Antibodies to the photosystem I chlorophyll a + b antenna cross-react with polypeptides of CP29 and LHCII

A chlorophyll (a + b)--protein complex associated with photosystem I (PSI) was isolated from a larger PSI complex (CPIa) produced by electrophoresis of barley thylakoids solubilized with 300 mM octyl glucoside. It had an apparent Mr of 35,000-43,000 on 7.5% and 10% acrylamide gels respectively, and a chlorophyll a/b ratio of 2.5 +/- 1.5. Denaturation released four polypeptides migrating between 21-24 kDa. They were well separated from the polypeptides of the two photosystem II chlorophyll a + b antenna complexes: LHCII (25-27 kDa) and CP29 (28-29 kDa). In order to study the PSI antenna complex, antibodies were raised against highly purified CPIa. The antigen appeared to be pure when electrophoresed, blotted and reacted with its antiserum, i.e. anti-CPIa detected only the 64-66-kDa CPI apoprotein and the four 21-24 kDa antenna polypeptides. However, when blotted against the whole spectrum of thylakoid proteins, it cross-reacted with both LHCII and CP29 apoproteins. Removal of anti-CPI activity from the anti-CPIa did not affect these cross-reactions, showing that they were not due to antibodies directed against CPI. To show that the same antibody population was reacting with both the photosystem I and photosystem II antenna polypeptides, anti-CPIa was adsorbed onto highly purified CPIa on nitrocellulose. The bound antibody was eluted and used again in a Western blot against whole thylakoid proteins. This selected antibody population showed the same relative strength of reaction with photosystem I and photosystem II antenna polypeptides as the original antibody population had. Similar observations have been made with antibodies to the two photosystem II antenna complexes. We therefore conclude that there are antigenic determinants in common among the chlorophyll a + b binding polypeptides, and predict that there could be amino acid sequence similarities.     

Photosystem II assembly in CP47 mutant of Synechocystis sp. PCC 6803 is dependent on the level of chlorophyll precursors regulated by ferrochelatase

Accumulation of chlorophyll and expression of the chlorophyll (Chl)-binding CP47 protein that serves as the core antenna of photosystem II are indispensable for the assembly of a functional photosystem II. We have characterized the CP47 mutant with an impaired photosystem II assembly and its two spontaneous pseudorevertants with their much improved photoautotrophic growth. The complementing mutations in these pseudorevertants were previously mapped to the ferrochelatase gene (1). We demonstrated that complementing mutations dramatically decrease ferrochelatase activity in pseudorevertants and that this decrease is responsible for their improved photoautotrophic growth. Photoautotrophic growth of the CP47 mutant was also restored by in vivo inhibition of ferrochelatase by a specific inhibitor. The decrease in ferrochelatase activity in pseudorevertants was followed by increased steady-state levels of Chl precursors and Chl, leading to CP47 accumulation and photosystem II assembly. Similarly, supplementation of the CP47 mutant with the Chl precursor Mg-protoporphyrin IX increased the number of active photosystem-II centers, suggesting that synthesis of the mutated CP47 protein is enhanced by an increased Chl availability in the cell. The probable role of ferrochelatase in the regulation of Chl biosynthesis is discussed.     

Energy transfer processes in the isolated core antenna complexes CP43 and CP47 of photosystem II

The energy equilibration and transfer processes in the isolated core antenna complexes CP43 and CP47 of photosystem II have been studied by steady-state and ultrafast (femto- to nanosecond) time-resolved spectroscopy at room temperature. The annihilation-free femtosecond absorption data can be described by surprisingly simple sequential kinetic models, in which the excitation energy transfer between blue and red states in both antenna complexes is dominated by sub-picosecond processes and is completed in less than 2 ps. The slowest energy transfer steps with lifetimes in the range of 1-2 ps are assigned to transfer steps between the chlorophyll layers located on the stromal and lumenal sides. We conclude that these ultrafast intra-antenna energy transfer steps do not represent a bottleneck in the rate of the primary processes in intact photosystem II. Since the experimental energy equilibration rates are up to a factor of 3-5 higher than concluded previously, our results challenge the conclusions drawn from theoretical modeling.     

Cold-Resistant and Cold-Sensitive Maize Lines Differ in the Phosphorylation of the Photosystem II Subunit,CP29

The effects of low temperature on the relative contributions of the reaction center and the antenna activities to photosystem II (PSII) electron transport were estimated by chlorophyll fluorescence. The inhibition of PSII photochemistry resulted from photo-damage to the reaction center and/or a reduced probability of excitation energy trapping by the reaction center. Although chill treatment did not modify the proportion of the dimeric to monomeric PSII, it destabilized its main light-harvesting complex. Full protection of the reaction center was achieved only in the presence of the phosphorylated PSII subunit, CP29. In a nonphosphorylating genotype the chill treatment led to photoinhibitory damage. The phosphorylation of CP29 modified neither its binding to the PSII core nor its pigment content. Phosphorylated CP29 was isolated by flat-bed isoelectric focusing. Its spectral characteristics indicated a depletion of the chlorophyll spectral forms with the highest excitation transfer efficiency to the reaction center. It is suggested that phosphorylated CP29 performs its regulatory function by an yet undescribed mechanism based on a shift of the equilibrium for the excitation energy toward the antenna.     

Phosphorylation of spinach chlorophyll-protein complexes. CPII, but not CP29, CP27, or CP24, is phosphorylated in vitro

Previous studies have indicated that the reversible phosphorylation of a population of antenna complexes that can donate energy to PS II ('mobile LHC II') plays a regulatory role in the state 1-state 2 transition in thylakoid membranes. The relationship of phosphorylated LHC II to the multiple PS II-associated chlorophyll a/b-proteins resolvable on green gels is currently unclear. We have used a high resolution gel system to analyze thylakoids phosphorylated in vitro. The only PS II-associated antenna complex to become phosphorylated is CPII, indicating that this complex represents the mobile LHC II. The other putative PS II antenna complexes, CP29, CP24, and the new complex designated CP27 which comigrates with CPII, are not phosphorylated and are probably components of the bound 'LHC II' antenna.     

Pathways for energy transfer in the core light-harvesting complexes CP43 and CP47 of photosystem II

The pigment-protein complexes CP43 and CP47 transfer excitation energy from the peripheral antenna of photosystem II toward the photochemical reaction center. We measured the excitation dynamics of the chlorophylls in isolated CP43 and CP47 complexes at 77 K by time-resolved absorbance-difference and fluorescence spectroscopy. The spectral relaxation appeared to occur with rates of 0.2-0.4 ps and 2-3 ps in both complexes, whereas an additional relaxation of 17 ps was observed only in CP47. Using the 3.8-A crystal structure of the photosystem II core complex from Synechococcus elongatus (A. Zouni, H.-T. Witt, J. Kern, P. Fromme, N. Krauss, W. Saenger, and P. Orth, 2001, Nature, 409:739-743), excitation energy transfer kinetics were calculated and a Monte Carlo simulation of the absorption spectra was performed. In both complexes, the rate of 0.2-0.4 ps can be ascribed to excitation energy transfer within a layer of chlorophylls near the stromal side of the membrane, and the slower 2-3-ps process to excitation energy transfer to the calculated lowest excitonic state. We conclude that excitation energy transfer within CP43 and CP47 is fast and does not contribute significantly to the well-known slow trapping of excitation energy in photosystem II.     

Chlorophyll-proteins of the photosystem II antenna system

The chlorophyll-protein complexes of purified maize photosystem II membranes were separated by a new mild gel electrophoresis system under conditions which maintained all of the major chlorophyll a/b-protein complex (LHCII) in the oligomeric form. This enabled the resolution of three chlorophyll a/b-proteins in the 26-31-kDa region which are normally obscured by monomeric LHCII. All chlorophyll a/b-proteins had unique polypeptide compositions and characteristic spectral properties. One of them (CP26) has not previously been described, and another (CP24) appeared to be identical to the connecting antenna of photosystem I (LHCI-680). Both CP24 and CP29 from maize had at least one epitope in common with the light-harvesting antennae of photosystem I, as shown by cross-reactivity with a monoclonal antibody raised against LHCI from barley thylakoids. A complex designated Chla.P2, which was capable of electron transport from diphenylcarbazide to 2,6-dichlorophenolindophenol, was isolated by nondenaturing gel electrophoresis. It lacked CP43, which therefore can be excluded as an essential component of the photosystem II reaction center core. Fractionation of octyl glucoside-solubilized photosystem II membranes in the presence and absence of Mg2+ enabled the isolation of the Chla . P2 complex and revealed the existence of a light-harvesting complex consisting of CP29, CP26, and CP24. This complex and the major light-harvesting system (LHCII) are postulated to transfer excitation energy independently to the photosystem II reaction center via CP43.     

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.     

A study of the relation between CP29 phosphorylation, zeaxanthin content and fluorescence quenching parameters in Zea mays leaves

The hypothesis that phosphorylation of the minor photosystem II antenna complex CP29 (CP34 formation) in Zea mays (cv. Dekalb DK300), under conditions of illumination and low temperature stress, may constitute a protective mechanism against photoinhibition, has been investigated. It is demonstrated that illumination at low temperature induces a marked increase in reversible non‐photochemical quenching yield of chlorophyll fluorescence, together with CP34 formation. These two parameters, however, are not related as CP34 dephosphorylates to CP29 in the dark, with a half‐time of about 10 min, while the enhanced non‐photochemical quenching yield is stable for many hours. The enhanced non‐photochemical quenching yield seems to correlate with zeaxanthin formation. The influence of CP34 formation on photoinhibition was also directly investigated. No measurable effect on this parameter could be observed after treatment with high light. It is concluded that CP34 is probably not directly involved in photoprotective processes.     

Photosynthetic acclimation: structural reorganisation of light harvesting antenna--role of redox-dependent phosphorylation of major and minor chlorophyll a/b binding proteins

In order to carry out photosynthesis, plants and algae rely on the co-operative interaction of two photosystems: photosystem I and photosystem II. For maximum efficiency, each photosystem should absorb the same amount of light. To achieve this, plants and green algae have a mobile pool of chlorophyll a/b-binding proteins that can switch between being light-harvesting antenna for photosystem I or photosystem II, in order to maintain an optimal excitation balance. This switch, termed state transitions, involves the reversible phosphorylation of the mobile chlorophyll a/b-binding proteins, which is regulated by the redox state of the plastoquinone-mediating electron transfer between photosystem I and photosystem II. In this review, we will present the data supporting the function of redox-dependent phosphorylation of the major and minor chlorophyll a/b-binding proteins by the specific thylakoid-bound kinases (Stt7, STN7, TAKs) providing a molecular switch for the structural remodelling of the light-harvesting complexes during state transitions. We will also overview the latest X-ray crystallographic and electron microscopy-derived models for structural re-arrangement of the light-harvesting antenna during State 1-to-State 2 transition, in which the minor chlorophyll a/b-binding protein, CP29, and the mobile light-harvesting complex II trimer detach from the light-harvesting complex II-photosystem II supercomplex and associate with the photosystem I core in the vicinity of the PsaH/L/O/P domain.     

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.     

Energy transfer of aromatic amino acids in photosystem 2 core antenna complexes CP43 and CP47

Energy transfer of aromatic amino acids in photosystem 2 (PS2) core antenna complexes CP43 and CP47 was studied using absorption spectroscopy, fluorescence spectroscopy, and the 0.35 nm crystal structure of PS2 core complex. The energy of tyrosines (Tyrs) was not effectively transferred to tryptophans (Trps) in CP43 and CP47. The fluorescence emission spectrum of CP43 and CP47 by excitation at 280 nm should be a superposition of the Tyr and Trp fluorescence emission spectra. The aromatic amino acids in CP43 and CP47 could transfer their energy to chlorophyll (Chl) a molecules by the Dexter mechanism and the Föster mechanism, and the energy transfer efficiency in CP47 was much higher than that in CP43. In CP47 the Föster mechanism must be the dominant energy transfer mechanism between aromatic amino acids and Chl a molecules, whereas in CP43 the Dexter mechanism must be the dominant one. Hence solar ultraviolet radiation brings not only damages but also benefits to plants.     

Spectroscopic characterization of the spinach Lhcb4 protein (CP29), a minor light-harvesting complex of photosystem II.

A spectroscopic characterization is presented of the minor photosystem II chlorophyll a/b-binding protein CP29 (or the Lhcb4 protein) from spinach, prepared by a modified form of a published protocol [Henrysson, T., Schroder, W. P., Spangfort, M. & Akerlund, H.-E. (1989) Biochim. Biophys. Acta 977, 301-308]. The isolation procedure represents a quicker, cheaper means of isolating this minor antenna protein to an equally high level of purity to that published previously. The pigment-binding protein shows similarities to other related light-harvesting complexes (LHCs), including the bulk complex LHCIIb but more particularly another minor antenna protein CP26 (Lhcb5). It is also, in the main, similar to other preparations of CP29, although some significant differences are discussed. In common with CP26, the protein binds about six chlorophyll a and two chlorophyll b molecules. Two chlorophyll b absorption bands are present at 638 and 650 nm and they are somewhat more pronounced than in a recent report [Giuffra, E., Zucchelli, G., Sandonà, D., Croce, R., Cugini, D., Garlaschi, F.M., Bassi, R. & Jennings, R.C. (1997) Biochem. 36, 12984-12993]. The bands give rise to positive and negative linear dichroism, respectively; both show negative CD bands (cf. bands with similar properties at 637 and 650 nm in CP26). Chlorophyll a absorption is dominated by a large contribution at 674 nm which also shows similarities to the major band in LHCIIb and CP26, while (as for CP26) a reduction in absorption around 670 nm is observed relative to the bulk complex. Principal differences from LHCIIb and CP26, and from other CP29 preparations, occur in the carotenoid region.     

Isolation of PS II reaction centre and its relationship to the minor chlorophyll-protein complexes

Evidence is presented for the identification of the chlorophyll- protein complex CPa-1 (CP 47) as the reaction centre of photosystem II (PS II). We have developed a simple, rapid method using octyl glucoside solubilization to obtain preparations from spinach and barley that are highly enriched in PS II reaction centre activity (measured as the light-driven reduction of diphenylcarbazide by 2,6-dichlorophenolindophenol). These preparations contain only the two minor chlorophyll-protein complexes CPa-1 and CPa-2. During centrifugation on a sucrose density gradient, there is a partial separation of the two CPa complexes from each other, and a complete separation from other chlorophyll-protein complexes. The PS II activity comigrates with CPa-1 but not CPa-2, strongly suggesting that the former is the reaction centre complex of PS II. Reaction centre preparations are sensitive to the herbicide 3(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU), but only at much higher concentrations than those required to inhibit intact thylakoid membranes. A model of PS II incorporating our current knowledge of the chlorophyll-protein complexes is presented. It is proposed that CPa-2 and the chlorophyll a + b complex CP 29 may function as internal antenna complexes surrounding the reaction centre, with the addition of variable amounts of the major chlorophyll a + b light-harvesting complex.     

Time-resolved fluorescence analysis of the recombinant photosystem II antenna complex CP29. Effects of zeaxanthin, pH and phosphorylation.

Nonradiative dissipation of excitation energy is the major photoprotective mechanism in plants. The formation of zeaxanthin in the antenna of photosystem II has been shown to correlate with the onset of nonphotochemical quenching in vivo. We have used recombinant CP29 protein, over-expressed in Escherichia coli and refolded in vitro with purified pigments, to obtain a protein indistinguishable from the native complex extracted from thylakoids, binding either violaxanthin or zeaxanthin together with lutein. These recombinant proteins and the native CP29 were used to measure steady-state chlorophyll fluorescence emission and fluorescence decay kinetics. We found that the presence of zeaxanthin bound to CP29 induces a approximately 35% decrease in fluorescence yield with respect to the control proteins (the native and zeaxanthin-free reconstituted proteins). Fluorescence decay kinetics showed that four components are always present but lifetimes (tau) as well as relative fluorescence quantum yields (rfqy) of the two long-lived components (tau3 and tau4) are modified by the presence of zeaxanthin. The most relevant changes are observed in the rfqy of tau3 and in the average lifetime ( approximately 2.4 ns with zeaxanthin and 3.2-3.4 ns in the control proteins). When studied in vitro, no significant effect of acidic pH (5.2-5.3) is observed on chlorophyll A fluorescence yield or kinetics. The data presented show that recombinant CP29 is able to bind zeaxanthin and this protein-bound zeaxanthin induces a significant quenching effect.     

Dynamic Interaction between the D1 protein,CP43 and OEC33 at the lumenal side of photosystem II in spinach chloroplasts: evidence from light-induced cross-Linking of the proteins in the donor-side photoinhibition

During the donor-side photoinhibition of spinach photosystem II, the reaction center D1 protein cross-linked with the antenna chlorophyll binding protein CP43 of photosystem II lacking the oxygen-evolving complex (OEC) subunit proteins. The cross-linking did not occur upon illumination of photosystem II samples that retained the OEC33, nor when OEC33-depleted photosystem II samples were reconstituted with the OEC33 prior to illumination. These results suggest that the D1 protein, CP43 and the OEC33 are located in close proximity at the lumenal side of photosystem II, and that the OEC33 suppresses the unnecessary contact between the D1 protein and CP43. Previously we presented data showing the D1 protein located adjacent to CP43 on the stromal side of photosystem II [Ishikawa et al. (1999) BIOCHIM: Biophys. Acta 1413: 147]. The present data suggest that the spatial arrangement of the D1 protein and CP43 at the lumenal side of photosystem II in spinach chloroplasts is similar to that at the stromal side of photosystem II and is consistent with the assignment of these proteins recently proposed on the crystal structures of the photosystem II complexes from cyanobacteria [Zouni et al. (2001) Nature 409: 739, Kamiya and Shen 2003 PROC: Natl. Acad. Sci. USA, 100: 98]. Moreover, the data suggest that the binding condition and positioning of the OEC33 in the photosystem II complex from higher plants may be different from those in cyanobacteria.     

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     

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。     

Structural characteristics of extra-membrane domains and guanidine hydrochloride-induced denaturation of photosystem 2 core antenna complexes CP43 and CP47

The structural characteristics of the extra-membrane domains and guanidine hydrochloride-induced denaturation of photosystem 2 (PS2) core antenna complexes CP43 and CP47 were investigated using fluorescence emission and circular dichroism (CD) spectra. The extra-membrane domains of CP43 and CP47 possessed a certain degree of secondary and tertiary structure and not a complete random coil conformation. The tertiary structure and the chlorophyll (Chl) a microenvironment of CP47 were more sensitive to guanidine hydrochloride (GuHCl) than that of CP43. Changes in energy transfer from β-carotene to Chl a corresponded well to changes in the tertiary structure while their correlation with changes in the secondary structure was rather poor. Unlike most of water-soluble proteins, both CP43 and CP47 are partly resistant to denaturation induced by guanidine hydrochloride (GuHCl); the denaturation of CP43 or CP47 is not a two-state process. Those features most probably reflect their character as intrinsic membrane proteins.     

Triplet and fluorescing states of the CP47 antenna complex of photosystem II studied as a function of temperature.

Fluorescence emission and triplet-minus-singlet (T-S) absorption difference spectra of the CP47 core antenna complex of photosystem II were measured as a function of temperature and compared to those of chlorophyll a in Triton X-100. Two spectral species were found in the chlorophyll T-S spectra of CP47, which may arise from a difference in ligation of the pigments or from an additional hydrogen bond, similar to what has been found for Chl molecules in a variety of solvents. The T-S spectra show that the lowest lying state in CP47 is at approximately 685 nm and gives rise to fluorescence at 690 nm at 4 K. The fluorescence quantum yield is 0.11 +/- 0.03 at 4 K, the chlorophyll triplet yield is 0.16 +/- 0.03. Carotenoid triplets are formed efficiently at 4 K through triplet transfer from chlorophyll with a yield of 0.15 +/- 0.02. The major decay channel of the lowest excited state in CP47 is internal conversion, with a quantum yield of about 0.58. Increase of the temperature results in a broadening and blue shift of the spectra due to the equilibration of the excitation over the antenna pigments. Upon increasing the temperature, a decrease of the fluorescence and triplet yields is observed to, at 270 K, a value of about 55% of the low temperature value. This decrease is significantly larger than of chlorophyll a in Triton X-100. Although the coupling to low-frequency phonon or vibration modes of the pigments is probably intermediate in CP47, the temperature dependence of the triplet and fluorescence quantum yield can be modeled using the energy gap law in the strong coupling limit of Englman and Jortner (1970. J. Mol. Phys. 18:145-164) for non-radiative decays. This yields for CP47 an average frequency of the promoting/accepting modes of 350 cm-1 with an activation energy of 650 cm-1 for internal conversion and activationless intersystem crossing to the triplet state through a promoting mode with a frequency of 180 cm-1. For chlorophyll a in Triton X-100 the average frequency of the promoting modes for non-radiative decay is very similar, but the activation energy (300 cm-1) is significantly smaller.