{"id":{"repo_id":"vt","oai_identifier":"oai:vtechworks.lib.vt.edu:10919/53908"},"canonical_url":"https://search.dev.ndltd.org/etd/vt/oai:vtechworks.lib.vt.edu:10919/53908","repository":{"repo_id":"vt","name":"Virginia Tech","base_url":"https://vtechworks.lib.vt.edu/oai/request"},"display":{"title":"A biochemical and physiological characterization of coenzyme F420-reducing hydrogenase from Methanobacterium formicicum","abstract":"The coenzyme F₄₂₀-reducing hydrogenase of Methanobacterium formicicum was purified 87-fold to electrophoretic homogeneity. The enzyme formed aggregates (1,000 kd) of a coenzyme F₄₂₀-active monomer (109 kd) composed of 1 each of a, β, and γ subunits (43.6, 36.7, xy and 28.8 kd, respectively). It contained 1 mol of FAD, 1 mol of nickel, 12-14 mols of iron, and 11 mols of acid-labile sulfide per mol of the 109 kd species, but no selenium. The amino acid sequence I---P--R-EGH-----EV was conserved in the N-terminus of a subunit of the enzyme and the largest subunits of nickel-containing hydrogenases from Methanobacterium thermoautotrophicum, Desulfovibrio baculatus, and Desulfovibrio gigag. FAD dissociated from the coenzyme F42O-reducing hydrogenase during reactivation with H2 and coenzyme F₄₂₀, unless KCl was present, yielding coenzyme F₄₂₀-inactive apoenzyme. The hydrogenase catalyzed H₂ production at a rate 3-fold less than that for H2 uptake. Specific antiserum inhibited the coenzyme F₄₂₀ dependent activity but not the methyl viologen-dependent activity of the purified enzyme. Cell extract of M. formicicum contained a coenzyme F₄₂₀-mediated formate hydrogenlyase system. Formate hydrogenlyase activity was reconstituted with coenzyme F₄₂₀-reducing hydrogenase, coenzyme F₄₂₀-reducing formate dehydrogenase, and coenzyme F₄₂₀, all purified from M. formicicum. The reconstituted system required FAD for maximal activity (kinetic Kd= 4 μM). without FAD, the formate dehydrogenase and hydrogenase rapidly lost coenzyme F₄₂₀-dependent activity relative to methyl viologen-dependent activity. Immunoadsorption of the formate dehydrogenase or hydrogenase from cell extract greatly reduced formate hydrogenlyase activity; addition of the purified enzymes restored activity. Formate hydrogenlyase activity of cell extract and the reconstituted system was reversible. The coenzyme F₄₂₀-reducing hydrogenase and formate dehydrogenase of M. formicicum were shown to be located at the cytoplasmic membrane using immunogold labeling of thin sectioned, Lowicryl-embedded cells. Neither enzyme was released from whole cells by osmotic shock treatment.","abstract_html":"The coenzyme F₄₂₀-reducing hydrogenase of Methanobacterium formicicum was purified 87-fold to electrophoretic homogeneity. The enzyme formed aggregates (1,000 kd) of a coenzyme F₄₂₀-active monomer (109 kd) composed of 1 each of a, β, and γ subunits (43.6, 36.7, xy and 28.8 kd, respectively). It contained 1 mol of FAD, 1 mol of nickel, 12-14 mols of iron, and 11 mols of acid-labile sulfide per mol of the 109 kd species, but no selenium. The amino acid sequence I---P--R-EGH-----EV was conserved in the N-terminus of a subunit of the enzyme and the largest subunits of nickel-containing hydrogenases from Methanobacterium thermoautotrophicum, Desulfovibrio baculatus, and Desulfovibrio gigag. FAD dissociated from the coenzyme F42O-reducing hydrogenase during reactivation with H2 and coenzyme F₄₂₀, unless KCl was present, yielding coenzyme F₄₂₀-inactive apoenzyme. The hydrogenase catalyzed H₂ production at a rate 3-fold less than that for H2 uptake. Specific antiserum inhibited the coenzyme F₄₂₀ dependent activity but not the methyl viologen-dependent activity of the purified enzyme. Cell extract of M. formicicum contained a coenzyme F₄₂₀-mediated formate hydrogenlyase system. Formate hydrogenlyase activity was reconstituted with coenzyme F₄₂₀-reducing hydrogenase, coenzyme F₄₂₀-reducing formate dehydrogenase, and coenzyme F₄₂₀, all purified from M. formicicum. The reconstituted system required FAD for maximal activity (kinetic Kd= 4 μM). without FAD, the formate dehydrogenase and hydrogenase rapidly lost coenzyme F₄₂₀-dependent activity relative to methyl viologen-dependent activity. Immunoadsorption of the formate dehydrogenase or hydrogenase from cell extract greatly reduced formate hydrogenlyase activity; addition of the purified enzymes restored activity. Formate hydrogenlyase activity of cell extract and the reconstituted system was reversible. The coenzyme F₄₂₀-reducing hydrogenase and formate dehydrogenase of M. formicicum were shown to be located at the cytoplasmic membrane using immunogold labeling of thin sectioned, Lowicryl-embedded cells. Neither enzyme was released from whole cells by osmotic shock treatment.","abstract_has_math":false,"creators":["Baron, Stephen Francis"],"institution":"Virginia Polytechnic Institute and State University","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":["Bevan, David R.","Anderson, Bruce M.","Wilkins, Tracy D.","Johnson, J.L."],"year":1988,"date_issued":"1988","date_published":"1988","updated_at":"2026-07-22T22:19:27Z","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/10919/53908","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":["Bevan, David R.","Anderson, Bruce M.","Wilkins, Tracy D.","Johnson, J.L."]},{"key":"dc:contributor.department","label":"Department","values":["Anaerobic Microbiology"]},{"key":"dc:creator","label":"Author","values":["Baron, Stephen Francis"]}]},{"id":"academic_context","label":"Academic Context","entries":[{"key":"dc:date.accessioned","label":"Dc Date Accessioned","values":["2015-06-29T22:07:06Z"]},{"key":"dc:date.available","label":"Dc Date Available","values":["2015-06-29T22:07:06Z"]},{"key":"dc:date.issued","label":"Date","values":["1988"]},{"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":["Anaerobic 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 formed aggregates (1,000 kd) of a coenzyme F₄₂₀-active monomer (109 kd) composed of 1 each of a, β, and γ subunits (43.6, 36.7, xy and 28.8 kd, respectively). It contained 1 mol of FAD, 1 mol of nickel, 12-14 mols of iron, and 11 mols of acid-labile sulfide per mol of the 109 kd species, but no selenium. The amino acid sequence I---P--R-EGH-----EV was conserved in the N-terminus of a subunit of the enzyme and the largest subunits of nickel-containing hydrogenases from Methanobacterium thermoautotrophicum, Desulfovibrio baculatus, and Desulfovibrio gigag. FAD dissociated from the coenzyme F42O-reducing hydrogenase during reactivation with H2 and coenzyme F₄₂₀, unless KCl was present, yielding coenzyme F₄₂₀-inactive apoenzyme. The hydrogenase catalyzed H₂ production at a rate 3-fold less than that for H2 uptake. Specific antiserum inhibited the coenzyme F₄₂₀ dependent activity but not the methyl viologen-dependent activity of the purified enzyme. Cell extract of M. formicicum contained a coenzyme F₄₂₀-mediated formate hydrogenlyase system. Formate hydrogenlyase activity was reconstituted with coenzyme F₄₂₀-reducing hydrogenase, coenzyme F₄₂₀-reducing formate dehydrogenase, and coenzyme F₄₂₀, all purified from M. formicicum. The reconstituted system required FAD for maximal activity (kinetic Kd= 4 μM). without FAD, the formate dehydrogenase and hydrogenase rapidly lost coenzyme F₄₂₀-dependent activity relative to methyl viologen-dependent activity. Immunoadsorption of the formate dehydrogenase or hydrogenase from cell extract greatly reduced formate hydrogenlyase activity; addition of the purified enzymes restored activity. Formate hydrogenlyase activity of cell extract and the reconstituted system was reversible. The coenzyme F₄₂₀-reducing hydrogenase and formate dehydrogenase of M. formicicum were shown to be located at the cytoplasmic membrane using immunogold labeling of thin sectioned, Lowicryl-embedded cells. Neither enzyme was released from whole cells by osmotic shock treatment."]},{"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":["A biochemical and physiological characterization of coenzyme F420-reducing hydrogenase from Methanobacterium formicicum"]}]}],"canonical_facts":{"dc:contributor.committeechair":["Ferry, James G."],"dc:contributor.committeemember":["Bevan, David R.","Anderson, Bruce M.","Wilkins, Tracy D.","Johnson, J.L."],"dc:contributor.department":["Anaerobic Microbiology"],"dc:creator":["Baron, Stephen Francis"],"dc:date.accessioned":["2015-06-29T22:07:06Z"],"dc:date.available":["2015-06-29T22:07:06Z"],"dc:date.issued":["1988"],"dc:description.abstract":["The coenzyme F₄₂₀-reducing hydrogenase of Methanobacterium formicicum was purified 87-fold to electrophoretic homogeneity. The enzyme formed aggregates (1,000 kd) of a coenzyme F₄₂₀-active monomer (109 kd) composed of 1 each of a, β, and γ subunits (43.6, 36.7, xy and 28.8 kd, respectively). It contained 1 mol of FAD, 1 mol of nickel, 12-14 mols of iron, and 11 mols of acid-labile sulfide per mol of the 109 kd species, but no selenium. The amino acid sequence I---P--R-EGH-----EV was conserved in the N-terminus of a subunit of the enzyme and the largest subunits of nickel-containing hydrogenases from Methanobacterium thermoautotrophicum, Desulfovibrio baculatus, and Desulfovibrio gigag. FAD dissociated from the coenzyme F42O-reducing hydrogenase during reactivation with H2 and coenzyme F₄₂₀, unless KCl was present, yielding coenzyme F₄₂₀-inactive apoenzyme. The hydrogenase catalyzed H₂ production at a rate 3-fold less than that for H2 uptake. Specific antiserum inhibited the coenzyme F₄₂₀ dependent activity but not the methyl viologen-dependent activity of the purified enzyme. Cell extract of M. formicicum contained a coenzyme F₄₂₀-mediated formate hydrogenlyase system. Formate hydrogenlyase activity was reconstituted with coenzyme F₄₂₀-reducing hydrogenase, coenzyme F₄₂₀-reducing formate dehydrogenase, and coenzyme F₄₂₀, all purified from M. formicicum. The reconstituted system required FAD for maximal activity (kinetic Kd= 4 μM). without FAD, the formate dehydrogenase and hydrogenase rapidly lost coenzyme F₄₂₀-dependent activity relative to methyl viologen-dependent activity. Immunoadsorption of the formate dehydrogenase or hydrogenase from cell extract greatly reduced formate hydrogenlyase activity; addition of the purified enzymes restored activity. Formate hydrogenlyase activity of cell extract and the reconstituted system was reversible. The coenzyme F₄₂₀-reducing hydrogenase and formate dehydrogenase of M. formicicum were shown to be located at the cytoplasmic membrane using immunogold labeling of thin sectioned, Lowicryl-embedded cells. Neither enzyme was released from whole cells by osmotic shock treatment."],"dc:description.degree":["Ph. D."],"dc:format.mimetype":["application/pdf"],"dc:identifier.uri":["http://hdl.handle.net/10919/53908"],"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":["A biochemical and physiological characterization of coenzyme F420-reducing hydrogenase from Methanobacterium formicicum"],"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":"2026-07-22T22:19:27Z"}