Room Temperature Characterization of the Dioxygen Intermediates of Cytochrome c Oxidase by Resonance Raman Spectroscopy

Resonance Raman spectroscopy was employed to investigate the heme structures of catalytic intermediates of cytochrome c oxidase at room temperature. The high-frequency resonance Raman spectra were obtained for compound C (the two-electron-reduced dioxygen intermediate), ferryl (the three-electron-reduced dioxygen intermediate), and the fully oxidized enzyme. Compound C was formed by photolyzing CO mixed-valence enzyme in the presence of O₂. The ferryl intermediate was formed by reoxidation of the fully reduced enzyme by an excess of H₂O₂. Two forms of the oxidized enzyme were prepared by reoxidizing the fully reduced enzyme with O₂. Our data indicate that, in compound C, cyt a₃ is either intermediate or low spin and is nonphotolabile and its oxidation state marker band, v₄, appears at a higher frequency than that of the resting form of the enzyme. The ferryl intermediate also displays a low-spin cyt a₃, which is nonphotolabile, and an even higher frequency for the oxidation state marker band, v₄. The reoxidized form of cytochrome c oxidase with a Soret absorption maximum at 420 nm has an oxidation state marker band (v₄) in a position similar to that of the resting form, while the spin-state region resembles that of compound C. This species subsequently decays to a second oxidized form of the enzyme, which displays a high-frequency resonance Raman spectrum identical with that of the original resting enzyme.