Suicide Inactivation in Rhodobacter Sphaeroides Cytochrome c Oxidase Lacking Subunit III Coincides with Release of CuB and Major Conformational Changes in Subunit I

Cytochrome c oxidase from Rhodobacter sphaeroides shares homology with the three subunit core of the mitochondrial form. Subunits I (SI) and II contain the redox centers (CuA, heme a, and the binuclear center oxygen binding site, heme a3 and CuB) of the enzyme. Subunit III (SIII) functions to inhibit turnover induced suicide inactivation by maintaining proton uptake into the D pathway and by stabilizing the heme a3-CuB active site. Metal analysis of I/II oxidase, as determined by ICP-OES, shows that suicide inactivation leads to the release of one copper; EPR spectroscopy indicates that the missing copper is CuB. Limited proteolysis of wild-type and I/II oxidase using α-chymotrypsin showed no differences in the pattern of proteolytic digestion, however, SI of I/II oxidase was digested at a faster rate. MALDI-TOF and protein sequencing showed that the cleavage sites are localized to the N and C-termini of SI. Suicide-inactivated I/II oxidase exhibits a completely different digestion pattern, including the release of a peptide (AA 237-258 in SI; identified by MS-MS) that is located in a region above CuB in the enzyme. In an attempt to identify the oxidative reaction which leads to suicide inactivation, the two-electron reduced PM and three-electron reduced F intermediates were generated chemically in I/II oxidase. After incubation, the I/II oxidase was assayed for O2 reduction activity and no inactivation was observed, suggesting that build up of strongly oxidizing chemical intermediates at the active site does not lead to suicide inactivation. These results suggest that suicide inactivation in I/II oxidase is triggered by a mechanism leading to CuB loss with concomitant conformation changes in SI.