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Strong light photoinhibition of electrontransport in Photosystem II. Impairment of the function of the first quinone acceptor, QA
Abstract
Electron paramagnetic resonance (EPR) spectroscopy has been applied in an investigation on the mechanism for photoinhibition of the electron transport in Photosystem II. The experiments were performed in vitro in thylakoid membranes and preparations of Photosystem-II-enriched membranes. Photoinhibition resulted in inhibition of the oxygen evolution and EPR measurements of the S2 state multiline EPR signal show that its induction by illumination at 198 K was decreased with the same kinetics as the oxygen evolution. Further EPR measurements show that the reduction of QA was inhibited with the same kinetics as the oxygen evolution. The amount of photoreducible pheophytin was estimated from photoaccumulation experiments under reducing conditions and the results show that the primary charge separation reaction was inhibited much slower than the oxygen evolution or the reduction of QA. These results indicate that photoinhibition inhibits the electron transfer between pheophytin and QA probably by impairment of the function of QA. In the inhibited centers the primary charge separation reaction is still operational. It is suggested that the event leading to photoinhibition of the electron transport is the double reduction of QA which then leaves its site. Photoinhibition also results in rapid oxidation of cytochrome b-559 and a change of cytochrome b-559 from its high potential form to its low potential form. The reaction is quantitative and proceeds with the same kinetics as the inhibition of oxygen evolution. The potential shift of cytochrome b-559 suggests that photoinhibition induces early conformational changes in Photosystem II.
... In that case, the Q B site remains nonfunctional due to the absence of oxidized plastoquinone, and the primary quinone acceptor remains in its reduced state (Q À A ) [10]. There is a probability that the latter becomes doubly reduced and protonated under conditions where the reoxidation is prevented [11]. The fully reduced and protonated Q A is supposed to leave its binding site [11], but even if it does not, the protonated state of Q A should prevent electrostatic repulsion between reduced pheophytin (Pheo À ) and reduced Q A [12]. ...
... There is a probability that the latter becomes doubly reduced and protonated under conditions where the reoxidation is prevented [11]. The fully reduced and protonated Q A is supposed to leave its binding site [11], but even if it does not, the protonated state of Q A should prevent electrostatic repulsion between reduced pheophytin (Pheo À ) and reduced Q A [12]. Under these conditions, charge separation between P680 and Pheo can still occur with a significant yield. ...
... The high-potential form of the cytochrome could be converted to the low-potential form following the reduction of the intermediates on the acceptor side of the photosystem by strong illumination as was shown to occur during photoinhibition [11]. The reverse conversion of the low-potential form of cyt b559 to its high-potential form was proposed to depend on the ambient redox system [146] and would lead to the reduction of P680 [144]. ...
... s -~ were compared (Tables 1, 2). In previous studies on the relationship between photoinhibition and loss of the D 1 protein, there are reports showing both good (Ohad et al. 1984) and poor (Cleland 1988;Virgin et al. 1988;Cleland et al. 1990;Styring et al. 1990;Hundal et al. 1990) correlations. Studies of photoinhibition at low temperatures have also shown that photoinhibition is not correlated with D 1 protein degradation either in vivo (Chow et al. 1989b;Gong and Nilsen 1989) or in vitro (Chow et al. ...
... This evidence does not unequivocally prove that such stable, inhibited PSII reaction centres necessarily confer protection to remaining functional centres by nonradiative dissipation of excitation energy among connected PSII units, but the fact that the PSII centres apparently are not degraded in phase with photoinhibition (Tables 1, 2) certainly strengthens the feasibility of the concept. From a theoretical viewpoint, PSII reaction centres inhibited in vitro appear unable to undergo charge stabilization, but maintain, through charge separation and recombination their trapping ability and non-photochemical dissipation of absorbed light (Cleland et al. 1986;Allakhverdiev et al. 1987;Setlik et al. 1990;Styring et al. 1990). Thus, such photoinhibited PSII reaction centres would be of physiological significance for the controlled dissipation of light energy absorbed in excess to the capacity of photosynthesis. ...
... Low temperatures supposedly slow down the PSII repair cycle as judged from a retarded D1 protein degradation upon photoinhibition (Chow et al. 1989b;Gong and Nilsen 1989;Aro et al. 1990), and also from the relative insensitivity of photoinhibition to chloroplast-encoded de-novo protein synthesis (Greer et al. 1991). Oquist and Huner (1991) therefore have proposed that under prevailing low-temperature conditions, photoinhibition represents a stable down-regulation of PSII reaction centres incapable of charge stabilization, but presumably still able to dissipate excitation energy nonphotochemically by primary charge separation and recombination (Cleland et al. 1986;Styring et al. 1990). amount of carotenoids in the light-harvesting antenna, and speeds up the repair cycle of PSII, all of these responses having the potential to protect against photoinhibition. ...
Leaf discs of the shade plant Tradescantia albiflora Kunth grown at 50 μmol · m(-2) · s(-1), and the facultative sun/shade plant Pisum sativum L. grown at 50 or 300 μmol · m(-2), s(-1), were photoinhibited for 4 h in 1700 μmol photons m(-2) · s(-1) at 22° C. The effects of photoinhibition on the following parameters were studied: i) photosystem II (PSII) function; ii) amount of D1 protein in the PSII reaction centre; iii) dependence of photoinhibition and its recovery on chloroplast-encoded protein synthesis; and, iv) the sensitivity of photosynthesis to photoinhibition in the presence or absence of the carotenoid zeaxanthin. We show that: i) despite different sensitivities to photoinhibition, photoinhibition in all three plants occurred at the reaction centre of PSII; ii) there was no correlation between the extent of photoinhibition and the degradation of the D1 protein; iii) the susceptibility to photoinhibition by blockage of chloroplas-tencoded protein synthesis was much less in shade plants than in plants acclimated to higher light; and iv) inhibition of zeaxanthin formation increased the sensitivity to photoinhibition in pea, but not in the shade plant Tradescantia. We suggest that there are mechanistic differences in photoinhibition of sun and shade plants. In sun plants, an active repair cycle of PSII replaces photoinhibited reaction centres with photochemically active ones, thereby conferring partial protection against photoinhibition. However, in shade plants, this repair cycle is less important for protection against photoinhibition; instead, photoinhibited PSII reaction centres may confer, as they accumulate, increased protection of the remaining connected, functional PSII centres by controlled, nonphotochemical dissipation of excess excitation energy.
... To regulate total energy production, chloroplasts partition light energy between photochemical processes which generate ATP and NADPH (LEF) and the energy dissipating process of "nonphotochemical quenching" (NPQ) [12][13][14][15][16][17]. When metabolic demand for energy is less than current supply, the major form of NPQ, termed qE (for 'energy dependent' quenching), is triggered by acidification of the lumen (i.e., by the ΔpH component of pmf), through activation of violaxanthin deepoxidase, which catalyzes the conversion of violaxanthin to antheraxanthin and zeaxanthin [18], and through protonation of the antenna protein PsbS [19,20]. ...
... To regulate total energy production, chloroplasts partition light energy between photochemical processes which generate ATP and NADPH (LEF) and the energy dissipating process of "non-photochemical quenching" (NPQ) [12][13][14][15][16][17]. When metabolic demand for energy is less than current supply, the major form of NPQ, termed q E (for 'energy dependent' quenching), is triggered by acidification of the lumen (i.e., by the ∆pH component of pmf ), through activation of violaxanthin deepoxidase, which catalyzes the conversion of violaxanthin to antheraxanthin and zeaxanthin [18], and through protonation of the antenna protein PsbS [19,20]. ...
Given their ability to harness chemical energy from the sun and generate the organic compounds necessary for life, photosynthetic organisms have the unique capacity to act simultaneously as their own power and manufacturing plant. This dual capacity presents many unique challenges, chiefly that energy supply must be perfectly balanced with energy demand to prevent photodamage and allow for optimal growth. From this perspective, we discuss the energy balancing network using recent studies and a quantitative framework for calculating metabolic ATP and NAD(P)H demand using measured leaf gas exchange and assumptions of metabolic demand. We focus on exploring how the energy balancing network itself is structured to allow safe and flexible energy supply. We discuss when the energy balancing network appears to operate optimally and when it favors high capacity instead. We also present the hypothesis that the energy balancing network itself can adapt over longer time scales to a given metabolic demand and how metabolism itself may participate in this energy balancing.
... Strong illuminations of light to oxygenic photosynthetic organisms results in decreased CO 2 fixation, inhibition of photosynthetic electron transport and oxygen evolution [4]. Photoinhibition causes mainly photoinactivation of photosystem (PS) II catalysed electron transport and irreversible damage to the reaction centres (RC) [5,6,7]. The 32 kDa, Q B binding protein of the PS II RC is known as D 1 protein. ...
In this present study the effect of high light (300-900 Wm 2) on photosynthetic electron transport activities of Synechococcus 6301 was studied by exposing the cells for 15 min. Our results clearly indicated that photosystem II is more susceptible when compare to that of photosystem I. The possible reason for the inhibition of photosystem II could be alterations at the level of D 1 protein of photosystem II complex. Thus high light shows differential effects on photosystems in the cyanobacterium, Synechococcus 6301.
... boldtianum displayed characteristics typical to that of shade-plant strategists Bertamini et al., 2004) and polar macroalgae (Lüning, 1990;Lobban & Harrison, 1994): (i) lower de-epoxidase activity at 21°C when compared to other Cosmarium strains (ii) low reliance on chloroplast-encoded protein synthesis to resist photoinhibition (iii) relatively slow rate of proteinsynthesis-dependent recovery. These facts are consistent with the hypothesis that shade-adapted plants in their photoinhibited state maintain a high level of photoinhibited, though presumably still physically intact PSII centres which serve for the controlled dissipation of excess light energy (Cleland et al., 1986;Allakhverdiev et al., 1987;Styring et al., 1990;, which may enable polar microalgae to survive in conditions of low temperature and occasionally high irradiance. The distinctly low protein synthesis rate, as noted in C. crenatum, is appropriate in habitats characterized by cold temperatures since low temperatures slow down the PSII repair cycle (Post et al., 1990;Ball et al., 1991;Ö quist & Huner, 1991). ...
Although conjugating algae are considered to have a cosmopolitan freshwater distribution, numerous ecological and taxonomic investigations revealed that many desmid taxa (at the level of genus, species and variety) are capable of occupying specific geographic zones, characterized by particular climatic attributes. Earlier studies have dealt with influences of temperature and irradiation (photosynthetically active radiation and ultraviolet radiation) on the physiology and ultrastructure of desmids. Yet, recent investigations demonstrated a clear relationship between these climatic factors and the distributional potential of conjugating algae, taking into account their photosynthetic, physiological and ultrastructural adaptations which had been revealed during and after certain temperature and irradiation treatments. Despite the fact that desmids can be considered as high light adapted algae, various species- and strain-specific characteristics and adaptations appeared in accordance with the light intensities predominating at their source localities, as estimated by their photosynthetic performance (obtained from PAM fluorometry and oxygen evolution measurements), pigment composition and morpho-anatomical characteristics. Interestingly, the high light adaptation of photosynthesis as well as the relatively high growth temperature optima for majority of the desmid species investigated may provide some support for Coesel’s hypothesis on the origin of desmids in the tropical zone.
... Photosynthesis is catalyzed by two major pigment-protein complexes, Photosystem I (PS I) and Photosystem II (PS II) (Nymark et al., 2009). It is well known that the photosynthetic apparatus, particularly the PSII complex, is sensitive to adverse environmental pressure such as strong light (Styring et al., 1990), and low and high temperature (Terzaghi et al., 1989). Therefore, the photosynthetic pigment contents were measured. ...
Harmful algal blooms (HABs) cause a variety of deleterious effects on aquatic ecosystems, especially the toxic dinoflagellate Alexandrium tamarense, which poses a serious threat to marine economic and human health based on releasing paralytic shellfish poison into the environment. The algicidal bacterium Deinococcus sp. Y35 which can induce growth inhibition on A. tamarense was used to investigate the functional mechanism. The growth status, reactive oxygen species (ROS) content, photosynthetic system and the nuclear system of algal cells were determined under algicidal activity. A culture of strain Y35 not only induced overproduction of ROS in algal cells within only 0.5 h of treatment, also decrease the total protein content as well as the response of the antioxidant enzyme. Meanwhile, lipid peroxidation was induced and cell membrane integrity was lost. Photosynthetic pigments including chlorophyll a and carotenoid decreased along with the photosynthetic efficiency being significantly inhibited. At the same time, photosynthesis-related gene expression showed down-regulation. More than, the destruction of cell nuclear structure and inhibition of proliferating cell nuclear antigen (PCNA) related gene expression were confirmed. The potential functional mechanism of the algicidal bacterium on A. tamarense was investigated and provided a novel viewpoint which could be used in HABs control.
... Together, the data suggests that the acceptor side of PSII did not promote a high singlet oxygen production implicating the formation of the P 680 triplet state. This detrimental reaction probably does not occur because the radical pair recombination becomes blocked by the double reduction of Q A (Vass et al., 1992) with its subsequent release from the binding site (Vass et al., 1988falta nas ref.;Styring et al., 1990). The non significant variation of the high and low potential cyt b 559 forms (Lidon et al., 2004) further suggest that the acceptor side of PSII is not photoinhibited. ...
... On the basis of in vitro studies, two major pathways have been implicated in the photodamage of PSII. The acceptor-side photoinhibition, typical under strong illumination, occurs at the level of the primary quinone electron acceptor Q A , which leaves its site in the D2 polypeptide after being double reduced (Styring et al., 1990; Vass et al., 1992). Such conditions lead to the recombination of the primary radical pair P680 Pheo and to the formation of chlorophyll triplets (Vass et al., 1992). ...
Several mutant strains ofSynechocystis sp. PCC 6803 with large deletions in the D-E loop of the photosystem II (PSII) reaction center polypeptide D1 were subjected to high light to investigate the role of this hydrophilic loop in the photoinhibition cascade of PSII. The tolerance of PSII to photoinhibition in the autotrophic mutant ΔR225-F239 (PD), when oxygen evolution was monitored with 2,6-dichloro-p-benzoquinone and the equal susceptibility compared with control when monitored with bicarbonate, suggested an inactivation of the QB-binding niche as the first event in the photoinhibition cascade in vivo. This step in PD was largely reversible at low light without the need for protein synthesis. Only the next event, inactivation of QA reduction, was irreversible and gave a signal for D1 polypeptide degradation. The heterotrophic deletion mutants ΔG240-V249 and ΔR225-V249 had severely modified QB pockets, yet exhibited high rates of 2,6-dichloro-p-benzoquinone-mediated oxygen evolution and less tolerance to photoinhibition than PD. Moreover, the protein-synthesis-dependent recovery of PSII from photoinhibition was impaired in the ΔG240-V249 and ΔR225-V249 mutants because of the effects of the mutations on the expression of the psbA-2gene. No specific sequences in the D-E loop were found to be essential for high rates of D1 polypeptide degradation.
... At the molecular level, one main target of photoinhibition is photosystem II (PSII), ~ whose electron transport activity is severely impaired by excess illumination. Two different photoinhibition mechanisms have been suggested to be operative under different experimental conditions: an acceptor side mechanism involving over-reduction of the plastoquinone acceptors which depends on the presence of oxygen (32,50,52), and a donor side mechanism, involving accumulation of the strongly oxidant species Tyrz + and/or P6s0 + and independent of the presence of oxygen (27,54). Irrespective of the particular mechanism for electron transport damage, photoinhibition is accompanied by an increased rate of degradation of the reaction center Dl-protein (6,7,24,26,51). ...
The structural and topological stability of thylakoid components under photoinhibitory conditions (4,500 microE.m-2.s-1 white light) was studied on Mn depleted thylakoids isolated from spinach leaves. After various exposures to photoinhibitory light, the chlorophyll-protein complexes of both photosystems I and II were separated by sucrose gradient centrifugation and analysed by Western blotting, using a set of polyclonals raised against various apoproteins of the photosynthetic apparatus. A series of events occurring during donor side photoinhibition are described for photosystem II, including: (a) lowering of the oligomerization state of the photosystem II core; (b) cleavage of 32-kD protein D1 at specific sites; (c) dissociation of chlorophyll-protein CP43 from the photosystem II core; and (d) migration of damaged photosystem II components from the grana to the stroma lamellae. A tentative scheme for the succession of these events is illustrated. Some effects of photoinhibition on photosystem I are also reported involving dissociation of antenna chlorophyll-proteins LHCI from the photosystem I reaction center.
... Although under intense research, the in vivo mechanism(s) of the light-induced irreversible damage of the electron transport in PSII and the actual proteolytic degradation of the D1 protein are still poorly understood. In vitro studies with various PSII preparations have given evidence for two different mechanisms of photoinhibition, the acceptor-side (Setlik et al., 1990;Styring et al., 1990;Vass et al., 1992) and the donor-side mechanisms (Theg et al., 1986;Jegerschold et al., 1990). Acceptor-side photoinhibition is induced when the forward electron flow from PSII is blocked because of complete reduction of the plastoquinone acceptors. ...
Photoinhibition-induced degradation of the D1 protein of the photosystem II reaction center was studied in intact pumpkin (Cucurbita pepo L.) leaves. Photoinhibition was observed to cause the cleavage of the D1 protein at two distinct sites. The main cleavage generated an 18-kD N-terminal and a 20-kD C-terminal degradation fragment of the D1 protein. this cleavage site was mapped to be located clearly N terminally of the DE loop. The other, less-frequent cleavage occurred at the DE loop and produced the well-documented 23-kD, N-terminal D1 degradation product. Furthermore, the 23-kD, N-terminal D1 fragment appears to be phosphorylated and can be detected only under severe photoinhibition in vivo. Comparison of the D1 degradation pattern after in vivo photoinhibition to that after in vitro acceptor-side and donor-side photoinhibition, performed with isolated photosystem II core particles, gives indirect evidence in support of donor-side photoinhibition in intact leaves.
... Excess photon exposure causes nonphysiological overreduction of the first quinone electron acceptor in PSII. Sequential modifications happen at the level of the QA and/or QB acceptors [19]. These conditions lead to the recombination of the radical pair, P680 Pheo [20] and the production of the triplet state of P680-3 P680 [21]. ...
... The acceptor side photoinibition occurs when the plastoquinone pool is highly reduced, i.e., under conditions that saturate the thylakoid transport chain and the Q B binding pocket in PSII remains unoccupied due to the limited availability of oxidized plastoquinones molecules. Since PQ diffusion is a relatively slow process (up to 10 ms for purely diffusion limited occupancy of Q B site) [320], this leads to a stabilization of Q A that in extreme reducing conditions can be doubly reduced and protonated to the quinol form and leave its binding site [321,322]. The absence of an electron acceptor at the photosystem acceptor side promotes the formation of the excited triplet state of P 680 ( 3 P 680 *) via charge recombination of the precursor radical pair (which is initially formed in a pure singlet state), according to the scheme: 1 [323][324][325][326]. ...
Oxygenic photosynthesis is indispensable both for the development and maintenance of life on earth by converting light energy into chemical energy and by producing molecular oxygen and consuming carbon dioxide. This latter process has been responsible for reducing the CO2 from its very high levels in the primitive atmosphere to the present low levels and thus reducing global temperatures to levels conducive to the development of life. Photosystem I and photosystem II are the two multi-protein complexes that contain the pigments necessary to harvest photons and use light energy to catalyse the primary photosynthetic endergonic reactions producing high energy compounds. Both photosystems are highly organised membrane supercomplexes composed of a core complex, containing the reaction centre where electron transport is initiated, and of a peripheral antenna system, which is important for light harvesting and photosynthetic activity regulation. If on the one hand both the chemical reactions catalysed by the two photosystems and their detailed structure are different, on the other hand they share many similarities. In this review we discuss and compare various aspects of the organisation, functioning and regulation of plant photosystems by comparing them for similarities and differences as obtained by structural, biochemical and spectroscopic investigations.
... If it is valid, such a conclusion has implications for any proposed mechanism of photoinactivation. In particular, any mechanism, such as the one proposed by Styring et al. (1990), which requires one photon absorption to reduce Qa and a second photon absorption to cause photodamage cannot be valid for pea leaf discs. The low value of the critical dosage for low-light leaf discs demonstrates that they have less capacity to protect themselves from photoinactivation than medium-and high-light leaf discs (Table 1). ...
Application of target theory to the photoinactivation of Photosystem II in pea leaf discs (Park et al. 1995, 1996a,b) reveals that there is a critical light dosage below which there is complete photoprotection and above which there is photoinactivation (i.e a light-induced loss of oxygen flash yield). The critical dosage is about 3 mol photons m(-2) for medium and high light-grown leaves and 0.36 mol photons m(-2) for low light-grown leaves. Photoinactivation is a one-hit process with an effective cross-section of 0.045 m(2) mol(-1) photons which does not vary with growth irradiance, unlike the cross-section for oxygen evolution which increases with decreasing growth irradiance. The cross-section for oxygen evolution increased by about 20% following exposure to 6.8 mol photons m(-2) which may be due to energy transfer from photoinactivated units to functional Photosystem II units. We propose that the photoinactivation of PS II begins when a small group of PS II pigment molecules whose structure is uninfluenced by growth irradiance, becomes uncoupled energetically from the rest of the photosynthetic unit and thus no longer transfers excitions to P680. De-excitation of this group of pigment molecules provides the energy which leads to the damage of Photosystem II. Treatment of pea leaves with dithiothreitol, an inhibitor of the xanthophyll cycle, decreases the critical dosage i.e. decreases photoprotection but has no effect on the PS II photoinactivation cross-section. Treatment with 1 μM nigericin increased the photoinactivation cross-section of PS II as did exposure to lincomycin which inhibits D1 protein synthesis and thus the repair of PS II reaction centres.
... Photosystem II-mediated oxygen evolution was measured according to Styring et al. (1990). ...
Modified forms of the D1 protein with deletions in lumen-exposed regions, were constructed in the cyanobacterium Synechocystis 6803 using site-directed mutagenesis. Integration and stability of the mutated D1 proteins in the thylakoid membrane were studied by immunoblot and pulse-chase analyses. It was found that in Δ(N325-E333), the D1 protein with a deletion in the C-terminal tail, could insert in the thylakoids to normal amounts but its stability in the membrane was dramatically reduced. Insertion of D1 in Δ(V58-D61) or Δ(D103-G109);G110R, with deletions in the A-B loop, was severely obstructed, For Δ(P350-T354), with a deletion in the processed region of the C-terminus of D1, no phenotypic effects were observed. The effects of failed D1 insertion or accumulation on Photosystem II assembly was monitored by immunoblot analysis. The conclusions from these experiments are that the extrinsic 33 kDa protein, CP43, and the β subunit of cytochrome b559 accumulate in the thylakoid membrane independently of the D1 protein, and that accumulation of the D2 protein and CP47 requires insertion but not necessarily accumulation of the D1 protein.
The cause of phytoplankton blooms has been extensively discussed and largely attributed to favorable external conditions such as nitrogen/phosphorus resources, pH and temperature. Here from the standpoint of hormesis response, we propose that phytoplankton blooms are initiated by stimulatory effects of low concentrations of herbicides as environmental contaminants spread over estuaries and lakes. The experimental results revealed general stimulations by herbicides on Microcystis aeruginosa and Selenastrum capricornutum, with the maximum stimulation in the 30-60% range, depending on the agent and experiment. In parallel with enhancing stimulation, the ratio of HP (high-potential) form to LP (low-potential) form of cytochrome b559 (RHL) was observed decreasing, while intracellular reactive oxygen species (ROS) were observed increasing. We propose that the ROS originated from the thermodynamic transformation of cytochrome b559, enhancing the stimulatory response. Furthermore, the results also proved that thermodynamic states of cytochrome b559 could be modulated by nitric oxide, thus affecting cellular equilibrium of oxidative stress (OS) and correspondingly causing the inhibitory effect of higher concentrations of herbicides on phytoplankton. This suggests that hormesis substantially derives from equilibrium shifting of OS. Moreover, it is reasonable to infer that phytoplankton blooms would be motivated by herbicides or other environmental pollutants. This study provides a new thought into global phytoplankton blooms from a contaminant perspective.
Numerous in vitro investigations have suggested that macroalgae exhibit regular geographic and depth distribution patterns in accordance with the light and temperature predominance at their habitats; however, there have been only a few similar studies concerning microalgae. We examined the potential influence of irradiance on patterns of distribution of four Cosmarium strains isolated from various climatic zones and cultured long term (>15 years) under a constant temperature–light regime. All the Cosmarium strains demonstrated physiological responses that were consistent with the light intensity prevailing at their source location, confirming that these responses are genetically preserved, as concluded from chlorophyll fluorescence and oxygen evolution rates measurements. Addition of inhibitors of chloroplast‐encoded protein synthesis (chloramphenicol and streptomycin) and violaxanthin de‐epoxidase (dithiothreitol) indicated that the Cosmarium strains developed “sun‐ or shade‐plant” protection strategies, in accordance with the climate at their sampling sites. The polar Cosmarium strains exhibited a “shade‐plant strategy”—to suffer some photoinhibition, but acquire increasing protection from photoinhibited PSII centers, whereas the tropical strains displayed a “sun‐plant strategy”—to counteract photoinhibition of PSII by a high rate of repair of photoinhibited PSII reaction centers and a high xanthophyll cycle turnover.
Cytochrome b559 is one of the ubiquitous constituents of the Photosystem II (PSII) reaction centre. Its presence is a prerequisite for the assembly of PSII, but its function is not understood. In contrast to other cytochromes b which participate in electron transfer reactions, no direct evidence is available to indicate that cytochrome b559 plays a functional role in the primary photosynthetic electron transfer processes [1]. Cytochrome b559 has been shown to assume two different redox-potential forms, a low potential (20 to 80 mV) and a remarkably high potential (330 to 400 mV) form [2]. The two subunits of cytochrome b559, termed α and β, are encoded by the plastid genes psbE and psbF, respectively. The genes are part of the psbE,F,L,J operon in higher plants and cyanobacteria [3,4], while in Chlamydomonas reinhardtii the two genes are found on different DNA strands and thus are transcribed separately [5].
Although conjugating algae are considered to have a cosmopolitan freshwater distribution, numerous ecological and taxonomic investigations revealed that many desmid taxa (at the level of genus, species and variety) are capable of occupying specific geographic zones, characterized by particular climatic attributes. Earlier studies have dealt with influences of temperature and irradiation (photosynthetically active radiation and ultraviolet radiation) on the physiology and ultrastructure of desmids. Yet, recent investigations demonstrated a clear relationship between these climatic factors and the distributional potential of conjugating algae, taking into account their photosynthetic, physiological and ultrastructural adaptations which had been revealed during and after certain temperature and irradiation treatments. Despite the fact that desmids can be considered as high-light-adapted algae, various species- and strain-specific characteristics and adaptations appeared in accordance with the light intensities predominating at their source localities, as estimated by their photosynthetic performance (obtained from PAM fluorometry and oxygen evolution measurements), pigment composition and morpho-anatomical characteristics. Interestingly, the high-light adaptation of photosynthesis as well as the relatively high growth temperature optima for majority of the desmid species investigated may provide some support for Coesel’s hypothesis on the origin of desmids in the tropical zone.
Algicidal bacteria have been turned out to be available for inhibiting Phaeocystis globosa which frequently caused harmful algal blooms and threatened to economic development and ecological balance. A marine bacterium Bacillus sp. Ts-12 exhibited significant algicidal activity against P. globosa by indirect attack. In present study, an algicidal compound was isolated by silica gel column, Sephadex G-15 column and HPLC, further identified as hexahydropyrrolo[1,2-a]pyrazine-1,4-dione, cyclo-(Pro-Gly), by GC-MS and 1H-NMR. Cyclo-(Pro-Gly) significantly increased the level of reactive oxygen species (ROS) within P. globosa cells, further activating the enzymatic and non-enzymatic antioxidant systems, including superoxide dismutase (SOD), catalase (CAT), glutathione (GSH) and ascorbic acid (AsA). The increase in methane dicarboxylic aldehyde (MDA) content showed that the surplus ROS induced lipid peroxidation on membrane system. Transmission electron microscope (TEM) and flow cytometry (FCM) analysis revealed that cyclo-(Pro-Gly) caused reduction of Chl-a content, destruction of cell membrane integrity, chloroplasts and nuclear structure. Real-time PCR assay showed that the transcriptions of photosynthesis related genes (psbA, psbD, rbcL) were significantly inhibited. This study indicated that cyclo-(Pro-Gly) from marine Bacillus sp. Ts-12 exerted photosynthetic inhibition and oxidative stress to P. globosa and eventually led to the algal cells lysis. This algicidal compound might be potential bio-agent for controlling P. globosa red tide.
To know more about the site of O-2((radical anion)) production in thylakoids, the F-v/F-m ratio was measured in parallel with the oxygen free-radical production detected by electron spin resonance (ESR) using Tiron as a probe in the isolated thylakoids of Scots pine ( Pinus sylvestris L.). As the light intensity was strong enough to induce photoinhibition leading to reduction in the F-v/F-m ratio, the intensity of the TH. signal representing the superoxide production was enhanced. The TH. signal was also stimulated hy 3-(3, 4-dichloropheneyl)-1, 1-dimethylurea but was suppressed by exogenous scavengers or by 2, 6-dichlorophenol indophenol. Therefore, the superoxide production is closely related to photoinhibition in PS II. The novel results may further promote research on the mechanism of photoinhibition and on the structure and function of PS II as well.
Kinetically-resolved absorbance measurements during extended, or steady-state illumination are typically hindered by large, light-induced changes in the light-scattering properties of the material. In this work, a new type of portable spectrophotometer, the Non-Focusing Optical Spectrophotometer (NoFOSpec), is introduced, which reduces interference from light-scattering changes and is in a form suitable for fieldwork. The instrument employs a non-focusing optical component, called a compound parabolic concentrator (CPC), to simultaneously concentrate and homogeneously diffuse measuring and actinic light (from light-emitting diode sources) onto the leaf sample. Light passing through the sample is then collected and processed using a subsequent series of CPCs leading to a photodiode detector. The instrument is designed to be compact, lightweight and rugged for field work. The pulsed measuring beam allows for high sensitivity (typically Photosystem I (PS I). In addition, the instrument can be used as a kinetic fluorimeter, e.g., to measure saturation-pulse fluorescence changes indicative of Photosystem II (PS II) quantum efficiency. The instrument is demonstrated by estimating electron and proton fluxes through the photosynthetic apparatus in an intact tobacco leaf, using respectively the saturation-pulse fluorescence changes and dark-interval relaxation kinetics (DIRK) of the electrochromic shift. A linear relationship was found, confirming our earlier results with the laboratory-based diffused-optics flash spectrophotometer, indicating a constant H+/e− stoichiometry for linear electron transfer, and suggesting that cyclic electron flow around PS I is either negligible or proportional to linear electron flow. This type of measurement should be useful under field conditions for estimating the extent of PS I cyclic electron transfer, which is proposed to operate under stressed conditions.
During the past years chlorophyll fluorescence has developed as one of the most frequently used measuring tools in plant science. This somewhat unexpected and remarkable development was triggered by recent progress in instrumentation for measuring fluorescence yield under ambient light conditions and by the increased awareness among plant scientists, as in the general public, of aspects of environmental and stress physiology. Since the discovery of the “Kautsky effect” in 1931, fluorescence had always served as a pioneer tool. However, for more than 50 years it was mainly used by biophysically oriented scientists for basic photosynthesis research. The phenomenology of fluorescence changes in intact cells was considered far too complex to provide more than qualitative information. Because the fluorescence characteristics were known to be strongly affected by preillumination, it appeared necessary to thoroughly dark-adapt a sample before recording dark-light induction curves. Additionally, as the complexity of fluorescence changes increased with increasing illumination time, it was mostly the rapid initial induction kinetics which were analyzed to assess the functioning of the primary reactions. Within less than a decade, a completely different situation has evolved. Along with the availability of new instrumentation and analytical methods for fluorescence analysis under normal daylight conditions, the interest has shifted from the primary reactions to the level of overall electron transport efficiency and photosynthesis regulation, and from induction kinetics to investigations of steady-state reactions. Chlorophyll fluorescence, which used to be a tool preferentially applied in dark laboratories, has made the step into the full sunlight, where in situ photosynthesis takes place with all its intricate and still poorly understood regulatory mechanisms in response to environmental factors.
This article describes about the multiple effects of high light intensity on photosynthetic electron transport activities in wheat thylakoid membranes. High light (305-615 Wm-2) affects preferentially photosynthetic electron transport depending on the nature of applied intensity. Between two photosystems, photosystem II is more susceptible as compared to photosystem I. The possible reason for the inhibition in photosystem II catatalysed electron transport activity is the alteration of light harvesting complex of photosystem II as evidenced from the electron transport measurements at different illuminating conditions.
The photoinhibition of photosystem II in vivo by analysis of diverse components -initial rate, steady state rate and lag phase-of photosynthetic O 2 evolution curves on greening wheat seedlings after illumination by excess white light (320 W/m 2) was investigated. A sharp reduction in the initial and steady state rates and a simultaneous intense rise in the lag phase of O 2 evolution were observed under the illumination of seedlings by excess light on the lag phase of chlorophyll a biosynthesis (less than 6 h of seedling greening) in comparison with the illumination of seedlings by excess light at the stage of substantial pigment synthesis (> 6 h of seedling greening). It is assumed that photosystem II proteins not completely integrated in thylakoid membranes as chlorophyll-protein complexes of reaction centres at the early stage of wheat seedling greening were more susceptible to excess light.
Photoinhibition is a state of physiological stress that occurs in all oxygen evolving photosynthetic organisms exposed to light. The primary damage occurs within the reaction center of Photosystem II (PS II). While irreversible photoinduced damage to PS II occurs at all light intensities, the efficiency of photosynthetic electron transfer decreases markedly only when the rate of damage exceeds the rate of its repair, which requires de novo PS II protein synthesis. Photoinhibition has been studied for over a century using a large variety of biochemical, biophysical and genetic methodologies. The discovery of the light induced turnover of a protein, encoded by the plastid psbA gene (the D1 protein), later identified as one of the photochemical reaction center II proteins, has led to the elucidation of the underlying mechanism of photoinhibition and to a deeper understanding of the PS II ‘life cycle.’
Plant experiences multiple abiotic stresses during the same growing season. The implications of submergence with and without saline water on growth and survival were investigated using four contrasting rice cultivars, such as FR13A (submergence-tolerant, salinity-susceptible), IR42 (susceptible to salinity and submergence), Rashpanjor and AC39416 (salinity-tolerant, submergence-susceptible). Though both FR13A and IR42 showed sensitivity to salinity, FR13A as the salinity-tolerant cultivar with higher initial biomass maintained greater dry mass under saline condition. Greater reduction of chlorophyll (Chl) content due to salinity was observed in susceptible cultivars including FR13A compared to salinitytolerant cultivars. Exposure of plants to salinity before submergence decreased the survival chance under submergence. Yet, survival percentage under submergence was greater in FR13A compared to other cultivars. Generally, the reduction in the Chl content and damage to PSII were higher under the submergence compared to salinity conditions. The submergence-tolerant cultivar maintained greater quantities of Chl during submergence compared to other cultivars. Quantification of the Chl a fluorescence transients (JIP-test) revealed large cultivar differences in the response of PSII to submergence in saline and nonsaline water. Submergence-tolerant cultivar maintained greater chloroplast structural integrity and functional ability irrespective of the quality of flooding water.
Photosynthesis involves the capture of the Sun's energy into biochemical energy that sustains all life on the Earth. It is a massive process that has radically changed the composition of the atmosphere. Its scale can be judged from the fact that man's entire fossil fuel use from 1860 to 1988 was equivalent to only 2 years’ global photosynthesis. Although photosynthesis occurs in a wide range of organisms (bacteria, cyanobacteria, algae, and plants), and about a third of it occurs in the oceans, this discussion concentrates on plants.
We have investigated the electron transfer from reduced tyrosine Y D (YDred) and cytochrome b559 to the S2 and S3 states of the water oxidizing complex (WOC) in Photosystem II. The EPR signal of oxidized cyt b559, the S 2 state multiline EPR signal and the EPR signal from Y D· were measured to follow the electron transfer to the S2 and S3 states at 245 and 275 K. The majority of the S2 centers was reduced directly from YDred but at 245 K we observed oxidation of cyt b559 in about 20% of the centers. Incubation of the YDredS3 state resulted in biphasic changes of the S2 multiline signal. The signal first increased due to reduction of the S3 state. Thereafter, the signal decreased due to decay of the S2 state. In contrast, the YD· signal increased with a monophasic kinetics at both temperatures. Again, we observed oxidation of cyt b559 in about 20% of the PSII centers at 245 K. This oxidation correlated with the decay of the S2 state. The complex changes can be explained by the conversion of YDredS3 centers (present initially) to YD·S1 centers, via the intermediate YD·S2 state. The early increase of the S2 state multiline signal involves electron transfer from Y Dred to the S3 state resulting in formation of YD·S2. This state is reduced by cyt b559 resulting in a single exponential oxidation of cyt b 559. Taken together, these results indicate that the electron donor to S2 is cyt b559 while cyt b559 is unable to compete with YDred in the reduction of the S3 state in the pre-reduced samples. We also followed the decay of the S 2 and S3 states and the oxidation of cyt b559 in samples where YD was oxidized from the start. In this case cyt b559 was able to reduce both the S2 and the S3 states suggesting that different pathways exist for the electron transfer from cyt b559 to the WOC. The activation energies for the Y DredS2→YD·S1 and YDredS 3→YD·S2 transformations are 0.57 and 0.67 eV, respectively, and the reason for these large activation energies is discussed.
The nature of interaction of cytochrome b-559 high potential (HP) with electron transport on the reducing side of photosystem II was investigated by measuring the susceptibility of cytochrome b-559HP to 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) under different conditions. Submicromolar DCMU concentrations decreased the rate of absorbance change corresponding to cytochrome b-559HP photoreduction while the amplitude was lowered at higher concentrations (up to 10 μM). Appreciable extents of cytochrome b-559HP photoreduction were observed at DCMU concentrations which completely abolished the electron transport from water to methyl viologen under the same experimental conditions. However, the susceptibility of cytochrome b-559HP to DCMU increased with the degree of cytochrome b-559HP oxidation, induced either by ferricyanide or by illumination of low intensity (2 W/m(2)) of red light in the presence of 2 μM carbonyl cyanide-m-chlorophenylhydrazone. Also, the DCMU inhibition was more severe when the pH increased from 6.5 to 8.5, indicating that the unprotonated form of cytochrome b-559HP is more susceptible to DCMU. These results demonstrate that cytochrome b-559HP can accept electrons prior to the QB site, probably via QA although both QA and QB can be involved to various extents in this reaction. We suggest that the redox state and the degree of protonation of cytochrome b-559HP alter its interaction with the reducing side of photosystem II.
Photoinhibition was analyzed in O2-evolving and in Tris-treated PS II membrane fragments by measuring flash-induced absorption changes at 830 nm reflecting the transient P680(+) formation and oxygen evolution. Irradiation by visible light affects the PS II electron transfer at two different sites: a) photoinhibition of site I eliminates the capability to perform a 'stable' charge separation between P680(+) and QA (-) within the reaction center (RC) and b) photoinhibition of site II blocks the electron transfer from YZ to P680(+). The quantum yield of site I photoinhibition (2-3×10(-7) inhibited RC/quantum) is independent of the functional integrity of the water oxidizing system. In contrast, the quantum yield of photoinhibition at site II depends strongly on the oxygen evolution capacity. In O2-evolving samples, the quantum yield of site II photoinhibition is about 10(-7) inhibited RC/quantum. After selective elimination of the O2-evolving capacity by Tris-treatment, the quantum yield of photoinhibition at site II depends on the light intensity. At low intensity (<3 W/m(2)), the quantum yield is 10(-4) inhibited RC/quantum (about 1000 times higher than in oxygen evolving samples). Based on these results it is inferred that the dominating deleterious effect of photoinhibition cannot be ascribed to an unique target site or a single mechanism because it depends on different experimental conditions (e.g., light intensity) and the functional status of the PS II complex.
A Photosystem two (PS II) core preparation containing the chlorophyll a binding proteins CP 47, CP 43, D1 and D2, and the non-chlorophyll binding cytochrome-b559 and 33 kDA polypeptides, has been isolated from PS II-enriched membranes of peas using the non-ionic detergent heptylthioglucopyranoside and elevated ionic strengths. The primary radical pair state, P680(+)Pheo(-), was studied by time-resolved absorption and fluorescence spectroscopy, under conditions where quinone reduction and water-splitting activities were inhibited. Charge recombination of the primary radical pair in PS II cores was found to have lifetimes of 17.5 ns measured by fluorescence and 21 ns measured by transient decay kinetics under anaerobic conditions. Transient absorption spectroscopy demonstrated that the activity of the particles, based on primary radical pair formation, was in excess of 70% (depending on the choice of kinetic model), while time-resolved fluorescence spectroscopy indicated that the particles were 91% active. These estimates of activity were further supported by steady-state measurements which quantified the amount of photoreducible pheophytin. It is concluded that the PS II core preparation we have isolated is ideal for studying primary radical pair formation and recombination as demonstrated by the correlation of our absorption and fluorescence transient data, which is the first of its kind to be reported in the literature for isolated PS II core complexes from higher plants.
Synopsis
We have studied the defences of Norway spruce seeds against pollutants during germination, using two different phenolic compounds, 5-OH-1,4-naphthoquinone (5-OH-NQ) ³ and 2,4,5-trichlorophenoxyacetic acid (2,4,5-T).
Only small effects of 5-OH-NQ on germination of seeds were observed at concentrations up to 200 μM, which could be explained by the formation of a less reactive metabolite of 5-OH-NQ. These results suggest that Norway spruce seeds have a very effective defence system against quinone and quinonederived reactive oxygen species.
The effect of 2,4,5-T on seed germination was, however, more pronounced, resulting in an abnormal growth of the seedlings. This behaviour was probably due to a strong increase in ethylene production (10-fold) in these seedlings. Also fatty acyl-CoA oxidase activity, a peroxisomal enzyme of β-oxidation that catalyses the formation of H 2 O 2 , was found to increase 6.9-fold in seedlings germinated in presence of 2,4,5-T. A strong decrease in the activity of photosystem II and an increase in lipid peroxidation in chloroplasts was also observed.
Characteristics of thermoluminescence (TL) glow curves were studied in thylakoids (isolated from pea leaves) or in intact pea leaves after an exposure to very high light for 2 min in the TL device. The inhibition of photosynthesis was detected as decreases of oxygen evolution rates and/or of variable fluorescence.
In thylakoids exposed to high light, then dark adapted for 5 min, a flash regime induced TL glow curves which can be interpreted as corresponding to special B bands since: 1) they can be fitted by a single B band (leaving a residual band at −5°C) with a lower activation energy and a shift of the peak maximum by −5 to −6°C and, 2) the pattern of oscillation of their amplitudes was normal with a period of 4 and maxima on flashes 2 and 6. During a 1 h dark adaptation, no recovery of PS II activity occurred but the shift of the peak maximum was decreased to −1 to −2°C, while the activation energy of B bands increased. It is supposed that centers which remained active after the photoinhibitory treatment were subjected to reversible and probably conformational changes.
Conversely, in intact leaves exposed to high light and kept only some minutes in the dark, TL bands induced by a flash regime were composite and could be deconvoluted into a special B band peaking near 30°C and a complex band with maximum at 2–5°C. In the case of charging bands by one flash, this low temperature band was largely decreased in size after a 10 min dark adaptation period; parallely, an increase of the B band type component appeared. Whatever was the flash number, bands at 2–5°C were suppressed by a short far red illumination given during the dark adaptation period and only remained a main band a 20°C; therefore, the origin of the low temperature band was tentatively ascribed to recombinations in centers blocked in state S2QA−QB2−. In vivo, the recovery of a moderately reduced state in the PQ pool, after an illumination, would be slow and under the dependence of a poising mechanism, probably involving an electron transfer between cytosol and chloroplasts or the so-called ‘chlororespiration’ process.
pH-dependent inactivation of Photosystem (PS) II and related quenching of chlorophyll-a-fluorescence have been investigated in isolated thylakoids and PS II-particles and related to calcium release at the donor side of PS II. The capacity of oxygen evolution (measured under light saturation) decreases when the ΔpH is high and the pH in the thylakoid lumen decreases below 5.5. Oxygen evolution recovers upon uncoupling. The pH-response of inactivation can be described by a 1 H(+)-transition with an apparent pK-value of about 4.7. The yield of variable fluorescence decreases in parallel to the inactivation of oxygen evolution. pH-dependent quenching requires light and can be inhibited by DCMU. In PS II-particles, inactivation is accompanied by a reversible release of Ca(2+)-ions (one Ca(2+) released per 200 Chl). In isolated thylakoids, where a ΔpH was created by ATP-hydrolysis, both inactivation of oxygen evolution (and related fluorescence quenching) by internal acidification and the recovery of that inactivation can be suppressed by calcium-channel blockers. In the presence of the Ca(2+)-ionophore A23187, recovery of Chl-fluorescence (after relaxation of the ΔpH) is stimulated by external Ca(2+) and retarded by EGTA. As shown previously (Krieger and Weis 1993), inactivation of oxygen evolution at low pH is accompanied by an upward shift of the midpoint redox-potential, Em, of QA. Here, we show that in isolated PS II particles the pH-dependent redox-shift (about 160 mV, as measured from redox titration of Chl-fluorescence) is suppressed by Ca(2+)-channel blockers and DCMU. When a redox potential of -80 to -120mV was established in a suspension of isolated thylakoids, the primary quinone acceptor, QA, was largely reduced in presence of a ΔpH (created by ATP-hydrolysis) but oxidized in presence of an uncoupler. Ca(2+)-binding at the lumen side seems to control redox processes at the lumen- and stroma-side of PS II. We discuss Ca(2+)-release to be involved in the physiological process of 'high energy quenching'.
Superoxide anion radical formation was studied with isolated spinach thylakoid membranes and oxygen evolving Photosystem II sub-thylakoid preparations using the reaction between superoxide and Tiron (1,2-dihydroxybenzene-3,5-disulphonate) which results in the formation of stable, EPR detectable Tiron radicals.
We found that superoxide was produced by illuminated thylakoids but not by Photosystem II preparations. The amount of the radicals was about 70% greater under photoinhibitory conditions than under moderate light intensity. Superoxide production was inhibited by DCMU and enhanced 4–5 times by methyl viologen. These observations suggest that the superoxide in illuminated thylakoids is from the Mehler reaction occurring in Photosystem I, and its formation is not primarily due to electron transport modifications brought about by photoinhibition.
Artificial generation of superoxide from riboflavin accelerated slightly the photoinduced degradation of the Photosystem II reaction centre protein D1 but did not accelerate the loss of oxygen evolution supported by a Photosystem II electron acceptor. However, analysis of the protein breakdown products demonstrated that this added superoxide did not increase the amount of fragments brought about by photoinhibition but introduced an additional pathway of damage.
On the basis of the above observations we propose that superoxide redicals are not the main promoters of acceptor-side-induced photoinhibition of Photosystem II.
Cytochrome b559 (Cyt b559) is a well-known intrinsic component of Photosystem II (PS II) reaction center in all photosynthetic oxygen-evolving organisms, but its physiological role remains unclear. This work reports the response of the two redox forms of Cyt b559 (i.e. the high- (HP) and low-potential (LP) forms) to inhibition of the donor or acceptor side of PS II. The photooxidation of HP Cyt b559 induced by red light at room temperature was pH-dependent under conditions in which electron flow from water was diminished. This photooxidation was observed only at pH values higher than 7.5. However, in the presence of 1 μM CCCP, a limited oxidation of HP Cyt b559 was observed at acidic pH, At pH 8.5 and in the presence of the protonophore, this photooxidation of the HP form was accompanied by its partial transformation into the LP form. On the other hand, a partial photoreduction of LP Cyt b559 was induced by red light under aerobic conditions when electron transfer through the primary quinone acceptor QA was impaired by strong irradiation in the presence of DCMU. This photoreduction was enhanced at acidic pH values. To the best of our knowledge, this is the first time that both photoreduction and photooxidation of Cyt b559 is described under inhibitory conditions using the same kind of membrane preparations. A model accommodating these findings is proposed.
The Photosystem II reaction center is rapidly inactivated by light, particularly at higher light intensity. One of the possible factors causing this phenomenon is the oxidized primary donor, P680(+), which may be harmful to Photosystem II because of its highly oxidizing nature. However, no direct evidence specificially implicating P680(+) in photoinhibition has been obtained yet. To investigate whether P680(+) is harmful to Photosystem II, turnover of the D1 protein and of the Photosystem II reaction center complex were measured in vivo in a mutant of the cyanobacterium Synechocystis sp. PCC 6803, in which the physiological donor to P680(+), Tyrz, was genetically deleted. In this mutant, D1 degradation in the light is an order of magnitude faster than in wild type. The most straightforward explanation of this phenomenon is that accumulation of P680(+) leads to an increased rate of turnover of the Photosystem II reaction center complex, which is compatible with the hypothesis of destructive oxidation by P680(+) that is damaging to the Photosystem II complex.
The mechanism of photoinhibition of photosystem II (PSII) was studied in intact leaf discs of Spinacia oleracea L. and detached leaves of Vigna unguiculata L. The leaf material was exposed to different photon flux densities (PFDs) for 100 min, while non-photochemical (qN) and photochemical quenching (qp) of chlorophyll fluorescence were monitored. The 'energy' and redox state of PSII were manipulated quite independently of the PFD by application of different temperatures (5-20° C), [CO2] and [O2] at different PFDs. A linear or curvilinear relationship between qp and photoinhibition of PSII was observed. When [CO2] and [O2] were both low (30 μl · l(-1) and 2%, respectively), PSII was less susceptible at a given qp than at ambient or higher [CO2] and photoinhibition became only substantial when qp decreased below 0.3. When high levels of energy-dependent quenching (qE) (between 0.6 and 0.8) were reached, a further increase of the PFD or a further decrease of the metabolic demand for ATP and NADPH led to a shift from qE to photoinhibitory quenching (qI). This shift indicated that photoinhibition was preceded by down-regulation through light-induced acidification of the lumen. We propose that photoinhibition took place in the centers down-regulated by qE. The shift from qE to qI occurred concomitant with qP decreasing to zero. The results clearly show that photoinhibition does not primarily depend on the photon density in the antenna, but that photoinhibition depends on the energy state of the membrane in combination with the redox balance of PSII. The results are discussed with regard to the mechanism of photoinhibition of PSII, considering, in particular, effects of light-induced acidification on the donor side of PSII. Interestingly, cold-acclimation of spinach leaves did not significantly affect the relationship between qP, qE and photoinhibition of PSII at low temperature.
The effects of potassium (K) deficiency on chlorophyll (Chl) content, photosynthetic gas exchange, and photosystem II (PSII) photochemistry during the seedling stage were investigated in two soybean [Glycine max (L.) Merr.] cultivars, low-K sensitive Tiefeng31 and low-K tolerant Shennong6. The cultivars were grown hydroponically in K-sufficient (KS) and K-deficient (KD) solutions. Photosynthetic gas exchange and Chl content in Tiefeng31 were severely affected by the low K condition, but were almost unaffected in Shennong6. This difference is in accordance with the PSII photochemistry in the plants, indicating that the photosynthetic apparatus of Shennong6 is more tolerant to low-K stress than that of Tiefeng31.
The effect of high light on the acceptor side of photosystem II of chloroplasts and core particles of spinach was studied. BothV
max and apparentK
m for DCIP were altered in photoinhibited photosystem II core particles. The double reciprocal plot analysis as a function of actinic light showed increased slope in chloroplasts photoinhibited in the presence of DCMU. Exposure of chloroplasts to high light in the presence of DCMU did not protect the chloroplast against high light induced decrease in Fm, level. Further the high light stress induced decrease inF
m level was not restored by the addition of DCMU. These results suggest that the high light stress induced damage to chloroplast involves alteration in the binding site forQ
B on the DI protein on the acceptor side of photosystem II
The effect of light (250 μmol m-2s-1) on the appearance of post-chilling symptoms was investigated in cold-treated maize (Zea mays L. hybrid Furio) seedlings using electrolyte leakage and chlorophyll fluorescence induction measurements as indicators. The longer the cold pretreatment (0.5 °C) in complete darkness, the more rapid the decrease in Fv/Fm and the increase in electrolyte leakage during cold treatment in the light. The most important difference in the changes in these two parameters is that the changes in Fv/Fm occur much earlier if the cold treatment is carried out in the light. These findings suggest that chilling stress in cold sensitive maize plants led to an increased susceptibility to photoinhibition at low temperatures. Fv/Fm and electrolyte leakage changed not only at low temperatures but also after a certain cold pretreatment period at normal temperature. When the seedlings were returned to 25 °C after various chilling periods in the dark both parameters showed that post-chilling symptoms appeared much more rapidly in the light than in the dark. By contrast to the change in Fv/Fm, where plants chilled for only two days exhibited differences in post-chilling changes in the light and dark, a substantial increase in electrolyte leakage was only observed after four days of cold pretreatment. These results suggest that photoinhibition has a role not only during the chilling period, but also in the appearance of post-chilling symptoms.
Isolated spinach thylakoids can be protected from photoinhibitory loss of electron transport capacity by the radical defense system composed of the enzymes SOD and catalase, as well as the antioxidants glutathione and ascorbate. With these compounds present at saturating concentrations, thylakoids not only retain a high photochemical capacity but also do not suffer D1-protein degradation during photoinhibition. However, a strong decrease in the quantum yield of oxygen evolution, ΦO2, occurs with the same thylakoids. These results support the view that the photochemical capacity and the quantum yield of oxygen evolution decline due to different mechanisms during photoinhibition. The mechanism underlying the loss of ΦO2, seems to be temperature-dependent, as ΦO2, did not decline below the electron transport capacity during photoinhibition at 0°C.
Dispersion of photosystem II (PS II) complex with phosphatidylcholines resulted in protection of strong light- and high temperature-induced inhibition of photosynthetic electron transport activity and variable chlorophyll fluorescence. Dispersion of the PS II complex with phosphatidylcholines with longer fatty acyl chains provided a greater extent of protection which indicates an interaction of the PS II components with the hydrophobic core of the lipid bilayer. This interaction might play an important role in the regulation of PS II photochemistry and determination of susceptibility of the PS II complex to environmental stress.
The role of semiquinone anion radicals in photoinhibition of isolated wheat (Triticum aestivum) chloroplasts was investigated by subjecting the chloroplasts to high light stress in the presence or absence of DCMU and DBMIB. The decrease in the efficiency of PS II photochemistry measured as Fv/Fm ratio and oxygen evolution after photoinhibition of isolated wheat chloroplasts was less in the presence of DCMU. We suggest that the protective effect of DCMU is due to its binding to the 32 kDa QB-binding protein and reducing the probability of formation of semiquinone anion and other free radical species that have been suggested to be involved in photoinhibition damage. The hypothesis was also tested by using DBMIB during photoinhibition. DBMIB is known to reduce the plastoquinone pool, resulting in an increase in the semiquinone ion population. A greater extent of reduction of Fv/Fm and oxygen evolution was observed when chloroplasts were photoinhibited in the presence of DBMIB. The results suggest an involvement of reduced semiquinones in the photoinhibition of wheat chloroplasts. A partial recovery of variable chlorophyll fluorescence in the presence of 20 mM hydroxylamine was also observed in chloroplasts subjected to light stress.
The effect of photoinhibition on the primary (Q A ) and secondary (Q B ) quinone acceptors of photosystem I I was investigated in isolated spinach thylakoids by the methods of thermoluminescence and delayed luminescence. The amplitudes of the Q (at about 2 °C) and B (at about 30 °C) thermoluminescence bands which are associated with the recombination of the S 2 Q A ⁻ and S 2 Q B charge pairs, respectively, exhibited parallel decay courses during photoinhibitory treatment. Similarly, the amplitudes of the flash-induced delayed luminescence components ascribed to the recombination of S20A and S2OB charge pairs and having half life-times of about 3 s and 30 s, respectively, declined in parallel with the amplitudes of the corresponding Q and B thermoluminescence bands. The course of inhibition of thermoluminescence and delayed luminescence intensity was parallel with that of the rate of oxygen evolution. The peak positions of the B and Q thermoluminescence bands as well as the half life-times of the corresponding delayed luminescence components were not affected by photoinhibition. These results indicate that in isolated thylakoids neither the amount nor the stability of the reduced OB acceptor is preferentially decreased by photoinhibition. We conclude that either the primary target of photodamage is located before the O b binding site in the reaction center of photosystem II or QA and OB undergo simultaneous damage.
Electron paramagnetic resonance (EPR) spectra of the reduced quinone-iron acceptor complex in reaction centers were measured in a variety of environments and compared with spectra calculated from a theoretical model. Spectra were obtained at microwave frequencies of 1, 9, and 35 GHz and at temperatures from 1.4 to 30 K. The spectra are characterized by a broad absorption peak centered at g = 1.8 with wings extending from g approximately equal to 5 to g less than 0.8. The peak is split with the low-field component increasing in amplitude with temperature. The theoretical model is based on a spin Hamiltonian, in which the reduced quinone, Q-, interacts magnetically with Fe2+. In this model the ground manifold of the interacting Q-Fe2+ system has two lowest doublets that are separated by approximately 3 K. Both perturbation analyses and exact numerical calculations were used to show how the observed spectrum arises from these two doublets. The following spin Hamiltonian parameters optimized the agreement between simulated and observed spectra: the electronic g tensor gFe, x = 2.16, gFe, y = 2.27, gFez = 2.04, the crystal field parameters D = 7.60 K and E/D = 0.25, and the antiferromagnetic magnetic interaction tensor, Jx = -0.13 K, Jy = -0.58 K, Jz = -0.58 K. The model accounts well for the g value (1.8) of the broad peak, the observed splitting of the peak, the high and low g value wings, and the observed temperature dependence of the shape of the spectra. The structural implications of the value of the magnetic interaction, J, and the influence of the environment on the spin Hamiltonian parameters are discussed. The similarity of spectra and relaxation times observed from the primary and secondary acceptor complexes Q-AFe2+ and Fe2+Q-B leads to the conclusion that the Fe2+ is approximately equidistant from QA and QB.
Photosynthetic oxygen evolution takes place in the thylakoid protein complex known as photosystem II. The reaction center core of this photosystem, where photochemistry occurs, is a heterodimer of homologous polypeptides called D1 and D2. Besides chlorophyll and quinone, photosystem II contains other organic cofactors, including two known as Z and D. Z transfers electrons from the site of water oxidation to the oxidized reaction center primary donor, P+.680, while D+. gives rise to the dark-stable EPR spectrum known as signal II. D+. has recently been shown to be a tyrosine radical. Z is probably a second tyrosine located in a similar environment. Indirect evidence indicates that Z and D are associated with the D1 and D2 polypeptides, respectively. To identify the specific tyrosine residue corresponding to D, we have changed Tyr-160 of the D2 polypeptide to phenylalanine by site-directed mutagenesis of a psbD gene in the cyanobacterium Synechocystis 6803. The resulting mutant grows photosynthetically, but it lacks the EPR signal of D+.. We conclude that D is Tyr-160 of the D2 polypeptide. We suggest that the C2 symmetry in photosystem II extends beyond P680 to its immediate electron donor and conclude that Z is Tyr-161 of the D1 polypeptide.
The 32 000-dalton Q(B)-protein of photosystem II (PS II) is rapidly damaged and removed from isolated pea thylakoids during incubation in the light resulting in a loss of photosynthetic electron flow through PS II. This in vitro photoinhibition is similar to that previously reported with intact Chlamydomonas cells. The damage occurs at a faster rate in vitro, however, due to the inability of isolated thylakoids to synthesize replacement Q(B)-protein. The removal of the damaged Q(B)-protein does not require any soluble components of the chloroplast stroma and is unaffected by the protease inhibitors phenyl-methylsulfonylfluoride or antipain. Unlike the effect of trypsin, no low mol. wt. membrane-bound or soluble fragments of the labelled Q(B)-protein could be identified either by autoradiography or immunologically using polyclonal antibodies specific for the Q(B)-protein. The lightinduced damage to the Q(B)-protein (indicated by a loss of Q(B) functional activity), preceded the removal of the protein from the membrane. We conclude that photodamage of the Q(B)-protein generates a conformational change which renders the protein susceptible to attack by a highly efficient, intrinsic membrane protease.
A loss of electron transport capacity in chloroplast membranes was induced by high-light intensities (photoinhibition). The primary site of inhibition was at the reducing side of photosystem II (PSII) with little damage to the oxidizing side or to the reaction center core of PSII. Addition of herbicides (atrazine or diuron) partially protected the membrane from photoinhibition; these compounds displace the bound plastoquinone (designated as Q(B)), which functions as the secondary electron acceptor on the reducing side of PSII. Loss of function of the 32-kilodalton Q(B) apoprotein was demonstrated by a loss of binding sites for [(14)C]atrazine. We suggest that quinone anions, which may interact with molecular oxygen to produce an oxygen radical, selectively damage the apoprotein of the secondary acceptor of PSII, thus rendering it inactive and thereby blocking photosynthetic electron flow under conditions of high photon flux densities.
A photosystem II reaction center complex consisting of D-1 and D-2 polypeptides and cytochrome b-559 was isolated from spinach grana thylakoids, treated with 4% (wt/vol) Triton X-100, by ion-exchange chromatography using DEAE-Toyopearl 650S. The isolated complex appears to contain five chlorophyll a, two pheophytin a, one beta-carotene, and one or two cytochrome b-559 heme(s) (molar ratio) and exhibits a reversible absorbance change attributable to the photochemical accumulation of reduced pheophytin typical for the intermediary electron acceptor of photosystem II reaction center. These results strongly suggest that the site of primary charge separation in photosystem II is located on the heterodimer composed of D-1 and D-2 subunits.
The oxidation‐reduction midpoint potential (E m) of the primary quinone (QA) of the acceptor quinone complex of bacterial photosynthetic reaction centers has been measured as a function of pH in the presence and absence of ubiquinone and o‐phenanthroline (o‐Phen). Reaction centers, isolated from Rhodopseudomonas sphaeroides, were incorporated into egg phosphatidylcholine vesicles. Contrary to earlier reports, the E m was found to exhibit a pH‐dependence very similar to that observed in chromatophores, with a slope of ‐ 60 mV/pH up to a p K for Q−A/Q−A(H+) at pH 9.5–10.0. In the presence of ubiquinone to reconstitute the secondary quinone (QB), the E m/pH curve of QA was shifted to lower potentials, indicating that the binding of Qn (actually QBH2) was suppressed by reduction of QA. o‐Phen, an inhibitor of electron transport from QA to QB, raised the pK of Q−A/Q−A(H+) and, at pH‐values below but not above this pK, reversed the effects of QB. In the absence of QB, o‐Phen lowered the E m of QA above the pK but had no effect below it. These results are discussed in terms of interactions between the binding sites for QA and QB (A‐ and B‐sites). It is suggested that ubiquinone and o‐Phen compete for the B‐site in a mutually exclusive fashion, and that their relative binding strengths are modulated by the redox and protonation state of QA. In preparations with low quinone content, o‐Phen inhibits photochemistry suggesting that it can also compete with ubiquinone at the A‐site. Competitive displacement of quinone from the B‐site by o‐Phen and other inhibitors is suggested as the primary mode of action of a broad class of herbicides active in Photosystem II of plants. The relative binding affinities of the various redox states of QB are also discussed and it is concluded that the order of binding strength is: Q−B → QB → QBH2. This accounts for the atypical stability of the semiquinone and the lower average E m for reduction to the quinol, compared to free ubiquinone in the quinone pool. It may also be significant in the functioning of quinones in communicating reducing equivalents from the reaction center to other electron transport complexes in the intact membrane.
Photoinhibition of photosynthesis is manifested at the level of the leaf as a loss of CO2 fixation and at the level of the chloroplast thylakoid membrane as a loss of photosystem II electron-transport capacity. At the photosystem II level, photoinhibition is manifested by a lowered chlorophyll a variable fluorescence yield, by a lowered amplitude of the light-induced absorbance change at 320 nm (ΔA320) and 540-minus-550 nm (ΔA540-550), attributed to inhibition of the photoreduction of the primary plastoquinone QA molecule. A correlation of the kinetics of variable fluorescence yield loss with the inhibition of QA photoreduction suggested that photoinhibited reaction centers are incapable of generating a stable charge separation but are highly efficient in the trapping and non-photochemical dissipation of absorbed light. The direct effect of photoinhibition on primary photochemical parameters of photosystem II suggested a permanent reaction center modification the nature of which remains to be determined.
Integrated intensities of EPR lines and the simulation of powder shapes in the presence of large anisotropy are discussed for field-sweep spectra. It is pointed out that the shape function, normalized by integration over the magnetic field, must be multiplied by a factor which in the case equals the inverse of the g-value. This factor seemingly has been omitted in previous calculations of intensities and shapes. As a consequence, the integrated intensity of an isotropic line is proportional to its g-value and not to g2. The total intensity of a powder spectrum is calculated, and examples of simulations of such spectra are given. A method for the determination of total intensities from the area under an “absorption” peak in a first derivative powder spectrum is also given.
In this paper, the evidence supporting two different models for the molecular mechanism of photoinhibition is discussed. One hypothesis centres around the suggestion that photoinhibition is due to the loss of the herbicide-binding Dl polypeptide of photosystem II. The other model suggests that damage to a functional group in the reaction centre is the primary cause of photoinhibition. In order to put the apparent controversy into context, recent developments in our understanding of the structure and function of the photosystem II reaction centre are described. Interpretation and judgement of all available evidence suggest primary photoinhibitory damage to be incurred by the reaction-centre chlorophyll P680 destabilising the apoprotein(s) and eventually resulting in their proteolytic degradation and removal from the photosystem II complex and the thylakoid membrane.
The decay kinetics for the S2 and S3 states of the oxygen-evolving complex in Photosystem II have been measured in the presence of an external electron acceptor. The S2- and S3-states decay monophasically with half-decay times at 18°C of 3–3.5 min and 3.5–4 min, respectively. The results also show that S3 decays via S2 under these circumstances. The temperature dependence of the individual S-state transitions has been measured in single flash experiments in which the multiline EPR signal originating from the S2 state has been used as spectroscopic probe. The half-inhibition temperatures are for S0 to S1 220–225 K, for S1 to S2 135–140 K, for S2 to S3 230 K and for the S3-to-S0 transition 235 K.
The high-affinity binding site for Mn2+ is characterized by a decrease in 1,5-diphenylcarbazide (DPC) to 2,6-dichlorophenolindophenol (DCIP) electron transport with NH2OH-treated spinach Photosystem II (PS II) membrane fragments when micromolar amounts of Mn2+ are present in the assay. This site is purported to be the binding site for Mn, functional in O2 evolution (Hsu, B.-D., Lee, J.-Y. and Pan, R.-L. (1987) Biochim. Biophys. Acta 890, 89–96). We have examined this site in PS II-enriched membranes from Scenedesmus obliquus wild-type (WT) and LF-1 mutant cells. LF-1 inserts an unprocessed D1 protein into the photosynthetic membrane, binds approx. 40% of the functional Mn as WT, and does not evolve O2 (Metz, J.G., Pakrasi, H.B., Seibert, M. and Arntzen, C.J. (1986) FEBS Lett. 205, 269–274). The dissociation constant for added high-affinity Mn2+ is about 0.3–0.4 μM in wheat, WT, and LF-1 PS II. However, the relative amount of available high-affinity Mn2+-binding site is about half as much in LF-1 PS II membranes compared to wheat, spinach, and WT PS II membranes. Despite the fact that LF-1 PS II can photoligate Mn, LF-1 cannot be photoactivateid as can NH2OH-treated WT PS II. LF-1 subjected to photoactivating conditions does not reach S2 as determined by thermoluminescence. This work indicates that the Hsu et al. high-affinity Mn2+ site is actually at least two sites, one of which is missing in LF-1, and that successful photoactivation potential requires the presence of all high-affinity Mn2+ site. The fact that the full complement of high-affinity Mn2+-binding site is observed in isolated spinach PS II reaction center (D1/D2/cytochrome b-559) complex demonstrates that other PS II core proteins do not affect the high-affinity site. Histidine chemical modifier experiments show that one component of the high-affinity site is probably associated with histidine(s) and that this component is missing in LF-1. We conclude that histidine(s) on the Dl protein provides ligand(s) for part of the Mn required for O2-evolution function and that the balance of the Mn is bound by other amino acids on the proteins composing the PS II reaction center.
We have investigated the irreversible inhibition produced in the photosynthetic electron-transport chain by illuminating Cl−-free or Tris-washed chloroplasts. Our data indicate that the site of this inhibition is on the oxidizing side of Photosystem II, either at the reaction center, P-680, or its immediate electron donor, Z; the possibility that the primary photoinduced lesion is at the second stable Photosystem II electron acceptor, QB, has been excluded. Comparison of our data with those in the literature lead us to postulate that the photoinactivation under study here is mechanistically the same as photoinhibition in living plants (Powles, S.B. (1984) Annu. Rev. Plant Physiol. 35, 15–44), the former occurring on an accelerated time scale.
The question of the primary site of photoinhibition was investigated using the light-induced absorbance change of pheophytin a (at 685 nm) in spinach chloroplasts and Chlamydomonas reinhardtii cells. Photoinhibition of spinach thylakoid membranes resulted in a parallel decrease in the amplitude of the pheophytin a (ΔA685) and QA (ΔA320) photoreduction signals. In intact Chlamydomonas reinhardtii cells the pheophytin photoreduction and oxygen evolution activity exhibited a similar decrease during photoinhibition. A complete recovery of both activities was attained within 60 min incubation in normal growth conditions. It is concluded that the primary site of photoinhibition involves the components (P-680 and/or pheophytin) of the primary charge separation.
Redox titrations of the photo-induced pheophytin EPR signal in Photosystem II show two transitions which reflect the redox state of Q. The high potential wave (Em ⋍ −50 mV) can be photo-induced at 5 K and 77 K. The low potential wave (Em ⋍ −275 mV) required illumination at 200 K. This indicates the presence of two kinds of PS-II reaction centres differing in terms of the competence of their donors at low temperature and the Em-values of their acceptors. Measurements of the semiquinone-iron acceptor also demonstrate functional heterogeneity at low temperature. This is the first observation of the semiquinone-iron acceptor in a non-mutant species.
The effects of selective removal of extrinsic proteins on donor side electron transport in oxygen-evolving PS II particles were examined by monitoring the decay time of the EPR signal from the oxidized secondary donor, Z+, and the amplitude of the multiline manganese EPR signal. Removal of the 16 and 24 kDa proteins by washing with 1 M NaCl inhibits oxygen evolution, but rapid electron transfer to Z+ still occurs as evidenced by the near absence of Signal IIf. The absence of a multiline EPR signal shows that NaCl washing induces a modification of the oxygen-evolving complex which prevents the formation of the S2 state. This modification is different from the one induced by chloride depletion of PS II particles, since in these a large multiline EPR signal is found. After removal of the 33 kDa protein with 1 M MgCl2, Signal IIf is generated after a light flash. Readdition of the 33 kDa component to the depleted membranes accelerates the reduction of Z+. Added calcium ions show a similar effect. These findings suggest that partial advancement through the oxygen-evolving cycle can occur in the absence of the 16 and 24 kDa proteins. The 33 kDa protein, on the other hand, may be necessary for such reactions to take place.
We have investigated the effects of temperature on the formation and decay of the light-induced multiline EPR signal species associated with photosynthetic oxygen evolution (Dismukes, G.C. and Siderer, Y. (1980) FEBS Lett. 121, 78–80). (1) The decay rate following illumination is temperature dependent: at 295 K the half-time of decay is about 40 s, at 253 K the half-time is approx. 40 min. (2) A single intense flash of light becomes progressively less effective in generating the multiline signal below about 240 K. (3) Continuous illumination is capable of generating the signal down to almost 160 K. (4) Continuous illumination after a preilluminating flash generates less signal above 200 K than at lower temperatures. Our results support the conclusion of Dismukes and Siderer that the S2 state gives rise to this multiline signal; we find that the S1 state can be fully advanced to the S2 state at temperatures as low as 160 K. The S2 state is capable of further advancement at temperatures above about 210 K, but not below that temperature.
— Using isolated chloroplasts and techniques as described by Joliot and Joliot[6] we studied the evolution of O2 in weak light and light flashes to analyze the interactions between light induced O2 precursors and their decay in darkness. The following observations and conclusions are reported: 1. Light flashes always produce the same number of oxidizing equivalents either as precursor or as O2. 2. The number of unstable precursor equivalents present during steady state photosynthesis is ∼ 1.2 per photochemical trapping center. 3. The cooperation of the four photochemically formed oxidizing equivalents occurs essentially in the individual reaction centers and the final O2 evolution step is a one quantum process. 4. The data are compatible with a linear four step mechanism in which a trapping center, or an associated catalyst, (S) successively accumulates four + charges. The S4+ state produces O2 and returns to the ground state S0. 5. Besides S0 also the first oxidized state S+ is stable in the dark, the two higher states, S2+ and S3+ are not. 6. The relaxation times of some of the photooxidation steps were estimated. The fastest reaction, presumably S*1←S2, has a (first) half time ≤ 200 μsec. The S*2 state and probably also the S*0 state are processed somewhat more slowly (˜ 300–400 μsec).
Freezing of spinach or barley chloroplasts during continuous illumination results in the trapping of a paramagnetic state or a mixture of such states characterized by a multiline EPR spectrum. Added Photosystem II electron acceptor enhances the signal intensity considerably. Treatments which abolish the ability of the chloroplasts to evolve oxygen, by extraction of the bound manganese, prevent the formation of the paramagnetic species. Restoration of Photosystem II electron transport in inhibited chloroplasts with an artificial electron donor (1,5-diphenylcarbazide) does not restore the multiline EPR spectrum. The presence of 3-(3,4-dichlorophenyl)-1, 1-dimethylurea (DCMU) results in a modified signal which may represent a second paramagnetic state. The paramagnetic forms appear to originate on the donor side in Photosystem II and are dependent on a functional oxygenevolving site and bound, intact manganese. It is suggested that magnetically interacting manganese ions in the oxygen-evolving site may be responsible for the EPR signals. This suggestion is supported by calculations.
Silicomolybdate and silicotungstate are unique among photosystem II electron acceptors in that they catalyze photosystem II-dependent electron transfer in thylakoid membranes that is resistant to inhibition by diuron. On this basis it is generally accepted that these compounds interact with the photosystem II electron transfer sequence prior to the site of diuron inhibition thereby creating an even more abbreviated partial reaction than is found with other photosystem II electron acceptors. The data presented here demonstrate that low concentrations of the silicomolybdate anion (10–30 μM) inhibit the binding of [14C]diuron to spinach thylakoid membranes. This effect of silicomolybdate is reversible and is a function of the redox state of silicomolybdate. Reduction of silicomolybdate leads to a loss of its ability to prevent herbicide binding. Thus, there is no reason to believe that silicomolybdate intercepts electrons from photosystem II prior to the site of diuron intervention or at a site different from any other photosystem II electron acceptor.
The microwave power for half-saturation (P1/2) for the radical in photosystem II giving rise to signal IIslow (SIIs) has been measured by EPR in samples illuminated by a series of flashes. The charge storage state of the oxygen-evolving complex (S0-S4) was monitored by measuring the multiline EPR signal arising from the S2 state. The following results were obtained: (1) SIIs becomes easier to saturate after tris(hydroxymethyl)aminomethane (Tris) washing, a treatment that partially removes the Mn cluster. (2) P1/2 for SIIs oscillates with the flash number. P1/2 is lower in S1 (in dark-adapted material and after four flashes) than in S2, S3, or S0. (3) P1/2(S2) = P1/2(S3). (4) At 8 K P1/2(S2) > P1/2(S2), but at 20 K P1/2(S0) < P1/2(S2). (5) P1/2 for SIIs increases with temperature (8-70 K) in the S1 state. SHs is more difficult to saturate in S2, S3, and S0 than in S1 over the investigated temperature range. In addition, the increase in P1/2 is complex around 20-30 K in S2, S3, and S0. (6) In S0, P1/2 for SIIs decreases with time (decay half-time 30-60 s) to a stable level significantly above the dark level. The data are explained in terms of cross relaxation between the radical giving rise to SIIs and an efficient relaxer, which is suggested to be the Mn cluster. This relaxes more slowly in S1 than in the other S states. Since it is known that a mixed-valence Mn cluster is present in S2, and because P1/2 of SIIs in S3 and S0 is comparable to that in S2, it is suggested that mixed-valence Mn clusters are present in the S3 and S0 states also. Different models with these features can be proposed, the simplest of which is the following: S0 [Mn(H)-Mn(III)], S1 [Mn(III)-Mn(III)], S2 [Mn(III)-Mn(IV)], and S3 [Mn(III)-Mn(IV)].
A study of electron paramagnetic resonance (EPR) signals from components on the electron donor side of photosystem II has been performed. By measurement of EPR signal IIslow (D+) it is shown that, after three flashes, D+ decays slowly in the dark at room temperature in the fraction of the centers that was in the S0 state (t1/2 of 20 min in thylakoid membranes and 50 min in photosystem II enriched membranes). This reaction is accompanied by a conversion of S0 to S1. The concentration of S1 was estimated from the amplitude of the S2-state multiline EPR signal that could be generated by illumination at 200 K. These observations indicate that D+ accepts an electron from S0 in a dark reaction in which D and S1 are formed. In addition, the reactions by which D donates an electron to S2 or S3 have been directly measured by monitoring both signal IIslow and the multiline signal. The redox interactions between the D/D+ couple and the S states are explained in terms of a model in which D/D+ has a midpoint potential between those of S0/S1, and S1/S2. In addition, this model provides explanations for a number of previously unrelated phenomena, and the proposal is put forward that the reaction between D+ and Mn2+ is involved in the so-called photoactivation process.
Oxygen-evolving photosystem II particles (DT 20) isolated from pea chloroplasts by digitonin-Triton X-100 fractionation were photoinhibited with 150 W. m(-2) white light, at 20°C under three conditions: aerobic, anaerobic and strongly reducing (E(h) poised to approx. -250 mV with dithionite). Hill reaction rate (H (20) + BQ)and variable fluorescence (Fv) declined in parallel in all three cases with shortening half times: 30, 10 and 2.5 min, respectively. Light-induced absorbance changes at 685 nm characteristic of reversible photo accumulation of reduced pheophytin (& z -2.50 mV) remained essentially unchanged. We conclude that the three types of photoinhibitory treatment do not impair the separation of charges between chlorophyll P-680 and pheophytin in the photosystem II reaction center.
Analysis of the early events occurring during photoinhibition of Chlamydomonas reinhardtii cells by the thermoluminescence technique shows a shift of the B-emission band at 30°C ascribed to charge recombination of S2Q−B to lower temperatures (15–17°C). The appearance of this modified emission band is gradual, affects the whole population of reaction centers and occurs already at relatively low light intensities and short periods of exposure (20–60 min, 300–1000 W · −2). Under these conditions a reduction of only 30–40% occurs in the intensity of the emission band ascribed to charge recombination of S2Q−A. The loss of the S2Q−B response at 30°C is interpreted as a destabilization of this state and seems to correlate with an increase in the value of the intrinsic fluorescence F0 while the reduction in the S2Q−A signal parallels the reduction of the maximal variable fluorescence in the presence of DCMU. Measurements of oxygen flash yield and oscillation under these conditions show that the S-states cycle is not impaired. Following more extensive photoinhibition the B-type signal was completely lost while the S2Q−A band emission persisted and remained at approx. 20% of its initial value. The light intensity, required for the complete shift of the B-emission band at 30°C to 15°–17°C, seems to be sufficient to accelerate the rate of D1 protein synthesis which continues for a while even if the cells are reexposed to low light intensity (recovery). These results indicate that during the initial stage of photoinhibition changes are induced in the reaction center which lead to some alteration of the D1 protein, resulting in a destabilisation of the S2Q−B charge recombination. These events may be connected with the light-dependent turnover of the D1 protein.
The iron-sulfur centers A and B of spinach and barley chloroplasts were studied using EPR spectroscopy. The spectrum of samples with both centers reduced is significantly different at the microwave frequencies 9 and 35 GHz. This shows that an interaction exists between the centers which is discussed in terms of exchange and dipolar effects. The orientation of the g tensors of centers A and B was studied in magnetically oriented chloroplasts. Changes were observed in going from the partially to the fully reduced sample, a fact which strengthens the interaction model. The existence of an interaction implies that the centers are situated close to each other, presumably in the same molecule and in the same electron-transport chain.
The 32 kDa QB protein of Chlamydomonas reinhardtii chloroplast membranes is rapidly and specifically degraded when a suspension of isolated thylakoids is exposed to high light intensity at 25°C, at pH 7.8. Loss of the 32 kDa QB protein correlates well with loss of QB-dependent electron-flow, does not require the addition of divalent cations, increases with light intensity and is enhanced by 650 nm light, is not inhibited by uncouplers but is partially inhibited by leupeptin and pepstatin when added in the presence of 0.15% () β-d-octylglucoside. The specificity of the light-dependent degradation toward the 32 kDa QB protein is gradually lost at increasing pH (8.5–9.5) or by the addition of β-d-octylglucose up to 1.0% (), a concentration at which the thylakoids are completely solubilized. The reaction is significantly accelerated at high pH and detergent concentrations, and a marked degree of activity is observed under these conditions also in the dark. Pretreatment of thylakoids with trypsin at pH 7.8 does not abolish the membrane-bound specific proteolytic activity toward the trypsin-generated hydrophobic fragment of 17.5 kDa obtained from the 32 kDa QB protein which still contains the amino acid sequence of the herbicide- and quinone-binding sites. The light-dependent specific degradation of the 32 kDa QB protein by isolated thylakoids at pH 7.8 mimics in vitro the photoinhibition phenomena occurring in vivo, as described before (Kyle, D.J., Ohad, I. and Arntzen, C.J. (1984) Proc. Natl. Acad. Sci. USA 81, 4070–4074).
An oxygen-evolving Photosystem (PS) II preparation was isolated after Triton X-100 treatment of spinach thylakoids in the presence of Mg2+. The structural and functional components of this preparation have been identified by SDS-polyacrylamide gel electrophoresis and sensitive spectrophotometric analysis. The main findings were: (1) The concentration of the primary acceptor Q of PS II was 1 per 230 chlorophyll molecules. (2) There are 6 to 7 plastoquinone molecules associated with a ‘quinone-pool’ reducible by Q. (3) The only cytochrome present in significant amounts (cytochrome b-559) occurred at a concentration of 1 per 125 chlorophyll molecules. (4) The only kind of photochemical reaction center complex present was identified by fluorescence induction kinetic analysis as PS IIα. (5) An Em = − 10 mV has been measured at pH 7.8 for the primary electron acceptor Qα of PS IIα. (6) With conventional SDS-polyacrylamide gel electrophoresis, the preparation was resolved into 13 prominent polypeptide bands with relative molecular masses of 63, 55, 51, 48, 37, 33, 28, 27, 25, 22, 15, 13 and 10 kDa. The 28 kDa band was identified as the PS II light-harvesting chlorophyll . In the presence of 2 M urea, however, SDS-polyacrylamide gel electrophoresis showed seven prominent polypeptides with molecular masses of 47, 39, 31, 29, 27, 26 and 13 kDa as well as several minor components. CP I under identical conditions had a molecular mass of 60–63 kDa.
The activity, the protein content and the manganese properties of photosystem II have been compared after photoinhibition of isolated thylakoid membranes. The results show a concomitant disappearance of the oxygen evolving activity and the ability to form the S2-state multiline EPR signal. The D1-protein is degraded in a subsequent event which closely correlates to release of manganese from the thylakoid membranes.
A study of signals, light-induced at 77 K in O2-evolving Photosystem II (PS II) membranes showed that the EPR signal that has been attributed to the semiquinone-iron form of the primary quinone acceptor, Q−AFe, at g = 1.82 was usually accompanied by a broad signal at g = 1.90. In some preparations, the usual g = 1.82 signal was almost completely absent, while the intensity of the g = 1.90 signal was significantly increased. The g = 1.90 signal is attributed to a second EPR form of the primary semiquinone-iron acceptor of PS II on the basis of the following evidence. (1) The signal is chemically and photochemically induced under the same conditions as the usual g = 1.82 signal. (2) The extent of the signal induced by the addition of chemical reducing agents is the same as that photochemically induced by illumination at 77 K. (3) When the g = 1.82 signal is absent and instead the g = 1.90 signal is present, illumination at 200 K of a sample containing a reducing agent results in formation of the characteristic split pheophytin− signal, which is thought to arise from an interaction between the photoreduced pheophytin acceptor and the semiquinone-iron complex. (4) Both the g = 1.82 and g = 1.90 signals disappear when illumination is given at room temperature in the presence of a reducing agent. This is thought to be due to a reduction of the semiquinone to the nonparamagnetic quinol form. (5) Both the g = 1.90 and g = 1.82 signals are affected by herbicides which block electron transfer between the primary and secondary quinone acceptors. It was found that increasing the pH results in an increase of the g = 1.90 form, while lowering the pH favours the g = 1.82 form. The change from the g = 1.82 form to the g = 1.90 form is accompanied by a splitting change in the split pheophytin− signal from approx. 42 to approx. 50 G. Results using chloroplasts suggest that the g = 1.90 signal could represent the form present in vivo.
The spin-lattice relaxation time of the EPR signal of the tyrosine radical D+ has been measured with electron spin echo spectroscopy in the range 5–30 K in Photosystem II preparations with intact oxygen evolving complex (OEC). The charge storage state of the OEC was set by illumination with a series of flashes and monitored by measuring the multiline EPR signal of the S2-state. The OEC was synchronized to 100% S1 initial state by dark-adaptation and one preflash. Agreeing with previous work (De Groot, A., Plijter, J.J., Evelo, R., Babcock, G.T. and Hoff, A.J. (1986) Biochim. Biophys. Acta 848, 8–15), the spin-lattice relaxation curves were foundto be bi-phasic. The average relaxation time τ¯ of each S-state was calculated from the data obtained for the 0—3 flash sample and the known S-state distribution,τ¯was found to be maximal in the S1-state. It decreased about 40% for the S2-state, was essentially the same for the S2- and the S3-states and decreased again by about 55% for the S0-state. These results are similar to those obtained earlier by cw EPR (Styring, S. and Rutherford, A.W. (1988) Biochemistry 27, 4915–4923). At 5 K the two exponentials describing the relaxation curves had characteristic timesτf, and τs that differed by an order of magnitude. Their amplitude was about equal, except for S0 where the faster process predominated. At 20 K the characteristic time of both the fast and the slow process was reduced by a factor of about five;their amplitudes were again about equal. The observed relaxation times τf and τs were deconvoluted as a function of S-state by an approximate method. At 5 K it was found that τf was about twice as fast for S0 and S3 than for S, and S2 (1.3 vs. 2.6 ms) and τ sabout twice as fast for S0, S2, S3 than for S1 (13.7–14.5 vs. 28.6 ms). The same trend was observed at higher temperatures. Interpreting the results with relaxation enhancement theory and integrating them with the results from cw EPR, NMR and EXAFS spectroscopy the following model for the OEC is presented, (i) To explain the biphasic relaxation of D+ it is suggested that two Mn are close to D+ at different distances, enhance the relaxation of D+and are not magnetically coupled. Their oxidation state differs by 1 unit, is probably Mn3+ and Mn4+, and does not change during the S0 → S3 sequence. It is postulated that at high temperature there is a charge resonance between the two Mn ions that is frozen out when cooling to cryogenic temperature, (ii) Two of the four Mn of the OEC form an antiferromagnetically coupled binuclear cluster in the oxidation state Mn2+·Mn3+, Mn3+·Mn3+, Mn3+·Mn4+, Mn3+·Mn4+ in the S0, S1, S2 and S3-sta respectively, (iii) From the temperature dependence of the relaxation of D+ in the S0-state, it is estimated that the distance between the Mn cluster and D+ is 30–40 .
(1) The re-reduction kinetics of chlorophyll a+II (P-680+) after the first, second, third etc. flash given to dark-adapted subchloroplasts have been monitored at 824 nm in the nanosecond range. After the first flash and, again, after the fifth flash, the re-reduction of chlorophyll a+II (Chl a+II) in the nanosecond range is nearly monophasic with . After the second and third flash, the re-reduction is significantly slower and biphasic; it can be well-adapted with and ≈260 ns. After the 4th flash, the re-reduction kinetics of Chl a+II are intermediate between the first/fifth and second/third flash. A similar dependence on flash number was obtained with a sample of oxygen-evolving Photosystem II particles from Synechococcus sp. (2) Considering the populations of the S-states of the O2-evolving complex before each flash, the following correlation of S-states to Chl a+II reduction kinetics and electron transfer times, respectively, is obtained: in state S0 as well as in state S1 Chl a+II is reduced with , whereas in state S2 as well as state S3 a biphasic reduction with and ≈260 ns (ratio of the amplitudes ≈1:1) occurs. (3) The observed multiphasic Chl a+II reduction under repetitive excitation is quantitatively explained by a superposition of the individual electron transfer times. (4) We suggest that the retardation of electron transfer to Chl a+II in states S2 and S3 as compared to S0 and S1 is caused by Coulomb attraction by one positive charge located in the O2-evolving complex. A positively charged O2-evolving complex in states S2 and S3 can be explained if the electron release pattern (1,1,1,1) is accompanied by a proton release pattern (1,0,1,2) for the transitions (S0 → S1, S1 → S2, S2 → S3, S3 → S0). (5) A kinetic model based on linear electron transfer from the O2-evolving complex (S) to Chl a+II via two carriers, D1 and D2, makes a quantitative description of the experimental results possible. (6) According to the kinetic model, the retardation of electron transfer to Chl a+II in states S2 and S3 is reflected by an increase in the change of standard free energy, ΔG0, of the reaction Chl from ΔG0 ≈ − 90 meV in states S0 and S1 to ΔG0 ≈ − 20 meV in states S2 and S3. (7) This increase by ≈ 70 meV can be quantitatively explained by the Coulomb potential of the positive charge in the O2-evolving complex, estimated by using the point charge approximation.
A correlation is demonstrated between the loss of the QA−Fe2+ EPR signal and the ability to photoinduce the radical-pair-recombination triplet state in Photosystem II. The QA−Fe2+ signal is diminished by procedures which are thought to reduce the semiquinone by a further electron: (1) low quantum yield photoreduction in the presence of sodium dithionite at room temperature; (2) chemical reduction in the dark by sodium dithionite at pH 7.0. The chemical reduction proeess is extremely slow (t1/2≈ 5 h) but can be accelerated (t1/2≈1.5 h) by the presence of the redox mediator, benzyl viologen. In redox titrations at pH. 7.0 the QA−Fe2+ signal disappears with an irreversible transition at potentials lower than −350 mV. The ability to observe the triplet signal shows a corresponding potential dependence. The variations in the amplitude of the triplet EPR signal match variations in triplet yield measured by flash absorption spectroscopy at low temperature. From these observations the following conclusions are drawn: (1) The redox titration data that led to the suggestion that an extra component functions between pheophytin and QA−Fe2+ (Evans, M.C.W., Atkinson, Y. E. and Ford, R. C. (1985) Biochim. Biophys. Acta 806, 247–254) can probably be explained instead by the second reduction of QA−Fe2+. (2) The variable yield of triplet and of P680+ Ph−, and possibly the lifetime of the latter, which have been reported in the literature probably reflect, at least in part, different amounts of native QA−Fe2+ remaining in the various preparations used. From considerations of the literature, and increase in quantum yield of charge separation is thought to occur upon the second reduction of QA−Fe2+. The most likely explanation for this is the disappearance of an electrostatic interaction between QA−Fe2+ and P680+Ph− as QA−Fe2+ becomes further reduced. Other factors which may influence or be responsible for these phenomena and comparisons with the primary photochemistry in purple bacteria are discussed. In addition the relevance of these observations to the lesions involved in photoinhibition is pointed out.
Preillumination of spinach thylakoid membranes under strictly anaerobic conditions (i.e. in the presence of glucose oxidase) and in the absence of an electron acceptor inactivates specifically photosystem (PS) II. Inhibition can be complete within 3 min depending on the glucose oxidase concentration and light intensity. Artificial donor or acceptor systems do not restore PS II. Atrazine binding affinity is not diminished. No degradation of peptide subunits is observed. Trypsinized PS II preparations, in which the QB-binding site has been destroyed, can also be inactivated by light. It is concluded that photoinhibition of thylakoid membranes under anaerobic conditions inactivates the reaction center of PS II. This photoinactivation does not depend on the degradation of a peptide subunit. Not only the QB site but also a modified (trypsinized) QA site can induce photoinhibition.
CO2 depletion leads to an approximately 10-fold increase in the light-induced EPR signal at g = 1.82, attributed to the QA− · Fe2+ complex, in Photosystem II-enriched thylakoid membrane fragments. Upon reconstitution with HCO3−the signal decreases to the size in control samples. The split pheophytin− signal is broader in control or reconstituted than in CO2-depleted samples. It is concluded that HCO2− strongly influences the localization and conformation of the QA− · Fe+ complex. The QA− · Fe2+ and split pheophytirr− EPR signals from triazine-resistant Brassica napus were virtually identical to those from triazine-susceptible samples, indicating that the change in the 32-kDa azidoatrazine-binding protein does not lead to a confonnational change of the Qa− · Fe2+ complex.
Counter-current distribution in an aqueous Dextran-polyethylene glycol two-phase system has been used to fractionate membrane fragments obtained by press treatment of Class II chloroplasts. By the counter-current distribution technique membrane particles are separated according to their surface properties such as charge and hydrophobicity. The fractions obtained were analysed with respect to photochemical activities, chlorophyll and P-700 contents. The Photosystem II enrichment after counter-current distribution was better than that obtained by differential centrifugation of the disrupted chloroplasts. However, the best separation of Photosystem I and II enriched particles could be achieved if differential centrifugation was combined with the counter-current distribution technique. Each centrifugal fraction could be further separated into Photosystems I and II enriched fractions since the Photosystem II particles preferred the dextran-rich bottom phase while the Photosystem I particles preferred the polyethylene glycol-rich top phase. By this procedure it was possible, without the use of detergents, to obtain vesicles which were more enriched in Photosystem II as compared to intact grana stacks. The partition behaviour of undisrupted Class II chloroplasts and the Photosystem I centrifugal fraction was the same. This similarity indicated that the membrane which is exposed to the surrounding polymers by the Class II chloroplasts is the Photosystem I rich membrane of the stroma lamellae.
The light-driven water-splitting/oxygen-evolving enzyme remains one of the great enigmas of plant biology. However, due to the recent expansion of research efforts on this enzyme, it is grudgingly giving up some of its secrets.
Optical, resonance Raman, and electron paramagnetic resonance spectroscopies have been used to characterize the ligands and spin state of the chloroplast cytochrome b-559. The protein was isolated from both maize and spinach in a low-potential form. The spectroscopic data indicate that the heme iron in both ferric and ferrous cytochrome b-559 is in its low-spin state and ligated in its fifth and sixth coordination positions by histidine nitrogens. Electron paramagnetic resonance data for the purified spinach cytochrome are in good agreement with those determined by Bergström and Vänngård [Bergström, J., & Vänngård, T. (1982) Biochim. Biophys. Acta 682, 452-456] for a low-potential membrane-bound form of cytochrome b-559. The g values of high-potential cytochrome b-559 are shifted from those of its low-potential forms; this shift is interpreted as arising from a deviation of the planes of the two axial histidine imidazole rings from a parallel orientation. The model is consistent with the physical data and may also account for the facility with which cytochrome b-559 can be converted between low- and high-potential forms. Recent biochemical and molecular biological data [Widger, W. R., Cramer, W. A., Hermodson, M., Meyer, D., & Gullifor, M. (1984) J. Biol. Chem. 259, 3870-3876; Herrmann, R. G., Alt, J., Schiller, D., Cramer, W. A., & Widger, W. R. (1984) FEBS Lett. 179, 239-244] have shown that two polypeptides, one with 83 residues and a second with 39 residues, most likely constitute the protein of the cytochrome.(ABSTRACT TRUNCATED AT 250 WORDS)
The photochemistry in photosystem II of spinach has been characterized by electron paramagnetic resonance (EPR) spectroscopy in the temperature range of 77-235 K, and the yields of the photooxidized species have been determined by integration of their EPR signals. In samples treated with 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU), a single stable charge separation occurred throughout the temperature range studied as reflected by the constant yield of the Fe(II)-QA-EPR signal. Three distinct electron donation pathways were observed, however. Below 100 K, one molecule of cytochrome b559 was photooxidized per reaction center. Between 100 and 200 K, cytochrome b559 and the S1 state competed for electron donation to P680+. Photooxidation of the S1 state occurred via two intermediates: the g = 4.1 EPR signal species first reported by Casey and Sauer [Casey, J. L., & Sauer, K. (1984) Biochim. Biophys. Acta 767, 21-28] was photooxidized between 100 and 160 K, and upon being warmed to 200 K in the dark, this EPR signal yielded the multiline EPR signal associated with the S2-state. Only the S1 state donated electrons to P680+ at 200 K or above, giving rise to the light-induced S2-state multiline EPR signal. These results demonstrate that the maximum S2-state multiline EPR signal accounts for 100% of the reaction center concentration. In samples where electron donation from cytochrome b559 was prevented by chemical oxidation, illumination at 77 K produced a radical, probably a chlorophyll cation, which accounted for 95% of the reaction center concentration. This electron donor competed with the S1 state for electron donation to P680+ below 100 K.(ABSTRACT TRUNCATED AT 250 WORDS)
Although cytochrome b-559 is an integral component of the photosystem II complex (PSII), its function is unknown. Because cytochrome b-559 has been shown to be both photooxidized and photoreduced in PSII, one of several proposals is that it mediates cyclic electron transfer around PSII, possibly as a protective mechanism. We have used electron paramagnetic resonance spectroscopy to investigate the pathway of photooxidation of cytochrome b-559 in PSII and have shown that it proceeds via photooxidation of chlorophyll. We propose that this photooxidation of chlorophyll is the first step in the photoinhibition of PSII. The unique susceptibility of PSII to photoinhibition is probably due to the fact that it is the only reaction center in photosynthesis which generates an oxidant with a reduction potential high enough to oxidize chlorophyll. We propose that the function of cytochrome b-559 is to mediate cyclic electron transfer to rereduce photooxidized chlorophyll and protect PSII from photoinhibition. We also suggest that the chlorophyll(s) which are susceptible to photooxidation are analogous to the monomer chlorophylls found in the bacterial photosynthetic reaction center complex.
Photosystem II contains two redox-active tyrosines. One of these, YZ, reduces the reaction center chlorophyll, P680, and transfers the oxidizing equivalent to the oxygen-evolving complex. The second, YD, has a long-lived free radical state of unknown function. We recently established that YD is Tyr-160 of the D2 polypeptide by site-directed mutagenesis of a psbD gene in the unicellular cyanobacterium Synechocystis 6803 [Debus, R. J., Barry, B. A., Babcock, G. T., & McIntosh, L. (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 427-430]. YZ is most likely the symmetry-related Tyr-161 of the D1 polypeptide. To test this hypothesis, we have changed Tyr-161 to phenylalanine by site-directed mutagenesis of a psbA gene in Synechocystis. The resulting mutant assembles PSII, as judged by its ability to produce the stable Y+D radical, but is unable to grow photosynthetically and exhibits altered fluorescence properties. The nature of the fluorescence change indicates that forward electron transfer to P+680 is disrupted in the mutant. These results provide strong support for our identification of Tyr-161 in the D1 polypeptide with YZ.
The fluorescence yield has been measured on spinach chloroplasts at low temperature (−30 to −60°C) for various dark times following a short saturating flash. A decrease in the fluorescence yield linked to the reoxidation of the Photosystem II electron acceptor Q is still observed at −60°C. Two reactions participate in this reoxidation: a back reaction or charge recombination and the transfer of an electron from Q− to Pool A. The relative competition between these two reactions at low temperature depends upon the oxidation state of the donor side of the Photosystem II center: 1.(1) In dark-adapted chloroplasts (i.e. in States S0+S1 according to Kok, B., Forbush, B. and McGloin, M. (1970) Photochem. Photobiol. 11, 457–475), Q, reduced by a flash at low temperature, is reoxidized by a secondary acceptor and the positive charge is stabilized on the Photosystem II donor Z. Although this reaction is strongly temperature dependent, it still occurs very slowly at −60°C.2.(2) When chloroplasts are placed in the S2+S3 states by a two-flash preillumination at room temperature, the reoxidation of Q− after a flash at low temperature is mainly due to a temperature-independent back reaction which occurs with non-exponential kinetics.3.(3) Long continuous illumination of a frozen sample at −30°C causes 6–7 reducing equivalents to be transferred to the pool. Thus, a sufficient number of oxidizing equivalents should have been generated to produce at least one O2 molecule.4.(4) A study of the back reaction in the presence of 3(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) shows the superposition of two distinct non-exponential reactions one temperature dependent, the other temperature independent.
The chemical nature of electron donor(s) in photosystem II in photosynthetic membranes was analyzed by site-directed mutagenesis of the gene encoding the protein D2 of the photosystem II reaction center. Mutation of the Tyr-160 residue of the D2 protein into phenylalanine results in the disappearance of the electron paramagnetic resonance signal II(S) originating from D(+), the oxidized form of the slow photosystem II electron donor D. Signal II(S) is still present if a neighboring residue in D2, Met-159, is mutated into arginine. Both mutants have normal rereduction kinetics of the oxidized primary electron donor, P680(+), in octyl glucoside-extracted thylakoids, indicating that D is not directly involved in P680(+) reduction. However, overall photosystem II activity appears to be impaired in the Tyr-160-Phe mutant: photosystem II-dependent growth of this mutant is slowed down by a factor of 3-4, whereas photoheterotrophic growth rates in wild type and mutant are essentially identical. Binding studies of diuron, a photosystem II herbicide, show that there is no appreciable decrease in the number of photosystem II centers in the Tyr-160-Phe mutant. The decrease in photosystem II activity in this mutant may be interpreted to indicate a role of D in photoactivation, rather than one as an important redox intermediate in the photosynthetic electron-transport chain.
Inactivation of the water splitting enzyme complex in leaves or isolated chloroplasts results in increased susceptibility of photosystem II (PSII) to damage by light. Photoinhibition under this condition occurs in very weak light. The site of damage is exclusive of the water splitting complex yet still on the oxidizing side of PSII, as the Q(B) locus is unaffected while photoreduction of silicomolybdate is inhibited. The kinetics of loss in PSII activity are more complex than apparent first-order, and the quantum efficiency is low. We observe no evidence of deletion from thylakoid membranes of any PSII polypeptide as a consequence of photoinhibition, although recovery from the photoinhibition is dependent upon both light and 70S protein synthesis. Enhanced synthesis of two proteins occurs during recovery, only one of which (D2) appears to be causally related to the recovery. We present a model which describes the relationship of weak light photoinhibition and its recovery to photoactivation of the S-state water oxidizing complex.