Studies on the ADRY agent-induced mechanism of the discharge of the holes trapped in the photosynthetic waterspliting enzyme system Y

The effect of 2-(3-chloro-4-trifluoromethyl)anilino-3,5-dinitrothiophene (ANT 2p) on the oxygen evolution, fluorescence and delayed light emission of spinach chloroplasts has been investigated. It was found that;

1. 1. ANT 2p strongly accelerates the deactivation of states S₂ and S₃ of the water-splitting enzyme system Y.

2. 2. In DCMU-poisoned chloroplasts ANT 2p prevents the back reaction of the electrons located at the primary acceptor, Q, with the holes (positive charges) stored in the water-splitting enzyme system Y.

3. 3. In chloroplast suspensions without artificial electron acceptors, the fluorescence rise in weak actinic light vanishes in the presence of ANT 2p. The fluorescence yield in DCMU-inhibited chloroplasts is not significantly changed by ANT 2p.

4. 4. The intensity of the delayed light emitted after excitation with one short flash is remarkably decreased by ANT 2p.

5. 5. In weak actinic light the reduction rate of the artificial electron acceptor methyl viologen is suppressed in the presence of ANT 2p.

From these experimental results it is concluded that ANT 2p induces a cycle within the electron transport chain, leading to a dissipative recombination of the holes stored in the water-splitting enzyme Y with the electrons of an as yet unknown donor.

Two possibilities for the mode of action of this cycle are discussed.     

The influence of cytochrome b559 on the fluorescence yield of chloroplasts at low temperature

The maximum light-induced fluorescence yield, F_M, of spinach chloroplasts at − 196 °C was less when the chloroplasts were oxidized with ferricyanide prior to freezing; the minimum fluorescence yield, F₀, of the dark-adapted chloroplasts at − 196 °C was unaffected. The ratio of the fluorescence yields, F_M/F₀, measured at 695 nm at low temperature was 4.5–5.0 for normal chloroplasts and 2.0–2.5 in the presence of ferricyanide. The oxidative titration curve of F_M followed a 1 electron Nernst equation with a midpoint potential of 365 mV and followed closely to the oxidation of cytochrome b₅₅₉. The photoreduction of C−550 at low temperature was the same at all redox potentials over the range of 200–500 mV. It is suggested that a relatively strong oxidant associated with the water-splitting side of Photosystem II, possibly the primary electron donor, can chlorophyll fluorescence of Photosystem II as well as the primary electron acceptor.     

A light-dependent oxygen consumption induced by photosystem II of isolated chloroplasts.

The photooxidants accumulated on the water-splitting side of photosystem II in chloroplasts are destabilized by certain membrane active chemicals. In the light and in the presence of oxygen, this destabilization results in a consumption of oxygen and in a lowering of the fluorescence emission from the chloroplasts. It is shown that a close correlation exists between the oxygen uptake and the fluorescence lowering, and that with some of the destabilizing agents photosystem I activity is not required for either process. When electron flow through photosystem I is blocked, the oxygen consumption appears to occur without formation of free oxygen-derived radicals. It is concluded that, in the light, a disturbed water-splitting enzyme may initiate oxygen-dependent photooxidations which the superoxide dismutase of chloroplasts cannot protect against. The fluorescence lowering is attributed to either direct quenching actions of oxygenated reaction products or to a cyclic electron flow between reduced electron carriers and such intermediates.     

Possible states of photosystem II reaction center and thermoluminescence of the higher plants. I. Analysis of thermoluminescence models and their applicability to the description of experimental data

A method for the deconvolution of experimental glow curves into overlapping bands and determination of the activation energies for these bands is proposed. The model includes the S-states of the water-splitting complex, tyrozine Z and P680 on the donor side, pheophytine, primary and secondary quinone acceptors on the acceptor side, and takes into account the connection between different states of the reaction center complex. The rate constants of forward electron transport and the activation energies of backward reactions of electron transfer in photosystem II reaction center, included into the model, are estimated from the known experimental data using the proposed approach.     

Multiple effects of cadmium on the photosynthetic apparatus of Avicennia germinans L. as probed by OJIP chlorophyll fluorescence measurements

The toxic effects of cadmium on the photosynthetic apparatus of Avicennia germinans were evaluated by means of the chlorophyll fluorescence transient O-J-I-P. The chlorophyll fluorescence transients were recorded in vivo with high time resolution and analyzed according to the OJIP-test that can quantify the performance of photosystem II. Cadmium-treated plants showed a decrease in yield for primary photochemistry, TR0/ABS. The performance index of photosystem II (PSII), PI(ABS), decreased due to cadmium treatment. This performance index is the combination of the indexes of three independent parameters: (1) total number of active reaction centers per absorption (RC/ABS), (2) yield of primary photochemistry (TR0/ABS), and (3) efficiency with which a trapped exciton can move an electron into the electron transport chain (ET0/TR0). Additionally, the F0/Fv registered the highest sensitivity to the metal, thus indicating that the water-splitting apparatus of the oxidizing side of PSII is the primary site of action of cadmium. In summary, cadmium affects several targets of photosystem II. More specifically the main targets of cadmium, according to the OJIP-test, can be listed as a decrease in the number of active reaction centers and damage to the activity of the water-splitting complex.     

Glu-69 of the D2 protein in photosystem II is a potential ligand to Mn involved in photosynthetic oxygen evolution

To probe the involvement of amino acid residues of the D2 protein in the water-splitting process in photosystem II, site-directed mutagenesis was applied to identify D2 residues that might contribute to binding the Mn cluster involved in oxygen evolution. Mutation of Glu-69 to Gln or Val in D2 of the cyanobacterium Synechocystis sp. PCC 6803 was found to lead to a loss of photoautotrophic growth. However, in cells of the Gln mutant (E69Q) a significant Hill reaction rate could be observed upon the start of illumination, but the oxygen evolution rate declined with a half-time of approximately 1 min. Addition of 1 mM Mn2+ stabilized oxygen evolution in E69Q thylakoids. Other divalent cations were ineffective in specific stabilization. When the water-splitting system was bypassed, the rate of electron transport remained stable during illumination, indicating that the inactivation of oxygen evolution is localized in the water-splitting complex. We interpret these observations to indicate that Glu-69 is a Mn ligand and that the loss of oxygen evolution in the E69Q mutant upon turnover of PS II is initiated by changes in the Mn cluster, possibly leading to Mn release from the water-splitting complex. The addition of exogenous Mn to E69Q thylakoids may help to keep the Mn cluster active for a longer time, perhaps by providing Mn to rebind in the cluster after release of one Mn and before the Mn cluster had disintegrated.(ABSTRACT TRUNCATED AT 250 WORDS)     

Chlorophyll a fluorescence rise induced by high light illumination of dark-adapted plant tissue studied by means of a model of photosystem II and considering photosystem II heterogeneity

Chlorophyll a fluorescence rise (FLR) measured in vivo in dark-adapted plant tissue immediately after the onset of high light continuous illumination shows complex O-K-J-I-P transient. The steps typically appear at about 400 micros (K), 2 ms (J), 30 ms (I), and 200 - 500 ms (P) and a transient decrease of fluorescence to local minima (dips D) can be observed after the K, J, and I steps. As the FLR reflects a function of photosystem II (PSII) and to more understand the FLR, a PSII reactions model was formulated comprising equilibrium of excited states among all light harvesting and reaction centre pigments and P680, reversible radical pair formation and the donor and acceptor side functions. Such a formulated model is the most detailed and complex model of PSII reactions used so far for simulations of the FLR. By varying of selected model parameters (rate constants and initial conditions) several conclusions can be made as for the origin of and changes in shape of the theoretical FLR and compare them with in-literature-reported results. For homogeneous population of PSII and using standard in-literature-reported values of the model parameters, the simulated FLR is characterized by reaching the minimal fluorescence F(0) at about 3 ns after the illumination is switched on lasting to about 1 micros, followed by fluorescence rise to a plateau located at about 2 ms and subsequent fluorescence rise to a global maximum that is reached at about 60 ms. Varying of the values of rate constants of fast processes that can compete for utilization of the excited states with fluorescence emission does not change qualitatively the shape of the FLR. However, primary photochemistry of PSII (the charge separation, recombination and stabilization), non-radiative loss of excited states in light harvesting antennae and excited states quenching by oxidized plastoquisnone (PQ) molecules from the PQ pool seem to be the main factors controlling the maximum quantum yield of PSII photochemistry as expressed by the F(V)/F(M) ratio. The appearance of the plateau at about 2 ms in the FLR is affected by several factors: the height of the plateau in the FLR increases when the fluorescence quenching by oxidized P680(+) is not considered in the simulations or when the electron transfer from Q(A)(-) to Q(B)((-)) is slowed down whereas the height of the plateau decreases and its position is shifted to shorter times when OEC is initially in higher S state. The plateau at about 2 ms is changed into the local fluorescence maximum followed by a dip when the fluorescence quenching by oxidized PQ molecules or the charge recombination between P680(+) and Q(A)(-) is not considered in the simulations or when all OEC is initially in the S(0) state or when the S -state transitions of OEC are slowed down. Slowing down of the S -state transitions of OEC as well as of the electron transfer from Q(A)(-) to Q(B)((-)) also causes a decrease of maximal fluorescence level. In the case of full inhibition of the S -state transitions of OEC as well as in the case of full inhibition of the electron donation to P680(+) by Y(Z), the local fluorescence maximum becomes the global fluorescence maximum. Assuming homogeneous PSII population, theoretical FLR curve that only far resembles experimentally measured O-J-I-P transient at room temperature can be simulated when slowly reducing PQ pool is considered. Assuming heterogeneous PSII population (i.e. the alpha/beta and the Q(B) -reducing/Q(B)-non-reducing heterogeneity and heterogeneity in size of the PQ pool and rate of its reduction) enables to simulate the FLR with two steps between minimal and maximal fluorescence whose relative heights are in agreement with the experiments but not their time positions. A cause of this discrepancy is discussed as well as different approaches to the definition of fluorescence signal during the FLR.     

A partial reaction in photosystem II: reduction of silicomolybdate prior to the site of dichlorophenyldimethylurea inhibition.

Silicomolybdate functions as an electron acceptor in a Photosystem II water oxidation (measured as O2 evolution) partial reaction that is 3-(3,4-dichlorophenyl)-1, 1-dimethylurea (DCMU) insensitive, that is, reduction os silicomolybdate occurs at or before the level of Q, the primary electron acceptor for Photosystem II. This report characterizes the partial reaction with the principal findings being as follows: 1. Electron transport to silicomolybdate significantly decreased room temperature Photosystem I side of the DCMU had no effect on the fluorescence level, consistent with silicomolybdate accepting electrons at or before Q. In the absence of DCMU, silicomolybdate is also reduced at a site on the Photosystem I side of the DCMU block, prior to or at plastoquinone, since the plastoquinone antagonist dibromothymoquinone (DBMIB) did not affect the electron transport rate. 3. Electron transport from water to silicomolybdate (+ DCMU) is not coupled to ATP formation, nor is there a measurable accumulation of protons within the membrane (measured by amine uptake). Silicomolybdate is not inhibitory to phosphorylation per se since neither cyclic nor post-illumination (XE) phosphorylation were inhibited. 4. Uncouplers stimulated electron transport from water to silicomolybdate in the pH range of 6 to 7, but inhibited at pH values near 8. These data are consistent with the view that when electron flow is through the abbreviated sequence of water to Photosystem II to silicomolybdate (+ DCMU), conditions are not established for the water protons to be deposited within the membrane. Experiments reported elsewhere (Fiaquinta, R.T., Dilley, R.A. and Horton, P.(19741 J. Bioenerg. 6, 167-177) and these data, are consistent with the hypothesis that electron transport between Q and plastoquinone energizes a membrane conformational change that is required to interact with the water oxication system so as to result in the deposition of water protons either within the membrane itself or within the inner oxmotic space.     

Evidence for a fluorescence yield change driven by a light-induced conformational change within photosystem II during the fast chlorophyll a fluorescence rise

Experiments were carried out to identify a process co-determining with Q(A) the fluorescence rise between F(0) and F(M). With 3-(3',4'-dichlorophenyl)-1,1-dimethylurea (DCMU), the fluorescence rise is sigmoidal, in its absence it is not. Lowering the temperature to -10°C the sigmoidicity is lost. It is shown that the sigmoidicity is due to the kinetic overlap between the reduction kinetics of Q(A) and a second process; an overlap that disappears at low temperature because the temperature dependences of the two processes differ. This second process can still relax at -60°C where recombination between Q(A)(-) and the donor side of photosystem (PS) II is blocked. This suggests that it is not a redox reaction but a conformational change can explain the data. Without DCMU, a reduced photosynthetic electron transport chain (ETC) is a pre-condition for reaching the F(M). About 40% of the variable fluorescence relaxes in 100ms. Re-induction while the ETC is still reduced takes a few ms and this is a photochemical process. The fact that the process can relax and be re-induced in the absence of changes in the redox state of the plastoquinone (PQ) pool implies that it is unrelated to the Q(B)-occupancy state and PQ-pool quenching. In both +/-DCMU the process studied represents ~30% of the fluorescence rise. The presented observations are best described within a conformational protein relaxation concept. In untreated leaves we assume that conformational changes are only induced when Q(A) is reduced and relax rapidly on re-oxidation. This would explain the relationship between the fluorescence rise and the ETC-reduction.     

Analysis of temperature-jump chlorophyll fluorescence induction in plants.

A newly observed general chlorophyll fluorescence induction effect in plants is described. Fluorescence yield can rise through as many as four different phases (alpha, beta, gamma, ) in the dark, when intact cells or leaves are rapidly heated (within approx. 2.5 s) from 20 to 40-50 degrees C. An analysis of this temperature-jump fluorescence induction in Scenedesmus obliquus leads to the following: 1. Phase alpha is due to removal of S-quenching and appears to be related to heat deactivation of the water-splitting enzyme system. With prolonged heating, irreversibility of alpha upon recooling reflects irreversible damage to the water-splitting enzyme system. 2. beta is independent of the S-states and of the redox state of primary System II acceptor Q. It is suggested that beta parallels functional separation of Q from the System II trapping centre. This effect is highly reversible. 3. gamma and beta reflect reduction of primary System II acceptor Q by a heat-induced endogenous reductant, which is probably identical to hydrogenase. Critical temperatures for pronounced alpha and beta phases differ markedly in different plants. Possible correlations between temperature-jump fluorescence inductio, thylakoid membrane lipid composition, lipid phase transition and lipid-protein interactions are discussed.     

Charge accumulation and recombination in Photosystem II studied by thermoluminescence. I. Participation of the primary acceptor Q and secondary acceptor B in the generation of thermoluminescence of chloroplasts

In the glow curves of chloroplasts excited by a series of flashes at +1°C the intensity of the main thermoluminescence band appearing at +30°C (B band; B, secondary acceptor of Photosystem II) exhibits a period-4 oscillation with maxima on the 2nd and 6th flashes indicating the participation of the S₃ state of the water-splitting system in the radiative charge recombination reaction. After long-term dark adaptation of chloroplasts (6 h), when the major part of the secondary acceptor pool (B pool) is oxidized, a period-2 contribution with maxima occurring at uneven flash numbers appears in the oscillation pattern. The B band can even be excited at ?160°C as well as by a single flash in which case the water-splitting system undergoes only one transition (S₁ → S₂). The experimental observations and computer simulation of the oscillatory patterns suggest that the B band originates from charge recombination of the S₂B^? and S₃B^? redox states. The half-time of charge recombination responsible for the B band is 48 s. When a major part of the plastoquinone pool is reduced due to prolonged excitation of the chloroplasts by continuous light, a second band (Q band; Q, primary acceptor of Photosystem II) appears in the glow curve at +10°C which overlaps with the B band. In chloroplasts excited by flashes prior to DCMU addition only the Q band can be observed showing maxima in the oscillation pattern at flash numbers 2, 6 and 10. The Q band can also be induced by flashes after DCMU addition which allows only one transition of the water-splitting system (S₁ → S₂). In the presence of DCMU, electrons accumulate on the primary acceptor Q, thus the Q band can be ascribed to the charge recombination of either the S₂Q^? or S₃Q^? states depending on whether the water-splitting system is in the S₂ or the S₃ state. The half-time of the back reaction of Q^? with the donor side of PS II (S₂ or S₃ states) is 3 s. It was also observed that in a sequence of flashes the peak positions of the Q and B bands do not depend on the advancement of the water-splitting system from the S₂ state to the S₃ state. This result implies that the midpoint potential of the water-splitting system remains unmodified during the S₂ → S₃ transition.     

农药的生态安全特性指标Q方程的研究

以北京地区冬小麦麦田的麦长管蚜Macrosiphumavenae (Fabricius)与七星瓢虫Coccinellaseptem punctataLinnaeus幼虫为实验材料 ,选择抗蚜威、乐果、氰戊菊酯 3种常用农药 ,改传统的死亡几率值为存活几率值 ,得到 6条剂量对数—存活几率值回归直线和 6个方程。定义特定农药的不同剂量下害虫与天敌存活率比值叫生态安全系数Q。利用Origin 5.0软件上机对 3个Q(x)方程绘图 ,直观地显示了不同农药种类的差异。田间实验证明 ,防治前后的害虫益虫比值与Q方程计算值基本吻合。因此可能有较大的实用价值     

Probing the turnover efficiency of photosystem II membrane fragments with different electron acceptors

In this study we employ isotope ratio membrane-inlet mass spectrometry to probe the turnover efficiency of photosystem II (PSII) membrane fragments isolated from spinach at flash frequencies between 1Hz and 50Hz in the presence of the commonly used exogenous electron acceptors potassium ferricyanide(III) (FeCy), 2,5-dichloro-p-benzoquinone (DCBQ), and 2-phenyl-p-benzoquinone (PPBQ). The data obtained clearly indicate that among the tested acceptors PPBQ is the best at high flash frequencies. If present at high enough concentration, the PSII turnover efficiency is unaffected by flash frequency of up to 30Hz, and at 40Hz and 50Hz only a slight decrease by about 5-7% is observed. In contrast, drastic reductions of the O(2) yields by about 40% and 65% were found at 50Hz for DCBQ and FeCy, respectively. Comparison with literature data reveals that PPBQ accepts electrons from Q(A)(-) in PSII membrane fragments with similar efficiency as plastoquinone in intact cells. Our data also confirm that at high flashing rates O(2) evolution is limited by the reactions on the electron-acceptor side of PSII. The relevance of these data to the evolutionary development of the water-splitting complex in PSII and with regard to the potential of artificial water-splitting catalysts is discussed. This article is part of a Special Issue entitled: Photosynthesis Research for Sustainability: from Natural to Artificial.     

Transfer and trapping of excitation energy in Photosystem II as studied by chlorophyll a2 fluorescence quenching by dinitrobenzene and carotenoid triplet. The matrix model

1. The curves representing the reciprocal fluorescence yield of chlorophyll a of Photosystem II (PS II) in Chlorella vulgaris as a function of the concentration of m-dinitrobenzene in the states P Q and P Q^-, are found to be straight parallel lines; P is the primary donor and Q the primary acceptor of PS II. In the weakly trapping state P Q^- the half-quenching of dinitrobenzene is about 0.2 mM, in vitro it is of the order of 10 mM. The fluorescence yield as a function of the concentration of a quencher is described for three models for the structure of pigment systems: the model of separate units, the model of limited energy transfer between the units, and the matrix model. If it is assumed that the rate constant of quenching by dinitrobenzene is high and thus the number of dinitrobenzene molecules per reaction center low, it can be concluded that the pigment system of PS II in C. vulgaris is a matrix of chlorophyll molecules in which the reaction centers are embedded. Theoretical and experimental evidence is consistent with such an assumption.

For Cyanidium caldarium the zero fluorescence yield Ф₀ and its quenching by dinitrobenzene were found to be much smaller than the corresponding quantities for C. vulgaris. Nevertheless, our measurements on C. caldarium could be interpreted by the assumption that the essential properties (rate constants, dinitrobenzene quenching) of PS II are the same for these two species belonging to such widely different groups.

2. The measured dinitrobenzene concentrations required for half-quenching in vivo and other observations are explained by (non-rate-limiting) energy transfer between the chlorophyll a molecules of PS II and by the assumptions that dinitrobenzene is approximately distributed at random in the membrane and does not diffuse during excitation.

3. The fluorescence kinetics of C. vulgaris during a 350 ns laser flash of variable intensity could be simulated on a computer using the matrix model. From the observed fluorescence quenching by the carotenoid triplet (C^T) and the measurement of the number of C^T per reaction center via difference absorption spectroscopy, the rate constant for quenching of C^T is calculated to be k_T = 3.3 · 10¹¹ s⁻¹ which is almost equal to the rate constant of trapping by an open reaction center (Duysens, L.N.M. (1979) CIBA Foundation Symposium 61 (New Series), pp. 323–340).

4. The fluorescence quenching by C^T in non-treated spinach chloroplasts after a 500 ns laser flash (Breton, J., Geacintov, N.E. and Swenberg, C.E. (1979) Biochim. Biophys. Acta 548, 616–635) could be explained within the framework of the matrix model when the value for k_T is used as given in point 3.

5. The observations mentioned under point 1 indicate that the fluorescence yield Ф₀ for centers in trapping state P Q is probably for a fraction exceeding 0.8 emitted by PS II.     

Charge recombination and thermoluminescence in photosystem II

In the recombination process of Photosystem II (S(2)Q(A)(-)-->S(1)Q(A)) the limiting step is the electron transfer from the reduced primary acceptor pheophytin Ph(-) to the oxidized primary donor P(+) and the rate depends on the equilibrium constant between states S(2)PPhQ(A)(-) and S(1)P(+)Ph(-)Q(A). Accordingly, mutations that affect the midpoint potential of Ph or of P result in a modified recombination rate. A strong correlation is observed between the effects on the recombination rate and on thermoluminescence (TL, the light emission from S(2)Q(A)(-) during a warming ramp): a slower recombination corresponds to a large enhancement and higher temperature of the TL peak. The current theory of TL does not account for these effects, because it is based on the assumption that the rate-limiting step coincides with the radiative process. When implementing the known fact that the radiative pathway represents a minor leak, the modified TL theory readily accounts qualitatively for the observed behavior. However, the peak temperature is still lower than predicted from the temperature-dependence of recombination. We argue that this reflects the heterogeneity of the recombination process combined with the enhanced sensitivity of TL to slower components. The recombination kinetics are accurately fitted as a sum of two exponentials and we show that this is not due to a progressive stabilization of the charge-separated state, but to a pre-existing conformational heterogeneity.     

Transfer and trapping of excitation energy in photosystem II as studied by chlorophyll alpha 2 fluorescence quenching by dinitrobenzene and carotenoid triplet. The matrix model

1. The curves representing the reciprocal fluorescence yield of chlorophyll alpha of Photosystem II (PS II) in Chlorella vulgaris as a function of the concentration of m-dinitrobenzene in the states P Q and P Q-, are found to be straight parallel lines; P is the primary donor and Q the primary acceptor of PS II. In the weakly trapping state P Q- the half-quenching of dinitrobenzene is about 0.2 mM, in vitro it is of the order of 10 mM. The fluorescence yield as a function of the concentration of a quencher is described for three models for the energy transfer between the units, and the matrix model. If it is assumed that the rate constant of quenching by dinitrobenzene is high and thus the number of dinitrobenzene molecules per reaction center low, it can be concluded that the pigment system of PS II in C. vulgaris is a matrix of chlorophyll molecules in which the reaction centers are embedded. Theoretical and experimental evidence is consistent with such an assumption. For Cyanidium caldarium the zero fluorescence yield phi 0 and its quenching by dinitrobenzene were found to be much smaller than the corresponding quantities for C. vulgaris. Nevertheless, our measurements on C. caldarium could be interpreted by the assumption that the essential properties (rate constants, dinitrobenzene quenching) of PS II are the same for these two species belonging to such widely different groups. 2. The measured dinitrobenzene concentrations required for half-quenching in vivo and other observations are explained by (non-rate-limiting) energy transfer between the chlorophyll alpha molecules of PS II and by the assumptions that dinitrobenzene is approximately distributed at random in the membrane and does not diffuse during excitation. 3. The fluorescence kinetics of C. vulgaris during a 350 ns laser flash of variable intensity could be simulated on a computer using the matrix model. From the observed fluorescence quenching by the carotenoid triplet (CT) and the measurement of the the number of CT per reaction center via difference absorption spectroscopy, the rate constant for quenching of CT is calculated to be kT = 3.3 . 10(11)s-1 which is almost equal to the rate constant of trapping by an open reaction center (Duysens, L.N.M. (1979) CIBA Foundation Symposium 61 (New Series), pp. 323--340). 4. The fluorescence quenching by CT in non-treated spinach chloroplasts after a 500 ns laser flash (Breton, J., Geacintov, N.E. and Swenberg, C.E. (1979) Biochim, Biophys. Acta 548, 616--635) could be explained within the framework of the matrix model when the value for kT is used as given in point 3. 5. The observations mentioned under point 1 indicate that the fluorescence yield phi 0 for centers in trapping state P Q is probably for a fraction exceeding 0.8 emitted by PS II.     

Phototropism of Pellia: Evidence for Mediation by Auxin-stimulated Acid Efflux

Flash induced 685 nm fluorescence emission of preilluminated and dark kept Chlamydobotrys stellata has been measured under conditions of CO₂-deprivation. The difference in fluorescence intensity between dark kept and preilluminated cells is taken as a measure for the reduced state of the primary stable electron acceptor of photosystem II, Q, at the given intensity of preillumination. CO₂ removal from growing cultures of this alga for 15 min diminishes photosynthetic electron transport at the oxidizing side of this photosystem. Prolonged CO₂-absence influences also its reducing side. Measurements of flash induced oxygen yields support the conclusion that both sides of photosystem II are affected in the absence of bicarbonate.     

Modulation of primary radical pair kinetics and energetics in photosystem II by the redox state of the quinone electron acceptor Q(A)

Time-resolved photovoltage measurements on destacked photosystem II membranes from spinach with the primary quinone electron acceptor Q(A) either singly or doubly reduced have been performed to monitor the time evolution of the primary radical pair P680(+)Pheo(-). The maximum transient concentration of the primary radical pair is about five times larger and its decay is about seven times slower with doubly reduced compared with singly reduced Q(A). The possible biological significance of these differences is discussed. On the basis of a simple reversible reaction scheme, the measured apparent rate constants and relative amplitudes allow determination of sets of molecular rate constants and energetic parameters for primary reactions in the reaction centers with doubly reduced Q(A) as well as with oxidized or singly reduced Q(A). The standard free energy difference DeltaG degrees between the charge-separated state P680(+)Pheo(-) and the equilibrated excited state (Chl(N)P680)* was found to be similar when Q(A) was oxidized or doubly reduced before the flash (approximately -50 meV). In contrast, single reduction of Q(A) led to a large change in DeltaG degrees (approximately +40 meV), demonstrating the importance of electrostatic interaction between the charge on Q(A) and the primary radical pair, and providing direct evidence that the doubly reduced Q(A) is an electrically neutral species, i.e., is doubly protonated. A comparison of the molecular rate constants shows that the rate of charge recombination is much more sensitive to the change in DeltaG degrees than the rate of primary charge separation.     

The kinetic relationship between the C-550 absorbance change, the reduction of Q(delta A320) and the variable fluorescence yield change in chloroplasts at room temperature.

The light minus dark difference spectrum and the kinetics of the indicator pigment C-550 have been measured at room temperature in isolate, envelope-free chloroplasts in the presence of 3-(3' ,4'-dichlorophenyl)-1,1-dimethylurea (DCMU). The C-550 spectrum indicates a band shift with peaks at 540 and 550 nm and has an isobestic point at 545 nm. On the assumption of 400 chlorophyll molecules per electron transfer chain the differentaial extinction coefficient delta epsilon (540-550) is calculated to be approximately 5 mM-1 . CM-1. The kinetics of the C-550 absorbance change, occurring upin the onset of continuous illumination, are shown to be biphasic and strictly correlated with the kinetics of the complementary area measured from the fluorescence induction curve under identical cinditions and with those of the absorbance increase at 320 nm due to photoreduction of Q. The lighted-induced change in these three parameters can be described as a function of the variable fluorescence yield change occurring under the same conditions. Such functions are non-linear and reveal a heterogeneous dependence of the variable fluorescence yield on the fraction of closed System II reaction centers. It is concluded that for every molecule of the primary electron acceptor Q of Photosystem II that is photochemically reduced there corresponds an equivalent change in the absorbance of the indicator pigment C-550 and in the size of the complementary area. Ths, C-550 and area are two valid parameters for monitoring the primary photochemical activity of System II at the room temperature.