The genes hoxF, -U, -Y, and -H which encode the four subunit polypeptides a, -y, 8, and 1 of the NADreducing hydrogenase (HoxS) ofAkaligenes eutrophus H16, were cloned, expressed in PseudomonasfacUis, and sequenced. On the basis of the nucleotide sequence, the predicted amino acid sequences, and the N-terminal amino acid sequences, it was concluded that the structural genes are tightly linked and presumably organized as an operon, denoted hoxS. Two pairs of -24 and -12 consensus sequences resembling RpoN-activatable promoters lie upstream of hoxF, the first of the four genes. Primer extension experiments indicate that the second promoter is responsible for hoxS transcription. hoxF and hoxU code for the flavin-containing dimer (a and y subunits) of HoxS which exhibits NADH:oxidoreductase activity. A putative flavin-binding region is discussed. The 26.0-kilodalton (kDa) y subunit contains two cysteine clusters which may participate in the coordination of two [4Fe-4S] centers. The genes hoxY and hoxH code for the small 22.9-kDa 8 subunit and the nickel-containing 54.8-kDa ,B subunit, respectively, of the hydrogenase dimer of HoxS. The latter dimer exhibits several conserved regions found in all nickel-containing hydrogenases. The roles of these regions in coordinating iron and nickel are discussed. Although the deduced amino acid sequences of the 8 and ,1 subunits share some conserved regions with the corresponding polypeptides of other [NiFe] hydrogenases, the overall amino acid homology is marginal. Nevertheless, significant sequence homology (35%) to the corresponding polypeptides of the soluble methylviologen-reducing hydrogenase of Methanobacterium thermoautotrophicum was found. Unlike the small subunits of the membrane-bound and soluble periplasmic hydrogenases, the HoxS protein does not appear to be synthesized with an N-terminal leader peptide.Alcaligenes eutrophus H16 is a gram-negative facultatively lithoautotrophic bacterium which can assimilate CO2 and utilize H2 as an energy source (reviewed in reference 5). Hydrogen oxidation is catalyzed by hydrogenases. These enzymes are found in phylogenetically diverse microorganisms, where they are responsible for both consumption and production of H2 (reviewed in reference 13). Two distinct hydrogenases have been purified and characterized from extracts of A. eutrophus (39, 44). These two enzymes differ in cellular localization, cofactor content, polypeptide composition, and apparent molecular weight. [NiFeSe]. An enzyme of this type has been described for Desulfovibrio and Methanococcus strains (reviewed in reference 13).The genes coding for the two hydrogenases of A. eutrophus H16 lie in a cluster of genes on a 450-kilobase-pair (kb) conjugative megaplasmid (15,24). Molecular cloning of megaplasmid DNA in Escherichia coli and screening of the resultant hybrid plasmids for complementation of hydrogenase-deficient mutants led to the identification of hydrogenase (hox) genes. Subsequent studies revealed that the two structural gene loci hoxS and hoxP coding for...