Fig. 5: The photoreduction and CTT release properties of the mutants in CTT.

A Illumination of the mutant that deleted CTT (dCT, 1-516) quickly photoreduced the oxidized FAD cofactor into a nearly pure asq state (left panel). Owing to the lack of CTT, the dCT mutant showed constant cleavage at Arg430 [middle and right panels. ▪, the ox state; ●, the asq state; ▲, the nsq state; ▼, the total of the redox states; the hq state was not detected; one representative result is shown. Orange stars, the intensity ratio of fragment d to the sum of fragments c’ (299-516) and d]. The red line represents the fitted curve of asq formation in dCT during photoreduction (k_pr1 ~ 0.03 s⁻¹, Supplementary Table 1). B Illumination of F534A also produced nearly pure asq state (left panel). The CTT release of F534A was impaired because of the central position of Phe534 (middle and right panels. The symbols are the same as above, except that orange stars represent the degree of CTT release according to the intensity ratio of fragment d to the sum of fragments c and d). The red line represents the fitted curve of asq formation in F534A during photoreduction (k_pr1 ~ 0.06 s⁻¹, Supplementary Table 1). C Illumination of W536A gave similar spectral results to those of dCT and F534A (left panel). However, the CTT release of W536A was enhanced (middle and right panels. The symbols are the same as above). The red line represents the fitted curve of asq formation at W536A during photoreduction (k_pr1 ~ 0.07 s⁻¹, Supplementary Table 1). D Structural representations of the middle tunnel that would form after CTT release (left panel), and the side tunnel through which protons might be transferred to FAD (middle and right panels). In the right panel, the side tunnel was depicted by CAVER 3.0³⁵, the entrance of which is formed by the phosphate binding loop^8,12 and α-helix 8 (α8). The side tunnel deeply accesses the FAD cofactor, and joins the middle tunnel (with a narrow bottleneck near the docked CTT) in a highly curved manner. E Molecular dynamics simulations revealed that α8 of the mutants in CTT (dCT, F534A, F535A, and W536A) shifted inward, making the entrances of the side tunnels narrower. The tunnels of the CTT mutants have lower curvatures than that of wild-type dCry, with different exits that correspond well with their mutation sites.