Engineering a Photoenzyme to Use Red Light

Photoenzymatic catalysis is an emerging platform for asymmetric synthesis. In most of these reactions, the protein templates a charge transfer complex between the cofactor and substrate, which absorbs in the blue region of the electromagnetic spectrum. Here, we report the engineering of a photoenzymatic ‘ene’-reductase to utilize red light (620 nm) for a radical cyclization reaction. Mechanistic studies indicate that red light ac-tivity is achieved by introducing a broadly absorbing shoulder off the previously identified cyan absorption feature. Molecular dynamics simulations, docking, and excited-state calculations suggest that red light absorption is a 𝜋→ 𝜋* transition from flavin to the substrate, while the cyan feature is the red-shift of the flavin 𝜋→ 𝜋* transition, which occurs upon substrate binding. Differences in the excitation event help to disfavor alkylation of the flavin cofactor, a pathway for catalyst decomposition observed with cyan light but not red.