Resonance Raman and optical spectroscopic monitoring of heme a redox states in cytochrome c oxidase during potentiometric titrations.

Resonance Raman spectroscopy is used to monitor the redox state of heme a and heme a3 centers in cyanide-inhibited and native cytochrome oxidase during potentiometric titrations. Specific vibrational modes are resolved for each reduced heme with 441.6-nm excitation while oxidized species show vanishingly small Raman intensities. The voltage dependencies of the Raman intensities of reduced heme a and reduced heme a3 modes are quantitatively measured and used to extract heme a and a3 midpoint potentials. In the cyanide-bound enzyme, in which heme a3 remains in the oxidized state, the Raman data indicate that heme a centers exhibit complex Nernstian behavior with two Em values near 350 and 260 mV. In the native enzyme, this resonance Raman-potentiometric method reveals significantly different redox behavior for the two hemes. Heme a centers are described by two effective Em values near 350 and 220 mV, while heme a3 centers have lower Em values near 260 and 200 mV. Singular value decomposition analysis of optical spectral changes supports the Raman data. These results are in contrast to models of cytochrome oxidase redox behavior in which heme a and heme a3 are thought to have essentially identical midpoint potentials.