Oxygen-evolving System Research Articles

Stabilisation of PS-II-mediated electron transport in oxygen-evolving PS II core preparations by the addition of compatible co-solutes

Addition of high concentrations of compatible co-solutes such as sugars, sugar alcohols and polyols has recently been shown to lead to marked increases in the thermal stability of oxygen-evolution in chloroplasts (Williams et al. (1992) Biochim. Biophys. Acta 1099, 137–144). In this paper, a similar stabilisation is demonstrated for oxygen-evolving PS II core preparations. The presence of such co-solutes appears, however, to have no ability to stabilise PS II reaction-centre preparations against heat-induced changes in their absorption spectrum. Nor do they protect electron transport from artificial electron donors in PS II core preparations lacking the extrinsic 33 kDa polypeptide of the oxygen-evolution system. Measurements performed on core preparations retaining the 33 kDa polypeptide but lacking the 17 kDa and 23 kDa polypetides indicate that the co-solutes protect PS-II-mediated electron transport by stabilising the binding of the 33 kDa polypeptide to the core complexes. These findings are discussed in terms of an extension of the general principles underlying the Hofmeister effect observed for soluble proteins to the stabilisation of photosynthetic membrane preparations.

Improved UV-visible spectra of the S-transitions in the photosynthetic oxygen-evolving system

(1) The first part of this paper is devoted to methodological tests pertaining to the deconvolution of the spectra of the S-transitions from flash sequence data, (i) The spectrum and decay kinetics of 'inactive PS II centers' were analyzed, showing similarity with normal centers inhibited on the QB site. When this contribution is subtracted, the change on the first flash agrees with estimate of the S1→S2 change derived from deconvolution. (ii) Preillumina-tion procedures in PS II (BBY) particles, aimed at modulating the initial S0/S1 distribution are confirmed to express deactivation towards the S1 state, with no involvement of an ‘S−1’ state, (iii) Elimination of semiquinone binary oscillations in BBY's is achieved by allowing total reoxidation by DCBQ after each flash, (iv) A deconvolution treatment is described, using the difference between two sequences. This method allows a determination of the initial S0/S1 distributions that cross-checks the conclusions (ii). (v) Oscillations of the amplitude of the ms-phase of the 295 nm absorption changes are shown to differ significantly from those corresponding to the S-transitions. This phase is expected to reflect predominantly the O2 release reaction, with slight deviations due to other transitions. Deconvolution results are in satisfactory agreement with this prediction. (2) The upshot of this work is to present improved spectra of the S-transitions. The basic features that were previously established with different material and deconvolution method are confirmed: negligible UV change on S0→S1, and significantly different spectra for the two other transitions. A shoulder around 350 nm on the S1→S2 spectrum and a secondary peak in the same region for the S2→S3 spectrum are now resolved. The S0→S1 step causes an electrochromic shift in the blue region, with direction opposite to S1→S2. Interpretation of these results is discussed.

Oxygen release time in leaf discs and thylakoids of peas and Photosystem II membrane fragments of spinach

The half-time of O 2 release in Chlorella, thylakoids and Photosystem II membranes has generally been found to be 0.9 to 3.0 ms, but recently Plijter et al. (Biochim. Biophys. Acta 935 (1988) 299–311) reported it to be between 30 to 130 ms. This work presents a new method, using a Clark electrode, for measuring O 2 release times in leaf discs, thylakoids and Photosystem II membranes. The sample is illuminated with saturating pulses of light of 3 μs to 200 ms duration given at 5 Hz. Presuming the Kok model of four charge transfers per O 2 evolved, the O 2 release time could be calculated from the time needed to observe the evolution of one O 2 less 4-times the turnover time of the charge transfer reaction. Upper bounds for the half times of O 2 release were found to be 6, 11, and 5 ms in thylakoids, leaf discs, and Photosystem II membrane fragments, respectively. In thylakoids, using a bare platinum electrode operated under weak bias conditions, the half-time of O 2 signal rise is 2.7 ms. The rate of rise of this O 2 signal could be slowed by treating the thylakoids with nitrate or formate. The turnover time of the oxygen evolution system, the minimum time between evolution of oxygen molecules, of Photosystem II that is necessary to support light saturated photosynthesis was measured to be 16, 28 and 22 ms in thylakoids, leaf discs and Photosystem II membrane fragments, respectively. The rate limitation for photosynthesis is not the O 2 release time but is the rate of charge transfer through or beyond the plastoquinone pool.

Cause for Dark, Chilling-Induced Inactivation of Photosynthetic Oxygen-Evolving System in Cucumber Leaves

Effects on oxygen evolution of the storage of detached cucumber (Cucumis sativus) leaves at 0 degrees C in the dark were investigated with thylakoids and oxygen-evolving photosystem II membranes isolated from stored leaves. The cold and dark treatment of leaves selectively inactivated electron transport on the oxidizing side of photosystem II. Photosystem II membranes isolated from treated leaves were largely depleted of two proteins of 20 and 14 kilodaltons, which correspond to the extrinsic 23- and 17- kilodalton proteins of spinach functioning in oxygen evolution. The manganese content of photosystem II membranes was also markedly reduced by the treatment. Thus, the inactivation of oxygen evolution induced by the dark, chilling treatment is ascribed to solubilization of the 20- and 14-kilodalton proteins and extraction of manganese.

Surface Charge Density Changes in Isolated Photosystem II Membranes Induced by Depletion of the Extrinsic Polypeptides of the Oxygen Evolving System

Abstract Treatment of PS II particles with either 1 M NaCl or alkaline Tris (1 M , pH 8.4) caused a considerable decrease in the average net negative surface charge density, concomitant with depletion of the extrinsic 17, 24 and 33 kDa proteins of the oxygen evolving complex from the membranes. The partial recovery of the values for surface charge in both NaCl- and Tris-treated membranes was registered after reconstitution experiments with the three proteins. These results are compared with the data for the charge densities of the thylakoid membranes, to examine the role of the three extrinsic proteins in the formation of heterogeneous arrangement of surface charge across the appressed (granal) thylakoids.

Water oxidation in photosystem II: from radical chemistry to multielectron chemistry.

This review article describes the progress that has been made in understanding the photosystem II/oxygen-evolving complex (PSII/OEC). Photosystem II forms the photochemical core of the system: upon light absorption PSII generates oxidizing equivalents or holes at sufficiently high potential to oxidize water. The OEC can exist in five redox states depending on the number of stored oxidizing equivalents. These redox states are designated S{sub 0}-S{sub 4}, with S{sub 4} being the most oxidizing and capable of oxidizing water. The rapid progress in researching this important membrane protein has been due to several factors. First, oxygen evolution has proven to be quite stable to biochemical manipulation. Second, the crystallization of the bacterial reaction center, the realization of the analogies between it and PSII, and the demonstration of the relevance of constructing experiments on the basis of the analogy have provided a shortcut to dissecting the oxygen-evolving system. Finally, because the water-splitting process, like all photosynthetic reactions, can be initiated by light, kinetic studies over the picosecond to second time regime have become possible. The majority of the current work is aimed at determining the structure of the complex.

Molecular organization of oxygen-evolution system in chloroplast

The main function of Photosystem II in chloroplast is to oxidize water molecules to produce oxygen. Strong oxidant produced by photoreaction at Photosystem II reaction center derives electrons from water and the electrons are transferred via Photosystem I to NADP+. The components required for water oxidation in Photosystem II were identified and their molecular properties as well as their roles in the oxygen evolution process were elucidated. The entity of the oxygen evolution system is a supramolecular complex of Photosystem II in the thylakoid membrane where reaction center binding polypeptides, three extrinsic polypeptides, managenese atoms, Ca2+ and Cl− ions are the essential components, and they constitute a specific catalytic domain for water oxidation.

The chloroplast reductase-binding protein is identical to the 16.5-kDa polypeptide described as a component of the oxygen-evolving complex

The N-terminal sequence of the spinach chloroplast reductase-binding protein was determined. The sequence is the same one of a 16.5-kDa polypeptide described as a component of the oxygen-evolving system. Antibodies against both proteins are equivalent as shown by immunoblots, Ouchterlony assays, precipitation of reductase-binding protein complex, and agglutination of thylakoids partially depleted of reductase. These results suggest both proteins are identical. Exposure of the binding protein on the stromal side of thylakoids is supported by agglutination of thylakoids partially depleted of reductase, proteolysis by trypsin, and by accessibility to Fab of anti-binding protein. The latter prevents rebinding of reductase supporting the functional role of the binding protein (16.5 kDa).

Assembly of photosystem II polypeptides and expression of oxygen evolution activity in the chloroplasts of Euglena gracilis Z during the dark-light transition

Euglena cells ( Euglena gracilis Z) grown heterotrophically in the dark were illuminated, and the assembly of the polypeptides of Photosystem II in the thylakoid membrane during greening of the cells was studied by sodium dodecylsulfate polyacrylamide gel electrophoresis and Western-blotting analysis with the antibodies against spinach Photosystem II polypeptides. In the dark-grown cells where the chloroplast is not developed, neither chlorophyll nor Photosystem-II-related polypeptide was detected. Upon illumination, chlorophylls and the polypeptides of Photosystem II began to be synthesized and accumulated in the thylakoid membrane. By comparing the amount of these membrane components in greening process of Euglena cells, it became evident that the extrinsic 30 kDa protein responsible for stabilization of Mn in oxygen-evolution system accumulated more slowly than the other components comprising the core complex of Photosystem II. In accordance with thiselectron transport activity from water to 2,6-dichloroin-dophenol in chloroplasts appeared later compared with that from 1,5-diphenylcarbazide to 2,6-dichloroindophenol, and the increase in the former activity was in parallel with the accumulation of the extrinsic 30 kDa protein in the thylakoid. These results suggest that accumulation and binding of the extrinsic 30 kDa protein to the core complex of Photosystem II is rate-limiting in the light-induced organization of Photosystem II and expression of the oxygen-evolution activity in Euglena . This feature is in contrast with that of higher plants and algae, where a considerable amount of extrinsic proteins of Photosystem II already exists in the dark growth condition and accumulation of the extrinsic proteins in the membrane itself is not a major determinant of the expression of oxygen evolution activity.

Removal of Ca by pH 3.0 treatment inhibits S 2 to S 3 transition in photosynthetic oxygen evolution system

Effects of Ca extraction by pH 3.0 treatment on the electron transport on donorside of Photosystem II (PS II) were investigated. The flash O 2 yieldwas abolished by the treatment, and was restored to normal by the addition of Ca 2+ with almost no change in the oscillation pattern. The light-induced fluorescence increase was lost by the treatment and was well restored by addition of Ca 2+ . Among the othercations tested, only Sr 2+ was effective. The treated membranes exhibited a light-dependent EPR Signal II fast , superimposed on dark-stable Signal II slow , but the Signal II fast was lost on addition of Ca 2+ , suggesting a Ca-dependent reversible conversion between Signal II very fast , and Signal II fast . The peak temperatures of thermoluminescence B- and Q-bands arising from S 2 Q − B and S 2 Q − A charge recombinations, respectively, were elevated to high temperatures after the treatment, and were largely reversed to theirrespective normal temperatures by the addition of Ca 2+ . When excited by a series of flashes, the amplitude of the modified B-band generated by the 1st flash did not change any more after the 2nd flash. These results were interpreted as indicating that extraction of one of two Ca from higher plant PS II by pH3.0 treatment induces an abnormal S 2 state and thereby inhibits S 2 to S 3 transition.

Isnitrogen liganded to manganese in the photosynthetic oxygen-evolving system? EPR studies after isotopic replacement with 15N

The nature of the ligands to manganese in the photosynthetic oxygen-evolving system was investigated by the effect of isotopic substitution with 15N on the spectral properties of the multiline EPR signal of the S2 state. No changes were observed in the general shape of the signal or in the linewidth. The results show that the resolved fine structure in the multiline signal cannot be explained as nitrogen superhyperfine structure but most likely arises from manganese hyperfine interaction. The large difference between the highest value for the nitrogen coupling constant consistent with the results and that earlier observed in a binuclear, antiferromagnetically coupled manganese model complex strongly favors non-nitrogen ligands such as oxygen in carboxylic amino acid side chains.

The interaction of ammonia with the photosynthetic oxygen-evolving system

The reaction of ammonia with the oxygen-evolving system was investigated using EPR. Two sites with distinct binding properties were found. One site, previously known to be responsible for the modification by ammonia of the multiline EPR signal from the S 2 state and believed to be accessible in this state only, was found to bind ammonia also in the S 1 state although weaker. The second binding site, identified by the effect of bound ammonia on the shape and position of the g = 4.1 EPR signal, was also found to be accessible in both the S 1 and S 2 states. The apparent dissociation constants for ammonia at the two sites in the S 1 and S 2 states were determined. In neither state did the binding the ammonia account for the observed inhibition of oxygen evolution, suggesting that binding to other S states plays an important role in the inhibition. Chloride, which is known to interfere with ammonia-induced inhibition of oxygen evolution, was found to compete with ammonia at the site associated with the modification of the g = 4.1 EPR signal. The broadening of the hyperfine lines of the multiline EPR signal, seen in the presence of 17O-labeled water, was still observed after the modification of the signal by ammonia. This indicates that ammonia has not completely displaced water bound to the catalytic site in the S 2 state. The results of the binding studies are interpreted in terms of a two state — two site model, where the two states are identified by their EPR signals, the multiline and the g = 4.1 signal, respectively, and the two sites identified by the effects of ammonia on these signals and where the equilibrium between the two states is regulated by the binding of ligands to the sites.

Oxygen evolution of synchronous Chlorella under continuous and flash illumination

We compared the relative changes in the rate of electron transport (measured as O 2 evolution in continuous light illumination) and the performance of the oxygen-evolving system (measured as O 2 pulses in flashing light) during the life cycle of a green alga. The two processes exhibit different time dependences. The decrease in continuous O 2 evolution observed in the second half of the light period is not caused by inefficiency in the oxygen-evolving system. The cause is a limited availability of reoxidized electron acceptors beyond Photosystem II during that period.

Deactivation kinetics and temperature dependence of the S-state transitions in the oxygen-evolving system of Photosystem II measured by EPR spectroscopy

The decay kinetics for the S 2 and S 3 states of the oxygen-evolving complex in Photosystem II have been measured in the presence of an external electron acceptor. The S 2- and S 3-states decay monophasically with half-decay times at 18°C of 3–3.5 min and 3.5–4 min, respectively. The results also show that S 3 decays via S 2 under these circumstances. The temperature dependence of the individual S-state transitions has been measured in single flash experiments in which the multiline EPR signal originating from the S 2 state has been used as spectroscopic probe. The half-inhibition temperatures are for S 0 to S 1 220–225 K, for S 1 to S 2 135–140 K, for S 2 to S 3 230 K and for the S 3-to-S 0 transition 235 K.

Site-directed mutagenesis identifies a tyrosine radical involved in the photosynthetic oxygen-evolving system.

Photosynthetic oxygen evolution takes place in the thylakoid protein complex known as photosystem II. The reaction center core of this photosystem, where photochemistry occurs, is a heterodimer of homologous polypeptides called D1 and D2. Besides chlorophyll and quinone, photosystem II contains other organic cofactors, including two known as Z and D. Z transfers electrons from the site of water oxidation to the oxidized reaction center primary donor, P+.680, while D+. gives rise to the dark-stable EPR spectrum known as signal II. D+. has recently been shown to be a tyrosine radical. Z is probably a second tyrosine located in a similar environment. Indirect evidence indicates that Z and D are associated with the D1 and D2 polypeptides, respectively. To identify the specific tyrosine residue corresponding to D, we have changed Tyr-160 of the D2 polypeptide to phenylalanine by site-directed mutagenesis of a psbD gene in the cyanobacterium Synechocystis 6803. The resulting mutant grows photosynthetically, but it lacks the EPR signal of D+.. We conclude that D is Tyr-160 of the D2 polypeptide. We suggest that the C2 symmetry in photosystem II extends beyond P680 to its immediate electron donor and conclude that Z is Tyr-161 of the D1 polypeptide.

Tyrosine radicals are involved in the photosynthetic oxygen-evolving system.

In addition to the reaction-center chlorophyll, at least two other organic cofactors are involved in the photosynthetic oxygen-evolution process. One of these cofactors, called "Z," transfers electrons from the site of water oxidation to the reaction center of photosystem II. The other species, "D," has an uncertain function but gives rise to the stable EPR signal known as signal II. Z+. and D+. have identical EPR spectra and are generally assumed to arise from species with the same chemical structure. Results from a variety of experiments have suggested that Z and D are plastoquinones or plastoquinone derivatives. In general, however, the evidence to support this assignment is indirect. To address this situation, we have developed more direct methods to assign the structure of the Z+./D+. radicals. By selective in vivo deuteration of the methyl groups of plastoquinone in cyanobacteria, we show that hyperfine couplings from the methyl protons cannot be responsible for the partially resolved structure seen in the D+. EPR spectrum. That is, we verify by extraction and mass spectrometry that quinones are labeled in algae fed deuterated methionine, but no change is observed in the line shape of signal II. Considering the spectral properties of the D+. radical, a tyrosine origin is a reasonable alternative. In a second series of experiments, we have found that deuteration of tyrosine does indeed narrow the D+. signal. Extraction and mass spectral analysis of the quinones in these cultures show that they are not labeled by tyrosine. These results eliminate a plastoquinone origin for D+.; we conclude instead that D+., and most likely Z+., are tyrosine radicals.

A comparison between the multiline EPR signals of spinach and Anacystis nidulans and their temperature dependence

The multiline EPR signals arising from manganese in the S 2 state of the oxygen-evolving system of spinach and the cyanobacterium Anacystis nidulans have very similar properties and are affected identically by NH 3, suggesting that the system is highly conserved. The temperature dependence of the signal amplitude follows Curie behavior down to sub-helium temperatures. This is in contrast to previous reports, which were taken as evidence for a tetrameric manganese cluster. Thus, it seems that it is not yet possible from EPR data alone to distinguish between this model and a dimeric structure.

Water binding to the oxygen-evolving system of chloroplasts; effects of isotope substitution on the S 2 state EPR spectrum

Replacement of H 2O by 2H 2O in oxygen-evolving Photosystem II preparations caused an increased resolution of the fine structure of the S 2 state EPR spectrum. In both 2H 2O and H 2O samples, comparison of the S 2 spectra generated by illumination at 200 and 283 K (10°C) showed a difference in the fine structure on the hyperfine lines. A reduction in the spacing of the outer hyperfine lines was also observed when samples illuminated at 283 K were compared to those where S 2 was formed by 200 K illumination. The observations are interpreted as due to proton binding, perhaps as water, at or near the manganese complex giving rise to the S 2 signal.

The Origin of the Multiline and g = 4.1 Electron Paramagnetic Resonance Signals from the Oxygen-Evolving System of Photosystem II

Continuous illumination at 200 K of photosystem (PS) II-enriched membranes generates two electron paramagnetic resonance (EPR) signals that both are connected with the S(2) state: a multiline signal at g 2 and a single line at g = 4.1. From measurements at three different X-band frequencies and at 34 GHz, the g tensor of the multiline species was found to be isotropic with g = 1.982. It has an excited spin multiplet at approximately 30 cm(-1), inferred from the temperature-dependence of the linewidth. The intensity ratio of the g = 4.1 signal to the multiline signal was found to be almost constant from 5 to 23 K. Based on these findings and on spin quantitation of the two signals in samples with and without 4% ethanol, it is concluded that they arise from the ground doublets of paramagnetic species in different PS II centers. It is suggested that the two signals originate from separate PS II electron donors that are in a redox equilibrium with each other in the S(2) state and that the g = 4.1 signal arises from monomeric Mn(IV).

Electron transfer in the water-oxidizing complex of Photosystem II.

An overview is presented of secondary electron transfer at the electron donor side of Photosystem II, at which ultimately two water molecules are oxidized to molecular oxygen, and the central role of manganese in catalyzing this process is discussed. A powerful technique for the analysis of manganese redox changes in the water-oxidizing mechanism is the measurement of ultraviolet absorbance changes, induced by single-turnover light flashes on dark-adapted PS II preparations. Various interpretations of these ultraviolet absorbance changes have been proposed. Here it is shown that these changes are due to a single spectral component, which presumably is caused by the oxidation of Mn(III) to Mn(IV), and which oscillates with a sequence +1, +1, +1, -3 during the so-called S0----S1----S2----S3----S0 redox transitions of the oxygen-evolving complex. This interpretation seems to be consistent with the results obtained with other techniques, such as those on the multiline EPR signal, the intervalence Mn(III)-Mn(IV) transition in the infrared, and EXAFS studies. The dark distribution of the S states and its modification by high pH and by the addition of low concentrations of certain water analogues are discussed. Finally, the patterns of proton release and of electrochromic absorbance changes, possibly reflecting the change of charge in the oxygen-evolving system, are discussed. It is concluded that nonstoichiometric patterns must be considered, and that the net electrical charge of the system probably is the highest in state S2 and the lowest in state S1.