光系统II核心天线CP43的纯化及性质

菠菜放氧的ＰＳＩ核心复合物经０．８ｍｏｌ／ＬＴｒｉｓ－ＨＣｌ（ｐＨ８．０）洗涤后,用温和的非离子去垢剂ＤＭ和高浓度的ＬｉＣｌＯ４增溶,再经ＤＥＡＥ－Ｔｏｙｏｐｅａｒｌ－６５０Ｓ离子交换柱层析分离,可得到ＰＳＩＩ核心天线４３ｋＤ叶绿素ａ结合蛋白（ＣＰ４３）。ＳＤＳ－ＰＡＧＥ显示一条４３ｋＤ蛋白质带。根据Ａｒｎｏｎ法和Ｍａｒｋｗｅｌ法的结果表明,每个蛋白质分子结合有２０～２１个分子的叶绿素ａ。室温条件下,ＣＰ４３在红光区具有６７１ｎｍ的最大吸收峰和６８３ｎｍ的荧光发射峰,以及Ｗ型的圆二色信号,表明其处于较为天然的状态。文中还制备并鉴定了对ＣＰ４３特异的抗血清。     

Three-Dimensional Electron Microscopy of the Photosystem II Core Complex

A three-dimensional image of the spinach photosystem II core complex composed of CP47, D1, D2, cytochromeb-559, andpsbI gene product was reconstructed at 20-Å resolution from the two-dimensional crystals negatively stained with phosphotungstate. Confirming the previous proposal, the crystal had ap22₁2₁symmetry. One PSII core complex was measured to be 80 × 80 Å in the membrane plane and 88 Å normal to it. The mass distribution was asymmetric about the lipid bilayer, consistent with predictions from the amino acid sequences. The lumenal mass consisted of three domains forming a characteristic triangular platform with another domain on top of it. Three stromal domains were smaller and linearly arranged. Due to strong stain exclusion in the hydrophobic core part of the lipid bilayer, the transmembrane region appeared to be imaged with a reversed contrast. Inverting the contrast resulted in a reasonable density distribution for that part. Thus, though the information on the transmembrane region is limited, the domain structure of the PSII core complex was revealed and allowed us to propose a model for the arrangement of subunits in the PSII core complex.     

Isolation and Characterization of an Oxygen Evolving Photosystem 2 Core Complex from the Thermophilic Cyanobacterium Mastigocladus laminosus

A novel purification procedure was developed for the isolation of oxygen evolving photosystem 2 (PS2) from Mastigocladus laminosus. The isolation procedure involves dodecyl maltoside extraction followed by column chromatography using anion exchange resins. The isolated PS2 reaction center (RC) was analyzed for its biochemical and biophysical characteristics. Analysis by SDS polyacrylamide gel electrophoresis revealed that the complex contained five intrinsic membrane proteins (CP 47, CP 43, D1, D2, and cyt b ₅₅₉) and at least three low molecular mass proteins. The complex exhibited high rates of oxygen evolution [333 mmol(O₂) kg^–1(Chl) s^–1] in the presence of 2.5 mM 2,6-dimethylbenzoquinone (DMBQ) as an artificial electron acceptor. The red chlorophyll a absorption peak of this complex was observed at 673.5±0.2 nm. The isolated PS2 core complex was free of photosystem 1 as inferred from its SDS-PAGE and fluorescence spectrum. The electron transfer properties of the Mastigocladus cells and the purified PS2 core complex were further probed by measuring thermoluminescence signals, which indicated the presence of a primary quinone electron acceptor (Q_A) in the purified PS2 core complex.     

Subunit Organization of a Synechocystis Hetero-Oligomeric Thylakoid FtsH Complex Involved in Photosystem II Repair

FtsH metalloproteases are key components of the photosystem II (PSII) repair cycle, which operates to maintain photosynthetic activity in the light. Despite their physiological importance, the structure and subunit composition of thylakoid FtsH complexes remain uncertain. Mutagenesis has previously revealed that the four FtsH homologs encoded by the cyanobacterium Synechocystis sp PCC 6803 are functionally different: FtsH1 and FtsH3 are required for cell viability, whereas FtsH2 and FtsH4 are dispensable. To gain insights into FtsH2, which is involved in selective D1 protein degradation during PSII repair, we used a strain of Synechocystis 6803 expressing a glutathione S-transferase (GST)–tagged derivative (FtsH2-GST) to isolate FtsH2-containing complexes. Biochemical analysis revealed that FtsH2-GST forms a hetero-oligomeric complex with FtsH3. FtsH2 also interacts with FtsH3 in the wild-type strain, and a mutant depleted in FtsH3, like ftsH2⁻ mutants, displays impaired D1 degradation. FtsH3 also forms a separate heterocomplex with FtsH1, thus explaining why FtsH3 is more important than FtsH2 for cell viability. We investigated the structure of the isolated FtsH2-GST/FtsH3 complex using transmission electron microscopy and single-particle analysis. The three-dimensional structural model obtained at a resolution of 26 Å revealed that the complex is hexameric and consists of alternating FtsH2/FtsH3 subunits.     

光系统II核心复合物的稳态荧光光谱(英文)

在 83K 和 160K 两个温度下,通过激发波长对荧光发射谱的影响研究了光系统II中核心复合物的荧光光谱特性。用不同波长的光激发,核心复合物的发射谱的最大发射峰值不变,用 480、489、495 和 507nm 的光分别激发核心复合物,其光谱最大峰值处的荧光强度随不同激发波长下β-胡萝卜素分子的吸收强度的增大而降低,在长波长区域光谱的变化依赖于首先被激发的色素分子。所以,激发波长的不同影响着核心复合物中能量传递的途径。通过高斯解析,分析出核心复合物中至少存在有 7组叶绿素a组分,它们是Chla660,Chla670,Chla680,Chla682,Chla684,Chla687和Chla690。     

Characterization of a 47-Kilodalton Chlorophyll-Binding Polypeptide (CP-47) Isolated from a Photosystem II Core Complex

The purified photosystem II core complex from spinach with aparticle size of about 480 kDa and containing five constituentpolypeptides was further resolved by octyl-rß-D-glucopyranosidetreatment followed by separation by high-performance liquidchromatography using a gel-permeation column. Of the four clearlyseparated, chlorophyll-containing fractions, one with a particlesize of 170–180 kDa was composed entirely of a single,47-kDa polypeptide. This chlorophyll a-polypeptide containsrß-carotene and pheophytin a, but no plastoquinone.The number of chlorophyll a associated with this polypeptidein situ was estimated to be 6–7 and an oligomeric structureof this polypeptide in vivo was proposed on the basis of itschlorophyll/protein ratio and the isolated particle size. Thecomplex exhibited F-695 emission, but was photochemically inactive.The amino acid composition of the apoprotein was also determined. (Received March 12, 1984; Accepted June 7, 1984)     

Fast Isolation of Highly Active Photosystem Ⅱ Core Complexes from Spinach

Purification of photosystem Ⅱ (PSII) core complexes is a time-consuming and low-efficiency process. In order to isolate pure and active PSII core complexes in large amounts, we have developed a fast method to isolate highly active monomeric and dimeric PSII core complexes from spinach leaves by using sucrose gradient ultracentrifugation. By using a vertical rotor the process was completed significantly faster compared with a swing-out rotor. In order to keep the core complexes in high activity, the whole isolation procedure was performed in the presence of glycine betain and pH at 6.3. The isolated pigment-protein complexes were characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, absorption spectroscopy, 77 K fluorescence spectroscopy and high performance liquid chromatography. Our results show that this method is a better choice for quick and efficient isolation of functionally active PSII core complexes.     

Biochemical Evidence that the Higher Plant Photosystem II Core Complex is Organized as a Dimer

The organization of the pigmented multiprotein core complexof higher plant PS II has been examined. Oxygen-evolving PSII particles or thylakoid membranes of wild-type and Chi b-lessbarley were extracted with various glycosidic surfactants andelectrophoretically fractionated. The predominant multiproteincore complex II (CC II) fractions had sizes on gel electrophoresisof M_r=230,000 and M_r= 140,000 and were photochemically active.Both fractions had identical absorption spectra, contained thebeta-carotene-chl a-proteins (Cp47 and Cp43), the PS II reactioncenter subunits (Dl and D2), and the two cytochrome b₅₅₉ subunitsin unit stoichiometry. The M_r=230,000 fraction could evolveoxygen in the light and contained an M_r=33,O0O oxygen evolutionenhancer (OEE 33) polypeptide, whereas the M_r= 140,000 fractionlacked OEE 33 and could not evolve oxygen. The apparent sizesof the two fractions were also estimated by gel filtration asM_r=490,000 and M_r=220,000, respectively; the estimates by gelfiltration more accurately reflect their predicted sizes. Furtheranalyses by nondenaturing gel electrophoresis indicated thatCp47, Cp43 and the three OEE gene products probably occur ashomodimers in situ. Our data suggest that phosphorylation ofCC II subunits occurs when they are located in the oligomericform. We propose that the native state of the PS II core complexin higher plants is dimeric, and that this state, which waspreviously observed only in thermophilic cyanobacteria, is probablythe form present in all oxygenic organisms. (Received August 9, 1991; Accepted September 26, 1991)     

Isolation and Characterization of a Light-Harvesting Chlorophyll a/b Protein Complex Associated with Photosystem I

A chlorophyll a/b protein complex has been isolated from a resolved native photosystem I complex by mildly dissociating sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The chlorophyll a/b protein contains a single polypeptide of molecular weight 20 kilodaltons, and has a chlorophyll a/b ratio of 3.5 to 4.0. The visible absorbance spectrum of the chlorophyll a/b protein complex showed a maximum at 667 nanometers in the red region and a 77 K fluorescence emission maximum at 681 nanometers. Alternatively, by treatment of the native photosystem I complex with lithium dodecyl sulfate and Triton, the chlorophyll a/b protein complex could be isolated by chromatography on Sephadex G-75. Immunological assays using antibodies to the P₇₀₀-chlorophyll a-protein and the photosystem II light-harvesting chlorophyll a/b protein show no cross-reaction between the photosystem I chlorophyll a/b protein and the other two chlorophyll-containing protein complexes.     

The Water Oxidation Complex of Chlamydomonas: Accumulation and Maturation of the Largest Subunit in Photosystem II Mutants

Photosystem II particles of Chlamydomonas reinhardtii contain three extrinsic polypeptides of 29, 20, and 16 kilodaltons, whose functions are incompletely defined. We prepared a monospecific polyclonal antibody against the 29 kilodalton protein and determined that it also specifically recognizes a protein of approximately 33 kilodaltons in thylakoid membrane fractions of several vascular plants, eukaryotic algae, and a cyanobacterium. The cross-reacting 33 kilodalton protein of pea was removed from inverted thylakoid vesicles by CaCl₂ washes demonstrating the structural relationship between the Chlamydomonas polypeptide and the largest subunit of the water oxidation complex of vascular plants. Functional identity of the Chlamydomonas polypeptide was confirmed by antibody inhibition of O₂ evolution in inverted pea vesicles. In contrast to wild-type cells, only low levels of the 29 kilodalton polypeptide are recovered with purified thylakoid membranes of the mutants examined. However, we show that the mature form of the 29 kilodalton polypeptide accumulates to wild-type levels in whole cell extracts of photosystem II deficient mutants and a water oxidation mutant of Chlamydomonas. Impaired membrane assembly has no effect on the maturation or stability of this component of the multi-subunit water oxidation complex.     

Photosystem II Complex in Vivo Is a Monomer

Photosystem II (PS II) complexes are membrane protein complexes that are composed of >20 distinct subunit proteins. Similar to many other membrane protein complexes, two PS II complexes are believed to form a homo-dimer whose molecular mass is ∼650 kDa. Contrary to this well known concept, we propose that the functional form of PS II in vivo is a monomer, based on the following observations. Deprivation of lipids caused the conversion of PS II from a monomeric form to a dimeric form. Only a monomeric PS II was detected in solubilized cyanobacterial and red algal thylakoids using blue-native polyacrylamide gel electrophoresis. Furthermore, energy transfer between PS II units, which was observed in the purified dimeric PS II, was not detected in vivo. Our proposal will lead to a re-evaluation of many crystallographic models of membrane protein complexes in terms of their oligomerization status.Photosystem II (PS II)3 complexes convert solar energy to biological redox energy. Through this reaction process, water molecules are oxidized and molecular oxygen is released as a byproduct (reviewed in Ref. 1), which is the only source of molecular oxygen upon which all aerobic organisms on earth rely. PS II core complexes are membrane protein complexes that are composed of >20 distinct subunit proteins and many functional cofactors, including chlorophylls (Chls), carotenoids, plastoquinone, and metal ions (2–5). Similar to many other membrane protein complexes (6–10), two PS II core complexes are believed to associate together to form a homo-dimer with a molecular mass of ∼650 kDa, as shown by crystallographic models (2, 3, 5).The PS II complex turns over dynamically, although it is quite an integrated complex; our current understanding is that the PS II complex that is damaged by high light is disintegrated into a monomeric form and is further dissociated to replace a degraded D1 protein with a de novo synthesized D1 (reviewed in Refs. 11 and 12). After the replacement, the PS II complex is integrated into a functional form as a dimer. It is supposed that PS II subunit proteins such as PsbI (13) or PsbTc (14) participate in the formation of the PS II dimer.Crystallographic models of PS II have enabled the determination of the accurate molecular architecture of PS II complexes, all of which are in a dimeric form. The most recent crystallographic model of the PS II dimer at 3.0-Å resolution revealed the presence of six detergent molecules located at the interface of the two monomers (5). Small structural fluctuations during the purification process might allow the invasion of those detergent molecules. However, it is also probable that the PS II complexes exist in the form of a monomer in vivo and the two distinct monomers become a dimer during the purification step incorporating detergents between their interfaces. This idea led us to investigate the actual form of PS II in vivo. Contrary to the above well known dimeric model of a functional PS II core complex, here we show that the PS II core complex functions and exists in a monomeric form in vivo.     

Putative Second Binding Site of DCMU on the Oxidizing Side of Photosystem II in Photosystem II Membranes Depleted of Functional Mn

The effects of DCMU on the oxidizing side of PS II were studiedwith Triton-solubilized PS II membranes depleted of functionalMn. 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) non-competitivelyinhibited the diphenylcarbazide-supported (DPC-supported) photoreductionof silicomolybdate (SiMo) at concentrations more than ten timeshigher than that required for inhibition of the DPC-supportedphotoreduction of 2,6-dichlorophenolindophenol (DCIP). The maximumfluorescence intensity was also reduced by DCMU at a similarconcentration to that required for the inhibition of the SiMophotoreduction. These findings suggest two inhibitory sitesof action of DCMU in PS II: one on the reducing side and oneon the oxidizing side of PS II. The inhibition constant forDCMU in the DPC-supported SiMo-photoreduction was 10 µMin every examination. The extent of inhibition was attenuatedby modifications of the PS II oxidizing side by the presenceof functional Mn, by photoinhibition and by chemical modificationsof histidine residues and acidic amino acid residues. Our resultssuggest that DCMU binds to the PS II oxidizing side near Z,D and the high-affinity Mn-binding sites. ¹ Present Address and address for all communications: NoriakiTamura (Dr.), Plant Physiology Laboratory Fukuoka Women's University,Kasumigaoka 1-1, Higashi-ku, Fukuoka, 813 Japan. FAX 092-661-2415.     

光系统II核心天线复合物CP43和CP47结构与功能研究进展

CP43和CP47是构成光合生物内周天线的两个重要的色素蛋白复合物,在生物体内主要起着传递激发能的作用。最近,大量研究证明,它们在放氧等过程中也起着重要作用。因此,近年来人们借助各种先进的研究技术对它们的结构进行了探讨,以揭示它们行使不同生理功能的分子机理。分子生物学技术可以使人们在整体水平上研究蛋白复合物的结构与功能,因此是一个非常有用的研究手段。本文即对近年来人们通过分子生物学手段,以蓝藻为转化材料,通过基因定点突变技术对CP43和CP47结构和功能的研究结果进行了全面综述,并进行了点评和分析,从而提出了一些新问题,为人们进行深入研究提供了详尽的研究资料和建设性的思路。     

光系统п核心天线复合物CP43和CP47结构与功能研究进展

ＣＰ４３和ＣＰ４７是构成光合生物内周天线的两个重要的色素蛋白复合物,在生物体内主要起着传递激发能的作用。最近,大量研究证明,它们在放氧等过程中也起着重要作用。因此,近年来人们借助各种先进的研究技术对它们的结构进行了探讨,以揭示它们行使不同生理功能的分子机理。分子生物学技术可以使人们在整体水平上研究蛋白复合物的结构与功能,因此是一个非常有用的研究手段。本文即对近年来人们通过分子生物手段,以蓝藻为转化     

Properties of Chlamydomonas Photosystem II Core Complex with a His-Tag at the C-Terminus of the D2 Protein

A His-tagged PSII core complex was purified from recombinantChlamydomonas reinhardtii D2-H thylakoids by single-step Ni²⁺-affinitycolumn chromatography and its properties were partially characterizedin terms of their PSII functions and chemical compositions.The PSII core complex that has a His-tag extension at the C-terminusof the D2 protein evolved oxygen at a high rate of 2,400 µmol(mg Chl)^–1h^–1 at the optimum pH of 6.5 with ferricyanideand 2,6-dichlorobenzoquinone as electron acceptors in the presenceof Ca²⁺ as an essential cofactor, and approximately 90% of theactivity was blocked by 10 µM DCMU. The core complex exhibitedthe thermoluminescence Q-band but not the B-band regardlessof the presence or absence of DCMU, although both bands wereobserved in the His-tagged thylakoids. The core complex wasfree from PSI and contained one Y_D, Tyr 160 of the D2 protein,four Mn atoms, two cytochrome b-559, about 46 Chl a molecules,and probably one Q_A, the primary acceptor quinone of PSII. Itwas inferred from these results that His-tagging at the C-terminusof the D2 protein does not affect the functional and structuralintegrity of the PSII core complex, and that the ‘His-tagstrategy’ is highly useful for biochemical, physicochemical,and structural studies of Chlamydomonas PSII. (Received October 22, 1998; Accepted December 25, 1998)     

Functional Analyses of the Plant Photosystem I-Light-Harvesting Complex II Supercomplex Reveal That Light-Harvesting Complex II Loosely Bound to Photosystem II Is a Very Efficient Antenna for Photosystem I in State II

State transitions are an important photosynthetic short-term response that allows energy distribution balancing between photosystems I (PSI) and II (PSII). In plants when PSII is preferentially excited compared with PSI (State II), part of the major light-harvesting complex LHCII migrates to PSI to form a PSI-LHCII supercomplex. So far, little is known about this complex, mainly due to purification problems. Here, a stable PSI-LHCII supercomplex is purified from Arabidopsis thaliana and maize (Zea mays) plants. It is demonstrated that LHCIIs loosely bound to PSII in State I are the trimers mainly involved in state transitions and become strongly bound to PSI in State II. Specific Lhcb1-3 isoforms are differently represented in the mobile LHCII compared with S and M trimers. Fluorescence analyses indicate that excitation energy migration from mobile LHCII to PSI is rapid and efficient, and the quantum yield of photochemical conversion of PSI-LHCII is substantially unaffected with respect to PSI, despite a sizable increase of the antenna size. An updated PSI-LHCII structural model suggests that the low-energy chlorophylls 611 and 612 in LHCII interact with the chlorophyll 11145 at the interface of PSI. In contrast with the common opinion, we suggest that the mobile pool of LHCII may be considered an intimate part of the PSI antenna system that is displaced to PSII in State I.     

An Oxygen-Evolving Photosystem II Complex Associated with the Core Substructure of the Phycobilisome from Synechococcus elongatus

Oxygen-evolving photosystem II (PS II) particles isolated fromthe thermophilic cyanobacterium Synechococcus elongatus consistedof about twenty polypeptides. Six polypeptides were identifiedby reaction with specific antisera as constituent subunit polypeptidesof oxygen-evolving PS II reaction center complexes. The mostabundant polypeptides were the and ß subunits ofallophycocyanin. Comparison with the polypeptide profile ofisolated phycobilisomes, as well as immunoblotting with an antiserumagainst the large linker polypeptide, showed that the largelinker polypeptide or some proteolytic fragments of it werepresent in the preparation. Thus, each PS II particle is, inessence, an oxygen-evolving PS II complex that is associatedwith the core substructure of the phycobilisome. Cross-linkingexperiments indicated that fragments of the large linker polypeptidesare closely associated with one another and that the Chl-carrying47- kDa polypeptide is located in close proximity to the D2protein and the extrinsic 33-kDa protein. (Received November 12, 1991; Accepted January 23, 1992)     

Effects of Light Stress on Redox Potential Forms of Cyt b-559 in Photosystem II Membranes Depleted of Water-Oxidizing Complex

Effects of photoinhibition on the redox properties of Cyt b-559were studied with NH₂OH treated PSII membranes, which are depletedof the water-oxidizing complex. The membranes contained threeredox forms (HP-, IP- and LP-forms) of Cyt b-559, with E_m valuesof +435, +237 and +45 mV, respectively. A novel intermediate-potentialform of Cyt b-559 was generated during photoinhibition on thedonor side of PSII: photoinhibitory illumination (7,000 µEm^–2 s^–1) for 1 min induced a 30% decrease in thelevel of the HP-form, with concomitant generation of the intermediate-potential(IP-) form whose E_m value was about +350mV. Prolonged illumination(10 min) resulted in complete loss of the HP-form and an apparentincrease in the level of the IPform. After further photoinhibitorytreatment (60 min), complete loss of the IP'-form was observedand levels of the IP- and LP-forms each increased to about 50%of the total amount of Cyt b-559. Kinetic analysis of thesedata led to the conclusion that the HP-form is converted tothe LP-form via two intermediate-potential forms (IP' and IP),and that IP'-form appears only at the early phase of photoinhibition. (Received March 30, 1994; Accepted February 27, 1995)