An extra residue in the electron transfer chain holds the key for the dual functions of an animal-like cryptochrome.

Homologous cryptochromes and photolyases have evolved divergently, with cryptochromes acting as photoreceptors to regulate circadian rhythms and photolyases serving as DNA repair enzymes. However, a group of these proteins from algae and fungi exhibit dual functions, including the animal-like cryptochrome from Chlamydomonas reinhardtii (CraCRY). In this study, we investigated the mechanism underlying the bifunctionality of CraCRY, using steady-state absorption kinetics, limited proteolysis, and mutagenesis, focusing on the roles of residues in the electron transfer chain beyond the well-known tryptophan triad. Site-directed mutagenesis of Y373, N395, and other residues revealed that charge separation in the electron transfer chain during photoreduction triggers conformational changes. Reducing environments promote the FADH- state required for DNA repair while suppressing the conformational changes needed for circadian regulation. Mutations at Y373 that either enhance or impair photoreduction result in increased dimerization. Our findings suggest that Y373 in the electron transfer chain regulates the dual roles of CraCRY, with the reducing microenvironment determining its functional outcome.