Response of the Sensory Animal-like Cryptochrome aCRY to Blue and Red Light As Revealed by Infrared Difference Spectroscopy

Abstract

Cryptochromes act as blue light sensors in plants, insects, fungi, and bacteria. Recently, an animal-like cryptochrome (aCRY) was identified in the green alga Chlamydomonas reinhardtii by which gene expression is altered in response to not only blue light but also yellow and red light. This unique response of a flavoprotein in vivo has been attributed to the fact that the neutral radical of the flavin chromophore acts as dark form of the sensor, which absorbs in almost the entire visible spectral range (<680 nm). Here, we investigated light-induced processes in the protein moiety of full-length aCRY by UV-vis and Fourier transform infrared spectroscopy. Findings are compared to published results on the homologous (6-4) photolyases, DNA repair enzymes. The oxidized state of aCRY is converted to the neutral radical by blue light. The recovery is strongly dependent on pH and might be catalyzed by a conserved histidine of the (6-4)/clock cluster. The decay is independent of oxygen concentration in contrast to that of other cryptochromes and (6-4) photolyases. This blue light reaction of the oxidized flavin is not accompanied by any detectable changes in secondary structure, in agreement with a role in vivo of an unphysiological preactivation. In contrast, the conversion by red light of the neutral radical to the anionic fully reduced state proceeds with conformational changes in turn elements, which most probably constitute a part of the signaling process. These changes have not been detected in the corresponding transition of (6-4) photolyase, which points to a decisive difference between the sensor and the enzyme.

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

“…The negative bands represent the dark state with bound FADH ⅐ in good agreement with the spectrum of the wild type (Fig. 6B), showing marker bands of FADH ⅐ at 1663, 1651, and 1534 cm Ϫ1 (16). The positive bands in the difference spectrum of aCRY⌬CCT are also in good agreement with those of the wild type, most of which can be assigned to FADH Ϫ .…”

“…However, the residue is buried deeply in a hydrophobic environment in ␣3Y without any hydrogen bonding partner or access to the solvent (38). Therefore, the surrounding protein matrix is rather different compared with the one present in aCRY, considering the relatively exposed position of Tyr-373 in a cavity on the surface of the protein, as derived from the modeled structure (16).…”

“…In PSII, arginine and aspartate close to Tyr D form a salt bridge, facilitating the proton transfer during oxidation (41). In the modeled structure of aCRY (16), arginine (Arg-485) and aspartate (Asp-321) also form a salt bridge close to Tyr-373, possibly creating a similar environment as in PSII. Still, it is unclear whether an amino acid residue or a coordinated water molecule is the proton acceptor in aCRY.…”

“…Accordingly, formation of FADH ⅐ requires a pre-illumination with blue light which was not applied in the in vivo experiments (13). However, FTIR spectroscopy revealed structural changes in the protein moiety only in the transition from FADH ⅐ to the anionic fully reduced state (FADH Ϫ ) of aCRY providing further support for FADH ⅐ as the dark state of the chromophore (16). Strikingly, these changes in turn structures were not detected in the closely related Xenopus laevis (6 -4) photolyase (17).…”

“…Red Light-induced FTIR Difference Spectroscopy on aCRY⌬CCT-The previously recorded FTIR difference spectrum of the wild type (16) showed changes in turn structures in the light-induced conversion from FADH ⅐ to FADH Ϫ that are not present in (6 -4) photolyase (17). To further investigate a possible involvement of the CCT in the red light response of aCRY, difference spectra of aCRY⌬CCT were taken after red light exposure.…”