Discovery of key intermediates for radical initiation in PFL-AE

Abstract

Members of the radical S-adenosyl-L-methionine (SAM) enzyme superfamily utilize a [4Fe-4S] cluster and the small molecule, SAM, to generate methionine and the 5'deoxyadenosyl radical (5'-dAdo*). Once formed, the 5'-dAdo* abstracts a hydrogen from substrate allowing for the catalyzation of a wide array of chemistry such as DNA repair, hydrogenase maturation, and anaerobic glucose metabolism. Originally, the 5'-dAdo* was thought to form directly through homolytic cleavage of the S-C5' bond on SAM. In 2016, this mechanism was called into question when a catalytically relevant organometallic intermediate (omega) was discovered in pyruvate formate-lyase activating enzyme (PFL-AE). This intermediate consisted of a 5'-dAdo moiety bound to the unique iron on the PFL-AE [4Fe-4S] cluster through an Fe-C5' bond. The work shown in this thesis provides novel insights into the RS enzyme mechanism considering the newly discovered omega species. Using rapid freeze quench (RFQ) in conjunction with electron paramagnetic resonance (EPR) spectroscopy, omega formation was observed in seven RS enzymes representing the totality of superfamily reaction types. Inspired by the idea that the Fe-C5' bond in omega could undergo photoinitiated homolysis, a unique procedure was developed to generate and capture the long elusive 5'-dAdo* through cryogenic photolysis of reduced PFL-AE and SAM. Isotopic labeling of SAM along with EPR spectroscopy confirmed definitely that this was the long sought after 5'-dAdo*. To better understand RS enzyme bond specificity and the order of intermediate formation, an analogue of SAM, S-3'4'-anhydroadenosyl-L-methionine (anSAM), was employed in RFQ and cryogenic photolysis experiments. By using anSAM, it was shown that the bond cleavage specificity of PFL-AE can changed under appropriate conditions and provided evidence that omega forms first in the radical initiation pathway of RS enzymes. These results have greatly increased our understanding of the RS enzyme mechanism and will help future work designed to utilize the incredible enzymatic potential of this diverse superfamily.