Ahmad, S.; Forouzandeh, A.; Togni, B.; Randloev, J. B.; Ogden, M.; Nielsen, T. K.

Per- and polyfluoroalkyl substances (PFAS) are persistent anthropogenic "forever chemicals" whose strong C-F bonds (~460-533 kJ mol-1) resist defluorination, and the recast EU Drinking Water Directive sets parametric values of 0.10 ug L-1 for the sum of 20 specified PFAS and 0.50 ug L-1 for total PFAS. Here, we present DeFluorX, a mechanism-first pipeline that uses Boltz-2 to co-fold 2,150 bacterial fluoroacetate dehalogenase (FAcD) variants with 26 PFAS and fluoroacetate, generating uniformly high model confidence (rho = +0.08) but poorly tracked catalytic competence. Thus, the complexes were ranked and tiered by SN2-defluorination geometry based on the known mechanism of control of FAcDs RPA1163 with fluoroacetate: Odelta...C-alpha distance and Odelta-C-alpha-F angle; relay; carboxylate clamp; F-stabiliser; F-cradle; and penalties for scissile bond strength, backside occlusion, beta-fluorination and bond multiplicity. The tiering identified eight complexes (med angle 173.2 degrees, distance 2.91 A) with a high probability of degradation, including Alphaproteobacterial enzymes, with mono-, di- and trifluoroacetate attacking at 172-178 degrees versus 163 degrees for the control. We performed WaterMap, restrained 1 us Desmond MD, MM-GBSA and scans of the three WaterMap-aware best MD frames per complex using B3LYP/6-31G** QM/MM. All 12 frames exhibited product-end C-F cleavage with barriers ranging from 1.1 to 11.8 kcal mol-1, and the departing fluoride carried -0.43 to -0.63 e. The Laboratory validation is ongoing.