P(M) and P(R) forms of cytochrome c oxidase have different spectral properties.

The reaction between bovine heart cytochrome c oxidase and dioxygen was monitored at room temperature in the visible and Soret regions following photolysis of the mixed-valence CO-bound enzyme. Time-resolved optical absorption difference spectra were collected between 50 ns and 1.7 ms by a gated multichannel analyzer. Singular value decomposition and global exponential fitting resolved three processes with apparent lifetimes of 2.2+/-0.5, 17+/-4 and 160+/-30 micros. The spectra of the intermediates were extracted based on a sequential kinetic mechanism and compared to the corresponding intermediate spectra observed during the reaction of the fully reduced enzyme with dioxygen. The first process is associated with a conformational change at heme a(3) upon dissociation of CO from Cu(B)(+) and concomitant back-electron transfer from heme a(3) to heme a. This is followed by O(2) binding to heme a(3) forming compound A (A(M)), with a spectrum identical to that observed upon O(2) binding to heme a(3) in the fully reduced enzyme (A(R)). Intermediate A(M) decays into P(M), the spectrum of which is equivalent to that of the 607 nm form, generated upon addition of H(2)O(2) to the oxidized enzyme at alkaline pH values (P(H)). However, the spectrum of P(M) is significantly different from the corresponding intermediate observed upon the reaction of dioxygen with the fully reduced enzyme (P(R)). The spectral differences between P(M) and P(R) may arise from the different number of redox equivalents at the binuclear site, with a tyrosine radical in the P(M) state, and tyrosine or tyrosinate in P(R), or may be the consequence of a more complex reaction mechanism in the case of the fully reduced enzyme.