Characterization of a HoxEFUYH type of [NiFe] hydrogenase from <Emphasis Type="Italic">Allochromatium vinosum</Emphasis> and some EPR and IR properties of the hydrogenase module

A soluble hydrogenase from Allochromatium vinosum was purified. It consisted of a large (M _r = 52 kDa) and a small (M _r = 23 kDa) subunit. The genes encoding for both subunits were identified. They belong to an open reading frame where they are preceded by three more genes. A DNA fragment containing all five genes was cloned and sequenced. The deduced amino acid sequences of the products characterized the complex as a member of the HoxEFUYH type of [NiFe] hydrogenases. Detailed sequence analyses revealed binding sites for eight Fe–S clusters, three [2Fe–2S] clusters and five [4Fe–4S] clusters, six of which are also present in homologous subunits of [FeFe] hydrogenases and NADH:ubiquione oxidoreductases (complex I). This makes the HoxEFUYH type of hydrogenases the one that is evolutionary closest to complex I. The relative positions of six of the potential Fe–S clusters are predicted on the basis of the X-ray structures of the Clostridium pasteurianum [FeFe] hydrogenase I and the hydrophilic domain of complex I from Thermus thermophilus. Although the HoxF subunit contains binding sites for flavin mononucleotide and NAD(H), cell-free extracts of A. vinosum did not catalyse a H₂-dependent reduction of NAD⁺. Only the hydrogenase module (HoxYH) could be purified. Its electron paramagnetic resonance (EPR) and IR spectral properties showed the presence of a Ni–Fe active site and a [4Fe–4S] cluster. Its activity was sensitive to carbon monoxide. No EPR signals from a light-sensitive Ni_a–C* state could be observed. This study presents the first IR spectroscopic data on the HoxYH module of a HoxEFUYH type of [NiFe] hydrogenase.     

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.     

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 role of Hox hydrogenase in the H2 metabolism of Thiocapsa roseopersicina

The purple sulfur phototrophic bacterium Thiocapsa roseopersicina BBS synthesizes at least three NiFe hydrogenases (Hox, Hup, Hyn). We characterized the physiological H₂ consumption/evolution reactions in mutants having deletions of the structural genes of two hydrogenases in various combinations. This made possible the separation of the functionally distinct roles of the three hydrogenases. Data showed that Hox hydrogenase (unlike the Hup and Hyn hydrogenases) catalyzed the dark fermentative H₂ evolution and the light-dependent H₂ production in the presence of thiosulfate. Both Hox⁺ and Hup⁺ mutants demonstrated light-dependent H₂ uptake stimulated by CO₂ but only the Hup⁺ mutant was able to mediate O₂-dependent H₂ consumption in the dark. The ability of the Hox⁺ mutant to evolve or consume hydrogen was found to depend on a number of interplaying factors including both growth and reaction conditions (availability of glucose, sulfur compounds, CO₂, H₂, light). The study of the redox properties of Hox hydrogenase supported the reversibility of its action. Based on the results a scheme is suggested to describe the role of Hox hydrogenase in light-dependent and dark hydrogen metabolism in T. roseopersicina BBS.     

The role of the bidirectional hydrogenase in cyanobacteria

Cyanobacteria have tremendous potential to produce clean, renewable fuel in the form of hydrogen gas derived from solar energy and water. Of the two cyanobacterial enzymes capable of evolving hydrogen gas (nitrogenase and the bidirectional hydrogenase), the hox-encoded bidirectional Ni-Fe hydrogenase has a high theoretical potential. The physiological role of this hydrogenase is a highly debated topic and is poorly understood relative to that of the nitrogenase. Here the structure, assembly, and expression of this enzyme, as well as its probable roles in metabolism, are discussed and analyzed to gain perspective on its physiological role. It is concluded that the bidirectional hydrogenase in cyanobacteria primarily functions as a redox regulator for maintaining a proper oxidation/reduction state in the cell. Recommendations for future research to test this hypothesis are discussed.     

HoxE--a subunit specific for the pentameric bidirectional hydrogenase complex (HoxEFUYH) of cyanobacteria

NAD(P)(+)-reducing hydrogenases have been described to be composed of a diaphorase (HoxFU) and a hydrogenase (HoxYH) moiety. This study presents for the first time experimental evidence that in cyanobacteria, a fifth subunit, HoxE, is part of this bidirectional hydrogenase. HoxE exhibits sequence identities to NuoE of respiratory complex I of Escherichia coli. The subunit composition of the cyanobacterial bidirectional hydrogenase has been investigated. The oxygen labile enzyme complex was purified to close homogeneity under anaerobic conditions from Synechocystis sp. PCC 6803 and Synechococcus sp. PCC 6301. The 647-fold and 1290-fold enriched purified enzyme has a specific activity of 46 micromol H(2) evolved (min mg protein)(-1) and 15 micromol H(2) evolved (min mg protein)(-1), respectively. H(2)-evolution of the purified enzyme of S. sp. PCC 6803 is highest at 60 degrees C and pH 6.3. Immunoblot experiments, using a polyclonal anti-HoxE antibody, demonstrate that HoxE co-purifies with the hydrogenase activity in S. sp. PCC 6301. SDS-PAGE gels of the purified enzymes revealed six proteins, which were partially sequenced and identified, besides one nonhydrogenase component, as HoxF, HoxU, HoxY, HoxH and, remarkably, HoxE. The molecular weight of the native protein (375 kDa) indicates a dimeric assembly of the enzyme complex, Hox(EFUYH)(2).     

A Second Soluble Hox-Type NiFe Enzyme Completes the Hydrogenase Set in Thiocapsa roseopersicina BBS

Three functional NiFe hydrogenases were previously characterized in Thiocapsa roseopersicina BBS: two of them are attached to the periplasmic membrane (HynSL and HupSL), and one is localized in the cytoplasm (HoxEFUYH). The ongoing genome sequencing project revealed the presence of genes coding for another soluble Hox-type hydrogenase enzyme (hox2FUYH). Hox2 is a heterotetrameric enzyme; no indication for an additional subunit was found. Detailed comparative in vivo and in vitro activity and expression analyses of HoxEFUYH (Hox1) and the newly discovered Hox2 enzyme were performed. Functional differences between the two soluble NiFe hydrogenases were disclosed. Hox1 seems to be connected to both sulfur metabolism and dark/photofermentative processes. The bidirectional Hox2 hydrogenase was shown to be metabolically active under specific conditions: it can evolve hydrogen in the presence of glucose at low sodium thiosulfate concentration. However, under nitrogen-fixing conditions, it can oxidize H₂ but less than the other hydrogenases in the cell.Hydrogenases are metalloenzymes involved in microbial hydrogen metabolism. A great variety of them have been identified and studied in various microorganisms and grouped on the basis of their metal content as NiFe, FeFe, and iron-sulfur cluster free hydrogenases (10, 42, 43). The basic protein structure of NiFe hydrogenases is heterodimeric, while FeFe hydrogenases are mostly composed of a single amino acid chain with multiple iron-sulfur clusters (28, 43, 44). Well-defined maturation proteins assist for the assembly and activation of hydrogenase enzymes; NiFe hydrogenases require a more complex accessory machinery than FeFe enzymes (2, 3, 24).Thiocapsa roseopersicina BBS is a photosynthetic purple sulfur bacterium belonging to the Chromatiaceae family (4). It prefers to utilize reduced sulfur compounds for anaerobic photochemolithoautotrophic growth, but simple organic substrates such as glucose or acetate can be also used as extra carbon, energy, and electron sources. It can be cultivated under aerobic (nonphotosynthetic) conditions in the presence of organic compounds. In the absence of other nitrogen sources, it is able to fix molecular nitrogen; this process is accompanied by H₂ production. T. roseopersicina was earlier shown to possess at least three NiFe hydrogenases varying in their in vivo functions, localizations, and compositions. Hyn and Hup hydrogenases are attached to the membrane facing the periplasmic side (6, 18, 30). Hyn is a bidirectional enzyme with extraordinary stability (17). Recent study has demonstrated that the HynSL subunits are physiologically connected to cellular redox processes via the Isp1 and Isp2 proteins, which play an essential role in electron transfer (27). The second membrane-associated enzyme, Hup, is involved in H₂ oxidation and shows homology to uptake hydrogenases, which recycle H₂ produced by the nitrogenase enzyme complex or present in the environment. Next to the hydrogenase small and large subunits (HupSL), a b-type cytochrome, HupC, was demonstrated to be part of the in vivo active enzyme as a transmitter of electrons to the quinone pool (27). In several bacteria, e.g., Rhodobacter capsulatus (7) and Ralstonia eutropha (15, 20), the expression of the hydrogenase(s) was shown to be regulated by the hydrogen level in the environment. The genes encoding the hydrogen-sensing system also exist in T. roseopersicina (hupUV, hupT, and hupR), but the hupTUV genes proved to be silent in the wild-type strain—only hupR is expressed—which is why expression of hupSL genes is constitutive (16).A Hox-type soluble hydrogenase was also identified in T. roseopersicina (31); it is a representative of the bidirectional heteromultimeric cytoplasmic NiFe hydrogenases (37, 39). Enzymes belonging to this group are basically composed of two moieties: hydrogenase (HoxYH) and diaphorase (HoxFU) heterodimers. Additional subunits were identified in few cases. In R. eutropha H16, two HoxI proteins completing the Hox complex were suggested to provide a binding domain for NADPH (5). HoxE has been identified as the fifth subunit of heteropentameric NAD⁺-reducing Hox hydrogenases in several cyanobacteria, Allochromatium vinosum and T. roseopersicina (21, 31, 37). In-frame deletion of the hoxE gene ceased both the H₂-producing and -oxidizing activities of Hox in vivo, but these were not affected in vitro. Consequently, an electron transfer role of the HoxE subunit was suggested (31, 32).The possibility of the presence of further hydrogenases in T. roseopersicina was noted few years ago (31). In the hynSL hupSL hoxH triple-mutant strain ({"type":"entrez-nucleotide","attrs":{"text":"GB112131","term_id":"336599610","term_text":"GB112131"}}GB112131), a small in vivo and in vitro hydrogenase activity could be measured under photomixotrophic growth conditions (both CO₂ and organic compounds are used for growth) at the late growth phase. This residual activity could not be detected in the hypF mutant strain (M539). Since HypF protein has an essential role in the maturation process of all NiFe hydrogenases (9), these results suggested the presence of a previously unknown hydrogenase. Here we describe the identification and characterization of the second Hox-type hydrogenase, emphasizing the functional similarities and differences between the two soluble enzymes of this bacterium. In order to distinguish between the two Hox-type enzymes unequivocally, the HoxEFUYH complex will be renamed Hox1 and the newly described Hox2FUYH enzyme is called Hox2.     

Computational analyses,molecular dynamics,and mutagenesis studies of unprocessed form of [NiFe] hydrogenase reveal the role of disorder for efficient enzyme maturation

Biological production and oxidation of hydrogen is mediated by hydrogenases, key enzymes for these energy-relevant reactions. Synthesis of [NiFe] hydrogenases involves a complex series of biochemical reactions to assemble protein subunits and metallic cofactors required for enzyme function. A final step in this biosynthetic pathway is the processing of a C-terminal tail (CTT) from its large subunit, thus allowing proper insertion of nickel in the unique NiFe(CN)₂CO cofactor present in these enzymes. In silico modelling and Molecular Dynamics (MD) analyses of processed vs. unprocessed forms of Rhizobium leguminosarum bv. viciae (Rlv) hydrogenase large subunit HupL showed that its CTT (residues 582-596) is an intrinsically disordered region (IDR) that likely provides the required flexibility to the protein for the final steps of proteolytic maturation. Prediction of pKa values of ionizable side chains in both forms of the enzyme's large subunit also revealed that the presence of the CTT strongly modify the protonation state of some key residues around the active site. Furthermore, MD simulations and mutant analyses revealed that two glutamate residues (E27 in the N-terminal region and E589 inside the CTT) likely contribute to the process of nickel incorporation into the enzyme. Computational analysis also revealed structural details on the interaction of Rlv hydrogenase LSU with the endoprotease HupD responsible for the removal of CTT.     

The Bidirectional NiFe-hydrogenase in Synechocystis sp. PCC 6803 Is Reduced by Flavodoxin and Ferredoxin and Is Essential under Mixotrophic,Nitrate-limiting Conditions

Cyanobacteria are able to use solar energy for the production of hydrogen. It is generally accepted that cyanobacterial NiFe-hydrogenases are reduced by NAD(P)H. This is in conflict with thermodynamic considerations, as the midpoint potentials of NAD(P)H do not suffice to support the measured hydrogen production under physiological conditions. We show that flavodoxin and ferredoxin directly reduce the bidirectional NiFe-hydrogenase of Synechocystis sp. PCC 6803 in vitro. A merodiploid ferredoxin-NADP reductase mutant produced correspondingly more photohydrogen. We furthermore found that the hydrogenase receives its electrons via pyruvate:flavodoxin/ferredoxin oxidoreductase (PFOR)-flavodoxin/ferredoxin under fermentative conditions, enabling the cells to gain ATP. These results strongly support that the bidirectional NiFe-hydrogenases in cyanobacteria function as electron sinks for low potential electrons from photosystem I and as a redox balancing device under fermentative conditions. However, the selective advantage of this enzyme is not known. No strong phenotype of mutants lacking the hydrogenase has been found. Because bidirectional hydrogenases are widespread in aquatic nutrient-rich environments that are capable of triggering phytoplankton blooms, we mimicked those conditions by growing cells in the presence of increased amounts of dissolved organic carbon and dissolved organic nitrogen. Under these conditions the hydrogenase was found to be essential. As these conditions close the two most important sinks for reduced flavodoxin/ferredoxin (CO₂-fixation and nitrate reduction), this discovery further substantiates the connection between flavodoxin/ferredoxin and the NiFe-hydrogenase.     

Genetic analysis of the parB+ locus of plasmid R1

Summary Megaplasmid DNA from mutants has been analysed physically for deletions and insertions in order to identify the location of hydrogenase (hox) genes in Alcaligenes eutrophus. Four classes of mutants have been examined: mutants defective in genes coding for soluble NAD-dependent hydrogenase (hoxS), mutants impaired in the membrane-bound hydrogenase (hoxP), mutants altered in the regulation of hox gene expression (hoxC) and mutants with lesions in the carbon dioxide fixing enzyme system (cfx). A comparison of the restriction patterns with EcoRI, BamHI and HindIII, complementation studies with cloned DNA and DNA - DNA hybridization experiments showed that genes coding for hox and cfx are clustered on a 100-kb region of the 450-kb plasmid pHG1.     

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.     

Role of HoxE subunit in Synechocystis PCC6803 hydrogenase

Cyanobacterial NAD(P)(+)-reducing reversible hydrogenases comprise five subunits. Four of them (HoxF, HoxU, HoxY, and HoxH) are also found in the well-described related enzyme from Ralstonia eutropha. The fifth one (HoxE) is not encoded in the R. eutropha genome, but shares homology with the N-terminal part of R. eutropha HoxF. However, in cyanobacteria, HoxE contains a 2Fe-2S cluster-binding motif that is not found in the related R. eutropha sequence. In order to obtain some insights into the role of HoxE in cyanobacteria, we deleted this subunit in Synechocystis PCC6803. Three types of interaction of the cyanobacterial hydrogenase with pyridine nucleotides were tested: (a) reductive activation of the NiFe site, for which NADPH was found to be more efficient than NADH; (b) H(2) production, for which NADH appeared to be a more efficient electron donor than NADPH; and (c) H(2) oxidation, for which NAD(+) was a much better electron acceptor than NADP(+). Upon hoxE deletion, the Synechocystis hydrogenase active site remained functional with artificial electron donors or acceptors, but the enzyme became unable to catalyze H(2) production or uptake with NADH/NAD(+). However, activation of the electron transfer-independent H/D exchange reaction by NADPH was still observed in the absence of HoxE, whereas activation of this reaction by NADH was lost. These data suggest different mechanisms for diaphorase-mediated electron donation and catalytic site activation in cyanobacterial hydrogenase.     

Diversity and transcription of proteases involved in the maturation of hydrogenases in Nostoc punctiforme ATCC 29133 and Nostoc sp. strain PCC 7120

Background  

The last step in the maturation process of the large subunit of [NiFe]-hydrogenases is a proteolytic cleavage of the C-terminal by a hydrogenase specific protease. Contrary to other accessory proteins these hydrogenase proteases are believed to be specific whereby one type of hydrogenases specific protease only cleaves one type of hydrogenase. In cyanobacteria this is achieved by the gene product of either hupW or hoxW, specific for the uptake or the bidirectional hydrogenase respectively. The filamentous cyanobacteria Nostoc punctiforme ATCC 29133 and Nostoc sp strain PCC 7120 may contain a single uptake hydrogenase or both an uptake and a bidirectional hydrogenase respectively.     

The effect of sulfur compounds on H<Subscript>2</Subscript> evolution/consumption reactions,mediated by various hydrogenases,in the purple sulfur bacterium, <Emphasis Type="Italic">Thiocapsa roseopersicina</Emphasis>

The influence of reduced sulfur compounds (including stored S⁰) on H₂ evolution/consumption reactions in the purple sulfur bacterium, Thiocapsa roseopersicina BBS, was studied using mutants containing only one of the three known [NiFe] hydrogenase enzymes: Hox, Hup or Hyn. The observed effects depended on the kind of hydrogenase involved. The mutant harbouring Hox hydrogenase was able to use S₂O₃²⁻, SO₃²⁻, S²⁻ and S⁰ as electron donors for light-dependent H₂ production. Dark H₂ evolution from organic substrates via Hox hydrogenase was inhibited by S⁰. Under light conditions, endogenous H₂ uptake by Hox or Hup hydrogenases was suppressed by S compounds. СО₂-dependent H₂ uptake by Hox hydrogenase in the light required the additional presence of S compounds, unlike the Hup-mediated process. Dark H₂ consumption via Hyn hydrogenase was connected to utilization of S⁰ as an electron acceptor and resulted in the accumulation of H₂S. In wild type BBS, with high levels of stored S⁰, dark H₂ production from organic substrates was significantly lower, but H₂S accumulation significantly higher, than in the mutant GB1121(Hox⁺). There is a possibility that H₂ produced via Hox hydrogenase is consumed by Hyn hydrogenase to reduce S⁰.     

Localization of hydrogenase in Desulfovibrio gigas cells

The localization of hydrogenase protein in Desulfovibrio gigas cells grown either in lactate-sulfate or hydrogen-sulfate media, has been investigated by subcellular fractionation with immunoblotting and by electron microscopic immunocytochemistry. Subcellular fractionation experiments suggest that no integral membrane-bound hydrogenase is present in D. gigas. About 40% of the hydrogenase activity could be extracted by treatment of D. gigas cells with Tris-EDTA buffer. The rest of the soluble hydrogenase activity (50%) was found in the soluble fraction which was obtained after disruption of Tris-EDTA extracted cells and high speed centrifugation. Both soluble hydrogenase fractions purified to homogeneity showed identical molecular properties including the N-terminal aminoacid sequences of their large and small subunits. Polyacrylamide gel electrophoresis of the proteins of the subcellular fractions revealed a single band of hydrogenase activity exhibiting the same mobility as purified D. gigas hydrogenase. Western blotting carried out on these subcellular fractions revealed crossreactivity with the antibodies raised against (NiFe) hydrogenase. The lack of crossreactivity with antibodies against (FE) or (NiFeSe) hydrogenases, indicated that only (NiFe) type hydrogenase is present in D. gigas.Immunocytolocalization in ultrathin frozen sections of D. gigas cells grown either in lactate-sulfate, pyruvate-sulfate or hydrogen-sulfate media showed only a (NiFe) hydrogenase located in the periplasmic space. The bioenergetics of D. gigas are discussed in the light of these findings.     

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.     

H2-Uptake and evolution in the unicellular cyanobacterium Chroococcidiopsis thermalis CALU 758

The unicellular cyanobacterium Chroococcidiopsis thermalis CALU 758 growing photoautotrophically synthesised a hydrogenase which catalysed an in vivo H₂ uptake in the oxyhydrogen reaction at a significant rate and showed only low level of in vitro MV-dependent H₂ evolution. The in vitro hydrogenase activity was not induced under microaerobic or nitrate-limiting conditions. Some correlation observed between the two activities indicated that the same enzyme may be involved in both H₂ uptake and H₂ evolution. Heterologous Southern hybridisations, using cyanobacterial hup and hox DNA fragments as probes, showed the presence of sequences similar to hox (encoding for a bidirectional hydrogenase) in C. thermalis CALU 758 with no indication for the presence of any sequences corresponding to an uptake hydrogenase. Further molecular experiments, using specific primers directed against different conserved regions of the large subunit (hoxH) of the bidirectional hydrogenase confirmed the presence of corresponding sequences in C. thermalis CALU 758. Low-stringency Southern hybridisations detected only one copy of hoxH within the genome of C. thermalis CALU 758.     

Structure-function relationships among the nickel-containing hydrogenases.

The enzymology of the heterodimeric (NiFe) and (NiFeSe) hydrogenases, the monomeric nickel-containing hydrogenases plus the multimeric F420-(NiFe) and NAD(+)-(NiFe) hydrogenases are summarized and discussed in terms of subunit localization of the redox-active nickel and non-heme iron clusters. It is proposed that nickel is ligated solely by amino acid residues of the large subunit and that the non-heme iron clusters are ligated by other cysteine-rich polypeptides encoded in the hydrogenase operons which are not necessarily homologous in either structure or function. Comparison of the hydrogenase operons or putative operons and their hydrogenase genes indicate that the arrangement, number and types of genes in these operons are not conserved among the various types of hydrogenases except for the gene encoding the large subunit. Thus, the presence of the gene for the large subunit is the sole feature common to all known nickel-containing hydrogenases and unites these hydrogenases into a large but diverse gene family. Although the different genes for the large subunits may possess only nominal general derived amino acid homology, all large subunit genes sequenced to date have the sequence R-X-C-X-X-C fully conserved in the amino terminal region of the polypeptide chain and the sequence of D-P-C-X-X-C fully conserved in the carboxyl terminal region. It is proposed that these conserved motifs of amino acids provide the ligands required for the binding of the redox-active nickel. The existing EXAFS (Extended X-ray Absorption Fine Structure) information is summarized and discussed in terms of the numbers and types of ligands to the nickel and the various redox species of nickel defined by EPR spectroscopy. New information concerning the ligands to nickel is presented based on site-directed mutagenesis of the gene encoding the large subunit of the (NiFe) hydrogenase-1 of Escherichia coli. Based on considerations of the biochemical, molecular and biophysical information, ligand environments of the nickel in different redox states of the (NiFe) hydrogenase are proposed.