Infrared Spectroscopy of Cytochrome C Oxidase Intermediate States
Nyquist, Rebecca Mary
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https://hdl.handle.net/2142/85431
Description
Title
Infrared Spectroscopy of Cytochrome C Oxidase Intermediate States
Author(s)
Nyquist, Rebecca Mary
Issue Date
2002
Doctoral Committee Chair(s)
Gennis, Robert
Department of Study
Biophysics and Computational Biology
Discipline
Biophysics and Computational Biology
Degree Granting Institution
University of Illinois at Urbana-Champaign
Degree Name
Ph.D.
Degree Level
Dissertation
Keyword(s)
Chemistry, Biochemistry
Language
eng
Abstract
Cytochrome c oxidase is a critical player in the process of cellular respiration, performing proton translocation coupled to the four-electron reduction of O2 to H2O. To accomplish this catalytic task, specific changes at the active site influence chemical and physical changes throughout the protein, altering amino acid side-chain orientations, hydrogen bond lengths, and protonation states. Infrared spectroscopy is capable of monitoring these changes. In this thesis work, cytochrome c oxidase was specially prepared for perfusion-induced infrared difference spectroscopy. The resulting infrared difference spectra demonstrate that the side-chain of a key glutamate, E286 from Rhodobacter sphaeroides, is protonated in both oxidized (O) and fully-reduced states with a p Ka higher than 9.5. Also presented in this work are the first infrared difference spectra for O2 bond-cleaved intermediate states P and F. In addition, time-resolved infrared spectroscopy was used to study vibrational differences between intermediate states preceding O 2 binding, the one- and two-electron reduced states (E and R2, respectively). Taken together, the infrared difference spectra presented here demonstrate that the E286 side-chain is deprotonated in E and P but protonated in O, R2, and F. This indicates that E286 transfers its proton in the O to E and R2 to P transitions; and that it accepts a proton in the E to R2 and P to F transitions. Also, a tyrosine residue, presumably the active site tyrosine Y288, was observed to be protonated in O and deprotonated in F. These results spark interpretation of mechanistic models as well as form the basis for future time-resolved infrared spectroscopic investigations.
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