Nitrogen ligation to the manganese cluster of Photosystem II in the absence of the extrinsic proteins and as a function of pH

Three extrinsic proteins (PsbO, PsbP and PsbQ), with apparent molecular weights of 33, 23 and 17 kDa, bind to the lumenal side of Photosystem II (PS II) and stabilize the manganese, calcium and chloride cofactors of the oxygen evolving complex (OEC). The effect of these proteins on the structure of the tetramanganese cluster, especially their possible involvement in manganese ligation, is investigated in this study by measuring the reported histidine-manganese coupling [Tang et al. (1994) Proc Natl Acad Sci USA 91: 704–708] of PS II membranes depleted of none, two or three of these proteins using ESEEM (electron spin echo envelope modulation) spectroscopy. The results show that neither of the three proteins influence the histidine ligation of manganese. From this, the conserved histidine of the 23 kDa protein can be ruled out as a manganese ligand. Whereas the 33 and 17 kDa proteins lack conserved histidines, the existence of a 33 kDa protein-derived carboxylate ligand has been posited; our results show no evidence for a change of the manganese co-ordination upon removal of this protein. Studies of the pH-dependence of the histidine–manganese coupling show that the histidine ligation is present in PS II centers showing the S₂ multiline EPR signal in the pH-range 4.2–9.5. This revised version was published online in June 2006 with corrections to the Cover Date.     

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     

The 33 kDa protein can be removed without affecting the association of the 23 and 17 kDa proteins with the luminal side of PS II of spinach

An earlier study shows that a 30 min incubation of spinach PS II submembrane fragments at pH 6.3 in the presence of 10 microM HgCl(2) induces a 40% depletion of the 33 kDa protein without the apparent release of the 17 and 23 kDa proteins [Bernier, M., and Carpentier, R. (1995) FEBS Lett. 360, 251-254]. Here we report that the photosystem II 33 kDa extrinsic protein is fully removed by HgCl(2) added at micromolar and higher concentrations (0.25, 20, and 50 microM), with the 17 and 23 kDa extrinsic proteins and other intrinsic proteins remaining bound to the reaction center. The data presented here put in doubt the "regulatory cap" model of PS II, which follows the OEC-33 kDa-23 kDa-17 kDa binding order, as these results directly demonstrate that the 33 kDa protein can be removed without affecting the binding of the 23 and 17 kDa proteins to the intrinsic subunits of PS II. This suggests that each extrinsic protein may possess its own binding site on PS II. A possible mechanism for HgCl(2) upon the release of the 33 kDa protein is discussed.     

Photosynthetic water oxidation: The protein framework

Approximately 20 protein subunits are associated with the PS II complex, not counting subunits of peripheral light-harvesting antenna complexes. However, it is not yet established which proteins specifically are involved in the water-oxidation process. Much evidence supports the concept that the D1/D2 reaction center heterodimer not only plays a central role in the primary photochemistry of Photosystem II, but also is involved in electron donation to P680 and in ligation of the manganese cluster. This evidence includes (a) the primary donor to P680 has been shown to be a redox-active tyrosyl residue (Tyr161) in the D1 protein, and (b) site-directed mutagenesis and computer-assisted modeling of the reaction center heterodimer have suggested several sites with a possible function in manganese ligation. These include Asp170, Gln165 and Gln189 of the D1 protein and Glu69 of the D2 protein as well as the C-terminal portion of the mature D1 protein. Also, hydrophilic loops of the chlorophyll-binding protein CP43 that are exposed at the inner thylakoid surface could be essential for the water-splitting process.In photosynthetic eukaryotes, three lumenal extrinsic proteins, PS II-O (33 kDa), PS II-P (23 kDa) and PS II-Q (16 kDa), influence the properties of the manganese cluster without being involved in the actual catalysis of water oxidation. The extrinsic proteins together may have multiple binding sites to the integral portion of PS II, which could be provided by the D1/D2 heterodimer and CP47. A major role for the PS II-O protein is to stabilize the manganese cluster. Most experimental evidence favors a connection of the PS II-P protein with binding of the Cl^- and Ca²⁺ ions required for the water oxidation, while the PS II-Q protein seems to be associated only with the Cl^- requirement. The two latter proteins are not present in PS II of prokaryotic organisms, where their functions may be replaced by a 10–12 kDa subunit and a newly discovered low-potential cytochrome c-550.Abbreviations PS II Photosystem II - PCC Pasteur Culture Collection     

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     

Evidence from Crosslinking for Nearest-Neighbor Relationships among the Three Extrinsic Proteins of Spinach Photosystem II Complexes that are Associated with Oxygen Evolution

Treatment of oxygen-evolving Photosystem II complexes, whichlack light-harvesting chlorophyll a/b proteins, with a seriesof disuccinimidyl esters with different chain lengths yieldeda crosslinked product which consisted of one molecule each ofthe extrinsic 33 kDa and 23 kDa proteins. In addition, crosslinkingbetween the 33 kDa protein and the chlorophyll-carrying 47 kDaprotein and between the 23 kDa and 17 kDa proteins was confirmed.Thus, the three extrinsic proteins are closely associated witheach other to form a complex which is attached to the PS IIreaction center complexes. (Received December 1, 1989; Accepted May 2, 1990)     

Structural and catalytic properties of the oxygen-evolving complex. Correlation of polypeptide and manganese release with the behavior of Z+ in chloroplasts and a highly resolved preparation of the PS II complex

Treatment of intact thylakoid membranes with Triton X-100 at pH 6 produces a preparation of the PS II complex capable of high rates of O₂ evolution. The preparation contains four managanese, one cytochrome b-559, one Signal II_f and one Signal II_s per 250 chlorophylls. By selective manipulation of the preparation polypeptides of approximate molecular weights of 33, 23 and 17 kDa can be removed from the complex. Release of 23 and 17 kDa polypeptides does not release functional manganese. Under these conditions Z⁺ is not readily and directly accessible to an added donor (benzidine) and it appears as if at least some of the S-state transitions occur. Evidence is presented which indicates that benzidine does have increased access to the oxygen-evolving complex in these polypeptide depleted preparations. Conditions which release the 33 kDa species along with Mn and the 23 and 17 kDa polypeptides generate an alteration in the structure of the oxidizing side of PS II, which becomes freely accessible to benzidine. These findings are examined in relationship to alterations of normal S-state behavior (induced by polypeptide release) and a model is proposed for the organization of functional manganese and polypeptides involved in the oxygen-evolving reaction.     

Structural organization of the oxidizing side of photosystem II. Exogenous reductants reduce and destroy the Mn-complex in photosystems II membranes depleted of the 17 and 23 kDa polypeptides

Removal of 23 and 17 kDa water-soluble polypeptides from PS II membranes causes a marked decrease in oxygen-evolution activity, exposes the oxidizing side of PS II to exogenous reductants (Ghanotakis, D.F., Babcock, G.T. and Yocum, C.F. (1984) Biochim. Biophys. Acta 765, 388–398) and alters a high-affinity binding site for Ca²⁺ in the oxygen-evolving complex (Ghanotakis, D.F., Topper, J.N., Babcock, G.T. and Yocum, C.F. (1984) FEBS Lett. 170, 169–173). We have examined further the state of the functional Mn complex in PS II membranes from which the 17 and 23 kDa species have been removed by high-salt treatment. These membranes contain a structurally altered Mn complex which is sensitive to destruction by low concentrations of NH₂OH which cannot, in native PS II membranes, cause extraction of functional Mn. In addition to NH₂OH, a wide range of other small (H₂O₂, NH₂NH₂, Fe²⁺) and bulky (benzidine, hydroquinone) electron donors extract Mn (up to 80%) from the polypeptide-depleted PS II preparations. This extraction is due to reduction of the functional Mn complex since light, which would generate higher oxidation states within the Mn complex, prevents Mn release by reductants. Release of Mn by reductants does not extract the 33 kDa water-soluble protein implicated in Mn binding to the oxidizing side of PS II, although the protein can be partially or totally extracted from Mn-depleted preparations by exposure to high ionic strength or to high (0.8 M) concentrations of Tris. We view our results as evidence for a shield around the Mn complex of the oxygen-evolving complex comprised of the 33 kDa polypeptide along with the 23 and 17 kDa proteins and tightly bound Ca²⁺.     

Stabilization of the water oxidizing polypeptide assembly on Photosystem II membranes by osmolytes and other solutes

The integrity of Photosystem II membranes isolated from chloroplast thylakoids is profoundly affected by the solute environment. Examples are given for stabilizing effects various solutes have on the binding of the 17 and 23 kDa extrinsic polypeptides under conditions conductive to their dissociation. It is concluded that these and many other solute effects on Photosystem II membranes can be accommodated readily in a concept developed by Timasheff and his coworkers according to which the responses of proteins to their solute environment are consequences of interaction preferences among the constituents of the solvent-protein-solute systems.Abbreviations Chl chlorophyll - MES 2-(N-morpholino)ethanesulfonic acid - MOPS (3-[N-morpholino]propanesulfonic acid) - PS II Photosystem II     

Identification of polypeptides associated with the 23 and 33 kDa proteins of photosynthetic oxygen evolution

An immunological approach was used for nearest-neighbor analyses for the 23 and 33 kDA proteins of the oxygen-evolving complex. Functional Photosystem II particles with a simple polypeptide composition were partly solubilized with detergent and incubated with monospecific antibodies against either the 23 or the 33 kDa protein. SDS-polyacrylamide gel electrophoresis revealed that the immunoprecipitates, apart from the antigenic proteins, also contained polypeptides at 24, 22 and 10 kDa. In contrast, polypeptides of the light-harvesting and Photosystem II core complexes showed very poor coprecipitation with the 23 and 33 kDa proteins. The 24, 22 and 10 kDa polypeptides were not precipitated by the antibodies if the 23 and 33 kDa proteins had been removed from the particles prior to solubilization. These observations demonstrate a close association between the 24, 22 and 10 kDa polypeptides and the 23 and 33 kDa proteins of the oxygen-evolving complex. None of these precipitated polypeptides contained any manganese. It is suggested that the 24, 22 and 10 kDa polypeptides are subunits of the oxygen-evolving complex and involved in the binding of the extrinsic 23 and 33 kDa proteins to the inner thylakoid surface.     

Influence of the 33 kDa manganese-stabilizing protein on the structure and substrate accessibility of the oxygen-evolving complex of photosystem II

The 33 kDa manganese-stabilizing extrinsic protein binds to the lumenal side of photosystem II (PS II) close to the Mn(4)Ca cluster of the oxygen-evolving complex, where it limits access of small molecules to the metal site. Our previous finding that the removal of this protein did not alter the magnetic coupling regime within the manganese cluster, measured by electron spin-echo envelope modulation [Gregor, W., and Britt, R. D. (2000) Photosynth. Res. 65, 175-185], prompted us to examine whether this accessibility control is also true for substrate water, using the same pulsed EPR technique. Comparing the deuteron modulation of the S(2)-state multiline signal of PS II membranes, equilibrated with deuterated water (D(2)O) after removal or retention of the 33 kDa protein, we observed no change in the number and the distance of deuterons magnetically coupled to manganese, indicating that the number and distance of water molecules bound to the manganese cluster are independent of bound 33 kDa protein in the S(1) state, in which the sample was poised prior to cryogenic illumination. A simple modulation depth analysis revealed a distance of 2.5-2.6 A between the closest deuteron and manganese. These results are in agreement with our refined X-ray absorption analysis. The manganese K-edge positions, reflecting their oxidation states, and the extended X-ray absorption fine structure amplitudes and distances between the manganese ions and their oxygen and nitrogen ligands (1.8, 2.7, and 3.3-3.4 A) were independent of bound 33 kDa protein.     

Low pH-Induced Dissociation of Three Extrinsic Proteins from O2-Evolving Photosystem II

Three extrinsic proteins involved in oxygen evolution reversiblydissociated from Photo-system II (PS II) membranes at acidicpHs showing distinctly different pH dependencies. The pHs forhalf dissociation of 17, 23 and 33 kDa extrinsic proteins weredetermined to be 5.0, 4.1 and 3.6, respectively. The half dissociationpHs of 17 and 23 kDa proteins were much lower than their respectiveisoelectric points, while that for 33 kDa protein was closeto its isoelectric point. It was suggested that protonationof the negatively charged binding domain on membrane proteinscauses dissociation of the former two extrinsic proteins, whereasprotonation of the extrinsic protein itself is responsible forthe dissociation of 33 kDa protein. Based on these, featuresof low pH-induced dissociation of extrinsic proteins and Mnfrom PS II were discussed. (Received September 25, 1990; Accepted February 13, 1991)     

Studies on the polypeptide composition of the cyanobacterial oxygen-evolving complex

Various approaches have been used to investigate the polypeptides required for oxygen evolution in cyanobacteria, in particular the thermophile Phormidium laminosum. Antibodies against the extrinsic 33 kDa protein from spinach Photosystem II cross-reacted clearly in immunoblotting experiments with a corresponding polypeptide in isolated thylakoids and Photosystem II particles from P. laminosum and with whole-cell homogenates of three species of cyanobacteria (Phormidium laminosum, Synechococcus leopoliensis and Anabaena variabilis). In contrast, no cyanobacterial proteins reacted with antibodies against the 23 and 16 kDa proteins of spinach Photosystem II. The lack of cross-reactivity and the absence of these polypeptides from highly active Photosystem II particles of Phormidium laminosum strongly suggest that cyanobacteria do not contain polypeptides corresponding to these two chloroplast proteins. Treatment of P. laminosum Photosystem II particles with 0.8 M alkaline Tris, 1 M NaCl, CaCl₂ or MgCl₂ inhibited O₂ evolution, and quantitatively removed a 9 kDa polypeptide from the particles. None of these treatments removed comparable amounts of the 33 kDa polypeptide, and only Tris treatment removed manganese. The release of the 9 kDa polypeptide upon NaCl treatment correlated well with the deactivation at the donor side of Photosystem II. A direct connection between the 33 kDa polypeptide and O₂ evolution was established by the finding that trypsin treatment digested this polypeptide and inhibited O₂ evolution in parallel.     

Further investigations on structural and catalytic properties of O2 evolving preparations from tobacco and two chlorophyll deficient tobacco mutants

Previous investigations (Specht, S., Pistorius, E.K. and Schmid, G.H.: Photosynthesis Res. 13, 47–56, 1987) of Photosystem II membranes from tobacco (Nicotiana tabacum L. cv. John William's Broadleaf) which contain normally stacked thylakoid membranes and from two chlorophyll deficient tobacco mutants (Su/su and Su/su var. Aurea) which have low stacked or essentially unstacked thylakoids with occasional membrane doublings, have been extended by using monospecific antisera raised against the three extrinsic polypeptides of 33,21 and 16 kDa. The results show that all three peptides are synthesized as well in wild type tobacco as in the two mutants to about the same level and that they are present in thylakoid membranes of all three plants. However, in the mutants the 16 and 21 kDa peptides (but not the 33 kDa peptide) are easily lost during solubilization of Photosystem II membranes. In the absence of the 16 and 21 kDa peptide Photosystem II membranes from the mutants have a higher O₂ evolving activity without addition of CaCl₂ than the wild type Photosystem II membranes. On the other hand, after removal of the 33 kDa peptide no significant differences in the binding of Mn could be detected among the three plants. The results also show that reaction center complexes from wild type tobacco and the mutant Su/su are almost identical to the Triton-solubilized Photosystem II membranes from the mutant Su/su var. Aurea.Abbreviations PS photosystem - chl chlorophyll - LHCP light harvesting chlorophyll a/b protein complex - WT wild type - OEE1, OEE2 and OEE3 oxygen evolution enhancing complex of 29–36 kDa, 21–24 kDa and 16–18 kDa, respectively     

Purification and characterization of photosystem I and photosystem II core complexes from wild-type and phycocyanin-deficient strains of the cyanobacterium Synechocystis PCC 6803

Highly photoactive Photosystem I (PS I) and Photosystem II (PS II) core complexes have been isolated from the cyanobacterium Synechocystis Pasteur Culture Collection (PCC) 6803 and a phycocyanin-deficient mutant, enriched in PS II. Cell breakage using glass beads was followed by sucrose density gradient centrifugation and two high-performance liquid chromatography steps involving anion-exchange and hydroxyapatite. The PS I core complex has an apparent molecular mass of 300 +/- 20 kDa (including a detergent shell of about 50 kDa) and contains subunits of approximately 60, approximately 60, 18.5, 18.5, 16, 15, 10.5, 9.5, and 6.5 kDa as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblots; its antenna size is 75 +/- 5 chlorophyll/P-700. The PS II core complex has an apparent molecular mass of 310 +/- 20 kDa (including the detergent shell); subunits of 43, 37, 33, 29, and 10-11 kDa were identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting. The antenna size of the average PS II complex is 45 +/- 5 chlorophyll/primary quinone electron acceptor (QA). This preparation procedure also yields, as a byproduct, a highly purified cytochrome b6f complex. This complex contains four subunits of 38, 24, 19, and 15 kDa and b- and c-type cytochromes in a ratio of 2:1. Its apparent molecular mass of 180 +/- 20 kDa (including the detergent shell) is consistent with a monomeric complex.     

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.     

NMR paramagnetic relaxation enhancements due to manganese in the S0 and S2 states of Photosystem II-enriched membrane fragments and in the detergent-solubilized Photosystem II complex

The NMR paramagnetic relaxation enhancement (NMR-PRE) produced in the solvent proton resonance by manganese in the S₀ and S₂ states of the oxygen evolving center (OEC) has been recorded for three Photosystem II (PS II)-enriched preparations: (1) PS II-enriched thylakoid membrane fragments (TMF-2 particles); (2) salt-washed (2M NaCl) TMF-2 particles; and (3) the octylglucopyranoside (OGP)-solubilized PS II complex. The second and third preparations, but not the first, are depleted of the peripheral 17 and 23 kD polypeptides associated with the OEC. It has been proposed that depletion of these polypeptides increases the exposure of OEC manganese to the aqueous phase. The NMR-PRE response measures the quantity (T_1m+_m)^-1, where T_1m is the spin relaxation time and _m is the mean residence time with respect to chemical exchange reactions of solvent protons in the manganese coordination sphere, and, thus, the NMR-PRE provides a direct measure of the solvent proton chemical exchange rate constant _m ^-1. This study tested whether the 17 and 23 kD polypeptides shield the OEC from the solvent phase and whether their depletion enhances the S₂ and S₀ NMR-PRE signals by removing a kinetic barrier to the solvent proton chemical exchange reaction. The amplitude of the S₂ NMR-PRE signal, measured in its chemical exchange-limited regime (_m>T_1m), is slightly decreased, rather than increased, in preparations (2) and (3) relative to (1), indicating that removal of the 17 and 23 kD polypeptides slightly slows, rather than accelerates, the rate-limiting steps of the solvent proton chemical exchange reactions. In addition, the lifetime of the S₂ state was shortened several-fold in the solubilized PS II complex and in salt-washed TMF-2 membranes relative to untreated TMF-2 control samples. The S₀ NMR-PRE signal, which is present in TMF-2 suspensions, was not detected in suspensions of the solubilized PS II complex, even though these samples contained high concentrations of active manganese centers (approximately double those of the TMF-2 control) and exhibited an S₂ NMR-PRE signal of comparable amplitude to that of the TMF-2 preparation. These results suggest that the 17 and 23 kD extrinsic polypeptides do not shield the NMR-visible water binding site in the OEC from the aqueous phase, although their removal substantially alters the proton relaxation efficiency by shortening T_1m.Abbreviations ADRY acceleration of the deactivation reactions of the water splitting enzyme Y - BBY Photosystem II-enriched membrane fragments prepared by the method of Berthold et al. (1981) - CCCP carbonyl cyanide m-chlorophenyl hydrazone - Chl chlorophyll - DCBQ 2,5-dichlorobenzoquinone - MES morpholinoethanesulfonate - NMR nuclear magnetic resonance - OEC oxygen evolving complex - OGP octylglucopyranoside - PRE paramagnetic relaxation enhancement - PS II Photosystem II - SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis - TMF-2 Photosystem II-enriched thylakoid membrane fragments prepared by the method of Radmer et al. (1986) - T₁, T₂ longitudinal and transverse nuclear spin relaxation times     

The effect of mono- and divalent salts on the rise and decay kinetics of EPR signal II in Photosystem II preparations from spinach

The rise and decay kinetics of EPR signal II have been used to probe the organization of the donor side of Photosystem II (PS II) before and after extraction of PS II preparations with high concentrations of salt. 800 mM NaCl or 500-800 mM NaBr substantially depletes the preparations of the 16 and 24 kDa proteins and decreases the steady-state rate of O2-evolution by 70-80% from control rates. These treatments do not largely alter the decay kinetics of Signal II; the rise kinetics remain in the instrument limited time range (2 microseconds or less) during the first 8-12 flashes. Treating PS II preparations with 800 mM CaCl2 removes the 16, 24 and 33 kDa proteins with at least 95% inhibition of the steady-state rates of O2 evolution. The additional removal of the 33 kDa polypeptide decreases the rates of oxidation and rereduction of Z, the species responsible for Signal II. Preparations treated with either mono- or divalent salts show a steady-state light-induced increase in Signal II similar to that seen in Tris-washed samples. Such a steady-state increase indicates that the rate of electron transport from water to Z is greatly decreased or blocked. The data are interpreted within a model in which there is an intermediate electron carrier between the O2 evolving complex and Z.     

Herbicide and plastoquinone-binding proteins of Photosystem II reaction center complexes from the thermophilic cyanobacterium,Synechococcus sp.

Photoaffinity labeling of Synechococcus Photosystem (PS) II preparations with radioactive azido-derivatives of three herbicides and of plastoquinone was carried out to identify herbicide and plastoquinone-binding proteins. [¹⁴C]Azido-atrazine and [¹⁴C]azido-monuron specifically labeled the 28 kDa polypeptide of the PS II reaction center complex, which is sensitive to 3-(3′,4′-dichlorophenyl)-1,1-dimethylurea (DCMU). No specific labeling of this polypeptide with azido-atrazine was found in CP2-b (PS II reaction center lacking the 40 kDa subunit) which is insensitive to DCMU. [³H]Azido-dinoseb reacted with the 28 kDa polypeptide and the 47 kDa chlorophyll-carrying protein. The labeling with [³H]azido-plastoquinone resulted in the incorporation of the radioactivity exclusively into the 47 kDa polypeptide. It is concluded that the 28 kDa polypeptide is the herbicide-binding protein of the cyanobacterium and that the 47 kDa polypeptide has a binding site for plastoquinone and for phenol-type herbicides.     

Importance of the N-terminal sequence of the extrinsic 23 kDa polypeptide in Photosystem II in ion retention in oxygen evolution

The function of the extrinsic 23 kDa polypeptide (OEC23) in Photosystem II (PS II) is to retain Ca(2+) and Cl(-) during the S-state turnover of the Mn cluster in photosynthetic oxygen evolution. Recombinant OEC23s from several plant species were produced in Escherichia coli to characterize the molecular mechanism of the OEC23 function then used in reconstitution experiments. One tobacco isoform, OEC23 (2AF), had much less oxygen-evolving activity than the spinach and cucumber OEC23s when PS II activities were reconstituted in salt-washed spinach PS II particles. The fact that the OEC23s had similar binding affinities for PS II particles suggests different ion-retention capacities for the individual OEC23s: The chimeric OEC23s produced between spinach and 2AF and those produced between cucumber and 2AF show that 58 N-terminal amino acid residues are important for PS II activity. Further dissection of the sequence and site-directed mutagenesis indicated the importance of 20 N-terminal amino acid residues for activity, in particular the asparagine at the 15th position. In spinach the N15D mutation decreased PS II activity, whereas in 2AF the D15N mutation increased it. This shows the importance of the N-terminal sequence of OEC23 in ion retention during the water-splitting process.