The Photocycle of the Red Light-Sensitive Plant-like Cryptochrome CryP from Phaeodactylum tricornutum.

Cryptochromes act as photoreceptors in diverse organisms and bind flavin adenine dinucleotide (FAD) as a chromophore. The plant-like cryptochrome CryP from the diatom Phaeodactylum tricornutum regulates in vivo the expression of light-harvesting proteins in response to blue light. CryP carries, in addition, 5,10-methenyltetrahydrofolate (MTHF) for light capturing. In contrast to most other cryptochromes, FAD in CryP is present as a stable flavin neutral radical in the dark, which undergoes a photoreduction to form the fully reduced state in the light. Here, we demonstrate by applying nanosecond-time-resolved UV-vis spectroscopy that the flavin neutral radical is photoreduced within 100 ns to the fully reduced state and subsequently recovers very fast with a time constant of 1.4 ms in the absence of reducing agents or in the presence of 1 mM dithiothreitol. Despite its short lifetime, the transient light state is sufficient to induce homo-oligomerization of CryP as shown by light-dependent size exclusion chromatography. A long-lived fully reduced flavin is only formed in the presence of external reducing agents by a second pathway that is lost after a single full conversion, likely by degradation. Then, the recovery to the neutral radical state takes hours with a time constant of 40 min. Moreover, we validate the existence of energy transfer between the two chromophores depending on the flavin redox state using fluorescence spectroscopy. We propose a detailed mechanism for the photocycle of CryP, highlighting the contribution of two separate reduction pathways.