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

Active-site water molecules are increasingly recognized as functionally involved species in enzymatic proton-coupled electron transfer (PCET), yet whether a single water molecule can support distinct catalytic roles within the same active site remains poorly understood. Here, using a multiscale computational approach we show that a previously unidentified water molecule (denoted here Wat3) in fatty acid photodecarboxylase (FAP), a natural photenzyme that converts fatty acids into alkanes, mediates radical quenching via two mechanistically distinct PCET pathways. In the lower-barrier pathway (∆A ‡ ≈ 23 kJ·mol -1 ), Wat3 acts as a hydrogen-bond donor, stabilizing the transition state of a direct proton transfer from cysteine C432 to the alkyl radical (water-assisted PCET). In an alternative, higher-barrier pathway (∆A ‡ ≈ 42 kJ·mol -1 ), the same water molecule acts as a proton shuttle, relaying the proton from C432 to the radical in a quasi-concerted two-proton transfer (water-relay PCET). In both scenarios, the flavin semiquinone provides the electron. The water-assisted route dominates the kinetics under ambient conditions and is consistent with the experimentally measured ∼100 ns timescale. The water-relay pathway, though less accessible in FAP, represents a mechanistic alternative that may become operative in engineered variants or in other enzymes. Additionally, we characterize the fate of the CO 2 coproduct, showing that it reacts with the nascent cysteine thiolate to form a transient thiocarbonate adduct, providing a structural interpretation of previously unassigned electron density in crystallographic data. These findings highlight the ability of a single active-site water molecule to modulate radical PCET through distinct functional roles, with implications for understanding and engineering light-driven biocatalysts.