Date of Award


Document Type

Open Access Dissertation


Chemistry and Biochemistry



First Advisor

F. Wayne Outten


The gram-negative bacterium E. coli encodes the Suf pathway to assemble iron-sulfur (Fe-S) clusters under iron starvation and oxidative stress conditions. The ATPase activity of SufC is critical for in vivo Fe-S cluster assembly by the Suf pathway. SufC shares homology with the nucleotide binding domain (NBD) of ATP-binding cassette (ABC) transporters and belongs to the AAA+ (ATPases Associated with diverse cellular Activities) ATPase superfamily, a family of proteins that utilizes energy from ATP hydrolysis to perform a variety of cellular functions. SufC forms a stable SufBC2D complex with partner proteins SufB and SufD. The SufBC2D complex serves as a novel scaffold where iron and sulfide are assembled into an Fe-S cluster prior to transfer to the SufA carrier protein. The detailed mechanism of the SufBC2D complex is unknown; however, it is known that all three proteins are required for in vivo Fe-S cluster assembly. The present research focuses on understanding the role of the SufC ATPase as a part of the SufBC2D complex during the Suf Fe-S cluster assembly process. We establish that the ATPase activity of SufC is significantly enhanced in SufBC2D compared to SufC alone. Steady state and pre-steady state kinetic analysis of SufBC2D provides evidence of the existence of two conformations of SufBC2D with different ATPase activities. We explore the specific step in the ATP cycle that contributes to the observed differences in ATPase activity using fluorescent nucleotides mantATP and mantADP. We also observe that SufC has a weaker affinity for SufB in the absence of SufD. The addition of ATP or ADP enhances the association of SufC with SufB to form a stable SufB2C2 complex. An affinity chromatography approach is used to investigate the in vivo protein-protein interactions between the Suf proteins. Chromosomally encoded polyhistidine-tagged SufC (SufCHis) interacts with partner proteins SufB and SufD and sulfur transferase proteins SufS and SufE in vivo. We discover that SufCHis is also present as a monomer in normal and H2O2 stressed cells. The data from these in vivo studies suggest a role for SufC alone that is separate from its role when associated with SufB and SufD. Because Suf is a stress-responsive pathway, post-translational regulation could be used to maintain Suf in an inactive state during normal growth conditions when Suf is not the predominant Fe-S cluster assembly pathway. Mass spectrometry analysis of recombinant SufC and SufCHis identifies phosphorylation sites at SufC residues Tyr241 and Ser10, respectively. Studies of the kinetics of the SufC ATPase in Fe-S scaffolds SufBC2D and SufB2C2, Suf protein-protein interactions in vivo and in vitro, and Suf posttranslational modifications provide insight into the functional roles of the SufC ATPase and identify phosphorylation as a mode of SufC regulation during Fe-S cluster assembly by the Suf pathway.

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