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

Rebecca M. Nyquist, Dirk Heitbrink, Carsten Bolwien, Todd A. Wells, Robert B. Gennis, Joachim Heberle

Research output: Contribution to journalArticlepeer-review


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.

Original languageEnglish (US)
Pages (from-to)63-67
Number of pages5
JournalFEBS Letters
Issue number1
StatePublished - Sep 7 2001


  • Bacteriorhodopsin
  • Electron transfer
  • Lipid
  • Membrane protein
  • Proton translocation
  • Respiration
  • Vibrational spectroscopy

ASJC Scopus subject areas

  • Biophysics
  • Structural Biology
  • Biochemistry
  • Molecular Biology
  • Genetics
  • Cell Biology


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