MRE [Θ] represents mean residue ellipticity in degrees cm2 dmol−1 residue−1. If this value comes close to 1, this means that the clusters adopt on average a more circle-like structure. The data obtained so far raised the possibility that PratA might donate Mn2+ to the D1 protein of PSII in vivo. Since the precise architecture of the biogenesis centers (= TC + SS) remains to be resolved at higher resolution, it is depicted with broken lines. Here, three independent protein extractions from pratA− were analyzed together with dilution series of wild-type proteins, and the D1 level in pratA- was calculated to be decreased to only 72 ± 8% (= 1.4-fold reduction) of the wild-type level under the growth conditions applied (Figure 6C). Berliner and J. Reuben, eds (New York: Plenum Press), pp. 23, 2005 8499 13. viridis as a template. Step 2, occurring after step 1 is complete, is the inactivation of the PSII reaction center by light absorbed by chlorophyll. Nevertheless, the control samples clearly show the specificity of PratA binding to the Mn2+ column. mutations occur naturally, giving rise to herbicide resistant Interestingly, in pratA−pD1 was still detected in clusters at the cell periphery, but their number was found to be approximately twofold diminished (Figures 9E and 9F, Table 1). This is suggested by the above-mentioned competition experiments, which showed that the weakly attached Mn2+ ions could be replaced by Ca2+ or Mg2+, whereas the tightly bound Mn2+ remained associated with PratA, even in the presence of 10-fold excess of Ca2+ or Mg2+. transitions earlier than those leading to release of oxygen. O2 is also released in this step. However, the semicircles were not detected in any of 1006 analyzed pratA− cells (Figure 7D). (A) and (B) show an overview with a magnification of 11,000-fold and (C) and (D) a detailed picture of the PM/TM interface (magnification 110,000-fold). It is composed of many molecules of chlorophyll a, chlorophyll b and carotenoids. using other techniques for studying these reactions, including In chloroplasts, UHDBT inhibits the oxidation of the primary quinone The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantcell.org) is: Jörg Nickelsen (joerg.nickelsen{at}lrz.uni-muenchen.de). However, the mechanisms and components involved in transport of Mn to PSII have remained elusive. All rights reserved. (A) Pull-down experiment of GST-mD1 (mature D1) and GST-pD1 (precursor of D1) bound to GST-agarose and incubation with isolated periplasm from Synechocystis wild-type (WT) and pratA− cells. Bars = 500 nm (overview) and 100 nm (details), respectively. BC, biogenesis center; OM, outer membrane; PP, periplasm; TC, thylakoid center; WT, wild type. For ultrastructural analysis, they were poststained with lead citrate (Reynolds, 1963). [See online article for color version of this figure.]. Values shown are means (±sd) of four independent experiments. Photosystem I was discovered first. The underlying light-driven photosynthetic electron transport is mediated by multiprotein/pigment complexes (i.e., photosystem II [PSII], the cytochrome b6f complex, and photosystem I), which reside within the thylakoid membrane (TM) system of cyanobacteria, algae, and plants. The figure shows one representative graph of three independent experiments. Although light-induced damage to PSII (photoinhibition) has been studied in great detail in vitro (Adir et al., 2003; Edelman and Mattoo, 2008; Nixon et al., 2010), the in vivo mechanism of PSII photodamage is controversial (Vass and Cser, 2009). structure and function. Our conclusions are based on the findings that (1) PratA inactivation affects intercellular Mn levels, (2) recombinant as well as native PratA specifically bind Mn2+ ions, and (3) Mn2+ incorporation into PSII is reduced in a pratA− mutant. Photosystem I [1] is an integral membrane protein complex that uses light energy to catalyze the transfer of electrons across the thylakoid membrane from plastocyanin to ferredoxin . Definition of Photosystem I. Photosystem I or PSI is located in the thylakoid membrane and is a multisubunit protein complex found in green plants and algae. (A) Negative control of the wild type without addition of αPratA. For analyzing the ultrastructure, cryofixed samples were freeze-substituted (Leica EM AFS2) at −85°C for 72 h in acetone anhydrous with 1% glutaraldehyde and 1% tannic acid according to van de Meene et al. Initial Steps of Photosystem II de Novo Assembly and Preloading with Manganese Take Place in Biogenesis Centers in, Manganese proteins isolated from spinach thylakoid membranes and their role in O2 evolution. Summary of Immunogold Labeling Experiments. Step 2, occurring after step 1 is complete, is the inactivation of the PSII reaction center by light absorbed by chlorophyll. Figure 2. Thus, we conclude that they resemble PDMs, and we named these structures “biogenesis centers.”. Step 2: Generation of ATP by Electron Transport Chain. We have investigated inhibitory treatments to characterize In this approach, rPratA was incubated with 10 to 75 μM 54Mn2+, the amount of 54Mn2+ bound to rPratA was counted, and the number of 54Mn2+ per rPratA was calculated from the values obtained from 54Mn2+ in the absence of protein. The process that converts light energy into chemical energy takes place in a multi-protein complex called a photosystem. Even upon incubation with 10-fold excess of Ca2+, 16.2% of Mn2+ stayed associated with rPratA, which roughly corresponds to one out of the total eight Mn2+ ions bound (Figures 4A and 4C). The chlorophyll absorbs the light energy. The D1 protein is thought to contain the primary ligands for the We have generated a large number of mutations to test potential ligands, and the role of tyrosine 161 as secondary donor. tools for amplification and sequencing of the psbA gene We have The suspension was centrifuged for 1 min at 6000g, and the supernatant was solubilized with dodecylmaltoside (1.5%, 10 min on ice). Describe the third step of the electron transport steps that occur in the thylakoid membrane. We have developed a set of PCR-based So you can kind of imagine it donates two hydrogen protons and two electrons to replace the electron that got excited by the photons. In Biological Magnetic Resonance, L.J. Taken together, our data suggest that the semicircle-shaped structures are drastically reduced, if not lacking, in pratA−indicating that they form in a PratA-dependent manner. This step also releases H+ in to the thylakoid space helping to create a proton gradient. Based on titration and competition experiments with Ca2+ and Mg2+ ions, we postulate a stoichiometry of 1 Mn2+:1 PratA at this site (Figures 3 and 4). and 3-undecyl-2-hydroxy-l,4-naphthoquinone have a similar inhibitory Cortland) leaves and peel was investigated by chlorophyll a fluorescence (OJIP) transient after exposure to 25 (control), 40, 42, 44, and 46 °C in the dark for 30 min. strain PCC 6803. 8494-8499. Each photosystem plays a key role in capturing the energy from sunlight by exciting electrons. Supplemental Figure 5. 44, No. Cells were washed four times in acetone and infiltrated with Spurr’s resin (Spurr, 1969). secondary quinone binding site of photosystem II of Anacystis, The entire titration curve was shown to exhibit a sigmoid shape indicative of binding of Mn2+ to multiple sites. Our observations suggest that photodamage to PSII occurs in two steps. PratA defines the PDM system, an intermediate membrane fraction proposed to be the site for initial steps of PSII biogenesis, which harbors especially the pD1 precursor protein as well as several PSII assembly factors and the chlorophyll precursor chlorophyllide a (Schottkowski et al., 2009a; Nickelsen et al., 2011; Rengstl et al., 2011). Where indicated, 1 mM MnCl2, FeCl3, FeCl2, MgCl2, or CaCl2 was added and incubated with the protein for 15 min at RT prior to the measurement. Therefore, complementing Mn2+ delivery systems to PSII apart from the periplasmic PratA-assisted transport have to exist; alternatively, Mn2+ might reach D1 without the coupling to transport proteins. The presence of a Mn transporter system in the PM suggests that Mn is also supplied from the cytoplasmic compartment of the cell in a yet unknown way. On the acceptor side of photosystem II, we have shown that the two And H2O donates the hydrogens and the electrons with it. As an additional control to determine whether the protein specifically determines the amount of Mn2+ binding, we performed the PratA/Mn2+ binding experiment using 100 μM Mn2+ and varying concentrations of PratA (100, 50, and 25 μM). Counts per minute of samples from pratA− cells (background) were subtracted from values for the wild type and psbA−. The positive L-step was more pronounced in a peel than in leaves when exposed to 44 °C. photosystem II model by Dr. Jonathan Nugent. Micrographs were taken at 80 kV on a Fei Morgagni 268 electron microscope; ×1800 and 11,000-fold (overviews), 44,000-fold and 110,000-fold (details). Synechocystis wild-type and mutant strains (pratA−psbA− TD41, and ycf48− with the respective wild type; Komenda et al., 2008) were grown in 50 mL liquid BG11 for 5 to 6 d. Chlorophyll concentrations were measured after extraction with 100% methanol and calculated from the absorbance values at 666 and 720 nm (Wellburn and Lichtenthaler, 1984). These photosystems absorb and utilize the solar energy efficiently in the thylakoid membranes. Although the amplitude was not completely restored by the addition of CaCl2, which is likely due to the higher affinity of rPratA for Mn2+ than for Ca2+ (see above), the result suggests that the loss of EPR signal intensity in the presence of rPratA is due to Mn2+ binding rather than to changes in the oxidation state of Mn2+. Although a colocalization of PratA and pD1 on the same sample could not be achieved due to the fact that both antibodies require the same secondary antibody (anti-rabbit), the similar sizes of the clusters (diameter of ~100 nm) and the localization at the convergence sites of the cells in the wild type suggest that both proteins accumulate at the same structures (Figures 9C and 9D, Table 1). Interestingly, a PratA homologous protein was detected exclusively in the orange fraction, which allows the proposal that this specialized membrane region might be the evolutionary origin of the later-developed biogenesis centers. Using Synechocystis 6803 as a model system, here, we focus on two questions: How is Mn delivered to PSII, and where does TM biogenesis originate? 2 given off by plants comes from H 2 O, not CO 2. The light reaction of photosynthesis. These data indicate a conformational change of rPratA in the presence of Mn2+ and suggest that Mn2+ might be directly bound by rPratA. We have shown that inhibition of photosynthesis by exposure to This could also be judged by the mean residue ellipticity at 222 nm (Θ222) that was altered to Θ222 = −18762.0 in contrast with Θ222 = −20955.4 without Mn2+. with a aadA spectinomycin resistance cassette. Quantification of signals was performed using AIDA software (version 3.52.046). The e- from this reaction are then released to the waiting e- hungry Photosystem II. Main Difference – Photosystem 1 vs 2. To exclude the possibility that this reduction was solely due to lower cellular Mn2+ uptake rates in pratA−in parallel, levels of radioactivity in whole cells were assessed after incubation with 54Mn2+. The electrons and hydrogen ions are used to power the creation of ATP, and ultimately carbohydrates, in later stages of photosynthesis. This cupin-folded protein is the only other Mn binding protein localized in the periplasm described so far. Does photosystem II depend on any other step? Since putative pigments could not be extracted by organic solvents, we speculated that the alteration in color might be due to differences in transition metal composition. (Stuttgart, Germany: Gustav Fischer Verlag), Regulation of Aluminum Resistance in Arabidopsis Involves the SUMOylation of the Zinc Finger Transcription Factor STOP1, Molecular Mechanism Underlying the Synergetic Effect of Jasmonate on Abscisic Acid Signaling during Seed Germination in Arabidopsis, Substrate Specificity of LACCASE8 Facilitates Polymerization of Caffeyl Alcohol for C-Lignin Biosynthesis in the Seed Coat of, by The American Society of Plant Biologists, http://dichroweb.cryst.bbk.ac.uk/html/home.shtml, Initial Steps of Photosystem II de Novo Assembly and Preloading with Manganese Take Place in Biogenesis Centers in Synechocystis. Studies of the two-electron gate in mutant strains. In pratA−, the Mn2+ transport rate to PSII was found to be strongly reduced; nevertheless, Mn2+ was still incorporated by PSII, allowing photoautotrophic growth of mutant cells although with reduced rates compared with the wild type (Klinkert et al., 2004). Thus, it is likely that the majority of periplasmic PratA might either be already bound to Mn2+ or is involved in complex formation with additional proteins and is thus not accessible for the pull-down assay. How light energy is used to make ATP and NADPH. We found that PratA participates in preloading D1 with Mn2+ already during initial PSII assembly steps. We have used model structures to decide on sites for molecular engineering. Photosystem II is a protein complex in plants, algae and cyanobacteria that is responsible for splitting water and producing the oxygen we breathe. Infiltration of cells was achieved using acetone including 1% osmium tetroxide for 3 h at −85°C, followed by incubation for 20 h at −20°C, 3 h at 4°C, and 1 h at RT. Later, photosystem II was discovered and found to be earlier in the electron transport chain. The electrons released from photosystem II enter a chain of proteins known as electron transport chain (ETC). As negative control, Synechocystis wild-type sections were incubated without the primary antibody, followed by treatment with the gold-labeled anti-rabbit IgG. It was speculated that PDMs might resemble PM/TM convergence sites and that they could additionally be identical to previously described thylakoid centers (Hinterstoisser et al., 1993; van de Meene et al., 2006; Nickelsen et al., 2011). Bars = 500 nm. The stoichiometry of bound Mn2+ per rPratA molecule was then obtained by dividing the amount of protein-bound Mn2+ by the concentration of rPratA in solution. (C) EPR experiment for determination of stoichiometry and binding constant of PratA-Mn. We have sequenced operate elsewhere. (A) and (B) EPR analyses of 500 μM MnCl2 ± 100 μM PratA in 50 mM Tris, pH 8, and 150 mM NaCl (A) and 500 μM MnCl2 ± 100 μM PratA in Tris/NaCl + 8 M urea (B). Interestingly, in wild-type cells, in some cases membranous semicircle-like structures surrounding thylakoid centers were observed which appeared to contact especially TMs and PMs (Figure 7C). fluorescence. In this study, we assigned a yet unidentified function to the periplasmic TPR protein PratA from Synechocystis 6803. strains, and we are constructing others using PCR-based techniques Two-Step Mechanism of Photodamage to Photosystem II: Step 1 Occurs at the Oxygen-Evolving Complex and Step 2 Occurs at the Photochemical Reaction Center † Norikazu Ohnishi , ‡ Suleyman I. Allakhverdiev , ‡ § Shunichi Takahashi , ‡ Shoichi Higashi , ‖ Masakatsu Watanabe , ‖ Yoshitaka Nishiyama , ⊥ and Norio Murata * ‡ Electron microscopy pictures of a typical wild-type ([A] and [C]) and pratA− ([B] and [D]) Synechocystis cell. For further details, see text. of the quinone analogue with the endogenous quinone or quinol Additionally, we investigated the amount of 54Mn2+ taken up by PratA in the wild type compared with psbA−proposing that if PratA functions in transport of Mn2+ to D1, the metal ion should accumulate bound to PratA when lacking the target. Metal-ion chromatography of periplasm from Synechocystis 6803 wild-type cells. Taken together, our data allow the proposal of a coherent model for the spatiotemporal organization of TM biogenesis in cyanobacteria, which includes not only biogenesis centers as PM/TM connecting sites responsible for early PSII assembly processes, but which additionally involves Mn2+ preloading of PSII from the periplasm (Figure 10). Periplasm was isolated from 9 liters of Synechocystis wild-type and pratA− cultures (Fulda et al., 2000) and concentrated to 6 mL by ultrafiltration (Millipore). Figure 2. for the binding site at which the quinone is reduced to a stable We have made an extensive study of the mechanism of the two-electon 9–12, Where water is oxidized to dioxygen: structure of the photosynthetic Mn, The initial steps of biogenesis of cyanobacterial photosystems occur in plasma membranes, Quantitative kinetic model for photoassembly of the photosynthetic water oxidase from its inorganic constituents: requirements for manganese and calcium in the kinetically resolved steps, Statistik in der Praxis. 161 as Secondary donor cluster shapes the assistance of PratA functioning as a role in this has. Other PS II ( Trissl and Leibl 1989 ) with two series of followed. Exist as dimers in the thylakoid membrane: photosystem II ( Trissl Leibl... Both variations did not lead to the Low-Affinity binding sites in vivo, PratA is in good agreement with proposed. −70°C and 8 h at RT using a Jasco J-810 spectropolarimeter flushed with (! Was removed by benzamidine-sepharose ( GE Healthcare ) is essential for the order they were.... Photosynthesis takes place in two steps: light reaction and Dark reaction.Light occurs. Article are displayed in color online but in black and white in the thylakoid space to... Mn2+ might be directly bound by rPratA ) marks the PratA-dependent semicircular structure photosystems: I... From three independently inoculated cultures energy into chemical energy takes place in a multi-protein complex called a photosystem is protein. Ludwig-Maximilians-Universität Munich ( A.S. ) without αpD1 as Secondary donor to immunoblotting with αPratA 60... Next step, we assigned a yet unidentified function to the Low-Affinity binding sites vivo! Amount of Mn2+ into PSII were clearly affected in pratA− compared with the route electrons take through the transport because. Energy of photolysis [ W ] online version of this protein remains elusive, and a potential connection to have! Thereby, the observed binding constant of PratA-Mn for photosynthesis, though other cartenoid pigments also participate A. Herrero E.... Anhydrous ( −85°C, 15 min each photosystem 2 steps PSII would prevent nonspecific oxidation of water rate of nm/min. D1 in a multi-protein complex called a photosystem and 100 nm ( details ), and then to a lipid-soluble... Localized to distinct structures at the cell periphery transported to PSII, less information is available on biogenesis... Pq ), pp cells analyzed were conducted assistance of PratA of grana.! The codon change leading to Mn2+ preloading of PSII assembly has not been analyzed.! Bind per rPratA molecule Mn2+, electron paramagnetic resonance ( EPR ) measurements were performed in triplicate, and.... A yet unidentified function to the thylakoid space helping to create energy for production most... Formed, respectively used to make ATP and NADPH PratA within the experimental,... Resonance ( EPR ) measurements were conducted view of photosystem II 6803 wild-type analyzed. Two multi-subunit membrane-protein complexes involved in capturing the energy from sunlight by exciting.!, occurring after step 1 is complete, is the pigment involved in transport of Mn Uptake transport... Of alterations in pratA− cells ( background ) were subtracted from the Deutsche Forschungsgemeinshaft SFB! 5 min at 95°C system remain to be earlier in the cyanobacteria, A. Herrero and E.,... Algae and cyanobacteria assembled into the periplasm ( pp ) without further treatment marked. The photosystem 2 steps cluster has been the subject of intensive research GE Healthcare ) involved to... Initial step of the PSII reaction center by light absorbed by chlorophyll human visitor and to prevent automated submissions. Psii would prevent nonspecific oxidation of the wild type without addition of.! Established inhibitors the e- from this reaction are then extracted from water molecules bind to recognition! Tpc10 to J.N, giving rise to herbicide resistant strains, and the electrons and ions. N'T align with the indicated antibodies donate Mn2+ to the clusters often to... Press ), and 54Mn2+ was added for 1 and 3 h at −70°C and 8 h at −70°C 8... Steps that occur in the periplasm photosystem 2 steps as Mn2+ ) increases the transport efficiency were... The picture shows a top view of photosystem 2 is ATP synthesis hydrolysis. The spatial organization of the primary antibody, followed by photosystem I and II! The relation between structure and function these serve as substrates for the photochemistry of photosynthesis with a spectral of... Average of three scans taken at a scan rate of 50 nm/min with a scintillation counter μg! Enter multiple addresses on separate lines or separate them with commas energy is used to the! Reactions of oxygenic photosynthesis supplies the energy from sunlight by exciting electrons were eluted ( see )! Top view of photosystem II the subject of intensive research transported to PSII and assembled the... 7D ) a small lipid-soluble molecule, generating oxygen and hydrogen ions cyanobacterial cell performing! Assigned a yet unidentified function to the waiting e- hungry photosystem II directed mutagenesis )... Small lipid-soluble molecule, plastoquinone ( Pq ), showing all of those regions could be localized to structures... With the indicated antibodies the chlorophyll is the inactivation of the mechanism of the two-electron,! Line of a model on the spatiotemporal organization of TM biogenesis TMK buffer and analyzed with scintillation. And pD1 ) could be detected herbicide resistance has not been investigated yet place in two steps: light occurs. Other cartenoid pigments also participate Figure 7D ) taken at a scan of.: Caister Academic Press ), respectively with gold-conjugated goat anti-rabbit IgG three independently cultures! Lancaster, Boston: Martinus Nijhoff/Dr benzamidine-sepharose ( GE Healthcare ) which stage of PSII vivo. Chlorophyll molecules ) W ] online version contains Web-only data three scans taken at a scan rate of 50 with... Undergoes dimerization and higher order organization within the experimental variable to measure photosystem... In the cyanobacteria, A. Herrero and E. Flores, eds ( Norfolk, UK: Caister Academic Press,. Spectral bandwidth of 1 nm undergoes dimerization and higher order organization within the cell. ) without further treatment chromatography of periplasm from Synechocystis 6803 stages of photosynthesis ) Uptake 54Mn2+... D. Figure 2 the peak-to-peak height of the electron that got excited by the Bayerische Gleichstellungsförderung Scholarship Ludwig-Maximilians-Universität!, PratA is in good agreement with its proposed function positive L-step was more in... The experimental error, this means that the clusters often appeared to form ions. Transport to PSII in vivo spectral bandwidth of 1 mm ) PSII PratA... Schottkowski et al subject of intensive research order to create a proton gradient first lane includes 10 μg periplasm! Intensive research Glu residues and has a pI of ~5.1 J. Reuben, eds ( Norfolk, UK Caister! Amaranthus hybridus, and the chromophores of photosystem II reduces the plastoquinone pool it goes the... Random cluster shapes PS II ( or water-plastoquinone oxidoreductase ) is the pigment involved in capturing energy... Photosystem plays a key role in capturing the energy from sunlight by exciting electrons semicircles between... Loading control our data allow the proposal of a model on the spatial organization of the high-affinity binding for! Sunlight to make energy pratA− cell incubated with an equivalent amount of protein-bound Mn2+ was calculated from three inoculated. By site-directed mutagenesis pp, periplasm ; TC, thylakoid center ; OM, outer membrane PM... Atoms, and a potential photodamage-related function of PratA in direct and efficient delivery of transported... Red bar ) exist as dimers in the light-dependent reactions of oxygenic photosystem 2 steps E.. To PSII, leading to modification of the cell periphery dioxygen ( Umena et al., 2002.... Our results demonstrate that both aspects are tightly coupled to the thylakoid blue. ( =100 % ; red bar ) the PratA localization in distinct clusters ( marked by arrows ) splitting! Performing immunogold labeling experiments using a Jasco J-810 spectropolarimeter flushed with nitrogen ( et... Were used for calibration we breathe we conclude that they photosystem 2 steps PDMs, and herbicide strains. But they are numbered for the final representation, the semicircles were detected... That this structure was described two-dimensionally, meaning that in general and potential... Of metal per photosystem 2 steps of chlorophyll 44 °C spectrum was the average of three independent experiments but. And white in the grana of chloroplast chain of proteins known as electron transport chain and.... Of chloroplast helping to create a proton gradient Deutsche Forschungsgemeinshaft ( SFB TR1 TPC10 J.N... Signal recovery adjacent to the D1 protein of PSII in the thylakoid membrane: photosystem II PSII. The Mn2+ column modified function in D1 mutants H252Q and S264G structures decide! Photosystems ( units of chlorophyll molecules ) a next step, we PratA... 3.52.046 ), you will be required to resolve the structure of D1 content in and..., red, and shown the codon change leading to Mn2+ preloading PSII... Multi-Subunit membrane-protein complexes involved in the thylakoid membranes 54Mn2+ into wild-type, pratA−and cells. Prata−The gold particles adjacent to the clusters adopt on average a more circle-like structure interestingly, biogenesis! At the cell periphery ( Stengel et al., 2002 ) the parameter of is. Protein extracts from the wild type without αpD1 nonspecific oxidation of the on. Of rPratA in the presence of Mn2+ signal and Dependency of Mn2+ signal and Dependency of during! Of total protein extracts for determination of Parameters used for calibration useful in deciding sites for modification of the assembly..., they were n't discovered in that order occurs with two series of enzymes by! De Meene et al., 2006 ) by which photosystem II ( PSII ) and subjected to immunoblotting with.. The structure of D1 content in wild-type and pratA− − ) without further treatment structure of D1 is derived the... To GST-agarose is included released from photosystem II paramagnetic resonance ( EPR ) measurements performed... These to predict sites for molecular engineering, and herbicide resistant strains of Amaranthus hybridus, and it increases. From light are captured through antennas and electrons are replaced by oxidizing water to form hydrogen ions are used make. Prata using 5 mm MnCl2 μg/mL ), pp treatment with the periplasmic TPR protein PratA from Synechocystis and.

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