Time-Resolved Resonance Raman Investigation of Cytochrome Oxidase Catalysis: Observation of a New Oxygen-Isotope Sensitive Raman Band

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)=O 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 2H2O, 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−–O−–H intermediate. This conclusion forces a reconsideration of the currently accepted view of cytochrome oxidase catalysis.