Hydrogenase Large Subunit Research Articles

The product of the hypB gene, which is required for nickel incorporation into hydrogenases, is a novel guanine nucleotide-binding protein

The products of the hyp operon genes are essential for the formation of catalytically active hydrogenases in Escherichia coli. At least one of these auxiliary proteins, HYPB, appears to be involved in nickel liganding to the hydrogenase apoprotein, since mutations in hypB can be phenotypically suppressed by high nickel concentrations in the medium (R. Waugh and D. H. Boxer, Biochimie 68:157-166, 1986). To approach the identification of the specific function of HYPB, we overexpressed the hypB gene and purified and characterized the gene product. HYPB is a homodimer of 31.6-kDa subunits, and it binds guanine nucleotides, with a Kd for GDP of 1.2 microM. The protein displays a low level of GTPase activity, with a kcat of 0.17 min-1. The apparent Km for GTP, as measured in the GTP hydrolysis reaction, was determined to be 4 microM. A chromatography system was established to measure nickel insertion into hydrogenase 3 from E. coli and to determine the effects of lesions in hypB. Nickel appears to be associated only with the processed large subunit of hydrogenase 3 in the wild type, and hypB mutants accumulate the precursor form of this subunit, which is devoid of nickel. The results are discussed in terms of a model in which HYPB is involved in nickel donation to the hydrogenase apoprotein and in which GTP hydrolysis is thought to reverse the interaction between either HYPB or another nickel-binding protein and the hydrogenase apoprotein after the nickel has been released.

Mutational analysis of the operon (hyc) determining hydrogenase 3 formation in Escherichia coli.

In-frame deletions were introduced into each of the eight genes of the hyc operon coding for products required for the formation of the formate hydrogenlyase (FHL) system. The deletions were transferred to the chromosome and the resulting mutants were analysed for development of formate dehydrogenase H and hydrogenase 1, 2 and 3 activity. It was found that hycA, the promoter-proximal gene, is a regulatory gene and that it codes for a product counteracting transcriptional activation by FhlA. Deletions within the hycB to hycH genes specifically affected formate dehydrogenase H activity or hydrogenase 3 activity, or both. None of the mutations affected hydrogenase 1 or 2 activity. A model is proposed for the functional interaction of the different hyc operon gene products in the formate hydrogenlyase complex, which is based on the results of the mutational analysis, on the determination of the subcellular localization of the FdhF, HycE, HycF and HycG polypeptides and on the similarity of hyc gene product sequences with those from other hydrogenase systems. HycH, the product of the most promoter-distal gene, does not seem to form part of the functional FHL complex but rather is required for the conversion of a precursor form of the large subunit of hydrogenase 3 into the mature form.

Rhizobitoxine inhibition of hydrogenase synthesis in free-living Bradyrhizobium japonicum

Rhizobitoxine produced by Bradyrhizobium species strongly prevented derepression of hydrogenase expression in free-living Bradyrhizobium japonicum, although the toxin had no effect on the activity of cells which had already synthesized hydrogenase protein. Dihydrorhizobitoxine, a structural analog of rhizobitoxine, proved to be a less potent inhibitor of hydrogenase derepression. Rhizobitoxine did not cause cell death at a concentration sufficient to eliminate hydrogenase expression. The large subunit of hydrogenase was not detectable with antibody after derepression in the presence of rhizobitoxine. The general pattern of proteins synthesized from 14C-labeled amino acids during derepression was not significantly different in the presence or absence of rhizobitoxine. These results indicated that rhizobitoxine inhibited hydrogenase synthesis in free-living B. japonicum. Cystathionine and methionine strongly prevented the inhibition of hydrogenase derepression by rhizobitoxine, suggesting that the inhibition involves the level of sulfur-containing amino acids in the cell.

Identification of a Locus Upstream from the Hydrogenase Structural Genes That Is Involved in Hydrogenase Expression in Bradyrhizobium japonicum

A locus involved in the expression of the uptake hydrogenase system of Bradyrhizobium japonicum was identified adjacent to genes encoding the hydrogenase subunits. A cloned fragment of DNA was used to complement to autotrophy a Hup putative regulatory mutant of B. japonicum. The mutant strain lacked hydrogenase activity and synthesized low levels of the large subunit of hydrogenase as determined by Western gels. Tn5-induced mutagenesis located the region within the fragment which was necessary for complementation of the mutant phenotype. The locus identified is adjacent to that encoding the small subunit of hydrogenase; its right border is <0.5 kilobase upstream from the hydrogenase transcriptional start site, and its left border is between 1 and 2.5 kilobases from that start site. However, the locus is outside the region previously shown to contain hup-related genes of B. japonicum. Thus, the localization of this gene describes a previously unidentified hup-related gene on a region of DNA not previously shown to contain hup-specific DNA.

Cloning and sequencing of the genes encoding the large and the small subunits of the H2 uptake hydrogenase (hup) of Rhodobacter capsulatus.

The structural genes (hup) of the H2 uptake hydrogenase of Rhodobacter capsulatus were isolated from a cosmid gene library of R. capsulatus DNA by hybridization of Bradyrhizobium japonicum. The R. capsulatus genes were localized on a 3.5 kb HindIII fragment. The fragment, cloned onto plasmid pAC76, restored hydrogenase activity and autotrophic growth of the R. capsulatus mutant JP91, deficient in hydrogenase activity (Hup-). The nucleotide sequence, determined by the dideoxy chain termination method, revealed the presence of two open reading frames. The gene encoding the large subunit of hydrogenase (hupL) was identified from the size of its protein product (68,108 dalton) and by alignment with the NH2 amino acid protein sequence determined by Edman degradation. Upstream and separated from the large subunit by only three nucleotides was a gene encoding a 34,256 dalton polypeptide. Its amino acid sequence showed 80% identity with the small subunit of the hydrogenase of B. japonicum. The gene was identified as the structural gene of the small subunit of R. capsulatus hydrogenase (hupS). The R. capsulatus hydrogenase also showed homology of Desulfovibrio baculatus and D. gigas. In the R. capsulatus hydrogenase the Cys residues (13 in the small subunit and 12 in the large subunit) were not arranged in the typical configuration found in [4Fe-4S] feredoxins.

Regulation of H2 oxidation activity and hydrogenase protein levels by H2, O2, and carbon substrates in Alcaligenes latus.

Regulation of H2 oxidation activity and hydrogenase protein levels in the free-living hydrogen bacterium Alcaligenes latus was investigated. Hydrogenase activity was induced when heterotrophically grown cells were transferred to chemolithoautotrophic conditions, i.e., in the presence of H2 and absence of carbon sources, with NH4Cl as the N source. Under these conditions, H2 oxidation activity was detectable after 30 min of incubation and reached near-maximal levels by 12 h. The levels of hydrogenase protein, as measured by a Western blot (immunoblot) assay of the hydrogenase large subunit, increased in parallel with activity. This increase suggested that the increased H2 oxidation activity was due to de novo synthesis of hydrogenase protein. H2 oxidation activity was controlled over a surprisingly wide range of H2 concentrations, between 0.001 and 30% in the gas phase. H2 oxidation activity was induced to high levels between 2 and 12.5% O2, and above 12.5% O2, H2 oxidation activity was inhibited. Almost all organic carbon sources studied inhibited the expression of hydrogenase, although none repressed hydrogenase synthesis completely. In all cases examined, hydrogenase protein, as detected by Western blot, paralleled the level of H2 oxidation activity, suggesting that control of hydrogenase activity was mediated through changes in hydrogenase protein levels.