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Properties of Photosystem II lacking the PsbJ subunit
Abstract
Photosystem II (PSII), the oxygen-evolving enzyme, consists of 17 trans-membrane and 3 extrinsic membrane proteins. Other subunits bind to PSII during assembly, like Psb27, Psb28, Tsl0063. The presence of Psb27 has been proposed (Zabret et al. 2021; Huang et al. 2021; Xiao et al. 2021) to prevent the binding of PsbJ, a single transmembrane α-helix close to the quinone QB binding site. Consequently, a PSII rid of Psb27, Psb28 and Tsl0034 prior to the binding of PsbJ would logically correspond to an assembly intermediate. The present work describes experiments aiming at further characterizing such a ΔPsbJ-PSII, purified from the thermophilic Thermosynechococcus elongatus, by means of MALDI-TOF spectroscopy, Thermoluminescence, EPR spectroscopy and UV-visible time-resolved spectroscopy. In the purified ΔPsbJ-PSII, an active Mn4CaO5 cluster is present in 60-70 % of the centers. In these centers, although the forward electron transfer seems not affected, the Em of the QB/QB- couple increases by ≈120 mV thus disfavoring the electron coming back on QA. The increase of the energy gap between QA/QA- and QB/QB- could contribute in a protection against the charge recombination between the donor side and QB-, identified at the origin of photoinhibition under low light (Keren et al. 1997), and possibly during the slow photoactivation process.
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Photosystem II (PS II) of oxygenic photosynthesis is found in the thylakoid membranes of plastids and cyanobacteria. The mature PS II complex comprises a central core of four membrane proteins that bind the majority of the redox-active cofactors. In cyanobacteria the central core is surrounded by 13 low-molecular-weight (LMW) subunits which each consist of one or two transmembrane helices. Three additional hydrophilic subunits known as PsbO, PsbU and PsbV are found associated with hydrophilic loops belonging to the core proteins protruding into the thylakoid lumen. During biogenesis the majority of the LMW subunits are known to initially associate with individual pre-assembly complexes consisting of one or more of the core proteins; however, the point at which the PsbJ LMW subunit binds to PS II is not known. The majority of models for PS II biogenesis propose that the three extrinsic proteins and PsbJ bind in the final stages of PS II assembly. We have investigated the impact of creating the double mutants ∆PsbJ:∆PsbO, ∆PsbJ:∆PsbU and ∆PsbJ:∆PsbV to investigate potential cooperation between these subunits in the final stages of biogenesis. Our results indicate that PsbJ can bind to PS II in the absence of any one of the extrinsic proteins. However, unlike their respective single mutants, the ∆PsbJ:∆PsbO and ∆PsbJ:∆PsbV strains were not photoautotrophic and were unable to support oxygen evolution suggesting a functional oxygen-evolving complex could not assemble in these strains. In contrast, the PS II centers formed in the ∆PsbJ:∆PsbU strain were capable of photoautotrophic growth and could support oxygen evolution when whole-chain electron transport was supported by the addition of bicarbonate.
Significance
Photosystem II (PSII) is a light-driven water:plastoquinone oxidoreductase in oxygenic photosynthetic organisms. Several inactive PSII intermediates are involved in the biogenesis of the multisubunit PSII complexes as well as in their repair after unavoidable oxidative damage targeted to PSII under light. Psb27 is one of the assembly factors associated with inactive PSII intermediate and plays important roles in the biogenesis/repair of PSII. Here, we report the structure of a dimeric Psb27-PSII from a thermophilic cyanobacterium Thermosynechococcus vulcanus by cryo-electron microscopy. Our results reveal the location and binding properties of Psb27 in the intermediate PSII and show the structural differences between the intermediate and native PSII. These results provide important clues for the roles of Psb27 in the biogenesis/repair of PSII.
The psbA gene family in cyanobacteria encodes different forms of the D1 protein that is part of the Photosystem II reaction centre. We have identified a phylogenetically distinct D1 group that is intermediate between previously identified G3-D1 and G4-D1 proteins (Cardona et al. Mol Biol Evol 32:1310–1328, 2015). This new group contained two subgroups: D1INT, which was frequently in the genomes of heterocystous cyanobacteria and D1FR that was part of the far-red light photoacclimation gene cluster of cyanobacteria. In addition, we have identified subgroups within G3, the micro-aerobically expressed D1 protein. There are amino acid changes associated with each of the subgroups that might affect the function of Photosystem II. We show a phylogenetically broad range of cyanobacteria have these D1 types, as well as the genes encoding the G2 protein and chlorophyll f synthase. We suggest identification of additional D1 isoforms and the presence of multiple D1 isoforms in phylogenetically diverse cyanobacteria supports the role of these proteins in conferring a selective advantage under specific conditions.
Significance
The E m values of the 2 Q B redox couples, E m (Q B /Q B •− ) and E m (Q B •− /Q B H 2 ), in Photosystem II are fundamental for understanding the enzyme function, but they remain vague. Using EPR signals arising from the semiquinone, Q B •− , we have now measured both redox couples. The semiquinone is thermodynamically stable and has a relatively high potential. This minimizes back-reactions and electrons leaking onto O 2 , explaining the remarkable stability of Q B •− . The release of Q B H 2 is thermodynamically favorable, and the binding of the substrate PQ is greatly favored over the product PQH 2 : This optimizes PSII function in the presence of a largely reduced plastoquinone pool. This work corrects and explains a recent report of anomalous values for the Q B redox couples.
Main conclusion
Recent investigations have provided important new insights into the structures and functions of the extrinsic proteins of Photosystem II.
This review is an update of the last major review on the extrinsic proteins of Photosystem II (Bricker et al., Biochemistry 31:4623–4628 2012). In this report, we will examine advances in our understanding of the structure and function of these components. These proteins include PsbO, which is uniformly present in all oxygenic organisms, the PsbU, PsbV, CyanoQ, and CyanoP proteins, found in the cyanobacteria, and the PsbP, PsbQ and PsbR proteins, found in the green plant lineage. These proteins serve to stabilize the Mn4CaO5 cluster and optimize oxygen evolution at physiological calcium and chloride concentrations. The mechanisms used to perform these functions, however, remain poorly understood. Recently, important new findings have significantly advanced our understanding of the structures, locations and functions of these important subunits. We will discuss the biochemical, structural and genetic studies that have been used to elucidate the roles played by these proteins within the photosystem and their locations within the photosynthetic complex. Additionally, we will examine open questions needing to be addressed to provide a coherent picture of the role of these components within the photosystem.
Photosynthetic water oxidation is catalyzed by the Mn4CaO5 cluster of photosystem II. The assembly of the Mn4O5Ca requires light and involves a sequential process called photoactivation. This process harnesses the charge-separation of the photochemical reaction center and the coordination environment provided by the amino acid side chains of the protein to oxidize and organize the incoming manganese ions to form the oxo-bridged metal cluster capable of H2O-oxidation. Although most aspects of this assembly process remain poorly understood, recent advances in the elucidation of the crystal structure of the fully assembled cyanobacterial PSII complex help in the interpretation of the rich history of experiments designed to understand this process. Moreover, recent insights on the structure and stability of the constituent ions of the Mn4CaO5 cluster may guide future experiments. Here we consider the literature and suggest possible models of assembly including one involving single Mn²⁺ oxidation site for all Mn but requiring ion relocation.
The main cofactors involved in the oxygen evolution activity of Photosystem II (PSII) are located in two proteins, D1 (PsbA) and D2 (PsbD). In Thermosynechococcus elongatus, a thermophilic cyanobacterium, the D1 protein is encoded by either the psbA(1) or the psbA(3) gene, the expression of which is dependent on environmental conditions. It has been shown that the energetic properties of the PsbA1-PSII and those of the PsbA3-PSII differ significantly (Sugiura, M., Kato, Y., Takahashi, R., Suzuki, H., Watanabe, T., Noguchi, T., Rappaport, F., and Boussac, A. (2010) Biochim. Biophys. Acta 1797, 1491-1499). In this work the structural stability of PSII upon a PsbA1/PsbA3 exchange was investigated. Two deletion mutants lacking another PSII subunit, PsbJ, were constructed in strains expressing either PsbA1 or PsbA3. The PsbJ subunit is a 4-kDa transmembrane polypeptide that is surrounded by D1 (i.e. PsbA1), PsbK, and cytochrome b(559) (Cyt b(559)) in existing three-dimensional models. It is shown that the structural properties of the PsbA3/ΔPsbJ-PSII are not significantly affected. The polypeptide contents, the Cyt b(559) properties, and the proportion of PSII dimer were similar to those found for PsbA3-PSII. In contrast, in PsbA1/ΔPsbJ-PSII the stability of the dimer is greatly diminished, the EPR properties of the Cyt b(559) likely indicates a decrease in its redox potential, and many other PSII subunits are lacking. These results shows that the 21-amino acid substitutions between PsbA1 and PsbA3, which appear to be mainly conservative, must include side chains that are involved in a network of interactions between PsbA and the other PSII subunits.
The membrane-embedded photosystem II core complex (PSIIcc) uses light energy to oxidize water in photosynthesis. Information about the spatial structure of PSIIcc obtained from x-ray crystallography was so far derived from homodimeric PSIIcc of thermophilic cyanobacteria. Here, we report the first crystallization and structural analysis of the monomeric form of PSIIcc with high oxygen evolution capacity, isolated from Thermosynechococcus elongatus. The crystals belong to the space group C222(1), contain one monomer per asymmetric unit, and diffract to a resolution of 3.6 A. The x-ray diffraction pattern of the PSIIcc-monomer crystals exhibit less anisotropy (dependence of resolution on crystal orientation) compared with crystals of dimeric PSIIcc, and the packing of the molecules within the unit cell is different. In the monomer, 19 protein subunits, 35 chlorophylls, two pheophytins, the non-heme iron, the primary plastoquinone Q(A), two heme groups, 11 beta-carotenes, 22 lipids, seven detergent molecules, and the Mn(4)Ca cluster of the water oxidizing complex could be assigned analogous to the dimer. Based on the new structural information, the roles of lipids and protein subunits in dimer formation of PSIIcc are discussed. Due to the lack of non-crystallographic symmetry and the orientation of the membrane normal of PSIIcc perpendicular ( approximately 87 degrees ) to the crystallographic b-axis, further information about the structure of the Mn(4)Ca cluster is expected to become available from orientation-dependent spectroscopy on this new crystal form.
PsbK is a small membrane protein of the PSII core complex and is highly conserved from cyanobacteria to plants. Here, we studied its role in the thermophilic cyanobacterium, Thermosynechococcus elongatus BP-1, by focusing on a psbK disruptant with hexahistidine-tagged CP47. The psbK disruptant showed photoautotrophic growth comparable with that of the wild type under a wide range of light conditions. The mutant PSII complex retained the oxygen-evolving activity with a unique modification of the acceptor Q(B) site. N-terminal sequencing revealed that Ycf12 and PsbZ proteins were lost in the PSII complex prepared from the mutant. Immunoblotting detected reduced accumulation of PsbZ in the mutant thylakoid. These results suggest that PsbK is required not only for association of PsbZ and Ycf12 with the isolated PSII complex but also for the stabilization of PsbZ in the thylakoid membrane.
Thermoluminesence measurements are useful for the study of Photosystem II electron transport in intact leaves, in algal and cyanobacterial cells, as well as in isolated membrane complexes. Here an overview of the experimental approaches is provided. In the present review, instruments and the experimental procedures for measuring thermoluminescence emission from photosynthetic systems of various origins are summarized and discussed. Major pitfalls frequently encountered in measurements with isolated membranes, suspensions of intact organisms or solid leaf samples are highlighted. Analytical and numeric methods for the analysis of measured thermoluminescence curves are also discussed.
PSII, the oxygen-evolving complex of photosynthetic organisms, contains an intriguingly large number of low molecular weight proteins. PsbX, one of these proteins, is ubiquitous in PSII complexes of cyanobacteria and plants. In previous studies, deletion of the PsbX protein in cyanobacteria has not resulted in clear phenotypic changes. Here we report the construction of an antisense (AS-PsbX) line in Arabidopsis thaliana with <10% of wild-type PsbX levels. AS-PsbX plants are capable of photoautotrophic growth, but biochemical, biophysical and immunological evidence demonstrates that reduction of PsbX contents leads to reduced levels of functional assembled PSII core complexes, while the light-harvesting antennae are not affected. In addition, levels of phosphorylation of the core proteins D1, D2 and CP43 are severely reduced in the antisense plants relative to their wild-type counterparts. We conclude that PsbX is important for accumulation of functional PSII.
The psbX gene (sml0002) coding for a 4.1 kDa protein in Photosystem II of plants and cyanobacteria was deleted in both wild type and in a Photosystem I-less mutant of the cyanobacterium Synechocystis sp. PCC 6803. Polymerase chain reaction and sequencing analysis showed that the mutants had completely segregated. Deletion of the PsbX protein does not seem to influence growth rate, electron transport or water oxidation ability. Whereas a high light induction of the psbX mRNA could be observed in wild type, deletion of the gene did not lead to high light sensibility. Light saturation measurements and 77K fluorescence measurements indicated a minor disconnection of the antenna in the deletion mutant. Furthermore, fluorescence induction measurements as well as immuno-staining of the D1 protein showed that the amount of Photosystem II complexes in the mutants was reduced by 30%. Therefore, PsbX does not seem to be necessary for the Photosystem II electron transport, but directly or indirectly involved in the regulation of the amount of functionally active Photosystem II centres in Synechocystis sp. PCC 6803.
The photosystem II (PSII) complex of photosynthetic oxygen evolving membranes comprises a number of small proteins whose functions remain unknown. Here we report that the low molecular weight protein encoded by the psbJ gene is an intrinsic component of the PSII complex. Fluorescence kinetics, oxygen flash yield, and thermoluminescence measurements indicate that inactivation of the psbJ gene in Synechocystis 6803 cells and tobacco chloroplasts lowers PSII-mediated oxygen evolution activity and increases the lifetime of the reduced primary acceptor Q(A)(-) (more than a 100-fold in the tobacco DeltapsbJ mutant). The decay of the oxidized S(2,3) states of the oxygen-evolving complex is considerably accelerated, and the oscillations of the Q(B)(-)/S(2,3) recombination with the number of exciting flashes are damped. Thus, PSII can be assembled in the absence of PsbJ. However, the forward electron flow from Q(A)(-) to plastoquinone and back electron flow to the oxidized Mn cluster of the donor side are deregulated in the absence of PsbJ, thereby affecting the efficiency of PSII electron flow following the charge separation process.
Redox properties of cytochrome b559 (Cyt b559) and cytochrome c550 (Cyt c550) have been studied by using highly stable photosystem II (PSII) core complex preparations from a mutant strain of the thermophilic cyanobacterium Thermosynechococcus
elongatus with a histidine tag on the CP43 protein of PSII. Two different redox potential forms for Cyt b559 are found in these preparations, with a midpoint redox potential (E′m) of +390 mV in about half of the centers and +275 mV in the other half. The high-potential form, whose E′m is pH independent, can be converted into the lower potential form by Tris washing, mild heating or alkaline pH incubation. The E′m of the low-potential form is significantly higher than that found in other photosynthetic organisms and is not affected by pH. The possibility that the heme of Cyt b559 in T. elongatus is in a more hydrophobic environment is discussed. Cyt c550 has a higher E′m when bound to the PSII core (–80 mV at pH 6.0) than after its extraction from the complex (–240 mV at pH 6.0). The E′m of Cyt c550 bound to PSII is pH independent, while in the purified state an increase of about 58 mV/pH unit is observed when the pH decreases below pH 9.0. Thus, Cyt c550 seems to have a single protonateable group which influences the redox properties of the heme. From these electrochemical measurements and from EPR controls it is proposed that important changes in the solvent accessibility to the heme and in the acid-base properties of that protonateable group could occur upon the release of Cyt c550 from PSII.
Luminescence from photosynthetic material observed in darkness following illumination is a delayed fluorescence produced by a recombination of charge pairs stored in photosystem II, i.e. the back-reaction of photosynthetic charge separation. Thermoluminescence (TL) is a technique consisting of a rapid cooling followed by the progressive warming of a preilluminated sample to reveal the different types of charge pairs as successive emission bands, which are resolved better than the corresponding decay phases recorded at constant temperatures. Progress in thermoelectric Peltier elements and in compact light detectors made the development of simple, affordable and transportable instruments possible. These instruments take advantage of multifurcated light guides for combined TL, fluorescence and absorbance/reflectance measurements. Meanwhile, experiments on unfrozen leaf discs, with excitation by single turn-over flashes or far red light and infiltration by specific inhibitors/uncouplers, have led to a better understanding of in vivo TL signals. Much like chlorophyll fluorescence and in a complementary way, TL in the 0-60 degrees C temperature range not only informs on the state of photosystem II in leaf tissues and its possible alterations, but also gives a broader insight into the energetic state inside the chloroplast by probing (1) the light-induced or dark-stable thylakoid proton gradient through the protonation of the Mn oxygen-evolving complex, (2) the induction of cyclic/chlororespiratory electron flow towards the plastoquinone pool, (3) the [NADPH+ATP] assimilatory potential. By a different mechanism, warming above 60 degrees C without preillumination reveals chemiluminescence high temperature thermoluminescence (HTL) bands due to the radiative thermolysis of peroxides, which are indicators of oxidative stress in leaves.
Site-directed mutagenesis in the photosystem II (PSII) oxygen-evolving enzyme was achieved in the thermophilic cyanobacterium Thermosynechococcus elongatus. PSII from this species is the focus of attention because its robustness makes it suitable for enzymological and biophysical studies. PSII, which lacks the redox-active tyrosine Tyr(D), was engineered by substituting a phenylalanine for tyrosine 160 of the D2 protein. An aim of this work was to engineer a mutant for spectroscopy, in particular, for EPR, on the active enzyme. The Tyr(D)(*) EPR signal was monitored in whole cells (i) to control the expression level of the two genes (psbD(1) and psbD(2)) encoding D2 and (ii) to assess the success of the mutagenesis. Both psbD(1) and psbD(2) could be expressed, and recombination occurred between them. The D2-Y160F mutation was introduced into psbD(1) after psbD(2) was deleted and a His-tag was attached to the CP43 protein. The effects of the Y160F mutation were characterized in cells, thylakoids, and isolated PSII. The efficiency of enzyme function under the conditions tested was unaffected. The distribution and lifetime of the redox states (S(n)() states) of the enzyme cycle were modified, with more S(0) in the dark and no rapid decay phase of S(3). Although not previously reported, these effects were expected because Tyr(D)(*) is able to oxidize S(0) and Tyr(D) is able to reduce S(2) and S(3). Slight changes in the difference spectra in the visible and infrared recorded upon the formation and reduction of the chlorophyll cation P(680)(+) and kinetic measurements of P(680)(+) reduction indicated minor structural perturbations, perhaps in the hydrogen-bonding network linking Tyr(D) and P(680), rather than electrostatic changes associated with the loss of a charge from Tyr(D)(*)(H(+)). We show here that this fully active preparation can provide spectra from the Mn(4)CaO(4) complex and associated radical species uncontaminated by Tyr(D)(*).
Photosystem II (PSII) performs one of the key reactions on our planet: the light-driven oxidation of water. This fundamental but very complex process requires PSII to act in a highly coordinated fashion. Despite detailed structural information on the fully assembled PSII complex, the dynamic aspects of formation, processing, turnover, and degradation of PSII with at least 19 subunits and various cofactors are still not fully understood. Transient complexes are especially difficult to characterize due to low abundance, potential heterogeneity, and instability. Here, we show that Psb27 is involved in the assembly of the water-splitting site of PSII and in the turnover of the complex. Psb27 is a bacterial lipoprotein with a specific lipid modification as shown by matrix-assisted laser-desorption ionization time of flight mass spectrometry. The combination of HPLC purification of four different PSII subcomplexes and (15)N pulse label experiments revealed that lipoprotein Psb27 is part of a preassembled PSII subcomplex that represents a distinct intermediate in the repair cycle of PSII.
The X-ray-derived Photosystem II (PS II) structure from the thermophilic cyanobacterium Thermosynechococcus vulcanus (Protein Data Bank entry 4UB6) indicates Phe239 of the DE loop of the D1 protein forms a hydrophobic interaction with Pro27 and Ile29 at the C-terminus of the 5 kDa PsbT protein found at the monomer-monomer interface of the PS II dimer. To investigate the importance of this interaction, we created the F239A and F239L mutants in Synechocystis sp. PCC 6803 through targeted mutagenesis of the D1:Phe239 residue into either an alanine or a leucine. Under moderate-light conditions, the F239A strain displayed reduced rates of oxygen evolution and impaired rates of fluorescence decay following a single-turnover actinic flash, while the F239L strain behaved like the control; however, under high-light conditions, the F239A and F239L strains grew more slowly than the control. Our results indicate the quinone-iron acceptor complex becomes more accessible to exogenously added electron acceptors in the F239A mutant and a ΔPsbT strain when compared with the control and F239L strains. This led to the hypothesis that the interaction between D1:Phe239 and the PsbT subunit is required to restrict movement of the DE loop of the D1 subunit, and we suggest disruption of this interaction may perturb the binding of bicarbonate to the non-heme iron and contribute to the signal for PS II to undergo repair following photodamage.
The investigation of water oxidation in photosynthesis has remained a central topic in biochemical research for the last few decades due to the importance of this catalytic process for technological applications. Significant progress has been made following the 2011 report of a high-resolution X-ray crystallographic structure resolving the site of catalysis, a protein-bound Mn 4 CaO x complex, which passes through ≥5 intermediate states in the water-splitting cycle. Spectroscopic techniques complemented by quantum chemical calculations aided in understanding the electronic structure of the cofactor in all (detectable) states of the enzymatic process. Together with isotope labeling, these techniques also revealed the binding of the two substrate water molecules to the cluster. These results are described in the context of recent progress using X-ray crystallography with free-electron lasers on these intermediates. The data are instrumental for developing a model for the biological water oxidation cycle.
Expected final online publication date for the Annual Review of Biochemistry, Volume 89 is June 22, 2020. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.
Photosynthesis is the natural process that converts solar photons into energy-rich products that are needed to drive the biochemistry of life. Two ultrafast processes form the basis of photosynthesis: excitation energy transfer and charge separation. Under optimal conditions, every photon that is absorbed is used by the photosynthetic organism. Fundamental quantum mechanics phenomena, including delocalization, underlie the speed, efficiency and directionality of the charge-separation process. At least four design principles are active in natural photosynthesis, and these can be applied practically to stimulate the development of bio-inspired, human-made energy conversion systems.
Oxygenic photosynthesis forms the basis of aerobic life on earth by converting light energy into biologically useful chemical energy and by splitting water to generate molecular oxygen. The water-splitting and oxygenevolving reaction is catalyzed by photosystem II (PSII), a huge, multisubunit membrane-protein complex located in the thylakoid membranes of organisms ranging from cyanobacteria to higher plants. The structure of PSII has been analyzed at 1.9-Å resolution by X-ray crystallography, revealing a clear picture of the Mn4CaO5 cluster, the catalytic center for water oxidation. This article provides an overview of the overall structure of PSII followed by detailed descriptions of the specific structure of the Mn4CaO5 cluster and its surrounding protein environment. Based on the geometric organization of the Mn4CaO5 cluster revealed by the crystallographic analysis, in combination with the results of a vast number of experimental studies involving spectroscopic and other techniques as well as various theoretical studies, the article also discusses possible mechanisms for water splitting that are currently under consideration. Expected final online publication date for the Annual Review of Plant Biology Volume 66 is April 29, 2015. Please see http://www.annualreviews.org/catalog/pubdates.aspx for revised estimates.
Photosynthesis converts light energy into biologically useful chemical energy vital to life on Earth. The initial reaction of photosynthesis takes place in photosystem II (PSII), a 700-kilodalton homodimeric membrane protein complex that catalyses photo-oxidation of water into dioxygen through an S-state cycle of the oxygen evolving complex (OEC). The structure of PSII has been solved by X-ray diffraction (XRD) at 1.9 ångström resolution, which revealed that the OEC is a Mn4CaO5-cluster coordinated by a well defined protein environment. However, extended X-ray absorption fine structure (EXAFS) studies showed that the manganese cations in the OEC are easily reduced by X-ray irradiation, and slight differences were found in the Mn-Mn distances determined by XRD, EXAFS and theoretical studies. Here we report a 'radiation-damage-free' structure of PSII from Thermosynechococcus vulcanus in the S1 state at a resolution of 1.95 ångströms using femtosecond X-ray pulses of the SPring-8 ångström compact free-electron laser (SACLA) and hundreds of large, highly isomorphous PSII crystals. Compared with the structure from XRD, the OEC in the X-ray free electron laser structure has Mn-Mn distances that are shorter by 0.1-0.2 ångströms. The valences of each manganese atom were tentatively assigned as Mn1D(iii), Mn2C(iv), Mn3B(iv) and Mn4A(iii), based on the average Mn-ligand distances and analysis of the Jahn-Teller axis on Mn(iii). One of the oxo-bridged oxygens, O5, has significantly longer distances to Mn than do the other oxo-oxygen atoms, suggesting that O5 is a hydroxide ion instead of a normal oxygen dianion and therefore may serve as one of the substrate oxygen atoms. These findings provide a structural basis for the mechanism of oxygen evolution, and we expect that this structure will provide a blueprint for the design of artificial catalysts for water oxidation.
Cyanobacteria have multiple psbA genes encoding PsbA, the D1 reaction center protein of the Photosystem II complex which bears together with PsbD, the D2 protein, most of the cofactors involved in electron transfer reactions. The thermophilic cyanobacterium Thermosynechococcus elongatus has three psbA genes differently expressed depending on the environmental conditions. Among the 344 residues constituting each of the 3 possible PsbA variants there are 21 substitutions between PsbA1 and PsbA3, 31 between PsbA1 and PsbA2 and 27 between PsbA2 and PsbA3. In this review, we summarize the changes already identified in the properties of the redox cofactors depending on the D1 variant constituting Photosystem II in T. elongatus. This article is part of a Special Issue entitled: Photosynthesis Research for Sustainability: Keys to Produce Clean Energy.
In Photosystem II (PSII) of the cyanobacterium Thermosynechococcus elongatus, glutamate 130 in the high-light variant of the D1-subunit (PsbA3) was changed to glutamine in a strain lacking the two other genes for D1, psbA1 and psbA2. The resulting PSII (PsbA3/Glu130Gln) was compared with those from the "native" high-light (PsbA3-PSII) and low-light (PsbA1-PSII) variants, which differ by 21 amino acid including Glu130Gln. H-bonding from D1-Glu130Gln to the primary electron acceptor, PheophytinD1 (PheoD1), is known to affect the Em of the PheoD1/PheoD1(-•) couple. The Gln130 mutation here had little effect on water splitting, charge accumulation and photosensitivity but did slow down S2QA(-●) charge recombination and up-shift the thermoluminescence while increasing its yield. These changes were consistent with a≈-30mV shift of the PheoD1/PheoD1(-●)Em, similar to earlier single site-mutation results from other species and double the≈-17mV shift seen for PsbA1-PSII versus PsbA3-PSII. This is attributed to the influence of the other 20 amino-acids that differ in PsbA3. A computational model for simulating S2QA(-●) recombination matched the experimental trend: the S2QA(-●) recombination rate in PsbA1-PSII differed only slightly from that in PsbA3-PSII, while in Glu130-PsbA3-PSII there was a more pronounced slowdown of the radical pair decay. The simulation predicted a major effect of the PheoD1/PheoD1(-●) potential on (1)O2 yield (~60 % in PsbA1-PSII, ~20% in PsbA3-PSII and ~7 % in Gln130-PsbA3-PSII), reflecting differential sensitivities to high light.
(1) The first part of this paper is devoted to methodological tests pertaining to the deconvolution of the spectra of the S-transitions from flash sequence data, (i) The spectrum and decay kinetics of 'inactive PS II centers' were analyzed, showing similarity with normal centers inhibited on the QB site. When this contribution is subtracted, the change on the first flash agrees with estimate of the S1→S2 change derived from deconvolution. (ii) Preillumina-tion procedures in PS II (BBY) particles, aimed at modulating the initial S0/S1 distribution are confirmed to express deactivation towards the S1 state, with no involvement of an ‘S−1’ state, (iii) Elimination of semiquinone binary oscillations in BBY's is achieved by allowing total reoxidation by DCBQ after each flash, (iv) A deconvolution treatment is described, using the difference between two sequences. This method allows a determination of the initial S0/S1 distributions that cross-checks the conclusions (ii). (v) Oscillations of the amplitude of the ms-phase of the 295 nm absorption changes are shown to differ significantly from those corresponding to the S-transitions. This phase is expected to reflect predominantly the O2 release reaction, with slight deviations due to other transitions. Deconvolution results are in satisfactory agreement with this prediction. (2) The upshot of this work is to present improved spectra of the S-transitions. The basic features that were previously established with different material and deconvolution method are confirmed: negligible UV change on S0→S1, and significantly different spectra for the two other transitions. A shoulder around 350 nm on the S1→S2 spectrum and a secondary peak in the same region for the S2→S3 spectrum are now resolved. The S0→S1 step causes an electrochromic shift in the blue region, with direction opposite to S1→S2. Interpretation of these results is discussed.
Incubation of PS II membranes with herbicides results in changes in EPR signals arising from reaction centre components. Dinoseb, a phenolic herbicide which binds to the reaction centre polypeptide, changes the width and form of the EPR signal arising from photoreduced Q−AFe. o-Phenanthroline slightly broadens the Q−AFe signal. These effects are attributed to changes in the interaction between the semi-quinone and the iron. DCMU, which binds to the 32 kDa protein, has virtually no effect on the width of the Q−AFe signal but does give rise to an increase in its amplitude. This could result from a change in redox state of an interacting component. Herbicide effects can also be seen when Q−AFe is chemically reduced and these seen to be reflected by changes in splitting and amplitude of the split pheophytin− signal. Dinoseb also results in the loss of ‘Signal II dark’, the conversion of reduced high-potential cytochrome b559 to its oxidized low-potential form and the presence of transiently photooxidized carotenoid after a flash at 25°C; these effects indicate that dinoseb may also act as an ADRY reagent.
A new spectrophotometric technique has been developed in which the absorption is sampled by short flashes produced by an optical parametric oscillator (OPO) pumped by the third harmonic of a Nd:YAG laser. Continuously tunable monochromatic flashes (6 ns duration, 1 nm bandwidth) are obtained in a domain ranging from 415 to 680 nm and 730 to 2000 nm. Light-induced absorption changes can be analyzed in a time window ranging from 10 ns to 100 s, with a 10−5 noise level. This new technique opens areas of research which are not accessible to previously existing spectrophotometers. Indeed, the high energy of the detecting flashes allows the analysis of samples of large optical density such as leaves, in the blue region of the spectrum. A few examples of application of this method to the analysis of electron transfer reactions in different types of photosynthetic apparatus (leaves, unicellular algae, and bacteria) are presented. This technique is also well suited to analyze the photodissociation of ligands associated with hemoproteins, in solution or in living cells. © 1999 American Institute of Physics.
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.
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 effect of photoactivation (the assembly of the Mn cluster involved in oxygen evolution) in Photosystem II (PS II), on the redox midpoint potential of the primary quinone electron acceptor, QA, has been investigated. Measurements of the redox state of QA were performed using chlorophyll fluorescence. Cells of Scenedesmus obliquus were grown in the dark to obtain PS II lacking the oxygen-evolving complex. Growth in the light leads to photoactivation. The midpoint potential of QA was shifted, upon photoactivation, from + 110 mV to −80 mV. In cells of a low-fluorescence mutant, LF1, that is unable to assemble the oxygen-evolving complex but that has an otherwise normal PS II, the higher potential form of QA was found. NH2OH treatment of spinach PS II, which releases the Mn and thus inactivates the oxygen-evolving complex, causes an upshift of the redox potential of QA (Krieger and Weis (1992) Photosynthetica 27, 89–98). Oxygen evolution can be reconstituted by incubation in the light in the presence of MnCl2 and CaCl2. Such photoactivation caused the midpoint potential of QA to be shifted back from around +55 mV to lower potentials (−80 mV), typical for active PS II. The above results indicate that the state of the donor side of PS II has a direct influence on the properties of the acceptor side. It is suggested that the change from the high- to the low-potential form of QA may represent a mechanism for protection of PS II during the assembly of the O2-evolving enzyme.
Charge-transfer reactions to secondary electron donors (Z, M) and acceptors (QA, QB) in Photosystem II particles isolated from a thermophilic cyanobacterium Synechococcus sp. (Schatz, G.H. and Witt H.T. (1984) Photobiochem. Photobiophys. 7, 1–14) were analyzed by measurements of fluorescence yield and absorbance changes in the millisecond time domain induced by repetitive flashes. (1) The electron-transfer reaction Q−AQB → QAQ−B was found to occur with kinetic phases of 0.2 ± 0.1 ms and 1.5 ± 0.5 ms half-time. At 10 ms after flashes an equilibrium distribution of of about in oxygen-evolving and of about in Tris-treated PS II particles was reached. (2) The absorbance difference spectra were determined for (Q−A - QA), (Q−B - QB), (Z+ - Z) and for (S4 - S0), the transition associated with oxygen evolution. In the ultraviolet region they show that these electron-acceptors and -donors are the same as in spinach PS II. In the visible region all the difference spectra contain major contributions by electrochromic bandshifts due to electrostatic interaction of the reduced acceptors or oxidized donors with nearby reaction center pigments. Upon electron transfer from Q−Ato QB electrochromic bandshifts due to interaction with pheophytin a disappeared almost completely. Bandshifts observed in the (Z+ - Z) and (S4 - S0) spectra were attributed to chlorophyll a.
The life cycle of Photosystem II (PSII) is embedded in a network of proteins that guides the complex through biogenesis, damage and repair. Some of these proteins, such as Psb27 and Psb28, are involved in cofactor assembly for which they are only transiently bound to the preassembled complex. In this work we isolated and analyzed PSII from a ΔpsbJ mutant of the thermophilic cyanobacterium Thermosynechococcus elongatus. From the four different PSII complexes that could be separated the most prominent one revealed a monomeric Psb27-Psb28 PSII complex with greatly diminished oxygen-evolving activity. The MALDI-ToF mass spectrometry analysis of intact low molecular weight subunits (<10kDa) depicted wild type PSII with the absence of PsbJ. Relative quantification of the PsbA1/PsbA3 ratio by LC-ESI mass spectrometry using (15)N labeled PsbA3-specific peptides indicated the complete replacement of PsbA1 by the stress copy PsbA3 in the mutant, even under standard growth conditions (50μmol photons m(-2) s(-1)). This article is part of a Special Issue entitled: Photosynthesis Research for Sustainability: from Natural to Artificial.
The photosystem II core complex is the water:plastoquinone oxidoreductase of oxygenic photosynthesis situated in the thylakoid membrane of cyanobacteria, algae and plants. It catalyzes the light-induced transfer of electrons from water to plastoquinone accompanied by the net transport of protons from the cytoplasm (stroma) to the lumen, the production of molecular oxygen and the release of plastoquinol into the membrane phase. In this review, we outline our present knowledge about the "acceptor side" of the photosystem II core complex covering the reaction center with focus on the primary (Q(A)) and secondary (Q(B)) quinones situated around the non-heme iron with bound (bi)carbonate and a comparison with the reaction center of purple bacteria. Related topics addressed are quinone diffusion channels for plastoquinone/plastoquinol exchange, the newly discovered third quinone Q(C), the relevance of lipids, the interactions of quinones with the still enigmatic cytochrome b559 and the role of Q(A) in photoinhibition and photoprotection mechanisms. This article is part of a Special Issue entitled: Photosystem II.
The quinone-iron complex of the electron acceptor complex of Photosystem II was studied by EPR spectroscopy in Thermosynechococcus elongatus. New g ∼ 2 features belonging to the EPR signal of the semiquinone forms of the primary and secondary quinone, i.e., Q(A)(•-)Fe(2+) and Q(B)(•-)Fe(2+), respectively, are reported. In previous studies, these signals were missed because they were obscured by the EPR signal arising from the stable tyrosyl radical, TyrD(•). When the TyrD(•) signal was removed, either by chemical reduction or by the use of a mutant lacking TyrD, the new signals dominated the spectrum. For Q(A)(•-)Fe(2+), the signal was formed by illumination at 77 K or by sodium dithionite reduction in the dark. For Q(B)(•-)Fe(2+), the signal showed the characteristic period-of-two variations in its intensity when generated by a series of laser flashes. The new features showed relaxation characteristics comparable to those of the well-known features of the semiquinone-iron complexes and showed a temperature dependence consistent with an assignment to the low-field edge of the ground state doublet of the spin system. Spectral simulations are consistent with this assignment and with the current model of the spin system. The signal was also present in Q(B)(•-)Fe(2+) in plant Photosystem II, but in plants, the signal was not detected in the Q(A)(•-)Fe(2+) state.
Photosystem II is the site of photosynthetic water oxidation and contains 20 subunits with a total molecular mass of 350 kDa. The structure of photosystem II has been reported at resolutions from 3.8 to 2.9 Å. These resolutions have provided much information on the arrangement of protein subunits and cofactors but are insufficient to reveal the detailed structure of the catalytic centre of water splitting. Here we report the crystal structure of photosystem II at a resolution of 1.9 Å. From our electron density map, we located all of the metal atoms of the Mn(4)CaO(5) cluster, together with all of their ligands. We found that five oxygen atoms served as oxo bridges linking the five metal atoms, and that four water molecules were bound to the Mn(4)CaO(5) cluster; some of them may therefore serve as substrates for dioxygen formation. We identified more than 1,300 water molecules in each photosystem II monomer. Some of them formed extensive hydrogen-bonding networks that may serve as channels for protons, water or oxygen molecules. The determination of the high-resolution structure of photosystem II will allow us to analyse and understand its functions in great detail.
A deletion mutant that lacks the Psb30 protein, one of the small subunits of Photosystem II, was constructed in a Thermosynechococcus elongatus strain in which the D1 protein is expressed from the psbA(3) gene (WT*). The DeltaPsb30 mutant appears more susceptible to photodamage, has a cytochrome b(559) that is converted into the low potential form, and probably also lacks the PsbY subunit. In the presence of an inhibitor of protein synthesis, the Psb30 lost more rapidly the water oxidation function than the WT* under the high light conditions. These results suggest that Psb30 contributes to structurally and functionally stabilise the Photosystem II complex in preventing the conversion of cytochrome b(559) into the low potential form. Structural reasons for such effects are discussed.
The primary electron acceptor pheophytin (Pheo(D1)) plays a crucial role in regulation of forward and backward electron transfer in photosystem II (PSII). It is known that some cyanobacteria control the Pheo(D1) potential in high-light acclimation by exchanging the D1 proteins from different copies of the psbA genes. To clarify the mechanism of the potential control of Pheo(D1), we studied the hydrogen bond interactions of Pheo(D1) in the neutral and anionic states using light-induced Fourier transform infrared (FTIR) difference spectroscopy. FTIR difference spectra of Pheo(D1) upon its photoreduction were obtained using three different PSII preparations, PSII core complexes from Thermosynechococcus elongatus possessing PsbA1 as a D1 subunit (PSII-PsbA1), those with PsbA3 (PSII-PsbA3), and PSII membranes from spinach. The D1-Gln130 side chain, which is hydrogen bonded to the 13(1)-keto C=O group of Pheo(D1) in PSII-PsbA1, is replaced by Glu in PSII-PsbA3 and spinach PSII. The spectrum of PSII-PsbA1 exhibited 13(1)-keto C=O bands at 1682 and 1605 cm(-1) in neutral Pheo(D1) and its anion, respectively, while the corresponding bands were observed at frequencies lower by 1-3 and 18-19 cm(-1), respectively, in the latter two preparations. This larger frequency shift in Pheo(D1)(-) than Pheo(D1) by the change of the hydrogen bond donor was well reproduced by density functional theory (DFT) calculations for the Pheo models hydrogen bonded with acetamide and acetic acid. The DFT calculations also exhibited a higher redox potential for Pheo reduction in the model with acetic acid than that with acetamide, consistent with previous observations for the D1-Gln130Glu mutant of Synechocystis. It is thus concluded that a stronger hydrogen bond effect on the Pheo(-) anion than the neutral Pheo causes the shift in the redox potential, which is utilized in the photoprotection mechanism of PSII.
Ycf12 (Psb30) and PsbZ are two low molecular weight subunits of photosystem II (PSII), with one and two trans-membrane helices, respectively. In order to study the functions of these two subunits from a structural point of view, we constructed deletion mutants lacking either Ycf12 or PsbZ from Thermosynechococcus elongatus, and purified, crystallized and analyzed the structure of PSII dimer from the two mutants. Our results showed that Ycf12 is located in the periphery of PSII, close to PsbK, PsbZ and PsbJ, and corresponded to the unassigned helix X1 reported previously, in agreement with the recent structure at 2.9 A resolution (A. Guskov, J. Kern, A. Gabdulkhakov, M. Broser, A. Zouni, W. Saenger, Cyanobacterial photosystem II at 2.9 A resolution: role of quinones, lipids, channels and chloride, Nat. Struct. Mol. Biol. 16 (2009) 334-342). On the other hand, crystals of PsbZ-deleted PSII showed a remarkably different unit cell constants from those of wild-type PSII, indicating a role of PsbZ in the interactions between PSII dimers within the crystal. This is the first example for a different arrangement of PSII dimers within the cyanobacterial PSII crystals. PSII dimers had a lower oxygen-evolving activity from both mutants than that from the wild type. In consistent with this, the relative content of PSII in the thylakoid membranes was lower in the two mutants than that in the wild type. These results suggested that deletion of both subunits affected the PSII activity, thereby destabilized PSII, leading to a decrease in the PSII content in vivo. While PsbZ was present in PSII purified from the Ycf12-deletion mutant, Ycf12 was present in crude PSII but absent in the finally purified PSII from the PsbZ-deletion mutant, indicating a preferential, stabilizing role of PsbZ for the binding of Ycf12 to PSII. These results were discussed in terms of the PSII crystal structure currently available.
Thermoluminescence (TL) probes the emission of luminescence associated with the de-trapping of a radical pair as the temperature is increased. This technique has proved useful for characterizing the energetic arrangement of cofactors in photosynthetic reaction centers. In the original TL theory, stemming from solid-state physics, the radical pair recombination was considered to coincide with the light-emitting process. In photosynthetic systems, however, recombination takes place through various routes among which the radiative pathway generally represents a relatively minor leak, and the theoretical framework must be modified accordingly. The radiative route is the one with the largest activation energy and is thus (still) more disfavored at low temperature, so that during the heating process, the TL peak tends to lag behind the decay of the radical pair. A consequence is that the integrated luminescence emission increases with the heating rate. In this article, we examine how the characteristics of the TL emission depend on the redox potentials of the cofactors, showing good agreement between theory and experimental studies on Photosystem (PS) II mutants. We also analyze the effect on (thermo-) luminescence of the connectivity of the light-harvesting pigment antenna, and show that while this should affect significantly luminescence kinetics at room temperature, the effect on TL is expected to be small.
The functions of cytochrome c-550 and a 12 kDa protein in cyanobacterial oxygen evolution were studied with directed deletion mutants delta psbV and delta psbU of Synechocystis sp. PCC 6803, and the following results were obtained. (1) In contrast to the delta psbU mutant which is capable of autotrophic growth in the absence of Ca2+ or Cl- at a reduced rate, the delta psbV mutant lacking cytochrome c-550 could not grow at all without Ca2+ or Cl-. (2) The delta psbV mutant had a significantly reduced thermoluminescence emission intensity and flash oxygen yield, whereas the delta psbU mutant showed slight decreases in thermoluminescence intensity and flash oxygen yield, indicating corresponding decreases in the concentrations of O2-evolving centers in these mutants. (3) The delta psbV and delta psbU mutants exhibited elevated peak temperature for the thermoluminescence B- and Q-bands indicative of more stable S2 states. (4) The rise time of the O2 signal during the S3-[S4]-S0 transition was increased slightly in the delta psbV mutant but not in the delta psbU mutant. (5) The oxygen evolution was inactivated in the dark rapidly in the delta psbV mutant with a half-time of 28 min, but this did not happen in the delta psbU mutant. (6) Photoactivation of the oxygen-evolving complex after removal of the manganese cluster by hydroxylamine showed a higher quantum yield in the delta psbV mutant than in the delta psbU mutant or wild type. Taken together, these results indicated that cytochrome c-550 plays a substantial role in maintaining the stability and function of the manganese cluster in algal photosystem II, whereas the 12 kDa protein plays primarily a regulatory role in maintaining normal S-state transitions. These functional features of cytochrome c-550 and the 12 kDa protein were compared with those of the 23 and 17 kDa proteins in higher plant photosystem II and of the 33 kDa protein in both algal and plant photosystem II.
Pigment-protein interactions play a significant role in determining the properties of photosynthetic complexes. Site-directed mutants of Synechocystis PCC 6803 have been prepared which modify the redox potential of the primary radical pair anion and cation. In one set of mutants, the environment of P680, the primary electron donor of Photosystem II, has been modified by altering the residue at D1-His198. It has been proposed that this residue is an axial ligand to the magnesium cation. In the other set, the D1-Gln130 residue, which is thought to interact with the C9-keto group of the pheophytin electron acceptor, has been changed. The effect of these mutations is to alter the free energy of the primary radical pair state, which causes a change in the equilibrium between excited singlet states and radical pair states. We show that the free energy of the primary radical pair can be increased or decreased by modifications at either the D1-His198 or the D1-Gln130 sites. This is demonstrated by using three independent measures of quantum yield and equilibrium constant, which exhibit a quantitative correlation. These data also indicate the presence of a fast nonradiative decay pathway that competes with primary charge separation. These results emphasize the sensitivity of the primary processes of PS II to small changes in the free energy of the primary radical pair.
The carboxyl terminus of the CP43 subunit of photosystem II (PSII) in the thermophilic cyanobacterium, Synechococcus elongatus, was genetically tagged with six consecutive histidine residues to create a metal binding site on the PSII supramolecular complex. The histidine-tagging enabled rapid isolation of an intact cyanobacterial PSII core complex from dodecyl maltoside-solubilized thylakoids by a simple one-step Ni(2+)-affinity column chromatography. The isolated core complex was in a dimeric form with a molecular mass of about 580 kDa, consisting of five major intrinsic membrane proteins (CP47, CP43, D1, D2 and cytochrome b-559), three extrinsic proteins (33 kDa, 12 kDa, and cytochrome c-550), and a few low molecular mass membrane proteins, and evolved oxygen at a rate as high as 3,400 mumol (mg Chl)-1 h-1 at 45 degrees C with ferricyanide as an electron acceptor. The core complex emitted thermoluminescence B2-, B1- and Q-bands arising from S2QB-, S3QB- and S2QA- charge recombinations at respective emission temperatures of 45, 38 and 20 degrees C, all of which were higher by about 15 degrees C as compared with those in mesophilic spinach BBY membranes. These results indicated that the isolated core complex well retained the intact properties of thermoluminescence of thermophilic cyanobacterial cells, the deeper stabilization of PSII charge pairs. The isolated complex was extremely stable in terms of both protein composition and function, exhibiting no release of extrinsic proteins, no proteolytic degradation in any of its subunits, accompanied by only a slight (less than 10%) loss in oxygen evolution, after dark-incubation at 20 degrees C for 8 d. These properties of the thermophilic PSII core complex are highly useful for various types of studies on PSII.
Some herbicides act by binding to the exchangeable quinone site in the photosystem II (PSII) reaction centre, thus blocking electron transfer. In this article, it is hypothesized that the plant is killed by light-induced oxidative stress initiated by damage caused by formation of singlet oxygen in the reaction centre itself. This occurs when light-induced charge pairs in herbicide-inhibited PSII decay by a charge recombination route involving the formation of a chlorophyll triplet state that is able to activate oxygen. The binding of phenolic herbicides favours this pathway, thus increasing the efficiency of photodamage in this class of herbicides.
The absorption properties of chlorophyll a (Chla) in active core complexes of photosystems I (PSI) and II (PSII) isolated in high purity from the thermophilic cyanobacterium Thermosynechococcus elongatus were correlated with those of extracts in 80% acetone to determine effective extinction coefficients of protein-bound Chla and molar extinction coefficients of core complexes and reaction centers (RC). These coefficients allow a quick determination of Chla and protein concentrations from steady-state absorption spectra of intact samples without the need for pigment extraction and protein destruction. In the visible range, epsilon(680)(p) = 57 mM(-1) cm(-1) for trimeric PSI (PSIt) and epsilon(674)(p) = 70 mM(-1) cm(-1) for dimeric (PSIId) and monomeric (PSIIm) PSII (error +/-6%; superscript "p" refers to Chla bound to intact protein, subscripts are the peak maxima in nm). The integral extinction coefficient phi(p) = 2.8 nm microM(-1) cm(-1) for the wavelength interval between 550 and 800 nm and the extinction coefficient epsilon(B)(p) = 14 mM(-1) cm(-1) for the smaller absorption maximum (B = 632 nm for PSI and 627 nm for PSII) were found to be essentially the same for both types of PS. The coefficients of PSIt are shown to remain unaltered when 65% (v/v) of the buffer is replaced with glycerol. Molar extinction coefficients of core complexes were determined using Chla/RC ratios of 96+/-1 for PSI and 35+/-2 for PSII based on X-ray data. In addition, the critical solubilisation concentration of n-dodecyl-beta-d-maltoside (betaDM), necessary to keep the core complexes in solution, was determined by turbidimetric titrations. It was found that at least approximately 500 betaDM molecules per PSIt ( approximately 2 betaDM per Chla) and 190 betaDM molecules per PSIIm ( approximately 5 betaDM per Chla, also for PSIId) in excess of the critical micelle concentration of 0.16 +/- 0.03 mM are necessary for a complete solubilisation of the core complexes.
The secondary quinone acceptor, Q(B), has been studied in photosystem II (PSII) isolated from Thermosynechococcus (T.) elongatus. Thermoluminescence indicated that Q(B) was present in this preparation. An EPR signal observed at low temperature at g = 1.9 was attributed to Fe2+ Q(B)- on the basis of the characteristic period-of-two variations in its intensity depending on the number of laser flashes given at 20 degrees C. When samples showing the Fe2+ Q(B)- signal were illuminated at 77 K, an EPR signal at g = 1.66 appeared with an amplitude proportional to that of the Fe2+ Q(B)- signal. This signal is attributed to the Q(A)- Fe2+ Q(B)- state. While these attributions have been made previously in PSII from other origins, they have remained relatively tentative since the characteristic period-of-two oscillations of Q(B) had not previously been observed. The flash experiments indicated that more than one exchangeable plastoquinone is associated with the isolated PSII. The g = 1.66 signal from the Q(A)- Fe2+ Q(B)- state was used to study the temperature dependence of electron transfer between the two quinones. Electron transfer occurred in half of the centers (after 30 s incubation) at -28 degrees C for Q(A)- to Q(B) but at -58 degrees C for Q(A)- to Q(B)-. This marked difference for the two electron transfer reactions indicates different types of rate-limiting reactions. In the better studied but homologous system, the purple bacterial reaction center, the Q(A)- to Q(B) step is limited by a gating process, while the Q(A)- to Q(B)- step is limited by protonation events. Similar reactions in PSII could give rise to the observed temperature dependence.
ATP has been shown to be a taste bud afferent transmitter, but the cells responsible for, and the mechanism of, its release have not been identified. Using CHO cells expressing high-affinity neurotransmitter receptors as biosensors, we show that gustatory stimuli cause receptor cells to secrete ATP through pannexin 1 hemichannels in mouse taste buds. ATP further stimulates other taste cells to release a second transmitter, serotonin. These results provide a mechanism to link intracellular Ca²⁺ release during taste transduction to secretion of afferent transmitter, ATP, from receptor cells. They also indicate a route for cell–cell communication and signal processing within the taste bud.
• afferent
• gustation
• serotonin
• synapses
The mechanism of charge recombination was studied in Photosystem II by using flash induced chlorophyll fluorescence and thermoluminescence measurements. The experiments were performed in intact cells of the cyanobacterium Synechocystis 6803 in which the redox properties of the primary pheophytin electron acceptor, Phe, the primary electron donor, P(680), and the first quinone electron acceptor, Q(A), were modified. In the D1Gln130Glu or D1His198Ala mutants, which shift the free energy of the primary radical pair to more positive values, charge recombination from the S(2)Q(A)(-) and S(2)Q(B)(-) states was accelerated relative to the wild type as shown by the faster decay of chlorophyll fluorescence yield, and the downshifted peak temperature of the thermoluminescence Q and B bands. The opposite effect, i.e. strong stabilization of charge recombination from both the S(2)Q(A)(-) and S(2)Q(B)(-) states was observed in the D1Gln130Leu or D1His198Lys mutants, which shift the free energy level of the primary radical pair to more negative values, as shown by the retarded decay of flash induced chlorophyll fluorescence and upshifted thermoluminescence peak temperatures. Importantly, these mutations caused a drastic change in the intensity of thermoluminescence, manifested by 8- and 22-fold increase in the D1Gln130Leu and D1His198Lys mutants, respectively, as well as by a 4- and 2.5-fold decrease in the D1Gln130Glu and D1His198Ala mutants, relative to the wild type, respectively. In the presence of the electron transport inhibitor bromoxynil, which decreases the redox potential of Q(A)/Q(A)(-) relative to that observed in the presence of DCMU, charge recombination from the S(2)Q(A)(-) state was accelerated in the wild type and all mutant strains. Our data confirm that in PSII the dominant pathway of charge recombination goes through the P(680)(+)Phe(-) radical pair. This indirect recombination is branched into radiative and non-radiative pathways, which proceed via repopulation of P(680)(*) from (1)[P(680)(+)Ph(-)] and direct recombination of the (3)[P(680)(+)Ph(-)] and (1)[P(680)(+)Ph(-)] radical states, respectively. An additional non-radiative pathway involves direct recombination of P(680)(+)Q(A)(-). The yield of these charge recombination pathways is affected by the free energy gaps between the Photosystem II electron transfer components in a complex way: Increase of DeltaG(P(680)(*)<-->P(680)(+)Phe(-)) decreases the yield of the indirect radiative pathway (in the 22-0.2% range). On the other hand, increase of DeltaG(P(680)(+)Phe(-)<-->P(680)(+)Q(A)(-)) increases the yield of the direct pathway (in the 2-50% range) and decreases the yield of the indirect non-radiative pathway (in the 97-37% range).