Stability of the cbb3-type cytochrome oxidase requires specific CcoQ-CcoP interactions.
Cytochrome cbb(3)-type oxidases are members of the heme copper oxidase superfamily and are composed of four subunits. CcoN contains the heme b-Cu(B) binuclear center where oxygen is reduced, while CcoP and CcoO are membrane-bound c-type cytochromes thought to channel electrons from the donor cytochrome into the binuclear center. Like many other bacterial members of this superfamily, the cytochrome cbb(3)-type oxidase contains a fourth, non-cofactor-containing subunit, which is termed CcoQ. In the present study, we analyzed the role of CcoQ on the stability and activity of Rhodobacter capsulatus cbb(3)-type oxidase. Our data showed that CcoQ is a single-spanning membrane protein with a N(out)-C(in) topology. In the absence of CcoQ, cbb(3)-type oxidase activity is significantly reduced, irrespective of the growth conditions. Blue native polyacrylamide gel electrophoresis analyses revealed that the lack of CcoQ specifically impaired the stable recruitment of CcoP into the cbb(3)-type oxidase complex. This suggested a specific CcoQ-CcoP interaction, which was confirmed by chemical cross-linking. Collectively, our data demonstrated that in R. capsulatus CcoQ was required for optimal cbb(3)-type oxidase activity because it stabilized the interaction of CcoP with the CcoNO core complex, leading subsequently to the formation of the active 230-kDa cbb(3)-type oxidase complex.
Zentrum für Biochemie und Molekulare Zellforschung, Institut für Biochemie und Molekularbiologie, Albert-Ludwigs-Universität Freiburg, Stefan-Maier-Str. 17, 79104 Freiburg, Germany.
Annette
A
Peters
Carmen
C
Kulajta
Grzegorz
G
Pawlik
Fevzi
F
Daldal
Hans-Georg
HG
Koch
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000378:metabolism*; D003576:Electron Transport Complex IV / Q000737:chemistry* / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability; D008565:Membrane Proteins / Q000302:isolation & purification / Q000378:metabolism; D008954:Models, Biological; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011485:Protein Binding; D021122:Protein Subunits / Q000378:metabolism*; D016354:Rhodobacter capsulatus / Q000201:enzymology*
D001426:Bacterial Proteins; D008565:Membrane Proteins; D021122:Protein Subunits; C414443:cbb3 oxidase; D003576:Electron Transport Complex IV