Coupling of electron transfer with proton transfer at heme a and Cu(A) (redox Bohr effects) in cytochrome c oxidase. Studies with the carbon monoxide inhibited enzyme

A study is presented on the coupling of electron transfer with proton transfer at heme a and CuA (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 a, CuA oxidized (H+/heme a, CuA 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 a, CuA, 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 a, CuA ratios varied between 0.65 and 0.90 in the pH range 6.0−8.5. The pH dependence of the H+/heme a, CuA ratios could be best-fitted by a function involving two redox-linked acid−base groups with pKo−pKr of 5.4−6.9 and 7.3−9.0, respectively. Redox titrations in the same samples of the CO-inhibited oxidase showed that CuA and heme a 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 model in which oxido−reduction of heme a and CuA are both linked to the pK shifts of the two acid−base groups, characterized by the analysis of the pH dependence of the H+/heme a, CuA ratios, provided a satisfactory fit for the pH dependence of the E‘m of heme a and CuA. The results presented are consistent with a primary involvement of the redox Bohr effects shared by heme a and CuA in the proton-pumping activity of cytochrome c oxidase.