The reduction of dioxygen by cytochrome oxidase has been investigated, in the time interval between 0.1 and 5.4 ms after initiation of the reaction, by using time-resolved resonance Raman spectroscopy combined with the Artificial Cardiovascular System [Ogura et al., Biochemistry, 28, 8022 (1989)]. At 0.5 ms we observed a new oxygen-isotope sensitive Raman band at 356 cm-1 for 16O2, which shifted down to 341 cm-1 for 18O2. The temporal behavior of this band was found to be very close to that of another oxygen-isotope sensitive band at 788 cm-1 which had been assigned previously to the Fe(IV)=0 stretching mode of the ferryloxo intermediate [Ogura et al., J. Biol. Chem., 265, 14721(1990)]. However, the fact that the 788 cm"1 band shifts to a higher frequency (802 cm"1) in 2H20, taken together with the present detection of two oxygen-isotope sensitive bands, prompts us to suggest an alternative assignment; namely, that these bands arise from the O--O- (788 cm-1) and Fe(III)-O- (356 cm-1) stretching modes of the Fe(III)-O--0--H intermediate. This conclusion forces a reconsideration of the currently accepted view of cytochrome oxidase catalysis.
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