Spectral characteristics of PS II reaction centres: as isolated preparations and when integral to PS II core complexes

The discovery that the native PS II enzyme undergoes charge separation via an absorption extending to 730 nm has led us to re-examine the low-temperature absorption spectra of Nanba-Satoh PS II reaction centre preparations with particular focus on the long wavelength region. It is shown that these preparations do not exhibit absorption in the 700-730 nm region at 1.7 K. Absorption in the Nanba-Satoh type preparations analogous to the 'red tail' as observed in functional PS II core complexes is likely shifted to higher energy by >20 nm. Spectral changes associated with the stable reduction of pheo(a) in chemically treated reaction centre preparations are also revisited. Dithionite treatment of PS II preparations in the dark leads to changes of pigment-pigment and/or pigment-protein interactions, as evidenced by changes in absorption and CD spectra. Absorption and CD changes associated with stable Pheo(D1) photo-reduction in PS II core complexes and Nanba-Satoh preparations are compared. For Nanba-Satoh preparations, Q(y) bleaches are approximately 3x broader than in PS II core complexes and are blue-shifted by approximately 4 nm. These data are discussed in terms of current models of PS II, and suggest a need to consider protein-induced changes of some electronic properties of reaction centre pigments.     

Characterisation of a PS II reaction centre isolated from the chloroplasts of Pisum sativum

A photosystem II reaction centre has been isolated from peas and found to consist of D1, D2 polypeptides and the apoproteins of cytochrome b-559, being similar to that reported for spinach by Nanba and Satoh [(1987) Proc. Natl. Acad. Sci. USA 84, 109–112]. The complex binds chlorophyll a, pheophytin and the haem of cytochrome b-559 in an approximate ratio of 4:2:1 and also contains about one molecule of β-carotene. It binds no plastoquinone-9 or manganese but does contain at least one non-haem iron. In addition to a light-induced signal due to Pheo⁻ seen under reducing conditions, a light-induced P680⁺ signal is seen when the reaction centre is incubated with silicomolybdate. In the presence of diphenylcarbazide, the P680⁺ signal is partially inhibited and net electron flow to silicomolybdate occurs. This net electron flow is insensitive to o-phenanthroline, 3-(3,4-dichlorophenyl)-1,1-dimethyl urea and 2-(3-chloro-4-trifluoromethyl)anilino-3,5-dinitrothiophene but is inhibited by proteolysis with trypsin and by other treatments. Fluorescence, from the complex, peaks at 682 nm at room temperature and at 685 nm at 77 K. This emission is significantly quenched when either the P680⁺Pheo or P680Pheo⁻ states are established indicating that the fluorescence emanates from the back reaction between P680⁺ and Pheo⁻.     

Widespread distribution of some minor chlorophyll-protein complexes in some plants and algae

The use of the non-ionic detergent octyl beta-d-glucoside has allowed the demonstration in spinach of a chlorophyll a+b-protein complex of apparent molecular weight 29 kilodaltons (Camm and Green, 1980, Plant Physiol 66: 428-432). Complexes analogous to this one also can be demonstrated in three grasses, in dicots of the Chenopodiaceae, Fabaceae, and Asteraceae, and in the siphonaceous green alga Acetabularia mediterranea. These complexes are clearly distinguishable from the light harvesting complex on the basis of the chlorophyll a/b ratio, apparent molecular weight, and polypeptide composition. In addition, most plants surveyed contained two, not one, minor chlorophyll a complexes as well as the chlorophyll a complex of Photosystem I.     

Isolation and characterisation of a photosystem II reaction centre lipoprotein complex

A photochemically active reaction centre complex has been isolated from photosystem II preparations of spinach chloroplasts by Triton X-100 solubilisation and sucrose gradient fractionation. Electrophoresis of the complex revealed 5 bands indicating polypeptides of apparent molecular masses of 47, 43, 33, 30 and 10 kDa. Lipid analyses showed that polar, as well as neutral, lipids are associated with the complex. For approx. 40 chlorophyll a molecules there were 3.4 plastoquinone-9, 3.3 pheophytin a, 2.9 β-carotene, 29.3 monogalactosyldiacylglycerol and 12.4 sulphoquinovosyldiacylglycerol molecules. These results suggest that this photosystem II reaction centre complex, which most likely represents a minimum photochemically active unit, is a lipoprotein complex. A striking feature of the associated polar lipids is their very low degree of unsaturation.     

The effect of herbicides on components of the PS II reaction centre measured by EPR

Incubation of PS II membranes with herbicides results in changes in EPR signals arising from reaction centre components. Dinoseb, a phenolic herbicide which binds to the reaction centre polypeptide, changes the width and form of the EPR signal arising from photoreduced Q^?_AFe. o-Phenanthroline slightly broadens the Q^?_AFe signal. These effects are attributed to changes in the interaction between the semi-quinone and the iron. DCMU, which binds to the 32 kDa protein, has virtually no effect on the width of the Q^?_AFe signal but does give rise to an increase in its amplitude. This could result from a change in redox state of an interacting component. Herbicide effects can also be seen when Q^?_AFe is chemically reduced and these seen to be reflected by changes in splitting and amplitude of the split pheophytin^? signal. Dinoseb also results in the loss of ‘Signal II dark’, the conversion of reduced high-potential cytochrome b₅₅₉ to its oxidized low-potential form and the presence of transiently photooxidized carotenoid after a flash at 25°C; these effects indicate that dinoseb may also act as an ADRY reagent.     

Isolation of chlorophyll-protein complexes and quantification of electron transport components in Synura petersenil and Tribonema aequale

The chlorophyll-protein complexes of the yellow alga Synura petersenii (Chrysophyceae) and the yellow-green alga Tribonema aequale (Xanthophyceae) were studied. The sodiumdodecylsulfate/sodiumdesoxycholate solubilized photosynthetic membranes of these species yielded three distinct pigment-protein complexes and a non-proteinous zone of free pigments, when subjected to SDS polyacrylamid gel electrophoresis. The slowest migrating protein was identical to complex I (CP I), the P-700 chlorophyll a-protein, which possessed 60 chlorophyll a molecules per reaction center in Tribonema and 108 in Synura. The zone of intermediate mobility contained chlorophyll a and carotenoids. The absorption spectrum of this complex was very similar to the chlorophyll a-protein of photosystem II (CP a), which is known from green plants. The fastest migrating pigment protein zone was identified as a light-harvesting chlorophyll-protein complex. In Synura this protein was characterized by the content of chlorophyll c and of fucoxanthin. Therefore this complex will be named as LH Chl a/c-fucocanthin protein. In addition to the separation of the chlorophyll-protein complexes the cellular contents of P-700, cytochrome f (bound cytochrome) and cytochrome c-553 (soluble cytochrome) were measured. The stoichiometry of cytochrome f: cytochrome c-553:P-700 was found to be 1:4:2.4 in Tribonema and 1:6:3.4 in Synurá.     

Isolation of chlorophyll-protein complexes and quantification of electron transport components in Synura petersenii and Tribonema aequale

The chlorophyll-protein complexes of the yellow alga Synura petersenii (Chrysophyceae) and the yellow-green alga Tribonema aequale (Xanthophyceae) were studied. The sodiumdodecylsulfate/sodiumdesoxycholate solubilized photosynthetic membranes of these species yielded three distinct pigment-protein complexes and a non-proteinuous zone of free pigments, when subjected to SDS polyacrylamid gel electrophoresis. The slowest migrating protein was identical to complex I (CP I), the P-700 chlorophyll a-protein, which possessed 60 chlorophyll a molecules per reaction center in Tribonema and 108 in Synura. The zone of intermediate mobility contained chlorophyll a and carotenoids. The absorption spectrum of this complex was very similar to the chlorophyll a-protein of photosystem II (CP a), which is known from green plants. The fastest migrating pigment protein zone was identified as a light-harvesting chlorophyll-protein complex. In Synura this protein was characterized by the content of chlorophyll c and of fucoxanthin. Therefore this complex will be named as LH Chl a/c-fucocanthin protein. In addition to the separation of the chlorophyll-protein complexes the cellular contents of P-700, cytochrome f (bound cytochrome) and cytochrome c-553 (soluble cytochrome) were measured. The stoichiometry of cytochrome f: cytochrome c-553:P-700 was found to be 1:4:2.4 in Tribonema and 1:6:3.4 in Synurá.Abbreviations CP a chlorophyll a-protein of photosystem II - CP I P-700 chlorophyll a-protein - FP free pigment - LH Chl a/c light-harvesting chlorophyll a/c-protein - PAGE polyacrylamidgelelectrophoresis - SDS Sodiumdodecylsulfate - SDOC sodium-desoxycholate     

Proton-induced grana formation in chloroplasts. Distribution of chlorophyll-protein complexes and Photosystem II photochemistry

Lowering the pH of the incubation medium to pH 5.4 leads to grana formation morphologically similar to that induced by metal cations. The same phenomenon is observed in EDTA-washed chloroplasts, indicating that it is not due in part to electrostatic ‘masking’ by residual cations associated with the membranes. Digitonin fractionation studies have indicated that the distribution of the major chlorophyll-protein complexes between granal and stromal membrane regions is similar at pH 5.4 in the absence of Mg²⁺, and at pH 7.4 in the presence of Mg²⁺. Chlorophyll fluorescence induction studies have indicated that the primary photochemistry of Photosystem II (PS II) is stimulated by lowering the pH to 5.4, just as it is upon metal cation addition at higher pH values. The failure to observe such an increase at pH 5.4 by measuring electron transport to ferricyanide is attributed to a combination of an inhibition by this pH of electron transport at a site after Q reduction and an increase in the number of PS II centres detached from the plastoquinone pool. We conclude that the stacked configuration of chloroplast membranes leads to increased PS II primary photochemistry, which is most simply explained in terms of a redistribution of excitation energy towards PS II.     

Transient states in the photoreactions of chlorophyll-protein complexes]

Transient short-lived species arising in chlorophyll-protein complexes of PS I on flash excitation were studied by means of flash-photolysis and luminescence methods. Complexes were isolated from chloroplasts by the solubilisation in SDS and subsequent electrophoresis. Three different types of reactions associated with: a) the triplet state of monomeric chlorophyll; b) redox reactions in reaction centres; c) photochemical reactions of monomeric chlorophyll were established on excitation. The arrangement of different forms of chlorophyll connected with the protein globule is discussed.     

Interaction of the high-spin Fe atom in the photosystem II reaction center with the quinones QA and QB in purified oxygen-evolving PS II reaction center complex and in PS II particles

EPR signals in the high-spin region were studied at 10 K in photosystem II (PS II) particles and in a purified oxygen-evolving PS II reaction center complex under oxidizing conditions. PS II particles showed EPR peaks at g = 8.0 and 5.6, confirming the recent report by Petrouleas and Diner [(1986) Biochim. Biophys. Acta 849, 264-275]. Addition of 3-(3',4'-dichlorophenyl)-1,1-dimethylurea (DCMU) or o-phenanthroline shifted the peaks to be closer to g = 6.0 depending on the medium pH. On the other hand, the PS II reaction center complex showed peaks at g = 6.1 and 7.8, and at g = 6.1 and 6.4, in the absence and presence of o-phenanthroline, respectively. All these peaks were found to be decreased by the illumination at 10 K. These results suggest that the high-spin signals are due to Q₄₀₀, Fe(III) atom interacting with the PS II primary electron acceptor quinone Q_A as reported and that the Fe atom also interacts with the secondary acceptor quinone Q_B. This interaction seems to induce the highly asymmetric ligand coordination of the Fe atom and to be affected by DCMU and o-phenanthroline in a somewhat different manner.     

Characteristics of the Photosystem II reaction centre. II. Electron donors

The properties of Photosystem II electron donation were investigated by EPR spectrometry at cryogenic temperatures. Using preparations from mutants which lacked Photosystem I, the main electron donor through the Photosystem II reaction centre to the quinone-iron acceptor was shown to be the component termed Signal II. A radical of 10 G line width observed as an electron donor at cryogenic temperatures under some conditions probably arises through modification of the normal pathway of electron donation. High-potential cytochrome b-559 was not observed on the main pathway of electron donation. Two types of PS II centres with identical EPR components but different electron-transport kinetics were identified, together with anomalies between preparations in the amount of Signal II compared to the quinone-iron acceptor. Results of experiments using cells from mutants of Scenedesmus obliquus confirm the involvement of the Signal II component, manganese and high-potential cytochrome b-559 in the physiological process leading to oxygen evolution.     

Effects of photoinhibition on the PS II acceptor side including the endogenous high spin Fe2+ in thylakoids,PS II-membrane fragments and PS II core complexes

Effects of photoinhibition at 0 °C on the PS II acceptor side have been analyzed by comparative studies in isolated thylakoids, PS II membrane fragments and PS II core complexes from spinach under conditions where degradation of polypeptide(s) D1(D2) is highly retarded. The following results were obtained by measurements of the transient fluorescence quantum and oxygen yield, respectively, induced by a train of short flashes in dark-adapted samples: (a) in the control the decay of the fluorescence quantum yield is very rapid after the first flash, if the dark incubation was performed in the presence of 300 M K₃[Fe(CN)₆]; whereas, a characteristic binary oscillation was observed in the presence of 100 M phenyl-p-benzoquinone with a very fast relaxation after the even flashes (2nd, 4th. . . ) of the sequence; (b) illumination of the samples in the presence of K₃[Fe(CN)₆] for only 5 min with white light (180 W m^-2) largely eliminates the very fast fluorescence decay after the first flash due to Q_A ^- reoxidation by preoxidized endogenous non-heme Fe³⁺, while a smaller effect arises on the relaxation kinetics of the fluorescence transients induced by the subsequent flashes; (c) the extent of the normalized variable fluorescence due to the second (and subsequent) flash(es) declines in all sample types with a biphasic time dependence at longer illumination. The decay times of the fast (6–9 min) and the slow degradation component (60–75 min) are practically independent of the absence or presence of K₃[Fe(CN)₆] and of anaerobic and aerobic conditions during the photo-inhibitory treatment, while the relative extent of the fast decay component is higher under anaerobic conditions. (d) The relaxation kinetics of the variable fluorescence induced by the second (and subsequent) flash(es) become retarded due to photoinhibition, and (e) the oscillation pattern of the oxygen yield caused by a flash train is not drastically changed due to photoinhibition.Based on these findings, it is concluded that photoinhibition modifies the reaction pattern of the PS II acceptor side prior to protein degradation. The endogenous high spin Fe²⁺ located between Q_A and Q_B is shown to become highly susceptible to modification by photoinhibition in the presence of K₃[Fe(CN)₆] (and other exogenous acceptors), while the rate constant of Q_A ^- reoxidation by Q_B(Q_B ^-) and other acceptors (except the special reaction via Fe³⁺) is markedly less affected by a short photoinhibition. The equilibrium constant between Q_A ^- and Q_B(Q_B ^-) is not drastically changed as reflected by the damping parameters of the oscillation pattern of oxygen evolution.     

Investigation of the structure of trimeric and monomeric photosystem I reaction centre complexes

Electron microscopy of monomeric and trimeric forms of the reaction centre of photosystem I from the thermophilic cyanobacterium Phormidium laminosum has allowed the construction of a three-dimensional model describing the shape of the complex. The trimeric form of the Photosystem I reaction centre complex was found to have a very regular shape corresponding to a rounded equilateral triangle with edges ˜18 nm long and a thickness of ˜6 nm. A distinctive chiral arrangement of the three reaction centres in the trimer could be observed on one face of the complex, whereas the opposing face appeared to be smooth with no distinctive internal features. The monomeric reaction centre is roughly pearshaped, with a length of ˜15 nm and a width of ˜9 nm. A thickness of 6 nm is assumed from comparison with the trimer. It is predicted to lie with its shortest axis spanning the membrane. A double-lobed structure, with one lobe larger than the other, was occasionally observed for the monomeric reaction centre. No experimental evidence could be obtained for the existence of the trimeric form in the membrane. The formation of the trimeric form after detergent extraction is suggested. The trimeric form was found to be more stable than the monomeric form in solutions containing anionic and non-ionic detergents.     

Hexane-solubilised reaction centre proteolipid complexes of Rhodopseudomonas sphaeroides

Reaction centres from Rhodopseudomonas sphaeroides, strain R-26, have been solubilised in hexane by the use of phospholipids and cations. Two procedures have been developed: (I) a two-step technique involving the formation of detergent-free proteoliposomes from detergent solubilised reaction centres and phospholipids and mixing these with hexane in the presence of cations; (II) directly sonicating detergent-solubilised reaction centres with phosopholipids before mixing with hexane and cations.The yield of the extracted complex varied with the ratios of protein, phospholipid and cations, species of phospholipid, and sonication time. The spectral characteristics of the complex in the organic phase were similar to those of detergent-solubilised reaction centres. The stability of the reaction centres appeared to be dependent on the presence of phospholipid and water in the hexane. The usefulness of the hexane solution as a model membrane system is discussed and its possible future applications are considered.     

Photosystem II reaction centre quenching: mechanisms and physiological role

Dissipation of excess absorbed light energy in eukaryotic photoautotrophs through zeaxanthin- and DeltapH-dependent photosystem II antenna quenching is considered the major mechanism for non-photochemical quenching and photoprotection. However, there is mounting evidence of a zeaxanthin-independent pathway for dissipation of excess light energy based within the PSII reaction centre that may also play a significant role in photoprotection. We summarize recent reports which indicate that this enigma can be explained, in part, by the fact that PSII reaction centres can be reversibly interconverted from photochemical energy transducers that convert light into ATP and NADPH to efficient, non-photochemical energy quenchers that protect the photosynthetic apparatus from photodamage. In our opinion, reaction centre quenching complements photoprotection through antenna quenching, and dynamic regulation of photosystem II reaction centre represents a general response to any environmental condition that predisposes the accumulation of reduced Q(A) in the photosystem II reaction centres of prokaryotic and eukaryotic photoautotrophs. Since the evolution of reaction centres preceded the evolution of light harvesting systems, reaction centre quenching may represent the oldest photoprotective mechanism.