{"id":{"repo_id":"uiuc","oai_identifier":"oai:www.ideals.illinois.edu:2142/30763"},"canonical_url":"https://search.dev.ndltd.org/etd/uiuc/oai:www.ideals.illinois.edu:2142/30763","repository":{"repo_id":"uiuc","name":"University of Illinois - Urbana-Champaign","base_url":"https://www.ideals.illinois.edu/oai-pmh"},"display":{"title":"A kinetic and equilibrium description of camphor hydroxylation by the P450cam monoxygenase system","abstract":"Camphor is hydroxylated in the soil bacterium Pseudomonas putida by a soluble three protein monoxygenase system consisting of (1) a NADH-specific FAD flavoprotein reductase, (2) a Fe2S*2 Cys4 iron-sulfur redoxin termed putidaredoxin (Pd) and (3) cytochrome P450cam, abbreviated cytochrome m for monoxygenase. The interaction of Pd and cytochrome m in the overall monoxygenase reaction is studied by equilibrium and kinetic techniques. Three compounds (termed effectors) are shown to react with oxygenated cytochrome m in the generation of product: Pdr , a modified putidaredoxin (Pdr.dTrp), and dihydrolipoic acid. In all cases a kinetic analysis of the product forming reaction indicates that an effector m02rs complex precedes camphor hydroxylation. Cytochrome m is shown by equilibrium methods to bind two molecules of putidaredoxin. Pd binding at one site induces a shift in the Soret absorption maximum of cytochrome m corresponding to an observed difference spectrum with peak at 420 nm and trough at 386 nm. The Pd-cytochrome m complex formed by ligation at this site is that indicated by kinetic methods to be the obligatory intermediate in the generation of product from m02rs. Pd binding at a second cytochrome m locus is observed by the quenching of fluorescence from a dye label attached to the external sulfhydryl group on cytochrome m. Regulation of oxidation/reduction potential is observed by ligation at this site and the data are presented in the form of a free energy diagram graphically indicating the coupling between redox and binding energies.","abstract_html":"Camphor is hydroxylated in the soil bacterium Pseudomonas putida by a soluble three protein monoxygenase system consisting of (1) a NADH-specific FAD flavoprotein reductase, (2) a Fe2S*2 Cys4 iron-sulfur redoxin termed putidaredoxin (Pd) and (3) cytochrome P450cam, abbreviated cytochrome m for monoxygenase. The interaction of Pd and cytochrome m in the overall monoxygenase reaction is studied by equilibrium and kinetic techniques. Three compounds (termed effectors) are shown to react with oxygenated cytochrome m in the generation of product: Pdr , a modified putidaredoxin (Pdr.dTrp), and dihydrolipoic acid. In all cases a kinetic analysis of the product forming reaction indicates that an effector m02rs complex precedes camphor hydroxylation. Cytochrome m is shown by equilibrium methods to bind two molecules of putidaredoxin. Pd binding at one site induces a shift in the Soret absorption maximum of cytochrome m corresponding to an observed difference spectrum with peak at 420 nm and trough at 386 nm. The Pd-cytochrome m complex formed by ligation at this site is that indicated by kinetic methods to be the obligatory intermediate in the generation of product from m02rs. Pd binding at a second cytochrome m locus is observed by the quenching of fluorescence from a dye label attached to the external sulfhydryl group on cytochrome m. Regulation of oxidation/reduction potential is observed by ligation at this site and the data are presented in the form of a free energy diagram graphically indicating the coupling between redox and binding energies.","abstract_has_math":false,"creators":["Sligar, Stephen Gary"],"institution":null,"degree_name":"Ph.D.","degree_level":"Dissertation","degree_discipline":"Physics","degree_department":null,"school":null,"contributors":["Debrunner, Peter G.","Gunsalus, I.C."],"advisors":[],"committee_chairs":[],"committee_members":[],"year":2012,"date_issued":"2012-04-26T18:10:33Z","date_published":"2012-04-26T18:10:33Z","updated_at":"2026-07-22T22:25:29Z","subjects":["camphor","Biochemistry","physics","catabolic pathway","enzymes","Pseudomonas putida"],"languages":["en"],"rights":["© 1975 Stephen Gary Sligar"],"rights_urls":[],"identifier_entries":[{"key":"dc:identifier","label":"Identifier","values":["2230997"],"render_values":[{"text":"2230997","href":null,"code":true}]}]},"links":{"outbound_url":"http://hdl.handle.net/2142/30763","outbound_label":"Handle","outbound_source":"dc:identifier"},"metadata_groups":[{"id":"people","label":"People","entries":[{"key":"dc:contributor","label":"Contributor","values":["Debrunner, Peter G.","Gunsalus, I.C."]},{"key":"dc:creator","label":"Author","values":["Sligar, Stephen Gary"]}]},{"id":"academic_context","label":"Academic Context","entries":[{"key":"dc:date","label":"Dc Date","values":["2012-04-26T18:10:33Z","10000-01-01","1975"]},{"key":"dc:type","label":"Dc Type","values":["Dissertation / Thesis","text"]},{"key":"thesis:degree_discipline","label":"Discipline","values":["Physics"]},{"key":"thesis:degree_level","label":"Degree Level","values":["Dissertation"]},{"key":"thesis:degree_name","label":"Degree Name","values":["Ph.D."]}]},{"id":"subjects_keywords","label":"Subjects and Keywords","entries":[{"key":"dc:subject","label":"Dc Subject","values":["camphor","Biochemistry","physics","catabolic pathway","enzymes","Pseudomonas putida"]}]},{"id":"language_rights","label":"Language and Rights","entries":[{"key":"dc:language","label":"Dc Language","values":["en"]},{"key":"dc:rights","label":"Dc Rights","values":["© 1975 Stephen Gary Sligar"]}]},{"id":"identifiers","label":"Identifiers","entries":[{"key":"dc:identifier","label":"Identifier","values":["2230997","http://hdl.handle.net/2142/30763"]}]},{"id":"additional","label":"Additional Metadata","entries":[{"key":"dc:description","label":"Description","values":["Camphor is hydroxylated in the soil bacterium Pseudomonas putida by a soluble three protein monoxygenase system consisting of (1) a NADH-specific FAD flavoprotein reductase, (2) a Fe2S*2 Cys4 iron-sulfur redoxin termed putidaredoxin (Pd) and (3) cytochrome P450cam, abbreviated cytochrome m for monoxygenase. The interaction of Pd and cytochrome m in the overall monoxygenase reaction is studied by equilibrium and kinetic techniques. Three compounds (termed effectors) are shown to react with oxygenated cytochrome m in the generation of product: Pdr , a modified putidaredoxin (Pdr.dTrp), and dihydrolipoic acid. In all cases a kinetic analysis of the product forming reaction indicates that an effector m02rs complex precedes camphor hydroxylation. Cytochrome m is shown by equilibrium methods to bind two molecules of putidaredoxin. Pd binding at one site induces a shift in the Soret absorption maximum of cytochrome m corresponding to an observed difference spectrum with peak at 420 nm and trough at 386 nm. The Pd-cytochrome m complex formed by ligation at this site is that indicated by kinetic methods to be the obligatory intermediate in the generation of product from m02rs. Pd binding at a second cytochrome m locus is observed by the quenching of fluorescence from a dye label attached to the external sulfhydryl group on cytochrome m. Regulation of oxidation/reduction potential is observed by ligation at this site and the data are presented in the form of a free energy diagram graphically indicating the coupling between redox and binding energies.","Submitted by Megan O'Donnell (mnodonn2@illinois.edu) on 2012-04-26T18:10:33Z No. of bitstreams: 1 1975_sligar.pdf: 4372418 bytes, checksum: 06bb435be7e59e8fdec530697e8a712f (MD5)","Made available in DSpace on 2012-04-26T18:10:33Z (GMT). No. of bitstreams: 1 1975_sligar.pdf: 4372418 bytes, checksum: 06bb435be7e59e8fdec530697e8a712f (MD5) Previous issue date: 1975","Item marked as restricted to the 'UIUC Users [automated]' Group (id=2) by Megan O'Donnell (mnodonn2@illinois.edu) on 2012-04-26T18:10:33Z Item is restricted indefinitely.","Restriction data tranferred 2014-07-01T11:33:14-05:00 Original Data Group with Access UIUC Users [automated] Release Date: none Reason: dissertation/thesis","dissertation/thesis","U of I Only"]},{"key":"dc:title","label":"Title","values":["A kinetic and equilibrium description of camphor hydroxylation by the P450cam monoxygenase system"]}]}],"canonical_facts":{"dc:contributor":["Debrunner, Peter G.","Gunsalus, I.C."],"dc:creator":["Sligar, Stephen Gary"],"dc:date":["2012-04-26T18:10:33Z","10000-01-01","1975"],"dc:description":["Camphor is hydroxylated in the soil bacterium Pseudomonas putida by a soluble three protein monoxygenase system consisting of (1) a NADH-specific FAD flavoprotein reductase, (2) a Fe2S*2 Cys4 iron-sulfur redoxin termed putidaredoxin (Pd) and (3) cytochrome P450cam, abbreviated cytochrome m for monoxygenase. The interaction of Pd and cytochrome m in the overall monoxygenase reaction is studied by equilibrium and kinetic techniques. Three compounds (termed effectors) are shown to react with oxygenated cytochrome m in the generation of product: Pdr , a modified putidaredoxin (Pdr.dTrp), and dihydrolipoic acid. In all cases a kinetic analysis of the product forming reaction indicates that an effector m02rs complex precedes camphor hydroxylation. Cytochrome m is shown by equilibrium methods to bind two molecules of putidaredoxin. Pd binding at one site induces a shift in the Soret absorption maximum of cytochrome m corresponding to an observed difference spectrum with peak at 420 nm and trough at 386 nm. The Pd-cytochrome m complex formed by ligation at this site is that indicated by kinetic methods to be the obligatory intermediate in the generation of product from m02rs. Pd binding at a second cytochrome m locus is observed by the quenching of fluorescence from a dye label attached to the external sulfhydryl group on cytochrome m. Regulation of oxidation/reduction potential is observed by ligation at this site and the data are presented in the form of a free energy diagram graphically indicating the coupling between redox and binding energies.","Submitted by Megan O'Donnell (mnodonn2@illinois.edu) on 2012-04-26T18:10:33Z No. of bitstreams: 1 1975_sligar.pdf: 4372418 bytes, checksum: 06bb435be7e59e8fdec530697e8a712f (MD5)","Made available in DSpace on 2012-04-26T18:10:33Z (GMT). No. of bitstreams: 1 1975_sligar.pdf: 4372418 bytes, checksum: 06bb435be7e59e8fdec530697e8a712f (MD5) Previous issue date: 1975","Item marked as restricted to the 'UIUC Users [automated]' Group (id=2) by Megan O'Donnell (mnodonn2@illinois.edu) on 2012-04-26T18:10:33Z Item is restricted indefinitely.","Restriction data tranferred 2014-07-01T11:33:14-05:00 Original Data Group with Access UIUC Users [automated] Release Date: none Reason: dissertation/thesis","dissertation/thesis","U of I Only"],"dc:identifier":["2230997","http://hdl.handle.net/2142/30763"],"dc:language":["en"],"dc:rights":["© 1975 Stephen Gary Sligar"],"dc:subject":["camphor","Biochemistry","physics","catabolic pathway","enzymes","Pseudomonas putida"],"dc:title":["A kinetic and equilibrium description of camphor hydroxylation by the P450cam monoxygenase system"],"dc:type":["Dissertation / Thesis","text"],"thesis:degree_discipline":["Physics"],"thesis:degree_level":["Dissertation"],"thesis:degree_name":["Ph.D."]},"updated_at":"2026-07-22T22:25:29Z"}