A search for radical intermediates in the photocycle of LOV domains

LOV domains are the light sensitive parts of phototropins and many other light-activated enzymes that regulate the response to blue light in plants and algae as well as some fungi and bacteria. Unlike all other biological photoreceptors known so far, the photocycle of LOV domains involves the excited triplet state of the chromophore. This chromophore is flavin mononucleotide (FMN) which forms a covalent adduct with a cysteine residue in the signaling state. Since the formation of this adduct from the triplet state involves breaking and forming of two bonds as well as a change from the triplet to the singlet spin state, various intermediates have been proposed, e.g. a protonated triplet state 3FMNH+, the radical anion 2FMN˙−, or the neutral semiquinone radical 2FMNH˙. We performed an extensive search for these intermediates by two-dimensional transient absorption (2D-TA) with a streak camera. However, no transient with a rate constant between the decay of fluorescence and the decay of the triplet state could be detected. Analysis of the decay associated difference spectra results in quantum yields for the formation of the adduct from the triplet of ΦA(LOV1) ≈ 0.75 and ΦA(LOV2) ≈ 0.80. This is lower than the values ΦA(LOV1) ≈ 0.95 and ΦA(LOV2) ≈ 0.99 calculated from the rate constants, giving indirect evidence of an intermediate that reacts either to form the adduct or to decay back to the ground state. Since there is no measurable delay between the decay of the triplet and the formation of the adduct, we conclude that this intermediate reacts much faster than it is formed. The LOV1-C57S mutant shows a weak and slowly decaying (τ > 100 μs) transient whose decay associated spectrum has bands at 375 and 500 nm, with a shoulder at 400 nm. This transient is insensitive to the pH change in the range 6.5–10.0 but increases on addition of β-mercaptoethanol as the reducing agent. We assign this intermediate to the radical anion which is protected from protonation by the protein. We propose that the adduct is formed via the same intermediate by combination of the radical ion pair

Combined pulsed electron double resonance EPR and molecular dynamics investigations of calmodulin suggest effects of crowding agents on protein structures

A.M.S. received Early Stage Research Funding from the European Union’s Seventh Framework Programme FP-7-PEOPLE-2013-ITN through the “MAGnetic Innovation in Catalysis” (MAGIC) Initial Training Network (grant agreement no. 606831). Part of this work was also supported by BBSRC grant: BB/M007065/1. J.L. thanks the Royal Society for a University Research Fellowship, the Carnegie Trust (RIG007510), and the Wellcome Trust for a Multi-User Equipment grant (099149/Z/12/Z).Calmodulin (CaM) is a highly dynamic Ca2+-binding protein that exhibits large conformational changes upon binding Ca2+ and target proteins. Although it is accepted that CaM exists in an equilibrium of conformational states in the absence of target protein, the physiological relevance of an elongated helical linker region in the Ca2+-replete form has been highly debated. In this study, we use PELDOR (pulsed electron–electron double resonance) EPR measurements of a doubly spin-labeled CaM variant to assess the conformational states of CaM in the apo-, Ca2+-bound, and Ca2+ plus target peptide-bound states. Our findings are consistent with a three-state conformational model of CaM, showing a semi-open apo-state, a highly extended Ca2+-replete state, and a compact target protein-bound state. Molecular dynamics simulations suggest that the presence of glycerol, and potentially other molecular crowding agents, has a profound effect on the relative stability of the different conformational states. Differing experimental conditions may explain the discrepancies in the literature regarding the observed conformational state(s) of CaM, and our PELDOR measurements show good evidence for an extended conformation of Ca2+-replete CaM similar to the one observed in early X-ray crystal structures.Publisher PDFPeer reviewe

Photochemically Induced Ring Opening of Spirocyclopropyl Oxindoles: Evidence for a Triplet 1,3‐Diradical Intermediate and Deracemization by a Chiral Sensitizer

The photochemical deracemization of spiro[cyclopropane-1,3 '-indolin]-2 '-ones (spirocyclopropyl oxindoles) was studied. The corresponding 2,2-dichloro compound is configurationally labile upon direct irradiation at lambda=350 nm and upon irradiation at lambda=405 nm in the presence of achiral thioxanthen-9-one as the sensitizer. The triplet 1,3-diradical intermediate generated in the latter reaction was detected by transient absorption spectroscopy and its lifetime determined (tau=22 mu s). Using a chiral thioxanthone or xanthone, with a lactam hydrogen bonding site as a photosensitizer, allowed the deracemization of differently substituted chiral spirocyclopropyl oxindoles with yields of 65-98 % and in 50-85 %ee(17 examples). Three mechanistic contributions were identified to co-act favorably for high enantioselectivity: the difference in binding constants to the chiral thioxanthone, the smaller molecular distance in the complex of the minor enantiomer, and the lifetime of the intermediate 1,3-diradical

Dual emissive dinuclear Pt(ii) complexes and application to singlet oxygen generation

Room-temperature dual emission consisting of spectrally separated fluorescence and phosphorescence is highly attractive as a design principle for ratiometric sensing materials, for example, for detection of dioxygen. Compounds susceptible to emission quenching by dioxygen, producing dioxygen in electronically excited states, are also used as photosensitizers for singlet oxygen generation. Combination of the dual emission behavior and efficient energy transfer from one of the emitting states (triplet state) of the dual emissive compound to molecular dioxygen can result in potent photosensitizers easily traceable by fluorescence spectroscopy, which may be advantageous for instance in biology studies. Herein, we present two Pt(II) complexes 1 and 2 of dinuclear structure which exhibit green fluorescence with sub-nanosecond lifetimes and near infrared (NIR) phosphorescence with microsecond lifetimes. Such properties are achieved via the design of a strongly pi-excessive ditopic ligand with a NC-CN coordinating mode that bridges the metal centers. The ligand centered character of the lowest excited singlet (S-1) and triplet (T-1) states leads to strong exchange interaction of the unpaired electrons and hence to large energy separation Delta E(S-1-T-1) amounting to 0.6 eV for 1 and 0.7 eV for 2, respectively. The large energy gap Delta E(S-1-T-1) and weak metal contribution to the states S-1 and T-1 results in unusually long intersystem crossing (ISC) times tau(ISC)(S-1 -> T-1) of 27.5 ps (1) and 65.2 ps (2), respectively, as determined by transient absorption spectroscopy. Owing to the slow ISC, the T-1 -> S-0 phosphorescence of both 1 and 2 is accompanied by S-1 -> S-0 fluorescence of comparable intensity. The large gap Delta E(S-1-T-1) provides also a good optical separation of the two emissions. The phosphorescence signal is efficiently quenched in the presence of dioxygen, which is manifested in both the lower relative intensity and shorter decay time of phosphorescence. Thus, the compounds show high potential as ratiometric dioxygen sensing materials. The singlet oxygen photogeneration efficiencies of complexes 1 and 2, measured in air saturated dichloromethane, are as high as phi(Delta) approximate to 0.77 +/- 0.1 and 0.57 +/- 0.1, respectively. Thus, the compounds represent efficient singlet oxygen photosensitizers

The photoinitiated reaction pathway of full-length cyanobacteriochrome Tlr0924 monitored over 12 orders of magnitude

The coupling of photochemistry to protein chemical and structural change is crucial to biological light-activated signaling mechanisms. This is typified by cyanobacteriochromes (CBCRs), members of the phytochrome superfamily of photoreceptors that exhibit a high degree of spectral diversity, collectively spanning the entire visible spectrum. CBCRs utilize a basic E/Z isomerization of the bilin chromophore as the primary step in their photocycle, which consists of reversible photoconversion between two photostates. Despite intense interest in these photoreceptors as signal transduction modules a complete description of light-activated chemical and structural changes has not been reported. The CBCR Tlr0924 contains both phycocyanobilin and phycoviolobilin chromophores, and these two species photoisomerize in parallel via spectrally and kinetically equivalent intermediates before the second step of the photoreaction where the reaction pathways diverge, the loss of a thioether linkage to a conserved cysteine residue occurs, and the phycocyanobilin reaction terminates in a red-absorbing state, whereas the phycoviolobilin reaction proceeds more rapidly to a final green-absorbing state. Here time-resolved visible transient absorption spectroscopy (femtosecond to second) has been used, in conjunction with time-resolved IR spectroscopy (femtosecond to nanosecond) and cryotrapping techniques, to follow the entire photoconversion of the blue-absorbing states to the green- and red-absorbing states of the full-length form of Tlr0924 CBCR. Our analysis shows that Tlr0924 undergoes an unprecedented long photoreaction that spans from picoseconds to seconds. We show that the thermally driven, long timescale changes are less complex than those reported for the red/far-red photocycles of the related phytochrome photoreceptors

Ligand design and nuclearity variation towards dual emissive Pt(ii) complexes for singlet oxygen generation, dual channel bioimaging, and theranostics

Organic ligands comprising thiophene ring(s) afford complexes of transition metals, such as Pt(II) and Ir(III), with photoluminescence readily tunable via ligand modifications. In this work we demonstrate the targeted design of a NC-CN type ditopic ligand with a central thiophene ring as the cyclometalating core and variation of the nuclearity of the complex to fine-tune the photophysical properties of the material. The mononuclear complex Pt-1 shows red T-1 -> S-0 phosphorescence with the emission maximum at lambda = 660 nm. The dinuclear complex Pt-2 shows near infrared (NIR) T-1 -> S-0 phosphorescence peaking at lambda = 710 nm. In both cases the phosphorescence is quenched by molecular oxygen generating singlet oxygen molecules with high efficiencies of phi(Delta) approximate to 83% (Pt-1) and phi(Delta)approximate to 70% (Pt-2, respectively) in air-equilibrated CH2Cl2 solutions under ambient conditions. The red phosphorescence of Pt-1 is accompanied by green S-1 -> S-0 fluorescence with the maximum at lambda = 495 nm. This makes Pt-1 a dual emissive material with two emissions stemming from a single chromophore moiety. Transient absorption studies revealed a relatively low rate of ISC from the S-1 state to the triplet manifold with a time constant tau(ISC) of about 4 ps. The slow ISC in Pt-1 is rationalized by a specific electronic structure with a relatively large energy gap Delta E(S-1 -> T-1) approximate to 0.63 eV and the higher triplet state T-2 being higher in energy than the singlet state S-1. In dinuclear Pt-2, state T-2 lies below S-1 opening fast T-2 -> S-1 ISC paths with a time constant tau of only approximate to 0.13 ps. The unique dual emission of Pt-1 was beneficial for its imaging in HeLa cells as it enabled switching between green fluorescence and red phosphorescence channels of detection in the time-span of the single confocal luminescence microscopy experiment. Pt-1 represents a prototype of new theranostic agents combining cytotoxic activity with a unique dual wavelength mode of detection

Design of amorphous thin films of azobenzene containing ruthenium acetylides for optical data storage

International audienceNovel photoresponsive materials based on ruthenium(II) σ-acetylides coupled to an azobenzene moiety in the main π-conjugated chain have been synthesized. The introduction of a metal acetylide fragment in the same conjugated chain as the azobenzene induces the trans-cis-trans isomerization of the azo unit, while the rate of the thermal cis → trans back isomerization increases with increasing overall electron richness of these compounds. These azobenzene-containing ruthenium(II) acetylides show satisfactory processability and give rise to spin-coated uniform thin films. Formation of surface-relief gratings on their amorphous thin films and in a PMMA polymer matrix using a picosecond pulsed laser at 532 nm results in instantaneous inscription: saturation of the first order diffraction efficiency and of the modulation amplitude of gratings were obtained in less than 1 s, while the orientation of these azodyes remains unchanged for up to 6 months

The photochemical mechanism of a B12-dependent photoreceptor protein

The coenzyme B(12)-dependent photoreceptor protein, CarH, is a bacterial transcriptional regulator that controls the biosynthesis of carotenoids in response to light. On binding of coenzyme B(12) the monomeric apoprotein forms tetramers in the dark, which bind operator DNA thus blocking transcription. Under illumination the CarH tetramer dissociates, weakening its affinity for DNA and allowing transcription. The mechanism by which this occurs is unknown. Here we describe the photochemistry in CarH that ultimately triggers tetramer dissociation; it proceeds via a cob(III)alamin intermediate, which then forms a stable adduct with the protein. This pathway is without precedent and our data suggest it is independent of the radical chemistry common to both coenzyme B(12) enzymology and its known photochemistry. It provides a mechanistic foundation for the emerging field of B(12) photobiology and will serve to inform the development of a new class of optogenetic tool for the control of gene expression

The Photoinitiated Reaction Pathway of Full-length Cyanobacteriochrome Tlr0924 Monitored Over 12 Orders of Magnitude

The coupling of photochemistry to protein chemical and structural change is crucial to biological light-activated signaling mechanisms. This is typified by cyanobacteriochromes (CBCRs), members of the phytochrome superfamily of photoreceptors that exhibit a high degree of spectral diversity, collectively spanning the entire visible spectrum. CBCRs utilize a basic E/Z isomerization of the bilin chromophore as the primary step in their photocycle, which consists of reversible photoconversion between two photostates. Despite intense interest in these photoreceptors as signal transduction modules a complete description of light-activated chemical and structural changes has not been reported. The CBCR Tlr0924 contains both phycocyanobilin and phycoviolobilin chromophores, and these two species photoisomerize in parallel via spectrally and kinetically equivalent intermediates before the second step of the photoreaction where the reaction pathways diverge, the loss of a thioether linkage to a conserved cysteine residue occurs, and the phycocyanobilin reaction terminates in a red-absorbing state, whereas the phycoviolobilin reaction proceeds more rapidly to a final green-absorbing state. Here time-resolved visible transient absorption spectroscopy (femtosecond to second) has been used, in conjunction with time-resolved IR spectroscopy (femtosecond to nanosecond) and cryotrapping techniques, to follow the entire photoconversion of the blue-absorbing states to the green- and red-absorbing states of the full-length form of Tlr0924 CBCR. Our analysis shows that Tlr0924 undergoes an unprecedented long photoreaction that spans from picoseconds to seconds. We show that the thermally driven, long timescale changes are less complex than those reported for the red/far-red photocycles of the related phytochrome photoreceptors. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc