Direct observation of protonation reactions during the catalytic cycle of cytochrome c oxidase

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

Research output: Contribution to journalArticlepeer-review


Cytochrome c oxidase, the terminal protein in the respiratory chain, converts oxygen into water and helps generate the electrochemical gradient used in the synthesis of ATP. The catalytic action of cytochrome c oxidase involves electron transfer, proton transfer, and O2 reduction. These events trigger specific molecular changes at the active site, which, in turn, influence changes throughout the protein, including alterations of amino acid side chain orientations, hydrogen bond patterns, and protonation states. We have used IR difference spectroscopy to investigate such modulations for the functional intermediate states E, R2, Pm, and F. These spectra reveal deprotonation of its key glutamic acid E286 in the E and in the Pm states. The consecutive deprotonation and reprotonation of E286 twice within one catalytic turnover illustrates the role of this residue as a proton shuttle. In addition, the spectra point toward deprotonation of a redox-active tyrosine, plausibly Y288, in the F intermediate. Structural insights into the molecular mechanism of catalysis based on the subtle molecular changes observed with IR difference spectroscopy are discussed.

Original languageEnglish (US)
Pages (from-to)8715-8720
Number of pages6
JournalProceedings of the National Academy of Sciences of the United States of America
Issue number15
StatePublished - Jul 22 2003


  • Bacteriorhodopsis
  • Electron transfer
  • Glutamic acid
  • Infrared attenuated total reflection
  • Membrane protein

ASJC Scopus subject areas

  • General


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