Flow-flash, time-resolved resonance Raman spectroscopy of the oxidation of reduced and of mixed valence cytochrome oxidase by dioxygen.

Time-resolved resonance Raman spectroscopy has been used to study the reduction of oxygen by both reduced and mixed valence cytochrome oxidase. Laser flash photodissociation of CO from the carbon monoxy complex of the enzyme, after this species had been rapidly mixed with oxygenated buffer, was used to initiate the reaction for both forms of the enzyme. The CO photolysis product of the mixed valence enzyme contains cytochrome a3+ and cytochrome a3(2+) in its unligated form. This species reacts with O2 in the first few microseconds to form a photolabile intermediate which has Raman frequencies characteristic of oxygenated heme. This indicates that an oxyhemoglobinlike complex of oxygen with a3(2+) is the precursor to oxygen reduction. A similar intermediate is detected in the fully reduced enzyme reaction. In the mixed valence oxidase system, the oxy intermediate is replaced by a nonphotolabile species in which a3 is oxidized with t1/2 approximately equal to 200 musec. These results demonstrate the feasibility of applying time-resolved vibrational techniques to irreversible electron transfer reactions and, in particular, elucidate some of the transient species in the cytochrome oxidase/O2 system.