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dc.contributor.authorZhang, Bo
dc.description.abstractThe objectives of this work were to investigate the properties and functions of iron-sulfur (Fe-S) cluster-bound forms of monothiol glutaredoxins with CGFS active-site (CGFS-Grxs), a ubiquitous family of proteins involved in Fe-S cluster biogenesis and Fe homeostasis, and to elucidate the oxygen-sensing mechanism of the E. coli fumarate and nitrate reduction transcriptional regulatory protein (FNR). The approach involved using a combination of biophysical techniques to elucidate the nature and properties of the Fe-S centers under steady-state conditions and/or during in vitro cluster transfer. Our results show that the recombinant S. cerevisiae mitochondrial Grx5 is a versatile Fe-S protein that is able to accommodate [2Fe-2S], linear [3Fe-4S], and [4Fe-4S] clusters in vitro depending on the reconstitution conditions and that the [4Fe-4S] cluster-bound Sc Grx5 is competent for in vitro activation of apo aconitase at physiological relevant rates. These results suggested potential roles of Sc Grx5 in scavenging linear [3Fe-4S] clusters released during protein unfolding under oxidative stress conditions and in the maturation of [4Fe-4S] cluster-containing proteins. Functional studies of [2Fe-2S] cluster-bound CGFS- Grxs were carried out using A. vinelandii Grx5 and Grx-nif by investigating in vitro cluster exchange of these two proteins with physiologically relevant partners from the ISC and NIF cluster assembly pathways, respectively. The results show that CGFS-Grxs can efficiently mediate [2Fe-2S] cluster trafficking from U-type primary scaffolds to apo acceptor proteins via intact cluster transfer. Moreover, Av Grx5 and Grx-nif have the potential to accept [2Fe-2S] clusters from [4Fe-4S] cluster-containing scaffolds and/or carrier proteins. These results provide further support for the proposed Fe-S cluster trafficking and storage functions of CGFS- Grxs. Spectroscopic and mass spectrometry investigation of the oxygen-induced [4Fe-4S]-to-[2Fe- 2S] cluster conversion in FNR revealed formation of atypical [2Fe-2S] clusters with one or two cysteine persulfide ligands that result from sulfur-based oxidation and retention of bridging sulfides. The cluster transformation can be reversed under anaerobic conditions upon incubation with DTT and Fe2+ ion. The observation of analogous oxygen-induced [4Fe-4S]-to-[2Fe-2S] cluster conversion in enzymes with oxygen-sensitive [4Fe-4S] clusters suggests that this novel type of cluster interconversion may represent a new mechanism for the assembly and repair of biological [4Fe-4S] clusters.
dc.subjectIron-sulfur cluster
dc.subjectIron-sulfur cluster biogenesis
dc.subjectMonothiol glutaredoxin
dc.subjectGene regulation
dc.subjectOxygen sensing
dc.subjectCluster conversion
dc.titleSpectroscopic and functional characterization of monothiol glutaredoxins and the fumarate nitrate reduction regulatory protein
dc.description.advisorMichael K. Johnson
dc.description.committeeMichael K. Johnson
dc.description.committeeTodd C. Harrop
dc.description.committeeMichael W.w. Adams

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