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     

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.     

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.     

Investigating the early stages of photosystem II assembly in Synechocystis sp. PCC 6803: isolation of CP47 and CP43 complexes

Biochemical characterization of intermediates involved in the assembly of the oxygen-evolving Photosystem II (PSII) complex is hampered by their low abundance in the membrane. Using the cyanobacterium Synechocystis sp. PCC 6803, we describe here the isolation of the CP47 and CP43 subunits, which, during biogenesis, attach to a reaction center assembly complex containing D1, D2, and cytochrome b(559), with CP47 binding first. Our experimental approach involved a combination of His tagging, the use of a D1 deletion mutant that blocks PSII assembly at an early stage, and, in the case of CP47, the additional inactivation of the FtsH2 protease involved in degrading unassembled PSII proteins. Absorption spectroscopy and pigment analyses revealed that both CP47-His and CP43-His bind chlorophyll a and β-carotene. A comparison of the low temperature absorption and fluorescence spectra in the Q(Y) region for CP47-His and CP43-His with those for CP47 and CP43 isolated by fragmentation of spinach PSII core complexes confirmed that the spectroscopic properties are similar but not identical. The measured fluorescence quantum yield was generally lower for the proteins isolated from Synechocystis sp. PCC 6803, and a 1-3-nm blue shift and a 2-nm red shift of the 77 K emission maximum could be observed for CP47-His and CP43-His, respectively. Immunoblotting and mass spectrometry revealed the co-purification of PsbH, PsbL, and PsbT with CP47-His and of PsbK and Psb30/Ycf12 with CP43-His. Overall, our data support the view that CP47 and CP43 form preassembled pigment-protein complexes in vivo before their incorporation into the PSII complex.     

Evidence for direct interaction between the chlorophyll-proteins CP29 and CP47 in photosystem II.

Fractionation by anionic-exchange chromatography of an oxygen-evolving photosystem II complex solubilized with 10 mM dodecyl maltoside shows the existence of a sovra-molecular complex between the internal chlorophyll a antenna CP47 and the chlorophyll a/b minor antenna CP29. The chromatographic result is confirmed by a cross-linking experiment which brings about a binary conjugate formed by CP47 and CP29. The sovra-molecular complex between the two chlorophyll protein-complexes has a low temperature fluorescence emission red shifted with respect to the two isolated antenna components. A possible two arms antenna topology for photosystem II is suggested.     

Cyanobacterial small chlorophyll-binding protein ScpD (HliB) is located on the periphery of photosystem II in the vicinity of PsbH and CP47 subunits

Cyanobacteria contain several genes coding for small one-helix proteins called SCPs or HLIPs with significant sequence similarity to chlorophyll a/b-binding proteins. To localize one of these proteins, ScpD, in the cells of the cyanobacterium Synechocystis sp. PCC 6803, we constructed several mutants in which ScpD was expressed as a His-tagged protein (ScpDHis). Using two-dimensional native-SDS electrophoresis of thylakoid membranes or isolated Photosystem II (PSII), we determined that after high-light treatment most of the ScpDHis protein in a cell is associated with PSII. The ScpDHis protein was present in both monomeric and dimeric PSII core complexes and also in the core subcomplex lacking CP43. However, the association with PSII was abolished in the mutant lacking the PSII subunit PsbH. In a PSII mutant lacking cytochrome b(559), which does not accumulate PSII, ScpDHis is associated with CP47. The interaction of ScpDHis with PsbH and CP47 was further confirmed by electron microscopy of PSII labeled with Ni-NTA Nanogold. Single particle image analysis identified the location of the labeled ScpDHis at the periphery of the PSII core complex in the vicinity of the PsbH and CP47. Because of the fact that ScpDHis did not form any large structures bound to PSII and because of its accumulation in PSII subcomplexes containing CP47 and PsbH we suggest that ScpD is involved in a process of PSII assembly/repair during the turnover of pigment-binding proteins, particularly CP47.     

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。     

Selective extraction of CP 26 and CP 29 proteins without affecting the binding of the extrinsic proteins (33, 23 and 17 kDa) and the DCMU sensitivity of a Photosystem II core complex

A highly purified oxygen evolving Photosystem II core complex was isolated from PS II membranes solubilized with the non-ionic detergent n-octyl--D-thioglucoside. The three extrinsic proteins (33, 23 and 17 kDa) were functionally bound to the PS II core complex. Selective extraction of the 22, 10 kDa, CP 26 and CP 29 proteins demonstrated that these species are not involved in the binding of the extrinsic proteins (33, 23 and 17 kDa) or the DCMU sensitivity of the Photosystem II complex.Abbreviations Chl chlorophyll - DCBQ 2,6-dichloro-p-benzoquinone - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - LHC light-harvesting complex - MES 2-(N-morpholino)ethanesulfonic acid - OGP n-octyl--d-glucoside - OTG n-octyl--d-thioglucoside - PAGE polyacrylamide gel electrophoresis - PS II Photosystem II - SDS sodium dodecyl sulfate     

Autotrophic cells of the <Emphasis Type="Italic">Synechocystis psbH</Emphasis> deletion mutant are deficient in synthesis of CP47 and accumulate inactive PS II core complexes

Cells of the psbH deletion mutant IC7 of the cyanobacterium Synechocystis PCC 6803 grown in the absence of glucose contain strongly reduced levels of chlorophyll when compared with cells grown in the presence of glucose, or compared with wild-type (WT) cells. Low-temperature fluorescence emission spectra revealed decreased content of both active PS II (Photosystem II) and PS I (Photosystem I) complexes. Analysis of thylakoid membrane complexes of IC7 by native electrophoresis showed a similar set of chlorophyll–proteins, namely a PS II core complex and trimeric and monomeric PS II complexes, as in WT. However, in contrast to WT, the ³⁵S-methionine protein labeling pattern of the mutant exhibited no preferential labeling of the D1 protein in the PS II core complexes, and the labeled D1 and D2 proteins accumulated predominantly in the PS II reaction center lacking CP47. The results show that in autotrophically grown cells of the psbH deletion mutant, selective D1 turnover is inhibited and synthesis of CP47 becomes a limiting step in the PS II assembly.     

Oligonucleotide-directed mutagenesis of psbB,the gene encoding CP47, employing a deletion mutant strain of the cyanobacterium Synechocystis sp. PCC 6803

A mutant strain of the cyanobacterium Synechocystis sp. PCC (Pasteur Culture Collection) 6803 has been developed in which psbB, the gene coding for the chlorophyl a-binding protein CP47 in Photosystem II (PSII), has been deleted. This deletion mutant can be used for the reintroduction of modified psbB into the cyanobacterium. To study the role of a large hydrophilic region in CP47, presumably located on the lumenal side of the thylakoid membrane between the fifth and sixth membrane-spanning regions, specific deletions have been introduced in psbB coding for regions within this domain. One psbB mutation leads to deletion of Gly-351 to Thr-365 in CP47, another psbB mutation was targeted towards deletion of Arg-384 to Val-392 in this protein. The deletion from Gly-351 to Thr-365 results in a loss of PSII activity and of photoautotrophic growth of the mutant, but the deletion between Arg-384 and Val-392 retains PSII activity and the ability to grow photoautotrophically. The mutant strain with the deletion from Gly-351 to Thr-365 does not assemble a stable PSII reaction center complex in its thylakoid membranes, and exhibits diminished levels of CP47 and of the reaction center proteins D1 and D2. In contrast to the Arg-384 to Val-392 portion of this domain, the region between Gly-351 and Thr-365 appears essential for the normal structure and function of photosystem II.     

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.     

The guanidine hydrochloride-induced denaturation of CP43 and CP47 studied by terahertz time-domain spectroscopy

Terahertz time-domain spectroscopy (THz-TDS) is a new technique in studying the conformational state of a molecule in recent years. In this work, we reported the first use of THz-TDS to examine the denaturation of two photosynthesis membrane proteins: CP43 and CP47. THz-TDS was proven to be useful in discriminating the different conformational states of given proteins with similar structure and in monitoring the denaturation process of proteins. Upon treatment with guanidine hydrochloride (GuHCl), a 1.8 THz peak appeared for CP47 and free chlorophyll a (Chl a). This peak was deemed to originate from the interaction between Chl a and GuHCl molecules. The Chl a molecules in CP47 interacted with GuHCl more easily than those in CP43.     

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 guanidine hydrochloride-induced denaturation of CP43 and CP47 studied by terahertz time-domain spectroscopy

Terahertz time-domain spectroscopy (THz-TDS) is a new technique in studying the conformational state of a molecule in recent years. In this work, we reported the first use of THz-TDS to examine the denaturation of two photosynthesis membrane proteins: CP43 and CP47. THz-TDS was proven to be useful in discriminating the different conformational states of given proteins with similar structure and in monitoring the denaturation process of proteins. Upon treatment with guanidine hydrochloride (GuHCl), a 1.8 THz peak appeared for CP47 and free chlorophyll a (Chl a). This peak was deemed to originate from the interaction between Chl a and GuHCl molecules. The Chl a molecules in CP47 interacted with GuHCl more easily than those in CP43. Supported by the National Natural Science Foundation of China (Grant No. 39890390)     

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.     

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.     

Chimaeric CP47 mutants of the cyanobacterium Synechocystis sp. PCC 6803 carrying spinach sequences: Construction and function

Chimaeric mutants of the cyanobacterium Synechocystis sp. PCC 6803 have been generated carrying part or all of the spinach psbB gene, encoding CP47 (one of the chlorophyll-binding core antenna proteins in Photosystem II). The mutant in which the entire psbB gene had been replaced by the homologous gene from spinach was an obligate photoheterotroph and lacked Photosystem II complexes in its thylakoid membranes. However, this strain could be transformed with plasmids carrying selected regions of Synechocystis psbB to give rise to photoautotrophs with a chimaeric spinach/cyanobacterial CP47 protein. This process involved heterologous recombination in the cyanobacterium between psbB sequences from spinach and Synechocystis 6803; which was found to be reasonably effective in Synechocystis. Also other approaches were used that can produce a broad spectrum of chimaeric mutants in a single experiment. Functional characterization of the chimaeric photoautotrophic mutants indicated that if a decrease in the photoautotrophic growth rates was observed, this was correlated with a decrease in the number of Photosystem II reaction centers (on a chlorophyll basis) in the thylakoid membrane and with a decrease in oxygen evolution rates. Remaining Photosystem II reaction centers in these chimaeric mutants appeared to function rather normally, but thermoluminescence and chlorophyll a fluorescence measurements provided evidence for a destabilization of Q_B ^–. This illustrates the sensitivity of the functional properties of the PS II reaction center to mild perturbations in a neighboring protein.Abbreviations diuron 3-(3,4-dichlorophenyl)-1,1-dimethylurea - F_v variable chlorophyll a fluorescence - HEPES N-(2-hydroxyethyl)piperazine-N-(2-ethanesulfonic acid) - (k)bp (kilo)base pairs - PS II Photosystem II - Q_A primary electron-accepting plastoquinone in Photosystem II - Q_B secondary electron-accepting plastoquinone in Photosystem II - SDS sodium dodecyl sulfate     

Identifying the lowest electronic states of the chlorophylls in the CP47 core antenna protein of photosystem II

CP47 is a pigment-protein complex in the core of photosystem II that tranfers excitation energy to the reaction center. Here we report on a spectroscopic investigation of the isolated CP47 complex. By deconvoluting the 77 K absorption and linear dichroism, red-most states at 683 and 690 nm have been identified with oscillator strengths corresponding to approximately 3 and approximately 1 chlorophyll, respectively. Both states contribute to the 4 K emission, and the Stark spectrum shows that they have a large value for the difference polarizability between their ground and excited states. From site-selective polarized triplet-minus-singlet spectra, an excitonic origin for the 683 nm state was found. The red shift of the 690 nm state is most probably due to strong hydrogen bonding to a protein ligand, as follows from the position of the stretch frequency of the chlorophyll 13(1) keto group (1633 cm(-)(1)) in the fluorescence line narrowing spectrum at 4 K upon red-most excitation. We discuss how the 683 and 690 nm states may be linked to specific chlorophylls in the crystal structure [Zouni, A., Witt, H.-T., Kern, J., Fromme, P., Krauss, N., Saenger, W., and Orth, P. (2001) Nature 409, 739-743].     

Highly purified thermo-stable oxygen-evolving photosystem II core complex from the thermophilic cyanobacterium Synechococcus elongatus having His-tagged CP43

The carboxyl terminus of the CP43 subunit of photosystem II (PSII) in the thermophilic cyanobacterium, Synechococcus elongatus, was genetically tagged with six consecutive histidine residues to create a metal binding site on the PSII supramolecular complex. The histidine-tagging enabled rapid isolation of an intact cyanobacterial PSII core complex from dodecyl maltoside-solubilized thylakoids by a simple one-step Ni(2+)-affinity column chromatography. The isolated core complex was in a dimeric form with a molecular mass of about 580 kDa, consisting of five major intrinsic membrane proteins (CP47, CP43, D1, D2 and cytochrome b-559), three extrinsic proteins (33 kDa, 12 kDa, and cytochrome c-550), and a few low molecular mass membrane proteins, and evolved oxygen at a rate as high as 3,400 mumol (mg Chl)-1 h-1 at 45 degrees C with ferricyanide as an electron acceptor. The core complex emitted thermoluminescence B2-, B1- and Q-bands arising from S2QB-, S3QB- and S2QA- charge recombinations at respective emission temperatures of 45, 38 and 20 degrees C, all of which were higher by about 15 degrees C as compared with those in mesophilic spinach BBY membranes. These results indicated that the isolated core complex well retained the intact properties of thermoluminescence of thermophilic cyanobacterial cells, the deeper stabilization of PSII charge pairs. The isolated complex was extremely stable in terms of both protein composition and function, exhibiting no release of extrinsic proteins, no proteolytic degradation in any of its subunits, accompanied by only a slight (less than 10%) loss in oxygen evolution, after dark-incubation at 20 degrees C for 8 d. These properties of the thermophilic PSII core complex are highly useful for various types of studies on PSII.