Unique Electron\nDonor–Acceptor Complex Conformation\nEnsures Both the Efficiency and Enantioselectivity of Photoinduced\nRadical Cyclization in a Non-natural Photoenzyme

Non-natural photoenzymatic catalysis exploits active\nsite tunability\nfor stereoselective radical reactions. In flavoproteins, light absorption\npromotes the excitation of an electron donor–acceptor (EDA)\ncomplex formed between the reduced flavin cofactor and a substrate\n(α-chloroacetamide in this case). This can trigger chloride\nmesolytic cleavage, leading to radical cyclization (forming a γ-lactam),\nor revert to the ground state. While this strategy is feasible using\na broad UV/visible/near-infrared spectrum, the low quantum yield presents\na significant challenge. Using a multiscale computational approach,\nwe elucidate the mechanisms of the light-driven radical initiation\nstep catalyzed by a Gluconobacter oxydans “ene”-reductase\nmutant (GluER-G6). The low experimental quantum yield stems from the\nlimited population (<10%) of EDA complexes with a charge transfer\nstate competent for mesolytic cleavage. Accessibility of this state\nrequires substrate bending positioning the chlorine atom near the\nstyrenic group. A subset of these reactive conformers exhibits enhanced\ncyan/red absorption due to the optimal C–Cl bond alignment\nwith the flavin. Engineering a GluER variant to stabilize this conformation\nis expected to significantly enhance catalytic efficiency when using\ncyan/red light. The identified reactive intermediates possess the\ncorrect prochirality for enantioselective cyclization. Our findings\nshow that ground-state conformational selection of these EDA complex\nconformers governs both light-activated mesolytic cleavage and enantioselectivity.