Fig. 2: The photoreduction, oxidation, and CTT release properties of the C416N mutant.

A Comparison of the structures of dCry (left panel) and Drosophila 6-4 photolyase (Dm6-4, right panel). Both dCry and Dm6-4 contain four conserved tryptophan residues (gray sticks), through which electrons can be transferred from the solvent to the FAD cofactor (yellow stick) during photoreduction. The residue facing N5 of FAD is a cysteine (Cys416) in dCry but is an asparagine (Asn403) in Dm6-4. Nevertheless, Leu401 and Leu405 provide solvent barriers that suppress the protonation of FAD in dCry. The homologous positions in Dm6-4 are Gln388 and Glu392, through which protons may be transferred to FAD to form nsq and/or hq states. Some important sites in dCry and Dm6-4 are depicted in stick representations. A possible alternative proton transfer pathway in dCry is also shown (top left corner in the left panel). B Short-term illumination of the C416N mutant converted the oxidized FAD cofactor into the asq state (left panel). The asq state FAD in C416N was quickly oxidized to the ox state (k_asqox ~ 0.21 min⁻¹) and protonated to the nsq state (k_pt ~ 0.41 min⁻¹, Supplementary Table 1) (middle and right panels. ▪, the ox state; ●, the asq state; ▲, the nsq state; ▼, the hq state; and ♦, the total of the redox states; one representative result is shown). The red line in the right panel represents the fitted curve of overall asq decomposition (~0.62 min⁻¹). C Illumination of the C416N mutant for several minutes converted most of the FAD cofactor into the nsq state (left panel). The nsq state of FAD in C416N was very stable toward oxidation, which needs more than 1000 min to be fully oxidized (middle and right panels. The symbols are the same as above). The red line in the right panel represents the fitted curve of nsq oxidation (k_nsqox ~ 0.001 min⁻¹, Supplementary Table 1). D Long-term illumination led to hq state formation in the C416N mutant (left panel). The oxidation of hq-state FAD in C416N was relatively fast that was completed within 80 min (middle and right panels. The symbols are the same as above). The red line in the right panel represents the fitted curve of hq oxidation (k_hqox ~ 0.03 min⁻¹, Supplementary Table 1). E Proteolysis assay of C416N to detect CTT release during photoreduction (for 3600 s) and redocking after oxidation for 1000 min. F Kinetics of the fractions of various redox states (the symbols are the same as above), and CTT release of C416N (orange stars, represented by the intensity ratio of fragment d to the sum of fragments c and d) during photoreduction. The red lines represent the fitted curves of asq protonation (k_pt ~ 6.5  × 10⁻³s⁻¹, i.e., ~0.39 min⁻¹) and hq formation (k_pr2 ~ 1.3 × 10^-4s⁻¹, Supplementary Table 1). The degree of CTT release was in line with asq and hq formation.