Charge translocation coupled to electron injection into oxidized cytochrome c oxidase from Paracoccus denitrificans

Electrons were discretely injected into oxidized cytochrome c oxidase in li posomes by laser flash excitation of bound ruthenium [II] bispyridlyl, and the membrane potential was recorded by time-resolved electrometry. Membrane potential is generated in a fast phase when an electron is transferred fro m the excited dye, via the Cu-A 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]. Sub sequently, membrane potential may develop further in a slower event, which is due to proton transfer into the enzyme from the opposite side of the mem brane [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 c onfirm that injection of the first electron into the fully oxidized cytochr ome c oxidase from Paracoccus denitrificans is associated with a fast elect rogenic 11 mus 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 Cts phase only becom es apparent and grows in amplitude upon increasing the number of light flas hes. This occurs in parallel with a decrease in amplitude of the 11 mus pha se and correlates with the number of enzyme molecules that are already redu ced by one electron before the flash. The electrogenic 135 mus phase does n ot appear with increasing flash number in the K354M mutant enzyme, where el ectron and proton transfer into the binuclear center is delayed. We conclud e that the 135 mus phase, and its associated proton uptake, take place on e lectron injection into enzyme molecules where the binuclear heme a(3)-Cu-B site is already reduced by one electron, and that it is accompanied by oxid ation of heme a with a similar time constant. Reduction of heme a is not as sociated with electrogenic proton uptake into the enzyme, neither in the fu lly oxidized nor in the one-electron-reduced enzyme. The extent of the elec trogenic 135 mus phase also rules out the possibility that reduction of the binuclear center by the second electron would be coupled to proton translo cation in addition to the electrogenic uptake of a proton.