Nucleotide sequence of the gene encoding the hydrogenase from Desulfovibrio vulgaris (Hildenborough)

Abstract

The nucleotide sequence of the 4.7‐kb Sa/I/EcoRI insert of plasmid pHV15 containing the hydrogenase gene from Desulfovibrio vulgaris (Hildenborough) has been determined with the dideoxy chain‐termination method. The structural gene for hydrogenase encodes a protein product of molecular mass 45820 Da. The NH₂‐terminal sequence of the enzyme deduced from the nucleic acid sequence corresponds exactly to the amino acid sequence determined by Edman degradation. The nucleic acid sequence indicates that a N‐formylmethionine residue precedes the NH₂‐terminal amino acid Ser‐1. There is no evidence for a leader sequence. The NH₂‐terminal part of the hydrogenase shows homology to the bacterial [8Fe‐8S] ferredoxins. The sequence Cys‐Ile‐Xaa‐Cys‐Xaa‐Xaa‐Cys‐Xaa‐Xaa‐Xaa‐Cys‐Pro‐Xaa‐Xaa‐Ala‐(Ile) occurs twice both in the hydrogenase and in [8Fe‐8S] ferredoxins, where the Cys residues have been shown to coordinate two [4Fe–4S] clusters [Adman, E. T., Sieker, L. C. and Jensen, L. H. (1973) J. Biol. Chem. 248, 3987–3996]. These results, therefore, suggest that two electron‐transferring ferredoxin‐like [4Fe‐4S] clusters are located in the NH₂‐terminal segment of the hydrogenase molecule. There are ten more Cys residues but it is not clear which four of these could participate in the formation of the third cluster, which is thought to be the hydrogen binding centre. Another gene, encoding a protein of molecular mass 13493 Da, was found immediately downstream from the gene for the 46‐kDa hydrogenase. The nucleic acid sequence suggests that the hydrogenase and the 13.5‐kDa protein belong to a single operon and are coordinately expressed. Since dodecylsulfate gel electrophoresis of purified hydrogenase indicates the presence of a 13.5‐kDa polypeptide in addition to the 46‐kDa component, it is proposed that the hydrogenase from D. vulgaris (Hildenborough) is a two‐subunit enzyme.