Cox11p is required for stable formation of the CuB and magnesium centers of cytochrome c oxidase

Assembly of the core subunits of the aa(3)-type cytochrome c oxidase in mitochondria and aerobic bacteria such as Rhodobacter sphaeroides requires the association of three subunits and the formation of five to seven metal centers. Several assembly proteins are required for the late stages of oxidase assembly in eukaryotes; some of these are also present in Rb. sphaeroides, To investigate the role of one of these proteins, Cox11p, the mitochondrial-like oxidase of Rb. sphaeroides was overexpressed and purified from cells that lacked cox11, the gene for Cox11p. The oxidase that assembled in the absence of Cox11p lacked Cu-B at the active site and contained greatly reduced amounts of metal at the magnesium/manganese-binding site between subunits I and II. This inactive oxidase, however, did contain hemes a and a(3), Cu-A, and all three subunits, These results indicate that Cox11p is required at a late, perhaps final, step in the assembly of cytochrome oxidase, most likely the insertion of Cu-B. Oxidase which assembled in a strain with a low copy number of cox11 appeared nearly wild type, suggesting that Cox11p is required in substoichiometric amounts for its role in oxidase assembly.