PM and PR 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 Sorel regions following photolysis of the mixed-valence CO-bound enzyme. Time-resolved optical absorption difference spectra were collected between 50 mus 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 mus. 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, upon dissociation of CO from Cu-B(+) and concomitant back-electron transfer from heme a, 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 H2O2 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. (C) 2002 Elsevier Science Inc. All rights reserved.