STRUCTURAL AND FUNCTIONAL-ANALYSIS OF AA(3)-TYPE AND CBB(3)-TYPE CYTOCHROME-C OXIDASES OF PARACOCCUS-DENITRIFICANS REVEALS SIGNIFICANT DIFFERENCES IN PROTON-PUMP DESIGN

In Paracoccus denitrificans the aa(3)-type cytochrome c oxidase and th e bb(3)-type quinol oxidase have previously been characterized in deta il, both biochemically and genetically. Here we report on the isolatio n of a genomic locus that harbours the gene cluster ccoNOQP, and demon strate that it encodes an alternative cbb(3)-type cytochrome c oxidase . This oxidase has previously been shown to be specifically induced at low oxygen tensions, suggesting that its expression is controlled by an oxygen-sensing mechanism, This view is corroborated by the observat ion that the ccoNOQP gene cluster is preceded by a gene that encodes a n FNR homologue and that its promoter region contains an FNR-binding m otif. Biochemical and physiological analyses of a set of oxidase mutan ts revealed that, at least under the conditions tested, cytochromes aa (3), bb(3) and cbb(3) make up the complete set of terminal oxidases in P. denitrificans, Proton-translocation measurements of these oxidase mutants indicate that all three oxidase types have the capacity to pum p protons, Previously, however, we have reported decreased H+/e(-) cou pling efficiencies of the cbb(3)-typeoxidase under certain conditions, Sequence alignment suggests that many residues that have been propose d to constitute the chemical and pumped proton channels in cytochrome aa(3) (and probably also in cytochrome bb(3)) are not conserved in cyt ochrome cbb(3). It is concluded that the design of the proton pump in cytochrome cbb(3) differs significantly from that in the other oxidase types.