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

• Published in: FEBS letters Vol. 505; no. 1; pp. 63 - 67
• Main Authors: Nyquist, Rebecca M., Heitbrink, Dirk, Bolwien, Carsten, Wells, Todd A., Gennis, Robert B., Heberle, Joachim
• Format: Journal Article
• Language: English
• Published: England Elsevier B.V 07-09-2001
• Subjects: Animals; ATR, attenuated total reflection; Bacteriorhodopsin; bR, bacteriorhodopsin; Cattle; CcO, cytochrome c oxidase; CuA, copper A; CuB, copper B; Electron transfer; Electron Transport Complex IV - chemistry; Electron Transport Complex IV - metabolism; Enzymes, Immobilized - chemistry; Enzymes, Immobilized - metabolism; Fea, iron of heme a; Fea3, iron of heme a 3; FT-IR, Fourier-transform infrared; IRE, internal reflection element; Lipid; Membrane protein; Mitochondria, Heart - enzymology; Oxidation-Reduction; Perfusion; Phospholipids - chemistry; Proton translocation; R4−O, fully-reduced minus oxidized; Respiration; Rhodobacter sphaeroides - enzymology; Spectroscopy, Fourier Transform Infrared - methods; Vibration; Vibrational spectroscopy; IRE, internal reflection element; Cu A, copper A; Membrane protein; Fe a3, iron of heme a 3; R 4−O, fully-reduced minus oxidized; Cu B, copper B; Electron transfer; ATR, attenuated total reflection; Proton translocation; Lipid; Vibrational spectroscopy; FT-IR, Fourier-transform infrared; Bacteriorhodopsin; Fe a, iron of heme a; bR, bacteriorhodopsin; Respiration; CcO, cytochrome c oxidase
• ISSN: 0014-5793; 1873-3468
• DOI: 10.1016/S0014-5793(01)02769-7

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.