The structural genes of cytochrome‐c oxidase in Bacillus subtilis have been isolated and sequenced. Five genes, ctaB–F, are closely spaced. ctaC, ctaD, ctaE and ctaF are the genes for subunits II, I, III and IVB, respectively. ctaB, which may encode an assembly factor, is separated and upstream from the others. In comparison to its mitochondrial counterparts, subunit I has an extended C‐terminus with two additional transmembrane segments, whereas subunit III has lost two such segments from its N‐terminus. The C‐terminal extension in subunit II is a covalent cytochrome‐c domain, previously characterized only in the thermophilic oxidases. Subunit IVB, a small hydrophobic protein, is a novel subunit. These predictions suggest that the B. subtilis cytochrome‐c oxidase is structurally more related to the four‐subunit Escherichia coli cytochrome‐bo complex than, for instance, to the Paracoccus denitrificans enzyme. Cytochrome aa3, which was previously isolated from B. subtilis [de Vrij, W., Azzi, A. & Konings, W. N. (1983) Eur. J. Biochem. 131, 97–103] is not encoded by the ctaC – F genes; thus, there seems to be two different cytochrome‐aa3‐type oxidases in this Gram‐positive bacterium.

COIII is one of the major subunits in the mitochondrial and a bacterial cytochrome c oxidase, cytochrome aa3. It does not contain any of the enzyme's redox‐active metal centres and can be removed from the enzyme without major changes in its established functions. We have deleted the COIII gene from Paracoccus denitrificans. The mutant still expresses spectroscopically detectable enzyme almost as the wild‐type, but its cytochrome c oxidase activity is much lower. From 50 to 80% of cytochrome a is reduced and its absorption maximum is 2‐3 nm blue‐shifted. The EPR signal of ferric cytochrome a is heterogeneous indicating the presence of multiple cytochrome a species. Proteolysis of the membrane‐bound oxidase shows new cleavage sites both in COI and COII. DEAE‐chromatography of solubilized enzyme yields fractions that contain a COI + COII complex and in addition haem‐binding, free COI as well as free COII. The mutant phenotype can be complemented by introducing the COIII gene back to cells in a plasmid vector. We conclude that cytochrome oxidase assembles inefficiently in the absence of COIII and that this subunit may facilitate a late step in the assembly. The different oxidase species in the mutant represent either accumulating intermediates of the assembly pathway or dissociation products of a labile COI + COII complex and its conformational variants.