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 chlorophyll-protein complexes of Acetabularia. A novel chlorophyll ab complex which forms oligomers

(1) Five minor chlorophyll-protein complexes were isolated from thylakoid membranes of the green alga Acetabularia by SDS-polyacrylamide gel electrophoresis, after SDS or octylglucoside solubilization. None of them were related to CP I (Photosystem I reaction center core) or CP II (chlorophyll $\text{a}\text{b}$ light-harvesting complex). (2) Two complexes (CPa-1 and CPa-2) contained only chlorophyll (Chl) a, with absorption maxima of 673 and 671 nm, and fluorescence emission maxima of 683 nm compared to 676 nm for CP II. The complexes had apparent molecular masses of 43–47 and 38–40 kDa, and contained a single polypeptide of 41 and 37 kDa, respectively. They each account for about 3% of the total chlorophyll. (3) Three complexes had identical spectra, with Chl $\text{a}\text{b}$ ratios of 3–4 compared to 2 for thylakoid membranes, and a pronounced shoulder around 485 nm indicating enrichment in carotenoids. One of them was the complex ‘CP 29’ (Camm, E.L. and Green, B.R. (1980) Plant Physiol. 66, 428–432) and the other two were slightly different oligomeric forms of CP 29. They could be formed from CP 29 during reelectrophoresis; but about half the complex was isolated originally in an oligomeric form. Together they account for at least 7% of the total chlorophyll. Their function is unknown.     

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     

Interaction of CPa-1 with the manganese-stabilizing protein of photosystem II: identification of domains cross-linked by 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide.

The structural organization of photosystem II proteins has been investigated by use of the zero-length protein cross-linking reagent 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide and monoclonal and polyclonal antibody reagents. Photosystem II membranes were treated with 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide which cross-links amino groups to carboxyl groups which are in van der Waals contact. This treatment did not affect the oxygen evolution rates of these membranes and increased the retention of oxygen evolution after CaCl2 washing. Analysis of the proteins cross-linked by this treatment indicated that two cross-linked species with apparent molecular masses of 95 and 110 kDa were formed which cross-reacted with antibodies against both the 33-kDa manganese-stabilizing protein and the chlorophyll protein CPa-1. Cleavage of the 110-kDa cross-linked species with cyanogen bromide followed by N-terminal sequence analysis was used to identify the peptide fragments of CPa-1 and the manganese-stabilizing protein which were cross-linked. Two cyanogen bromide fragments were identified with apparent molecular masses of 50 and 25 kDa. N-Terminal sequence analysis of the 50-kDa cyanogen bromide fragment indicates that this consists of the C-terminal 16.7-kDa fragment of CPa-1 and the intact manganese-stabilizing protein. This strongly suggests that the manganese-stabilizing protein is cross-linked to the large extrinsic loop domain of CPa-1. N-Terminal analysis of the 25-kDa cyanogen bromide fragment indicates that this consists of the C-terminal 16.7-kDa peptide of CPa-1 and the N-terminal 8-kDa peptide of the manganese-stabilizing protein.(ABSTRACT TRUNCATED AT 250 WORDS)     

A new evaluation of chlorophyll absorption in photosynthetic membranes

The three major chlorophyll-proteins of spinach chloroplasts were solubilized with digitonin and isolated by electrophoresis with deoxycholate. The gel bands were identified from their absorption and fluorescence spectra measured at 77 K. The slowest moving band was a Photosystem I complex (CPI); the second, a Photosystem II complex (Cpa); and the third, a chlorophyll a-b, antenna complex (LHCP). When absorption spectra (630–730 nm) of the bands were added in the proportions found in the gel, the sum closely matched the absorption of the chloroplasts both before and after solubilization. Thus these spectra represent the native absorption of the major antenna chlorophyll-proteins of green plants. Each of these spectra was resolved with a computer assisted, curve-fitting program into 8 mixed Gaussian-Lorentzian shaped components. The major, Chl a components in the 3 fractions were different both in peak positions and bandwidths. This result suggests that each chlorophyll-protein has its own unique set of chlorophyll a spectral forms or components.Abbreviations Chl chlorophyll - CPI Photosystem I Chl-protein - CPa Photosystem II Chl-protein - LHCP light-harvesting Chl a-b protein - DOC sodium deoxycholate - SDS sodium dodecylsulfate CIW-DPB No. 819     

Functional discrimination between Photosystem-II associated chlorophyll a proteins in Zea mays

Three minor Chl a proteins were detected in electrophoretic profiles from wild-type maize thylakoids. The spectral characteristics of these Chl proteins and the apparent molecular weights of their constituent apoproteins suggested that they were associated with the Photosystem-II reaction center. One of these Chl a-proteins, CPa-1, was present in wild-type thylakoids and a photochemically active Photosystem-II particle, but was missing from thylakoids of a mutant-lacking Photosystem-II reaction center. CPa-2, on the other hand, was enriched in mutant thylakoids but was completely missing from the Photosystem-II particles. We conclude that CPa-1 is most likely to contain the photoactive chlorophyll of Photosystem II, while CPa-2 is not required for Photosystem-II activity. The apparent molecular weights of the major CPa-1 and CPa-2 apoproteins were 48 000 and 42 000, respectively. The third minor Chl protein seems most likely to be an electrophoretic variant of CPa-1 and has been designated CPa-11. Seven other Chl proteins were detected in wild-type profiles. Many of these Chl proteins appeared to be oligomers or highly order complexes of LHCP and CP-1.     

Interaction of CPa-1 with the manganese-stabilizing protein of photosystem II: identification of domains on CPa-1 which are shielded from N-hydroxysuccinimide biotinylation by the manganese-stabilizing protein.

The structural organization of photosystem II proteins has been investigated by use of the amino group-labeling reagent N-hydroxysuccinimidobiotin (NHS-biotin) and calcium chloride-washed photosystem II membranes. We have previously shown that the presence of the extrinsic, manganese-stabilizing protein on photosystem II membranes prevents the modification of lysyl residues located on the chlorophyll protein CPa-1 (CP-47) by NHS-biotin [Bricker, T. M., Odom, W. R., & Queirolo, C. B. (1988) FEBS Lett. 231, 111-117]. Upon removal of the manganese-stabilizing protein by calcium chloride-washing, CPa-1 can be specifically modified by treatment with NHS-biotin. Preparative quantities of biotinylated CPa-1 were subjected to chemical cleavage with cyanogen bromide. Two major biotinylated peptides were identified with apparent molecular masses of 11.8 and 15.7 kDa. N-terminal sequence analysis of these peptides indicated that the 11.8-kDa peptide was 232G-330M and that the 15.7-kDa peptide was 360P-508V. The 15.7-kDa CNBr peptide was subjected to limited tryptic digestion. The two smallest tryptic fragments identified migrated at apparent molecular masses of 9.1 (nonbiotinylated) and 7.5 kDa (biotinylated). N-terminal sequence analysis and examination of the predicted amino acid sequences of these peptides suggest that the 9.1-kDa fragment was 422R-508V and that the 7.5-kDa fragment was 360P-421A. These results strongly suggest that two NHS-biotinylated domains, 304K-321K and 389K-419K, become exposed on CPa-1 when the manganese-stabilizing protein is removed by CaCl2 treatment. Both of these domains lie in the large extrinsic loop E of CPa-1.     

Pigment and protein composition of reconstituted light-harvesting complexes and effects of some protein modifications

The structure and heterogeneity of LHC II were studied by in vitro reconstitution of apoproteins with pigments (Plumley and Schmidt 1987, Proc Natl Acad Sci 84: 146–150). Reconstituted CP 2 complexes purified by LDS-PAGE were subsequently characterized and shown to have spectroscopic properties and pigment-protein compositions and stoichiometries similar to those of authentic complexes. Heterologous reconstitutions utilizing pigments and light-harvesting proteins from spinach, pea and Chlamydomonas reinhardtii reveal no evidence of specialized binding sites for the unique C. reinhardtii xanthophyll loroxanthin: lutein and loroxanthin are interchangeable for in vitro reconstitution. Proteins modified by the presence of a transit peptide, phosphorylation, or proteolytic removal of the NH₂-terminus could be reconstituted. Evidence suggests that post-translational modification are not responsible for the presence of six electrophoretic variants of C. reinhardtii CP 2. Reconstitution is blocked by iodoacetamide pre-treatment of the apoproteins suggesting a role for cysteine in pigment ligation and/or proper folding of the pigment-protein complex. Finally, no effect of divalent cations on pigment reassembly could be detected.Abbreviations cab chlorophyll a/b-binding protein genes - Chl chlorophyll - CP2 light-harvesting chlorophyll A+b-protein complex fractionated by mildly denaturing LDS-PAGE from Photosystem II in thylakoids - CP 43 and CP 47 chlorophyll a-antenna complexes fractionated from Photosystem II in thylakoids by mildly denaturing LDS-PAGE at 4°C - IgG gamma immunoglobulin - LDS lithium dodecyl sulfate - LDS-PAGE lithium dodecyl sulfate polyacrylamide gel electrophoresis at 4°C - LHC I and LHC II thylakoid light-harvesting chlorophyll a+b-protein holocomplexes associated with Photosystems I and II, respectively - PS II Photosystem II - TX100 Triton X-100 - TX100-derived LHC light-harvesting complexes enriched in LHC II following fractionation of thylakoids by TX100     

COOH-terminal residues of D1 and the 44 kDa CPa-2 at spinach photosystem II core complex

The COOH-termini of the 32 kDa D1 and 44 kDa CPa-2 were determined by protein sequencing of peptides from trypsinized photosystem II core complexes. COOH-terminal fragments were isolated by affinity chromatography using anhydrotrypsin-agarose. One peptide had a sequence corresponding to the segment from Asn at position 335 to Ala at position 344 of the sequence deduced from the psbA gene coding for D1. Nine amino acids may be cleaved from the COOH-terminus of pre-D1 during maturation. In contrast, CPa-2 was not modified at its COOH-terminus.     

Isolation and crystallization of CP47, a Photosystem II chlorophyll binding protein. Degradation of CP47 upon dissociation from the core complex

The CP47 protein was isolated from Photosystem II membranes by using a combination of the detergents n-dodecyl-β-D-maltoside and octyl-β-D-thioglucoside. The purified CP47 was used in a series of crystallization experiments, which yielded highly reproducible hexagonal crystals. Immunoblot analysis revealed that the isolated CP47 undergoes degradation even under dim light conditions. This degradation takes place after the protein has been dissociated from the core complex. Proteolysis experiments with trypsin demonstrated that the dissociation of the CP47 from the PS II core complex results in changes that render the protein sensitive to proteolysis. This revised version was published online in June 2006 with corrections to the Cover Date.     

The distribution of Photosystem I and Photosystem II polypeptides between the cytoplasmic and thylakoid membranes of cyanobacteria

Abstract In a previous study we found that the 33 kDa extrinsic polypeptide of Photosystem II is present in both the cytoplasmic and thylakoid membranes of cyanobacteria, but forms part of a functional complex only in the latter [Smith et al. (1987) Mol. Microbiol. 6, 1821–1827]. In order to determine if this phenomenon is restricted to the 33 kDa polypeptide we have extended this study in Anacystis nidulans to include a number of other polypeptides of Photosystem I and Photosystem II. We have found that D1 and possibly PsaC are present in both membranes, CP43 and CP47 are confined to the thylakoid membranes, and the distribution of PsaD and PsaE is dependent upon the growth stage of the cyanobacteria.     

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

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

A new evaluation of chlorophyll absorption in photosynthetic membranes

The three major chlorophyll-proteins of spinach chloroplasts were solubilized with digitonin and isolated by electrophoresis with deoxycholate. The gel bands were identified from their absorption and fluorescence spectra measured at 77 K. The slowest moving band was a Photosystem I complex (CPI); the second, a Photosystem II complex (Cpa); and the third, a chlorophyll a-b, antenna complex (LHCP). When absorption spectra (630–730 nm) of the bands were added in the proportions found in the gel, the sum closely matched the absorption of the chloroplasts both before and after solubilization. Thus these spectra represent the native absorption of the major antenna chlorophyll-proteins of green plants. Each of these spectra was resolved with a computer assisted, curve-fitting program into 8 mixed Gaussian-Lorentzian shaped components. The major, Chl a components in the 3 fractions were different both in peak positions and bandwidths. This result suggests that each chlorophyll-protein has its own unique set of chlorophyll a spectral forms or components.     

Energy transfer for low temperature fluorescence in PS II mutant thylakoids

The Chl-protein complexes of three maize (Zea mays L.) mutants and one barley (Hordeum vulgare L.) mutant were analyzed using low temperature Chl fluorescence emissions spectroscopy and LDS-polyacrylamide gel electrophoresis. The maize mutants hcf-3, hcf-19, and hcf-114 all exhibited a high Chl fluorescence (hcf) phenotype indicating a disruption of the energy transfer within the photosynthetic apparatus. The mutations in each of these maize mutants affects Photosystem II. The barley mutant analyzed was the well characterized Chl b-less mutant chlorina-f2, which did not exhibit the hcf phenotype. Chlorina-f2 was used because no complete Chl b-less mutant of maize is available. Analysis of hcf-3, hcf-19, and hcf-114 revealed that in the absence of CP43, LHC II can still transfer excitation energy to CP47. These results suggest that in mutant membranes LHC II can interact with CP47 as well as CP43. This functional interaction of LHC II with CP47 may only occur in the absence of CP43, however, it is possible that LHC II is positioned in the thylakoid membranes in a manner which allows association with both CP43 and CP47.Abbreviations hcf high chlorophyll fluorescence - LDS lithium dodecyl sulfate - LHC II light-harvesting complex of Photosystem II - LHC I light-harvesting complex of Photosystem I - CPIa chlorophyll-protein complex consisting of LHC I and the PS I core complex - CPI chlorophyll-protein complex consisting of the PS I core complex - CP47 47 kDa chlorophyll-protein of the Photosystem II core - CP43 43 kDa chlorophyll-protein of the Photosystem II core - CP29 29 kDa chlorophyll-protein of Photosystem II - CP26 26 kDa chlorophyll-protein of Photosystem II - CP24 24 kDa chlorophyll-protein of Photosystem II - fp free pigments     

Multiple forms of chlorophyll-protein complexes from a thermophilic cyanobacterium Synechococcus sp

Eight chlorophyll-proteins were resolved from the thylakoid membranes, or digitonin particles, of a thermophilic cyanobacterium Synechococcus sp. by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Six chlorophyll-proteins with slower electrophoretic mobilities were shown to be P700-chlorophyll a-protein complexes (CP1), whereas faster-moving proteins (CP2) were related to photosystem 2. Extraction of CP1 complexes from the membranes with different detergent/chlorophyll ratios and reelectrophoresis of extracted CP1 complexes indicated that the chlorophyll-proteins are closely interrelated with each other; any CP1 complex could be transformed to other CP1 complexes with faster electrophoretic mobilities. This, together with the Ferguson plot and the polypeptide composition, showed that six CP1 complexes are different in terms of polypeptide composition, oligomerization, SDS-binding, or conformation of the proteins but represent, in the order of increasing electrophoretic mobility, increasing degree of modification of the native P700-chlorophyll a-protein.     

Isolation and characterization of chloroplast Photosystem II antenna of spinach by reversed-phase liquid chromatography

The protein components of the Photosystem II antenna system, isolated from spinach thylakoids, have been resolved by reversed-phase high performance liquid chromatography (RP-HPLC) using a butyl-silica stationary phase packed either into analytical or semi-preparative columns. Peak identification has been accomplished by a combination of various SDS–PAGE systems employing either Comassie (or silver) staining or immunological detection using polyclonal antibodies raised against LHC II and against CP29, CP26 and CP24 proteins and by aminoacid microsequence. Moreover, peak identification is consistent with the molecular masses determined by Electrospray Ionization Mass Spectrometry (HPLC-ESI-MS). The developed RP-HPLC method allows the resolution of all the protein components of the Photosystem II major Light Harvesting Complex (LHC II) and minor PS II antenna complex (CP24, CP26 and CP29) from grana membranes (BBY) and estimation of their relative stoichiometry in natural and stressed conditions, avoiding the expensive and time consuming separation procedure by sucrose-gradient ultracentrifugation and isoelectrofocusing.     

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     

Characterization of a spinach photosystem II core preparation isolated by a simplified method

A photosystem II core complex from spinach exhibiting high rates of electron transport was obtained rapidly and in high yield by treatment of a Tris-extracted, O2-evolving photosystem II preparation with the detergent dodecyl-beta-D-maltoside. The core complex was essentially free of light-harvesting chlorophyll-protein and photosystem I polypeptides, and was highly enriched in the polypeptides associated with the photosystem II reaction center (45 and 49 kDa), cytochrome b559, and three polypeptides in the region 32-34 kDa. The photosystem II core complex contained two chlorophyll-proteins which had a slightly higher apparent molecular mass than CPa-1 and CPa-2. Additionally, a high-molecular-mass chlorophyll-protein complex termed CPa* was observed, which exhibited a low fluorescence yield when illuminated with ultraviolet light. This observation suggests that CPa* contains a functionally efficient quencher of chlorophyll fluorescence, possibly P680.     

Site-directed mutagenesis of the CPa-1 protein of photosystem II: alteration of the basic residue pair 384,385R to 384,385G leads to a defect associated with the oxygen-evolving complex.

The psbB gene encodes the intrinsic chlorophyll-a binding protein CPa-1 (CP-47), a component of photosystem II in higher plants, algae, and cyanobacteria. Oligonucleotide-directed mutagenesis was used to introduce mutations into a segment of the psbB gene encoding the large extrinsic loop region of CPa-1 in the cyanobacterium Synechocystis sp. PCC 6803. Altered psbB genes were introduced into a mutant recipient strain (DEL-1) of Synechocystis in which the genomic psbB gene had been partially deleted. Initial target sites for mutagenesis were absolutely conserved basic residue pairs occurring within the large extrinsic loop. One mutation, RR384385GG, produced a strain with impaired photosystem II activity. This strain exhibited growth characteristics comparable to controls. However, at saturating light intensities this mutant strain evolved oxygen at only 50% of the rate of the control strains. Quantum yield measurements at low light intensities indicated that the mutant had 30% fewer fully functional photosystem II centers than do control strains of Synechocystis. Immunological analysis of a number of photosystem II protein components indicated that the mutant accumulates normal quantities of photosystem II proteins and that the ratio of photosystem II to photosystem I proteins is comparable to that found in control strains. Upon exposure to high light intensities the mutant cells exhibited a markedly increased susceptibility to photoinactivation. However, Tris-treated thylakoid membranes from both the mutant and wild-type exhibited comparable rates of photoinactivation. Thylakoid membranes isolated from RR384385GG exhibited only 15% of the H2O to 2,6-dichlorophenolindophenol electron transport rate observed in wild-type strains.(ABSTRACT TRUNCATED AT 250 WORDS)     

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