Iron sulfur (Fe-S) clusters are essential cofactors that function in electron transport, catalyzing substrate turnover, environmental sensing, and initiating radical chemistry. Elaborate multi-component systems have evolved to protect organisms from the toxic effects of free iron and sulfide ions while promoting the efficient biosynthesis of these cofactors. The in vivo loss of frataxin (FXN) function results in depleted activity of Fe-S enzymes and is directly linked to the fatal and incurable neurodegenerative disease Friedreich's ataxia (FRDA). Previously, our lab discovered the cysteine desulfurase and Fe-S assembly activities of the human Fe-S assembly complex (SDU), which consists of the cysteine desulfurase complex NFS1-ISD11 and scaffold protein ISCU2, are greatly stimulated by FXN binding and forming the SDUF complex. This dissertation's objectives were to identify critical FXN interactions for binding and activation of the SDU complex, investigate the interprotein sulfur transfer reaction between NFS1 and ISCU2, and provide mechanistic details of Fe-S assembly on the SDUF complex. First, surface residues on FXN were substituted with alanine or glycine and the ability of each variant to bind and activate the SDU complex was assessed. These experiments revealed a localized "hotspot" of critical residues on FXN, which could aid in designing small peptide mimics for FRDA therapeutics. Second, ^(35)S-radiolabeling experiments indicated FXN accelerates the accumulation of persulfide species on NFS1 and ISCU2. The ISCU2 persulfide species was established as a viable intermediate in Fe-S cluster biosynthesis by tracking the ^(35)S-radiolabel as it converts from a persulfide species to a [2Fe-2S] cluster. Additional mutagenic, enzymatic, and spectroscopic studies suggest conserved ISCU2 residue C104 is critical for FXN-based activation, whereas C35, C61, and C104, are all essential for Fe-S cluster biosynthesis. These results lead to an activation model in which FXN facilitates sulfur transfer from NFS1 to ISCU2 as an initial step in Fe-S cluster biosynthesis and favors helix-to-coil interconversion on ISCU2. Third, UV-visible, circular dichroism, and Mossbauer spectroscopic studies indicated the SDUF complex synthesizes transient [2Fe-2S] clusters that readily transfer to thiol-containing acceptor molecules. Moreover, these studies revealed competing DTT-mediated transfer and mineralization chemistry that cause complications when studying the mechanism of Fe-S cluster biosynthesis.
