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oai:www.ideals.illinois.edu:2142/85431 2023-07-11 col_2142_5131 col_2142_14828 com_2142_5130 com_2142_14827 com_2142_14793 com_2142_8903

Gennis, Robert Nyquist, Rebecca Mary 2015-09-25T22:46:04Z 2015-09-25T22:46:04Z 10000-01-01 2002 2002 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. Made available in DSpace on 2015-09-25T22:46:04Z (GMT). No. of bitstreams: 2 license.txt: 4848 bytes, checksum: 96035ab3f5e1c23cc7138a224ce498bd (MD5) 3070397.pdf: 8111147 bytes, checksum: 85942d69916ec794bc7541202db080f3 (MD5) Previous issue date: 2002 Embargo set by: Seth Robbins for item 86712 Lift date: Forever Reason: Restricted to the U of I community idenfinitely during batch ingest of legacy ETDs Restricted to the U of I community idenfinitely during batch ingest of legacy ETDs U of I Only 179 p. Thesis (Ph.D.)--University of Illinois at Urbana-Champaign, 2002. http://hdl.handle.net/2142/85431 (MiAaPQ)AAI3070397 eng Chemistry, Biochemistry Infrared Spectroscopy of Cytochrome C Oxidase Intermediate States text Biophysics and Computational Biology Biophysics and Computational Biology University of Illinois at Urbana-Champaign Dissertation Ph.D.