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Access Type

WSU Access

Date of Award

1-1-2016

Degree Type

Dissertation

Degree Name

Ph.D.

Department

Biochemistry and Molecular Biology

First Advisor

Timothy L. Stemmler

Abstract

Fe-S clusters are iron-containing cofactors utilized by numerous proteins within several biological pathways essential to life. In eukaryotes, the primary pathway for Fe-S cluster production is the iron-sulfur cluster (ISC) pathway. The eukaryotic ISC pathway, localized primarily within the mitochondria, has been best characterized within Saccharomyces cerevisiae. In yeast, de novo Fe-S cluster formation is accomplished through coordinated assembly of the substrates iron and sulfur on the primary scaffold assembly protein “Isu1”. The sulfur used for cluster assembly is provided by the cysteine desulfurase “Nfs1”, a protein that works in union with its accessory protein “Isd11”. Frataxin “Yfh1” helps direct cluster assembly by serving as a modulator of Nfs1 activity, by assisting in the delivery of Fe(II) to Isu1, or through a combination of roles.

This work describes and optimizes an in vitro method for generating an Fe-S cluster on the scaffold Isu1. Further in vitro studies on the yeast ISC system have been limited, however, due to the inherent instability of recombinant Isu1, a molecule prone to degradation and aggregation. To circumvent Isu1 stability issues, this research demonstrates successful cross-reactivity between the fly Isu1 ortholog, “fIscU”, and yeast ISC in vitro and in vivo. This strategy could facilitate novel experimentation of the yeast ISC pathway in the future. This work also isolates complexes formed between recombinant yeast Nfs1-Isd11, Nfs1-Isd11-Isu1, and Nfs1-Isd11-Isu1-Yfh1, and characterizes their activity related to substrate binding and cluster assembly. Complexed Isu1, as isolated within Nfs1-Isd11-Isu1 and Nfs1-Isd11-Isu1-Yfh1 complexes, demonstrates inhibited Fe-S cluster biogenesis without the addition of exogenous apo-Isu1. This suggests a mechanism for de novo Fe-S cluster formation on Isu1 that is similar to recent observations from bacterial ISC. Lastly, evidence is provided that suggests that frataxin can function as an allosteric activator of Nfs1 in vitro.

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