Perfusion-induced redox differences in cytochrome c oxidase: ATR/FT-IR spectroscopy

Attenuated total reflection (ATR) spectroscopy brings an added dimension to studies of structural changes of cytochrome c oxidase (CcO) because it ena bles the recording of reaction-induced infrared difference spectra under a wide variety of controlled conditions (e.g. pH and chemical composition), w ithout relying on light or potentiometric changes to trigger the reaction. We have used the ATR method to record vibrational difference spectra of CcO with reduction induced by flow-exchange of the aqueous buffer. Films of Cc O prepared from Rhodobacter sphaeroides and beef heart mitochondria by reco nstitution with lipid were adhered to the internal reflection element of th e ATR device and retained their full functionality as evidenced by visible spectroscopy and time-resolved vibrational spectroscopy. These results demo nstrate that the technique of perfusion-induced Fourier-transform infrared difference spectroscopy can be successfully applied to a large, complex enz yme, such as CcO, with sufficient signal/noise to probe vibrational changes in individual residues of the enzyme under various conditions. (C) 2001 Fe deration of European Biochemical Societies. Published by Elsevier Science B .V. All rights reserved.