title: Multi-step assembly pathway of the cbb3-type cytochrome c oxidase complex.
abstract: The cbb3-type cytochrome c oxidases as members of the heme-copper oxidase superfamily are involved in microaerobic respiration in both pathogenic and non-pathogenic proteobacteria. The biogenesis of these multisubunit enzymes, encoded by the ccoNOQP operon, depends on the ccoGHIS gene products, which are proposed to be specifically required for co-factor insertion and maturation of cbb3-type cytochrome c oxidases. Here, the assembly of the cbb3-type cytochrome c oxidase from the facultative photosynthetic model organism Rhodobacter capsulatus was investigated using blue-native polyacrylamide gel electrophoresis. This process involves the formation of a stable but inactive 210 kDa sub-complex consisting of the subunits CcoNOQ and the assembly proteins CcoH and CcoS. By recruiting monomeric CcoP, this sub-complex is converted into an active 230 kDa CcoNOQP complex. Formation of these complexes and the stability of the monomeric CcoP are impaired drastically upon deletion of ccoGHIS. In a ccoI deletion strain, the 230 kDa complex was absent, although monomeric CcoP was still detectable. In contrast, neither of the complexes nor the monomeric CcoP was found in a ccoH deletion strain. In the absence of CcoS, the 230 kDa complex was assembled. However, it exhibited no enzymatic activity, suggesting that CcoS might be involved in a late step of biogenesis. Based on these data, we propose that CcoN, CcoO and CcoQ assemble first into an inactive 210 kDa sub-complex, which is stabilized via its interactions with CcoH and CcoS. Binding of CcoP, and probably subsequent dissociation of CcoH and CcoS, then generates the active 230 kDa complex. The insertion of the heme cofactors into the c-type cytochromes CcoP and CcoO precedes sub-complex formation, while the cofactor insertion into CcoN could occur either before or after the 210 kDa sub-complex formation during the assembly of the cbb3-type cytochrome c oxidase.
authors.affiliation: Institute for Biochemistry and Molecular Biology, Faculty of Medicine, University of Freiburg, D-79104 Freiburg, Germany.
authors.forename: Carmen
authors.initials: C
authors.lastname: Kulajta
authors.forename: Jörg Oliver
authors.initials: JO
authors.lastname: Thumfart
authors.forename: Sybille
authors.initials: S
authors.lastname: Haid
authors.forename: Fevzi
authors.initials: F
authors.lastname: Daldal
authors.forename: Hans-Georg
authors.initials: HG
authors.lastname: Koch
mesh_terms: D001426:Bacterial Proteins / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism; D019069:Cell Respiration / Q000502:physiology; D003067:Coenzymes; D003576:Electron Transport Complex IV / Q000378:metabolism*; D009097:Multienzyme Complexes; D021122:Protein Subunits / Q000378:metabolism; D016354:Rhodobacter capsulatus / Q000166:cytology / Q000201:enzymology*
chemical_list: D001426:Bacterial Proteins; D003067:Coenzymes; D009097:Multienzyme Complexes; D021122:Protein Subunits; C414443:cbb3 oxidase; D003576:Electron Transport Complex IV