Partial degradation of the extrinsic 23-kDa protein of the Photosystem II complex of spinach

Chymotrypsin eliminated nine amino acid residues at the amino-terminal side of the extrinsic 23-kDa protein of the oxygen-evolving Photosystem II complex of spinach. The resultant 22-kDa fragment was able to bind to the Photosystem II complex but with lowered binding affinity. However, once the 22-kDa fragment bound to the complex, it retained most functions of the 23-kDa protein; the fragment provided a binding site for the extrinsic 18-kDa protein, preserved a tight trap for Ca²⁺ in the complex, and shifted the optimum Cl⁻ concentration for oxygen evolution from 30 to 10 mM, although it was less effective in sustaining oxygen evolution at Cl⁻ concentrations below 10 mM. These observations suggest that the elimination of nine amino acid residues at the amino-terminal region of the 23-kDa protein does not significantly alter the conformation of the protein, except for partial modification of its binding site and its interaction with Cl⁻.     

The structure and function of the 33 kDa extrinsic protein of Photosystem II: A critical assessment

In this review the structure and function of the 33 kDa protein of Photosystem II is examined. Significant controversies exist concerning the solution secondary structure of the protein, the location of its binding site(s) within Photosystem II, the amino acid residues of the 33 kDa protein required for binding and its stoichiometry within the photosystem. The studies which examine these topics are considered from a critical perspective. A hypothetical model of the folding of the 33 kDa extrinsic protein which is supported by site-specific labeling studies and site-directed mutagenesis experiments is presented. Additionally, the function of the protein within the photosystem is unclear. We present a hypothesis that the 33 kDa protein is involved in maintaining the chloride associated with photosynthetic oxygen evolution in close proximity to the oxygen-evolving site.     

Manganese binding to the 23 kDa extrinsic protein of Photosystem II

The recombinant form of the extrinsic 23 kDa protein (psbP) of Photosystem II (PSII) was studied with respect to its capability to bind Mn. The stoichiometry was determined to be one manganese bound per protein. A very high binding constant, K(A)=10(-17) M(-1), was determined by dialysis of the Mn containing protein against increasing EDTA concentration. High Field EPR spectroscopy was used to distinguish between specific symmetrically ligated Mn(II) from those non-specifically Mn(II) attached to the protein surface. Upon Mn binding PsbP exhibited fluorescence emission with maxima at 415 and 435 nm when tryptophan residues were excited. The yield of this blue fluorescence was variable from sample to sample. It was likely that different conformational states of the protein were responsible for this variability. The importance of Mn binding to PsbP in the context of photoactivation of PSII is discussed.     

On the role of the N-terminus of the extrinsic 33 kDa protein of Photosystem II

The role of the N-terminus of the extrinsic 33 kDa protein of Photosystem II has been investigated by means of site-directed mutagenesis and cross-linking. Replacement of Asp-9 resulted in a dramatic increase in proteolytic sensitivity leading to the degradation of the protein forming a 31 kDa fragment with an undefined N-terminus. This fragment was unable to restore oxygen evolution. However, the variants of the 33 kDa protein which remained intact could reconstitute oxygen evolution as effectively as the wild-type protein. Cross-linking experiments with a water-soluble carbodiimide revealed that mutagenesis of residue D9 led to the disruption of an intramolecular salt bridge. Therefore we suggest that the N-terminus of the 33 kDa protein is necessary for maintaining the binding ability of the protein to Photosystem II but might not be involved in binding itself.     

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

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

Effect of the manganese complex on the binding of the extrinsic proteins (17, 23 and 33 kDa) of Photosystem II

Selective extraction-reconstitution experiments with the extrinsic Photosystem II polypeptides (33 kDa, 23 kDa and 17 kDa) have demonstrated that the manganese complex and the 33 kDa polypeptide are both necessary structural elements for the tight binding of the water soluble 17 and 23 kDa species. When the manganese complex is intact the 33 kDa protein interacts strongly with the rest of the photosynthetic complex. Destruction of the Mn-complex has two dramatic effects: i) The binding of the 33 kDa polypeptide is weaker, since it can be removed by exposure of the PS II system to 2 M NaCl, and ii) the 17 and 23 kDa species do not rebind to Mn-depleted Photosystem II membranes that retain the 33 kDa protein.Abbreviations Chl chlorophyll - HQ hydroquinone - MES 2(N-morpholino)ethanesulfonic acid - PS II Photosystem II - Tris 2-amino-2-hydroxymethylpropane-1,3-diol     

Fluorescence and Fourier-transform infrared spectroscopic studies on the role of disulfide bond in the calcium binding in the 33 kDa protein of Photosystem II

The 33 kDa protein of Photosystem II has one intrachain disulfide bond. Fluorescence spectroscopy shows that the major groups in the protein that bind to Ca²⁺ should be the carboxylic side groups of glutamic acid and/or aspartic acid. Fluorescence and Fourier-transform infrared (FTIR) spectroscopic studies indicate that the conformation of the 33 kDa protein is altered upon reduction, while the reduced protein still retains the secondary structure. FTIR spectroscopy also shows that the metal ions induce a relative decrease of unordered structure and -sheet, and a substantial increase of -helix in both the intact and the reduced 33 kDa protein. This indicates that the addition of cations results in a much more compact structure and that both the intact and the reduced 33 kDa proteins have the ability to bind calcium. The above results may suggest that the disulfide bridge is not essential for calcium binding.Abbreviations CD circular dichroism - FTIR Fourier transform infrared - La lanthanum - PS photosystem - Tb terbium     

Function of the 23 kDa extrinsic protein of Photosystem II as a manganese binding protein and its role in photoactivation

The function of the extrinsic 23 kDa protein of Photosystem II (PSII) was studied with respect to Mn binding and its ability to supply Mn to PSII during photoactivation, i.e. the light-dependent assembly of the tetramanganese cluster. The extrinsic proteins and the Mn cluster were removed by TRIS treatment from PSII-enriched membrane fragments and purified by anion exchange chromatography. Room temperature EPR spectra of the purified 23 kDa protein demonstrated the presence of Mn. Photoactivation was successful with low Mn concentrations when the 23 kDa protein was present, while in its absence a higher Mn concentration was needed to reach the same level of oxygen evolution activity. In addition, the rate of photoactivation was significantly accelerated in the presence of the 23 kDa protein. It is proposed that the 23 kDa protein plays an important role in providing Mn during the process of PSII assembly and that it acquires Mn during the light-induced turnover of D1 in the PSII damage-repair cycle and delivers Mn to repaired PSII.     

The extrinsic proteins of Photosystem II

Years of genetic, biochemical, and structural work have provided a number of insights into the oxygen evolving complex (OEC) of Photosystem II (PSII) for a variety of photosynthetic organisms. However, questions still remain about the functions and interactions among the various subunits that make up the OEC. After a brief introduction to the individual subunits Psb27, PsbP, PsbQ, PsbR, PsbU, and PsbV, a current picture of the OEC as a whole in cyanobacteria, red algae, green algae, and higher plants will be presented. Additionally, the role that these proteins play in the dynamic life cycle of PSII will be discussed.     

The action of mercury on the binding of the extrinsic polypeptides associated with the water oxidizing complex of photosystem II

Mercury (Hg²⁺), a sulfhydryl group reactant, wasused to probe structure-function relationships in photosystem II (PSII). In the present work, we investigated the impact of mercury on the polypeptide composition of PSII submembrane preparations. Electrophoretic analysis revealed that the incubation of the membranes in the presence of mercury produces the depletion of a polypeptide of molecular weight of 33 kDa. This polypeptide corresponds to the extrinsic protein EP33 of the oxygen evolving complex removed following urea treatment. However, the two closely related extrinsic polypeptides of 16 and 23 kDa, usually removed concomitantly after urea treatment, remained unaffected after the mercury treatment. These data demonstrated the existence of an intrinsic binding site for EP23. The molecular mode of action of mercury in the oxygen evolving complex of PSII is discussed.     

CI-NMR measurement of chloride binding to the oxygen-evolving complex of spinach Photosystem II

The mechanism by which Cl⁻ activates the oxygen-evolving complex (OEC) of Photosystem II (PS II) in spinach was studied by ³⁵Cl-NMR spectroscopy and steady-state measurements of oxygen evolution. Measurements of the excess ³⁵Cl-NMR linewidth in dark-adapted, Cl⁻-depleted thylakoid and Photosystem II membranes show an overall hyperbolic decrease which is interrupted by sharp increases in linewidth (linewidth maxima) at approx. 0.3 mM, 0.75 mM, 3.25 mM (2.0 mM in PS II membranes), and 7.0 mM Cl⁻. The rate of the Hill reaction (H₂O → 2,6-dichlorophenolindophenol) at low light intensities (5% of saturation) as a function of [Cl⁻] in thylakoids shows three intermediary plateaus in the concentration range between 0.1 and 10 mM Cl⁻ indicating kinetic cooperativity with respect to Cl⁻. The presence of linewidth maxima in the ³⁵Cl-NMR binding curve indicates that Cl⁻ addition exposes four types of Cl⁻ binding site that were previously inaccessible to exchange with Cl⁻ in the bulk solution. These results are best explained by proposing that Cl⁻ binds to four sequestered (salt-bridged) domains within the oxygen-evolving complex. Binding of Cl⁻ is facilitated by the presence of H⁺ and vice versa. The pH dependence of the excess ³⁵Cl-NMR linewidth at 0.75 mM Cl⁻ shows that Cl⁻ binding has a maximum at pH 6.0 and two smaller maxima at pH 5.4 and 6.5 which may suggest that as many as three groups (perhaps histidine) with pK_a values in the region may control the binding.     

The tetranuclear manganese complex of Photosystem II

A polynuclear manganese complex functions in Photosystem II both to accumulate oxidizing equivalents and to bind water and catalyze its four-electron oxidation. Recent electron paramagnetic resonance (EPR) spectroscopic studies of the manganese complex show that four manganese ions are required to account for its magnetic properties. The exchange couplings between manganese ions in the S₂ state are characteristic of a Mn₄O₄ cubane-like structure. Based on this structure for the manganese complex in the S₂ state, as well as a consideration of the known properties of the manganese complex in Photosystem II and the coordination chemistry of manganese, structures are proposed for the five intermediate oxidation states of the manganese complex. A molecular mechanism for the formation of an O-O bond and the displacement of O₂ from the S₄ state is suggested.     

Structural predictions on the 33 kDa extrinsic protein associated to the oxygen evolving complex of photosynthetic organisms

Modern computational methods for protein structure prediction have been used to study the structure of the 33 kDa extrinsic membrane protein, associated to the oxygen evolving complex of photosynthetic organisms. A multiple alignment of 14 sequences of this protein from cyanobacteria, algae and plants is presented. The alignment allows the identification of fully conserved residues and the recognition of one deletion and one insertion present in the plant sequences but not in cyanobacteria. A tree of similarity, deduced from pair-wise comparison and cluster analysis of the sequences, is also presented. The alignment and the consensus sequence derived are used for prediction the secondary structure of the protein. This prediction indicates that it is a mainly-beta protein (25–38% of -strands) with no more than 4% of -helix. Fold recognition by threading is applied to obtain a topological 2D model of the protein. In this model the secondary structure elements are located, including several highly conserved loops. Some of these conserved loops are suggested to be important for the binding of the 33 kDa protein to Photosystem II and for the stability of the manganese cluster. These structural predictions are in good agreement with experimental data reported by several authors.     

Requirements for different combinations of the extrinsic proteins in specific cyanobacterial Photosystem II mutants

The crystallographic data available for Photosystem II (PS II) in cyanobacteria has now provided complete structures for loop E from CP43 and CP47 as well as the extrinsic subunits PsbO, PsbU and PsbV. Protein interactions between these subunits are essential for stable water splitting and there is evidence that the binding of PsbU facilitates optimal energy transfer from the phycobilisome. Interactions between PsbO and CP47 may also play a role in dimer stabilization while loop E of CP43 contributes directly to the water-splitting reaction. Recent evidence also suggests that homologs of PsbP and PsbQ play key roles in cyanobacterial PS II, and under nutrient-deficient conditions PsbQ appears essential for photoautotrophic growth.     

Function of the 23 kDa extrinsic protein of Photosystem II as a manganese binding protein and its role in photoactivation

The function of the extrinsic 23 kDa protein of Photosystem II (PSII) was studied with respect to Mn binding and its ability to supply Mn to PSII during photoactivation, i.e. the light-dependent assembly of the tetramanganese cluster. The extrinsic proteins and the Mn cluster were removed by TRIS treatment from PSII-enriched membrane fragments and purified by anion exchange chromatography. Room temperature EPR spectra of the purified 23 kDa protein demonstrated the presence of Mn. Photoactivation was successful with low Mn concentrations when the 23 kDa protein was present, while in its absence a higher Mn concentration was needed to reach the same level of oxygen evolution activity. In addition, the rate of photoactivation was significantly accelerated in the presence of the 23 kDa protein. It is proposed that the 23 kDa protein plays an important role in providing Mn during the process of PSII assembly and that it acquires Mn during the light-induced turnover of D1 in the PSII damage-repair cycle and delivers Mn to repaired PSII.     

Modification of the thermoluminescence properties of Ca2+ depleted Photosystem II membranes by the 23 kDa extrinsic polypeptide and by oligocarboxylic acids

Photosystem II membranes were isolated from chloroplasts of pokeweed (Phytolacca americana) and rendered deficient in Ca²⁺, an inorganic cofactor of photosynthetic water oxidation. The thermoluminescence properties of such membranes were found to depend on the Ca²⁺-depleting method used. This feature was analyzed with respect to the thermoluminescence emission that accompanied the recombination reaction between the reduced acceptor Q_A ^– and the oxidant of the S₂ state. It was determined that the differences observed among various preparations of Ca²⁺-depleted membranes were attributable to the presence or absence of the extrinsic 23 kDa polypeptide on the membranes. The binding of this polypeptide to Ca²⁺-depleted membranes devoid of the 17 and 23 kDa extrinsic polypeptides caused the thermoluminescence to be emitted at a higher temperature due to a further stabilization of an already abnormally stable S₂ state. Addition of the chelators EDTA or EGTA and of citrate brought about a similar response. The conditions required for the upshift of the emission temperature of thermoluminescence strongly resembled those identified by Boussac et al. (FEBS Lett. 277 (1990) 69–74) as responsible for modifying the EPR multiline signal from the S₂ state of Ca²⁺-depleted PS II membranes. Consistent with the authors' interpretation of the reason for this modification, we conclude that the elevated emission temperature of the thermoluminescence emission reflects an abnormal ligand environment of the Mn-center in PS II that may be created by a direct ligation of the added agents to Mn. Evidence is also presented that the return to a normal S₂ after an addition of Ca²⁺ occurs via yet another condition of S₂ which, in terms of its thermoluminescence properties, resembles that of Ca²⁺-depleted membranes before addition of modifying agents, but is not identical to it.     

Characterization of cDNA clones encoding the extrinsic 23 kDa polypeptide of the oxygen-evolving complex of photosystem II in pea

The 23 kDa polypeptide of the oxygen-evolving complex of photosystem II has been extracted from pea photosystem II particles by washing with 1 M NaCl and purified by anion-exchange chromatography. The N-terminal amino acid sequence has been determined and specific antisera have been raised in rabbits and used to screen a pea-leaf cDNA library in gt11. Determination of the nucleotide sequence of two clones provided the nucleotide sequence for the full 23 kDa polypeptide. The deduced amino acid sequence showed it to code for a mature protein of 186 amino acid residues with an N-terminal presequence of 73 amino acid residues showing a high degree of conservation with previously reported 23 kDa sequences from spinach and Chlamydomonas. Southern blots of genomic DNA from pea probed with the labelled cDNA gave rise to only one band suggesting that the protein is encoded by a single gene. Northern blots of RNA extracted from various organs indicated a message of approximately 1.1 kb, in good agreement with the size of the cDNA, in all chlorophyll-containing tissues. Western blots of protein extracted from the same organs indicated that the 23 kDa polypeptide was present in all major organs of the plant except the roots.Abbreviations bis-Tris bis (2-hydroxyethyl) imino-tris (hydroxymethyl)-methane - pfu plaque-forming units