Cellular Metabolites Enhance the Light Sensitivity of Arabidopsis Cryptochrome through Alternate Electron Transfer Pathways

Cryptochromes are blue light receptors with multiple signaling roles in plants and animals. Plant cryptochrome (cry1 and cry2) biological activity has been linked to flavin photoreduction via an electron transport chain comprising three evolutionarily conserved tryptophan residues known as the Trp triad. Recently, it has been reported that cry2 Trp triad mutants, which fail to undergo photoreduction in vitro, nonetheless show biological activity in vivo, raising the possibility of alternate signaling pathways. Here, we show that Arabidopsis thaliana cry2 proteins containing Trp triad mutations indeed undergo robust photoreduction in living cultured insect cells. UV/Vis and electron paramagnetic resonance spectroscopy resolves the discrepancy between in vivo and in vitro photochemical activity, as small metabolites, including NADPH, NADH, and ATP, were found to promote cry photoreduction even in mutants lacking the classic Trp triad electron transfer chain. These metabolites facilitate alternate electron transfer pathways and increase light-induced radical pair formation. We conclude that cryptochrome activation is consistent with a mechanism of light-induced electron transfer followed by flavin photoreduction in vivo. We further conclude that in vivo modulation by cellular compounds represents a feature of the cryptochrome signaling mechanism that has important consequences for light responsivity and activation.     

Mechanism of Photosignaling by Drosophila Cryptochrome: ROLE OF THE REDOX STATUS OF THE FLAVIN CHROMOPHORE*?

Cryptochrome (CRY) is the primary circadian photoreceptor in Drosophila. Upon light absorption, dCRY undergoes a conformational change that enables it to bind to Timeless (dTIM), as well as to two different E3 ligases that ubiquitylate dTIM and dCRY, respectively, resulting in their proteolysis and resetting the phase of the circadian rhythm. Purified dCRY contains oxidized flavin (FAD_ox), which is readily photoreduced to the anionic semiquinone through a set of 3 highly conserved Trp residues (Trp triad). The crystal structure of dCRY has revealed a fourth Trp (Trp-536) as a potential electron donor. Previously, we reported that the Trp triad played no role in photoinduced proteolysis of dCRY in Drosophila cells. Here we investigated the role of the Trp triad and Trp-536, and the redox status of the flavin on light-induced proteolysis of both dCRY and dTIM and resetting of the clock. We found that both oxidized (FAD_ox) and reduced (FAD^⨪) forms of dCRY undergo light-induced conformational change in vitro that enable dCRY to bind JET and that Trp triad and Trp-536 mutations that block known or presumed intraprotein electron transfer reactions do not affect dCRY phototransduction under bright or dim light in vivo as measured by light-induced proteolysis of dCRY and dTIM in Drosophila S2R+ cells. We conclude that both oxidized and reduced forms of dCRY are capable of photosignaling.     

Formation and function of flavin anion radical in cryptochrome 1 blue-light photoreceptor of monarch butterfly

The monarch butterfly (Danaus plexippus) cryptochrome 1 (DpCry1) belongs in the class of photosensitive insect cryptochromes. Here we purified DpCry1 expressed in a bacterial host and obtained the protein with a stoichiometric amount of the flavin cofactor in the two-electron oxidized, FAD(ox), form. Exposure of the purified protein to light converts the FAD(ox) to the FAD*(-) flavin anion radical by intraprotein electron transfer from a Trp residue in the apoenzyme. To test whether this novel photoreduction reaction is part of the DpCry1 physiological photocycle, we mutated the Trp residue that acts as the ultimate electron donor in flavin photoreduction. The mutation, W328F, blocked the photoreduction entirely but had no measurable effect on the light-induced degradation of DpCry1 in vivo. In light of this finding and the recently published action spectrum of this class of Crys, we conclude that DpCry1 and similar insect cryptochromes do not contain flavin in the FAD(ox) form in vivo and that, most likely, the [see text] photoreduction reaction is not part of the insect cryptochrome photoreaction that results in proteolytic degradation of the photopigment.     

Conformational change induced by ATP binding correlates with enhanced biological function of Arabidopsis cryptochrome

Cryptochromes are widely distributed blue light photoreceptors involved in numerous signaling functions in plants and animals. Both plant and animal-type cryptochromes are found to bind ATP and display intrinsic autokinase activity; however the functional significance of this activity remains a matter of speculation. Here we show in purified preparations of Arabidopsis cry1 that ATP binding induces conformational change independently of light and increases the amount and stability of light-induced flavin radical formation. Nucleotide binding may thereby provide a mechanism whereby light responsivity in organisms can be regulated through modulation of cryptochrome photoreceptor conformation.     

Cryptochrome blue light photoreceptors are activated through interconversion of flavin redox states

Cryptochromes are blue light-sensing photoreceptors found in plants, animals, and humans. They are known to play key roles in the regulation of the circadian clock and in development. However, despite striking structural similarities to photolyase DNA repair enzymes, cryptochromes do not repair double-stranded DNA, and their mechanism of action is unknown. Recently, a blue light-dependent intramolecular electron transfer to the excited state flavin was characterized and proposed as the primary mechanism of light activation. The resulting formation of a stable neutral flavin semiquinone intermediate enables the photoreceptor to absorb green/yellow light (500-630 nm) in addition to blue light in vitro. Here, we demonstrate that Arabidopsis cryptochrome activation by blue light can be inhibited by green light in vivo consistent with a change of the cofactor redox state. We further characterize light-dependent changes in the cryptochrome1 (cry1) protein in living cells, which match photoreduction of the purified cry1 in vitro. These experiments were performed using fluorescence absorption/emission and EPR on whole cells and thereby represent one of the few examples of the active state of a known photoreceptor being monitored in vivo. These results indicate that cry1 activation via blue light initiates formation of a flavosemiquinone signaling state that can be converted by green light to an inactive form. In summary, cryptochrome activation via flavin photoreduction is a reversible mechanism novel to blue light photoreceptors. This photocycle may have adaptive significance for sensing the quality of the light environment in multiple organisms.     

The Class III Cyclobutane Pyrimidine Dimer Photolyase Structure Reveals a New Antenna Chromophore Binding Site and Alternative Photoreduction Pathways

Photolyases are proteins with an FAD chromophore that repair UV-induced pyrimidine dimers on the DNA in a light-dependent manner. The cyclobutane pyrimidine dimer class III photolyases are structurally unknown but closely related to plant cryptochromes, which serve as blue-light photoreceptors. Here we present the crystal structure of a class III photolyase termed photolyase-related protein A (PhrA) of Agrobacterium tumefaciens at 1.67-Å resolution. PhrA contains 5,10-methenyltetrahydrofolate (MTHF) as an antenna chromophore with a unique binding site and mode. Two Trp residues play pivotal roles for stabilizing MTHF by a double π-stacking sandwich. Plant cryptochrome I forms a pocket at the same site that could accommodate MTHF or a similar molecule. The PhrA structure and mutant studies showed that electrons flow during FAD photoreduction proceeds via two Trp triads. The structural studies on PhrA give a clearer picture on the evolutionary transition from photolyase to photoreceptor.     

Exploring the possibilities for radical pair effects in cryptochrome

The ability of some animals to sense magnetic fields has long captured the human imagination. In our recent paper, we explored how radical pair effects in the protein cryptochrome may underlie the magnetic orientation sense of migratory birds. Here we explain our model and discuss its relationship to experimental results on plant cryptochromes, as well as discuss the next steps in refining our model, and explore alternate but related possibilities for modeling and understanding cryptochrome as a magnetic sensor.Key words: cryptochrome, radical pair machanism, avian orientation, magnetic field effect, Arabidopsis thaliana, avian magnetoreception, magnetic sensorThe ability of some animals to sense magnetic fields is a long-standing open problem in biology. Over the past 50 years, scientific studies have shown that a wide variety of living organisms have the ability to perceive magnetic fields and can use information from the earth''s magnetic field in orientation behavior. The best-studied example of animal magnetoreception is the case of migratory birds, who use the earth''s magnetic field, as well as a variety of other environmental cues, to find their way during migration.The two prevailing hypotheses for the mechanism of avian magnetoreception are an iron-mineral-based explanation, wherein birds use small deposits of magnetic iron minerals^1,2,12 in the base of their beaks for magnetic orientation, and a radical-pair-based explanation, in which a magnetically sensitive chemical reaction in the eye of the bird enables perception of the magnetic field via its effects on reaction products. The latter hypothesis is based on the idea that a radical pair reaction may take place in the protein cryptochrome in the retina of the bird.^3,4 Cryptochrome contains a blue-light-absorbing chromophore, flavin adenine dinucleotide (FAD); this FAD cofactor is reduced via a series of light-induced electron transfers from a chain of three tryptophans that bridge the space between FAD and the protein surface (see Fig. 1). The hypothesis explored in our paper⁴ is that a radical pair reaction takes place between FAD and the tryptophans in the photoreduction pathway which modulates the signaling activity of cryptochrome. The specifics of this idea are outlined in Figure 1.Open in a separate windowFigure 1Right: Cryptochrome internally binds the FAD cofactor and contains a three-tryptophan photoreduction pathway conserved from photolyase, consisting of Trp400, Trp377, and Trp324, with Trp400 nearest the FAD and Trp324 closest to the protein surface. After the FAD cofactor absorbs a photon, bringing it into an excited state, it is protonated from a nearby acidic residue, and then electron transfer proceeds from Trp400. At this stage, the semireduced FADH and Trp400⁺ comprise a radical pair—that is, each partner has an unpaired electron, and the spins of those electrons are in a correlated state. Cryptochrome is thought to be in its active, signaling state when the FAD cofactor is in this semireduced FADH form. An electron is then transferred from Trp377 to Trp400 and from Trp324 to Trp377, forming radical pairs FADH + Trp377⁺ and FADH + Trp324⁺ in the process. The Trp324 radical is then deprotonated. Before this final deprotonation, it is possible for the electron to back transfer from the tryptophan to FADH. If this occurs, FADH reverts to the oxidized FAD form, and cryptochrome is no longer in its active state. Left: This schematic of the electron transfer pathway in cryptochrome shows the estimated lifetimes of each of the radical pair states. The system spends most of its time in the FADH + Trp324 radical pair state. Also shown are the electron and nuclear spins on the FADH and Trp324 radicals. Each nuclear spin adds a small contribution to the local magnetic field. The unpaired electron spins are shown here in the singlet (antiparallel) state. They precess around the local magnetic field, which consists of contributions from the external field and from each of the nuclear spins, causing interconversion to the triplet (parallel) state and back again. This singlet-triplet interconversion is the basis of the radical pair effect in the following sense. Electron back-transfer from Trp324 to FADH proceeds only when the unpaired electrons on each radical are in the singlet state. Cryptochrome remains in its active state so long as this back-transfer is impeded. Therefore, singlet-triplet interconversion influences the time cryptochrome can spend in its active state, and so this magnetic-field-driven effect can alter the protein''s signaling behavior.That magnetic field effects do occur in cryptochrome is supported indirectly by experiments done by Margaret Ahmad and co-workers, as reported in their recent paper⁵ on the effects of magnetic fields on cryptochrome-dependent responses in Arabidopsis thaliana seedlings. In our paper, Magnetic Field Effects in Arabidopsis thaliana Cryptochrome-1 (4), we sought to evaluate this possibility computationally, to see whether a magnetic field effect in the FADH - tryptophan radical pair is reasonable. We found that it is possible to see a change in cryptochrome activation yield (the amount of time cryptochrome stays in its active state) of about 10%.Unfortunately, the magnetic field dependence of cryptochrome activation seen in our calculations cannot be taken as exact because of several limitations. Chief among these are that the models of the radical pair did not include all nuclei, and the hyperfine coupling constants were taken from DNA photolyase, which is a protein highly similar to cryptochrome in structure, but which does not necessarily have precisely the same hyperfine coupling for the FAD cofactor and the tryptophans in the photoreduction pathway as does cryptochrome. However, the suggested theory is general and with the knowledge of correct hyperfine coupling constants for the radical pair partners it can be used to calculate the activation yield precisely. Although it would be ideal to obtain hyperfine parameters from experiment, it is also possible to calculate the hyperfine coupling constants with advanced ab initio techniques using the Gaussian package.⁶ Our preliminary calculations of the hyperfine couplings in tryptophan radicals compare well with the values used in our paper.⁴ This sort of calculation creates the opportunity not only to refine our current picture of the radical pair mechanism in cryptochrome, but also to explore other possible radical pairs in the system.In light of work being done by Margaret Ahmad and co-workers (not yet published), it has been suggested recently that the radical pair reaction in cryptochrome may not occur between the FAD cofactor and tryptophan, but in some other radical pair within the protein. It is possible that rather than occurring in the FAD photoreduction process, the radical pair reaction actually takes place in the reoxidation reaction wherein the semireduced FADH is brought back to the oxidized FAD form. One possible radical pair in the back reaction is between FAD and an oxygen molecule which is thought to be involved in the reoxidization process. This radical pair is of particular interest because an oxygen radical would be devoid of hyperfine interactions. Such a radical pair, where one radical has no hyperfine coupling, would be consistent with studies on the effects of weak radio-frequency oscillating magnetic fields on migratory bird orientation. Thorsten Ritz and co-workers found that appropriate orientation behavior depended not only on the strength and angle of the oscillating field, but also that the minimum field strength necessary to disrupt orientation depended on the frequency of the oscillating field in a resonance-like behavior that would be predicted by just such a radical pair^7–9 (personal communication with T. Ritz).The scientific community is still a long way from a complete understanding of avian magnetoreception. The best that may be said of our understanding of it is that birds do demonstrably perceive and use magnetic field information, and that their responses to magnetic fields under different conditions—light intensity and color, magnetic field strength and presence and frequency of oscillating fields—belies a complex phenomenon which is probably the result of multiple receptors which interact in unknown ways.^10,11 However, disorientation responses to low-intensity oscillating magnetic fields are strongly suggestive of the involvement of a radical-pair mechanism, making the exploration of radical pair effects in cryptochrome a useful endeavor. Much remains to be done. Even if cryptochrome is confirmed as magnetoreceptor, it remains for biologists to determine how its signaling modulation enters into a bird''s sensory perception and ultimately its orientation behavior. Nevertheless, radical pair effects in cryptochrome seem promising as a possible source of magnetoreception in birds, and continued investigation may yet shed light on this complex behavior.     

Lifetimes of Arabidopsis cryptochrome signaling states in vivo

One crucial component in light signaling is the quantity of photoreceptor present in the active signaling state. The lifetime of the signaling state of a photoreceptor is limited because of thermal or otherwise back reversion of the chromophore to the ground state, and/or degradation of the photoreceptor in the light‐activated state. It was previously shown that the lit state of plant cryptochromes contains flavin‐neutral semiquinone, and that the half‐lives of the lit state were in the range of 3–4 min in vitro. However, it was unknown how long‐lived the signaling states of plant cryptochromes are in situ. Based on the loss of degradation of cry2 after prolonged dark incubation and loss of reversibility of photoactivated cry1 by a pulse of green light, we estimate the in vivo half‐lives of the signaling states of cry1 and cry2 to be in the range of 5 and 16 min, respectively. Based on electron paramagnetic resonance measurements, the lifetime of the Arabidopsis cry1 lit state in insect cells was found to be ~6 min, and thus very similar to the lifetime of the signaling state in planta. Thus, the signaling state lifetimes of plant cryptochromes are not, or are only moderately, stabilized in planta.     

Novel ATP-binding and autophosphorylation activity associated with Arabidopsis and human cryptochrome-1.

Cryptochromes are blue-light photoreceptors sharing sequence similarity to photolyases, a class of flavoenzymes catalyzing repair of UV-damaged DNA via electron transfer mechanisms. Despite significant amino acid sequence similarity in both catalytic and cofactor-binding domains, cryptochromes lack DNA repair functions associated with photolyases, and the molecular mechanism involved in cryptochrome signaling remains obscure. Here, we report a novel ATP binding and autophosphorylation activity associated with Arabidopsis cry1 protein purified from a baculovirus expression system. Autophosphorylation occurs on serine residue(s) and is absent in preparations of cryptochrome depleted in flavin and/or misfolded. Autophosphorylation is stimulated by light in vitro and oxidizing agents that act as flavin antagonists prevent this stimulation. Human cry1 expressed in baculovirus likewise shows ATP binding and autophosphorylation activity, suggesting this novel enzymatic activity may be important to the mechanism of action of both plant and animal cryptochromes.     

Light-induced electron transfer in Arabidopsis cryptochrome-1 correlates with in vivo function

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 light-induced flavin reduction via the tryptophan chain is the primary step in the signaling pathway of plant cryptochrome.     

Photoreduction of methemoglobin by irradiation in the near-ultraviolet region

Ferric metHb can be photoreduced to the ferrous state by direct photoexcitation in the near-ultraviolet region. In this research, we studied the mechanism and facilitating conditions for the photoreduction and the resulting restoration of O(2) binding. MetHb in phosphate-buffered saline or pure water in a CO atmosphere was photoreduced to form HbCO by illuminating the N band (365 nm), one of the porphyrin pi --> pi transitions, whereas the photoreduction did not occur in Ar, N(2), or O(2). The transient absorption spectrum exhibited the generation of deoxyHb within 30 ns in both the CO and Ar atmospheres; however, only in CO did the subsequent CO binding inhibit the back reaction. The photoreduction rate was dependent on the pH and ligand anions, showing that aquametHb in the high-spin state was predominant for the photoreduction. Axial ligand-to-metal charge-transfer (LMCT) bands overlap with the Soret and Q bands in metHb; however, the excitation of these bands showed little photoreduction, indicating that the contribution of these LMCT bands is minimal. Excitation of the N band significantly contributes to the photoreduction, and this is facilitated by the external addition of mannitol, hyaluronic acid, Trp, Tyr, etc. Especially, Trp allowed the photoreduction even in an Ar atmosphere, and the reduced Hb can be converted to HbO(2) by O(2) bubbling. One mechanism of the metHb photoreduction that is proposed on the basis of these results consists of a charge transfer from the porphyrin ring to the central ferric iron to form the porphyrin pi cation radical and ferrous iron by the N band excitation, and the contribution of the amino acid residues in the globin chain as an electron donor or an electron pathway.     

Photoreduction of the Folate Cofactor in Members of the Photolyase Family

Cryptochromes and DNA photolyases are related flavoproteins with flavin adenine dinucleotide as the common cofactor. Whereas photolyases repair DNA lesions caused by UV radiation, cryptochromes generally lack repair activity but act as UV-A/blue light photoreceptors. Two distinct electron transfer (ET) pathways have been identified in DNA photolyases. One pathway uses within its catalytic cycle, light-driven electron transfer from FADH⁻* to the DNA lesion and electron back-transfer to semireduced FADH^o after photoproduct cleavage. This cyclic ET pathway seems to be unique for the photolyase subfamily. The second ET pathway mediates photoreduction of semireduced or fully oxidized FAD via a triad of aromatic residues that is conserved in photolyases and cryptochromes. The 5,10-methenyltetrahydrofolate (5,10-methenylTHF) antenna cofactor in members of the photolyase family is bleached upon light excitation. This process has been described as photodecomposition of 5,10-methenylTHF. We show that photobleaching of 5,10-methenylTHF in Arabidopsis cry3, a member of the cryptochrome DASH family, with repair activity for cyclobutane pyrimidine dimer lesions in single-stranded DNA and in Escherichia coli photolyase results from reduction of 5,10-methenylTHF to 5,10-methyleneTHF that requires the intact tryptophan triad. Thus, a third ET pathway exists in members of the photolyase family that remained undiscovered so far.DNA photolyases and cryptochromes (cry)² form a large family of related flavoproteins with DNA repair activity and photoreceptor function, respectively. Members of this protein family were identified in all kingdoms of life and can be grouped in at least nine subclades (1). DNA photolyases repair cytotoxic and mutagenic DNA lesions that are formed during exposure of DNA to UV-B. These DNA lesions are cyclobutane pyrimidine dimers (CPDs) or pyrimidine-pyrimidone (6-4) photoproducts. According to their substrate specificity, DNA photolyases are designated as CPD photolyases or (6-4) photolyases (2). The repair of both types of DNA lesions by photolyase requires the catalytic fully reduced and anionic flavin cofactor FADH⁻ that, when photoexcited, injects an electron directly into the DNA lesion (1) as shown in Fig. 1A (electron transfer pathway 1). During extraction from the cell and purification under aerobic conditions the flavin cofactor is usually oxidized to the semireduced and eventually to the fully oxidized form. Reduction of these flavin species to FADH⁻ in vitro can be achieved by illumination of the enzyme in the presence of reducing agents such as dithiothreitol or β-mercaptoethanol. This process is named photoactivation (1). Photoactivation in vitro requires photoexcitation of the flavin and a triad of redox-active residues in the protein moiety that is highly conserved in DNA photolyases (3, 4) as shown in Fig. 1A (electron transfer pathway 2). These residues are generally tryptophans that allow transport of an electron from the protein surface to the U-shaped flavin cofactor, which is buried within the C-terminal α-helical domain (5–9). Whether the same mechanism is used by photolyase to photoreduce FAD in vivo is a matter of debate (10). Photoreduction of the flavin cofactor was also observed in cryptochrome blue/UV-A photoreceptors. However, instead of fully reduced flavin, semireduced flavin species (either anionic flavin semiquinone radical or neutral semiquinone radical) accumulate. This form of the photoreceptor is considered as the signaling state (11–14).Open in a separate windowFIGURE 1.Electron transfer pathways in cry3 and structures of folates. A, indicated are the distances of the tryptophans in the tryptophan triad (Trp-356, -409, -432) of Trp-432 to FADH⁻ and of FADH⁻ to the 5,10-methenylTHF (MTHF) cofactor in cry3. Shown are also the two established routes of electrons from FADH⁻ to the DNA lesion (Route 1) and within the tryptophan triad to FAD (Route 2). The third electron transfer pathway from FADH⁻ to 5,10-methenylTHF (Route 3) is the subject of this study. B, chemical structures of folates and their molecular masses. Folypolyglutamate molecules have a pteridin and a p-aminobenzoate moiety linked with a glutamate chain with a variable number of glutamic acids. The various THF species differ in their oxidation state of the C1 unit that is attached at the N-5 or N-10 position or form a bridge between both.A recently discovered subclade of the DNA photolyase/cryptochrome family are DASH cryptochromes, which have members in plants, bacteria, and aquatic animals (6, 15–17). Because DASH cryptochromes were found to lack repair activity for CPDs in double-stranded DNA, they were considered as cryptochrome-type photoreceptors (6, 16). However, it was recently shown that DASH cryptochromes repair CPDs in single-stranded DNA (18) and loop structures of double-stranded DNA (19) and, thus, belong to the CPD photolyase group. In contrast to conventional CPD photolyases, DASH cryptochromes are unable to flip the CPD lesion out of the DNA duplex (7).Besides the flavin cofactor that is essential for enzymatic activity, DNA photolyases and most likely all cryptochromes contain a second chromophore (1). Like the catalytic flavin, the second chromophore is non-covalently attached to the protein moiety. The majority of DNA photolyases and, as far as studied, the cryptochromes including the DASH-type like cry3 from Arabidopsis thaliana contain polyglutamated 5,10-methenyltetrahydrofolate (5,10-methenylTHF) as the second chromophore (1, 12, 17, 20, 21) (see Fig. 1B for folate structures). Several organisms like the cyanobacterium Anacystis nidulans (Synechococcus elongatus) produce deazariboflavins (7,8-didemethyl-8-hydroxy-5-deazariboflavin) and utilize them as second cofactor (22). In photolyases of thermophilic bacteria and Archaea of the genus Sulfolobus, FMN and FAD, respectively, were found as second cofactors (23, 24). The sole function of the second cofactors demonstrated at present is transfer of excitation energy to the catalytic flavin cofactor via a Förster-type mechanism. The crystal structures of DNA photolyases and DASH cryptochromes revealed that the second chromophores are located in a cleft between the N-terminal α/β domain and the C-terminal α domain (7–9). The centroid distances between the catalytic FAD and the second chomophore are in the range of 15–18 Å. The close distances and the angles between the transition dipole moments of the two cofactors are favorable for efficient energy transfer. Indeed, energy transfer efficiencies are about 70% for Escherichia coli photolyase (25), close to 100% for A. nidulans photolyase (26), and between 78% (dark-adapted) and 87% (light-adapted) for Arabidopsis cry3 (27). Although the second cofactors are not essential for catalysis (28, 29), they increase the efficiency of repair and possibly of photoactivation by having higher extinction coefficients than FADH⁻ in the near UV and blue region (30). The spectral overlap between 5,10-methenylTHF emission and the absorption of the different flavin redox states is on the order FADH^o > FAD_ox > FADH⁻ (31).Illumination in vitro of photolyase that contains fully oxidized or semireduced flavin results in light-induced absorbance changes. The decrease in absorption in the 450–470-nm region reflects a decrease in the amount of fully oxidized FAD concomitant with transient increase in absorption above 500 nm, which indicates the formation of a neutral semiquinone radical. Excitation of the 5,10-methenylTHF antenna chromophore at its absorption peak at 380 nm causes a likewise photoreduction of the catalytic FAD (1, 27, 28, 30, 31). However, irreversible bleaching of the 380-nm peak is observed under high irradiance UV-A or camera flash illumination (28, 30). This irreversible bleaching goes along with release of the folate cofactor from the protein moiety (30) and was named photodecomposition of 5,10-methenylTHF (28). However, the identity of the formed folate species remained unknown (30). In our previous spectroscopic characterization of Arabidopsis cry3, a similar bleaching of the 380-nm peak was observed (27).Here we show that a third electron transfer pathway exists in photolyase and DASH cryptochome, where the 5,10-methenylTHF cofactor is photoreduced to 5,10-methyleneTHF. Thus, bleaching at 380 nm does not simply reflect destruction but is a specific chemical conversion of the second chromophore.     

Effect of tryptophan mutation on the structure of LOV1 domain of phototropin1 protein of Ostreococcus tauri: A combined molecular dynamics simulation and biophysical approach

BackgroundLight, oxygen and voltage (LOV) proteins detect blue light by formation of a covalent ‘photoadduct’ between the flavin chromophore and the neighboring conserved cysteine residue. LOV proteins devoid of this conserved photoactive cysteine are unable to form this ‘photoadduct’ upon light illumination, but they can still elicit functional response via the formation of neutral flavin radical. Recently, tryptophan residue has been shown to be the primary electron donors to the flavin excited state.MethodsPhotoactive cysteine (Cys42) and tryptophan (Trp68) residues in the LOV1 domain of phototropin1 of Ostreococcus tauri (OtLOV1) was mutated to alanine and threonine respectively. Effect of these mutations have been studied using molecular dynamics simulation and spectroscopic techniques.ResultsMolecular dynamics simulation indicated that W68T did not affect the structure of OtLOV1 protein, but C42A leads to some structural changes. An increase in the fluorescence lifetime and quantum yield values was observed for the Trp68 mutant.ConclusionsAn increase in the fluorescence lifetime and quantum yield of Trp68 mutant compared to the wild type protein suggests that Trp68 residue participates in quenching of the flavin excited state followed by photoexcitation.General significanceEnhanced photo-physical properties of Trp68 OtLOV1 mutant might enable its use for the optogenetic and microscopic applications.     

Analysis of the role of intraprotein electron transfer in photoreactivation by DNA photolyase in vivo

Escherichia coli DNA photolyase contains FADH(-) as the catalytic cofactor. The cofactor becomes oxidized to the FADH(*) blue neutral radical during purification. The E-FADH(*) form of the enzyme is catalytically inert but can be converted to the active E-FADH(-) form by a photoreduction reaction that involves intraprotein electron transfer from Trp306. It is thought that the E-FADH(*) form is also transiently generated during pyrimidine dimer repair by photoinduced electron transfer, and it has been suggested that the FADH(*) that is generated after each round of catalysis must be photoreduced before the enzyme can engage in subsequent rounds of repair. In this study, we introduced the Trp306Phe mutation into the chromosomal gene and tested the non-photoreducible W306F mutant for photorepair in vivo. We find that both wild-type and W306F mutant photolyases carry out at least 25 rounds of photorepair at the same rate. We conclude that photoreduction by intraprotein electron transfer is not part of the photolyase photocycle under physiological conditions.     

Human and Drosophila cryptochromes are light activated by flavin photoreduction in living cells

Cryptochromes are a class of flavoprotein blue-light signaling receptors found in plants, animals, and humans that control plant development and the entrainment of circadian rhythms. In plant cryptochromes, light activation is proposed to result from photoreduction of a protein-bound flavin chromophore through intramolecular electron transfer. However, although similar in structure to plant cryptochromes, the light-response mechanism of animal cryptochromes remains entirely unknown. To complicate matters further, there is currently a debate on whether mammalian cryptochromes respond to light at all or are instead activated by non-light-dependent mechanisms. To resolve these questions, we have expressed both human and Drosophila cryptochrome proteins to high levels in living Sf21 insect cells using a baculovirus-derived expression system. Intact cells are irradiated with blue light, and the resulting cryptochrome photoconversion is monitored by fluorescence and electron paramagnetic resonance spectroscopic techniques. We demonstrate that light induces a change in the redox state of flavin bound to the receptor in both human and Drosophila cryptochromes. Photoreduction from oxidized flavin and subsequent accumulation of a semiquinone intermediate signaling state occurs by a conserved mechanism that has been previously identified for plant cryptochromes. These results provide the first evidence of how animal-type cryptochromes are activated by light in living cells. Furthermore, human cryptochrome is also shown to undergo this light response. Therefore, human cryptochromes in exposed peripheral and/or visual tissues may have novel light-sensing roles that remain to be elucidated.     

Searching for a photocycle of the cryptochrome photoreceptors

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.     

Electron-nuclear double resonance and hyperfine sublevel correlation spectroscopic studies of flavodoxin mutants from Anabaena sp. PCC 7119.

The influence of the amino acid residues surrounding the flavin ring in the flavodoxin of the cyanobacterium Anabaena PCC 7119 on the electron spin density distribution of the flavin semiquinone was examined in mutants of the key residues Trp(57) and Tyr(94) at the FMN binding site. Neutral semiquinone radicals of the proteins were obtained by photoreduction and examined by electron-nuclear double resonance (ENDOR) and hyperfine sublevel correlation (HYSCORE) spectroscopies. Significant differences in electron density distribution were observed in the flavodoxin mutants Trp(57) --> Ala and Tyr(94) --> Ala. The results indicate that the presence of a bulky residue (either aromatic or aliphatic) at position 57, as compared with an alanine, decreases the electron spin density in the nuclei of the benzene flavin ring, whereas an aromatic residue at position 94 increases the electron spin density at positions N(5) and C(6) of the flavin ring. The influence of the FMN ribityl and phosphate on the flavin semiquinone was determined by reconstituting apoflavodoxin samples with riboflavin and with lumiflavin. The coupling parameters of the different nuclei of the isoalloxazine group, as detected by ENDOR and HYSCORE, were very similar to those of the native flavodoxin. This indicates that the protein conformation around the flavin ring and the electron density distribution in the semiquinone form are not influenced by the phosphate and the ribityl of FMN.     

Effect of jasmonic acid on morphogenesis and photosynthetic pigment level in Arabidopsis seedlings grown under green light

The role of jasmonic acid (JA) in plant photomorhogenesis under green light (GL) was studied. The effect of GL of different intensity (8.1 and 18.1 W/m²) with or without 1 μM JA treatment on growth of plants and photosynthetic pigment level was compared for two types of Arabidopsis thaliana (L.) Heynh. Landsberg erecta ecotype plants: Ler, the wild type, and hy4, a mutant. A much more pronounced growth of hypocotyls and cotyledons of Ler plants in GL was observed compared to that of hy4 with suppressed cryptochrome 1 (cry1), a GL photoreceptor. Treatment with JA in GL caused retardation of hypocotyl and cotyledon growth of Ler plants; however, it stimulated their growth in hy4 plants. JA reduced the chlorophyll a and total carotenoids levels in cotyledons of Arabidopsis plants in GL. Blocked GL signal transduction due to the absence of cry1 in hy4, as well as the higher intensity of GL reduced the negative effect of exogenous JA on growth of cotyledons and photosynthetic pigments. The data obtained provide a basis for discussion of interaction between the JA and GL signals in the growth regulation controlled by cry1. Original Russian Text ? I.F. Golovatskaya, R.A. Karnachuk, 2008, published in Fiziologiya Rastenii, 2008, Vol. 55, No. 2, pp. 240–244.     

Variable Electron Transfer Pathways in an Amphibian Cryptochrome: TRYPTOPHAN VERSUS TYROSINE-BASED RADICAL PAIRS*

Electron transfer reactions play vital roles in many biological processes. Very often the transfer of charge(s) proceeds stepwise over large distances involving several amino acid residues. By using time-resolved electron paramagnetic resonance and optical spectroscopy, we have studied the mechanism of light-induced reduction of the FAD cofactor of cryptochrome/photolyase family proteins. In this study, we demonstrate that electron abstraction from a nearby amino acid by the excited FAD triggers further electron transfer steps even if the conserved chain of three tryptophans, known to be an effective electron transfer pathway in these proteins, is blocked. Furthermore, we were able to characterize this secondary electron transfer pathway and identify the amino acid partner of the resulting flavin-amino acid radical pair as a tyrosine located at the protein surface. This alternative electron transfer pathway could explain why interrupting the conserved tryptophan triad does not necessarily alter photoreactions of cryptochromes in vivo. Taken together, our results demonstrate that light-induced electron transfer is a robust property of cryptochromes and more intricate than commonly anticipated.