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

Abstract

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 À1 , which we assigned to the n7 0 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 p-p 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.

Cryptochrome magnetoreception: four tryptophans could be better than three

“…Tyr319 (figure 1) has an edge-to-edge distance to Trp D of 3.9 Å (electronic supplementary material, table S1), and appears well placed to be oxidized by Trp D H •+ . In several members of the photolyase-Cry superfamily there is a tyrosine at the far end of the Trp-triad that donates an electron to the terminal Trp C H •+ radical, thus extending the electron transfer chain and stabilizing the FAD •− radical against back electron transfer [40,53,54,[62][63][64][65][66]. The tyrosine radical (TyrO • ) so formed is solvent-exposed and therefore able to be reduced by exogenous electron donors, potentially allowing the efficient formation of a SS containing FADH • as the only radical.…”

Cryptochromes: Photochemical and structural insight into magnetoreception

“…Specifically, in several systems the Trp-triad chain can be extended to include a fourth, or even fifth redox active amino acid that impacts photochemical yield (Figure 1(c)). 60 In several cases Tyr residues substitute for Trp species, enabling a long-lived terminal radical, 13,[61][62][63][64][65][66][67] or enhancement of photochemical yield. 14 In addition, alternatives to the canonical Trp-Triad pathway have been identified that can likely substitute in vivo.…”

Interconnection of the Antenna Pigment 8-HDF and Flavin Facilitates Red-Light Reception in a Bifunctional Animal-like Cryptochrome

“…DFT calculations of 8-HDF with different protonation states and hydrogen bonding scenarios at the oxygen of the 8hydroxyl and the carbonyl and amine groups were performed and used for band assignment due to very good agreement of calculated and experimentally obtained spectra in previous studies. 23,32,[48][49][50] The difference spectrum of the deprotonated 8-HDF accepting two hydrogen bonds from two water molecules at the 8-hydroxyl group and each a hydrogen bond at the 1-amine and 4-carbonyl group, model 2, minus the protonated 8-HDF accepting a hydrogen bond at the 2-and 4-carbonyl group and donating one at the 3-amine group, model 1, (Fig. 4C) yielded the best agreement with the experimental data and were used for the band assignment (Fig.…”