Iron-sulfur (Fe-S) clusters are critical protein cofactors found in all life forms. In eukaryotes, a well-conserved biosynthetic pathway located in the mitochondria is used to assemble Fe-S clusters. Although proteins required for Fe-S cluster biosynthesis have been identified, their precise function and mechanism remain elusive. In this study, biochemical and biophysical methods are applied to understand molecular details for the core components of the human Fe-S cluster biosynthesis: Nfs1, Isd11, Isu2, and frataxin (Fxn). Nfs1 is a cysteine desulfurase that converts cysteine into alanine and transfers the sulfur to a scaffold protein Isu2 for Fe-S clusters. Fxn depletion is associated with the neurodegenerative disease Friedreich’s ataxia (FRDA), and results in a complicated phenotype that includes loss of Fe-S clusters. The results presented here provide the first in vitro evidence for a stable protein complex that exists in at least two forms: an inactive complex with Nfs1, Isd11, and Isu2 (SDU) components and an active form that also includes Fxn (SDUF). Fxn binding dramatically changes the catalytic efficiency (kcat/KM) of Nfs1 from 25 to 10,100 M-1s-1 and enhances the rate of Fe-S cluster biosynthesis 25 fold. Oxidizing conditions diminish the levels of both complex formation and Fxn-based activation, whereas Fe2 further stimulates Nfs1 activity. Mutagenesis coupled to enzyme kinetics indicate that one of the three conserved cysteines (C104) on Isu2 accepts the sulfane sulfur from Nfs1 and that this transfer event likely requires prior binding of Fxn. In vitro interrogation of FRDA I154F and W155R and related Fxn variants revealed the binding affinity to SDU followed the trend Fxn ~ I154F > W155F > W155A ~ W155R. The Fxn variants also have diminished ability to facilitate both sulfur transfer and Fe-S cluster assembly. Fxn crystallographic structures reveal specific rearrangements associated with the loss of function. Importantly, the weaker binding and lower activity of the W155R variant compared to I154F explains the earlier onset and more severe disease progression. Finally, these experimental results coupled with computational docking studies suggest a model for how human Fxn functions as an allosteric activator and triggers sulfur transfer and Fe-S cluster assembly.

abstract: It has been well established that mitochondria play a critical role in the pathology of Friedreich's Ataxia. This disease is believed to be caused by a deficiency of frataxin, which research suggests is responsible for iron sulfur cluster assembly. This incomplete assembly of iron sulfur clusters is believed to be linked with dysfunctional complexes in the mitochondrial respiratory chain, increased oxidative stress, and potential cell death. Increased understanding of the pathophysiology of this disease has enabled the development of various therapeutic strategies aimed at restoring mitochondrial respiration. This thesis contains an analysis of the biological activity of several classes of antioxidants against oxidative stress induced by diethyl maleate in Friedreich's Ataxia lymphocytes and CEM leukemia cells. Analogues of vitamin E α-tocopherol have been shown to protect cells under oxidative stress. However, these same analogues show various levels of inhibition towards the electron transport chain complex I. Bicyclic pyridinols containing a ten carbon substituent provided favorable cytoprotection. N-hydroxy-4-pyridone compounds were observed to provide little protection. Similarly, analogues of CoQ10 in the form of pyridinol and pyrimidinol compounds also preserved cell viability at low concentrations. / Dissertation/Thesis / M.S. Biochemistry 2012

Friedreich ataxia (FRDA) is a progressive neurodegenerative disease with primary sites of pathology in the large sensory neurons of the dorsal root ganglia (DRG) and dentate nucleus of the cerebellum. FRDA is also often accompanied by severe cardiomyopathy and diabetes mellitus. FRDA is caused by loss of frataxin (FXN) expression, which is due to GAA repeat expansion in intron 1 of the FXN gene. Frataxin is a mitochondrial protein important in iron-sulphur cluster (ISC) biogenesis and in the electron transport chain (ETC). As a consequence of impaired mitochondrial energy metabolism, FRDA cells show increased levels of and sensitivity to oxidative stress, which is known to be associated with genome instability. In this study, we investigated DNA damage/repair in relation to FXN expression via immunostaining of γ-H2AX, a nuclear protein that is recruited to DNA double strand breaks (DSBs). We found FRDA patient and YG8sR FRDA mouse model fibroblasts to have inherently elevated DNA DSBs (1.8 and 0.9 foci/nucleus) compared to normal fibroblasts (0.6 and 0.2 foci/nucleus, in each case P < 0.001). By delivering the FXN gene to these cells with a lentivirus vector (LV) at a copy number of ~1/cell, FXN mRNA levels reached 48 fold (patient cells) and 42 fold (YG8sR cells) and protein levels reached 20 fold (patient cells) and 3.5 fold (YG8sR cells) that of untreated fibroblasts, without observable cytotoxicity. This resulted in a reduction in DNA DSB foci to 0.7 and 0.43 (in each case P < 0.001) in human and YG8sR fibroblasts, respectively and an increase in cell survival to that found for normal fibroblasts. We next irradiated the FRDA fibroblasts (2Gy) and measured their DSB repair profiles. Both human and mouse FRDA fibroblasts were unable to repair damaged DNA. However, repair returned to near normal levels following LV FXN gene transfer. Our data suggest frataxin may be important for genome stability and cell survival by ensuring ISC for DNA damage repair enzymes or may be required directly for DNA DSB repair.

In 1996, scientists discovered a connection between the gene for the human protein frataxin (FXN) and the neurodegenerative disease Friedreich’s ataxia (FRDA). Decreased FXN levels result in a variety of aberrant phenotypes including loss of activity for iron-sulfur containing enzymes, mitochondrial iron accumulation, and susceptibility to oxidative stress. These symptoms are the primary focus of current therapeutic efforts. In contrast our group is pursuing an alternate strategy of first defining FXN function at a molecular level then using this information to identify small molecule functional replacements. Recently, our group has discovered that FXN functions as an allosteric activator for the human Fe-S cluster assembly complex. The work presented here helps to further define molecular details of FXN activation and explain how FRDA missense mutants are functionally compromised. First, the FRDA missense mutants L182H and L182F were investigated. Unlike other characterized FRDA missense mutants, the L182F variant was not compromised in its ability to bind and activate the Fe-S assembly complex. The L182H variant exhibited an altered circular dichroism signature; suggesting a change in secondary structure relative to native FXN, and rapidly degraded. Together these studies suggest that L182 variants are less stable than native FXN and are likely prone to degradation in FRDA patients. Second, as a regulatory role of FXN suggests that its function is likely controlled by environmental stimuli, different maturation forms of FXN as well as post-translational modification mimics were tested as mechanisms to control FXN regulation. Here experiments were designed to test if a larger polypeptide form of FXN represents a functional form of the protein. Kinetic and analytical ultracentrifugation studies revealed a complex heterogeneous mixture of species some of which can activate the Fe-S assembly complex. A previously identified acetylation site was also tested using mutants that mimic acetylation. These mutants had little effect on the ability of FXN to bind and activate the assembly complex. Third, mutagenesis experiments were designed in which the FXN surface residues were replaced with alanine and the resulting variants were tested in binding and activity assays. These experiments revealed a localized “hot-spot” on the surface of FXN that suggests small cyclic peptide mimics might be able to replace FXN and function as FRDA therapeutics. Unexpectedly, one of the FXN variants exhibited significantly tighter binding and could have relevance for therapeutic development.