Nytrosyl cytochrome c oxidase: Formation and properties of mixed-valence enzyme.

We report the first resonance Raman scattering studies of NO- bound cytochrome c oxidase. Resonance Raman scattering and optical absorption spectra have been obtained on the fully reduced enzyme (a sup (2+,) a sup (2+ over 3 NO) and the mixed valence enzyme (a sup (3+,) a sup (2+ over 3 NO). Clear vibrational frequency shifts are detected in the lines associated with cytochrome a in comparing the two redox states. With 441.6 nm excitation the fully reduced preparation yields a spectrum similar to that of carbon monoxide bound cytochrome c oxidase and is dominated by the spectrum of reduced cytochrome a. In contrast, in the mixed valence preparation no contributions from reduced cytochrome a are evident in the spectrum, verifying that this heme is no longer in the Fe sup (+2) state. In the mixed valence NO-bound samples a line appears at ~545cm sup (-1), a frequency similar to that found in NO-bound hemoglobin and myoglobin and assigned as an Fe-N-O bending mode in those proteins. We do not detect this line in the spectrum of the fully reduced NO-bound enzyme.