Our radical clock experiment with a cyclopropyl substrate indicated the formation of cyclopropane ring-opening product, consistent with the proposed radical mechanism (see the SI for details). To gain further insights into the mechanism and the roles of active site residues of this new-to-nature biocatalytic C(sp 3 )-H fluorination, we performed computational studies using classical molecular dynamics (MD) simulations and density functional theory (DFT) calculations (see the SI for computational details). First, classical MD simulations of the complexes of Nfluoroamide 1a with both wt ACCO and ACCOCHF variants were carried out to study the preferred substrate binding modes and to identify key active site residues involved in substrate binding (Fig. ). In order to model the substrate near-attack-conformations 58 that promote the fluorine atom transfer step, the Fe-F distance was restrained to be within 3.0-3.2 Å using a harmonic potential of 100 kcal•mol -1 •Å -2 . The restrained MD simulations showed that in order to minimise unfavourable steric repulsions with the protein scaffold, substrate 1a approaches the Fe centre from the coordination site trans-to H234. Furthermore, these MD studies indicated that mutations K158I, F91L, and T89A widen the substrate entrance tunnel, providing more space to facilitate the transport of the bulky N-fluoroamide substrate 1a into the active site. In particular, mutations K158I and F91L greatly reduce the bulkiness of these tunnel bottleneck residues, increasing the tunnel bottleneck radius from 3.12 Å in wt ACCO to 4.17 Å in ACCOCHF (see Fig. ). In addition, the I184A mutation opens up more space to accommodate 1a, allowing the fluorine atom in 1a to approach the Fe centre while circumventing unfavourable steric clashes between the adjacent N-tBu group of 1a and active site residues. Next, to elucidate the reactivities of the unnatural fluorine atom transfer and fluorine rebound steps, we performed DFT calculations of the reaction energy profile of this biocatalytic C-H fluorination. We constructed a truncated model based on the active site geometry of the ACCO enzymes, in which imidazole (Im) and acetate (AcO) groups are used to model aspartate and histidine residues, respectively . Our DFT calculations indicate that all the intermediates and transition states in the catalytic cycle feature a high-spin quintet Fe(II) and sextet Fe(III) 9 centres (see Fig. for computed energy profile at the less favorable intermediate-spin state). Previous studies indicated that fluorine atom transfer to a transition metal centre is kinetically more challenging than the analogous chlorine and bromine atom transfer, due to the higher bond dissociation energy (BDE) of the fluorine atom donor. In addition, fluorine atom rebound from a transition metal fluoride is also slower than other halogens because of the strong metal fluoride bond . In contrast, the computed reaction energy profile (Fig. ) reveals a relatively low barrier of 11.8 kcal/mol for the initial fluorine atom transfer (TS-1). Here, the high exothermicity (-14.3 kcal/mol) in converting an activated N-F bond in 4 to a stronger Fe(III)-F bond provides the thermodynamic driving force for substrate activation. In addition, the Fe(II) centre in 4 has a relatively high HOMO energy of -7.03 eV, promoting the charge transfer (0.31 e) from the Fe centre to the N-fluoroamide substrate 1a at the transition state (TS-1). After the fluorine atom transfer, the nitrogen-centred radical in complex 5 undergoes rapid 1,5-hydrogen atom transfer via TS-2 to provide the benzyl radical intermediate 6. Finally, radical rebound with the Fe(III)-F species leading to C-F bond formation via TS-3 is found to be highly kinetically facile, displaying an activation barrier of 3.4 kcal/mol with respect to 6. Unlike other transition metal fluorides featuring a strong M-F bond with BDEs higher than 100 kcal/mol 61 , the Fe(III)-F bond in 6 has a weaker BDE of 81.4 kcal/mol, allowing thermodynamically downhill fluorine rebound to form the benzylic C-F bond with a BDE of 98.1 kcal/mol. In addition, the heterolytic dissociation of F - from Fe(III)-F in 5 and 6 was found to be thermodynamically unfavourable (Fig. ), allowing for productive C-F bond formation via the radical rebound pathway. We note that the low