From bioavailability scarcity to energy barriers: limitations of anaerobic microbial reductive defluorination.

Reductive dehalogenases in organohalide-respiring bacteria underpin anaerobic bioremediation of chlorinated pollutants but are rarely effective for reductive defluorination of per- and polyfluoroalkyl substances. However, the physicochemical basis for this selectivity remains unclear. Here, we integrate quantum chemistry and molecular dynamics to evaluate constraints on microbial reductive defluorination. The scarcity of naturally occurring organofluorines has imposed limited evolutionary selective pressure, explaining the absence of robust defluorination pathways. Using quantum mechanical calculations, we show that organofluorines have low bioavailability, due to increasingly unfavorable solvation free energies of fluorinated ethenes in both polar and nonpolar solvents, impeding cellular uptake. Using molecular dynamics simulations, we show that the substrate recognition by reductive dehalogenases is compromised, due to progressively weaker van der Waals energies as chlorines are replaced by fluorines. A tetrafluorinated ligand can form hydrogen bonds with polar residues and is preferentially stabilised in a sub-pocket away from the catalytic site. Using quantum mechanics calculations with a cluster model of the active site, we show that the reductive cleavage of the C-F bond has prohibitively high energy barriers. Together, these results explain the limited anaerobic microbial reductive defluorination of linear per- and polyfluoroalkyl substances and highlight why engineering applications are unlikely to succeed. The workflow provides a screening framework for assessing biodegradability of new organofluorines prior to industrial deployment.