Herein, we describe the successful implementation of this cooperative photometallobiocatalysis design through the repurposing and directed evolution of metapyrocatechase (MPC), a nonheme Fe extradiol dioxygenase. As a nonheme Fe enzyme that does not require a co-substrate such as a-ketoglutarate for its native function, MPC and related extradiol dioxygenases have not yet been studied in new-to-nature biocatalysis. During the finalization of this manuscript, elegant related study on the engineering of 4-hydroxyphenylpyruvate dioxygenase (HPPD) from the Huang group appeared in the literature. We note that our MPC is phylogenetically distal and functionally distinct to HPPD with a sequence similarity (percent identity) of only 16%. Additionally, the organic azide and thiocyanate products prepared from our engineered MPC are the opposite enantiomer of those obtained with Huang's HPPD. Furthermore, our MPC-catalyzed asymmetric isocyanation with OCN - for unsymmetrical urea synthesis remains elusive with other nonheme Fe enzymes in photobiocatalysis. We initiated this investigation by evaluating an in-house collection of ca. 200 nonheme Fe enzymes and enzyme variants generated from our prior study 9c for the targeted asymmetric decarboxylative azidation of redox-active N-hydroxyphthalimide esters using fluorescein 8d as the photocatalyst (Table ). Using racemic N-hydroxyphthalimide esters 1a as the model substrate, in the prescence of 5 mol% fluorescein, 5 mol% added (NH4)2Fe(II)(SO4)2 (Mohr's salt) and 3.0 equiv NaN3 (2), the use of 1 mol% 2-hydroxypropylphosphonate epoxidase (HPPE) from Streptomyces wedmorensis (Uniprot ID: Q56185) did not lead to the desired organic azide product 3a (Table , entry 1). When 4-hydroxyphenylpyruvate dioxygenase (HPPD) from Streptomyces avermitilis (Uniprot ID: Q53586) was used, organic azide 3a formed in 11% yield and 33:67 enantiomeric ratio (e.r., entry 2). Quercetin 2,3-dioxygenase(QueD) from Bacillus subtilis (Uniprot ID: P42106) was also found to be a good nonheme Fe enzyme candidate for this transformation, furnishing 3a in 8% yield and 45:55 e.r. (entry 3). 1-Aminocyclopropane-1-carboxylate oxidase (ACCO) from Petunia hydrida (Uniprot ID: Q08506) 30 , a nonheme Fe enzyme we previously engineered for C-F bond formation, was also effective, albeit with lower activity and enantioselectivity (4% yield and 55:45 e.r., entry 4). Other nonheme Fe enzymes, including biphenyl-2,3-diol 1,2-dioxygenase (BDDO) from Pseudomonas aeruginosa (Uniprot ID: Q79F01) and an ergothioneine biosynthetic enzyme EgtB (a hercynine oxygenase) from Mycolicibacterium thermoresistibile (Uniprot ID: G7CFI3) 32 provided 3a in 4% yield, 75:25 e.r. (entry 5) and 7% yield, 46:54 e.r., respectively. Finally, metapyrocatechase (MPC) form Pseudomonas putida (Uniprot ID: P06622), an extradiol dioxygenase whose native function is to convert catechol into 2-hydroxy-6-oxohexa-2,4-dienoate under O2 dependent conditions, provided the secondary alkyl azide product (3a) with the highest activity and enantioselectivity (28% yield, 77:23 e.r., entry 6). In light of the substantially higher initial activity of PpMPC relative to all the other nonheme Fe enzymes we examined, MPC was selected as the template for further enzyme engineering. A detailed phylogenetic analysis of nonheme Fe enzymes described in Table Next, we performed further reaction condition optimization to improve the efficiency of this dual photobiocatalytic azidation with wild-type (wt) PpMPC (Table ). A wide range of transition-metal and organic photoredox catalysts was evaluated. It was found that transition-metal-based photosensitizers were inferior to organic dyes. For example, the use of 3 mol% [Ru(bpy)3]Cl2 in lieu of fluorescein only furnished 3a in1% yield and 74:26 e.r. (Table 2). The employment of fac-Ir(ppy)3 only led to trace amount of 3a (<1% yield, 66:34 e.r., entry 3). Among organic photoredox catalysts, Fukuzumi's acridinium salt showed a low degree of activity (2% yield, 66:34 e.r., entry 4). Rhodamine dyes such as rhodamine B (RhB) exhibited improved activity, resulting in the formation of 3a in 12% yield and 76:24 e.r. (entry 5). Organic photosensitizers from the fluorescein family in general displayed the highest activity. For example, Eosin Y was found to be able to facilitate the formation of 3a in 21% yield and 77:23 e.r. (entry 6). After surveying additional organic photocatalysts (see Table in the SI for full details), the structurally simplest fluorescein remained the best photocatalyst (entry 1). The use of 3 mol% added ferrous Mohr's salt (NH4)2Fe(II)(SO4)2 was critical to achieving good efficiency, as replacing Mohr's salt with ferric salt (NH4)Fe(III)(SO4)2 provided much lower efficiency (24% yield, entry 7). Omitting (NH4)2Fe(II)(SO4)2 under the standard reaction conditions led to 21% yield of 3a along with 77:23 e.r. (entry 8). Together, these results are consistent with previous biochemical studies showing MPC's strong binding affinity towards Fe(II) and attenuated binding affinity towards Fe(III) 25a . Irradiation at 525 nm using green LED led to reduced yield (24%) of 3a under otherwise identical reaction conditions (entry 9). The use of N-hydroxytetrachlorophthalimide ester 1a' previously used in radical coupling reactions in place of 1a also resulted in drastically reduced yield of 3a (2%, entry 10). Finally, omitting the nonheme enzyme PpMPC (entry 11), the photocatalyst fluorescein (entry 12) and visible light source (440 nm, entry 13) under the standard reaction conditions led to no product 3a formation, confirming the dual photometallobiocatalytic nature of the present transformation. With chemically optimal conditions in hand, we carried out the directed evolution of PpMPC to further improve its activity and enantioselectivity in this asymmetric decarboxylative azidation (Table ). To aid in this enzyme engineering campaign, we first build a substrate tunnel model using CAVER (Figure ). Due to the tetrameric aggregation state of PpMPC 36 , the substrate tunnel with the highest priority score identified by CAVER has its entrance blocked by the neighboring polypeptide chain. After careful examination, the substrate tunnel revealed by CAVER with the second highest priority score was consistent with previous structural studies with It was thus identified as the key channel for the radical intermediate to enter the active site and approach the nonheme Fe center. As a notable feature, a series of hydrophobic amino acid residues including L155, V188, F191, I204, L248, L287, I291, F302 and M303 flanking the entrance of this substrate tunnel, thus creating a hydrophobic lid of the substrate binding pocket. Equipped with this understanding, we used site-saturation mutagenesis (SSM) and screening to direct the evolution of PpMPC by targeting residues from the substrate tunnel (Table ). We first focused on residues from the substrate tunnel which are closer to the nonheme Fe center and then turned our attention to residues further outside closer to the entrance of the substrate tunnel. To facilitate this engineering campaign, we assembled an in-house setup for high throughput photochemistry with temperature control. Nonheme enzyme expression was carried out in 24-well plate and for each SSM library constructed using the 22c trick method , 88 clones per library were selected for screening. In the first round of SSM and evaluation, an isoleucine (I) to leucine (L) displacement at residue 291 (I291L) was found to be a key beneficial mutation, resulting in almost two-fold enhancement in activity and significantly better enantioselectivity (Table , entries 1 and 2). In subsequent enzyme engineering, L155F was discovered as another important mutation, allowing the further enhancement of both the yield and enantioselectivity of azide product 3a (72% yield, 97.5:2.5 e.r., entry 3). A final round of engineering afforded triple mutant PpMPC I291L L155F F302Y. At 1 mol% loading of this triple mutant enzyme, 3a formed in 74% yield and 99.5:0.5 e.r. (entry 5). Furthermore, by further lowering the enzyme loading to 0.25 mol%, 3a could be prepared in a comparable yield and identical enantioselectivity (62% yield and 99.5:0.5 e.r., entry 6), indicating the enhanced efficiency and enantiocontrol of this final PpMPC azidase variant. The absolute stereochemistry of 3a was determined by comparison with authentic samples prepared by other methods (see the SI for details). With the newly evolved nonheme Fe azidase PpMPC I291L L155F F302Y, we examined its substrates scope (Table ). N-hydroxyphthalimide esters with a methyl substituent attached to the ortho-(3b), meta-(3c) and para-(3d) positions of the aromatic ring were readily accommodated by the PpMPC triple mutant, giving rise to the corresponding secondary alkyl azide products with excellent enantioselectivity. Additionally, halogen substituents including a fluorine (3e), a chlorine (3f), and a bromine (3g) were also compatible with this process. The availability of a halogen functional group handle permitted further functionalization of the aromatic ring via cross-coupling technologies. Furthermore, a wide range of electron-withdrawing substituents, including a para-trifluoromethyl (3h), a para-trifluoromethoxy (3i), a meta-methoxy (3k) and a meta-cyano (3l) group, were readily tolerated by this radical azidation reaction. Sensitive functional groups such as a 4-methylthio (3k) and a cyano (3l) as well as heterocycles including thiophenes (3n and 3q) and a pyridine (3o) were also readily accommodated. Furthermore, redox-active esters bearing an extended a-aliphatic chain including an a-ethyl (3p) and an a-propyl (3q) could also be transformed with excellent yield and varying enantioselectivity. Cyclic Nhydroxyphthalimide esters such as an indane moiety (3r and 3t) and a tetrahydronaphthalene moiety (3s) were also accepted by our evolved dual photometallobiocatalytic system, giving rise to the corresponding cyclic organic azide products with good enantiocontrol. Finally, without additional enzyme engineering, racemic tertiary alkyl radical precursors (3t) could also be applied, delivering the corresponding tertiary alkyl azide product with modest enantioselectivity. Together, these results demonstrate the potential utility of this nonheme Fe enzyme-catalyzed radical azidation in preparing enantioenriched products.