Molecular Origins of Simultaneous Chemo-, Enantio-, and Substrate Selectivity in Non-Natural Photoenzymatic Radical...

Selective radical chemistry poses fundamental challenges for modern catalysis. Non-natural photoenzymes, most prominently flavin-dependent "ene"-reductases, have recently emerged as appealing systems to address these challenges by offering unmatched control over chemo-, enantio-, and substrate selectivity, yet their underlying photocatalytic mechanisms remain unclear. Here, we reveal the complete molecular basis of the triple selectivity control in the photoenzymatic radical reactions by the flavin-dependent "ene"-reductase GkOYE-G7 through computational simulations based on multiscale multireference-quantum-mechanics/molecular-mechanics modeling and bias-exchange metadynamics. Our findings demonstrate that control emerges from reaction-level mechanisms rather than binding preferences. We discover that productive photochemistry requires a previously unknown preactivation step involving bond elongation. Stereochemical outcomes likely result from reaction barrier differences, while chemoselectivity, is controlled by crossing points between the ground and excited electronic states around the conical intersection that channel the reaction before competing pathways activate. Substrate scope follows predictable electronic-steric rules, establishing fundamental principles for engineering next-generation photoenzymes with predictable selectivity profiles.