A biochemical and physiological characterization of coenzyme F420-reducing hydrogenase from Methanobacterium formicicum

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dc.contributor.authorBaron, Stephen Francisen
dc.contributor.committeechairFerry, James G.en
dc.contributor.committeememberBevan, David R.en
dc.contributor.committeememberAnderson, Bruce M.en
dc.contributor.committeememberWilkins, Tracy D.en
dc.contributor.committeememberJohnson, J.L.en
dc.contributor.departmentAnaerobic Microbiologyen
dc.date.accessioned2015-06-29T22:07:06Zen
dc.date.available2015-06-29T22:07:06Zen
dc.date.issued1988en
dc.description.abstractThe 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.en
dc.description.degreePh. D.en
dc.format.extentxv, 157 leavesen
dc.format.mimetypeapplication/pdfen
dc.identifier.urihttp://hdl.handle.net/10919/53908en
dc.language.isoen_USen
dc.publisherVirginia Polytechnic Institute and State Universityen
dc.relation.isformatofOCLC# 19730724en
dc.rightsIn Copyrighten
dc.rights.urihttp://rightsstatements.org/vocab/InC/1.0/en
dc.subject.lccLD5655.V856 1988.B3785en
dc.subject.lcshMethanobacteriumen
dc.subject.lcshHydrogenaseen
dc.titleA biochemical and physiological characterization of coenzyme F420-reducing hydrogenase from Methanobacterium formicicumen
dc.typeDissertationen
dc.type.dcmitypeTexten
thesis.degree.disciplineAnaerobic Microbiologyen
thesis.degree.grantorVirginia Polytechnic Institute and State Universityen
thesis.degree.leveldoctoralen
thesis.degree.namePh. D.en

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