Key residues Tyr293, Val360, and Tyr399 regulate flavin stability in the plant cryptochrome from Chlamydomonas reinhardtii.

Cryptochromes (CRYs) evolved from cyclobutane pyrimidine dimer (CPD) photolyases (PLs), losing DNA repair activity while gaining novel functions as blue-light photoreceptors. The substrate binding pocket has undergone significant evolutionary changes, including three highly conserved residues in plant CRYs specially. However, the functional contributions of these substrate binding site residues to the distinct properties of CRYs remain poorly understood. Here, we systematically investigated the roles of three critical substrate binding site residues in Chlamydomonas reinhardtii cryptochrome (pCRY): Tyr293, Val360 and Tyr399 (corresponding to Escherichia coli CPD photolyase, EcCPD Trp277, Met345 and Trp384). In this study, single-point mutations Y293W, V360M, and Y399W were generated, and their effects on photoreduction kinetics, oxidation kinetics, and ATP binding were characterized. Our results demonstrate that all three mutations significantly increased the photoreduction rate constants and decreased the oxidation rate constants compared to wild-type pCRY, indicating enhanced FAD stability and reduced accessibility to molecular oxygen. Importantly, these mutations did not impair ATP binding, suggesting that substrate binding and ATP binding are functionally separable. These findings reveal that evolutionary divergence at substrate binding sites fundamentally alters the redox properties of the FAD cofactor and provide mechanistic insights into the functional transition from DNA repair to light signaling.