2025 Volume 73 Issue 8 Pages 678-686
Oxyluciferin is the key photon emitter in the firefly/beetle luciferase-catalyzed bioluminescence reaction, and elucidating its chemical form within the active site of luciferase is essential for understanding and modulating emission wavelength. In this study, we focused on aminoluciferin (AL), a d-luciferin (d-LH2) analog in which the hydroxy group is substituted with an amino group. AL has been widely used as an alternative substrate to d-LH2 and in the development of bioluminescence probes. To identify the emissive form of AL in bioluminescence, we synthesized OxyAL (the emitter derived from AL) and 5-methyl-OxyAL, and measured their fluorescence spectra under various conditions. We assigned the range of emission wavelengths of keto-, enol- and enolate-OxyAL using 5,5-dimethyl-OxyAL, fixed in the keto form, as a reference. Based on spectral assignments and comparison with the bioluminescence spectra of AL, we conclude that enolate-OxyAL is the most probable light-emitting species in the active site of luciferase. These findings provide new insight into AL-based bioluminescence.
