Show simple item record

dc.contributor.advisorBarondeau, David P.
dc.creatorRabb, Jennifer
dc.date.accessioned2012-07-16T15:58:03Z
dc.date.accessioned2012-07-16T20:29:50Z
dc.date.available2014-09-16T07:28:20Z
dc.date.created2012-05
dc.date.issued2012-07-16
dc.date.submittedMay 2012
dc.identifier.urihttps://hdl.handle.net/1969.1/ETD-TAMU-2012-05-10907
dc.description.abstractIron-sulfur clusters are protein cofactors that are critical for all life forms. Elaborate multi-component systems have evolved for the biosynthesis of these cofactors to protect organisms from the toxic effects of free iron and sulfide ions. In eukaryotes, the Fe-S cluster assembly machinery operates in the matrix space of the mitochondria and contains a myriad of proteins that mediate sulfur, iron, and electron transfer to assemble Fe-S clusters on the scaffold protein ISCU2 and then distribute these clusters to target proteins. Our lab has recently described stable 3, and 4-protein complexes composed of the cysteine desulfurase NFS1, the co-chaperone ISD11, and ISCU2 (SDU), and NFS1, ISD11, ISCU2, and FXN (SDUF) subunits. In the latter, SDUF, FXN functions as an allosteric activator switching this assembly complex on for Fe-S cluster biosynthesis. Insufficient expression of the mitochondrial protein FXN leads to a progressive neurodegenerative disease, Friedreich's Ataxia (FRDA). In ~2% of patients, FRDA is caused by one of 15 known missense mutations on one allele accompanied by the GAA repeat on the other leading to a complicated phenotype that includes loss of Fe-S clusters. Here we present in vitro evidence that FRDA FXN variants are deficient in their ability to bind the SDU complex, their ability to stimulate the sulfur transfer reaction from NFS1 to ISCU2, and in their ability to stimulate the rate of cluster assembly on ISCU2. Here, in vitro evidence is presented that FXN accelerates the sulfur transfer reaction from NFS1 to ISCU2. Additionally, we present kinetic evidence that identifies the most buried cysteine residue, C104 on ISCU2 as the sulfur acceptor residue suggesting, FXN stabilizes a conformational change to facilitate sulfur delivery. Subsequent mutational studies suggest FXN binding to SDU results in a helix to coil transition in ISCU2 exposing C104 to accept the persulfide sulfur and thereby accelerating the rate of sulfur transfer. We further provide the first biochemical evidence that the persulfide transferred to ISCU2 from NFS1 is viable in Fe-S cluster formation. In contrast to human FXN, the Escherichia coli FXN homolog CyaY has been reported to inhibit Fe-S cluster biosynthesis. To resolve this discrepancy, a series of inter-species enzyme kinetic experiments were performed. Surprisingly, our results reveal that activation or inhibition by the frataxin homolog is determined by which cysteine desulfurase is present and not by the identity of the frataxin homolog. These data are consistent with a model in which the frataxin-less Fe-S assembly complex exists as a mixture of functional and nonfunctional states, which are stabilized by binding of frataxin homologs. Intriguingly, this appears to be an unusual example in which modifications to an enzyme during evolution inverts or reverses the mode of control imparted by a regulatory molecule.en
dc.format.mimetypeapplication/pdf
dc.language.isoen_US
dc.subjectFrataxinen
dc.subjectFe-S Cluster Assemblyen
dc.subjectFriedreich's ataxiaen
dc.titleFrataxin (FXN) Based Regulation of the Iron-Sulfur Cluster Assembly Complexen
dc.typeThesisen
thesis.degree.departmentChemistryen
thesis.degree.disciplineChemistryen
thesis.degree.grantorTexas A&M Universityen
thesis.degree.nameDoctor of Philosophyen
thesis.degree.levelDoctoralen
dc.contributor.committeeMemberBegley, Tadhg P.
dc.contributor.committeeMemberBell-Pedersen, Deborah
dc.contributor.committeeMemberRaushel, Frank M.
dc.type.genrethesisen
dc.type.materialtexten
local.embargo.terms2014-07-16


Files in this item

Thumbnail

This item appears in the following Collection(s)

Show simple item record