title: Proton transfer to flavin stabilizes the signaling state of the blue light receptor plant cryptochrome.
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.
authors.affiliation: From the Physical and Biophysical Chemistry, Department of Chemistry, Bielefeld University, Universitätsstrasse 25, 33615 Bielefeld, Germany.
authors.forename: Anika
authors.initials: A
authors.lastname: Hense
authors.affiliation: From the Physical and Biophysical Chemistry, Department of Chemistry, Bielefeld University, Universitätsstrasse 25, 33615 Bielefeld, Germany.
authors.forename: Elena
authors.initials: E
authors.lastname: Herman
authors.affiliation: From the Physical and Biophysical Chemistry, Department of Chemistry, Bielefeld University, Universitätsstrasse 25, 33615 Bielefeld, Germany.
authors.forename: Sabine
authors.initials: S
authors.lastname: Oldemeyer
authors.affiliation: From the Physical and Biophysical Chemistry, Department of Chemistry, Bielefeld University, Universitätsstrasse 25, 33615 Bielefeld, Germany tilman.kottke@uni-bielefeld.de.
authors.forename: Tilman
authors.initials: T
authors.lastname: Kottke
mesh_terms: D000255:Adenosine Triphosphate / Q000737:chemistry; D020816:Amino Acid Motifs; D000838:Anions; D001224:Aspartic Acid / Q000737:chemistry*; D016825:Chlamydomonas reinhardtii / Q000737:chemistry*; D056931:Cryptochromes / Q000737:chemistry*; D004138:Dinitrocresols / Q000737:chemistry*; D008027:Light; D009154:Mutation; D010940:Plant Proteins / Q000737:chemistry*; D017434:Protein Structure, Tertiary; D011522:Protons; D015398:Signal Transduction; D013053:Spectrophotometry; D013056:Spectrophotometry, Ultraviolet; D017550:Spectroscopy, Fourier Transform Infrared
chemical_list: D000838:Anions; D056931:Cryptochromes; D004138:Dinitrocresols; D010940:Plant Proteins; D011522:Protons; C024132:4,6-dinitro-o-cresol; D001224:Aspartic Acid; D000255:Adenosine Triphosphate
keywords: Chlamydomonas; IR Spectroscopy; Photoreceptor; Radical; cryptochrome; flavoprotein