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1.
Krauss U Losi A Gärtner W Jaeger KE Eggert T 《Physical chemistry chemical physics : PCCP》2005,7(14):2804-2811
The open reading frame PP2739 from Pseudomonas putida KT2440 encodes a 151 amino acid protein with sequence similarity to the LOV domains of the blue-light sensitive protein YtvA from Bacillus subtilis and to the phototropins (phot) from plants. This sensory box LOV protein, PpSB2-LOV, comprises a LOV core, followed by a C-terminal segment predicted to form an alpha-helix, thus constituting a naturally occurring paradigm for an extended LOV construct. The recombinant PpSB2-LOV shows a photochemistry very similar to that of YtvA and phot-LOV domains, yet the lifetime for the recovery dark reaction, taurec=114 s at 20 degrees C, resembles that of phot-LOV domains (5-300 s) and is much faster than that of YtvA or YtvA-LOV (>3000 s). Time-resolved optoacoustics reveals phot-like, light-driven reactions on the ns-micros time window with the sub-nanosecond formation of a flavin triplet state (PhiT=0.46) that decays into the flavin-cysteine photoadduct with 2 micros lifetime (Phi390=0.42). The fluorescence spectrum and lifetime of the conserved W97 resembles the corresponding W103 in full-length YtvA, although the quantum yield, PhiF, is smaller (about 55% of YtvA) due to an enhanced static quenching efficiency. The anisotropy of W97 is the same as for W103 in YtvA (0.1), and considerably larger than the value of 0.06, found for W103 in YtvA-LOV. Different to YtvA and YtvA-LOV, the fluorescence for W97 becomes larger upon photoproduct formation. These data indicate that W97 is located in a similar environment as W103 in full-length YtvA, but undergoes larger light-driven changes. It is concluded that the protein segment located C-terminally to the LOV core (analogous to an interdomain linker) is enough to confer to the conserved tryptophan the fluorescence characteristics typical of full-length YtvA. The larger changes experienced by W97 upon light activation may reflect a larger conformational freedom of this protein segment in the absence of a second domain. 相似文献
2.
Song SH Dick B Zirak P Penzkofer A Schiereis T Hegemann P 《Journal of photochemistry and photobiology. B, Biology》2005,81(1):55-65
An absorption and emission spectroscopic characterisation of the combined wild-type LOV1-LOV2 domain string (abbreviated LOV1/2) of phot from the green alga Chlamydomonas reinhardtii is carried out at pH 8. A LOV1/2-MBP fusion protein (MBP=maltose binding protein) and LOV1/2 with a His-tag at the C-terminus (LOV1/2-His) expressed in an Escherichia coli strain are investigated. Blue-light photo-excitation generates a non-fluorescent intermediate photoproduct (flavin-C(4a)-cysteinyl adduct with absorption peak at 390 nm). The photo-cycle dynamics is studied by dark-state absorption and fluorescence measurement, by following the temporal absorption and emission changes under blue and violet light exposure, and by measuring the temporal absorption and fluorescence recovery after light exposure. The fluorescence quantum yield, phi(F), of the dark adapted samples is phi(F)(LOV1/2-His) approximately 0.15 and phi(F)(LOV1/2-MBP) approximately 0.17. A bi-exponential absorption recovery after light exposure with a fast (in the several 10-s range) and a slow component (in the near 10-min range) are resolved. The quantum yield of photo-adduct formation, phi(Ad), is extracted from excitation intensity dependent absorption measurements. It decreases somewhat with rising excitation intensity. The behaviour of the combined wildtype LOV1-LOV2 double domains is compared with the behaviour of the separate LOV1 and LOV2 domains. 相似文献
3.
The Bacillus subtilis protein YtvA, related to plant phototropins (phot), binds flavin mononucleotide (FMN) within the N‐terminal light, oxygen and voltage (LOV) domain. The blue light‐triggered photocycle of YtvA and phot involves the reversible formation of a covalent photoadduct between FMN and a cysteine (cys) residue. YtvA contains a single tryptophan, W103, localized on the LOV domain and conserved in all phot‐LOV domains. In this study, we show that the fluorescence parameters of W103 in YtvA‐LOV are markedly different from those observed in the full‐length YtvA. The fluorescence quantum yields are ca 0.03 and 0.08, respectively. In YtvA‐LOV, the maximum is redshifted (ca 345 vs 335 nm) and the average fluorescence lifetime shorter (2.7 vs 4.7 ns). These data indicate that W103 is located in a site of tight contact between the two domains of YtvA. In the FMN‐cys adduct, selective excitation of W103 at 295 nm results in minimal changes of the fluorescence parameters with respect to the dark state. On 280 nm excitation, however, there is a detectable decrease in the fluorescence emitted from tyrosines, with concomitant increase in W103 fluorescence. This effect is reversible in the dark and might arise from a light‐regulated energy transfer process from a yet unidentified tyrosine to W103. 相似文献
4.
Metagenomes from various environments were screened for sequences homologous to light, oxygen, voltage (LOV)-domain proteins. LOV domains are flavin binding, blue-light (BL)-sensitive photoreceptors present in 10-15% of deposited prokaryotic genomes. The LOV domain has been selected, since BL is an ever present and sometimes harmful environmental factor for microbial communities. The majority of the metagenome material originated from the Sargasso Sea Project and from open-ocean sampling. In total, more than 40 million open reading frames were investigated for LOV-domain sequences. Most sequences were identified from aquatic material, but they were also found in metagenomes from soil and extreme environments, e.g. hypersaline ponds, acidic mine drainage or wastewater treatment facilities. A total of 578 LOV domains was assigned by three criteria: (1) the highly conserved core region, (2) the presence of minimally 14 essential amino acids and (3) a minimal length of 80 amino acids. More than three quarters of these identified genes showed a sequence divergence of more than 20% from database-deposited LOV domains from known organisms, indicating the large variation of this photoreceptor motif. The broad occurrence of LOV domains in metagenomes emphasizes their important physiological role for light-induced signal transduction, stress adaptation and survival mechanisms. 相似文献
5.
Suzuki H Okajima K Ikeuchi M Noguchi T 《Journal of the American Chemical Society》2008,130(39):12884-12885
BLUF and LOV are blue-light sensor domains that possess flavin as a common chromophore but exhibit distinct photoreactions. Ile66 located in the BLUF domain of a cyanobacterial photosensor protein, TePixD, was replaced with Cys to mimic the LOV domain. Light-induced Fourier transform infrared spectra of the I66C TePixD showed that a flavin-Cys adduct, typical of the photoinduced intermediates of LOV domains, was formed in the I66C BLUF domain. This result demonstrates that different types of flavin photoreactions can be realized in the same domain if key amino acids are properly arranged near the flavin and the domain structure itself is not a crucial factor to determine the photoreaction type. 相似文献
6.
van Stokkum IH Gauden M Crosson S van Grondelle R Moffat K Kennis JT 《Photochemistry and photobiology》2011,87(3):534-541
The phototropins are blue-light receptors that base their light-dependent action on the reversible formation of a covalent bond between a flavin mononucleotide (FMN) cofactor and a conserved cysteine in light, oxygen or voltage (LOV) domains. The primary reactions of the Avena sativa phototropin 1 LOV2 domain were investigated by means of time-resolved and low-temperature fluorescence spectroscopy. Synchroscan streak camera experiments revealed a fluorescence lifetime of 2.2 ns in LOV2. A weak long-lived component with emission intensity from 600 to 650 nm was assigned to phosphorescence from the reactive FMN triplet state. This observation allowed determination of the LOV2 triplet state energy level at physiological temperature at 16600 cm(-1). FMN dissolved in aqueous solution showed pH-dependent fluorescence lifetimes of 2.7 ns at pH 2 and 3.9-4.1 ns at pH 3-8. Here, too, a weak phosphorescence band was observed. The fluorescence quantum yield of LOV2 increased from 0.13 to 0.41 upon cooling the sample from 293 to 77 K. A pronounced phosphorescence emission around 600 nm was observed in the LOV2 domain between 77 and 120 K in the steady-state emission. 相似文献
7.
Matsuoka D Iwata T Zikihara K Kandori H Tokutomi S 《Photochemistry and photobiology》2007,83(1):122-130
Phototropin is a blue-light photoreceptor in plants that mediates phototropism, chloroplast relocation, stomata opening and leaf expansion. Phototropin molecule has two photoreceptive domains named LOV1 (light-oxygen-voltage) and LOV2 in the N-terminus and a serine/threonine kinase domain in the C-terminus, and acts as a blue light-regulated kinase. Each LOV domain binds a flavin mononucleotide as a chromophore and undergoes unique cyclic reactions upon blue-light absorption that comprises a cysteinyl-flavin adduct formation through a triplet-excited state and a successive adduct break to revert to the initial ground state. The molecular reactions underlying the photocycle are reviewed and one of the probable molecular schemes is presented. Adduct formation alters the secondary protein structure of the LOV domains. This structural change could be transferred to the linker between the kinase domain and involved in the photoregulation of the kinase activity. The structural changes as well as the oligomeric structures seem to differ between LOV1 and LOV2, which may explain the proposed roles of each domain in the photoregulation of the kinase activity. The photoregulation mechanism of phototropin kinase is reviewed and discussed in reference to the regulation mechanism of protein kinase A, which it resembles. 相似文献
8.
Kennis JT van Stokkum IH Crosson S Gauden M Moffat K van Grondelle R 《Journal of the American Chemical Society》2004,126(14):4512-4513
Light, oxygen, or voltage (LOV) domains constitute a new class of photoreceptor proteins that are sensitive to blue light through a noncovalently bound flavin chromophore. Blue-light absorption by the LOV2 domain initiates a photochemical reaction that results in formation of a long-lived covalent adduct between a cysteine and the flavin cofactor. We have applied ultrafast spectroscopy on the photoaccumulated covalent adduct state of LOV2 and find that, upon absorption of a near-UV photon by the adduct state, the covalent bond between the flavin and the cysteine is broken and the blue-light-sensitive ground state is regained on an ultrafast time scale of 100 ps. We thus demonstrate that the LOV2 domain is a reversible photochromic switch, which can be activated by blue light and deactivated by near-UV light. 相似文献
9.
Alexandre MT van Grondelle R Hellingwerf KJ Robert B Kennis JT 《Physical chemistry chemical physics : PCCP》2008,10(44):6693-6702
In LOV2, the blue-light sensitive domain of phototropin, the primary photophysical event involves intersystem crossing (ISC) from the singlet-excited state to the triplet state. The ISC rate is enhanced in LOV2 as compared to flavin mononucleotide (FMN) in solution, which likely results from a heavy-atom effect of a nearby conserved cysteine, C450. Here, we applied fluorescence line narrowing (FLN), resonance Raman (RR) and Fourier-transform infrared (FTIR) spectroscopy to investigate the electronic structure of FMN bound to Avena sativa LOV2 (AsLOV2), its C450A mutant and Adiantum LOV2 (Phy3LOV2). We demonstrate that FLN is the method of choice to obtain accurate vibrational spectra on highly fluorescent flavoproteins. The vibrational spectrum of AsLOV2-C450A showed small but significant shifts with respect to those of wild type AsLOV2 and Phy3LOV2, with a systematic down-shift of Ring I vibrations, upshifts of Ring II and III vibrations and an upshift of the C2=O mode. These trends are similar to those in FMN model systems with an electron-donating group substituted at Ring I, known to induce a quinoid character to the electronic structure of oxidized flavin. Thus, enhancement of the ISC rate in LOV2 is induced through weak electron donation by the cysteine which mixes the FMN pi-electrons with the heavy sulfur orbitals, manifesting itself in a quinoid character of the ground electronic state of oxidized FMN. The proximity of the cysteine to FMN thus not only enables formation of a covalent adduct between FMN and cysteine, but also facilitates the rapid electronic formation of the reactive FMN triplet state. 相似文献
10.
Abstract— The photoreduction of cytochrome c and the photostimulation of oxygen uptake were studied in solutions of flavin and cytochrome as a possible model system for similar photoreactions which have been observed in vivo. Light causes the photoreduction of the flavin. Under aerobic conditions the photoreduced flavin reacts with oxygen to form the superoxide anion which in turn can reduce cytochrome c. Dismutation of the superoxide anions forms hydrogen peroxide which mediates the dark oxidation of the photoreduced cytochrome. Superoxide formation and dismutation also account for the light-induced oxygen uptake. Action spectra confirm the role of flavin in the photoreduction of cytochrome c and the photostimulation of oxygen uptake. Under anaerobic conditions the photoreduced flavin reduces cytochrome c directly. In the presence of an electron donor only catalytic amounts of flavin are required. In the absence of an added electron donor flavin itself can act as the electron donor if substrate amounts are present. Azide inhibits all of these flavin-mediated photoresponses. Azide also inhibits the photoreduction of cytochrome b which occurs in the mycelium of Newospora. 相似文献
11.
Raffelberg S Mansurova M Gärtner W Losi A 《Journal of the American Chemical Society》2011,133(14):5346-5356
An extended hydrogen-bonding (HB) network stabilizes the isoalloxazine ring of the flavin mononucleotide (FMN) chromophore within the photosensing LOV domain of blue-light protein receptors, via interactions between the C(2)═O, N(3)H, C(4)═O, and N(5) groups and conserved glutamine and asparagine residues. In this work we studied the influence of the HB network on the efficiency, kinetics, and energetics of a LOV protein photocycle, involving the reversible formation of a FMN-cysteine covalent adduct. The following results were found for mutations of the conserved amino acids N94, N104, and Q123 in the Bacillus subtilis LOV protein YtvA: (i) Increased (N104D, N94D) or strongly reduced (N94A) rate of adduct formation; this latter mutation extends the lifetime of the flavin triplet state, i.e., adduct formation, more than 60-fold, from 2 μs for the wild-type (WT) protein to 129 μs. (ii) Acceleration of the overall photocycle for N94S, N94A, and Q123N, with recovery lifetimes 20, 45, and 85 times faster than for YtvA-WT, respectively. (iii) Slight modifications of FMN spectral features, correlated with the polarization of low-energy transitions. (iv) Strongly reduced (N94S) or suppressed (Q123N) structural volume changes accompanying adduct formation, as determined by optoacoustic spectroscopy. (v) Minor effects on the quantum yield, with the exception of a considerable reduction for Q123N, i.e., 0.22 vs 0.49 for YtvA-WT. The data stress the importance of the HB network in modulating the photocycle of LOV domains, while at the same time establishing a link with functional responses. 相似文献
12.
Distinctive Properties of Dark Reversion Kinetics between Two Red/Green‐Type Cyanobacteriochromes and their Application in the Photoregulation of cAMP Synthesis
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Keiji Fushimi Gen Enomoto Masahiko Ikeuchi Rei Narikawa 《Photochemistry and photobiology》2017,93(3):681-691
Cyanobacteriochromes (CBCRs) are photoreceptors that bind to a linear tetrapyrrole within a conserved cGMP‐phosphodiesterase/adenylate cyclase/FhlA (GAF) domain and exhibit reversible photoconversion. Red/green‐type CBCR GAF domains that photoconvert between red‐ (Pr) and green‐absorbing (Pg) forms occur widely in various cyanobacteria. A putative phototaxis regulator, AnPixJ, contains multiple red/green‐type CBCR GAF domains. We previously reported that AnPixJ's second domain (AnPixJg2) but not its fourth domain (AnPixJg4) shows red/green reversible photoconversion. Herein, we found that AnPixJg4 showed Pr‐to‐Pg photoconversion and rapid Pg‐to‐Pr dark reversion, whereas AnPixJg2 showed a barely detectable dark reversion. Site‐directed mutagenesis revealed the involvement of six residues in Pg stability. Replacement at the Leu294/Ile660 positions of AnPixJg2/AnPixJg4 showed the highest influence on dark reversion kinetics. AnPixJg2_DR6, wherein the six residues of AnPixJg2 were entirely replaced with those of AnPixJg4, showed a 300‐fold faster dark reversion than that of the wild type. We constructed chimeric proteins by fusing the GAF domains with adenylate cyclase catalytic regions, such as AnPixJg2‐AC, AnPixJg4‐AC and AnPixJg2_DR6‐AC. We detected successful enzymatic activation under red light for both AnPixJg2‐AC and AnPixJg2_DR6‐AC, and repression under green light for AnPixJg2‐AC and under dark incubation for AnPixJg2_DR6‐AC. These results provide platforms to develop cAMP synthetic optogenetic tools. 相似文献
13.
The blue light photoreceptor phototropin mediates crucial processes in plants leading to optimization of photosynthesis. Phototropin comprises two flavin mononucleotide-binding LOV (light-, oxygen-, or voltage-sensitive) domains. The LOV domains undergo a photocycle upon illumination, in which two intermediates have been detected by UV/Vis spectroscopy. The triplet excited state of flavin is formed and decays within a few microseconds into a photoadduct with an adjacent cysteine, which represents the signaling state of the LOV domain. For bond formation of the photoadduct, several reaction pathways have been proposed, but evidence for an intermediate at ambient conditions has not been found. Here, we performed nanosecond time-resolved UV/Vis spectroscopy on the phototropin-LOV1 domain from Chlamydomonas reinhardtii. We designed a flow cell which was used to efficiently replace the sample after each photoexcitation because the cycling time is in the order of hundreds of seconds. The comparison of difference spectra of the wild type with those of the C57S mutant that produces only the triplet excited state revealed the existence of an additional intermediate between the triplet and the adduct state. This intermediate exhibits spectral properties similar to a neutral flavin radical. This finding supports a reaction mechanism involving a neutral radical pair. 相似文献
14.
Song SH Dick B Penzkofer A Hegemann P 《Journal of photochemistry and photobiology. B, Biology》2007,87(1):37-48
The photo-excitation dynamics of the mutants LOV1-C57S and LOV2-C250S of the LOV-domains of the phototropin photoreceptor phot from the green alga Chlamydomonas reinhardtii is investigated by absorption and fluorescence studies. The LOV domains fused to a maltose binding protein (MBP) are expressed in Escherichia coli. The mutants were studied under aerobic conditions in aqueous solution at pH 8. Blue-light exposure reduced the fully oxidized flavin mononucleotide, FMN(ox), to FMN semiquinone, FMNH*, (quantum efficiency around 1%) which further reduced to FMN hydroquinone, FMN(red)H(2) or FMN(red)H(-) (quantum efficiency ca. 3 x 10(-5)). In the dark both reduced forms recovered back to the oxidized form on a minute timescale. Besides photoreduction, blue-light photo-excitation of the mutants resulted in photoproduct formation (efficiency in the 2 x 10(-4) - 10(-3) range). Photo-reaction schemes for the mutants are discussed. 相似文献
15.
Anne-Kathrin Hendrischk Julia Moldt Sebastian Walter Frühwirth Gabriele Klug 《Photochemistry and photobiology》2009,85(5):1254-1259
The facultatively phototrophic purple bacterium Rhodobacter sphaeroides 2.4.1 harbors a LOV (light, oxygen and voltage) domain protein, which shows a particular structure. LOV domains perceive blue light by a noncovalently bound flavin and transmit the signal to various coupled output domains. Proteins, that harbor a LOV core, function e.g. as phototropins or circadian clock regulators. Jα helices, which act as linker between the LOV core and the output domain, were shown to be involved in the light-dependent activation of the output domain. Like PpSB2 from Pseudomonas putida , the LOV domain protein of R. sphaeroides is not coupled to an effector domain and harbors an extended C-terminal α helix. We expressed the R. sphaeroides LOV domain recombinantly in Escherichia coli . The protein binds an FMN as a cofactor and shows a photocycle typical for LOV domain containing proteins. In R. sphaeroides , we detected the protein as well in the cytoplasm as in the membrane fraction, which was not reported for other bacterial LOV domain proteins. 相似文献
16.
Phy3-LOV2 is the chromophore domain of a blue-light photoreceptor for tropic responses of plants. FMN is noncovalently bound to phy3-LOV2, and the protein structure is characteristic of the LOV (light-oxygen-voltage) domain. Primary photoreaction is considered to be adduct formation between FMN and a cysteine, while deprotonation of the cysteine S-H group was suggested. On the basis of the infrared spectral analysis, however, we have shown that the cysteine of phy3-LOV2 is in the protonated S-H form, and not in the thiolate form in the ground state. Upon formation of S390, the S-H group disappears, presumably forming the adduct between FMN and Cys966. S390 can be trapped at 150 K, and the protein structure of S390 may not be changed drastically at 295 K. 相似文献
17.
《Angewandte Chemie (Weinheim an der Bergstrasse, Germany)》2017,129(32):9469-9473
The dark‐ and light‐adapted states of YtvA LOV domains exhibit distinct excited‐state behavior. We have employed high‐level QM(MS‐CASPT2)/MM calculations to study the photochemical reactions of the dark‐ and light‐adapted states. The photoreaction from the dark‐adapted state starts with an S1→T1 intersystem crossing followed by a triplet‐state hydrogen transfer from the thiol to the flavin moiety that produces a diradical intermediate, and a subsequent internal conversion that triggers a barrierless C−S bond formation in the S0 state. The energy profiles for these transformations are different for the four conformers of the dark‐adapted state considered. The photochemistry of the light‐adapted state does not involve the triplet state: photoexcitation to the S1 state triggers C−S bond cleavage followed by recombination in the S0 state; both these processes are essentially barrierless and thus ultrafast. The present work offers new mechanistic insights into the photoresponse of flavin‐containing blue‐light photoreceptors. 相似文献
18.
Flavin‐based photoreceptor proteins of the LOV (light, oxygen and voltage) superfamily are ubiquitous and appear to be essential blue‐light sensing systems not only in plants, algae and fungi, but also in prokaryotes, where they are represented in more than 10% of known species. Despite their broad occurrence, only in few cases LOV proteins have been correlated with important phenomena such as bacterial infectivity, selective growth patterns or/and stress responses; nevertheless these few known roles are helping us understand the multiple ways by which prokaryotes can exploit these soluble blue‐light photoreceptors. Given the large number of sequences now deposited in databases, it becomes meaningful to define a signature for bona fide LOV domains, a procedure that facilitates identification of proteins with new properties and phylogenetic analysis. The latter clearly evidences that a class of LOV proteins from alpha‐proteobacteria is the closest prokaryotic relative of eukaryotic LOV domains, whereas cyanobacterial sequences cluster with the archaeal and the other bacterial LOV domains. Distance trees built for LOV domains suggest complex evolutionary patterns, possibly involving multiple horizontal gene transfer events. Based on available data, the in vivo relevance and evolution of prokaryotic LOV is discussed. 相似文献
19.
Eisenreich W Joshi M Weber S Bacher A Fischer M 《Journal of the American Chemical Society》2008,130(41):13544-13545
Strongly spin-polarized 13C NMR lines have been observed upon photoexcitation of FMN-binding LOV domains from the blue-light receptor phototropin. Their origin can be rationalized in terms of intermediate radical-pair spin chemistry. Due to hyperfine-selective branching into singlet and triplet products of different lifetime, nuclear spin polarization builds up on nuclei that possess high electron-spin density in the radical state. By examining point-mutated LOV domains of phototropin, spin-polarized 13C NMR signals in emission arising from 13C nuclei at natural abundance in the apoprotein can be unambiguously assigned to a tryptophan residue that is located at a distance of about 14 A from the FMN cofactor and that undergoes photoinduced electron transfer to the flavin. This demonstrates the potential of photo-CIDNP in unraveling reactive intermediates in protein function. 相似文献