Date of Award

Summer 2019

Document Type

Open Access Dissertation

Department

Chemistry and Biochemistry

First Advisor

Franklin W. Outten

Abstract

The regulation of transition metals within living organisms is essential to the persistence of life. Of particular interest to our lab is iron due to the prevalence of this metal within the cell in addition to the numerous functions it performs. Fe-S cofactors are a significant prosthetic group consisting of iron that are necessary for the function of various metalloproteins. Generation of Fe-S clusters depends on several biosynthesis pathways varying between organisms with Isc and Suf both utilized in E. coli. While Isc controls Fe- S manufacturing under normal conditions, Suf assumes control when the cell is exposed to iron deplete conditions or oxidative stress. Consisting of six proteins, cluster synthesis cannot begin without the sulfur obtained from free cellular cysteine via a cysteine desulfurase reaction. As one of the six proteins coded for by suf, the PLP-bound SufS protein operates as a type II cysteine desulfurase capable of interacting with cysteine to produce alanine and persulfide. This process, however, requires a sulfur shuttle in the form of SufE capable of increasing desulfurase activity up to 50-fold when added to SufS. While the steady state kinetics of this mechanism have been investigated, the exact pre-steady state kinetics as well as the intermediates involved offer critical targets for analysis due to the lack of the Suf pathway in humans. In this dissertation, we investigated the mechanism by which E. coli SufS performs the cysteine desulfurase process alone successfully assigning several intermediates while determining the pre-steady state kinetics. Furthermore, the addition of SufE reveals a novel effector role beyond the ability to solely remove sulfur from SufS and instead alter the reaction mechanism. Other studies have hinted at this allosteric enhancement, but this is the first instance in which the cysteine desulfurase process shows this impact. Finally, we also investigate two other proteins associated with iron regulation through regulation of iron reduction (YqjI), which revealed a link between iron regulation and nickel levels, and potential iron storage (FtnB). The combined importance of iron and Suf in pathogenic bacteria along with the absence of Suf in humans offer optimal therapeutic drug targets.

Rights

© 2019, Matthew Ryan Blahut

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