Slow proton transfer through the pathways for pumped protons in cytochrome c oxidase induces suicide inactivation of the enzyme.

In the absence of subunit III the aa(3)-type cytochrome c oxidase exhibits a shortened catalytic life span (total number of turnovers) due to an increased probability of undergoing irreversible inactivation during steady-state turnover. Inactivation results from structural alteration of the heme a(3)-Cu(B) active site in subunit I [Hosler (2004) Biochim. Biophys. Acta 1655, 332-339]. The absence of subunit III also dramatically slows proton uptake to the active site via the D proton pathway, as well as inhibiting the proton backflow/exit pathway that connects the active site/proton pump with the outer surface of the oxidase complex. Here we demonstrate that these phenomena are linked: slow proton delivery to the active site through these pathways induces suicide inactivation, thus shortening the catalytic life span of the enzyme. Mutations that inhibit the D pathway, but not the K pathway, increase the probability of suicide inactivation. Strong inhibition of the D pathway allows suicide inactivation to occur even in the presence of subunit III. Arachidonic acid, which stimulates proton uptake by the D pathway, retards suicide inactivation. Steady-state turnover in the presence of DeltaPsi and DeltapH, which inhibits proton uptake from the inner surface of the protein, enhances suicide inactivation. Simultaneous inhibition of proton uptake from both sides of the protein by a double mutation affecting the D pathway and the proton backflow/exit pathway greatly shortens the catalytic life span of the oxidase even in the presence of subunit III. Thus, maintenance of rapid proton transfer through the D pathway and the backflow/exit pathway is one mechanism by which subunit III normally functions to prevent suicide inactivation of cytochrome c oxidase. The experiments suggest that increased lifetimes of the heme a(3) oxoferryl intermediates as well as the anionic form of Glu286 of the D pathway cause suicide inactivation in the active site.