Cytochrome c oxidase from the bacterium Paracoccus denitrcjicans, while being related to the mitochondrial enzyme in many ways, consists of only two to three different subunits. For the identification of its genes, a Paracoccus DNA library was constructed and screened with specific antibodies for expression of cloned inserts in E. coli. A positive clone expressing immunoreactive products in the molecular mass region of authentic subunit I1 revealed a high homology of its DNA-deduced amino acid sequence with subunit I1 sequences of the mitochondrial oxidases; several typical features, such as the transmembrane folding pattern and the presumed copper-binding site, are highly conserved between prokaryotic and mitochondrial polypeptides. A comparison with peptide sequencing data of the purified subunit established the presence of a characteristic N-terminal extension as well as a longer C terminus in the initial translation product of the Paracoccus subunit; by mass spectroscopy, the first N-terminally blocked residue of the mature polypeptide was identified as a pyroglutamate. No code abnormalities, but a highly specific codon usage were observed; no evidence for a localization of the subunit I gene directly adjacent to this gene has been obtained.Cytochrome c oxidase is a key enzyme in the electrontransport chains of mitochondria and many bacteria. It catalyzes the transfer of electrons from the one-electron donor cytochrome c to the four-electron acceptor dioxygen; this reaction is coupled to the extrusion of protons from the mitochondrial matrix (or the cytoplasma in the case of bacteria) to generate an electrochemical gradient across the membrane (for recent reviews on the mitochondrial enzyme, see [l -61). Interest in the bacterial enzymes has been stimulated by a number of reports (reviewed in [7 -91) showing that the structure of the prokaryotic oxidases is far less complex than that of the mitochondrial enzymes which, depending on the source, are composed of from 6 (lower eukaryotes) to up to 13 subunits (mammalian mitochondria). Typically only two to three different subunits are seen in isolated bacterial heme aa3-type oxidases; such preparations show full activity in electron transport, and several of them are also active in proton translocation (see [9]). Since evidence for homology of subunits from mitochondrial and prokaryotic oxidases has been presented, these findings have greatly supported the notion that the three largest, mitochondrially coded subunits constitute the catalytic core also in the mitochondrial enzyme.The oxidase from the bacterium Paracoccus denitrificans [lo -121 is among the best-studied prokaryotic oxidases and may be viewed as a structurally simple model system for the mitochondrial enzyme: as isolated, the Paracoccus enzyme is composed of two different subunits of 45 kDa and 28-30 kDa, efficiently oxidizes reduced bacterial or mitochon- drial cytochrome c, and acts as a redox-coupled proton pump both in proteoliposomes and in whole cells [13 -151. Apart from considerable furthe...

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