Doctor of Philosophy, The Ohio State University, 2011, Biochemistry Program, Ohio State

Fe/S cluster proteins are key players in diverse pathways, such as mitochondrial respiration, gene regulation, and DNA/RNA metabolism. It is generally believed that an Fe/S cluster is synthesized inside the mitochondria before it is exported out to the cytosol. But the details of this export pathway are still unclear; especially what is the substrate that is exported. This substance should be essentially small enough to go through the exporter Atm1p, stable enough to survive the transport process and labile enough to deliver the cluster to cytosolic proteins. It has been shown that glutathione (GSH) or its derivatives might be involved in this pathway. Several Glutaredoxin protein (Grx) structures revealed that Grx is able to coordinate a Fe/S cluster along with two molecules of GSH. The cluster transfer between Grx and scaffold protein ISU was found to be reversible, which indicates Grx may deliver the cluster to the export pathway and plays important role in Fe/S cluster biosynthesis. However, the role of this cluster is still not well understood. A complex of four GSH-coordinated Fe/S cluster was successfully synthesized and characterized and found to be stable under physiological conditions, but undergoes a reversible exchange with scaffold protein ISU. Considering the high cellular concentration of GSH and the stability of this complex under physiological conditions, this complex may contribute to a labile cellular Fe/S cluster pool. The GSH cluster complex is a very intriguing candidate for the substrate of mitochondrial Fe/S cluster exporter. A liposome system was constructed onto which transporter was successfully reconstituted. The stimulation of proteoliposome ATPase activity by GSH cluster complex indicates that the GSH cluster complex is very likely to be the exported substance. Evidence indicates frataxin to be the general iron donor protein in Fe/S cluster biosynthesis. However, a thorough search of the genomic database shows that based on sequence (open full item for complete abstract)

Committee: James Cowan PhD (Advisor); Ross Dalbey PhD (Committee Member); Karin Musier-Forsyth PhD (Committee Member); Richard Swenson PhD (Committee Member) Subjects: Biochemistry; Biophysics; Cellular Biology; Chemistry

Doctor of Philosophy, The Ohio State University, 2004, Chemistry

In this research, we primarily focus on the structure and function of both the nucleotide binding domain, and the full length membrane-spanning transporter. First, the soluble nucleotide binding domain of Atm1 (Atm1-C), an ABC transporter in yeast mitochondria, that has previously been implicated in the maturation of cytosolic iron-sulfur cluster proteins, has been overexpressed in E. coli, purified, and characterized. The full length version of Atm1 from Saccharomyces cerevisiae has been cloned, over-expressed, purified from a yeast expression system, and characterized. A fluorescent assay of liposome-loaded reconstituted Atm1p suggested that Atm1p only allowed small molecules and/or metal complexes to cross the channel. Both pH gradient and fluorescent assays also indicated that ADP-bound Atm1p existed in an open state that is different from the closed state for ATP-bound Atm1p. The further discovery of an iron carrying peptide, hepcidin, provides the first step toward understanding iron trafficking in living cells. With eight, well-conserved cysteine residues in the sequence, hepcidin may not only be a signal peptide, but could potentially serve as an iron carrier. The iron binding properties have been determined by UV-vis spectroscopy, mass spectroscopy, and isothermal titration calorimetry (ITC). The iron binding affinity has been determined in the micromolar range. Studies by circular dichroism (CD) reveal varying degrees of secondary structure within an apparent dynamic tertiary fold. Taken together, hepcidin clearly binds iron, and the secondary structure change induced by iron binding may be required for the full function of the peptide in iron homeostasis and antimicrobial activity. In Part II, a novel fluorescent assay has been developed for monitoring the cleavage of a target RNA by cooper kanamycin in vivo. However, demonstration of the efficacy of such reagents in vitro is only a first step. To demonstrate in vivo cleavage chemistry we have designed a (open full item for complete abstract)

Committee: James Cowan (Advisor) Subjects: Chemistry, Biochemistry