Time Evolution of the Intermediates Formed in the Reaction of Oxygen with Mixed-Valence Cytochrome c Oxidase

The intermediates formed in the reaction of oxygen with mixed-valence cytochrome c oxidase were followed as a function of time by monitoring the resonance Raman and optical absorption spectra with a flow-flash-probe technique. At early times (50 ms), the optical absorption spectra confirm the presence of the primary intermediate (I_m). This intermediate has an Fe-O₂ stretching mode in the resonance Raman spectrum at 568 cm^-1 that was used as a marker for its population. By following the change in intensity of this mode, the first-order rate constant for the decay of I_m was measured to be 4.5 × 103 s^-1. The formation of the next intermediate (II_m) of the enzyme with a 607-nm peak in the optical absorption difference spectrum was monitored by its optical absorption spectrum and displays a first-order rate constant for its formation, which is the same as the rate constant for the decay I_m. The resonance Raman spectrum of I_m and II_m are nearly identical, indicating that the π-electron depletion of the porphyrin macrocycle in cytochrome a₃ is the same for these two intermediates. The absorption spectrum and the resonance Raman spectrum of an intermediate formed by mixing oxygen with CO-bound mixed-valence cytochrome c oxidase and allowing O₂ to spontaneously replace CO are the same as those of II_m formed in the photolysis experiments. II_m, formed in this way, decays biphasically to the resting form of the enzyme.