Excited state annihilation in the photosynthetic unit

The kinetics of the in vivo fluorescence decays and fluorescence yields, as a function of excitation intensity, have been analysed with a model using excited state annihilation and time-dependent quenching processes. Triplet states, formed in the singlet-singlet annihilation processes, account for additional quenching of singlet states and the persistence of annihilation at longer times than the fluorescence lifetime. Together these processes give a satisfactory account of existing experimental data of the intensity dependence of fluorescence in vivo.     

Picosecond processes in chromatophores at various excitation intensities

The aim of this paper is to review and discuss the results obtained by fluorescence and absorption spectroscopy of bacterial chromatophores excited with picosecond pulses of varying power and intensity. It was inferred that spectral and kinetic characteristics depend essentially on the intensity, the repetition rate of the picosecond excitation pulses as well as on the optical density of the samples used. Taking the different experimental conditions properly into account, most of the discrepancies between the fluorescence and absorption measurements can be solved. At high pulse repetition rate (>10⁶ Hz), even at moderate excitation intensities (10¹⁰–10¹¹ photons/cm² per pulse), relatively long-lived triplet states start accumulating in the system. These are efficient (as compared to the reaction centers) quenchers of mobile singlet excitations due to singlet-triplet annihilation. The singlet-triplet annihilation rate constant in Rhodospirillum rubrum was determined to be equal to 10^-9 cm³ s^-1. At fluences >10¹² photons/cm² per pulse singlet-singlet annihilation must be taken into account. Furthermore, in the case of high pulse repetition rates, triplet-triplet annihilation must be considered as well. From an analysis of experimental data it was inferred that the singlet-singlet annihilation process is probably migration-limited. If this is the case, one has to conclude that the rate of excitation decay in light-harvesting antenna at low pumping intensities is limited by the efficiency of excitation trapping by the reaction center. The influence of annihilation processes on spectral changes is also discussed as is the potential of a local heating caused by annihilation processes. The manifestation of spectral inhomogeneity of light-harvesting antenna in picosecond fluorescence and absorption kinetics is analyzed.Abbreviations LHA light-harvesting antenna - RC reaction center     

两种不同红藻的R－藻红蛋白激发强度相关的皮秒（10~（－12）秒）荧光动力学研究

本文研究了分别从红藻多管藻（Ｐｏｌｙｓｉｐｈｏｎｉａｕｒｃｅｏｌａｔｅ）和条斑紫菜（Ｐｏｒｐｈｙｒａｙｅｚｏｅｎｓｉｓ）中提取的两种不同光谱类型的Ｒ－藻红蛋白Ｒ－ｐｈｙｃｏｅｒｙｔｈｒｉｎ激发强度相关的皮秒（１０－１２秒）荧光衰减动力学过程。结果发现：随激发光强增大,单重态－单重态激子湮灭发生（其衰减过程约为６０～８０皮秒）,并引起荧光量子产率下降。这两种Ｂ－藻红蛋白在相同光强激发下,表现出不同的单重态一单重态激子湮灭过程,主要因它们处于激发态的发色团数目不同所致。     

Bimolecular quenching of excitons and fluorescence in the photosynthetic unit.

The recent results of Campillo et al. and Mauzerall on the quenching of the fluorescence of chlorophyll a in Chlorella pyrenoidosa as a function of the intensity of the laser excitation pulses are rationalized by applying a model invoking singlet-singlet exciton annihilation.     

Exciton Annihilation in the Two Photosystems in Chloroplasts at 100°K

The fluorescence yield (F) of spinach chloroplasts at 100°K measured at 735 nm (photosystem I fluorescence—F 735) and at 685 nm (photosystem II fluorescence—F 685) has been determined with different modes of laser excitation. The modes of excitation included a single picosecond pulse, sequences of picosecond pulses (4, 22, and 300 pulses spaced 5 ns apart) and a single nonmode-locked 2-μs pulse (MP mode). The F 735/F 685 intensity ratios decrease from 1.62 to 0.61 when a single picosecond pulse (or low-power continuous helium-neon laser) is replaced by excitation with the 300-ps pulse train (PPT mode) or MP mode. In the PPT mode of excitation, the 735-nm fluorescence band is quenched by a factor of 45 as the intensity is increased from 10¹⁵ to 10¹⁸ photons/cm² per pulse train and the 685-nm fluorescence is quenched by a factor of 10. In the MP mode, the quenching factors are 25 and 7, respectively, in the same intensity range. Fluorescence quantum yield measurements with different picosecond pulse sequences indicate that relatively long-lived quenching species are operative, which survive from one picosecond pulse to another within the pulse train. The excitonic processes possible in the photosynthetic units are discussed in detail. The differences in the quenching factors between the MP and PPT modes of excitation are attributed to singlet-singlet annihilation, possible when picosecond pulses are utilized, but minimized in the MP mode of excitation. The long-lived quenchers are identified as triplets and/or bulk chlorophyll ions formed by singlet-singlet annihilation. The preferential quenching in photosystem I is attributed to triplet excitons. The influence of heating effects, photochemistry, bleaching, and two-photon processes is also considered and is shown to be negligible.     

Theory of exciton annihilation in complexes of a finite number of molecular sites

A theory of the kinematics of singlet exciton annihilation in complexes of a finite number of molecular sites is developed. The theory is based on a specific scheme suggested earlier by Gülen, Wittmershaus, and Knox [Biophys J. 49:469-477 (1986)]. It is adequate to address the excitation kinetics and dynamics in such systems, especially under high excitation intensities. A Pauli master equation is formulated and is solved to give explicit expressions for observables such as quantum yield and fluorescence intensity. The excitation intensity dependence of the observables is taken into account by introducing Poisson statistics. Details relevant to its application to the annihilation of excitons in photosynthetic systems and its connection to earlier theories are presented.     

Fluorescence lifetime heterogeneity in aggregates of LHCII revealed by time-resolved microscopy.

Two-photon excitation, time-resolved fluorescence microscopy was used to investigate the fluorescence quenching mechanisms in aggregates of light-harvesting chlorophyll a/b pigment protein complexes of photosystem II from green plants (LHCII). Time-gated microscopy images show the presence of large heterogeneity in fluorescence lifetimes not only for different LHCII aggregates, but also within a single aggregate. Thus, the fluorescence decay traces obtained from macroscopic measurements reflect an average over a large distribution of local fluorescence kinetics. This opens the possibility to resolve spatially different structural/functional units in chloroplasts and other heterogeneous photosynthetic systems in vivo, and gives the opportunity to investigate individually the excited states dynamics of each unit. We show that the lifetime distribution is sensitive to the concentration of quenchers contained in the system. Triplets, which are generated at high pulse repetition rates of excitation (>1 MHz), preferentially quench domains with initially shorter fluorescence lifetimes. This proves our previous prediction from singlet-singlet annihilation investigations (Barzda, V., V. Gulbinas, R. Kananavicius, V. Cervinskas, H. van Amerongen, R. van Grondelle, and L. Valkunas. 2001. Biophys. J. 80:2409-2421) that shorter fluorescence lifetimes originate from larger domains in LHCII aggregates. We found that singlet-singlet annihilation has a strong effect in time-resolved fluorescence microscopy of connective systems and has to be taken into consideration. Despite that, clear differences in fluorescence decays can be detected that can also qualitatively be understood.     

Theory of Picosecond-Laser-Induced Fluorescence from Highly Excited Complexes with Small Numbers of Chromophores

The problem of singlet excitation kinetics and dynamics, especially at high excitation intensities, among a small number of chromophores of a given system has been addressed. A specific scheme for the kinetics is suggested and applied to CPII, a small chlorophyll (Chl)a/b antenna complex the fluorescence lifetime of which has been reported to be independent of excitation intensity over a wide intensity range of picosecond pulses. We have modeled the kinetics from the point of view that Chla molecules in CPII are Förster coupled so that a second excitation received by the group of Chla's either creates a state with two localized excitons or raises the first one to a doubly excited state. The data on CPII can be understood on the basis of a kinetic model that does not exclude exciton annihilation during the excitation pulse. The implied annihilation rate is consistent with our theoretical estimates of that rate obtained by applying excitation transfer theory to pairs of molecules both initially excited.     

Nonlinear annihilation of excitations in photosynthetic systems.

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.     

Singlet and triplet excited carotenoid and antenna bacteriochlorophyll of the photosynthetic purple bacterium Rhodospirillum rubrum as studied by picosecond absorbance difference spectroscopy

Picosecond absorbance difference spectra at a number of delay times after a 35 ps excitation pulse and kinetics of absorbance changes were measured in chromatophores of the photosynthetic purple bacterium Rhodospirillum rubrum after chemical oxidation of the primary electron donor P-875. Kinetics and spectra were measured of the excited singlet states of carotenoid and bacteriochlorophyll a and also of the triplet state of the carotenoid. The excited singlet state of carotenoid, produced by direct excitation at 532 nm, is characterized by a bleaching of the ground state absorption bands in the region 450–490 nm and by an absorbance increase with a maximum near 570 nm. Its lifetime was calculated to be 0.6 ± 0.1 ps in vitro and less than 1 ps in vivo. The triplet state of carotenoid in vivo is formed within 100 ps after direct carotenoid excitation via a pathway that does not involve excited states of bacteriochlorophyll. Singlet excitation of a bacteriochlorophyll a molecule causes the bleaching of its Q_x and Q_y absorption bands, and is probably associated with blue shifts of the Q_y absorption band of about six neighboring bacteriochlorophyll molecules. Upon increasing the excitation density, the average lifetime of the singlet excitations on bacteriochlorophyll decreased from about 350 ps to about 10 ps or less. The results are in quantitative agreement with the known effect of singlet-singlet annihilation upon the fluorescence yield, and furthermore show that no bacteriochlorophyll or carotenoid triplet formation is associated with this annihilation.     

Singlet-singlet annihilation kinetics in aggregates and trimers of LHCII

Singlet-singlet annihilation experiments have been performed on trimeric and aggregated light-harvesting complex II (LHCII) using picosecond spectroscopy to study spatial equilibration times in LHCII preparations, complementing the large amount of data on spectral equilibration available in literature. The annihilation kinetics for trimers can well be described by a statistical approach, and an annihilation rate of (24 ps)(-1) is obtained. In contrast, the annihilation kinetics for aggregates can well be described by a kinetic approach over many hundreds of picoseconds, and it is shown that there is no clear distinction between inter- and intratrimer transfer of excitation energy. With this approach, an annihilation rate of (16 ps)(-1) is obtained after normalization of the annihilation rate per trimer. It is shown that the spatial equilibration in trimeric LHCII between chlorophyll a molecules occurs on a time scale that is an order of magnitude longer than in Photosystem I-core, after correcting for the different number of chlorophyll a molecules in both systems. The slow transfer in LHCII is possibly an important factor in determining excitation trapping in Photosystem II, because it contributes significantly to the overall trapping time.     

Low-Intensity picosecond fluorescence kinetics and excitation dynamics in barley chloroplasts

The fluorescence decays of barley chloroplasts have been measured by single-photon counting with tunable picosecond dye laser excitation. The fluorescence decays of dark-adapted chloroplasts are best fitted to a sum of three exponential lifetime components with lifetimes of 112, 380 and 2214 ps. The relative magnitude of each component is shown to be dependent on the excitation wavelength and collected emission wavelength. The excitation wavelength dependence is correlated with the Photosystem (PS) I and PS II action study of Ried [36] and with the measured pigment distributions in the photosynthetic unit [37,41]. Experiments varying the single excitation pulse intensity from 10⁸ to 10¹² photons/cm² pulse show that our results are not distorted by singlet-singlet annihilation. Unflowed samples where the cloroplasts are under constant illumination show 2-fold increases in quantum yield of fluorescence primarily in the two longer lifetime components. Theoretical calculations of Shipman [31] on an isolated reaction center with a homogeneous antenna are discussed and the principles extended to discussion of the measured barley chloroplast fluorescence decay components in terms of photosynthetic unit light-harvesting array models and earlier experimental work. Our data support a photosynthetic unit model in which 70–90% of the photons absorbed are quenched by either PS I or efficiently quenching PS II in a process where the fluorescence lifetime is 100 ps. The origin of the intermediate 380 ps. component is probably due to excitation transfer to a PS II reaction center in a redox state which quenches less efficiently.     

The structure of monoacylglycerol lipase from Bacillus sp. H257 reveals unexpected conservation of the cap architecture between bacterial and human enzymes

In order to prevent photodestruction by high light, photosynthetic organisms have evolved a number of mechanisms, known as non-photochemical quenching (NPQ), that deactivate the excited states of light harvesting pigments. Here we investigate the NPQ mechanism in the cyanobacterium Synechocystis sp. PCC 6803 mutant deficient in both photosystems. Using non-linear laser fluorimetry, we have determined molecular photophysical characteristics of phycocyanin and spectrally distinct forms of allophycocyanin for the cells in non-quenched and quenched states. Our analysis of non-linear fluorescence characteristics revealed that NPQ activation leads to an ~2-fold decrease in the relaxation times of both allophycocyanin fluorescence components, F660 and F680, and a 5-fold decrease in the effective excitation cross-section of F680, suggesting an emergence of a pathway of energy dissipation for both types of allophycocyanin. In contrast, NPQ does not affect the rates of singlet-singlet exciton annihilation. This indicates that, upon NPQ activation, the excess excitation energy is transferred from allophycocyanins to quencher molecules (presumably 3'hydroxyechinenone in the orange carotenoid protein), rather than being dissipated due to conformational changes of chromophores within the phycobilisome core. Kinetic measurements of fluorescence quenching in the Synechocystis mutant revealed the presence of several stages in NPQ development, as previously observed in the wild type. However, the lack of photosystems in the mutant enhanced the magnitude of NPQ as compared to the wild type, and allowed us to better characterize this process. Our results suggest a more complex kinetics of the NPQ process, thus clarifying a multistep model for the formation of the quenching center.     

Taking account of macromolecule conformation dynamics in the analysis of luminescent probe signals

A mathematical model of electron excitation energy transport between molecular probes sorbed on the polymeric chain in solution was proposed. The kinetics of the process was described in terms of the conception of stochastic changes in macromolecule conformation. The results of computer simulation and the analytical expressions obtained in the framework of the perturbation theory for the cases of low transfer rate and/or fast conformation motion of the macrochain are presented. A channel of nonlinear deactivation as a result of binary annihilation of closely-spaced excited centers was considered. Expressions for the effective rate of mutual quenching and delayed annihilation fluorescence of the probe were obtained. Time dependencies of typical luminescent signals and parameteric curves of relative fluorescence quantum yield are presented.     

Tuning FlaSh: redesign of the dynamics,voltage range,and color of the genetically encoded optical sensor of membrane potential

The optical voltage sensor FlaSh, made from a fusion of a GFP "reporter domain" and a voltage-gated Shaker K(+) channel "detector domain," has been mutagenically tuned in both the GFP reporter and channel detector domains. This has produced sensors with improved folding at 37 degrees C, enabling use in mammalian preparations, and yielded variants with distinct spectra, kinetics, and voltage dependence, thus expanding the types of electrical signals that can be detected. The optical readout of FlaSh has also been expanded from single wavelength fluorescence intensity changes to dual wavelength measurements based on both voltage-dependent spectral shifts and changes in FRET. Different versions of FlaSh can now be chosen to optimize the detection of either action potentials or synaptic potentials, to follow high versus low rates of activity, and to best reflect electrical activity in cell types with distinct voltages of operation.     

Singlet-singlet annihilation at low temperatures in the antenna of purple bacteria

Chromatophores of the purple photosynthetic bacteria Rhodospirillum rubrum and Rhodobacter (Rhodopseudomonas) sphaeroides were excited by means of 35-ps flashes at 532 nm of varying intensities, both at room temperature and at 4 K. With increasing exciting energy densities the integrated yield of fluorescence produced by these flashes was found to decrease considerably due to singlet-singlet annihilation. An analysis of the results showed that in R. rubrum the number of connected antenna molecules between which energy transfer is possible decreases from about 1000 to about 150 when the temperature is lowered from 298 to 4 K. In Rb. sphaeroides the B875 light-harvesting complex appears to contain about 100 connected bacteriochlorophyll (BChl) 875 molecules at 4 K, while the B800–850 complex contains about 45 BChl 850 molecules. The data are explained by a model for the antenna of Rb. sphaeroides in which units of B875, containing about four reaction centres, are separated by an array of B800–850 units that surrounds B875. By applying a random walk model we found that in both species the rate of energy transfer between neighbouring antenna molecules decreased about 10-fold upon lowering the temperature. The rate of energy transfer from antenna molecules to either open or closed reaction centres decreased only 3- to 4-fold in R. rubrum and remained approximately constant in Rb. sphaeroides upon cooling. A blue shift of the emission spectra at 4 K of both species was observed when the excitation energy density was increased to a level where singlet-singlet annihilation plays a significant role. This observation appears to support the notion that an additional long-wave pigment exists in the antenna of these bacteria.     

Low-temperature energy transfer in FMO trimers from the green photosynthetic bacterium Chlorobium tepidum

The pump-probe kinetics of the slowest spectral equilibrations between inequivalent BChl a Q_y states in FMO trimers from Chlorobium tepidum are decelerated by nearly two orders of magnitude when the temperature is lowered from 300 K to 19 K. The pump-probe anisotropy decays are also markedly slower at 19 K than at 300 K. Singlet-singlet annihilation in FMO trimers is negligible at the laser powers used here. However, reduced temperatures greatly accentuate the probability of singlet-triplet annihilation, due to accumulation of metastable BChl a states under high laser repetition rates.Abbreviations BChl bacteriochlorophyll - FMO Fenna-Matthews-Olson - fwhm full width at half maximum - PB photobleaching - SE stimulated emission     

Short-pulse pump-and-probe technique for airborne laser assessment of Photosystem II photochemical characteristics

The development of a technique for laser measurement of fPhotosystem II (PS II) photochemical characteristics of phytoplankton and terrestrial vegetation from an airborne platform is described. Results of theoretical analysis and experimental study of pump-and-probe measurement of the PS II functional absorption cross-section and photochemical quantum yield are presented. The use of 10 ns probe pulses of PS II sub-saturating intensity provides a significant, up to 150-fold, increase in the fluorescence signal compared to conventional `weak-probe' protocol. Little effect on the fluorescence yield from the probe-induced closure of PS II reaction centers is expected over the short pulse duration, and thus a relatively intense probe pulse can be used. On the other hand, a correction must be made for the probe-induced carotenoid triplet quenching and singlet-singlet annihilation. A Stern-Volmer model developed for this correction assumes a linear dependence of the quenching rate on the laser pulse fluence, which was experimentally validated. The PS II saturating pump pulse fluence (532 nm excitation) was found to be 10 and 40 μmol quanta m⁻² for phytoplankton samples and leaves of higher plants, respectively. Thirty μs was determined as the optimal delay in the pump-probe pair. Our results indicate that the short-pulse pump-and-probe measurement of PS II photochemical characteristics can be implemented from an airborne platform using existing laser and LIDAR technologies. This revised version was published online in June 2006 with corrections to the Cover Date.     

Photoelectric study on the kinetics of trapping and charge stabilization in oriented PS II membranes

Excitation energy trapping and charge separation in Photosystem II were studied by kinetic analysis of the fast photovoltage detected in membrane fragments from peas with picosecond excitation. With the primary quinone acceptor oxidized the photovoltage displayed a biphasic rise with apparent time constants of 100–300 ps and 550±50 ps. The first phase was dependent on the excitation energy whereas the second phase was not. We attribute these two phases to trapping (formation of P-680⁺ Phe^-) and charge stabilization (formation of P-680⁺ Q_A ^-), respectively. A reversibility of the trapping process was demonstrated by the effect of the fluorescence quencher DNB and of artificial quinone acceptors on the apparent rate constants and amplitudes. With the primary quinone acceptor reduced a transient photoelectric signal was observed and attributed to the formation and decay of the primary radical pair. The maximum concentration of the radical pair formed with reduced Q_A was about 30% of that measured with oxidized Q_A. The recombination time was 0.8–1.2 ns.The competition between trapping and annihilation was estimated by comparison of the photovoltage induced by short (30 ps) and long (12 ns) flashes. These data and the energy dependence of the kinetics were analyzed by a reversible reaction scheme which takes into account singlet-singlet annihilation and progressive closure of reaction centers by bimolecular interaction between excitons and the trap. To put on firmer grounds the evaluation of the molecular rate constants and the relative electrogenicity of the primary reactions in PS II, fluorescence decay data of our preparation were also included in the analysis. Evidence is given that the rates of radical pair formation and charge stabilization are influenced by the membrane potential. The implications of the results for the quantum yield are discussed.Abbreviations DCBQ 2,6-dichloro-p-benzoquinone - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - DNB m-dinitrobenzene - PPBQ phenyl-p-benzoquinone - PS I photosystem I of green plants - PS II photosystem II of green plants - PSU photosynthetic unit - P-680 primary donor of PS II - Phe intermediary pheophytin acceptor of PS II - Q_A primary quinone acceptor of PS II - RC reaction center     

Fluorescence characteristics and photophysical parameters of light-harvesting chlorophyll <Emphasis Type="Italic">a/b</Emphasis> complex aggregates

Fluorescence characteristics and molecular photophysical parameters of light-harvesting chlorophyll a/b complexes isolated from pea were studied in relation to their aggregation state. The aggregate size was varied by changing the Triton X-100 concentration from 0 to 0.23 mM at a chlorophyll concentration of 2.45 μg/ml. Molecular photophysical parameters were determined with laser fluorimetry. Dispersion of large aggregates into smaller ones drastically decreased the intensity of low-temperature (77 K) fluorescence at 700 nm, reduced the singlet-singlet annihilation rate by more than two orders of magnitude, and prolonged the fluorescence lifetime from 0.16 to 3.2 ns.