CONTROL OF ELECTRON DELIVERY TO THE OXYGEN REDUCTION SITE OF CYTOCHROME-C-OXIDASE - A ROLE FOR PROTONS

We have studied the reaction of oxidized ''pulsed'' cytochrome c oxidase with reduced cytochrome c and with ruthenium(II) hexaammine using stopped-flow mixing. The rate of reduction of Fe-alpha 3 (the oxygen-binding heme) is not a linear function of the population of reduced Fe-alpha (the low-spin heme), as would be expected if electron transfer between these sites is rate-limiting. Instead, the rate can be increased significantly by increasing the driving force of the reductant (lowering of E(h)) even after Fe-alpha is almost completely reduced. Reduction of Fe-alpha 3 becomes slower as the pH is raised, and consumption of protons can be seen simultaneously with electron entry into Fe-alpha 3. Both the reduction of Fe-alpha 3 and the proton uptake are biphasic. To explain these findings, we propose a model in which (1) intramolecular heme-heme electron transfer is fast, and has an essentially constant rate; (2) when reduction begins, the midpoint potentials of Fe-alpha 3 and Cu-B are initially low, and only a small fraction of these centers become reduced; and (3) this reduced population is then stabilized by the uptake of protons. Thus, net reduction of Fe-alpha 3 and Cu-B is controlled by the amount of the low-potential population which becomes reduced together with the rate of proton uptake by this reduced low-potential species. Important consequences of this mechanism for the function of the enzyme and for the respiratory chain as a whole are discussed.