PM and PR forms of cytochrome c oxidase have different spectral properties

Abstract

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 μs. 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 a3 upon dissociation of CO from CuB+ and concomitant back-electron transfer from heme a3 to heme a. This is followed by O2 binding to heme a3 forming compound A (AM), with a spectrum identical to that observed upon O2 binding to heme a3 in the fully reduced enzyme (AR). Intermediate AM decays into PM, 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 (PH). However, the spectrum of PM is significantly different from the corresponding intermediate observed upon the reaction of dioxygen with the fully reduced enzyme (PR). The spectral differences between PM and PR may arise from the different number of redox equivalents at the binuclear site, with a tyrosine radical in the PM state, and tyrosine or tyrosinate in PR, or may be the consequence of a more complex reaction mechanism in the case of the fully reduced enzyme.