The Role of auxiliary iron-sulfur clusters in radical SAM catalyzed carbon-sulfur bond formation

Members of the 4Fe-4S-containing radical S-adenosyl-l-methionine (rSAM) superfamily of enzymes catalyze a diverse array of reactions, and a recent boom in studies on the SPASM/Twitch domain-containing subgroup has revealed even more types of chemistry. At the heart of rSAM catalysis is the formation of a radical intermediate following H-atom abstraction on substrate. How these enzymes shepherd the substratebased radical is still an active area of research. Auxiliary clusters bound by SPASM/Twitch domains are widely speculated to assist this process via electron transfer or other more complicated mechanisms. This dissertation's scope is limited to the role of SPASM/Twitch auxiliary clusters in the maturation of ribosomally synthesized and post-translationally modified peptide natural products (RiPPs). Two related enzymes served as model systems for my studies, SkfB and TteB. More information on these proteins is provided in Chapter 1, where I comprehensively review the current understanding of all rSAM enzymes involved in the biogenesis RiPP natural products. Sections 1.1 and 1.6 give ample background for the remaining chapters in the dissertation. Chapter 1's entirety highlights how little is known about the mechanisms of this massive family of proteins. Chapter 2 reports investigations into the reaction catalyzed by SkfB using a radical clock. This unnatural amino acid allowed for the uncoupling of H-atom abstraction from iv overall turnover, yielding valuable insights into the mechanistic role of the auxiliary cluster. The findings in Chapter 3 demonstrate that a SPASM domain RiPP maturase can tolerate a selenocysteine-substituted substrate, which permits the enzymatic transformation to a selenoether. Spectroscopic analyses showed that the selenium interacts with an iron atom of an auxiliary cluster-a groundbreaking observation. Chapter 4 reports investigations on a 4Fe-4S chaperone protein having an observable effect on SkfB's cofactor content and its observed activity. This section provides a starting point for future studies on the nature and assembly of the various 4Fe- 4S clusters found in rSAM enzymes. In Appendices A and B, I have included publications that I have contributed to that have had a significant impact on the field of 4Fe-4S-dependent enzyme chemistry.