Room temperature characterization of the dioxygen intermediates of cytochrome c oxidase by resonance Raman spectroscopy

Abstract

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 02. The ferryl intermediate was formed by reoxidation of the fully reduced enzyme by an excess of H202. Two forms of the oxidized enzyme were prepared by reoxidizing the fully reduced enzyme with 02. 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 u3, 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 (u4) 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.

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