A Single Active-Site Water Molecule Encodes Two Distinct Proton-Coupled Electron Transfer Pathways in Fatty Acid Photodecarboxylase

Abstract Water molecules are key actors in enzymatic proton-coupled electron transfer (PCET), yet their mechanistic versatility remains underexplored. Using a multiscale computational approach, we show that a previously unidentified water molecule (Wat3) in fatty acid photodecarboxylase (FAP), a natural photoenzyme converting fatty acids into alkanes, mediates radical quenching via two PCET pathways: in the dominant one, which qualitatively agrees with the 100 ns experimental time scale, Wat3 stabilizes the transition state for direct proton transfer from Cys432 to the alkyl radical; in the alternative, the same molecule acts as a proton shuttle in a quasi-concerted two-proton transfer, with the flavin semiquinone providing the electron in both cases. The minor pathway, though less accessible in wild-type FAP, may become operative in engineered variants or related enzymes. Additionally, CO2 reacts with the thiolate to yield a thiocarbonate adduct, explaining unassigned crystallographic electron density. These results show how a single water molecule encodes mechanistic diversity in enzymatic PCET.