Fig. 6: His378 acts as a proton interceptor during photoreduction.
A Dissection of the dCry structure shows the details of the side tunnel, which joins the binding pocket, and accesses the FAD cofactor in a side direction. His378 is located at the end of the side tunnel, in the vicinity of Phe534 and the FAD cofactor. B Illumination of the H378A mutant slowly converted the oxidized FAD cofactor into asq and nsq. The fractions of nsq were greater than those of wild-type dCry under neutral conditions. Prolonged illumination converted some nsq into hq (left panel). Proteolysis analysis of H378A showed that CTT binding was less tight in the dark and that CTT release was less sufficient in light than in wild-type dCry, but still increased in line with asq and hq formation (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. Orange stars, the degree of CTT release represented by the intensity ratio of fragment d to the sum of fragments c and d). C Photoreduction of the H378K mutant was even slower, but significant amounts of nsq still formed (left panel). The proteolysis of H378K was similar to that of H378A (middle and right panels. The symbols are the same as above). D Illumination of the H378D mutant resulted in rapid formation of nearly pure asq (left panel). Proteolysis of H378D showed that the CTT remained in a half-release state both in the dark and in the light (middle and right panels. The symbols are the same as above). The red line represents the fitted curve of asq formation in H378D during photoreduction (kpr1 ~ 0.05 s−1, Supplementary Table 1).
