Charge Translocation Coupled to Electron Injection into Oxidized CytochromecOxidase fromParacoccus denitrificans

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Abstract
Electrons were discretely injected into oxidized cytochrome c oxidase in liposomes by laser flash excitation of bound ruthenium [II] bispyridyl, and the membrane potential was recorded by time-resolved electrometry. Membrane potential is generated in a fast phase when an electron is transferred from the excited dye, via the CuA center, to heme a at a relative dielectric depth d inside the membrane [Zaslavsky, D., Kaulen, A. D., Smirnova, I. A., Vygodina, T., and Konstantinov, A. A. (1993) FEBS Lett. 336, 389−393]. Subsequently, membrane potential may develop further in a slower event, which is due to proton transfer into the enzyme from the opposite side of the membrane [Ruitenberg, M., Kannt, A., Bamberg, E., Ludwig, B., Michel, H., and Fendler, K. (2000) Proc. Natl. Acad. Sci. U.S.A. 97, 4632−4636]. Here, we confirm that injection of the first electron into the fully oxidized cytochrome c oxidase from Paracoccus denitrificans is associated with a fast electrogenic 11 μs phase, but there is no further electrogenic phase up to 100 milliseconds when special care is taken to ensure that only fully oxidized enzyme is present initially. A slower electrogenic 135 μs phase only becomes apparent and grows in amplitude upon increasing the number of light flashes. This occurs in parallel with a decrease in amplitude of the 11 μs phase and correlates with the number of enzyme molecules that are already reduced by one electron before the flash. The electrogenic 135 μs phase does not appear with increasing flash number in the K354M mutant enzyme, where electron and proton transfer into the binuclear center is delayed. We conclude that the 135 μs phase, and its associated proton uptake, take place on electron injection into enzyme molecules where the binuclear heme a3−CuB site is already reduced by one electron, and that it is accompanied by oxidation of heme a with a similar time constant. Reduction of heme a is not associated with electrogenic proton uptake into the enzyme, neither in the fully oxidized nor in the one-electron-reduced enzyme. The extent of the electrogenic 135 μs phase also rules out the possibility that reduction of the binuclear center by the second electron would be coupled to proton translocation in addition to the electrogenic uptake of a proton.