Abstract 1281 Interrogating thiyl radical chemistry in Pyruvate Formate lyase through Mechanism-based inhibitors and genetic code expansion

Cysteine thiyl radicals are reactive cofactors in challenging biochemical reactions across all domains of life. Despite their ubiquity, characterizing thiyl radical generation, energetics and control is challenging due to their transient nature. Pyruvate Formate Lyase (PFL) is a paradigm of thiyl radical enzymes, it catalyzes acetyl transfer from pyruvate to coenzyme A during anaerobic metabolism by a mechanism involving multiple amino acid radicals: A post-translationally installed glycyl radical (G734· in Escherichia coli) and two cysteines (C418 and C419) are essential for enzyme activity. C418 and C419 are proposed to form thiyl radicals during turnover, however, their roles in the mechanism of PFL have not been directly demonstrated with structural and electronic resolution. We employed genetic code expansion, substrates analogs and time-resolved freeze-quench EPR techniques to gain insight into the mechanism of PFL. By substituting C418 or C419 for selenocysteine, which is expected to stabilize the corresponding selenyl radical, we observed a decrease in PFL kinetic constants. Although, direct observation of a selenyl radical remained elusive. Nevertheless, by using methacrylate, an isostructural analog of pyruvate and an irreversible inhibitor of PFL, we were able to shed light over the role of the cysteines in the enzyme mechanism. Freeze-quench time resolved EPR of reactions of PFL with methacrylate showed the conversion of the G734· to a new radical specie, in concert with enzyme inhibition. We identified the new radical as a C2 tertiary methacryl radical located in C418. The methacryl radical decays over time with a H/D solvent isotope effect of 3.4, suggesting its quenching by H-atom transfer presumably by C419. Together, the results support unique roles of the two active site cysteines of PFL and allowed us to estimate the C419 S-H bond dissociation energy for the first time. The research reported here used shared facilities of the UC Santa Barbara Materials Research Science and Engineering Center (MRSEC, NSF DMR-1720256). J.C.C. thanks Anid/Subdirección de Capital Humano/Beca de Doctorado Becas Chile/72200442 for their support. B.L.G thanks the Hellman Faculty Fellowship and a UCSB Faculty Research Grant for funding.