Net proton uptake is preceded by multiple proton transfer steps upon electron injection into cytochrome c oxidase.

Cytochrome c oxidase (COX), the last enzyme of the respiratory chain of aerobic organisms, catalyzes the reduction of molecular oxygen to water. It is a redox-linked proton pump, whose mechanism of proton pumping has been controversially discussed, and the coupling of proton and electron transfer is still not understood. Here, we investigated the kinetics of proton transfer reactions following the injection of a single electron into the fully oxidized enzyme and its transfer to the hemes using time-resolved absorption spectroscopy and pH indicator dyes. By comparison of proton uptake and release kinetics observed for solubilized COX and COX-containing liposomes, we conclude that the 1-μs electron injection into Cu(A), close to the positive membrane side (P-side) of the enzyme, already results in proton uptake from both the P-side and the N (negative)-side (1.5 H(+)/COX and 1 H(+)/COX, respectively). The subsequent 10-μs transfer of the electron to heme a is accompanied by the release of 1 proton from the P-side to the aqueous bulk phase, leaving ∼0.5 H(+)/COX at this side to electrostatically compensate the charge of the electron. With ∼200 μs, all but 0.4 H(+) at the N-side are released to the bulk phase, and the remaining proton is transferred toward the hemes to a so-called "pump site." Thus, this proton may already be taken up by the enzyme as early as during the first electron transfer to Cu(A). These results support the idea of a proton-collecting antenna, switched on by electron injection.