Nitrosyl 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 (a²⁺, a₃²⁺ NO) and the mixed valence enzyme (a³⁺, a₃²⁺ 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 Fe²⁺ state. In the mixed valence NO-bound samples, a line appears at ~545 cm^-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. The carbonyl line of the cytochrome a₃ heme formyl group in the fully reduced NO-bound enzyme appears at ≃1666 cm^-1 in the resonance Raman spectrum. In the mixed valence NO-bound preparation the frequency of the carbonyl line increases by 1.2 cm^-1 to ≃1667 cm^-1. Thus, modes in cytochrome a₃^2- NO are sensitive to the redox state of the cytochrome a and/or Cu(A) centers. We propose that the redox sensitivity of the formyl mode and the Fe-N-O mode results from an interaction between cytochrome a₃²⁺ (NO) and the cytochrome a-Cu(A) pair, and is linked to the cytochrome a₃ (NO)) by the coupling between Cu(B) and the NO-bound cytochrome a₃ heme.