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A Highly Resolved Oxygen-Evolving Photosystem II Preparation from Spinach Thylakoid Membranes
... PS II membranes have been instrumental in expanding our understanding of the structure and function of PS II. These are typically isolated by treatment of stacked thylakoid membranes with TX-100 followed by differential centrifugation (Berthold et al. 1981). These membranes exhibit high rates of oxygen evolution (typically, 400-600 μmoles O 2 mg chl hr), and are highly enriched in PS II membrane proteins and LHC II, being almost fully devoid of PS I, the cytochrome b 6 f complex, and CF 1 -CF o . ...
... Photosystem II membranes and PS I-LHC I-LHC II-enriched membranes were prepared as previously described (Berthold et al. 1981;Bell et al. 2015). LiDS-PAGE (Delepelaire and Chua 1979) was performed using a non-oxidizing gel system (Rabilloud et al. 1995). ...
... PS II membranes represent the population of PS II which is found in the core regions of grana stacks and these lack grana margins. These are very similar regardless of isolation by either detergent treatment (Berthold et al. 1981;Dunahay et al. 1984) or mechanical isolation (Veerman et al. 2007;Danielsson and Albertsson 2009). The PS II located in this region produce a large amount of oxygen and, since PS II is the principal source of ROS in the chloroplast, would be expected to be a primary site of ROS-induced oxidative damage. ...
Under aerobic conditions the production of Reactive Oxygen Species (ROS) by electron transport chains is unavoidable, and occurs in both autotrophic and heterotrophic organisms. In photosynthetic organisms both Photosystem II (PS II) and Photosystem I (PS I), in addition to the cytochrome b6/f complex, are demonstrated sources of ROS. All of these membrane protein complexes exhibit oxidative damage when isolated from field-grown plant material. An additional possible source of ROS in PS I and PS II is the distal, chlorophyll-containing light-harvesting array LHC II, which is present in both photosystems. These serve as possible sources of ¹O2 produced by the interaction of ³O2 with ³chl* produced by intersystem crossing. We have hypothesized that amino acid residues close to the sites of ROS generation will be more susceptible to oxidative modification than distant residues. In this study, we have identified oxidized amino acid residues in a subset of the spinach LHC II proteins (Lhcb1 and Lhcb2) that were associated with either PS II membranes (i.e. BBYs) or PS I-LHC I-LHC II membranes, both of which were isolated from field-grown spinach. We identified oxidatively modified residues by high-resolution tandem mass spectrometry. Interestingly, two different patterns of oxidative modification were evident for the Lhcb1 and Lhcb2 proteins from these different sources. In the LHC II associated with PS II membranes, oxidized residues were identified to be located on the stromal surface of Lhcb1 and, to a much lesser extent, Lhcb2. Relatively few oxidized residues were identified as buried in the hydrophobic core of these proteins. The LHC II associated with PS I-LHC I-LHC II membranes, however, exhibited fewer surface-oxidized residues but, rather a large number of oxidative modifications buried in the hydrophobic core regions of both Lhcb1 and Lhcb2, adjacent to the chlorophyll prosthetic groups. These results appear to indicate that ROS, specifically ¹O2, can modify the Lhcb proteins associated with both photosystems and that the LHC II associated with PS II membranes represent a different population from the LHC II associated with PS I-LHC I-LHC II membranes.
... PS II membranes have been instrumental in expanding our understanding of the structure and function of PS II. These are typically isolated by treatment of stacked thylakoid membranes with TX-100 followed by differential centrifugation (Berthold et al. 1981). These membranes exhibit high rates of oxygen evolution (generally, 400-600 µmoles O2•mg chl•hr), are highly enriched in PS II membrane proteins and LHC II, being almost fully devoid of PS I, the cytochrome b6f complex, and CF1-CFo. ...
... Photosystem II membranes and PS I-LHC I-LHC II-enriched membranes were prepared as previously described (Berthold et al. 1981;Bell et al. 2015). LiDS-PAGE (Delepelaire and Chua 1979) was performed using a non-oxidizing gel system (Rabilloud et al. 1995). ...
... /2021 in the core regions of grana stacks and these lack grana margins. These are very similar regardless of isolation by either detergent treatment (Berthold et al. 1981;Dunahay et al. 1984) or mechanical isolation (Veerman et al. 2007;Danielsson and Albertsson 2009). The PS II located in this region produce a large amount of oxygen and, since PS II is the principal source of ROS in the chloroplast, would be expected to be a primary site of ROS-induced oxidative damage. ...
Under aerobic conditions the production of Reactive Oxygen Species (ROS) by electron transport chains is unavoidable, and occurs in both autotrophic and heterotrophic organisms. In photosynthetic organisms both Photosystem II (PS II) and Photosystem I (PS I), in addition to the cytochrome b 6 /f complex, are demonstrated sources of ROS. All of these membrane protein complexes exhibit oxidative damage when isolated from field-grown plant material. An additional possible source of ROS in PS I and PS II is the distal, chlorophyll-containing light-harvesting array LHC II, which is present in both photosystems. These serve as possible sources of ¹ O 2 produced by the interaction of ³ O 2 with ³ chl* produced by intersystem crossing. We have hypothesized that amino acid residues close to the sites of ROS generation will be more susceptible to oxidative modification than distant residues. In this study, we have identified oxidized amino acid residues in a subset of the spinach LHC II proteins (Lhcb1 and Lhcb2) that were associated with either PS II membranes (i.e. BBYs) or PS I-LHC I-LHC II membranes, both of which were isolated from field-grown spinach. We identified oxidatively modified residues by high-resolution tandem mass spectrometry. Interestingly, two different patterns of oxidative modification were evident for the Lhcb1 and Lhcb2 proteins from these different sources. In the LHC II associated with PS II membranes, oxidized residues were identified to be located on the stromal surface of Lhcb1 and, to a much lesser extent, Lhcb2. Relatively few oxidized residues were identified as buried in the hydrophobic core of these proteins. The LHC II associated with PS I-LHC I-LHC II membranes, however, exhibited fewer surface-oxidized residues but, rather a large number of oxidative modifications buried in the hydrophobic core regions of both Lhcb1 and Lhcb2, adjacent to the chlorophyll prosthetic groups. These results appear to indicate that ROS, specifically ¹ O 2 , can modify the Lhcb proteins associated with both photosystems and that the LHC II associated with PS II membranes represent a different population from the LHC II associated with PS I-LHC I-LHC II membranes.
... The role of PSII as the initiator of photosynthesis has made it an object of intense study with much interest focused on the roles of the various complexes, specifically in identifying the RC as the complex that performs charge separation. Isolation of the RC was first reported by Nanba and Satoh [21] with several variations on their original method developed by Berthold et al. [22] and van Leeuwen et al. [23]. ...
... The procedure begins by following the method of Berthold, Babcock and Yocum [22] (colloquially referred to as the BBY prep as per the authors' names), whereby de-stemmed spinach leaves are first ground by a blender and then subjected to a series of pelletings and buffer washes. Thylakoid membrane fragments containing PSII complexes (of BBY particles) are extracted and can be stored long term in a -80 • C freezer. ...
... This procedure serves as a more detailed supplement to the procedure detailed in Appendix A of Anton Loukianov's thesis, which is based off the work in references [22,23,113]. It is included in this thesis in the interest of posterity and includes equipment and language particular to our lab at the time of writing that may not be universally relevant. ...
Many aspects of energy and charge transfer in oxygenic photosynthesis are still poorly understood at both the level of single pigments and multi-pigment complexes. At the single pigment level, chlorophylls and chlorophyll-like pigments (including bacteriochlorophyll, pheophytin, etc.) exhibit similar structure in the Q-band absorption region that has long been accepted to have correspondingly similar electronic structure. However, evidence has been mounting that suggests that despite the qualitative similarities between chlorophyll-like pigments they are in fact unique in their electronic structure. On larger length scales, key processes in photosynthesis such as stable charge separation, are accomplished via multiple pigments embedded within a protein matrix acting in concert. Understanding all the intermediate states that occur during charge separation in oxygenic photosynthesis, particularly in the photosystem II complex, is challenging due to the large degree of spectral overlap between chlorophyll and chlorophyll-like pigments. This has made it nearly impossible to disentangle individual pigment contributions from spectroscopic signatures and understand the structure-function relationship in this important system. This thesis presents my work that capitalizes on the advantages of two-dimensional electronic spectroscopy (2DES), particularly its ability to maintain simultaneous high temporal and spectral resolution, and incorporates further modifications to the technique that allow for studying oxygenic photosynthesis at the single pigment and multi-pigment regimes. By integrating polarization control among the multi-laser pulse experiment I was able to compare the underlying electronic and vibrational energy level structure of bacteriochlorophyll a and chlorophyll a to show how the structure of chlorophyll a deviates from the simple Gouterman model framework and lends support to the argument for vibronic models. Results of this work were supported by theoretical calculations performed by our collaborators from the group of Eitan Geva in the Chemistry Department at the University of Michigan and the group of Barry Dunietz at Kent State University. Futhermore, I used a multi-spectral 2DES technique, exciting across the Q$_y$ band and probing the higher energy Q$_x$ and carotenoid transitions in the photosystem II reaction center. The 2DES spectra reveal cross peaks between the highly congested Q$_y$ band and Q$_x$ and carotenoid transitions, providing insight into the contributions of the individual pigments to the absorption in the Q$_y$ region. Analysis of the kinetics of the 2DES data allows us to test the proposed two-pathway model of charge separation in the photosystem II reaction center. The results of these studies emphasize the importance of the feed-back between experiment and theory in building and refining an overall understanding of oxygenic photosynthesis. We anticipate that the information obtained in these studies will contribute to building the new models of the underlying energy structure of single pigments and excitonic interactions that lead to energy and charge transfer in the reaction center of oxygenic photosynthetic complexes.
... Photosynthetic membranes, thylakoids, enriched in photosystem II (PSII BBY) were isolated from Spinacia oleracea, purchased from the local market, according to the procedure described in (Berthold et al., 1981) omitting the second Tris-washing. The freshly isolated thylakoids were suspended in the Hepes II buffer (15 mM NaCl, 5 mM MgCl 2 , 20 mM Hepes, 400 mM sucrose), frozen in liquid nitrogen, and stored at À80 C. Before measurements, they were thawed out on ice and kept at 4 C in the darkness. ...
... Nevertheless, the highest rate of oxygen release recorded for these systems, which they achieve in their initial phase of operation, converted per gram of catalyst, that is, PSII supercomplex remains 2-3 orders of magnitude lower than that exhibited by isolated PSII systems under their native conditions (the highest initial activity for the hybrid systems ranged between 2 and 30 mmol O 2 g CAT À1 h À1 (Kato et al., 2012;Mersch et al., 2015;Müh & Zouni, 2020;Sokol et al., 2018;Tian et al., 2021) while thylakoids enriched in PSII (PSII BBY) usually show an O 2 yield $1950 mmol O 2 g cat À1 h À1 , 1 (Berthold et al., 1981;Ghanotakis & Yocum, 1986;Ikeuchi et al., 1985). In this work, an approach to constructing hybrid systems self-organized, based on wild natural PSII BBY has been demonstrated. ...
In this work, a new approach to construct self‐assembled hybrid systems based on natural PSII‐enriched thylakoid membranes (PSII BBY) is demonstrated. Superfine m‐WO 3 NPs (≈1–2 nm) are introduced into PSII BBY. Transmission electron microscopy (TEM) measurements showed that even the highest concentrations of NPs used did not degrade the PSII BBY membranes. Using atomic force microscopy (AFM), it is shown that the organization of PSII BBY depends strongly on the concentration of NPs applied. This proved that the superfine NPs can easily penetrate the thylakoid membrane and interact with its components. These changes are also related to the modified energy transfer between the external light‐harvesting antennas and the PSII reaction center, shown by absorption and fluorescence experiments. The biohybrid system shows stability at pH 6.5, the native operating environment of PSII, so a high rate of O 2 evolution is expected. In addition, the light‐induced water‐splitting process can be further stimulated by the direct interaction of superfine WO 3 NPs with the donor and acceptor sides of PSII. The water‐splitting activity and stability of this colloidal system are under investigation.
Research Highlights
The phenomenon of the self‐organization of a biohybrid system composed of thylakoid membranes enriched in photosystem II and superfine WO 3 nanoparticles is studied using AFM and TEM.
A strong dependence of the organization of PSII complexes within PSII BBY membranes on the concentration of NPs applied is observed.
This observation turns out to be crucial to understand the complexity of the mechanism of the action of WO 3 NPs on modifications of energy transfer from external antenna complexes to the PSII reaction center.
... Historically, the partial detergent solubilization method of Berthold, Babcock, and Yocum (Berthold, Babcock, & Yocum, 1981), the so-called BBY preparation, has been well suited for various types of spectroscopic, biochemical, structural investigations. The use of other additives, such as glycinebetaine in various buffers in the modified protocol, results in particularly high rates of PSII activity (Pi et al., 2019;Schiller & Dau, 2000). ...
... The PSII particle containing all LHC antenna complexes can be prepared by using market spinach (no baby spinach!) (Berthold et al., 1981). The following protocol is adapted from the aforementioned reference. ...
Protein cross-linking is the process of chemically joining two amino acids in a protein or protein complex by a covalent bond. When combined with mass spectrometry, it becomes one of the structural mass spectrometry techniques gaining in importance for deriving valuable three-dimensional structural information on proteins and protein complexes. This platform complements existing structural methods, such as NMR spectroscopy, X-ray crystallography, and cryo-EM. Photosynthetic pigment protein complexes serve as light-energy harvesting systems and perform photochemical conversion as part of the “early events” of photosynthesis. This chapter outlines how to prepare cross-linking pigment protein complex samples for LC-MS/MS analysis, including identification of the cross-linked species, network analysis in a protein complex, and structural modeling and justification.
... Thylakoids, i.e., photosynthetic membranes containing PSII complexes, were isolated from fresh leaves of spinach (Spinacia oleracea L.) according to the method described in [47] with minor modifications. The major steps of the preparation procedure are summarized in Fig. 3 (left side) and described in more detail in Appendix. ...
... Thylakoids were isolated from fresh leaves of spinach (Spinacia oleracea L.) according to the method described in [47] with minor modifications. Briefly, after removing stems from the spinach, leaves were homogenized in wash medium (pH=7.8) ...
Chlorophylls and carotenoids, key components of photosynthetic systems, are proposed for molecular communications at the nanoscale with the mechanism of resonance energy transfer. Both types of pigments are introduced focusing on their exceptional properties like energy harvesting, ultra-low energy consumption during transmissions, picoseconds delays, an ability for signal conversions, and bio-compatibility. The theoretical considerations are supported by two spectroscopic experiments on the photosystem II complex. The first experiment aims at the description of the photosystem II including calculation of the energy transfer efficiency between carotenoids and chlorophylls. In the second one, the photochemical efficiency is estimated, showing how effective the chlorophylls are in further energy processing. With the experimentally determined values, communication and energetic performance are analyzed, the probability of channel blockage is calculated and the energy consumption per bit is estimated. Treating carotenoid molecules as transmitters, chlorophylls as receivers, and the energy transfer between them as a way to encode information, a throughput up to 1 Gbit/s is achievable with a bit error rate below 10-3, average transmission delays about 20 ps, and energy consumption c.a. 2.0×10-18 J/bit. These results indicate a high potential of photosynthetic systems for nanocommunications and other related applications, due to suitable energetic characteristics in terms of energy harvesting abilities and low consumption for data transmissions.
... PSII-enriched thylakoid membranes were prepared from fresh market spinach by extraction with Triton X-100 as described previously (Berthold et al. 1981) with modifications (Ford and Evans 1983;Franzén et al. 1985). The final preparation was stored in liquid nitrogen in buffer containing 20 mM MES-NaOH, pH 6.3, 0.4 M sucrose, and 15 mM NaCl. ...
Calcium and chloride are activators of oxygen evolution in photosystem II (PSII), the light-absorbing water oxidase of higher plants, algae, and cyanobacteria. Calcium is an essential part of the catalytic Mn4CaO5 cluster that carries out water oxidation and chloride has two nearby binding sites, one of which is associated with a major water channel. The co-activation of oxygen evolution by the two ions is examined in higher plant PSII lacking the extrinsic PsbP and PsbQ subunits using a bisubstrate enzyme kinetics approach. Analysis of three different preparations at pH 6.3 indicates that the Michaelis constant, KM, for each ion is less than the dissociation constant, KS, and that the affinity of PSII for Ca²⁺ is about ten-fold greater than for Cl⁻, in agreement with previous studies. Results are consistent with a sequential binding model in which either ion can bind first and each promotes the activation by the second ion. At pH 5.5, similar results are found, except with a higher affinity for Cl⁻ and lower affinity for Ca²⁺. Observation of the slow-decaying Tyr Z radical, YZ•, at 77 K and the coupled S2YZ• radical at 10 K, which are both associated with Ca²⁺ depletion, shows that Cl⁻ is necessary for their observation. Given the order of electron and proton transfer events, this indicates that chloride is required to reach the S3 state preceding Ca²⁺ loss and possibly for stabilization of YZ• after it forms. Interdependence through hydrogen bonding is considered in the context of the water environment that intervenes between Cl⁻ at the Cl−1 site and the Ca²⁺/Tyr Z region.
... PSII and other photosynthetic complexes are generally conserved from cyanobacteria to algae to plants, and researchers in this field use all three groups as model species. For example, many biophysical studies of PSII have used membrane preparations from market spinach [18][19][20][21]. The cyanobacterium Synechocystis sp. ...
Oxygenic photosynthetic organisms use Photosystem II (PSII) to oxidize water and reduce plastoquinone. Here, we review the mechanisms by which PSII is assembled and turned over in the model green alga Chlamydomonas reinhardtii. This species has been used to make key discoveries in PSII research due to its metabolic flexibility and amenability to genetic approaches. PSII subunits originate from both nuclear and chloroplastic gene products in Chlamydomonas. Nuclear-encoded PSII subunits are transported into the chloroplast and chloroplast-encoded PSII subunits are translated by a coordinated mechanism. Active PSII dimers are built from discrete reaction center complexes in a process facilitated by assembly factors. The phosphorylation of core subunits affects supercomplex formation and localization within the thylakoid network. Proteolysis primarily targets the D1 subunit, which when replaced, allows PSII to be reactivated and completes a repair cycle. While PSII has been extensively studied using Chlamydomonas as a model species, important questions remain about its assembly and repair which are presented here.
... Thylakoid membranes were solubilized with 0.6% dodecyl-d-maltoside (DDM) at a final chlorophyll concentration of 0.5 mg/ml. The sucrose density ultracentrifugation was used to obtain PSII core particles as described in (47)(48)(49)(50). The purification of PSII RCs from PSII core particles proceeds as follows: The PSII core particles were diluted in BTS200 buffer [20 mM bis-tris (pH 6.5), 20 mM MgCl 2 , 5 mM CaCl 2 , 10 mM MgSO 4 , 0.03% DDM, and 0.2 M sucrose] to a chlorophyll concentration of 0.15 mg/ml and solubilized with an equal volume of 10% Triton X-100 in BTS200 buffer for 20 min; then, the material was loaded on a HiTrap Q Sepharose HP 1-ml column (GE Healthcare) and washed with a BTS buffer until the eluate became colorless. ...
Photosystem II (PSII) reaction center (RC) is a unique complex that is capable of efficiently separating electronic charges across the membrane. The primary energy-and charge-transfer (CT) processes occur on comparable ul-trafast timescales, which makes it extremely challenging to understand the fundamental mechanism responsible for the near-unity quantum efficiency of the transfer. Here, we elucidate the role of quantum coherences in the ultrafast energy and CT in the PSII RC by performing two-dimensional (2D) electronic spectroscopy at the cryo-genic temperature of 20 kelvin, which captures the distinct underlying quantum coherences. Specifically, we uncover the electronic and vibrational coherences along with their lifetimes during the primary ultrafast processes of energy and CT. We construct an excitonic model that provides evidence for coherent energy and CT at low temperature in the 2D electronic spectra. The principles could provide valuable guidelines for creating artificial photosystems with exploitation of system-bath coupling and control of coherences to optimize the photon conversion efficiency to specific functions.
... PSII membrane particles were prepared from spinach leaves as described previously (Schiller and Dau 2000), based on a protocol developed by Berthold, Babcock, and Yocum (Berthold et al. 1981). The oxygen evolution activity (> 1100 μmol O 2 per mg chlorophyll and hour) was measured with a Clarke-type electrode at 28 °C using 10 μg of Chl in 1 M betaine, 25 mM MES, 15 mM NaCl, 5 mM CaCl 2 (pH 6.2) buffer, 1 mM K 3 [Fe(CN) 6 ] and 0.25 mM DCBQ (2,6 dichloro-1,4-benzoquinone), of which the latter two ingredients serve as artificial electron acceptors. ...
In oxygen-evolving photosystem II (PSII), the multi-phasic electron transfer from a redox-active tyrosine residue (TyrZ) to a chlorophyll cation radical (P680 ⁺ ) precedes the water-oxidation chemistry of the S-state cycle of the Mn 4 Ca cluster. Here we investigate these early events, observable within about 10 ns to 10 ms after laser-flash excitation, by time-resolved single-frequency infrared (IR) spectroscopy in the spectral range of 1310–1890 cm ⁻¹ for oxygen-evolving PSII membrane particles from spinach. Comparing the IR difference spectra at 80 ns, 500 ns, and 10 µs allowed for the identification of quinone, P680 and TyrZ contributions. A broad electronic absorption band assignable P680 ⁺ was used to trace largely specifically the P680 ⁺ reduction kinetics. The experimental time resolution was taken into account in least-square fits of P680 ⁺ transients with a sum of four exponentials, revealing two nanosecond phases (30–46 ns and 690–1110 ns) and two microsecond phases (4.5–8.3 µs and 42 µs), which mostly exhibit a clear S-state dependence, in agreement with results obtained by other methods. Our investigation paves the road for further insight in the early events associated with TyrZ oxidation and their role in the preparing the PSII donor side for the subsequent water oxidation chemistry.
... Sample preparation: The preparation of PSIImf was described in detail earlier [18]. Briefly, PSIImf were isolated from spinach (Spinacea oleracea) following the procedure described by Berthold et al. [27]. PSIImf with modifications according to Völker et al. [28]. ...
A detailed comprehension of protein function requires information on the spatial structure of the protein, which is often gathered from X-ray crystallography. However, conformational dynamics often also plays an important functional role in proteins and can be directly investigated by complementary quasielastic neutron scattering. A classic example for dynamics-function correlations is Photosystem II, which is a multimeric pigment-protein complex responsible for catalyzing the light-induced photosynthetic water splitting into protons and oxygen. Several functional subprocesses of photosynthetic electron transfer and water splitting are strongly dependent on temperature and hydration, two factors also known to affect protein dynamics. Photosystem II is often investigated in the form of membrane fragments, where the protein complex remains embedded into its native lipid environment. However, experiments on protein function are often carried out in solution state, while direct investigations of molecular dynamics by quasielastic neutron scattering are mainly performed using specifically hydrated membrane fragments only. The present study provides the first quasielas-tic neutron scattering investigation of the molecular dynamics of Photosystem II membrane fragments (PSIImf) in solution over a wide temperature range from 50 to 300 K. At physiological temperatures above the melting point of water, we observed that the dynamics of PSIImf are significantly activated, leading to larger atomic mean square displacement values compared to those of specifically hydrated membrane stacks. The QENS data can be described by two dynamical components: a fast one, most probably corresponding to methyl group rotation; and a slower one, representing localized conformational dynamics. The latter component could be fitted by a jump-diffusion model at 300 K. The dynamics observed characterize the level of flexibility necessary for the proper PS II functionality under physiological conditions. In contrast, we observe a severe restriction of molecular dynamics upon freezing of the solvent below~276 K. We associate this unexpected suppression of dynamics with a substantial aggregation of PSIImf caused by ice formation.
... Sample preparation PSII membrane particles were prepared from spinach leaves as described previously (Schiller and Dau 2000), based on a protocol developed by Berthold, Babcock, and Yocum (Berthold et al. 1981). The oxygen evolution activity (>1,100 μmol O2 per mg chlorophyll and hour) was measured with a Clarke-type electrode at 28 °C using 10 μg of Chl in 1 M Betaine, 25 mM MES, 15 mM NaCl ,5 mM CaCl2 (pH 6.2) buffer, 1 mM K3[Fe (CN)6] and 0.25 mM DCBQ (2,6 Dichloro-1,4-benzoquinone), of which the latter two ingredients serve as artificial electron acceptors. ...
In oxygen-evolving photosystem II (PSII), the multi-phasic electron transfer from a redox-active tyrosine residue (TyrZ) to a chlorophyll cation radical (P680 ⁺ ) precedes the water-oxidation chemistry of the S-state cycle of the Mn 4 Ca cluster. Here we investigate these early events, observable within about 10 nanoseconds to 10 microseconds after laser-flash excitation, by time-resolved single-frequency infrared (IR) spectroscopy in the spectral range of 1310–1890 cm − 1 for oxygen-evolving PSII membrane particles from spinach. Comparing the IR difference spectra at 80 ns, 500 ns, and 10 µs allowed for the identification of quinone, P680 and TyrZ contributions. A broad electronic absorption band assignable P680 ⁺ was used to trace specifically the P680 ⁺ reduction kinetics. The experimental time resolution was taken into account in least-square fits of P680 ⁺ transients with a sum of four exponentials, revealing two nanosecond phases (30–46 ns and 690–1110 ns) and two microsecond phases (4.5–8.3 µs and 42 µs), which mostly exhibit a clear S-state dependence, in agreement with results obtained by other methods. Our investigation paves the road for further insight in the early events associated with TyrZ oxidation and their role in the preparing the PSII donor side for the subsequent water oxidation chemistry.
... The oxygen-evolving PSII-containing membranes were isolated from the leaves of the greenhouse spinach (Spinacia oleracea L.), according to [35], with a little modification as in [36]. These PSII-containing membranes contain about 200 chlorophyll molecules per RC (per one molecule of photoactive pheophytin) [37] and, when illuminated with red light (λ ≥ 650 nm) of saturating intensity, they release oxygen in the presence of two artificial electron acceptors (0.1 mM 2,5-dichloro-p-benzoquinone and 1 mM K 3 Fe(CN) 6 ) at a rate of 450-500 µmol O 2 (mg Chl) −1 h −1 . ...
Modern agricultural cultivation relies heavily on genetically modified plants that survive after exposure to herbicides that kill weeds. Despite this biotechnology, there is a growing need for new sustainable, environmentally friendly, and biodegradable herbicides. We developed a novel [CuL2]Br2 complex (L = bis{4H-1,3,5-triazino[2,1-b]benzothiazole-2-amine,4-(2-imidazole) that is active on PSII by inhibiting photosynthetic oxygen evolution on the micromolar level. [CuL2]Br2 reduces the FV of PSII fluorescence. Artificial electron donors do not rescind the effect of [CuL2]Br2. The inhibitory mechanism of [CuL2]Br2 remains unclear. To explore this mechanism, we investigated the effect of [CuL2]Br2 in the presence/absence of the well-studied inhibitor DCMU on PSII-containing membranes by OJIP Chl fluorescence transient measurements. [CuL2]Br2 has two effects on Chl fluorescence transients: (1) a substantial decrease of the Chl fluorescence intensity throughout the entire kinetics, and (2) an auxiliary “diuron-like” effect. The initial decrease dominates and is observed both with and without DCMU. In contrast, the “diuron-like” effect is small and is observed only without DCMU. We propose that [CuL2]Br2 has two binding sites for PSII with different affinities. At the high-affinity site, [CuL2]Br2 produces effects similar to PSII reaction center inhibition, while at the low-affinity site, [CuL2]Br2 produces effects identical to those of DCMU. These results are compared with other PSII-specific classes of herbicides.
... When used, Arabidopsis seedlings immersed in water were incubated in the dark at RT in presence of 400 μM radulanin A, 10 μM DCMU or 100 μM bromoxynil for the indicated amount of time before the measurements. PSII-enriched membrane fragments from spinach, prepared according to Berthold et al. 16 were diluted to a final chlorophyll concentration of 10 μg mL −1 in ice-cold resuspension buffer (50 mM MES-NaOH pH 6.5, 5 mM CaCl 2 , 10 mM MgCl 2 , 1.2 M betaine, 20% w/v glycerol). The samples were pre-illuminated for 10 s with white light (neon bulbs; 10 micromol photons s-1 m-1) and then kept in the dark on ice for at least 1 h before measurement. ...
BACKGROUND
Radulanin A is a natural 2,5‐dihydrobenzoxepin synthesized by several liverworts of the Radula genus. Breakthroughs in the total synthesis of radulanin A paved the way for the discovery of its phytotoxic activity. Nevertheless, its mode‐of‐action (MoA) has remained unknown so far and thus was investigated, in Arabidopsis thaliana.
RESULTS
Radulanin A phytotoxicity was associated with cell death and partially depended on light exposure. Photosynthesis measurements based on chlorophyll‐a fluorescence evidenced that radulanin A and a Radula chromene inhibited photosynthetic electron transport with IC50 of 95 and 100 μm, respectively. We established a strong correlation between inhibition of photosynthesis and phytotoxicity for a range of radulanin A analogs. Based on these data, we also determined that radulanin A phytotoxicity was abolished when the hydroxyl group was modified, and was modulated by the presence of the heterocycle and its aliphatic chain. Thermoluminescence studies highlighted that radulanin A targeted the QB site of the Photosystem II (PSII) with a similar MoA as 3‐(3,4‐dichloropheny)‐1,1‐dimethylurea (DCMU).
CONCLUSION
We establish that radulanin A targets PSII, expanding QB sites inhibitors to bibenzyl compounds. The identification of an easy‐to‐synthesize analog of radulanin A with similar MoA and efficiency might be useful for future herbicide development. © 2023 Society of Chemical Industry.
... The oxygen-evolving membranes of PSII (BBY) were prepared from the pea (Pisum sativum L.) chloroplasts using Triton X-100 according to [8]. The oxygen-evolving complex (OEC) and the RC preparations were isolated from the membranes using our protocol [9]. ...
Interaction of water-soluble and immobilized (on BrCN-activated agarose) manganese-stabilizing
protein PsbO with Mn2+ and Mn3+ cations and with preparations of D1/D2/cyt b559 reaction center (RC) of
photosystem II was studied. By native electrophoresis, the formation of dimeric and aggregated forms of
PsbO protein were found in the presence of Mn2+, Mg2+, or Fe2+ ions. The dimerization of PsbO occurred
after ultraviolet irradiation of the protein preparation. The presence of protein-bound Mn3+ cations increased
the electrostatic interaction of the immobilized PsbO with the RC. This was evidenced to by higher amounts
of CaCl2 that were required for dissociation of the PsbO–RC complex. It was first demonstrated that the protein
exhibited superoxide dismutase (SOD) activity after an electrophoresis in PAAG upon incubation of the
gel in an Mn2+-containing solution. Tetrazolium-reductase activity was also ascertained in the protein after
its electrophoresis in a mixture with preparations of the oxygen-evolving complex (OEC). It is suggested that the
protein interaction with Mn ions and superoxide radicals, as well as short-term UV irradiation, reduces tyrosine
and a disulfide bond in the PsbO protein. This yields tyrosil radical and SH-groups participating in redox reactions
with ETC components. The interactions of PsbO with Mn cations and UV light, taking place in the chloroplast
thylakoids, may regulate the protein binding to RC, modify structural organization of the protein, and
promote its participation in alternative pathways of electron transport under the influence of stress factors. The
hypothetical scheme of interaction of the immobilized PsbO protein with Mn ions and RC is discussed.
... The D1-D2-Cytb559 reaction center (PSII RC) complexes were extracted from spinach following a method based on the work of Berthold et al. (56) and van Leeuwen et al. (57). We verified purity of the sample and removal of CP47 and CP43 via a ratio of 1.2 for the linear absorption at 417:435 nm as discussed by Eijckelhoff et al. (58). ...
The photosystem II reaction center (PSII RC) performs the primary energy conversion steps of oxygenic photosynthesis. While the PSII RC has been studied extensively, the similar time scales of energy transfer and charge separation and the severely overlapping pigment transitions in the Qy region have led to multiple models of its charge separation mechanism and excitonic structure. Here, we combine two-dimensional electronic spectroscopy (2DES) with a continuum probe and two-dimensional electronic vibrational spectroscopy (2DEV) to study the cyt b559-D1D2 PSII RC at 77 K. This multispectral combination correlates the overlapping Qy excitons with distinct anion and pigment-specific Qx and mid-infrared transitions to resolve the charge separation mechanism and excitonic structure. Through extensive simultaneous analysis of the multispectral 2D data, we find that charge separation proceeds on multiple time scales from a delocalized excited state via a single pathway in which PheoD1 is the primary electron acceptor, while ChlD1 and PD1 act in concert as the primary electron donor.
... PS I electrons were transferred through plastoquinones by PS II, which produced ATP and NADPH simultaneously [28]. PsbS was first identified in spinach [29], and the amino acid sequence showed that this protein belonged to the light-harvesting complex (LHC) protein superfamily [30,31]. Different from other LHC proteins, with three transmembrane helixes (TMN), four TMNs have been observed in PsbS [32]. ...
Sugar metabolism influences the quality of sweet corn (Zea mays var. saccharate Sturt) kernels, which is a major goal for maize breeding. In this study, the genome-wide transcriptomes from two supersweet corn cultivars (cv. Xuetian 7401 and Zhetian 11) with a nearly two-fold difference in kernel sugar content were carried out to explore the genes related to kernel sugar metabolism. In total, 45,748 differentially expressed genes (DEGs) in kernels and 596 DEGs in leaves were identified. PsbS, photosynthetic system II subunit S, showed two isoforms with different expression levels in leaf tissue between two cultivars, indicating that this gene might influence sugar accumulation in the kernel. On the other hand, hexokinases and beta-glucosidase genes involved in glycolysis, starch and sucrose metabolism were found in developing kernels with a genome-wide transcriptome analysis of developing kernels, which might contribute to the overaccumulation of water-soluble polysaccharides and an increase in the sweetness in the kernels of Xuetian 7401. These results indicated that kernel sugar accumulation in sweet corn might be influenced by both photosynthesis efficiency and the sugar metabolism rate. Our study supplied a new insight for breeding new cultivars with high sugar content and laid the foundation for exploring the regulatory mechanisms of kernel sugar content in corn.
... The PSII core complex sample was prepared by two-step detergent treatment. First, the PSII membrane fragments was isolated from fresh spinach leaves according to a reported procedure [20]. Second, the PSII core complex was collected by treating PSII membrane fragments with 0.4% (w/v) n-octyl-β-D-thioglucoside (OTG) solution [21]. ...
For natural oxygenic photosynthesis, there is a consensus that H2O is oxidized to molecular oxygen by photosystem II (PSII) in the grana, while CO2 assimilation takes place, long after oxygen evolution, in light-independent reactions, for example, through the Calvin-Benson Cycle in the stroma. Here, we report, for the first time, light-driven CO2 assimilation by the PSII core complex, where the formation of methanol, along with the oxygen evolution, is validated by in-situ mass spectrometry, gas chromatography and isotopic labeling experiments. Such an unusual CO2 assimilation is likely to be a simultaneous event along with the usual electron transfer occurring in normal light–independent assimilation. This discovery is extraordinary and is of great significance as it may substantially modify our understanding of the mechanism of photosynthesis.
... All procedures for sample preparation were performed in the dark to minimize exposure to light as much as possible. We prepared PSII-enriched membranes according to the previous literature (66,67) with some modifications as described previously (45). We isolated PSII-CC according to the previous literature (68) with some modifications as follows. ...
The photosystem II core complex (PSII-CC) is the smallest subunit of the oxygenic photosynthetic apparatus that contains core antennas and a reaction center, which together allow for rapid energy transfer and charge separation, ultimately leading to efficient solar energy conversion. However, there is a lack of consensus on the interplay between the energy transfer and charge separation dynamics of the core complex. Here, we report the application of two-dimensional electronic-vibrational (2DEV) spectroscopy to the spinach PSII-CC at 77 K. The simultaneous temporal and spectral resolution afforded by 2DEV spectroscopy facilitates the separation and direct assignment of coexisting dynamical processes. Our results show that the dominant dynamics of the PSII-CC are distinct in different excitation energy regions. By separating the excitation regions, we are able to distinguish the intraprotein dynamics and interprotein energy transfer. Additionally, with the improved resolution, we are able to identify the key pigments involved in the pathways, allowing for a direct connection between dynamical and structural information. Specifically, we show that C505 in CP43 and the peripheral chlorophyll ChlzD1 in the reaction center are most likely responsible for energy transfer from CP43 to the reaction center.
... For detailed effects of various pre-treatments, see . Shen et al. (1990) compared polypeptides of thylakoids and those of PSII particles (Berthold et al. 1981) and found that, after the dark-chilling treatment of leaves, two (PsbP and PsbQ) out of the three extrinsic proteins (PsbO) were dissociated from the inner surface of the thylakoid membrane. These proteins were not degraded but remained in the thylakoid lumen. ...
Light is indispensable for plants to photosynthesize organic matters. Almost all the organisms including animals on our planet eventually rely on this plant function for their energy as well as the materials forming their bodies. Paradoxically, light often damages the photosynthetic apparatus. This phenomenon is called photoinhibition and has been attracting attention of many photosynthesis researchers. Although the term photoinhibition had been used almost synonymously to refer to photoinhibition of photosystem II (PSII), it was recently shown that PSI is susceptible to fluctuating light. First, we compare two PSII photoinhibition hypotheses: the Mn/(Two-step) hypothesis and Excess-Y(NO) hypothesis. The former claims that the oxygen-evolving complex (OEC) is primarily damaged, while the latter claims excess excitation energy in PSII directly damages D1 function. Both can be induced in the laboratory and may parallelly occur in the same leaf. Because OEC is damaged by ultraviolet (UV) or blue light, UV screening substances in the leaf epidermis plays a crucial role. It is also indicated that the rate of PSII repair in PSII damaged by the Mn/(Two-step) mechanism is much slower than that by the Excess-Y(NO) mechanism. It appears then plants should avoid PSII photoinhibition by the Mn/(Two-step) hypothesis. Photoinhibition of photosystem I (PSI) in cucumber, a model chilling sensitive plant, and that in the mutant of PROTON GRADIENT 5 are compared. The effects of fluctuating light, which naturally occurs in the field, on PSI photoinhibition are also discussed. The PSI photoinhibition in these three cases can be explained by similar scenarios. When reduced P700 donates electrons to O2, oxidative damage is induced. The mechanisms that protect PSI from photoinhibition all contribute to oxidation of P700 to P700+, a safe quencher. When a leaf is suddenly exposed to strong light, spillover of excitation energy from PSII to PSI protects both PSII and PSI from photoinhibition. In all these situations, far-red (FR) light plays essential roles in PSI protection. As FR light not only protects PSI but also enhances photosynthesis, especially in low light phases in the fluctuating light, the roles of FR light in photosynthesis should be fully examined. Other ecologically important problems that should be solved in the future are also pointed out.KeywordsExcess-Y(NO) mechanismFluctuating lightMn/(Two-step) hypothesisNon-photochemical quenchingPhotooxidative stressRepairSpillover
... The D1-D2-Cytb559 reaction center (PSII-RC) complexes were extracted from spinach following a method based on the work of Berthold et al. (56) and van Leeuwen et al. (57). We verified purity of the sample and removal of CP47 and CP43 via a ratio of 1.2 for the linear absorption at 417:435 nm as discussed by Eijckelhoff et al. (58). ...
The photosystem II reaction center (PSII-RC) performs the primary energy conversion steps of oxygenic photosynthesis. While the PSII-RC has been studied extensively, the similar timescales of energy transfer and charge separation, and the severely overlapping pigment transitions in the Qy region have led to multiple models of its charge separation mechanism and excitonic structure. Here we combine two-dimensional electronic spectroscopy (2DES) with a continuum probe and two-dimensional electronic vibrational spectroscopy (2DEV) to study the cyt b559-D1D2 PSII-RC at 77K. This multispectral combination correlates the overlapping Qy excitons with distinct anion and pigment-specific Qx and mid-IR transitions to resolve the charge separation mechanism and excitonic structure. Through extensive simultaneous analysis of the multispectral 2D data we find that charge separation proceeds on multiple timescales from a highly delocalized excited state via a single pathway in which PheoD1 is the primary electron acceptor, while ChlD1 and PD1 act in concert as the primary electron donor.
... Photochemically active thylakoid membrane preparations were prepared from leaves of pea plants (Pisum sativum) grown for 2-3 weeks by the method described in [38], with modifications, as in [39]. ...
Photosystem II (PSII) is the unique pigment–protein complex that is capable of evolving molecular oxygen using solar energy. The activity of PSII determines the overall productivity of all oxygenic photosynthetic organisms. It is well known that the absence of HCO3− induces a drop in the activity of PSII. However, it is not yet clear what type of photochemical reaction, single turn-over or multiple turn-over, HCO3− is involved in. Kinetic parameters of this (these) involvement(s) are almost unexplored now. This work addresses these issues. Using the JIP test, being the perspective noninvasive method for measuring PSII activity in plants, this paper describes how HCO3− deficiency affects the electron transfer on the oxidizing as well as the reducing sides of PSII in thylakoids and in PSII preparations from the leaves of pea plants. HCO3− was found to be simultaneously involved both in single turn-over and in multiple turn-over events (“dynamical processes”). Moreover, the involvement of HCO3− in dynamical photochemical processes was revealed to be associated with both sides of PSII, being the rate limiting on the reducing side, which follows from obtained kinetic parameters. The involvement of HCO3− in dynamical processes as the constant exchangeable ligand is discussed for both the electron donor and acceptor sides of PSII.
... The preparation of PSII monomeric and dimeric core complexes was as previously described [29], adapted with small modifications for the extraction of seedlings [51]. Isolated PSII particles were solubilized with DDM using the same chlorophyll to detergent ratio of 2:3 (1 mg Chl in 0.3 mL 0.5% DDM). ...
The photosystem II (PSII) reaction centre is the critical supramolecular pigment–protein complex in the chloroplast which catalyses the light-induced transfer of electrons from water to plastoquinone. Structural studies have demonstrated the existence of an oligomeric PSII. We carried out radiation inactivation target analysis (RTA), together with sucrose gradient ultracentrifugation (SGU) of PSII, to study the functional size of PSII in diverse plant species under physiological and stress conditions. Two PSII populations, made of dimeric and monomeric core particles, were revealed in Pisum sativum, Spinacea oleracea, Phaseulus vulgaris, Medicago sativa, Zea mais and Triticum durum. However, this core pattern was not ubiquitous in the higher plants since we found one monomeric core population in Vicia faba and a dimeric core in the Triticum durum yellow-green strain, respectively. The PSII functional sizes measured in the plant seedlings in vivo, as a decay of the maximum quantum yield of PSII for primary photochemistry, were in the range of 75–101 ± 18 kDa, 2 to 3 times lower than those determined in vitro. Two abiotic stresses, heat and drought, imposed individually on Pisum sativum, increased the content of the dimeric core in SGU and the minimum functional size determined by RTA in vivo. These data suggest that PSII can also function as a monomer in vivo, while under heat and drought stress conditions, the dimeric PSII structure is predominant.
... The oxygen-evolving PSII-enriched thylakoid membrane fragments (PSII membranes) were isolated from the leaves of the greenhouse spinach (Spinacia oleracea L.), as described earlier [52], with a little modification as in [53]. A similar PSII membrane ratio of RC (determined from pheophytin photoreduction)-to-chlorophyll molecules is about 1/200-220 was used [54]. ...
The effects of the novel [CuL2]Br2 complex (L = bis{4H-1,3,5-triazino [2,1-b]benzothiazole-2-amine,4-(2-imidazole)}copper(II) bromide complex) on the photosystem II (PSII) activity of PSII membranes isolated from spinach were studied. The absence of photosynthetic oxygen evolution by PSII membranes without artificial electron acceptors, but in the presence of [CuL2]Br2, has shown that it is not able to act as a PSII electron acceptor. In the presence of artificial electron acceptors, [CuL2]Br2 inhibits photosynthetic oxygen evolution. [CuL2]Br2 also suppresses the photoinduced changes of the PSII chlorophyll fluorescence yield (FV) related to the photoreduction of the primary quinone electron acceptor, QA. The inhibition of both characteristic PSII reactions depends on [CuL2]Br2 concentration. At all studied concentrations of [CuL2]Br2, the decrease in the FM level occurs exclusively due to a decrease in Fv. [CuL2]Br2 causes neither changes in the F0 level nor the retardation of the photoinduced rise in FM, which characterizes the efficiency of the electron supply from the donor-side components to QA through the PSII reaction center (RC). Artificial electron donors (sodium ascorbate, DPC, Mn2+) do not cancel the inhibitory effect of [CuL2]Br2. The dependences of the inhibitory efficiency of the studied reactions of PSII on [CuL2]Br2 complex concentration practically coincide. The inhibition constant Ki is about 16 µM, and logKi is 4.8. As [CuL2]Br2 does not change the aromatic amino acids’ intrinsic fluorescence of the PSII protein components, it can be proposed that [CuL2]Br2 has no significant effect on the native state of PSII proteins. The results obtained in the present study are compared to the literature data concerning the inhibitory effects of PSII Cu(II) aqua ions and Cu(II)-organic complexes.
... Thylakoid membrane preparation and solubilisation were performed as described previously, with modifications (Berthold et al., 1981). Here, c. 10 g of gametophore was blended in 50 ml of buffer B1 (0.4 M NaCl, 2 mM MgCl 2 , 20 mM tricine KOH pH 7.8, 0.2 mM benzamidine, 1 mM hexanoic acid) and passed through a 30-μM filter. ...
The nucleotides guanosine tetraphosphate and pentaphosphate (or (p)ppGpp) are implicated in the regulation of chloroplast function in plants. (p)ppGpp signalling is best understood in the model vascular plant Arabidopsis thaliana in which it acts to regulate plastid gene expression to influence photosynthesis, plant development and immunity. However, little information is known about the conservation or diversity of (p)ppGpp signalling in other land plants.
We studied the function of ppGpp in the moss Physcomitrium (previously Physcomitrella) patens using an inducible system for triggering ppGpp accumulation. We used this approach to investigate the effects of ppGpp on chloroplast function, photosynthesis and growth.
We demonstrate that ppGpp accumulation causes a dramatic drop in photosynthetic capacity by inhibiting chloroplast gene expression. This was accompanied by the unexpected reorganisation of the thylakoid system into super grana. Surprisingly, these changes did not affect gametophore growth, suggesting that bryophytes and vascular plants may have different tolerances to defects in photosynthesis.
Our findings point to the existence of both highly conserved and more specific targets of (p)ppGpp signalling in the land plants that may reflect different growth strategies.
... PSII membrane preparations (BBY particles) were used in the study (Berthold et al., 1981;Völker et al., 1985). The experiment was carried out in the medium containing 50 mM MES-NaOH (pH6.1), ...
The photochemical activity of photosystem II (PSII) illuminated at different temperatures (20-55℃) has been studied. The photochemical activity of PSII preparations incubated at different temperatures in the dark decreased sharply in the range of 40-55°C, and was relatively stable at temperatures 20-40°C. The photochemical activity of PSII decreased with relatively monotonous kinetics in preparations processed at different temperatures and exposed to light, but in the end, it was higher than the activity observed in samples incubated in the dark. The effects of glycerin and sucrose on the PSII inhibition due to the exposure to different temperatures in darkness, and after temperature-light treatment were studied. The photochemical activity of PSII was measured after the incubation of the samples in a solution containing 50% glycerol (volume) or 1 M sucrose for 5 minutes in the dark at a certain temperature or under high light intensity at the same temperature. The photochemical activity of the PSII complex was found to be partially preserved in samples incubated in a glycerin-containing solution, at high temperatures, in both dark and light. The addition of sucrose to the solution resulted in a higher degree of protection of the photochemical activity of PSII.
... All procedures for sample preparation were performed in the dark to minimize exposure to light as much as possible. We first isolated PSIIenriched membranes according to the previous literature with some modifications as follows 60,61 . We obtained spinach leaves (Spinacia oleracea) from a local store and kept in the dark overnight at 4°C. ...
Photosystem II is crucial for life on Earth as it provides oxygen as a result of photoinduced electron transfer and water splitting reactions. The excited state dynamics of the photosystem II-reaction center (PSII-RC) has been a matter of vivid debate because the absorption spectra of the embedded chromophores significantly overlap and hence it is extremely difficult to distinguish transients. Here, we report the two-dimensional electronic-vibrational spectroscopic study of the PSII-RC. The simultaneous resolution along both the visible excitation and infrared detection axis is crucial in allowing for the character of the excitonic states and interplay between them to be clearly distinguished. In particular, this work demonstrates that the mixed exciton-charge transfer state, previously proposed to be responsible for the far-red light operation of photosynthesis, is characterized by the Chl D1 ⁺ Phe radical pair and can be directly prepared upon photoexcitation. Further, we find that the initial electron acceptor in the PSII-RC is Phe, rather than P D1 , regardless of excitation wavelength.
... The author's generation of PS2 researchers were confronted by a series of experimental results that were, in some instances, puzzling. For example, in the early 1980's, the simplicity with which pure, active PS2 preparations could be obtained (Berthold et al. 1981;Kuwabara and Murata 1982) went against the opinions of a number of senior investigators who believed this couldn't be accomplished. (A historical note: the BBY and K&M notations for these preparations were not an invention of the authors of these papers on PS2 isolation (see Dunahay et al. 1984)). ...
These special issues of photosynthesis research present papers documenting progress in revealing the many aspects of photosystem 2, a unique, one-of-a-kind complex system that can reduce a plastoquinone to a plastoquinol on every second flash of light and oxidize 2 H2O to an O2 on every fourth flash. This overview is a brief personal assessment of the progress observed by the author over a four-decade research career, including a discussion of some remaining unsolved issues. It will come as no surprise to readers that there are remaining questions given the complexity of PS2, and the efforts that have been needed so far to uncover its secrets. In fact, most readers will have their own lists of outstanding questions.
... PS I-LHC I-LHC II-enriched membranes were prepared as previously described (1,6). ...
During proteomic investigations examining the mobile LHC II chlorophyll antenna complex associated with Photosystem I (PS I), we identified, with very high confidence, the association of Rubisco Activase with PS I-LHC I-LHC II membranes. Using very rigorous criteria (p-values ≤ 10 ⁻⁵ ), a total of fifty-three high-quality Rubisco Activase peptides were identified by high resolution tandem mass spectrometry in two biological replicates, each digested with either trypsin or chymotrypsin, independently. Using these criteria, and searching the entire spinach proteome, only proteins previously known to be associated with PS I (the PS I subunits PsaD and PsaL, Lhca1-4, Lhcb1-3), the monomeric Lhcb proteins, Lhcb4-6 (which will be discussed elsewhere), and Rubisco Activase were identified. The presence of Rubisco Activase closely associated with PS I has important implications with respect to the activation of this enzyme and, consequently, the Calvin cycle by PS I, which will be discussed.
... State 1 and State 2 were induced as in Bressan et al. (2018) with slight modifications; leaves from overnight darkadapted plants were placed on wet paper for 45 min with either PSI light (a combination of far-red LEDs with a peak at 730 nm and 850 nm) or PSII light (30-W warm white fluorescent lamps filtered with Lee 105 orange filters). Thylakoids were isolated as previously described (Berthold et al., 1981). Leaves treated with State 1 or State 2 light were directly homogenized using ice-cold buffer B1 (20-mM tricine-KOH, pH 7.8, 0.4-M NaCl, 2-mM MgCl 2 , 0.5% milk powder) and filtered with a nylon mesh before centrifugation at 1,500g, 4 C for 12 min. ...
Photosynthesis powers nearly all life on Earth. Light absorbed by photosystems drives the conversion of water and carbon dioxide into sugars. In plants, photosystem I (PSI) and photosystem II (PSII) work in series to drive the electron transport from water to NADP+. As both photosystems largely work in series, a balanced excitation pressure is required for optimal photosynthetic performance. Both photosystems are composed of a core and light-harvesting complexes: LHCI for PSI and LHCII for PSII. When the light conditions favor the excitation of one photosystem over the other, a mobile pool of trimeric LHCII moves between both photosystems thus tuning their antenna cross-section in a process called state transitions. When PSII is over-excited multiple LHCIIs can associate with PSI. A trimeric LHCII binds to PSI at the PsaH/L/O site to form a well characterized PSI-LHCI-LHCII supercomplex. The binding site(s) of the “additional” LHCII is still unclear, although a mediating role for LHCI has been proposed. In this work we measured the PSI antenna size and trapping kinetics of photosynthetic membranes from Arabidopsis (Arabidopsis thaliana) plants. Membranes from WT plants were compared to those of the ΔLhca mutant that completely lacks the LHCI antenna. The results showed that “additional” LHCII complexes can transfer energy directly to the PSI core in the absence of LHCI. However, the transfer is about two times faster, and therefore more efficient, when LHCI is present. This suggests LHCI mediates excitation-energy transfer from loosely bound LHCII to PSI in WT plants.
... PS II membranes were prepared as described previously (Berthold et al. 1981) with some modification (Asada and Mino 2015). For the study of the leftover Mn 2+ site on the deactivation process, the membranes of 0.5 mgChl/ml were incubated for 1 min at 4 °C in a buffer containing 1 mM NH 2 OH, 400 mM sucrose, 20 mM NaCl, and 20 mM Mes/ NaOH (pH 6.5), and centrifuged at 35,000 × g for 20 min. ...
In this study, we identified two Mn2+ sites in apo-Photosystem II (PSII) using the pulsed electron–electron double resonance (PELDOR). A Mn2+ ion was bound to apo-PSII on the deactivation of the oxygen-evolving complex. The electron–electron magnetic dipole interaction of the Mn2+ to YD· was estimated to be 2.4 MHz. The site was assigned at the position between His332 and Glu189 in the D1 polypeptide, which is close to the Mn1 site in mature PS II. Using recent structures observed under electron microscopes (EM), the location of the Mn2+ site on photoactivation was reevaluated. The position between Asp170 and Glu189 in the D1 polypeptide is a good candidate for the initial high-affinity site for photoactivation. Based on a comparison with the PELDOR results, the two EM structures were evaluated.
... PSII-enriched thylakoid membranes were prepared from spinach, following standard methods [45,46]. The samples were suspended in 400 mM sucrose, 15 mM NaCl, 5 mM MgCl 2 , 40 mM MES at pH 6.5, at about 6-8 mg Chl/mL. ...
The biological water oxidation takes place in Photosystem II (PSII), a multi-subunit protein located in thylakoid membranes of higher plant chloroplasts and cyanobacteria. The catalytic site of PSII is a Mn4Ca cluster and is known as the oxygen evolving complex (OEC) of PSII. Two tyrosine residues D1-Tyr161 (YZ) and D2-Tyr160 (YD) are symmetrically placed in the two core subunits D1 and D2 and participate in proton coupled electron transfer reactions. YZ of PSII is near the OEC and mediates electron coupled proton transfer from Mn4Ca to the photooxidizable chlorophyll species P680+. YD does not directly interact with OEC, but is crucial for modulating the various S oxidation states of the OEC. In PSII from higher plants the environment of YD• radical has been extensively characterized only in spinach (Spinacia oleracea) Mn- depleted non functional PSII membranes. Here, we present a 2D-HYSCORE investigation in functional PSII of spinach to determine the electronic structure of YD• radical. The hyperfine couplings of the protons that interact with the YD• radical are determined and the relevant assignment is provided. A discussion on the similarities and differences between the present results and the results from studies performed in non functional PSII membranes from higher plants and PSII preparations from other organisms is given.
... The bands at (+)689 and (+)510 nm, the amplitudes of which varied from batch to batch, are attributed to residual psi-type bands reflecting the remaining long-range order of the pigment PPCs in the granum TM preparations. In purified PSII membranes-BBY [37] and grana patches [38], lacking multilamellar organization-these bands are absent [39]. The excitonic CD signal is a highly sensitive marker of the molecular organization of the PPCs. ...
In Part I, by using 31P-NMR spectroscopy, we have shown that isolated granum and stroma thylakoid membranes (TMs), in addition to the bilayer, display two isotropic phases and an inverted hexagonal (HII) phase; saturation transfer experiments and selective effects of lipase and thermal treatments have shown that these phases arise from distinct, yet interconnectable structural entities. To obtain information on the functional roles and origin of the different lipid phases, here we performed spectroscopic measurements and inspected the ultrastructure of these TM fragments. Circular dichroism, 77 K fluorescence emission spectroscopy, and variable chlorophyll-a fluorescence measurements revealed only minor lipase- or thermally induced changes in the photosynthetic machinery. Electrochromic absorbance transients showed that the TM fragments were re-sealed, and the vesicles largely retained their impermeabilities after lipase treatments—in line with the low susceptibility of the bilayer against the same treatment, as reflected by our 31P-NMR spectroscopy. Signatures of HII-phase could not be discerned with small-angle X-ray scattering—but traces of HII structures, without long-range order, were found by freeze-fracture electron microscopy (FF-EM) and cryo-electron tomography (CET). EM and CET images also revealed the presence of small vesicles and fusion of membrane particles, which might account for one of the isotropic phases. Interaction of VDE (violaxanthin de-epoxidase, detected by Western blot technique in both membrane fragments) with TM lipids might account for the other isotropic phase. In general, non-bilayer lipids are proposed to play role in the self-assembly of the highly organized yet dynamic TM network in chloroplasts.
... The overall structure of the Arabidopsis Photosystem II complex. The imaged Photosystem II C 2 S 2 M 2 -type complexes were isolated from wild-type Arabidopsis BBY membrane fragments 42 solubilised with a mixture of digitonin and β-DDM detergents. Visual inspection of the obtained micrographs suggested a high level of sample homogeneity. ...
In higher plants, the photosynthetic process is performed and regulated by Photosystem II (PSII). Arabidopsis thaliana was the first higher plant with a fully sequenced genome, conferring it the status of a model organism; nonetheless, a high-resolution structure of its Photosystem II is missing. We present the first Cryo-EM high-resolution structure of Arabidopsis PSII supercomplex with average resolution of 2.79 Å, an important model for future PSII studies. The digitonin extracted PSII complexes demonstrate the importance of: the LHG2630-lipid-headgroup in the trimerization of the light-harvesting complex II; the stabilization of the PsbJ subunit and the CP43-loop E by DGD520-lipid; the choice of detergent for the integrity of membrane protein complexes. Furthermore, our data shows at the anticipated Mn4CaO5-site a single metal ion density as a reminiscent early stage of Photosystem II photoactivation.
... Photosynthetic material PS II-membranes were prepared from spinach according to (Berthold et al. 1981) with some minor modifications. The chlorophyll concentration was determined according to Arnon (1949). ...
The reactivity of the S3 and S2 states towards NO and NH2OH was studied and compared using the period-4 oscillations in the F0-value induced by a train of single turnover Xenon flashes spaced 100 ms apart to monitor the reaction kinetics. The flash frequency also determined the time resolution of the assay, i.e. 100 ms. The S2 and S3-states were created by one and two single turnover pre-flashes, respectively. The NO concentration-dependence of the S3-decay indicated that at low NO-concentrations an S2-state was formed as an intermediate, whereas at higher concentrations a seemingly monophasic decay to the S1-state was observed. The sigmoidal concentration dependence indicated that a fast interaction of the S3-state with (at least) two NO-molecules is necessary for the fast S3 to S1 decay (τ ~0.4 s at 1.2 mM NO). The pH-dependence of the S3-decay suggests that a protonation reaction (pK ~6.9) is involved in the S3 to S1 decay. At intermediate NO-concentrations the protonation is only partially rate limiting, since the pH effect is more pronounced at high compared to intermediate NO-concentrations. A comparison of the reactivity of NO and hydroxylamine suggests that hydroxylamine reacts more efficiently with the S1 and S2 states, whereas NO reacts more efficiently with the S3-state. Based on our present knowledge of the oxygen evolving complex a possible reaction mechanism is proposed for the interaction between NO and the S3 state.
Interaction of water-soluble and immobilized (on BrCN-activated agarose) manganese-stabilizing protein PsbO with Mn2+ and Mn3+ cations and with preparations of D1/D2/cyt b559 reaction center (RC) of photosystem II was studied. By native electrophoresis, the formation of dimeric and aggregated forms of PsbO protein were found in the presence of Mn2+, Mg2+, or Fe2+ ions. The dimerization of PsbO occurred after ultraviolet irradiation of the protein preparation. The presence of protein-bound Mn3+ cations increased the electrostatic interaction of the immobilized PsbO with the RC. This was evidenced to by higher amounts of CaCl2 that were required for dissociation of the PsbORC complex. It was first demonstrated that the protein exhibited superoxide dismutase (SOD) activity after an electrophoresis in PAAG upon incubation of the gel in an Mn2+-containing solution. Tetrazolium-reductase activity was also ascertained in the protein after its electrophoresis in a mixture with preparations of the oxygen-evolving complex (OEC). It is suggested that the protein interaction with Mn ions and superoxide radicals, as well as short-term UV irradiation, reduces tyrosine and a disulfide bond in the PsbO protein. This yields tyrosil radical and SH-groups participating in redox reactions with ETC components. The interactions of PsbO with Mn cations and UV light, taking place in the chloroplast thylakoids, may regulate the protein binding to RC, modify structural organization of the protein, and promote its participation in alternative pathways of electron transport under the influence of stress factors. The hypothetical scheme of interaction of the immobilized PsbO protein with Mn ions and RC is discussed.
Nanomaterials that generate reactive oxygen species (ROS) upon light irradiation have significant applications in various fields, including photodynamic therapy (PDT) that is widely recognized as a highly momentous strategy for the eradication of cancer cells. However, the ROS production rate of photosensitizers, as well as the tumor hypoxia environment, are two major challenges that restrict the widespread application of PDT. In this study, a cancer‐thylakoid hybrid membrane‐camouflaged thulium oxide nanoparticles (Tm2O3) for tumor‐homing phototherapy through dual‐stage‐light‐guided ROS generation and oxygen self‐supply is developed. Tm2O3 as a type II photosensitizer are viable for NIR‐stimulated ROS generation due to the unique energy levels, large absorption cross section, and long lifetime of the 3H4 state of Tm ions. The thylakoid membrane (TK) plays a catalase‐like role in converting hydrogen peroxide into oxygen and also acts as a natural photosensitizer that can generate lethal ROS through electron transfer when exposed to light. In addition, fluorescence dye DiR is embedded in the hybrid membrane for in vivo tracing as well as photothermal therapy. Results show that tumors in Tm2O3@TK‐M/DiR group are effectively ablated following dual‐stage‐light irradiation, highlighting the promising potential of rare‐earth element‐based type II photosensitizers in various applications.
It was established that in a heterogeneous model system, which consisted of two types of complexes: reaction center or core complex of photosystem 2 of higher plants and LH2 complex of the sulfur bacterium Alc. vinosum, BChl850 oxidation of the LH2 complex could be observed under illumination by the light at a wavelength of 662 nm, which is the red absorption band of Chl. It has been shown that this process induces release of singlet oxygen, which is generated in photosystem II complexes and then partially diffuses into LH2 complex, where it oxidizes BChl850. It was established by HPLC that this results in formation of a product of BChl oxidation, 3-acetylchlorophyll. The process of BChl850 oxidation is inhibited by singlet oxygen quenchers (Trolox and Na ascorbate). It is suggested that the LH2 complex from the sulfur bacterium Alc. vinosum could be used to detect generation of singlet oxygen by the chlorophyll containing samples.
Photosystem II (PSII) is a multi-subunit membrane protein complex that catalyzes light-driven oxidation of water to molecular oxygen. The chloride ion (Cl−) has long been known as an essential cofactor for oxygen evolution by PSII, and two Cl− ions (Cl-1 and Cl-2) have been found to specifically bind near the Mn4CaO5 cluster within the oxygen-evolving center (OEC). However, despite intensive studies on these Cl− ions, little is known about the function of Cl-2, the Cl− ion that is associated with the backbone nitrogens of D1-Asn338, D1-Phe339, and CP43-Glu354. In green plant PSII, the membrane extrinsic subunits—PsbP and PsbQ—are responsible for Cl− retention within the OEC. The Loop 4 region of PsbP, consisting of highly conserved residues Thr135–Gly142, is inserted close to Cl-2, but its importance has not been examined to date. Here, we investigated the importance of PsbP-Loop 4 using spinach PSII membranes reconstituted with spinach PsbP proteins harboring mutations in this region. Mutations in PsbP-Loop 4 had remarkable effects on the rate of oxygen evolution by PSII. Moreover, we found that a specific mutation, PsbP-D139N, significantly enhanced the oxygen-evolving activity in the absence of PsbQ, but not significantly in its presence. The D139N mutation increased the Cl− retention ability of PsbP and induced a unique structural change in the OEC, as indicated by light-induced Fourier transform infrared (FTIR) difference spectroscopy and theoretical calculations. Our findings provide insight into the functional significance of Cl-2 in the water-oxidizing reaction of PSII.
The major photosystem II light-harvesting antenna (LHCII) is the most abundant membrane protein in nature and plays an indispensable role in light harvesting and photoprotection in the plant thylakoid. Here, we show that "pseudothylakoid characteristics" can be observed in artificial LHCII membranes. In our proteoliposomal system, at high LHCII densities, the liposomes become stacked, mimicking the in vivo thylakoid grana membranes. Furthermore, an unexpected, unstructured emission peak at ∼730 nm appears, similar in appearance to photosystem I emission, but with a clear excimeric character that has never been previously reported. These states correlate with the increasing density of LHCII in the membrane and a decrease in its average fluorescence lifetime. The appearance of these low-energy states can also occur in natural plant membrane structures, which has unique consequences for the interpretation of the spectroscopic and physiological properties of the photosynthetic membrane.
Semi-artificial photosynthesis interfacing catalytic protein machinery with synthetic photocatalysts exhibits great potential in solar-to-chemical energy conversion. However, characterizing and manipulating the molecular integration structure at the biotic-abiotic interface remain a challenging task. Herein, the biointerface molecular integration details of photosystem II (PSII)-semiconductor hybrids, including the PSII orientation, interfacial microdomains, and overall structure modulation, are systematically interrogated by lysine reactivity profiling mass spectrometry. We demonstrate the semiconductor surface biocompatibility is essential to the PSII self-assembly with uniform orientation and electroactive structure. Highly directional localization of PSII onto more hydrophilic Ru/SrTiO3:Rh surface exhibits less disturbance on PSII structure and electron transfer chain, beneficial to the high water splitting activity. Further, rational modification of hydrophobic Ru2S3/CdS surface with biocompatible protamine can improve the hybrid O2-evolving activity 83.3%. Our results provide the mechanistic understanding to the structure-activity relationship of PSII-semiconductor hybrids and contribute to their rational design in the future.
The Photosystem II (PSII) water oxidation to molecular oxygen process is catalyzed by an oxygen-bridged Mn4CaO5 cluster, known as Oxygen Evolving Complex (OEC) of PSII. The Mn4CaO5 cluster undergoes periodically four one-electron oxidation steps, S0 → S1, S1 → S2, S2 → S3, S3 → S0. The O2 release takes place during the S3-[S4]-S0 transition, S4 being a transient. The intermediates of the S-state transitions are known as metalloradical intermediate states (SiYz) and involve the free radical of Tyrosine Z (Yz, TyrZ). In most metalloradical states it was established that Yz interacts magnetically with the Mn4CaO5 cluster. However, in Ca²⁺- depleted PSII preparations the spin-spin interaction between the Mn4O5 and Yz, during the formation of the S2Yz intermediate state was not strongly supported experimentally. In our effort to investigate the existence of the aforementioned interaction, we took advantance of the NIR sensitivity of the S2 state of Mn4 and examined whether the S2Yz EPR spectrum can be modified. Our EPR experiments, combined with the simulation analysis of the spectra show that reversible modification of the S2 EPR spectrum by NIR irradiation results in a reversible change of the Yz EPR signal shape. These observations strongly support the idea of the magnetic interaction between the Mn4 and tyrosyl radical, upon the formation of the S2Yz metalloradical state in Ca²⁺- depleted PSII membranes.
The mechanism of water oxidation by the Photosystem II (PSII) protein-cofactor complex is of high interest, but specifically, the crucial coupling of protonation dynamics to electron transfer (ET) and dioxygen chemistry remains insufficiently understood. We drove spinach-PSII membranes by nanosecond-laser flashes synchronously through the water-oxidation cycle and traced the PSII processes by time-resolved single-frequency infrared (IR) spectroscopy in the spectral range of symmetric carboxylate vibrations of protein side chains. After the collection of IR-transients from 100 ns to 1 s, we analyzed the proton-removal step in the S 2 ⇒ S 3 transition, which precedes the ET that oxidizes the Mn 4 CaOx-cluster. Around 1400 cm −1 , pronounced changes in the IR-transients reflect this pre-ET process (∼40 μs at 20 ○ C) and the ET step (∼300 μs at 20 ○ C). For transients collected at various temperatures, unconstrained multi-exponential simulations did not provide a coherent set of time constants, but constraining the ET time constants to previously determined values solved the parameter correlation problem and resulted in an exceptionally high activation energy of 540 ± 30 meV for the pre-ET step. We assign the pre-ET step to deprotonation of a group that is re-protonated by accepting a proton from the substrate-water, which binds concurrently with the ET step. The analyzed IR-transients disfavor carboxylic-acid deprotonation in the pre-ET step. Temperature-dependent amplitudes suggest thermal equilibria that determine how strongly the proton-removal step is reflected in the IR-transients.
The detergent Triton X-100 effectively fragments the membranes of photosynthetic systems and has been used extensively for this purpose. This detergent behaves as other nonionic detergents in that it removes a small particle containing the components of photosystem I, leaving a membrane residuum containing photosystem II. Recent data indicate that Triton X-100 produces a cleaner fractionation, in terms of separation of photosystem I and photosystem II activities, than do other detergents. The photosystem I particle so obtained, called the “Triton subchloroplast fraction l” (TSF-1) particle, is enriched in chlorophyll α and β-carotene and has a lower content of the xanthophylls. The ratio of Chl:P700 in the TSF-1 particle is approximately 100:1. Application of the detergent to membrane systems, which do not contain carotenoids, however, solubilizes a particle, which is enriched in P700 and has a ratio of Chl:P700 of approximately 1:30. The preparation of both types of particles is described in the chapter.
An analysis of electron paramagnetic resonance Signal II in spinach chloroplasts has been made using both continuous and flashing light techniques. In order to perform the experiments we developed a method which allows us to obtain fresh, untreated chloroplasts with low dark levels of Signal II. Under these conditions a single 10-μs flash is sufficient to generate greater than 80% of the possible light-induced increase in Signal II spin concentration. The risetime for this flash-induced increase in Signal II is approx. 1 s. The close association of Signal II with Photo-system II is confirmed by the observations that red light is more effective than is far red light in generating Signal II, and that 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) does not inhibit the formation of the radical. Single flash saturation curves for the flash-induced increase in Signal I and Signal II indicate that the quantum efficiency for Signal II formation is close to that for Signal I. While one or two flashes (spaced 10 ms apart) are quite efficient in generating Signal II, three or four flashes are much less effective. However, if this spacing is decreased to 100 μs, three or four flashes become as efficient as one or two flashes. From observations of a deficiency of O2 evolved during the initial flashes of dark-adapted chloroplasts, we conclude that the species which gives rise to Signal II is able to compete with water for oxidizing equivalents generated by Photosystem II. On the basis of these results we postulate a model in which Signal II arises from an oxidized radical which is produced by a slow electron transfer to the specific states S2 and S3 on the water side of Photo-system II.
Rapid light-induced transients in EPR Signal IIf (F-+) are observed in 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU)-treated, Tris-washed chloroplasts until the state F P680 Q minus is reached. In the absence of exogenous redox mediators several flashes are required to saturate this photoinactive state. However, the Signal IIf transient is observed on only the first flash following DCMU addition if an efficient donor to Signal IIf, phenylenediamine or hydroquinone, is present. Complementary polarographic measurements show that under these conditions oxidized phenylenediamine is produced only on the first flash of a series. The DCMU inhibition of Signal IIf can be completely relieved by oxidative titration of a one-electron reductant with E'Os.o equals to + 480 mV. At high reduction potentials the decay time of Signal IIf is constant at about 300 ms, whereas in the absence of DCMU the decay time is longer and increases with increasing reduction potential. A model is proposed in which Q minus, the reduced Photosystem II primary acceptor, and D, a one-electron 480 mV donor endogenous to the chloroplast suspension, compete in the reduction of Signal IIf (F-+). At high potentials D is oxidized in the dark, and the (Q-+F-+) back reaction regenerates the photoactive F P680 Q state. The electrochemical and kinetic evidence is consistent with the hypothesis that the Signal IIf species, F, is identical with Z, the physiological donor to P680.
Transient electron paramagnetic resonance (EPR) methods are used to examine the spin populations of the light-induced radicals produced in spinach chloroplasts, photosystem I particles, and Chlorella pyrenoidosa. We observe both emission and enhanced absorption within the hyperfine structure of the EPR spectrum of P700+, the photooxidized reaction-center chlorophyll radical (Signal I). By using flow gradients or magnetic fields to orient the chloroplasts in the Zeeman field, we are able to influence both the magnitude and sign of the spin polarization. Identification of the polarized radical and P700+ is consistent with the effects of inhibitors, excitation light intensity and wavelength, redox potential, and fractionation of the membranes. The EPR signal of the polarized P700+ radical displays a 30% narrower line width than P700+ after spin relaxation. This suggests a magnetic interaction between P700+ and its reduced (paramagnetic) acceptor, which leads to a collapse of the P700+ hyperfine structure. Narrowing of the spectrum is evident only in the spectrum of polarized P700+, because prompt electron transfer rapidly separates the radical pair. Evidence of cross-relaxation between the adjacent radicals suggests the existence of an exchange interaction. The results indicate that polarization is produced by a radical pair mechanism between P700+ and the reduced primary acceptor of photosystem I. The orientation dependence of the spin polarization of P700+ is due to the g-tensor anisotropy of the acceptor radical to which it is exchange-coupled. The EPR spectrum of P700+ is virtually isotropic once the adjacent acceptor radical has passed the photoionized electron to a later, more remote acceptor molecule. This interpretation implies that the acceptor radical has g-tensor anisotropy significantly greater than the width of the hyperfine field on P700+ and that the acceptor is oriented with its smallest g-tensor axis along the normal to the thylakoid membranes. Both the ferredoxin-like iron-sulfur centers and the X- species observed directly by EPR at low temperatures have g-tensor anisotropy large enough to produce the observed spin polarization; however, studies on oriented chloroplasts show that the bound ferredoxin centers do not have this orientation of their g tensors. In contrast, X- is aligned with its smallest g-tensor axis predominantly normal to the plane of the thylakoid membranes. This is the same orientation predicted for the acceptor radical based on analysis of the spin polarization of P700+, and indicates that the species responsible for the anisotropy of the polarized P700+ spectrum is probably X-. The dark EPR Signal II is shown to possess anisotropic hyperfine structure (and possibly g-tensor anisotropy), which serves as a good indicator of the extent of membrane alignment.
A manganese electron paramagnetic resonance (EPR) signal not present in untreated chloroplasts is observed in chloroplasts treated by washing in Tris buffer (0.8 M, pH 8.0) for 15 min. Centrifugation indicates that the Mn responsible for the EPR signal is localized in the chloroplasts, not free in the supernatant liquid. Atomic absorption analysis demonstrates that less than 10% of total chloroplast Mn is lost upon Tris treatment. Tris treatment converts 60% of the total chloroplast Mn pool to an EPR-detectable state. Sonication causes the Mn EPR signal to be divided proportionately between the chloroplast pellet and the supernatant liquid.The EPR spectrum of Mn in Tris-washed chloroplasts is identical with that of a divalent aqueous Mn spectrum in g factor, hyperfine splitting, and linewidth temperature dependence. It is concluded that upon Tris treatment, Mn is released into the interior space of the thylakoid membrane.Transport of Mn and anionic chelating agents across the thylakoid membrane was investigated using EPR. The rate of Mn diffusion through the thylakoid membrane is slow, with a of 2.5 h. The rate of transfer of chelating agents such as EDTA is much faster, with of 750 ms.Tris washing also destroys a weak Mn-binding site on the exterior of the thylakoid membrane.
We have used the decay kinetics of Signal IIf in Tris-washed chloroplasts as a direct probe to reactions on the oxidizing side of Photosystem II. A study of the salt concentration dependence of the rate of reduction of Z . + by the ascorbate monoanion has been interpreted by using the Gouy-Chapman diffuse double layer model and allows the calculation of an inner membrane surface charge density of -3.4 +/- 0.3 microC . cm-2 at pH = 8.0 in the vicinity of Photosystem II. We have also measured the outer membrane surface charge density at this pH in Tris- and sucrose-washed chloroplasts by monitoring the rate of potassium ferricyanide oxidation of Q-, and arrive at values of -2.2 +/- 0.3 microC . cm-2 and -2.1 microC . cm-2, respectively. From these experiments we conclude that in dark-adapted chloroplasts at pH 8.0 there exists a transmembrane electric field in the vicinity of Photosystem II which arises from this surface charge asymmetry. In the presence of 10 mM monovalent salts, the transmembrane potential difference is of the order of 20 mV, corresponding to a field of 4 . 10(4) V . cm-1 (negative inside) for a 50A membrane. It is both smaller in magnitude and in the opposite direction compared to the photoinduced transmembrane field which gives rise to the 515 nm absorption change. We have also found non-double layer Ca2+ effects on the decay kinetics of Signal IIf with both charged (ascorbate monoanion) and neutral (diphenylcarbazide) donors. These results suggest a change in the environment of Z from lipophilic to hydrophilic upon specific binding of Ca2+.
The EPR characteristics of oxygen evolving particles prepared from Phormidium laminosum are described. These particles are enriched in Photosystem II allowing EPR investigation of signals which were previously small or masked by those from Photosystem I in other preparations. EPR signals from a Signal II species and high potential cytochrome beta-559 appear as they are photooxidised at cryogenic temperatures by Photosystem II. The Signal II species is a donor close to the Photosystem II reaction centre and may represent part of the charge accumulation system of water oxidation. An EPR signal from an iron-sulphur centre which may represent an unidentified component of photosynthetic electron transport is also described. The properties of the oxygen evolving particles show that the preparation is superior to chloroplasts or unfractionated alga membranes for the study of Photosystem II with a functional water oxidation system.
Field dispersion profiles of the proton spin-lattice relaxation rate, T1−1, in chloroplast suspensions show a local maximum near 20 MHz, probably due to bound Mn(II); EDTA extraction eliminates, and MnCl2 addition restores, the paramagnetic relaxivity. Since neither treatment affects water oxidation, the Mn(II) site monitored appears to lie outside the water-splitting enzyme. Intense illumination almost totally suppresses the paramagnetic relaxivity through an electron-transport-dependent mechanism. Previous reports that chloroplast nuclear magnetic relaxivity varies cyclically in flash experiments require reevaluation in terms of the probable role of Mn(II) that is nonfunctional in water oxidation.
1. A cell-free preparation of membrane fragments was prepared from the thermophilic blue-green alga Phormidium laminosum by lysozyme treatment of the cells followed by osmotic shock to lyse the spheroplasts. The membrane fragments showed high rates of photosynthetic electron transport and O2 evolution (180-250 mumol of O2/h per mg of chlorophyll a with 2,6-dimethyl-1,4-benzoquinone as electron acceptor). O2-evolution activity was stable provided that cations (e.g. 10mM-Mg2+ or 100mM-Na+) or glycerol (25%, v/v) were present in the suspending medium. 2. The components of the electron-transport chain in P. laminosum were similar to those of other blue-green algae: the cells contained Pigment P700, plastocyanin, soluble high-potential cytochrome c-553, soluble low-potential cytochrome c-54 and membrane-bound cytochromes f, b-563 and b-559 (both low- and high-potential forms). The amounts and midpoint potentials of the membrane-bound cytochromes were similar to those in higher-plant chloroplasts. 3. Although O2 evolution in P. laminosum spheroplasts was resistant to high temperatures, thermal stability was not retained in the cell-free preparation. However, in contrast with higher plants, O2 evolution in P. laminosum membrane fragments was remarkably resistant to the non-ionic detergent Triton X-100.
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