Attenuated total reflection Fourier transform infrared studies of redox changes in bovine cytochrome c oxidase: resolution of the redox Fourier transform infrared difference spectrum of heme a(3).

Attenuated total reflection Fourier transform infrared (ATR-FTIR) difference spectroscopy has been performed on samples of bovine cytochrome c oxidase that have been deposited as a thin film on the surface of a silicon microprism. The technique has several advantages over transmission methods in terms of amount of material required, the time required to reach sufficient optical stability, and the range of reactants that can be repetitively added and removed. The ATR-FTIR method has been used to record redox difference spectra of cytochrome c oxidase in the unligated and cyanide-ligated states. By subtraction of the spectra, the redox FTIR difference spectrum of heme a(3) can be resolved from those of the other metal centers. This difference spectrum is compared with available vibrational and Raman data on homologous oxidases and on heme A model compounds.