A highly resolved, oxygen‐evolving photosystem II preparation from spinach thylakoid membranes

Berthold, Deborah A.; Babcock, Gerald T.; Yocum, Charles F.

doi:10.1016/0014-5793(81)80608-4

Cited by 1,874 publications

(1,003 citation statements)

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“…The Journal of Physical Chemistry B isolated from intact thylakoid membranes as described in ref 29. Chlorophyll concentrations were determined spectroscopically in 80% (v/v) acetone.…”

Section: ■ Experimental Methodsmentioning

confidence: 99%

Carotenoid–Chlorophyll Coupling and Fluorescence Quenching Correlate with Protein Packing Density in Grana-Thylakoids

et al. 2013

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ABSTRACT:The regulation of light-harvesting in photosynthesis under conditions of varying solar light irradiation is essential for the survival and fitness of plants and algae. It has been proposed that rearrangements of protein distribution in the stacked grana region of thylakoid membranes connected to changes in the electronic pigment-interaction play a key role for this regulation. In particular, carotenoid−chlorophyll interactions seem to be crucial for the downregulation of photosynthetic light-harvesting. So far, it has been difficult to determine the influence of the dense protein packing found in native photosynthetic membrane on these interactions. We investigated the changes of the electronic couplings between carotenoids and chlorophylls and the quenching in grana thylakoids of varying protein packing density by two-photon spectroscopy, conventional chlorophyll fluorometry, low-temperature fluorescence spectroscopy, and electron micrographs of freeze-fracture membranes. We observed an increasing carotenoid−chlorophyll coupling and fluorescence quenching with increasing packing density. Simultaneously, the antennas size and excitonic connectivity of Photosystem II increased with increasing quenching and carotenoid−chlorophyll coupling whereas isolated, decoupled LHCII trimers decreased. Two distinct quenching data regimes could be identified that show up at different protein packing densities. In the regime corresponding to higher protein packing densities, quenching is strongly correlated to carotenoid−chlorophyll interactions whereas in the second regime, a weak correlation is apparent with low protein packing densities. Native membranes are in the strong-coupling data regime. Consequently, PSII and LHCII in grana membranes of plants are already quenched by protein crowding. We concluded that this ensures efficient electronic connection of all pigment−protein complexes for intermolecular energy transfer to the reaction centers and allows simultaneously sensitive regulation of light harvesting by only small changes in the protein packaging. ■ INTRODUCTIONPhotosynthesis in higher plants and algae is initiated by the harvesting of sunlight through a series of pigment−protein complexes located in the thylakoid membranes of chloroplasts. Starting from these light harvesting complexes (LHCs), the absorbed energy is efficiently transferred from one complex to the other. These transfer processes end up in the photosystem reaction centers where the energy is converted into redox energy. However, plants are exposed to changing light intensities on a daily basis that can vary over several orders of magnitude. 1 Thus, a regulation mechanism is necessary that assures effective usage of sunlight energy under low-light conditions but avoids photo-oxidative damage to the reaction centers or other parts of the photosynthetic apparatus if the absorbed light exceeds its capacity. A main mechanism that evolved to minimize photo-oxidative damage in plants is called high-energy quenching (qE). qE enables the dissipation ...

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“…The Journal of Physical Chemistry B isolated from intact thylakoid membranes as described in ref 29. Chlorophyll concentrations were determined spectroscopically in 80% (v/v) acetone.…”

Section: ■ Experimental Methodsmentioning

confidence: 99%

Carotenoid–Chlorophyll Coupling and Fluorescence Quenching Correlate with Protein Packing Density in Grana-Thylakoids

et al. 2013

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“…At room temperature, 1 mM ferricyanide was added to keep the PSII reaction centers open. Photosystem II membranes or BBY particles were isolated from thylakoid membranes as described by (Berthold et al 1981) with some modifications as described by (Van Leeuwen et al 1991) and dissolved in the same buffer as the native membranes. Additionally GY particles (named after Ghanotakis and Yocum who where the first ones to isolate this particle), which consist of the PSII core complex and the minor antenna complexes CP29 and CP26 were prepared as in Ghanotakis et al (1987).…”

Section: Methodsmentioning

confidence: 99%

Excitation energy transfer in native and unstacked thylakoid membranes studied by low temperature and ultrafast fluorescence spectroscopy

et al. 2007

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In this work, the transfer of excitation energy was studied in native and cation-depletion induced, unstacked thylakoid membranes of spinach by steady-state and time-resolved fluorescence spectroscopy. Fluorescence emission spectra at 5 K show an increase in photosystem I (PSI) emission upon unstacking, which suggests an increase of its antenna size. Fluorescence excitation measurements at 77 K indicate that the increase of PSI emission upon unstacking is caused both by a direct spillover from the photosystem II (PSII) core antenna and by a functional association of light-harvesting complex II (LHCII) to PSI, which is most likely caused by the formation of LHCII-LHCI-PSI supercomplexes. Time-resolved fluorescence measurements, both at room temperature and at 77 K, reveal differences in the fluorescence decay kinetics of stacked and unstacked membranes. Energy transfer between LHCII and PSI is observed to take place within 25 ps at room temperature and within 38 ps at 77 K, consistent with the formation of LHCII-LHCI-PSI supercomplexes. At the 150-160 ps timescale, both energy transfer from LHCII to PSI as well as spillover from the core antenna of PSII to PSI is shown to occur at 77 K. At room temperature the spillover and energy transfer to PSI is less clear at the 150 ps timescale, because these processes compete with charge separation in the PSII reaction center, which also takes place at a timescale of about 150 ps.

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“…PSII-enriched membrane fragments were prepared from market spinach by the method described elsewhere (41,42). Under saturating continuous illumination, the oxygen evolution rate of the PSII membranes was 400-500 µmol of O 2 (mg of Chl) -1 h -1 in the presence of 1 mM K 3 Fe(CN) 6 and 0.25 mM 2,5-dichloro-p-benzoquinone (DCBQ) as electron acceptors.…”

Section: Methodsmentioning

confidence: 99%

Spectroscopic Evidence for Ca²⁺Involvement in the Assembly of the Mn₄Ca Cluster in the Photosynthetic Water-Oxidizing Complex

et al. 2006

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Biogenesis and repair of the inorganic core (Mn 4 CaO x Cl y ), in the water-oxidizing complex of photosystem II (WOC-PSII), occurs through the light-induced (re)assembly of its free elementary ions and the apo-WOC-PSII protein, a reaction known as photoactivation. Herein, we use electron paramagnetic resonance (EPR) spectroscopy to characterize changes in the ligand coordination environment of the first photoactivation intermediate, the photo-oxidized Mn 3+ bound to apo-WOC-PSII. On the basis of the observed changes in electron Zeeman (g eff ), 55 Mn hyperfine (A Z ) interaction, and the EPR transition probabilities, the photogenerated Mn 3+ is shown to exist in two pH-dependent forms, differing in terms of strength and symmetry of their ligand fields. The transition from an EPR-invisible low-pH form to an EPR-active high-pH form occurs by deprotonation of an ionizable ligand bound to Mn 3+ , implicated to be a water molecule: [Mn 3+ (OH 2 In the absence of Ca 2+ , the EPR-active Mn 3+ exhibits a strong pH dependence (pH ∼6.5-9) of its ligand-field symmetry (rhombicity ∆δ ) 10%, derived from g eff ) and A Z (∆A Z ) 22%), attributable to a protein conformational change. Binding of Ca 2+ to its effector site eliminates this pH dependence and locks both g eff and A Z at values observed in the absence of Ca 2+ at alkaline pH. Thus, Ca 2+ directly controls the coordination environment and binds close to the highaffinity Mn 3+ , probably sharing a bridging ligand. This Ca 2+ effect and the pH-induced changes are consistent with the ionization of the bridging water molecule, predicting that [ A single tight-binding Ca 2+ ion is essential for photosynthethic O 2 evolution activity in ViVo (1-5). Calcium is located within the water-oxidizing complex of photosystem II (PSII-WOC), 1 comprised of an oxo/aquo-bridged inorganic core whose stoichiometry, Mn 4 CaO x , is based on several lines of evidence, including X-ray absorption (6-9), electron paramagnetic resonance (EPR) spectroscopy (10,11), and the recent X-ray diffraction (XRD) data (12, 13). Mn-, Sr-, and Ca-extended X-ray absorption fine structure (EXAFS) spectroscopies accurately place the calcium effector site at 3.3-3.5 Å from Mn atoms. However, the relative position of Ca 2+ within the Mn 4 O x cluster differs according to which type of data is emphasized and the assumptions of the models used to interpret the raw data. Spectroscopic and XRD data have constrained the possible core topologies to only a few types, with the structures given in Chart 1 proposed on the basis of one or more lines of evidence (9)(10)(11)13). These structural proposals have led to a number of mechanistic proposals for the role of calcium in O 2 evolution reviewed elsewhere (11,(14)(15)(16)(17).Dissection of the functional roles of the inorganic cofactors has come from in Vitro studies of the photoactivation process, which is defined as the assembly of the inorganic core during biogenesis and repair of the WOC. In Vitro photoactivation of the WOC is described by the following ...

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A highly resolved, oxygen‐evolving photosystem II preparation from spinach thylakoid membranes

Cited by 1,874 publications

References 11 publications

Carotenoid–Chlorophyll Coupling and Fluorescence Quenching Correlate with Protein Packing Density in Grana-Thylakoids

Carotenoid–Chlorophyll Coupling and Fluorescence Quenching Correlate with Protein Packing Density in Grana-Thylakoids

Excitation energy transfer in native and unstacked thylakoid membranes studied by low temperature and ultrafast fluorescence spectroscopy

Spectroscopic Evidence for Ca²⁺Involvement in the Assembly of the Mn₄Ca Cluster in the Photosynthetic Water-Oxidizing Complex

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A highly resolved, oxygen‐evolving photosystem II preparation from spinach thylakoid membranes

Cited by 1,874 publications

References 11 publications

Carotenoid–Chlorophyll Coupling and Fluorescence Quenching Correlate with Protein Packing Density in Grana-Thylakoids

Carotenoid–Chlorophyll Coupling and Fluorescence Quenching Correlate with Protein Packing Density in Grana-Thylakoids

Excitation energy transfer in native and unstacked thylakoid membranes studied by low temperature and ultrafast fluorescence spectroscopy

Spectroscopic Evidence for Ca2+Involvement in the Assembly of the Mn4Ca Cluster in the Photosynthetic Water-Oxidizing Complex

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Spectroscopic Evidence for Ca²⁺Involvement in the Assembly of the Mn₄Ca Cluster in the Photosynthetic Water-Oxidizing Complex