Light-induced Electron Transfer in Arabidopsis Cryptochrome-1 Correlates with in Vivo Function

Abstract

Cryptochromes are blue light-activated photoreceptors found in multiple organisms with significant similarity to photolyases, a class of light-dependent DNA repair enzymes. Unlike photolyases, cryptochromes do not repair DNA and instead mediate blue light-dependent developmental, growth, and/or circadian responses by an as yet unknown mechanism of action. It has recently been shown that Arabidopsis cryptochrome-1 retains photolyase-like photoreduction of its flavin cofactor FAD by intraprotein electron transfer from tryptophan and tyrosine residues. Here we demonstrate that substitution of two conserved tryptophans that are constituents of the flavin-reducing electron transfer chain in Escherichia coli photolyase impairs light-induced electron transfer in the Arabidopsis cryptochrome-1 photoreceptor in vitro. Furthermore, we show that these substitutions result in marked reduction of light-activated autophosphorylation of cryptochrome-1 in vitro and of its photoreceptor function in vivo, consistent with biological relevance of the electron transfer reaction. These data support the possibility that lightinduced flavin reduction via the tryptophan chain is the primary step in the signaling pathway of plant cryptochrome.Cryptochromes are found in plants, animals, and microbial systems, where they mediate numerous blue light-dependent developmental, growth, and/or circadian reponses (1-4). Cryptochrome-type photoreceptors are distinguished by their significant similarity to photolyases, a class of DNA repair enzymes (4) that removes lesions in UV-damaged DNA via a lightactivated electron transfer mechanism. Despite their similarity to photolyases, and the fact that they bind the same flavin cofactor, FAD, the cryptochromes do not repair DNA and appear to function by interaction with downstream cellular signaling intermediates of the various response pathways (1, 2). The mechanism whereby light activates the cryptochrome photoreceptors, and the significance of their marked structural similarity to photolyases (5-7), is currently unknown.Photolyases can undergo two distinct light-induced electron transfer reactions upon excitation of their FAD cofactor (4,8,9). The first reaction initiates DNA repair and requires the flavin in its fully reduced form. In the second reaction, known as photoactivation, the semi-reduced flavin is converted to the fully reduced form by an electron ultimately provided by an extrinsic reductant. An intraprotein electron transfer pathway connecting the buried flavin to the protein surface has been derived for this photoactivation reaction in Escherichia coli photolyase based on crystallographic structural information and on a combination of site-directed mutagenesis and spectroscopy (10 -12). This pathway comprises a chain of three tryptophan residues (Trp 382 -Trp 359 -Trp 306 ) that are conserved throughout the photolyase/cryptochrome family. Recently, a study with purified Arabidopsis cryptochrome-1 (cry1) 1 demonstrated occurrence of a similar photoreaction, starting from the fully oxidiz...

Analysis of Autophosphorylating Kinase Activities of Arabidopsis and Human Cryptochromes

“…Furthermore, contrary to earlier reports (18,19) we find no correlation between the presence of FAD and the autokinase activity nor do we observe any effect of blue light on the kinase activity of AtCry1 which contains near stoichiometric amount of FAD. Finally, it was reported that photoreduction of AtCry1 by illuminating with blue light increased the AtCry1 kinase activity and that nonphotoreducible mutants failed to exhibit such an effect and therefore it was concluded that the photoreduction was the primary photophysical reaction in AtCry1 (19). We not only fail to observe any increase in AtCry1 kinase activity under blue light but we also fail to observe any measurable change in activity when the flavin of AtCry1 is chemically reduced by dithionite.…”

“…A previous study found that preillumination of the protein, which reduces the flavin cofactor (19), increases the subsequent light-stimulated kinase activity of the pigment (18). Conversely, it was reported that the presence of I 2 that quenches the excited state and of H 2 O 2 that oxidizes the flavin, abolished the light stimulation of the kinase activity (18).…”

Hydrogen-bond switching through a radical pair mechanism in a flavin-binding photoreceptor

“…The present results show that the hydrogen-bond switch in BLUF domains is driven by initial ET from aromatic residues to the oxidized flavin. In the cryptochromes, similar ET processes appear to underlie their activity, with Trps as likely electron donors (10,33). The question remains whether in LOV domains, ET or PT from a conserved Cys to the FMN chromophore constitutes the primary photochemical event (9,(34)(35)(36).…”

Resolving cryptic aspects of cryptochrome signaling

“…These conflicts are further exacerbated by apparent differences within the CPF family. For instance, the Arabidopsis thaliana AtCRY1 and AtCRY2 proteins reportedly differ in the requirement of the Trp triad for function: the Trp triad is not required for signaling in CRY2 (5), but was reported to be required for CRY1 function in vivo (6). In PNAS, Gao et al elegantly demonstrate that the Trp triad is indeed not required for in vivo function of AtCRY1 and that photochemical activation of Trp triad mutants is not dependent upon ATP or other metabolites (7).…”