Thermophilic Cyanobacterium Synechococcus Elongatus Research Articles

Physiological and spectroscopical changes of the thermophilic cyanobacterium Synechococcus elongatus under iron stress and recovery culture

The iron in cyanobacterial cells contributes to energy conversion via electron transport chains, so its availability directly influences all metabolic pathways. Besides exploring cell adaptations and changes occurring with iron insufficient stress, the present work examines profoundly for the first time the metabolic changes accompanied with iron recovery for the thermophilic cyanobacterium Synechococcus elongatus (Nag., var. thermalis Geitl. Strain Kovrov 1972/8). S. elongatus cells were cultivated on two iron deprivation levels, iron-limited (45 nM Fe) and iron-deficient (4.5 nM Fe). Growth, carotenoids/Chlorophyll-a (Car/Chl-a) ratio, transmitting electron images, pigments fractionation analysis, and cell activity were checked. The obtained results demonstrated an astounding decrease in Chl-a content, an increment in β-carotene content, leading to rising the Car/Chl-a ratio, a reduction of phycobilins content, a reduction in cell diameters, a decrease of energy transfer, a depletion of the electron transport chain, and a reduction of photosynthesis and respiration processes under inadequate iron condition. Likewise, the photochemical activity of photosystem II (PSII), determined by Fv/Fm ratio and thermoluminescence estimations, demonstrated that PSII was disabled under iron pressure. With iron recovery by 200 nM Fe, cells regained full metabolic activity within 24 h. Despite the fact that photosynthesis and respiration activity exhibited nearly a similar behavior, S. elongatus under iron-deficiency began to recuperate the activity of the photosynthetic apparatus quicker than the respiratory machinery by regaining their pigment content and activity.

Equal-quantum Action Spectra Indicate Fluence-rate-selective Action of Multiple Photoreceptors for Photomovement of the Thermophilic Cyanobacterium Synechococcus elongatus¶

Unicellular thermophilic cyanobacterium Synechococcus elongatus displayed phototaxis on agar plate at 55°C. Equal-quantum action spectra for phototactic migration were determined at various fluence rates using the Okazaki Large Spectrograph as the light source. The shapes of the action spectra drastically changed depending on the fluence rate of the unilateral monochromatic irradiation: at a low fluence rate (3 μmol/m2/s), only lights in the red region had significant effect; at a medium fluence rate (10 μmol/m2/s), four major action peaks were observed at 530 nm (green), 570 nm (yellow), 640 nm (red) and 680 nm (red). At high fluence rates (30–90 μmol/m2/s), the former two peaks remained, while red peaks at 640 nm and 680 nm disappeared and, interestingly, an action peak around 700–740 nm (far-red) newly appeared. These results indicate that two or more distinct photoreceptors are involved in the phototaxis and that suitable photoreceptors are selectively active in response to the stimulus of light fluence rates. Far-red or red background lights irradiated vertically from above drastically inhibited phototaxis toward red light or far-red light, respectively. These results indicate involvement of some phytochrome(s).

Reversed-phase HPLC determination of chlorophyll a' and naphthoquinones in photosystem I of red algae: existence of two menaquinone-4 molecules in photosystem I of Cyanidium caldarium.

Chlorophyll (Chl) a', the C13(2)-epimer of Chl a, is one of the two Chl molecules constituting the primary electron donor (P700) of photosystem (PS) I of a thermophilic cyanobacterium Synechococcus elongatus. To examine whether PS I of other oxygenic photosynthetic organisms in general contain one Chl a' molecule in P700, the pigment composition of thylakoid membranes and PS I preparations isolated from red algae Porphyridium purpureum and Cyanidium caldarium was examined by reversed-phase HPLC with particular attention to Chl a' and phylloquinone (PhQ), the secondary electron acceptor of PS I. The two red algae contained one Chl a' molecule at the core part of PS I. In PS I of C. caldarium, two menaquinone-4 (MQ-4) molecules were detected in place of PhQ used by higher plants and cyanobacteria. The 1:2:1 stoichiometry among Chl a', PhQ (MQ-4) and P700 in PS I of the red algae indicates that one Chl a' molecule universally exists in PS I of oxygenic photosynthetic organisms, and two MQ-4 molecules are associated with PS I of C. caldarium.

Photosystem II single crystals studied by transient EPR: the light-induced triplet state

Transient electron paramagnetic resonance (TR EPR) at 9.8 GHz has been used to study the light-induced triplet state in single crystals of Photosystem II (PS II). The crystals were grown from a solution of PS II core complexes from the thermophilic cyanobacterium Synechococcus elongatus. The core complexes contain at least 17 subunits, including the water-oxidizing complex, and 32 chlorophyll a molecules per PS II complex. The PS II complexes are active in light-induced electron transfer and water oxidation. The crystals belong to the orthorhombic space group P2 12 12 1, with four dimers of PS II complexes per unit cell. Laser excitation was used to generate the recombination triplet state in PS II which was then studied by EPR at low temperatures (10 K). The crystal spectra show the same magnitude of the zero-field splitting (ZFS) values D, E as spectra obtained earlier for the triplet state of PS II in frozen solution. The orientation of the ZFS tensor D of the triplet state with respect to the crystallographic axes has been deduced from the analysis of angular-dependent EPR spectra. Knowledge of the orientation of the D tensor component perpendicular to the plane of the chlorophyll ( D Z ) allows an assignment on which chlorophyll of the reaction centre the triplet state is localized at low temperatures. Furthermore, the orientation of the D X and D Y components of the D tensor yielded the in-plane orientation of the respective chlorophyll in the reaction centre providing first experimental evidence for the orientation of this molecule in the PS II.

Reversed-phase HPLC determination of chlorophyll a' and phylloquinone in Photosystem I of oxygenic photosynthetic organisms. Universal existence of one chlorophyll a' molecule in Photosystem I.

Chlorophyll (Chl) a', the C132-epimer of Chl a, is a constituent of the primary electron donor (P700) of Photosystem (PS) I of a thermophilic cyanobacterium Synechococcus (Thermosynechococcus) elongatus, as was recently demonstrated by X-ray crystallography. To determine whether PS I of oxygenic photosynthetic organisms universally contains one molecule of Chl a', pigment compositions of thylakoid membranes and PS I complexes isolated from the cyanobacteria T. elongatus and Synechocystis sp. PCC 6803, the green alga Chlamydomonas reinhardtii, and the green plant spinach, were examined by simultaneous detection of phylloquinone (the secondary electron acceptor of PS I) and Chl a' by reversed-phase HPLC. The results were compared with the Chl a/P700 ratio determined spectrophotometrically. The Chl a'/PS I ratios of thylakoid membranes and PS I were about 1 for all the organisms examined, and one Chl a' molecule was found in PS I even after most of the peripheral subunits were removed. Chl a' showed a characteristic extraction behaviour significantly different from the bulk Chl a in acetone/methanol extraction upon varying the mixing ratio. These findings confirm that a single Chl a' molecule in P700 is the universal feature of PS I of the Chl a-based oxygenic photosynthetic organisms.

Functional Role of C α–H⋯O Hydrogen Bonds Between Transmembrane α-Helices in Photosystem I

The crystal structure at 2.5 Å resolution of the membrane-intrinsic, homotrimeric photosystem I (PSI) isolated from the thermophilic cyanobacterium Synechococcus elongatus shows that each monomer is composed of 12 protein subunits of which nine are embedded in the membrane and feature a total of 34 transmembrane α-helices (TMH). Hence, PSI provides an ideal case to study “conventional” and C α–H⋯O hydrogen bonds between TMH engaged in intra- and intersubunit interactions. Of the total of 75 C α–H⋯O hydrogen bonds between TMHs, 72 are intrasubunit and only three are intersubunit. The two largest subunits PsaA and PsaB are each folded into 11 TMHs showing 29 and 24 intrasubunit C α–H⋯O hydrogen bonds, respectively, that are not distributed randomly but many of them flank chlorophyll a (Chl a) co-ordinating amino acids, suggesting stabilisation of these structural segments. As major constituent of the trimerisation domain, subunit PsaL is located next to the 3-fold axis relating the three monomers of PSI. PsaL features a unique number of 19 intrasubunit C α–H⋯O hydrogen bonds that connect two of its three TMHs but there are no intersubunit C α–H⋯O hydrogen bonds between the three PsaL. Of the three intersubunit C α–H⋯O hydrogen bonds, two are formed between PsaA and PsaB and one between PsaB and PsaM. The large number of 75 C α–H⋯O hydrogen bonds contrasts the 49 conventional hydrogen bonds, indicating that the former and van der Waals contacts determine association and orientation of TMHs in PSI.

The 1.45 Å three-dimensional structure of C-phycocyanin from the thermophilic cyanobacterium Synechococcus elongatus

The conversion of solar radiation to chemical energy by photosynthetic organisms provides the primary driving force for life on earth. Light energy is captured by a variety of pigments, usually bound to proteins, which vary with different types of organisms. We report here the 1.45 Å resolution three-dimensional structure of one such pigment protein, C-phycocyanin, from Synechococcus elongatus. The structure is at the highest resolution achieved for any such phycobiliprotein. This level of resolution was made possible by implementing a novel crystallization method whereby nucleation is decoupled from subsequent growth, by incubating crystallizing drops for 7 h under nucleation conditions and then transferring them to metastable conditions for growth. This is done without touching the crystallization drops throughout the process.

Immobilisation of engineered molecules on electrodes and optical surfaces

Monolayers of genetically modified proteins with an hexahistidine tag, (His) 6, were obtained by using a Ni–NTA chelator synthesized on gold-sputtered surfaces (via sulphide bonds), or on gold and graphite (via sililating agents) working electrodes of screen-printed devices. Two kinds of proteins were produced and purified for this study: (a) a recombinant antibody, derived from the ‘single-chain Fv’ (scFv) format, and (b) a photosystem II (PSII) core complex isolated from the mutant strain CP43-H of the thermophilic cyanobacterium Synechococcus elongatus. An scFv previously isolated from a synthetic ‘phage display’ library was further engineered with an alkaline phosphatase activity genetically added between the carboxy-terminal of the scFvs and the (His) 6 to allow direct measurement of immobilisation. Renewable specific binding of (His) 6 proteins to gold and graphite surfaces and fast and sensitive electrochemical or optical detection of analytes were obtained. Additionally, “on chip” protein preconcentration was conveniently achieved for biosensing purposes, starting from crude unpurified extracts and avoiding protein purification steps.

Light Harvesting in Photosystem I: Modeling Based on the 2.5-Å Structure of Photosystem I from Synechococcus elongatus

The structure of photosystem I from the thermophilic cyanobacterium Synechococcus elongatus has been recently resolved by x-ray crystallography to 2.5-Å resolution. Besides the reaction center, photosystem I consists also of a core antenna containing 90 chlorophyll and 22 carotenoid molecules. It is their function to harvest solar energy and to transfer this energy to the reaction center (RC) where the excitation energy is converted into a charge separated state. Methods of steady-state optical spectroscopy such as absorption, linear, and circular dichroism have been applied to obtain information on the spectral properties of the complex, whereas transient absorption and fluorescence studies reported in the literature provide information on the dynamics of the excitation energy transfer. On the basis of the structure, the spectral properties and the energy transfer kinetics are simultaneously modeled by application of excitonic coupling theory to reveal relationships between structure and function. A spectral assignment of the 96 chlorophylls is suggested that allows us to reproduce both optical spectra and transfer and emission spectra and lifetimes of the photosystem I complex from S. elongatus. The model calculation allowed to study the influence of the following parameters on the excited state dynamics: the orientation factor, the heterogeneous site energies, the modifications arising from excitonic coupling (redistribution of oscillator strength, energetic splitting, reorientation of transition dipoles), and presence or absence of the linker cluster chlorophylls between antenna and reaction center. For the Förster radius and the intrinsic primary charge separation rate, the following values have been obtained: R0=7.8 nm and kCS=0.9ps−1. Variations of these parameters indicate that the excited state dynamics is neither pure trap limited, nor pure transfer (to-the-trap) limited but seems to be rather balanced.

Crystallization and preliminary X-ray diffraction analysis of the light-harvesting protein phycocyanin from the thermophilic cyanobacterium Synechococcus elongatus.

The crystallization and preliminary crystallographic study of phycocyanin from the thermophilic cyanobacterium Synechococcus elongatus is reported. Phycocyanin is composed of alpha- and beta-subunits consisting of 162 and 172 amino-acid residues, respectively. These associate to form an alphabeta heterodimer, which further associates to give a ring-shaped trimer (alphabeta)(3). Two trimers bind head-to-head to form a hexamer (alphabeta)(6). Phycocyanin crystals have been obtained by the sitting-drop vapour-diffusion method with a precipitant solution containing 30%(w/v) PEG 4000 and 100 mM MES pH 7.5-8.0. Using synchrotron radiation, the crystals diffract to 2.0 A resolution. They belong to the trigonal space group R32, with unit-cell parameters a = b = 186.75 (3), c = 59.75 (4) A, alpha = beta = 90, gamma = 120 degrees. Assuming that the crystallographic triad is identical to the threefold axis of the hexamer and with three (alphabeta)(6) molecules in a unit cell, the calculated molar volume (V(M)) is 2.64 A(3) Da(-1). This value corresponds to a solvent content of approximately 53%, with one alphabeta heterodimer occupying the asymmetric unit.

Fluorescent probes for non-invasive bioenergetic studies of whole cyanobacterial cells

Fluorescent ΔpH and Δ Ψ indicators have been screened for the non-invasive monitoring of bioenergetic processes in whole cells of the cyanobacterium Synechocystis sp. PCC 6803. Acridine yellow and Acridine orange proved to be the best ΔpH indicators for the investigation of thylakoid and cytoplasmic membrane energization: While Acridine yellow indicated only cytosolic energization, Acridine orange showed signals from both the thylakoid lumen and the cytosol that could be separated kinetically. Both indicators were applied successfully to monitor cellular energetics, such as the interplay of linear and cyclic photosynthetic electron transport, osmotic adaptation and solute transport across the cytoplasmic membrane. In contrast, useful membrane potential indicators were more difficult to find, with Di-4-ANEPPS and Brilliant cresyl blue being the only promising candidates for further studies. Finally, Acridine yellow and Acridine orange could also be applied successfully for the thermophilic cyanobacterium Synechococcus elongatus. Different from Synechocystis sp. PCC 6803, where both respiration and ATP hydrolysis could be utilized for cytoplasmic membrane energization, proton extrusion at the cytoplasmic membrane in Synechococcus elongatus was preferentially driven by ATP hydrolysis.

Photosystem II single crystals studied by EPR spectroscopy at 94 GHz: the tyrosine radical Y(D)(*).

Electron paramagnetic resonance (EPR) spectroscopy at 94 GHz is used to study the dark-stable tyrosine radical Y(D)(*) in single crystals of photosystem II core complexes (cc) isolated from the thermophilic cyanobacterium Synechococcus elongatus. These complexes contain at least 17 subunits, including the water-oxidizing complex (WOC), and 32 chlorophyll a molecules/PS II; they are active in light-induced electron transfer and water oxidation. The crystals belong to the orthorhombic space group P2(1)2(1)2(1), with four PS II dimers per unit cell. High-frequency EPR is used for enhancing the sensitivity of experiments performed on small single crystals as well as for increasing the spectral resolution of the g tensor components and of the different crystal sites. Magnitude and orientation of the g tensor of Y(D)(*) and related information on several proton hyperfine tensors are deduced from analysis of angular-dependent EPR spectra. The precise orientation of tyrosine Y(D)(*) in PS II is obtained as a first step in the EPR characterization of paramagnetic species in these single crystals.

Targeted disruption of psbX and biochemical characterization of photosystem II complex in the thermophilic cyanobacterium Synechococcus elongatus.

PSII-X is a small hydrophobic protein, which is universally present in photosystem II (PSII) core complex among cyanobacteria and plants. The role of PSII-X was studied by directed mutagenesis and biochemical analysis in the thermophilic cyanobacterium Synechococcus elongatus. The psbX-disrupted mutant could grow photoautotrophically indicative of non-essential function, while it showed growth defect under low CO(2) conditions. An active O(2)-evolving PSII complex was successfully isolated from the mutant and wild type. Protein composition of the isolated PSII complex was the same as wild type except for the absence of PSII-X. O(2) evolution supported by artificial quinones was affected in the psbX-disrupted mutant. At high concentration of 2,6-dichlorobenzoquinone or 2,6-dimethylbenzoquinone, the mutant showed much lower activity than wild type, while not much difference was found at low concentration. These results imply that binding or turnover of quinones at the Q(B) site depends, at least in part, on PSII-X protein in the PSII complex. Gel filtration chromatography of the PSII complex revealed that the dimeric structure of the complex was not greatly affected in the psbX-disrupted mutant.

Orientation and Electronic Structure of the Primary Donor Radical Cation in Photosystem I: A Single Crystals EPR and ENDOR Study

The primary donor in single crystals of photosystem I (PS I) obtained from the thermophilic cyanobacterium Synechococcus elongatus is investigated by EPR and ENDOR techniques. The cation radical was generated chemically or photolytically in the single crystals, and angular dependent spectra were obtained at a temperature of 100 K in the frozen state. The principal values and corresponding axes orientations were determined for three different proton hyperfine coupling (hfc) tensors. On the basis of the tensor magnitudes, their symmetry, and the relative orientations of their axes, they were assigned to the protons of three methyl groups of a single chlorophyll (Chl) a molecule. From X-ray crystallography of PS I single crystals, it is known that P700 is structurally a chlorophyll dimer (Klukas et al., J. Biol. Chem. 1999, 274, 7361). The EPR/ENDOR experiments show that the second chlorophyll half carries no or very little (≤15%) spin density. The molecular plane of the spin-carrying Chl a molecule in is pa...

X-Ray Crystallographic Structure Analysis of Cyanobacterial Photosystem I at 2.5 A Resolution

Photosystem I isolated from the thermophilic cyanobacterium Synechococcus elongatus was crystallized in a form suitable for X-ray structure analysis at 2.5 A resolution, resulting in a structural model at atomic detail which forms the basis for understanding the numerous interactions between 12 protein subunits, 96 chlorophyll, 22 carotenoid, two phylloquinone, four lipid molecules and three [4Fe4S] clusters. Six chlorophylls and two phylloquinones involved in transmembrane charge separation are arranged in two branches of pronounced twofold pseudo-symmetry, most significantly broken at the primary donor P700 consisting of a chlorophylla/chlorophylla' heterodimer with an asymmetric hydrogen-bonding environment. The dominating ligands to the central Mg2+ ions of the chlorophylls are histidine side chains, but other ligands not observed in the structures of chlorophyll-binding proteins so far, among these methionine side chains and the phosphodiester group of a phospholipid, have also been found in the structure. Each of the carotenoids (17 in all-trans- and five in different cis-configurations) is in close contact with at least one chlorophyll, in agreement with their two major functions in photoprotection and light-harvesting.

Comparative study of thermal degradation of iron-sulfur proteins in spinach chloroplasts and membranes of thermophilic cyanobacteria: mössbauer spectroscopy.

Mössbauer spectra of chloroplasts isolated from spinach plants grown in a mineral medium enriched with 57Fe and Mössbauer spectra of native membranes of the thermophilic cyanobacterium Synechococcus elongatus contain a broad asymmetric doublet typical of the iron-sulfur proteins of Photosystem (PS) I. Exposure of chloroplasts to temperatures of 20-70 degrees C significantly modifies the central part of the spectra. This spectral change is evidence of decreased magnitude of the quadrupole splitting. However, the thermally induced doublet (DeltaQ = 3.10 mm/sec and delta = 1.28 mm/sec) typical of hydrated forms of reduced (divalent) inorganic iron is not observed in spinach chloroplasts. This doublet is usually associated with degradation of active centers of ferredoxin, a surface-exposed protein of PS I. The Mössbauer spectra of photosynthetic membranes of spinach chloroplasts and cyanobacteria were compared using the probability distribution function of quadrupole shift (1/2 quadrupole splitting DeltaQ) of trivalent iron. The results of calculation of these functions for the two preparations showed that upon increasing the heating temperature there was a decrease in the probability of the presence of native iron-sulfur centers FX, FA, and FB (quadrupole shift range, 0.43-0.67 mm/sec) in heated preparations. This process was also accompanied by an increase in the probability of appearance of clusters of trivalent iron. This increase was found to be either gradual and continuous or abrupt and discrete in photosynthetic membranes of cyanobacteria or spinach chloroplasts, respectively. The probability of the presence of the iron-sulfur centers FX, FA, and FB in chloroplasts abruptly decreases to virtually to zero within the temperature range critical for inhibition of electron transport through PS I to oxygen. In cyanobacteria, both thermal destruction of iron-sulfur centers of PS I and functional degradation of PS I are shifted toward a higher temperature. The results of this study suggest that the same mechanism of thermal destruction of the PS I core occurs in both thermophilic and mesophilic organisms: destruction of iron-sulfur centers FX, FA, and FB, release of oxidized (trivalent) iron, and its accumulation in membrane-bound iron-oxo clusters.

HPLC determination of chlorophyll a' and phylloquinone in photosystem I of higher plants and cyanobacteria

Stoichiometries of Chlorophyll (Chl) a?, C132-epimer of Chl a, within Photosystem (PS) I of higher plants and cyanobacteria were determined by simultaneous detection of Chl a? and phylloquinone, the secondary electron acceptor of PS I (two molecules per PS I), with reversed-phase HPLC analyses, and spectrophotometric determination of P700, the primary donor of PS I. For thermophilic cyanobacterium Synechococcus elongatus, the Chl a?/PhQ ratio was about 0.5 for cell, thylakoid membranes, and PS I trimer. The Chl a?/P700 ratio and the PhQ/P700 ratio were about 1 and 2, respectively, for thylakoid membranes and PS I trimer. Theses results showed that a 1:1:2 stoichiometry is established among Chl a?, P700, and PhQ within PS I of S. elongatus. PS I trimer isolated from Synechocystis PCC 6803 also showed the same stoichiometry as that found for S. elongatus. Native PS I complexes of spinach and barley also showed the 1:1:2 stoichiometry among Chl a?, P700, and PhQ. Treatments of PS I with water-saturated/dry diethyl ether mixtures enriched Chl a? up to the Chl a/Chl a? ratio of 9-10 with keeping the Chl a?/P700 ratio at about 1. These findings show that higher plant PS I contains one molecule of Chl a? in close association with the primary donor, P700. Pigment composition analyses with reversed-phase HPLC indicate that one molecule of Chl a? is universally present in PS I of oxygenic photo-autotroph as the indispensable ingredient, possibly as the constituent of the primary donor, P700.

Equal-quantum action spectra indicate fluence-rate-selective action of multiple photoreceptors for photomovement of the thermophilic cyanobacterium Synechococcus elongatus.

Unicellular thermophilic cyanobacterium Synechococcus elongatus displayed phototaxis on agar plate at 55 degrees C. Equal-quantum action spectra for phototactic migration were determined at various fluence rates using the Okazaki Large Spectrograph as the light source. The shapes of the action spectra drastically changed depending on the fluence rate of the unilateral monochromatic irradiation: at a low fluence rate (3 mumol/m2/s), only lights in the red region had significant effect; at a medium fluence rate (10 mumol/m2/s), four major action peaks were observed at 530 nm (green), 570 nm (yellow), 640 nm (red) and 680 nm (red). At high fluence rates (30-90 mumol/m2/s), the former two peaks remained, while red peaks at 640 nm and 680 nm disappeared and, interestingly, an action peak around 700-740 nm (far-red) newly appeared. These results indicate that two or more distinct photoreceptors are involved in the phototaxis and that suitable photoreceptors are selectively active in response to the stimulus of light fluence rates. Far-red or red background lights irradiated vertically from above drastically inhibited phototaxis toward red light or far-red light, respectively. These results indicate involvement of some phytochrome(s).

Three-dimensional Structure of Chlamydomonas reinhardtii and Synechococcus elongatus Photosystem II Complexes Allows for Comparison of Their Oxygen-evolving Complex Organization

Electron microscopy and single-particle analyses have been carried out on negatively stained photosystem II (PSII) complexes isolated from the green alga Chlamydomonas reinhardtii and the thermophilic cyanobacterium Synechococcus elongatus. The analyses have yielded three-dimensional structures at 30-A resolution. Biochemical analysis of the C. reinhardtii particle suggested it to be very similar to the light-harvesting complex II (LHCII).PSII supercomplex of spinach, a conclusion borne out by its three-dimensional structure. Not only was the C. reinhardtii LHCII.PSII supercomplex dimeric and of comparable size and shape to that of spinach, but the structural features for the extrinsic OEC subunits bound to the lumenal surface were also similar thus allowing identification of the PsbO, PsbP, and PsbQ OEC proteins. The particle isolated from S. elongatus was also dimeric and retained its OEC proteins, PsbO, PsbU, and PsbV (cytochrome c(550)), which were again visualized as protrusions on the lumenal surface of the complex. The overall size and shape of the cyanobacterial particle was similar to that of a PSII dimeric core complex isolated from spinach for which higher resolution structural data are known from electron crystallography. By building the higher resolution structural model into the projection maps it has been possible to relate the positioning of the OEC proteins of C. reinhardtii and S. elongatus with the underlying transmembrane helices of other major intrinsic subunits of the core complex, D1, D2, CP47, and CP43 proteins. It is concluded that the PsbO protein is located over the CP47 and D2 side of the reaction center core complex, whereas the PsbP/PsbQ and PsbV/PsbU are positioned over the lumenal surface of the N-terminal region of the D1 protein. However, the mass attributed to PsbV/PsbU seems to bridge across to the PsbO, whereas the PsbP/PsbQ proteins protrude out more from the lumenal surface. Nevertheless, within the resolution and quality of the data, the relative positions of the center of masses for OEC proteins of C. reinhardtii and S. elongatus are similar and consistent with those determined previously for the OEC proteins of spinach.

Towards Structural Determination of the Water-splitting Enzyme: PURIFICATION, CRYSTALLIZATION, AND PRELIMINARY CRYSTALLOGRAPHIC STUDIES OF PHOTOSYSTEM II FROM A THERMOPHILIC CYANOBACTERIUM

A photosystem II preparation from the thermophilic cyanobacterium Synechococcus elongatus, which is especially suitable for three-dimensional crystallization in a fully active form was developed. The efficient purification method applied here yielded 10 mg of protein of a homogenous dimeric complex of about 500 kDa within 2 days. Detailed characterization of the preparation demonstrated a fully active electron transport chain from the manganese cluster to plastoquinone in the Q(B) binding site. The oxygen-evolving activity, 5000-6000 micromol of O(2)/(h.mg of chlorophyll), was the highest so far reported and is maintained even at temperatures as high as 50 degrees C. The crystals obtained by the vapor diffusion method diffracted to a resolution of 4.3 A. The space group was determined to be P2(1)2(1)2(1) with four photosystem II dimers per unit cell. Analysis of the redissolved crystals revealed that activity, supramolecular organization, and subunit composition were maintained during crystallization.