Fluorescence decay kinetics of chlorophyll in photosynthetic membranes

The absorption of light by the pigments of photosynthetic organisms results in electronic excitation that provides the energy to drive the energy-storing light reactions. A small fraction of this excitation gives rise to fluorescence emission, which serves as a sensitive probe of the energetics and kinetics of the excited states. The wavelength dependence of the excitation and emission spectra can be used to characterize the nature of the absorbing and fluorescing molecules and to monitor the process of sensitization of the excitation transfer from one pigment to another. This excitation transfer process can also be followed by the progressive depolarization of the emitted radiation. Using time-resolved fluorescence rise and decay kinetics, measurements of these processes can now be characterized to as short as a few picoseconds. Typically, excitation transfer among the antenna or light harvesting pigments occurs within 100 psec, whereupon the excitation has reached a photosynthetic reaction center capable of initiating electron transport. When this trap is functional and capable of charge separation, the fluorescence intensity is quenched and only rapidly decaying kinetic components resulting from the loss of excitation in transit in the antenna pigment bed are observed. When the reaction centers are blocked or saturated by high light intensities, the photochemical quenching is relieved, the fluorescence intensity rises severalfold, and an additional slower decay component appears and eventually dominates the decay kinetics. This slower (1-2 nsec) decay results from initial charge separation followed by recombination in the blocked reaction centers and repopulation of the excited electronic state, leading to a rapid delayed fluorescence component that is the origin of variable fluorescence. Recent growth in the literature in this area is reviewed here, with an emphasis on new information obtained on excitation transfer, trapping, and communication between different portions of the photosynthetic membranes.     

高等植物体内激子相干迁移与俘获

依据所建立的色素分子排列和取向的新型结构模型,利用激发能传递的广义主方程理论,提出了高等植物体内激子相干迁移与俘获的点阵理论,研究了静态荧光量子产额、定态能量传递速率和荧光强度的变化规律。指出激子相干迁移有助于活体的激发能转移与俘获,并且它有可能是活体内激子寿命的限制因素之一。     

Chlorophyll fluorescence phenomena in chloroplasts on subnanosecond time-scales probed by picosecond pulse pairs

Fluorescence enhancement phenomena and quenching by exciton-exciton annihilation on subnanosecond and nanosecond time-scales were investigated in spinach chloroplasts utilizing picosecond laser pulse pairs (530 nm, 30 ps wide) of equal intensity, spaced apart in time by variable delays of Δt = 0−6 ns. This new method was devised to study the effect of pulse energies (1·10¹⁰–2·10¹⁵ photons per cm²) on the overall fluorescence yield in order to deduce the degree of correlation between the two pulses as a function of Δt. In the case of open reaction centers (F₀ state) in Photosystem II (PS II), it is shown that the quenching effect of excitons generated by the first pulse on the fluorescence yield of the second pulse diminishes with increasing Δt with a characteristic decorrelation time of 140 ± 60 ps. This effect is attributed to either (1) the decay of mobile excitons in the light-harvesting antenna pigment bed as these excitons migrate towards the PS II reaction centers and the associated smaller core antenna pigment pools, or (2) the decay of a quenching state of the reaction center (and/or core antenna) which appears following a rapid (less than 140 ps) trapping of the excitons initially created in the antenna pigment bed. The absence of a significant decay component of exciton quenchers with a lifetime comparable to the 300–600 ps intermediate phase of fluorescence decay kinetics suggests that this phase, although contributing to more than half of the integrated fluorescence emission signal, is not caused by freely mobile exitons migrating in a lake of pigments, but originates instead from smaller pigment pools to which the excitons have migrated. It is proposed that bimolecular exciton-exciton annihilation in these smaller domains dominates annihilation in the larger antenna pigment bed. In the case of closed reaction centers (F_max state), the decorrelation time between the two pulses is increased to 400 ± 100 ps, which is also attributed to either a mobile exciton component or to the decay of a quenching state of the reaction center. At low pulse intensities (below approx. 2 · 10¹² photons per cm²) anomalous fluorescence enhancement effects are noted, which are clearly linked to the existence of initially open PS II reaction centers. These enhancement effects are different from the well-known fluorescence induction phenomena which occur on longer time-scales, and are tentatively attributed to variations in the quenching efficiencies of transitory photochemical states of PS II reaction centers.     

Theory of fluorescence induction in photosystem II: derivation of analytical expressions in a model including exciton-radical-pair equilibrium and restricted energy transfer between photosynthetic units.

The theoretical relationships between the fluorescence and photochemical yields of PS II and the fraction of open reaction centers are examined in a general model endowed with the following features: i) a homogeneous, infinite PS II domain; ii) exciton-radical-pair equilibrium; and iii) different rates of exciton transfer between core and peripheral antenna beds. Simple analytical relations are derived for the yields and their time courses in induction experiments. The introduction of the exciton-radical-pair equilibrium, for both the open and closed states of the trap, is shown to be equivalent to an irreversible trapping scheme with modified parameters. Variation of the interunit transfer rate allows continuous modulation from the case of separated units to the pure lake model. Broadly used relations for estimating the relative amount of reaction centers from the complementary area of the fluorescence kinetics or the photochemical yield from fluorescence levels are examined in this framework. Their dependence on parameters controlling exciton decay is discussed, allowing assessment of their range of applicability. An experimental induction curve is analyzed, with a discussion of its decomposition into alpha and beta contributions. The sigmoidicity of the induction kinetics is characterized by a single parameter J related to Joliot's p, which is shown to depend on both the connectivity of the photosynthetic units and reaction center parameters. On the other hand, the relation between J and the extreme fluorescence levels (or the deviation from the linear Stern-Volmer dependence of 1/phi f on the fraction of open traps) is controlled only by antenna connectivity. Experimental data are consistent with a model of connected units for PS II alpha, intermediate between the pure lake model of unrestricted exciton transfer and the isolated units model.     

Energy migration and exciton trapping in green plant photosynthesis

The possible origins of the different fluorescence decay components in green plants are discussed in terms of a random walk and Butler's bipartite model. The interaction of the excitations with the photosystem II reaction centers and, specifically, the regeneration of theses excitations by charge recombination within the reaction centers, are considered. Based on comparisons between fluorescence decay profiles, time-dependent exciton annihilation and photoelectric phenomena, it appears that the fast 200 ps decay component corresponds to primary energy transport from the antenna to the reaction centers and is dominant in filling the photosystem II reaction centers.     

A theory of excitation transfer in photosynthetic units

A theory of the excitation kinetics in the bacteria photosynthetic unit with regard to its globular structure is presented. It assumes that the excitation transfer between globulae is carried out by means of the mechanism of incoherent excitons, at the same time considering the finite time of the excitation fixation in the reaction center. A method of local perturbations is used with a view to finding a solution to the given problem. The expressions obtained for the fluorescence decay time and its quantum yield are discussed in connection with the multiple experiments considering the cubic as well as the hexagonal probable structure of the photosynthetic unit. The analysis given shows that the period of the excitation transfer between globulae equals 10 to 100 psec and the number of the globulae is less than 35. These conclusions fall in with the initial assumption of the energy transfer between globulae by incoherent excitons. Without considering the globularity, the consistency of the theory with experimental data becomes difficult.     

A perturbed two-level model for exciton trapping in small photosynthetic systems.

The study of exciton trapping in photosynthetic systems provides significant information about migration kinetics within the light harvesting antenna (LHA) and the reaction center (RC). We discuss two random walk models for systems with weakly coupled pigments, with a focus on the application to small systems (10-40 pigments/RC). Details of the exciton transfer to and from the RC are taken into consideration, as well as migration within the LHA and quenching in the RC. The first model is obtained by adapting earlier local trap models for application to small systems. The exciton lifetime is approximated by the sum of three contributions related to migration in the LHA, trapping by the RC, and quenching within the RC. The second model is more suitable for small systems and regards the finite rate of migration within the LHA as a perturbation of the simplified model, where the LHA and the RC are each represented by a single pigment level. In this approximation, the exciton lifetime is the sum of a migration component and a single nonlinear expression for the trapping and quenching of the excitons. Numerical simulations demonstrate that both models provide accurate estimates of the exciton lifetime in the intermediate range of 20-50 sites/RC. In combination, they cover the entire range of very small to very large photosynthetic systems. Although initially intended for regular LHA lattices, the models can also be applied to less regular systems. This becomes essential as more details of the structure of these systems become available. Analysis with these models indicates that the excited state decay in LH1 is limited by the average rate at which excitons transfer to the RC from neighboring sites in the LHA. By comparing this to the average rate of transfer within the LHA, various structural models that have been proposed for the LH1 core antenna are discussed.     

Coupling of exciton motion in the core antenna and primary charge separation in the reaction center

The relation between exciton motion in the LH1 antenna and primary charge separation in the reaction center of purple bacteria is briefly reviewed. It is argued that in models based on hopping excitons described strictly by Förster theory, transfer-to-trap-limited kinetics is quite unlikely according to the relation between the exciton trapping kinetics and N, the size of the photosynthetic unit in such models. Because the results of several recent experiments have been interpreted in terms of transfer-to-trap limited kinetics, this presents a conflict between these experimental interpretations and strictly Förster-based theoretical models. Two possible resolutions are proposed. One arises from the random phase-redistribution trapping kinetics of partially coherent excitons, a kinetics uniquely independent of both N and the rate constant for primary charge separation in the reaction center. The other comes from multiple-pathways models of the multipicosecond nonexponentiality of the decay of P^*, the electronically excited primary electron donor in the reaction center. In these models, because it depends only on a certain averaged electron-transfer time constant, the exciton lifetime may be relatively insensivive to variations of individual electrontransfer rate constants-thereby undercutting the argument appearing in recent literature that by default the exciton kinetics must be transfer-to-trap limited.     

Effect of photosystem II reaction center closure on nanosecond fluorescence relaxation kinetics

The fluorescence decay of chlorophyll in spinach thylakoids was measured as a function of the degree of closure of Photosystem II reaction centers, which was set for the flowed sample by varying either the preillumination by actinic light or the exposure of the sample to the exciting pulsed laser light. Three exponential kinetic components originating in Photosystem II were fitted to the decays; a fourth component arising from Photosystem I was determined to be negligible at the emission wavelength of 685 nm at which the fluorescence decays were measured. Both the lifetimes and the amplitudes of the components vary with reaction center closure. A fast (170–330 ps) component reflects the trapping kinetics of open Photosystem II reaction centers capable of reducing the plastoquinone pool; its amplitude decreases gradually with trap closure, which is incompatible with the concept of photosynthetic unit connectivity where excitation energy which encounters a closed trap can find a different, possibly open one. For a connected system, the amplitude of the fast fluorescence component is expected to remain constant. The slow component (1.7–3.0 ns) is virtually absent when the reaction centers are open, and its growth is attributable to the appearance of closed centers. The middle component (0.4–1.7 ns) with approximately constant amplitude may originate from centers that are not functionally linked to the plastoquinone pool. To explain the continuous increase in the lifetimes of all three components upon reaction center closure, we propose that the transmembrane electric field generated by photosynthetic turnover modulates the trapping kinetics in Photosystem II and thereby affects the excited state lifetime in the antenna in the trap-limited case.Abbreviations DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - HEPES 4-(2-hydroxyethyl)-1-piperazineethane sulfonic acid - PQ plastoquinone - PSI and PSII Photosystem I and II - Q_A and Q_B primary and secondary quinone acceptor of PSII     

The site of regulation of light capture in Symbiodinium: Does the peridinin–chlorophyll a–protein detach to regulate light capture?

Dinoflagellates from the genus Symbiodinium form symbiotic associations with cnidarians including corals and anemones. The photosynthetic apparatuses of these dinoflagellates possess a unique photosynthetic antenna system incorporating the peridinin–chlorophyll a–protein (PCP). It has been proposed that the appearance of a PCP-specific 77 K fluorescence emission band around 672–675 nm indicates that high light treatment results in PCP dissociation from intrinsic membrane antenna complexes, blocking excitation transfer to the intrinsic membrane-bound antenna complexes, chlorophyll a–chlorophyll c₂–peridinin–protein-complex (acpPC) and associated photosystems (Reynolds et al., 2008 Proc Natl Acad Sci USA 105:13674–13678).We have tested this model using time-resolved fluorescence decay kinetics in conjunction with global fitting to compare the time-evolution of the PCP spectral bands before and after high light exposure. Our results show that no long-lived PCP fluorescence emission components appear either before or after high light treatment, indicating that the efficiency of excitation transfer from PCP to membrane antenna systems remains efficient and rapid even after exposure to high light. The apparent increased relative emission at around 675 nm was, instead, caused by strong preferential exciton quenching of the membrane antenna complexes associated with acpPC and reaction centers. This strong non-photochemical quenching (NPQ) is consistent with the activation of xanthophyll-associated quenching mechanisms and the generally-observed avoidance in nature of long-lived photoexcited states that can lead to oxidative damage. The acpPC component appears to be the most strongly quenched under high light exposure suggesting that it houses the photoprotective exciton quencher.     

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     

Energy trapping and detrapping by wild type and mutant reaction centers of purple non-sulfur bacteria

Time-correlated single photon counting was used to study energy trapping and detrapping kinetics at 295 K in Rhodobacter sphaeroides chromatophore membranes containing mutant reaction centers. The mutant reaction centers were expressed in a background strain of Rb. sphaeroides which contained only B880 antenna complexes and no B800-850 antenna complexes. The excited state decay times in the isolated reaction centers from these strains were previously shown to vary by roughly 15-fold, from 3.4 to 52 ps, due to differences in the charge separation rates in the different mutants (Allen and Williams (1995) J Bioenerg Biomembr 27: 275–283). In this study, measurements were also performed on wild type Rhodospirillum rubrum and Rb. sphaeroides B880 antenna-only mutant chromatophores for comparison. The emission kinetics in membranes containing mutant reaction centers was complex. The experimental data were analyzed in terms of a kinetic model that involved fast excitation migration between antenna complexes followed by reversible energy transfer to the reaction center and charge separation. Three emission time constants were identified by fitting the data to a sum of exponential decay components. They were assigned to trapping/quenching of antenna excitations by the reaction center, recombination of the P⁺H^– charge-separated state of the reaction center reforming an emitting state, and emission from uncoupled antenna pigment-protein complexes. The first varied from 60 to 160 ps, depending on the reaction center mutation; the second was 200–300 ps, and the third was about 700 ps. The observed weak linear dependence of the trapping time on the primary charge separation time, together with the known sub-picosecond exciton migration time within the antenna, supports the concept that it is energy transfer from the antenna to the reaction center, rather than charge separation, that limits the overall energy trapping time in wild type chromatophores. The component due to charge recombination reforming the excited state is minor in wild type membranes, but increases substantially in mutants due to the decreasing free energy gap between the states P^* and P⁺H^–.Abbreviations PSU photosynthetic unit - Bchl bacteriochlorophyll - Bphe bacteriopheophytin - P reaction center primary electron donor - RC reaction center - Rb. Rhodobacter - Rs. Rhodospirillum - EDTA (ethylenediamine)tetraacetic acid - Tris tris(hydroxymethyl)aminomethane Author for correspondence     

Mechanisms of chlorophyll fluorescence revisited: Prompt or delayed emission from photosystem II with closed reaction centers?

This paper proposes a model which correlates the exciton decay kinetics observed in picosecond fluorescence studies with the primary processes of charge separation in the reaction center of photosystem II. We conclude that the experimental results from green algae and chloroplasts from higher plants are inconsistent with the concept that delayed luminescence after charge recombination should account for the long-lived (approx. 2 ns) fluorescence decay component of closed photosystem II centers. Instead, we show that the experimental data are in agreement with a model in which the long-lived fluorescence is also prompt fluorescence. The model suggests furthermore that the rate constant of primary charge separation is regulated by the oxidation state of the quinone acceptor Q_A.     

A master equation theory of fluorescence induction, photochemical yield, and singlet-triplet exciton quenching in photosynthetic systems.

A master equation theory is formulated to describe the dependence of the fluorescence yield (phi) in photosynthetic systems on the number of photons (Y) absorbed per photosynthetic unit (or domain). This theory is applied to the calculation of the dependence of the fluorescence yield on Y in (a) fluorescence induction, and (b) singlet exciton-triplet excited-state quenching experiments. In both cases, the fluorescence yield depends on the number of previously absorbed photons per domain, and thus evolves in a nonlinear manner with increasing Y. In case a, excitons transform the photosynthetic reaction centers from a quenching state to a nonquenching state, or a lower efficiency of quenching state; subsequently, absorbed photons have a higher probability of decaying by radiative pathways and phi increases as Y increases. In case b, ground-state carotenoid molecules are converted to long-lived triplet excited-state quenchers, and phi decreases as Y increases. It is shown that both types of processes are formally described by the same theoretical equations that relate phi to Y. The calculated phi (Y) curves depend on two parameters m and R, where m is the number of reaction centers (or ground-state carotenoid molecules that can be converted to triplets), and R is the ratio phi (Y leads to infinity)/(Y leads to 0). The finiteness of the photosynthetic units is thus taken into account. The m = 1 case corresponds to the "puddle" model, and m leads to infinity to the "lake," or matrix, model. It is shown that the experimental phi (Y) curves for both fluorescence induction and singlet-triplet exciton quenching experiments are better described by the m leads to infinity cases than the m = 1 case.     

Band Structure and Local Dynamics of Excitons in Bacterial Light-harvesting Complexes Revealed by Spectrally Selective Spectroscopy

Hole-burned absorption and line-narrowed fluorescence spectra are studied at 5 K in wild type and mutant LH1 and LH2 antenna preparations from the photosynthetic purple bacterium Rhodobacter sphaeroides. Evidence was found in all samples, even in intact membranes, of the presence of a broad distribution of bacteriochlorophyll species that are unable to communicate energy between each other and to the exciton states of functional antenna complexes. The distribution maximum of these localized species determined by zero phonon hole action spectroscopy is at 783.5 nm in purified LH1 complexes and at 786.8 nm in B850-only mutant LH2 complexes. A well-resolved peak at 807 nm in LH1 complexes is assigned to the exciton band structure of functional core antenna complexes. Similar structure in LH2 complexes overlaps with the distribution of localized species. Off-diagonal (structural) disorder may be responsible for this exciton band structure. Our data also imply that pair-wise inter-chlorophyll couplings determine the resonance fluorescence lineshape of excitonic polarons.     

Addition of lipid to the photosynthetic membrane: effects on membrane structure and energy transfer

We have carried out a series of experiments in which the lipid composition of the photosynthetic membrane has been altered by the addition of lipid from a defined source under experimental conditions. Liposomes prepared by sonication are mixed with purified photosynthetic membranes obtained from spinach chloroplasts and are taken through cycles of freezing and thawing. Several lines of evidence, including gel electrophoresis and freeze-fracture electron microscopy, indicate that an actual addition of lipid has taken place. Structural analysis by freeze-fracture shows that intramembrane particles are widely separated after the addition of large amounts of lipid, with one exception: large hexagonal lattices of particles appear in some regions of the membrane. These lattices are identical in appearance with lattices formed from a single purified component of the membrane known as chlorophyll-protein complex II. The suggestion that the presence of such lattices in lipid-enriched membranes reflects a profound rearrangement of photosynthetic structures has been confirmed by analysis of the fluorescence emission spectra of natural and lipid- enriched membranes. Specifically, lipid addition in each of the cases we have studied results in the apparent detachment of chlorophyll- protein complex II from photosynthetic reaction centers. It is concluded that specific arrangements of components in the photosynthetic membrane, necessary for the normal functioning of the membrane in the light reaction of photosynthesis, can be regulated to a large extent by the lipid content of the membrane.     

Chirality-based signatures of local protein environments in two-dimensional optical spectroscopy of two species photosynthetic complexes of green sulfur bacteria: simulation study

Two-dimensional electronic chirality-induced signals of excitons in the photosynthetic Fenna-Matthews-Olson complex from two species of green sulfur bacteria (Chlorobium tepidum and Prosthecochloris aestuarii) are compared. The spectra are predicted to provide sensitive probes of local protein environment of the constituent bacteriochlorophyll a chromophores and reflect electronic structure variations (site energies and couplings) of the two complexes. Pulse polarization configurations are designed that can separate the coherent and incoherent exciton dynamics contributions to the two-dimensional spectra.     

Functional and structural organization of chlorophyll in the developing photosynthetic membranes of Euglena gracilis Z. II. Formation of system II photosynthetic units during greening under optimal light intensity.

The relationships between light-harvesting chlorophyll and reaction centers in Photosystem II were analyzed during the chloroplast development of dark-grown, non-dividing Euglena gracilis Z. Comparative measurements included light saturation of photosynthesis, oxygen evolution under flashing-light and fluorescence induction. The results obtained can be summarized as follows: (1) Photosystem II photocenters are formed in parallel with chlorophyll synthesis, but after a long lag phase. (2) As a consequence, the chlorophyll reaction center ratio (Emerson's type photosynthetic unit) decreases during greening. (3) This decrease is accompanied by considerable changes in the energy transfer and trapping properties of Photosystem II. Most of the initially synthesized chlorophylls are inactive in the transfer of excitations to active photochemical centers and are shared among newly formed Photosystem II photocenters; as a consequence, the number of chlorophylls functionally connected to each Photosystem II photocenter decreases and cooperatively between these centers appears. Results are discussed in terms of chlorophyll organization in developing photosynthetic membranes with reference to the lake or puddle models of photosynthetic unit organization.     

Functional and structural organization of chlorophyll in the developing photosynthetic membranes of Euglena gracilis Z. I. Formation of System II photosynthetic units during greening under optimal light intensity

The relationships between light-harvesting chlorophyll and reaction centers in Photosystem II were analyzed during the chloroplast development of dark-grown, non-dividing Euglena gracilis Z. Comparative measurements included light saturation of photosynthesis, oxygen evolution under flashing-light and fluorescence induction. The results obtained can be summarized as follows: (1) Photosystem II photocenters are formed in parallel with chlorophyll synthesis, but after a longer lag phase. (2) As a consequence, the chlorophyll: reaction center ratio (Emerson's type photosynthetic unit) decreases during greening. (3) This decrease is accompanied by considerable changes in the energy transfer and trapping properties of Photosystem II. Most of the initially synthesized chlorophylls are inactive in the transfer of excitations to active photochemical centers and are shared among newly formed Photosystem II photocenters; as a consequence, the number of chlorophylls functionally connected to each Photosystem II photocenter decreases and cooperativity between these centers appears. Results are discussed in terms of chlorophyll organization in developing photosynthetic membranes with reference to the lake or puddle models of photosynthetic unit organization.