Comparative study of the water oxidizing reactions and the millisecond delayed chlorophyll fluorescence in photosystem II at different pH

Water splitting activity, the multiline EPR signal associated with S(2)-state of the CaMn(4)-cluster and the fast and slow phases of the induction curve of the millisecond delayed chlorophyll fluorescence from photosystem II (PSII) in the pH range of 4.5-8.5 were studied in the thylakoid membranes and purified PSII particles. It has been found that O(2) evolution and the multiline EPR signal were inhibited at acidic (pK approximately 5.3) and alkaline (pK approximately 8.1) pH values, and were maximal at pH 6.0-7.0. Our results indicate that the loss of O(2) evolution and the S(2)-state multiline EPR signal associated with the decrease of the millisecond delayed chlorophyll fluorescence only in alkaline region (pH 7.0-8.5). Possible correlations of the millisecond delayed chlorophyll fluorescence components with the donor side reactions in PSII are discussed.     

Isolation of the first ferromagnetically coupled Mn(III/IV) complex

Binuclear manganese complexes Mn2(III/IV)(dtsalpn)2DCBI, 1, Mn2(III/III)(dtsalpn)2HDCBI, 2, containing the ligand dicarboxyimidazole (DCBI) have been prepared in order to address the issue of imidazole bridged and ferromagnetically coupled Mn sites in high oxidation states of the OEC in Photosystem II (PS II). Temperature dependent magnetic susceptibility studies of 1 indicates that the interaction between the two Mn(III)/Mn(IV) ions is ferromagnetic (J = +1.4 cm(-1)). Variable temperature EPR spectra of 1 shows that a g = 2 multiline is as an excited state signal corresponding to S = 1/2.     

Novel EPR characterization of the antioxidant activity of tea leaves

Electron paramagnetic resonance (EPR) spectroscopy is utilized to investigate several categories of green and black tea: Twining green tea (TGT), Chinese green tea (CGT), Red-labels black tea (RBT). Basically, two EPR signals from all the studied samples are observed: One of them is a very weak sharp EPR signal with deltaHpp approximately 10 G and g-factor = 2.00023 superimposed on the other broad signal with deltaHpp approximately 550 G and g-factor = 2.02489. The broad signal is a characteristic one of manganese(II) complex, while the sharp signal is related to a stable radical of aromatic origin exist in a powder condition. The feature of the manganese EPR signal is attributed to manganese(II) complex and reflected the molecular behavior of Mn(II) in the protein system of the natural leaves. The sharp signal, which is most probably due to a semiquinones radicals, is observed at room temperature and its intensity is remarkably affected by photo degradation of the studied samples. The intensity of manganese(II) EPR signal is found to be related to ageing and disintegration of the tea leaves. Moreover, direct relation between the relative intensity of the semiquinones radical signal and antioxidant activity of the studied samples was also correlated.     

An electron paramagnetic resonance study of Mn2(H2O)(OAc)4(tmeda)2 (tmeda = N,N,N',N'-tetramethylethylenediamine): a model for dinuclear manganese enzyme active sites

The complex Mn2(H2O)(OAc)4(tmeda)2 (tmeda = N,N,N',N'-tetramethylethylenediamine) is a model for the active site of hydrolase enzymes containing acetate-bridged dimanganese cores. The two high-spin Mn(II) ions are antiferromagnetically coupled, as determined by previous magnetic susceptibility studies (Yu, S.-B; Lippard, S. J.; Shweky, I; Bino, A. Inorg. Chem. 1992, 31, 3502-3504) to yield a spin "ladder" with total spin S = 0, 1, 2, ..., 5 in increasing energy. In this study, the complex was characterized by Q-band and X-band EPR spectroscopy in frozen solution. Analysis of the temperature dependence of these EPR spectra indicates that the primary spectral contribution is from the S = 2 manifold. The EPR spectra were simulated using a full spin Hamiltonian for this manifold of a coupled spin system, which provided the fit parameters J = -2.9 cm-1, g = 2.00, and D2 = -0.060 +/- 0.003 cm-1. An additional multiline EPR signal is observed which is proposed to arise from the total spin S = 5/2 ground state of a Mn(II) trimer of the type Mn3(OAc)6(tmeda)2.     

Listening to PS II: enthalpy, entropy, and volume changes

Photosystem II, located in the thylakoid membranes of green plants, algae, and cyanobacteria, uses sunlight to split water into protons, electrons, and a dioxygen molecule. The mechanism of its electron transfers and oxygen evolution including the structure of the protein and rates of the S-state cycle has been extensively investigated. Substantial progress has been made; however, the thermodynamics of PS II electron transfer and of the oxygen cycle are poorly understood. Recent progress in thermodynamic measurements in photosynthesis provides novel insights on the enthalpic and entropic contribution to electron transfer in proteins. In this review the thermodynamic parameters including quantum yield, enthalpy, entropy, and volume changes of PS II photochemistry determined by photoacoustics and other laser techniques are summarized and evaluated. Light-driven volume changes via electrostriction are directly related to the photoreaction in PS II and thus can be a useful measurement of PS II activity and function. The enthalpy changes of the reactions observed can be directly measured by photoacoustics. The apparent reaction entropy can also be estimated when the free energy is known. Dissecting the free energy of a photoreaction into enthalpic and entropic components provides critical information about mechanisms of PS II function. Potential limitations and future direction of the study of the thermodynamics of PS II electron transfer and oxygen evolution are presented.     

The peroxidase activity of a hemin--DNA oligonucleotide complex: free radical damage to specific guanine bases of the DNA.

A specific DNA oligonucleotide--hemin complex (PS2.M--hemin complex) that exhibits DNA-enhanced peroxidative activity was studied by EPR and UV--visible spectroscopy and by chemical probing analysis. EPR data obtained from low-temperature experiments on the PS2.M--hemin complex showed both a low-field g approximately 6 and a high-field g approximately 2 signal. These EPR signals are typical of high-spin ferric heme with axial symmetry as judged by the EPR spectrum of six-coordinate heme iron in acidic Fe(III)-myoglobin. This similarity is consistent with the presence of two axial ligands to the heme iron within the PS2.M--hemin complex, one of which is a water molecule. Optical analyses of the acid-base transition for the hemin complex yielded a pK(a) value for the water ligand of 8.70 +/- 0.03 (mean +/- SD). Low-temperature EPR analysis coupled with parallel spin-trapping investigations following the reaction of the PS2.M--hemin complex and hydrogen peroxide (H(2)O(2)) indicated the formation of a carbon-centered radical, most likely on the PS2.M oligonucleotide. Chemical probing analysis identified specific guanine bases within the PS2.M sequence that underwent oxidative damage upon reaction with H(2)O(2). These and other experimental findings support the hypothesis that the interaction of specific guanines of PS2.M with the bound hemin cofactor might contribute to the superior peroxidative activity of the PS2.M--hemin complex.     

An EPR and 1H NMR active mixed-valence manganese (III/II/III) trinuclear compound

A mixed-valence Mn(III)-Mn(II)-Mn(III) trinuclear complex of stoichiometry MnIIIMnIIMnIII(Hsaladhp)2(Sal)4.2CH3CN (1), where H3saladhp is a tridentate Schiff-base ligand, has been structurally characterized with X-ray crystallography. The Mn(III)Mn(II)Mn(III) angles are strictly 180 degrees as required by crystallographic inversion symmetry. The complex is valence-trapped with two terminal Mn(III) ions in a distorted square pyramidal geometry. The Mn(III)...Mn(II) separation is 3.495 A. The trinuclear complex shows small antiferromagnetic exchange J coupling. The magnetic parameters obtained from the fitting procedure in the temperature range 10-300 K are J1 = -5.7 cm-1, g = 2.02, zJ = -0.19 cm-1, and R = 0.004. The EPR spectrum was obtained at 4 K in CHCl3 and in tetrahydrofuran glasses. The low-field EPR signal is a superposition of two signals, one centered around g = 3.6 and the other, for which hyperfine structure is observed, centered around g = 4.1 indicating an S = 3/2 state. In addition, there is a 19-line signal at g = 2.0. The multiline signal compares well with that observed for the S2 or S0* states of the oxygen-evolving complex. 1H NMR data reveal that the trinuclear compound keeps its integrity into the CHCl3 solution. Crystal data for complex 1: [C54H52N4O18Mn3], M = 1209.82, triclinic, space group P1, a = 10.367(6) A, b = 11.369(6) A, c = 13.967(8) A; alpha = 112.56(1) degree, beta = 93.42(2) degrees, gamma = 115.43(1) degree, Z = 1.     

Effect of Nano-anatase TiO2 on Spectral Characterization of Photosystem Ⅱ Particles from Spinach

The photosystem I (PS Ⅱ) particles were purified by means of nano-anatase TiO2 treatment of spinach and studied by spectroscopy. The results show that the electron transport and the oxygen-evolving rate of PS I are accelerated after it has been treated with nano-anatase TiO2; the UV-Vis absorption spectrum of PS I particles is increased; the red shift of fluorescence emission peak of PS I is 2 nm; the peak intensity is decreased; the PS Ⅱ signal I s of low temperature electron paramagnetic resonanace(EPR) spectrum is intensified under light, and the PS I circular dichroism(CD) spectrum is similar to that of control. It is suggested that nano-anatase TiO2 might bind to the PS I reaction center complex and intensify the function of the PS I electron donor, however, nano-anatase TiO2 treatment does not change the configuration of the PS Ⅱ reaction center complex.     

g-Anisotropy of the S2-state manganese cluster in single crystals of cyanobacterial photosystem II studied by W-band electron paramagnetic resonance spectroscopy

The multiline signal from the S2-state manganese cluster in the oxygen evolving complex of photosystem II (PSII) was observed in single crystals of a thermophilic cyanobacterium Thermosynechococcus vulcanus for the first time by W-band (94 GHz) electron paramagnetic resonance (EPR). At W-band, spectra were characterized by the g-anisotropy, which enabled the precise determination of the tensor. Distinct hyperfine splittings (hfs's) as seen in frozen solutions of PSII at X-band (9.5 GHz) were detected in most of the crystal orientations relative to the magnetic field. In some orientations, however, the hfs's disappeared due to overlapping of a large number of EPR lines from eight crystallographic symmetry-related sites of the manganese cluster within the unit cell of the crystal. Analysis of the orientation-dependent spectral features yielded the following g-tensor components: g(x) = 1.988, g(y) = 1.981, g(z) = 1.965. The principal values suggested an approximate axial symmetry around the Mn(III) ion in the cluster.     

Comparative Study of Biosorption of Heavy Metals Using Living Green Algae Scenedesmus quadricauda and Neochloris pseudoalveolaris: Equilibrium and Kinetics

The biosorption of several heavy metals such as cobalt(II), chromium(III), lead(II), cadmium(II), nickel(II), and manganese(II) from aqueous systems on living microalgae cultures, Scenedesmus quadricauda and Neochloris pseudoalveolaris were studied under laboratories conditions. The kinetic and statistical parameters were calculated by using the data obtained from batch cultivation and well fitted a pseudo-first-order rate equation. The initial metal concentrations in solution were about 5–40 mg · L^?1. According to the pseudo-second-order model, the biosorption capacities of Scenedesmus quadricauda for Co(II), Cr(III), Pb(II), Cd(II), Ni(II), and Mn(II) ions were found in the ranges of 2.14–52.48, 1.98–81.98, 8.05–4.26, 7.81–24.96, 2.17–55.71, and 3.54–75.20 mg g^?1, respectively. Kinetic studies revealed that the metal uptake capacity of each living green algae was rather fast. It was also observed that the biosorption kinetic rate decreased with increasing concentration for both microalgae. The application of diffusion-controlled models to the experimental results indicated that the contribution of intraparticle diffusion to the overall sorption kinetics was not very important. Results showed that Co(II), Cr(III), Pb(II), Cd(II), Ni(II), and Mn(II) ions could effectively be absorbed by using living microalga cultures from aqueous solutions.     

A carbonic anhydrase inhibitor induces bicarbonate-reversible suppression of electron transfer in pea photosystem 2 membrane fragments

The effects of suppression of the carbonic anhydrase (CA) activity by a CA-inhibitor, acetazolamide (AA), on the photosynthetic activities of photosystem II (PS II) particles from higher plants were investigated. AA along with CA-activity inhibits the PS II photosynthetic electron transfer and the AA-induced suppression is totally reversed by the addition of bicarbonate (3-5 mM). Similar effect of recovery in the PS II photosynthetic activity was also revealed upon the addition of known artificial electron donors (potassium ferrocyanide and TMPD). Significance and possible functions of CA for the PS II donor side are discussed.     

Shape-shifting tetranuclear oxo-bridged manganese cluster: relevance to photosystem II water oxidase active site

The redox properties of the "dimer-of-dimers" complex, [{Mn2(mu-O)2(tphpn)}2]4+ (1) (where Htphpn = N,N,N',N'-tetra(2-methylpyridyl)-2-hydroxypropane-diamine) were investigated. The structure changes dramatically to an adamantane-shaped core upon one-electron oxidation. On the other hand, the one-electron reduced product of 1, [Mn4O4(tphpn)2]3+, exhibits a hyperfine-structured multiline EPR signal very similar to the so-called S0 state of the tetramanganese cluster, which resides at the Photosystem II water oxidase active site.     

Dried biomass of green algae and its matrix matching with green parts of higher plants

Literature data for the content of matrix elements in dry biomass of green algae have been compared with those for green parts of higher plants. The data were evaluated by means of frequency histograms and box-and-whisker plots. With two exceptions (Fe, P), the spread of the data values for a given element was broader for higher plants than for algae. Distribution of the data was asymmetric, in most cases with long right-hand tails. All outliers found (average 5.6% for algae, 9.1% for higher plants) lay above the bulk of the data values. In all cases, there was either partial or (mostly) complete overlap of the ranges for algae with those for higher plants. While the question of matrix-matching of reference materials prepared from algal biomass with samples from green parts of higher plants can exhaustively be answered only after a similar comparison of the composition of matrix compounds, the content of the main matrix elements is certainly not doubtful.     

THE ROLE OF CYCLIC ELECTRON FLOW AROUND PHOTOSYSTEM I and EXCITATION ENERGY DISTRIBUTION BETWEEN THE PHOTOSYSTEMS UPON ACCLIMATION TO HIGH IONIC STRESS IN Dunaliella salina

Abstract— The distribution of excitation energy between the two photosystems in the halophylic alga Dunaliella salina has been analyzed under ionic stress. In the transition from state 1 to state 2, it was found that a, the absorption cross-section of photosystem (PS) I increased from 42 to 49% until an equal distribution between PS I and PS II was obtained in state 2. Acclimation of the algae to different salt concentrations did not change the fractions of light absorbed in PS II and PS I, but slowed down the transition time from state 1 to state 2. A large increase in ΔpH induced fluorescence quenching was observed which was abolished by the uncoupler nigericin. Photoacoustic quantum yield spectra of energy storage indicated a larger energy storage at 700 nm induced upon stress. The additional ΔpH quenching of fluorescence and the additional quantum yield of energy storage at 700 nm, in the stressed algae, are consistent with the operation of a cyclic, energy-storing pathway in PS I which is uncoupler sensitive.     

Evidence for PSII donor-side damage and photoinhibition induced by cadmium treatment on rice (Oryza sativa L.)

The effects of cadmium (from 7.5 to 75 microM) on chloroplasts of rice were studied at the structural and biochemical level. Loss of pigments, reduction of thylakoids and decrease in oxygen evolution and Fv/Fm ratio occur in leaves following cadmium treatment. However, the amount of photosystem II reaction center proteins and that of its light harvesting complex is not affected, indicating that cadmium does not adversely influence the structural organization of this photosystem. In thylakoids isolated from cadmium-treated plants a loss in the capability to reduce 2,6-dichlorophenolindophenol is observed, which is partially restored if diphenylcarbazide is used as an electron donor, indicating that cadmium affects water splitting activity. In thylakoids isolated from control plants and treated with cadmium, diphenylcarbazide preserves most of the photosystem II activity lost after incubation with cadmium; most of the S(2) multiline electron paramagnetic resonance signal from the manganese cluster is lost, whereas the TyrD(+) and other signals are retained. Light-induced photosystem II damage, in vitro, is promoted by Cd-treatment as deduced from the mobility shift of the D1 protein observed by immunoblot.     

Reversed-phase HPLC determination of chlorophyll a' and naphthoquinones in photosystem I of red algae: existence of two menaquinone-4 molecules in photosystem I of Cyanidium caldarium.

Chlorophyll (Chl) a', the C13(2)-epimer of Chl a, is one of the two Chl molecules constituting the primary electron donor (P700) of photosystem (PS) I of a thermophilic cyanobacterium Synechococcus elongatus. To examine whether PS I of other oxygenic photosynthetic organisms in general contain one Chl a' molecule in P700, the pigment composition of thylakoid membranes and PS I preparations isolated from red algae Porphyridium purpureum and Cyanidium caldarium was examined by reversed-phase HPLC with particular attention to Chl a' and phylloquinone (PhQ), the secondary electron acceptor of PS I. The two red algae contained one Chl a' molecule at the core part of PS I. In PS I of C. caldarium, two menaquinone-4 (MQ-4) molecules were detected in place of PhQ used by higher plants and cyanobacteria. The 1:2:1 stoichiometry among Chl a', PhQ (MQ-4) and P700 in PS I of the red algae indicates that one Chl a' molecule universally exists in PS I of oxygenic photosynthetic organisms, and two MQ-4 molecules are associated with PS I of C. caldarium.     

INACTIVATION OF THE ACCEPTOR SIDE AND DEGRADATION OF THE D1 PROTEIN OF PHOTOSYSTEM II BY SINGLET OXYGEN PHOTOGENERATED FROM THE OUTSIDE

Abstract— The possible association of photodynamic sensitization with photoinhibition damage to the photosystem II complex (PS II) has been investigated using isolated intact thylakoids from pea leaves. For this study singlet oxygen (¹O₂), photoproduced by endogenous chromophores that are independent of the function of PS II, was assumed to be the major reactive intermediate involved in the photoinhibition process. When thylakoid samples preincubated with rose bengal were subjected to exposure to relatively weak green light (500–600 nm) under aerobic conditions, PS II was severely damaged. The pattern of the rose bengal-sensitized inhibition of PS II was similar to that of high light-induced damage to PS II: (1) the secondary quinone (Q_B)-dependent electron transfer through PS II is inactivated much faster than the Q_B-independent electron flow, (2) PS II activity is lost prior to degradation of the D1 protein, (3) diuron, an herbicide that binds to the Q_B domain on the D1 protein, prevents D1 degradation, and (4) PS II is damaged to a greater extent by the deuteration of thylakoid suspensions but to a lesser extent by the presence of histidine. Furthermore, it was observed that destroying thylakoid Fe-S centers resulted in a marked reduction of high light-induced PS II damage. These results may suggest that the primary processes of photoinhibition are mediated by ¹O₂ and that Fe-S centers, which are located in some membrane components, but not in PS II, play an important role in photogenerating the activated oxygen immediately responsible for the initiation of photodamage to PS II.     

KINETIC STUDIES ON THE STABILIZATION OF THE PRIMARY RADICAL PAIR P680+ Pheo- IN DIFFERENT PHOTOSYSTEM II PREPARATIONS FROM HIGHER PLANTS*

Abstract— The stabilization of the primary radical pair P680+ pheophytin (Pheo)^- through rapid electron transfer from Pheo to the special plastoquinone of photosystem II (PS II), Q_A, was analyzed on the basis of time-resolved (40 ps) UV-absorption changes detected in different PS II preparations from higher plants. Lifetime measurements of¹Chi* fluorescence by single photon counting and a numerical analysis of the redox reactions revealed (1) at exciton densities required for light saturation of the stable charge separation, annihilation processes dominate the excited state decay leading to very similar lifetimes of ¹Chi* in systems with open and closed reaction centers and (2) the difference of absorption changes induced by actinic flashes of comparatively high photon density in samples with open and photochemically closed reaction centers, respectively, provides a suitable measure of the rate constant of QA formation. Conclusion 2 was confirmed in PS II membrane fragments by measurements at three wavelengths (280 nm, 292 nm and 325 nm) where the difference spectrum of Q^-_A formation exhibits characteristic features. The numerical evaluation of the experimental data led to the following results: (1) the rate constant of Q^-_A formation was found to be (300 ± 100 ps)^-1 in PS II membrane fragments and PS II core complexes deprived of the distal and proximal antenna and (2) an iron depletion treatment of membrane fragments does not affect these kinetics. The implications of these results are briefly discussed in terms of the PS II reaction pattern.     

Molecular functions of PsbP and PsbQ proteins in the photosystem II supercomplex

The PsbP and PsbQ proteins are extrinsic subunits of the photosystem II (PSII) supercomplex, which are found in green plants including higher plants and green algae. These proteins are thought to have evolved from their cyanobacterial homologs; cyanoP and cyanoQ respectively. It has been suggested that the functions of PsbP and PsbQ have largely changed from those of cyanoP and cyanoQ. In addition, multiple isoforms and homologs of PsbP and PsbQ were found in green plants, indicating that the acquisition of PsbP and PsbQ in PSII is not a direct path but a result of intensive functional divergence during evolution from cyanobacterial endosymbiont to chloroplast. In this review, we highlight newly introduced topics related to the functions and structures of both PsbP and PsbQ proteins. The present data suggest that PsbP together with PsbQ have specific and important roles in coordinating the activity of the donor and acceptor sides of PSII and stabilizing the active form of the PSII-light-harvesting complex II (LHCII) supercomplex.     

ACTION OF DETERGENTS AND ELECTROPHORESIS ON SPINACH CHLOROPLASTS UNDER HIGH ALKALINE CONDITIONS

Abstract. The alkaline solubilization treatment (AUT) procedure followed by column electrophoresis is an easy and effective method for the preparation and isolation of PS I complexes. The subchloroplast particles prepared by the action of the detergents Triton or Triton/SDS differ in that, in the latter case, only low molecular weight polypeptides are observed. Similarly, the liquid He EPR spectra are almost identical except for the absence of a g ˜ 2.03 line in the case of the Triton/SDS treated sample and the significant absence of 1.86 and 1.89 inflections in either spectra.