RAMAN DETECTION OF A PEROXY INTERMEDIATE IN THE HYDROQUINONE-OXIDIZING CYTOCHROME AA(3) OF BACILLUS-SUBTILIS

When the mixed valence, carbon monoxide-bound form of the hydroquinone -oxidizing cytochrome aa(3)-600 of Bacillus subtilis is illuminated in the presence of O-2, it forms a species that corresponds to 'Compound C', first described for the mitochondrial cytochrome c oxidase by Cha nce, Saronio and Leigh (J. Biol. Chem. 250 (1975) 9226-9237). Resonanc e Raman spectra of the this species show a mode at 366 cm(-1) that shi fts to 342 cm(-1) when the experiment is repeated with O-18(2). The ap pearance of this mode is insensitive to deuteration exchange within th e limits of resolution. High- (1200-1700 cm(-1)) and low-frequency (20 0-500 cm(-1)) data, allow us to assign the 366 cm(-1) mode to the Fe3-O stretching vibration of a peroxide adduct where the iron is either low or intermediate spin. This is to our knowledge the first time an O -18(2)-sensitive iron-oxygen stretching: mode has been reported for 'C ompound C', providing strong support for the notion that this species is a peroxide adduct. The observed 366 cm(-1) v(Fe3+-O--O-) frequency is 8 cm(-1) higher than that previously found for a transient peroxy i ntermediate in the reaction between the fully reduced mitochondrial en zyme and O-2. Our observation indicates that, while similar, the metas table peroxyheme a, species reported here differs in the fine details of geometry, protonation state, and/or hydrogen bond status.