The proton/electron coupling ratio at heme a and Cu(A) in bovine heart cytochrome c oxidase

Measurements of the H(+)/heme a, Cu(A) ratios for proton-electron coupling at these centers (redox Bohr effect) in CO-inhibited cytochrome c oxidase purified from bovine heart mitochondria, both in the soluble state and reconstituted in liposomes, are presented. In the soluble oxidase, the H(+)/heme a, Cu(A) ratios were experimentally determined upon oxidation by ferricyanide of these centers as well as upon their reduction by hexammineruthenium(II). These measurements showed that in order to obtain H(+)/heme a, Cu(A) ratios approaching 1, one-step full oxidation of both metal centers by ferricyanide had to be induced by a stoicheiometric amount of the oxidant. Partial stepwise oxidation or reduction of heme a and Cu(A) did produce H(+)/heme a, Cu(A) ratios significantly lower or higher than 1, respectively. The experimental H(+)/heme a, Cu(A) ratios measured upon stepwise reduction/oxidation of the metals were reproduced by mathematical simulation based on the coupling of oxido-reduction of both heme a and Cu(A) to pK shifts of common acid-base groups. The vectorial nature of the proton-electron coupling at heme a/Cu(A) was analyzed by measuring pH changes in the external bulk phase associated with oxido-reduction of these redox centers in the CO-inhibited oxidase reconstituted in liposomes. The results show that the proton release associated with the oxidation of heme a and Cu(A) takes place in the external aqueous phase. Protons taken up by the oxidase upon rereduction of the centers derive, on the other hand, from the inner space. These results provide evidence supporting the view that cooperative proton-electron coupling at heme a/Cu(A) is involved in the proton pump of the oxidase.