Engineering a photoenzyme to use red light

Photoenzymatic reactions involving flavin-dependent “ene”-reductases (EREDs) rely on protein-templated charge transfer (CT) complexes between the cofactor and substrate for radical initiation. These complexes typically absorb in the blue region of the electromagnetic spectrum. Here, we engineered an ERED to form CT complexes that absorb red light. Mechanistic studies indicate that red-light activity is due to the growth of a red-absorbing shoulder off the previously identified cyan absorption feature. Molecular dynamics simulations, docking, and excited-state calculations suggest that the cyan feature involves a π→π∗ transition on flavin, whereas the red-light absorption is a π→π∗ transition between flavin and the substrate. Differences in the electronic transition are due to changes in the substrate-binding conformation and allosteric tuning of the electronic structure of the cofactor-substrate complex. Microenvironment tuning of the CT complex for red-light activity is observed with other engineered photoenzymatic reactions, highlighting this effect’s generality. [Display omitted] •Spectral tuning of enzyme-templated charge transfer complexes using directed evolution•Computational studies show a different electron transition for cyan and red light•Mutations at the protein surface allosterically tune the active site complex•Engineered enzymes for other radical reactions improve red-light performance Photoenzymes are a class of biocatalysts that use photonic energy to drive a chemical transformation. Although nature only has three known photoenzymes, over the past decade, chemists have found that some established enzyme platforms have latent photochemical functions that were previously unknown. In this field, there is a need for general strategies to spectrally tune the photoenzymatic chromophore to enhance the stability of the enzyme and scalability of these reactions. Inspired by this goal, we engineered a flavin-dependent “ene”-reductase to use red light for a photoenzymatic radical cyclization previously reported with cyan light. By targeting residues located throughout the protein, we optimized the enzyme activity with red light, enabling the transformation to be run on up to a 10-g scale. Our mechanistic studies revealed that protein engineering changes the substrate-binding conformation, resulting in red absorptions. Importantly, mutations at the protein's surface tune the light-absorbing complex, indicating allostery in artificial photoenzymes, a p