Iron-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.

The organization of metabolism is an essential feature of cellular biochemistry. Metabolism does not occur as a linear assembly of freely diffusing enzymes, but as a complex web in which multiple interactions are possible. Because of the crowded environment of the cell, there must be structured and ordered mechanisms that control metabolic pathways. The following work will examine two metabolic pathways, one that is ubiquitous among living organisms and another that is entirely unique to plants, and examine the organization of each in an attempt to further define mechanisms that are fundamental features of metabolic control. One study offers some of the first characterizations of genes involved in [Fe-S] cluster assembly in Arabidopsis. The other explores the mechanisms that control localization of an enzyme that is part of the well-characterized flavonoid biosynthetic pathway. These two distinct pathways serve as unique models for genetic and biochemical studies that contribute to our overall understanding of plant metabolism. / Ph. D.

In the search for the mitochondrion of oxymonads, DNA of Monocercomonoides exilis - an oxymonad isolated from the gut of Chinchilla, was isolated and its genome was sequenced. Sequencing resulted in a fairly complete genome which was extensively searched or genes for mitochondrion related proteins, but no reliable candidate for such gene was identified. Even genes for the ISC pathway, which is responsible for Fe-S cluster assembly and considered to be the only essential function of reduced mitochondrion-like organelles (MROs), were absent. Instead, we were able to detect the presence of a SUF pathway which functionally replaced the ISC pathway. Closer examination of the SUF pathway based on heterologous localisation revealed that this pathway localised in the cytosol. In silico analysis showed that SUF genes are highly conserved at the level of secondary and tertiary structure and most catalytic residues and motifs are present in their sequences. The functionality of these proteins was further indirectly confirmed by complementation experiments in Escherichia coli where SUF proteins of M. exilis were able to restore at least partially Fe-S cluster assembly of strains deficient in the SUF and ISC pathways. We also proved by bacterial adenylate cyclase two-hybrid system that SufB and SufC can form...