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dc.contributor.authorDos Santos, Patricia Coutinhoen_US
dc.date.accessioned2014-03-14T20:19:33Z
dc.date.available2014-03-14T20:19:33Z
dc.date.issued2004-11-29en_US
dc.identifier.otheretd-12012004-151629en_US
dc.identifier.urihttp://hdl.handle.net/10919/29851
dc.description.abstractNitrogenase catalyzes the biological reduction of N2 to ammonia (nitrogen fixation). The metalloclusters associated with the nitrogenase components include the [4Fe-4S] cluster of the Fe protein, and the P-cluster [8Fe7S] and FeMo-cofactor [7Fe-9S-Mo-X-homocitrate], both contained within the MoFe protein. These metal-complexes play a vital role in enzyme activity during electron transport and substrate reduction. It is known that the FeMo-cofactor provides the site of substrate reduction, but the exact site of substrate binding remains a topic of intense debate. Some models for the substrate binding location favor the molybdenum atom, while other models favor one or more iron atoms within FeMo-cofactor. We have shown that the a-70 residue of the MoFe protein plays a significant role in defining substrate access to the active site: a-70 approaches one 4Fe-4S face of the FeMo-cofactor. Substitutions at this position alter enzyme specificity for reduction of alternative alkyne substrates. These altered MoFe proteins and alternative alkyne substrates, such as propargyl alcohol, were used to trap an intermediate during substrate reduction. Further studies involving the effect of pH on substrate reduction of these altered MoFe proteins pinpointed the location of the bound substrate-derived intermediate on the FeMo-cofactor to a specific Fe atom, designated Fe6. In addition to understanding how substrates are bound and reduced at the active site, understanding how these clusters are biologically assembled is a second point of interest. Inactivation of NifU or NifS has been shown to affect the activity of both nitrogenase components. NifS is a cysteine desulfurase that provides the sulfur for cluster formation and NifU serves as a molecular scaffold during [Fe-S] cluster assembly. Genetic and biochemical experiments involving amino acid substitutions within the N-terminal and C-terminal domains of NifU indicate that both domains can separately participate in nitrogenase-specific [Fe-S] cluster formation. Furthermore, the NifU and NifS protein appear to have specialized functions in the maturation of metalloclusters of nitrogenase and cannot functionally replace the isc [Fe-S] cluster system used for the maturation of other [Fe-S] proteins. These results indicate that, in certain cases, [Fe-S] cluster biosynthetic machineries have evolved to perform only specialized functions.en_US
dc.publisherVirginia Techen_US
dc.relation.haspartCONTENTS.pdfen_US
dc.relation.haspartChapter6&7.pdfen_US
dc.relation.haspartSumRefApenVita.pdfen_US
dc.relation.haspartChapter4&5.pdfen_US
dc.relation.haspartChapter3.pdfen_US
dc.relation.haspartChapter1&2.pdfen_US
dc.rightsIn Copyrighten
dc.rights.urihttp://rightsstatements.org/vocab/InC/1.0/en
dc.subjectFeMo-cofactoren_US
dc.subjectnifSen_US
dc.subjectnifUen_US
dc.subjectnitrogenaseen_US
dc.subjectmetalloclustersen_US
dc.titleThe Biosynthesis and Function of Nitrogenase Metalloclustersen_US
dc.typeDissertationen_US
dc.contributor.departmentBiochemistryen_US
dc.description.degreePh. D.en_US
thesis.degree.namePh. D.en_US
thesis.degree.leveldoctoralen_US
thesis.degree.grantorVirginia Polytechnic Institute and State Universityen_US
thesis.degree.disciplineBiochemistryen_US
dc.contributor.committeechairDean, Dennis R.en_US
dc.contributor.committeememberBevan, David R.en_US
dc.contributor.committeememberLarson, Timothy J.en_US
dc.contributor.committeememberKim, Sunyoungen_US
dc.contributor.committeememberLuckhart, Shirleyen_US
dc.identifier.sourceurlhttp://scholar.lib.vt.edu/theses/available/etd-12012004-151629/en_US
dc.date.sdate2004-12-01en_US
dc.date.rdate2004-12-03
dc.date.adate2004-12-03en_US


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