Two open reading frames (ORFs) were identified immediately downstream of the four structural genes for the soluble hydrogenase (SH) of Alcaligenes eutrophus H16. While a mutation in ORF2 had no obvious effect on hydrogen metabolism, an in-frame deletion in ORF1, subsequently designated hoxW, led to a complete loss of SH activity and hence a significant retardation of autotrophic growth on hydrogen. Hydrogen oxidation in the hoxW mutant was catalyzed by the second hydrogenase, a membrane-bound enzyme. Assembly of the four subunits of the SH was blocked in mutant cells, and HoxH, the hydrogen-activating subunit, accumulated as a precursor which was still capable of binding nickel. Protein sequencing revealed that HoxH isolated from the wild type terminates at His-464, whereas the C-terminal amino acid sequence of HoxH from the hoxW mutant is colinear with the deduced sequence. Processing of the HoxH precursor was restored in vitro by a cell extract containing HoxW. These results indicate that HoxW is a highly specific carboxyl-terminal protease which releases a 24-amino-acid peptide from HoxH prior to progression of subunit assembly.The facultative lithoautotroph Alcaligenes eutrophus can use molecular hydrogen as the sole energy source. Hydrogen oxidation in this proteobacterium is mediated by two (NiFe)-containing hydrogenases. The membrane-bound hydrogenase (MBH) is composed of a large and a small subunit, which are coupled to electron transport phosphorylation via a cytochrome b-like anchor protein. While the MBH is a representative of the more common type of (NiFe)-hydrogenases, the second, soluble hydrogenase (SH) belongs to a family of less abundant multimeric (NiFe)-hydrogenases (reviewed in reference 7). Multimeric hydrogenases are present in a few Alcaligenes species, in the gram-positive Rhodococcus sp. strain 1b (formerly Nocardia opaca) (4), and in the cyanobacterium Anabaena variabilis (32).The cytoplasmic SH reacts with NAD as the physiological electron carrier. The enzyme is composed of four heterologous subunits (35) encoded by the megaplasmid-borne genes hoxF, hoxU, hoxY, and hoxH (39). The products of hoxF and hoxU form the flavononheme iron protein of the SH which possesses NADH oxidoreductase (diaphorase) activity (33). The diaphorase dimer is closely related to three peripheral subunits of the NADH:ubiquinone oxidoreductase (complex I) of mitochondria and of some bacterial species (42, 43). The hoxH and hoxY gene products show features of a hydrogen-activating site and an intramolecular electron transfer pathway (7). The first crystallographic analysis of a (NiFe)-hydrogenase carried out for the periplasmic hydrogenase of Desulfovibrio gigas was recently completed. According to this study, the active site appears to be a binuclear cluster of nickel and probably iron, buried deep inside the protein (41).Genetic studies on various bacteria uncovered a series of accessory genes which code for functions involved in the biogenesis of (NiFe)-hydrogenases. Several specific gene products are required...