Fig. 3: The photoreduction, oxidation, and CTT release properties of the L405E/C416N mutant.
A Short-term illumination of the L405E/C416N mutant converted the oxidized FAD cofactor into the nsq state (left panel). The nsq state of FAD in L405E/C416N was extremely stable toward oxidation, which needs more than 2000 min to be fully oxidized (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 nsq oxidation (knsqox ~ 3.3 × 10−4 min−1, Supplementary Table 1). B Long-term illumination led to hq state formation in the L405E/C416N mutant (left panel). The oxidation of hq state FAD in L405E/C416N was relatively fast that was completed within 60 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 (khqox ~ 0.08 min-1, Supplementary Table 1). C Proteolysis assay of L405E/C416N to detect CTT release during photoreduction (for 3600 s) and redocking after oxidation for 2000 min (D) Kinetics of the fractions of various redox states (the symbols are the same as above), and CTT release of L405E/C416N (orange stars, represented by the intensity ratio of fragment d to the sum of fragments c and d) during photoreduction. The red line represents the fitted curve of hq formation (kpr2 ~ 2.2 × 10−3 s−1, Supplementary Table 1). The degree of CTT release was in line with hq formation.
