PHOTOLYTIC ACTIVITY OF EARLY INTERMEDIATES IN DIOXYGEN ACTIVATION ANDREDUCTION BY CYTOCHROME-OXIDASE

Time-resolved resonance Raman spectra have been recorded during the re action of fully reduced (a(2+)a(3)(2+)) cytochrome oxidase with dioxyg en at room temperature. We have monitored the Fe2+-O-2 vibration at 57 1 cm(-1) and the time course of reaction photolability. Our results in dicate that, in addition to the a(3)(2+)-O-2 species, the following in termediate in the reaction sequence, which can be described as a perox y species with a total of three reducing equivalents in the binuclear center, is also photolabile and can be photolyzed to regenerate the fu lly reduced enzyme. The apparent rate constant that we observe for the decay of photolytic activity is similar to 10(4) s(-1), which correla tes with other relaxation phenomena that have been observed in the O-2 reduction reaction, We suggest that the underlying process that gover ns these phenomena is an input/output configurational transition assoc iated with the proton-pumping activity of the enzyme. These results on the kinetics of the photolytic activity of the early intermediates in the cytochrome/O-2 reaction resolve apparent differences between our earlier results and interpretation of the oxidase/O-2 reaction time co urse and those of Blackmore, Greenwood, and Gibson (J. Biol. Chem. 199 1, 266, 19245). We have also recorded Raman spectra in the low-frequen cy (200-500 cm(-1)) region during the oxidase/O-2 reaction that show t he v(Fe2+-his) stretching vibration in photoproducts that result from CO, O-2, and peroxy adduct photolysis. The photolysis products that ca n be generated during the O-2 reduction reaction have vibrational prop erties similar to those of the CO photolysis product, which suggests t hat the relaxation dynamics of heme a(3) following ligand photolysis a re independent of ligand.