INTRAMOLECULAR ELECTRON-TRANSFER IN CYTOCHROME-C-OXIDASE - A CASCADE OF EQUILIBRIA

Intramolecular electron redistribution in cytochrome c oxidase after photolysis of the partially reduced CO-bound enzyme was followed at a number of different wavelengths by absorption spectroscopy. Spectra were constructed for the first two phases of this process. The first phase (tau = 3 mus) has a spectrum essentially identical to the difference between the Fe(a) and Fe(a3), reduced-minus-oxidized spectra, indicating a 1:1 stoichiometry between the amount of Fe(a3) oxidized and Fe(a) reduced. It is not necessary to invoke reduction or oxidation of other redox carriers in this phase. The second phase (tau = 35 mus) spectrum appears to be a linear combination of the Fe(a3) and Fe(a) reduced-minus-oxidized difference spectra, reflecting the oxidation of four parts of Fe(a3) for every part of Fe(a) oxidized. This process can be described in terms of transfer to Cu(A) of electrons from the Fe(a3) <-> Fe(a) equilibrium system established in the first phase. The relative contributions of Fe(a3) and Fe(a) in the second phase allow us to calculate the equilibrium constant for Fe(a3) <-> Fe(a) electron exchange, which yields a DELTAE(m) of 36 mV for the two centers (Fe(a3) more positive). Together with the apparent rate constant for the fast phase, this equilibrium constant yields, in turn, the forward (k(f)) and reverse (k(r)) rates for electron transfer from Fe(a) to Fe(a3) as follows: k(f) = 2.4 X 10(5) s-1 and k(r) = 6 X 10(4) s-1. k(f) is much faster than any observed step in the reaction of the reduced enzyme with O2. Thus, the catalytic mechanism of O2 reduction to water is not rate-limited by electron transfer from Fe(a) to the binuclear Fe(a3)/Cu(B) site.