Ca. Varotsis et Gt. Babcock, PHOTOLYTIC ACTIVITY OF EARLY INTERMEDIATES IN DIOXYGEN ACTIVATION ANDREDUCTION BY CYTOCHROME-OXIDASE, Journal of the American Chemical Society, 117(45), 1995, pp. 11260-11269
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.