Integrity and Activity of Photosystem 2 Complexes Isolated from the Thermophilic Cyanobacterium Synechococcus Elongatus Using Various Detergents

The efficiency in selective extraction of photosystem (PS) 2 oxygen evolving complexes was compared among seven detergents. These were applied to thylakoid membranes of the thermophilic cyanobacterium Synechococcus elongatus. Used were five non-ionic detergents with one ionic and one zwitterionic for comparison. To compare the suitability and efficiency of the detergents the following properties of the extracts were examined: maximum rate of oxygen evolution with various electron acceptors, the relative variable fluorescence (F_V/F_M), the contamination of the extract with photosystem (PS) 1, and the status of the electron acceptor side of PS2 reaction centre. None of the detergents yielded a highly selective extraction of the PS2 complexes (negligible contamination with PS1) which would simultaneously display a high photochemical activity and high structural intactness. Heptylthioglucoside and dodecylmaltoside yielded the nearest approximation to the optimum result. Kinetic fluorometry was applied here for the first time to characterize the functional and structural properties of PS2 particles from cyanobacteria. This revised version was published online in August 2006 with corrections to the Cover Date.     

New Bistriazole Derivatives and the Biological Activity of the Thermophilic Cyanobacterium Mastigocladus laminosus Cohn.

The conditions of cultivation and the composition of medium for Desulfovibrio vulgaris Miyazaki F (DvMF) were examined to obtain cytochrome c₃ labeled with a stable isotope. The growth of DvMF was steady and reproducible under purging with N₂ and under pH control. DvMF was able to grow on a defined medium without natural products. The composition of medium containing a small amount of NH₄Cl as sole nitrogen source was established. Then, [U-¹⁵N]cytochrome c₃ was obtained during the culture of DvMF in a defined medium with ¹⁵NH₄Cl; it was confirmed by ¹H?¹⁵N HMQC. This is the first report of [U-¹⁵N]cytochrome c₃.     

Isolation and biliprotein characterization of phycobilisomes from the thermophilic cyanobacterium Mastigocladus laminosus Cohn

A method for the effective isolation of functionally intact phycobilisomes from the thermophilic cyanobacterium M. laminosus is presented, using an unconventional high buffer molarity for stabilizing the aggregates and introducing a DNAse treatment of the disrupted cells to obtain sharp banding of the phycobilisomes in the linear sucrose density gradients.The structural integrity of the isolated phycobilisomes is demonstrated by a fluorescence emission maximum at 673 nm of aggregated allophycocyanin and by electron microscopy.Besides C-phycocyanin and allophycocyanin, phycoerythrocyanin is a constituent pigment of the phycobilisomes. These pigments indicated in the absorption spectrum of phycobilisomes with a maximum at 610 nm and two shoulders at 650 and 580 nm, respectively, were characterized by spectral data and isoelectric points.     

Ascorbate accumulation during sulphur deprivation and its effects on photosystem II activity and H2 production of the green alga Chlamydomonas reinhardtii

In nature, H₂ production in Chlamydomonas reinhardtii serves as a safety valve during the induction of photosynthesis in anoxia, and it prevents the over‐reduction of the photosynthetic electron transport chain. Sulphur deprivation of C. reinhardtii also triggers a complex metabolic response resulting in the induction of various stress‐related genes, down‐regulation of photosynthesis, the establishment of anaerobiosis and expression of active hydrogenase. Photosystem II (PSII) plays dual role in H₂ production because it supplies electrons but the evolved O₂ inhibits the hydrogenase. Here, we show that upon sulphur deprivation, the ascorbate content in C. reinhardtii increases about 50‐fold, reaching the mM range; at this concentration, ascorbate inactivates the Mn‐cluster of PSII, and afterwards, it can donate electrons to tyrozin Z⁺ at a slow rate. This stage is followed by donor‐side‐induced photoinhibition, leading to the loss of charge separation activity in PSII and reaction centre degradation. The time point at which maximum ascorbate concentration is reached in the cell is critical for the establishment of anaerobiosis and initiation of H₂ production. We also show that ascorbate influenced H₂ evolution via altering the photosynthetic electron transport rather than hydrogenase activity and starch degradation.     

Preparation and Characterization of Heat-Stable and Very Active Oxygen-Evolving Photosystem II Particles from the Thermophilic Cyanobacterium, Synechococcus elongatu

Very active and heat-stable oxygen-evolving photosystem II particleswere isolated from the thermophilic cyanobacterium Synechococcuselongatus by treatment of thylakoid membranes with a non-ionicdetergent, sucrose monolaurate (SML). The particles were analyzedin a comparison with photosystem II particles prepared withß-octylglucoside (OG). The two preparations had similarpolypeptide compositions, which were caracterized by high levelsof polypeptides from phycobilisomes. The ratio of chlorophylla to QA was 45 and there were four Mn atoms and one tightlybound Ca²⁺ ion per QA in the particles prepared with SML. Thepreparations were thermophilic, showing substantial rates ofoxygen evolution at temperatures up to 60°C. The maximumrates attained at 45°C were as high as 6.0 mmoles O₂ mg^–1Chl h^–1. PS II particles prepared with OG were similarlythermostable but were less active in oxygen evolution at alltemperatures examined. Kinetic analysis of flash-induced absorptiontransients revealed that about 22% and 28% of photosystem IIreaction centers were not associated with the functional QBsite in the SML- and OG-particles, respectively. When correctedfor the inactive reaction centers, the maximum rates of oxygenevolution by SML- and OG-particles were 7.7 and 7.0 mmoles O₂mg^–1 Chl h^–1, which correspond to half times of1.9 and 2.1 ms for the first-order electron transfer, respectively.Comparison of these half times with those of the S-state transitionand the release of oxygen indicates that the overall photosystemII electron transport is limited by the reduction of added electronacceptors and not by release of oxygen. ³On leave from National Chemical Laboratory for Industry, Higashi1-1, Tsukuba, Ibaraki 305     

Isolation and Characterization of a Photosystem II Core Complex Depleted in the 43 kDa-Chlorophyll-Binding Subunit

The photosystem II core complex purified from digitonin extractsof spinach chloroplasts was resolved into two chlorophyll-proteincomplexes by digitonin polyacrylamide gel electrophoresis aftertreatment with 1 M potassium thiocyanate. One of the chlorophyll-proteincomplexes resolved consisted of 47, 32, 30 and 9 kDa polypeptidesand the other was complementally composed of only the 43 kDapolypeptide. The former complex was highly active in the photoreductionof 2, 6-dichlorophenol indophenol by 1,5-diphenylcarbazide andretained all of the components responsible for the electrontransport from the secondary electron donor (Z) to the primaryelectron acceptor (Q_A). EPR signal II_fast and II_slow were alsopreserved in this complex although their hyperfine structureswere largely modified. The complex was estimated to contain1.8 molecules of plastoquinone A as well as 1.5, 3.7 and 3.9molecules of cytochrome b559, pheophytin and ß-carotene,respectively, per Q_A. These results indicate that potassiumthiocyanate specifically removes the 43 kDa polypeptide fromthe PS II core complex leaving the electron transport systemin an almost intact state. (Received June 17, 1987; Accepted October 23, 1987)     

植物光系统II放氧复合体外周蛋白结构和功能的研究进展

光合放氧是植物光系统II(PSII)的重要功能之一。PSII的放氧反应主要是由PSII氧化侧的 4个锰原子组成的锰簇催化的。在类囊体膜的囊腔侧还结合有若干个外周蛋白 ,对放氧反应起着重要作用。文章总结了植物光系统II外周蛋白的结构和功能研究方面的最新进展     

Proximity Labeling Facilitates Defining the Proteome Neighborhood of Photosystem II Oxygen Evolution Complex in a Model Cyanobacterium

Ascorbate peroxidase (APEX)-based proximity labeling coupled with mass spectrometry has a great potential for spatiotemporal identification of proteins proximal to a protein complex of interest. Using this approach is feasible to define the proteome neighborhood of important protein complexes in a popular photosynthetic model cyanobacterium Synechocystis sp. PCC6803 (hereafter named as Synechocystis). To this end, we developed a robust workflow for APEX2-based proximity labeling in Synechocystis and used the workflow to identify proteins proximal to the photosystem II (PS II) oxygen evolution complex (OEC) through fusion APEX2 with a luminal OEC subunit, PsbO. In total, 38 integral membrane proteins (IMPs) and 93 luminal proteins were identified as proximal to the OEC. A significant portion of these proteins are involved in PS II assembly, maturation, and repair, while the majority of the rest were not previously implicated with PS II. The IMPs include subunits of PS II and cytochrome b₆/f, but not of photosystem I (except for PsaL) and ATP synthases, suggesting that the latter two complexes are spatially separated from the OEC with a distance longer than the APEX2 labeling radius. Besides, the topologies of six IMPs were successfully predicted because their lumen-facing regions exclusively contain potential APEX2 labeling sites. The luminal proteins include 66 proteins with a predicted signal peptide and 57 proteins localized also in periplasm, providing important targets to study the regulation and selectivity of protein translocation. Together, we not only developed a robust workflow for the application of APEX2-based proximity labeling in Synechocystis and showcased the feasibility to define the neighborhood proteome of an important protein complex with a short radius but also discovered a set of the proteins that potentially interact with and regulate PS II structure and function.     

Effects of azide on the S2 state EPR signals from Photosystem II

The anion azide, N₃ ^-, has been previously found to be an inhibitor of oxygen evolution by Photosystem II (PS II) of higher plants. With respect to chloride activation, azide acts primarily as a competitive inhibitor but uncompetitive inhibition also occurs [Haddy A, Hatchell JA, Kimel RA and Thomas R (1999) Biochemistry 38: 6104–6110]. In this study, the effects of azide on PS II-enriched thylakoid membranes were characterized by electron paramagnetic resonance (EPR) spectroscopy. Azide showed two distinguishable effects on the S₂ state EPR signals. In the presence of chloride, which prevented competitive binding, azide suppressed the formation of the multiline and g = 4.1 signals concurrently, indicating that the normal S₂ state was not reached. Signal suppression showed an azide concentration dependence that correlated with the fraction of PS II centers calculated to bind azide at the uncompetitive site, based on the previously determined inhibition constant. No evidence was found for an effect of azide on the Fe(II)Q_A ^- signals at the concentrations used. This result is consistent with placement of the uncompetitive site on the donor side of PS II as suggested in the previous study. In chloride-depleted PS II-enriched membranes azide and fluoride showed similar effects on the S₂ state EPR signals, including a notable increase and narrowing of the g = 4.1 signal. Comparable effects of other anions have been described previously and apparently take place through the chloride-competitive site. The two azide binding sites described here correlate with the results of other studies of Lewis base inhibitors.This revised version was published online in October 2005 with corrections to the Cover Date.     

The Isolation and Characterization of a Cytochrome b6f Complex from the Cyanobacterium Spirulina sp.

A cytochrome b₆f complex was isolated and purified from Spirulinasp. The complex was solubilized with n-heptyl ß-D-thioglucosideand chromatographed on a DEAE-Toyopearl 650M column. The purifiedcomplex contained a small amount of chlorophyll and carotenoid.At least four polypeptides were present in the complex: cytochromef (29 kDa), cytochrome b₆(23 kDa), iron-sulfur protein (ISP,23 kDa), and a 17 kDa polypeptide. Each polypeptide was separatedfrom the complex treated with 2-mercaptoethanol or urea. Theabsorption spectra of cytochrome b₆ and cytochrome f were similarto those of Anabaena and spinach as expected. The complex wasactive in supporting ubiquinol-cytochrome c oxidoreductase activity.Fifty percent inhibition of the activity was accomplished by1 µM dibromothymoquinone (DBMIB). The K_m values for ubiquinol-2and cytochrome c (horse heart) were 5.7 µM and 7.4 µM,respectively. (Received August 15, 1988; Accepted November 14, 1988)     

Isolation and characterization of the immunoglobulin-binding protein from Yersinia pseudotuberculosis

A high molecular weight immunoglobulin-binding protein localized on the surface of bacterial cells has been isolated from the protein fraction of the outer membrane of Yersinia pseudotuberculosis, and its properties are described. The immunoglobulin-binding protein is a trypsin-resistant and temperature-sensitive -structured protein. As shown by MALDI-TOF mass spectrometry, after heating at 100°C the molecular weight of the protein constituted 37.5 kD. The native protein is capable of interacting with human and rabbit IgG but looses the ability to bind the immunoglobulins after the temperature denaturation. The immunoglobulin-binding protein binds to the Fc-fragments of the immunoglobulins and binding depends on the presence of calcium ions.     

Isolation and properties of particles containing the reactioncenter complex of Photosystem II from spinach chloroplasts

By density gradient centrifugation of the 80000 × g supernatant of digitonintreated spinach chloroplasts two main green bands and one minor green band were obtained. The purification and properties of the particles present in the main bands, which were shown to be derived from Photosystem I and Photosystem II, have been described previously; those of the particles in the minor fraction will be described in the present paper.

After purification, these particles show Photosystem II activity but are devoid of Photosystem I activity. They have a high chlorophyll a/chlorophyll b ratio and are enriched in β-carotene and cytochrome b₅₅₉. At liquid nitrogen temperature, photoreduction of C550 and photooxidation of cytochrome-b₅₅₉ can be observed. At room temperature, cytochrome b₅₅₉ undergoes slight photooxidation.

These properties indicate that this particle may be the reaction-center complex of Photosystem II. It is suggested that, in vivo, the Photosystem II unit is made up of a reaction-center complex and an accessory complex, the latter being found in one of the main green bands of the density gradient.     

嗜热蓝藻优雅粘囊藻藻胆体的特性和别藻蓝蛋白的亚基组成

研究了优雅粘囊藻藻胆体（ＰＢＳ）的特性。从聚丙烯酰胺凝胶电泳（ＰＡＧＥ）、吸收光谱和二阶导数图谱可证明,它的ＰＢＳ含有一种红色藻胆蛋白,两种Ｃ－藻蓝蛋白和三种别藻蓝蛋白。但是,用ＰＡＧＥ和羟基磷灰石柱层析分离和纯化所得藻胆蛋白,一般仅得到一种Ｃ－ＰＣ和一种亚基组成特殊的别藻蓝蛋白ＡＰＣ。这种ＡＰＣ吸收光谱和荧光发射相同于已报告的ＡＦＣ６６０ｎｍ。但是它的亚基组成是（αα′ββ′）２而不是（α′α２β２β′）Ｌｃ１０或（αβ）３。     

Experimental and theoretical studies on the excess capacity of Photosystem II

It has been recently suggested that compensatory changes in Photosystem II (PS II) electron turnover rates can protect photosynthesis from photoinhibition [Behrenfeld et al. (1998) Photosynth Res 58: 259–268]. We have further explored this feature of PS II using a rate electrode for simultaneous measurements of the steady-state rate of oxygen evolution and the oxygen flash yield depending on the background irradiance in both control and photoinhibited algal cells of Chlorella Böhm. Theoretical simulations based on the two-electron gate model agree qualitatively with experimental data if we assume an increase of the electron turnover rate in the remaining functional PS II centers of the photoinhibited sample. Our results confirm the hypothesis that the compensatory effect enables cells to maintain the maximal rates of photosynthesis even in the presence of moderate photoinhibition (decrease of up to 50% in the number of functional centers) and that the effect originates from the inner capacity of electron transport through PS II. The origin of the compensatory effect is briefly discussed.     

Selective extraction of 22 kDa and 10 kDa polypeptides from Photosystem II without removal of 23 kDa and 17 kDa extrinsic proteins

Selective solubilization of Photosystem II membranes with the non-ionic detergent octyl thioglucopyranoside has allowed the isolation of a PS II system which has been depleted of the 22 and 10 kDa polypeptides but retains all three extrinsic proteins (33, 23 and 17 kDa). The PS II membranes which have been depleted of the 22 and 10 kDa species show high rates of oxygen evolution activity, external calcium is not required for activity and the manganese complex is not destroyed by exogenous reductants. When we compared this system to control PS II membranes, we observed a minor modification of the reducing side, and a conversion of the high-potential to the low-potential form of cytochrome b ₅₅₉.Abbreviations Chl- chlorophyll - DCBQ- 2,5-dichloro-p-benzoquinone - DCMU- 3-(3,4-dichlorophenyl)-1,1-dimethylurea - ESR- electron spin resonance - MES- 2-(N-morpholino)ethanesulfonic acid - OTG- octyl--d-thioglucopyranoside - PS II- Photosystem II - PEG- polyethylene glycol, M_r=6000 - Tris- 2-amino-2-hydroxyethylpropane-1,3-diol     

Chemical oxidation and reduction of the O2-evolution center in Photosystem II

Treatment of Photosystem II (PS II) with low concentrations of hydroxylamine is known to cause a two-flash delay in the O₂-evolution pattern, and in the formation of the S₂-state multiline EPR signal, due to the two-electron reduction of the S₁-state by hydroxylamine to form the S_-1-state. Past work has shown that these delays are not reversed by washing out the hydroxylamine nor by adding DCBQ or ferricyanide to oxidize the residual hydroxylamine, but are reversed by illumination with two saturating flashes followed by a 30-min dark incubation. We have examined the effects of treatments aimed at restoring the normal flash-induced O₂-evolution pattern and S₂-state multiline EPR signal after treatment of PS II with 40 M hydroxylamine. In agreement with past work, we find that the two-flash delay in O₂ evolution is not reversed when the hydroxylamine is removed by three cycles of centrifugation and resuspension in hydroxylamine-free buffer nor by adding ferricyanide or DCBQ to oxidize the unreacted hydroxylamine. However, the normal flash-induced O₂-evolution pattern is restored by illumination with two saturating flashes followed by a 30-min dark incubation (after the sample was first treated with 40 M hydroxylamine and the unreacted hydroxylamine was removed); illumination with one saturating flash followed by a 30-min dark incubation is only partially effective. These results show that ferricyanide and DCBQ are not effective at oxidizing the S_-1-state to the S₁-state. In contrast, adding hypochlorite (OCl^-) after treatment with hydroxylamine restored the normal flash-induced O₂-evolution pattern and also restored the formation of the S₂-state multiline EPR signal by illumination at 200 K. We conclude that hypochlorite is capable of oxidizing the S_-1-state to the S₁-state. This is the first example of a chemical treatment that advances the delayed flash-induced O₂ evolution pattern.Abbreviations DCBQ 2,5-dichloro-p-benzoquinone - OEC O₂-evolving center     

The Release of Extrinsic Polypeptides and Manganese Cluster from Photosystem 2 Membranes under High Hydrostatic Pressure

Three extrinsic polypeptides and manganese cluster were sequentially released from the membrane when photosystem 2 (PS2) membranes were kept under high hydrostatic pressure. The 17 kDa polypeptide was the most sensitive, while the 33 kDa polypeptide was the most reluctant to the treatment with high pressure. The release of manganese was not simply correlated with the loss of 33 kDa polypeptide. The losing of oxygen-evolving activity of PS2 was synchronised with the releasing of extrinsic polypeptides and manganese.     

Oxygen diffusion-concentration in erythrocyte plasma membranes studied by the fluorescence quenching of anionic and cationic pyrene derivatives

Fluorescence quenching by oxygen of cationic [pyrene-(CH₂) _n N(CH₃) ₃ ⁺ ;n=1, 4, and 11] and anionic [pyrene-(CH₂) _n CO ₂ ^– ,n=3, 8, 11, and 15] probes was investigated in erythrocyte plasma membranes (leaky) in order to assess the ability of oxygen molecules to interact with solutes located at different positions in the membrane. The pseudounimolecular quenching rate constants measured increase, both for cationic and anionic probes, whenn increases. These results are interpreted in terms of an increased oxygen solubility toward the center of the membrane interior, and imply that lateral diffusion contributes more than transverse diffusion to total oxygen mobility. For all of the probes considered, quenching rates increase whenn-alkanols are added. The effect observed increases whenn decreases and when the size of then-alkanol alkyl chain increases. Arrhenius-type plots for the quenching rate constants show noticeable downward curvatures. Average (0–40°C) activation energies are 6 kcal/mol.Abbreviations EPM erythrocyte plasma membrane - PMTMA (1-pyrenyl)methyltrimethyl-ammonium - PBTMA 4-(1-pyrenyl)butyltrimethylammonium - PUTMA 11-(1-pyrenyl)-undecyltrimethylammonium - PB 4-(1-pyrenyl)butanoate - PN 9-(1-pyrenyl)nonanoate - PD 12-(1-pyrenyl)dodecanoate - PHD 16-(1-pyrenyl)hexadecnoate     

Isolation and Characterization of the Small Subunit of the Uptake Hydrogenase from the Cyanobacterium Nostoc punctiforme

In nitrogen-fixing cyanobacteria, hydrogen evolution is associated with hydrogenases and nitrogenase, making these enzymes interesting targets for genetic engineering aimed at increased hydrogen production. Nostoc punctiforme ATCC 29133 is a filamentous cyanobacterium that expresses the uptake hydrogenase HupSL in heterocysts under nitrogen-fixing conditions. Little is known about the structural and biophysical properties of HupSL. The small subunit, HupS, has been postulated to contain three iron-sulfur clusters, but the details regarding their nature have been unclear due to unusual cluster binding motifs in the amino acid sequence. We now report the cloning and heterologous expression of Nostoc punctiforme HupS as a fusion protein, f-HupS. We have characterized the anaerobically purified protein by UV-visible and EPR spectroscopies. Our results show that f-HupS contains three iron-sulfur clusters. UV-visible absorption of f-HupS has bands ∼340 and 420 nm, typical for iron-sulfur clusters. The EPR spectrum of the oxidized f-HupS shows a narrow g = 2.023 resonance, characteristic of a low-spin (S = ½) [3Fe-4S] cluster. The reduced f-HupS presents complex EPR spectra with overlapping resonances centered on g = 1.94, g = 1.91, and g = 1.88, typical of low-spin (S = ½) [4Fe-4S] clusters. Analysis of the spectroscopic data allowed us to distinguish between two species attributable to two distinct [4Fe-4S] clusters, in addition to the [3Fe-4S] cluster. This indicates that f-HupS binds [4Fe-4S] clusters despite the presence of unusual coordinating amino acids. Furthermore, our expression and purification of what seems to be an intact HupS protein allows future studies on the significance of ligand nature on redox properties of the iron-sulfur clusters of HupS.