Non-photochemical quenching of chlorophyll a fluorescence by oxidised plastoquinone: new evidences based on modulation of the redox state of the endogenous plastoquinone pool in broken spinach chloroplasts

Twenty-five years ago, non-photochemical quenching of chlorophyll fluorescence by oxidised plastoquinone (PQ) was proposed to be responsible for the lowering of the maximum fluorescence yield reported to occur when leaves or chloroplasts were treated in the dark with 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU), an inhibitor of electron flow beyond the primary quinone electron acceptor (Q(A)) of photosystem (PS) II. Since then, the notion of PQ-quenching has received support but has also been put in doubt, due to inconsistent experimental findings. In the present study, the possible role of the native PQ-pool as a non-photochemical quencher was reinvestigated, employing measurements of the fast chlorophyll a fluorescence kinetics (from 50 micros to 5 s). The about 20% lowering of the maximum fluorescence yield F(M), observed in osmotically broken spinach chloroplasts treated with DCMU, was eliminated when the oxidised PQ-pool was non-photochemically reduced to PQH(2) by dark incubation of the samples in the presence of NAD(P)H, both under anaerobic and aerobic conditions. Incubation under anaerobic conditions in the absence of NAD(P)H had comparatively minor effects. In DCMU-treated samples incubated in the presence of NAD(P)H fluorescence quenching started to develop again after 20-30 ms of illumination, i.e., the time when PQH(2) starts getting reoxidized by PS I activity. NAD(P)H-dependent restoration of F(M) was largely, if not completely, eliminated when the samples were briefly (5 s) pre-illuminated with red or far-red light. Addition to the incubation medium of HgCl(2) that inhibits dark reduction of PQ by NAD(P)H also abolished NAD(P)H-dependent restoration of F(M). Collectively, our results provide strong new evidence for the occurrence of PQ-quenching. The finding that DCMU alone did not affect the minimum fluorescence yield F(0) allowed us to calculate, for different redox states of the native PQ-pool, the fractional quenching at the F(0) level (Q(0)) and to compare it with the fractional quenching at the F(M) level (Q(M)). The experimentally determined Q(0)/Q(M) ratios were found to be equal to the corresponding F(0)/F(M) ratios, demonstrating that PQ-quenching is solely exerted on the excited state of antenna chlorophylls.     

In intact leaves, the maximum fluorescence level (F(M)) is independent of the redox state of the plastoquinone pool: a DCMU-inhibition study

The effects of DCMU (3-(3',4'-dichlorophenyl)-1,1-dimethylurea) on the fluorescence induction transient (OJIP) in higher plants were re-investigated. We found that the initial (F(0)) and maximum (F(M)) fluorescence levels of DCMU-treated leaves do not change relative to controls when the treatment is done in complete darkness and DCMU is allowed to diffuse slowly into the leaves either by submersion or by application via the stem. Simultaneous 820 nm transmission measurements (a measure of electron flow through Photosystem I) showed that in the DCMU-treated samples, the plastoquinone pool remained oxidized during the light pulses whereas in uninhibited leaves, the F(M) level coincided with a fully reduced electron transport chain. The identical F(M) values with and without DCMU indicate that in intact leaves, the F(M) value is independent of the redox state of the plastoquinone pool. We also show that (i) the generally observed F(0) increase is probably due to the presence of (even very weak) light during the DCMU treatment, (ii) vacuum infiltration of leaf discs leads to a drastic decrease of the fluorescence yield, and in DCMU-treated samples, the F(M) decreases to the I-level of their control (leaves vacuum infiltrated with 1% ethanol), (iii) and in thylakoid membranes, the addition of DCMU lowers the F(M) relative to that of a control sample.     

The rapid component of electron paramagnetic resonance signal II: a candidate for the physiological donor to photosystem II in spinach chloroplasts.

Rapid light-induced transients in EPR Signal IIf (F-+) are observed in 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU)-treated, Tris-washed chloroplasts until the state F P680 Q minus is reached. In the absence of exogenous redox mediators several flashes are required to saturate this photoinactive state. However, the Signal IIf transient is observed on only the first flash following DCMU addition if an efficient donor to Signal IIf, phenylenediamine or hydroquinone, is present. Complementary polarographic measurements show that under these conditions oxidized phenylenediamine is produced only on the first flash of a series. The DCMU inhibition of Signal IIf can be completely relieved by oxidative titration of a one-electron reductant with E'Os.o equals to + 480 mV. At high reduction potentials the decay time of Signal IIf is constant at about 300 ms, whereas in the absence of DCMU the decay time is longer and increases with increasing reduction potential. A model is proposed in which Q minus, the reduced Photosystem II primary acceptor, and D, a one-electron 480 mV donor endogenous to the chloroplast suspension, compete in the reduction of Signal IIf (F-+). At high potentials D is oxidized in the dark, and the (Q-+F-+) back reaction regenerates the photoactive F P680 Q state. The electrochemical and kinetic evidence is consistent with the hypothesis that the Signal IIf species, F, is identical with Z, the physiological donor to P680.     

Cytochrome c oxidase exhibits a rapid conformational change upon reduction of CuA: a tryptophan fluorescence study

When cytochrome c oxidase is reduced, it undergoes a conformational change that shifts its tryptophan fluorescence maximum from 329 to 345 nm. Studies of ligand-bound, mixed-valence forms of the enzyme show that this conformational change is dependent on the redox state of the low-potential metal centers, cytochrome a and CuA. The intrinsic fluorescence of oxidized cytochrome c oxidase is not effectively quenched by Cs+; however, marked quenching is observed for the reduced enzyme with a Stern-Volmer constant of 0.69. These observations, together with the significant red shift of the emission maximum, suggest that the emitting tryptophan residues are becoming more solvent accessible in the reduced enzyme. Stopped-flow spectra show that this conformational transition occurs rapidly upon reduction of the low-potential sites with a pseudo-first-order rate constant of 4.07 +/- 0.40 s-1. The conformational change monitored by tryptophan fluorescence is suggested to be related to the previously proposed "open-closed" transition of cytochrome c oxidase. Reductive titration of the cyanide-inhibited enzyme with ferrocytochrome c shows a nonlinear response of the fluorescence shift to added electron equivalents. A theoretical treatment of the reduction of the two interacting sites of the cyanide-inhibited enzyme has been developed that gives the population of each redox state as a function of the total number of electrons accepted by the enzyme. This treatment depends on two parameters: the difference in redox potential between the two metals and the redox interaction between the redox centers.(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.     

Studies on the Component Responsible for the Oxidation of the Primary Electron Acceptor of Photosystem II in the Presence of 3-(3',4'-Dichlorophenyl)-1,1-Dimethyl Urea: Reactivity to External Reductants

The dark reoxidation of the photochemically reduced primaryelectron acceptor of photosystem II, Q., in the presence of3-(3',4'-dichlorophenyl)-l,l-dimethyl urea (DCMU) by the redoxcounterpart (here designated Z) of Q, was studied by monitoringthe dark recovery of the induction of chlorophyll fluorescence. In normal chloroplasts, the dark reoxidation of reduced Q inthe presence of DCMU was not affected by the externally addedhydrophilic reductants; ascorbate, hydroquinone, hydrogen peroxide,manganous chloride, potassium iodide and potassium ferrocyanide.In chloroplasts whose oxidizing side of photosystem II had beeninactivated by heat- or Tris-treatments, reoxidation was inhibitedpartially. This inhibition increased on the addition of hydrophilicreductants, but was relieved by increasing the redox potentialof the suspension medium with the chloroplasts. We concluded that the redox counterpart, Z, of Q, in the presenceof DCMU is located in a hydrophobic environment which can bedenatured by heat- or Tris-treatments to allow the access ofnormally extruded hydrophilic electron donors. (Received January 10, 1981; Accepted March 12, 1981)     

High-Temperature Induced Chlorophyll Fluorescence Rise in Plants at 40–50 °C: Experimental and Theoretical Approach

We studied the temperature dependence of chlorophyll fluorescence intensity in barley leaves under weak and actinic light excitation during linear heating from room temperature to 50 degrees C. The heat-induced fluorescence rise usually appearing at around 40-50 degrees C under weak light excitation was also found in leaves treated with 3-(3',4'-dichlorophenyl)-1,1-dimethylurea (DCMU) or hydroxylamine (NH(2)OH). However, simultaneous treatment with both these compounds caused a disappearance of the fluorescence rise. We have suggested that the mechanism of the heat-induced fluorescence rise in DCMU-treated leaves is different than that in untreated or NH(2)OH-treated leaves. In DCMU-treated leaves, the heat-induced fluorescence rise reflects an accumulation of Q(A) (-) even under weak light excitation due to the thermal inhibition of the S(2)Q(A) (-) recombination as was further documented by a decrease in the intensity of the thermoluminescence Q band. Mathematical model simulating this experimental data also supports our interpretation. In the case of DCMU-untreated leaves, our model simulations suggest that the heat-induced fluorescence rise is caused by both the light-induced reduction of Q(A) and enhanced back electron transfer from Q(B) to Q(A). The simulations also revealed the importance of other processes occurring during the heat-induced fluorescence rise, which are discussed with respect to experimental data.     

Evidence for a double hit process in photosystem II based on fluorescence studies

1. The amplitudes of the fast (0-20 microseconds) and slow (20 microseconds-2 ms) fluorescence rise induced by a 2 microseconds flash have been measured as a function of the energy of the flash in chloroplasts inhibited by 3(3,4-dichlorophenyl)-1, 1-dimethylurea. The saturation curve for the slow rise shows a characteristic lag which is not observed for the fast fluorescence rise. This lag indicates that Photosystem II centers undergo a double hit process which implies that (a), each photocenter includes two acceptors Q1 and Q2; (B), after the first hit, oxidized chlorophyll Chl+ is reduced by a secondary acceptor Y in a time shor compared to the duration of the flash; (c), after the second hit, Chl+ is reduced by another secondary donor, D. 2. According to Den Haan et al. (1974) Biochim. Biophys. Acta 368, 409-421), hydroxylamine destroys the secondary donor responsible for the fast reduction of Chl+. In the presence of 3 mM hydroxylamine, only the secondary donor D is functional and a flash induses mainly a single hit process. 3. The saturation curves for the fast and the slow rises have been studied in the presence of 3(3,4-dichlorophenyl)-1, 1-dimethylurea for a second actinic flash given 2.5 s after a first saturating one. The large decrease in the half-saturating energy indicates the existence of efficient energy transfer occuring between potosynthetic units. 4. Two alternate hypotheses are discussed (a) in which D is an auxiliary donor and (b) in which D is included in the main electron transfer chain.     

A kinetic model structure for delayed fluorescence from plants

In this research, we demonstrated that the plastoquinone-related electron-transport kinetics in photosynthesis could be sufficiently described with as few as three state variables, Q(A)(-), Q(B)(-), and Q(B)(2-). A third-order kinetic model structure was developed with delayed fluorescence as the measurable output. Delayed fluorescence emissions from drought-stressed, DCMU [3-(3,4-dichlorophenyl)-1,1-dimethylurea] treated, or healthy plants were measured with a photon-counting system and used to verify the model structure through nonlinear least-squares optimization. While there were no visible differences between the healthy and the stressed plants, the model showed an obvious decrease of Q(A) reduction rate in the drought-stressed samples and a clear decline of functional Q(A)Q(B) pairs in the DCMU-treated samples. The changes were consistent with the known mechanisms by which water and DCMU affect electron transport in photosynthetic plants. The results proved that the three-state formulation was a compact and practically useful model structure for describing delayed fluorescence from plants.     

Photoinduced quenching of chlorophyll fluorescence in intact chloroplasts and algae. Resolution into two components

The light-induced decline of chlorophyll a fluorescence from a peak (P) to a low stationary level (S) in intact, physiologically active isolated chloroplasts and in intact Chlorella cells is shown to be predominantly composed of two components: (1) fluorescence quenching by partial reoxidation of the quencher Q, the primary acceptor of Photosystem II and (2) energy-dependent fluorescence quenching related to the photoinduced acidification of the intrathylakoid space. These two mechanisms of fluorescence quenching can be distinguished by the different kinetics of the relaxation of quenching observed upon addition of 3-(3′,4′-dichlorophenyl)-1,1-dimethylurea (DCMU). The relaxation of quenching by addition of DCMU is biphasic. The fast phase with a half-time of about 1 s is attributed to the reversal of Q-dependent quenching. The slow phase with a half-time of about 15 s in chloroplasts and 5 s in Chlorella cells is ascribed to relaxation of energy-dependent quenching. As shown by fluorescence spectroscopy at 77 K, the energy-dependent fluorescence quenching essentially is not caused by increased transfer of excitation energy to Photosystem I. By analyzing the energy- and Q-dependent components of quenching, information on the energy state of the thylakoid membranes and on the redox state of Q under various physiological conditions is obtained.     

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.     

The effect of redox potential on the kinetics of fluorescence induction in pea chloroplasts. II. Sigmoidicity

The shape of the fluorescence induction curve in chloroplasts inhibited by 3-(3′,4′-dichlorophenyl)-1,1-dimethylurea has been determined at different redox potentials. At ?10 mV a monophasic and sigmoidal curve is seen which is transformed into an exponential curve when the potential is poised at ?150 mV. At this potential, the quencher with high midpoint, Q_H, is reduced but that with low midpoint, Q_L, is oxidized. Thus, a sigmoidal induction is observed during photoreduction of Q_L and Q_H but photoreduction of Q_L proceeds with exponential kinetics. A correlation between the relative proportions of Q_L and Q_H observed in redox titration and the sigmoidicity of induction is also seen upon depletion of Mg²⁺ and after alkalinization to pH 9.5. Several models are discussed to explain the relationship between Photosystem II interactions and Q heterogeneity.     

D1-arginine257 mutants (R257E, K, and Q) of Chlamydomonas reinhardtii have a lowered QB redox potential: analysis of thermoluminescence and fluorescence measurements

Arginine257 (R257), in the de-helix that caps the Q(B) site of the D1 protein, has been shown by mutational studies to play a key role in the sensitivity of Photosystem II (PS II) to bicarbonate-reversible binding of the formate anion. In this article, the role of this residue has been further investigated through D1 mutations (R257E, R257Q, and R257K) in Chlamydomonas reinhardtii. We have investigated the activity of the Q(B) site by studying differences from wild type on the steady-state turnover of PS II, as assayed through chlorophyll (Chl) a fluorescence yield decay after flash excitation. The effects of p-benzoquinone (BQ, which oxidizes reduced Q(B), Q(B)(-) ) and 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU, which blocks electron flow from Q(A)(-) to Q(B)) were measured. The equilibrium constants of the two-electron gate were obtained through thermoluminescence measurements. The thermoluminescence properties were changed in the mutants, especially when observed after pretreatment with 100 microM BQ. A theoretical analysis of the thermoluminescence data, based mainly on the recombination pathways model of Rappaport et al. (2005), led to the conclusion that the free-energy difference for the recombination of Q(B)(-) with S(2) was reduced by 20-40 mV in the three mutants (D1-R257K, D1-R257Q, and D1-R257E); this was interpreted to be due to a lowering of the redox potential of Q(B)/Q(B)(-). Further, since the recombination of Q(A)(-) with S(2) was unaffected, we suggest that no significant change in redox potential of Q(A)/Q(A)(-) occurred in these three mutants. The maximum variable Chl a fluorescence yield is lowered in the mutants, in the order R257K > R257Q > R257E, compared to wild type. Our analysis of the binary oscillations in Chl a fluorescence following pretreatment of cells with BQ showed that turnover of the Q(B) site was relatively unaffected in the three mutants. The mutant D1-R257E had the lowest growth rate and steady-state activity and showed the weakest binary oscillations. We conclude that the size and the charge of the amino acid at the position D1-257 play a role in PS II function by modulating the effective redox potential of the Q(B)/Q(B)(-) pair. We discuss an indirect mechanism mediated through electrostatic and/or surface charge effects and the possibility of more pleiotropic effects arising from decreased stability of the D1/D2 and D1/CP47 interfaces.     

pH dependence of the tryptophan fluorescence in cytochrome c oxidase: further evidence for a redox-linked conformational change associated with CuA

When the low-potential metal centers of cytochrome c oxidase are reduced, the enzyme undergoes a conformational transition that shifts the fluorescence maximum of the emitting tryptophan residues from 329 to 345 nm. At pH 7.4, the change in this tryptophan fluorescence intensity is a nonlinear function of the electron equivalents added to the cyanide-inhibited enzyme. This nonlinear behavior is a result of the difference in redox potential between cytochrome a and CuA, which, at equilibrium, favors electron occupancy at cytochrome a. Studies on the cyanide-inhibited enzyme suggest that the conformational change is associated with reduction of CuA [Copeland, R. A., Smith, P. A., & Chan, S. I. (1987) Biochemistry 26, 7311-7316]. In this work we present tryptophan fluorescence data for the cyanide-inhibited enzyme at pH 8.9. Because of the pH dependence of the midpoint potential of cytochrome a in this form of the enzyme, the two low-potential centers become virtually isopotential at pH 8.9. The results obtained confirm our earlier conclusion that the observed conformational change is linked to the reduction of CuA only, rather than to the redox activity of both low-potential metal centers. We find that, in partially reduced cyanide-inhibited oxidase, raising the pH from 7.4 to 8.9 results in an intensification and red shift of the enzyme's tryptophan emission as the electron occupancy redistributes from cytochrome a to CuA. Moreover, when the fluorescence change is plotted as a function of the number of electrons added to the enzyme at pH 8.9, the data fit the nearly linear function expected for a conformational change triggered by reduction of CuA exclusively.     

The role of carbon dioxide and oxygen in determining chlorophyll fluorescence quenching during leaf development

Chlorophyll fluorescence emission at 680 nm (F680) and the rate of CO₂ fixation were measured simultaneously in sections along the length of wheat and maize leaves. These leaves possess a basal meristem and show a gradation in development towards the leaf tip. The redox state of the primary electron acceptor, Q, of photosystem II was estimated using a non-invasive method. Distal mature leaf sections displayed typical F680 induction curves which were generally anti-parallel with CO₂ fixation and during which Q became gradually oxidised. In leaf-base sections net assimilation of CO₂ was not detectable, F680 quenched slowly and monotonously without displaying any of the oscillations typical of mature tissue and Q remained relatively reduced. Sections cut from mid-regions of the leaf showed intermediate characteristics. There were no major differences between the wheat and maize leaf in the parameters measured. The results support the hypothesis that generation of the transthylakoid proton gradient and associated ATP production is not a major limitation to photosynthesis during leaf development in either C₃ or C₄ plants. Removal of CO₂ from the mature leaf sections caused little change in steady-state F680 and produced about 50% reduction of Q. When O₂ was then removed, F680 rose sharply and Q became almost totally reduced. In immature tissue unable to assimilate CO₂, removal of O₂ alone caused a similar large rise in F680 and reduction of Q whilst removal of CO₂ had negligible effects on F680 and the redox state of Q. It is concluded that in leaf tissue unable to assimilate CO₂, either because CO₂ is absent or the tissue is immature, O₂ acts as an electron acceptor and maintains Q in a partially oxidised state. The important implication that O₂ may have a role in the prevention of photoinhibition of the photochemical apparatus in the developing leaf is discussed.Abbreviations F680 chlorophyll fluorescence emission at 680 nm - PSI photosystem I - PSII photosystem II - Q PSII primary electron acceptor - pH transthylakoid proton gradient     

UV-B radiation-induced donor- and acceptor-side modifications of photosystem II in the cyanobacterium Synechocystis sp. PCC 6803.

We studied the effect of UV-B radiation (280-320 nm) on the donor- and acceptor-side components of photosystem II in the cyanobacterium Synechocystis sp. PCC 6803 by measuring the relaxation of flash-induced variable chlorophyll fluorescence. UV-B irradiation increases the t(1/2) of the decay components assigned to reoxidation of Q(A)(-) by Q(B) from 220 to 330 micros in centers which have the Q(B) site occupied, and from 3 to 6 ms in centers with the Q(B) site empty. In contrast, the t(1/2) of the slow component arising from recombination of the Q(A)Q(B)(-) state with the S(2) state of the water-oxidizing complex decreases from 13 to 1-2 s. In the presence of DCMU, fluorescence relaxation in nonirradiated cells is dominated by a 0.5-0.6 s component, which reflects Q(A)(-) recombination with the S(2) state. After UV-B irradiation, this is partially replaced by much faster components (t(1/2) approximately 800-900 micros and 8-10 ms) arising from recombination of Q(A)(-) with stabilized intermediate photosystem II donors, P680(+) and Tyr-Z(+). Measurement of fluorescence relaxation in the presence of different concentrations of DCMU revealed a 4-6-fold increase in the half-inhibitory concentration for electron transfer from Q(A) to Q(B). UV-B irradiation in the presence of DCMU reduces Q(A) in the majority (60%) of centers, but does not enhance the extent of UV-B damage beyond the level seen in the absence of DCMU, when Q(A) is mostly oxidized. Illumination with white light during UV-B treatment retards the inactivation of PSII. However, this ameliorating effect is not observed if de novo protein synthesis is blocked by lincomycin. We conclude that in intact cyanobacterium cells UV-B light impairs electron transfer from the Mn cluster of water oxidation to Tyr-Z(+) and P680(+) in the same way that has been observed in isolated systems. The donor-side damage of PSII is accompanied by a modification of the Q(B) site, which affects the binding of plastoquinone and electron transport inhibitors, but is not related to the presence of Q(A)(-). White light, at the intensity applied for culturing the cells, provides protection against UV-B-induced damage by enhancing protein synthesis-dependent repair of PSII.     

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.