Abstract
Internal electron transfer in bovine cytochrome c oxidase was initiated by CO photolysis of the CO-bound mixed-valence form of the enzyme. Transient absorption spectroscopy was used to monitor changes in the redox states of the metal centers in the enzyme brought about by electron re-equilibration. Upon CO photodissociation, reduced high spin cytochrome a3 was generated in less than 0.1 μsec, and a portion of the reduced cytochrome a3 was reoxidized with biphasic rate constants of k1 = 1.0 × 106 s−1 and k2 = 7.8 × 104 s−1. Concomitant reduction of cytochrome a was also observed with biphasic rate constants of k1 = 1.6 × 106 s−1 and k2 = 9 × 104 s−1. The stoichiometry of cytochrome a3 oxidized to cytochrome a reduced was found to be close to 1:1. Contrary to similar studies in the literature, no reduction of CuA was observed. As a control, no transient absorption changes corresponding to electron transfer was observed in the CO-inhibited fully reduced form of the enzyme. These results indicate that there is significant electron reequilibration only between cytochrome a3 and cytochrome a upon photolysis of the CO-bound mixed-valence enzyme.