Ultrafast Oxidation of a Tyrosine by Proton-Coupled Electron Transfer Promotes Light Activation of an Animal-like Cryptochrome.

The animal-like cryptochrome of Chlamydomonas reinhardtii (CraCRY) is a recently discovered photoreceptor that controls the transcriptional profile and sexual life cycle of this alga by both blue and red light. CraCRY has the uncommon feature of efficient formation and longevity of the semireduced neutral form of its FAD cofactor upon blue light illumination. Tyrosine Y373 plays a crucial role by elongating , as fourth member, the electron transfer (ET) chain found in most other cryptochromes and DNA photolyases, which comprises a conserved tryptophan triad. Here, we report the full mechanism of light-induced FADH• formation in CraCRY using transient absorption spectroscopy from hundreds of femtoseconds to seconds. Electron transfer starts from ultrafast reduction of excited FAD to FAD•- by the proximal tryptophan (0.4 ps) and is followed by delocalized migration of the produced WH•+ radical along the tryptophan triad (∼4 and ∼50 ps). Oxidation of Y373 by coupled ET to WH•+ and deprotonation then proceeds in ∼800 ps, without any significant kinetic isotope effect, nor a pH effect between pH 6.5 and 9.0. The FAD•-/Y373• pair is formed with high quantum yield (∼60%); its intrinsic decay by recombination is slow (∼50 ms), favoring reduction of Y373• by extrinsic agents and protonation of FAD•- to form the long-lived, red-light absorbing FADH• species. Possible mechanisms of tyrosine oxidation by ultrafast proton-coupled ET in CraCRY, a process about 40 times faster than the archetypal tyrosine-Z oxidation in photosystem II, are discussed in detail.

Redox transients of P680 associated with the incremental chlorophyll-a fluorescence yield rises elicited by a series of saturating flashes in diuron-treated photosystem II core complex of Thermosynechococcus vulcanus.

Recent chlorophyll-a fluorescence yield measurements, using single-turnover saturating flashes (STSFs), have revealed the involvement of a rate-limiting step in the reactions following the charge separation induced by the first flash. As also shown here, in diuron-inhibited PSII core complexes isolated from Thermosynechococcus vulcanus the fluorescence maximum could only be reached by a train of STSFs. In order to elucidate the origin of the fluorescence yield increments in STSF series, we performed transient absorption measurements at 819 nm, reflecting the photooxidation and re-reduction kinetics of the primary electron donor P680. Upon single flash excitation of the dark-adapted sample, the decay kinetics could be described with lifetimes of 17 ns (∼50%) and 167 ns (∼30%), and a longer-lived component (∼20%). This kinetics are attributed to re-reduction of P680•+ by the donor side of PSII. In contrast, upon second-flash (with Δt between 5 µs and 100 ms) or repetitive excitation, the 819 nm absorption changes decayed with lifetimes of about 2 ns (∼60%) and 10 ns (∼30%), attributed to recombination of the primary radical pair P680•+ Pheo•- , and a small longer-lived component (∼10%). These data confirm that only the first STSF is capable of generating stable charge separation - leading to the reduction of QA ; and thus, the fluorescence yield increments elicited by the consecutive flashes must have a different physical origin. Our double-flash experiments indicate that the rate-limiting steps, detected by chlorophyll-a fluorescence, are not correlated with the turnover of P680.

Sub-nanosecond tryptophan radical deprotonation mediated by a protein-bound water cluster in class II DNA photolyases.

Class II DNA photolyases are flavoenzymes occurring in both prokaryotes and eukaryotes including higher plants and animals. Despite considerable structural deviations from the well-studied class I DNA photolyases, they share the main biological function, namely light-driven repair of the most common UV-induced lesions in DNA, the cyclobutane pyrimidine dimers (CPDs). For DNA repair activity, photolyases require the fully reduced flavin adenine dinucleotide cofactor, FADH-, which can be obtained from oxidized or semi-reduced FAD by a process called photoactivation. Using transient absorption spectroscopy, we have examined the initial electron and proton transfer reactions leading to photoactivation of the class II DNA photolyase from Methanosarcina mazei. Upon photoexcitation, FAD is reduced via a distinct (class II-specific) chain of three tryptophans, giving rise to an FAD˙- TrpH˙+ radical pair. The distal Trp388H˙+ deprotonates to Trp388˙ in 350 ps, i.e., by three orders of magnitude faster than TrpH˙+ in aqueous solution or in any previously studied photolyase. We identified a class II-specific cluster of protein-bound water molecules ideally positioned to serve as the primary proton acceptor. The high rate of Trp388H˙+ deprotonation counters futile radical pair recombination and ensures efficient photoactivation.

A Long-Lived Triplet State Is the Entrance Gateway to Oxidative Photochemistry in Green Fluorescent Proteins.

Though ubiquitously used as selective fluorescence markers in cellular biology, fluorescent proteins (FPs) still have not disclosed all of their surprising properties. One important issue, notably for single-molecule applications, is the nature of the triplet state, suggested to be the starting point for many possible photochemical reactions leading to phenomena such as blinking or bleaching. Here, we applied transient absorption spectroscopy to characterize dark states in the prototypical enhanced green fluorescent protein (EGFP) of hydrozoan origin and, for comparison, in IrisFP, a representative phototransformable FP of anthozoan origin. We identified a long-lived (approximately 5 ms) dark state that is formed with a quantum yield of approximately 1% and has pronounced absorption throughout the visible-NIR range (peak at around 900 nm). Detection of phosphorescence emission with identical kinetics and excitation spectrum allowed unambiguous identification of this state as the first excited triplet state of the deprotonated chromophore. This triplet state was further characterized by determining its phosphorescence emission spectrum, the temperature dependence of its decay kinetics and its reactivity toward oxygen and electron acceptors and donors. It is suggested that it is this triplet state that lies at the origin of oxidative photochemistry in green FPs, leading to phenomena such as so-called "oxidative redding", "primed photoconversion", or, in a manner similar to that previously observed for organic dyes, redox induced blinking control with the reducing and oxidizing system ("ROXS").

An algal photoenzyme converts fatty acids to hydrocarbons.

Although many organisms capture or respond to sunlight, few enzymes are known to be driven by light. Among these are DNA photolyases and the photosynthetic reaction centers. Here, we show that the microalga Chlorella variabilis NC64A harbors a photoenzyme that acts in lipid metabolism. This enzyme belongs to an algae-specific clade of the glucose-methanol-choline oxidoreductase family and catalyzes the decarboxylation of free fatty acids to n-alkanes or -alkenes in response to blue light. Crystal structure of the protein reveals a fatty acid-binding site in a hydrophobic tunnel leading to the light-capturing flavin adenine dinucleotide (FAD) cofactor. The decarboxylation is initiated through electron abstraction from the fatty acid by the photoexcited FAD with a quantum yield >80%. This photoenzyme, which we name fatty acid photodecarboxylase, may be useful in light-driven, bio-based production of hydrocarbons.

Ultrafast flavin photoreduction in an oxidized animal (6-4) photolyase through an unconventional tryptophan tetrad.

Photolyases are flavoenzymes repairing UV-induced lesions in DNA, which may be activated by a photoreduction of their FAD cofactor. In most photolyases, this photoreduction proceeds by electron transfer along a chain of three tryptophan (Trp) residues, connecting the flavin to the protein surface. Much less studied, animal (6-4) photolyases (repairing pyrimidine-pyrimidone (6-4) photoproducts) are particularly interesting as they were recently shown to have a longer electron transfer chain, counting four Trp residues. Using femtosecond polarized transient absorption spectroscopy, we performed a detailed analysis of the photoactivation reaction in the (6-4) photolyase of Xenopus laevis with oxidized FAD. We showed that the excited flavin is very quickly reduced (∼0.5 ps) by a nearby tryptophan residue, yielding FAD˙- and WH˙+ radicals. Subsequent kinetic steps in the picosecond regime were assigned to the migration of the positive charge along the Trp tetrad, in competition with charge recombination. We propose that the positive charge is actually delocalized over various Trp residues during most of the dynamics and that charge recombination essentially occurs through the proximal tryptophanyl radical. Oxidation of the fourth tryptophan is thought to be reached about as fast as that of the third one (∼40 ps), based on a comparison with a mutant protein lacking the distal Trp, implying ultrafast electron transfer between these two residues. This unusual mechanism sheds light on the rich diversity of electron transfer pathways found in various photolyases, and evolution-related cryptochromes alike.

Repair of (6-4) Lesions in DNA by (6-4) Photolyase: 20 Years of Quest for the Photoreaction Mechanism.

Exposure of DNA to ultraviolet (UV) light from the Sun or from other sources causes the formation of harmful and carcinogenic crosslinks between adjacent pyrimidine nucleobases, namely cyclobutane pyrimidine dimers and pyrimidine(6-4)pyrimidone photoproducts. Nature has developed unique flavoenzymes, called DNA photolyases, that utilize blue light, that is photons of lower energy than those of the damaging light, to repair these lesions. In this review, we focus on the chemically challenging repair of the (6-4) photoproducts by (6-4) photolyase and describe the major events along the quest for the reaction mechanisms, over the 20 years since the discovery of (6-4) photolyase.

Weak temperature dependence of P (+) H A (-) recombination in mutant Rhodobacter sphaeroides reaction centers.

In contrast with findings on the wild-type Rhodobacter sphaeroides reaction center, biexponential P (+) H A (-)  â†’ PH A charge recombination is shown to be weakly dependent on temperature between 78 and 298 K in three variants with single amino acids exchanged in the vicinity of primary electron acceptors. These mutated reaction centers have diverse overall kinetics of charge recombination, spanning an average lifetime from ~2 to ~20 ns. Despite these differences a protein relaxation model applied previously to wild-type reaction centers was successfully used to relate the observed kinetics to the temporal evolution of the free energy level of the state P (+) H A (-) relative to P (+) B A (-) . We conclude that the observed variety in the kinetics of charge recombination, together with their weak temperature dependence, is caused by a combination of factors that are each affected to a different extent by the point mutations in a particular mutant complex. These are as follows: (1) the initial free energy gap between the states P (+) B A (-) and P (+) H A (-) , (2) the intrinsic rate of P (+) B A (-)  â†’ PB A charge recombination, and (3) the rate of protein relaxation in response to the appearance of the charge separated states. In the case of a mutant which displays rapid P (+) H A (-) recombination (ELL), most of this recombination occurs in an unrelaxed protein in which P (+) B A (-) and P (+) H A (-) are almost isoenergetic. In contrast, in a mutant in which P (+) H A (-) recombination is relatively slow (GML), most of the recombination occurs in a relaxed protein in which P (+) H A (-) is much lower in energy than P (+) H A (-) . The weak temperature dependence in the ELL reaction center and a YLH mutant was modeled in two ways: (1) by assuming that the initial P (+) B A (-) and P (+) H A (-) states in an unrelaxed protein are isoenergetic, whereas the final free energy gap between these states following the protein relaxation is large (~250 meV or more), independent of temperature and (2) by assuming that the initial and final free energy gaps between P (+) B A (-) and P (+) H A (-) are moderate and temperature dependent. In the case of the GML mutant, it was concluded that the free energy gap between P (+) B A (-) and P (+) H A (-) is large at all times.

Photochemistry of Wild-Type and N378D Mutant E. coli DNA Photolyase with Oxidized FAD Cofactor Studied by Transient Absorption Spectroscopy.

DNA photolyases (PLs) and evolutionarily related cryptochrome (CRY) blue-light receptors form a widespread superfamily of flavoproteins involved in DNA photorepair and signaling functions. They share a flavin adenine dinucleotide (FAD) cofactor and an electron-transfer (ET) chain composed typically of three tryptophan residues that connect the flavin to the protein surface. Four redox states of FAD are relevant for the various functions of PLs and CRYs: fully reduced FADH(-) (required for DNA photorepair), fully oxidized FADox (blue-light-absorbing dark state of CRYs), and the two semireduced radical states FAD(.-) and FADH(.) formed in ET reactions. The PL of Escherichia coli (EcPL) has been studied for a long time and is often used as a reference system; however, EcPL containing FADox has so far not been investigated on all relevant timescales. Herein, a detailed transient absorption study of EcPL on timescales from nanoseconds to seconds after excitation of FADox is presented. Wild-type EcPL and its N378D mutant, in which the asparagine facing the N5 of the FAD isoalloxazine is replaced by aspartic acid, known to protonate FAD(.-) (formed by ET from the tryptophan chain) in plant CRYs in about 1.5 µs, are characterized. Surprisingly, the mutant protein does not show this protonation. Instead, FAD(.-) is converted in 3.3 µs into a state with spectral features that are different from both FADH(.) and FAD(.-) . Such a conversion does not occur in wild-type EcPL. The chemical nature and formation mechanism of the atypical FAD radical in N378D mutant EcPL are discussed.

Discovery and functional analysis of a 4th electron-transferring tryptophan conserved exclusively in animal cryptochromes and (6-4) photolyases.

A 4th electron transferring tryptophan in animal cryptochromes and (6-4) photolyases is discovered and functionally analyzed by transient absorption. It yields a much longer-lived flavin-tryptophan radical pair than the mere tryptophan triad in related flavoproteins, questioning the putative role of the primary light reaction of cryptochrome in animal magnetoreception.

ATP binding turns plant cryptochrome into an efficient natural photoswitch.

Cryptochromes are flavoproteins that drive diverse developmental light-responses in plants and participate in the circadian clock in animals. Plant cryptochromes have found application as photoswitches in optogenetics. We have studied effects of pH and ATP on the functionally relevant photoreduction of the oxidized FAD cofactor to the semi-reduced FADH(·) radical in isolated Arabidopsis cryptochrome 1 by transient absorption spectroscopy on nanosecond to millisecond timescales. In the absence of ATP, the yield of light-induced radicals strongly decreased with increasing pH from 6.5 to 8.5. With ATP present, these yields were significantly higher and virtually pH-independent up to pH 9. Analysis of our data in light of the crystallographic structure suggests that ATP-binding shifts the pKa of aspartic acid D396, the putative proton donor to FAD·(-), from ~7.4 to >9, and favours a reaction pathway yielding long-lived aspartate D396(-). Its negative charge could trigger conformational changes necessary for signal transduction.

Analysis of the temperature-dependence of P(+)HA(-) charge recombination in the Rhodobacter sphaeroides reaction center suggests nanosecond temperature-independent protein relaxation.

The temperature dependence of charge recombination of the pair P(+)HA(-) in isolated reaction centers from the purple bacterium Rhodobacter sphaeroides with prereduced quinone QA was studied by sub-nanosecond to microsecond time-scale transient absorption. Overall, the kinetics slowed down substantially upon cooling from room temperature to ∼200 K, and then remained virtually unchanged down to 77 K, indicating the coexistence of two competitive pathways of charge recombination, a thermally-activated pathway appearing only above ~200 K and a temperature-independent pathway. In our modelling, the thermally activated pathway includes an uphill electron transfer from HA(-) to BA(-) leading to transient formation of the state P(+)BA(-), whereas the temperature-independent pathway is due to direct downhill electron transfer from HA(-) to P(+). At all temperatures studied, the kinetics could be approximated by a four-component decay. Detailed analysis of the lifetimes and amplitudes of particular phases over the range of temperatures suggests that the kinetically resolved phases reveal the consecutive appearance of three conformational states characterized by an increasing free energy gap between the states P(+)BA(-) and P(+)HA(-). The initial gap between these states was estimated to be only ~8 meV, the intermediate gap being ~92 meV, and the final gap ~135 meV, with no dependence on temperature. It was also calculated through a very straightforward approach that the relaxation process from the initial to the intermediate state occurs within 0.6 ± 0.1 ns, whereas the second step of relaxation from the intermediate to the final state takes 11 ± 2 ns. Both phases of the protein relaxation process are essentially temperature-independent. Possible alternative models to describe the experimental data that cannot be definitely excluded are also discussed.

Repair of the (6-4) photoproduct by DNA photolyase requires two photons.

It takes two (photons) to tango: Single-turnover flash experiments showed that the flavoenzyme (6-4) photolyase uses a successive two-photon mechanism to repair the UV-induced T(6-4)T lesion in DNA (see picture). The intermediate (X) formed by the first photoreaction is likely to be the oxetane-bridged dimer T(ox)T. The enzyme could stabilize the normally short-lived T(ox)T, allowing repair to be completed by the second photoreaction.

Charge recombination in S(n)Tyr(Z)(•)Q(A)(-•) radical pairs in D1 protein variants of Photosystem II: long range electron transfer in the Marcus inverted region.

Charge recombination in the light-induced radical pair SnTyrZ(•)QA(-•) in Photosystem II (PSII) from Thermosynechococcus elongatus has been studied at cryogenic temperatures by time-resolved EPR for different configurations of PSII that are expected to affect the driving force of the reaction (oxidation states S0, S1, or S2 of the Mn4CaO5 cluster; PsbA1, PsbA2, or PsbA3 as D1 protein). The kinetics were independent of temperature in the studied range from 4.2 to 50 K and were not affected by exchange of H2O for D2O, consistent with single-step electron tunneling over the distance of ∼32 Å without any repopulation through Boltzmann equilibration of intermediates lying higher in energy. In PsbA1-PSII, the charge recombinations in the radical pairs SnTyrZ(•)QA(-•) (ket = 3.4 × 10(-3) s(-1) for S1) were slower than in PsbA3-PSII despite an expected lower driving force owing to a downshifted Em(QA/QA(-•)) in PsbA1-PSII. Conversely, the reaction was slower in the presence of S2 than in the presence of S1, despite an expected larger driving force due to an upshifted Em(TyrZ(•)/TyrZ) in S2. These observations indicate that the charge recombination occurs in the Marcus inverted region. Assuming that the driving force of the reaction (-ΔG(0) ≈ 1.2 eV at room temperature for S1) does not vary strongly with temperature, the data indicate an optimal electron transfer rate (for a hypothetical -ΔG(0) = λ) substantially faster than would be predicted from extrapolation of room temperature intraprotein ET rates over shorter distances. Possible origins of this deviation are discussed, including a possible enhancement of the electronic coupling of TyrZ(•) and QA(-•) by aromatic cofactors located in between. Observed similar S1TyrZ(•)QA(-•) charge recombinations in PsbA2-PSII and PsbA3-PSII predict that Em(QA/QA(-•)) in PsbA2-PSII is similar to that in PsbA3-PSII.

Analysis of the kinetics of P+ HA- recombination in membrane-embedded wild-type and mutant Rhodobacter sphaeroides reaction centers between 298 and 77 K indicates that the adjacent negatively charged QA ubiquinone modulates the free energy of P+ HA- and may influence the rate of the protein dielectric response.

Time-resolved spectroscopic studies of recombination of the P(+)HA(-) radical pair in photosynthetic reaction centers (RCs) from Rhodobacter sphaeroides give an opportunity to study protein dynamics triggered by light and occurring over the lifetime of P(+)HA(-). The state P(+)HA(-) is formed after the ultrafast light-induced electron transfer from the primary donor pair of bacteriochlorophylls (P) to the acceptor bacteriopheophytin (HA). In order to increase the lifetime of this state, and thus increase the temporal window for the examination of protein dynamics, it is possible to block forward electron transfer from HA(-) to the secondary electron acceptor QA. In this contribution, the dynamics of P(+)HA(-) recombination were compared at a range of temperatures from 77 K to room temperature, electron transfer from HA(-) to QA being blocked either by prereduction of QA or by genetic removal of QA. The observed P(+)HA(-) charge recombination was significantly slower in the QA-deficient RCs, and in both types of complexes, lowering the temperature from RT to 77 K led to a slowing of charge recombination. The effects are explained in the frame of a model in which charge recombination occurs via competing pathways, one of which is thermally activated and includes transient formation of a higher-energy state, P(+)BA(-). An internal electrostatic field supplied by the negative charge on QA increases the free energy levels of the state P(+)HA(-), thus decreasing its energetic distance to the state P(+)BA(-). In addition, the dielectric response of the protein environment to the appearance of the state P(+)HA(-) is accelerated from ∼50-100 ns in the QA-deficient mutant RCs to ∼1-16 ns in WT RCs with a negatively charged QA(-). In both cases, the temperature dependence of the protein dynamics is weak.

[Ru(bpy)(3)](2+) as a reference in transient absorption spectroscopy: differential absorption coefficients for formation of the long-lived (3)MLCT excited state.

Transient absorption spectroscopy and other time-resolved methods are commonly used to study chemical reactions and biological processes induced by absorption of light. In order to scale the signal amplitude or to compare results obtained under different conditions, it is advisable to use a reference system, a standard of convenient and well-defined properties. Finding Tris(bipyridine)ruthenium(ii), [Ru(bpy)(3)](2+), a suitable candidate for a transient-absorption spectroscopy reference due to its favourable photochemical properties, we have determined accurate relative values of differential molar absorption coefficients (Δε) for light-induced formation of the metal-to-ligand charge transfer (MLCT) excited triplet state at several relevant wavelengths (wavelengths of commercially available lasers) in the UV and visible regions. We have also attempted to determine the absolute value of Δε close to the wavelength of maximum bleaching (∼450 nm) and we propose to narrow down the interval of conceivable values for Δε(450) from the broad range of published values (-0.88 × 10(4) M(-1)cm(-1) to -1.36 × 10(4) M(-1)cm(-1)) to -1.1 × 10(4) M(-1)cm(-1)± 15%. Having ourselves successfully applied [Ru(bpy)(3)](2+) as a standard in a recent time-resolved study of enzymatic DNA repair, we would like to encourage other scientists to use this convenient tool as a reference in their future spectroscopic studies on time scales from picoseconds to hundreds of nanoseconds.

Mechanism of recombination of the P+H(A)- radical pair in mutant Rhodobacter sphaeroides reaction centers with modified free energy gaps between P+B(A)- and P+H(A)-.

The kinetics of recombination of the P(+)H(A)(-) radical pair were compared in wild-type reaction centers from Rhodobacter sphaeroides and in seven mutants in which the free energy gap, ΔG, between the charge separated states P(+)B(A)(-) and P(+)H(A)(-) was either increased or decreased. Five of the mutant RCs had been described previously, and X-ray crystal structures of two newly constructed complexes were determined by X-ray crystallography. The charge recombination reaction was accelerated in all mutants with a smaller ΔG than in the wild-type, and was slowed in a mutant having a larger ΔG. The free energy difference between the state P(+)H(A)(-) and the PH(A) ground state was unaffected by most of these mutations. These observations were consistent with a model in which the P(+)H(A)(-) → PH(A) charge recombination is thermally activated and occurs via the intermediate state P(+)B(A)(-), with a mean rate related to the size of the ΔG between the states P(+)B(A)(-) and P(+)H(A)(-) and not the ΔG between P(+)H(A)(-) and the ground state. A more detailed analysis of charge recombination in the mutants showed that the kinetics of the reaction were multiexponential, and characterized by ~0.5, ~1-3, and 7-17 ns lifetimes, similar to those measured for wild-type reaction centers. The exact lifetimes and relative amplitudes of the three components were strongly modulated by the mutations. Two models were considered in order to explain the observed multiexponentiality and modulation, involving heterogeneity or relaxation of P(+)H(A)(-) states, with the latter model giving a better fit to the experimental results.

Kinetics of cyclobutane thymine dimer splitting by DNA photolyase directly monitored in the UV.

CPD photolyase uses light to repair cyclobutane pyrimidine dimers (CPDs) formed between adjacent pyrimidines in UV-irradiated DNA. The enzyme harbors an FAD cofactor in fully reduced state (FADH(-)). The CPD repair mechanism involves electron transfer from photoexcited FADH(-) to the CPD, splitting of its intradimer bonds, and electron return to restore catalytically active FADH(-). The two electron transfer processes occur on time scales of 10(-10) and 10(-9) s, respectively. Until now, CPD splitting itself has only been poorly characterized by experiments. Using a previously unreported transient absorption setup, we succeeded in monitoring cyclobutane thymine dimer repair in the main UV absorption band of intact thymine at 266 nm. Flavin transitions that overlay DNA-based absorption changes at 266 nm were monitored independently in the visible and subtracted to obtain the true repair kinetics. Restoration of intact thymine showed a short lag and a biexponential rise with time constants of 0.2 and 1.5 ns. We assign these two time constants to splitting of the intradimer bonds (creating one intact thymine and one thymine anion radical T(∘-)) and electron return from T(∘-) to the FAD cofactor with recovery of the second thymine, respectively. Previous model studies and computer simulations yielded various CPD splitting times between < 1 ps and < 100 ns. Our experimental results should serve as a benchmark for future efforts to model enzymatic photorepair. The technique and methods developed here may be applied to monitor other photoreactions involving DNA.

The cryptochromes: blue light photoreceptors in plants and animals.

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.