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Incorporation of membrane proteins into reconstituted lipid membranes is a common approach for studying their structure and function relationship in a native-like environment. In this work, we investigated fluorescence properties of liposome-reconstituted major light-harvesting complexes of plants (LHCII). By utilizing liposome labelling with the fluorescent dye molecules and single-molecule microscopy techniques, we were able to study truly liposome-reconstituted LHCII and compare them with bulk measurements and liposome-free LHCII aggregates bound to the surface. Our results showed that fluorescence lifetime obtained in bulk and in single liposome measurements were correlated. The fluorescence lifetimes of LHCII were shorter for liposome-free LHCII than for reconstituted LHCII. In the case of liposome-reconstituted LHCII, fluorescence lifetime showed dependence on the protein density reminiscent to concentration quenching. The dependence of fluorescence lifetime of LHCII on the liposome size was not significant. Our results demonstrated that fluorescence quenching can be induced by LHCII - LHCII interactions in reconstituted membranes, most likely occurring via the same mechanism as photoprotective non-photochemical quenching in vivo.

Incorporation of membrane proteins into reconstituted lipid membranes is a common approach for studying their structure and function relationship in a native-like environment. In this work, we investigated fluorescence properties of liposome-reconstituted LHCII. By utilizing liposome labelling with the fluorescent dye molecules and single-molecule microscopy techniques, we were able to study truly liposome-reconstituted LHCII and compare them with bulk measurements and liposome-free LHCII aggregates on bound surface. Our results showed that fluorescence lifetime in bulk and of that for single liposome measurements were correlated. The fluorescence lifetimes of LHCII were shorter for liposome-free LHCII than for reconstituted LHCII. In the case of liposome-reconstituted LHCII, fluorescence lifetime showed dependence on the protein density reminiscent to concentration quenching. The dependence of fluorescence lifetime of LHCII on the liposome size was not significant. Our results demonstrated that fluorescence quenching can be induced by LHCII-LHCII interactions in reconstituted membranes, most likely occurring via the same mechanism as photoprotective non-photochemical quenching in vivo.

Photosystem II of higher plants is protected against light damage by thermal dissipation of excess excitation energy, a process that can be monitored through non-photochemical quenching of chlorophyll fluorescence. When the light intensity is lowered, non-photochemical quenching largely disappears on a time scale ranging from tens of seconds to many minutes. With the use of picosecond fluorescence spectroscopy, we demonstrate that one of the underlying mechanisms is only functional when the reaction centre of photosystem II is closed, that is when electron transfer is blocked and the risk of photodamage is high. This is accompanied by the appearance of a long-wavelength fluorescence band. As soon as the reaction centre reopens, this quenching, together with the long-wavelength fluorescence, disappears instantaneously. This allows plants to maintain a high level of photosynthetic efficiency even in dangerous high-light conditions.

Reconstitution of transmembrane proteins into liposomes is a widely used method to study their behavior under conditions closely resembling the natural ones. However, this approach does not allow precise control of the liposome size, reconstitution efficiency, and the actual protein-to-lipid ratio in the formed proteoliposomes, which might be critical for some applications and/or interpretation of data acquired during the spectroscopic measurements. Here, we present a novel strategy employing methods of proteoliposome preparation, fluorescent labeling, purification, and surface immobilization that allow us to quantify these properties using fluorescence microscopy at the single-liposome level and for the first time apply it to study photosynthetic pigment-protein complexes LHCII. We show that LHCII proteoliposome samples, even after purification with a density gradient, always contain a fraction of nonreconstituted protein and are extremely heterogeneous in both protein density and liposome sizes. This strategy enables quantitative analysis of the reconstitution efficiency of different protocols and precise fluorescence spectroscopic study of various transmembrane proteins in a controlled nativelike environment.

We have compared picosecond fluorescence decay kinetics for stacked and unstacked photosystem II membranes in order to evaluate the efficiency of excitation energy transfer between the neighboring layers. The measured kinetics were analyzed in terms of a recently developed fluctuating antenna model that provides information about the dimensionality of the studied system. Independently of the stacking state, all preparations exhibited virtually the same value of the apparent dimensionality, d = 1.6. Thus, we conclude that membrane stacking does not affect the efficiency of the delivery of excitation energy toward the reaction centers but ensures a more compact organization of the thylakoid membranes within the chloroplast and separation of photosystems I and II. (Figure Presented).

Complex multi-exponential fluorescence decay kinetics observed in various photosynthetic systems like photosystem II (PSII) have often been explained by the reversible quenching mechanism of the charge separation taking place in the reaction center (RC) of PSII. However, this description does not account for the intrinsic dynamic disorder of the light-harvesting proteins as well as their fluctuating dislocations within the antenna, which also facilitate the repair of RCs, state transitions, and the process of non-photochemical quenching. Since dynamic fluctuations result in varying connectivity between pigment-protein complexes, they can also lead to non-exponential excitation decay kinetics. Based on this presumption, we have recently proposed a simple conceptual model describing excitation diffusion in a continuous medium and accounting for possible variations of the excitation transfer pathways. In the current work, this model is further developed and then applied to describe fluorescence kinetics originating from very diverse antenna systems, ranging from PSII of various sizes to LHCII aggregates and even the entire thylakoid membrane. In all cases, complex multi-exponential fluorescence kinetics are perfectly reproduced on the entire relevant time scale without assuming any radical pair equilibration at the side of the excitation quencher, but using just a few parameters reflecting the mean excitation energy transfer rate as well as the overall average organization of the photosynthetic antenna.

Photosynthetic organisms are famously adaptable to environmental variances, yet the underlying physical mechanisms are seldom known. Here, we study the mechanisms that are responsible for the very large (∼50-nm) blue shift of the lowest-energy absorption band in the stressed LH3 antenna form from the photosynthetic bacterium Rhodopseudomonas acidophila compared to its position in regular LH2 antenna. By analyses of the antenna absorption and polarized fluorescence excitation spectra measured at 5 K, essential modifications of the antenna excitons have been revealed that involve both relative narrowing and shifting of the exciton band.

The role of individual photosynthetic antenna complexes of Photosystem II (PSII) both in membrane organization and excitation energy transfer have been investigated. Thylakoid membranes from wild-type Arabidopsis thaliana, and three mutants lacking light-harvesting complexes CP24, CP26, or CP29, respectively, were studied by picosecond-fluorescence spectroscopy. By using different excitation/detection wavelength combinations it was possible for the first time, to our knowledge, to separate PSI and PSII fluorescence kinetics. The sub-100 ps component, previously ascribed entirely to PSI, turns out to be due partly to PSII. Moreover, the migration time of excitations from antenna to PSII reaction center (RC) was determined for the first time, to our knowledge, for thylakoid membranes. It is four times longer than for PSII-only membranes, due to additional antenna complexes, which are less well connected to the RC. The results in the absence of CP26 are very similar to those of wild-type, demonstrating that the PSII organization is not disturbed. However, the kinetics in the absence of CP29 and, especially, of CP24 show that a large fraction of the light-harvesting complexes becomes badly connected to the RCs. Interestingly, the excited-state lifetimes of the disconnected light-harvesting complexes seem to be substantially quenched.

Spectral characteristics of the optically excited states in the ring-shaped quasi-one-dimensional aggregates comprising 18 and 32 tightly coupled bacteriochlorophyll a molecules have been investigated using selective spectroscopy methods and theoretical modelling of the data. Distinguished by the lowest electronic transition energies in the LH2 and LH1 antenna complexes these aggregates govern the functionally important ultrafast funneling of solar excitation energy in the photosynthetic membranes of purple bacteria. It was found by using a sophisticated differential fluorescence line narrowing method that exciton–phonon coupling in terms of the dimensionless Huang–Rhys factor is strong in these systems, justifying an excitonic polaron theoretical approach for the data analysis. Although we reached this qualitative conclusion already previously, in this work essential dependence of the exciton–phonon coupling strength and reorganization energy on excitation wavelength as well as on excitation light fluence has been established. We then show that these results corroborate with the properties of excitonic polarons in diagonally disordered ensembles of the aggregates. Furthermore, the weighted density of states of the phonon modes, which is an important characteristic of dynamical systems interacting with their surroundings, was derived. Its shape, being similar for all studied circular aggregates, deviates significantly from a reference profile describing local response of a protein to the Qy electronic transition in a single bacteriochlorophyll a molecule. Similarities of the data for regular and B800 deficient mutant LH2 complexes indicate that the B800 pigments have no direct influence on the electronic states of the B850 aggregate system. Consistent set of model parameters was determined, unambiguously implying that excitonic polarons, rather than bare excitons are proper lowest-energy optical excitations in the LH1 and LH2 antenna complexes.

We have characterized the influence of the protein environment on the spectral properties of the bacteriochlorophyll (Bchl) molecules of the peripheral light-harvesting (or LH2) complex from Rhodobacter sphaeroides . The spectral density functions of the pigments responsible for the 800 and 850nm electronic transitions were determined from the temperature dependence of the Bchl absorption spectra in different environments (detergent micelles and native membranes). The spectral density function is virtually independent of the hydrophobic support that the protein experiences. The reorganization energy for the B850 Bchls is 220cm −1 , which is almost twice that of the B800 Bchls, and its Huang-Rhys factor reaches 8.4. Around the transition point temperature, and at higher temperatures, both the static spectral inhomogeneity and the resonance interactions become temperature-dependent. The inhomogeneous distribution function of the transitions exhibits less temperature dependence when LH2 is embedded in membranes, suggesting that the lipid phase protects the protein. However, the temperature dependence of the fluorescence spectra of LH2 cannot be fitted using the same parameters determined from the analysis of the absorption spectra. Correct fitting requires the lowest exciton states to be additionally shifted to the red, suggesting the reorganization of the exciton spectrum.

We have performed time-resolved fluorescence measurements on photosystem II (PSII) containing membranes (BBY particles) from spinach with open reaction centers. The decay kinetics can be fitted with two main decay components with an average decay time of 150 ps. Comparison with recent kinetic exciton annihilation data on the major light-harvesting complex of PSII (LHCII) suggests that excitation diffusion within the antenna contributes significantly to the overall charge separation time in PSII, which disagrees with previously proposed trap-limited models. To establish to which extent excitation diffusion contributes to the overall charge separation time, we propose a simple coarse-grained method, based on the supramolecular organization of PSII and LHCII in grana membranes, to model the energy migration and charge separation processes in PSII simultaneously in a transparent way. All simulations have in common that the charge separation is fast and nearly irreversible, corresponding to a significant drop in free energy upon primary charge separation, and that in PSII membranes energy migration imposes a larger kinetic barrier for the overall process than primary charge separation. © 2006 by the Biophysical Society.

Fluorescent base analogues such as 2-aminopurine report DNA dynamics on the scale of single bases. We find that the time-dependent fluorescence of various 2-aminopurine-containing dinucleotides can be described by only two components: a fast (∼20 ps) exponential decay and a much slower (∼1 ns) stretched exponential. This is much simpler than previously proposed models. The fast component reflects quenching in the stacked equilibrium conformation. The slow stretched exponential indicates diffusive dynamics towards the equilibrium conformation. Depending on the dinucleotide, this migration effectively takes place in a one- or two-dimensional manifold. Molecular-dynamics simulations indicate that it involves twisting and sliding with parallel base planes. Our very simple representation of the data provides a powerful tool to study DNA fluorescence quenching and diffusive dynamics independently.

Excitonic polarons in antenna complexes are subject to static lattice disorder. A model has been developed to analyze polarized fluorescence excitation spectra of circular light-harvesting complexes from purple photosynthetic bacteria containing bacteriochlorophyll as the main photoactive pigment that includes both diagonal (energetic) and off-diagonal (structural) disorders. Essential differences of disorder realizations seem to exist between the core LH1 and peripheral LH2 complexes from the bacterium Rhodobacter sphaeroides. The disorder in LH1 appears to be dominated by the structural disorder, while that in LH2, by energetic one. These differences may be due to relatively bigger size of the LH1 complex and, consequently, with its enhanced structural flexibility.

In order to determine the relationship between the pigment-protein and the pigment-pigment interactions, the measurements of absorption spectra of the peripheral light-harvesting complex LH2 from the purple bacteria Rhodobacter sphaeroides solvated in glycerol/buffer solution were carried out in a wide temperature range, from 4 to 250 K. The SDFs used for simulating the temperature dependence of B800 and B850 bands were determined in a parametric form. To fit experimental spectra the overall exciton-phonon coupling had to be assumed to be weak for B850 (lambda/2V approximately 0.3, where lambda is the reorganization energy and V is the nearest-neighbor dipole-dipole coupling for bacteriochlorophylls). At physiological temperatures the intermediate nuclear bath dynamics compares with the magnitude of energy gap fluctuations. Slower dynamics with kappa approximately 0.39, where kappa is the ratio of the nuclear relaxation rate and the line width parameter, determines the spectral shape of B850 whilst faster modulations characterize B800 (kappa approximately 2.39). The static disorder for the B800 band is relatively high with the characteristic value of the inhomogeneous bandwidth Gamma(inh) approximately 120 cm-1, while for the B850 band this value is almost equal to the dipole-dipole coupling strength (Gamma(inh) approximately 360 cm-1). It has been found that the LH2 absorption spectrum is likely to be influenced by the temperature dependence of the dielectric constant of the solution in the high temperature range, when the glycerol/buffer solution is in the liquid state.

The structure of the exciton manifold in the core bacteriochlorophyll antenna complexes from the purple photosynthetic bacterium Rhodobacter sphaeroides has been studied at 5 K using conventional and polarized fluorescence excitation spectroscopy supported with model simulations. Exciton bandwidths in the range of 2100–2200 cm −1 , depending on sample integrity, have been determined in correlation with the largest mean coupling energy between the nearest-neighbor bacteriochlorophyll molecules close to 600 cm −1 . These numbers are considerably bigger than those in any other native antenna complex known. Another noteworthy feature of these complexes is dominance of structural (off-diagonal) static disorder over the energetic (diagonal) disorder.

We show that in finite, disordered one-dimensional aggregates the exciton self-trapping takes place at significantly weaker exciton–lattice coupling than it is known for the regular infinite lattices. A series of abrupt changes of self-trapped exciton energy and other parameters can take place at multiple critical couplings, depending on initial exciton amplitude distributions. These states vary by the wave function localization at different segments of the aggregate. The observed energy splitting suggests a coexistence of two or more types of self-trapped excitons with altered lattice reorganization energy. This provides a general explanation for several recent spectroscopic observations on photosynthetic antenna complexes.

The bandwidth of the exciton manifold in LH2 peripheral antenna complexes from the photosynthetic bacterium Rhodobacter sphaeroides has been determined using polarized fluorescence excitation spectroscopy and model simulations. It is shown that the fluorescence anisotropy spectra reveal a hidden structure of the exciton band that is correlated with its boundaries. The estimated exciton coupling energy and exciton bandwidth in the native complex is ∼360 and ∼1620 cm −1 , respectively. The corresponding numbers in a mutant with the B800 bacteriochlorophyll molecules absent are somewhat larger (∼420 and ∼1890 cm −1 ), probably due to tightening of the protein structure.

A dual nature of fluorescence from LH2 pigment–protein complexes, which is a part of the light harvesting system of purple bacteria, is confirmed by fluorescence-lifetime dependence on recording wavelength and spectrally selective spectroscopy. An analysis based on the Holstein molecular crystal model, modified by allowing diagonal disorder, suggests coexistence of large- and small-radius self-trapped excitons, which serve as the origin of the dual fluorescence.

High-spectral-resolution hole-burning and fluorescence line-narrowing spectra of excitons in LH2 complexes from the photosynthetic purple bacterium Rhodobacter sphaeroides have been investigated together with conventional broadband fluorescence spectra and their temperature dependence. The steady-state spectroscopy has been complemented by fluorescence lifetime measurements. The experimental results are discussed on the basis of the adiabatic Holstein exciton polaron model, modified by including diagonal disorder. As a result, a new interpretation for the LH2 antenna optical spectra is provided. The exciton when optically excited becomes localized after relaxation. The LH2 fluorescence is mainly due to self-trapped excitons not only at low temperature, as previously suggested (Timpmann, K.; Katiliene, Z.; Woodbury, N. W.; Freiberg, A. J. Phys. Chem. B 2001, 105, 12223), but also over the whole temperature range up to physiological temperatures because the self-trapped exciton binding energy is of the same...

Fluorescence line narrowing and hole-burning spectroscopic studies of excitons in the LH2 pigment-protein complex, which is a part of the light harvesting system of purple bacteria, are combined with straightforward numerical simulations of the emission spectra based on exciton polaron model. The analysis provides evidence for self-trapping of all the excitons, except the lowest one.

The absorption and emission spectra of excitons in LH2 antenna complexes from the photosynthetic purple bacterium Rhodobacter sphaeroides have been studied under hydrostatic pressure. The measurements made between ambient pressure and 6 kbar over a broad temperature range reveal largely different rates of the pressure-induced shifts for the absorption and emission bands. Numerical calculations based on exciton polaron model provide evidence for the exciton self-trapping at ambient pressure as well as for the pressure stabilization of the self-trapped exciton states responsible for the emission, whereas the light absorbing states belong to nearly free excitons over the whole pressure and temperature ranges studied.

One of the major players in oxygenic photosynthesis, photosystem II (PSII), exhibits complex multiexponential fluorescence decay kinetics that for decades has been ascribed to reversible charge separation taking place in the reaction center (RC). However, in this description the protein dynamics is not taken into consideration. The intrinsic dynamic disorder of the light-harvesting proteins along with their fluctuating dislocations within the antenna inevitably result in varying connectivity between pigment-protein complexes and therefore can also lead to nonexponential excitation decay kinetics. On the basis of this presumption, we propose a simple conceptual model describing excitation diffusion in a continuous medium and accounting for possible variations of the excitation transfer rates. Recently observed fluorescence kinetics of PSII of different sizes are perfectly reproduced with only two adjustable parameters instead of the many decay times and amplitudes required in standard analysis procedures; no charge recombination in the RC is required. The model is also able to provide valuable information about the structural and functional organization of the photosynthetic antenna and in a straightforward way solves various contradictions currently existing in the literature.

The excited state mixing effect is taken into account considering the difference spectra of dimers. Both the degenerate (homo) dimer as well as the nondegenerate (hetero) dimer are considered. Due to the higher excited state mixing with the two-exciton states in the homodimer, the excited state absorption (or the difference spectrum) can be strongly affected in comparison with the results obtained in the Heitler–London approximation. The difference spectrum of the heterodimer is influenced by two resonance effects (i) mixing of the ground state optical transitions of both monomers in the dimer and (ii) mixing of the excited state absorption of the excited monomer with the ground state optical transition in the nonexcited monomer. These effects have been tested by simulating the difference absorption spectra of the light-harvesting complex of photosystem II (LHC II) experimentally obtained with the 60 fs excitation pulses at zero delay times and various excitation wavelengths. The pairs of coupled chloroph...

A model for the spectral characteristics, the transition dipole moment orientations, and the energy transfer properties of chlorophyll (Chl) a and b molecules in the light-harvesting complex (LHC) ...

A model for the protein control of energy transfer in photosynthetic light-harvesting antenna is proposed. Thermal fluctuations of the energy gap between the excited donor and acceptor molecules for the pairwise energy transfer step are assumed when the latter is a bottleneck for the overall transfer from the antenna to the reaction center. The model is motivated by fluorescence decay experiments on photosystem I particles. It emphasizes the dynamical aspect of spectral heterogeneity rather than the conventionally employed static picture. A comparison and discussion between experimental data and theoretical predictions are given.

The theory of the singlet-singlet annihilation in quasi-homogeneous photosynthetic antenna systems is developed further. In the new model, the following important contributions are taken into account: 1) the finite excitation pulse duration, 2) the occupation of higher excited states during the annihilation, 3) excitation correlation effects, and 4) the effect of local heating. The main emphasis is concentrated on the analysis of pump-probe kinetic measurements demonstrating the first two above possible contributions. The difference with the results obtained from low-intensity fluorescence kinetic measurements is highlighted. The experimental data with picosecond time resolution obtained for the photosynthetic bacterium Rhodospirillum rubrum at room temperature are discussed on the basis of this theory.

The variability of the exciton spectra of bacteriochlorophyll molecules in light-harvesting (LH) complexes of photosynthetic bacteria ensures the excitation energy funneling trend toward the reaction center. The decisive shift of the energies is achieved due to exciton spectra formation caused by the resonance interaction between the pigments. The possibility to resolve the upper Davydov sub-band corresponding to the B850 ring and, thus, to estimate the exciton bandwidth by analyzing the temperature dependence of the steady-state absorption spectra of the LH2 complexes is demonstrated. For this purpose a self-modeling curve resolution approach was applied for analysis of the temperature dependence of the absorption spectra of LH2 complexes from the photosynthetic bacteria Rhodobacter (Rba.) sphaeroides and Rhodoblastus (Rbl.) acidophilus. Estimations of the intradimer resonance interaction values as follows directly from obtained estimations of the exciton bandwidths at room temperature give 385 and 397 cm(-1) for the LH2 complexes from the photosynthetic bacteria Rba. sphaeroides and Rhl. acidophilus, respectively. At 4 K the corresponding couplings are slightly higher (391 and 435 cm(-1), respectively). The retained exciton bandwidth at physiological conditions supports the decisive role of the exciton coherence determining light absorption in bacterial light-harvesting antenna complexes.

Excitation energy transfer and quenching in LHCII aggregates is considered in terms of a coarse-grained model. The model assumes that the excitation energy transfer within a pigment-protein complex is much faster than the intercomplex excitation energy transfer, whereas the quenching ability is attributed to a specific pigment-protein complex responsible for the nonphotochemical quenching (NPQ). It is demonstrated that the pump-probe experimental data obtained at low excitation intensities for LHCII aggregates under NPQ conditions can be equally well explained at two limiting cases, either describing the excitation kinetics in the migration-limited or in the trap-limited regime. Thus, it is concluded that low excitation conditions do not allow one to unambiguously define the relationship between the mean times of excitation migration and trapping. However, this could be achieved by using high excitation conditions when exciton-exciton annihilation is dominant. In this case it was found that in the trap-limited regime the excitation kinetics in the aggregate should be almost insensitive to the excitation density, meaning that singlet-singlet annihilation has little effect on the NPQ decay kinetics, whereas in the migration-limited case there is a clear intensity dependence. In order to account for the random distribution of the NPQ-traps within the LHCII aggregates, excitation diffusion in a continuous medium with random static traps was considered. This description demonstrates a very good correspondence to the experimental fluorescence kinetics assuming a lamellar (quasi-3D) structure of the antenna characterized by the dimension d = 2.4 and therefore justifying the diffusion-limited approach on which the model is based. Using the coarse-grained model to describe the aggregate we estimate one NPQ-trap per 100 monomeric LHCII complexes. Finally we discuss the origin of the traps responsible for excitation quenching under NPQ conditions. © 2011 American Chemical Society.

The nature of electronic excitations created by photon absorption in the cyclic B850 aggregates of 18 bacteriochlorophyll molecules of LH2 antenna complexes of photosynthetic bacteria is studied over a broad temperature range using absorption, fluorescence, and fluorescence anisotropy spectra. The latter technique has been proved to be suitable for revealing the hidden structure of excitons in inhomogeneously broadened spectra of cyclic aggregates. A theoretical model that accounts for differences of absorbing excitons in undeformed and emitting exciton polarons in deformed antenna lattices is also developed. Only a slight decrease of the exciton bandwidth and exciton coupling energy with temperature is observed. Survival of excitons in the whole temperature span from cryogenic to nearly ambient temperatures strongly suggests that collective, coherent electronic excitations might play a role in the functional light-harvesting process taking place at physiological temperatures.

Excitation energy transfer and trapping by the artificially postulated traps in photosystem II (PSII) were modeled in terms of a coarse-grained model. The model is based on the assumption that the excitation energy transfer within a pigment–protein complex is much faster than the intercomplex excitation energy transfer. As a result, the excitation energy transfer and trapping rates by the reaction center (RC) were rescaled by the relevant entropic factors and an additional trapping rate for a specific pigment–protein complex responsible for the non-photochemical quenching (NPQ) had to be included into the theoretical framework. For the analysis, dimeric models of PSII were considered. The efficiency of the excitation quenching by the NPQ traps, depending on their positioning and on the trapping rate, was analyzed. It was concluded that the highest efficiency of the NPQ quencher could be achieved when they are localized in the major light-harvesting complexes, LHCII, and the excitation relaxation in this state is fast, of the order of picoseconds and even faster. The origin of the state responsible for NPQ is discussed.

A comparative analysis of the temperature dependence of the absorption spectra of the LH2 complexes from different species of photosynthetic bacteria, i.e., Rhodobacter sphaeroides, Rhodoblastus acidophilus, and Phaeospirillum molischianum, was performed in the temperature range from 4 to 300 K. Qualitatively, the temperature dependence is similar for all of the species studied. The spectral bandwidths of both B800 and B850 bands increases with temperature while the band positions shift in opposite directions: the B800 band shifts slightly to the red while the B850 band to the blue. These results were analyzed using the modified Redfield theory based on the exciton model. The main conclusion drawn from the analysis was that the spectral density function (SDF) is the main factor underlying the strength of the temperature dependence of the bandwidths for the B800 and B850 electronic transitions, while the bandwidths themselves are defined by the corresponding inhomogeneous distribution function (IDF). Slight variation of the slope of the temperature dependence of the bandwidths between species can be attributed to the changes of the values of the reorganization energies and characteristic frequencies determining the SDF. To explain the shift of the B850 band position with temperature, which is unusual for the conventional exciton model, a temperature dependence of the IDF must be postulated. This dependence can be achieved within the framework of the modified (dichotomous) exciton model. The slope of the temperature dependence of the B850 bandwidth is then defined by the value of the reorganization energy and by the difference between the transition energies of the dichotomous states of the pigment molecules. The equilibration factor between these dichotomous states mainly determines the temperature dependence of the peak shift.

The fluorescence decay kinetics of Photosystem II (PSII) membranes from spinach with open reaction centers (RCs), were compared after exciting at 420 and 484 nm. These wavelengths lead to preferential excitation of chlorophyll (Chl) a and Chl b, respectively, which causes different initial excited-state populations in the inner and outer antenna system. The non-exponential fluorescence decay appears to be 4.3+/-1.8 ps slower upon 484 nm excitation for preparations that contain on average 2.45 LHCII (light-harvesting complex II) trimers per reaction center. Using a recently introduced coarse-grained model it can be concluded that the average migration time of an electronic excitation towards the RC contributes approximately 23% to the overall average trapping time. The migration time appears to be approximately two times faster than expected based on previous ultrafast transient absorption and fluorescence measurements. It is concluded that excitation energy transfer in PSII follows specific energy transfer pathways that require an optimized organization of the antenna complexes with respect to each other. Within the context of the coarse-grained model it can be calculated that the rate of primary charge separation of the RC is (5.5+/-0.4 ps)(-1), the rate of secondary charge separation is (137+/-5 ps)(-1) and the drop in free energy upon primary charge separation is 826+/-30 cm(-1). These parameters are in rather good agreement with recently published results on isolated core complexes [Y. Miloslavina, M. Szczepaniak, M.G. Muller, J. Sander, M. Nowaczyk, M. Rögner, A.R. Holzwarth, Charge separation kinetics in intact Photosystem II core particles is trap-limited. A picosecond fluorescence study, Biochemistry 45 (2006) 2436-2442].

Structural arrangement of pigment molecules of Photosystem I of photosynthetic cyanobacterium Synechococcus elongatus is used for theoretical modeling of the excitation energy spectrum. It is demonstrated that a straightforward application of the exciton theory with the assumption of the same molecular transition energy does not describe the red side of the absorption spectrum. Since the inhomogeneity in the molecular transition energies caused by a dispersive interaction with the molecular surrounding cannot be identified directly from the structural model, the evolutionary search procedure is used for fitting the low temperature absorption and circular dichroism spectra. As a result, one dimer, three trimers and one tetramer of chlorophyll molecules responsible for the red side of the absorption spectrum with their assignment to the spectroscopically established three bands at 708, 714 and 719 nm are determined. All of them are found to be situated not in the very close vicinity of the reaction center but are encircling it almost at the same distance. In order to explain the unusual broadening on the red side of the spectrum the exciton state mixing with the charge transfer (CT) states is considered. It is shown that two effects can be distinguished as caused by mixing of those states: (i) the oscillator strength borrowing by the CT state from the exciton transition and (ii) the borrowing of the high density of the CT state by the exciton state. The intermolecular vibrations between two counter-charged molecules determine the high density in the CT state. From the broad red absorption wing it is concluded that the CT state should be the lowest state in the complexes under consideration. Such mixing effect enables resolving the diversity in the molecular transition energies as determined by different theoretical approaches.

The absorption and emission spectra of excitons in LH1 and LH2 antenna complexes from the photosynthetic purple bacterium Rhodobacter sphaeroides have been studied under hydrostatic pressure varying between ambient pressure and 6 kbar. The observations performed at low (80 K) and physiological temperatures reveal distinctive differences in spectral response of LH1 and LH2 excitons to compression. Analysis shows that unlike in LH2 where small-radius self-trapped excitons exist, the self-trapping is much less developed in LH1. This is probably because the resonant exciton coupling in the LH1 complex is relatively stronger at similar exciton-lattice coupling energy.

It is shown that the excitation annihilation kinetics of a finite molecular assembly asymptotically approaches the steady state corresponding to the last excitation remaining in the system. The excitation decay kinetics after eliminating the steady state signal reveals the asymptotic dominance of the slowest annihilation mode. The corresponding annihilation time can be easily estimated from the experimental kinetic traces to give a measure of the system size. The annihilation time estimations for the light-harvesting complex LH2, C60 structures and a few poly(arylene vinylenes) are presented.© (2001) COPYRIGHT SPIE--The International Society for Optical Engineering. Downloading of the abstract is permitted for personal use only.

Singlet-singlet annihilation is used to study exciton delocalization in the light harvesting antenna complex LH2 (B800-B850) from the photosynthetic purple bacterium Rhodobacter sphaeroides. The characteristic femtosecond decay constants of the high intensity isotropic and the low intensity anisotropy kinetics of the B850 ring are related to the hopping time tau(h) and the coherence length N(coh) of the exciton. Our analysis yields N(coh) = 2.8+/-0.4 and tau(h) = 0.27+/-0.05 ps. This approach can be seen as an extension to the concept of the spectroscopic ruler.

Energy transfer and charge separation kinetics in the photosystem I complex was investigated under low excitation intensities to characterize the functional organization of the complex.

In our recent work on modelling of the excitation dynamics in the light-harvesting complex (LHC) II from higher plants [I] the Chi a and b spectral type and transition dipole moment orientation distributions in the LHC II structure have been obtained satisfactorily reproducing the low intensity femtosecond transient as well as steady state spectra, namely, fluorescence and linear dichroism (LD) data. It suggested a swap of two Chi a with Chi b locations as compared to Chl identities proposed up to now [2]. This property is well in line with recently obtained evidence that Chi b molecules are the primary excitation acceptors from the excited Luteins [3]. The determined Chl transition dipole moment orientations, removing the uncertainty in a current chromophore resolution, resulted in a weak coupling between the Chis a stronger coupling (V≤100 cm-1) between the pairs of Chls a and b in the LHC II monomer and weak energy transfer coupling between the monomers in the trimer. This implied that energy transfer between Chls can be well described by the Forster mechanism supporting also our approach in approximating the absorption spectrum of LHC II by a sum of noninteracting CM spectra [1]. The Chl-Chl coupling effects are important, however, at very initial times as it comes from the detailed analysis of the initial difference absorption spectra at various excitation wavelengths (see contribution of Valkunas et al. to proceedings).

A procedure is described to generate and optimize the lattice models for spectrally inhomogeneous photosynthetic antenna/reaction center (RC) particles. It is based on the genetic algorithm search for the pigment spectral type distributions on the lattice by making use of steady-state and time-resolved spectroscopic input data. Upon a proper fitness definition, a family of excitation energy transfer models can be tested for their compatibility with the availability experimental data. For the case of the photosystem I core antenna (99 chlorophyll + primary electron donor pigment (P700)), three spectrally inhomogeneous three-dimensional lattice models, differing in their excitation transfer conditions, were tested. The relevant fit parameters were the pigment distribution on the lattice, the average lattice spacing of the main pool pigments, the distance of P700 and of long wavelength-absorbing (LWA) pigments to their nearest-neighbor main pool pigments, and the rate constant of charge separation from P700. For cyanobacterial PS I antenna/RC particles containing a substantial amount of LWA pigments, it is shown that the currently available experimental fluorescence data are consistent both with more migration-limited, and with more trap-limited excitation energy transfer models. A final decision between these different models requires more detailed experimental data. From all search runs about 30 different relative arrangements of P700 and LWA pigments were found. Several general features of all these different models can be noticed: 1) The reddest LWA pigment never appears next to P700. 2) The LWA pigments in most cases are spread on the surface of the lattice not far away from P700, with a pronounced tendency toward clustering of the LWA pigments. 3) The rate constant kP700 of charge separation is substantially higher than 1.2 ps-1, i.e., it exceeds the corresponding rate constant of purple bacterial RCs by at least a factor of four. 4) The excitation transfer within the main antenna pool is very rapid (less than 1 ps equilibration time), and only the equilibration with the LWA pigments is slow (about 10–12 ps). The conclusions from this extended study on three-dimensional lattices are in general agreement with the tendencies and limitations reported previously for a simpler two-dimensional array. Once more detailed experimental data are available, the procedure can be used to determine the relevant rate-limiting processes in the excitation transfer in such spectrally inhomogeneous antenna systems.

This report addresses the energy migration problem in the light-harvesting antenna systems of photosynthetic objects. Both linear and nonlinear relaxation cases are discussed. In the linear case, the spectral inhomogeneity is taken into account by averaging the Master equation for the excitation over the spectrally inhomogeneous distribution of pigment molecules. That reformulates the Master equation as the excitation diffusion in the energy space. The stationary solution of this equation enables the determination of the Stokes shift dependence on homogeneous and inhomogeneous spectral bandwidths. The nonlinear relaxation is considered at the singlet-singlet annihilation conditions. The analysis of the fluorescence quantum yield, the excitation delay kinetics as well as the changes of spectral properties of the object under consideration as a function of the excitation intensity, demonstrates the possibility to determine the energy migration parameters. An important conclusion of this analysis is that the real pulse-shape of the excitation as well as the population of higher excited states during the nonlinear annihilation have to be taken into account.

Kinetic modeling of the exciton migration in the cyanobacterial photosystem I core complex from Synechococcus sp. was performed by an exact solution of the Pauli master equation for exciton motion. A square two-dimensional 10 x 10 pigment lattice and a Forster dipole-dipole coupling between chromophores was assumed. We calculated decay-associated spectra and lifetimes and compared them to the corresponding experimental data from picosecond fluorescence and transient absorption obtained by global analysis. Seven spectral chlorophyll(Chl) forms, identical in shape but shifted in their absorption maximums, were used to describe the non-homogeneous broadening of the PS I-100 particle absorption spectrum. The optimized Chl lattice arrangement best reproducing the experimental decay-associated spectra as well as the steady-state fluorescence spectrum indicated the long-wavelength-absorbing Chls forming a cluster in the corner of the lattice with the reaction center (RC) placed apart at a distance of two lattice constants. The variable parameters, i.e., the charge separation rate in the RC and the lattice constant a, were found to be optimal at kRC = 2.3 ps-1 and a = 1.14 nm, respectively. The surprising conclusions of the simulations is that Chls with absorption maxima as long a 724 nm have to be taken into account to describe the time-resolved spectra of this PS I particle properly. The dependencies of the exciton decay in the model PS I particle on the excitation wavelength and on the temperature are discussed. We also show that the excited state decay of similar PS I particles that lack the long-wavelength absorbing Chls is nearly mono-exponential. Various critical factors that limit the general reliability of the conclusions of such simulations are discussed in detail.

The main 137Cs accumulation zone in the study area was foundto be located at depths below the 50-metre isobath, i.e. below the layer of hydrodynamic activity.In coastal waters not influenced by the fresh water discharge from the Curonian Lagoon,137Cs occurs mostly in soluble form. The particulate 137Cs activity concentrationin the marine area affected by Curonian Lagoon water can make up 10% of the total 137Csactivity concentration. The circulation model was developed to assess the distributionof artificial radionuclides in Lithuanian territorial waters. The model was validated on the basis of data acquired during the measurement campaign in the Lithuanian part of the Baltic Sea and the CuronianLagoon in the years 1999-2001. The model enables the 137Cs activity concentration to besimulated as a passive admixture (error within c. 15%).

Photosystem II is a supercomplex composed of 27-28 different subunits and it represents the most important machinery of the plants photosynthetic appara- tus, having the ability to split water into oxygen, protons and electrons. In the last few years the structures of most of the photosynthetic complexes have been resolved, allowing to organize in a ‘‘visual framework’’ the large body of information obtained by genetics, biochemical and spectroscopic methods about the function and organization of the complexes. Only the struc- ture of PSII-LHCII from higher plants is still lacking due to the impossibility to obtain a homogeneous and stable preparation of the supercomplex, which has also prevented functional and spectroscopic studies. In this work homogeneous and stable Photosystem II supercomplexes with dif- ferent antenna size were isolated. A full gallery of complexes, from the core to the largest C2S2M2, was characterized by electron microscopy and biochemi- cal and spectroscopic methods, allowing to relate for the first time the supramo- lecular organization to the protein and pigment content and the energy transfer processes. A new complex containing a monomeric core, a trimeric LHCII (S) and a monomeric CP26 was isolated, showing that the antenna proteins can bind to the monomeric core in contrast to the current belief. The comparison of the supercomplexes obtained from WT plants and knock out mutants of sev- eral Lhcb proteins allowed determining the hierarchy of the assembly and to suggest a role for the individual subunits. The data also provides information about the organization of the oxygen evolving complex. For the first time it was possible to study the energy transfer process in the supercomplexes with the use of picosecond fluorescence spectroscopy. The functional implication of these results on photoinhibition, state transition and energy transfer are discussed.