Spectroscopic properties of the reaction center and of the 47 kDa chlorophyll protein of Photosystem II

The D1-D2-cytochrome b-559 reaction center complex and the 47 kDa antenna chlorophyll protein isolated from spinach Photosystem II were characterized by means of low temperature absorption and fluorescence spectroscopy. The low temperature absorption spectrum of the D1-D2-cytochrome b-559 complex showed two bands in the Q_y region located at 670 and 680 nm. On the basis of its absorption maximum and orientation the latter component may be attributed at least in part to P-680, the primary electron donor of Photosystem II. The 47 kDa antenna complex showed absorption bands at 660, 668 and 677 nm and a minor component at 690 nm. The latter transition appeared to be associated with the characteristic low temperature 695 nm fluorescence band of Photosystem II. The 695 nm emission band was absent in the D1-D2 complex, which indicates that it does not originate from the reaction center pheophytin, as earlier proposed. The transition dipole responsible for the main fluorescence at 684 nm from this complex had a parallel orientation with respect to the membrane plane in the native structure. The reaction center preparation contains two spectrally distinct carotenoids with different orientations.     

Rapid and simple isolation of pure photosystem II core and reaction center particles from spinach

Pure and active oxygen-evolving PS II core particles containing 35 Chl per reaction center were isolated with 75% yield from spinach PS II membrane fragments by incubation with n-dodecyl--D-maltoside and a rapid one step anion-exchange separation. By Triton X-100 treatment on the column these particles could be converted with 55% yield to pure and active PS II reaction center particles, which contained 6 Chl per reaction center.Abbreviations Bis-Tris bis[2-hydroxyethyl]imino-tris[hydroxymethyl]methane - Chl chlorophyll - CP29 Chl a/b protein of 29 kDa - Cyt b ₅₅₉ cytochrome b ₅₅₉ - DCBQ 2,5-dichloro-p-benzo-quinone - LHC II light-harvesting complex II, predominant Chl a/b protein - MES 2-[N-Morpholino]ethanesulfonic acid - Pheo pheophytin - PS H photosystem II - Q_A bound plastoquinone, serving as the secondary electron acceptor in PS II (after Pheo) - SDS sodiumdodecylsulfate     

Thermoluminescence properties of the isolated photosystem two reaction centre

Formation of thermoluminescence signals is characteristics of energy- and charge storage in Photosystem II. In isolated D1/D2/cytochrome b-559 Photosystem II reaction centre preparation four thermoluminescence components were found. These appear at -180 (Z band), between -80 and -50 (Z_v band), at -30 and at +35°C. The Z band arises from pigment molecules but not correlated with photosynthetic activity. The Z_v and -30°C bands arise from the recombination of charge pairs stabilized in the Photosystem II reaction centre complex. The +35°C band probably corresponds to the artefact glow peak resulting from a pigment-protein-detergent interaction in subchloroplast preparations (Rózsa Zs, Droppa M and Horváth G (1989) Biochim Biophys Acta 973, 350–353).Abbreviations Chl chlorophyll - Cyt cytochrome - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - D1 psbA gene product - D2 psbD gene product - P680 primary electron donor of PS II - Pheo pheophytin - PS II Photosystem II - Q_A primary quinone acceptor of PS II - Q_B secondary quinone acceptor of PS II - RC reaction centre of PS II - TL thermoluminescence     

pH-dependent photoreactions of the high- and low-potential forms of cytochrome b559 in spinach PS II-enriched membranes

Cytochrome b559 (Cyt b559) is a well-known intrinsic component of Photosystem II (PS II) reaction center in all photosynthetic oxygen-evolving organisms, but its physiological role remains unclear. This work reports the response of the two redox forms of Cyt b559 (i.e. the high- (HP) and low-potential (LP) forms) to inhibition of the donor or acceptor side of PS II. The photooxidation of HP Cyt b559 induced by red light at room temperature was pH-dependent under conditions in which electron flow from water was diminished. This photooxidation was observed only at pH values higher than 7.5. However, in the presence of 1 M CCCP, a limited oxidation of HP Cyt b559 was observed at acidic pH, At pH 8.5 and in the presence of the protonophore, this photooxidation of the HP form was accompanied by its partial transformation into the LP form. On the other hand, a partial photoreduction of LP Cyt b559 was induced by red light under aerobic conditions when electron transfer through the primary quinone acceptor Q_A was impaired by strong irradiation in the presence of DCMU. This photoreduction was enhanced at acidic pH values. To the best of our knowledge, this is the first time that both photoreduction and photooxidation of Cyt b559 is described under inhibitory conditions using the same kind of membrane preparations. A model accommodating these findings is proposed.Abbreviations CCCP carbonylcyanide 3-chlorophenylhydrazone - Cyt cytochrome - DCBQ 2,5-dichloro-p-benzoquinone - DCMU dichlorophenyldimethylurea - E _m midpoint redox potential - HP and LP high- and low-potential forms of Cyt b559 - P680 primary donor - I_A acceptor side inhibition - I_D donor side inhibition - Pheo pheophytin - PS II photosystem II - Q_A primary quinone acceptor of PS II - Q_B secondary quinone acceptor of PS II     

The photochemical activities and electron carriers of developing barley leaves

The development of photochemical activities in isolated barley plastids during illumination of dark-grown plants has been studied and compared with the behaviour of plastocyanin, cytochromes f, b-559_LP, b-563 and b-559_HP and pigments P546 (C550) and P700. Electron-transport activity dependent on Photosystem 1 and cyclic photophosphorylation dependent on N-methylphenazonium methosulphate (phenazine methosulphate) were very active relative to the chlorophyll content after only a few minutes of illumination of etiolated leaves, and then rapidly declined during the first few hours of greening. By contrast, Photosystem 2 activity (measured with ferricyanide as electron acceptor) and non-cyclic photophosphorylation were not detectable during the first 2½h of greening, but then increased in total amount in parallel with chlorophyll. The behaviour of the electron carriers suggested their association with either Photosystem 1 or 2 respectively. In the first group were plastocyanin, cytochrome f and cytochrome b-563, whose concentrations in the leaf did not change during greening, and cytochrome b-559_LP whose concentration fell to one-half its original value, and in the second group were cytochrome b-559_HP and pigment P546, the concentrations of which closely followed the activities of Photosystem 2. Pigment P700 could not be detected during the first hour, during which time some other form of chlorophyll may take its place in the reaction centre of Photosystem 1. The plastids started to develop grana at about the time that Photosystem 2 activity became detectable.     

Functional subunit structure of photosystem 1 reaction center in Synechococcus sp

Photochemical activities of six different P700-chlorophyll a-proteins (CP1-a, -b₁, -b₂, -c, -d, and -e) separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis from digitonin particles of a thermophilic cyanobacterium Synechococcus sp. were examined. CP1-a, -b₁, -b₂, and -c contain the competent reaction center of photosystem 1: They were highly active in photooxidation of cytochrome c-553, the physiological electron donor to P700 in the organism, with methyl viologen as electron acceptor and showed flash-induced absorption changes indicating the charge separation between P700 and the secondary electron acceptors, P430 and A₂. The cytochrome photooxidation and P430 and A₂ photoresponses were significantly suppressed in CP1-d. CP1-e which lacks P430 and A₂ was least active in the cytochrome photooxidation. A₁, the primary electron acceptor of P700, is present in CP1-e as well as in other CP1 complexes. Comparison of the results with the polypeptide composition of CP1 complexes (Y. Takahashi, H. Koike, and S. Katoh, 1982, Arch. Biochem. Biophys.219, 209–218). indicates that CP1-c which contains four polypeptides with molecular weights of 62,000, 60,000, 14,000, and 10,000 represents the functional core of the photosystem 1 reaction center. P700, A₁, and antenna chlorophyll are associated with 62,000- and 60,000-dalton polypeptides, whereas 14,000- and 10,000-dalton polypeptides are assumed to carry P430 and A₂. The 13,000-dalton polypeptide which is associated with CP1-a, -b₁, and -b₂ is not required for the functioning of the reaction center.     

Photosystem II characteristics of a constructedSynechocystis 6803 mutant lacking synthesis of the D1 polypeptide

Photosystem II (PSII) composition was studied in a mutant of the cyanobacteriumSynechosystis 6803 in which synthesis of the reaction center polypeptide D1 has been inactivated. The mutant thylakoids had lost also the other reaction center polypeptide D2 and the chlorophylla-binding protein CP47. Cytochromeb559 and the chlorophylla-binding protein CP43 accumulated to almost wild-type amounts in mutant thylakoids. Also the 33 kDa polypeptide involved in water oxidation was present and membrane-bound in mutant thylakoids. The intrinsic 22 kDa polypeptide, so far known only from plants, was detected both in wild-type and mutant thylakoids.     

Effects of trypsin upon EPR signals arising from components of the donor side of Photosystem II

EPR signals from components functioning on the electron donor side of Photosystem II (PS II) have been monitored in PS II membranes isolated from spinach chloroplasts after treatment with trypsin at pH 7.5 and pH 6.0. The following information has been obtained. (1) The multiline manganese signal, the g = 4.1 signal and Signal II_slow are lost with trypsin treatment at pH 7.5, but not at pH 6.0. (2) At pH 7.5 the multiline S₂ signal and the g = 4.1 signal are lost with approximately the same dependency on the incubation time with trypsin. At pH 6.0 trypsin treatment is known to block electron transfer between Q_A and Q_B (the first and the second quinone electron acceptors, respectively) allowing only a single turnover to occur. Under these conditions both the g = 4.1 signal and the multiline signal are induced by illumination at 200 K and their amplitudes are almost the same as in untreated samples. These results are interpreted as indicating that the g = 4.1 signal arises from a side path donor or from S₂ itself rather than a carrier functioning between the S states and the reaction center as previously suggested. (3) Cytochrome b-559 is converted to its oxidized low-potential form by trypsin treatment at both values of pH. At pH 6.0 the S-state turnover still occurs indicating that the presence of reduced high-potential cytochrome b-559 is not necessary for this process.     

pH sensitivity of the redox state of cytochrome b559 may regulate its function as a protectant against donor and acceptor side photoinhibition

A series of experiments have been conducted with isolated reaction centers of photosystem two (PS II) with the aim to elucidate the functional role of cytochrome (Cyt b ₅₅₉). At pH 6.5 it was found that Cyt b ₅₅₉ was reversibly photoreduced by red actinic light when Mn²⁺ was present as an electron donor while at pH 8.5 a photo-oxidation was observed under the same lighting conditions, which was dark reversible in the presence of hydroquinone. These pH dependent light induced changes were measured under anaerobic conditions and correlated with changes in the relative levels of high (HP) and low (LP) potential forms of the cytochrome. At pH 6.5 the cytochrome was mainly in its LP form while at pH 8.5 a significant proportion was converted to the HP form as detected by dark titrations with hydroquinone. This pH dependent difference in the levels of HP and LP Cyt b ₅₅₉ was also detected when bright white light was used to monitor the level of the LP form using a novel reaction involving direct electron donation from the flavin of glucose oxidase (present in the medium and used together with glucose and catalase as an oxygen trap). The results suggest that PS II directly oxidises and reduces the HP and LP forms, respectively and that the extent of these photo-reactions is dependent on the relative levels of the two forms, which are in turn governed by the pH. This conclusion is interpreted in terms of the model presented previously (Barber J and De Las Rivas J (1993) Proc Natl Acad Sci USA 90: 10942–10946) whereby the pH induced effect is considered as a possible mechanism by which interconversion of LP and HP forms of Cyt b ₅₅₉ is achieved. In agreement with this was the finding that as the extent of photo-oxidisable HPCyt b ₅₅₉ increases, with increasing pH, the rate of irreversible photo-oxidation of -carotene decreases, a result expected if the HP form protects against donor side photoinhibition.Abbreviations -car -carotene - CCCP carbonylcyanide m-chloro-phenylhydrazone - Chl chlorophyll - Cyt b ₅₅₉ cytochrome b ₅₅₉ - HPCyt b ₅₅₉ high potential form of cytochrome b ₅₅₉ which is reducible by hydroquinone - LPCyt b ₅₅₉ low potential form of cytochrome b ₅₅₉ which is non-reducible by hydroquinone - D1 and D2 products of the psbA and psbD genes, respectively - LHC II light-harvesting chlorophyll protein complex associated with PS II - Mes 2-(N-morpholino) ethanesulphonic acid - P680 primary electron donor of PS II - Pheo pheophytin - PQ plastoquinone - PS II Photosystem II - Q_A first stable quinone electron acceptor of PS II - Q_B second stable quinone electron acceptor of PS II - RC reaction center - SDS sodium dodecyl sulphate - SiMo silicomolybdate - Tris tris(hydroxymethyl) amino methane - Y_Z and Y_D tyrosine residues 161 in D1 and D2 proteins of the PS II RC which act as secondary electron donors to P680     

Dynamic metabolism of photosystem II reaction center proteins and pigments

Photosystem II (PSII) reaction center is an intrinsic membrane-protein complex in the chloroplast that catalyzes primary charge separation between P680, a chlorophyll a dimer, and the primary quinone acceptor Q_A. This supramolecular protein complex consists of D1, D2, α and β subunits of cytochrome b₅₅₉, the psbI gene product, and a few low molecular mass proteins. Ligated to this complex are pigments: chlorophyll a, pheophytin a, β-carotenes, and non-heme iron. One of the major outcomes of light-mediated photochemistry is the fact that in the light, D1 protein is rapidly turned over compared to the other proteins of the reaction center; the relative lability of proteins being: D1?D2>Cyt b₅₅₉. D1 degradation in visible light exhibits complex, multiphasic kinetics. D1 degradation can be uncoupled from photosynthetic electron transport, which suggests that degradation may perform some separate function(s) beyond maintaining photosynthetic activity. The presence of a physiologically relevant level of ultraviolet-B (UV-B) radiation in a background of photosynthetically active radiation stimulates D1/D2 heterodimer degradation in a synergistic manner. D1 undergoes several post-translational modifications including N-acetylation, phosphorylation, and palmitoylation. Light-dependent phosphorylation of D1 occurs in all flowering plants but not in the green alga Chlamydomonas or in cyanobacteria, and the same may be true for D2. The roles of these modifications in D1/D2 assembly, turnover, or function are still a matter of conjecture. Nor do we yet know about the fate of the liganded pigments, such as the chlorophyll and carotenoids bound to the reaction center proteins. Environmental extremes that negatively impact photosynthesis seem to involve D1 metabolism. Thus, D1 protein is a major factor of PSII instability, and its replacement after its degradation is a primary component of the PSII repair cycle.     

Histidine residue 252 of the Photosystem II D1 polypeptide is involved in a light-induced cross-linking of the polypeptide with the α subunit of cytochrome b-559: study of a site-directed mutant of Synechocystis PCC 6803

Properties of the Photosystem II (PSII) complex were examined in the wild-type (control) strain of the cyanobacterium Synechocystis PCC 6803 and its site-directed mutant D1-His252Leu in which the histidine residue 252 of the D1 polypeptide was replaced by leucine. This mutation caused a severe blockage of electron transfer between the PSII electron acceptors Q_A and Q_B and largely inhibited PSII oxygen evolving activity. Strong illumination induced formation of a D1-cytochrome b-559 adduct in isolated, detergent-solubilized thylakoid membranes from the control but not the mutant strain. The light-induced generation of the adduct was suppressed after prior modification of thylakoid proteins either with the histidine modifier platinum-terpyridine-chloride or with primary amino group modifiers. Anaerobic conditions and the presence of radical scavengers also inhibited the appearance of the adduct. The data suggest that the D1-cytochrome adduct is the product of a reaction between the oxidized residue His²⁵² of the D1 polypeptide and the N-terminal amino group of the cytochrome α subunit. As the rate of the D1 degradation in the control and mutant strains is similar, formation of the adduct does not seem to represent a required intermediary step in the D1 degradation pathway.     

Gel electrophoresis of chloroplast membranes of mutants of Chlamydomonas reinhardii which have impaired Photosystem II function and lack photosynthetic cytochromes

Photosystem (PS) II-enriched particles or chloroplast fragments of the wild type and of three nonphotosynthetic mutants of Chlamydomonas reinhardii, which lack chloroplast cytochromes, were analyzed by lithium dodecyl sulfate polyacrylamide gel electrophoresis at 4°C to locate which chlorophyll complexes and which proteins are associated with cytochrome b-559. Two mutants, Fl 39 and Fl 50, have previously been shown to contain, respectively, 3.6- and 2.7-times less hydroquinone-reducible high-potential cytochrome b-559 than the wild type. They have impaired PS II functions. In the presence of ADRY agents: carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP), 2-(3-chloro-4-trifluoromethyl)anilino-3,5-dinitrothiophene (ANT 2p) or 2-(3,4,5-trichloro)-anilino-3,5-dinitrothiophene (ANT 2s), Fl 50 carried out photo-oxidation of cytochrome b-559 with half the amplitude of that of the wild type. No photo-oxidation was observed with Fl 39. We show here that in both these mutants chlorophyll-protein complexes CP III, CP IV and CP V were missing. There were traces of the corresponding apoproteins (45 000, 42 000 and 33 000 daltons, respectively) in Fl 50 but none in Fl 39. In addition, a 19 000 dalton protein was missing in Fl 39 and present in a very small amount in Fl 50. In another mutant, Fl 9, previously characterized as lacking both cytochromes b-563 and c-553 with a normal cytochrome b-559 content, CP III-CP V and the 19 000 dalton protein were detected. CP I (110 000 daltons) and CP II (24 000 daltons) were present in all strains. These observations confirmed the close relationship between deficiencies in cytochrome b-559, lack of CP III and CP IV and anomalies in the photochemistry of PS II. They provided additional evidence that CP V and a 19 000 dalton protein are also involved in this PS II photochemistry. Staining of the gels with 3,3′,5,5′-tetramethylbenzidine and H₂O₂ allowed us to distinguish clearly four heme protein bands having peroxidase activity. Three of these bands (45 000, 42 000 and 19 000 daltons), which were shown in wild-type, Fl 39 and Fl 50 preparations but not in Fl 9, appeared related to cytochromes b-563 and c-553. The fourth heme protein (14 000 daltons) occurred in wild type and Fl 9 but was missing in Fl 39 and Fl 50; it appeared related to cytochrome b-559.     

Two-dimensional crystals of photosystem II: biochemical characterization, cryoelectron microscopy and localization of the D1 and cytochrome b559 polypeptides

Photosystem II (PS II) is a photosynthetic reaction center found in higher plants which has the unique ability to evolve oxygen from water. Several groups have formed two-dimensional PS II crystals or have isolated PS II complexes and studied them by electron microscopy and image analysis. The majority of these specimens have not been well characterized biochemically and have yielded relatively low resolution two-dimensional projection maps with a variety of unit cell sizes. We report the characterization of the polypeptide and lipid content of tubular crystals of PS II. The crystals contain the reaction center core polypeptides D1, D2, cytochrome b559, as well as the chlorophyll- binding polypeptides (CP) CP47, CP43, CP29, CP26, CP24, and CP22. The lipid composition was similar to the lipids found in the stacked portion of thylakoids. We also report a 2.0-nm resolution projection map determined by electron microscopy and image analysis of frozen, hydrated PS II crystals. This projection map includes information on the portion of the complex buried in the lipid bilayer. The unit cell is a dimer with unit vectors of 17.0 and 11.4 nm separated by an angle of 106.6 degrees. In addition, Fab fragments against D1 and cytochrome b559 were used to localize those two polypeptides, and thus the reaction center, within the PS II complex. The results indicate that D1 and cytochrome b559 are found within one of the heaviest densities of the monomeric unit.     

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.     

Inhibition of Photosystem 2 primary photochemistry by photogenerated protons

Photosystem 2 photochemical efficiency, measured as the rate of Qa reduction, was observed to be inhibited by preillumination with single turnover flashes, whilst F_o and F_m were not affected. Such inhibition was reversed by the uncoupler nigericin or by incubating the thylakoids in the dark for ca. 2 min after the preillumination. The presence of ATP in micromolar concentrations increased the time of dark recovery from the inhibition. The inhibition of fluorescence rise was not changed when 70% of the excitation energy available in the antenna was quenched by dinitrobenzene. Quantitative analysis of the observed fluorescence induction indicates that this phenomenon is due to the inhibition of the photochemical reaction itself. Uncouplers such NH₄Cl were unable to reverse the inhibition and only a few flashes of saturating intensity (10 or less) were required for the onset of it. This suggests that protons localised in domains rather than a pH gradient between the thylakoid lumen bulk solution and the external one are involved in this regulation of PS 2 efficiency.Abbreviations Chl- chlorophyll - cyt b ₅₅₉- cytochrome b ₅₅₉ - DCMU- 3-(3,4 dichlorophenyl)-1, 1 dimethylurea - DMBQ- dimethylbenzoquinone - DNB- dinitrobenzene - - electric potential difference - F_o- minimal fluorescence level measured with open reaction centres - F_m- maximal fluorescence level measured with closed reaction centres - F_v- variable fluorescence, defined as F_m-F_o - FWHM- full width at half maximum transmission - HA- hydroxylamine - MV- methylviologen - P680- pigment involved in the charge separation in Photosystem 2 - pheo- pheophytin - PS 1- Photosystem 1 - PS 2- Photosystem 2 - Q_a- primary quinone acceptor of Photosystem 2 - Q_b- secondary quinone acceptor of Photosystem 2     

The relationship between the activity of chloroplast Photosystem II and the midpoint oxidation-reduction potential of cytochrome b-559

The role of cytochrome b-559 in Photosystem II reactions has been investigated using hydroxylamine treatment of chloroplast membranes. Incubation of chloroplasts with hydroxylamine in darkness resulted in inhibition of water oxidation and a decrease in the amplitude of cytochrome b-559 reducible by hydroquinone. The loss of water oxidizing activity perfectly correlated with the decrease in amplitude of cytochrome b-559 reduction. Potentiometric titration of cytochrome b-559 after hydroxylamine treatment revealed a component with E_m7.8 at +240 mV in addition to a lower potential species at +90 mV. This compared to control chloroplasts in which cytochrome b-559 exists in the typical high potential state, E_m7.8 = +383 mV, in addition to some of the low potential (E_m7.8 = +77 mV) form. Photosystem II activity could be further inhibited by incubation with hydroxylamine in the light. In these chloroplasts only low rates of photooxidation of artificial electron donors were observed compared to ‘dark’ chloroplasts. In addition, the hydroxylamine light treatment caused a further change in cytochrome b-559 redox properties; a single component, E_m7.8 = 90 mV is seen in titration curves. The role of cytochrome b-559 in Photosystem II functioning is discussed on the basis of these observations which suggest a dependence of photooxidizing ability of Photosystem II on the redox properties of this cytochrome.     

EPR and optical changes of the photosystem II reaction center produced by low temperature illumination

Electron paramagnetic resonance (EPR) and absorption spectroscopy have been used to study the low temperature photochemical behavior of the Photosystem II D-1/D-2/ cytochrome b559 reaction center complex. The reaction center displays large triplet state EPR signals which are attenuated after actinic illumination at low temperatures in the presence of sodium dithionite. Concomitant with the triplet attenuation is the buildup of a structured radical signal with an effective g value of 2.0046 and a peak-to-peak width of 11.9 G. The structure in the signal is suggestive of it being comprised in part of the anion radical of pheophytin a. This assignment is corroborated by low temperature optical absorbance measurements carried out after actinic illumination at the low temperatures which show absorption bleachings at 681 nm, 544 nm and 422 nm and an absorbance buildup at 446 nm indicating the formation of reduced pheophytin.Abbreviations EPR electron paramagnetic resonance     

Enhanced susceptibility of the oxidized and unprotonated forms of high potential cytochrome b-559 toward DCMU

The nature of interaction of cytochrome b-559 high potential (HP) with electron transport on the reducing side of photosystem II was investigated by measuring the susceptibility of cytochrome b-559HP to 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) under different conditions. Submicromolar DCMU concentrations decreased the rate of absorbance change corresponding to cytochrome b-559HP photoreduction while the amplitude was lowered at higher concentrations (up to 10 M). Appreciable extents of cytochrome b-559HP photoreduction were observed at DCMU concentrations which completely abolished the electron transport from water to methyl viologen under the same experimental conditions. However, the susceptibility of cytochrome b-559HP to DCMU increased with the degree of cytochrome b-559HP oxidation, induced either by ferricyanide or by illumination of low intensity (2 W/m²) of red light in the presence of 2 M carbonyl cyanide-m-chlorophenylhydrazone. Also, the DCMU inhibition was more severe when the pH increased from 6.5 to 8.5, indicating that the unprotonated form of cytochrome b-559HP is more susceptible to DCMU. These results demonstrate that cytochrome b-559HP can accept electrons prior to the Q_B site, probably via Q_A although both Q_A and Q_B can be involved to various extents in this reaction. We suggest that the redox state and the degree of protonation of cytochrome b-559HP alter its interaction with the reducing side of photosystem II.Abbreviations ADRY acceleration of the deactivation reactions of the water-splitting system Y - CCCP carbonylcyanide m-chlorophenylhydrazone - FeCN ferricyanide - HP high potential - MV methylviologen CIW-DPB Publication No.1096.     

Comparative EPR and thermoluminescence study of anoxic photoinhibition in Photosystem II particles

Photosystem II particles were exposed to 800 W m^–2 white light at 20 °C under anoxic conditions. The F_o level of fluorescence was considerably enhanced indicating formation of stable-reduced forms of the primary quinone electron acceptor, Q_A. The F_m level of fluorescence declined only a little. The g=1.9 and g=1.82 EPR forms characteristic of the bicarbonate-bound and bicarbonate-depleted semiquinone-iron complex, Q_A ^–Fe²⁺, respectively, exhibited differential sensitivity against photoinhibition. The large g=1.9 signal was rapidly diminished but the small g=1.82 signal decreased more slowly. The S₂-state multiline signal, the oxygen evolution and photooxidation of the high potential form of cytochrome b-559 were inhibited approximately with the same kinetics as the g=1.9 signal. The low potential form of oxidized cytochrome b-559 and Signal II_slow arising from Tyr_D ⁺ decreased considerably slower than the g=1.9 semiquinone-iron signal. The high potential form of oxidized cytochrome b-559 was diminished faster than the low potential form. Photoinhibition of the g=1.9 and g=1.82 forms of Q_A was accompanied with the appearance and gradual saturation of the spin-polarized triplet signal of P 680. The amplitude of the radical signal from photoreducible pheophytin remained constant during the 3 hour illumination period. In the thermoluminescence glow curves of particles the Q band (S₂Q_A ^– charge recombination) was almost completely abolished. To the contrary, the C band (Tyr_D ⁺Q_A ^– charge recombination) increased a little upon illumination. The EPR and thermoluminescence observations suggest that the Photosystem II reaction centers can be classified into two groups with different susceptibility against photoinhibition.Abbreviations C band thermoluminescence band associated with Tyr-D⁺Q _a ^– charge recombination - Chl chlorophyll - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - EPR electron paramagnetic resonance - F_o initial fluorescence - F_m maximum fluorescence - Q band thermoluminescence band originating from S₂Q _a -charge recombination - Q _a the primary quinone electron acceptor of PS II - P 680 the primary electron donor chlorophyll of PS II - S₂ oxidation state of the water-splitting system - Phe pheophytin - TL thermoluminescence - Tyr _d redox active tyrosine-160 of the D₂ protein     

The role of the high potential form of the cytochrome b559: Study of Thermosynechococcus elongatus mutants

Cytochrome b559 is an essential component of the photosystem II reaction center in photosynthetic oxygen-evolving organisms, but its function still remains unclear. The use of photosystem II preparations from Thermosynechococcus elongatus of high integrity and activity allowed us to measure for the first time the influence of cytochrome b559 mutations on its midpoint redox potential and on the reduction of the cytochrome b559 by the plastoquinone pool (or Q_B). In this work, five mutants having a mutation in the α-subunit (I14A, I14S, R18S, I27A and I27T) and one in the β-subunit (F32Y) of cytochrome b559 have been investigated. All the mutations led to a destabilization of the high potential form of the cytochrome b559. The midpoint redox potential of the high potential form was significantly altered in the αR18S and αI27T mutant strains. The αR18S strain also showed a high sensitivity to photoinhibitory illumination and an altered oxidase activity. This was suggested by measurements of light induced oxidation and dark re-reduction of the cytochrome b559 showing that under conditions of a non-functional water oxidation system, once the cytochrome is oxidized by P680⁺, the yield of its reduction by Q_B or the PQ pool was smaller and the kinetic slower in the αR18S mutant than in the wild-type strain. Thus, the extremely positive redox potential of the high potential form of cytochrome b559 could be necessary to ensure efficient oxidation of the PQ pool and to function as an electron reservoir replacing the water oxidation system when it is not operating.