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 trans fer at heme a and Cu-A (redox Bohr effects) in carbon monoxide inhibited cy tochrome c oxidase isolated from bovine heart mitochondria. Detailed analys is 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 reduc ed COX, the equivalents of heme a, Cu-A, and added cytochrome c oxidized an d 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 su ccinate plus a trace of mitochondrial membranes (providing a source of succ inate-c reductase) or hexaammineruthenium(II) as the chloride salt. The exp erimental H+/heme a, CuA ratios varied between 0.65 and 0.90 in the pH rang e 6.0-8.5. The pH dependence of the H+/heme a, CUA ratios could be best-fit ted 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 samp les of the GO-inhibited oxidase showed that CUA and heme a exhibited superi mposed E'(m) values, which decreased, for both metals, by around 20 mV/pH u nit 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, ch aracterized by the analysis of the pH dependence of the H+/heme a, CUA rati os, 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 ac tivity of cytochrome c oxidase.