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.

The structure of photosystem I from the thermophilic cyanobacterium Synechococcus elongatus has been recently resolved by x-ray crystallography to 2.5-A resolution. Besides the reaction center, photosystem I consists also of a core antenna containing 90 chlorophyll and 22 carotenoid molecules. It is their function to harvest solar energy and to transfer this energy to the reaction center (RC) where the excitation energy is converted into a charge separated state. Methods of steady-state optical spectroscopy such as absorption, linear, and circular dichroism have been applied to obtain information on the spectral properties of the complex, whereas transient absorption and fluorescence studies reported in the literature provide information on the dynamics of the excitation energy transfer. On the basis of the structure, the spectral properties and the energy transfer kinetics are simultaneously modeled by application of excitonic coupling theory to reveal relationships between structure and function. A spectral assignment of the 96 chlorophylls is suggested that allows us to reproduce both optical spectra and transfer and emission spectra and lifetimes of the photosystem I complex from S. elongatus. The model calculation allowed to study the influence of the following parameters on the excited state dynamics: the orientation factor, the heterogeneous site energies, the modifications arising from excitonic coupling (redistribution of oscillator strength, energetic splitting, reorientation of transition dipoles), and presence or absence of the linker cluster chlorophylls between antenna and reaction center. For the Förster radius and the intrinsic primary charge separation rate, the following values have been obtained: R(0) = 7.8 nm and k(CS) = 0.9 ps(-1). Variations of these parameters indicate that the excited state dynamics is neither pure trap limited, nor pure transfer (to-the-trap) limited but seems to be rather balanced.

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.

Chromophores absorb light in photosensitive proteins and thereby initiate fundamental biological processes such as photosynthesis, vision and biofluorescence. An important goal in their understanding is the provision of detailed structural descriptions of the ultrafast photochemical events that they undergo, in particular of the excited states that connect chemistry to biological function. Here we report on the structures of two excited states in the reversibly photoswitchable fluorescent protein rsEGFP2. We populated the states through femtosecond illumination of rsEGFP2 in its non-fluorescent off state and observed their build-up (within less than one picosecond) and decay (on the several picosecond timescale). Using an X-ray free-electron laser, we performed picosecond time-resolved crystallography and show that the hydroxybenzylidene imidazolinone chromophore in one of the excited states assumes a near-canonical twisted configuration halfway between the trans and cis isomers. This is in line with excited-state quantum mechanics/molecular mechanics and classical molecular dynamics simulations. Our new understanding of the structure around the twisted chromophore enabled the design of a mutant that displays a twofold increase in its off-to-on photoswitching quantum yield.

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...

Fatty acid photodecarboxylase (FAP) is a photoenzyme with potential green chemistry applications. By combining static, time-resolved, and cryotrapping spectroscopy and crystallography as well as computation, we characterized Chlorella variabilis FAP reaction intermediates on time scales from subpicoseconds to milliseconds. High-resolution crystal structures from synchrotron and free electron laser x-ray sources highlighted an unusual bent shape of the oxidized flavin chromophore. We demonstrate that decarboxylation occurs directly upon reduction of the excited flavin by the fatty acid substrate. Along with flavin reoxidation by the alkyl radical intermediate, a major fraction of the cleaved carbon dioxide unexpectedly transformed in 100 nanoseconds, most likely into bicarbonate. This reaction is orders of magnitude faster than in solution. Two strictly conserved residues, R451 and C432, are essential for substrate stabilization and functional charge transfer.

In Escherichia coli photolyase, excitation of the FAD cofactor in its semireduced radical state (FADH • ) induces an electron transfer over Ϸ15 Å from tryptophan W306 to the flavin. It has been suggested that two additional tryptophans are involved in an electron transfer chain FADH • 4 W382 4 W359 4 W306. To test this hypothesis, we have mutated W382 into redox inert phenylalanine. Ultrafast transient absorption studies showed that, in WT photolyase, excited FADH • decayed with a time constant Ϸ 26 ps to fully reduced flavin and a tryptophan cation radical. In W382F mutant photolyase, the excited flavin was much longer lived ( Ϸ 80 ps), and no significant amount of product was detected. We conclude that, in WT photolyase, excited FADH • is quenched by electron transfer from W382. On a millisecond scale, a product state with extremely low yield (Ϸ0.5% of WT) was detected in W382F mutant photolyase. Its spectral and kinetic features were similar to the fully reduced flavin͞neutral tryptophan radical state in WT photolyase. We suggest that, in W382F mutant photolyase, excited FADH • is reduced by W359 at a rate that competes only poorly with the intrinsic decay of excited FADH • ( Ϸ 80 ps), explaining the low product yield. Subsequently, the W359 cation radical is reduced by W306. The rate constants of electron transfer from W382 to excited FADH • in WT and from W359 to excited FADH • in W382F mutant photolyase were estimated and related to the donor-acceptor distances.

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