Date of Award
Campus Access Dissertation
Chemistry and Biochemistry
Franklin W Outten
Iron-sulfur (Fe-S) clusters are essential cofactors in various metalloproteins and are best known for their roles in electron transport as well as redox and non-redox catalysis. Three Fe-S cluster assembly systems: Nif, Isc, and Suf have been identified. The sufABCDSE operon is activated in E. coli to build Fe-S clusters under oxidative stress and iron starvation conditions. Despite the impressive progress made in this field, the molecular details of Fe-S cluster biogenesis, including the identity of the in vivo iron source and the mechanism of Fe-S cluster transfer from the scaffold proteins to target proteins, are still poorly understood. The present research is focused on understanding the relative roles of SufB and SufA in Fe-S cluster trafficking in the Suf pathway. We studied the role of SufA both as a potential Fe-S scaffold and as a Fe-S cluster carrier protein. We established that SufA binds a [2Fe-2S] cluster in vivo and transfers it to target metalloproteins. We compared the cluster transfer abilities of SufA and SufBC2D and found that [4Fe-4S]-SufBC2D can load a [2Fe-2S] cluster into SufA, whereas SufA can not transfer cluster to SufBC2D. This uni-directional cluster transfer suggests that SufBC2D is an Fe-S scaffold while SufA is an Fe-S cluster transfer protein. To test this hypothesis, the relative scaffold and cluster transfer abilities of SufA and SufBC2D to the target [2Fe-2S]-Fdx protein were compared. We observed that SufBC2D is a scaffold and uses SufA as an intermediate carrier to load [2Fe-2S] target proteins like Fdx. The role of the SufC protein, a member of ABC-ATPases family, in Fe-S cluster transfer was also investigated. SufC exhibits low basal ATPase activity which is enhanced by SufB and SufD. The effect of ATP addition on cluster transfer from SufBC subcomplexes to SufA and to Fdx resulted in reduced cluster transfer to [2Fe-2S] target proteins. We also found some differential regulation of Suf proteins under heat-shock and cold-shock suggesting that they may function independently under some stress conditions. These studies have expanded our understanding of Suf pathway by providing insight into the roles of the SufB and SufA proteins in this pathway.
Chahal, H. K.(2011). Characterizing the Roles of Sufb and Sufa in Fe-S Cluster Assembly and Trafficking in Suf Pathway. (Doctoral dissertation). Retrieved from http://scholarcommons.sc.edu/etd/665