Mechanism of cytochrome c oxidase-catalyzed reduction of dioxygen to water: evidence for peroxy and ferryl intermediates at room temperature.

Abstract

The reaction between bovine heart cytochrome oxidase and dioxygen was investigated at room temperature following photolysis of the fully reduced CO-bound enzyme. Time-resolved optical absorption difference spectra were collected by a gated multichannel analyzer in the visible region (lambda = 460-720 nm) from 50 ns to 50 ms after photolysis. Singular value decomposition (SVD) analysis indicated the presence of at least seven intermediates. Multiexponential fitting gave the following apparent lifetimes: 1.2 microseconds, 10 microseconds, 25 microseconds, 32 microseconds, 86 microseconds, and 1.3 ms. On the basis of the SVD results and a double difference map, a sequential kinetic mechanism is proposed from which the spectra and time-dependent populations of the reaction intermediates were determined. The ferrous-oxy complex (compound A), with a peak at 595 nm and a trough at 612 nm versus the reduced enzyme, reaches a maximum concentration approximately 30 microseconds after photolysis. It decays to a 1:6 mixture of peroxy species (a3(3+)-O(-)-O-) in which cytochrome a is reduced and oxidized. Cytochrome a3 in both species has a peak at 606 nm versus its oxidized form. The peroxy species decay to a ferryl intermediate, with a peak at 578 nm versus the oxidized enzyme, followed by electron redistribution between CuA and cytochrome a. The two ferryl species reach a maximum concentration approximately 310 microseconds after photolysis. The excellent agreement between the experimental and theoretical spectra of the intermediates provides unequivocal evidence for the presence of peroxy and ferryl species during dioxygen reduction by cytochrome oxidase at room temperature.