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dc.contributor.authorYu, Michelle Annette
dc.date.accessioned2018-07-12T17:36:11Z
dc.date.available2018-07-12T17:36:11Z
dc.date.issued2009
dc.identifier.citationSource: Dissertation Abstracts International, Volume: 70-11, Section: B, page: 6763.;Advisors: Denis L. Rousseau.
dc.identifier.urihttp://gateway.proquest.com/openurl?url_ver=Z39.88-2004&rft_val_fmt=info:ofi/fmt:kev:mtx:dissertation&res_dat=xri:pqm&rft_dat=xri:pqdiss:3385443
dc.identifier.urihttps://hdl.handle.net/20.500.12202/1104
dc.description.abstractCytochrome c oxidase (CcO) reduces O2 to water, harnessing the energy released to translocate protons against an electrochemical gradient. The pumping of protons produces a chemiosmotic gradient that drives the synthesis of adenosine 5'-triphosphate (ATP). This thesis examines the intermediates, populated during the oxygen reaction of bovine heart CcO, whose structures are integral to understanding how CcO stores energy for proton pumping. Samples are prepared by manual-mixing and a customized rapid freeze-quench device. Two tyrosyl radicals are characterized by multi-frequency EPR in the oxygen reaction with the two-electron reduced CcO, produced by H2O 2 treatment. The wide radical, trapped at pH 6, is most consistent with Tyr129. This finding resolves a historically controversial assignment, in which the radical had been assigned as lying on a tryptophan residue in bovine CcO (Fabian, M. and G. Palmer (1995). Biochemistry . 34(42): 13802-10; Rich, P. R., S. E. Rigby, et al. (2002). Biochim Biophys Acta. 1554(3): 137-46; Rigby, S. E., S. Junemann, et al. (2000). Biochemistry. 39(20): 5921-8.). The narrow radical, trapped at pH 8, has features unique to a modified tyrosine, which is consistent with Tyr244. In a separate series of work, intermediates of the oxygen-reaction of the four-electron reduced CcO are examined. No protein-based radicals are observed. Instead, ascorbyl and sulfur dioxide anion radicals, resulting from reductants, are characterized by multi-frequency EPR. These radicals possess features similar to "unknown" radicals noted by past CcO studies (Wilson, M. T., P. Jensen, et al. (1982). Biochem J. 203(2): 483-92). Finally, the relationship between a key gating glutamate and the hemes is examined through resonance Raman spectroscopy and optical characterization of site-directed mutants of Rhodobacter sphaeroides CcO. In E286C and E286A (RS numbering), perturbations of the spin and formyl markers indicate a hydrogen-bonding network between E286 and the hemes. This thesis provides a synthesis of optical absorption, resonance Raman, and EPR data, which cultivates our understanding of the oxygen chemistry underlying how CcO pumps protons.
dc.publisherProQuest Dissertations & Theses
dc.subjectBiophysics.
dc.subjectPhysiology.
dc.titleSpectroscopic studies on the structure and function of bovine heart and Rhodobacter sphaeroides cytochrome c oxidase
dc.typeDissertation


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