Stability of the cbb 3 -Type Cytochrome Oxidase Requires Specific CcoQ-CcoP Interactions

Cytochromecbb 3 -type oxidases are members of the heme copper oxidase superfamily and are composed of four subunits. CcoN contains the hemeb -CuB binuclear center where oxygen is reduced, while CcoP and CcoO are membrane-boundc -type cytochromes thought to channel electrons from the donor cytochrome into the binuclear center. Like many other bacterial members of this superfamily, the cytochromecbb 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 ofRhodobacter capsulatus cbb 3 -type oxidase. Our data showed that CcoQ is a single-spanning membrane protein with a Nout -Cin 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 thecbb 3 -type oxidase complex. This suggested a specific CcoQ-CcoP interaction, which was confirmed by chemical cross-linking. Collectively, our data demonstrated that inR. capsulatus CcoQ was required for optimalcbb 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-kDacbb 3 -type oxidase complex.