Characterization of an altered MoFe protein from a nifV- strain from Azotobacter vinelandii

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dc.contributor.authorComaratta, Leonard M.en
dc.contributor.committeechairDean, Dennis R.en
dc.contributor.committeememberLarson, Timothy J.en
dc.contributor.committeememberGregory, Eugene M.en
dc.contributor.committeememberChen, Jiann-Shinen
dc.contributor.committeememberClaus, George Williamen
dc.contributor.departmentBiochemistry and Anaerobic Microbiologyen
dc.date.accessioned2014-03-14T20:50:01Zen
dc.date.adate1998-11-13en
dc.date.available2014-03-14T20:50:01Zen
dc.date.issued1998-12-03en
dc.date.rdate1999-11-13en
dc.date.sdate1998-12-15en
dc.description.abstractThe site of substrate binding and reduction for the nitrogenase complex is located on the iron molybdenum cofactor (FeMo-co) which is contained within the a-subunit of the molybdenum iron protein. FeMo co consists of a metal sulfur core composed of an FeS cluster bridged by three inorganic sulfides to a MoFeS cluster. An organic acid, homocitrate, is coordinated to the Mo atom through its 2-carboxy and 2-hydroxy groups. Homocitrate is formed by the condensation of acetyl-CoA and a-ketoglutarate, which is catalyzed by a homocitrate synthase encoded by nifV. By deleting the nifV gene from Azotobacter vinelandii we were able to study the role of homocitrate in nitrogenase catalysis. A poly-histidine tail was incorporated into the C-termini of the a-subunit permitting isolation of the homocitrateless MoFe protein by using metal affinity chromatography. We have found that the addition of a poly-histidine tag does not alter the catalytic behavior of the native enzyme. In NifV- strains of Klebsiella pneumoniae, citrate has been found to replace homocitrate as the organic constituent of FeMo-co. We have found no evidence this is so in A. vinelandii. Gas chromatography mass spectrophotometry studies indicate little or no organic acids are associated with FeMo-co. We examined the catalytic properties of the NifV- MoFe protein In the mutant, H2 evolution is inhibited by the addition of CO, unlike in the wild type. We have found that the NifV- MoFe protein from A. vinelandii is able to catalyze the reduction of acetylene to both ethylene and ethane.en
dc.description.degreeMaster of Scienceen
dc.identifier.otheretd-121598-113534en
dc.identifier.sourceurlhttp://scholar.lib.vt.edu/theses/available/etd-121598-113534/en
dc.identifier.urihttp://hdl.handle.net/10919/36177en
dc.publisherVirginia Techen
dc.relation.haspartALLthesisrefs2.pdfen
dc.relation.haspartETDTITLEABTRSTBLCON.pdfen
dc.rightsIn Copyrighten
dc.rights.urihttp://rightsstatements.org/vocab/InC/1.0/en
dc.subjectMoFeen
dc.subjectAzotobacter vinelandiien
dc.subjectNitrogenaseen
dc.titleCharacterization of an altered MoFe protein from a nifV- strain from Azotobacter vinelandiien
dc.typeThesisen
thesis.degree.disciplineBiochemistry and Anaerobic Microbiologyen
thesis.degree.grantorVirginia Polytechnic Institute and State Universityen
thesis.degree.levelmastersen
thesis.degree.nameMaster of Scienceen

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