Resonance Raman studies on the CuA site of cytochrome c oxidase using a multichannel scanning Raman spectrometer with a CCD detector.

A new Raman measurement system with a CCD (charge coupled device) detector was constructed and applied successfully to observe far-red excited Raman spectra of bovine cytochrome c oxidase. In resonance with the 830-nm absorption band, Raman bands were observed at around 330 cm-1 and assigned to the CuA-S(Cys) and CuA-N(His) stretching vibrations. Although the CuA center has been believed to be coupled with the other redox center and to serve as the electron acceptor from cytochrome c, the frequency and intensity of these bands were influenced by neither the redox and ligation changes in cytochrome alpha 3-CuB center nor the binding of cytochrome c, indicating the highly independent nature of the CuA center. In the higher frequency region of the far-red excited Raman spectra, some protein vibrations which could not be observed in resonance Raman spectra of heme proteins upon visible excitation were observed together with some preresonant heme modes. The amide I and III frequencies indicated a predominance of alpha-helix in the enzyme, and the amino hydrogens were scarcely exchanged with deuterons in D2O, while some Trp side chain bands clearly displayed the deuteration shift.