One of the key enzymes in the chemolithoautotrophic metabolism ofAlcaligenes eutrophus H16 is a dimeric, membrane-associated hydrogenase. The genetic determinants of this enzyme are located on the endogenous megaplasmid pHG1 (G. Eberz, C. Hogrefe, C. Kortluke, A. Kamienski, and B. Friedrich, J. Bacteriol. 168:636-641, 1986). Complementation studies showed that the information required for the formation of active membrane-bound hydrogenase occupies more than 7.5 kb of megaplasmid DNA. We cloned and sequenced this region and identified the genes encoding the two hydrogenase subunits (hoxK and hoxG). (3,18). The cytoplasmic hydrogenase (SH) is a tetramer composed of four heterologous polypeptides. This NAD-reducing enzyme provides reducing equivalents primarily for autotrophic carbon dioxide fixation (3). The membrane-bound hydrogenase (MBH) ofA. eutrophus is a dimer consisting of two heterologous polypeptides, the 31-kDa small subunit (SSU) and the 62-kDa large subunit (LSU) (51). The oxidation of hydrogen catalyzed by the MBH is coupled to the respiratory chain and thus contributes to the generation of ATP. The A. eutrophus MBH is a member of the more widespread family of membrane-associated hydrogenases, whereas the soluble NADreducing type has been found only in Alcaligenes and Nocardia strains (3).Previous studies on deletion and insertion mutants of A. In this communication we report the cloning, nucleotide sequence, and organization of the hoxP locus, which encodes the MBH. We present evidence that this region contains two structural genes and at least eight accessory genes organized in at least three transcriptional units. We also describe results indicating that one of the accessory genes is involved in the process leading to the attachment of the MBH enzyme to the membrane. MATERIALS AND METHODSStrains and plasmids. The bacterial strains, phages, and plasmids used in this study are listed in

. 174:6277-6289, 1992). Other genes located in the adjacent pleiotropic region are also required. In the absence of these genes, MBH is synthesized but is catalytically inactive. Immunological analyses revealed that cells containing active MBH produced the small and large subunits of the enzyme in two distinct conformations each; only one of each, presumably the immature form, occurred in cells devoid of MBH activity. The results suggest that the conversion of the two subunits into the catalytically active membraneassociated heterodimer depends on specific maturation processes mediated by hox genes.The gram-negative, facultative chemolithoautotroph Alcaligenes eutrophus H16 (ATCC 17699) contains two hydrogenases. These enzymes catalyze the oxidation of hydrogen coupled to the reduction of various electron acceptors via an energy-conserving mechanism (reviewed in reference 2).Both enzymes are encoded in the cluster of hox genes on the 450-kb megaplasmid pHG1 (reviewed in reference 9). The membrane-bound hydrogenase (MBH) is a heterodimer composed of a small subunit (SSU; apparent molecular mass, 31 kDa) and a large subunit (LSU; apparent molecular mass, 62 kDa) and contains 10 iron atoms and 1 atom of nickel per mol of enzyme (19). The genes encoding the SSU and the LSU (designated hoxK and hoxG, respectively) belong to a complex locus consisting of 11 open reading frames. The derived amino acid sequences of the two subunits of theA. eutrophus MBH show clear-cut homology (60 to 80% identity) to the respective sequences of [NiFe] hydrogenases from various aerobic hydrogen bacteria. Arrays of cysteines are typical of [NiFe] hydrogenases and probably coordinate metal complexes in the redox-active centers of these enzymes (17).Comparison of the derived amino acid sequence of the SSU of the A. eutrophus MBH with the experimentally determined NH2-terminal sequence indicates the presence of a 43-amino-acid leader peptide (17,19). The experimentally determined NH2-terminal sequence of the LSU is-except for the missing initial methionine-colinear with the sequence deduced from the coding region, ruling out a leader peptide for this protein (17,19).To elucidate the functions of the other genes of the hox cluster in the biosynthesis of MBH, mutants defective for lithoautotrophic growth were generated and subjected to biochemical and genetic analyses. These mutants fell into three major classes: (i) mutants that produce catalytically * Corresponding author.

The activities of NAD+-photoreduction and NADH/decyl-ubiquinone reductase in membrane preparations of Rhodobacter capsulatus changed to the same extent under different conditions. These results indicated that NADH:ubiquinone oxidoreductase (complex I) catalyzes the electron transport in the downhill direction (respiratory chain) and in the uphill direction (reverted electron flow). This conclusion was confirmed by the characterization of a complex-I-deficient mutant of R. capsulatus. The mutant was not able to reduce NAD+ in the light. Since this mutant was not able to grow photoautotrophically, we concluded that complex I is the enzyme that catalyzes the reverted electron flow to NAD+ to provide reduction equivalents for CO2 fixation. Complex I is not essential for the reverted electron flow to nitrogenase since the mutant grew under nitrogen-fixing conditions. As shown by immunological means, NuoE, a subunit of complex I from R. capsulatus having an extended C-terminus, was modified depending on the nitrogen source present in the growth medium. When the organism used N2 instead of NH4+, a smaller NuoE polypeptide was synthesized. The complex-I-deficient mutant was not able to modify NuoE. The function of the modification is discussed.

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Hydrogenase plays a major role in lithoautotrophic metabolism of aerobic H2-utilizing bacteria. It catalyzes the hydrogen uptake reaction H2 ± 2H+ + 2e-.