Abstract
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 Ki of ∼1 µM, close to Kd of calcium binding with the oxidase determined spectrophotometrically. The inhibition is observed only at low, but physiologically relevant, turnover rates of the enzyme (∼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.
Highlights
Cytochrome c oxidase (COX) is a terminal enzyme of the mitochondrial and bacterial respiratory chains
Our conjecture was that the putative regulatory effect of Ca2+ on COX activity, if in existence, would be more pronounced under the conditions of the enzyme turnover close to those in the respiring mitochondria
In the standard assays, turnover rate of cytochrome oxidase is close to Vmax, whereas in the mitochondria respiring on succinate or NADHdependent substrates, COX turns over much slower, ca. 10 s21 or less, even in the fully uncoupled state
Summary
Cytochrome c oxidase (COX) is a terminal enzyme of the mitochondrial and bacterial respiratory chains. The electron transfer in the oxidase is mediated by four metal redox centers: two A-type hemes, low-spin a and high-spin a3, and two copper centers, a binuclear CuA and a mononuclear CuB. The high-spin heme a3 iron and CuB are located within ,5 A from each other and form a di-nuclear site of oxygen reduction to water. In addition to the redox centers, cytochrome oxidases from mitochondria and many bacteria contain non-redox metal ions, revealed by chemical analysis [4] and identified later on in the crystal structure of the enzyme [5,6]. There is Mg2+ (or Mn2+) ion which holds together subunits I and II and may be part of the exit pathway for the pumped protons and for water formed in the active site [7,8,9]. A novel metal cation binding site (CBS) was resolved in the 3D structure of COX from both mitochondria and bacteria (refs. [10,11] and PDB entry 1M56) that can harbour Ca2+ or Na+ ion
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