Biochemical characterization of a novel iron-sulfur flavoprotein from Methanosarcina thermophila strain TM-1

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dc.contributor.authorLeartsakulpanich, Ubolsreeen
dc.contributor.committeechairFerry, James G.en
dc.contributor.committeememberWhite, Robert H.en
dc.contributor.committeememberDean, Dennis R.en
dc.contributor.committeememberLarson, Timothy J.en
dc.contributor.committeememberGregory, Eugene M.en
dc.contributor.committeememberHess, John L.en
dc.contributor.departmentBiochemistry and Anaerobic Microbiologyen
dc.date.accessioned2014-03-14T20:13:36Zen
dc.date.adate1999-06-30en
dc.date.available2014-03-14T20:13:36Zen
dc.date.issued1999-06-11en
dc.date.rdate2000-06-30en
dc.date.sdate1999-06-29en
dc.description.abstractThe iron-sulfur flavoprotein (Isf) from the acetate utilizing methanoarchaeon Methanosarcina thermophila was heterologously produced in Escherichia coli, purified to homogeneity, and characterized to determine the properties of the iron-sulfur cluster and FMN. Chemical and spectroscopic analyses indicated that Isf contained one 4Fe-4S cluster and one FMN per monomer. The midpoint potentials of the [4Fe-4S]2+/1+ center and FMN/FMNH2 redox couple were -394 and -277 mV respectively. The deduced amino acid sequence of Isf revealed high identity with Isf homologues from the CO2 reducing methanoarchaea Methanococcus jannaschii and Methanobacterium thermoautotrophicum. Extracts of H2-CO2-grown M. thermoautotrophicum cells were able to reduce Isf from M. thermophila using either H2 or CO as the reductant. Addition of ferredoxin A to the reaction further stimulated the rate of Isf reduction. These results suggest that Isf homologues are coupled to ferredoxin in electron transfer chains in methanoarchaea with diverse metabolic pathways. Reconstituted systems containing carbon monoxide dehydrogenase/acetyl-CoA synthase complex (CODH/ACS), ferredoxin A, Isf, and the designated electron carriers (NAD, NADP, F420, and 2-hydroxyphenazine) were used in an attempt to determine the electron acceptor for Isf. Isf was unable to reduce any of these compounds. Furthermore, 2-hydroxyphenazine competed with Isf to accept electrons from ferredoxin A indicating that ferredoxin A is a more favorable electron partner for 2-hydroxyphenazine. Thus, the physiological electron acceptor for Isf is unknown. Amino acid sequence alignment of Isf sequences revealed a conserved atypical cysteine motif with the potential to ligate the 4Fe-4S cluster. Site-directed mutagenesis of the cysteine residues in this motif, and the two additional cysteines in the sequence, was used to investigate these cysteine residue as ligands for coordinating the 4Fe-4S center of Isf. Spectroscopic and biochemical analyses were consistent with the conserved cysteine motif functioning as ligating the 4Fe-4S center. Redox properties of the 4Fe-4S and FMN centers revealed a role for the 4Fe-4S center in the transfer of electrons from ferredoxin A to FMN.en
dc.description.degreePh. D.en
dc.identifier.otheretd-062999-160810en
dc.identifier.sourceurlhttp://scholar.lib.vt.edu/theses/available/etd-062999-160810/en
dc.identifier.urihttp://hdl.handle.net/10919/28161en
dc.publisherVirginia Techen
dc.relation.haspartulthesis.pdfen
dc.rightsIn Copyrighten
dc.rights.urihttp://rightsstatements.org/vocab/InC/1.0/en
dc.subjectIron-sulfur flavoproteinen
dc.subjectMethanosarcina thermophilaen
dc.titleBiochemical characterization of a novel iron-sulfur flavoprotein from Methanosarcina thermophila strain TM-1en
dc.typeDissertationen
thesis.degree.disciplineBiochemistry and Anaerobic Microbiologyen
thesis.degree.grantorVirginia Polytechnic Institute and State Universityen
thesis.degree.leveldoctoralen
thesis.degree.namePh. D.en

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