Electronic Excited States of the CP29 Antenna Complex of Green Plants: A Model Based on Exciton Calculations

We have suggested a model for the electronic excited states of the minorplant antenna, CP29, by incorporating a considerable part of the currentinformation offered by structure determination, site-directed mutagenesis,and spectroscopy in the modeling.We have assumed that the electronic excited states of the complex havebeen decided by the chlorophyll-chlorophyll (Chl) and Chl-proteininteractions and have modeled the Coulombic interaction between a pairof Chls in the point-dipole approximation and the Chl-protein interactionsare treated as empirical fit parameters.We have suggested the Q_y dipole moment orientations and the siteenergies for all the chlorophylls in the complex through a simultaneoussimulation of the absorption and linear dichroism spectra.The assignments proposed have been discussed to yield a satisfactoryreproduction of all prominent features of the absorption, linear and circulardichroism spectra as well as the key spectral and temporal characteristics ofthe energy transfer processes among the chlorophylls.The orientations and the spectral assignments obtained by relatively simpleexciton calculations have been necessary to provide a good point ofdeparture for more detailed treatments of structure-function relationship inCP29. Moreover, it has been discussed that the CP29 model suggested canguide the studies for a better understanding of the structure-functionrelationship in the major plant antenna, LHCII.     

Energy transfer for low temperature fluorescence in PS II mutant thylakoids

The Chl-protein complexes of three maize (Zea mays L.) mutants and one barley (Hordeum vulgare L.) mutant were analyzed using low temperature Chl fluorescence emissions spectroscopy and LDS-polyacrylamide gel electrophoresis. The maize mutants hcf-3, hcf-19, and hcf-114 all exhibited a high Chl fluorescence (hcf) phenotype indicating a disruption of the energy transfer within the photosynthetic apparatus. The mutations in each of these maize mutants affects Photosystem II. The barley mutant analyzed was the well characterized Chl b-less mutant chlorina-f2, which did not exhibit the hcf phenotype. Chlorina-f2 was used because no complete Chl b-less mutant of maize is available. Analysis of hcf-3, hcf-19, and hcf-114 revealed that in the absence of CP43, LHC II can still transfer excitation energy to CP47. These results suggest that in mutant membranes LHC II can interact with CP47 as well as CP43. This functional interaction of LHC II with CP47 may only occur in the absence of CP43, however, it is possible that LHC II is positioned in the thylakoid membranes in a manner which allows association with both CP43 and CP47.Abbreviations hcf high chlorophyll fluorescence - LDS lithium dodecyl sulfate - LHC II light-harvesting complex of Photosystem II - LHC I light-harvesting complex of Photosystem I - CPIa chlorophyll-protein complex consisting of LHC I and the PS I core complex - CPI chlorophyll-protein complex consisting of the PS I core complex - CP47 47 kDa chlorophyll-protein of the Photosystem II core - CP43 43 kDa chlorophyll-protein of the Photosystem II core - CP29 29 kDa chlorophyll-protein of Photosystem II - CP26 26 kDa chlorophyll-protein of Photosystem II - CP24 24 kDa chlorophyll-protein of Photosystem II - fp free pigments     

The roles of low temperature and light in accumulation of a 31-kDa polypeptide in the light-harvesting apparatus of maize leaves

Abstract Previously, accumulation of a 31-kDa polypeptide had been observed in the light-harvesting apparatus of thylakoids of maize leaves exposed to 5°C and high light (Hayden et al., 1986). The accumulation and disappearance of this 31-kDa polypeptide in thylakoids of maize leaves are examined as a function of photon flux density and temperature. The accumulation of large amounts of the polypeptide at 5°C was light-dependent during a 6-h chill period, with 50% of maximal accumulation occurring at a photon flux density of 60 μmol m^?2 s^?1.Some polypeptide accumulation did occur in leaves kept in the dark at 5°C for 6 h, i.e. ca. 18% of that accumulating at a photon flux density of 1500 μmol m^?2 s^?1. The temperature optimum for polypeptide accumulation was ca. 9°C with greater than 50% of maximal accumulation being achieved between 5 and 11°C. The breakdown of maximally accumulated polypeptide on returning leaves to 25°C was complete after 1 h, had a half-time of ca. 20 min and was independent of light. Breakdown of the polypeptide was also observed when thylakoids isolated from chilled leaves were incubated at 25°C. Reductions of thylakoid incubation temperature between 13 and 5°C markedly reduced the rate of polypeptide disappearance. The accumulation of the polypeptide is discussed in relation to temperature and light effects on the rate of the polypeptide synthesis and of peptidase activities. The results are also discussed in the context of accumulation of the polypeptide in maize leaves in the field and consideration is given to the possible physiological significance.     

Photosystem II antenna complexes CP26 and CP29 are essential for nonphotochemical quenching in Chlamydomonas reinhardtii

Photosystems must balance between light harvesting to fuel the photosynthetic process for CO₂ fixation and mitigating the risk of photodamage due to absorption of light energy in excess. Eukaryotic photosynthetic organisms evolved an array of pigment-binding proteins called light harvesting complexes constituting the external antenna system in the photosystems, where both light harvesting and activation of photoprotective mechanisms occur. In this work, the balancing role of CP29 and CP26 photosystem II antenna subunits was investigated in Chlamydomonas reinhardtii using CRISPR-Cas9 technology to obtain single and double mutants depleted of monomeric antennas. Absence of CP26 and CP29 impaired both photosynthetic efficiency and photoprotection: Excitation energy transfer from external antenna to reaction centre was reduced, and state transitions were completely impaired. Moreover, differently from higher plants, photosystem II monomeric antenna proteins resulted to be essential for photoprotective thermal dissipation of excitation energy by nonphotochemical quenching.     

The 28 kDa apoprotein of CP 26 in PS II binds copper

Photosystem II (PS II) particles isolated from spinach in the presence of 10 M CuSO₄ contained 1.2 copper/300 Chl that was resistant to EDTA. When CuSO₄ was not added during the isolation, PS II particles contained variable amounts of copper resistant to EDTA (0.1–1.1 copper/300 Chl). No correlation was found between copper content and oxygen evolving capacity of the PS II particles. To identify the copper binding protein, we developed a fractionation procedure which included solubilisation of PS II particles followed by precipitation with polyethylene glycol. A 22-fold purification of copper with respect to protein was achieved for a 28 kDa protein. Partial amino acid sequence of a 13 kDa fragment, obtained after V8 (endo Glu-C) protease treatment, showed identity with CP 26 over a 14 amino acid stretch. EPR measurements on the purified protein suggest oxygen and/or nitrogen as ligands for copper but tend to exclude sulfur. We conclude that the 28 kDa apoprotein of CP 26 from spinach binds one copper per molecule of CP 26. A possible function for this copper protein in the xanthophyll cycle is discussed.Abbreviations CP 26 and CP 29 chlorophyll a/b protein complex 26 and 29 - LHC II light-harvesting chlorophyll a/b protein complex of Photosystem II - SB14 sulfobetaine 14 A preliminary report of these results was presented at the IX Int. Congress on Photosynthesis, Nagoya, Japan, 1992.     

A specific binding site for neoxanthin in the monomeric antenna proteins CP26 and CP29 of Photosystem II

The location of the neoxanthin binding site in CP26 and CP29 was investigated by site-directed mutagenesis. The crystallographic structure of LHCII shows that the binding of neoxanthin to the N1 site is stabilised by an H bond with a tyrosine in the lumenal loop. This residue is conserved in CP26 and CP29. Mutation of this tyrosine into phenylalanine induced specific loss of neoxanthin without affecting violaxanthin binding. In contrast to previous proposals, it is thus concluded that also in these minor antenna complexes neoxanthin is accommodated in the N1 site. The characteristics of this binding site in the different antenna complexes are discussed.     

The structure and function of CP47 and CP43 in Photosystem II

This Minireview presents a summary of recent investigations examining the structure and functions of the Photosystem II chlorophyll-proteins CP47 and CP43, updating our previous review which appeared in 1990 (TM Bricker, Photosynth Res 24: 1–13). Since this time, numerous studies have clarified the roles of these chlorophyll-proteins within the photosystem. Biochemical, molecular and structural studies (electron and X-ray diffraction) have demonstrated the close association of these components with the photochemical reaction center of the photosystem and with the extrinsic oxygen evolution enhancer proteins. This revised version was published online in June 2006 with corrections to the Cover Date.     

The structure of photosystem II in Arabidopsis: localization of the CP26 and CP29 antenna complexes

A genetic approach has been adopted to investigate the organization of the light-harvesting proteins in the photosystem II (PSII) complex in plants. PSII membrane fragments were prepared from wild-type Arabidopis thaliana and plants expressing antisense constructs to Lhcb4 and Lhcb5 genes, lacking CP29 and CP26, respectively (Andersson et al. (2001) Plant Cell 13, 1193-1204). Ordered PS II arrays and PS II supercomplexes were isolated from the membranes of plants lacking CP26 but could not be prepared from those lacking CP29. Membranes and supercomplexes lacking CP26 were less stable than those prepared from the wild type. Transmission electron microscopy aided by single-particle image analysis was applied to the ordered arrays and the isolated PSII complexes. The difference between the images obtained from wild type and antisense plants showed the location of CP26 to be near CP43 and one of the light-harvesting complex trimers. Therefore, the location of the CP26 within PSII was directly established for the first time, and the location of the CP29 complex was determined by elimination. Alterations in the packing of the PSII complexes in the thylakoid membrane also resulted from the absence of CP26. The minor light-harvesting complexes each have a unique location and important roles in the stabilization of the oligomeric PSII structure.     

ΔpH-Dependent Fluorescence Quenching and Its Photoprotective Role in the Unicellular Red Alga Rhodella Violacea

Plants have developed various photoprotective mechanisms to resist irradiation stress. One of the photoprotective mechanisms described in the literature for LHC2-containing organisms involves a down-regulation of photosystem (PS) 2 occurring simultaneously with the build-up of a proton gradient across the thylakoid membrane (ΔpH). It is often correlated with deepoxidation of xanthophylls located in LHC2. In Rhodophyta instead of LHC2, the peripheral antenna of PS2 consists of a large extramembrane complex, the phycobilisome (PBS), which transfers its excitation to the core antennae of PS2 composed of the CP43 and CP47 protein-chlorophyll complexes and there is no xanthophyll cycle. In the red alga Rhodella violacea a ΔpH-dependent chlorophyll (Chl) a fluorescence quenching can be formed. We characterised this quenching, studied the effects of various irradiances and inhibitors. Under photoinhibitory conditions, the ΔpH-dependent Chl fluorescence quenching exerts a photoprotective role and delays the kinetics of photoinhibition. It is the first time that such a photoprotective mechanism is described in PBS-containing organisms. This revised version was published online in August 2006 with corrections to the Cover Date.     

Site-Directed Spin-Labeling Study of the Light-Harvesting Complex CP29

The topology of the long N-terminal domain (∼100 amino-acid residues) of the photosynthetic Lhc CP29 was studied using electron spin resonance. Wild-type protein containing a single cysteine at position 108 and nine single-cysteine mutants were produced, allowing to label different parts of the domain with a nitroxide spin label. In all cases, the apoproteins were either solubilized in detergent or they were reconstituted with their native pigments (holoproteins) in vitro. The spin-label electron spin resonance spectra were analyzed in terms of a multicomponent spectral simulation approach, based on hybrid evolutionary optimization and solution condensation. These results permit to trace the structural organization of the long N-terminal domain of CP29. Amino-acid residues 97 and 108 are located in the transmembrane pigment-containing protein body of the protein. Positions 65, 81, and 90 are located in a flexible loop that is proposed to extend out of the protein from the stromal surface. This loop also contains a phosphorylation site at Thr81, suggesting that the flexibility of this loop might play a role in the regulatory mechanisms of the light-harvesting process. Positions 4, 33, 40, and 56 are found to be located in a relatively rigid environment, close to the transmembrane protein body. On the other hand, position 15 is located in a flexible region, relatively far away from the transmembrane domain.     

Variability in the synthesis of cytochromes f and b-563 during the cell cycle of Euglena gracilis

The study of the successive formation of the photosynthetic apparatus in Euglena gracilis (Brandt, P. and Von Kessel, B. (1983) Plant Physiol. 72, 616–619) was extended to the determination of the stage-specific synthesis of cytochrome $\text{b}\text{f}$ complex during the cell cycle of this alga. Most of the cytochrome f (33 kDa) has properties of an intrinsic membrane protein, but part of it is soluble. Cytochrome b-563 (18 kDa) is only intrinsic. The intensity of binding the intrinsic cytochromes in the thylakoids depends on the developmental stage of the organism. The light-independent synthesis of cytochrome f takes place prior to the assembly of the chlorophyll-protein complex I (CP I). Immediately after this assembly of CP I, cytochrome b-563 is synthesized in the light. Hence, the ratio cytochrome b-563/cytochrome f changes during the cell cycle of E. gracilis. The physiological implication of presumably non-complexed cytochrome f and of complex-bound cytochromes f and b-563 on the stage-specific efficiency of photosynthesis of E. gracilis is discussed.     

Tissue-specific secretory proteins of the salivary glands of Chironomus thummi An electrophoretic and immunochemical analysis

The salivary proteins of Chironomus thummi larvae were separated by SDS gel electrophoresis and characterized by immunological techniques. As a result, five protein fractions (sp-220, sp-180, sp-35, sp-18, and sp-16) with molecular weights Mr=220000, 180000, 35000, 18000 and 16000 were identified in 6%–20% polyacrylamide gradient gels. In addition, three giant proteins fractions (molecular weights exceeding Mr=800000) were detected in composite polyacrylamide-agarose gels. Crossed immunoelectrophoresis allowed us to identify five immunochemically dissimilar organ-specific antigen fractions in the salivary gland secretion. Data were obtained indicating that the protein fractions, sp-220, sp-180, sp-35, sp-18, and sp-16, are immunochemically and structurally similar. The giant secretory proteins and the secretory fractions with low molecular weights were found to be immunochemically unrelated.     

Properties of zeaxanthin and its radical cation bound to the minor light-harvesting complexes CP24, CP26 and CP29

Nonphotochemical quenching (NPQ) is a fundamental mechanism in photosynthesis by which plants protect themselves against excess excitation energy and which is thus of crucial importance for plant survival and fitness. Recently, carotenoid radical cation (Car^•+) formation has been discovered to be a key step in the feedback deexcitation quenching component (qE) of NPQ, whose molecular mechanism and location remains elusive. A recent model for qE suggests that the replacement of violaxanthin (Vio) by zeaxanthin (Zea) in photosynthetic pigment binding pockets can in principle result in qE via the so-called “gear-shift” or electron transfer quenching mechanisms. We performed pump-probe measurements on individual antenna complexes of photosystem II (CP24, CP26 and CP29) upon excitation of the chlorophylls (Chl) into their first excited Q_y state at 660 nm when either Vio or Zea was bound to those complexes. The Chl lifetime was then probed by measuring the decay kinetics of the Chl excited state absorption (ESA) at 900 nm. The charge-transfer quenching mechanism, which is characterized by a spectral signature of the transiently formed Zea radical cation (Zea^•+) in the near-IR, has also been addressed, both in solution and in light-harvesting complexes of photosystem II (LHC-II). Applying resonant two-photon two-color ionization (R2P2CI) spectroscopy we show here the formation of β-Car^•+ in solution, which occurs on a femtosecond time-scale by direct electron transfer to the solvent. The β-Car^•+ maxima strongly depend on the solvent polarity. Moreover, our two-color two-photon spectroscopy on CP29 reveals the spectral position of Zea^•+ in the near-IR region at 980 nm.     

Antisense inhibition of the photosynthetic antenna proteins CP29 and CP26: implications for the mechanism of protective energy dissipation

The specific roles of the chlorophyll a/b binding proteins CP29 and CP26 in light harvesting and energy dissipation within the photosynthetic apparatus have been investigated. Arabidopsis was transformed with antisense constructs against the genes encoding the CP29 or CP26 apoprotein, which gave rise to several transgenic lines with remarkably low amounts of the antisense target proteins. The decrease in the level of CP24 protein in the CP29 antisense lines indicates a physical interaction between these complexes. Analysis of chlorophyll fluorescence showed that removal of the proteins affected photosystem II function, probably as a result of changes in the organization of the light-harvesting antenna. However, whole plant measurements showed that overall photosynthetic rates were similar to those in the wild type. Both antisense lines were capable of the qE type of nonphotochemical fluorescence quenching, although there were minor changes in the capacity for quenching and in its induction kinetics. High-light-induced violaxanthin deepoxidation to zeaxanthin was not affected, although the pool size of these pigments was decreased slightly. We conclude that CP29 and CP26 are unlikely to be sites for nonphotochemical quenching.     

The role of CP 47 in the evolution of oxygen and the binding of the extrinsic 33-kDa protein to the core complex of Photosystem II as determined by limited proteolysis

In order to identify the domain within Photosystem II complexes that functions in the evolution of oxygen, we performed limited proteolysis with lysylendopeptidase of the core complex of Photosystem II which had been depleted of the extrinsic 33-kDa protein (Mn-stabilizing protein). The cleavage sites were estimated from the amino-terminal sequences of the degradation fragments, their apparent molecular masses and amino-acid compositions. Under certain conditions, the D2 protein was cleaved at Lys13; and a chlorophyll a-binding protein, CP 47, was cleaved at Lys227 and Lys389. Another chlorophyll a-binding protein, CP 43, was degraded more rapidly than CP 47. The oxygen-evolving activity and the capacity for rebinding of the 33-kDa protein to the core complex of Photosystem II decreased in parallel, with kinetics very similar to those of the cleavage of CP 47 at Lys389. These observations strongly suggest that the hydrophilic domain around Lys389 of CP 47, which are located on the lumenal side, is important in the binding of the 33-kDa protein and in maintaining the oxygen-evolving activity of the Photosystem II complex.Abbreviations CP 47 and CP 43- intrinsic chlorophyll a-binding proteins with apparent molecular masses of 47 and 43 kDa, respectively - PBQ- phenyl-p-benzoquinone - TLCK- N--p-tosyl-L-lysine chloromethyl ketone     

从假根羽藻类囊体膜直接分离纯化主要捕光叶绿素a／b蛋白复合体

应用液相色谱层析技术从海生绿藻,即假根羽藻(Bryopsis corticulans Setch.)的类囊体膜直接分离纯化获得了主要捕光叶绿素a/b蛋白质复合体(LHC II)。类囊体膜提取物经3% n-Octyl-b-D-glucopyranoside去垢剂处理后,其中的LHC II蛋白质获得与Q型阴离子交换层析柱的特异亲和力,因此LHC II从类囊体膜中被高选择性分离出来。经过蔗糖密度梯度离心,获得了LHC II单体、三聚体和聚集体。多肽组分的SDS-PAGE分析证明纯化获得的LHC II单体、三聚体和寡聚体具有很高的纯度。该LHC II制备物的吸收光谱也说明了其结构的完整性。首次提出了通过少数简单步骤从类囊体膜直接分离、纯化LHC II是可以实现的。     

从假根羽藻类囊体膜直接分离纯化主要捕光叶绿素a/b蛋白复合体

应用液相色谱层析技术从海生绿藻,即假根羽藻(Bryopsis corticulans Setch.)的类囊体膜直接分离纯化获得了主要捕光叶绿素a/b蛋白质复合体(LHCⅡ).类囊体膜提取物经3％n-Octyl-b-D-glucopyranoside去垢剂处理后,其中的LHCⅡ蛋白质获得与Q型阴离子交换层析柱的特异亲和力,因此LHCⅡ从类囊体膜中被高选择性分离出来.经过蔗糖密度梯度离心,获得了LHCⅡ单体、三聚体和聚集体.多肽组分的SDS-PAGE分析证明纯化获得的LHCⅡ单体、三聚体和寡聚体具有很高的纯度.该LHCⅡ制备物的吸收光谱也说明了其结构的完整性.首次提出了通过少数简单步骤从类囊体膜直接分离、纯化LHCⅡ是可以实现的.     

Molecular characterization and gene expression of lhcb5 gene encoding CP26 in the light-harvesting complex II of Chlamydomonas reinhardtii

We isolated and sequenced a cDNA clone encoding a minor chlorophyll a/b-binding protein, CP26, which is associated with the light-harvesting complex II of Chlamydomonas reinhardtii. Protein sequences of internal peptide fragments from purified CP26 were determined and used to identify a cDNA clone. The 1.1 kb lhcb5 gene codes for a polypeptide of 289 amino acids with a predicted molecular weight of 30713. The lhcb5 gene product could reconstitute with chlorophylls and xanthophylls to form a green band on a gel. Although the expression of many lhcb genes are strictly regulated by light, the lhcb5 gene was only loosely regulated. We propose that a plant acclimatizes itself to the light environment by quantitatively and qualitatively modulating the light-harvesting complex. Characterization of the primary structure and the implications of its unique expression are discussed.     

A study of molecular interactions in light-harvesting complexes LHCIIb, CP29, CP26 and CP24 by Stark effect spectroscopy

Electric field-induced absorption changes (electrochromism or Stark effect) of the light-harvesting PSII pigment-protein complexes LHCIIb, CP29, CP26 and CP24 were investigated. The results indicate the lack of strong intermolecular interactions in the chlorophyll a (Chl a) pools of all complexes. Characteristic features occur in the electronic spectrum of Chl b, which reflect the increased values of dipole moment and polarizability differences between the ground and excited states of interacting pigment systems. The strong Stark signal recorded for LHCIIb at 650-655 nm is much weaker in CP29, where it is replaced by a unique Stark band at 639 nm. Electrochromism of Chl b in CP26 and CP24 is significantly weaker but increased electrochromic parameters were also noticed for the Chl b transition at 650 nm. The spectra in the blue region are dominated by xanthophylls. The differences in Stark spectra of Chl b are linked to differences in pigment content and organization in individual complexes and point to the possibility of electron exchange interactions between energetically similar and closely spaced Chl b molecules.     

A study of molecular interactions in light-harvesting complexes LHCIIb, CP29, CP26 and CP24 by Stark effect spectroscopy

Electric field-induced absorption changes (electrochromism or Stark effect) of the light-harvesting PSII pigment-protein complexes LHCIIb, CP29, CP26 and CP24 were investigated. The results indicate the lack of strong intermolecular interactions in the chlorophyll a (Chl a) pools of all complexes. Characteristic features occur in the electronic spectrum of Chl b, which reflect the increased values of dipole moment and polarizability differences between the ground and excited states of interacting pigment systems. The strong Stark signal recorded for LHCIIb at 650-655 nm is much weaker in CP29, where it is replaced by a unique Stark band at 639 nm. Electrochromism of Chl b in CP26 and CP24 is significantly weaker but increased electrochromic parameters were also noticed for the Chl b transition at 650 nm. The spectra in the blue region are dominated by xanthophylls. The differences in Stark spectra of Chl b are linked to differences in pigment content and organization in individual complexes and point to the possibility of electron exchange interactions between energetically similar and closely spaced Chl b molecules.