Absorption changes during the O2 reaction of reduced bovine cytochrome c oxidase were investigated by the rapid-reaction technique of flow-flash spectrophotometry in the Soret, visible and near-i.r. spectral regions. New features in the time courses of absorption change were observed relative to the earlier findings reported by Greenwood & Gibson [(1967) J. Biol. Chem. 242, 1782-1787]. These new features arise in the Soret and near-i.r. regions and allow the reaction to be described at all wavelengths as a composite of three exponential processes. There is a rapid O2-sensitive phase detectable in the Soret and visible region. The second phase has a rate that is somewhat less dependent on O2 concentration than is the fastest phase rate and is detectable in all three spectral regions. The rate of the third phase is almost independent of the O2 concentration and is also detectable in all spectral regions. Analysis of the three phases gives their rates and absorption amplitudes. The fast phase reaches a rate of 2.5 × 10(4) s-1 at the highest O2 concentration available at 20 degrees C, whereas the phase of intermediate rate is limited at a value of 7 × 10(3) s-1 and the slow phase rate is limited at 700 s-1. The ratios of the kinetic difference spectra for the fast phase and the slow phase do not correspond to the spectra of the individual haem centres. A branched mechanism is advanced that is able to reconcile the kinetic and static difference spectra. This mechanism suggests that some of the cytochrome a is oxidized along with cytochrome a3 in the initial O2-sensitive phase. In addition, the model requires that CuA is oxidized heterogeneously. This fits with the complex time course of oxidation observed at 830 nm while retaining CuA as virtually the sole contributor to absorbance at this wavelength.

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