Suicide Inactivation of Cytochrome c Oxidase: Catalytic Turnover in the Absence of Subunit III Alters the Active Site

The catalytic core of cytochrome c oxidase is composed of three subunits: I, II, and III. Subunit III is a highly hydrophobic membrane protein that contains no redox centers; its role in cytochrome oxidase function is not obvious. Here, subunit III has been removed from the three-subunit mitochondrial-like oxidase of Rhodobacter sphaeroides by detergent washing. The resulting two-subunit oxidase, subunit III (−), is highly active. Ligand-binding analyses and resonance Raman spectroscopy show that its heme a3−CuB active site is normal. However, subunit III (−) spontaneously and irreversibly inactivates during O2 reduction. At pH 7.5, its catalytic lifetime is only 2% that of the normal oxidase. This suicide inactivation event primarily alters the active site. Its ability to form specific O2 reduction intermediates is lost, and CO binding experiments suggest that the access of O2 to reduced heme a3 is inhibited. Reduced heme a accumulates in response to a decrease in the redox potential of heme a3; electron transfer between the hemes is inhibited. Ligand-binding experiments and resonance Raman analysis show that increased flexibility in the structure of the active site accompanies inactivation. CuB is partially lost. It is proposed that suicide inactivation results from the dissociation of a ligand of CuB and that subunit III functions to prevent suicide inactivation by maintaining the structural integrity of the CuB center via long-range interactions.