Probing the origin of the metabolic precursor of the CO ligand in the catalytic center of [NiFe] hydrogenase

The O(2)-tolerant [NiFe] hydrogenases of Ralstonia eutropha are capable of H(2) conversion in the presence of ambient O(2). Oxygen represents not only a challenge for catalysis but also for the complex assembling process of the [NiFe] active site. Apart from nickel and iron, the catalytic center contains unusual diatomic ligands, namely two cyanides (CN(-)) and one carbon monoxide (CO), which are coordinated to the iron. One of the open questions of the maturation process concerns the origin and biosynthesis of the CO group. Isotope labeling in combination with infrared spectroscopy revealed that externally supplied gaseous (13)CO serves as precursor of the carbonyl group of the regulatory [NiFe] hydrogenase in R. eutropha. Corresponding (13)CO titration experiments showed that a concentration 130-fold higher than ambient CO (0.1 ppmv) caused a 50% labeling of the carbonyl ligand in the [NiFe] hydrogenase, leading to the conclusion that the carbonyl ligand originates from an intracellular metabolite. A novel setup allowed us to the study effects of CO depletion on maturation in vivo. Upon induction of CO depletion by addition of the CO scavenger PdCl(2), cells cultivated on H(2), CO(2), and O(2) showed severe growth retardation at low cell concentrations, which was on the basis of partially arrested hydrogenase maturation, leading to reduced hydrogenase activity. This suggests gaseous CO as a metabolic precursor under these conditions. The addition of PdCl(2) to cells cultivated heterotrophically on organic substrates had no effect on hydrogenase maturation. These results indicate at least two different pathways for biosynthesis of the CO ligand of [NiFe] hydrogenase.     

The CO and CN(-) ligands to the active site Fe in [NiFe]-hydrogenase of Escherichia coli have different metabolic origins

The Fe atom in the bimetallic active site of [NiFe]-hydrogenases has one CO and two cyanide ligands. To determine their metabolic origin, [NiFe]-hydrogenase-2 was isolated from Escherichia coli grown in the presence of L-[ureido-(13)C]citrulline, purified and analyzed by infrared spectroscopy. The spectra indicate incorporation of (13)C only into the cyanide ligands and not into the CO, showing that cyanide and CO have different metabolic origins. After growth of E. coli in the presence of (13)CO only the CO ligand was labelled with (13)C. Labelling did not result from an exchange of the intrinsic CO ligand with the exogenous CO.     

A Universal Scaffold for Synthesis of the Fe(CN)2(CO) Moiety of [NiFe] Hydrogenase

Hydrogen-cycling [NiFe] hydrogenases harbor a dinuclear catalytic center composed of nickel and iron ions, which are coordinated by four cysteine residues. Three unusual diatomic ligands in the form of two cyanides (CN⁻) and one carbon monoxide (CO) are bound to the iron and apparently account for the complexity of the cofactor assembly process, which involves the function of at least six auxiliary proteins, designated HypA, -B, -C, -D, -E, and -F. It has been demonstrated previously that the HypC, -D, -E, and -F proteins participate in cyanide synthesis and transfer. Here, we show by infrared spectroscopic analysis that the purified HypCD complexes from Ralstonia eutropha and Escherichia coli carry in addition to both cyanides the CO ligand. We present experimental evidence that in vivo the attachment of the CN⁻ ligands is a prerequisite for subsequent CO binding. With the aid of genetic engineering and subsequent mutant analysis, the functional role of conserved cysteine residues in HypD from R. eutropha was investigated. Our results demonstrate that the HypCD complex serves as a scaffold for the assembly of the Fe(CN)₂(CO) entity of [NiFe] hydrogenase.     

The biosynthetic routes for carbon monoxide and cyanide in the Ni-Fe active site of hydrogenases are different

The incorporation of carbon into the carbon monoxide and cyanide ligands of [NiFe]-hydrogenases has been investigated by using (13)C labelling in infrared studies of the Allochromatium vinosum enzyme and by (14)C labelling experiments with overproduced Hyp proteins from Escherichia coli. The results suggest that the biosynthetic routes of the carbon monoxide and cyanide ligands in [NiFe]-hydrogenases are different.     

The presence of a SO molecule in [NiFe] hydrogenase from Desulfovibrio vulgaris Miyazaki as detected by mass spectrometry

The active site of [NiFe] hydrogenase is a binuclear metal complex composed of Fe and Ni atoms and is called the Ni–Fe site, where the Fe atom is known to be coordinated to three diatomic ligands. Two mass spectrometric techniques, pyrolysis-MS (pyrolysis-mass spectrometry) and TOF-SIMS (time-of-flight secondary ion mass spectrometry), were applied to several proteins, including native and denatured forms of [NiFe] hydrogenase from Desulfovibrio vulgaris Miyazaki F, [Fe₄S₄]₂-ferredoxin from Clostridium pasteurianum, [Fe₂S₂]-ferredoxin from Spirulina platensis, and porcine pepsin. Pyrolysis-MS revealed that only native hydrogenase liberated SO/SO₂ (ions of m/z 48 and 64 at an equilibrium ratio of SO and SO₂) at relatively low temperatures before the covalent bonds in the polypeptide moiety started to decompose. TOF-SIMS indicated that native Miyazaki hydrogenase released SO/SO₂ (m/z 47.97 and 63.96) as secondary ions when irradiated with a high-energy Ga⁺ beam. Denatured hydrogenase, clostridial ferredoxin, and pepsin did not release SO as a secondary ion. The FT-IR spectrum of the enzyme suggested the presence of CO and CN. These lines of evidence suggest that the three diatomic ligands coordinated to the Fe atom at the Ni–Fe site in Miyazaki hydrogenase are SO, CO, and CN. The role of the SO ligand in helping to cleave H₂ molecules at the active site and stabilizing the Fe atom in the diamagnetic Fe(II) state in the redox cycle of this enzyme is discussed.     

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.     

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)     

Characterization and in vitro interaction study of a [NiFe] hydrogenase large subunit from the hyperthermophilic archaeon Thermococcus kodakarensis KOD1

The large subunit of the [NiFe] hydrogenases harbors a NiFe(CN)(2)(CO) cluster. Maturation proteins HypA, B, C, D, E, and F are required for the NiFe cluster biosynthesis. While the maturation machinery has been hitherto studied intensively, little is known about interactions between the Hyp proteins and the large subunit of the [NiFe] hydrogenase. In this study, we have purified and characterized the cytosolic [NiFe] hydrogenase large subunit HyhL from Thermococcus kodakarensis (Tk-HyhL). Tk-HyhL exists in equilibrium between monomeric and dimeric forms. In vitro interaction analyses showed that Tk-HyhL monomer forms a tight complex with Tk-HypA and weakly interacts with Tk-HypC. The expected ternary complex formation was not detected. These observations reflect a diversity in the mechanism of Ni insertion in [NiFe] hydrogenase maturation depending on the organism.     

The Crystal Structure of the [NiFe] Hydrogenase from the Photosynthetic Bacterium Allochromatium vinosum: Characterization of the Oxidized Enzyme (Ni-A State)

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 (> 90%). 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.     

Carbamoylphosphate requirement for synthesis of the active center of [NiFe]-hydrogenases

The iron of the binuclear active center of [NiFe]-hydrogenases carries two CN and one CO ligands which are thought to confer to the metal a low oxidation and/or spin state essential for activity. Based on the observation that one of the seven auxiliary proteins required for the synthesis and insertion of the [NiFe] cluster contains a sequence motif characteristic of O-carbamoyl-transferases it was discovered that carbamoyl phosphate is essential for formation of active [NiFe]-hydrogenases in vivo and is specifically required for metal center synthesis suggesting that it is the source of the CO and CN ligands. A chemical path for conversion of a carbamoyl group into cyano and carbonyl moieties is postulated     

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.     

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.     

Cloning and sequencing of a [NiFe] hydrogenase operon from Desulfovibrio vulgaris Miyazaki F

A hydrogenase operon was cloned from chromosomal DNA isolated from Desulfovibrio vulgaris Miyazaki F with the use of probes derived from the genes encoding [NiFe] hydrogenase from Desulfovibrio vulgaris Hildenborough. The nucleic acid sequence of the cloned DNA indicates this hydrogenase to be a two-subunit enzyme: the gene for the small subunit (267 residues; molecular mass = 28763 Da) precedes that for the large subunit (566 residues; molecular mass = 62495 Da), as in other [NiFe] and [NiFeSe] hydrogenase operons. The amino acid sequences of the small and large subunits of the Miyazaki hydrogenase share 80% homology with those of the [NiFe] hydrogenase from Desulfovibrio gigas. Fourteen cysteine residues, ten in the small and four in the large subunit, which are thought to co-ordinate the iron-sulphur clusters and the active-site nickel in [NiFe] hydrogenases, are found to be conserved in the Miyazaki hydrogenase. The subunit molecular masses and amino acid composition derived from the gene sequence are very similar to the data reported for the periplasmic, membrane-bound hydrogenase isolated by Yagi and coworkers, suggesting that this hydrogenase belongs to the general class of [NiFe] hydrogenases, despite its low nickel content and apparently anomalous spectral properties.     

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.     

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.     

Maturation of Rhizobium leguminosarum Hydrogenase in the Presence of Oxygen Requires the Interaction of the Chaperone HypC and the Scaffolding Protein HupK

[NiFe] hydrogenases are key enzymes for the energy and redox metabolisms of different microorganisms. Synthesis of these metalloenzymes involves a complex series of biochemical reactions catalyzed by a plethora of accessory proteins, many of them required to synthesize and insert the unique NiFe(CN)₂CO cofactor. HypC is an accessory protein conserved in all [NiFe] hydrogenase systems and involved in the synthesis and transfer of the Fe(CN)₂CO cofactor precursor. Hydrogenase accessory proteins from bacteria-synthesizing hydrogenase in the presence of oxygen include HupK, a scaffolding protein with a moderate sequence similarity to the hydrogenase large subunit and proposed to participate as an intermediate chaperone in the synthesis of the NiFe cofactor. The endosymbiotic bacterium Rhizobium leguminosarum contains a single hydrogenase system that can be expressed under two different physiological conditions: free-living microaerobic cells (∼12 μm O₂) and bacteroids from legume nodules (∼10–100 nm O₂). We have used bioinformatic tools to model HupK structure and interaction of this protein with HypC. Site-directed mutagenesis at positions predicted as critical by the structural analysis have allowed the identification of HupK and HypC residues relevant for the maturation of hydrogenase. Mutant proteins altered in some of these residues show a different phenotype depending on the physiological condition tested. Modeling of HypC also predicts the existence of a stable HypC dimer whose presence was also demonstrated by immunoblot analysis. This study widens our understanding on the mechanisms for metalloenzyme biosynthesis in the presence of oxygen.     

The exchange activities of [Fe] hydrogenase (iron–sulfur-cluster-free hydrogenase) from methanogenic archaea in comparison with the exchange activities of [FeFe] and [NiFe] hydrogenases

[Fe] hydrogenase (iron–sulfur-cluster-free hydrogenase) catalyzes the reversible reduction of methenyltetrahydromethanopterin (methenyl-H₄MPT⁺) with H₂ to methylene-H₄MPT, a reaction involved in methanogenesis from H₂ and CO₂ in many methanogenic archaea. The enzyme harbors an iron-containing cofactor, in which a low-spin iron is complexed by a pyridone, two CO and a cysteine sulfur. [Fe] hydrogenase is thus similar to [NiFe] and [FeFe] hydrogenases, in which a low-spin iron carbonyl complex, albeit in a dinuclear metal center, is also involved in H₂ activation. Like the [NiFe] and [FeFe] hydrogenases, [Fe] hydrogenase catalyzes an active exchange of H₂ with protons of water; however, this activity is dependent on the presence of the hydride-accepting methenyl-H₄MPT⁺. In its absence the exchange activity is only 0.01% of that in its presence. The residual activity has been attributed to the presence of traces of methenyl-H₄MPT⁺ in the enzyme preparations, but it could also reflect a weak binding of H₂ to the iron in the absence of methenyl-H₄MPT⁺. To test this we reinvestigated the exchange activity with [Fe] hydrogenase reconstituted from apoprotein heterologously produced in Escherichia coli and highly purified iron-containing cofactor and found that in the absence of added methenyl-H₄MPT⁺ the exchange activity was below the detection limit of the tritium method employed (0.1 nmol min⁻¹ mg⁻¹). The finding reiterates that for H₂ activation by [Fe] hydrogenase the presence of the hydride-accepting methenyl-H₄MPT⁺ is essentially required. This differentiates [Fe] hydrogenase from [FeFe] and [NiFe] hydrogenases, which actively catalyze H₂/H₂O exchange in the absence of exogenous electron acceptors.     

Cyanide inactivation of hydrogenase from Azotobacter vinelandii.

The effects of cyanide on membrane-associated and purified hydrogenase from Azotobacter vinelandii were characterized. Inactivation of hydrogenase by cyanide was dependent on the activity (oxidation) state of the enzyme. Active (reduced) hydrogenase showed no inactivation when treated with cyanide over several hours. Treatment of reversibly inactive (oxidized) states of both membrane-associated and purified hydrogenase, however, resulted in a time-dependent, irreversible loss of hydrogenase activity. The rate of cyanide inactivation was dependent on the cyanide concentration and was an apparent first-order process for purified enzyme (bimolecular rate constant, 23.1 M-1 min-1 for CN-). The rate of inactivation decreased with decreasing pH. [14C]cyanide remained associated with cyanide-inactivated hydrogenase after gel filtration chromatography, with a stoichiometry of 1.7 mol of cyanide bound per mol of inactive enzyme. The presence of saturating concentrations of CO had no effect on the rate or extent of cyanide inactivation of hydrogenases. The results indicate that cyanide can cause a time-dependent, irreversible inactivation of hydrogenase in the oxidized, activatable state but has no effect when hydrogenase is in the reduced, active state.     

Involvement of hyp Gene Products in Maturation of the H2-Sensing [NiFe] Hydrogenase of Ralstonia eutropha

The biosynthesis of [NiFe] hydrogenases is a complex process that requires the function of the Hyp proteins HypA, HypB, HypC, HypD, HypE, HypF, and HypX for assembly of the H(2)-activating [NiFe] site. In this study we examined the maturation of the regulatory hydrogenase (RH) of Ralstonia eutropha. The RH is a H(2)-sensing [NiFe] hydrogenase and is required as a constituent of a signal transduction chain for the expression of two energy-linked [NiFe] hydrogenases. Here we demonstrate that the RH regulatory activity was barely affected by mutations in hypA, hypB, hypC, and hypX and was not substantially diminished in hypD- and hypE-deficient strains. The lack of HypF, however, resulted in a 90% decrease of the RH regulatory activity. Fourier transform infrared spectroscopy and the incorporation of (63)Ni into the RH from overproducing cells revealed that the assembly of the [NiFe] active site is dependent on all Hyp functions, with the exception of HypX. We conclude that the entire Hyp apparatus (HypA, HypB, HypC, HypD, HypE, and HypF) is involved in an efficient incorporation of the [NiFe] center into the RH.