Photolytic activity of early intermediates in dioxygen activation and reduction by cytochrome oxidase

Abstract

Time-resolved resonance Raman spectra have been recorded during the reaction of fully reduced (a2+a32+) cytochrome oxidase with dioxygen at room temperature. We have monitored the Fe2+-O2 vibration at 571 cm-1 and the time course of reaction photolability. Our results indicate that, in addition to the a32+-O2 species, the following intermediate in the reaction sequence, which can be described as a peroxy species with a total of three reducing equivalents in the binuclear center, is also photolabile and can be photolyzed to regenerate the fully reduced enzyme. The apparent rate constant that we observe for the decay of photolytic activity is ∼104 s-1, which correlates with other relaxation phenomena that have been observed in the O2 reduction reaction. We suggest that the underlying process that governs these phenomena is an input/output configurational transition associated with the proton-pumping activity of the enzyme. These results on the kinetics of the photolytic activity of the early intermediates in the cytochrome/O2 reaction resolve apparent differences between our earlier results and interpretation of the oxidase/O2 reaction time course and those of Blackmore, Greenwood, and Gibson (J. Biol. Chem. 1991, 266, 19245). We have also recorded Raman spectra in the low-frequency (200-500 cm-1) region during the oxidase/O2 reaction that show the V(Fe2+-his) stretching vibration in photoproducts that result from CO, O2, and peroxy adduct photolysis. The photolysis products that can be generated during the Ch reduction reaction have vibrational properties similar to those of the CO photolysis product, which suggests that the relaxation dynamics of heme O3 following ligand photolysis are independent of ligand