{"id":{"repo_id":"vt","oai_identifier":"oai:vtechworks.lib.vt.edu:10919/54440"},"canonical_url":"https://search.dev.ndltd.org/etd/vt/oai:vtechworks.lib.vt.edu:10919/54440","repository":{"repo_id":"vt","name":"Virginia Tech","base_url":"https://vtechworks.lib.vt.edu/oai/request"},"display":{"title":"Studies on the carbon monoxide dehydrogenase enzyme complex present in acetate-grown Methanosarcina thermophila strain TM-1","abstract":"The carbon monoxide dehydrogenase complex was purified from acetate-grown Methanosarcina thermophila. This complex made up greater than 10% of the cellular protein and the native enzyme formed aggregates with a Mr of approximately 1,000,000. The enzyme contained five subunits of different molecular weight suggesting a multifunctional enzyme complex. Nickel, iron, cobalt, zinc, inorganic sulfide, and a corrinoid were present in the complex. The electron paramagnetic resonance spectrum of CO-reduced enzyme at 113K contained g values of 2.073, 2.049, and 2.028. Isotopic substitution with ⁶¹Ni, ⁵⁷Fe, or ¹³Co resulted in broadening of the spectrum consistent with a Ni-Fe-C spin-coupled complex. Acetyl-CoA caused a perturbation of the signal that was not caused by acetyl-phosphate or mercaptoethanol indicating acetyl-CoA is a physiological substrate. Cell extracts from acetate-grown M. thermophila contained CO-oxidizing:H₂-evolving activity 16-fold greater than extracts of methanol-grown cells. CO-oxidizing:H₂-evolving activity was reconstituted upon combination of: (i) CO dehydrogenase complex, (ii) a ferredoxin, and (iii) purified membranes with associated hydrogenase and b-type cytochrome. The ferredoxin was a direct electron acceptor for the CO dehydrogenase complex. The molecular weight of the isolated protein was 16,400, and the apparent minimum molecular weight was 4,900. The ferredoxin contained 2.8 ± 0.56 Fe atoms and 1.98 ± 0.12 acid-labile sulfide. UV-visible absorption maxima were 395 and 295 nm with a A₃₉₅/A₂₉₅ ratio range of 0.80 to 0.88. The N-terminal amino acid sequence revealed a 4-cysteine cluster, similar to other Fe:S centers that coordinate a Fe:S center. A CH₃-B₁₂:HS-CoM methyltransferase activity was characterized in extracts of acetate- and methanol-grown cells. The activity from extracts of acetate-grown M. thermophila was stable at 70°C for 30 minutes. The activity in cell extracts of acetate- and methanol-grown cells was fractionated with ammonium sulfate treatment and FPLC phenyl superose chromatography. Two peaks of methyltransferase activity were observed in each cell extract sample following phenyl superose fractionation.","abstract_html":"The carbon monoxide dehydrogenase complex was purified from acetate-grown Methanosarcina thermophila. This complex made up greater than 10% of the cellular protein and the native enzyme formed aggregates with a Mr of approximately 1,000,000. The enzyme contained five subunits of different molecular weight suggesting a multifunctional enzyme complex. Nickel, iron, cobalt, zinc, inorganic sulfide, and a corrinoid were present in the complex. The electron paramagnetic resonance spectrum of CO-reduced enzyme at 113K contained g values of 2.073, 2.049, and 2.028. Isotopic substitution with ⁶¹Ni, ⁵⁷Fe, or ¹³Co resulted in broadening of the spectrum consistent with a Ni-Fe-C spin-coupled complex. Acetyl-CoA caused a perturbation of the signal that was not caused by acetyl-phosphate or mercaptoethanol indicating acetyl-CoA is a physiological substrate. Cell extracts from acetate-grown M. thermophila contained CO-oxidizing:H₂-evolving activity 16-fold greater than extracts of methanol-grown cells. CO-oxidizing:H₂-evolving activity was reconstituted upon combination of: (i) CO dehydrogenase complex, (ii) a ferredoxin, and (iii) purified membranes with associated hydrogenase and b-type cytochrome. The ferredoxin was a direct electron acceptor for the CO dehydrogenase complex. The molecular weight of the isolated protein was 16,400, and the apparent minimum molecular weight was 4,900. The ferredoxin contained 2.8 ± 0.56 Fe atoms and 1.98 ± 0.12 acid-labile sulfide. UV-visible absorption maxima were 395 and 295 nm with a A₃₉₅/A₂₉₅ ratio range of 0.80 to 0.88. The N-terminal amino acid sequence revealed a 4-cysteine cluster, similar to other Fe:S centers that coordinate a Fe:S center. A CH₃-B₁₂:HS-CoM methyltransferase activity was characterized in extracts of acetate- and methanol-grown cells. The activity from extracts of acetate-grown M. thermophila was stable at 70°C for 30 minutes. The activity in cell extracts of acetate- and methanol-grown cells was fractionated with ammonium sulfate treatment and FPLC phenyl superose chromatography. Two peaks of methyltransferase activity were observed in each cell extract sample following phenyl superose fractionation.","abstract_has_math":false,"creators":["Terlesky, Katherine C."],"institution":"Virginia Polytechnic Institute and State University","degree_name":"Ph. D.","degree_level":"doctoral","degree_discipline":"Microbiology","degree_department":"Microbiology","school":null,"contributors":[],"advisors":[],"committee_chairs":["Ferry, James G.","Gregory, E.M."],"committee_members":["Ebel, Richard E.","Wilkins, Tracy D.","Johnson, J.L."],"year":1989,"date_issued":"1989","date_published":"1989","updated_at":"2026-07-22T22:19:54Z","subjects":[],"languages":["en_US"],"rights":["In Copyright"],"rights_urls":["http://rightsstatements.org/vocab/InC/1.0/"],"identifier_entries":[]},"links":{"outbound_url":"http://hdl.handle.net/10919/54440","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.","Gregory, E.M."]},{"key":"dc:contributor.committeemember","label":"Committee Member","values":["Ebel, Richard E.","Wilkins, Tracy D.","Johnson, J.L."]},{"key":"dc:contributor.department","label":"Department","values":["Microbiology"]},{"key":"dc:creator","label":"Author","values":["Terlesky, Katherine C."]}]},{"id":"academic_context","label":"Academic Context","entries":[{"key":"dc:date.accessioned","label":"Dc Date Accessioned","values":["2015-07-10T20:00:06Z"]},{"key":"dc:date.available","label":"Dc Date Available","values":["2015-07-10T20:00:06Z"]},{"key":"dc:date.issued","label":"Date","values":["1989"]},{"key":"dc:publisher","label":"Institution","values":["Virginia Polytechnic Institute and State University"]},{"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":["Microbiology"]},{"key":"thesis:degree_level","label":"Degree Level","values":["doctoral"]},{"key":"thesis:degree_name","label":"Degree Name","values":["Ph. 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The enzyme contained five subunits of different molecular weight suggesting a multifunctional enzyme complex. Nickel, iron, cobalt, zinc, inorganic sulfide, and a corrinoid were present in the complex. The electron paramagnetic resonance spectrum of CO-reduced enzyme at 113K contained g values of 2.073, 2.049, and 2.028. Isotopic substitution with ⁶¹Ni, ⁵⁷Fe, or ¹³Co resulted in broadening of the spectrum consistent with a Ni-Fe-C spin-coupled complex. Acetyl-CoA caused a perturbation of the signal that was not caused by acetyl-phosphate or mercaptoethanol indicating acetyl-CoA is a physiological substrate. Cell extracts from acetate-grown M. thermophila contained CO-oxidizing:H₂-evolving activity 16-fold greater than extracts of methanol-grown cells. CO-oxidizing:H₂-evolving activity was reconstituted upon combination of: (i) CO dehydrogenase complex, (ii) a ferredoxin, and (iii) purified membranes with associated hydrogenase and b-type cytochrome. The ferredoxin was a direct electron acceptor for the CO dehydrogenase complex. The molecular weight of the isolated protein was 16,400, and the apparent minimum molecular weight was 4,900. The ferredoxin contained 2.8 ± 0.56 Fe atoms and 1.98 ± 0.12 acid-labile sulfide. UV-visible absorption maxima were 395 and 295 nm with a A₃₉₅/A₂₉₅ ratio range of 0.80 to 0.88. The N-terminal amino acid sequence revealed a 4-cysteine cluster, similar to other Fe:S centers that coordinate a Fe:S center. A CH₃-B₁₂:HS-CoM methyltransferase activity was characterized in extracts of acetate- and methanol-grown cells. The activity from extracts of acetate-grown M. thermophila was stable at 70°C for 30 minutes. The activity in cell extracts of acetate- and methanol-grown cells was fractionated with ammonium sulfate treatment and FPLC phenyl superose chromatography. Two peaks of methyltransferase activity were observed in each cell extract sample following phenyl superose fractionation."]},{"key":"dc:description.degree","label":"Dc Description Degree","values":["Ph. D."]},{"key":"dc:format.mimetype","label":"Dc Format Mimetype","values":["application/pdf"]},{"key":"dc:title","label":"Title","values":["Studies on the carbon monoxide dehydrogenase enzyme complex present in acetate-grown Methanosarcina thermophila strain TM-1"]}]}],"canonical_facts":{"dc:contributor.committeechair":["Ferry, James G.","Gregory, E.M."],"dc:contributor.committeemember":["Ebel, Richard E.","Wilkins, Tracy D.","Johnson, J.L."],"dc:contributor.department":["Microbiology"],"dc:creator":["Terlesky, Katherine C."],"dc:date.accessioned":["2015-07-10T20:00:06Z"],"dc:date.available":["2015-07-10T20:00:06Z"],"dc:date.issued":["1989"],"dc:description.abstract":["The carbon monoxide dehydrogenase complex was purified from acetate-grown Methanosarcina thermophila. This complex made up greater than 10% of the cellular protein and the native enzyme formed aggregates with a Mr of approximately 1,000,000. The enzyme contained five subunits of different molecular weight suggesting a multifunctional enzyme complex. Nickel, iron, cobalt, zinc, inorganic sulfide, and a corrinoid were present in the complex. The electron paramagnetic resonance spectrum of CO-reduced enzyme at 113K contained g values of 2.073, 2.049, and 2.028. Isotopic substitution with ⁶¹Ni, ⁵⁷Fe, or ¹³Co resulted in broadening of the spectrum consistent with a Ni-Fe-C spin-coupled complex. Acetyl-CoA caused a perturbation of the signal that was not caused by acetyl-phosphate or mercaptoethanol indicating acetyl-CoA is a physiological substrate. Cell extracts from acetate-grown M. thermophila contained CO-oxidizing:H₂-evolving activity 16-fold greater than extracts of methanol-grown cells. CO-oxidizing:H₂-evolving activity was reconstituted upon combination of: (i) CO dehydrogenase complex, (ii) a ferredoxin, and (iii) purified membranes with associated hydrogenase and b-type cytochrome. The ferredoxin was a direct electron acceptor for the CO dehydrogenase complex. The molecular weight of the isolated protein was 16,400, and the apparent minimum molecular weight was 4,900. The ferredoxin contained 2.8 ± 0.56 Fe atoms and 1.98 ± 0.12 acid-labile sulfide. UV-visible absorption maxima were 395 and 295 nm with a A₃₉₅/A₂₉₅ ratio range of 0.80 to 0.88. The N-terminal amino acid sequence revealed a 4-cysteine cluster, similar to other Fe:S centers that coordinate a Fe:S center. A CH₃-B₁₂:HS-CoM methyltransferase activity was characterized in extracts of acetate- and methanol-grown cells. The activity from extracts of acetate-grown M. thermophila was stable at 70°C for 30 minutes. The activity in cell extracts of acetate- and methanol-grown cells was fractionated with ammonium sulfate treatment and FPLC phenyl superose chromatography. Two peaks of methyltransferase activity were observed in each cell extract sample following phenyl superose fractionation."],"dc:description.degree":["Ph. D."],"dc:format.mimetype":["application/pdf"],"dc:identifier.uri":["http://hdl.handle.net/10919/54440"],"dc:language.iso":["en_US"],"dc:publisher":["Virginia Polytechnic Institute and State University"],"dc:rights":["In Copyright"],"dc:rights.uri":["http://rightsstatements.org/vocab/InC/1.0/"],"dc:title":["Studies on the carbon monoxide dehydrogenase enzyme complex present in acetate-grown Methanosarcina thermophila strain TM-1"],"dc:type":["Dissertation"],"dc:type.dcmitype":["Text"],"thesis:degree_discipline":["Microbiology"],"thesis:degree_level":["doctoral"],"thesis:degree_name":["Ph. D."],"thesis:institution_name":["Virginia Polytechnic Institute and State University"]},"updated_at":"2026-07-22T22:19:54Z"}