Phototropins and Their LOV Domains：Versatile Plant Blue-Light Receptors

The phototropins phot1 and phot2 are plant blue-light receptors that mediate phototropism, chloroplast movements, stomatal opening, leaf expansion, the rapid Inhibition of hypocotyl growth in etiolated seedlings, and possibly solar tracking by leaves in those species in which It occurs. The phototroplns are plasma membrane-associated hydrophilic proteins with two chromophore domains （designated LOV1 and LOV2 for their resemblance to domains In other signaling proteins that detect light, oxygen, or voltage） in their Nterminal half and a classic serine/threonlne kinase domain in their C-terminal half. Both chromophore domains bind flavin mononucleotide （FMN） and both undergo light-activated formation of a covalent bond between a nearby cystelne and the C（4a） carbon of the FMN to form the signaling state. LOV2-cystelnyl adduct formation leads to the release downstream of a tightly bound amphlpathlc α-helix, a step required for activation of the klnase function. This cysteinyl adduct then slowly decays over a matter of seconds or minutes to return the photoreceptor chromophore modules to their ground state. Functional LOV2 is required for light-activated phosphorylation and for various blue-light responses mediated by the phototroplns. The function of LOV1 is still unknown, although It may serve to modulate the signal generated by LOV2. The LOV domain Is an ancient chromophore module found In a wide range of otherwise unrelated proteins In fungi and prokaryotes, the latter Including cyanobacterla, eubacterla, and archaea. Further general reviews on the phototropins are those by Celaya and Liscum （2005） and Christie and Briggs （2005）.     

A LOV story: the signaling state of the phot1 LOV2 photocycle involves chromophore-triggered protein structure relaxation, as probed by far-UV time-resolved optical rotatory dispersion spectroscopy

Light-, oxygen-, or voltage-regulated (LOV1 and LOV2) domains bind flavin mononucleotide (FMN) and activate the phototropism photoreceptors phototropin 1 (phot1) and phototropin 2 (phot2) by using energy from absorbed blue light. Upon absorption of blue light, chromophore and protein conformational changes trigger the kinase domain for subsequent autophosphorylation and presumed downstream signal transduction. To date, the light-induced photocycle of the phot1 LOV2 protein is known to involve formation of a triplet flavin mononucleotide (FMN) chromophore followed by the appearance of a FMN adduct within 4 micros [Swartz, T. E., Corchnoy, S. B., Christie, J. M., Lewis, J. W., Szundi, I., Briggs, W. R., and Bogomolni, R. A. (2001) J. Biol. Chem. 276, 36493-36500] before thermal decay back to the dark state. To probe the mechanism by which the blue light information is relayed from the chromophore to the protein, nanosecond time-resolved optical rotatory dispersion (TRORD) spectroscopy, which is a direct probe of global secondary structure, was used to study the phot1 LOV2 protein in the far-UV region. These TRORD experiments reveal a previously unobserved intermediate species (tau approximately 90 micros) that is characterized by a FMN adduct chromophore and partially unfolded secondary structure (LOV390(S2)). This intermediate appears shortly after the formation of the FMN adduct. For LOV2, formation of a long-lived species that is ready to interact with a receptor domain for downstream signaling is much faster by comparison with formation of a similar species in other light-sensing proteins.     

Mutational analysis of phototropin 1 provides insights into the mechanism underlying LOV2 signal transmission

Phototropins (phot1 and phot2) are blue light-activated serine/threonine protein kinases that elicit a variety of photoresponses in plants. Light sensing by the phototropins is mediated by two flavin mononucleotide (FMN)-binding domains, designated LOV1 and LOV2, located in the N-terminal region of the protein. Exposure to light results in the formation of a covalent adduct between the FMN chromophore and a conserved cysteine residue within the LOV domain. LOV2 photoexcitation is essential for phot1 function in Arabidopsis and is necessary to activate phot1 kinase activity through light-induced structural changes within a conserved alpha-helix situated C-terminal to LOV2. Here we have used site-directed mutagenesis to identify further amino acid residues that are important for phot1 activation by light. Mutagenesis of bacterially expressed LOV2 and full-length phot1 expressed in insect cells indicates that perturbation of the conserved salt bridge on the surface of LOV2 does not play a role in receptor activation. However, mutation of a conserved glutamine residue (Gln(575)) within LOV2, reported previously to be required to propagate structural changes at the LOV2 surface, attenuates light-induced autophosphorylation of phot1 expressed in insect cells without compromising FMN binding. These findings, in combination with double mutant analyses, indicate that Gln(575) plays an important role in coupling light-driven cysteinyl adduct formation from within LOV2 to structural changes at the LOV2 surface that lead to activation of the C-terminal kinase domain.     

Phototropin LOV domains exhibit distinct roles in regulating photoreceptor function

Phototropins (phot1 and phot2) are autophosphorylating serine/threonine kinases that function as photoreceptors for phototropism, light-induced chloroplast movement, and stomatal opening in Arabidopsis. The N-terminal region of phot1 and phot2 contains two specialized PAS domains, designated LOV1 and LOV2, which function as binding sites for the chromophore flavin mononucleotide (FMN). Both LOV1 and LOV2 undergo a self-contained photocycle, which involves the formation of a covalent adduct between the FMN chromophore and a conserved active-site cysteine residue (Cys39). Replacement of Cys39 with alanine abolishes the light-induced photochemical reaction of LOV1 and LOV2. Here we have used the Cys39Ala mutation to investigate the role of LOV1 and LOV2 in regulating phototropin function. Photochemical analysis of a bacterially expressed LOV1 + LOV2 fusion protein indicates that LOV2 functions as the predominant light-sensing domain for phot1. LOV2 also plays a major role in mediating light-dependent autophosphorylation of full-length phot1 expressed in insect cells and transgenic Arabidopsis. Moreover, photochemically active LOV2 alone in full-length phot1 is sufficient to elicit hypocotyl phototropism in transgenic Arabidopsis, whereas photochemically active LOV1 alone is not. Further photochemical and biochemical analyses also indicate that the LOV1 and LOV2 domains of phot2 exhibit distinct roles. The significance for the different roles of the phototropin LOV domains is discussed.     

The photochemistry of the light-, oxygen-, and voltage-sensitive domains in the algal blue light receptor phot

Phot proteins are blue light photoreceptors in plants and algae that mainly regulate photomovement responses. They contain two light-, oxygen-, and voltage-sensitive (LOV) domains and a serine/threonine kinase domain. Both LOV domains noncovalently bind a flavin mononucleotide (FMN) as chromophore. Upon blue light illumination, the LOV domains undergo a photocycle, transiently forming a covalent adduct of the FMN moiety with a nearby cysteine residue. The presence of two light-sensitive domains in the photoreceptor raises the question about the differences in properties and function between LOV1 and LOV2. As a model system, the photocycles of the LOV1 and LOV2 domains from phot of the green alga Chlamydomonas reinhardtii have been studied in detail, both separately and in a tandem construct. Here we give an overview about the results on the individual behavior of the domains and their interaction. Furthermore, the current status in the understanding of the role of LOV1 in phot in general is presented.     

Steric interactions stabilize the signaling state of the LOV2 domain of phototropin 1

Phototropins (phot1 and phot2) are blue light receptor kinases that control a range of photoresponses that serve to optimize the photosynthetic efficiency of plants. Light sensing by the phototropins is mediated by a repeated motif at the N-terminal region of the protein known as the LOV domain. Bacterially expressed LOV domains bind flavin mononucleotide noncovalently and are photochemically active in solution. Irradiation of the LOV domain results in the formation of a flavin-cysteinyl adduct (LOV390) which thermally relaxes back to the ground state in the dark, effectively completing a photocycle that serves as a molecular switch to control receptor kinase activity. We have employed a random mutagenesis approach to identify further amino acid residues involved in LOV-domain photochemistry. Escherichia coli colonies expressing a mutagenized population of LOV2 derived from Avena sativa (oat) phot1 were screened for variants that showed altered photochemical reactivity in response to blue light excitation. One variant showed slower rates of LOV390 formation but exhibited adduct decay times 1 order of magnitude faster than wild type. A single Ile --> Val substitution was responsible for the effects observed, which removes a single methyl group found in van der Waals contact with the cysteine sulfur involved in adduct formation. A kinetic acceleration trend was observed for adduct decay by decreasing the size of the isoleucine side chain. Our findings therefore indicate that the steric nature of this amino acid side chain contributes to stabilization of the C-S cysteinyl adduct.     

Phototropin receptor kinase activation by blue light

Phototropins (phot1 and phot2) are blue light-activated serine/threonine protein kinases that function to mediate a variety of adaptive processes that serve to optimize the photosynthetic efficiency of plants and thereby promote their growth. Light sensing by the phototropins is mediated by a repeated motif located within the N-terminal region of the protein designated the LOV domain. Although phototropins possess two LOV photosensors (LOV1 and LOV2), recent biophysical and structure-function analyses clearly indicate that the LOV2 domain plays a predominant role in regulating phototropin kinase activity owing to specific protein changes that occur in response to LOV2 photoexcitation. In particular, the central β-sheet scaffold plays a role in propagating the photochemical signal generated from within LOV2 to protein changes at the surface that are necessary for kinase activation.Key words: phototropin, LOV domain, FMN, cysteinyl adduct, amphipathic helix, receptor autophosphoryation     

FMN Binding and Photochemical Properties of Plant Putative Photoreceptors Containing Two LOV Domains,LOV/LOV Proteins

LOV domains function as blue light-sensing modules in various photoreceptors in plants, fungi, algae, and bacteria. A LOV/LOV protein (LLP) has been found from Arabidopsis thaliana (AtLLP) as a two LOV domain-containing protein. However, its function remains unknown. We isolated cDNA clones coding for an LLP homolog from tomato (Solanum lycopersicum) and two homologs from the moss Physcomitrella patens. The tomato LLP (SlLLP) contains two LOV domains (LOV1 and LOV2 domains), as in AtLLP. Most of the amino acids required for association with chromophore are conserved in both LOV domains, except that the amino acid at the position equivalent to the cysteine essential for cysteinyl adduct formation is glycine in the LOV1 domain as in AtLLP. When expressed in Escherichia coli, SlLLP binds FMN and undergoes a self-contained photocycle upon irradiation of blue light. Analyses using mutant SlLLPs revealed that SlLLP binds FMN in both LOV domains, although the LOV1 domain does not show spectral changes on irradiation. However, when Gly⁶⁶ in the LOV1 domain, which is located at the position equivalent to the essential cysteine of LOV domains, is replaced by cysteine, the mutated LOV1 domain shows light-induced spectral changes. In addition, all four LOV domains of P. patens LLPs (PpLLP1 and PpLLP2) show the typical features of LOV domains, including the reactive cysteine in each. This study shows that plants have a new LOV domain-containing protein family with the typical biochemical and photochemical properties of other LOV domain-containing proteins such as the phototropins.     

The phototropin family as photoreceptors for blue light-induced chloroplast relocation

Blue light-induced chloroplast accumulation and avoidance relocation movements are controlled by the blue light photoreceptor phototropin. The Arabidopsis thaliana genome has two phototropin genes encoding phot1 and phot2. Each of these photoreceptors contains two LOV (light oxygen and voltage) domains and a kinase domain. The LOV domains absorb blue light though an associated flavin mononucleotide chromophore, while the kinase domain is thought to be associated with signal transduction. The phototropins control not only chloroplast relocation movement, but also blue light-induced phototropic responses, leaf expansion and stomatal opening. Here I review the role of phototropin as a photoreceptor for chloroplast photorelocation movement. Electronic Publication     

Vibration spectroscopy reveals light-induced chromophore and protein structural changes in the LOV2 domain of the plant blue-light receptor phototropin 1

Phototropins (phot1 and phot2), the plant blue-light receptors for phototropism, chloroplast movement, and stomatal opening, are flavoproteins that contain two approximately 12 kDa FMN-binding domains, LOV1 and LOV2, at their N-terminus, and a serine/threonine protein kinase domain at their C-terminus. The light-activated LOV2 domain forms a metastable intermediate which has been shown to be a protein-chromophore cysteinyl adduct (Cys39) at C(4a) of FMN. This species thermally relaxes back to the ground state in the dark. We measured the light-minus-dark FTIR difference spectra for the LOV2 domain of oat phot1. These spectra show the disappearance of bands at 1580, 1550, and 1350 cm(-1) that originate from, or are strongly coupled to, the N5=C(4a) stretching vibrations, consistent with the perturbations expected upon C(4a) adduct formation. Assignment of these negative difference FTIR bands to native chromophore vibrations is based on the alignment with resonance Raman bands of FMN. Prominent positive bands include a doublet at 1516 and 1536 cm(-1) and one at 1375 and 1298 cm(-1). Normal-mode vibrational-frequency calculations for both lumiflavin and lumiflavin with a sulfur attached at the C(4a) position agree with many of the positive and negative bands observed in the difference spectra. Both calculated and experimental difference FTIR spectra for deuterium isotope substitutions at exchangeable positions in the flavin chromophore are consistent with the assignment of the above positive bands to vibrational modes involving both the newly formed tetrahedral geometry of C(4a) and the N5-H bond in the long-lived LOV2(S)(390) cysteinyl species.     

13C Isotopologue editing of FMN bound to phototropin domains

The plant blue light receptor phototropin comprises a protein kinase domain and two FMN-binding LOV domains (LOV1 and LOV2). Blue light irradiation of recombinant LOV domains is conducive to the addition of a cysteinyl thiolate group to carbon 4a of the FMN chromophore, and spontaneous cleavage of that photoadduct completes the photocycle of the receptor. The present study is based on (13)C NMR signal modulation observed after reconstitution of LOV domains of different origins with random libraries of (13)C-labeled FMN isotopologues. Using this approach, all (13)C signals of FMN bound to LOV1 and LOV2 domains of Avena sativa and to the LOV2 domain of the fern, Adiantum capillus-veneris, could be unequivocally assigned under dark and under blue light irradiation conditions. (13)C Chemical shifts of FMN are shown to be differently modulated by complexation with the LOV domains under study, indicating slight differences in the binding interactions of FMN and the apoproteins.     

Domain Swapping to Assess the Mechanistic Basis of Arabidopsis Phototropin 1 Receptor Kinase Activation and Endocytosis by Blue Light

Phototropins (phot1 and phot2) are plasma membrane–associated receptor kinases that respond specifically to blue and UV wavelengths. In addition to a C-terminal Ser/Thr kinase domain, phototropins contain two N-terminal chromophore binding LOV domains that function as photoswitches to regulate a wide range of enzymatic activities in prokaryotes and eukaryotes. Through domain swapping, we show that the photochemical properties of Arabidopsis thaliana phot1 rely on interactions between LOV1 and LOV2, which are facilitated by their intervening linker sequence. Functional analysis of domain-swap proteins supports a mechanism whereby LOV2 acts as a dark-state repressor of phot1 activity both in vitro and in vivo. Moreover, we find a photoactive role for LOV1 in arresting chloroplast accumulation at high light intensities. Unlike LOV2, LOV1 cannot operate as a dark-state repressor, resulting in constitutive receptor autophosphorylation and accelerated internalization from the plasma membrane. Coexpression of active and inactive forms of phot1 demonstrates that autophosphorylation can occur intermolecularly, independent of LOV1, via light-dependent receptor dimerization in vivo. Indeed, transphosphorylation is sufficient to promote phot1 internalization through a clathrin-dependent endocytic pathway triggered primarily by phosphorylation of Ser-851 within the kinase activation loop. The mechanistic implications of these findings in regard to light-driven receptor activation and trafficking are discussed.     

Chromophore exchange in the LOV2 domain of the plant photoreceptor phototropin1 from oat

Phototropins are a family of plant photoreceptors mediating blue light responses such as phototropism, leaf expansion, chloroplast relocation, and stomatal opening. Characteristic for phototropins are two LOV domains which, when expressed in heterologous systems, each carry a single flavin mononucleotide (FMN) chromophore. Here we describe removal of FMN from the LOV2 domain of Avena sativa using a hydrophobic matrix and successful incorporation of flavin adenine dinucleotide (FAD), riboflavin, and 5'-malonyl-riboflavin into the resulting apoprotein; 5-deaza-FMN was not incorporated under the applied conditions. The chromoproteins reconstituted with the various flavins showed absorption spectra and photocycle almost identical to those of the native LOV2 domain and that reconstituted with FMN except for the kinetics: LOV2-riboflavin and LOV2-5'-malonyl-riboflavin showed more rapid regeneration in the dark. LOV2-FAD can be hydrolyzed to LOV2-FMN with phosphodiesterase, indicating that the adenosine part extrudes from the protein. Together with the data from the X-ray structure (Crosson, S., and Moffat, K. (2001) Proc. Natl. Acad. Sci. U.S.A. 98, 2995-3000), the results allow us to decide which of the chromophore-protein interactions are essential for the reconstitution process.     

Physiological roles of the light, oxygen, or voltage domains of phototropin 1 and phototropin 2 in Arabidopsis

Phototropins (phot1 and phot2) are plant blue-light receptors that mediate phototropism, chloroplast movement, stomatal opening, rapid inhibition of growth of etiolated seedlings, and leaf expansion in Arabidopsis (Arabidopsis thaliana). Their N-terminal region contains two light, oxygen, or voltage (LOV) domains, which bind flavin mononucleotide and form a covalent adduct between a conserved cysteine and the flavin mononucleotide chromophore upon photoexcitation. The C-terminal region contains a serine/threonine kinase domain that catalyzes blue-light-activated autophosphorylation. Here, we have transformed the phot1 phot2 (phot1-5 phot2-1) double mutant with PHOT expression constructs driven by the cauliflower mosaic virus 35S promoter. These constructs encode either wild-type phototropin or phototropin with one or both LOV-domain cysteines mutated to block their photochemistry. We selected multiple lines in each of the eight resulting categories of transformants for further physiological analyses. Specifically, we investigated whether LOV1 and LOV2 serve the same or different functions for phototropism and leaf expansion. Our results show that the LOV2 domain of phot1 plays a major role in phototropism and leaf expansion, as does the LOV2 domain of phot2. No complementation of phototropism or leaf expansion was observed for the LOV1 domain of phot1. However, phot2 LOV1 was unexpectedly found to complement phototropism to a considerable level. Similarly, transformants carrying a PHOT transgene with both LOV domains inactivated developed strong curvatures toward high fluence rate blue light. However, we found that the phot2-1 mutant is leaky and produces a small level of full-length phot2 protein. In vitro experiments indicate that cross phosphorylation can occur between functional phot2 and inactivated phot1 molecules. Such a mechanism may occur in vivo and therefore account for the functional activities observed in the PHOT transgenics with both lov domains inactivated. The implications of this mechanism with respect to phototropin function are discussed.     

Photochemical and mutational analysis of the FMN-binding domains of the plant blue light receptor, phototropin

The plant photoreceptor phototropin is an autophosphorylating serine-threonine protein kinase activated by UV-A/blue light. Two domains, LOV1 and LOV2, members of the PAS domain superfamily, mediate light sensing by phototropin. Heterologous expression studies have shown that both domains function as FMN-binding sites. Although three plant blue light photoreceptors, cry1, cry2, and phototropin, have been identified to date, the photochemical reactions underlying photoactivation of these light sensors have not been described so far. Herein, we demonstrate that the LOV domains of Avena sativa phototropin undergo a self-contained photocycle characterized by a loss of blue light absorbance in response to light and a spontaneous recovery of the blue light-absorbing form in the dark. Rate constants and quantum efficiencies for the photoreactions indicate that LOV1 exhibits a lower photosensitivity than LOV2. The spectral properties of the photoproduct produced for both LOV domains are unrelated to those found for photoreduced flavins and flavoproteins, but are consistent with those of a flavin-cysteinyl adduct. Flavin-thiol adducts are generally short-lifetime reaction intermediates formed during the flavoprotein-catalyzed reduction of protein disulfides. By site-directed mutagenesis, we have identified several amino acid residues within the putative chromophore binding site of LOV1 and LOV2 that appear to be important for FMN binding and/or the photochemical reactivity. Among those is Cys39, which plays an important role in the photochemical reaction of the LOV domains. Replacement of Cys39 with Ala abolished the photochemical reactions of both LOV domains. We therefore propose that light sensing by the phototropin LOV domains occurs via the formation of a stable adduct between the FMN chromophore and Cys39.     

Primary reactions of the LOV2 domain of phototropin,a plant blue-light photoreceptor

The phototropins constitute an important class of plant photoreceptor kinases that control a range of physiological responses, including phototropism, light-directed chloroplast movement, and light-induced stomatal opening. The LOV2 domain of phototropin binds a molecule of flavin mononucleotide (FMN) and undergoes a photocycle involving light-driven covalent adduct formation between a conserved cysteine residue and the C(4a) atom of FMN. This product state promotes C-terminal kinase activation and downstream signal transduction. Here, we report the primary photophysics and photochemistry of LOV2 domains of phototropin 1 of Avena sativa (oat) and of the phy3 photoreceptor of Adiantum capillus-veneris (maidenhair fern). In agreement with earlier reports [Swartz, T. E., et al. (2001) J. Biol. Chem. 276, 36493-36500], we find that the FMN triplet state is the reactive species from which the photoreaction occurs. We demonstrate that the triplet state is the primary photoproduct in the LOV2 photocycle, generated at 60% efficiency. No spectroscopically distinguishable intermediates precede the FMN triplet on the femtosecond to nanosecond time scale, indicating that it is formed directly via intersystem crossing (ISC) from the singlet state. Our results indicate that the majority of the FMN triplets in the LOV2 domain exist in the protonated form. We propose a reaction mechanism that involves excited-state proton transfer, on the nanosecond time scale or faster, from the sulfhydryl group of the conserved cysteine to the N5 atom of FMN. This event promotes adduct formation by increasing the electrophilicity of C(4a) and subsequent nucleophilic attack by the cysteine's thiolate anion. Comparison to free FMN in solution shows that the protein environment of LOV2 increases the ISC rate of FMN by a factor of 2.4, thus improving the yield of the cysteinyl-flavin adduct and the efficiency of phototropin-mediated signaling processes.     

Intramolecular proton transfers and structural changes during the photocycle of the LOV2 domain of phototropin 1

The phototropins are a family of membrane-associated flavoproteins that function as the primary blue light receptors regulating phototropism, chloroplast movements, stomatal opening, and leaf expansion in plants. Phot1, a member of this family, contains two FMN-binding domains, LOV1 and LOV2, within the N-terminal region and a C-terminal serine-threonine protein kinase domain. Light irradiation of oat phot1 LOV2 produces a cysteinyl adduct (Cys-39) at the flavin C(4a) position, which decays thermally back to the dark state. We measured pH and isotope effects on the photocycle. Between pH 3.7 and 9.5, adduct formation showed minimal pH dependence, and adduct decay showed only slight pH dependence, indicating that the pK values of mechanistically relevant groups are outside this range. LOV2 showed a nearly 5-fold slowing of adduct formation in D(2)O relative to H(2)O, indicating that the rate-limiting step involves proton transfer(s). Light-induced changes in the far UV CD spectrum of LOV2 revealed putative protein structural perturbations. The light minus dark CD difference spectrum resembles an inverted alpha-helix spectrum, suggesting that alpha-helicity is reversibly lost upon light irradiation. Decay kinetics for CD spectral changes in the far UV region occur at the same rate as those in the visible region, indicating synchronous relaxation of protein and chromophore structures.     

Comparative investigation of the LOV1 and LOV2 domains in Adiantum phytochrome3

Phototropin (phot) is a blue-light photoreceptor for phototropic responses, relocation of chloroplasts, and stomata opening in plants. Phototropin has two chromophore-binding domains named LOV1 and LOV2 in its N-terminal half, each of which binds a flavin mononucleotide (FMN) noncovalently. The C-terminal half is a Ser/Thr kinase. A transgenic study of Arabidopsis suggested that only LOV2 domain is necessary for the kinase activity, whereas X-ray crystallographic structures of LOV1 and LOV2 domains are almost identical. These facts imply that the detailed structures and/or structural changes are different between LOV1 and LOV2 domains. In this study, we compared light-induced structural changes of the LOV1 and LOV2 domains of a phototropin, Adiantum phytochrome3 (phy3), by means of UV-visible and Fourier transform infrared (FTIR) spectroscopy. Photochemical properties of an adduct formation between FMN and a cysteine are essentially similar between phy3-LOV1 and phy3-LOV2. On the other hand, the S-H group of the reactive cysteine forms a hydrogen bond in phy3-LOV1, which is strengthened at low temperatures. This is possibly correlated with the fact that no adduct formation takes place for phy3-LOV1 at 77 K as revealed by the UV-visible absorption spectra. The most prominent difference was seen in the amide-I vibration that monitors the secondary structure of peptide backbone. Protein structural changes in phy3-LOV2 involve the regions of loops, alpha-helices, and beta-sheets, which differ significantly among various temperatures. Extended protein structural changes are probably correlated with the signal transduction activity of LOV2. In contrast, protein structural changes were very small in phy3-LOV1, and they were almost temperature independent. The photocycle of phy3-LOV1 takes 3.1 h, being more than 100 times longer than that of phy3-LOV2. These facts suggest that Adiantum phy3-LOV1 does not work for light sensing, being consistent with the previous transgenic study of Arabidopsis. It is likely that plants utilize a unique protein architecture (LOV domain) for different functions by regulating their protein structural changes.     

A base-catalyzed mechanism for dark state recovery in the Avena sativa phototropin-1 LOV2 domain

Phototropins are autophosphorylating serine/threonine kinases responsible for blue-light perception in plants; their action gives rise to phototropism, chloroplast relocation, and opening of stomatal guard cells. The kinase domain constitutes the C-terminal part of Avena sativa phototropin 1. The N-terminal part contains two light, oxygen, or voltage (LOV) sensing domains, LOV1 and LOV2; each binds a flavin mononucleotide (FMN) chromophore (lambdamax = 447 nm, termed D447) and forms the light-sensitive domains, of which LOV2 is the principal component. Blue-light absorption produces a covalent adduct between a very conserved nearby cysteine residue and the C(4a) atom of the FMN moiety via the triplet state of the flavin. The covalent adduct thermally decays to regenerate the D447 dark state, with a rate that may vary by several orders of magnitude between different species. We report that the imidazole base can act as a very efficient enhancer of the dark recovery of A. sativa phot1 LOV2 (AsLOV2) and some other well-characterized LOV domains. Imidazole accelerates the thermal decay of AsLOV2 by 3 orders of magnitude in the submolar concentration range, via a base-catalyzed mechanism involving base abstraction of the FMN N(5)-H adduct state and subsequent reprotonation of the reactive cysteine. The LOV2 crystal structure suggests that the imidazole molecules may act from a cavity located in the vicinity of the FMN, explaining its high efficiency, populated through a channel connecting the cavity to the protein surface. Use of pH titration and chemical inactivation by diethyl pyrocarbonate (DEPC) suggests that histidines located at the surface of the LOV domain act as base catalysts via an as yet unidentified H-bond network, operating at a rate of (55 s)-1 at pH 8. In addition, molecular processes other than histidine-mediated base catalysis contibute significantly to the total thermal decay rate of the adduct and operate at a rate constant of (65 s)-1, leading to a net adduct decay time constant of 30 s at pH 8.