Searching for a photocycle of the cryptochrome photoreceptors

Abstract

The initial photochemistry of plant cryptochromes has been extensively investigated in recent years. It is hypothesized that cryptochrome photoexcitation involves a Trp-triad-dependent photoreduction. According to this hypothesis, cryptochromes in the resting state contain oxidized FAD; light triggers a sequential electron transfer from three tryptophan residues to reduce FAD to a neutral semiquinone (FADH*); FADH* is the presumed signaling state and it is re-oxidized to complete the photocycle. However, this photoreduction hypothesis is currently under debate. An alternative model argues that the initial photochemistry of cryptochromes involves a photolyase-like cyclic electron shuttle without a bona fide redox reaction mediated by the Trp-triad residues, leading to conformational changes, signal propagation, and physiological responses.

Fig. 1. Oxidoreduction of flavins

A. Five possible redox forms of flavins are shown. R indicates different side groups in different flavins. The two different forms of semiquinone radicals: anion radical (e.g. FAD^{• −}) and neutral radical (e.g. FADH^•), and two forms of reduced flavins: protonated hydroquinone (e.g. FADH₂) and anionic hydroquinone (e.g. FADH⁻) are shown. B. Absorption spectra and extinction coefficients (ε) of different redox forms of selected cryptochrome/photolyase [4]. Mosquito AgCRY1 (Anopheles gambiae) containing oxidized FAD (black line) or anion radical semiquinone (FAD^{• −}, blue line), and E. coli photolyase containing neutral radical semiquinone (FADH^•, green line) or fully reduced flavin (FADH⁻, red line). The absorption spectra of Arabidopsis cryptochromes with the respective redox forms of flavin are similar to those shown here.

Fig. 1. Oxidoreduction of flavins

A. Five possible redox forms of flavins are shown. R indicates different side groups in different flavins. The two different forms of semiquinone radicals: anion radical (e.g. FAD^{• −}) and neutral radical (e.g. FADH^•), and two forms of reduced flavins: protonated hydroquinone (e.g. FADH₂) and anionic hydroquinone (e.g. FADH⁻) are shown. B. Absorption spectra and extinction coefficients (ε) of different redox forms of selected cryptochrome/photolyase [4]. Mosquito AgCRY1 (Anopheles gambiae) containing oxidized FAD (black line) or anion radical semiquinone (FAD^{• −}, blue line), and E. coli photolyase containing neutral radical semiquinone (FADH^•, green line) or fully reduced flavin (FADH⁻, red line). The absorption spectra of Arabidopsis cryptochromes with the respective redox forms of flavin are similar to those shown here.

Fig. 2. The Trp-triad of photolyases and cryptochromes

A. A partial sequence alignment of the region of photolyases and cryptochromes highly conserved and important for FAD-binding. Synechococcus elongatus (SePhotolyse) and Escherichia coli photolyases (Ecphotolyase), Arapidopsis cryptochrome 1 and 2 (AtCRY1, AtCRY2), human cryptochrome 1 and 2 (HsCRY1, HsCRY2), and Drosophila cryptochrome (dCRY) are compared. Symbols above indicate secondary structures, with dashed-lines representing loops and “H” representing alpha-helices. Numbers beneath indicate positions of the tryptophan residues of Arabidopsis CRY1. The three tryptophan residues of the Trp-triad are highlighted in red, the two tryptophan residues not part of Trp-triad are highlighted in blue. B–C. The structure of FAD–binding pocket, FAD (yellow), the tryptophan residues of the Trp-triad (pink), and the path of electron movement from Trp-triad to FAD by photoreduction (blue dashed-line) of E. coli photolyase (B) and the PHR domain of Arabidopsis CRY1 (C) are indicated by blue dashed lines.

Fig. 3. Alternative models of cryptochrome photochemistry

A. The Trp-triad-dependent photoreduction model. In this model, the resting state of a cryptochrome contains the oxidized FAD. Upon photon absorption, the excited FAD* gains an electron from tryptophan residues of Trp-triad (see Fig. 2) and a proton from an unknown source to become the active neutral radical semiquinone, FADH^•. How the Trp-triad-dependent photoreduction triggers autophosphorylation and conformational change is not clear. B. The photolyase-like cyclic electron shuttle model. In this model, the resting state of a cryptochrome contains the anion radical semiquinone (FAD⁻). Upon photon absorption, the excited FAD⁻* transfers an electron to ATP, triggering phosphotransfer and autophosphorylation of the cryptochrome. The electron is subsequently transferred back to flavin to complete the cycle. Autophosphorylation of cryptochromes facilitates further phosphorylation by an unidentified kinase (kinase X), resulting in separation of the CCE domain from the PHR domain and formation of the open conformation. Cryptochromes with the open conformation interacts with signaling partners to trigger signal transduction. The locations of FAD and ATP are indicated by the yellow and red structures, respectively. The putative locations of phosphorous group (red circle) and electron transfer path (red arrows) are indicated.