Date of Award

5-10-2014

Document Type

Open Access Dissertation

Department

Chemistry and Biochemistry

Sub-Department

Chemistry

First Advisor

Franklin W Outten

Abstract

Fe-S clusters are critical metallocofactors required for cell function. Because of the toxicity of ferrous iron and sulfide to the cell, in vivo Fe-S cluster assembly is carried out by multiprotein biosynthetic pathways. Escherichia coli contains a stress-responsive Fe-S cluster assembly system, the SufABCDSE pathway, working under iron starvation and oxidative stress conditions. The cysteine desulfurase SufS and its accessory protein SufE work together to mobilize persulfide from L-cysteine. We collaborated with Dr. Laura S. Busenlehner to use hydrogen/deuterium exchange mass spectrometry (HDX-MS) to characterize SufS-SufE interactions and protein dynamics. HDX-MS analysis shows that SufE binds near the SufS active site to accept persulfide and initiates allosteric changes in other parts of the SufS structure. SufE enhances the initial L-cysteine substrate binding to SufS and formation of the external aldimine required for early steps in SufS catalysis. HDX-MS analysis suggests a more active role for SufE in promoting SufS \reaction for Fe-S cluster assembly and provides a new picture of the SufS-SufE sulfur transferase pathway, which is different from IscS-IscU sulfur system in Isc pathway working under normal conditions. To determine why the Suf pathway is favored under stress conditions, we directly compared the stress response SufS-SufE sulfur transfer pathway and the basal housekeeping IscS-IscU pathway. We found that SufS-SufE cysteine desulfurase activity is significantly higher than IscS-IscU at physiological cysteine concentrations and after exposure to H2O2. Mass spectrometry analysis demonstrated that IscS-IscU is more susceptible thant SufS-SufE to oxidative modification by H2O2. These results provide biochemical insight into the stress resistance of the Suf pathway. We also found an interesting mutant SufE(D74R), which can interacts stronger with SufS and better enhance SufS activity compared to SufE. Besides the SufS-SufE system, there are two cluster scaffold candidates in Suf pathway, SufBC2D complex and SufA. Both of them can be purified and reconstituted with Fe-S cluster in vivo and in vitro respectively. To distinguish their relative roles, we used a combination of protein-protein interaction and in vitro Fe-S cluster assembly assays and found that SufA works as a shuttle protein to accept Fe-S clusters formed de novo on the SufBC2D complex.

Included in

Chemistry Commons

Share

COinS