title: Expression of cytochrome o in hydrogen uptake constitutive mutants of Rhizobium japonicum.
abstract: Mutant strains of Rhizobium japonicum constitutive for H2 uptake activity (Hupc) contained significantly more membrane-bound b-type cytochrome than did the wild type when grown heterotrophically. The Hupc strains contained approximately three times more dithionite- and NADH-reducible CO-reactive b-type cytochrome than did the wild type; the absorption features of the CO spectra were characteristic of cytochrome o. This component, designated cytochrome b', was not reduced by NADH in the presence of cyanide. Cytochrome o from the wild type (SR) and cytochrome b' from mutants SR476 and SR481 bound to CO with similar dissociation constants of 5.4, 7.4, and 5.6 microM, respectively. NADH-dependent reduction of cytochrome b' from SR476 and SR481 and the cytochrome o from SR followed pseudo-first-order kinetics with similar rate constants. Based on these spectral, ligand-binding, and kinetic measurements, it was concluded that cytochrome b' expressed by the Hupc mutants is equivalent to cytochrome o found in the wild type. H2, NADH, and succinate each reduced the same amount of total b-type cytochrome in membranes from SR481, and the rate of H2-dependent cytochrome o reduction was significantly less than with succinate or NADH as the reductants. It was concluded that neither cytochrome o nor any b-type cytochrome expressed by the Hupc mutants was unique to the H2 oxidation system. At low O2 concentrations, the inhibition of H2 and NADH oxidase activities by CO closely paralleled the binding of CO to cytochrome o rather than cytochromes a3 or c'. This suggested that NADH and H2 oxidation involved primarily cytochrome o as the terminal oxidase at low O2 tensions.
mesh_terms: D002248:Carbon Monoxide / Q000378:metabolism / Q000494:pharmacology; D003486:Cyanides / Q000494:pharmacology; D003573:Cytochrome b Group / Q000378:metabolism; D003580:Cytochromes / Q000032:analysis*; D029968:Escherichia coli Proteins; D006859:Hydrogen / Q000378:metabolism*; D007700:Kinetics; D009154:Mutation; D010084:Oxidation-Reduction; D012231:Rhizobium / Q000201:enzymology* / Q000235:genetics
chemical_list: D003486:Cyanides; D003573:Cytochrome b Group; D003580:Cytochromes; D029968:Escherichia coli Proteins; D002248:Carbon Monoxide; D006859:Hydrogen; C017168:cytochrome bo, E coli