Coupling of electron transfer with proton transfer at heme a and CuA (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 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 a, CUA oxidized (H+/ heme a, Cu-A ratio) was based on direct measurement of the balance between the oxidizing equivalents added as ferricyanide to the GO-inhibited fully reduced COX, the equivalents of heme a, 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 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 pK(o)-pK(r) of 5.4-6.9 and 7.3-9.0, respectively. Redox titrations in the same samples of the GO-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 oxide-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.