Essential Role of an Unusually Long-lived Tyrosyl Radical in the Response to Red Light of the Animal-like Cryptochrome aCRY

• Published in: The Journal of biological chemistry Vol. 291; no. 27; pp. 14062 - 14071
• Main Authors: Oldemeyer, Sabine, Franz, Sophie, Wenzel, Sandra, Essen, Lars-Oliver, Mittag, Maria, Kottke, Tilman
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 01-07-2016; American Society for Biochemistry and Molecular Biology
• Subjects: algae; Animals; biophysics; Chlamydomonas reinhardtii - metabolism; cryptochrome; Cryptochromes - genetics; Cryptochromes - metabolism; flavoprotein; infrared spectroscopy (IR spectroscopy); Light; Molecular Biophysics; photoreceptor; Spectrophotometry, Ultraviolet; Spectroscopy, Fourier Transform Infrared; Tyrosine - metabolism; ultraviolet-visible spectroscopy (UV-Vis spectroscopy); infrared spectroscopy (IR spectroscopy); flavoprotein; cryptochrome; photoreceptor; algae; ultraviolet-visible spectroscopy (UV-Vis spectroscopy); biophysics
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M116.726976

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.