dc.contributor.author	Dos Santos, Patricia C.	en
dc.contributor.committeechair	Dean, Dennis R.	en
dc.contributor.committeemember	Bevan, David R.	en
dc.contributor.committeemember	Larson, Timothy J.	en
dc.contributor.committeemember	Kim, Sunyoung	en
dc.contributor.committeemember	Luckhart, Shirley	en
dc.contributor.department	Biochemistry	en
dc.date.accessioned	2014-03-14T20:19:33Z	en
dc.date.adate	2004-12-03	en
dc.date.available	2014-03-14T20:19:33Z	en
dc.date.issued	2004-11-29	en
dc.date.rdate	2004-12-03	en
dc.date.sdate	2004-12-01	en
dc.description.abstract	Nitrogenase 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
dc.description.degree	Ph. D.	en
dc.identifier.other	etd-12012004-151629	en
dc.identifier.sourceurl	http://scholar.lib.vt.edu/theses/available/etd-12012004-151629/	en
dc.identifier.uri	http://hdl.handle.net/10919/29851	en
dc.publisher	Virginia Tech	en
dc.relation.haspart	CONTENTS.pdf	en
dc.relation.haspart	Chapter6&7.pdf	en
dc.relation.haspart	SumRefApenVita.pdf	en
dc.relation.haspart	Chapter4&5.pdf	en
dc.relation.haspart	Chapter3.pdf	en
dc.relation.haspart	Chapter1&2.pdf	en
dc.rights	In Copyright	en
dc.rights.uri	http://rightsstatements.org/vocab/InC/1.0/	en
dc.subject	FeMo-cofactor	en
dc.subject	nifS	en
dc.subject	nifU	en
dc.subject	nitrogenase	en
dc.subject	metalloclusters	en
dc.title	The Biosynthesis and Function of Nitrogenase Metalloclusters	en
dc.type	Dissertation	en
thesis.degree.discipline	Biochemistry	en
thesis.degree.grantor	Virginia Polytechnic Institute and State University	en
thesis.degree.level	doctoral	en
thesis.degree.name	Ph. D.	en

The Biosynthesis and Function of Nitrogenase Metalloclusters

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