Time-Resolved Infrared and Visible Spectroscopy on Cryptochrome aCRY: Basis for Red Light Reception.

Cryptochromes function as flavin-binding photoreceptors in bacteria, fungi, algae, land plants, and insects. The discovery of an animal-like cryptochrome in the green alga Chlamydomonas reinhardtii has expanded the spectral range of sensitivity of these receptors from ultraviolet A/blue light to almost the complete visible spectrum. The broadened light response has been explained by the presence of the flavin neutral radical as a chromophore in the dark. Concomitant with photoconversion of the flavin, an unusually long-lived tyrosyl radical with a red-shifted ultraviolet-visible spectrum is formed, which is essential for the function of the receptor. In this study, the microenvironment of this key residue, tyrosine 373, was scrutinized using time-resolved Fourier transform infrared spectroscopy on several variants of animal-like cryptochrome and density functional theory for band assignment. The reduced tyrosine takes on distinct hydrogen bond scenarios depending on the presence of the C-terminal extension and of a neighboring cysteine. Upon radical formation, all variants showed a signal at 1400 cm, which we assigned to the ν7'a marker band of the CO stretching mode. The exceptionally strong downshift of this band cannot be attributed to a loss of hydrogen bonding only. Time-resolved ultraviolet-visible spectroscopy on W322F, a mutant of the neighboring tryptophan residue, revealed a decrease of the tyrosyl radical lifetime by almost two orders of magnitude, along with a shift of the absorbance maximum from 416 to 398 nm. These findings strongly support the concept of a π-π stacking as an apolar interaction between Y373 and W322 to be responsible for the characteristics of the tyrosyl radical. This concept of radical stabilization has been unknown to cryptochromes so far but might be highly relevant for other homologs with a tetrad of tryptophans and tyrosines as electron donors.