Factors affecting the H+/e- stoichiometry in mitochondrial cytochrome c oxidase: Influence of the rate of electron flow and transmembrane ΔpH

A study is presented of the factors affecting the H+/e- stoichiometry of the proton pump of mitochondrial cytochrome c oxidase, isolated and reconstituted in phospholipid vesicles (COV). Under lever flow conditions, i.e., in the absence of a transmembrane ΔμH+, the H+/e- ratio, obtained from spectrophotometric measurements of the initial rates of electron flow and H+ release specifically elicited by cytochrome c, varied from around 0 to 1, depending on the actual rate of electron flow through the oxidase. At steady state the H+/e- ratio for the oxidase was specifically depressed by the transmembrane ΔpH. The study of the H+/e- ratio of the pump was complemented by an analysis of the redox pattern of cytochrome c, Cu(A), and heme a. From both sets of results and recent structural data from other groups, it is concluded that the dependence of the H+/e- ratio on the rate of electron flow through the oxidase and transmembrane ΔpH is associated with the possible occurrence of two electron transfer pathways in cytochrome c oxidase, a coupled one (cyt c → Cu(A) → heme a → heme a3-Cu(B)) and a decoupled one (cyt c → Cu(A) → heme a3-Cu(B)). The contributions of the two pathways, differently affected by kinetics and thermodynamic factors, will determine the actual H+/e- ratio of the pump. A possible role of heme a in the proton pump and the physiological implication of the variable H+/e- ratio in the oxidase are discussed.