First Mechanistic Insights into Mammalian P450-Catalyzed Reductive Defluorination and Bioactivation of Cl-PFAS: From Halothane to a QSAR Model

Chlorinated polyfluoroalkyl substances (Cl-PFAS) are widely used alternatives to legacy PFAS, but the molecular mechanism of their metabolic defluorination in mammals remains unknown. Here, we report the first atomic-level mechanistic study of P450-mediated reductive defluorination of Cl-PFAS in mammalian systems. Using halothane as a model, DFT calculations show under aerobic conditions, P450 Compound I catalyzes H-abstraction/OH-rebound followed by elimination and hydrolysis to form trifluoroacetic acid. Under hypoxic conditions (e.g., liver centrilobular regions), we elucidate the hepatotoxicity-associated reductive pathway by the Fe 2+ form of P450: rate-determining concerted electron-transfer with C–Br cleavage generates •CF 3 CHCl in a kinetic well between the first and second electron transfers; its prolonged lifetime initiates lipid peroxidation, while subsequent defluorination yields alkenes. The computed carbon kinetic isotope effect for the reductive pathway (1.076) is larger than for the oxidative pathway (1.020), providing an experimental signature for distinction. A QSAR model using vertical electron affinity and molar refractivity predicts the rate-determining electron-transfer barrier for diverse compounds, particularly long-chain Cl-PFAS (R 2 = 0.95), suggesting this reductive mode generalizes to other Cl-PFAS, consistent with their emerging hepatotoxicity. These findings reveal, for the first time, a molecular-level reductive defluorination pathway for Cl-PFAS in mammals, providing a mechanistic basis for bioactivation risk assessment.