Widespread Distribution and Functional Specificity of the Copper Importer CcoA: Distinct Cu Uptake Routes for Bacterial Cytochrome c Oxidases

Cytochrome c oxidases are members of the heme-copper oxidase superfamily. These enzymes have different subunits, cofactors, and primary electron acceptors, yet they all contain identical heme-copper (Cu(B)) binuclear centers within their catalytic subunits. The uptake and delivery pathways of the Cu(B) atom incorporated into this active site, where oxygen is reduced to water, are not well understood. Our previous work with the facultative phototrophic bacterium Rhodobacter capsulatus indicated that the copper atom needed for the Cu(B) site of cbb(3)-type cytochrome c oxidase (cbb(3)-Cox) is imported to the cytoplasm by a major facilitator superfamily-type transporter, CcoA. In this study, a comparative genomic analysis of CcoA orthologs in alphaproteobacterial genomes showed that CcoA is widespread among organisms and frequently co-occurs with cytochrome c oxidases. To define the specificity of CcoA activity, we investigated its function in Rhodobacter sphaeroides, a close relative of R. capsulatus that contains both cbb(3)- and aa(3)-Cox. Phenotypic, genetic, and biochemical characterization of mutants lacking CcoA showed that in its absence, or even in the presence of its bypass suppressors, only the production of cbb(3)-Cox and not that of aa(3)-Cox was affected. We therefore concluded that CcoA is dedicated solely to cbb(3)-Cox biogenesis, establishing that distinct copper uptake systems provide the Cu(B) atoms to the catalytic sites of these two similar cytochrome c oxidases. These findings illustrate the large variety of strategies that organisms employ to ensure homeostasis and fine control of copper trafficking and delivery to the target cuproproteins under different physiological conditions.