Proton Transfer to Flavin Stabilizes the Signaling State of the Blue Light Receptor Plant Cryptochrome [Plant Biology]

December 3rd, 2014 by Hense, A., Herman, E., Oldemeyer, S., Kottke, T.

Plant cryptochromes regulate circadian rhythm, flowering time, and photomorphogenesis in higher plants as responses to blue light. In the dark, these photoreceptors bind oxidized FAD in the photolyase homology region (PHR). Upon blue light absorption, FAD is converted to the neutral radical state, the likely signaling state, by electron transfer via a conserved tryptophan triad and proton transfer from a nearby aspartic acid. Here we demonstrate by infrared and time-resolved UV/Vis spectroscopy on the PHR domain that replacement of the aspartic acid D396 with cysteine prevents proton transfer. The lifetime of the radical is decreased by six orders of magnitude. This lifetime does not permit anymore to drive conformational changes in the C-terminal extension, which have been associated with signal transduction. Only in the presence of ATP, both wild type and mutant form a long-lived radical state. However, in the mutant an anion radical is formed instead of the neutral radical as it has been previously found in animal type I cryptochromes. Infrared spectroscopic experiments demonstrate that the light-induced conformational changes of the PHR domain are conserved in the mutant despite the lack of proton transfer. These changes are not detected in the photoreduction of the non-photosensory D-amino acid oxidase to the anion radical. In conclusion, formation of the anion radical is sufficient to generate a protein response in plant cryptochrome. Moreover, the intrinsic proton transfer is required for stabilization of the signaling state in the absence of ATP.