Raman detection of a peroxy intermediate in the hydroquinone-oxidizing cytochrome aa₃ of Bacillus subtilis.

C. Varotsis, G.T. Babcock, M. Lauraeus, and M. Wikstršm.Biochim. Biophys. Acta 1231, 111-116, 1995.

SUMMARY: When the mixed valence, carbon monoxide-bound form of the hydroquinone-oxidizing cytochrome aa₃-600 of Bacillus subtilis is illuminated in the presence of O₂, it forms a species that corresponds to ÔCompound CÕ, first described for the mitochondrial cytochrome c oxidase by Chance, Saronio and Leigh (J. Biol. Chem. 250, 9226-9237, 1975). Resonance Raman spectra of this species show a mode at 366 cm^-1 that shifts to 342 cm^-1 when the experiment is repeated with ¹⁸O₂. The appearance of this mode is insensitive to deuteration exchange within the limits of resolution. High- (1200-1700 cm^-1) and low- frequency (200-500 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 ¹⁸O₂-sensitive iron-oxygen stretching mode has been reported for ÔCompound CÕ, providing strong support for the notion that this species is a peroxide adduct. The observed 366 cm^-1 u(Fe³⁺-O^-- O^-) frequency is 8 cm^-1 higher than that previously found for a transient peroxy intermediate in the reaction between the fully reduced mitochondrial enzyme and O₂. Our observation indicates that, while similar, the metastable peroxyheme a₃ species reported here differs in the fine details of geometry, protonation state, and/or hydrogen bond status.