Identification of a Ferryl Intermediate of Escherichia coli Cytochrome d Terminal Oxidase by Resonance Raman Spectroscopy

The 680-nm-absorbing “peroxide state” of the Escherichia coli cytochrome d terminal oxidase complex, obtained by addition of excess hydrogen peroxide to the enzyme, is shown to be a ferryl intermediate in the catalytic cycle of the enzyme. This ferryl intermediate is also created by aerobic oxidation of the fully reduced enzyme. Resonance Raman spectra with 647.1-nm excitation show an Fe^IV=O stretching band at 815 cm^-1, a higher frequency than noted in any other ferrylcontaining enzyme to date. The band shows an ¹⁶O/¹⁸O frequency shift of -46 cm^-1, larger than that observed for any porphyrin ferryl species. The Fe^IV=O formulation was unambiguously established by oxidations of the reduced enzyme with ¹⁶O₂, ¹⁸O₂, and ¹⁶O¹⁸O. Only the use of a mixed-isotope gas permitted discrimination between a ferryl and a peroxo structure. A catalytic cycle for the cytochrome d terminal oxidase complex is proposed, and possible reasons for the high v(Fe=O) frequency are discussed.