Ground-state heterogeneity and vibrational energy redistribution in bacterial phytochrome observed with femtosecond 2D IR spectroscopy.

Phytochromes belong to a group of photoreceptor proteins containing a covalently bound biliverdin chromophore that inter-converts between two isomeric forms upon photoexcitation. The existence and stability of the photocycle products are largely determined by the protein sequence and the presence of conserved hydrogen-bonding interactions in the vicinity of the chromophore. The vibrational signatures of biliverdin, however, are often weak and obscured under more intense protein bands, limiting spectroscopic studies of its non-transient signals. In this study, we apply isotope-labeling techniques to isolate the vibrational bands from the protein-bound chromophore of the bacterial phytochrome from Deinococcus radiodurans. We elucidate the structure and ultrafast dynamics of the chromophore with 2D infra-red (IR) spectroscopy and molecular dynamics simulations. The carbonyl stretch vibrations of the pyrrole rings show the heterogeneous distribution of hydrogen-bonding structures, which exhibit distinct ultrafast relaxation dynamics. Moreover, we resolve a previously undetected 1678 cm-1 band that is strongly coupled to the A- and D-ring of biliverdin and demonstrate the presence of complex vibrational redistribution pathways between the biliverdin modes with relaxation-assisted measurements of 2D IR cross peaks. In summary, we expect 2D IR spectroscopy to be useful in explaining how point mutations in the protein sequence affect the hydrogen-bonding structure around the chromophore and consequently its ability to photoisomerize to the light-activated states.

The three-dimensional structure of Drosophila melanogaster (6-4) photolyase at room temperature.

(6-4) photolyases are flavoproteins that belong to the photolyase/cryptochrome family. Their function is to repair DNA lesions using visible light. Here, crystal structures of Drosophila melanogaster (6-4) photolyase [Dm(6-4)photolyase] at room and cryogenic temperatures are reported. The room-temperature structure was solved to 2.27 Šresolution and was obtained by serial femtosecond crystallography (SFX) using an X-ray free-electron laser. The crystallization and preparation conditions are also reported. The cryogenic structure was solved to 1.79 Šresolution using conventional X-ray crystallography. The structures agree with each other, indicating that the structural information obtained from crystallography at cryogenic temperature also applies at room temperature. Furthermore, UV-Vis absorption spectroscopy confirms that Dm(6-4)photolyase is photoactive in the crystals, giving a green light to time-resolved SFX studies on the protein, which can reveal the structural mechanism of the photoactivated protein in DNA repair.

Ultrafast structural changes within a photosynthetic reaction centre.

Photosynthetic reaction centres harvest the energy content of sunlight by transporting electrons across an energy-transducing biological membrane. Here we use time-resolved serial femtosecond crystallography1 using an X-ray free-electron laser2 to observe light-induced structural changes in the photosynthetic reaction centre of Blastochloris viridis on a timescale of picoseconds. Structural perturbations first occur at the special pair of chlorophyll molecules of the photosynthetic reaction centre that are photo-oxidized by light. Electron transfer to the menaquinone acceptor on the opposite side of the membrane induces a movement of this cofactor together with lower amplitude protein rearrangements. These observations reveal how proteins use conformational dynamics to stabilize the charge-separation steps of electron-transfer reactions.

Observing the Structural Evolution in the Photodissociation of Diiodomethane with Femtosecond Solution X-Ray Scattering.

Resolving the structural dynamics of the initial steps of chemical reactions is challenging. We report the femtosecond time-resolved wide-angle x-ray scattering of the photodissociation of diiodomethane in cyclohexane. The data reveal with structural detail how the molecule dissociates into radicals, how the radicals collide with the solvent, and how they form the photoisomer. We extract how translational and rotational kinetic energy is dispersed into the solvent. We also find that 85% of the primary radical pairs are confined to their original solvent cage and discuss how this influences the downstream recombination reactions.

The primary structural photoresponse of phytochrome proteins captured by a femtosecond X-ray laser.

Phytochrome proteins control the growth, reproduction, and photosynthesis of plants, fungi, and bacteria. Light is detected by a bilin cofactor, but it remains elusive how this leads to activation of the protein through structural changes. We present serial femtosecond X-ray crystallographic data of the chromophore-binding domains of a bacterial phytochrome at delay times of 1 ps and 10 ps after photoexcitation. The data reveal a twist of the D-ring, which leads to partial detachment of the chromophore from the protein. Unexpectedly, the conserved so-called pyrrole water is photodissociated from the chromophore, concomitant with movement of the A-ring and a key signaling aspartate. The changes are wired together by ultrafast backbone and water movements around the chromophore, channeling them into signal transduction towards the output domains. We suggest that the observed collective changes are important for the phytochrome photoresponse, explaining the earliest steps of how plants, fungi and bacteria sense red light.

Plants adapt to the availability of light throughout their lives because it regulates so many aspects of their growth and reproduction. To detect the level of light, plant cells use proteins called phytochromes, which are also found in some bacteria and fungi. Phytochrome proteins change shape when they are exposed to red light, and this change alters the behaviour of the cell. The red light is absorbed by a molecule known as chromophore, which is connected to a region of the phytochrome called the PHY-tongue. This region undergoes one of the key structural changes that occur when the phytochrome protein absorbs light, turning from a flat sheet into a helix. Claesson, Wahlgren, Takala et al. studied the structure of a bacterial phytochrome protein almost immediately after shining a very brief flash of red light using a laser. The experiments revealed that the structure of the protein begins to change within a trillionth of a second: specifically, the chromophore twists, which disrupts its attachment to the protein, freeing the protein to change shape. Claesson, Wahlgren, Takala et al. note that this structure is likely a very short-lived intermediate state, which however triggers more changes in the overall shape change of the protein. One feature of the rearrangement is the disappearance of a particular water molecule. This molecule can be found at the core of many different phytochrome structures and interacts with several parts of the chromophore and the phytochrome protein. It is unclear why the water molecule is lost, but given how quickly this happens after the red light is applied it is likely that this disappearance is an integral part of the reshaping process. Together these events disrupt the interactions between the chromophore and the PHY-tongue, enabling the PHY-tongue to change shape and alter the structure of the phytochrome protein. Understanding and controlling this process could allow scientists to alter growth patterns in plants, such as crops or weeds.

Transient IR spectroscopy identifies key interactions and unravels new intermediates in the photocycle of a bacterial phytochrome.

Phytochromes are photosensory proteins in plants, fungi, and bacteria, which detect red- and far-red light. They undergo a transition between the resting (Pr) and photoactivated (Pfr) states. In bacterial phytochromes, the Pr-to-Pfr transition is facilitated by two intermediate states, called Lumi-R and Meta-R. The molecular structures of the protein in these states are not known and the molecular mechanism of photoconversion is not understood. Here, we apply transient infrared absorption spectroscopy to study the photocycle of the wild-type and Y263F mutant of the phytochrome from Deinococcus radiodurans (DrBphP) from nano- to milliseconds. We identify two sequentially forming Lumi-R states which differ in the local structure surrounding the carbonyl group of the biliverdin D-ring. We also find that the tyrosine at position 263 alters local structure and dynamics around the D-ring and causes an increased rate of Pfr formation. The results shed new light on the mechanism of light-signalling in phytochrome proteins.

Ultrafast Chemical Exchange Dynamics of Hydrogen Bonds Observed via Isonitrile Infrared Sensors: Implications for Biomolecular Studies.

Local probes are indispensable to study protein structure and dynamics with site-specificity. The isonitrile functional group is a highly sensitive and H-bonding interaction-specific probe. Isonitriles exhibit large spectral shifts and transition dipole moment changes upon H-bonding while being weakly affected by solvent polarity. These unique properties allow a clear separation of distinct subpopulations of interacting species and an elucidation of their ultrafast dynamics with two-dimensional infrared (2D-IR) spectroscopy. Here, we apply 2D-IR to quantify the picosecond chemical exchange dynamics of solute-solvent complexes forming between isonitrile-derivatized alanine and fluorinated ethanol, where the degree of fluorination controls their H-bond-donating ability. We show that the molecules undergo faster exchange in the presence of more acidic H-bond donors, indicating that the exchange process is primarily dependent on the nature of solvent-solvent interactions. We foresee isonitrile as a highly promising probe for studying of H-bonds dynamics in the active site of enzymes.

Excitation Power Modulates Energy-Transfer Dynamics in a Supramolecular RuII -FeII -RuII Triad.

Multichromophoric arrays are key to light harvesting in natural and artificial photosynthesis. A trinuclear, symmetric RuII -FeII -RuII triad may resemble a light-harvesting model system in which excitation energy from donor units (Ru-terpyridine fragments) is efficiently transferred to the acceptor (the Fe-terpyridine fragment). The photoinduced dynamics after simultaneous excitation of more than a single chromophoric unit (donor/acceptor) at varying excitation fluence is investigated in this contribution. Data suggests that energy transfer is decelerated if the acceptor states (on the FeII unit) are not depopulated fast enough. As a consequence, the lifetime of a high-lying excited state (centered on either of the RuII units) is prolonged. A kinetic model is suggested to account for this effect. Although the proposed model is specifically adopted to account for the experimental data reported here, it might be generalized to other situations in which multiple energy or electron donors are covalently linked to a single acceptor site, a situation of interest in contemporary artificial photosynthesis.

Tailoring Cellular Uptake and Fluorescence of Poly(2-oxazoline)-Based Nanogels.

Controlling the size and charge of nanometer-sized objects is of upmost importance for their interactions with cells. We herein present the synthesis of poly(2-oxazoline) based nanogels comprising a hydrophilic shell and an amine containing core compartment. Amine groups were cross-linked using glutaraldehyde resulting in imine based nanogels. As a drug model, amino fluorescein was covalently immobilized within the core, quenching excessive aldehyde functions. By varying the amount of cross-linker, the zeta potential and, hence, the cellular uptake could be adjusted. The fluorescence of the nanogels was found to be dependent on the cross-linking density. Finally, the hemocompatibility of the described systems was studied by hemolysis and erythrocyte aggregation assays. While cellular uptake was shown to be dependent on the zeta potential of the nanogel, no harmful effects to red blood cells was observed, rendering the present system as an interesting toolbox for the production of nanomaterials with a defined biological interaction profile.

Covalent Photosensitizer-Polyoxometalate-Catalyst Dyads for Visible-Light-Driven Hydrogen Evolution.

A general concept for the covalent linkage of coordination compounds to bipyridine-functionalized polyoxometalates is presented. The new route is used to link an iridium photosensitizer to an Anderson-type hydrogen-evolution catalyst. This covalent dyad catalyzes the visible-light-driven hydrogen evolution reaction (HER) and shows superior HER activity compared with the non-covalent reference. Hydrogen evolution is observed over periods >1 week. Spectroscopic, photophysical, and electrochemical analyses give initial insight into the stability, electronic structure, and reactivity of the dyad. The results demonstrate that the proposed linkage concept allows synergistic covalent interactions between functional coordination compounds and reactive molecular metal oxides.

Efficient Energy Transfer and Metal Coupling in Cyanide-Bridged Heterodinuclear Complexes Based on (Bipyridine)(terpyridine)ruthenium(II) and (Phenylpyridine)iridium(III) Complexes.

We report a series of cyanide-bridged, heterodinuclear iridium(III)-ruthenium(II) complexes with the generalized formula [Ir((R2)2-ppy)2(CN)(µ-CN)Ru(bpy)(tpy-R1)]PF6 (ppy = 2-phenylpyridine, bpy = 2,2'-bipyridine, and tpy = 2,2':6',2″-terpyridine). The structural, spectroscopic, and electrochemical properties were analyzed in the context of variation of the electron-withdrawing (e.g., -F, -Br, -CHO) and -donating (e.g., -Me) and extended π-conjugated groups at several positions. In total, ten dinuclear complexes and the appropriate model complexes have been prepared. The iridium(III)-based emission is almost fully quenched in these complexes, and only the ruthenium(II)-based emission is observed, which indicates an efficient energy transfer toward the Ru center. Upon oxidation of the Ru center, the fluorinated complexes 2 exhibit a broad intervalence charge-transfer transition in the near-infrared region. The complexes are assigned to a weakly coupled class II system according to the Robin-Day classification. The electronic structure was evaluated by density functional theory (DFT) and time-dependent DFT calculations to corroborate the experimental data.

Supramolecular macrocycles reversibly assembled by Te( )O chalcogen bonding.

Organic molecules with heavy main-group elements frequently form supramolecular links to electron-rich centres. One particular case of such interactions is halogen bonding. Most studies of this phenomenon have been concerned with either dimers or infinitely extended structures (polymers and lattices) but well-defined cyclic structures remain elusive. Here we present oligomeric aggregates of heterocycles that are linked by chalcogen-centered interactions and behave as genuine macrocyclic species. The molecules of 3-methyl-5-phenyl-1,2-tellurazole 2-oxide assemble a variety of supramolecular aggregates that includes cyclic tetramers and hexamers, as well as a helical polymer. In all these aggregates, the building blocks are connected by Te(…)O-N bridges. Nuclear magnetic resonance spectroscopic experiments demonstrate that the two types of annular aggregates are persistent in solution. These self-assembled structures form coordination complexes with transition-metal ions, act as fullerene receptors and host small molecules in a crystal.

Energy transfer and formation of long-lived (3)MLCT states in multimetallic complexes with extended highly conjugated bis-terpyridyl ligands.

Multimetallic complexes with extended and highly conjugated bis-2,2':6',2''-terpyridyl bridging ligands, which present building blocks for coordination polymers, are investigated with respect to their ability to act as light-harvesting antennae. The investigated species combine Ru(II)- with Os(II)- and Fe(II)-terpyridyl chromophores, the latter acting as energy sinks. Due to the extended conjugated system the ligands are able to prolong the lifetime of the (3)MLCT states compared to unsubstituted terpyridyl species by delocalization and energetic stabilization of the (3)MLCT states. This concept is applied for the first time to Fe(II) terpyridyl species and results in an exceptionally long lifetime of 23 ps for the Fe(II) (3)MLCT state. While partial energy (>80%) transfer is observed between the Ru(II) and Fe(II) centers with a time-constant of 15 ps, excitation energy is transferred completely from the Ru(II) to the Os(II) center within the first 200 fs after excitation.

New ruthenium bis(terpyridine) methanofullerene and pyrrolidinofullerene complexes: synthesis and electrochemical and photophysical properties.

A series of terpyridine (tpy) methanofullerene and pyrrolidinofullerene dyads linked via p-phenylene or p-phenyleneethynylenephenylene (PEP) units is presented. The coordination to ruthenium(II) yields donor-bridge-acceptor assemblies with different lengths. Cyclic voltammetry and UV-vis and luminescence spectroscopy are applied to study the electronic interactions between the active moieties. It is shown that, upon light excitation of the ruthenium(II)-based (1)MLCT transition, the formed (3)MLCT state is readily quenched in the presence of C60. The photoinduced dynamics have been studied by transient absorption spectroscopy, which reveals fast depopulation of the (3)MLCT (73-406 ps). As a consequence, energy transfer occurs, populating a long-lived triplet state, which could be assigned to the (3)C60* state.

Towards hydrogen evolution initiated by LED light: 2-(1H-1,2,3-Triazol-4-yl)pyridine-containing polymers as photocatalyst.

Two- and three-component polymethacrylates, featuring a 2-(1-substituted-1H-1,2,3-triazol-4-yl)pyridine-based metal complex as photosensitizer, a viologen-type electron mediator, and a triethylene glycol methyl ether as solubilizing part are synthesized by statistical reversible addition-fragmentation chain transfer (RAFT) radical polymerization allowing the construction of well-defined copolymers. Thereby, heteroleptic ruthenium(II) and iridium(III) complexes serve as charged photosensitizers. In hydrogen evolution experiments, as proof-of-concept, triethylamine is utilized as a sacrificial donor and colloidal platinum as hydrogen evolving catalyst. The macromolecules bearing heteroleptic iridium(III) complexes of the general formula [Ir(ppy)2 (trzpy)]PF6 (ppy: 2-phenylpyridine; trzpy: 2-(1-substituted-1H-1,2,3-triazol-4-yl)pyridine) and [Ir(btac)2 (trzpy)]PF6 (btac: 3-(2-benzothiazolyl)-7-(diethylamino)coumarin) are photocatalytically active producing molecular hydrogen in water upon illumination at 470 nm. By changing the cyclometalating ligand from ppy to btac, the photocatalytic performance of the copolymer as reflected in the turnover number increases by two orders of magnitude.

Hole injection dynamics from two structurally related Ru-bipyridine complexes into NiO(x) is determined by the substitution pattern of the ligands.

The dyes bis[2,2'-bipyridine][4,4'-dicarboxy-2,2'-bipyridine]ruthenium(II) dihexafluorophosphate, [Ru(bpy)2dcb](PF6)2 (Ru1), and tris[4,4'-bis(ethylcarboxy)-2,2'-bipyridine]ruthenium(II) dihexafluorophosphate, [Ru(dceb)3](PF6)2 (Ru2), attached to NiOx nanoparticle films were investigated using transient absorption and luminescence spectroscopy. In acetonitrile solution the dyes reveal very similar physical and chemical properties, i.e. both dyes exhibit comparable ground state and long-lived, broad excited state absorption. However, when immobilized onto a NiOx surface the photophysical properties of the two dyes differ significantly. For Ru1 luminescence is observed, which decays within 18 ns and ultrafast transient absorption measurements do not show qualitative differences from the photophysics of Ru1 in solution. In contrast to this the luminescence of photoexcited Ru2 on NiOx is efficiently quenched and the ultrafast transient absorption spectra reveal the formation of oxidized nickel centres overlaid by the absorption of the reduced dye Ru2 with a characteristic time-constant of 18 ps. These findings are attributed to the different localization of the initially photoexcited state in Ru1 and Ru2. Due to the inductive effect (−I) of the carboxylic groups, the lowest energy excited state in Ru1 is localized on the dicarboxy-bipyridine ligand, which is bound to the NiOx surface. In Ru2, on the other hand, the initially populated excited state is localized on the ester-substituted ligands, which are not bound to the semiconductor surface. Hence, the excess charge density that is abstracted from the Ru-ion in the metal-to-ligand charge-transfer transition is shifted away from the NiOx surface, which ultimately facilitates hole transfer into the semiconductor.

And yet they glow: thiazole based push-pull fluorophores containing nitro groups and the influence of regioisomerism.

Reported is a study on the influence of regioisomerism on the photophysical properties in 4-hydroxy-1,3-thiazole-based push-pull-chromophores/fluorophores to evaluate the molecular structure-property relationship as a basic foundation for future design strategies concerning this class of dyes. Surprisingly, the nitro groups used as acceptors do not act as a fluorescence quencher, instead the derivatives synthesized exhibit quantum yields of 37-40%. Two 4-ethoxy-1,3-thiazole derivatives which differ only in the positioning of their electron donating (methoxy) and electron withdrawing (nitro) groups have been synthesized and examined in terms of their photophysical properties, i.e. UV/Vis absorption and fluorescence emission spectra. Additionally, quantum chemical calculations have been performed to unravel the underlying fundamental transitions and to explain the experimental results.

Structure-property relationships in an iridium(III) bis(terpyridine) complex with extended conjugated side chains.

Iridium(III) bis(terpyridine) complexes are known as excellent triplet emitters with emission lifetimes in the order of microseconds. We report the homoleptic complex [IrL2](3+) (L = 4'-(4-2,5-bis(octyloxy)-4-styrylphenyl)ethinyl)phenyl)-2,2':6',2″-terpyridine) that shows no detectable phosphorescence at room temperature but shows fluorescence. Emission spectra of [IrL2](3+) depend on the excitation wavelength. The origin of this behavior is studied with the help of results from (TD-)DFT calculations and is attributed to the selective excitation of different rotamers and isomers. Femtosecond-transient absorption experiments give further support for this interpretation as the specific excited-state absorption features of Z- and E-stilbene motives can be identified.

Physicochemical analysis of ruthenium(II) sensitizers of 1,2,3-triazole-derived mesoionic carbene and cyclometalating ligands.

A series of heteroleptic bis(tridentate) ruthenium(II) complexes bearing ligands featuring 1,2,3-triazolide and 1,2,3-triazolylidene units are presented. The synthesis of the C^N^N-coordinated ruthenium(II) triazolide complex is achieved by direct C-H activation, which is enabled by the use of a 1,5-disubstituted triazole. By postcomplexation alkylation, the ruthenium(II) 1,2,3-triazolide complex can be converted to the corresponding 1,2,3-triazolylidene complex. Additionally, a ruthenium(II) complex featuring a C^N^C-coordinating bis(1,2,3-triazolylidene)pyridine ligand is prepared via transmetalation from a silver(I) triazolylidene precursor. The electronic consequences of the carbanion and mesoionic carbene donors are studied both experimentally and computationally. The presented complexes exhibit a broad absorption in the visible region as well as long lifetimes of the charge-separated excited state suggesting their application in photoredox catalysis and photovoltaics. Testing of the dyes in a conventional dye-sensitized solar cell (DSSC) generates, however, only modest power conversion efficiencies (PCEs).

Synthesis and characterization of poly(phenylacetylene)s with Ru(II) bis-terpyridine complexes in the side-chain.

An alkyne-functionalized ruthenium(II) bis-terpyridine complex is directly copolymerized with phenylacetylene by alkyne polymerization. The polymer is characterized by size-exclusion chromatography (SEC), (1) H NMR spectroscopy, cyclic voltammetry (CV) measurements, and thermal analysis. The photophysical properties of the polymer are studied by UV-vis absorption spectroscopy. In addition, spectro-electrochemical measurements are carried out. Time-resolved luminescence lifetime decay curves show an enhanced lifetime of the metal complex attached to the conjugated polymer backbone compared with the Ru(tpy)2 (2+) model complex.