Reconciling Catalytic Mechanism of Fatty Acid Photodecarboxylase: Role of a Newly Identified Water in the Hydrocarbon Formation Step

Fatty acid photodecarboxylase (FAP) is a unique photoenzyme that converts fatty acids into alkanes. After light-induced electron transfer and decarboxylation, alkane formation is proposed to proceed via proton-coupled electron transfer (PCET), yet its molecular details and the fate of CO 2 remain unclear, particularly at room temperature. Here, using a multiscale computational strategy, we propose a mechanistic picture operating at ambient conditions. The PCET step involves a previously unidentified water molecule (Wat3) that stabilizes the transition state for direct proton transfer from cysteine C432 to the alkyl radical, while the flavin semiquinone provides the electron. The calculated activation freeenergy (∆A ‡ ≈ 25 kJ•mol −1) is consistent with the experimentally-measured 100 ns timescale. Subsequently, CO 2 can either be released from the active site or react with the nascent cysteine thiolate to form a transient thiocarbonate adduct, here characterized at the molecular level for the first time. These findings reconcile available spectroscopic and crystallographic data and provide a coherent mechanistic picture of FAP catalysis under physiological conditions.