The location of the chloride binding sites in the oxygen-evolving complex of spinach Photosystem II

³⁵Cl-NMR studies are presented here for spinach Photosystem II membranes inhibited by hydroxylamine (to remove Mn), Tris (to remove Mn and 18, 24 and 33 kDa polypeptides), and salt-washing (to remove 18 and 24 kDa; and 33 kDa polypeptides). Removal of Mn affects the ³⁵Cl-NMR binding curve only slightly, indicating that not all of the bound Mn is directly required for Cl-binding. Removal of both Mn and extrinsic polypeptides eliminates almost all of the Cl⁻-specific binding observable by NMR. Removal of the extrinsic 18 and 24 kDa polypeptides drastically changes the ³⁵Cl-NMR binding pattern; this effect is partially restored by the addition of 2 mM CaSO₄, and, to a lesser extent, by the partial rebinding of the polypeptides. Existence of Cl⁻ binding to the intrinsic polypeptides (e.g., D₁/D₂), with a peak at 0.5 mM Cl⁻, is shown in samples lacking 18, 24 and 33 kDa polypeptides. Thus, both intrinsic (i.e., on the D₁/D₂ membrane protein) and extrinsic (i.e., on the 33 kDa protein) binding sites for Cl⁻ are suggested to exist.     

Partial degradation of the extrinsic 23-kDa protein of the Photosystem II complex of spinach

Chymotrypsin eliminated nine amino acid residues at the amino-terminal side of the extrinsic 23-kDa protein of the oxygen-evolving Photosystem II complex of spinach. The resultant 22-kDa fragment was able to bind to the Photosystem II complex but with lowered binding affinity. However, once the 22-kDa fragment bound to the complex, it retained most functions of the 23-kDa protein; the fragment provided a binding site for the extrinsic 18-kDa protein, preserved a tight trap for Ca²⁺ in the complex, and shifted the optimum Cl⁻ concentration for oxygen evolution from 30 to 10 mM, although it was less effective in sustaining oxygen evolution at Cl⁻ concentrations below 10 mM. These observations suggest that the elimination of nine amino acid residues at the amino-terminal region of the 23-kDa protein does not significantly alter the conformation of the protein, except for partial modification of its binding site and its interaction with Cl⁻.     

Intactness of the oxygen-evolving system in thylakoids and Photosystem II particles

Photosystem II activity of oxygen-evolving membranes can be quantified by their capacity to do charge separation or their capacity to transport electrons. In this study using flash excitation of saturating intensity, charge separation is measured by absorption changes in the ultraviolet region of the spectra associated with primary-quinone reduction, and electron transport is measured by oxygen flash yield. These methods are applied to thylakoids and three different types of Photosystem II particles. In thylakoids electron-transport activity is 75–85% of charge separation activity. In Photosystem II particles this percentage is 60–70%, except for the BBY type (Berthold, D.A., Babcock, G.T. and Yocum, C.F. (1981) FEBS Lett. 135, 231–234), in which it is only 29%. These estimates of non-functional oxygen-evolving centers agree within experimental error, except for the BBY particle, with the quantum requirement for oxygen evolution measured under light-limited conditions. These reaction centers that are non-functional in oxygen evolution occur during sample preparation and are not a result of inhibition by ferricyanide or quinone acceptor systems. In thylakoids on the first flash, absorption changes at 325 nm do not show significant contributions from oxygen evolution S-state transitions. In the presence of ferricyanide the absorption change at 325 nm does have a significant contribution from Q₄₀₀ in thylakoids, but considerably less in Photosystem II particles.     

Spin-label studies of the lipid regions of spinach thylakoids and a detergent-derived oxygen-evolving Photosystem II preparation

We have compared the fluidity of thylakoid membranes with the membrane present in a Triton X-100-derived, oxygen-evolving Photosystem II (PS II) preparation using two different spin labels. Data obtained with 2,2,6,6-tetramethylpipiridine-N-oxyl (TEMPO) shows that the PS II preparation contains less fluid membrane than the thylakoid. The TEMPO partition parameter (f) is about 2.5-times greater for the thylakoids at 6 mg chlorophyll/ml than for the PS II preparation at the same chlorophyll concentration. Similarly, the rotational correlation time, τ, of TEMPO residing in the membrane of the PS II preparation is about 2-times longer than the τ for TEMPO in the thylakoid membrane. A spin label which partitions more completely into the bilayer, 2-heptyl-2-hexyl-5,5-dimethyloxazolidine-N-oxyl (7N14), indicates a much greater fluidity in the thylakoid membrane than the membrane of the PS II preparation. The PS II preparation appears to have a hydrocarbon phase which approaches the rigid limit of EPR detectable motion. These results are discussed in terms of possible lipid depletion in the PS II preparation and in terms of lateral heterogeneity of hydrocarbon fluidity in the thylakoid membrane caused by the lateral heterogeneity in protein components.     

Detection of oxygen-evolving Photosystem II centers inactive in plastoquinone reduction

Quite different estimates of the number of Photosystem II centers present in thylakoid membranes are obtained depending on the technique used in making the determination. By using brief saturating light flashes and measuring the electron transport per flash, we have obtained two values for the number of functional centers. When the electrons produced reduce the intersystem plastoquinone pool, there are about 1.7 mmol of active Photosystem II centers per mol chlorophyll, whereas there are at least 3 mmol of active centers per mol chlorophyll when certain halogenated benzoquinones are being reduced. There are also at least 3 mmol of terbutryn binding sites per mol of chlorophyll when this tightly binding herbicide is employed as a specific inhibitor of Photosystem II. Thus only about 60% of the membrane's total complement of Photosystem II centers are able to transfer electrons to Photosystem I at appreciable rates. Many functional assays requiring significant rates of turnover sample only this more active pool, whereas herbicide-binding studies and measurements of changes in the Photosystem II electron donor Z and electron acceptor Q_A performed by other investigators reveal, in addition, a large population of Photosystem II reaction centers that normally have negligible turnover numbers. However, these normally inactive centers readily transfer electrons to the halogenated benzoquinones and are then counted among the active centers. Therefore, it can be concluded that all of herbicide-binding sites represent centers with operative water-oxidizing reactions. It can also be concluded that there are few, if any, centers capable of binding more than a single herbicide molecule.     

Kinetics of the oxygen-evolving complex in salt-washed photosystem II preparations

The kinetics of flash-induced electron transport were investigated in oxygen-evolving Photosystem II preparations, depleted of the 23 and 17 kDa polypeptides by washing with 2 M NaCl. After dark-adaptation and addition of the electron acceptor 2,5-dichloro-p-benzoquinone, in such preparations approx. 75% of the reaction centers still exhibited a period 4 oscillation in the absorbance changes of the oxygen-evolving complex at 350 nm. In comparison to the control preparations, three main effects of NaCl-washing could be observed: the half-time of the oxygen-evolving reaction was slowed down to about 5 ms, the misses and double hits parameters of the period 4 oscillation had changed, and the two-electron gating mechanism of the acceptor side could not be detected anymore. EPR-measurements on the oxidized secondary donor Z⁺ confirmed the slower kinetics of the oxygen-releasing reaction. These phenomena could not be restored by readdition of the released polypeptides nor by the addition of CaCl₂, and are ascribed to deleterious action of the highly concentrated NaCl. Otherwise, the functional coupling of Photosystem II and the oxygen-evolving complex was intact in the majority of the reaction centers. Repetitive flash measurements, however, revealed P⁺Q⁻ recombination and a slow Z⁺ decay in a considerable fraction of the centers. The flash-number dependency of the recombination indicated that this reaction only appeared after prolonged illumination, and disappeared again after the addition of 20 mM CaCl₂. These results are interpreted as a light-induced release of strongly bound Ca²⁺ in the salt-washed preparations, resulting in uncoupling of the oxygen-evolving system and the Photosystem II reaction center, which can be reversed by the addition of a relatively high concentration of Ca²⁺.     

Interaction of 1,4-benzoquinones with Photosystem II in thylakoids and Photosystem II membrane fragments from spinach

The interaction of exogenous quinones with the Photosystem II (PS II) acceptor side has been analyzed by measurements of flash-induced 320 nm absorption changes, transient flash-induced variable fluorescence changes, thermoluminescence emission and oxygen yield in dark-adapted thylakoids and PS II membrane fragments. Two classes of 1,4-benzoquinones were shown to give rise to remarkably different reaction patterns. (A) Phenyl-p-benzoquinone (Ph-p-BQ) -type compounds give rise to a marked binary oscillation of the initial amplitudes of 320 nm absorption changes induced by a flash train in dark-adapted PS II membrane fragments and a retardation of the decay kinetics of the flash-induced variable fluorescence. The electron transfer reactions to these type of quinones are severely inhibited by 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU). (B) In the presence of tribromotoluquinone (TBTQ) a different oscillation pattern of the 320 nm absorption changes is observed characterized by a marked relaxation after the first flash in the 5 ms domain. This relaxation is insensitive to 10 μM DCMU. Likewise the decay of the flash-induced variable fluorescence in TBTQ-treated samples is much less sensitive to DCMU than in control. The thermoluminescence emission exhibits an oscillation in samples incubated for 5 min with TBTQ before addition of 30 μM DCMU. Under the same conditions a significant flash-induced oxygen evolution is observed only after the third and fourth flash, respectively, whereas in the presence of TBTQ alone a normal oscillation pattern is observed. The different functional patterns of PS II caused by the two types of classes of exogenous quinones are interpreted by their binding properties: a noncovalent association with the Q_B-site of Ph-p-BQ-type quinones versus a tight (covalent?) binding in the vicinity of Q_A (possibly also at the Q_B-site) in the case of halogenated 1,4-benzoquinones. The mechanistic implications of these findings are discussed.     

Quantitative analysis of membrane components in a highly active O2-evolving photosystem II preparation from spinach chloroplasts

Stoichiometry of membrane components associated with Photosystem II was determined in a highly active O₂-evolving Photosystem II preparation isolated from spinach chloroplasts by the treatment with digitonin and Triton X-100. From the analysis with sodium dodecyl sulfate polyacrylamide gel electrophoresis and Triton X-114 phase partitioning, the preparation was shown to contain the reaction center protein (43 kDa), the light-harvesting chlorophyll-protein complex (the main band, 27 kDa), the herbicide-binding protein (32 kDa) and cytochrome b-559 (10 kDa) as hydrophobic proteins, and three proteins (33, 24 and 18 kDa) which probably constitute the O₂-evolution enzyme complex as hydrophilic proteins. These proteins were associated stoichiometrically with the Photosystem II reaction center: one Photosystem II reaction center, approx. 200 chlorophyll, one high-potential form of cytochrome b-559, one low-potential form of cytochrome b-559, one 33 kDa protein, one (to two) 24 kDa protein and one (to two) 18 kDa protein. Measurement of fluorescence induction showed the presence of three electron equivalents in the electron acceptor pool on the reducing side of Photosystem II in our preparation. Three molecules of plastoquinone A were detected per 200 chlorophyll molecules with high-performance liquid chromatography. The Photosystem II preparation contained four managanese atoms per 200 chlorophyll molecules.     

EPR studies of the oxygen-evolving enzyme of Photosystem II

The light-induced EPR multiline signal is studied in O₂-evolving PS II membranes. The following results are reported: (1) Its amplitude is shown to oscillate with a period of 4, with respect to the number of flashes given at room temperature (maxima on the first and fifth flashes). (2) Glycerol enhances the signal intensity. This effect is shown to come from changes in relaxation properties rather than an increase in spin concentration. (3) Deactivation experiments clearly indicate an association with the S₂ state of the water-oxidizing enzyme. A signal at g = 4.1 with a linewidth of 360 G is also reported and it is suggested that this arises from an intermediate donor between the S states and the reaction centre. This suggestion is based on the following observations: (1) The g = 4.1 signal is formed by illumination at 200 K and not by flash excitation at room temperature, suggesting that it arises from an intermediate unstable under physiological conditions. (2) The formation of the g = 4.1 signal at 200 K does not occur in the presence of DCMU, indicating that more than one turnover is required for its maximum formation. (3) The g = 4.1 signal decreases in the dark at 220 K probably by recombination with Q^?_AFe. This recombination occurs before the multiline signal decreases, indicating that the g = 4.1 species is less stable than S₂. (4) At short times, the decay of the g = 4.1 signal corresponds with a slight increase in the multiline S₂ signal, suggesting that the loss of the g = 4.1 signal results in the disappearance of a magnetic interaction which diminishes the multiline signal intensity. (5) Tris-washed PS II membranes illuminated at 200 K do not exhibit the signal.     

Characterization of a resolved oxygen-evolving Photosystem II preparation from spinach thylakoids

An oxygen-evolving Photosystem (PS) II preparation was isolated after Triton X-100 treatment of spinach thylakoids in the presence of Mg²⁺. The structural and functional components of this preparation have been identified by SDS-polyacrylamide gel electrophoresis and sensitive spectrophotometric analysis. The main findings were: (1) The concentration of the primary acceptor Q of PS II was 1 per 230 chlorophyll molecules. (2) There are 6 to 7 plastoquinone molecules associated with a ‘quinone-pool’ reducible by Q. (3) The only cytochrome present in significant amounts (cytochrome b-559) occurred at a concentration of 1 per 125 chlorophyll molecules. (4) The only kind of photochemical reaction center complex present was identified by fluorescence induction kinetic analysis as PS II_α. (5) An E_m = ? 10 mV has been measured at pH 7.8 for the primary electron acceptor Q_α of PS II_α. (6) With conventional SDS-polyacrylamide gel electrophoresis, the preparation was resolved into 13 prominent polypeptide bands with relative molecular masses of 63, 55, 51, 48, 37, 33, 28, 27, 25, 22, 15, 13 and 10 kDa. The 28 kDa band was identified as the PS II light-harvesting chlorophyll $\text{a}\text{b-}\text{protein}$. In the presence of 2 M urea, however, SDS-polyacrylamide gel electrophoresis showed seven prominent polypeptides with molecular masses of 47, 39, 31, 29, 27, 26 and 13 kDa as well as several minor components. CP I under identical conditions had a molecular mass of 60–63 kDa.     

Flash-induced redox changes in oxygen-evolving spinach Photosystem II core particles

Flash-induced redox reactions in spinach PS II core particles were investigated with absorbance difference spectroscopy in the UV-region and EPR spectroscopy. In the absence of artificial electron acceptors, electron transport was limited to a single turnover. Addition of the electron acceptors DCBQ and ferricyanide restored the characteristic period-four oscillation in the UV absorbance associated with the S-state cycle, but not the period-two oscillation indicative of the alternating appearance and disappearance of a semiquinone at the Q_B-site. In contrast to PS II membranes, all active centers were in state S₁ after dark adaptation. The absorbance increase associated with the S-state transitions on the first two flashes, attributed to the Z⁺S₁ZS₂ and Z⁺S₂ZS₃ transitions, respectively, had half-times of 95 and 380 s, similar to those reported for PS II membrane fragments. The decrease due to the Z⁺S₃ZS₀ transition on the third flash had a half-time of 4.5 ms, as in salt-washed PS II membrane fragments. On the fourth flash a small, unresolved, increase of less than 3 s was observed, which might be due to the Z⁺S₀ZS₁ transition. The deactivation of the higher S-states was unusually fast and occurred within a few seconds and so was the oxidation of S₀ to S₁ in the dark, which had a half-time of 2–3 min. The same lifetime was found for tyrosine D⁺, which appeared to be formed within milliseconds after the first flash in about 10% inactive centers and after the third and later flashes by active centers in Z⁺S₃.Abbreviations Bis-Tris (bis[2-hydroxyethyl]imino-tris[hydroxymethyl]methane) - D secondary electron donor of PS II - DCBQ 2,5-dichloro-p-benzoquinone - DCMU 3-(3,4dichlorophenyl)-1,1-dimethylurea - PS II Photosystem II - Q_A secondary electron acceptor of PS II - S_0–3 redox state of the oxygen-evolving complex - Z secondary electron donor of PS II     

Oxygen-evolution patterns from spinach Photosystem II preparations

Patterns of O₂ evolution resulting from sequences of short flashes are reported for Photosystem (PS) II preparations isolated from spinach and containing an active, O₂-evolving system. The results can be interpreted in terms of the S-state model developed to explain the process of photosynthetic water splitting in chloroplasts and algae. The PS II samples display damped, oscillating patterns of O₂ evolution with a period of four flashes. Unlike chloroplasts, the flash yields of the preparations decay with increasing flash number due to the limited plastoquinone acceptor pool on the reducing side of PS II. The optimal pH for O₂ evolution in this system (pH 5.5–6.5) is more acidic than in chloroplasts (pH 6.5–8.0). The O₂-evolution, inactivation half-time of dark-adapted preparations was 91 min (on the rate electrode) at room temperature. Dark-inactivation half-times of 14 h were observed if the samples were aged off the electrode at room temperature. Under our conditions (experimental conditions can influence flash-sequence results), deactivation of S₃ was first order with a half-time of 105 s while that of S₂ was biphasic. The half-times for the first-order rapid phase were 17 s (one preflash) and 23 s (two preflashes). The longer S₂ phase deactivated very slowly (the minimum half-time observed was 265 s). These results indicate that deactivation from S₃ → S₂ → S₁, thought to be the dominant pathway in chloroplasts, is not the case for PS II preparations. Finally, it was demonstrated that the ratio of S₁ to S₀ can be set by previously developed techniques, that S₀ is formed mostly from activated S₃ (S₄), and that both S₀ and S₁ are stable in the dark.     

Thermoluminescence as a probe of Photosystem II photochemistry. The origin of the flash-induced glow peaks

A single flash given at − 15°C to chloroplasts results in charge separation in Photosystem II to form a stable state which, upon warming, recombines giving rise to luminescence. This recombination occurs at 25°C in untreated chloroplasts but is shifted to 0°C in the presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea or weak concentrations of a reducing agent. The luminescence at 0°C is attributed to recombination of the S₂Q⁻_A state while that at 25°C is attributed to recombination of S₂Q_AQ⁻_B (and S₃Q_AQ⁻_B upon further flash illumination). The identification of the thermoluminescence at 25°C is based upon the following experimental evidence: (1) illumination of chloroplasts in the presence of methyl viologen with 710 nm light before and after flash illumination has no effect on the extent or temperature of the thermoluminescence. This is taken as evidence that the plastoquinone pool is not involved in the recombination reaction. (2) Calculations of the extent of thermoluminescence expected after a number of flashes, assuming that S₂Q_AQ⁻_B and S₃Q_AQ⁻_B are the thermoluminescent reactants, give a good fit to the experimental results. (3) The effect of continuous illumination at 77 K (i.e., donation from cytochrome b-559 to Q_A and thence to Q_B or Q⁻_B) results in predictable changes in the extent of flash-induced thermoluminescence.     

The S-state dependence of Cl binding to plant Photosystem II

A combined single-turnover flash and ³⁵Cl NMR technique has been used to monitor S-state dependence of Cl⁻ binding to PS-II particles derived from mangrove (Avicennia marina). No detectable high-affinity binding was found to particles in the S₀ and S₁ states, but binding with an affinity comparable to that which activates O₂ evolution was found in the S₂ and S₃ states.     

Chloride binding proteins: mechanistic implications for the oxygen-evolving complex of Photosystem II

Chloride plays a key role in activating the photosynethetic oxygen-evolving complex (OEC) of Photosystem II, but the OEC is only one of many enzymes affected by this anion. Some of the mechanistic features of Cl^– involvement in water-splitting resemble those of other proteins whose structure and chemistry are known in detail. An overview of the similarities and differences between these Cl^–-binding systems is presented.The literature survey for this Minireview was, for the most part, completed in 1987.     

Polypeptide composition of the purified Photosystem II pigment-protein complex from spinach

The Photosystem II pigment-protein complex, the chlorophyll α-protein comprising the reaction center of Photosystem II, was prepared from EDTA-treated spinach chloroplasts by digitonin extraction, sucrose-gradient centrifugation, DEAE-cellulose column chromatography, and isoelectrofocussing on Ampholine.The dissociated pigment-protein complex exhibits two polypeptide subunits that migrate in SDS-polyacrylamide gel with electrophoretic mobilities corresponding to molecular weights of approximately 43 000 and 27 000. The chlorophyll was always found in the free pigment zone at the completion of the electrophoresis. Heat-treatment of the sample (100°C, 90 s) for electrophoresis caused association of the two polypeptides into large aggregates. It is concluded that these two polypeptides, 43 000 and 27 000, are valid structural or functional components of Photosystem II pigment-protein complex.     

Assignment of the g = 4.1 ERP signal to manganese in the S2 state of the photosynthetic oxygen-evolving complex: An X-ray absorption edge spectroscopy study

X-ray absorption spectroscopy at the Mn K-edge has been utilized to study the origin of the g = 4.1 EPR signal associated with the Mn-containing photosynthetic O₂-evolving complex. Formation of the g = 4.1 signal by illumination of Photosystem II preparations at 140 K is associated with a shift of the Mn edge inflection point to higher energy. This shift is similar to that observed upon formation of the S₂ multiline EPR signal by 190 K illumination. The g = 4.1 signal is assigned to the Mn complex in the S₂ state.     

The influence of anions on oxygen evolution by isolated spinach chloroplasts

A study has been made of the onset of chloride deprivation on the oxygen-evolving characteristics of isolated spinach chloroplasts. Using a modulated oxygen electrode it is found that the type of inhibition depends on the anion replacing chloride in the bathing medium. With nitrate a large increase in phase lag accompanies a relatively small inhibition which can be shown to be consistent with a decrease in the rate constant of the reaction which limits the rate of electron transport between water and Photosystem II. With sulphate there is a very small phase change but a larger inhibition which suggests that replacing chloride with sulphate in an electron-transport chain shuts off that chain. With acetate there is a moderate increase in phase lag and the largest inhibitory effect. The phase-lag increase suggests that acetate is affecting the same chloride-sensitive site as nitrate. However, the inhibition cannot be explained by this effect alone and points to the existence of a second chloride-sensitive site. Of the four forward reactions associated with the Kok model of oxygen evolution (Kok, B., Forbush, B. and McGloin, M. (1970) Photochem. Photobiol. 11, 457–475) only S1₃ → S₀ is slowed down when chloride is replaced by nitrate. This reaction is not slowed down by replacing chloride with sulphate.     

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.