Measurements of the H+/heme a, CuA 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, CuA 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, CuA 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 CuA did produce H+/heme a, CuA ratios significantly lower or higher than 1, respectively. The experimental H+/heme a, CuA 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 CuA to pK shifts of common acid−base groups. The vectorial nature of the proton−electron coupling at heme a/CuA 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 CuA 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/CuA is involved in the proton pump of the oxidase.
The proton/electron coupling ratio at heme a and CuA in bovine heart cytochrome c oxidase
CAPITANIO, Giuseppe;
2000-01-01
Abstract
Measurements of the H+/heme a, CuA 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, CuA 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, CuA 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 CuA did produce H+/heme a, CuA ratios significantly lower or higher than 1, respectively. The experimental H+/heme a, CuA 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 CuA to pK shifts of common acid−base groups. The vectorial nature of the proton−electron coupling at heme a/CuA 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 CuA 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/CuA is involved in the proton pump of the oxidase.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
