Heterogenous lipid distribution among chlorophyll-binding proteins of photosystem II in maize mesophyll chloroplasts

Photosystem II membrane fractions from dark-adapted mesophyll chloroplasts of maize were solubilized in different concentrations of dodecyl beta-D-maltoside. Chlorophyll-binding proteins from photosystem II were isolated either by ultracentrifugation on a sucrose gradient, or by flat bed isoelectric focusing and identified by gel electrophoresis analysis for their polypeptide composition. Lipid and fatty acid compositions were determined in complexes prepared by both methods and also in purified light-harvesting complex II, in minor chlorophyll a/b binding complexes 29, 26, 24, in photosystem II antennae (chlorophyll-protein complexes 43, 47) and in the photosystem II reaction centers chlorophyll-protein complexes. Comparative analysis of the results suggests that a true heterogeneity exists in the lipid class distribution among the different chlorophyll-protein complexes in this region of the photosynthetic membrane. Photosystem II core fractions prepared either by ultra-centrifugation on a sucrose gradient or by isoelectric focusing were found significantly enriched in monogalactosyldiacylglycerol; fractionation of the photosystem II core in its components showed that it was the chlorophyll-protein complexes 43 and 47 which were mainly responsible for this enrichment. One of them, the chlorophyll-protein complex 47, was found containing monogalactosyldiacylglycerol and having a very high level of saturated fatty acids. The minor chlorophyll a/b binding linkers (chlorophyll-protein complexes 24, 26 and 29) retain a largely higher amount of lipids than all other complexes and especially of highly unsaturated galactolipids. Concerning the main light-harvesting antenna (LHCII), it is demonstrated that phosphatidylglycerol is strongly linked to the complex if it cannot be detached at high detergent concentration, while many galactolipids (which nevertheless represent the major lipid classes) are lost. This main light-harvesting complex has been fractionated into several families by isoelectric focusing showing a marked difference in lipid and polypeptide composition. A spectacular increase in the phosphatidylglycerol content was observed in the fraction migrating near the anode and enriched in a 26-kDa polypeptide; but this result is difficult to interpret in physiological terms as it was shown that phosphatidylglycerol alone, because of its negative charge, also migrates toward the anode in isoelectric focusing.     

Effects of cations upon chloroplast membrane subunit. Interactions and excitation energy distribution

When isolated chloroplasts from mature pea (Pisum sativum) leaves were treated with digitonin under "low salt" conditions, the membranes were extensively solubilized into small subunits (as evidenced by analysis with small pore ultrafilters). From this solubilized preparation, a photochemically inactive chlorophyll - protein complex (chlorophyll alpha/beta ratio, 1.3) was isolated. We suggest that the detergent-derived membrane fragment from mature membranes is a structural complex within the membrane which contains the light-harvesting chlorophyll alpha/beta protein and which acts as a light-harvesting antenna primarily for Photosystem II. Cations dramatically alter the structural interaction of the light-harvesting complex with the photochemically active system II complex. This interaction has been measured by determining the amount of protein-bound chlorophyll beta and Photosystem II activity which can be released into dispersed subunits by digitonin treatment of chloroplast lamellae. When cations are present to cause interaction between the Photosystem II complex and the light-harvesting pigment - protein, the combined complexes pellet as a "heavy" membranous fraction during differential centrifugation of detergent treated lamellae. In the absence of cations, the two complexes dissociate and can be isolated in a "light" submembrane preparation from which the light-harvesting complex can be purified by sucrose gradient centrifugation. Cation effects on excitation energy distribution between Photosystems I and II have been monitored by following Photosystem II fluorescence changes under chloroplast incubation conditions identical to those used for detergent treatment (with the exception of chlorophyll concentration differences and omission of detergents). The cation dependency of the pigment - protein complex and Photosystem II reaction center interactions measured by detergent fractionation, and regulation of excitation energy distribution as measured by fluorescence changes, were identical. We conclude that changes in substructural organization of intact membranes, involving cation induced changes in the interaction of intramembranous subunits, are the primary factors regulating the distribution of excitation energy between Photosystems II and I.     

Spatial relationship of photosystem I, photosystem II, and the light-harvesting complex in chloroplast membranes

We have previously demonstrated (Armond, P. A., C. J. Arntzen, J.-M. Briantais, and C. Vernotte. 1976. Arch. Biochem. Biophys. 175:54-63; and Davis, D. J., P. A. Armond, E. L. Gross, and C. J. Arntzen. 1976. Arch. Biochem. Biophys. 175:64-70) that pea seedlings which were exposed to intermittent illumination contained incompletely developed chloroplasts. These plastids were photosynthetically competent, but did not contain grana. We now demonstrate that the incompletely developed plastids have a smaller photosynthetic unit size; this is primarily due to the absence of a major light-harvesting pigment-protein complex which is present in the mature membranes. Upon exposure of intermittent-light seedlings to continuous white light for periods up to 48 h, a ligh-harvesting chlorophyll-protein complex was inserted into the chloroplast membrane with a concomitant appearance of grana stacks and an increase in photosynthetic unit size. Plastid membranes from plants grown under intermediate light were examined by freeze-fracture electron microscopy. The membrane particles on both the outer (PF) and inner (EF) leaflets of the thylakoid membrane were found to be randomly distributed. The particle density of the PF fracture face was approx. four times that of the EF fracture face. While only small changes in particle density were observed during the greening process under continuous light, major changes in particle size were noted, particularly in the EF particles of stacked regions (EFs) of the chloroplast membrane. Both the changes in particle size and an observed aggregation of the EF particles into the newly stacked regions of the membrane were correlated with the insertion of light-harvesting pigment-protein into the membrane. Evidence is presented for identification of the EF particles as the morphological equivalent of a "complete" photosystem II complex, consisting of a phosochemically active "core" complex surrounded by discrete aggregates of the light-harvesting pigment protein. A model demonstrating the spatial relationships of photosystem I, photosystem II, and the light-harvesting complex in the chloroplast membrane is presented.     

Supramolecular organization of the photosynthetic apparatus of Rhodobacter sphaeroides

Native tubular membranes were purified from the purple non-sulfur bacterium Rhodobacter sphaeroides. These tubular structures contain all the membrane components of the photosynthetic apparatus, in the relative ratio of one cytochrome bc1 complex to two reaction centers, and approximately 24 bacteriochlorophyll molecules per reaction center. Electron micrographs of negative-stained membranes diffract up to 25 A and allow the calculation of a projection map at 20 A. The unit cell (a = 198 A, b = 120 A and gamma = 103 degrees) contains an elongated S-shaped supercomplex presenting a pseudo-2-fold symmetry. Comparison with density maps of isolated reaction center and light-harvesting complexes allowed interpretation of the projection map. Each supercomplex is composed of light-harvesting 1 complexes that take the form of two C-shaped structures of approximately 112 A in external diameter, facing each other on the open side and enclosing the two reaction centers. The remaining positive density is tentatively attributed to one cytochrome bc1 complex. These features shed new light on the association of the reaction center and the light-harvesting complexes. In particular, the organization of the light-harvesting complexes in C-shaped structures ensures an efficient exchange of ubihydroquinone/ubiquinone between the reaction center and the cytochrome bc1 complex.     

Biochemical and functional properties of photosystem II in agranal membranes from maize mesophyll and bundle sheath chloroplasts

We have studied the occurrence and organization of photosystem II (PSII) in bundle sheath thylakoids and stroma lamellae from maize. As shown by non-denaturing lauryl beta-D- iminopropionidate (Deriphat)/PAGE, PSII exists in a dimeric form in grana membranes. In bundle sheath and stroma lamellae, however, only a monomeric form was found. Based on immunotitration data, we estimated the stoichiometry of the individual components of the PSII core complex and antenna systems. In stroma lamellae, all PSII antenna complexes had a stoichiometry similar to that in grana membranes, with the exception of light-harvesting complex II (LHCII) that was somewhat over-represented, while the minor antenna complexes CP26 and CP29 were under-represented. In bundle sheath, the amount of LHCII was approximately eight times higher than expected with respect to D1. The 33-kDa protein of the oxygen-evolving enhancer polypeptides was not detectable nor was the ferredoxin-NADP+ reductase, thus strongly suggesting that no significant linear electron transport occurs in bundle sheath thylakoids. Fluorescence induction data suggest that most of the PSII reaction centers in bundle sheath and stroma lamellae sustain electron transport towards a secondary acceptor pool. Stromal PSII centers are only weakly inhibited by 3-(3,4-dichlorophenyl)-1,1-dimethylurea (diuron), whereas, unexpectedly, dichlorobenzoquinone and methyl viologen had a pronounced inhibitory effect of the QA- reoxidation. An additional specificity of these centers is the slow rate (50-ms range) of the QA to QB electron transfer. The amplitude of variable fluorescence found in stroma lamellae can only account for a small fraction (1-2%) of the variable fluorescence of whole thylakoids. This suggests that stromal PSII cannot be solely responsible for the slow beta-phase of the induction kinetics.     

Close vicinity of Lhc b1 and Lhc b4 in Photosystem II--membrane fragments as verified by chemical cross-linking

The nearest neighbourhood of pigment-protein complexes within Photosystem II (PSII) membrane fragments has been studied by means of chemical cross-linking with o-phthalaldehyde (OPA) in conjunction with protein-chemical techniques. By means of OPA-induced cross-linking a major conjugate of about 60 kDa has been identified. This conjugate was shown to consist of two pigment-protein complexes of light-harvesting complex II (LHC II), Lhc b1 (CP27) and Lhc b4 (CP29) by means of SDS/PAGE in combination with an immunological analysis using mAbs directed against Lhc b4 and by matrix-assisted-laser-desorption-ionization mass spectrometry (MALDI-MS) and sequence analysis of peptides derived from a proteolytic digest of the conjugate. Domains of Lhc bl and Lhc b4 have been localized to a distance of not more than 5 A within LHC II. Our results are discussed in the light of recent models on the topography of the two subunits within the antenna system of Photosystem II.     

The peculiarities of pigment-protein composition of intergranular thylakoid fragments from maize chloroplasts

The pigment-protein composition of two fractions of intergrana fragments from maize inbred lines F 7 and II 346 chloroplast had been investigated. It was shown that under electrophoretic separation of fraction 70,000 g from both lines and fraction 100,000 g from line F 7 the new band of pigment-protein complexes had been observed. It was determined that its polypeptide composition is the same as light-harvesting complex of PS II one, but it differs by low electrophoretical mobility. The conclusion was made that this is a new form of light-harvesting complex of PS II.     

Purification and characterization of oxygen-evolving photosystem II core complexes from the green alga Chlamydomonas reinhardtii

Oxygen-evolving photosystem II complexes were isolated from the green alga Chlamydomonas reinhardtii by selective solubilization of thylakoid membranes with dodecyl maltoside followed by density gradient centrifugation and anion-exchange chromatography. In the presence of CaCl2 and K3[Fe(CN)6] the complexes evolved oxygen at rates exceeding 1000 mumol (mg of chl)-1 h-1. The particles contained 40 chlorophylls a and had properties very similar to those of PSII isolated from higher plants. Chlamydomonas reinhardtii is now the first organism which can be used for both site-directed mutagenesis and detailed biochemical and biophysical characterization of oxygen-evolving photosystem II. It seems therefore to be an ideal model organism for investigation of structure-function relationships in photosynthetic oxygen evolution.     

Involvement of active oxygen species in degradation of the D1 protein under strong illumination in isolated subcomplexes of photosystem II

The effects of strong illumination on the proteins in photosystem II (PSII) were investigated using three different isolated subcomplexes of PSII, namely, the PSII complex depleted of major light-harvesting proteins, the core complex, and the reaction center complex. Under illumination, not only the D1 protein of the reaction center but also other intrinsic proteins sustained some damage in all three subcomplexes: Coomassie blue-stained bands after polyacrylamide gel electrophoresis were smeared, and their migration distances on the gel were reduced with increasing duration of illumination. Such damage occurred first in the D1 and D2 proteins and subsequently in the 43- and 47-kDa proteins of the core antenna and the subunit of cytochrome b559. Immunoblot analysis using an antibody specific to the D1 protein showed that the D1 protein was degraded to major fragments of about 23 and 16 kDa during illumination. The smearing and changes in mobility of protein bands, as well as the fragmentation of the D1 protein, were greatly suppressed by scavengers of active oxygen species. From the effectiveness of scavengers, it appeared that superoxide anions participate in the protein damage in the PSII complex, hydrogen peroxide in the PSII and core complexes, and singlet oxygen, hydroxyl, and alkoxyl radicals in all three subcomplexes. We also found that fragments of the D1 protein of 23 and 16 kDa were formed even when PSII complexes that had been completely solubilized with sodium dodecyl sulfate were illuminated. This fragmentation was also suppressed by active oxygen scavengers.(ABSTRACT TRUNCATED AT 250 WORDS)     

Reconstitution and pigment-binding properties of recombinant CP29

The minor light-harvesting chlorophyll-a/b-binding protein CP29 (Lhcb4), overexpressed in Escherichia coli, has been reconstituted in vitro with pigments. The recombinant pigment-protein complexes show biochemical and spectral properties identical to the native CP29 purified from maize thylakoids. The xanthophyll lutein is the only carotenoid necessary for reconstitution, a finding consistent with the structural role of two lutein molecules/polypeptide suggested by the crystallographic data for the homologous protein light-harvesting chlorophyll-a/b-binding protein of photosystem II (LHCII). The CP29 protein scaffold can accommodate different chromophores. This conclusion was deduced by the observation that the pigment composition of the reconstituted protein depends on the pigments present in the reconstitution mixture. Thus, in addition to a recombinant CP29 identical to the native one, two additional forms of the complex could be obtained by increasing chlorophyll b content. This finding is typical of CP29 because the major LHCII complex shows an absolute selectivity for chromophore binding [Plumley, F. G. & Schmidt, G. W. (1987) Proc. Natl Acad. Sci. USA 84, 146-150; Paulsen, H., Rümler, U. & Rüdiger, W. (1990) Planta (Heidelb.) 181, 204-211], and it is consistent with the higher stability of CP29 during greening and in chlorophyll b mutants compared with LHCII.     

Role of the RCII-D1 protein in the reversible association of the oxygen-evolving complex proteins with the lumenal side of photosystem II

The nuclear-encoded proteins of the oxygen-evolving complex (OEC) of photosystem II are bound on the lumenal side of the thylakoid membrane and stabilize the manganese ion cluster forming the photosystem II electron donor side. The OEC proteins are released from their binding site(s) following light-induced degradation of reaction center II (RCII)-D1 protein in Chlamydomonas reinhardtii. The kinetics of OEC proteins release correlates with that of RCII-D1 protein degradation. Only a limited amount of RCII-D2 protein is degraded during the process, and no loss of the core proteins CP43 and CP47 is detected. The release of the OEC proteins is prevented when the photoinactivated RCII-D1 protein degradation is retarded by addition of 3-(3,5-dichlorophenyl)-1,1-dimethylurea or by a high PQH2/PQ ratio prevailing in membranes of the plastocyanin-deficient mutant Ac208. The released proteins are not degraded but persist in the thylakoid lumen for up to 8 h and reassociate with photosystem II when new D1 protein is synthesized in cells exposed to low light, thus allowing recovery of photosystem II function. Reassociation also occurs following D1 protein synthesis in darkness when RCII activity is only partially recovered. These results indicate that (i) the D1 protein participates in the formation of the lumenal OEC proteins binding site(s) and (ii) the photoinactivation of RCII-D1 protein does not alter the conformation of the donor side of photosystem II required for the binding of the OEC proteins.     

Site-directed photosystem II mutants with perturbed oxygen-evolving properties. 1. Instability or inefficient assembly of the manganese cluster in vivo

Several site-directed photosystem II mutants with substitutions at Asp-170 of the D1 polypeptide were characterized by noninvasive methods in vivo. In several mutants, including some that evolve oxygen, a significant fraction of photosystem II reaction centers are shown to lack photooxidizable Mn ions. In this fraction of reaction centers, either the high-affinity site from which Mn ions rapidly reduce the oxidized secondary electron donor, YZ+, is devoid of Mn ions or the Mn ion(s) bound at this site are unable to reduce YZ+. It is concluded that the Mn clusters in these mutants are unstable or are assembled inefficiently in vivo. Mutants were constructed in the unicellular cyanobacterium Synechocystis sp. PCC 6803. The in vivo characterization procedures employed in this study involved measuring changes in the yield of variable chlorophyll a fluorescence following a saturating flash or brief illumination given in the presence of the electron transfer inhibitor 3-(3,4-dichlorophenyl)-1,1-dimethylurea, or following each of a series of saturating flashes given in the absence of this inhibitor. These procedures are easily applied to mutants that evolve little or no oxygen, facilitate the characterization of mutants with labile oxygen-evolving complexes, permit photosystem II isolation efforts to be concentrated on mutants having the stablest Mn clusters, and guide systematic spectroscopic studies of isolated photosystem II particles to mutants of particular interest.     

Investigation of the structure of spinach photosystem II reaction center complex

The photosystem II (PSII) reaction center (RC) complex was isolated from spinach and characterized by gel electrophoresis, gel filtration and analytical ultracentrifugation. The purified complex contained the PsbA, PsbD, PsbE, PsbF and PsbI subunits. Gel filtration and analytical ultracentrifugation indicated the presence of a homogeneous complex. The mass of the RC complexes was found to be 107 kDa by analytical ultracentrifugation and 132 kDa by scanning transmission electron microscopy (STEM). The mass obtained showed the isolated complex to exist as a monomer and only one cytochrome b559 (cyt b559) to be associated with the RC complex. Digital images of negatively stained RC complexes were recorded by STEM and analyzed by single-particle averaging. The complex was 9 nm long and 5 nm wide, and exhibited a pronounced quasi-twofold symmetry. This supports the symmetric organization of the PSII complex, with the PsbA and the PsbD proteins in the center and symmetrically arranged PsbB and PsbC proteins at the periphery of the monomeric complex.     

Regulation of SNARE complex assembly by an N-terminal domain of the t-SNARE Sso1p

The fusion of intracellular transport vesicles with their target membranes requires the assembly of SNARE proteins anchored in the apposed membranes. Here we use recombinant cytoplasmic domains of the yeast SNAREs involved in Golgi to plasma membrane trafficking to examine this assembly process in vitro. Binary complexes form between the target membrane SNAREs Sso1p and Sec9p; these binary complexes can subsequently bind to the vesicle SNARE Snc2p to form ternary complexes. Binary and ternary complex assembly are accompanied by large increases in alpha-helical structure, indicating that folding and complex formation are linked. Surprisingly, we find that binary complex formation is extremely slow, with a second-order rate constant of approximately 3 M(-1) s(-1). An N-terminal regulatory domain of Sso1p accounts for slow assembly, since in its absence complexes assemble 2,000-fold more rapidly. Once binary complexes form, ternary complex formation is rapid and is not affected by the presence of the regulatory domain. Our results imply that proteins that accelerate SNARE assembly in vivo act by relieving inhibition by this regulatory domain.     

Reversible particle movements associated with unstacking and restacking of chloroplast membranes in vitro

Freeze-fracture and freeze-etch techniques have been employed to study the supramolecular structure of isolated spinach chloroplast membranes and to monitor structural changes associated with in vitro unstacking and restacking of these membranes. High-resolution particle size histograms prepared from the four fracture faces of normal chloroplast membranes reveal the presence of four distinct categories of intramembranous particles that are nonrandomly distributed between grana and stroma membranes. The large surface particles show a one to one relationship with the EF-face particles. Since the distribution of these particles between grana and stroma membranes coincides with the distribution of photosystem II (PS II) activity, it is argued that they could be structural equivalents of PS II complexes. An interpretative model depicting the structural relationship between all categories of particles is presented. Experimental unstacking of chloroplast membranes in low-salt medium for at least 45 min leads to a reorganization of the lamellae and to a concomitant intermixing of the different categories of membrane particles by means of translational movements in the plane of the membrane. In vitro restacking of such experimentally unstacked chloroplast membranes can be achieved by adding 2-20 mM MgCl2 or 100-200 mM NaCl to the membrane suspension. Membranes allowed to restack for at least 1 h at room temperature demonstrate a resegregation of the EF-face particles into the newly formed stacked membrane regions to yield a pattern and a size distribution nearly indistinguishable from the normally stacked controls. Restacking occurs in two steps: a rapid adhesion of adjoining stromal membrane surfaces with little particle movement, and a slower diffusion of additional large intramembranous particles into the stacked regions where they become trapped. Chlorophyll a:chlorophyll b ratios of membrane fraction obtained from normal, unstacked, and restacked membranes show that the particle movements are paralleled by movements of pigment molecules. The directed and reversible movements of membrane particles in isolated chloroplasts are compared with those reported for particles of plasma membranes.     

A chloroplast processing enzyme functions as the general stromal processing peptidase

A highly specific stromal processing activity is thought to cleave a large diversity of precursors targeted to the chloroplast, removing an N-terminal transit peptide. The identity of this key component of the import machinery has not been unequivocally established. We have previously characterized a chloroplast processing enzyme (CPE) that cleaves the precursor of the light-harvesting chlorophyll a/b binding protein of photosystem II (LHCPII). Here we report the overexpression of active CPE in Escherichia coli. Examination of the recombinant enzyme in vitro revealed that it cleaves not only preLHCPII, but also the precursors for an array of proteins essential for different reactions and destined for different compartments of the organelle. CPE also processes its own precursor in trans. Neither the recombinant CPE nor the native CPE of chloroplasts process a preLHCPII mutant with an altered cleavage site demonstrating that both forms of the enzyme are sensitive to the same structural modification of the substrate. The transit peptide of the precursor of ferredoxin is released by a single cleavage event and found intact after processing by recombinant CPE and a chloroplast extract as well. These results provide the first direct demonstration that CPE is the general stromal processing peptidase that acts as an endopeptidase. Significantly, recombinant CPE cleaves in the absence of other chloroplast proteins, and this activity depends on metal cations, such as zinc.     

Spectral and photochemical properties of borohydride-treated D1-D2-cytochrome b-559 complex of photosystem II

The D1-D2-cytochrome b-559 reaction center complex of photosystem II with an altered pigment composition was prepared from the original complex by treatment with sodium borohydride (BH4-). The absorption spectra of the modified and original complexes were compared to each other and to the spectra of purified chlorophyll a and pheophytin a (Pheo a) treated with BH4- in methanolic solution. The results of these comparisons are consistent with the presence in the modified complex of an irreversibly reduced Pheo a molecule, most likely 13(1)-deoxo-13(1)-hydroxy-Pheo a, replacing one of the two native Pheo a molecules present in the original complex. Similar to the original preparation, the modified complex was capable of a steady-state photoaccumulation of Pheo- and P680+. It is concluded that the pheophytin a molecule which undergoes borohydride reduction is not involved in the primary charge separation and seems to represent a previously postulated photochemically inactive Pheo a molecule. The Qy and Qx transitions of this molecule were determined to be located at 5 degrees C at 679.5-680 nm and 542 nm, respectively.     

Light-harvesting chlorophyll a/b-binding protein stably inserts into etioplast membranes supplemented with Zn-pheophytin a/b

Light-harvesting chlorophyll a/b-binding protein, LHCP, or its precursor, pLHCP, cannot be stably inserted into barley etioplast membranes in vitro. However, when these etioplast membranes are supplemented with the chlorophyll analogs Zn-pheophytin a/b, synthesized in situ from Zn-pheophorbide a/b and digeranyl pyrophosphate, pLHCP is inserted into a protease-resistant state. This proves that chlorophyll is the only component lacking in etioplast membranes that is necessary for stable LHCP insertion. Synthesis of Zn-pheophytin b alone promotes insertion of LHCP in vitro into a protease-resistant state, whereas synthesis of Zn-pheophytin a alone does not. Insertion of pLHCP into etioplast membranes can also be stimulated by adding chlorophyll a and chlorophyll b to the membranes, albeit at a significantly lower efficiency as compared with Zn-pheophytin a/b synthesized in situ. When pLHCP is inserted into chlorophyll- or Zn-pheophytin-supplemented etioplast membranes and then assayed with protease, only the protease digestion product indicative of the monomeric major light-harvesting chlorophyll a/b complex (LHCII) is found but not the one indicating trimeric complexes. In this respect, chlorophyll- or Zn-pheophytin-supplemented etioplast membranes resemble thylakoid membranes at an early greening stage: pLHCP inserted into plastid membranes from greening barley is assembled into trimeric LHCII only after more than 1 h of greening.     

S-state dependence of chloride binding affinities and exchange dynamics in the intact and polypeptide-depleted O2 evolving complex of photosystem II

The Cl- binding properties in the successive oxidation states of the O2 evolving complex of photosystem II were investigated by measurements of UV absorbance changes, induced by a series of saturating flashes, that monitor manganese oxidation state transitions. In dark-adapted, intact photosystem II, Cl- can be replaced by NO3- in minutes, in an exchange reaction that depends on the NO3- concentration and that is not rate-limited by dissociation of Cl- from its binding site. Preillumination of dark-adapted photosystem II by one or two flashes accelerated the NO3- substitution reaction by an order of magnitude. A quantitative analysis of the Cl- concentration dependence of UV absorbance changes, measured in photosystem II preparations depleted of extrinsic 17 and 23 kDa polypeptides, shows that the Cl- binding properties of photosystem II change with the oxidation state of the oxygen evolving complex. Although the affinity for the individual S-states could not be determined with precision, it is shown that the affinity is an order of magnitude lower in the S2 state than in the S1 state. Comparison of the results obtained using intact photosystem II and preparations depleted of the 17 and 23 kDa extrinsic polypeptides suggests that these proteins constitute a diffusion barrier, which prevents fast equilibration of the Cl- binding site with the medium, but does not change the Cl- affinity of the binding site.