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.

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...