Biochemical characterization of the HydE and HydG iron-only hydrogenase maturation enzymes from Thermatoga maritima

Fe-only hydrogenases contain a di-iron active site complex, in which the two Fe atoms have carbon monoxide and cyanide ligands and are linked together by a putative di(thiomethyl)amine molecule. We have cloned, purified and characterized the HydE and HydG proteins, thought to be involved in the biosynthesis of this peculiar metal site, from the thermophilic organism Thermotoga maritima. The HydE protein anaerobically reconstituted with iron and sulfide binds two [4Fe-4S] clusters, as characterized by UV and EPR spectroscopy. The HydG protein binds one [4Fe-4S] cluster, and probably an additional one. Both enzymes are able to reductively cleave S-adenosylmethionine (SAM) when reduced by dithionite, confirming that they are Radical-SAM enzymes.     

Fe-only hydrogenases: structure,function and evolution

Hydrogenases are enzymes capable of catalyzing the oxidation of molecular hydrogen or its production from protons and electrons according to the reversible reaction: H(2)<==>2H(+)+2e(-). Most of these enzymes fall into to major classes: NiFe and Fe-only hydrogenases. Extensive spectroscopic, electrochemical and structural studies have shed appreciable light on the catalytic mechanism of hydrogenases. Although evolutionarily unrelated, NiFe and Fe-hydrogenases share a common, unusual feature: an active site low-spin Fe center with CO and CN coordination. We have recently focused our attention on Fe-hydrogenases because from structural studies by us and others, it appears to be a simpler system than the NiFe counterpart. Thus the primary hydrogen binding site has been identified and plausible, electron, proton and hydrogen pathways from and to the buried active site may be proposed from the structural data. The extensive genome sequencing effort currently under way has shown that eukaryotic organisms contain putatively gene coding sequences that display significant homology to Fe-hydrogenases. Here, we summarize the available evidence concerning the mechanism of these enzymes and carry out a structural comparison between Fe-hydrogenases and related proteins of unknown metal content from yeast, plant, worm, insect and mammals.     

Reduction of unusual iron-sulfur clusters in the H2-sensing regulatory Ni-Fe hydrogenase from Ralstonia eutropha H16

The regulatory Ni-Fe hydrogenase (RH) from Ralstonia eutropha functions as a hydrogen sensor. The RH consists of the large subunit HoxC housing the Ni-Fe active site and the small subunit HoxB containing Fe-S clusters. The heterolytic cleavage of H(2) at the Ni-Fe active site leads to the EPR-detectable Ni-C state of the protein. For the first time, the simultaneous but EPR-invisible reduction of Fe-S clusters during Ni-C state formation was demonstrated by changes in the UV-visible absorption spectrum as well as by shifts of the iron K-edge from x-ray absorption spectroscopy in the wild-type double dimeric RH(WT) [HoxBC](2) and in a monodimeric derivative designated RH(stop) lacking the C-terminal 55 amino acids of HoxB. According to the analysis of iron EXAFS spectra, the Fe-S clusters of HoxB pronouncedly differ from the three Fe-S clusters in the small subunits of crystallized standard Ni-Fe hydrogenases. Each HoxBC unit of RH(WT) seems to harbor two [2Fe-2S] clusters in addition to a 4Fe species, which may be a [4Fe-3S-3O] cluster. The additional 4Fe-cluster was absent in RH(stop). Reduction of Fe-S clusters in the hydrogen sensor RH may be a first step in the signal transduction chain, which involves complex formation between [HoxBC](2) and tetrameric HoxJ protein, leading to the expression of the energy converting Ni-Fe hydrogenases in R. eutropha.     

Immunological relationship among hydrogenases.

We examined the immunological cross-reactions of 11 different hydrogenase antigens with 9 different hydrogenase antibodies. Included were antibodies and antigens of both subunits of the hydrogenases of Bradyrhizobium japonicum and Thiocapsa roseopersicina. The results showed a strong relationship among the Ni-Fe dimeric hydrogenases. The two subunits of Ni-Fe dimeric hydrogenases appeared immunologically distinct: specific interactions occurred only when antibodies to the 60- and 30-kilodalton subunits reacted with the 60- and 30-kilodalton-subunit antigens. The interspecies cross-reactions suggested that at least one conserved protein region exists among the large subunits of these enzymes, whereas the small subunits are less conserved. Antibodies to the Fe-only bidirectional hydrogenase of Clostridium pasteurianum reacted with the Desulfovibrio vulgaris bidirectional hydrogenase. Surprisingly, antibodies to the clostridial uptake hydrogenase did not react with any of the Fe-only bidirectional hydrogenases but did react with several of the Ni-Fe dimeric hydrogenases. The two hydrogenases from C. pasteurianum were found to be quite different immunologically. The possible relationship of these findings to the structure and catalytic functions of hydrogenase are discussed.     

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.     

Bias from H2 cleavage to production and coordination changes at the Ni-Fe active site in the NAD+-reducing hydrogenase from Ralstonia eutropha

The soluble NAD+-reducing Ni-Fe hydrogenase (SH) from Ralstonia eutropha H16 is remarkable because it cleaves hydrogen in the presence of dioxygen at a unique Ni-Fe active site (Burgdorf et al. (2005) J. Am. Chem. Soc. 127, 576). By X-ray absorption (XAS), FTIR, and EPR spectroscopy, we monitored the structure and oxidation state of its metal centers during H2 turnover. In NADH-activated protein, a change occurred from the (CN)O2Ni(II)(mu-S)2Fe(II)(CN)3(CO) site dominant in the wild-type SH to a standard-like S2Ni(II)(mu-S)2Fe(II)(CN)2(CO) site as the prevailing species in a specific mutant protein, HoxH-H16L. The wild-type SH primarily was active in H2 cleavage. The nonstandard reaction mechanism does not involve stable EPR-detectable trivalent Ni oxidation states, namely, the Ni-A,B,C states as observed in standard hydrogenases. In the HoxH-mutant protein H16L, H2 oxidation was impaired, but H2 production occurred via a stable Ni-C state (Ni(III)-H(-)-Fe(II)), suggesting a reaction sequence similar to that of standard hydrogenases. It is proposed that reductive activation by NADH of both wild-type and H16L proteins causes the release of an oxygen species from Ni and is initiated by electron transfer from a [2Fe-2S] cluster in the HoxU subunit that at first becomes reduced by electrons from NADH. Electrons derived from H2 cleavage, on the other hand, are transferred to NAD+ via a different pathway involving a [4Fe-4S] cluster in HoxY, which is reducible only in wild-type SH but not in the H16L variant.     

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.     

Structural differences between the ready and unready oxidized states of [NiFe] hydrogenases

[NiFe] hydrogenases catalyze the reversible heterolytic cleavage of molecular hydrogen. Several oxidized, inactive states of these enzymes are known that are distinguishable by their very different activation properties. So far, the structural basis for this difference has not been understood because of lack of relevant crystallographic data. Here, we present the crystal structure of the ready Ni-B state of Desulfovibrio fructosovorans [NiFe] hydrogenase and show it to have a putative -hydroxo Ni–Fe bridging ligand at the active site. On the other hand, a new, improved refinement procedure of the X-ray diffraction data obtained for putative unready Ni-A/Ni-SU states resulted in a more elongated electron density for the bridging ligand, suggesting that it is a diatomic species. The slow activation of the Ni-A state, compared with the rapid activation of the Ni-B state, is therefore proposed to result from the different chemical nature of the ligands in the two oxidized species. Our results along with very recent electrochemical studies suggest that the diatomic ligand could be hydro–peroxide.An erratum to this article can be found at     

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 A resolution.

BACKGROUND: The active site of [NiFe] hydrogenase, a heterodimeric protein, is suggested to be a binuclear Ni-Fe complex having three diatomic ligands to the Fe atom and three bridging ligands between the Fe and Ni atoms in the oxidized form of the enzyme. Two of the bridging ligands are thiolate sidechains of cysteinyl residues of the large subunit, but the third bridging ligand was assigned as a non-protein monatomic sulfur species in Desulfovibrio vulgaris Miyazaki F hydrogenase. RESULTS: The X-ray crystal structure of the reduced form of D. vulgaris Miyazaki F [NiFe] hydrogenase has been solved at 1.4 A resolution and refined to a crystallographic R factor of 21.8%. The overall structure is very similar to that of the oxidized form, with the exception that the third monatomic bridge observed at the Ni-Fe site in the oxidized enzyme is absent, leaving this site unoccupied in the reduced form. CONCLUSIONS: The unusual ligand structure found in the oxidized form of D. vulgaris Miyazaki F [NiFe] hydrogenase was confirmed in the reduced form of the enzyme, with the exception that the electron density assigned to the monatomic sulfur bridge had almost disappeared. On the basis of this finding, as well as the observation that H2S is liberated from the oxidized enzyme under an atmosphere of H2 in the presence of its electron carrier, it was postulated that the monatomic sulfur bridge must be removed for the enzyme to be activated. A possible mechanism for the catalytic action of the hydrogenase is proposed.     

Liberation of hydrogen sulfide during the catalytic action of Desulfovibrio hydrogenase under the atmosphere of hydrogen

The active site of [NiFe] hydrogenase from Desulfovibrio species is composed of a binuclear Ni-Fe complex bearing three diatomic nonprotein ligands to Fe and three bridges between the two metals, two of which are thiolate side chains of the protein moiety. The third bridging atom in the enzyme isolated from D. vulgaris Miyazaki F was suggested to be sulfur species, but was suggested to be oxygen species in D. gigas enzyme. When the hydrogenase from D. vulgaris Miyazaki F was incubated under the atmosphere of H2, H2S was liberated from the enzyme only in the presence of its electron carrier, cytochrome c3 or methylviologen. The amount of H2S liberation was little in the absence of electron carrier or essentially null when the enzyme was incubated under N2. The amount of H2S liberated was about 37% of the hydrogenase contained in the reaction vial in molar basis. These observations are in agreement with the recent observation that the third bridging site at the Ni-Fe active site is vacant in the reduced form of the enzyme revealed by X-ray crystallography.     

A model system for [NiFe] hydrogenase maturation studies: Purification of an active site-containing hydrogenase large subunit without small subunit

The large subunit HoxC of the H2-sensing [NiFe] hydrogenase from Ralstonia eutropha was purified without its small subunit. Two forms of HoxC were identified. Both forms contained iron but only substoichiometric amounts of nickel. One form was a homodimer of HoxC whereas the second also contained the Ni-Fe site maturation proteins HypC and HypB. Despite the presence of the Ni-Fe active site in some of the proteins, both forms, which lack the Fe-S clusters normally present in hydrogenases, cannot activate hydrogen. The incomplete insertion of nickel into the Ni-Fe site provides direct evidence that Fe precedes Ni in the course of metal center assembly.     

Insights into [FeFe]-hydrogenase structure, mechanism, and maturation

Hydrogenases are metalloenzymes that are key to energy metabolism in a variety of microbial communities. Divided into three classes based on their metal content, the [Fe]-, [FeFe]-, and [NiFe]-hydrogenases are evolutionarily unrelated but share similar nonprotein ligand assemblies at their active site metal centers that are not observed elsewhere in biology. These nonprotein ligands are critical in tuning enzyme reactivity, and their synthesis and incorporation into the active site clusters require a number of specific maturation enzymes. The wealth of structural information on different classes and different states of hydrogenase enzymes, biosynthetic intermediates, and maturation enzymes has contributed significantly to understanding the biochemistry of hydrogen metabolism. This review highlights the unique structural features of hydrogenases and emphasizes the recent biochemical and structural work that has created a clearer picture of the [FeFe]-hydrogenase maturation pathway.     

Spectroscopic and kinetic characterization of active site mutants of Desulfovibrio fructosovorans Ni-Fe hydrogenase

Site-directed mutagenesis of amino acid residues proximate to the active site of the Ni-Fe hydrogenase of Desulfovibrio fructosovorans has been done. The different mutants have been analyzed by FTIR spectroscopy and compared with wild type enzyme. The changes observed in the spectra confirm that hydrogen bonds between the CN(-) ligands of the active site's Fe atom and certain neighbor amino acid residues stabilize the active center within the protein matrix. However, kinetic analysis of the mutants indicates that none of the replaced residues have an important role in the catalytic mechanism of the hydrogenase.     

A novel FeS cluster in Fe-only hydrogenases

Many microorganisms can use molecular hydrogen as a source of electrons or generate it by reducing protons. These reactions are catalysed by metalloenzymes of two types: NiFe and Fe-only hydrogenases. Here, we review recent structural results concerning the latter, putting special emphasis on the characteristics of the active site.     

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.     

Maturation of [NiFe]-hydrogenases in Escherichia coli

Hydrogenases catalyze the reversible oxidation of dihydrogen. Catalysis occurs at bimetallic active sites that contain either nickel and iron or only iron and the nature of these active sites forms the basis of categorizing the enzymes into three classes, the [NiFe]-hydrogenases, the [FeFe]-hydrogenases and the iron sulfur cluster-free [Fe]-hydrogenases. The [NiFe]-hydrogenases and the [FeFe]-hydrogenases are unrelated at the amino acid sequence level but the active sites share the unusual feature of having diatomic ligands associated with the Fe atoms in the these enzymes. Combined structural and spectroscopic studies of [NiFe]-hydrogenases identified these diatomic ligands as CN- and CO groups. Major advances in our understanding of the biosynthesis of these ligands have been achieved primarily through the study of the membrane-associated [NiFe]-hydrogenases of Escherichia coli. A complex biosynthetic machinery is involved in synthesis and attachment of these ligands to the iron atom, insertion of the Fe(CN)2CO group into the apo-hydrogenase, introduction of the nickel atom into the pre-formed active site and ensuring that the holoenzyme is correctly folded prior to delivery to the membrane. Although much remains to be uncovered regarding each of the individual biochemical steps on the pathway to synthesis of a fully functional enzyme, our understanding of the initial steps in CN- synthesis have revealed that it is generated from carbamoyl phosphate. What is becoming increasingly clear is that the metabolic origins of the carbonyl group may be different.     

The three classes of hydrogenases from sulfate-reducing bacteria of the genus Desulfovibrio

Three types of hydrogenases have been isolated from the sulfate-reducing bacteria of the genus Desulfovibrio. They differ in their subunit and metal compositions, physico-chemical characteristics, amino acid sequences, immunological reactivities, gene structures and their catalytic properties. Broadly, the hydrogenases can be considered as 'iron only' hydrogenases and nickel-containing hydrogenases. The iron-sulfur-containing hydrogenase ([Fe] hydrogenase) contains two ferredoxin-type (4Fe-4S) clusters and an atypical iron-sulfur center believed to be involved in the activation of H2. The [Fe] hydrogenase has the highest specific activity in the evolution and consumption of hydrogen and in the proton-deuterium exchange reaction and this enzyme is the most sensitive to CO and NO2-. It is not present in all species of Desulfovibrio. The nickel-(iron-sulfur)-containing hydrogenases [( NiFe] hydrogenases) possess two (4Fe-4S) centers and one (3Fe-xS) cluster in addition to nickel and have been found in all species of Desulfovibrio so far investigated. The redox active nickel is ligated by at least two cysteinyl thiolate residues and the [NiFe] hydrogenases are particularly resistant to inhibitors such as CO and NO2-. The genes encoding the large and small subunits of a periplasmic and a membrane-bound species of the [NiFe] hydrogenase have been cloned in Escherichia (E.) coli and sequenced. Their derived amino acid sequences exhibit a high degree of homology (70%); however, they show no obvious metal-binding sites or homology with the derived amino acid sequence of the [Fe] hydrogenase. The third class is represented by the nickel-(iron-sulfur)-selenium-containing hydrogenases [( NiFe-Se] hydrogenases) which contain nickel and selenium in equimolecular amounts plus (4Fe-4S) centers and are only found in some species of Desulfovibrio. The genes encoding the large and small subunits of the periplasmic hydrogenase from Desulfovibrio (D.) baculatus (DSM 1743) have been cloned in E. coli and sequenced. The derived amino acid sequence exhibits homology (40%) with the sequence of the [NiFe] hydrogenase and the carboxy-terminus of the gene for the large subunit contains a codon (TGA) for selenocysteine in a position homologous to a codon (TGC) for cysteine in the large subunit of the [NiFe] hydrogenase. EXAFS and EPR studies with the 77Se-enriched D. baculatus hydrogenase indicate that selenium is a ligand to nickel and suggest that the redox active nickel is ligated by at least two cysteinyl thiolate and one selenocysteine selenolate residues.(ABSTRACT TRUNCATED AT 400 WORDS)     

Interaction of potassium cyanide with the [Ni-4Fe-5S] active site cluster of CO dehydrogenase from Carboxydothermus hydrogenoformans

The Ni-Fe carbon monoxide (CO) dehydrogenase II (CODHII(Ch)) from the anaerobic CO-utilizing hydrogenogenic bacterium Carboxydothermus hydrogenoformans catalyzes the oxidation of CO, presumably at the Ni-(micro(2)S)-Fe1 subsite of the [Ni-4S-5S] cluster in the active site. The CO oxidation mechanism proposed on the basis of several CODHII(Ch) crystal structures involved the apical binding of CO at the nickel ion and the activation of water at the Fe1 ion of the cluster. To understand how CO interacts with the active site, we have studied the reactivity of the cluster with potassium cyanide and analyzed the resulting type of nickel coordination by x-ray absorption spectroscopy. Cyanide acts as a competitive inhibitor of reduced CODHII(Ch) with respect to the substrate CO and is therefore expected to mimic the substrate. It inhibits the enzyme reversibly, forming a nickel cyanide. In this reaction, one of the four square-planar sulfur ligands of nickel is replaced by the carbon atom of cyanide, suggesting removal of the micro(2)S from the Ni-(micro(2)S)-Fe1 subsite. Upon reactivation of the inhibited enzyme, cyanide is released, and the square-planar coordination of nickel by 4S ligands is recovered, which includes the reformation of the Ni-(micro(2)S)-Fe1 bridge. The results are summarized in a model of the CO oxidation mechanism at the [Ni-4Fe-5S] active site cluster of CODHII(Ch) from C. hydrogenoformans.     

Spectroelectrochemical characterization of the [NiFe] hydrogenase of Desulfovibrio vulgaris Miyazaki F

The active site in the [NiFe] hydrogenase of Desulfovibrio vulgaris Miyazaki F has been investigated by Fourier transform infrared (FTIR) spectroscopy. Analysis of the spectra allowed the three diatomic inorganic ligands to Fe in this enzyme to be identified as one CO molecule and two CN(-) molecules. Furthermore, pH-dependent redox titrations were performed to determine the midpoint potentials as well as the pK value of the respective reactions and revealed that each single-electron redox transition is accompanied by a single-proton transfer step. The comparison of these spectra with those published for other [NiFe] hydrogenases shows that the electronic structure of the active sites of these enzymes and their redox processes are essentially the same. Nevertheless, differences with respect to the frequency of the CO band and the pH dependence of the Ni-R states have been observed. Finally, the frequency shifts of the bands in the IR spectra were interpreted with respect to the electronic configuration of the redox intermediates in the catalytic cycle.     

Enzymes of hydrogen metabolism in Pyrococcus furiosus.

The genome of Pyrococcus furiosus contains the putative mbhABCDEFGHIJKLMN operon for a 14-subunit transmembrane complex associated with a Ni-Fe hydrogenase. Ten ORFs (mbhA-I and mbhM) encode hydrophobic, membrane-spanning subunits. Four ORFs (mbhJKL and mbhN) encode putative soluble proteins. Two of these correspond to the canonical small and large subunit of Ni-Fe hydrogenase, however, the small subunit can coordinate only a single iron-sulfur cluster, corresponding to the proximal [4Fe-4S] cubane. The structural genes for the small and the large subunits, mbhJ and mbhL, are separated in the genome by a third ORF, mbhK, encoding a protein of unknown function without Fe/S binding. The fourth ORF, mbhN, encodes a 2[4Fe-4S] protein. With P. furiosus soluble [4Fe-4S] ferredoxin as the electron donor the membranes produce H2, and this activity is retained in an extracted core complex of the mbh operon when solubilized and partially purified under mild conditions. The properties of this membrane-bound hydrogenase are unique. It is rather resistant to inhibition by carbon monoxide. It also exhibits an extremely high ratio of H2 evolution to H2 uptake activity compared with other hydrogenases. The activity is sensitive to inhibition by dicyclohexylcarbodiimide, an inhibitor of NADH dehydrogenase (complex I). EPR of the reduced core complex is characteristic for interacting iron-sulfur clusters with Em approximately -0.33 V. The genome contains a second putative operon, mbxABCDFGHH'MJKLN, for a multisubunit transmembrane complex with strong homology to the mbh operon, however, with a highly unusual putative binding motif for the Ni-Fe-cluster in the large hydrogenase subunit. Kinetic studies of membrane-bound hydrogenase, soluble hydrogenase and sulfide dehydrogenase activities allow the formulation of a comprehensive working hypothesis of H2 metabolism in P. furiosus in terms of three pools of reducing equivalents (ferredoxin, NADPH, H2) connected by devices for transduction, transfer, recovery and safety-valving of energy.