Direct Regulation of Cytochrome c Oxidase by Calcium Ions

Cytochrome c oxidase from bovine heart binds Ca2+ reversibly at a specific Cation Binding Site located near the outer face of the mitochondrial membrane. Ca2+ shifts the absorption spectrum of heme a, which allowed previously to determine the kinetics and equilibrium characteristics of the binding. However, no effect of Ca2+ on the functional characteristics of cytochrome oxidase was revealed earlier. Here we report that Ca2+ inhibits cytochrome oxidase activity of isolated bovine heart enzyme by 50-60% with K-i of similar to 1 mu M, close to K-d of calcium binding with the oxidase determined spectrophotometrically. The inhibition is observed only at low, but physiologically relevant, turnover rates of the enzyme (similar to 10 s(-1) or less). No inhibitory effect of Ca2+ is observed under conventional conditions of cytochrome c oxidase activity assays (turnover number >100 s(-1) at pH 8), which may explain why the effect was not noticed earlier. The inhibition is specific for Ca2+ and is reversed by EGTA. Na+ ions that compete with Ca2+ for binding with the Cation Binding Site, do not affect significantly activity of the enzyme but counteract the inhibitory effect of Ca2+. The Ca2+-induced inhibition of cytochrome c oxidase is observed also with the uncoupled mitochondria from several rat tissues. At the same time, calcium ions do not inhibit activity of the homologous bacterial cytochrome oxidases. Possible mechanisms of the inhibition are discussed as well as potential physiological role of Ca2+ binding with cytochrome oxidase. Ca2+-binding at the Cation Binding Site is proposed to inhibit proton-transfer through the exit part of the proton conducting pathway H in the mammalian oxidases.