Dependence of Cytochrome a3-NO Spectra on the Enzyme Redox Level in Nitrosyl Cytochrome Oxidase

We report the first resonance Raman studies of NO-bound crytochrome oxidase. Resonance Raman scattering and optical absorption spectra have been obtained on the fully reduced enzyme (a2+a23+NO) and the mixed valence enxyme (a3+ a23+ NO). Clear vibrational frequency shifts are detected in the lines associated with cytochrome a in comparing the two redox levels. For example, in the resonance Raman spectra of the fully reduced enzyme obtained with 406.7 nm excitation, a strong line appears at approximately 1611 cm-1 from cytochrome a2+ whereas in the mixed valence preparation yields a spectrum similar cytrochrome a3+ line appears at approximately 1647 cm-1. Similarly, with 441.6 nm excitation the fully reduced preparation yields a spectrum similar to that of carbon monoxide bound cytochrome oxidase and is dominated by the spectrum of reduced cytochrome a. In contrast, in the mixed valence preparation the spectrum has no reduced cytochrome a lines. For both types of NO-bound sample a line appears at approximately 545 cm-1, a frequency similar to that found in NO-bound enzyme appears at equivalent or equal to 1666 cm-1 in the stretching or bending mode. The carbonyl line of the formyl group in the fully reduced NO-bound enzyme appears at ~-1666 cm-1 in the resonance Raman spectrum. In the mixed valence preparation the frequency of the carbonyl line increases by 1.2 cm-1 to ~-1667 cm-1. Thus, this mode in cytochrome a23+ NO is sensitive to the enzyme redox state. In the absence of any direct evidence about the oxidation state of the CuB, we conclude that this sensitivity may result from either a heme-heme interaction, or a copper-heme interaction.