Proton uptake controls electron transfer in cytochrome c oxidase

In cytochrome c oxidase, a requirement for proton pumping is a tight coupling between electron and proton transfer, which could be accomplished if internal electron-transfer rates were controlled by uptake of protons. During reaction of the fully reduced enzyme with oxygen, concomitant with the “peroxy” to “oxoferryl” transition, internal transfer of the fourth electron from Cu(A) to heme a has the same rate as proton uptake from the bulk solution (8,000 s(−1)). The question was therefore raised whether the proton uptake controls electron transfer or vice versa. To resolve this question, we have studied a site-specific mutant of the Rhodobacter sphaeroides enzyme in which methionine 263 (SU II), a Cu(A) ligand, was replaced by leucine, which resulted in an increased redox potential of Cu(A). During reaction of the reduced mutant enzyme with O(2), a proton was taken up at the same rate as in the wild-type enzyme (8,000 s(−1)), whereas electron transfer from Cu(A) to heme a was impaired. Together with results from studies of the EQ(I-286) mutant enzyme, in which both proton uptake and electron transfer from Cu(A) to heme a were blocked, the results from this study show that the Cu(A) → heme a electron transfer is controlled by the proton uptake and not vice versa. This mechanism prevents further electron transfer to heme a(3)–Cu(B) before a proton is taken up, which assures a tight coupling of electron transfer to proton pumping.