The cryptochrome/photolyase protein family possesses a conserved triad of tryptophans that may act as a molecular wire to transport electrons from the protein surface to the FAD cofactor for activation and/or signaling-state formation. Members from the animal (and animal-like) cryptochrome subclade use this process in a light-induced fashion in a number of exciting responses, such as the (re-)setting of circadian rhythms or magnetoreception; however, electron-transfer pathways have not been explored in detail yet. Therefore, we present an in-depth time-resolved optical and electron-paramagnetic resonance spectroscopic study of two cryptochromes from Chlamydomonas reinhardtii and Drosophila melanogaster. The results do not only reveal the existence of a fourth, more distant aromatic amino acid that serves as a terminal electron donor in both proteins, but also show that a tyrosine is able to fulfill this very role in Chlamydomonas reinhardtii cryptochrome. Additionally, exchange of the respective fourth aromatic amino acid to redox-inactive phenylalanines still leads to light-induced radical pair formation; however, the lifetimes of these species are drastically reduced from the ms- to the μs-range. The results presented in this study open up a new chapter, to our knowledge, in the diversity of electron-transfer pathways in cryptochromes. Moreover, they could explain unique functions of animal cryptochromes, in particular their potential roles in magnetoreception because magnetic-field effects of light-induced radical pairs strongly depend on distance and orientation parameters.

Photolyases and cryptochromes form an almost ubiquitous family of blue light photoreceptors involved in the repair and maintenance of DNA integrity or regulatory control. We found that one cryptochrome from the green alga Chlamydomonas reinhardtii (CraCRY) is capable of both, control of transcript levels and the sexual cycle of the alga in a positive (germination) and negative manner (mating ability), as well as catalyzing the repair of UV-DNA lesions. Its 1.6 Å crystal structure shows besides the FAD chromophore an aromatic tetrad that is indispensable in animal-like type I cryptochromes for light-driven change of their signaling-active redox state and formation of a stable radical pair. Given CraCRY’s catalytic activity as (6-4) photolyase in vivo and in vitro, we present the first co-crystal structure of a cryptochrome with duplex DNA comprising a (6-4) pyrimidine–pyrimidone lesion. This 2.9 Å structure reveals a distinct conformation for the catalytic histidine His1, H357, that challenges previous models of a single-photon driven (6-4) photolyase mechanism.

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 UVvisible 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 s 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.Cryptochromes represent a group of diverse sensory photoreceptors present in all kingdoms of life (1, 2). Together with the UV-light-dependent DNA repair enzymes, the photolyases (3), they constitute the cryptochrome/photolyase family. Members of this family share a highly conserved photolyase homology region (PHR) 2 , which comprises ϳ500 amino acids and carries a non-covalently bound flavin adenine dinucleotide (FAD) as a chromophore. The C-terminal extension (CCT) present in many cryptochromes and photolyases is strongly variable in amino acid composition and length and has been shown to be crucial for signal transduction in the Arabidopsis cryptochrome AtCRY1 (4). The diverse subfamilies of cryptochromes comprise proteins acting as central blue light sensors in bacteria, fungi, plants, and insects (animal type I CRY) (1). Moreover, CRYs are also found as the light-independent, central part of the oscillator of the biological clock in mammals (animal type II CRY) (5) and as a mediator for light-dependent magnetosensitivity in flies (6). Opposed to these findings, DASH cryptochromes have been found to repair lesions in single-stranded DNA and double-stranded loop-structured DNA in vitro (7,8). Therefore, DASH cryptochromes are more similar in terms of functionality to photolyases than cryptochromes. Among the photolyases, two different types are separated dependin...

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