Expression of cytochrome o in hydrogen uptake constitutive mutants of Rhizobium japonicum

Mutant strains of Rhizobium japonicum constitutive for H₂ uptake activity (Hup(c)) contained significantly more membrane-bound b-type cytochrome than did the wild type when grown heterotrophically. The Hup(c) strains contained approximately 3 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 μM, 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 Hup(c) 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 Hup(c) 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.