Synthetic oligonucleotide probes were used to clone two loci from the chromosomal DNA of Paracoccus denitrificans that contain the genes for cytochrome c oxidase (cytochrome aa3). One locus seems to contain four or five genes probably forming an operon. Two of these code for the oxidase subunits II and III. Three open reading frames are found between the COII and COIII genes. The other locus codes for the subunit I. A short open reading frame is found upstream of this gene. All three subunits of the Paracoccus enzyme show remarkable homology to the corresponding subunits of the mitochondrial cytochrome oxidase. Possible protein products of the open reading frames have not yet been identified.

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.

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The region of Paracoccus denitrificans chromosome where the genes coding for cytochrome oxidase (cytochrome aa 3) subunits are located has been cloned. DNA sequencing revealed an open reading frame that codes for a protein homologous to the subunit III of the eukaryotic, mitochondrial enzyme. This subunit is absent from the isolated Paracoccus oxidase. It now seems that it is part of the native enzyme in the bacterial cytoplasmic membrane. This may explain the observed discrepancies in the function of the isolated enzyme.

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