Abstract
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.
Highlights
The coupling mechanism of proton and electron transfer in the redox-linked proton pump cytochrome c oxidase (COX) is still not understood
By comparison of proton uptake and release kinetics observed for solubilized COX and COX-containing liposomes, we conclude that the 1-s electron injection into CuA, close to the positive membrane side (P-side) of the enzyme, already results in proton uptake from both the P-side and the negatively charged side (N-side) (1.5 H؉/COX and 1 H؉/COX, respectively)
Time-resolved Measurements of Electron Transfer—To observe the kinetics of electrons transferred to heme a in a single-electron photochemical reduction of the enzyme from the light-reactive electron donor Ru2D, the latter was excited with a single laser flash, and the absorbance change at 605 nm was recorded (Fig. 2B, inset)
Summary
The coupling mechanism of proton and electron transfer in the redox-linked proton pump cytochrome c oxidase (COX) is still not understood. 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. Injection of 1 electron into the fully oxidized O state leads to formation of the 1 electron-reduced E state During this step of the catalytic cycle, proton uptake was proposed to take place from the N-side of the membrane via the K-pathway and to be linked to the reduction of heme a [23]. We present a model that contains important aspects of each of the previous models and is, in a sense, a unification of these hypotheses
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