Pair Equilibrium Model Research Articles

Variable fluorescence of closed photochemical reaction centers.

Chlorophyll fluorescence induction during 0.4 to 200ms multiple-turnover pulses (MTP) was measured in parallel with O2 evolution induced by the MTP light. Additionally, a saturating single-turnover flash (STF) was applied at the end of each MTP and the total MTP +STF O2 evolution was measured. Quantum yield of O2 evolution during the MTP transients was calculated and related to the number of open PSII centers, found from the STF O2 evolution. Proportionality between the number of open PSII and their running photochemical activity showed the quantum yield of open PSII remained constant independent of the closure of adjacent centers. During the induction, total fluorescence was partitioned between Fo of all the open centers and Fc of all the closed centers. The fluorescence yield of a closed center was 0.55 of the final Fm while less than a half of the centers were closed, but later increased, approaching Fm to the end of the induction. In the framework of the antenna/radical pair equilibrium model, the collective rise of the fluorescence of centers closed earlier during the induction is explained by an electric field, facilitating return of excitation energy from the Pheo- P680+ radical pair to the antenna.

On the relationship between non-photochemical quenching and photoprotection of Photosystem II

Non-photochemical quenching (NPQ) of chlorophyll fluorescence is thought to be an indicator of an essential regulation and photoprotection mechanism against high-light stress in photosynthetic organisms. NPQ is typically characterized by modulated pulse fluorometry and it is often assumed implicitly to be a good proxy for the actual physiological photoprotection capacity of the organism. Using the results of previously published ultrafast fluorescence measurements on intact leaves of w.t. and mutants of Arabidopsis (Holzwarth et al. 2009) we have developed exact relationships for the fluorescence quenching and the corresponding Photosystem II acceptor side photoprotection effects under NPQ conditions. The approach based on the exciton–radical pair equilibrium model assumes that photodamage results from triplet states generated in the reaction center. The derived relationships allow one to distinguish and determine the individual and combined quenching as well as photoprotection contributions of each of the multiple NPQ mechanisms. Our analysis shows inter alia that quenching and photoprotection are not linearly related and that antenna detachment, which can be identified with the so-called qE mechanism, contributes largely to the measured fluorescence quenching but does not correspond to the most efficient photoprotective response. Conditions are formulated which allow simultaneously the maximal photosynthetic electron flow as well as maximal acceptor side photoprotection. It is shown that maximal photoprotection can be achieved if NPQ is regulated in such a way that PSII reaction centers are open under given light conditions. The results are of fundamental importance for a proper interpretation of the physiological relevance of fluorescence-based NPQ data.

Light emission originating from photosystem II radical pair recombination is sensitive to zeaxanthin related non-photochemical quenching (NPQ)

We have used chlorophyll fluorescence, delayed luminescence and thermoluminescence measurements to study the influence of an artificial ΔpH in the presence or absence of zeaxanthin on photosystem II reactions. Energization of the pea thylakoid membranes induced non-photochemical fluorescence quenching and an increase in the overall luminescence emission of PSII during delayed luminescence and thermoluminescence measurements. This ΔpH-induced overall luminescence increase was caused by a strongly enhanced delayed luminescence in the seconds range before sample heating. In the subsequent thermoluminescence measurements the intensity of the B-band decreased after one and increased after two or more single turnover flashes. We propose that strong membrane energization shifted the redox potential of photosystem II radical pairs to more negative values causing the high delayed luminescence. The zeaxanthin-dependent non-photochemical fluorescence quenching component, however, did not alter thermoluminescence B-bands but decreased the delayed luminescence intensity by 30%. To our knowledge this is the first report that the radiative radical pair recombination, exhibited as delayed luminescence but not thermoluminescence emission, is sensitive to the antenna located zeaxanthin related non-photochemical fluorescence quenching. Our data can be interpreted within the frame of the exciton/radical pair equilibrium model that describes photosystem II as a shallow trap and incorporates the transfer of energy from the re-excitated reaction centre to the antenna of photosystem II.

Charge Separation Kinetics in Intact Photosystem II Core Particles Is Trap-Limited. A Picosecond Fluorescence Study

The fluorescence kinetics in intact photosystem II core particles from the cyanobacterium Thermosynechococcus elongatus have been measured with picosecond resolution at room temperature in open reaction centers. At least two new lifetime components of approximately 2 and 9 ps have been resolved in the kinetics by global analysis in addition to several known longer-lived components (from 42 ps to approximately 2 ns). Kinetic compartment modeling yields a kinetic description in full agreement with the one found recently by femtosecond transient absorption spectroscopy [Holzwarth et al. (2005) submitted to Proc. Natl. Acad. Sci. U.S.A.]. We have for the first time resolved directly the fluorescence spectrum and the kinetics of the equilibrated excited reaction center in intact photosystem II and have found two early radical pairs before the electron is transferred to the quinone Q(A). The apparent lifetime for primary charge separation is 7 ps, that is, by a factor of 8-12 faster than assumed on the basis of earlier analyses. The main component of excited-state decay is 42 ps. The effective primary charge separation rate constant is 170 ns(-)(1), and the secondary electron-transfer rate constant is 112 ns(-)(1). Both electron-transfer steps are reversible. Electron transfer from pheophytin to Q(A) occurs with an apparent overall lifetime of 350 ps. The energy equilibration between the CP43/CP47 antenna and the reaction center occurs with a main apparent lifetime of approximately 1.5 ps and a minor 10 ps lifetime component. Analysis of the overall trapping kinetics based on the theory of energy migration and trapping on lattices shows that the charge separation kinetics in photosystem II is extremely trap-limited and not diffusion-to-the-trap-limited as claimed in several recent papers. These findings support the validity of the assumptions made in deriving the earlier exciton radical pair equilibrium model [Schatz, G. H., Brock, H., and Holzwarth, A. R. (1988) Biophys. J. 54, 397-405].

An evaluation of the potential triggers of photoinactivation of photosystem II in the context of a Stern-Volmer model for downregulation and the reversible radical pair equilibrium model.

Photoinactivation of photosystem II (PS II) is a light-dependent process that frequently leads to break-down and replacement of the D1 polypeptide. Photoinhibition occurs when the rate of photoinactivation is greater than the rate at which D1 is replaced and results in a decrease in the maximum efficiency of PS II photochemistry. Downregulation, which increases non-radiative decay within PS II, also decreases the maximum efficiency of PS II photochemistry and plays an important role in protecting against photoinhibition by reducing the yield of photoinactivation. The yield of photoinactivation has been shown to be relatively insensitive to photosynthetically active photon flux density (PPFD). Formation of the P680 radical (P680+), through charge separation at PS II, generation of triplet-state P680 (3P680*), through intersystem crossing and charge recombination, and double reduction of the primary stable electron acceptor of PS II (the plastoquinone, Q(A)) are all potentially critical steps in the triggering of photoinactivation. In this paper, these processes are assessed using fluorescence data from attached leaves of higher plant species, in the context of a Stern-Volmer model for downregulation and the reversible radical pair equilibrium model. It is shown that the yield of P680+ is very sensitive to PPFD and that downregulation has very little effect on its production. Consequently, it is unlikely to be the trigger for photoinactivation. The yields of 3P680* generated through charge recombination or intersystem crossing are both less sensitive to PPFD than the yield of P680+ and are both decreased by down regulation. The yield of doubly reduced Q(A) increases with incident photon flux density at low levels, but is relatively insensitive at moderate to high levels, and is greatly decreased by downregulation. Consequently, 3P680* and doubly reduced Q(A) are both viable as triggers of photoinactivation.

Characterization of the fast and slow reversible components of non-photochemical quenching in isolated pea thylakoids by picosecond time-resolved chlorophyll fluorescence analysis.

The fast and slow reversible components of non-photochemical chlorophyll fluorescence quenching commonly assigned to the qE and the qI mechanism have been studied in isolated pea thylakoids which were prepared from leaves after a moderate photoinhibitory treatment. Chlorophyll fluorescence decays were measured at picosecond resolution and analyzed on the basis of the heterogeneous exciton/radical pair equilibrium model. Our results show that the fast reversible non-photochemical quenching is completely assigned to the PS II antenna and is related to zeaxanthin. The slow reversible qI type quenching is located at the PS II reaction center and involves enhanced nonradiative decay of the primary charge separated state to its ground state and/or triplet excited state. Apart from its independence from the proton gradient, the qI quenching shows striking similarities to a particular form of qE quenching which is also located at the PS II reaction center and has resently been resolved in isolated thylakoids from dark-adapted leaves [Wagner, B., et al. (1996) J. Photochem. Photobiol., B 36, 339-350]. Our data suggest that during exposure to the supersaturating light the reaction center qE component was replaced by qI quenching. This qE to qI transition is supposed to be part of the mechanism of the long-term downregulation of PS II during photoinhibition. It is also evident that under the conditions used in our study zeaxanthin-dependent antenna quenching is not involved in the slow reversible downregulation of PS II but that it retains its dependence on the proton gradient during exposure to strong light.

Nonphotochemical quenching of excitation energy in photosystem II. A picosecond time-resolved study of the low yield of chlorophyll a fluorescence induced by single-turnover flash in isolated spinach thylakoids.

Chlorophyll a fluorescence emission is widely used as a noninvasive measure of a number of parameters related to photosynthetic efficiency in oxygenic photosynthetic organisms. The most important component for the estimation of photochemistry is the relative increase in fluorescence yield between dark-adapted samples which have a maximal capacity for photochemistry and a minimal fluorescence yield (F0) and light-saturated samples where photochemistry is saturated and fluorescence yield is maximal (Fm). However, when photosynthesis is saturated with a short (less than 50 micro(s)) flash of light, which induces only one photochemical turnover of photosystem II, the maximal fluorescence yield is significantly lower (Fsat) than when saturation is achieved with a millisecond duration multiturnover flash (Fm). To investigate the origins of the difference in fluorescence yield between these two conditions, our time-resolved fluorescence apparatus was modified to allow collection of picosecond time-resolved decay kinetics over a short time window immediately following a saturating single-turnover flash (Fsat) as well as after a multiturnover saturating pulse (Fm). Our data were analyzed with a global kinetic model based on an exciton radical pair equilibrium model for photosystem II. The difference between Fm and Fsat was modeled well by changing only the rate constant for quenching of excitation energy in the antenna of photosystem II. An antenna-based origin for the quenching was verified experimentally by the observation that addition of the antenna quencher 5-hydroxy-1,4-naphthoquinone to thylakoids under Fm conditions resulted in decay kinetics and modeled kinetic parameters very similar to those observed under Fsat conditions in the absence of added quinone. Our data strongly support the origin of low fluorescence yield at Fsat to be an antenna-based nonphotochemical quenching of excitation energy in photosystem II which has not usually been considered explicitly in calculations of photochemical and nonphotochemical quenching parameters. The implications of our data with respect to kinetic models for the excited-state dynamics of photosystem II and the practical applications of the fluorescence yield parameters Fm and Fsat to calculations of photochemical yield are discussed.

Unifying model for the photoinactivation of Photosystem II in vivo under steady-state photosynthesis

We present a unifying mechanism for photoinhibition based on current obsevations from in vivo studies rather than from in vitro studies with isolated thylakoids or PS II membranes. In vitro studies have limited relevance for in vivo photoinhibition because very high light is used with photon exposures rarely encountered in nature, and most of the multiple, interacting, protective strategies of PS II regulation in living cells are not functional. It is now established that the photoinactivation of Photosystem II in vivo is a probability and light-dosage event which depends on the photons absorbed and not the irradiance per se. As the reciprocity law is obeyed and target theory analysis strongly suggests that only one photon is required, we propose that a single dominant molecular mechanism occurs in vivo with one photon inactivating PS II under limiting, saturating or sustained high light. Two mechanisms have been proposed for photoinhibition under high light, acceptor-side and donor-side photoinhibition [see Aro et al. (1994) Biochim Biophys Acta 1143: 113–134], and another mechanism for very low light, the low-light syndrome [Keren et al. (1995) J Biol Chem 270: 806–814]. Based on the exciton-radical pair equilibrium model of exciton dynamics, we propose a unifying mechanism for the photoinactivation of PS II in vivo under steady-state photosynthesis that depends on the generation and maintenance of increased concentrations of the primary radical pair, P680+Pheo−, and the different ways charge recombination is regulated under varying environmental conditions [Anderson et al. (1997) Physiol Plant 100: 214–223]. We suggest that the primary cause of damage to D1 protein is P680+, rather than singlet O2 formed from triplet P680, or other reactive oxygen species.

Picosecond time-resolved study on the nature of high-energy-state quenching in isolated pea thylakoids different localization of zeaxanthin dependent and independent quenching mechanisms

The influence of the transthylakoid proton gradient on the kinetics of picosecond fluorescence decay was examined using isolated pea thylakoids having high or low zeaxanthin contents. Fluorescence lifetime measurements were performed with open (Fo) and closed (Fm) PS II reaction centers. Zeaxanthin formation in membrane energized isolated thylakoids led to a marked decrease of the average fluorescence lifetime at both Fm and Fo. In contrast, when zeaxanthin synthesis was blocked by the inhibitor DTT, the fluorescence lifetime decrease was less pronounced in the Fm state and totally missing in the Fo state. Samples containing the uncoupler ammonium chloride did not exhinit any zeaxanthin influence on the fluorescence decay kinetics. By detailed kinetic analysis of the fluorescence data based on the exciton/radical pair equilibrium model it was possible to separately locate and quantify the effects of zeaxanthin, on the one hand, and the proton gradient, on the other hand, in terms of rate constants of individual primary processes within PS II. It is shown that the enhanced non-photochemical fluorescence quenching (NPQ) in the presence of zeaxanthin mainly originates in the antenna, while without zeaxanthin smaller changes in the Fm state are owing to changes in processes located at the reaction center. Possible mechanisms of zeaxanthin dependent and independent nonphotochemical fluorescence quenching in open and closed PS II are discussed.

On the role of exchangeable hydrogen bonds for the kinetics of P680 +. Q A−. formation and P680 +. Pheo −. recombination in photosystem II

Possible effects of the hydrogen bond network on the reactions leading to the stabilized ion-radical pair P680 +. Q A −. formation and P680 +. Pheo −. recombination in PS II were analyzed by introducing modifications due to replacement of H 2O by D 2O or the addition of cryoprotectants. The rate constants of primary charge separation and subsequent stabilization were derived from measurements of time resolved fluorescence decay curves in PS II membrane fragments with reaction centers kept in the open state by addition of K 3[Fe(CN) 6]. The numerical analysis of the experimental data was performed on the basis of the exciton radical pair equilibrium model proposed by Holzwarth and coworkers (Schatz, G. et al. (1988) Biophys. J. 54, 397–405). Recombination kinetics of the radical pair P680 +. Pheo −. were measured using D1-D2-cyt b559 preparations resuspensed in either H 2O or D 2O. It was found that: (a) the molecular rate constants k PC of the primary charge separation and that of the subsequent stabilization step, k stab, were about (3 ps) −1 and (300 ps) −1, respectively; (b) these rate constants were only slightly affected by D 2O or cryoprotectants; (c) small H/D isotope exchange effects were also found in PS II core complexes and Tris-washed PS II membrane fragments; (d) the recombination kinetics of the P680 +. Pheo −. radical pair in D1-D2-cyt b559 preparations is almost invariant to replacement of H 2O by D 2O. The results obtained reveal that in PS II the charge separation and recombination processes are only weakly dependent on the network of hydrogen bridges with exchangeable protons, in contrast to pronounced effects observed in anoxygenic purple bacteria (Paschenko et al. (1987) FEBS Lett. 214, 28–34). The measured differences between both types of reaction centers are discussed.

Cu(II)-inhibitory effect on photosystem II from higher plants. A picosecond time-resolved fluorescence study.

The influence of Cu(II) inhibition on the primary reactions of photosystem II (PSII) electron transport was studied by picosecond time-resolved fluorescence on isolated PSII membranes. The fluorescence decay from Cu(II)-inhibited PSII centers showed a dominant amplitude of a fast phase (100-300 ps) similar to PSII centers in the uninhibited "open state" and minor contributions of components around 600 ps and 2.6 ns. These data indicate efficient primary charge separation in PSII membranes incubated with Cu(II). The quantum yield of primary reactions in the inhibited PSII centers was similar to that of "open" PSII centers. Kinetic analysis of the decay curves in the framework of the exciton/radical pair equilibrium model showed no significant changes in the rate constants associated with the charge separation/recombination equilibrium. However, in closed centers (QA reduced), a decrease in the rate constant K23, associated with the back-reaction of a relaxed radical pair, by a factor of 4 was calculated. The free energy losses upon primary charge separation (delta G1) and during subsequent radical pair relaxation (delta G2) were also determined in Cu(II)-inhibited centers and were compared with uninhibited centers. No changes in the delta G1 values and a significant decrease in the delta G2 values were found as compared with those of control PSII centers in the "closed" state. These data indicate that Cu(II) does not affect primary radical pair formation, but strongly affects the formation of a relaxed radical pair, by neutralizing the negative charge on QA- and eliminating the repulsive interaction between Pheo- and QA- and/or by modifying the general dielectric properties of the protein region, surrounding these cofactors. Moreover, a close attractive interaction between Pheo-, QA-, and Cu2+ can be proposed. Our results are in good agreement with very recent EPR results indicating an additional effect of Cu2+ on the acceptor side [Jegerschöld et al. (1995) Biochemistry 34, 12747-12758].

Picosecond time-resolved fluorescence studies on photoinhibition and double reduction of Q A in photosystem II

The influence of double reduction of Q A, the secondary electron acceptor in Photosystem (PS) II, on the primary events of PS II electron transport was studied by picosecond time-resolved fluorescence measurements in isolated PS II membranes. The double reduction was achieved either by chemical treatment or by strong anaerobic illumination. In the presence of doubly reduced Q A fluorescence decay from the PS II reaction centres showed a dominant amount of a fast phase (150–250 ps) similar to open PS II. In addition to two further components of 600 ps and 2–3 ns, a long-lived component of about 10 ns was observed which is characteristic of the doubly reduced state only. The data indicate efficient primaru charge separation as well as the presence of a long-lived radical pair when Q A is doubly reduced. From these results it is concluded that the electrostatic effect of the two electrons on Q A 2−, which is expected to strongly suppress primary charge separation, is neutralized, most likely by protonation at or near the Q A site. Analysis of the decay curves in the framework of the exciton/radical pair equilibrium model indicates the formation of a relaxed radical pair state. Fluorescence quenching due to aerobic photoinhibition was shown to arise from the increase of a fast decaying (300–320 ps) component at the expense of the more slowly decaying components during the photoinhibitory treatment. No long-lived component in the range of about 10 ns was observed in case of aerobic photoinhibition. It is concluded that, in contrast to the anaerobic photoinhibition, no doubly reduced Q A is accumulated during the aerobic photoinhibitory treatment.

Global target analysis of picosecond chlorophyll fluorescence kinetics from pea chloroplasts: A new approach to the characterization of the primary processes in photosystem II α- and β-units

In this study, we have used the method of target analysis to analyze the ps fluorescence kinetics of pea chloroplasts with open (F(0)) and closed (F(max)) photosystem II (PS II) centers. Extending the exciton/radical pair equilibrium model (Schatz, G. H., H. Brock, and A. R. Holzwarth. 1988. Biophys. J. 54:397-405) to allow for PS II heterogeneity, we show that two types of PS II (labeled alpha and beta) must be accounted for, each pool being characterized by its own set of molecular rate constants within the model. Simultaneous global target analysis of the data at F(0) and F(max) results in a detailed description of the molecular kinetics and energetics of the primary processes in both types of PS II units. This characterization revealed that the PS IIalpha pool accounts for twice as many Chl molecules as PS IIbeta, which suggests a PSIIalpha/PSIIbeta reaction center stoichiometry of close to unity. By extrapolation it is shown that the primary charge separation in hypothetical "isolated" beta reaction centers is slower than in isolated alpha reaction centers: in open centers by a factor of 4 (1/k(1) (int) = 11 vs 2.9 ps), in closed centers by a factor of 2 (1/k(1) (int) = 34 vs 19 ps). Despite this slower charge separation process in PS IIbeta, the quantum efficiency of the charge separation process is hardly affected: a charge stabilization yield at F(0), (i.e., P(+)IQ(A) (-)) of 86% (as compared to 90% in PS IIalpha). Reduction of Q(A) (closing PS II) has distinctly different effects on the primary kinetics of PS IIbeta, as compared to PS IIalpha. In PS IIalpha the charge separation rate drops by a factor of 6, whereas the charge recombination process is hardly affected. In PS IIbeta the charge separation is slowed down by a factor of 3, whereas the charge recombination rate increases by a factor of 5. In terms of changes in standard free energy, the reduction to Q(A) (-) lifts the free energy of the radical pair P(+)I(-), relative to the excited state (Chl(n)/P)(*), by 47 meV in PS IIalpha and by 67 meV in PS IIbeta. The concomitant increase in fluorescence quantum yield is the same for both types of PS II. These results show that PS IIalpha and PS IIbeta exhibit a different molecular functioning with respect to the primary processes, which might have its origin in a different molecular structure of the reaction centers and/or a different local environment of these centers. Location in different parts of the thylakoid membrane might be involved. We also applied different error analysis procedures to determine the error ranges of the values found for the molecular rate constants. It is shown that the commonly used standard error has very little meaning, as it assumes independence of the fit parameters. Instead, an exhaustive search procedure, accounting for all possible correlations between the fit parameters, gives a more realistic view on the accuracy of the fit parameters.