We commenced our investigation by evaluating an in-house collection of ca. 200 metalloproteins and their variants, including diverse heme and nonheme Fe proteins, by high throughput experimentation using 24-or 96-well plates. All the identified active biocatalysts were then validated, and representative results are summarized in Fig. . Among all the heme proteins we evaluated, only reduced amide 3a derived from the N-fluoroamide substrate 1a was observed in varying yields (Table ). For example, our previously evolved P450 atom transfer radical cyclases P450ATRCase1 possessing a Fe-binding serine residue (Table , entry 1) and P450ATRCase2 lacking an Fe-binding residue (Table , entry 2) 6 provided 3a in 14% and 17% yield, respectively, with no measurable 2a formation. In contrast to heme proteins, a set of fluorination biocatalysts emerged from the nonheme Fe enzyme superfamily. Among these, isopenicillin N synthase (IPNS) from Emericella nidulans with a two-histidine-one-carboxylate facial triad 41 provided the desired C-H fluorination product 2a in 0.2% yield and 10:90 enantiomeric ratio (e.r.) (Fig. , see Tables and for further details). Anthocyanidin synthase from Arabidopsis thaliana furnished 2a in 0.4% yield and 37:63 e.r.. Additionally, quercetin 2,3-dioxygenase from Bacillus subtilis (BsuQueD) with a three-histidine-one-carboxylate coordination sphere 43 also afforded 1a in 0.2% yield and 29:71 e.r.. Furthermore, 1-aminocyclopropane-1-carboxylic acid oxidase (ACCO) from Petunia hybrida , a plant-derived nonheme Fe enzyme with a two-histidine-one carboxylate facial triad whose native function is to produce ethylene from 1-aminocyclopropane-1-carboxylic acid , furnished fluorinated 2a in 0.9% yield and 10:90 e.r.. Finally, natural Fe-and aKGdependent C-H halogenases, including SyrB2 (Table , entry 7), WelO5 (Table , entry 8), and BesD (Table , entry 9), were found to be ineffective in facilitating this unnatural C(sp 3 )-H fluorination. atom transfer activity among Fe-dependent enzymes. b, Directed evolution of ACCOCHF as a highly chemo-and enantioselective C-H fluorinating enzyme. Active-site illustration of ACCO was made from PDB ID: 1W9Y.