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

Abstract

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.