State Multiline Signal Research Articles

Far-red photosynthesis: Two charge separation pathways exist in plant Photosystem II reaction center

An alternative charge separation pathway in Photosystem II under the far-red light was proposed by us on the basis of electron transfer properties at 295 K and 5 K. Here we extend these studies to the temperature range of 77–295 K with help of electron paramagnetic resonance spectroscopy. Induction of the S2 state multiline signal, oxidation of Cytochrome b559 and ChlorophyllZ was studied in Photosystem II membrane preparations from spinach after application of a laser flashes in visible (532 nm) or far-red (730–750 nm) spectral regions. Temperature dependence of the S2 state signal induction after single flash at 730–750 nm (Tinhibition ~ 240 K) was found to be different than that at 532 nm (Tinhibition ~ 157 K). No contaminant oxidation of the secondary electron donors cytochrome b559 or chlorophyllZ was observed. Photoaccumulation experiments with extensive flashing at 77 K showed similar results, with no or very little induction of the secondary electron donors. Thus, the partition ratio defined as (yield of YZ/CaMn4O5-cluster oxidation):(yield of Cytb559/ChlZ/CarD2 oxidation) was found to be 0.4 at under visible light and 1.7 at under far-red light at 77 K. Our data indicate that different products of charge separation after far-red light exists in the wide temperature range which further support the model of the different primary photochemistry in Photosystem II with localization of hole on the ChlD1 molecule.

An EPR study of the pH dependence of formate effects on Photosystem II

Effects of formate on rates of O 2 evolution and electron paramagnetic resonance (EPR) signals were observed in the oxygen evolving PS II membranes as a function of pH. In formate treated PS II membranes, decrease in pH value resulted in the inhibition of the O 2 evolving activity, a decrease in the intensity of S 2 state multiline signal but an increase in the intensity of the Q A –Fe 2+ EPR signal. Time-resolved EPR study of the Y Z ● decay kinetics showed that the light-induced intensity of Y Z ● EPR signal was proportional to the formate concentration. The change in the pH affected both the light-induced intensities and the decay rates of Y Z ●, which was found to be faster at lower pH. At 253 K, t 1/e value of Y Z ● decay kinetics was found to be 8-10 s at pH 6.0 and 18-21 s at pH 5.0. The results presented here indicate that the extent of inhibition at the donor and the acceptor side of PS II due to formate is pH dependent, being more effective at lower pH.

EPR characteristics of chloride-depleted photosystem II membranes in the presence of other anions

Chloride is an essential cofactor for the oxidation of water to oxygen. Anion substitution (Br(-), I(-), NO(2)(-), F(-)) in Cl(-)-depleted PS II membranes brings out significant changes in the EPR signals arising from the S(2) state and from the iron-quinone complex of PS II. On the basis of the changes observed in the S(2) state multiline signal and the Q(A)Fe(3+) EPR signal in Cl(-)-depleted PS II membranes after substituting with various anions, we report a possible binding site of anions such as chloride and bromide at the PS II donor side as well as at the acceptor side.

The origin of split EPR signals in the Ca2+-depleted Photosystem II

A light-driven reaction model for the Ca2+-depleted Photosystem (PS) II is proposed to explain the split signal observed in electron paramagnetic resonance (EPR) spectra based on a comparison of EPR assignments with recent x-ray structural data. The split signal has a splitting linewidth of 160 G at around g = 2 and is seen upon illumination of the Ca2+-depleted PS II in the S2 state associated with complete or partial disappearance of the S2 state multiline signal. Another g=2 broad ESR signal with a 110 G linewidth was produced by 245 K illumination for a short period in the Ca2+-depleted PS II in S1 state. At the same time a normal YZ . radical signal was also efficiently trapped. The g=2 broad signal is attributed to an intermediate S1X. state in equilibrium with the trapped YZ . radical. Comparison with x-ray structural data suggests that one of the split signals (doublet signal) is attributable to interaction between His 190 and the YZ . radical, and other signals is attributable to interaction between His 337 and the manganese cluster, providing further clues as to the mechanism of water oxidation in photosynthetic oxygen evolution.

Influence of the 33 kDa manganese-stabilizing protein on the structure and substrate accessibility of the oxygen-evolving complex of photosystem II.

The 33 kDa manganese-stabilizing extrinsic protein binds to the lumenal side of photosystem II (PS II) close to the Mn(4)Ca cluster of the oxygen-evolving complex, where it limits access of small molecules to the metal site. Our previous finding that the removal of this protein did not alter the magnetic coupling regime within the manganese cluster, measured by electron spin-echo envelope modulation [Gregor, W., and Britt, R. D. (2000) Photosynth. Res. 65, 175-185], prompted us to examine whether this accessibility control is also true for substrate water, using the same pulsed EPR technique. Comparing the deuteron modulation of the S(2)-state multiline signal of PS II membranes, equilibrated with deuterated water (D(2)O) after removal or retention of the 33 kDa protein, we observed no change in the number and the distance of deuterons magnetically coupled to manganese, indicating that the number and distance of water molecules bound to the manganese cluster are independent of bound 33 kDa protein in the S(1) state, in which the sample was poised prior to cryogenic illumination. A simple modulation depth analysis revealed a distance of 2.5-2.6 A between the closest deuteron and manganese. These results are in agreement with our refined X-ray absorption analysis. The manganese K-edge positions, reflecting their oxidation states, and the extended X-ray absorption fine structure amplitudes and distances between the manganese ions and their oxygen and nitrogen ligands (1.8, 2.7, and 3.3-3.4 A) were independent of bound 33 kDa protein.

The two forms of the S 2 state multiline signal in Photosystem II: effect of methanol and ethanol

The characteristic Mn hyperfine ‘multiline’ signal exhibited in the S 2 state of the oxygen-evolving complex (OEC) complex of Photosystem II (PSII) has been shown to be heterogeneous in character. In this study, we have explored the effects that influence the proportions of the two forms of the S 2 state multiline signal present in any sample. The narrow form of the signal is lost upon storage (weeks) at 77 K, whereas the broad form remains. In particular, we explore the roles of ethanol and methanol as well as effects of the second turnover of the enzyme on storage of the sample at 77 K. We find that in samples containing methanol, the narrow form may predominate upon the first flash, but the broad form predominates on the fifth flash and also in samples containing ethanol.

Electron transfer from cytochrome b559 and tyrosineD to the S2 and S3 states of the water oxidizing complex in photosystem II

We have investigated the electron transfer from reduced tyrosine YD (YDred) and cytochrome b559 to the S2 and S3 states of the water oxidizing complex (WOC) in Photosystem II. The EPR signal of oxidized cyt b559, the S2 state multiline EPR signal and the EPR signal from YD@? were measured to follow the electron transfer to the S2 and S3 states at 245 and 275 K. The majority of the S2 centers was reduced directly from YDred but at 245 K we observed oxidation of cyt b559 in about 20% of the centers. Incubation of the YDredS3 state resulted in biphasic changes of the S2 multiline signal. The signal first increased due to reduction of the S3 state. Thereafter, the signal decreased due to decay of the S2 state. In contrast, the YD@? signal increased with a monophasic kinetics at both temperatures. Again, we observed oxidation of cyt b559 in about 20% of the PSII centers at 245 K. This oxidation correlated with the decay of the S2 state. The complex changes can be explained by the conversion of YDredS3 centers (present initially) to YD@?S1 centers, via the intermediate YD@?S2 state. The early increase of the S2 state multiline signal involves electron transfer from YDred to the S3 state resulting in formation of YD@?S2. This state is reduced by cyt b559 resulting in a single exponential oxidation of cyt b559. Taken together, these results indicate that the electron donor to S2 is cyt b559 while cyt b559 is unable to compete with YDred in the reduction of the S3 state in the pre-reduced samples. We also followed the decay of the S2 and S3 states and the oxidation of cyt b559 in samples where YD was oxidized from the start. In this case cyt b559 was able to reduce both the S2 and the S3 states suggesting that different pathways exist for the electron transfer from cyt b559 to the WOC. The activation energies for the YDredS2->YD@?S1 and YDredS3->YD@?S2 transformations are 0.57 and 0.67 eV, respectively, and the reason for these large activation energies is discussed. (Less)

Near-IR irradiation of the S2 state of the water oxidizing complex of photosystem II at liquid helium temperatures produces the metalloradical intermediate attributed to S1Y(Z*).

Near-IR (NIR) excitation at liquid He temperatures of photosystem II (PSII) membranes from the cyanobacterium Synechococcus vulcanus or from spinach poised in the S2 state results in the production of a g = 2.035 EPR resonance, reminiscent of metalloradical signals. The signal is smaller in the spinach preparations, but it is significantly enhanced by the addition of exogenous quinones. Ethanol (2-3%, v/v) eliminates the ability to trap the signal. The g = 2.035 signal is identical to the one recently obtained by Nugent et al. by visible-light illumination of the S1 state, and preferably assigned to S1Y(Z*) [Nugent, J. H. A., Muhiuddin, I. P., and Evans, M. C. W. (2002) Biochemistry 41, 4117-4126]. The production of the g = 2.035 signal by liquid He temperature NIR excitation of the S2 state is paralleled by a significant reduction (typically 40-45% in S. vulcanus) of the S2 state multiline signal. This is in part due to the conversion of the Mn cluster to higher spin states, an effect documented by Boussac et al. [Boussac, A., Un, S., Horner, O., and Rutherford, A. W. (1998) Biochemistry 37, 4001-4007], and in part due to the conversion to the g = 2.035 configuration. Following the decay of the g = 2.035 signal at liquid helium temperatures (decay halftimes in the time range of a few to tens of minutes depending on the preparation), annealing at elevated temperatures (-80 degrees C) results in only partial restoration of the S2 state multiline signal. The full size of the signal can be restored by visible-light illumination at -80 degrees C, implying that during the near-IR excitation and subsequent storage at liquid helium temperatures recombination with Q(A-) (and therefore decay of the S2 state to the S1 state) occurred in a fraction of centers. In support of this conclusion, the g = 2.035 signal remains stable for several hours (at 11 K) in centers poised in the S2...Q(A) configuration before the NIR excitation. The extended stability of the signal under these conditions has allowed the measurement of the microwave power saturation and the temperature dependence in the temperature range of 3.8-11 K. The signal intensity follows Curie law temperature dependence, which suggests that it arises from a ground spin state, or a very low-lying excited spin state. The P1/2 (microwave power at half-saturation) value is 1.7 mW at 3.8 K and increases to 96 mW at 11 K. The large width of the g = 2.035 signal and its relatively fast relaxation support the assignment to a radical species in the proximity of the Mn cluster. The whole phenomenology of the g = 2.035 signal production is analogous to the effects of NIR excitation on the S3 state [Ioannidis, N., Nugent, J. H. A., and Petrouleas, V. (2002) Biochemistry 41, 9589-9600] producing an S2'Y(Z*) intermediate. In the present case, the intermediate is assigned to S1Y(Z*). The NIR-induced increase in the oxidative capability of the Mn cluster is discussed in relation to the photochemical properties of a Mn(III) ion that exists in both S2 and S3 states. The EPR properties of the S1Y(Z*) intermediate cannot be reconciled easily with our current understanding of the magnetic properties of the S1 state. It is suggested that oxidation of tyr Z alters the magnetic properties of the Mn cluster via exchange of a proton.

The function of the chloride ion in photosynthetic oxygen evolution.

The involvement of Cl(-) and several other monovalent anions in photosynthetic oxygen evolution was studied using photosystem II membranes depleted of Cl(-) by dialysis. The results of these studies differ significantly from results obtained using other depletion methods. Binding studies with glycerol as a cryoprotectant confirm our previous observations with sucrose of two interconvertible binding states of photosystem II with similar activities and with slow or fast exchange, respectively, of the bound ion. With glycerol, Cl(-) depletion decreased the oxygen evolution rate to 55% of that with Cl(-) present without decreasing the quantum efficiency of the reaction, supporting our previous conclusion that oxygen evolution can proceed at high rates in the absence of Cl(-). Further, after Cl(-) depletion the S(2) state multiline signal displayed the same periodic appearance with the same signal yield after consecutive laser flashes as with Cl(-) present. Br(-), I(-), and NO(3)(-), although with different capacities to reactivate oxygen evolution, also showed two binding modes. I(-) inhibited when bound in the low-affinity, fast-exchange mode but activated in the high-affinity mode. A comparison of the EPR properties of the S(2) state with these anions suggests that the nature of the ion or the binding mode only has a minor influence on the environment of the manganese. In contrast, F(-) completely inhibited oxygen evolution by preventing the S(2) to S(3) transition and shifted the equilibrium between the g = 4.1 and multiline S(2) forms toward the former, which suggests a considerable perturbation of the manganese cluster. To explain these and earlier observations, we propose that the role of chloride in the water-splitting mechanism is to participate together with charged amino acid side chains in a proton-relay network, which facilitates proton transfer from the manganese cluster to the medium. The structural requirements likely to be involved may explain the sensitivity of oxygen evolution to Cl(-) depletion or other perturbations.

Influence of protein phosphorylation on the electron-transport properties of Photosystem II.

Many of the core proteins in Photosystem II (PS II) undergo reversible phosphorylation. It is known that protein phosphorylation controls the repair cycle of Photosystem II. However, it is not known how protein phosphorylation affects the partial electron transport reactions in PS II. Here we have applied variable fluorescence measurements and EPR spectroscopy to probe the status of the quinone acceptors, the Mn cluster and other electron transfer components in PS II with controlled levels of protein phosphorylation. Protein phosphorylation was induced in vivo by varying illumination regimes. The phosphorylation level of the D1 protein varied from 10 to 58% in PS II membranes isolated from pre-illuminated spinach leaves. The oxygen evolution and Q(A) (-) to Q(B)(Q(B) (-)) electron transfer measured by flash-induced fluorescence decay remained similar in all samples studied. Similar measurements in the presence of DCMU, which reports on the status of the donor side in PS II, also indicated that the integrity of the oxygen-evolving complex was preserved in PS II with different levels of D1 protein phosphorylation. With EPR spectroscopy we examined individual redox cofactors in PS II. Both the maximal amplitude of the charge separation reaction (measured as photo-accumulated pheophytin(-)) and the EPR signal from the Q(A) (-) Fe(2+) complex were unaffected by the phosphorylation of the D1 protein, indicating that the acceptor side of PS II was not modified. Also the shape of the S(2) state multiline signal was similar, suggesting that the structure of the Mn-cluster in Photosystem II did not change. However, the amplitude of the S(2) multiline signal was reduced by 35% in PS II, where 58% of the D1 protein was phosphorylated, as compared to the S(2) multiline in PS II, where only 10% of the D1 protein was phosphorylated. In addition, the fraction of low potential Cyt b (559) was twice as high in phosphorylated PS II. Implications from these findings, were precise quantification of D1 protein phosphorylation is, for the first time, combined with high-resolution biophysical measurements, are discussed.

Comparative studies of the S 0 and S 2 multiline electron paramagnetic resonance signals from the manganese cluster in Photosystem II

Electron paramagnetic resonance (EPR) spectroscopy is one of the major techniques used to analyse the structure and function of the water oxidising complex (WOC) in Photosystem II. The discovery of an EPR signal from the S 0 state has opened the way for new experiments, aiming to characterise the S 0 state and elucidate the differences between the different S states. We present a review of the biochemical and biophysical characterisation of the S 0 state multiline signal that has evolved since its discovery, and compare these results to previous and recent data from the S 2 multiline signal. We also present some new data from the S 2 state reached on the second turnover of the enzyme.

Formate-induced inhibition of the water-oxidizing complex of photosystem II studied by EPR.

The effects of various formate concentrations on both the donor and the acceptor sides in oxygen-evolving PS II membranes (BBY particles) were examined. EPR, oxygen evolution and variable chlorophyll fluorescence have been observed. It was found that formate inhibits the formation of the S(2) state multiline signal concomitant with stimulation of the Q(A)(-)Fe(2+) signal at g = 1.82. The decrease and the increase in intensities of the multiline and Q(A)(-)Fe(2+) signals, respectively, had a linear relation for formate concentrations between 5 and 500 mM. The g = 4.1 signal formation measured in the absence of methanol was not inhibited by formate up to 250 mM in the buffer. In the presence of 3% methanol the g = 4.1 signal evolved as formate concentration increased. The evolved signal could be ascribed to the inhibited centers. Oxygen evolution measured in the presence of an electron acceptor, phenyl-p-benzoquinone, was also inhibited by formate proportionally to the decrease in the multiline signal intensity. The inhibition seemed to be due to a retarded electron transfer from the water-oxidizing complex to Y(Z)(+), which was observed in the decay kinetics of the Y(Z)(+) signal induced by illumination above 250 K. These results show that formate induces inhibition of water oxidation reactions as well as electron transfer on the PS II acceptor side. The inhibition effects of formate in PS II were found to be reversible, indicating no destructive effect on the reaction center induced by formate.

An oscillating manganese electron paramagnetic resonance signal from the S0 state of the oxygen evolving complex in photosystem II.

Photosynthesis produces the oxygen necessary for all aerobic life. During this process, the manganese-containing oxygen evolving complex (OEC) in photosystem II (PSII), cycles through five oxidation states, S0-S4. One of these, S2, is known to be paramagnetic and gives rise to electron paramagnetic resonance (EPR) signals used to probe the catalytic structure and function of the OEC. The S0 states has long been thought to be paramagnetic. We report here a Mn EPR signal from the previously EPR invisible S0 state. The new signal oscillates with a period of four, indicating that it originates from fully active PSII centers. Although similar to the S2 state multiline signal, the new signal is wider (2200 gauss compared with 1850 gauss in samples produced by flashing), with different peak intensity and separation (82 gauss compared with 89 gauss). These characteristics are consistent with the S0 state EPR signal arising from a coupled MnII-MnIII intermediate. The new signal is more stable than the S2 state signal and its decay in tens of minutes is indicative of it originating from the S0 state. The S0 state signal will provide invaluable information toward the understanding of oxygen evolution in plants.

EPR and ENDOR studies of the water oxidizing complex of Photosystem II

A comparative study of X-band EPR and ENDOR of the S2 state of photosystem II membrane fragments and core complexes in the frozen state is presented. The S2 state was generated either by continuous illumination at T=200 K or by a single turn-over light flash at T=273 K yielding entirely the same S2 state EPR signals at 10 K. In membrane fragments and core complex preparations both the multiline and the g=4.1 signals were detected with comparable relative intensity. The absence of the 17 and 23 kDa proteins in the core complex preparation has no effect on the appearance of the EPR signals. (1)H-ENDOR experiments performed at two different field positions of the S2 state multiline signal of core complexes permitted the resolution of four hyperfine (hf) splittings. The hf coupling constants obtained are 4.0, 2.3, 1.1 and 0.6 MHz, in good agreement with results that were previously reported (Tang et al. (1993) J Am Chem Soc 115: 2382-2389). The intensities of all four line pairs belonging to these hf couplings are diminished in D2O. A novel model is presented and on the basis of the two largest hfc's distances between the manganese ions and the exchangeable protons are deduced. The interpretation of the ENDOR data indicates that these hf couplings might arise from water which is directly ligated to the manganese of the water oxidizing complex in redox state S2.

Temperature-dependent pulsed electron paramagnetic resonance studies of the S2 state multiline signal of the photosynthetic oxygen-evolving complex.

The electron spin-lattice relaxation rate (1/T1) of the g = 2 "multiline" manganese electron paramagnetic resonance (EPR) signal arising from the photosystem II oxygen-evolving complex poised in the S2 state has been directly measured over the temperature range of 4.2-11 K via the inversion-recovery pulsed EPR technique. The electron spin echo amplitude of the g = 2 "multiline" signal varies inversely with temperature over this range, indicating a ground spin state Curie law behavior in agreement with our previously reported work [Britt et al. (1992) Biochim. Biophys. Acta 1140, 95-101]. Results of a plot of the natural log of the electron spin-lattice relaxation rate versus reciprocal temperature are consistent with an Orbach mechanism serving as the dominant relaxation pathway for the "multiline" signal in this temperature range. The slope of the plot indicates that an excited spin state manifold exists 36.5 cm-1 above the ground-state manifold that gives rise to the "multiline" signal.

Protein and substrate coordination to the manganese cluster in the photosynthetic water oxidizing complex: nitrogen 15 and proton ENDOR spectroscopy of the S2 state multiline signal in the thermophilic cyanobacterium Synechococcus elongatus

ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTProtein and substrate coordination to the manganese cluster in the photosynthetic water oxidizing complex: nitrogen 15 and proton ENDOR spectroscopy of the S2 state multiline signal in the thermophilic cyanobacterium Synechococcus elongatusX. S. Tang, M. Sivaraja, and G. C. DismukesCite this: J. Am. Chem. Soc. 1993, 115, 6, 2382–2389Publication Date (Print):March 1, 1993Publication History Published online1 May 2002Published inissue 1 March 1993https://doi.org/10.1021/ja00059a037RIGHTS & PERMISSIONSArticle Views139Altmetric-Citations47LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InReddit PDF (2 MB) Get e-AlertsSupporting Info (2)»Supporting Information Supporting Information Get e-Alerts

Isolation and characterization of oxygen-evolving Photosystem II membranes from the cyanobacterium Synechocystis 6803

A simple and rapid procedure has been designed for isolation of a highly active Photosystem II (PS II) subfraction from the cyanobacterium Synechocystis 6803. The thylakoid membranes used as starting material were isolated from cells treated with red light to increase the relative PS II content. The cell walls were broken in a glass-bead cell disruptor. The isolated thylakoids were pretreated with 0.01% dodecyl β-maltoside, followed by solubilization in 2.5% Triton X-100 to selectively extract photosystem I. The membrane residue, still containing PS II, was collected by a rapid differential centrifugation. The isolation PS II membranes possess rates of oxygen evolution as high as 2800–3200 μmol O 2/mg Chl h, and a low proportion of contaminating photosystem I. The yield of PS II is 30–35% on a PS II activity basis. Typically, PS II membranes corresponding to 2 mg of chlorophyll could be obtained within 6 h of preparative work, including the cell breakage. The purity and high intactness of the material allowed EPR analysis of various PS II parameters, including signal II slow and the S 2 state multiline signal. Considering the simplicity of the subfractionation procedure, the high yield and functional intactness, this preparation should have the potential of playing the same important experimental role for studying PS II in Synechocystis 6803 as the so called BBY-preparation has played for studies on PS II from plants. In particular, the preparation should be essential for studies on structure-function relations after site-directed mutagenesis of various PS II subunits in Synechocystis 6803 cells.