ATR-FTIR difference spectroscopy of the PM intermediate of bovine cytochrome c oxidase

Perfusion-induced attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopy was used to investigate changes induced in protein and cofactors of bovine cytochrome c oxidase when it was converted from the oxidised state to the catalytic Pm intermediate. The transition was induced in a film of detergent-depleted 'fast' oxidase with a buffer containing CO and O-2. The extent of formation of the Pm state was quantitated simultaneously by monitoring formation of its characteristic 607-nm band with a scanned visible beam reflected off the top surface of the prism. The Pm minus 0 FTIR difference spectrum is distinctly different from the redox spectra reported to date and includes features that can be assigned to changes of haem a(3) and surrounding protein. Tentative assignments are made based on vibrational data of related proteins and model compounds. (C) 2002 Elsevier Science B.V All rights reserved.