PccA and SenC are periplasmic copper chaperones required for the biogenesis of cbb3-type cytochrome c oxidase ( cbb3-Cox) in Rhodobacter capsulatus at physiological Cu concentrations. However, both proteins are dispensable for cbb3-Cox assembly when the external Cu concentration is high. PccA and SenC bind Cu using Met and His residues and Cys and His residues as ligands, respectively, and both proteins form a complex during cbb3-Cox biogenesis. SenC also interacts directly with cbb3-Cox, as shown by chemical cross-linking. Here we determined the periplasmic concentrations of both proteins in vivo and analyzed their Cu binding stoichiometries and their Cu(I) and Cu(II) binding affinity constants ( KD) in vitro. Our data show that both proteins bind a single Cu atom with high affinity. In vitro Cu transfer assays demonstrate Cu transfer both from PccA to SenC and from SenC to PccA at similar levels. We conclude that PccA and SenC constitute a Cu relay system that facilitates Cu delivery to cbb3-Cox.