| dc.description.abstract | Iron-sulfur (Fe-S) clusters are ubiquitous protein cofactors that are required for important biological processes including oxidative respiration, nitrogen fixation, and photosynthesis. Defects on Fe-S cluster assembly lead to lethal diseases such as Friedreich’s Ataxia. Despite extensive research on Fe-S cluster biogenesis, key molecular mechanisms remain elusive due to the complicated nature of the system. This dissertation contains pioneering works employing advanced mass spectrometry (MS) methods, especially native ion-mobility MS (native IM-MS), to interrogate Fe-S cluster biogenesis. Recent advances in native IM-MS have allowed proteins to be preserved in native-like structures and support applications in the investigation of protein structure, dynamics, ligand interactions, and the identification of protein-associated intermediates. In our mechanistic study of E. coli ISC Fe-S cluster assembly, samples were prepared under anaerobic conditions and high-resolution native MS methods were applied to probe the intermediates. This approach was validated by the agreement between native MS and circular dichroism spectroscopic assays. Time-dependent native MS experiments revealed potential iron- and sulfur-based intermediates that decay as the [2Fe–2S] cluster signal developed. Additional experiments establish that Fe(II) ions bind to the scaffold protein IscU active site cysteine residues and promote the intermolecular sulfur transfer reaction from the cysteine desulfurase IscS to the Fe-S scaffold protein IscU. These results together support an iron-first model for Fe-S cluster synthesis. In addition, to elaborate the controversial role of the bacterial frataxin CyaY and its relation to cell antioxidation, acid-quench denaturing MS and native IM-MS were utilized to respectively monitor sulfur trafficking and protein-protein interactions. A thorough stepwise analysis of CyaY effect on the ISC machinery suggests CyaY specifically inhibits the sulfur transfer step from IscS to IscU. Investigation of sulfur trafficking from IscS to protein acceptors IscU and TusA and glutathione indicates CyaY switches sulfur transfer from protein acceptors to GSH, leading to generation of antioxidant GSSH which helps relieve oxidative stress. Native IM-MS methods were also employed for monitoring the conformation landscape of the human ISC machinery. IM results suggest the NFS1-ISD11-ACP complex is converted from an inactive extended architecture into an active compact architecture upon binding of the allosteric activator FXN in a morpheein-like mechanism. Overall, the crucial insights shown in this dissertation highlight the power and potential of mass spectrometry methods in exploring enzyme mechanisms. | en |
