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 LU 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 a(3)-Cu-B active site is normal. However, subunit III (-) spontaneously and irreversibly inactivates during O-2 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 O-2 reduction intermediates is lost, and CO binding experiments suggest that the access of O-2 to reduced heme a(3) is inhibited. Reduced heme a accumulates in response to a decrease in the redox potential of heme a(3); 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. Cu-B is partially lost. It is proposed that suicide inactivation results from the dissociation of a ligand of Cu-B and that subunit III functions to prevent suicide inactivation by maintaining the structural integrity of the Cu-B center via long-range interactions.