Equilibrium between quenched and nonquenched conformations of the major plant light-harvesting complex studied with high-pressure time-resolved fluorescence

Nonphotochemical quenching (NPQ) of chlorophyll fluorescence plays an important role in the protection of plants against excessive light. Fluorescence quenching of the major light-harvesting complex (LHCII) provides a model system to study the mechanism of NPQ. The existence of both quenched and nonquenched states of LHCII has been postulated. We used time-resolved fluorescence and hydrostatic pressure to study differences between these states. Pressure shifts the thermodynamic equilibrium between the two states. The estimated volume difference was 5 mL/mol, indicating a local conformational switch. The estimated free energy difference was 7.0 kJ/mol: high enough to keep the quenched state population low under normal conditions, but low enough to switch in a controlled way. These properties are physiologically relevant properties, because they guarantee efficient light harvesting, while at the same time maintaining the capacity to switch to a quenched state. These results indicate that conformational changes of LHCII can play an important role in NPQ.     

Theoretical investigation of the role of strongly coupled chlorophyll dimers in photoprotection of LHCII

Nonphotochemical quenching is the photoprotection mechanism by which the excess excitation energy absorbed by the light harvesting complex LHCII is dissipated through the protein scaffold as heat. Using the quenched structure of LHCII obtained from crystallographic experiments, the potential quenching of photoexcited excitons by aggregates of chlorophylls is theoretically investigated. In monomeric LHCII there is a hierarchy of length scales resulting in a hierarchy of energy scales that determine the interpigment direct Coulomb coupling. We propose a model whereby eight chlorophylls are coupled quantum mechanically into four dimers, with exciton transfer between these dimers and the remaining six single chlorophylls proceeding incoherently via Forster transfer. The chlorophyll dimer Chl a604-Chl b606 possesses a quasi-parallel geometry, resulting in a weakly dipole-allowed low-lying excited state. This weakly allowed state is accessible via exciton transfer to a higher, strongly allowed state followed by fast vibrational relaxation. This parallel, H-type aggregate can potentially function as an exciton trap. Calculated Forster transfer rates between single chlorophylls and chlorophyll dimers are used in a simulation of exciton transfer in monomeric LHCII to explore this possibility. It is found that Chl a604-Chl b606 has a short-lived enhanced population (on the time scale of approximately picoseconds), but not a long-time resident population. The fluorescence quantum yield of the model was calculated to be phi F = 0.38. Comparison of this result with phi F approximately 0.26 for unquenched LHCII in dilute solution and phi F approximately 0.06 for the highly quenched LHCII crystal reveals that the proposed model does not account for the quenching observed in the LHCII crystal. We therefore conclude that the formation of chlorophyll dimers is not the main cause of excitonic NPQ in LHCII.     

Kinetic Analysis of the Light-induced Fluorescence Quenching in Light-harvesting Chlorophyll a/b Pigment-Protein Complex of Photosystem II

We carried out a kinetic analysis of the light-induced fluorescence quenching (AF) of the light-harvesting chlorophyll a/b pigment-protein complex of photosystem II (LHCII) that was first observed by Jennings et at (Pho-tosynth. Res. 27, 57–64, 1991). We show that during a 2 min light, 2 min dark cycle, both the light and dark phases exhibit biexponential kinetics; this is tentatively explained by the presence of two types of light-induced quenchers in different domains of aggregated LHCII. Quantitative analysis could be carried out on the faster kinetic component; the slower component that was not completed during the measurement was not amenable for quantitative analysis. Our analysis revealed that the rate of the light-induced decrease of the fluorescence yield depended linearly on the light intensity, which shows that the generation of the quencher originates from a reaction that is first order with respect to the concentration of the excited domains. As shown by the estimated rate constant, pho-togeneration of the quencher is a fast reaction that can compete with other excitation-relaxation pathways. Both the decay and the recovery time constants of AF depended strongly on the temperature. Thermodynamic analysis showed that the fast light-induced decline in the fluorescence was determined by a low fraction of the excited states. Recovery was associated with large decrease in the entropy of activation that indicated the involvement of large structural rearrangements. Macroaggregated LHCII exhibited larger ΔF than small aggregates, which is consistent with the proposed role of aggregated LHCII in thy-lakoid membranes in nonphotochemical quenching.     

Different crystal morphologies lead to slightly different conformations of light-harvesting complex II as monitored by variations of the intrinsic fluorescence lifetime

In 2005, it was found that the fluorescence of crystals of the major light-harvesting complex LHCII of green plants is significantly quenched when compared to the fluorescence of isolated LHCII (A. A. Pascal et al., Nature, 2005, 436, 134-137). The Raman spectrum of crystallized LHCII was also found to be different from that of isolated LHCII but very similar to that of aggregated LHCII, which has often been considered a good model system for studying nonphotochemical quenching (NPQ), the major protection mechanism of plants against photodamage in high light. It was proposed that in the crystal LHCII adopts a similar (quenching) conformation as during NPQ and indeed similar changes in the Raman spectrum were observed during NPQ in vivo (A. V. Ruban et al., Nature, 2007, 450, 575-579). We now compared the fluorescence of various types of crystals, differing in morphology and age. Each type gave rise to its own characteristic mono-exponential fluorescence lifetime, which was 5 to 10 times shorter than that of isolated LHCII. This indicates that fluorescence is not quenched by random impurities and packing defects (as proposed recently by T. Barros et al., EMBO Journal, 2009, 28, 298-306), but that LHCII adopts a particular structure in each crystal type, that leads to fluorescence quenching. Most interestingly, the extent of quenching appears to depend on the crystal morphology, indicating that also the crystal structure depends on this crystal morphology but at the moment no data are available to correlate the crystals' structural changes to changes in fluorescence lifetime.     

Role of LHCII organization in the interaction of substituted 1,4-anthraquinones with thylakoid membranes

The chlorophyll fluorescence, photochemical activity and surface electric properties of thylakoid membranes with different stoichiometry of pigment-protein complexes and organization of the light-harvesting chlorophyll a/b protein complex of photosystem II (LHCII) were studied in the presence of substituted 1,4-anthraquinones. Data show strong dependence of the quenching of the chlorophyll fluorescence on the structural organization of LHCII. The increase of the LHCII oligomerization, which is associated with significant reduction of the transmembrane electric charge asymmetry and electric polarizability of the membrane, correlates with enhanced quenching effect of substituted 1,4-athraquinones. Crucial for the large quinone-induced changes in the membrane electric dipole moments is the structure of the quinone molecule. The strongest reduction in the values of the dipole moments is observed after interaction of thylakoids with 3-chloro-9-hydroxy-1,4-anthraquinone (TF33) which has the highest quenching efficiency. The quinone induced changes in the photochemical activity of photosystem II (PSII) correlate with the total amount of the supramolecular LHCII-PSII complex and depend on the number of substituents in the 1,4-anthraquinone molecule.     

Mimicking the role of the antenna in photosynthetic photoprotection

One mechanism used by plants to protect against damage from excess sunlight is called nonphotochemical quenching (NPQ). Triggered by low pH in the thylakoid lumen, NPQ leads to conversion of excess excitation energy in the antenna system to heat before it can initiate production of harmful chemical species by photosynthetic reaction centers. Here we report a synthetic hexad molecule that functionally mimics the role of the antenna in NPQ. When the hexad is dissolved in an organic solvent, five zinc porphyrin antenna moieties absorb light, exchange excitation energy, and ultimately decay by normal photophysical processes. Their excited-state lifetimes are long enough to permit harvesting of the excitation energy for photoinduced charge separation or other work. However, when acid is added, a pH-sensitive dye moiety is converted to a form that rapidly quenches the first excited singlet states of all five porphyrins, converting the excitation energy to heat and rendering the porphyrins kinetically incompetent to readily perform useful photochemistry.     

Magnetic field effect on fluorescence in a mixture of N-ethylcarbazole and dimethyl terephthalate in a polymer film in the presence of electric fields

Magnetic field effects on the fluorescence spectrum and on the electrofluorescence spectrum (plots of the electric field-induced change in fluorescence intensity as a function of wavelength) have been examined in electron donor and acceptor pairs of N-ethylcarbazole (ECZ) and dimethyl terephthalate (DMTP) in polymer films at different ratios of donor/acceptor concentration. In the mixture having a high concentration of ECZ, electric field-induced quenching of the exciplex fluorescence originating from the photoinduced electron transfer becomes less efficient in the presence of a magnetic field. In the mixture having a low concentration of ECZ, on the other hand, no magnetic field effect was observed in the electrofluorescence spectrum, indicating that the hole carrier plays an important role in synergy effects of magnetic and electric field effects on exciplex fluorescence. In the absence of the applied electric field, the magnetic field does not affect either exciplex fluorescence with a peak at 450 nm or LE fluorescence emitted from the locally excited state of ECZ but enhances the broad emission with a peak at approximately 380 nm, probably assigned to the fluorescence of another type of exciplex between ECZ and DMTP. Thus, two kinds of magnetic field effects on fluorescence have been observed in a mixture of ECZ and DMTP in a polymer film.     

Synthesis and Functional Reconstitution of Light‐Harvesting Complex II into Polymeric Membrane Architectures

One of most important processes in nature is the harvesting and dissipation of solar energy with the help of light‐harvesting complex II (LHCII). This protein, along with its associated pigments, is the main solar‐energy collector in higher plants. We aimed to generate stable, highly controllable, and sustainable polymer‐based membrane systems containing LHCII–pigment complexes ready for light harvesting. LHCII was produced by cell‐free protein synthesis based on wheat‐germ extract, and the successful integration of LHCII and its pigments into different membrane architectures was monitored. The unidirectionality of LHCII insertion was investigated by protease digestion assays. Fluorescence measurements indicated chlorophyll integration in the presence of LHCII in spherical as well as planar bilayer architectures. Surface plasmon enhanced fluorescence spectroscopy (SPFS) was used to reveal energy transfer from chlorophyll b to chlorophyll a, which indicates native folding of the LHCII proteins.     

Quenching of exciplex and charge-transfer complex fluorescence of poly(N-vinylcarbazole) – benzanthrone system

When benzanthrone (Bt), a weak electron acceptor, is doped into poly(N-vinylcarbazole) (PVCz) solution or film, an excited carbazole chromophore (D*) interacts with Bt to form a new exciplex state, which gives a broad fluorescence band (λ_max = 440 nm) in solution and a new state, which gives broad fluorescence (λ_max = 550 nm) in the film. In order to elucidate the origin of these new states, we have studied the results of experiments for absorption, concentration dependence of the excimer and exciplex fluorescence quenching, both in solution and in the film, and electric field-induced fluorescence quenching in the film. Taking into account that (i) the new state formation in the PVCz film containing small amounts of Bt enhances the photocurrent in the absorption region, where the photon energy is insufficient to excite polymer molecules directly into the conduction state, (ii) the 550 nm fluorescence of the PVCz - Bt system in film is only partly quenched by electric field, (iii) the appearance of structureless tail in the fluorescence excitation spectrum, the charge transfer interaction model of the PVCz - Bt system in film is proposed.     

Photophysics of untethered ZnTPP-fullerene complexes in solution

The spectroscopy and dynamic behavior of the self-assembled, Soret-excited zinc tetraphenylporphyrin (ZnTPP) plus fullerene (C(60)) model system in solution has been examined using steady state fluorescence quenching, nanosecond time-correlated single photon counting, picosecond fluorescence upconversion, and picosecond transient absorption methods. Evidence of ground state complexation is presented. Steady-state quenching of the S(2) and S(1) fluorescence of ZnTPP by C(60) reveals that the quenching processes only occur in the excited complexes, are ultrafast, and proceed at different rates in the two states. Only uncomplexed ZnTPP is observed by fluorescence lifetime methods; the locally excited complexes are either dark or, more likely, rapidly relax to products that do not radiate strongly. Both short-range (Dexter) energy transfer and electron transfer relaxation mechanisms are evaluated. Picosecond transient absorption data obtained from the subtle differences between the spectra of Soret-excited ZnTPP with and without a large excess of added C(60) reveal the formation, on a subpicosecond time scale, of relatively long-lived charge-separated species. Soret excitation of ZnTPP···C(60) does not produce a quantitative yield of species in the lower S(1) excited state.     

Bio serves nano: biological light-harvesting complex as energy donor for semiconductor quantum dots

Light-harvesting complex (LHCII) of the photosynthetic apparatus in plants is attached to type-II core-shell CdTe/CdSe/ZnS nanocrystals (quantum dots, QD) exhibiting an absorption band at 710 nm and carrying a dihydrolipoic acid coating for water solubility. LHCII stays functional upon binding to the QD surface and enhances the light utilization of the QDs significantly, similar to its light-harvesting function in photosynthesis. Electronic excitation energy transfer of about 50% efficiency is shown by donor (LHCII) fluorescence quenching as well as sensitized acceptor (QD) emission and corroborated by time-resolved fluorescence measurements. The energy transfer efficiency is commensurable with the expected efficiency calculated according to F?rster theory on the basis of the estimated donor-acceptor separation. Light harvesting is particularly efficient in the red spectral domain where QD absorption is relatively low. Excitation over the entire visible spectrum is further improved by complementing the biological pigments in LHCII with a dye attached to the apoprotein; the dye has been chosen to absorb in the "green gap" of the LHCII absorption spectrum and transfers its excitation energy ultimately to QD. This is the first report of a biological light-harvesting complex serving an inorganic semiconductor nanocrystal. Due to the charge separation between the core and the shell in type-II QDs the presented LHCII-QD hybrid complexes are potentially interesting for sensitized charge-transfer and photovoltaic applications.     

Changes in the room-temperature emission spectrum of chlorophyll during fast and slow phases of the Kautsky effect in intact leaves

Changes in the room-temperature emission spectrum of chlorophyll (Chl) were analyzed using fast diode-array recordings during the Kautsky effect in mature and in greening barley leaves. In mature leaves, the comparison of F(O) (basal level of fluorescence yield at transient O) and F(M) (maximum level of fluorescence yield at transient M) spectra showed that the relative amplitude of total variable fluorescence was maximal for the 684 nm Photosystem II (PSII) band and minimal for the 725 nm Photosystem I band. During the increase from F(O) to F(M), a progressive redshift of the spectrum of variable fluorescence occurred. This shift reflected the different fluorescence rise kinetics of different layers of chloroplasts inside the leaf. This was verified by simulating the effect of screening on the emission spectrum of isolated chloroplasts and by experiments on greening leaves with low Chl content. In addition, experiments performed at different greening stages showed that the presence of uncoupled Chl at early-greening stages and light-harvesting complex II (LHCII) at later stages have detectable but minor effects on the shape of room-temperature emission spectra. When strong actinic light was applied to mature green leaves, the slow fluorescence yield, which declined from F(M) to F(T) (steady-state level of fluorescence yield at transient T), was accompanied by a slight redshift of the 684 nm PSII band because of nonphotochemical quenching of short-wavelength-emitting Chl ascribed to LHCII.     

Designing systems for a multiple use of light signals.

The photochemical and photophysical behavior of two dendrimers consisting of a benzophenone core and branches that contain dimethoxybenzene units has been investigated. Such dendrimers can undergo a variety of photochemical and photophysical processes, namely: 1) quenching of the fluorescence and phosphorescence of the dimethoxybenzene units by energy transfer to the benzophenone core (antenna effect), 2) direct and sensitized phosphorescence (and delayed fluorescence) of the benzophenone core, 3) hydrogen abstraction by the triplet excited state of the benzophenone core from solvent molecules, 4) intramolecular hydrogen abstraction by the triplet excited state of the benzophenone core from the dendrimer branches, 5) quenching of the phosphorescence and hydrogen abstraction reaction of the benzophenone core by energy transfer to terbium ions and dioxygen; 6) conversion of the UV light absorbed by the dendrimer branches into visible (Tb3+) or near infrared (O2) emission via the sequence of processes 1) and 5). The results obtained emphasize the great potential of suitably designed dendrimers for a multiple use of light signals.     

INFLUENCE OF ELECTRIC FIELDS ON FLUORESCENCE QUENCHING OF CHLOROPHYLL a IN NEMATIC LIQUID CRYSTAL MATRIX

Abstract— An electric field enhances the yield of fluorescence of chlorophyll in a liquid crystal solvent. The presented data suggest that the electric field effect is caused by the decrease in the efficiency of fluorescence quenching by ions. Quenching of each polarized component of fluorescence of chlorophyll molecules oriented by the liquid crystal matrix is different. The relative increase in fluorescence yield due to the applied electric field is stronger for a fluorescence component polarized parallel to the direction of liquid crystal orientation than for the perpendicular component.     

外电场作用下甲基乙烯基硅酮分子结构和电子光谱

采用密度泛函B3P86方法在6-311++G(d, p)基组水平上优化得到了沿分子轴方向不同外电场(0-0.04 a.u.)作用下, 甲基乙烯基硅酮分子的基态电子状态、几何结构、电偶极矩和分子总能量. 在优化构型下利用杂化CIS-DFT方法(CIS-B3P86)研究了同样外电场条件下对甲基乙烯基硅酮的激发能和振子强度的影响. 计算结果表明, 分子几何构型与电场大小呈现强烈的依赖关系, 分子偶极矩μ随电场的增加先减小后急剧增大. 电场为零时, 分子总能量为-483.5532137 a.u., 随着电场增加, 能量升高, 在F=0.02 a.u.时达到最大值-483.5393952 a.u., 此后, 继续增大电场系统总能量则开始降低. 激发能随电场增加急剧减小, 表明在电场作用下, 分子易于激发和离解.     

Site, rate, and mechanism of photoprotective quenching in cyanobacteria

In cyanobacteria, activation of the Orange Carotenoid Protein (OCP) by intense blue-green light triggers photoprotective thermal dissipation of excess absorbed energy leading to a decrease (quenching) of fluorescence of the light harvesting phycobilisomes and, concomitantly, of the energy arriving to the reaction centers. Using spectrally resolved picosecond fluorescence, we have studied cells of wild-type Synechocystis sp. PCC 6803 and of mutants without and with extra OCP (ΔOCP and OverOCP) both in the unquenched and quenched state. With the use of target analysis, we managed to spectrally resolve seven different pigment pools in the phycobilisomes and photosystems I and II, and to determine the rates of excitation energy transfer between them. In addition, the fraction of quenched phycobilisomes and the rates of charge separation and quenching were resolved. Under our illumination conditions, ～72% of the phycobilisomes in OverOCP appeared to be substantially quenched. For wild-type cells, this number was only ～29%. It is revealed that upon OCP activation, a bilin chromophore in the core of the phycobilisome, here called APC(Q)(660), with fluorescence maximum at 660 nm becomes an effective quencher that prevents more than 80% of the excitations in the phycobilisome to reach Photosystems I and II. The quenching rate of its excited state is extremely fast, that is, at least (～240 ± 60 fs)(-1). It is concluded that the quenching is most likely caused by charge transfer between APC(Q)(660) and the OCP carotenoid hECN in its activated form.     

Excitation decay pathways of Lhca proteins: a time-resolved fluorescence study

Light-harvesting complex I (LHCI), which serves as a peripheral antenna for photosystem I (PSI) in green plants, consists mainly of four polypeptides, Lhca1-4. We report room temperature emission properties of individual reconstituted monomeric Lhca proteins (Lhca1, -2, -3, and -4) and dimeric Lhca1/4, performed by steady-state and time-resolved fluorescence techniques. The emission quantum yields of the samples are approximately 0.12, 0.085, 0.081, 0.041, and 0.063 for Lhca1, -2, -3, -4, and the -1/4 dimer, respectively, which is considerably lower than the value of 0.22 found for light-harvesting complex II (LHCII), the main peripheral antenna complex of photosystem II in green plants. The decay components of LHCI proteins can be divided in two categories: Lhca1 and Lhca3 have decay times of 1.1-1.6 ns and 3.3-3.6 ns, and Lhca2 and Lhca4 have decay times of 0.7-0.9 ns and 3.1-3.2 ns. These categories seem to correlate with the pigment composition of the samples. All decay times are faster than that observed previously for LHCII. When the absolute emission yields and the lifetimes of the Lhca samples are combined, the overall emission properties of the individual Lhca proteins are expressed in terms of their emitting dipole moment strength. In the samples without extreme red states, that is, Lhca1 and Lhca2, the emitting dipole moment has a value close to unity (relative to monomeric chlorophyll in acetone), which is similar to that for LHCII, whereas, in the samples with the red-most state (F-730), that is, Lhca3, -4, and the -1/4 dimer, the emitting dipole moment has a value less than unity (0.6-0.8), which can be explained by mixing the red-most (exciton) state with a dark charge-transfer state, as suggested in previous PSI red pigment studies. In addition, we find a lifetime component of approximately 50-150 ps in all red-pigment-containing samples, which cannot be due to "slow" energy transfer, but is instead assigned to an unrelaxed state of the pigment-protein, which, on this time-scale, is converted into the final emitting state.     

External electric field effects on state energy and photoexcitation dynamics of diphenylpolyenes

External electric field effects on state energy and photoexcitation dynamics have been examined for para-substituted and unsubstituted all-trans-diphenylpolyenes doped in a film, based on the steady-state and picosecond time-resolved measurements of the field effects on absorption and fluorescence. The substitution dependence of the electroabsorption spectra shows that the dipole moment of the substituted stilbene in the Franck-Condon excited state becomes larger with increasing difference between the Hammet constants of the substituents. Fluorescence quantum yields of 4-(dimethylamino)-4'-nitrostilbene and 4-(dimethylamino)-4'-nitrodiphenylbutadiene are markedly reduced by an electric field, suggesting that the rates of the intramolecular charge transfer (CT) from the fluorescent state to the nonradiative CT state are accelerated by an external electric field. The magnitude of the field-induced decrease in fluorescence lifetime has been evaluated. The isomerization of the unsubstituted all-trans-diphenylpolyenes to the cis forms is shown to be a significant nonradiative pathway even in a film. Field-induced quenching of their fluorescence as well as field-induced decrease in fluorescence lifetime suggests that the trans to cis photoisomerization is enhanced by an electric field.     

强外电场作用下二甲基硅酮的分子结构和激发态

The ground states of dimethyl siloxane under different intense electric fields ranging from - 0. 04 to 0. 04 a. u. are optimized using density functional theory DFT / B3P86 at 6-311 ++ G（d,p）level. The excitation energies and oscillator strengths under the same intense applied electric fields are calculated employing the revised hybrid CIS-DFT method. The result shows that the electronic state,molecular geometry,total energy,dipole moment and excitation energy are strongly dependent on the field strength and behave asymmetry to the direction of the applied electric field. As the electric field changes from - 0. 04 to 0. 04 a. u. ,the bond length of Si-O increases whereas the bond length of Si-C decreases because of the charge transfer induced by the applied electric field. The dipole moment of the ground state decreases linearly with the applied field strength. However,the dipole moment of molecule changes from positive to negative as the inverse electric field increase to - 0. 03 a. u. Further increase of the inverse electric field results in an increase of the total energy of the molecule. The dependence of the calculated excitation energies on the applied electric field strength is fitting well to the relationship proposed by Grozema. The excitation energies of the first five excited states of dimethyl siloxane decrease as the applied electric filed increases because the energy gap between the HOMO and LUMO become close with the field,which shows that the molecule is easy to be excited under electric field and hence can be easily dissociated.     

Highly Efficient Singlet–Singlet Energy Transfer in Light‐Harvesting [60,70]Fullerene–4‐Amino‐1,8‐naphthalimide Dyads

New C₆₀ and C₇₀ fullerene dyads formed with 4‐amino‐1,8‐naphthalimide chromophores have been prepared by the Bingel cyclopropanation reaction. The resulting monoadducts were investigated with respect to their fluorescence properties (quantum yields and lifetimes) to unravel the role of the charge‐transfer naphthalimide chromophore as a light‐absorbing antenna and excited‐singlet‐state sensitizer of fullerenes. The underlying intramolecular singlet–singlet energy transfer (EnT) process was fully characterized and found to proceed quantitatively (Φ_EnT≈1) for all dyads. Thus, these conjugates are of considerable interest for applications in which fullerene excited states have to be created and photonic energy loss should be minimized. In polar solvents (tetrahydrofuran and benzonitrile), fluorescence quenching of the fullerene by electron transfer from the ground‐state aminonaphthalimide was postulated as an additional path.