Iron sulfur (FeS) clusters are ubiquitous cofactors required for numerous fundamental biochemical processes, including DNA replication and repair, transcription, and translation. In the cell, these metallocofactors require a dedicated protein pathway for assembly. The Cytosolic Iron Sulfur Cluster Assembly (CIA) pathway is conserved across higher-level eukaryotes and is responsible for building and inserting these cofactors into the FeS proteins that need them. A major unsolved problem in the FeS cluster biogenesis field is how so many diverse FeS proteins are identified for cluster insertion. Several studies have identified a multiprotein complex containing Cia1, Cia2, and Met18 as the CIA targeting complex responsible for FeS cluster recognition and target maturation. The CIA targeting complex has been shown to associate with an FeS cluster protein, Nar1. Nar1 is a CIA factor that plays an unknown role in cluster transfer. Little information is known about the structure of the CIA targeting complex its mechanism of FeS cluster protein recognition. In this thesis, I investigate the architecture of the CIA targeting complex as well as the role each subunit plays in identification of apo-proteins and iron-sulfur cluster insertion. Previous proteomic and cell biological studies from the Lill lab propose that the CIA targeting complex exists as a mixture of discrete complexes in vivo. Each of these complexes is responsible for recognizing a distinct subset of targets. Herein, we utilize affinity co-purification and size exclusion chromatography investigate connectivity of the targeting complex, identify stable subcomplexes, and define their roles in recognizing our two model targets Rad3 and Leu1. We determine the CIA targeting complex contains one Met18, two Cia1, and four Cia2 polypepides. This complex is required to recognize Leu1. Our experiments reveal the formation of the stable subcomplexes Cia1-Cia2 and Met18-Cia2, which is sufficient to identify to Rad3. We also interrogate the role of Nar1 in binding to targets and cluster transfer, excluding the model that it acts as an adapter for cluster transfer. Furthermore, using site directed mutagenesis, combined with our co-purification and in vivo assays, we map the key interfaces required to form the targeting complex and investigate how their mutations impacts CIA function in vivo. We identify the binding site of Cia1 on Cia2, as well as the general region in which Cia2 binds to Met18. Through these experiments, we shed light on the role these subunits of CIA targeting complex and Nar1 play in FeS target recognition and FeS cluster transfer.

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AgaR, a protein in <i>Escherichia coli</i> thought to control the metabolism of N-acetylgalactosamine, is a member of the DeoR family of transcriptional regulators. Three transcriptional promoters within a cluster of genes containing the gene for AgaR were identified, specific for <i>agaR, agaZ</i> and <i>agaS</i>, and the transcription start sites mapped. Transcription from these promoters was specifically induced by N-acetylgalactosamine or galactosamine, though K-12 strains lacked the ability to utilize these as sole sources of carbon. The activity of these promoters was constitutively elevated in a strain in which <i>agaR</i> had been disrupted confirming that the promoters are subject to negative regulation by AgaR. AgaR-His6, purified using immobilized metal affinity chromatography, was used for DNase I footprint analysis of the promoter regions. Four operator sites bound by AgaR were identified. A putative consensus binding sequence for AgaR was proposed based on these four sites. <i>In vivo</i> and <i>in vitro</i> analysis of the <i>agaZ</i> promoter indicated that this promoter was activated by the cAMP-cAMP receptor protein (CRP). Expression from the <i>aga</i> promoters was less sensitive to catabolite repression in revertants capable of <i>N</i>-acetylgalactosamine utilization, suggesting that these revertants have mutation(s) that result in an elevated level of inducer for AgaR. A cluster of genes at minute 87.7 of the <i>E. coli</i> genome contains a gene that encodes another member of the DeoR family of transcriptional regulators. This protein, YihW, is more similar to GlpR, transcriptional regulator of <i>sn</i>-glycerol 3-phosphate metabolism in <i>E. coli</i>, than other members of the DeoR family. Despite the high degree of similarity, YihW lacked the ability to repress P_glpK, a promoter known to be controlled by GlpR. A variant of YihW containing substitutions in the putative recognition helix to more closely match the recognition helix of GlpR was also unable to repress P_glpK. Transcriptional promoters identified in this cluster of genes were negatively regulated by YihW. Regulation of genes involved in the metabolism of <i>sn</i>-glycerol 3-phosphate in <i>E. coli</i> by GlpR has been well characterized. However, the function of a protein (GlpE) encoded by a gene cotranscribed with that for GlpR was unknown prior to this work. GlpE was identified as a single-domain, 12-kDa rhodanese (thiosulfate:cyanide sulfurtransferase). The enzyme was purified to near homogeneity and characterized. As shown for other characterized rhodaneses, kinetic analysis revealed that catalysis occurs via an enzyme-sulfur intermediate utilizing a double-displacement mechanism requiring an active-site cysteine. K_m (SSO₃²⁻) and K_m (CN⁻) were determined to be 78 mM and 17 mM, respectively. The native molecular mass of GlpE was 22.5 kDa indicating that GlpE functions as a dimer. GlpE exhibited a kcat of 230 s-1. Thioredoxin, a small multifunctional dithiol protein, served as sulfur-acceptor substrate for GlpE with an apparent K_m of 34 mM when thiosulfate was near its K_m, suggesting thioredoxin may be a physiological substrate. / Ph. D.

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.

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.

Congenital sideroblastic anemias (CSA) are inherited diseases resulting from defects in heme biosynthesis, mitochondrial iron-sulfur cluster (ISC) assembly, or mitochondrial translation. CSAs are characterized by pathological iron deposits in the mitochondria of bone marrow erythroblasts. Recently the Fleming Lab at Boston Children’s Hospital has reported mutations in HSPA9, a chaperone involved in ISC assembly, as a cause of nonsyndromic CSA. Here we identified a CSA patient harboring two variants in HSCB, encoding a binding partner of HSPA9: a paternally inherited promoter variant (c-134C>A) and a maternally inherited frameshift variant (T87fs) predicted to result in a truncated protein. To better understand the pathophysiology of these variants, we investigated HSCB protein expression and function in patient-derived skin fibroblasts. Patient fibroblasts show evidence of decreased HSCB protein levels. shRNA targeting HSCB was employed to specifically suppress HSCB expression in the K562 erythroid-like cell line model. shRNA-infected K562 cells presented with perturbed iron homeostasis, a shift to glycolytic energy production, and diminished hemoglobinization. Targeted deletion of murine Hscb is embryonic lethal prior day E7.0. Tissue-specific lox-Cre transgenic lines, including Vav-, EpoR- and Mx-Cre demonstrate that Hscb is essential for hematopoiesis and erythropoiesis. Mutant mice present with hematopoietic defects similar to the index patient. Vav-Cre animals die prior to post-natal day 9 with decreased red cell counts, white cell counts, and decreased hemoglobin compared to wild-type animals. Floxed-null EpoR-Cre animals die before embryonic day 13. To excise Hscb specifically in the hematopoietic compartment of adult animals, conditional Mx-Cre animals were generated through bone marrow transplantation and temporally induced with polyinosinic-polycytidylic acid treatment. The animals died 22 days post-injection with decreased red blood cells, white blood cells, hemoglobin, and an overall decline in hematopoiesis of the bone marrow. These data demonstrate that HSCB is required for erythropoiesis and hematopoiesis and that the patient mutations are a pathogenic cause of CSA.

abstract: Redox enzymes represent a big group of proteins and they serve as catalysts for biological processes that involve electron transfer. These proteins contain a redox center that determines their functional properties, and hence, altering this center or incorporating non-biological redox cofactor to proteins has been used as a means to generate redox proteins with desirable activities for biological and chemical applications. Porphyrins and Fe-S clusters are among the most common cofactors that biology employs for electron transfer processes and there have been many studies on potential activities that they offer in redox reactions. In this dissertation, redox activity of Fe-S clusters and catalytic activity of porphyrins have been explored with regard to protein scaffolds. In the first part, modular property of repeat proteins along with previously established protein design principles have been used to incorporate multiple Fe-S clusters within the repeat protein scaffold. This study is the first example of exploiting a single scaffold to assemble a determined number of clusters. In exploring the catalytic activity of transmetallated porphyrins, a cobalt-porphyrin binding protein known as cytochrome c was employed in a water oxidation photoelectrochemical cell. This system can be further coupled to a hydrogen production electrode to achieve a full water splitting tandem cell. Finally, a cobalt-porphyrin binding protein known as cytochrome b562 was employed to design a whole cell catalysis system, and the activity of the surface-displayed protein for hydrogen production was explored photochemically. This system can further be expanded for directed evolution studies and high-throughput screening. / Dissertation/Thesis / Doctoral Dissertation Chemistry 2019