Date of Award

1-1-2011

Document Type

Campus Access Dissertation

Department

Chemistry and Biochemistry

Sub-Department

Chemistry

First Advisor

Franklin W Outten

Abstract

In vivo biogenesis of iron-sulfur (Fe-S) cluster cofactors requires complex biosynthetic machinery to limit release of iron and sulfide, to protect the Fe-S cluster from oxidation, and to target the Fe-S cluster to the correct apo-enzyme. The SufABCDSE pathway for Fe-S cluster assembly in E. coli accomplishes these tasks under iron starvation and oxidative stress conditions that disrupt Fe-S cluster metabolism. Although SufB, SufC, and SufD are all required for in vivo Suf function, their exact roles are unclear. Here we show that the SufBC2D complex can assemble a [4Fe-4S] cluster that can be transferred to the Aconitase A (AcnA) apo-enzyme in vitro. In addition, in vivo studies show that SufB, SufC, and SufD, co-expressed with the SufS-SufE sulfur transfer pair, purify as two distinct complexes (SufBC2D and SufB2C2) that contain Fe-S clusters and FADH2. These studies also show that SufC and SufD are required for in vivo Fe-S cluster formation on SufB. Furthermore, while SufD is dispensable for in vivo sulfur transfer, it is absolutely required for in vivo iron acquisition. Finally, we demonstrate for the first time that the ATPase activity of SufC is necessary for in vivo iron acquisition during Fe-S cluster assembly. Previous studies showed that suf transcription is controlled by OxyR (an H2O2 stress response transcription factor), the iron metalloregulatory protein Fur, and the Fe-S transcription factor IscR. While transcriptional regulation of the suf genes has been well characterized, almost nothing is known about Suf post-transcriptional or post- translational regulation. We show that recombinant SufBC2D proteins over-expressed in E. coli are phosphorylated at serine and tyrosine residues. Protein phosphorylation is one of the most common covalent modifications used to regulate protein activity at the post-translational level. Together these studies will further our understanding of this important pathway for Fe-S cluster biogenesis.

Rights

© 2011, Avneesh Kumar Saini

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