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

FEBS Letters

Wells, Todd A.¹  Heberle, Joachim²  Gennis, Robert B.¹  Bolwien, Carsten²  Heitbrink, Dirk²  Nyquist, Rebecca M.² 

[1] Center for Biophysics and Computational Biology, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA;Forschungszentrum Jülich, IBI-2: Biologische Strukturforschung, 52425 Jülich, Germany

关键词: Vibrational spectroscopy;    Lipid;    Membrane protein;    Respiration;    Proton translocation;    Electron transfer;    Bacteriorhodopsin;    CcO;    cytochrome c oxidase;    bR;    bacteriorhodopsin;    ATR;    attenuated total reflection;    IRE;    internal reflection element;    FT-IR;    Fourier-transform infrared;    R4−O;    fully-reduced minus oxidized;    CuA;    copper A;    CuB;    copper B;    Fea;    iron of heme a;    Fea3;    iron of heme a 3;   

DOI  :  10.1016/S0014-5793(01)02769-7

学科分类：生物化学/生物物理

来源: John Wiley & Sons Ltd.

【 摘 要 】

Attenuated total reflection (ATR) spectroscopy brings an added dimension to studies of structural changes of cytochrome c oxidase (CcO) because it enables the recording of reaction-induced infrared difference spectra under a wide variety of controlled conditions (e.g. pH and chemical composition), without 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 CcO prepared from Rhodobacter sphaeroides and beef heart mitochondria by reconstitution with lipid were adhered to the internal reflection element of the ATR device and retained their full functionality as evidenced by visible spectroscopy and time-resolved vibrational spectroscopy. These results demonstrate that the technique of perfusion-induced Fourier-transform infrared difference spectroscopy can be successfully applied to a large, complex enzyme, such as CcO, with sufficient signal/noise to probe vibrational changes in individual residues of the enzyme under various conditions.

【 授权许可】

Unknown