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• W2012076237 abstract "Using the detergents n-dodecyl β-d-maltoside and heptyl thioglycopyranoside, a subcore complex of photosystem II (PSII) has been isolated that contains the chlorophyll-binding protein, CP47, and the reaction center components, D1, D2, and cytochrome b 559. We have found, by using sucrose density centrifugation, that the resulting preparation consisted of a mixture of dimeric and monomeric forms of the CP47 reaction center (RC) complex, having molecular masses of 410 ± 30 and 200 ± 28 kDa, respectively, as estimated by size exclusion chromatography. The level of the dimer in the preparation is significantly higher than the monomeric form. Both the monomer and dimer contain the proteins CP47, D1, and D2 and the α- and β-subunits of cytochrome b 559. Analyses by mass spectrometry and N-terminal sequencing showed that both forms of the CP47-RC complex contain the products of the psbI,psbT c (chloroplast gene), and psbWwith molecular masses of 4195.5, 3849.6, and 5927.4 Da, respectively. In contrast to the monomeric form, the CP47-RC dimer contained two extra proteins with low molecular weights, identified as the products of the psbL and psbK genes having molecular masses of 4365.5 and 4292.1, respectively. It was also found that the dimer contained slightly more molecules of chlorophyll a(21 ± 2.5) than the monomer (18 ± 1.5), a characteristic also observed in the room temperature absorption spectrum by comparing the ratio of absorption at 416 and 435 nm. Of particular note was the finding that the dimer, but not the monomer, contained plastoquinone-9 (estimated to be 1.5 ± 0.3 molecules per RC). The results indicate that the CP47-RC monomer is derived from the dimeric form of the complex, and therefore the latter is likely to represent anin vivo conformation. The PsbTc as well as the PsbI and PsbW proteins are identified as being intimately associated with the D1 and D2 proteins, and in the case of the dimer, importance is placed on the PsbL and PsbK proteins in sustaining plastoquinone binding and maintenance of the dimeric organization. Assuming only one copy of the α- and β-subunits of cytochromeb 559, the monomeric and dimeric forms of the complex would be expected to contain 21 and 23 × 2 transmembrane helices, respectively. Using the detergents n-dodecyl β-d-maltoside and heptyl thioglycopyranoside, a subcore complex of photosystem II (PSII) has been isolated that contains the chlorophyll-binding protein, CP47, and the reaction center components, D1, D2, and cytochrome b 559. We have found, by using sucrose density centrifugation, that the resulting preparation consisted of a mixture of dimeric and monomeric forms of the CP47 reaction center (RC) complex, having molecular masses of 410 ± 30 and 200 ± 28 kDa, respectively, as estimated by size exclusion chromatography. The level of the dimer in the preparation is significantly higher than the monomeric form. Both the monomer and dimer contain the proteins CP47, D1, and D2 and the α- and β-subunits of cytochrome b 559. Analyses by mass spectrometry and N-terminal sequencing showed that both forms of the CP47-RC complex contain the products of the psbI,psbT c (chloroplast gene), and psbWwith molecular masses of 4195.5, 3849.6, and 5927.4 Da, respectively. In contrast to the monomeric form, the CP47-RC dimer contained two extra proteins with low molecular weights, identified as the products of the psbL and psbK genes having molecular masses of 4365.5 and 4292.1, respectively. It was also found that the dimer contained slightly more molecules of chlorophyll a(21 ± 2.5) than the monomer (18 ± 1.5), a characteristic also observed in the room temperature absorption spectrum by comparing the ratio of absorption at 416 and 435 nm. Of particular note was the finding that the dimer, but not the monomer, contained plastoquinone-9 (estimated to be 1.5 ± 0.3 molecules per RC). The results indicate that the CP47-RC monomer is derived from the dimeric form of the complex, and therefore the latter is likely to represent anin vivo conformation. The PsbTc as well as the PsbI and PsbW proteins are identified as being intimately associated with the D1 and D2 proteins, and in the case of the dimer, importance is placed on the PsbL and PsbK proteins in sustaining plastoquinone binding and maintenance of the dimeric organization. Assuming only one copy of the α- and β-subunits of cytochromeb 559, the monomeric and dimeric forms of the complex would be expected to contain 21 and 23 × 2 transmembrane helices, respectively. The light-induced splitting of water and the consequential release of dioxygen is a fundamental reaction in photosynthesis taking place in all higher plants, algae, and cyanobacteria. This reaction is catalyzed by a complex known as photosystem II (PSII) 1The abbreviations and protein names used are: PSII, photosystem II; RC, reaction center; CP47, chlorophyll-binding protein encoded by psbB gene; CP43, chlorophyll-binding protein encoded by psbC gene; D1 and D2 proteins, products of the psbA and psbD genes, respectively; DM,n-dodecyl β-d-maltoside; HTG, heptyl thioglycopyranoside; QA, the first PSII plastoquinone electron acceptor; QX and QY, optical absorptions with their transitions approximately parallel to thex and y axes of the chromophore; LHCII, light-harvesting complex of PSII; Mes, 4-morpholineethanesulfonic acid; HPLC, high pressure liquid chromatography; PAGE, polyacrylamide gel electrophoresis; Chl, chlorophyll; Pheo, pheophytin; BBY, membrane fraction enriched in PSII and isolated according to Ref. 32Berthold D.A. Babcock G.T. Yocum C.F. FEBS Lett. 1981; 134: 231-234Crossref Scopus (1647) Google Scholar; PQ, plastoquinone; PQ-9, plastoquinone-9. 1The abbreviations and protein names used are: PSII, photosystem II; RC, reaction center; CP47, chlorophyll-binding protein encoded by psbB gene; CP43, chlorophyll-binding protein encoded by psbC gene; D1 and D2 proteins, products of the psbA and psbD genes, respectively; DM,n-dodecyl β-d-maltoside; HTG, heptyl thioglycopyranoside; QA, the first PSII plastoquinone electron acceptor; QX and QY, optical absorptions with their transitions approximately parallel to thex and y axes of the chromophore; LHCII, light-harvesting complex of PSII; Mes, 4-morpholineethanesulfonic acid; HPLC, high pressure liquid chromatography; PAGE, polyacrylamide gel electrophoresis; Chl, chlorophyll; Pheo, pheophytin; BBY, membrane fraction enriched in PSII and isolated according to Ref. 32Berthold D.A. Babcock G.T. Yocum C.F. FEBS Lett. 1981; 134: 231-234Crossref Scopus (1647) Google Scholar; PQ, plastoquinone; PQ-9, plastoquinone-9. which is embedded in the lipid bilayer of the thylakoid membrane. The PSII complex is composed of at least 25 different proteins and binds a large number of pigments (1Barber J. Miflin B.J. Oxford Surveys of Plant Molecular and Cellular Biology. 6. Oxford University Press, Oxford1989: 115-162Google Scholar). At the heart of this complex is the reaction center (RC), consisting of the D1 and D2 proteins, where primary charge separation occurs (2Nanba O. Satoh K. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 109-112Crossref PubMed Google Scholar). Closely associated with the D1 and D2 heterodimer are the two chlorophyll a-binding proteins, CP47 and CP43, and a range of small hydrophobic polypeptides including the α- and β-subunits of cytochrome b 559. CP47 and CP43 act both as an “inner” light harvesting system and as a conduit for the transfer of energy captured by an “outer” light harvesting system composed either of proteins that bind chlorophyll aand b (in the case of higher plants and green algae) or phycobilisomes (in the case of cyanobacteria and red algae) (3Bricker T.M. Photosynth. Res. 1990; 24: 1-113Crossref PubMed Scopus (192) Google Scholar). The outer antennae can be removed during isolation procedures to obtain a “core” preparation consisting of CP47, CP43, D1, and D2 proteins and other polypeptides including those that are extrinsically bound to the luminal surface and required for stabilizing and optimizing the water splitting system and its catalytic cluster of manganese atoms (4Hankamer B. Barber J. Boekema E.J. Annu. Rev. Plant Physiol. Mol. Biol. 1997; 48: 641-671Crossref PubMed Scopus (290) Google Scholar). Further solubilization of PSII to produce a “CP47-RC subcore” results in the loss of water splitting activity, in part due to the removal of extrinsic proteins and the manganese cluster but may also be due to the stripping away of CP43 (5Ghanotakis D.F. de Paula J.C. Demetriou D.M. Bowlby N.R. Petersen J. Babcock G.T. Yocum C.F. Biochim. Biophys. Acta. 1989; 974: 44-53Crossref PubMed Scopus (104) Google Scholar).Recently, the structures of several isolated forms of PSII have been reported (6Boekema D.J. Hankamer B. Bald D. Kruip J. Nield J. Boonstra A.F. Barber J. Rögner M. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 175-179Crossref PubMed Scopus (272) Google Scholar, 7Hankamer B. Nield J. Zheleva D. Boekema E.J. Jansson S. Barber J. Eur. J. Biochem. 1997; 243: 422-429Crossref PubMed Scopus (171) Google Scholar, 8Morris E.P. Hankamer B. Zheleva D. Friso G. Barber J. Structure. 1997; 5: 837-849Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar, 9Rhee K.-H. Morris E.P Zheleva D. Hankamer B. Kühlbrandt W. Barber J. Nature. 1997; 389: 522-527Crossref Scopus (126) Google Scholar). Using single particle analysis by electron microscopy, the top and side views of a large (725-kDa) PSII particle isolated from spinach have been described at about 25-Å resolution (6Boekema D.J. Hankamer B. Bald D. Kruip J. Nield J. Boonstra A.F. Barber J. Rögner M. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 175-179Crossref PubMed Scopus (272) Google Scholar). This particle, which is dimeric, contains the PSII core and a complement of chlorophyll a/b-binding proteins (LHCII (Lhcb1 and -2), CP29 (Lhcb6), and CP26 (Lhcb5)), totaling about 100 chlorophyll molecules/RC (7Hankamer B. Nield J. Zheleva D. Boekema E.J. Jansson S. Barber J. Eur. J. Biochem. 1997; 243: 422-429Crossref PubMed Scopus (171) Google Scholar). Since this LHCII-PSII supercomplex is isolated using a mild detergent treatment and maintains high oxygen evolving activity, it seems highly likely that it represents anin vivo form of PSII (7Hankamer B. Nield J. Zheleva D. Boekema E.J. Jansson S. Barber J. Eur. J. Biochem. 1997; 243: 422-429Crossref PubMed Scopus (171) Google Scholar). This would therefore suggest that functional PSII is dimeric in vivo and would account for the fact that the core complex can also be isolated in a dimeric form by stripping away the chlorophyll a/b proteins (8Morris E.P. Hankamer B. Zheleva D. Friso G. Barber J. Structure. 1997; 5: 837-849Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar). Indeed the dimeric form of the PSII core complex isolated from spinach is more functionally active and stable than the monomeric form (7Hankamer B. Nield J. Zheleva D. Boekema E.J. Jansson S. Barber J. Eur. J. Biochem. 1997; 243: 422-429Crossref PubMed Scopus (171) Google Scholar).A PSII structure that has recently been revealed to 8-Å resolution is that of the CP47-RC subcore complex (9Rhee K.-H. Morris E.P Zheleva D. Hankamer B. Kühlbrandt W. Barber J. Nature. 1997; 389: 522-527Crossref Scopus (126) Google Scholar). This structure has been obtained by two-dimensional crystallization and cryoelectron microscopy, and the published work was in the form of a projection map. By comparison with x-ray crystallography data of the reaction center of purple bacteria and of photosystem one, it was possible to suggest that some density in the map was due to the transmembrane helices of the D1 and D2 proteins and CP47, while the remaining density results from other proteins present. It was also found that the unit cell in the two-dimensional crystals contained two dimers of the CP47-RC complex and that the size of the dimer was compatible with a central location in the LHCII·PSII supercomplex (9Rhee K.-H. Morris E.P Zheleva D. Hankamer B. Kühlbrandt W. Barber J. Nature. 1997; 389: 522-527Crossref Scopus (126) Google Scholar).In order to complement the structural analyses of the two-dimensional crystals of CP47-RC, we have undertaken studies to determine the composition and properties of the isolated solubilized CP47-RC complex.MATERIALS AND METHODSIsolation of CP47-RC ComplexsCP47-RC complexes were obtained from BBY-type PSII-enriched membranes isolated from market spinach with modifications described by Hankamer and co-workers (6Boekema D.J. Hankamer B. Bald D. Kruip J. Nield J. Boonstra A.F. Barber J. Rögner M. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 175-179Crossref PubMed Scopus (272) Google Scholar). After washing with 50 mmTris-HCl (pH 9.0), 1 mg of Chl ml−1 of BBYs were solubilized with n-dodecyl β- d-maltoside (DM; 1% final concentration) and heptyl thioglycopyranoside (HTG; 2.7% final concentration) in the dark with constant stirring for 30 min at room temperature. The nonsolubilized material was removed by a 30-min centrifugation at 40,000 × g at 4 °C. For purification the solubilized BBYs were loaded onto a Fractogel ion exchange column (DEAE-Toyopearl 650S, TSK) that had been equilibrated with 50 mm Tris-HCl (pH 7.3), 10 mm NaCl, and 2 mm DM. The sample was washed with 50 mmTris-HCl (pH 7.3), 2 mm DM, and 40 mm NaCl and was eluted with the same buffer but using a 40–200 mm NaCl gradient. The ion exchange chromatography was carried out at 4 °C under dim light conditions.Isolation of Monomeric and Dimeric CP47-RC ComplexesMonomeric and dimeric CP47-RC complexes were separated using sucrose gradient centrifugation. The sucrose gradient mix solution consisted of 25 mm Mes, pH 6.5, 0.5 msucrose, 10 mm NaCl, 5 mm CaCl2 and 0.03% DM. The sucrose density gradients were prepared after freezing at −20 °C and slow thawing at 4 °C. The ion exchange chromatography-purified CP47-RC complex (300 μg of Chl) was loaded onto the gradients and centrifuged at 90,000 × g for 16 h at 4 °C in a Beckman SW41 swing out rotor and separated into two different fractions.Room Temperature Absorption SpectraThese were measured using an SLM Aminco (Urbana, IL) model DW2000 spectrophotometer.HPLC Size Exclusion AnalysisHPLC size exclusion analysis was carried out using a Zorbax GF-450 column 9.4/250-mm (Jones Chromatography). The mobile phase consisted of 50 mmTris-HCl, pH 7.2, 0.03% DM, 0.3 m sucrose and was passed through the column at a rate of 0.5 ml min−1. The 20-μl samples injected contained 5 μg of Chl, and the profiles were monitored at 418 nm. The molecular weights of the complexes were determined by using a gel filtration calibration kit (Amersham Pharmacia Biotech).SDS-PAGE and Western BlottingThe polypeptide compositions of the isolated PSII preparations were analyzed by gradient SDS-PAGE (10–17% polyacrylamide) containing 6 m urea, essentially using the method of Laemmli (10Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (206024) Google Scholar). The gels were stained with Coomassie Blue R-250.The protein profiles resolved by SDS-PAGE were transferred onto nitrocellulose (11Dunn S.D. Anal. Biochem. 1986; 157: 144-153Crossref PubMed Scopus (418) Google Scholar) and immunolabeled with CP47- or CP43-specific antibodies raised against electrophoretically purified spinach CP47, CP43, D2 (a kind gift from Dr. R. Barbato), or C-terminal D1-specific antibody (Dupont 304) raised against a synthetic polypeptide, homologous to the 29 amino acids of the C-terminal pea D1 precursor (a kind gift from Dr. P. Nixon). Biotinylated anti-rabbit IgG was used as a secondary antibody and was labeled with Extravidine-alkaline phosphatase conjugate (Sigma). The chromogenic substrates were 5-bromo-4-chloroindolyl phosphate ρ-toluidine salt and nitro blue tetrazolium chloride.Reverse Phase HPLC Pigment AnalysisPigment AnalysisPigments (chlorophyll a, pheophytin a, and β-carotene) were extracted into 80% (v/v) acetone/H2O at 4 °C in dim light conditions. The samples were vortexed for 30 s, centrifuged for 2 min in a microcentrifuge in order to separate out proteinaceous materials, and filtered through a 0.2-μm (pore size) membrane (polyvinylidene difluoride; Vatman) before injection (injection volume was 20 μl). The pigments were resolved using an ODS-1 Spherisorb column (Anachem) and isocratic elution with methanol/ethyl acetate/water 68:30:2 (v/v/v), at a flow rate of 1 ml/min. We used a variable wavelength detector (Kontron 30), detecting simultaneously at 663 nm (for Chla and Pheo a) and 453 nm (for β-carotene) or 255 nm (for PQ). Chl a and β-carotene quantification was performed after calibration of the corresponding peaks with pigment standards (purchased from Sigma), whose concentrations were determined using extinction coefficients 76.79 mm−1cm−1 at 663.6 nm in 80% acetone for Chla (12Porra R.J. Thompson W.A. Kriedmann P.E. Biochim. Biophys. Acta. 1989; 975: 384-394Crossref Scopus (4580) Google Scholar) and 139 mm−1cm−1 at 452 nm in 100% hexane for β-carotene (13Zechmeister L. Polgar A. J. Am. Chem. Soc. 1943; 65: 1522-1528Crossref Scopus (83) Google Scholar). The Pheo a standard used for calibration was produced by acidifying the Chl a standard with 2 mm HCl (14Zheleva D. Hankamer B. Barber J. Biochemistry. 1996; 35: 15074-15079Crossref PubMed Scopus (23) Google Scholar). PQ-9 (obtained from Sigma) was dissolved in 100% ethanol, and its concentration was determined using the extinction coefficient of 15.2 m−1 cm−1 at 255 nm (15Redfearn E.R. Friend J. Phytochemistry. 1962; 1: 147-151Crossref Scopus (33) Google Scholar).Separation of ProteinsPartial separation of the protein subunits in the CP47-RC monomer and dimer complexes was carried out for subsequent mass spectrometric measurements and was achieved using a Spherisorb Aquapore RP-300 (220 × 4.6-mm) column fitted to a Kontron HPLC system (Datasystem 450, HPLC pump 420, detector 430 and mixer M800). The PSII preparations were dialyzed against 2 × 2.0 liters of aqueous 5% acetic acid at 4 °C for 16–24 h and were loaded directly onto the column. The components were eluted with a solvent system of A (aqueous 0.1% trifluoroacetic acid) and B (90% acetonitrile in aqueous 0.1% trifluoroacetic acid), using a linear gradient from 100% A to 40% B in 10 min followed by an increase to 100% B over 60 min at a flow rate of 1.0 ml min−1. Elution was monitored at 214 and 280 nm, and fractions were collected at 1-min intervals.Gas Phase Edman SequencingPeptides were Edman-degraded using a CI 4000 gas phase protein sequencer, and the standard protein sequencing method was used (16Sharma J. Panico M. Barber J. Morris H.R. J. Biol. Chem. 1997; 272: 3935-3943Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar). Samples that failed to give sequence information were treated on the glass fiber disc with 0.6 n HCl at 25 °C for 24 h.Electrospray Mass Spectrometry AnalysisIntact proteins of the CP47-RC complexes and their digestion products were also analyzed by electrospray mass spectrometry. Spectra were obtained by direct injection of 10-μl aliquots of HPLC-purified proteins into the ion source of a VG Bio-Q triple quadrupole electrospray mass spectrometer. The instrument was operated using a carrier flow buffer of 90% acetonitrile in aqueous 0.1% trifluoroacetic acid, propan-1-ol and 2-methoxyethanol in a 1:1:1:1 (v/v/v/v) ratio at a flow rate of 5 μl min−1. The mass spectrometer was calibrated using a solution of horse heart myoglobin (1 pm ml−1) to give an average chemical molecular mass for the signals observed.Matrix-assisted Laser Desorption Ionization Mass Spectrometry AnalysisMatrix-assisted laser desorption ionization mass spectra were acquired using a Fisons VG ZAB 2SE 2FPD mass spectrometer, fitted with a UV laser (337 nm). The instrument was calibrated using CsI clusters. Data acquisition and processing were performed using VG Analytical Opus software. The samples were dissolved in 60% propan-1-ol in 5% aqueous acetic acid, and 1-μl aliquots were loaded onto a probe that had been treated with a saturated solution of 2,5-dihydroxybenzoic acid made up in a 70:30 (v/v) solution of 90% acetonitrile in aqueous 0.1% trifluoroacetic acid and aqueous 0.1% trifluoroacetic acid, respectively.DISCUSSIONThe results presented here show that CP43 is more readily removed from the PSII core than CP47 despite the fact that both proteins are likely to be structurally rather similar based on the homologies between their amino acid sequences (3Bricker T.M. Photosynth. Res. 1990; 24: 1-113Crossref PubMed Scopus (192) Google Scholar). Of particular importance is that we have discovered by our isolation procedure that the resulting CP47-RC complex is mainly in a dimeric state with a molecular mass estimated to be 410 ± 30 kDa. We present several lines of evidence that the monomeric form of the complex is derived by dissociation of the dimer. This dissociation gives rise to the loss of PsbL and PsbK proteins and some chlorophyll. Of particular note is that the dimer to monomer conversion also results in the loss of bound plastoquinone-9.The fact that the CP47-RC dimer was significantly more abundant than the monomeric form in our preparation, contained more protein subunits, and bound plastoquinone suggests that it represents a more native form of the complex. This conclusion is consistent with the finding that the largest and functionally most active forms of isolated PSII from spinach are dimeric (7Hankamer B. Nield J. Zheleva D. Boekema E.J. Jansson S. Barber J. Eur. J. Biochem. 1997; 243: 422-429Crossref PubMed Scopus (171) Google Scholar). Moreover, the most up to date mapping of subunit positioning within PSII places CP43 toward the outer side of the central core dimer (4Hankamer B. Barber J. Boekema E.J. Annu. Rev. Plant Physiol. Mol. Biol. 1997; 48: 641-671Crossref PubMed Scopus (290) Google Scholar, 8Morris E.P. Hankamer B. Zheleva D. Friso G. Barber J. Structure. 1997; 5: 837-849Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar). Thus, its removal need not disturb the dimeric subcore complex composed of CP47-RC. It is noteworthy that a dimeric organization of CP47-RC was also observed in two-dimensional crystals, which have been analyzed recently by electron microscopy to 8 Å (9Rhee K.-H. Morris E.P Zheleva D. Hankamer B. Kühlbrandt W. Barber J. Nature. 1997; 389: 522-527Crossref Scopus (126) Google Scholar).We have shown by a combination of reverse phase HPLC, mass spectrometry, and N-terminal sequencing that the monomeric and dimeric forms of CP47-RC contain a number of small polypeptides in addition to CP47 and the D1 and D2 proteins. Present in both forms of the complex are the α- and β-subunits of cytochrome b 559and proteins that are the products of the psbI,psbT c, and psbW genes. The presence of cytochrome b 559 polypeptides and the PsbI protein is expected, given that these subunits occur in the isolated reaction center complex, which is depleted of CP47 (16Sharma J. Panico M. Barber J. Morris H.R. J. Biol. Chem. 1997; 272: 3935-3943Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar, 17Ikeuchi M. Inoue Y. FEBS Lett. 1989; 241: 99-104Crossref Scopus (147) Google Scholar, 25Webber A.N. Packman L. Chapman D.J. Barber J. Gray J.C. FEBS Lett. 1989; 242: 251-262Google Scholar). That PsbW would be present is also expected based on the work of Schroeder and colleagues (19Lorkovic Z.J. Schroeder W.P. Pakrasi H.B. Irrgang K.D. Herrmann R.G. Oelmuller R. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 8930-8934Crossref PubMed Scopus (83) Google Scholar, 26Irrgang K.D. Shi L.-X. Funk C. Schroeder W.P. J. Biol. Chem. 1995; 270: 17588-17593Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar). This is a nuclear encoded gene not found in the genome of Synechocystis 6803. ThepsbT c gene is located on the same operon as thepsbB gene (which encodes CP47) in the chloroplast genome. It had previously been known as the ycf8 gene, and its identification as a PSII gene has only recently been made using antibodies raised against its product (27Monod C. Takahashi Y. Goldschmidt-Clermont M. Rochaix J.-D. EMBO J. 1994; 13: 2747-2754Crossref PubMed Scopus (54) Google Scholar). Here we confirm that this protein is indeed a PSII component and further show that it is in the CP47-RC complex and therefore located close to the D1-D2 heterodimer.The fact that the dimer also contains the products of thepsbL and psbK genes but that the monomer does not could be important for explaining the difference in their ability to bind functionally active plastoquinone as well as accounting for the slightly higher chlorophyll content. It has previously been argued that PsbL is involved in the functioning of QA, possibly by stabilizing its binding to the D2 protein (28Nagatsuka T. Fukuhara S. Akabori K. Toyoshima Y. Biochim. Biophys. Acta. 1991; 1057: 223-231Crossref Scopus (29) Google Scholar). In terms of the recent structural analysis of the CP47-RC complex (9Rhee K.-H. Morris E.P Zheleva D. Hankamer B. Kühlbrandt W. Barber J. Nature. 1997; 389: 522-527Crossref Scopus (126) Google Scholar), it is possible to estimate the number of transmembrane helices likely to be present. Assuming only one copy of the α- and β-subunits of cytochromeb 559 (29Miyazaki A. Shina T. Toyoshima Y. Gounaris K. Barber J. Biochim. Biophys. Acta. 1989; 933: 423-431Google Scholar) and given that the small subunits, PsbI, PsbTc, PsbW, PsbK, and PsbL, are all predicted to have one transmembrane helix, the monomer would contain 21 transmembrane helices and the dimer would contain 23. These numbers rely on the likelihood that the D1-D2 heterodimer and CP47 contain 10 and 6 transmembrane helices, respectively (1Barber J. Miflin B.J. Oxford Surveys of Plant Molecular and Cellular Biology. 6. Oxford University Press, Oxford1989: 115-162Google Scholar, 4Hankamer B. Barber J. Boekema E.J. Annu. Rev. Plant Physiol. Mol. Biol. 1997; 48: 641-671Crossref PubMed Scopus (290) Google Scholar). If there are two copies of the cytochrome b 559 subunits as some advocate (30Lam E. Baltimore B. Ortiz W. Chollar S. Melis A. Malkin R. Biochim. Biophys. Acta. 1983; 724: 201-211Crossref Scopus (98) Google Scholar), then the total number of membrane-spanning segments would be 23 for the monomer and 25 for the dimer. In the two-dimensional crystals of CP47-RC recently analyzed to 8 Å by electron crystallography (9Rhee K.-H. Morris E.P Zheleva D. Hankamer B. Kühlbrandt W. Barber J. Nature. 1997; 389: 522-527Crossref Scopus (126) Google Scholar), the complex seems to be dimeric within the unit cell. Whether the dimer in the crystal is identical to that studied here is not certain, but if it is then the projection map obtained for the CP47-RC complex would be expected to contain at least 23 transmembrane helices. The light-induced splitting of water and the consequential release of dioxygen is a fundamental reaction in photosynthesis taking place in all higher plants, algae, and cyanobacteria. This reaction is catalyzed by a complex known as photosystem II (PSII) 1The abbreviations and protein names used are: PSII, photosystem II; RC, reaction center; CP47, chlorophyll-binding protein encoded by psbB gene; CP43, chlorophyll-binding protein encoded by psbC gene; D1 and D2 proteins, products of the psbA and psbD genes, respectively; DM,n-dodecyl β-d-maltoside; HTG, heptyl thioglycopyranoside; QA, the first PSII plastoquinone electron acceptor; QX and QY, optical absorptions with their transitions approximately parallel to thex and y axes of the chromophore; LHCII, light-harvesting complex of PSII; Mes, 4-morpholineethanesulfonic acid; HPLC, high pressure liquid chromatography; PAGE, polyacrylamide gel electrophoresis; Chl, chlorophyll; Pheo, pheophytin; BBY, membrane fraction enriched in PSII and isolated according to Ref. 32Berthold D.A. Babcock G.T. Yocum C.F. FEBS Lett. 1981; 134: 231-234Crossref Scopus (1647) Google Scholar; PQ, plastoquinone; PQ-9, plastoquinone-9. 1The abbreviations and protein names used are: PSII, photosystem II; RC, reaction center; CP47, chlorophyll-binding protein encoded by psbB gene; CP43, chlorophyll-binding protein encoded by psbC gene; D1 and D2 proteins, products of the psbA and psbD genes, respectively; DM,n-dodecyl β-d-maltoside; HTG, heptyl thioglycopyranoside; QA, the first PSII plastoquinone electron acceptor; QX and QY, optical absorptions with their transitions approximately parallel to thex and y axes of the chromophore; LHCII, light-harvesting complex of PSII; Mes, 4-morpholineethanesulfonic acid; HPLC, high pressure liquid chromatography; PAGE, polyacrylamide gel electrophoresis; Chl, chlorophyll; Pheo, pheophytin; BBY, membrane fraction enriched in PSII and isolated according to Ref. 32Berthold D.A. Babcock G.T. Yocum C.F. FEBS Lett. 1981; 134: 231-234Crossref Scopus (1647) Google Scholar; PQ, plastoquinone; PQ-9, plastoquinone-9. which is embedded in the lipid bilayer of the thylakoid membrane. The PSII complex is composed of at least 25 different proteins and binds a large number of pigments (1Barber J. Miflin B.J. Oxford Surveys of Plant Molecular and Cellular Biology. 6. Oxford University Press, Oxford1989: 115-162Google Scholar). At the heart of this complex is the reaction center (RC), consisting of the D1 and D2 proteins, where primary charge separation occurs (2Nanba O. Satoh K. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 109-112Crossref PubMed Google Scholar). Closely associated with the D1 and D2 heterodimer are the two chlorophyll a-binding proteins, CP47 and CP43, and a range of small hydrophobic polypeptides including the α- and β-subunits of cytochrome b 559. CP47 and CP43 act both as an “inner” light harvesting system and as a conduit for the transfer of energy captured by an “outer” light harvesting system composed either of proteins that bind chlorophyll aand" @default.
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