The ionic and protonation states of flavin control the activation and recovery of Drosophila cryptochrome

Drosophila cryptochrome (dCry) is a flavin-containing photoreceptor that entrains the circadian clock with light. The C-terminal tail (CTT) release upon illumination is a crucial step for light signal transduction of dCry. Here, we demonstrated that both anionic semiquinone (asq) and anionic hydroquinone (hq) formed during illumination (the latter only obtained in dCry mutants) could trigger CTT release. Meanwhile, neutral semiquinone (nsq) formation suppressed CTT release. However, it was observed that a fraction of nsq was formed during photoreduction of dCry at neutral conditions. Nevertheless, the nsq fraction of dCry was increased during photoreduction under acidic pH conditions, accompanied with inhibition of CTT release. Evidence suggested that the proton required for nsq formation was transferred to flavin through a side tunnel, which is a structural feature that does not exist in the homologs of dCry, eukaryotic 6 − 4 photolyases. The nsq formation was minimized in dCry under basic conditions, or the mutants in CTT, which resulted enhanced CTT release, but slower oxidation after photoreduction. Therefore, forming a proper fraction of nsq is important for fast recovery of dCry after light sensing. Nevertheless, a key residue at the side tunnel, His378, was found to be a proton interceptor that adjusted the nsq formation.