Biosynthesis of the NiFe hydrogenase active site is a complex process involving the action of the Hyp proteins: HypA-HypF. Here we investigate the mechanism of NiFe site biosynthesis in Ralstonia eutropha by examining the interactions between HypC, HypD, HypE, and HypF1. Using an affinity purification procedure based on the Strep-tag II, we purified HypC and HypE from different genetic backgrounds as complexes with other hydrogenase-related proteins and characterized them using immunological analysis. Copurification of HypC and HoxH, the active site-containing subunit of the soluble hydrogenase in R. eutropha, from several different genetic backgrounds suggests that this complex forms early in the maturation process. With respect to the Hyp proteins, it is shown that HypE and HypF1 formed a stable complex both in vivo and in vitro. Furthermore, HypC and HypD functioned as a unit. Together, they were able to interact with HypE to form a range of complexes probably varying in stoichiometry. The HypC/HypD/HypE complexes did not involve HypF1 but appeared to be more stable when HypF1 was also present in the cells. We hypothesize that HypF1 is able to modify some component of the HypC/HypD/HypE complex. Since we have also seen that HypF1 and HypE form a complex, it is likely that HypF1 modifies HypE. On the basis of these results, we propose a complete catalytic cycle for HypE. First, it is modified by HypF1, and then it can form a complex with HypC/HypD. This activated HypE/HypC/HypD complex could then decompose by donating active site components to the immature hydrogenase and regenerate unmodified HypE.

Heterologous complementation studies using Alcaligenes eutrophus H16 as a recipient identified a hydrogenase-specific regulatory DNA region on megaplasmid pHG21-a of the related species Alcaligenes hydrogenophilus. Nucleotide sequence analysis revealed four open reading frames on the subcloned DNA, designated hoxA, hoxB, hoxC, and hoxJ. The product of hoxA is homologous to a transcriptional activator of the family of twocomponent regulatory systems present in a number of H 2 -oxidizing bacteria. hoxB and hoxC predict polypeptides of 34.5 and 52.5 kDa, respectively, which resemble the small and the large subunits of [NiFe] hydrogenases and correlate with putative regulatory proteins of Bradyrhizobium japonicum (HupU and HupV) and Rhodobacter capsulatus (HupU). hoxJ encodes a protein with typical consensus motifs of histidine protein kinases. Introduction of the complete set of genes on a broad-host-range plasmid into A. eutrophus H16 caused severe repression of soluble and membrane-bound hydrogenase (SH and MBH, respectively) synthesis in the absence of H 2 . This repression was released by truncation of hoxJ. H 2 -dependent hydrogenase gene transcription is a typical feature of A. hydrogenophilus and differs from the energy and carbon source-responding, H 2 -independent mode of control characteristic of A. eutrophus H16. Disruption of the A. hydrogenophilus hoxJ gene by an in-frame deletion on megaplasmid pHG21-a led to conversion of the regulatory phenotype: SH and MBH of the mutant were expressed in the absence of H 2 in response to the availability of the carbon and energy source. RNA dot blot analysis showed that HoxJ functions on the transcriptional level. These results suggest that the putative histidine protein kinase HoxJ is involved in sensing molecular hydrogen, possibly in conjunction with the hydrogenase-like polypeptides HoxB and HoxC.Alcaligenes hydrogenophilus is an aerobic, facultatively lithoautotrophic proteobacterium capable of obtaining energy from the oxidation of molecular hydrogen (5). Hydrogen oxidation in this strain is catalyzed by two hydrogenases (18). The soluble hydrogenase (SH) is closely related to the NAD-reducing hydrogenases present in Alcaligenes eutrophus, in the gram-positive Rhodococcus sp. strain 1b (5), and in the cyanobacterium Anabaena variabilis (42). The second hydrogenase of A. hydrogenophilus is a membrane-bound hydrogenase (MBH) coupled to the respiratory chain and involved in electron transportdependent phosphorylation (18). The large and the small subunits of this enzyme show 75 and 90% identity, respectively, to the corresponding polypeptides of the A. eutrophus MBH (28,53). Moreover, in both Alcaligenes species H 2 -oxidizing ability is genetically linked to a megaplasmid (18) and dependent on the function of an RpoN-like sigma factor of RNA polymerase which is encoded on the chromosome (32, 38).Despite the high degree of similarity, hydrogenase regulation in A. hydrogenophilus appears to be quite different from the mode of control observed in A. eutrophus. Whi...

[NiFe] hydrogenases catalyze the reversible conversion of H 2 into protons and electrons. The reaction takes place at the active site, which is composed of a nickel and an iron atom and three diatomic ligands, two cyanides and one carbon monoxide, bound to the iron. The NiFe(CN ؊ ) 2 CO cofactor is synthesized by an intricate posttranslational maturation process, which is mediated by a set of six conserved Hyp proteins. Depending on the cellular location and the physiological function, additional auxiliary proteins are involved in hydrogenase biosynthesis. Depending on the physiological conditions, hydrogenases catalyze either heterolytic cleavage of H 2 into protons and electrons or the reduction of protons, yielding dihydrogen (1). Three convergently evolved groups of hydrogenases are distributed in nature. They have been classified according to their metal content in di-iron [FeFe], nickel-iron [NiFe], and monoiron [Fe] hydrogenases (2). This study focuses on one member of the group of [NiFe] hydrogenases, which consists of a Ni-Fe active site-containing large subunit, a Fe-S cluster-accommodating, electron-transferring small subunit, and a membrane-spanning cytochrome b. The Ni-Fe cofactor is coordinated to the protein via thiol groups originating from four invariant cysteine residues, two of which form a bridge between nickel and iron. The iron is additionally equipped with two CN Ϫ groups and one CO ligand, which confer a low spin state on the iron (3, 4).Biosynthesis of intricate metal cofactors usually involves protein-assisted maturation processes (5-7). Maturation of [NiFe] hydrogenases underlies posttranslational reactions that implicate a set of at least six conserved auxiliary proteins (HypA, -B, -C, -D, -E, and -F), which mediate assembly and insertion of the Ni-Fe cofactor (5,8,9). According to the current model, predominantly based on studies with hydrogenase 3 from Escherichia coli, a carbamoyl group is transferred from carbamoylphosphate via HypF to the thiol group of the C-terminal cysteine in HypE. Upon dehydration, a HypE-thiocyanate complex is formed, and subsequently the CN Ϫ ligand is transmitted to an iron exposed on a HypC-D complex (10 -13). It has been proven experimentally that carbamoylphosphate is the substrate of the CN Ϫ ligands, whereas the origin of the CO ligand remains an open question (14 -17).Once the Fe(CN Ϫ ) 2 CO moiety is inserted, nickel is incorporated into the hydrogenase precursor mediated by HypA and HypB (18,19). Folding and oligomerization of the catalytic subunit are triggered by removal of a C-terminal peptide catalyzed by a specific endoprotease and dissociation of the chaperone HypC (20).The basic module of the membrane-bound hydrogenase (MBH) 2 of Ralstonia eutropha H16 (Re) is composed of the large subunit HoxG (67.1 kDa) containing the Ni-Fe active site and a small subunit HoxK (34.6 kDa) harboring three Fe-S clusters (21-24). Physiologically active MBH is exposed to the periplasm and anchored to the membrane via a hydrophobic C terminus of HoxK and the...

By taking advantage of the tightly clustered genes for the membrane-bound [NiFe] hydrogenase of Ralstonia eutropha H16, broad-host-range recombinant plasmids were constructed carrying the entire membrane-bound hydrogenase (MBH) operon encompassing 21 genes. We demonstrate that the complex MBH biosynthetic apparatus is actively produced in hydrogenase-free hosts yielding fully assembled and functional MBH protein.Synthesis of metalloenzymes, such as nitrogenase, urease, and hydrogenase, relies on specific protein-based machineries which assist the incorporation of the catalytic metal center into the apoprotein. In addition, scaffold proteins, specific proteases, and regulatory proteins are often involved in this process (15,24,41,49). Among metalloenzymes, the periplasmically oriented membrane-bound [NiFe] hydrogenases, which catalyze the reversible cleavage of H 2 into protons and electrons, undergo one of the most complex maturation pathways (10,12,41,49). Crystal structure analysis and spectroscopic studies revealed an extraordinary architecture of the Ni-Fe active site deeply buried in the large subunit of the heterodimeric enzyme (17). Ni and Fe are coordinated to the protein via four thiol groups donated by cysteine residues. Three additional diatomic molecules, two cyanides (CN Ϫ ) and one carbon monoxide (CO), are bound to the iron and probably maintaining the metal in a low-spin state (2, 50). An electrogenic coupling between the Ni-Fe active site and three Fe-S clusters accommodated in the small subunit exists.In many organisms, the genes coding for the hydrogenase subunits are tightly linked with clusters of hydrogenase-related accessory genes pointing to a complex biosynthetic apparatus implicating nickel uptake, metallocenter insertion, proteolytic maturation, membrane translocation, and coordinated transcriptional regulation (41, 49). For hydrogenase-3 of Escherichia coli, it was shown that six hyp gene products participate in a stepwise insertion of the Ni-Fe site into the large subunit precursor (10). Based on the current model, first, Fe and its diatomic ligands are incorporated by protein complexes of HypC, HypD, HypE, and HypF, followed by the insertion of Ni, mediated by HypA and HypB (8,9,20). Once the metal center is completed, a hydrogenase-specific protease removes a peptide from the C terminus of the large subunit which triggers folding and oligomerization to the holoenzyme (28).Previous attempts to express active [NiFe] hydrogenase in E. coli failed presumably because of incompatibility of the maturation systems (30). On the other hand, transfer of H 2 -oxidizing ability by conjugal transfer of entire megaplasmids that harbor the complete set of genetic information for [NiFe] hydrogenase biosynthesis was successful but restricted to a narrow host range (18,48). A more straightforward approach was the transfer of relatively small transposable elements, carrying the hydrogenase gene cluster of Rhizobium leguminosarum, into closely related, hydrogenase-free recipients. The recombinants w...