Kinetic and Energetic Model for the Primary Processes in Photosystem II

A detailed model for the kinetics and energetics of the exciton trapping, charge separation, charge recombination, and charge stabilization processes in photosystem (PS) II is presented. The rate constants describing these processes in open and closed reaction centers (RC) are calculated on the basis of picosecond data (Schatz, G. H., H. Brock, and A. R. Holzwarth. 1987. Proc. Natl. Acad. Sci. USA. 84:8414-8418) obtained for oxygen-evolving PS II particles from Synechococcus sp. with ~80 chlorophylls/P₆₈₀. The analysis gives the following results. (a) The PS II reaction center donor chlorophyll P₆₈₀ constitutes a shallow trap, and charge separation is overall trap limited. (b) The rate constant of charge separation drops by a factor of ~6 when going from open (Q-oxidized) to closed (Q-reduced) reaction centers. Thus the redox state of Q controls the yield of radical pair formation and the exciton lifetime in the Chl antenna. (c) The intrinsic rate constant of charge separation in open PS II reaction centers is calculated to be ~2.7 ps^-1. (d) In particles with open RC the charge separation step is exergonic with a decrease in standard free energy of ~38 meV. (e) In particles with closed RC the radical pair formation is endergonic by ~12 meV. We conclude on the basis of these results that the long-lived (nanoseconds) fluorescence generally observed with closed PS II reaction centers is prompt fluorescence and that the amount of primary radical pair formation is decreased significantly upon closing of the RC.     

Tryptophan at position 181 of the D2 protein of photosystem II confers quenching of variable fluorescence of chlorophyll: implications for the mechanism of energy-dependent quenching.

The lumenal CD loop region of the D2 protein of photosystem II contains residues that interact with a reaction center chlorophyll and the redox-active Tyr(D). Using combinatorial mutagenesis, photoautotrophic mutants of Synechocystis sp. PCC 6803 have been generated with multiple amino acid changes in this region. The CD loop mutations were transferred into a photosystem I-less Synechocystis strain to facilitate characterization of photosystem II properties in the mutants. Most of the combinatorial photosystem I-less mutants obtained had a high yield of variable fluorescence, F(V). However, in three mutants, which shared a replacement of Phe181 by Trp, the F(V) yield was dramatically reduced although a high rate of oxygen evolution was maintained. A site-directed F181W D2 mutant shared similar properties. Picosecond time-resolved fluorescence measurements revealed that in the combinatorial F181W mutants the fluorescence lifetimes in closed and open photosystem II centers were essentially identical and were similar to the fluorescence lifetime in open centers of the control strain. These results are explained by quenching of variable fluorescence in the mutants by charge separation between Trp181 and excited reaction center chlorophyll. This reaction competes efficiently with fluorescence and nonradiative decay in closed photosystem II centers, where the lifetime of the excitation in the chlorophyll antenna is long. Thermodynamic considerations favor the formation of oxidized tryptophan and reduced chlorophyll in the quenching reaction, presumably followed by charge recombination. A possible role of tryptophan-chlorophyll charge separation in the mechanism of energy-dependent quenching of excitations in photosynthesis is discussed.     

Delayed fluorescence observed in the nanosecond time region at 77 K originates directly from the photosystem II reaction center

The excited-state dynamics of delayed fluorescence in photosystem (PS) II at 77 K were studied by time-resolved fluorescence spectroscopy and decay analysis on three samples with different antenna sizes: PS II particles and the PS II reaction center from spinach, and the PS II core complexes from Synechocystis sp. PCC 6803. Delayed fluorescence in the nanosecond time region originated from the 683-nm component in all three samples, even though a slight variation in lifetimes was detected from 15 to 25 ns. The relative amplitude of the delayed fluorescence was higher when the antenna size was smaller. Energy transfer from the 683-nm pigment responsible for delayed fluorescence to antenna pigment(s) at a lower energy level was not observed in any of the samples examined. This indicated that the excited state generated by charge recombination was not shared with antenna pigments under the low-temperature condition, and that delayed fluorescence originates directly from the PS II reaction center, either from chlorophyll a(D1) or P680. Supplemental data on delayed fluorescence from spinach PS I complexes are included.     

Functional assembly in vitro of phycobilisomes with isolated photosystem II particles of eukaryotic chloroplasts

Phycobiliproteins obtained by dissociation of phycobilisomes were reassociated in vitro with intact thylakoids or isolated photosystems I and II preparations obtained from cyanophytes (prokaryotes) or green algae (eukaryotes) to form bound phycobilisome complexes. Energy transfer from Fremyella diplosiphon phycobiliproteins to chlorophyll a of reaction centers I and II was measured in: complexes containing intact thylakoids of the cyanophytes F. diplosiphon or Anacystis nidulans and the eukaryotic algae Euglena gracilis and mutants of Chlamydomonas reinhardtii; complexes containing isolated photosystem II particles of A. nidulans or C. reinhardtii; and complexes containing reaction center I of F. diplosiphon or C. reinhardtii. Energy transfer from phycoerythrin to chlorophyll a of photosystem II could be demonstrated in complexes containing phycobilisomes bound to cyanophyte thylakoids or isolated photosystem II particles of A. nidulans or C. reinhardtii. Bound phycobilisomes did not transfer energy to photosystem II within green algae thylakoids containing altered forms of light-harvesting chlorophyll a/b-protein complex (LHC) II antenna, reduced amounts of LHC II, or chlorophyll b, or chlorophyll b-less mutants, nor to chlorophyll a of photosystem I of intact thylakoids or isolated reaction centers. We conclude that phycobilisomes can form a specific and functional association with photosystem II particles of both cyanophytes and eukaryotic thylakoids. This interaction appears to be hindered by the presence of LHC II antenna in the eukaryotic thylakoids.     

The photosystem I trimer of cyanobacteria: molecular organization, excitation dynamics and physiological significance

The photosystem I complex organized in cyanobacterial membranes preferentially in trimeric form participates in electron transport and is also involved in dissipation of excess energy thus protecting the complex against photodamage. A small number of longwave chlorophylls in the core antenna of photosystem I are not located in the close vicinity of P700, but at the periphery, and increase the absorption cross-section substantially. The picosecond fluorescence kinetics of trimers resolved the fastest energy transfer components reflecting the equilibration processes in the core antenna at different redox states of P700. Excitation kinetics in the photosystem I bulk antenna is nearly trap-limited, whereas excitation trapping from longwave chlorophyll pools is diffusion-limited and occurs via the bulk antenna. Charge separation in the photosystem I reaction center is the fastest of all known reaction centers.     

Delayed fluorescence observed in the nanosecond time region at 77 K originates directly from the photosystem II reaction center

The excited-state dynamics of delayed fluorescence in photosystem (PS) II at 77 K were studied by time-resolved fluorescence spectroscopy and decay analysis on three samples with different antenna sizes: PS II particles and the PS II reaction center from spinach, and the PS II core complexes from Synechocystis sp. PCC 6803. Delayed fluorescence in the nanosecond time region originated from the 683-nm component in all three samples, even though a slight variation in lifetimes was detected from 15 to 25 ns. The relative amplitude of the delayed fluorescence was higher when the antenna size was smaller. Energy transfer from the 683-nm pigment responsible for delayed fluorescence to antenna pigment(s) at a lower energy level was not observed in any of the samples examined. This indicated that the excited state generated by charge recombination was not shared with antenna pigments under the low-temperature condition, and that delayed fluorescence originates directly from the PS II reaction center, either from chlorophyll a_D1 or P680. Supplemental data on delayed fluorescence from spinach PS I complexes are included.     

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     

Excitation energy transfer and charge separation in photosystem II membranes revisited

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.     

Pigment organization and energy transfer dynamics in isolated photosystem I (PSI) complexes from Arabidopsis thaliana depleted of the PSI-G, PSI-K, PSI-L, or PSI-N subunit

Green plant photosystem I (PSI) consists of at least 18 different protein subunits. The roles of some of these protein subunits are not well known, in particular those that do not occur in the well characterized PSI complexes from cyanobacteria. We investigated the spectroscopic properties and excited-state dynamics of isolated PSI-200 particles from wild-type and mutant Arabidopsis thaliana plants devoid of the PSI-G, PSI-K, PSI-L, or PSI-N subunit. Pigment analysis and a comparison of the 5 K absorption spectra of the various particles suggests that the PSI-L and PSI-H subunits together bind approximately five chlorophyll a molecules with absorption maxima near 688 and 667 nm, that the PSI-G subunit binds approximately two red-shifted beta-carotene molecules, that PSI-200 particles without PSI-K lack a part of the peripheral antenna, and that the PSI-N subunit does not bind pigments. Measurements of fluorescence decay kinetics at room temperature with picosecond time resolution revealed lifetimes of ~0.6, 5, 15, 50, 120, and 5000 ps in all particles. The 5- and 15-ps phases could, at least in part, be attributed to the excitation equilibration between bulk and red chlorophyll forms, though the 15-ps phase also contains a contribution from trapping by charge separation. The 50- and 120-ps phases predominantly reflect trapping by charge separation. We suggest that contributions from the core antenna dominate the 15-ps trapping phase, that those from the peripheral antenna proteins Lhca2 and Lhca3 dominate the 50-ps phase, and that those from Lhca1 and Lhca4 dominate the 120-ps phase. In the PSI-200 particles without PSI-K or PSI-G protein, more excitations are trapped in the 15-ps phase and less in 50- and 120-ps phases, which is in agreement with the notion that these subunits are involved in the interaction between the core and peripheral antenna proteins.     

Electron transfer in photosystem II

The picture presently emerging from studies on the mechanism of photosystem II electron transport is discussed. The reactions involved in excitation trapping, charge separation and stabilization of the charge pair in the reaction center, followed by the reactions with the substrates, plastoquinone reduction and water oxidation, are described successively. Finally, a brief discussion on photosystem II heterogeneity is presented.     

Influence of the diadinoxanthin pool size on photoprotection in the marine planktonic diatom Phaeodactylum tricornutum

The pool size of the xanthophyll cycle pigment diadinoxanthin (DD) in the diatom Phaeodactylum tricornutum depends on illumination conditions during culture. Intermittent light caused a doubling of the DD pool without significant change in other pigment contents and photosynthetic parameters, including the photosystem II (PSII) antenna size. On exposure to high-light intensity, extensive de-epoxidation of DD to diatoxanthin (DT) rapidly caused a very strong quenching of the maximum chlorophyll fluorescence yield (F(m), PSII reaction centers closed), which was fully reversed in the dark. The non-photochemical quenching of the minimum fluorescence yield (F(o), PSII centers open) decreased the quantum efficiency of PSII proportionally. For both F(m) and F(o), the non-photochemical quenching expressed as F/F' - 1 (with F' the quenched level) was proportional to the DT concentration. However, the quenching of F(o) relative to that of F(m) was much stronger than random quenching in a homogeneous antenna could explain, showing that the rate of photochemical excitation trapping was limited by energy transfer to the reaction center rather than by charge separation. The cells can increase not only the amount of DT they can produce, but also its efficiency in competing with the PSII reaction center for excitation. The combined effect allowed intermittent light grown cells to down-regulate PSII by 90% and virtually eliminated photoinhibition by saturating light. The unusually rapid and effective photoprotection by the xanthophyll cycle in diatoms may help to explain their dominance in turbulent waters.     

Determination of the excitation migration time in Photosystem II consequences for the membrane organization and charge separation parameters

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. R?gner, A.R. Holzwarth, Charge separation kinetics in intact Photosystem II core particles is trap-limited. A picosecond fluorescence study, Biochemistry 45 (2006) 2436-2442].     

Energy and electron transfer in photosystem II of a chlorophyll b-containing Synechocystis sp. PCC 6803 mutant

Using a Synechocystis sp. PCC 6803 mutant strain that lacks photosystem (PS) I and that synthesizes chlorophyll (Chl) b, a pigment that is not naturally present in the wild-type cyanobacterium, the functional consequences of incorporation of this pigment into the PS II core complex were investigated. Despite substitution of up to 75% of the Chl a in the PS II core complex by Chl b, the modified PS II centers remained essentially functional and were able to oxidize water and reduce Q(A), even upon selective excitation of Chl b at 460 nm. Time-resolved fluorescence decay measurements upon Chl excitation showed a significant reduction in the amplitude of the 60-70 ps component of fluorescence decay in open Chl b-containing PS II centers. This may indicate slower energy transfer from the PS II core antenna to the reaction center pigments or slower energy trapping. Chl b and pheophytin b were present in isolated PS II reaction centers. Pheophytin b can be reversibly photoreduced, as evidenced from the absorption bleaching at approximately 440 and 650 nm upon illumination in the presence of dithionite. Upon excitation at 685 nm, transient absorption measurements using PS II particles showed some bleaching at 650 nm together with a major decrease in absorption around 678 nm. The 650 nm bleaching that developed within approximately 10 ps after the flash and then remained virtually unchanged for up to 1 ns was attributed to formation of reduced pheophytin b and oxidized Chl b in some PS II reaction centers. Chl b-containing PS II had a lower rate of charge recombination of Q(A)(-) with the donor side and a significantly decreased yield of delayed luminescence in the presence of DCMU. Taken together, the data suggest that Chl b and pheophytin b participate in electron-transfer reactions in PS II reaction centers of Chl b-containing mutant of Synechocystis without significant impairment of PS II function.     

Trap-limited charge separation kinetics in higher plant photosystem I complexes

Time-resolved fluorescence measurements were performed on isolated core and intact Photosystem I (PS I) particles and stroma membranes from Arabidopsis thaliana to characterize the type of energy-trapping kinetics in higher plant PS I. Target analysis confirms the previously proposed “charge recombination” model. No bottleneck in the energy flow from the bulk antenna compartments to the reaction center has been found. For both particles a trap-limited kinetics is realized, with an apparent charge separation lifetime of ∼6 ps. No red chlorophylls (Chls) are found in the PS I-core complex from A. thaliana. Rather, the observed red-shifted fluorescence (700-710 nm range) originates from the reaction center. In contrast, two red Chl compartments, located in the peripheral light-harvesting complexes, are resolved in the intact PS I particles (decay lifetimes 33 and 95 ps, respectively). These two red states have been attributed to the two red states found in Lhca 3 and Lhca 4, respectively. The influence of the red Chls on the slowing of the overall trapping kinetics in the intact PS I complex is estimated to be approximately four times larger than the effect of the bulk antenna enlargement.     

Kinetics and spectra of photo-induced changes in the absorption of pigment-protein complexes of photosystem 1 in a picosecond range

The energy transfer from the light-harvesting antenna chlorophylls to the reaction center molecules and subsequent charge separation were investigated using a difference picosecond spectrophotometer with selective excitation. The objects were the pigment-protein complexes of photosystem 1 (Chl/P700 = 60) isolated from bean leaves. The difference absorption spectra of the excited states of light-harvesting antenna chlorophylls and the P700 photooxidation were measured. It was shown that the excited states of antenna chlorophylls were generated within 10 ps and deactivated with three-component kinetics: tau 1 = 20--45 ps, tau 2 = 100--300 ps, tau 3 greater than 500 ps. The process of the P700 photooxidation induced by the 650 nm exciting pulse was approximately monoexponential with tau equal to 15--30 ps. It is established that the P700 photooxidation is due to the efficient transfer of excitation energy from antenna chlorophylls to reaction centers.     

Excited state dynamics in photosystem I: effects of detergent and excitation wavelength.

Femtosecond transient absorption spectroscopy has been used to investigate the energy transfer and trapping processes in both intact membranes and purified detergent-isolated particles from a photosystem II deletion mutant of the cyanobacterium Synechocystis sp. PCC 6803, which contains only the photosystem I reaction center. Processes with similar lifetimes and spectra are observed in both the membrane fragments and the detergent-isolated particles, suggesting little disruption of the core antenna resulting from the detergent treatment. For the detergent-isolated particles, three different excitation wavelengths were used to excite different distributions of pigments in the spectrally heterogeneous core antenna. Only two lifetimes of 2.7-4.3 ps and 24-28 ps, and a nondecaying component are required to describe all the data. The 24-28 ps component is associated with trapping. The trapping process gives rise to a nondecaying spectrum that is due to oxidation of the primary electron donor. The lifetimes and spectra associated with trapping and radical pair formation are independent of excitation wavelength, suggesting that trapping proceeds from an equilibrated excited state. The 2.7-4.3 ps component characterizes the evolution from the initially excited distribution of pigments to the equilibrated excited state distribution. The spectrum associated with the 2.7-4.3 ps component is therefore strongly excitation wavelength dependent. Comparison of the difference spectra associated with the spectrally equilibrated state and the radical pair state suggests that the pigments in the photosystem I core antenna display some degree of excitonic coupling.     

Slow exciton trapping in Photosystem II: A possible physiological role

Photosystem II, which has a primary photochemical charge separation time of about 300 ps, is the slowest trapping of all photosystems. On the basis of an analysis of data from the literature this is shown to be due to a number of partly independent factors: a shallow energy funnel in the antenna, an energetically shallow trap, exciton dynamics which are partly trap limited and a large antenna. It is argued that the first three of these properties of Photosystem II can be understood in terms of protective mechanisms against photoinhibition. These protective mechanisms, based on the generation of non photochemical quenching states mostly in the peripheral antenna, are able to decrease pheophytin reduction under conditions in which the primary quinone, Q_A, is already reduced, due to the slow trapping properties. The shallow antenna funnel is important in allowing quenching state-protective mechanisms in the peripheral antenna.Abbreviations chl chlorophyll - PS I Photosystem I - PS II Photosystem II - Q_A the primary quinone acceptor - RC reaction centre - RT room temperature     

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.     

Dynamics of energy transfer and trapping in the light-harvesting antenna of Rhodopseudomonas viridis.

By low intensity picosecond absorption spectroscopy it is shown that the exciton lifetime in the light-harvesting antenna of Rhodopseudomonas (Rps.) viridis membranes with photochemically active reaction centers at room temperature is 60 +/- 10 ps. This lifetime reflects the overall trapping rate of the excitation energy by the reaction center. With photochemically inactive reaction centers, in the presence of P+, the exciton lifetime increases to 150 +/- 15 ps. Prereducing the secondary electron acceptor QA does not prevent primary charge separation, but slows it down from 60 to 90 +/- 10 ps. Picosecond kinetics measured at 77 K with inactive reaction centers indicates that the light-harvesting antenna is spectrally homogeneous. Picosecond absorption anisotropy measurements show that energy transfer between identical Bchlb molecules occurs on the subpicosecond time scale. Using these experimental results as input to a random-walk model, results in strict requirements for the antenna-RC coupling. The model analysis prescribes fast trapping (approximately 1 ps) and an approximately 0.5 escape probability from the reaction center, which requires a more tightly coupled RC and antenna, as compared with the Bchla-containing bacteria Rhodospirillum (R.) rubrum and Rhodobacter (Rb.) sphaeroides.