Unique Electron Donor-Acceptor Complex Conformation Ensures Both the Efficiency and Enantioselectivity of Photoinduced Radical Cyclization in a Non-natural Photoenzyme

Non-natural photoenzymatic catalysis exploits active site tunability for stereoselective radical reactions. In flavoproteins, light absorption promotes the excitation of an electron donor-acceptor (EDA) complex formed between the reduced flavin cofactor and a substrate (α-chloroacetamide in this case). This can trigger chloride mesolytic cleavage, leading to radical cyclization (forming a γ-lactam), or revert to the ground state. While this strategy is feasible using a broad UV/visible/near-infrared spectrum, the low quantum yield presents a significant challenge. Using a multiscale computational approach, we elucidate the mechanisms of the light-driven radical initiation step catalyzed by a Gluconobacter oxydans “ene”-reductase mutant (GluER-G6). The low experimental quantum yield stems from the limited population (<10%) of EDA complexes with a charge transfer state competent for mesolytic cleavage. Accessibility of this state requires substrate bending positioning the chlorine atom near the styrenic group. A subset of these reactive conformers exhibits enhanced cyan/red absorption due to the optimal C-Cl bond alignment with the flavin. Engineering a GluER variant to stabilize this conformation is expected to significantly enhance catalytic efficiency when using cyan/red light. The identified reactive intermediates possess the correct prochirality for enantioselective cyclization. Our findings show that ground-state conformational selection of these EDA complex conformers governs both light-activated mesolytic cleavage and enantioselectivity.