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J Biol Chem. 2015 Jan 16;290(3):1743-51. doi: 10.1074/jbc.M114.606327. Epub 2014 Dec 3.

Proton transfer to flavin stabilizes the signaling state of the blue light receptor plant cryptochrome.

Author information

1
From the Physical and Biophysical Chemistry, Department of Chemistry, Bielefeld University, Universitätsstrasse 25, 33615 Bielefeld, Germany.
2
From the Physical and Biophysical Chemistry, Department of Chemistry, Bielefeld University, Universitätsstrasse 25, 33615 Bielefeld, Germany tilman.kottke@uni-bielefeld.de.

Abstract

Plant cryptochromes regulate the 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-visible spectroscopy on the PHR domain, that replacement of the aspartic acid Asp-396 with cysteine prevents proton transfer. The lifetime of the radical is decreased by 6 orders of magnitude. This short lifetime does not permit to drive conformational changes in the C-terminal extension that have been associated with signal transduction. Only in the presence of ATP do both the 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 found previously 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 cryptochromes. Moreover, the intrinsic proton transfer is required for stabilization of the signaling state in the absence of ATP.

KEYWORDS:

Chlamydomonas; IR Spectroscopy; Photoreceptor; Radical; cryptochrome; flavoprotein

PMID:
25471375
PMCID:
PMC4340416
DOI:
10.1074/jbc.M114.606327
[Indexed for MEDLINE]
Free PMC Article

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