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^? 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′_08.0 = +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^?, 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.     

Two electron donation sites for exogenous reductants in chloroplast photosystem II.

Two sites are distinguished for the oxidation of exogenous donors by Photosystem II in non-oxygen evolving chloroplasts. In the presence of lipophilic donors (e.g. phenylenediamine, benzidine, diphenylcarbazide), the rate for Signal IIf rereduction following a flash increases as the concentration of exogenous reductant increases. There is a decrease (20-40%) in Signal IIf magnitude accompanying donor addition at low (smaller than 10(-%) M) concentrations, but the extent of the decrease does not change further with increasing donor concentrations. Complementary polarographic experiments monitoring donor (phenylenediamine) oxidation show an increase in oxidation rate with increasing donor concentration. In the presence of the hydrophilic donor, Mn-2+, the Signal IIf decay halftime remains constant with increasing Mn-2+ concentration. However, the flash-induced Signal IIf magnitude pregressively decreases with increasing Mn-2+ concentration. These results are interpreted in terms of two competing paths for the reduction of P680+. In one path P680+ reduction is accompanied by the appearance of Signal IIf, and lipophilic donors subsequently rereduce the Signal IIf species in a series reaction. This reduction follows pseudo-first order kinetics as a function of donor concentration. In the second path Mn-2+ reduces P680+ in a parallel reaction that competes with the formation of the Signal IIf species. This results in a decrease in the magnitude of Signal IIf, but no change in its decay time.     

Two electron donation sites for exogenous reductants in chloroplast Photosystem II

Two sites are distinguished for the oxidation of exogenous donors by Photosystem II in non-oxygen evolving chloroplasts. In the presence of lipophilic donors (e.g. phenylenediamine, benzidine, diphenylcarbazide), the rate for Signal IIf rereduction following a flash increases as the concentration of exogenous reductant increases. There is a decrease (20–40%) in Signal IIf magnitude accompanying donor addition at low (< 10^?5M) concentrations, but the extent of the decrease does not change further with increasing donor concentration. Complementary polarographic experiments monitoring donor (phenylenediamine) oxidation show an increase in oxidation rate with increasing donor concentration.In the presence of the hydrophilic donor, Mn²⁺, the Signal IIf decay halftime remains constant with increasing Mn²⁺ concentration. However, the flash-induced Signal IIf magnitude progressively decreases with increasing Mn²⁺ concentration.These results are interpreted in terms of two competing paths for the reduction of P680⁺. In one path P680⁺ reduction is accompanied by the appearance of Signal IIf, and lipophilic donors subsequently rereduce the Signal IIf species in a series reaction. This reduction follows pseudo-first order kinetics as a function of donor concentration. In the second path Mn²⁺ reduces P680⁺ in a parallel reaction that competes with the formation of the Signal IIf species. This results in a decrease in the magnitude of Signal IIf, but no change in its decay time.     

Exogenous versus endogenous acceptors in photosystem ii in inhibited chloroplasts

The conditions for steady-state Signal IIf formation in response to single turnover flashes in Tris-treated, 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU)-inhibited chloroplasts have been investigated. DCMU inhibits Signal IIf generation as the photoinactive state, Z P680 Q-A, accumulates. Potassium ferricyanide relieves this inhibition so that Signal IIf can be fully developed on each flash in a flash series. The effectiveness of ferricyanide in stimulating Signal IIf formation is dependent on its concentration, the flash repetition rate, and the salt composition of the chloroplast suspension. There are two models in the literature for Q-A oxidation under these inhibitory conditions: direct oxidation of Q-A by exogenous acceptors like ferricyanide or oxidation of Q-A by an endogenous acceptor, AH, which has a midpoint potential of approximately 400 mV. It is found that the direct exogenous acceptor model accounts well for these data, whereas the AH model does not explain several of these results. The apparent rate constant for the direct oxidation of Q-A by ferricyanide at various concentrations of salt has been calculated from our electron paramagnetic resonance (EPR) data and compared with the corresponding rate constant determined by S. Itoh from fluorescence data (Biochim, Biophys. Acta 504, 324-340, 1978); good agreement is found for the two different experimental approaches.     

Interaction of linolenic acid with bound quinone molecules in Photosystem II. Time-resolved optical and electron spin resonance studies

Time-resolved spectroscopic techniques, including optical flash photolysis and electron spin resonance spectroscopy, have been utilized to monitor electron-transport activity in Photosystem II subchloroplast particles. These studies have indicated that in the presence of 100 microM linolenic acid (1) a high initial fluorescence yield (Fi) is observed upon steady-state illumination of the dark-adapted sample; (2) flash-induced absorption transients (t greater than 10 mus) in the region of 820 nm, attributed to P-680+, are first slowed, then abolished; and (3) electron spin resonance Signal IIs and Signal IIf (Z+) are not detectable. Upon reversal of linolenic acid inhibition by washing with bovine serum albumin, optical and electron spin resonance transients originating from the photooxidation of P-680 are restored. Similarly, the variable component of fluorescence is recovered with an accompanying restoration of Signal IIs and Signal IIf. The data indicate that linolenic acid affects two inhibition sites in Photosystem II: one located between pheophytin and QA on the reducing side, and the other between electron donor Z and P-680 on the oxidizing side. Since both sites are associated with bound quinone molecules, we suggest that linolenic acid interacts at the level of quinone binding proteins in Photosystem II.     

The rate of charge recombination in Photosystem II

Loss by recombination of the charge separated state P(680+)Q(A-) limits the performance of Photosystem II (PS II) as a photochemical energy converter. Time constants reported in literature for this process are mostly either near 0.17 ms or near 1.4 ms. The shorter time is found in plant PS II when reduction of P(680+) by the secondary electron donor Tyrosine Z cannot occur because Y(Z) is already oxidized. The 1.4 ms recombination is seen in Y(Z)-less mutants of the cyanobacterium Synechocystis. However, the rate of P(680+)Q(A-) recombination that actually competes with the stabilization of the charge separation has not been previously reported. We have measured the kinetics of the flash-induced fluorescence yield changes in the microsecond time domain in Tris-washed spinach chloroplasts. In this way the kinetics and yield of P(680+) reduction by Y(Z) were obtained, and the rate of the competing P(680+)Q(A-) recombination could be evaluated. The recombination time was less than 0.5 ms; the best-fitting time constant was 0.1 ms. The presence of Y(Z)(ox) slightly decreased the efficiency of excitation trapping but did not seem to accelerate P(680+)Q(A-) recombination. The two P(680+)Q(A-) lifetimes in the literature probably reflect a significant difference between plant and cyanobacterial PS II.     

Flash photolysis-electron spin resonance studies of photosystem I. A fast reduction of component of P-700+.

A 300 mus decay component of ESR Signal I (P-700+) in chloroplasts is observed following a 10 mus actinic xenon flash. This transient is inhibited by treatments which block electron transfer from Photosystem II to Photosystem I (e.g. 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU), 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone (DBMIB), KCN and HgCl2). The fast transient reduction of P-700+ can be restored in the case of DCMU or DBMIB inhibition by addition of an electron donor couple (2,6-dichlorophenol indophenol (Cl2Ind)/ascorbate) which supplies electrons to cytochrome f. However, this donor couple is inefficient in restoring electron transport in chloroplasts which have been inhibited with the plastocyanin inactivators, KCN and HgCl2. Oxidation-reduction measurements reveal that the fast P-700+ reduction component reflects electron transfer from a component with Em = 375 +/- 10 mV (pH = 7.5). These data suggest the assignment of the 300-mus decay kinetics to electron transfer from cytochrome f (Fe2+) to P-700+, thus confirming the recent observations of Haehnel et al. (Z. Naturforsch. 26b, 1171-1174 (1971)).     

Primary and secondary electron donors in photosystem II of chloroplasts. Rates of electron transfer and location in the membrane.

Absorption changes at 820 or 515 nm after a short laser flash were studied comparatively in untreated chloroplasts and in chloroplasts in which oxygen evolution is inhibited. In chloroplasts pre-treated with Tris, the primary donor of Photosystem II (P-680) is oxidized by the flash it is re-reduced in a biphasic manner with half-times of 6 microseconds (major phase) and 22 microseconds. After the second flash, the 6 microseconds phase is nearly absent and P-680+ decays with half-times of 130 microseconds (major phase) and 22 microseconds. Exogenous electron donors (MnCl2 or reduced phenylenediamine) have no direct influence on the kinetics of P-680+. In untreated chloroplasts the 6 and 22 microseconds phases are of very small amplitude, either at the 1st, 2nd or 3rd flash given after dark-adaptation. They are observed, however, after incubation with 10 mM hydroxylamine. These results are interpreted in terms of multiple pathways for the reduction of P-680+: a rapid reduction (less than 1 microseconds) by the physiological donor D1; a slower reduction (6 and 22 microseconds) by donor D'1, operative when O2 evolution is inhibited; a back-reaction (130 microseconds) when D'1 is oxidized by the pre-illumination in inhibited chloroplasts. In Tris-treated chloroplasts the donor system to P-680+ has the capacity to deliver only one electron. The absorption change at 515 nm (electrochromic absorption shift) has been measured in parallel. It is shown that the change linked to Photosystem II activity has nearly the same magnitude in untreated chloroplasts or in chloroplasts treated with hydroxylamine or with Tris (first and subsequent flashes). Thus we conclude that all the donors (P-680, D1, D'1) are located at the internal side of the thylakoid membrane.     

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.     

Investigations on the electron transfer processes in Photosystem II: New insights by chlorophyll fluorescence decay measurements with additional saturating light pulses

Electron transfer processes in leaves were investigated by chlorophyll fluorescence decay measurements. A fast chlorophyll fluorescence decay was observed in the intact state, reflecting normal electron transfer in Photosystem II. After treatment with DCMU a slow chlorophyll fluorescence decay was measured due to blocked electron transfer after the primary quinone Q_A. Additional saturating light pulses, one between each two measuring pulses, were used to completely reduce Q_A of the intact leaf: the chlorophyll fluorescence decay became similar to that of a DCMU treated leaf. A decreased electron donation rate to the reaction centre P680 was obtained after treatment with hydroxylamine. The intensity of the additional saturating light pulses was not sufficient to reduce all Q_A under this condition and only a small increase of the average chlorophyll fluorescence decay time occurred. Following our previous paper [Berg et al. (1997) Photosynthetica 34, in press], we investigated the effects of water stress with the additional saturating light pulses. An almost complete reduction of Q_A was possible after water stress started. A small, but systematic shortening of the slow chlorophyll fluorescence decay followed, up to a relative loss of leaf mass of 80%. At this time a rapid shortening of the chlorophyll fluorescence decay occurred, caused by an electron deficiency at the donor site of PS II. Additional saturating light pulses had no effects on the chlorophyll fluorescence decay any more, revealing a radiationless recombination between the reduced primary quinone Q and the oxidized reaction centre P680⁺.     

Radiative and non-radiative charge recombination pathways in Photosystem II studied by thermoluminescence and chlorophyll fluorescence in the cyanobacterium Synechocystis 6803

The mechanism of charge recombination was studied in Photosystem II by using flash induced chlorophyll fluorescence and thermoluminescence measurements. The experiments were performed in intact cells of the cyanobacterium Synechocystis 6803 in which the redox properties of the primary pheophytin electron acceptor, Phe, the primary electron donor, P(680), and the first quinone electron acceptor, Q(A), were modified. In the D1Gln130Glu or D1His198Ala mutants, which shift the free energy of the primary radical pair to more positive values, charge recombination from the S(2)Q(A)(-) and S(2)Q(B)(-) states was accelerated relative to the wild type as shown by the faster decay of chlorophyll fluorescence yield, and the downshifted peak temperature of the thermoluminescence Q and B bands. The opposite effect, i.e. strong stabilization of charge recombination from both the S(2)Q(A)(-) and S(2)Q(B)(-) states was observed in the D1Gln130Leu or D1His198Lys mutants, which shift the free energy level of the primary radical pair to more negative values, as shown by the retarded decay of flash induced chlorophyll fluorescence and upshifted thermoluminescence peak temperatures. Importantly, these mutations caused a drastic change in the intensity of thermoluminescence, manifested by 8- and 22-fold increase in the D1Gln130Leu and D1His198Lys mutants, respectively, as well as by a 4- and 2.5-fold decrease in the D1Gln130Glu and D1His198Ala mutants, relative to the wild type, respectively. In the presence of the electron transport inhibitor bromoxynil, which decreases the redox potential of Q(A)/Q(A)(-) relative to that observed in the presence of DCMU, charge recombination from the S(2)Q(A)(-) state was accelerated in the wild type and all mutant strains. Our data confirm that in PSII the dominant pathway of charge recombination goes through the P(680)(+)Phe(-) radical pair. This indirect recombination is branched into radiative and non-radiative pathways, which proceed via repopulation of P(680)(*) from (1)[P(680)(+)Ph(-)] and direct recombination of the (3)[P(680)(+)Ph(-)] and (1)[P(680)(+)Ph(-)] radical states, respectively. An additional non-radiative pathway involves direct recombination of P(680)(+)Q(A)(-). The yield of these charge recombination pathways is affected by the free energy gaps between the Photosystem II electron transfer components in a complex way: Increase of DeltaG(P(680)(*)<-->P(680)(+)Phe(-)) decreases the yield of the indirect radiative pathway (in the 22-0.2% range). On the other hand, increase of DeltaG(P(680)(+)Phe(-)<-->P(680)(+)Q(A)(-)) increases the yield of the direct pathway (in the 2-50% range) and decreases the yield of the indirect non-radiative pathway (in the 97-37% range).     

Site of salicylaldoxime interaction with photosystem II

The reversible inhibition of Photosystem II by salicylaldoxime was studied in spinach D-10 particles by fluorescence, optical absorption, and electron spin resonance spectroscopy. In the presence of 15 mM salicylaldoxime, the initial fluorescence yield was raised to the level of the maximum fluorescence, indicating efficient charge recombination between reduced pheophytin (Ph) and P680⁺. In agreement with the rapid (ns) backreaction expected between Ph^– and P680⁺, the optical absorption transient at 820 mm was not observed. When the particles were washed free of salicylaldoxime, the optical absorption transient resulting from the rereduction of P680⁺ was restored to the µs timescale. These results, along with the previously observed inhibition of electron transport reactions and diminution of the 515-nm absorption change in chloroplasts [Golbeck, J.H. (1980) Arch Biochem Biophys 202, 458–466], are consistent with a site of inhibition between Ph and QA in Photosystem II. ESR Signal IIf and Signal Its were abolished in the presence of 25 mM salicylaldoxime, but both signals could be recovered by washing the D-10 particles free of the inhibitor. The loss of Signal Ilf is most likely a consequence of the inhibition between Ph and Q_A; the rapid charge recombination between Ph^– and P680⁺ would preclude electron transfer from an electron donor on the oxidizing side of Photosystem II. The loss of Signal Its may be due to a change in the environment of the donor complex such that the semiquinone radical giving rise to Signal Its interacts with a nearby reductant.Abbreviations D₁ electron donor to P680⁺ in oxygen-inhibited chloroplasts - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - F₀ prompt chlorophyll a fluorescence yield - F_i initial chlorophyll a fluorescence yield - F_max maximum chlorophyll a fluorescence yield - F_var variable chlorophyll a fluorescence yield - FWHM full width at half maximum - Mes 2-(N-morpholino) ethanesulfonic acid - P680 reaction center chlorophyll a of photosystem II - Ph pheophytin intermediate electron acceptor - Q_A primary quinone electron acceptor - Q_B secondary quinone electron acceptor - Tris tris(hydroxymethyl)aminomethane - Z electron donor to P680⁺     

Photooxidation of cytochrome b559 in oxygen-evolving photosystem II.

Cytochrome b559 (cyt b559) is an intrinsic and essential component of the photosystem II (PSII) protein complex, but its function, stoichiometry, and electron-transfer kinetics in the physiological system are not well-defined. In this study, we have used flash-detection optical spectroscopy to measure the kinetics and yields of photooxidation and dark reduction of cyt b559 in untreated, O2-evolving PSII-enriched membranes at room temperature. The dark redox states of cyt b559 and the primary electron acceptor, QA, were determined over the pH range 5.0-8.5. Both the fraction of dark-oxidized cyt b559 and dark-reduced QA increased with increasing acidity. Consistent with these results, an acid-induced drop in pH from 8.5 to 4.9 in a dark-adapted sample caused the oxidation of cyt b559, indicating a shift in the redox state during the dark reequilibration. As expected from the dark redox state of cyt b559, the rate and extent of photooxidation of cyt b559 during continuous illumination decreased toward more acidic pH values. After a single, saturating flash, the rate of photooxidation of cyt b559 was of the same order of magnitude as the rate of S2QA- charge recombination. In untreated PSII samples at pH 8.0 with 42% of cyt b559 oxidized and 15% of QA reduced in the dark, 4.7% of one copy of cyt b559 was photooxidized after one flash with a t1/2 of 540 +/- 90 ms. On the basis of our previous work [Buser, C. A., Thompson, L. K., Diner, B. A., & Brudvig, G. W (1990) Biochemistry 29, 8977] and the data presented here, we conclude that Sn+1, YZ., and P680+ are in redox equilibrium and cyt b559 (and YD) are oxidized via P680+. After a period of illumination sufficient to fully reduce the plastoquinone pool, we also observed the pH-dependent dark reduction of photooxidized cyt b559, where the rate of reduction decreased with decreasing pH and was not observed at pH < 6.4. To determine the direct source of reductant to oxidized cyt b559, we studied the dark reduction of cyt b559 and the reduction of the PQ pool as a function of 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) concentration. We find that DCMU inhibits the reduction of cyt b559 under conditions where the plastoquinone pool and QA are reduced. We conclude that QB-. (H+) or QBH2 is the most likely source of the electron required for the reduction of oxidized cyt b559.(ABSTRACT TRUNCATED AT 400 WORDS)     

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.     

Electron-transfer reactions in manganese-depleted photosystem II

We have used flash-detection optical and electron paramagnetic resonance spectroscopy to measure the kinetics and yield per flash of the photooxidation of cytochrome b559 and the yield per flash of the photooxidation of the tyrosine residue YD in Mn-depleted photosystem II (PSII) membranes at room temperature. The initial charge separation forms YZ+ QA-. Following this, cytochrome b559 is oxidized on a time scale of the same order and with the same pH dependence as is observed for the decay of YZ+; under the conditions of our experiments, the decay of YZ+ is determined by the lifetime of YZ+ QA-. In order to explain this observation, we have constructed a model for electron donation in which YZ+ and P680+ are in redox equilibrium and cytochrome b559 and YD are oxidized via P680+. Using our results, together with data from earlier investigations of the kinetics of electron transfer from YZ to P680+ and charge recombination of YZ+ QA-, we have obtained the first global fit for electron donation in Mn-depleted PSII that accounts for the data over the pH range from 5 to 7.5. From these calculations, we have obtained the intrinsic rate constants of all the electron-donation reactions in Mn-depleted PSII. These rate constants allow us to calculate the free energy difference between YZ+ P680 and YZ P680+, which is found to increase by 47 +/- 4 mV/pH from pH 5 to 6 and is observed to increase more slowly per pH unit for pH greater than 6. An important conclusion of our experimental work is that the rates of photooxidation of cytochrome b559 and YD are determined by the lifetime of the oxidizing equivalent on YZ/P680. Extension of our model to oxygen-evolving PSII samples leads to the prediction that the kinetics and yields of electron donation from cytochrome b559 and YD to P680+ will depend on the S2- or S3-state lifetime.     

The mechanism of UV-A radiation-induced inhibition of photosystem II electron transport studied by EPR and chlorophyll fluorescence

The UV-A (320-400 nm) component of sunlight is a significant damaging factor of plant photosynthesis, which targets the photosystem II complex. Here we performed a detailed characterization of UV-A-induced damage in photosystem II membrane particles using EPR spectroscopy and chlorophyll fluorescence measurements. UV-A irradiation results in the rapid inhibition of oxygen evolution accompanied by the loss of the multiline EPR signal from the S(2) state of the water-oxidizing complex. Gradual decrease of EPR signals arising from the Q(A)(-)Fe(2+) acceptor complex, Tyr-D degrees, and the ferricyanide-induced oxidation of the non-heme Fe(2+) to Fe(3+) is also observed, but at a significantly slower rate than the inhibition of oxygen evolution and of the multiline signal. The amplitude of Signal II(fast), arising from Tyr-Z degrees in the absence of fast electron donation from the Mn cluster, was gradually increased during the course of UV-A treatment. However, the amount of functional Tyr-Z decreased to a similar extent as Tyr-D as shown by the loss of amplitude of Signal II(fast) that could be measured in the UV-A-treated particles after Tris washing. UV-A irradiation also affects the relaxation of flash-induced variable chlorophyll fluorescence. The amplitudes of the fast (600 micros) and slow (2 s) decaying components, assigned to reoxidation of Q(A)(-) by Q(B) and by recombination of (Q(A)Q(B))(-) with donor side components, respectively, decrease in favor of the 15-20 ms component, which reflects PQ binding to the Q(B) site. In the presence of DCMU, the fluorescence relaxation is dominated by a 1 s component due to recombination of Q(A)(-) with the S(2) state. After UV-A radiation, this is partially replaced by a much faster component (30-70 ms) arising from recombination of Q(A)(-) with a stabilized intermediate PSII donor, most likely Tyr-Z degrees. It is concluded that the primary damage site of UV-A irradiation is the catalytic manganese cluster of the water-oxidizing complex, where electron transfer to Tyr-Z degrees and P(680)(+) becomes inhibited. Modification and/or inactivation of the redox-active tyrosines and the Q(A)Fe(2+) acceptor complex are subsequent events. This damaging mechanism is very similar to that induced by the shorter wavelength UV-B (280-320) radiation, but different from that induced by the longer wavelength photosynthetically active light (400-700 nm).     

Photosystem II oxidation of charged electron donors. Surface charge effects

Reactions occurring on the oxidizing side of Photosystem II have been studied in Tris-washed chloroplasts by monitoring the decay kinetics of EPR signal IIf, arising from the photoinduced oxidation of Z, an intermediate in the electron transport chain between P-680 and the water-splitting enzyme. Upon addition of electron donors, signal IIf follows pseudo-first order decay kinetics with rates dependent on the chemical nature of the donor. Negatively charged donors (I-, Fe(CN)6(4-), W(CN)8(4-) are poor reducing agents for Z.+ whereas neutral donors (benzidine, hydroquinone, diphenylcarbazide) are more efficient, their effectiveness paralleling their lipophilicity. The slow signal IIf reduction observed with the charged donors is consistent with the non-polar nature of the thylakoid membrane and a location for Z toward the inner membrane surface. It most probably exists in a hydrophobic site as indicated by the positive correlation between rate constant and lipophilicity for the neutral donors. A detailed study of the mechanism of Photosystem II reduction by ascorbic acid has been carried out. The pH dependence of the decay kinetics of signal IIf in the presence of this donor is consistent with a model in which both the neutral acid and the ascorbate mono-anion serve as reducing agents to Z.+. The second-order rate constant for reduction by the mono-anion is less than that of the neutral acid and is found to vary with the suspension pH. This observation is interpreted to indicate the occurrence of negative charge on the inner membrane surface in the vicinity of Z. Additional experiments, which assessed the effect of mono- and divalent cations and of cationic detergents on the signal IIf reaction rate constants, support both the presence of negative surface charge and its location on the membrane inner surface.     

Oxido-reduction kinetics of Signal II slow in tris-washed chloroplasts

In this report, we characterize the relationship between species “Z” (giving rise to EPR Signal II fast) and “D” (EPR Signal II slow) in triswashed chloroplasts.At pH 8.5 an externally added donor phenylenediamine competes with D for Z⁺ reduction after its oxidation by a flash. The reduction of Z⁺ by D occurs within some milliseconds. In a subsequent dark period, D⁺ is reduced by PD, the reaction rate being independant of phenylenediamine concentration. These results are consistent with the hypothesis of an equilibrium between Z⁺D and ZD⁺, the reduction of D by phenylenediamine occuring via Z. At lower pH's, the connection between Z and D is looser: a high concentration of phenylenediamine which reduces rapidly Z⁺, is very slow in reducing D⁺ and the subsequent photooxidation of D is less efficient.     

Photoreduction of pheophytin in reaction centers of the photosystem II in intact cells of green algae and cyanobacteria under anaerobic conditions

Reversible photoreduction of pheophytin (Pheo) accompanied by a decrease in the chlorophyll fluorescence yield is observed in Photosystem 2 of the intact cells of green algae and cyanobacteria under anaerobic conditions. The photoreaction is inhibited by DCMU and reactivated upon subsequent addition of either ascorbate of dithionite. It is suggested that as a result of electron donation from the water splitting system being in the state S(3), to the reaction centre of Photosystem 2 in the state [P(+)(680)Pheo(-)] Q(-) after the primary photoreaction there occurs formation of the long-living state [P(680)Pheo(-)] Q(-). It was found that oxidized NADP, benzyl viologen and methyl viologen accelerate oxidation of Pheo reduced int he Photosystem 2 in the light indicating that these electron acceptors (typical for Photosystem 1) can accept an election from Pheo in Photosystem 2.     

Primary and secondary electron donors in Photosystem II of chloroplasts. Rates of electron transfer and location in the membrane

Absorption changes at 820 or 515 nm after a short laser flash were studied comparatively in untreated chloroplasts and in chloroplasts in which oxygen evolution is inhibited.In chloroplasts pre-treated with Tris, the primary donor of Photosystem II (P-680) is oxidized by the flash, as observed by an absorption increase at 820 nm. After the first flash it is re-reduced in a biphasic manner with half-times of 6 μs (major phase) and 22 μs. After the second flash, the 6 μs phase is nearly absent and P-680⁺ decays with half-times of 130 μs (major phase) and 22 μs. Exogenous electron donors (MnCl₂ or reduced phenylenediamine) have no direct influence on the kinetics of P-680⁺.In untreated chloroplasts the 6 and 22 μs phases are of very small amplitude, either at the 1st, 2nd or 3rd flash given after dark-adaptation. They are observed, however, after incubation with 10 mM hydroxylamine.These results are interpreted in terms of multiple pathways for the reduction of P-680⁺: a rapid reduction (<1 μs) by the physiological donor D₁; a slower reduction (6 and 22 μs) by donor D′₁, operative when O₂ evolution is inhibited; a back-reaction (130 μs) when D′₁ is oxidized by the pre-illumination in inhibited chloroplasts. In Tris-treated chloroplasts the donor system to P-680⁺ has the capacity to deliver only one electron.The absorption change at 515 nm (electrochromic absorption shift) has been measured in parallel. It is shown that the change linked to Photosystem II activity has nearly the same magnitude in untreated chloroplasts or in chloroplasts treated with hydroxylamine or with Tris (first and subsequent flashes). Thus we conclude that all the donors (P-680, D₁, D′₁) are located at the internal side of the thylakoid membrane.