Specific responses to blue light are found throughout the biological kingdom. These responses--which in higher plants include phototropism, inhibition of hypocotyl elongation, and stomatal opening--are in many cases thought to be mediated by flavin-type photoreceptors. But no such blue-light photoreceptor has yet been identified or isolated, although blue-light responses in plants were reported by Darwin over a century ago, long before the discovery of the now relatively well characterized red/far-red light photoreceptor, phytochrome. Here we describe the isolation of a gene corresponding to the HY4 locus of Arabidopsis thaliana. The hy4 mutant is one of several mutants that are selectively insensitive to blue light during the blue-light-dependent inhibition of hypocotyl elongation response, which suggests that they lack an essential component of the cryptochrome-associated light-sensing pathway. The HY4 gene, isolated by gene tagging, was shown to encode a protein with significant homology to microbial DNA photolyases. As photolyases are a rare class of flavoprotein that catalyse blue-light-dependent reactions, the protein encoded by HY4 has a structure consistent with that of a flavin-type blue-light photoreceptor.

Cryptochromes are flavoprotein photoreceptors first identified in Arabidopsis thaliana, where they play key roles in growth and development. Subsequently identified in prokaryotes, archaea, and many eukaryotes, cryptochromes function in the animal circadian clock and are proposed as magnetoreceptors in migratory birds. Cryptochromes are closely structurally related to photolyases, evolutionarily ancient flavoproteins that catalyze light-dependent DNA repair. Here, we review the structural, photochemical, and molecular properties of cry-DASH, plant, and animal cryptochromes in relation to biological signaling mechanisms and uncover common features that may contribute to better understanding the function of cryptochromes in diverse systems including in man.

Cryptochromes are blue light-sensing photoreceptors found in plants, animals, and humans. They are known to play key roles in the regulation of the circadian clock and in development. However, despite striking structural similarities to photolyase DNA repair enzymes, cryptochromes do not repair double-stranded DNA, and their mechanism of action is unknown. Recently, a blue light-dependent intramolecular electron transfer to the excited state flavin was characterized and proposed as the primary mechanism of light activation. The resulting formation of a stable neutral flavin semiquinone intermediate enables the photoreceptor to absorb green/yellow light (500 -630 nm) in addition to blue light in vitro. Here, we demonstrate that Arabidopsis cryptochrome activation by blue light can be inhibited by green light in vivo consistent with a change of the cofactor redox state. We further characterize light-dependent changes in the cryptochrome1 (cry1) protein in living cells, which match photoreduction of the purified cry1 in vitro. These experiments were performed using fluorescence absorption/emission and EPR on whole cells and thereby represent one of the few examples of the active state of a known photoreceptor being monitored in vivo. These results indicate that cry1 activation via blue light initiates formation of a flavosemiquinone signaling state that can be converted by green light to an inactive form. In summary, cryptochrome activation via flavin photoreduction is a reversible mechanism novel to blue light photoreceptors. This photocycle may have adaptive significance for sensing the quality of the light environment in multiple organisms.

Among the biological phenomena that fall within the emerging field of “quantum biology” is the suggestion that magnetically sensitive chemical reactions are responsible for the magnetic compass of migratory birds. It has been proposed that transient radical pairs are formed by photo-induced electron transfer reactions in cryptochrome proteins and that their coherent spin dynamics are influenced by the geomagnetic field leading to changes in the quantum yield of the signaling state of the protein. Despite a variety of supporting evidence, it is still not clear whether cryptochromes have the properties required to respond to magnetic interactions orders of magnitude weaker than the thermal energy, k B T . Here we demonstrate that the kinetics and quantum yields of photo-induced flavin—tryptophan radical pairs in cryptochrome are indeed magnetically sensitive. The mechanistic origin of the magnetic field effect is clarified, its dependence on the strength of the magnetic field measured, and the rates of relevant spin-dependent, spin-independent, and spin-decoherence processes determined. We argue that cryptochrome is fit for purpose as a chemical magnetoreceptor.

The arabidopsis thaliana HY4 gene encodes CRY1, a 75-kilodalton flavoprotein mediating blue light-dependent regulation of seedling development. CRY1 is demonstrated here to noncovalently bind stoichiometric amounts of flavin adenine dinucleotide (FAD). The redox properties of FAD bound by CRY1 include an unexpected stability of the neutral radical flavosemiquinone (FADH.). The absorption properties of this flavosemiquinone provide a likely explanation for the additional sensitivity exhibited by CRY1-mediated responses in the green region of the visible spectrum. Despite the sequence homology to microbial DNA photolyases, CRY1 was found to have no detectable photolyase activity.

Cryptochromes are flavoproteins implicated in multiple blue light-dependent signaling pathways regulating, for example, photomorphogenesis in plants or circadian clocks in animals. Using transient absorption spectroscopy, it is demonstrated that the primary light reactions in isolated Arabidopsis thaliana cryptochrome-1 involve intraprotein electron transfer from tryptophan and tyrosine residues to the excited flavin adenine dinucleotide cofactor.

Cryptochrome (Cry) photoreceptors share high sequence and structural similarity with DNA repair enzyme DNA-photolyase and carry the same flavin cofactor. Accordingly, DNA-photolyase was considered a model system for the light activation process of cryptochromes. In line with this view were recent spectroscopic studies on cryptochromes of the CryDASH subfamily that showed photoreduction of the flavin adenine dinucleotide (FAD) cofactor to its fully reduced form. However, CryDASH members were recently shown to have photolyase activity for cyclobutane pyrimidine dimers in single-stranded DNA, which is absent for other members of the cryptochrome/photolyase family. Thus, CryDASH may have functions different from cryptochromes. The photocycle of other members of the cryptochrome family, such as Arabidopsis Cry1 and Cry2, which lack DNA repair activity but control photomorphogenesis and flowering time, remained elusive. Here we have shown that Arabidopsis Cry2 undergoes a photocycle in which semireduced flavin (FADH ⅐ ) accumulates upon blue light irradiation. Green light irradiation of Cry2 causes a change in the equilibrium of flavin oxidation states and attenuates Cry2-controlled responses such as flowering. These results demonstrate that the active form of Cry2 contains FADH ⅐ (whereas catalytically active photolyase requires fully reduced flavin (FADH ؊ )) and suggest that cryptochromes could represent photoreceptors using flavin redox states for signaling differently from DNA-photolyase for photorepair.Blue light signaling has been historically of great interest in plants, because distinct blue light receptors are involved in such key processes as photomorphogenesis, phototropism, and initiation of flowering (1). Cryptochromes (Crys) 2 are UV-A/blue light photoreceptors (also found in animals and bacteria (2)) and are implicated in several of these responses. Because of their high sequence and structural similarity (3-6) and identical flavin cofactor content as DNA-photolyase (7, 8), it was hypothesized that cryptochromes might use the same mechanism for signaling as does photolyase for catalysis, which is light-driven electron transfer from the fully reduced flavin FADH Ϫ (2, 4, 7-9). Indeed, spectroscopic studies on Vibrio cholerae Cry1 (10) and Arabidopsis Cry3 (11) have shown that blue light illumination results in the formation of fully reduced flavin similar to the photoactivation process of DNA-photolyase (2). However, CryDASH members were recently shown to have photolyase activity for cyclobutane pyrimidine dimers in single-stranded DNA (12), which is absent for other members of the cryptochrome/photolyase family.The photoreduction of flavin in DNA-photolyase involves several aromatic amino acid residues (mostly tryptophans) that transfer electrons from the protein surface to the flavin adenine dinucleotide (FAD), which is buried in a U-shaped conformation in the ␣-helical domain of the protein (13-16). The cryptochrome structures solved so far, namely Synechocystis Cry-DASH (3), the photolyase-related d...

Cryptochromes are a class of flavoprotein blue-light signaling receptors found in plants, animals, and humans that control plant development and the entrainment of circadian rhythms. In plant cryptochromes, light activation is proposed to result from photoreduction of a protein-bound flavin chromophore through intramolecular electron transfer. However, although similar in structure to plant cryptochromes, the light-response mechanism of animal cryptochromes remains entirely unknown. To complicate matters further, there is currently a debate on whether mammalian cryptochromes respond to light at all or are instead activated by non–light-dependent mechanisms. To resolve these questions, we have expressed both human and Drosophila cryptochrome proteins to high levels in living Sf21 insect cells using a baculovirus-derived expression system. Intact cells are irradiated with blue light, and the resulting cryptochrome photoconversion is monitored by fluorescence and electron paramagnetic resonance spectroscopic techniques. We demonstrate that light induces a change in the redox state of flavin bound to the receptor in both human and Drosophila cryptochromes. Photoreduction from oxidized flavin and subsequent accumulation of a semiquinone intermediate signaling state occurs by a conserved mechanism that has been previously identified for plant cryptochromes. These results provide the first evidence of how animal-type cryptochromes are activated by light in living cells. Furthermore, human cryptochrome is also shown to undergo this light response. Therefore, human cryptochromes in exposed peripheral and/or visual tissues may have novel light-sensing roles that remain to be elucidated.