They are generally heterodimeric, contain three iron-sulfur clusters in their small subunit and a nickel-iron-containing active site in their large subunit that includes a selenocysteine (SeCys) ligand. RESULTS: We report here the X-ray structure at 2.15 A resolution of the periplasmic [NiFeSe] hydrogenase from Desulfomicrobium baculatum in its reduced, active form. A comparison of active sites of the oxidized, as-prepared, Desulfovibrio gigas and the reduced D. baculatum hydrogenases shows that in the reduced enzyme the nickel-iron distance is 0.4 A shorter than in the oxidized enzyme. In addition, the putative oxo ligand, detected in the as-prepared D. gigas enzyme, is absent from the D. baculatum hydrogenase. We also observe higher-than-average temperature factors for both the active site nickel-selenocysteine ligand and the neighboring Glu18 residue, suggesting that both these moieties are involved in proton transfer between the active site and the molecular surface. Other differences between [NiFeSe] and [NiFe] hydrogenases are the presence of a third [4Fe4S] cluster replacing the [3Fe4S] cluster found in the D. gigas enzyme, and a putative iron center that substitutes the magnesium ion that has already been described at the C terminus of the large subunit of two [NiFe] hydrogenases. CONCLUSIONS: The heterolytic cleavage of molecular hydrogen seems to be mediated by the nickel center and the selenocysteine residue. Beside modifying the catalytic properties of the enzyme, the selenium ligand might protect the nickel atom from oxidation. We conclude that the putative oxo ligand is a signature of inactive 'unready' [NiFe] hydrogenases.