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Title:Infrared Spectroscopy of Cytochrome C Oxidase Intermediate States
Author(s):Nyquist, Rebecca Mary
Doctoral Committee Chair(s):Gennis, Robert
Department / Program:Biophysics and Computational Biology
Discipline:Biophysics and Computational Biology
Degree Granting Institution:University of Illinois at Urbana-Champaign
Subject(s):Chemistry, Biochemistry
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.
Issue Date:2002
Description:179 p.
Thesis (Ph.D.)--University of Illinois at Urbana-Champaign, 2002.
Other Identifier(s):(MiAaPQ)AAI3070397
Date Available in IDEALS:2015-09-25
Date Deposited:2002

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