title: Essential Role of an Unusually Long-lived Tyrosyl Radical in the Response to Red Light of the Animal-like Cryptochrome aCRY.
abstract: Cryptochromes constitute a group of flavin-binding blue light receptors in bacteria, fungi, plants, and insects. Recently, the response of cryptochromes to light was extended to nearly the entire visible spectral region on the basis of the activity of the animal-like cryptochrome aCRY in the green alga Chlamydomonas reinhardtii This finding was explained by the absorption of red light by the flavin neutral radical as the dark state of the receptor, which then forms the anionic fully reduced state. In this study, time-resolved UV-visible spectroscopy on the full-length aCRY revealed an unusually long-lived tyrosyl radical with a lifetime of 2.6 s, which is present already 1 μs after red light illumination of the flavin radical. Mutational studies disclosed the tyrosine 373 close to the surface to form the long-lived radical and to be essential for photoreduction. This residue is conserved exclusively in the sequences of other putative aCRY proteins distinguishing them from conventional (6-4) photolyases. Size exclusion chromatography showed the full-length aCRY to be a dimer in the dark at 0.5 mm injected concentration with the C-terminal extension as the dimerization site. Upon illumination, partial oligomerization was observed via disulfide bridge formation at cysteine 482 in close proximity to tyrosine 373. The lack of any light response in the C-terminal extension as evidenced by FTIR spectroscopy differentiates aCRY from plant and Drosophila cryptochromes. These findings imply that aCRY might have evolved a different signaling mechanism via a light-triggered redox cascade culminating in photooxidation of a yet unknown substrate or binding partner.
authors.affiliation: Physical and Biophysical Chemistry, Department of Chemistry, Bielefeld University, Universitätsstr. 25, 33615 Bielefeld.
authors.forename: Sabine
authors.initials: S
authors.lastname: Oldemeyer
authors.affiliation: Structural Biochemistry, Department of Chemistry, Philipps University Marburg, Hans-Meerwein Straβe 4, 35039 Marburg.
authors.forename: Sophie
authors.initials: S
authors.lastname: Franz
authors.affiliation: Institute of General Botany and Plant Physiology, Friedrich Schiller University, Am Planetarium 1, 07743 Jena, Germany.
authors.forename: Sandra
authors.initials: S
authors.lastname: Wenzel
authors.affiliation: Structural Biochemistry, Department of Chemistry, Philipps University Marburg, Hans-Meerwein Straβe 4, 35039 Marburg.
authors.forename: Lars-Oliver
authors.initials: LO
authors.lastname: Essen
authors.affiliation: Institute of General Botany and Plant Physiology, Friedrich Schiller University, Am Planetarium 1, 07743 Jena, Germany.
authors.forename: Maria
authors.initials: M
authors.lastname: Mittag
authors.affiliation: Physical and Biophysical Chemistry, Department of Chemistry, Bielefeld University, Universitätsstr. 25, 33615 Bielefeld,. Electronic address: tilman.kottke@uni-bielefeld.de.
authors.forename: Tilman
authors.initials: T
authors.lastname: Kottke
mesh_terms: D000818:Animals; D016825:Chlamydomonas reinhardtii / Q000378:metabolism*; D056931:Cryptochromes / Q000235:genetics / Q000378:metabolism*; D008027:Light; D013056:Spectrophotometry, Ultraviolet; D017550:Spectroscopy, Fourier Transform Infrared; D014443:Tyrosine / Q000378:metabolism*
chemical_list: D056931:Cryptochromes; D014443:Tyrosine
keywords: algae; biophysics; cryptochrome; flavoprotein; infrared spectroscopy (IR spectroscopy); photoreceptor; ultraviolet-visible spectroscopy (UV-Vis spectroscopy)