Coupling of Electron Transfer with Proton Transfer at Heme #alpha# and Cu_A (Redox Bohr Effects) in Cytochrome c Oxidase. Studies with the Carbon Monoxide Inhibited Enzyme

摘要

A study is presented on the coupling of electron transfer with proton trnasfer at heme #alpha# and Cu_A (redox Bohr effects) in carbon monoxide inhibited cytochrome c oxidase isolated from bovine heart mitochondria. Detailed analysis of the coupling number for H~+ release per heme #alpha#, Cu_A oxidized (H~+/heme #alpha#, Cu_A ratio) was based on direct measurement of the balance between the oxidizing equivalents added as ferricyanide to the CO-inhibited fully reduced COX, the equivalents of heme #alpha#, Cu_A, and added cytochrome c oxidized and the H~+ released upon oxidation and all taken up back by the oxidase upon rereduction of the metal centers. One of two reductants was used, either succinate plus a trace of mitochondrial membranes (providing a source of succinate-c reductase) or hexaammineruthenium(II) as the chloride salt. The experimental H~+/heme #alpha#, Cu_A ratios varied between 0.65 and 0.90 in the pH range 6.0-8.5. the pH dependence of the H~+/heme #alpha#, Cu_A ratios could be best-fitted by a function involving two redox-linked acid-base groups with pK_0-pK_r of 5.4-6.9 and 7.3-9.0, respectively. Redox titrations in the same samples of the CO-inhibited oxidase showed that Cu_A and heem #alpha# exhibited superimposed E'_m values, which decreased, for both metals, by around 20 mV/pH unit increase in the range 6.0-8.5. A modle in which oxido-reduction of heme #alpha# and Cu_A are both linked to the pK shifts of the two acid-base groups, characterized by the analysis of the E'_m of heme #alpha# and Cu_A. The results presented are consistent with a primary involvement of the redox Bohr effects shared by heme #alpha# and Cu_A in the proton-pumping activity of cytochrome c oxidase.