Instantly Reversible Enzyme Regulation via Dichromatic Irradiation of Incorporated Azobenzene

Azobenzene is a widely recognized tool for achieving artificial spatiotemporal control of enzyme activity using light. Photocontrol reversibility is typically based on photostationary states with varying E and Z isomer compositions achieved through irradiation at specific wavelengths. Here, we report an alternative mechanism for azobenzene based enzyme regulation, discovered through simultaneous irradiation with two wavelengths. Using two engineered variants of imidazole glycerol phosphate synthase, in which azobenzene was incorporated as an unnatural amino acid to enable reversible control under monochromatic irradiation, we uncovered unique behavior under dichromatic irradiation. Notably, a distinct spectroscopic signal from the azobenzene moiety emerged during simultaneous irradiation at 365 nm and 420 nm and vanished upon return to the dark. Intriguingly, dichromatic irradiation triggered a reproducible two-fold increase in catalytic activity and an instantaneous return to baseline activity in the dark for one variant. In contrast, the other variant and the wild-type enzyme maintained their baseline activity under the same conditions. These findings reveal an unexplored avenue for azobenzene photoswitching, offering a novel approach to photocontrol with potential applications in sequentially regulating multiple enzymes, especially when combined with monochromatic irradiation strategies.