{"id":{"repo_id":"uiuc","oai_identifier":"oai:www.ideals.illinois.edu:2142/85431"},"canonical_url":"https://search.dev.ndltd.org/etd/uiuc/oai:www.ideals.illinois.edu:2142/85431","repository":{"repo_id":"uiuc","name":"University of Illinois - Urbana-Champaign","base_url":"https://www.ideals.illinois.edu/oai-pmh"},"display":{"title":"Infrared Spectroscopy of Cytochrome C Oxidase Intermediate States","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.","abstract_html":"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.","abstract_has_math":false,"creators":["Nyquist, Rebecca Mary"],"institution":"University of Illinois at Urbana-Champaign","degree_name":"Ph.D.","degree_level":"Dissertation","degree_discipline":"Biophysics and Computational Biology","degree_department":null,"school":null,"contributors":["Gennis, Robert"],"advisors":[],"committee_chairs":[],"committee_members":[],"year":2015,"date_issued":"2015-09-25T22:46:04Z","date_published":"2015-09-25T22:46:04Z","updated_at":"2026-07-22T22:26:25Z","subjects":["Chemistry, Biochemistry"],"languages":["eng"],"rights":[],"rights_urls":[],"identifier_entries":[{"key":"dc:identifier","label":"Identifier","values":["(MiAaPQ)AAI3070397"],"render_values":[{"text":"(MiAaPQ)AAI3070397","href":null,"code":true}]}]},"links":{"outbound_url":"http://hdl.handle.net/2142/85431","outbound_label":"Handle","outbound_source":"dc:identifier"},"metadata_groups":[{"id":"people","label":"People","entries":[{"key":"dc:contributor","label":"Contributor","values":["Gennis, Robert"]},{"key":"dc:creator","label":"Author","values":["Nyquist, Rebecca Mary"]}]},{"id":"academic_context","label":"Academic Context","entries":[{"key":"dc:date","label":"Dc Date","values":["2015-09-25T22:46:04Z","10000-01-01","2002"]},{"key":"dc:type","label":"Dc Type","values":["text"]},{"key":"thesis:degree_discipline","label":"Discipline","values":["Biophysics and Computational Biology"]},{"key":"thesis:degree_level","label":"Degree Level","values":["Dissertation"]},{"key":"thesis:degree_name","label":"Degree Name","values":["Ph.D."]},{"key":"thesis:institution_name","label":"Thesis Institution Name","values":["University of Illinois at Urbana-Champaign"]}]},{"id":"subjects_keywords","label":"Subjects and Keywords","entries":[{"key":"dc:subject","label":"Dc Subject","values":["Chemistry, Biochemistry"]}]},{"id":"language_rights","label":"Language and Rights","entries":[{"key":"dc:language","label":"Dc Language","values":["eng"]}]},{"id":"identifiers","label":"Identifiers","entries":[{"key":"dc:identifier","label":"Identifier","values":["http://hdl.handle.net/2142/85431","(MiAaPQ)AAI3070397"]}]},{"id":"additional","label":"Additional Metadata","entries":[{"key":"dc:description","label":"Description","values":["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."]},{"key":"dc:title","label":"Title","values":["Infrared Spectroscopy of Cytochrome C Oxidase Intermediate States"]}]}],"canonical_facts":{"dc:contributor":["Gennis, Robert"],"dc:creator":["Nyquist, Rebecca Mary"],"dc:date":["2015-09-25T22:46:04Z","10000-01-01","2002"],"dc:description":["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."],"dc:identifier":["http://hdl.handle.net/2142/85431","(MiAaPQ)AAI3070397"],"dc:language":["eng"],"dc:subject":["Chemistry, Biochemistry"],"dc:title":["Infrared Spectroscopy of Cytochrome C Oxidase Intermediate States"],"dc:type":["text"],"thesis:degree_discipline":["Biophysics and Computational Biology"],"thesis:degree_level":["Dissertation"],"thesis:degree_name":["Ph.D."],"thesis:institution_name":["University of Illinois at Urbana-Champaign"]},"updated_at":"2026-07-22T22:26:25Z"}