{"id":{"repo_id":"vt","oai_identifier":"oai:vtechworks.lib.vt.edu:10919/38835"},"canonical_url":"https://search.dev.ndltd.org/etd/vt/oai:vtechworks.lib.vt.edu:10919/38835","repository":{"repo_id":"vt","name":"Virginia Tech","base_url":"https://vtechworks.lib.vt.edu/oai/request"},"display":{"title":"Studies on two nickel-containing enzymes from Methanosarcina thermophila TM-1","abstract":"The cell extract protein content of acetate- and methanol-grown Methanosarcina thermophila was examined by two-dimensional polyacrylamide gel electrophoresis to determine the extent of regulation by the growth substrate. More than 100 mutually-exclusive spots were present in acetate- and methanol-grown cells suggesting a high degree of regulation. Spots corresponding to acetate kinase, phosphotransacetylase, and the five subunits of the nickel-containing carbon monoxide dehydrogenase (CODH) complex were identified in acetate-grown cells. The nickel-containing methyl coenzyme M methylreductase from acetate-grown M. thermophila was purified 16-fold from a cell extract to apparent homogeneity. The enzyme had a native molecular weight of between 132,000 and 141,000 and contained three subunits with a configuration of a1B1y1-. The as-isolated enzyme was inactive, but could be reductively reactivated by either titanium (III) citrate or reduced ferredoxin. Reactivation with ferredoxin was a simplification over previously reported reactivation systems. ATP stimulated, but was not required for reactivation. The CO dehydrogenase enzyme complex from M. thermophila was purified and separated into its respective components: the CO-oxidizing nickel/iron-sulfur (Ni/Fe-S) component and the cobalt-containing corrinoid/iron sulfur (Co/Fe-S) component. EPR spectroscopy and spectroelectrochemical titration of the Fe-S centers of the Ni/Fe-S component indicated the presence of two low-potential [4Fe-4S]2+/1+ centers and third high-potential center whose Fe-S configuration is unknown. When reduced with CO, the NilFe-S component exhibited a previously unobserved Ni-Fe-C EPR signal. The Co/Fe-S component contained one [4Fe-4S]2+/1+ cluster, and the as-isolated corrinoid in the component was in the base-off conformation suggesting that modulation of the electron density of the cobalt ion may result in a modified reactivity of the active site of the corrin. The CODH enzyme complex and isolated Co/Fe-S component reductively dechlorinated trichloroethylene to cis-dichloroethylene, trans-dichloroethylene, 1,1-dichloroethylene, vinyl chloride, and ethylene. Factor III also catalyzed the dechlorination of trichloroethylene when in the presence of titanium (III) citrate. Reconstitution of the Co/Fe-S component with the CO-reduced NilFe-S component also allowed dechlorination demonstrating an electron transfer from the reduced Ni/Fe-S component to the Co/Fe-S component.","abstract_html":"The cell extract protein content of acetate- and methanol-grown Methanosarcina thermophila was examined by two-dimensional polyacrylamide gel electrophoresis to determine the extent of regulation by the growth substrate. More than 100 mutually-exclusive spots were present in acetate- and methanol-grown cells suggesting a high degree of regulation. Spots corresponding to acetate kinase, phosphotransacetylase, and the five subunits of the nickel-containing carbon monoxide dehydrogenase (CODH) complex were identified in acetate-grown cells. The nickel-containing methyl coenzyme M methylreductase from acetate-grown M. thermophila was purified 16-fold from a cell extract to apparent homogeneity. The enzyme had a native molecular weight of between 132,000 and 141,000 and contained three subunits with a configuration of a1B1y1-. The as-isolated enzyme was inactive, but could be reductively reactivated by either titanium (III) citrate or reduced ferredoxin. Reactivation with ferredoxin was a simplification over previously reported reactivation systems. ATP stimulated, but was not required for reactivation. The CO dehydrogenase enzyme complex from M. thermophila was purified and separated into its respective components: the CO-oxidizing nickel/iron-sulfur (Ni/Fe-S) component and the cobalt-containing corrinoid/iron sulfur (Co/Fe-S) component. EPR spectroscopy and spectroelectrochemical titration of the Fe-S centers of the Ni/Fe-S component indicated the presence of two low-potential [4Fe-4S]2+/1+ centers and third high-potential center whose Fe-S configuration is unknown. When reduced with CO, the NilFe-S component exhibited a previously unobserved Ni-Fe-C EPR signal. The Co/Fe-S component contained one [4Fe-4S]2+/1+ cluster, and the as-isolated corrinoid in the component was in the base-off conformation suggesting that modulation of the electron density of the cobalt ion may result in a modified reactivity of the active site of the corrin. The CODH enzyme complex and isolated Co/Fe-S component reductively dechlorinated trichloroethylene to cis-dichloroethylene, trans-dichloroethylene, 1,1-dichloroethylene, vinyl chloride, and ethylene. Factor III also catalyzed the dechlorination of trichloroethylene when in the presence of titanium (III) citrate. Reconstitution of the Co/Fe-S component with the CO-reduced NilFe-S component also allowed dechlorination demonstrating an electron transfer from the reduced Ni/Fe-S component to the Co/Fe-S component.","abstract_has_math":false,"creators":["Jablonski, Peter Edward"],"institution":"Virginia Tech","degree_name":"Ph. D.","degree_level":"doctoral","degree_discipline":"Anaerobic Microbiology","degree_department":"Anaerobic Microbiology","school":null,"contributors":[],"advisors":[],"committee_chairs":["Ferry, James G."],"committee_members":["Dean, Dennis R.","Johnson, John L.","Krieg, Noel R.","Larson, Timothy J."],"year":1992,"date_issued":"1992-08-15","date_published":"1992-08-15","updated_at":"2026-07-22T22:20:05Z","subjects":[],"languages":["en"],"rights":["In Copyright"],"rights_urls":["http://rightsstatements.org/vocab/InC/1.0/"],"identifier_entries":[{"key":"dc:identifier.other","label":"Dc Identifier Other","values":["etd-07282008-134014"],"render_values":[{"text":"etd-07282008-134014","href":null,"code":true}]}]},"links":{"outbound_url":"http://hdl.handle.net/10919/38835","outbound_label":"Handle","outbound_source":"dc:identifier.uri"},"metadata_groups":[{"id":"people","label":"People","entries":[{"key":"dc:contributor.committeechair","label":"Committee Chair","values":["Ferry, James G."]},{"key":"dc:contributor.committeemember","label":"Committee Member","values":["Dean, Dennis R.","Johnson, John L.","Krieg, Noel R.","Larson, Timothy J."]},{"key":"dc:contributor.department","label":"Department","values":["Anaerobic Microbiology"]},{"key":"dc:creator","label":"Author","values":["Jablonski, Peter Edward"]}]},{"id":"academic_context","label":"Academic Context","entries":[{"key":"dc:date.accessioned","label":"Dc Date Accessioned","values":["2014-03-14T21:16:16Z"]},{"key":"dc:date.available","label":"Dc Date Available","values":["2014-03-14T21:16:16Z","2008-07-28"]},{"key":"dc:date.issued","label":"Date","values":["1992-08-15"]},{"key":"dc:publisher","label":"Institution","values":["Virginia Tech"]},{"key":"dc:type","label":"Dc Type","values":["Dissertation"]},{"key":"dc:type.dcmitype","label":"Dc Type Dcmitype","values":["Text"]},{"key":"thesis:degree_discipline","label":"Discipline","values":["Anaerobic Microbiology"]},{"key":"thesis:degree_level","label":"Degree Level","values":["doctoral"]},{"key":"thesis:degree_name","label":"Degree Name","values":["Ph. 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More than 100 mutually-exclusive spots were present in acetate- and methanol-grown cells suggesting a high degree of regulation. Spots corresponding to acetate kinase, phosphotransacetylase, and the five subunits of the nickel-containing carbon monoxide dehydrogenase (CODH) complex were identified in acetate-grown cells. The nickel-containing methyl coenzyme M methylreductase from acetate-grown M. thermophila was purified 16-fold from a cell extract to apparent homogeneity. The enzyme had a native molecular weight of between 132,000 and 141,000 and contained three subunits with a configuration of a1B1y1-. The as-isolated enzyme was inactive, but could be reductively reactivated by either titanium (III) citrate or reduced ferredoxin. Reactivation with ferredoxin was a simplification over previously reported reactivation systems. ATP stimulated, but was not required for reactivation. The CO dehydrogenase enzyme complex from M. thermophila was purified and separated into its respective components: the CO-oxidizing nickel/iron-sulfur (Ni/Fe-S) component and the cobalt-containing corrinoid/iron sulfur (Co/Fe-S) component. EPR spectroscopy and spectroelectrochemical titration of the Fe-S centers of the Ni/Fe-S component indicated the presence of two low-potential [4Fe-4S]2+/1+ centers and third high-potential center whose Fe-S configuration is unknown. When reduced with CO, the NilFe-S component exhibited a previously unobserved Ni-Fe-C EPR signal. The Co/Fe-S component contained one [4Fe-4S]2+/1+ cluster, and the as-isolated corrinoid in the component was in the base-off conformation suggesting that modulation of the electron density of the cobalt ion may result in a modified reactivity of the active site of the corrin. The CODH enzyme complex and isolated Co/Fe-S component reductively dechlorinated trichloroethylene to cis-dichloroethylene, trans-dichloroethylene, 1,1-dichloroethylene, vinyl chloride, and ethylene. Factor III also catalyzed the dechlorination of trichloroethylene when in the presence of titanium (III) citrate. Reconstitution of the Co/Fe-S component with the CO-reduced NilFe-S component also allowed dechlorination demonstrating an electron transfer from the reduced Ni/Fe-S component to the Co/Fe-S component."]},{"key":"dc:description.degree","label":"Dc Description Degree","values":["Ph. D."]},{"key":"dc:format.medium","label":"Dc Format Medium","values":["BTD"]},{"key":"dc:format.mimetype","label":"Dc Format Mimetype","values":["application/pdf"]},{"key":"dc:title","label":"Title","values":["Studies on two nickel-containing enzymes from Methanosarcina thermophila TM-1"]}]}],"canonical_facts":{"dc:contributor.committeechair":["Ferry, James G."],"dc:contributor.committeemember":["Dean, Dennis R.","Johnson, John L.","Krieg, Noel R.","Larson, Timothy J."],"dc:contributor.department":["Anaerobic Microbiology"],"dc:creator":["Jablonski, Peter Edward"],"dc:date.accessioned":["2014-03-14T21:16:16Z"],"dc:date.available":["2014-03-14T21:16:16Z","2008-07-28"],"dc:date.issued":["1992-08-15"],"dc:description.abstract":["The cell extract protein content of acetate- and methanol-grown Methanosarcina thermophila was examined by two-dimensional polyacrylamide gel electrophoresis to determine the extent of regulation by the growth substrate. More than 100 mutually-exclusive spots were present in acetate- and methanol-grown cells suggesting a high degree of regulation. Spots corresponding to acetate kinase, phosphotransacetylase, and the five subunits of the nickel-containing carbon monoxide dehydrogenase (CODH) complex were identified in acetate-grown cells. The nickel-containing methyl coenzyme M methylreductase from acetate-grown M. thermophila was purified 16-fold from a cell extract to apparent homogeneity. The enzyme had a native molecular weight of between 132,000 and 141,000 and contained three subunits with a configuration of a1B1y1-. The as-isolated enzyme was inactive, but could be reductively reactivated by either titanium (III) citrate or reduced ferredoxin. Reactivation with ferredoxin was a simplification over previously reported reactivation systems. ATP stimulated, but was not required for reactivation. The CO dehydrogenase enzyme complex from M. thermophila was purified and separated into its respective components: the CO-oxidizing nickel/iron-sulfur (Ni/Fe-S) component and the cobalt-containing corrinoid/iron sulfur (Co/Fe-S) component. EPR spectroscopy and spectroelectrochemical titration of the Fe-S centers of the Ni/Fe-S component indicated the presence of two low-potential [4Fe-4S]2+/1+ centers and third high-potential center whose Fe-S configuration is unknown. When reduced with CO, the NilFe-S component exhibited a previously unobserved Ni-Fe-C EPR signal. The Co/Fe-S component contained one [4Fe-4S]2+/1+ cluster, and the as-isolated corrinoid in the component was in the base-off conformation suggesting that modulation of the electron density of the cobalt ion may result in a modified reactivity of the active site of the corrin. The CODH enzyme complex and isolated Co/Fe-S component reductively dechlorinated trichloroethylene to cis-dichloroethylene, trans-dichloroethylene, 1,1-dichloroethylene, vinyl chloride, and ethylene. Factor III also catalyzed the dechlorination of trichloroethylene when in the presence of titanium (III) citrate. Reconstitution of the Co/Fe-S component with the CO-reduced NilFe-S component also allowed dechlorination demonstrating an electron transfer from the reduced Ni/Fe-S component to the Co/Fe-S component."],"dc:description.degree":["Ph. D."],"dc:format.medium":["BTD"],"dc:format.mimetype":["application/pdf"],"dc:identifier.other":["etd-07282008-134014"],"dc:identifier.uri":["http://hdl.handle.net/10919/38835"],"dc:language.iso":["en"],"dc:publisher":["Virginia Tech"],"dc:rights":["In Copyright"],"dc:rights.uri":["http://rightsstatements.org/vocab/InC/1.0/"],"dc:title":["Studies on two nickel-containing enzymes from Methanosarcina thermophila TM-1"],"dc:type":["Dissertation"],"dc:type.dcmitype":["Text"],"thesis:degree_discipline":["Anaerobic Microbiology"],"thesis:degree_level":["doctoral"],"thesis:degree_name":["Ph. D."],"thesis:institution_name":["Virginia Polytechnic Institute and State University"]},"updated_at":"2026-07-22T22:20:05Z"}