Analysis of chlorophyll fluorescence quenching by DBMIB as a means of investigating the consequences of thylakoid membrane phosphorylation

The effect of Mg²⁺ concentration and phosphorylation of the light harvesting chlorophyll $\text{a}\text{b}$ protein on the ability of DBMIB to quench chlorophyll fluorescence of isolated pea thylakoids has been studied. Over a wide range of Mg²⁺ concentrations (5?0.33 mM), the observed changes in fluorescence yield are mirrored by similar changes in the quenching ability of DBMIB, indicating that the cation-induced phenomenon involves alterations in radiative lifetimes. In contrast, phosphorylation at 10 mM Mg²⁺ brings about a lowering of the chlorophyll fluorescence yield, while having no effect on the quenching capacity of DBMIB. This result can be interpreted as a phosphorylation-induced decrease in PS II absorption cross-section. At Mg²⁺ levels between 5 and 1 mM, phosphorylation leads to a change in the quenching of fluorescence by DBMIB, when compared with non-phosphorylated thylakoids. At these cation levels, the degree of DBMIB-induced quenching cannot wholly account for the observed changes in chlorophyll fluorescence due to phosphorylation. It is concluded that the phosphorylation- and Mg²⁺-induced changes in fluorescence yield are independent but inter-related processes which involve surface charge screening as emphasised by the change in cation sensitivity of the DBMIB quenching before and after phosphorylation.     

Analysis of chlorophyll fluorescence induction curves in the presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea as a function of magnesium concentration and NADPH-activated light-harvesting chlorophyll ab-protein phosphorylation

The effect of Mg²⁺ concentration and phosphorylation of light-harvesting chlorophyll $\text{a}\text{b-}\text{protein}$ on various chlorophyll fluorescence induction parameters of isolated pea thylakoids has been studied. (1) Lowering the Mg²⁺ concentration from 3 to 0.4 mM decreases only the variable fluorescence (F_v) and the area above the induction curve while at the same time increasing the slow exponential component of the rise (β_max). (2) A further decrease in Mg²⁺ concentration from 0.4 to 0 mM decreases the initial (F₀) fluorescence level such that the ratio $\text{F}{}_{\mathrm{<mtext>v</mtext>}}\text{F}{}_{\mathrm{<mtext>m</mtext>}}$ increases slightly as does the area above the induction curve and β_max. (3) Thylakoid membranes, phosphorylated at 5 mM Mg²⁺, show an equal decrease in F_v and F₀, no change in the area above the induction curve and an increase in β_max. At 2 mM Mg²⁺, however, phosphorylation induced a more extensive quenching of F_v so that the $\text{F}{}_{\mathrm{<mtext>v</mtext>}}\text{F}{}_{\mathrm{<mtext>m</mtext>}}$ ratio was lowered and the area above the induction curve decreased while β_max increased. (4) When phosphorylated membranes were subsequently suspended in an Mg²⁺-free medium the effect on F₀ due to phosphorylation was found to be additive to that due to the absence of Mg²⁺. The effect of membrane phosphorylation on fluorescence is discussed in relation to the control of excitation energy distribution and shows that different mechanisms operate depending on the background Mg²⁺ levels. At high Mg²⁺ the phosphorylation seems to affect the absorption cross-section of Photosystem II while at lower Mg²⁺ levels there is an additional effect of increased spillover from Photosystem II to I.     

Changes in the redox state of the secondary acceptor of Photosystem II associated with light-induced thylakoid protein phosphorylation

Thylakoid membrane protein phosphorylation affects photochemical reactions of Photosystem II. Incubation of thylakoids in the light with ATP leads to: (1) an increase in the amplitude of three components (4–6, 25–45 and 280–300 μs) of delayed light emission after a single flash without any change in their kinetics; (2) a reduction of the flash-dependent binary oscillations of chlorophyll a fluorescence yield associated with electron transfer from the primary quinone acceptor, Q, to the secondary quinone acceptor, B; (3) an increase in the $\text{B}{}^{\mathrm{?}}\text{B}$ ratio resulting from an increase in stability of the semiquinone anion during dark adaptation; and (4) no change in the redox state of the plastoquinone pool as determined by flash-induced photooxidation of the Photosystem I reaction center, P-700. All the above observations are reversible upon dephosphorylation of the thylakoid membranes. These data are explained by a protein phosphorylation-induced stabilization of the bound semiquinone anion, B^?. It is proposed that this increased stability may be due to an alteration in the accessibility of an endogenous reductant to B, or to an increase in dissipative cycling of charge around Photosystem II.     

A study on the lateral distribution of the plastoquinone pool with respect to Photosystem II in stacked and unstacked spinach chloroplasts

The quenching of Photosystem II (PS II) chlorophyll fluorescence by oxidised plastoquinone has been used in an attempt to determine their relative distribution in the partition zone and stroma-exposed thylakoid membranes. Thus, the PS II-plastoquinone interaction was determined in stacked (2.5 mM MgCl₂) and largely unstacked (0.25 mM MgCl₂) membranes. A method to correct for spillover or other quenching changes at the different MgCl₂ concentrations, which would compete with the plastoquinone-induced quenching, was devised utilising the quinone dibromothymoquinone. This compound is demonstrated to behave as an ideal (theoretically) PS II quencher at both high and low MgCl₂ concentrations, which indicates that it distributes itself homogeneously between partition zone and stroma-exposed membrane regions. In passing from the stacked to the unstacked configuration, the PS II-plastoquinone interaction decreases less than the PS II-dibromothymoquinone interaction. This is interpreted to mean that plastoquinone is present in both the partition zone and stroma-exposed membranes, with somewhat higher concentrations in the stroma-exposed membranes. Thus, plastoquinone is well placed to transport reducing equivalents from the partition zones to the stroma-exposed membranes.     

Measurement of degree of chlorophyll fluorescence polarization in relation to the regulation of excitation energy transfer between Photosystems I and II in pea chloroplasts

The degree of chlorophyll fluorescence polarization (p) at 740 nm was measured at room temperature for pea chloroplasts subjected to various conditions. (1) In agreement with previous published observations, p decreased when the Photosystem (PS) II traps were closed by illumination in the presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea. (2) Under these conditions, the magnitude of p was also sensitive to the presence of salts. Under conditions when ‘spillover’ of the excitation energy from PS II to PS I was low, p was also low, being consistent with increased migration of energy between the PS II light-harvesting chlorophylls. In contrast, when spillover was at a maximum p increased. (3) The change in p in the presence of salts was dependent on the concentration and valency of the cations in such a way as to suggest the changes were mediated through electrostatic forces. The dependency of p on ionic composition of the experimental medium was closely related to the associated changes in fluorescence yield. (4) Membrane stacking, caused by lowering pH of the chloroplast suspension, did not induce a significant change in p, suggesting that this pH-induced process is different from the membrane stacking brought about by manipulating the salt levels. (5) Incubation of thylakoids with ATP induces light-dependent phosphorylation of the light-harvesting chlorophyll-protein complexes, and regulates excitation energy transfer between PS I and PS II (Bennett, J., Steinback, K.R. and Arntzen, C.J. (1980) Proc. Natl. Acad. Sci. U.S.A. 77, 5253–5257). Under conditions which bring about this phosphorylation it was found that p increased to a value indicative of spillover.     

Thylakoid conformational changes accompanying membrane protein phosphorylation

The effect of reversible membrane phosphorylation on the room temperature linear dichroism signal of magneto-oriented pea thylakoids was investigated. Membrane phosphorylation, induced by photoreduction of the plastoquinone pool, resulted in a change in the linear dichroism signal in the region of the red absorption band of chlorophyll. The optical change was due to modifications in selective polarized light scattering which have been shown to be indicative of alterations in the degree of membrane stacking. No changes in linear dichroism due to a reorientation of pigments were observed. It is concluded that phosphorylation of the membrane results in about a 10% destacking of the thylakoids and that this conformational change is implicated in energy redistribution between the two photosystems.     

Multiple effects of linolenic acid addition to pea thylakoids

The addition of linolenic acid to thylakoids produces various pH-dependent effects. We have demonstrated a binding site near the Photosystem (PS) II center with a pK_a of 6.5: when linolenic acid is unprotonated it induces in the dark a rise of the initial fluorescence level, the latter being similar to the maximum fluorescence obtained during illumination of untreated thylakoids. The comparison of the fluorescence lifetimes in the presence and absence of linolenic acid leads us to conclude that the charge stabilisation on the primary acceptor, Q, is prevented by linolenic acid. A second binding site on the protein carrying B, the secondary acceptor of PS II, has also been demonstrated for linolenic acid. It has a 3-(3,4-dichlorophenyl)-1,1-dimethylurea-type effect both in the protonated and unprotonated forms. Finally, measurements of electrophoretic mobility of the thylakoids indicate several other sites of linolenic acid inclusion with an average pK_a of 5.7. At alkaline pH the presence of unprotonated linolenic acid increases the charge density on the membrane. As a result a higher concentration of divalent cations is needed to obtain fluorescence and stacking changes than for untreated thylakoids. The presence, at acidic pH values, of the unprotonated form of linolenic acid leads to the inhibition of cation-induced fluorescence changes, probably by preventing the movement of chlorophyll-protein complexes in the membrane.     

The effect of low concentrations of uncouplers on the detectability of proton deposition in thylakoids. Evidence for subcompartmentation and preexisting pH differences in the dark

Flash-induced pH changes inside thylakoids were measured with neutral red as an indicator in the presence and absence of low concentrations of uncouplers. We found that both carrier-type and pore-forming uncouplers caused the rapidly rising phase of the neutral red signal, previously attributed to proton deposition by water oxidation, to disappear. Gramicidin was particularly efficient in this respect, requiring only one molecule of uncoupler per 10⁴ chlorophyll molecules to render the rapid proton deposition undectectable. This suggests that gramicidin did not act on each water-oxidizing enzyme individually, but rather at the level of the thylakoid membrane. In contrast to the effect on water-derived protons, the appearance of protons from plastoquinol was unaffected by gramicidin. Nor did gramicidin affect the rise of the neutral red signal due to proton deposition during two Photosystem I partial reactions with artificial donors. At the low gramicidin concentrations used, its effect on the neutral red signal could not be attributed to a general increase in proton permeability of the thylakoid membrane (acceleration of half decay from 9 to 0.8 s). The extent of alkalinization of the external medium during the first few hundred milliseconds following a light flash was unaltered by gramicidin, and we did not observe a kinetic correlation between the disappearance of the water proton and the decay of the transmembrane electric field. The last two findings suggest that the undetected protons had not crossed the thylakoid membrane, but instead were buffered away by some gramicidin-induced extra buffering power. pH titration of this extra buffering power revealed an apparent pK ranging between 7.2 and 7.7 and a stoichiometry of $\text{2}\text{H}{}^{\mathrm{+}}\text{site}$. The rapid phase of the neutral red signal regained 90% of its original amplitude after seven flashes were applied at 6.7 Hz repetition rate to a sample containing gramicidin. This suggests limits to the extra buffering power. One possible interpretation of our experiments is the following: Protons derived from water oxidation are initially deposited into extended and highly buffering special domains, and only escape into the common internal phase when the buffering capacity of the domains is saturated. As an alternative one may consider that the thylakoid lumen is partitioned into at least two domains, each dominated by different photosystems and with slow proton equilibration between them. Either view requires internal subcompartmentation. The consequences of such subcompartmentation for chloroplast bioenergetics are still obscure.     

Interactions of herbicides and azidoquinones at a Photosystem II binding site in the thylakoid membrane

6-Azido-5-decyl-2,3-dimethoxy-p-benzoquinone (6-azido-Q₀C₁₀) was found to replace the native plastoquinone at B (the second stable electron acceptor to Photosystem II (PS II)). The 6-azido-Q₁₀C₁₀ would accept electrons from the primary electron-accepting quinone, Q, thus allowing electron transport through PS II to the plastoquinone pool in thylakoids. The synthetic azidoquinone also competes with the PS II herbicides ioxynil and atrazine for binding. This observation strongly favors the hypothesis that PS II herbicides block electron transport by replacing the native quinone which acts as the second electron carrier on the reducing side of PS II (termed B). Covalent linkage of 6-azido-Q₀C₁₀ to its binding environment by ultraviolet irradiation greatly reduces herbicide-binding affinity but does not lead to a loss in herbicide-binding sites. We take this as evidence that covalent attachment of 6-azido-Q₀C₁₀ allows some freedom of quinone head-group movement such that the herbicides can enter the binding site. This indicates that the protein determinants which regulate quinone and herbicide binding are very closely related, but not identical. A compound somewhat related to 6-azido-Q₀C₁₀ is 2-azido-3-methoxy-5-geranyl-6-methyl-p-benzoquinone (2-azido-Q₂). This compound was found to be an ineffective competitor with respect to herbicide binding. Thus, interactions with protein-binding determinants are highly dependent on the molecular structure of quinones. The 2-azido-Q₂ was an inhibitor of electron flow in the intersystem portion of the chain.     

Oxygen evolution in the thylakoid-lacking cyanobacterium Gloeobacter violaceus PCC 7421

The oxygen-evolving reactions of the thylakoid-lacking cyanobacterium Gloeobacter violaceus PCC 7421 were compared with those of Synechocystis sp. PCC 6803. Four aspects were considered: sequence conservation in three extrinsic proteins for oxygen evolution, steady-state oxygen-evolving activity, charge recombination reactions, i.e., thermoluminescence and oscillation patterns of delayed luminescence on a second time scale and delayed fluorescence on the nanosecond time scale at − 196 °C. Even though there were significant differences between the amino acid sequences of extrinsic proteins in G. violaceus and Synechocystis sp. PCC 6803, the oxygen-evolving activities were similar. The delayed luminescence oscillation patterns and glow curves of thermoluminescence were essentially identical between the two species, and the nanosecond delayed fluorescence spectral profiles and lifetimes were also very similar. These results indicate clearly that even though the oxygen-evolving reactions are carried out in the periplasm by components with altered amino acid sequences, the essential reaction processes for water oxidation are highly conserved. In contrast, we observed significant changes on the reduction side of photosystem II. Based on these data, we discuss the oxygen-evolving activity of G. violaceus.     

Light-, pH- and uncoupler-dependent association of chloride with chloroplast thylakoids

Studies of the association of Cl^? with Photosystem (PS) II in CF₁-containing thylakoid membranes revealed that photosynthetically active Cl^? is retained in a Cl^?-free medium unless it is sufficiently alkaline, uncoupling conditions are established and light is excluded. After treatment under such conditions, electron transport from water became dependent on added Cl^? under all conditions. Quantitative measurements of ³⁶Cl^? retention in the light revealed that there were about five Cl^? anions present in Cl^?-sufficient chloroplasts per PS II reaction center, and one-fourth of that in Cl^?-deficient samples. Uncouplers representing three different types of uncoupling mechanism were found to be effective mediators of Cl^? release from thylakoids. Since the ability to collapse a proton gradient probably is the only property shared by all the tested uncouplers, a proton gradient may be involved in the retention of Cl^?. As uncoupler-mediated Cl^? release did not depend on preillumination of our samples, a long-lived proton gradient must exist in dark-adapted chloroplasts which may not span the whole thickness of the thylakoid membrane. It is postulated that the Cl^? active in PS II reactions resides in a special membrane domain from which protons slowly equilibrate with those in the bulk solutions. Cl^? is thought to be released to the bulk phases only when the pH of the membrane domain is raised above a certain threshold by the action of uncouplers. This domain may be identical to the intramembranous compartment which has been postulated to be associated with PS II (Prochaska, L.J. and Dilley, R.A., (1978) Front. Biol. Res. Energ. 1, 265–274).     

The yield of chlorophyll a fluorescence as a means to test the various deexcitation mechanisms in the antenna system of green plants

With the aid of measurements of the fluorescence yield, the efficiency of the various deexcitation mechanisms of an exciton in the light-harvesting system has been determined. For this purpose, the fluorescence of dark-adapted as well as of 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU)-treated and preilluminated leaves of Zea mays L. was excited by single ultrashort laser pulses of different energies. The experimental results have served for the fitting of solutions of rate equations, which describe the deexcitation by linear relaxation processes like fluorescence and radiationless transitions, by annihiation of excitons, and by traps both in the ground state and in an excited state. We have obtained the following results: a ratio of antenna chlorophyll molecules to Photosystem II traps of 600:1, an annihilation constant γ = 2·10^?8 cm³·s^?1, a mean trapping time of $\text{t}\text{?}\text{=0.5}\text{ns}$, a trapping probability for traps in the ground state of 2·10^?8 cm³·s^?1, and 6·10^?9 cm³·s^?1 for traps in an excited state.     

Changes in composition and function of thylakoid membranes as a result of photosynthetic adaptation of chloroplasts from pea plants grown under different light conditions

The hypothesis that chloroplasts having different light-saturated rates of photosynthesis will have different proportions of the intrinsic thylakoid complexes engaged in light-harvesting and electron transport (Anderson, J.M. (1982) Mol. Cell. Biochem. 46, 161–172) has been tested. Peas were grown in light regimes which varied in light intensity, quality and time of irradiance, and ranged from sunlight through red to blue-enriched light of very low radiation. The electron-transport capacity at saturating light of Photosystem I and Photosystem II of chloroplasts isolated from light-adapted peas was 2-fold and 5–6-fold lower, respectively, in the lowest radiation compared to sunlight. There was a marked increase in the amount of total chlorophyll associated with the main chlorophyll $\text{a}\text{b-}\text{proteins}$ (LHCP¹, LHCP² and LHCP³) and a 2-fold decrease in the core reaction centre complex of Photosystem II (CP a) as the radiation decreased; the $\text{LHCP}{}^{\mathrm{1–3}}\text{CP}$ a ratio changed from 3.5 to 9.0. The amount of chlorophyll associated with Photosystem I varied from 34% in sunlight to 27% in the lowest radiation, but the antenna size of Photosystem I was not markedly different; there was a 2-fold decrease in the amount of cytochrome f on a chlorophyll basis, which partly accounted for the decreased electron-transport capacity of Photosystem I. Since the increases or decreases in the levels of each of the components correlated with decreasing radiation, it is clear that the light-adaptation of both light-harvesting and electron-transport components is indeed closely co-ordinated.     

The accumulation of neutral red in illuminated thylakoids

Thylakoids isolated from spinach (Spinacia oleracea L.) bind only a small fraction of neutral red in the dark whereas they accumulate large amounts of the protonated dye in their inner space under light. Light-induced neutral red uptake depends on the size of the proton gradient across the thylakoid membrane but does not follow the mechanism established for amines. Instead, the correlation between pH gradient and neutral red uptake can be predicted quantitatively assuming that protonated neutral red is accumulated mainly as dimer species.Under appropriate conditions, accumulation of protonated neutral red in the inner thylakoid space is proportional to an absorbance increase at 520 nm. This 520-nm change can be used for the continuous measurement of pH changes in thylakoids during steady-state illumination.     

The 520 nm absorbance changes in Scenedesmus obliquus and its relation to Photosystem I

The kinetics (region of seconds) of the light-induced 520 nm absorbance change and its dark reversal have been studied in detail in the wild type and in some pigment and photosynthetic mutants of Scenedesmus obliquus. The following 5 lines of evidence led us to conclude that the signal is entirely due to the photosystem I reaction modified by electron flow from Photosystem II.Gradual blocking of the electron transport with 3(3,4-dichlorophenyl)-1,1-dimethylurea resulted in diminution and ultimate elimination of the biphasic nature of the signal without reducing the extent of the absorbance change or of the dark kinetics. On the contrary, blocking electron flow at the oxidizing side of plastoquinone with 2, $\text{5-}\text{dibromo}\text{-3-}\text{methyl}\text{-6-}\text{isoprophyl}\text{-p-}\text{benzoquinone}$ or inactivating the plastocyanin with KCN, prolonged the dark reversal of the absorbance change apart from abolishing the biphasic nature of the signal.Action spectra clearly indicate that the main signal (I) is due to electron flow in Photosystem I and that its modification (Signal II) is due to the action of Photosystem II.Signal I is pH independent, whereas Signal II demonstrates a strong pH dependence, parallel to the O₂-evolving capacity of the cells.Chloroplast particles isolated from the wild type Scenedesmus cells demonstrated in the absence of any added artificial electron donor or acceptor and also under non-phosphorylation conditions the 520 nm absorbance change with approximately the same magnitude as whole cells. The dark kinetics of the particles were comparatively slower. Removal of plastocyanin and other electron carriers by washing with Triton X-100 slowed down the kinetics of the dark reversal reaction to a greater extent. A similar positive absorbance change at 520 nm and slow dark reversal was also observed in the Photosystem I particles prepared by the Triton method.Mutant C-6E, which contains neither carotenoids nor chlorophyll $\text{b}$ and lacks Photosystem II activity, demonstrates a normal signal I of the 520 nm absorbance change. This latter result contradicts the postulate that carotenoids are the possible cause of the 520 nm absorbance change.     

The role of phospholipids in the molecular organisation of pea chloroplast membranes. Effect of phospholipid depletion on photosynthetic activities

Pea chloroplasts were treated with phospholipase A₂ which hydrolysed approx. 75% phosphatidylglycerol and 60% phosphatidylcholine. The major effect of the treatment was an inhibition of Photosystem (PS) II electron transport together with an (approx. 30%) increase of initial chlorophyll fluorescence (F₀) and a subsequent loss of variable fluorescence during induction, as well as an inhibition of the cation-induced rise in steady-state chlorophyll fluorescence. In contrast to the effects upon PS II activities, PS I activity was not depressed and increased slightly under certain conditions, while the coupling factor for photophosphorylation was inhibited to some extent. No significant increase in spillover was observed following the treatment with phospholipase A₂. These results are discussed in relation to the ways in which phospholipid depletion may lead to the various effects observed. It is proposed that the site of PS II inhibition after phospholipase A₂ treatment may be at the electron transfer from pheophytin to Q, the first quinone-type electron acceptor.     

Picosecond fluorescence kinetics in spinach chloroplasts at room temperature. Effects of phosphorylation

We have used single-photon timing with picosecond resolution to investigate the effect of phosphorylation on the fluorescence decay from broken spinach chloroplasts. Phosphorylation of spinach thylakoids causes a quenching of the slow decay phase (equivalent to a quenching of variable fluorescence) and an increase in the yield of the middle phase decay component. In addition, phosphorylation alters the intensity dependence of fluorescence in a manner which indicates a decreased antenna size of Photosystem II. The observed changes are indicative of a State 1-State 2 transition and show a clear reversal when the membranes are dephosphorylated.     

The role of pH and membrane potential in the reactions of Photosystem II as measured by effects on delayed fluorescence

(1) If DCMU is added to chloroplasts which have been preilluminated (0–8 flashes) the turnover of the water-splitting enzyme is limited to one further transition upon continuous illumination. (2) The intensity of millisecond delayed fluorescence measured in the presence of mediators of cyclic electron transport around Photosystem I and of DCMU added after pre-flashing is stimulated above the level in the presence of DCMU alone and varies according to the number of pre-flashes (Bowes, J.M. and Crofts, A.R. (1978) Z. Naturforsch 33c, 271–275). (3) Separate contributions of the following energetic terms to the induction kinetics and extent of millisecond delayed fluorescence under these conditions have been examined with a view to assessing their involvement in and the mechanism of the stimulation of the emission above the level in dark-adapted chloroplasts in the presence of DCMU: (a) the initial pH of the phase in equilibrium with the water-splitting enzyme; (b) the change in internal pH which occurred when Photosystem I acted as a proton pump; (c) the electrical potential difference across the membrane resulting from rapid charging of the membrane capacitance. (4) It was confirmed that delayed light was stimulated as a result of the interaction of the intrathylakoid pH (3a and b) with the equilibria of the S-states involving proton release according to the model in which this occurs on all except the transition S₁ → S₂; the stimulation was qualitatively proportional to the number of protons released. (5) There was no marked variation of the membrane potential as a function of the number of pre-flashes.     

Metastable proton pools in thylakoids and their importance for the stability of Photosystem II

After isolated chloroplast thylakoids have been transferred to a medium which is more alkaline than their storage medium, they retain considerable amounts of unequilibrated protons for often longer than 10 min. Essentially all of these protons are released upon uncoupler addition when the thylakoids are osmotically swollen, but only a portion of them when they are in a shrunken state. Osmotic swelling also greatly accelerates the inactivation of the water-oxidizing system enzyme of Photosystem II, and its depletion of functional Cl^?, at alkaline pH. Analyses of the mestable proton gradient in terms of stoichiometry, temperature dependence, and effect on fluorescent amine probes, suggest that most of the protons involved are bound and exchange readily with the bulk phases only when the thylakoids are swollen. It is concluded that, in shrunken thylakoids, the water-oxidizing enzymes are buried in special H⁺-sequestering domains which probably are formed by cavities in the inner surface of the thylakoid membrane. An observed cooperative action of alkaline pH and divalent cations during Cl^?-extraction from Photosystem II is interpreted as revealing an involvement of both a negatively charged surface region and positively charged groups in maintaining the functional integrity of the site of water oxidization.