The monoheme and diheme c-type cytochromes FixO and FixP are two of the subunits of the respiratory cbb3-type oxidase of Bradyrhizobium japonicum. The cysteines of the respective heme C binding motifs CXXCH were changed to serines by site-directed mutagenesis, which led to inactive oxidases in all mutants. Western blot analyses showed that an intact heme binding site in the FixO polypeptide is a prerequisite not only for the synthesis of holo-FixO protein but also for the formation of the entire cbb3-type oxidase complex. Both heme binding sites of FixP were essential for maturation and assembly of this subunit. It was not possible to create stable FixP variants that contained only one heme C.