Access Type

Open Access Dissertation

Date of Award

January 2019

Degree Type

Dissertation

Degree Name

Ph.D.

Department

Pharmaceutical Sciences

First Advisor

Timothy L. Stemmler

Abstract

Mitochondrial Fe-S cluster biosynthesis is accomplished within yeast utilizing the biophysical characteristics of the “Isu1” scaffold protein. As a member of a highly homologous protein family, Isu1 has sequence conservation with orthologs and a conserved ability to assemble [2Fe-2S] clusters. Regardless of species, scaffold orthologs can exist in both “disordered” and “structured” conformations and is directly related to conformations utilized during Fe-cofactor assembly. During assembly, the scaffold directs the delivery and the utilization of both Fe(II) and sulfide substrates in order to produce [2Fe-2S] clusters, however Zn(II) binding can alter the activity of the scaffold with stabilizing the protein in the structured state. Understanding the interplay between Fe(II) and Zn(II) binding in vitro may help clarify metal loading events that occur during Fe-S cluster assembly in vivo.

Here we determine the metal:protein stoichiometry for Isu1 Zn and Fe binding is 1:1 and 2:1, respectively. As expected, while Zn binding shifts the Isu1 to its structured state, folding is not influenced by Fe(II) binding alone. X-ray absorption spectroscopy (XAS) confirms Zn(II) binds to the scaffold’s cysteine rich cluster assembly active site and Fe(II) binds at a location distinct from the active site. XAS results show Isu1 binding of either Fe(II) or Zn(II) does not perturb the structure of the alternatively bound metal. XAS and Mössbauer spectroscopies combined confirm that several scaffold orthologs bind iron as high-spin Fe(II) at a site composed of ca 6 oxygen and nitrogen only nearest neighbor ligands. Finally, Zn binding dramatically reduces the Fe-S cluster assembly activity of Isu1, even in the presence of frataxin. Given the metal binding activity of Isu1, a mechanism for Fe(II) transport during cluster assembly and a possible role for Zn for the protein have been considered in this report.

We also present evidence for the significance of the C-terminal alpha helix of Isu1. Despite the importance of the pathway, very little is known about the molecular details of Isu1, especially in the protein’s C-terminal helical region. Data shows that a loss of the 10 C-terminal residues in Isu1 causes a complete loss of cluster assembly activity and impairs cellular growth without while the proteins structure and iron binding activity remain unchanged. One possible role for the C-terminus is that it provides structural anchor which positions the LPPVK domain and active site to make contacts with key ISC members. This report shows the C-terminus is essential for cluster assembly emphasizing a direct relationship to the pathogenesis of ISCU myopathy.

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