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A guide to electron paramagnetic resonance spectroscopy of Photosystem II membranes
Abstract
This guide is intended to aid in the detection and identification of paramagnetic species in Photosystem II membranes, by electron paramagnetic resonance spectroscopy. The spectral features and occurrence of each of the electron paramagnetic resonance signals from Photosystem II are discussed, in relation to the nature of the moiety giving rise to the signal and the role of that species in photosynthetic electron transport. Examples of most of the signals discussed are shown. The electron paramagnetic resonance signals produced by the cytochrome b6f and Photosystem I complexes, as well as the signals from other common contaminants, are also reviewed. Furthermore, references to seminal experiments on bacterial reaction centers are included. By reviewing both the spectroscopic and biochemical bases for the electron paramagnetic resonance signals of the cofactors that mediate photosynthetic electron transport, this paper provides an introduction to the use and interpretation of electron paramagnetic resonance spectroscopy in the study of Photosystem II.
... Variations in the content and character of manganese ions, occurring upon oxidative stress, were also studied, based on EPR spectra [5]. Characteristic hyperfine structure of manganese signals visible in the spectra of inorganic compounds of Mn (II) in oxide environments, for example in [Mn(H 2 O) 6 ] 2+ aqua-complexes allowed for differentiating these species from organic structures containing interacting manganese ions [6,7]. Moreover, signal intensities of these manganese aqua-complexes were different in the spectra of each genotype, which allowed us to distinguish them from one another [5]. ...
... Variations in the content and character of manganese ions, occurring upon oxidative stress, were also studied, based on EPR spectra [5]. Characteristic hyperfine structure of manganese signals visible in the spectra of inorganic compounds of Mn (II) in oxide environments, for example in [Mn(H 2 O) 6 ] 2+ aqua-complexes allowed for differentiating these species from organic structures containing interacting manganese ions [6,7]. Moreover, signal intensities of these manganese aqua-complexes were different in the spectra of each genotype, which allowed us to distinguish them from one another [5]. ...
... The signal of six hyperfine lines overlapping a broad one was ascribed to Mn species. The well resolved hyperfine structure observed at room temperature originated from freely rotating aqua -complex of Mn (II) and is often found in EPR spectra of various plants [6,10], while the broad line was ascribed to dipole-dipole interacting Mn (II) ions situated mainly in protein matrix [17]. Signals observed in g range between 2.2 and 2.5 were attributed to inorganic antiferromagnetically coupled paramagnetic Fe (III) ions forming Fe-O-Fe clusters, ferric oxides, oxyhydroxides and/or phosphates that were accumulated in the "iron-core" of ferritin [18,19], whereas broad signals at g = 2.4 and 2.6, appearing at 77 K, were ascribed to Fe (III) ions bonded to protein matrix in the ferritin protein shell, containing ferric and ferrous ions [20,21]. ...
Background
Mycotoxins are among the environmental stressors whose oxidative action is currently widely studied. The aim of this paper was to investigate the response of seedling leaves to zearalenone (ZEA) applied to the leaves (directly) and to the grains (indirectly) in tolerant and sensitive wheat cultivars.
Results
Biochemical analyses of antioxidant activity were performed for chloroplasts and showed a similar decrease in this activity irrespective of plant sensitivity and the way of ZEA application. On the other hand, higher amounts of superoxide radical (microscopic observations) were generated in the leaves of plants grown from the grains incubated in ZEA solution and in the sensitive cultivar. Electron paramagnetic resonance (EPR) studies showed that upon ZEA treatment greater numbers of Mn - aqua complexes were formed in the leaves of the tolerant wheat cultivar than in those of the sensitive one, whereas the degradation of Fe-protein complexes occurred independently of the cultivar sensitivity.
Conclusion
The changes in the quantity of stable, organic radicals formed by stabilizing reactive oxygen species on biochemical macromolecules, indicated greater potential for their generation in leaf tissues subjected to foliar ZEA treatment. This suggested an important role of these radical species in protective mechanisms mainly against direct toxin action. The way the defense mechanisms were activated depended on the method of the toxin application.
... Variations in the content and character of manganese ions, occurring upon oxidative stress, were also studied, based on EPR spectra [5]. Characteristic hyper ne structure of manganese signals visible in the spectra of inorganic compounds of Mn(II) in oxide environments, for example in the [Mn(H 2 O) 6 ] 2+ aqua-complexes allowed differentiating these species from organic structures containing interacting manganese ions [6,7]. Moreover, the signal intensities of these manganese aqua-complexes were different in the spectra of both genotypes which allowed them to be distinguished [5]. ...
... The signal of six hyper ne lines overlapping a broad one was ascribed to Mn species. Well resolved hyper ne structure observed at room temperature originated from freely rotating aqua -complex of Mn(II) and was often found in EPR spectra of various plants [6,10], while the broad line was ascribed to dipole -dipole interacting Mn(II) ions situated mainly in protein matrix [17]. Signals observed in g range between 2.2-2.5 were attributed to inorganic antiferromagnetically coupled paramagnetic Fe(III) ions forming Fe-O-Fe clusters, ferric oxides, oxyhydroxides and/or phosphates which were accumulated in the "iron-core" of ferritin [18,19], whereas broad signals at g = 2.4 and 2.6, appearing at 77 K, were ascribed to Fe(III) ions bonded to protein matrix in the ferritin protein shell, containing ferric and ferrous ions [20,21]. ...
... Signals observed in g range between 2.2-2.5 were attributed to inorganic antiferromagnetically coupled paramagnetic Fe(III) ions forming Fe-O-Fe clusters, ferric oxides, oxyhydroxides and/or phosphates which were accumulated in the "iron-core" of ferritin [18,19], whereas broad signals at g = 2.4 and 2.6, appearing at 77 K, were ascribed to Fe(III) ions bonded to protein matrix in the ferritin protein shell, containing ferric and ferrous ions [20,21]. The small line with g = 4.26, observed in all spectra recorded at 77 K, was attributed to non-hem high spin Fe(III) with rhombic symetry [6]. ...
BACKGROUND
Among the environmental stressors, which oxidative action is currently widely studied, are mycotoxins. The aim of presented studies was to investigate the response of leaves of seedlings to zearalenone (ZEN) applied directly to leaves and to grains (indirectly) of tolerant and sensitive wheat cultivars.
RESULTS
Biochemical analyses of antioxidative activity were performed for chloroplasts and showed the similar decrease of this activity independently of plant sensitivity and the way of ZEN application. On the other hand, higher amounts of superoxide radicals (microscopic observation) were formed in leaves of plants grown from grains incubated in solution of ZEN and in sensitive variety. Electron paramagnetic resonance (EPR) studies showed that upon ZEN treatment larger amount of Mn - aqua complexes was formed in leaves of tolerant wheat cultivar than in those of sensitive one whereas the degradation of Fe-protein complexes occurred independently of the sensitivity of plant varieties.
CONCLUSION
The changes in the quantity of organic stable radicals, which were formed by stabilizing reactive oxygen species on biochemical macromolecules, pointed to the higher ability to their generation in leaf tissues subjected to foliar ZEN treatment indicating the important role of these radical species in protective mechanism mainly against direct toxin action. The way of activation the defense mechanisms depended on the method of applications toxin.
... The EPR signals associated with PS II paramagnetic components have been reviewed recently by Miller and Brudvig (1991). ...
... Cytochrome 6 5 5 9 has several EPR signals depending on the conditions of sample preparation and sample illumination conditions (reviewed in Miller and Brudvig, 1991). Various PS II preparations were analyzed for their cyt bss9 EPR signals. ...
... It is difficult to separate the effects of the pH and the slightly different polypeptide composition of the preparations. The effect of the removal of PS II polypeptides on the cyt 6 5 5 g EPR signal has been reviewed (Miller and Brudvig, 1991). ...
A three dimensional model of the Photosystem II (PS II) reaction centre has been generated by computer modelling based on the x-ray crystallographic derived structures of the reaction centres from two purple non-sulphur bacteria, following sequence alignment and secondary structure analysis. Analysis of the evolutionary relationship between the different Q-type reaction centre proteins has suggested that the ancestral complex was a homodimer. The model of the basic protein structure of PS II has allowed modelling experiments to be carried out to predict binding of bicarbonate, the water oxidizing complex and the herbicide DCMU. Several models of bicarbonate binding have been proposed to agree with the current biochemical and biophysical observations. Two orientations of DCMU in the QB binding site have been identified and a model has been proposed that links the two orientations with the DCMU effect on the QA-Fe2+ EPR signal. Computer generated structures have been modelled for the predicted transmembrane spans of the gene products psbE, F and I. PsbE and F gene products compose the cytochrome b559 complex, an integral part of PS II. Analysis of the sequence and secondary structure allows the construction of a variety of multimers some of which are presented. A method for isolating cytochrome b559 is presented and the effect of purification on the associated EPR signals is discussed. The effect of photoinhibition on PS II activity under aerobic, anaerobic and anoxic conditions has been studied in PS II samples prepared with the detergent heptyl thioglucopyranoside (HTG). The absence of oxygen predisposes the preparations to a loss of PS II activity under high light conditions. HTG preparations without bound bicarbonate are shown to be more sensitive to photodamage.
... The main signal of all spectra was composed of a broad line with overlapping well-resolved hyperfine structure, which was attributed to the manganese species. The hyperfine structure originated from the freely rotating aqua-complex of Mn(II), and was often detected in the EPR spectra of plant materials [19,31,32]. The broad line was attributed to dipole-dipole interacting manganese ions situated in the protein matrix. ...
... As this effect was more noticeable in the sensitive genotype, this pointed to a stronger susceptibility of the latter genotype to damage of the protein-complex structure after ZEA treatment ( Figure 3C). Another signal of an iron species was observed in all spectra at 77 K with g = 4.30, and was attributed to the non-haem, high spin Fe(III) with rhombic symmetry [20,32]. In some spectra, a line at g = 2.05 could also be seen. ...
... In some spectra, a line at g = 2.05 could also be seen. Tentatively, this signal might be described as a perpendicular g component of the signal of copper ions in the square planar complexes of proteins [32,36]. ...
These studies concentrate on the possibility of using seleniumions and/or 24-epibrassinolide at non-toxic levels as protectors of wheat plants against zearalenone, which is a common and widespread mycotoxin. Analysis using the UHPLC-MS technique allowed for identification of grains having the stress-tolerant and stress-sensitive wheat genotype. When germinating in the presence of 30 μM of zearalenone, this mycotoxin can accumulate in both grains and hypocotyls germinating from these grains. Selenium ions (10 μM) and 24-epibrassinolide (0.1 μM) introduced together with zearalenone decreased the uptake of zearalenone from about 295 to 200 ng/g and from about 350 to 300 ng/g in the grains of tolerant and sensitive genotypes, respectively. As a consequence, this also resulted in a reduction in the uptake of zearalenone from about 100 to 80 ng/g and from about 155 to 128 ng/g in the hypocotyls from the germinated grains of tolerant and sensitive wheat, respectively. In the mechanism of protection against the zearalenone-induced oxidative stress, the antioxidative enzymes—mainly superoxide dismutase (SOD) and catalase (CAT)—were engaged, especially in the sensitive genotype. Electron paramagnetic resonance (EPR) studies allowed for a description of the chemical character of the long-lived organic radicals formed in biomolecular structures which are able to stabilize electrons released from reactive oxygen species as well as the changes in the status of transition paramagnetic metal ions. The presence of zearalenone drastically decreased the amount of paramagnetic metal ions—mainly Mn(II) and Fe(III)—bonded in the organic matrix. This effect was particularly found in the sensitive genotype, in which these species were found at a smaller level. The protective effect of selenium ions and 24-epibrassinolide originated from their ability to inhibit the destruction of biomolecules by reactive oxygen species. An increased ability to defend biomolecules against zearalenone action was observed for 24-epibrassinolide.
... Reduced cyt b 559 is a potential electron donor to the higher S-states or Y ox D , and we therefore followed the redox-state of cyt b 559 during incubation of the one-and two-flash samples. As judged from the line shape the EPR signal [41,51,58], the remaining oxidized cyt b 559 in our samples arises from the LP-form of cyt b 559 while the HP-form of cyt b 559 is completely reduced (not shown). Fig. 6A shows the EPR spectra from oxidized cyt b 559 , recorded in two-flash samples incubated at 245 K for different times. ...
... Fig. 6A shows the EPR spectra from oxidized cyt b 559 , recorded in two-flash samples incubated at 245 K for different times. The signal recorded immediately after the two flashes (Fig. 6A, spectrum 1) is dominated by the LP form of cyt b 559 as is evident from the g-value of the signal (right bar in Fig. 6A) [58]. The signal intensity increased slowly during prolonged incubation at 245 K (Fig. 6, spectra 2 and 3), implying oxidation of cyt b 559 . ...
... Due to our chemical reduction protocol a large fraction of cyt b 559 is present in the LP-form which remains oxidized (Fig. 6A, spectrum 1). This represented 65-70% of the total signal obtained after illumination at 77 K (Fig. 6A, spectrum ''ill'') which is known to oxidize HP-cyt b 559 [41,48,58]. This was confirmed by optical measurements (not shown) which demonstrated that 33% of cyt b 559 was oxidized by ferricyanide in our samples. ...
We have investigated the electron transfer from reduced tyrosine Y D (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 S 2 state multiline EPR signal and the EPR signal from Y D· 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 Y Dred 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 b 559. 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 S 2 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 Y DredS2→YD·S1 and YDredS 3→YD·S2 transformations are 0.57 and 0.67 eV, respectively, and the reason for these large activation energies is discussed.
... The EPR spectrum of dark-adapted D1-D170E PSII core complexes measured with continuous wave X-band (9.40 GHz) at 5.0 K shows a signal at g = 4.3 and multiple signals between g = 5.9 and 6.7 (Fig. 2, blue trace). These signals correspond to free and protein-bound ferric ions normally observed in PS II (Miller and Brudvig 1991;Haddy et al. 2004). The signal at g = 4.3 corresponds to rhombic iron whereas the features between g = 5.9 and g = 6.7 arise in part from the oxidation of the non-heme iron in the Q A Fe complex from Fe 2+ to Fe 3+ induced by the presence of DCMU in a fraction of PSII reaction centers (Boussac et al. 2011). ...
... The trough at approximately 100 mT are caused by the reduction of nonheme Fe 3+ in Q A Fe complexes in which the iron atom had been oxidized to Fe 3+ through the action of DCMU (Boussac 2019). The troughs at approximately 150 mT correspond to an alteration of the background signal of rhombic iron caused by minute changes in temperature (Miller and Brudvig 1991). ...
The residue D1-D170 bridges Mn4 with the Ca ion in the O2-evolving Mn4CaO5 cluster of Photosystem II. Recently, the D1-D170E mutation was shown to substantially alter the Sn+1-minus-Sn FTIR difference spectra [Debus RJ (2021) Biochemistry 60:3841-3855]. The mutation was proposed to alter the equilibrium between different Jahn–Teller conformers of the S1 state such that (i) a different S1 state conformer is stabilized in D1-D170E than in wild-type and (ii) the S1 to S2 transition in D1-D170E produces a high-spin form of the S2 state rather than the low-spin form that is produced in wild-type. In this study, we employed EPR spectroscopy to test if a high-spin form of the S2 state is formed preferentially in D1-D170E PSII. Our data show that illumination of dark-adapted D1-D170E PSII core complexes does indeed produce a high-spin form of the S2 state rather than the low-spin multiline form that is produced in wild-type. This observation provides further experimental support for a change in the equilibrium between S state conformers in both the S1 and S2 states in a site-directed mutant that retains substantial O2 evolving activity.
... 10 K 下 PSⅡ中电子转移中间体的 原 位 可 见 光 诱 导 实 验 依 照 文 献 [25,26] 进 行 . 具 体 Mn 2+ 的 6 重峰信号[27] . 如果给体侧 Mn 簇发生破坏, 就难以产生 S 2 态 Mn 簇的特征信号, 并会导致 Mn 2+ 的形成[27] . ...
... 具 体 Mn 2+ 的 6 重峰信号[27] . 如果给体侧 Mn 簇发生破坏, 就难以产生 S 2 态 Mn 簇的特征信号, 并会导致 Mn 2+ 的形成[27] . 在我们的样品中可以看到清晰的 S 2 态 Mn 簇的特征 EPR 信号, 但未观察到任何 Mn 2+ 的 6 重峰 信号(图 1), 这充分说明样品中 Mn 簇保持完好. ...
... Chlorophyll triplet states, in addition to being highly reactive, serve as chemical probes to investigate primary electron transfer pathways and characterize the chemical environment of photosynthetic reaction center pigments. 44 Electron paramagnetic resonance (EPR) and electron-nuclear double resonance (ENDOR) spectroscopies 29,37,39,44,[56][57][58][59][60][61][62][63][64][65][66][67][68][69] and other spectroscopic approaches including Fourier-transform infrared (FTIR) and optically detected magnetic resonance (ODMR) 36,38,39,51,54,[70][71][72][73][74][75][76] suggest that the "primary donor" triplet is located on an individual accessory chlorophyll (Chl D1 or Chl D2 ) at cryogenic temperatures. 30,[56][57][58]61,77 It has also been suggested that the triplet is partially shared with other chlorophylls at the RC at higher temperatures, but this has not been well characterized. ...
In oxygenic photosynthesis sunlight is harvested and funneled as excitation energy into the reaction center (RC) of Photosystem II (PSII), the site of primary charge separation that initiates the photosynthetic electron transfer chain. The chlorophyll ChlD1 pigment of the RC is the primary electron donor, forming a charge-separated radical pair with the vicinal pheophytin PheoD1 (ChlD1⁺PheoD1⁻). To avert charge recombination, the electron is further transferred to plastoquinone QA, whereas the hole relaxes to a central pair of chlorophylls (PD1PD2), subsequently driving water oxidation. Spin-triplet states can form within the RC when forward electron transfer is inhibited or back reactions are favored. This can lead to formation of singlet dioxygen, with potential deleterious effects. Here we investigate the nature and properties of triplet states within the PSII RC using a multiscale quantum-mechanics/molecular-mechanics (QM/MM) approach. The low-energy spectrum of excited singlet and triplet states, of both local and charge-transfer nature, is compared using range-separated time-dependent density functional theory (TD-DFT). We further compute electron paramagnetic resonance properties (zero-field splitting parameters and hyperfine coupling constants) of relaxed triplet states and compare them with available experimental data. Moreover, the electrostatic modulation of excited state energetics and redox properties of RC pigments by the semiquinone QA⁻ is described. The results provide a detailed electronic-level understanding of triplet states within the PSII RC and form a refined basis for discussing primary and secondary electron transfer, charge recombination pathways, and possible photoprotection mechanisms in PSII.
... These discoveries joined many others in expanding research on PS2 in a number of areas. Electron paramagnetic resonance spectroscopy (EPR) was used to characterize a number of PS2 signals (Miller and Brudvig 1991). Optical absorbance changes from Mn oxidation state advancements were reported (Dekker et al.1984), and X-ray absorption spectroscopy became an important technique for probing Mn oxidation states and Mn-Mn, Mn-Ca 2+ and Mn-ligand distances (Sauer et al. 2005). ...
These special issues of photosynthesis research present papers documenting progress in revealing the many aspects of photosystem 2, a unique, one-of-a-kind complex system that can reduce a plastoquinone to a plastoquinol on every second flash of light and oxidize 2 H2O to an O2 on every fourth flash. This overview is a brief personal assessment of the progress observed by the author over a four-decade research career, including a discussion of some remaining unsolved issues. It will come as no surprise to readers that there are remaining questions given the complexity of PS2, and the efforts that have been needed so far to uncover its secrets. In fact, most readers will have their own lists of outstanding questions.
... The source of this copper is unlikely to be from plastocyanin, as digitonin treatment removes any bound plastocyanin (Hanley et al., 1992). Copper signals have been reported which arise from the light-harvesting complex of PSn (Miller and Brudvig, 1991). A light-harvesting protein present in the digitonin preparation may be the source of this contamination. ...
This thesis reports structural, biochemical and biophysical analyses of photosystem I (PSI), using site-directed mutants and second-site revertants of Chlamydomonas reinhardtii. The principal techniques used were non-denaturing gel electrophoresis, biochemical assays of electron transport, continuous wave and pulsed electron paramagnetic resonance spectroscopy (EPR) and electron-nuclear double resonance spectroscopy (ENDOR). Site-directed mutants of the conserved region of PsaA which is thought to form the FX binding site have been previously generated (Hallahan et al., 1995): C575D, C575H, C575S and D576L, all of which are non-photosynthetic. Photosynthetic second-site revertants have been generated from D576L (Evans ct al., 1999). The secondary mutations are in nuclear genes. Non-denaturing polyacrylamide gel electrophoresis of thylakoid membranes indicated that PSI did not assemble in C575D. C575H, C575S and D576L assembled PSI at reduced levels. Continuous wave EPR showed no photoreduction of iron-sulphur centres in C575H. Spectra of FA/FB in D576L and the revertants showed an altered electron distribution. NADP+ photoreduction was abolished in the site-directed mutants and restored in the revertants. Photoreduction of methyl viologen took place with thylakoid membranes of C575S and D576L. Photoreduction of neutral red but not of methyl viologen took place with C575H. EPR and ENDOR spectra of A1- indicated no significant differences in electronic structure or binding site structure between wild type and D576L, and only very small differences between C. reinhardtii and spinach. Rates of electron transfer were determined using time-resolved pulsed EPR. Forward electron transfer from A1 did not take place in C575H and C575S. The rate of forward electron transfer from A1 was considerably slower in D576L than in wild type. The revertants showed rates similar to wild type. C575H and C575S do not bind FX. In D576L, PsaC is incorrectly bound. The structure of the FX binding site is incorrect in D576L, and is corrected in the revertants.
... Õâ¸öµ¥·å EPR ÐźÅÊÇÔ´ÓÚÑõ»¯Ì¬µÄ¹âϵͳ ·´Ó¦ÖÐÐÄÒ ¶ÂÌËØË«·Ö×Ó + 700 P µÄ Signal [3,8] . ÍêÈ«±£ÁôÏÂÀ´,ÍêÈ«±£ÁôÏÂÀ´, ÇÒʹAEä¾ßÓкܺõݵÎÈ ¶¨ÐÔ . ...
... Fe, Mn, or Cu. 16 These metals are directly engaged in photosynthesis and indirectly in carbohydrate metabolism. Changes in their oxidation indicate appearance of reactive oxygen species (ROS). ...
BACKGROUND
Biofortification with selenium (Se) elevates its concentration in feed and fodder plants and helps to prevent health problems in animals and humans. The aim of this study was to describe Se‐induced modifications in the accumulation of elements important for the proper functioning of wheat, one of the most popular cereals. The presence of Se correlated with carbohydrate synthesis and electron paramagnetic resonance (EPR). This explained the mechanisms of Se's antioxidant activity.
RESULTS
Selenium accumulation in vegetative and generative leaves, and in the grains of three wheat genotypes (cv. Parabola, cv. Raweta and cv. Manu), differing in their stress tolerance and grown hydroponically in the presence of 10 or 20 μM Na2SeO4,, was proportional to its content in the medium. Stronger Se accumulation was typical of a stress‐sensitive genotype. Selenium generally promoted the uptake of macronutrients and micronutrients but their distribution depended on tissue and genotype. Changes in the Se‐induced EPR signals of paramagnetic metals and organic radicals corresponded with stress tolerance of the tested genotypes.
CONCLUSIONS
Se application increased the accumulation of nutrients and carbohydrates that are vital for proper plant growth and development. Accelerated uptake of molybdenum (Mo), an element improving dietary properties of grains, may be an additional advantage of Se fertilization. The mechanisms of Se‐induced changes in removing Mn and iron (Fe) ions from macromolecules may be one of the factors that differentiate plant tolerance to oxidative stress. © 2019 Society of Chemical Industry
... Distance distributions were fitted by a Gaussian model. In order to oxidize Y D fully, the samples were illuminated for 1 min at 0°C to form Y D radicals and then the samples were incubated on ice for 1 min in dark before the measurements [26]. The EPR S 2 multiline signal of the sample was not observed after the incubation (data not shown). ...
The binding site of the extrinsic protein PsbP in plant photosystem II was mapped by pulsed electron-electron double resonance, using mutant spinach PsbP (Pro20Cys, Ser82Cys, Ala111Cys, and Ala186Cys) labeled with 4-maleimido-TEMPO (MSL) spin label. The distances between the spin label and the Tyr160 neutral radical (YD) in PsbD, the D2 subunit of plant photosystem II, were 50.8 ± 3.5 Å, 54.9 ± 4.0 Å, 57.8 ± 4.9 Å, and 58.4 ± 14.1 Å, respectively. The geometry inferred from these distances was fitted to the PsbP crystal structure (PDB: 4RTI) to obtain the coordinates of YDrelative to PsbP. These coordinates were then fitted under boundary conditions to the structure of cyanobacterial photosystem II (PDB: 4UB6), by rotating on Euler angles centered at fixed YDcoordinates. The result proposed two models which show possible acidic amino acid residues in CP43, CP47 and D2 that can bind the basic amino acids Arg48, Lys143, and Lys160 in PsbP.
... Crucial to the functional organization of the OEC as well as the tuning and control of the redox properties of the charge-transfer cofactors is the role of the surrounding protein environment. Over the years, a wealth of information on the structure and function of the charge-transfer cofactors has been provided by X-ray spectroscopy [10], electron paramagnetic resonance (EPR) spectroscopy [11] , optical spectros- copy [12][13][14][15][16], vibrational spectroscopy [17], and quantum mechanical calculations [18]. For example, X-ray absorption near-edge structure (XANES) has been used to address the oxidation state and symmetry of the manganese ions in the Mn 4 Ca-oxo cluster in the different S state intermediates of the OEC and X-ray absorption fine structure (EXAFS) has provided information on the number, type, and distances of the amino acid ligands and neighboring manganese atoms in the Mn 4 Ca-oxo cluster in the OEC of PSII [19]. ...
The solar water-splitting protein complex, photosystem II (PSII), catalyzes one of the most energetically demanding reactions in nature by using light energy to drive the catalytic oxidation of water to dioxygen. Light-driven electron and proton-coupled electron transfer (PCET) reactions, which are exquisitely tuned by smart protein matrix effects, are central to this water-splitting chemistry. PSII contains a series of charge-transfer cofactors, such as the special chlorophylls, pheophytin, primary and secondary plastoquinones, tetranuclear manganese-calcium-oxo cluster, and two symmetrically placed redox-active tyrosine residues, YD and YZ, that participate in the charge-transfer reactions. These cofactors are functionally very distinct and the versatility is provided by their distinct local environments in PSII. This chapter focuses on providing the reader with an outline of the primary electron transfer reactions of PSII and a description of the structure and function of the charge-transfer cofactors that participate in the primary electron transfer pathway.
... Because the overall concentration of PSII is too low at these early stages of de-etiolation to probe the presence of the Mn 4 CaO 5 cluster directly via, for example, the S 2 -EPR-multiline signal, we utilized the unique temperature sensitivity of the Mn 4 CaO 5 cluster in PSII to detect it indirectly. For this, we monitored the increase of the characteristic six-line EPR signal of hexa-aquo Mn 2+ (Miller and Brudvig 1991) in etiochloroplasts in response to a heat treatment (70°C for 10 min) above the background level normally present in thylakoids (Nash et al. 1985;Coleman et al. 1988;Shevela et al. 2008). Figure 3 (insert) shows the temperature-induced signals for various times of de-etiolation [for original background signals of free Mn 2+ ions in the (etio-)chloroplasts before the heat treatment, see Supporting Information Fig. S2]. ...
Etioplasts lack thylakoid membranes and photosystem complexes. Light triggers differentiation of etioplasts into mature chloroplasts, and photosystem complexes assemble in parallel with thylakoid membrane development. Plastids isolated at various time-points of de-etiolation are ideal to study the kinetic biogenesis of photosystem complexes during chloroplast development. Here, we investigated the chronology of photosystem II (PSII) biogenesis by monitoring assembly status of chlorophyll-binding protein complexes and development of water-splitting via O2 production in plastids (etiochloroplasts) isolated during de-etiolation of barley (Hordeum vulgare L.). Assembly of PSII monomers, dimers, and of complexes binding outer light-harvesting antenna (PSII-LHCII supercomplexes) were identified after 1, 2, and 4 h of de-etiolation, respectively. Water-splitting was detected in parallel with assembly of PSII monomers and its development correlated with an increase of bound Mn in the samples. After 4 h of de-etiolation, etiochloroplasts revealed the same water-splitting efficiency as mature chloroplasts. We conclude that the capability of PSII to split water during de-etiolation precedes assembly of the PSII-LHCII supercomplexes. Taken together, data show a rapid establishment of water-splitting activity during etioplast-to-chloroplast transition, and emphasize that assembly of the functional water-splitting site of PSII is not the rate limiting step in the formation of photoactive thylakoid membranes.
... The dark incubation time, prior to methylamine addition, was 2.5 h. This is a long incubation time compared with the life-time of oxidized and reduced species in PSII-3 (32,33). ...
Photosynthetic oxygen evolution is catalyzed at the manganese-containing active site of photosystem II (PSII). Amines are
analogs of substrate water and inhibitors of oxygen evolution. Recently, the covalent incorporation of 14C from [14C]methylamine and benzylamine into PSII subunits has been demonstrated (Ouellette, A. J. A., Anderson, L. B., and Barry, B.
A. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 2204–2209). To obtain more information concerning these labeling reactions, t-[14C]butylamine and phenylhydrazine were employed as probes. Neither compound can be oxidized by a transamination or addition/elimination
mechanism, but both can react with activated carbonyl groups, produced as a result of posttranslational modification of amino
acid residues, to give amine-derived adducts. 14C incorporation into the PSII subunits D2/D1 and CP47 was obtained upon treatment of PSII with eithert-[14C]butylamine or [14C]phenylhydrazine. For t-butylamine and methylamine, the amount of labeling increased when PSII was treated with denaturing agents. Labeling of CP47,
D2, and D1 with methylamine and phenylhydrazine approached a one-to-one stoichiometry, assuming that D2 and D1 each have one
binding site. Evidence was obtained suggesting that reductive stabilization and/or access are modulated by PSII light reactions.
These results support the proposal that PSII subunits D2, D1, and CP47 contain quinocofactors and that access to these sites
is sterically limited.
... Crucial to the functional organization of the OEC as well as the tuning and control of the redox properties of the charge-transfer cofactors is the role of the surrounding protein environment. Over the years, a wealth of information on the structure and function of the charge-transfer cofactors has been provided by X-ray spectroscopy [10], electron paramagnetic resonance (EPR) spectroscopy [11], optical spectroscopy [12][13][14][15][16], vibrational spectroscopy [17], and quantum mechanical calculations [18]. For example, X-ray absorption near-edge structure (XANES) has been used to address the oxidation state and symmetry of the manganese ions in the Mn 4 Ca-oxo cluster in the different S state intermediates of the OEC and X-ray absorption fine structure (EXAFS) has provided information on the number, type, and distances of the amino acid ligands and neighboring manganese atoms in the Mn 4 Ca-oxo cluster in the OEC of PSII [19]. ...
The solar water-splitting protein complex, photosystem II (PSII), catalyzes one of the most energetically demanding reactions in nature by using light energy to drive the catalytic oxidation of water to dioxygen. Light-driven electron and proton-coupled electron transfer (PCET) reactions, which are exquisitely tuned by smart protein matrix effects, are central to this water-splitting chemistry. PSII contains a series of charge-transfer cofactors, such as the special chlorophylls, pheophytin, primary and secondary plastoquinones, tetranuclear manganese-calcium-oxo cluster, and two symmetrically placed redox-active tyrosine residues, Y D and Y Z , that participate in the charge-transfer reactions. These cofactors are functionally very distinct and the versatility is provided by their distinct local environments in PSII.
... OEC, including the S 0 (spin S = 1/2) multiline 33,36,[41][42][43][44][45][46][47] and S 2 (S = 1/2) multiline signal, 11,[31][32][33]48,49 the two parallel-mode signals of the S 1 state of higher plants (S = 1) 50,51 and cyanobacteria (S = 2), 52 and the split signals that result from coupling of the Y z • radical to the paramagnetic forms of S 0 , S 1 , and S 3 . 35 There appear to be differences in the concentration dependence of the effects of methanol on these signals that are S-state dependent, indicating that the binding affinity of MeOH at the Mn 4 CaO 5 cluster may change as a function of the oxidation state of the cluster. ...
The binding of the substrate analogue methanol to the catalytic Mn4CaO5 cluster of the water-oxidizing enzyme photosystem II is known to alter the electronic structure properties of the oxygen-evolving complex without retarding O2-evolution under steady-state illumination conditions. We report the binding mode of 13C-labeled methanol determined using 9.4 GHz (X-band) hyperfine sublevel-correlation (HYSCORE) and 34 GHz (Q-band) electron spin-echo electron nuclear double resonance (ESE-ENDOR) spectroscopies. These results are compared to analogous experiments on a mixed-valence Mn(III)Mn(IV) complex (2-OH-3,5-Cl2-salpn)2Mn(III)Mn(IV) (salpn = N,N'-bis(3,5-dichlorosalicylidene)-1,3-diamino-2-hydroxypropane) in which methanol ligates to the Mn(III) ion (Larson, et al. 1992 J. Am. Chem. Soc. 114:6263).1 In the mixed-valence Mn(III,IV) complex, the hyperfine coupling to the 13C of the bound methanol (Aiso = 0.65 MHz, T = 1.25 MHz) is appreciably larger than that observed for 13C methanol associated with the Mn4CaO5 cluster poised in the S2 state, where only a weak dipolar hyperfine interaction (Aiso = 0.05 MHz, T = 0.27 MHz) is observed. An evaluation of the 13C hyperfine interaction using the x-ray structure coordinates of the Mn4CaO5 cluster indicates that methanol does not bind as a terminal ligand to any of the manganese ions in the OEC. We favor methanol binding in place of a water ligand to the Ca2+ in the Mn4CaO5 cluster or in place of one of the waters that form hydrogen bonds with the oxygen bridges of the cluster.
... While the method has been applied to microcrystals of PSII in the S 1 and S 2 states, no significant changes in the structure of the OEC have been detected upon the S 1 → S 2 transition. 7 The S-state transitions have also been extensively studied by a variety of other experimental techniques, including timeresolved mass spectrometry, 16,17 electron paramagnetic resonance (EPR) spectroscopy, 18 and Fourier transform infrared (FTIR) spectroscopy. 19−21 In particular, FTIR has been instrumental in detecting changes induced by the S 1 → S 2 transition in the properties of the carboxylate ligands. ...
The S1 → S2 transition of the oxygen-evolving
complex (OEC) of photosystem II does not involve the transfer of a
proton to the lumen and occurs at cryogenic temperatures. Therefore,
it is commonly thought to involve only Mn oxidation without any significant
change in the structure of the OEC. Here, we analyze structural changes
upon the S1 → S2 transition, as revealed
by quantum mechanics/molecular mechanics methods and the isomorphous
difference Fourier method applied to serial femtosecond X-ray diffraction
data. We find that the main structural change in the OEC is in the
position of the dangling Mn and its coordination environment.
... The EPR signal (Figure 2A, spectrum d) was too small to integrate, and we estimate it reflects oxidation of Cyt b 559 in <3% of the PS II centers (Table 1). Figure 2C shows the radical EPR spectra from Chl Z + (10 G wide, g value of 2.0026) 57 induced by flash illumination at 5 K. The signal was deconvoluted to obtain clean Chl Z + radical (see the Supporting Information). ...
We have earlier shown that all electron transfer reactions in Photosystem II are operational up to 800 nm at room temperature [Thapper et al. (2009), Plant Cell 21, 2391-2401]. This led us to suggest an alternative charge separation pathway for far-red excitation. Here we extend these studies to very low temperature (5 K). Illumination of photosystem II (PS II) with visible light at 5 K is known to result in oxidation of almost similar amounts of YZ and the Cyt b559/ChlZ/CarD2 pathway. This is reproduced here using laser flashes at 532 nm and we find the partition ratio between the two pathways to be 1:0.8 at 5 K (the partition ratio is here defined as (yield of YZ/CaMn4 oxidation):(yield of Cyt b559/ChlZ/CarD2 oxidation)). The result using far red laser flashes is very different. We find partition ratios of 1.8 at 730 nm; 2.7 at 740 nm and >2.7 at 750 nm. No photochemistry involving these pathways is observed above 750 nm at this temperature. Thus, far-red illumination preferentially oxidizes YZ while the Cyt b559/ChlZ/CarD2 pathway is hardly touched. We propose that the difference in the partition ratio between visible and far-red light at 5 K reflects the formation of different first stable charge pair. In visible light, the first stable charge pair is considered to be PD1+Qa-. In contrast, we propose that the electron hole is residing on the ChlD1 molecule after illumination by far red at light 5 K resulting in the first stable charge pair being ChlD1+QA-. ChlD1 is much closer to YZ (11.3 Å) than to any component in Cyt b559/ChlZ/CarD2 pathway (closest distance is ChlD1 - CarD2 is 28.8 Å). This would then explain that far-red illumination preferentially drives efficient electron transfer from YZ. We also discuss mechanisms to account for the absorption of the far-red light and the existence of a hitherto unobserved charge transfer states. The involvement of two or more of the porphyrin molecules in the core of the Photosystem II reaction center is proposed.
We address the protonation state of the water-derived ligands in the oxygen-evolving complex (OEC) of photosystem II (PSII), prepared in the S2 state of the Kok cycle. We perform quantum mechanics/molecular mechanics calculations of isotropic proton hyperfine coupling constants, with direct comparisons to experimental data from two-dimensional hyperfine sublevel correlation (HYSCORE) spectroscopy and extended X-ray absorption fine structure (EXAFS). We find a low-barrier hydrogen bond with significant delocalization of the proton shared by the water-derived ligand, W1, and the aspartic acid residue D1–D61 of the D1 polypeptide. The lowering of the zero-point energy of a shared proton due to quantum delocalization precludes its release to the lumen during the S1→ S2 transition. Retention of the proton facilitates the shuttling of a proton during the isomerization of the tetranuclear manganese–calcium–oxo (Mn4Ca–oxo) cluster, from the “open” to “closed” conformation, a step suggested to be necessary for oxygen evolution from previous studies. Our findings suggest that quantum-delocalized protons, stabilized by low-barrier hydrogen bonds in model catalytic systems, can facilitate the accumulation of multiple oxidizing equivalents at low overpotentials.
The temperature stability of electron transfer to the artificial electron acceptor 2,6-dichlorophenolindophenol in preparations of native photosystem II and photosystem II without the calcium cation in an oxygen-evolving complex was studied. The thermal stability of the processes of oxygen evolution and electron transfer from the oxygen-evolving complex to 2,6-dichlorophenolindophenol in photosystem II were significantly different: the reduction of 2,6-dichlorophenolindophenol was more resistant to temperature than oxygen evolution. The reaction of 2,6-dichlorophenolindophenol reduction in the Ca2+-depleted preparations of photosystem II was less resistant to heating than in the preparations of native photosystem II. The thermal inactivation of the photosystem II in the Ca2+-depleted membrane preparations was inhibited by cytochrome c at a concentration of 50 cytochrome c molecules per a photosystem II reaction center. The activity of this preparation (the rate of the 2,6-dichlorophenolindophenol reduction) increased by 19%, approaching the activity of the native photosystem II. The protective effect of cytochrome c appears to be determined by its protein nature, rather than its redox activity, since an equal protective effect was observed upon the addition of albumin at a similar concentration. Almost complete inactivation of the 2,6-dichlorophenolindophenol reduction reaction in the native and Ca2+-depleted preparations of photosystem II was observed at the same temperature (50°C). According to the EPR data, photosystem II in the Ca2+-depleted preparation after incubation at this temperature lacked a manganese cluster, while a peripheral protein of 33 kDa was present.
World demand for energy is rapidly increasing and finding sufficient supplies of clean energy for the future is one of the major scientific challenges of today. This book presents the latest knowledge and chemical prospects in developing hydrogen as a solar fuel. Using oxygenic photosynthesis and hydrogenase enzymes for bio-inspiration, it explores strategies for developing photocatalysts to produce a molecular solar fuel. The book begins with perspective of solar energy utilization and the role that synthetic photocatalysts can play in producing solar fuels. It then summarizes current knowledge with respect to light capture, photochemical conversion, and energy storage in chemical bonds. Following chapters on the natural systems, the book then summarizes the latest developments in synthetic chemistry of photo- and reductive catalysts. Finally, important future research goals for the practical utilization of solar energy are discussed. The book is written by experts from various fields working on the biological and synthetic chemical side of molecular solar fuels to facilitate advancement in this area of research.
Aerobic respiration is the indispensable physiology of higher organism living on earth, since the required energy for biological activities is supplied mainly by this metabolism. It is well established that mitochondria plays an important role in converting the stable chemical energy stored in foods into the active and utilizable energy supplying to life activities. Upon the process, the energy conversion is carried out by the respiratory electron transport chain, which consists of five distinct and coupled protein complexes (termed complex I- V) embedded in the mitochondria intima. Among them, complex I (NADH-ubiquinone oxidoreductase), III (cytochrome bc1) and IV (cytochrome c oxidase) are the electron-coupled proton-pumping enzymes. Except for the complex V (i.e., ATP synthase), each complex has several redox cofactors to promote the electron transfer within themselves, and the transfer from complex I and II to complex III is shuttled by membrane-embedded ubiquinones, from complex III to IV by the soluble cytochrome c. When the reductive electron is donated by the initial donors NADH or FADH2 to the final acceptor molecular oxygen O2, a transmembrane proton motive force is formed simultaneously. The latter is the driving force for the ATP synthesis via ATP synthase with a ratio of three protons per ATP. All the aforementioned processes are also known as oxidative phosphorylation, and the arrangement of the electron carriers therein is called as respiratory electron transport chain. In the stepwise electron relay, these carriers undergo the redox changes, giving rise to the respective paramagnetic and diamagnetic spin states. These single-electron relays are prone to the electron paramagnetic resonance spectroscopy (EPR), which is a powerful and noninvasive technique to monitor the unpaired electron in situ. As shown in the overwhelming literatures, EPR technique has been adopted to reveal the electron transfer pathway in different biological activities. Herein, the application of EPR to study the dynamic electron transfer in mitochondria is reviewed briefly. However, this conventional application required rather high concentration of the active center at a level of 1-10 μmol/L. Actually, most of the intermediates of the electron carriers are very active and short-life. In the human body, only a few tissues can accumulate such required concentration of paramagnetic substances, e.g., in blood, heart and liver. For the past decade, a more sensitive single-molecule magnetic resonance technique based on the nitrogen-vacancy (NV) center of diamond has been developing dramatically. In principle, this advanced EPR technique requires the less sample or concentration, even down to the single biomolecule, and provides the time-resolved dynamic information. Lately, the studies of single-biomolecule EPR at room temperature or in aqueous solution have been reported consecutively in single protein (MAD2/mitotic arrest deficient-2) and single DNA (tethered DNA duplexes). Finally, a perspective of single- biomolecule magnetic resonance technique is given for the further study on mitochondrion and relative physiological activities. The single molecule EPR technique is also helpful to unveil the other specific information of biological processes, for example, drug screening, personalized medicine and other major applications.
In photosystem II (PSII), photosynthetic water oxidation occurs at the tetramanganese-calcium cluster that cycles through light-induced intermediates (S0 - S4) to produce oxygen from two substrate waters. The surrounding hydrogen-bonded amino-acid residues and waters form channels that facilitate proton transfer and substrate water delivery, thereby ensuring efficient water oxidation. The residue D1-S169 lies in the “narrow” channel and forms hydrogen bonds with the Mn4CaO5 cluster via waters W1 and Wx. To probe the role of the narrow channel in substrate-water binding, we studied the D1-S169A mutation. PSII core-complexes isolated from mutant cells exhibit inefficient S-state cycling and delayed oxygen evolution. The S2 state multiline EPR spectrum of D1-S169A PSII core-complexes differed significantly from that of wild-type and FTIR difference spectra showed that the mutation strongly perturbs the extensive network of hydrogen bonds that extends at least from D1-Y161 (YZ) to D1-D61. These results imply a possible role of D1-S169 in proton egress or substrate water delivery.
The evolution of aerobic life on earth is depended on proceeding water splitting accomplished through photosynthesis in plants, algae, and cyanobacteria. Photosystem II (PSII), with a catalytic center CaMn4O5 located on the lumenal surface, is responsible for water splitting and generating molecular oxygen through a four-step photocatalytic cycle. So far, the structure of the catalytic center and its ligation environments have been studied by different methods mostly relied on various spectroscopic techniques, disclosing unknowing aspects of the PSII components. Over the last half-decade, the experimental methods have extensively been coupled with quantum mechanics/molecular mechanics (QM/MM) methods to explore diverse aspects of PSII structure and water oxidation mechanism. However, despite the progress made in the past years, distinguishing a generally accepted mechanism on the O–O bond formation is still a challenge. This substantial challenge, if resolved, would provide a widespread criterion for development of globally deployable biomimetic model systems for water splitting catalysts. Here, we highlight some latest studies performed on the structure and function of PSII, the information that tells us how to establish new artificial catalytic systems to deliver maximum performance through water splitting in research labs.
Background:
UV irradiation has ionization character and leads to the generation of reactive oxygen species (ROS). Destructive character of ROS was observed among others during interaction of cereal grains with ozone and was caused by changes in structures of biomolecules leading to the formation of stable organic radicals. That effect was more evident for stress sensitive genotypes. In this study we investigated the influence of UV irradiation on cereal grains originating from genotypes with different tolerance to oxidative stress.
Results:
Grains and their parts (endosperm, embryo and seed coat) of barley, wheat and oat were subjected to short term UV irradiation. It was found that UV caused the appearance of various kinds of reactive species (O2(-) , H2 O2 ) and stable radicals (semiquinone, phenoxyl and carbon-centered). Simultaneously, lipid peroxidation occurred and the organic structure of Mn(II) and Fe(III) complexes become disturbed.
Conclusions:
UV irradiation causes damage of main biochemical structures of plant tissues, the effect is more significant in sensitive genotypes. In comparison with ozone treatment, UV irradiation leads to stronger destruction of biomolecules in grains and their parts. It is caused by high energy of UV light, facilitating easier breakage of molecular bonds in biochemical compounds.
Leaves of Urtica dioica collected from two areas of different environmental pollution were analysed by fourier transform infrared spectroscopy (FTIR) and electron paramagnetic resonance (EPR) spectroscopy.
Analysis of FTIR spectra allows to describe main component of plant like proteins, lipids and carbohydrates. Although the FTIR spectra of plants from these two geographical locations of different environmental pollution appear to be relatively similar, 2D correlation shows completely different patterns. Synchronous and asynchronous correlation maps showed sequences of changes occurring during development of plant, manly in Amide I and Amide II, lignin, lipids and cellulose. In addition, 2D analysis revealed another sequence of changes as the function of plant growth depending on the degree of the environmental pollution.
Two various kinds of paramagnetic species, transition metal ions (Mn(II), Fe(III)) and stable organic radicals (chlorophyll, semiquinone, tyrosyl and carbon centered) were found in leaves of nettle collected at different stages of development and growing in clean and polluted environment. In plants growing in polluted area the injuries of protein molecules bonding metal ions and the disturbances of photosynthesis and redox equilibrium in cells, as well as instability of polysaccharide structure of cell walls were observed.
Photosystem II (PS II) core complex is the structural minimum required for evolution of molecular oxygen from water, and consists of more than 10 subunit proteins including the heterodimer of D1 and D2 proteins, CM-binding inner antenna proteins of CP43 and CP47, a hemoprotein of Cyt b-559 and several low-molecular-weight membrane proteins. Aiming at elucidation of the mechanism of oxygen evolution, several types of oxygen-evolving PS II core complexes have been purified from thylakoids of higher plants, green alga, and cyanobacteria, by density-gradient centrifugation of detergent-solubilized thylakoids, followed by anion exchange and size-exclusion column chromatographies. The oxygen-evolving activities retained in these preparations differed variously, due probably to denaturation during the time-consuming purification steps required for eliminating LHC and PS I complex, so that development of a rapid and simple protocol for isolating the oxygen-evolving PS II core complex has been desired.
Cytb559 is an integral part of the PSII reaction center. It consists of the membrane spanning α and β subunits [1], both of which are required for the stable assembly and function of PSII [2]. Despite the numerous investigations concerning Cytb559 [for a review see 3] its function, structural arrangement and content (one or two heme group per PSII) is not clear [3]. Cytb559 can exist in several different thermodynamic forms: high-potential (HP. Em≈360 mV) [4], intermediate potential (IP, Em≈230–270 mV) [5], low potential (LP, E.m≈20–80 mV) [4] and very low potential (VLP, Em<45 mV) [6].
The cyanobacterium Synechocystis PCC 6803 is especially useful in site-directed mutagenesis studies of photosystem II (PSII). Unfortunately, methods which rely on ion-exchange chromatography for recovery of the mutant PSII (1–4) are lengthy and can even be inadequate for the generation of material in sufficient yield and purity for biophysical study when the level of PSII expression in the mutant is low. Here we present the use of an engineered hexahistidine tag fused to the carboxy-terminus of the CP47 subunit for the rapid purification of PSII core complexes from Synechocystis PCC 6803 by Ni²⁺-affinity chromatography. A recent paper also reported purification of PSII from Chlamydomonas using a His-tagged D2 subunit (5).
Photosystem II (PSII) is conserved in all oxygenic photosynthetic organisms and is important for its unique ability to use energy from light to split water, generate molecular oxygen in the Earth’s atmosphere and drive electrons into the photosynthetic electron transport chain by reducing the plastoquinone (PQ) pool in the thylakoid membrane. The focus of this chapter is on alternative electron-transfer pathways on the acceptor side of PSII. Upon close examination of the literature there is evidence of exogenous electron acceptors that are reduced directly by the primary PQ electron acceptor (QA), bypassing the canonical terminal PQ-reduction (QB) site. These herbicide-insensitive electron-acceptor molecules include but are not limited to ferricyanide, synthetic cobalt coordination complexes, and cytochrome c. We also discuss experimental treatments to PSII such as cation exchange and herbicide treatment that have been shown to alter the redox midpoint potential (Em) of QA and impact electron transfer from QA to QB. The results described in this chapter provide a platform for understanding how electrons generated in PSII by photochemical water oxidation can be extracted from the electron-acceptor side of PSII for energy applications.
Efficient photoelectrochemical water oxidation may open a way to produce energy from renewable solar power. In biology, generation of fuel due to water oxidation happens efficiently on an immense scale during the light reactions of photosynthesis. To oxidize water, photosynthetic organisms have evolved a highly conserved protein complex, Photosystem II. Within that complex, water oxidation happens at the CaMn4O5 inorganic catalytic cluster, the so-called oxygen-evolving complex (OEC), which cycles through storage “S” states as it accumulates oxidizing equivalents and produces molecular oxygen. In recent years, there has been significant progress in understanding the OEC as it evolves through the catalytic cycle. Studies have combined conventional and femtosecond X-ray crystallography with extended X-ray absorption fine structure (EXAFS) and quantum mechanics/molecular mechanics (QM/MM) methods and have addressed changes in protonation states of μ-oxo bridges and the coordination of substrate water through the analysis of ammonia binding as a chemical analog of water. These advances are thought to be critical to understanding the catalytic cycle since protonation states regulate the relative stability of different redox states and the geometry of the OEC. Therefore, establishing the mechanism for substrate water binding and the nature of protonation/redox state transitions in the OEC is essential for understanding the catalytic cycle of O2 evolution.
The aim of this research was to characterize the changes of structural organization of chloroplasts of sensitive (Maresi) and tolerant (Cam/B1) barley genotypes upon soil drought (10 days), which was applied in two stages of plant growth, i.e. seedlings and flag leaves. The electron paramagnetic resonance (EPR) technique was used for the determination of changes in the concentration and nature of long-lived radicals and metal ions (Mn, Fe), measured directly in the structures of fresh leaves, occurring after stress treatment. Stronger variations of EPR parameters were found after drought stress application in the flag-leaf phase and for sensitive genotype. Chloroplasts of Cam/B1 were characterized by a larger surface area and less degradation of their structure during drought stress in comparison to Maresi. The data obtained from Raman spectra showed that better stress tolerance of the genotype was accompanied by greater accumulation of carotenoids in chloroplasts and was correlated with an increase in carotenoid radicals. The increase of the value of the electrokinetic potential (relative to control), which was slightly larger for the chloroplasts of Maresi than of Cam/B1, indicated the chemical reconstruction of the membrane leading to a reduction of their polarity during drought action.
An introduction to electron magnetic resonance (EMR) with applications in biophysical studies is presented at the level of nonspecialist or beginning graduate student. The first half of the chapter briefly introduces the resonance phenomenon, a typical EMR spectrum and its interpretation, and describes fundamental applications of electron resonance spectroscopy in free radical research, identification and characterization of metalloproteins and reaction intermediates, spin probes, and imaging. The second half of the chapter describes the magnetochemical origins of resonance spectroscopy and the steps that have led to modern EMR techniques.
This chapter provides an overview of oxygenic photosynthesis, with primary focus on the oxygen-evolving complex (OEC) of photosystem II (PSII). The introduction includes a general discussion of photosynthesis and a brief overview of the metals involved. This is followed by an in-depth description of the metal centers within PSII, in which the structural and electronic characterizations of the OEC are reviewed in detail. The role of chloride in PSII and the proton exit pathway are discussed. Several mechanisms proposed for the O. O bond formation in the OEC are presented, along with a few detailed mechanisms for the complete catalytic cycle obtained from quantum mechanics/molecular mechanics and density functional theory calculations. Model complexes that are functional mimics of the OEC are also briefly discussed. General discussion on plastocyanin, iron-sulfur centers, [FeFe] and [NiFe] hydrogenases, and ribulose-1,5-bisphosphate carboxylase-oxygenase is included. The chapter concludes with the implications of natural photosynthesis on the development of an artificial photosynthetic framework.
The increase of the concentration of ozone in the atmosphere, being the direct source of reactive oxygen species, results in the yield loss of agronomic crops. On the other hand, ozone is also used as a protector against microorganisms, living in plants and present in materials obtained from them, dangerous for human and animal health. In this work it has been studied if ozone in doses similar to those used for removal of microorganisms can have significant influence on the generation of stable organic radicals and changes in the character of transition metal ions and in the antioxidative biochemical parameters of cereal grains. The aim of this work was to find if the response of grains of three cereals (wheat, oat and barley) to ozone depended on their oxidative stress tolerance. The influence of direct short-term ozone application on grains of these cereals, each represented by two genotypes with different oxidative stress tolerance, was studied by biochemical analyses and by electron paramagnetic resonance (EPR) technique. Whole grains as well as their parts: embryo, endosperm and seed coat were subjected to ozone treatment for 30min. Biochemical investigation of control samples showed that their antioxidant activity increased in order: wheat<oat<barley. EPR method revealed that character and the number of paramagnetic species (transition metal ions: Fe(III), Cu(II), Mn(II) and stable organic radicals) changed upon ozone exposure, depending on the kind of cereal, stress tolerance of particular genotype and the part of grain. The control samples of whole grains and their parts originating from sensitive genotypes contained higher amounts of stable organic radicals (semiquinone, phenoxyl and carbohydrate types) than those from tolerant ones. In embryos of grains from sensitive genotypes their amount increased upon ozone treatment stronger than in embryos from grains of tolerant cultivars. In seed coats and endosperms such relation was not found and the changes in the content of the radicals during ozone application were correlated with the amount of transition metal ions and were more intensive in parts of grains richer in easily oxidized iron species Fe(II), located in inorganic structures. On the contrary, Fe(II) ions situated in embryos were stabilized by organic matrix and did not undergo oxidation by ozone.
A frequent challenge when dealing with multinuclear transition metal clusters in biology is to determine the absolute oxidation states of the individual metal ions and to identify how they evolve during catalytic turnover. The oxygen-evolving complex of biological photosynthesis, an active site that harbors an oxo-bridged Mn4Ca cluster as the water-oxidizing species, offers a prime example of such a challenge that withstood satisfactory resolution for decades. A multitude of experimental studies have approached this question and have offered insights from different angles, but they were also accompanied by incomplete or inconclusive interpretations. Only very recently, through a combination of experiment and theory, has a definitive assignment of the individual Mn oxidation states been achieved for all observable catalytic states of the complex. Here we review the information obtained by structural and spectroscopic methods, describe the interpretation and synthesis achieved through quantum chemistry, and summarize our current understanding of the electronic structure of nature’s water splitting catalyst.
The aim of this study was to uncover the specific species in grains that might differentiate the wheat genotypes according to their tolerance to oxidative stress. Measurements by EPR and Raman spectroscopy techniques were used to examine whole grains and their parts (embryo, endosperm, seed coat) originating from four wheat genotypes with differing tolerance to drought stress. Raman spectra showed that, in spite of the similar amounts of proteins in whole grains from tolerant and sensitive genotypes, in tolerant ones they were accumulated mainly in embryos. Moreover, in embryos from these grains, a higher content of unsaturated fatty acids was observed. Endosperm of grains from the tolerant genotype, richer with starch than that of sensitive one, exhibited higher content of amylopectin. Detailed analysis of EPR signals and simulation procedures of the spectra allowed the estimation of the nature of interactions of Fe(III) and Mn(II) with organic and inorganic structures of grains and the character of organic stable radicals. Three types of these radicals: carbohydrate, semiquinone and phenoxyl, were identified. The amounts of these radicals were higher in grains of sensitive genotypes, mostly because of differences in carbohydrate radical content in endosperm. Taking into account the level of radical concentration and greater capacity for radical formation in grains from plants of lower tolerance to stress, the content of radicals, especially of a carbohydrate nature, was considered as a marker of the plant resistance to stress conditions.
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Photosystem II, the photosynthetic water oxidizing complex, contains two well characterized redox active tyrosines, D and Z. D forms a stable radical of unknown function. Z is an electron carrier between the primary chlorophyll donor and the manganese catalytic site. The vibrational difference spectra associated with the oxidation of tyrosines Z and D have been obtained through the use of infrared spectroscopy (MacDonald, G. M., Bixby, K. A., and Barry, B. A.(1993) Proc. Natl. Acad. Sci. U. S. A. 90, 11024-11028). Here, we examine the effect of deuterium exchange on these vibrational difference spectra. While the putative C-O vibration of stable tyrosine radical D downshifts in 2H2O, the putative C-O vibration of tyrosine radical Z does not. This result is consistent with the existence of a hydrogen bond to the phenol oxygen of the D radical; we conclude that a hydrogen bond is not formed to the Z radical. In an effort to identify the amino acid residue that is the proton acceptor for Z, we have performed global N labeling. While significant N shifts are observed in the vibrational difference spectrum, substitution of a glutamine for a histidine that is predicted to lie in the environment of tyrosine Z has little or no effect on the difference infrared spectrum. There is also no significant change in the yield or lineshape of the Z EPR signal under continuous illumination in this mutant. Our results are inconsistent with the possibility that this residue, histidine 190 of the D1 polypeptide, acts as the sole proton acceptor for tyrosine Z.
Current energy resources largely rely on fossil fuels that are expected to be depleted in
50-200 years. On a global scale, the intensive use of this energy source has resulted in
highly detrimental effects to the environment. Hydrogen production by water splitting,
with sunlight as the main energy source, is a promising way to augment the production
of renewable energy. However, the development of an efficient and stable wateroxidizing
catalyst remains a key task before a technological breakthrough based on water
splitting can be realized. A main issue hampering the development of commercially
viable, non-precious metal-based catalysts is their susceptibility to degradation. To
efficiently address this major drawback, self-healing catalysts, that can repair their
structure without human intervention, will be necessary. In this review, we focus on
water oxidation by natural and artificial Mn-, Co- and Ni-based catalysts and then
discuss the self-healing properties that contribute to sustaining their catalytic activity.
This study was aimed at establishing the effect of saturating starches from different botanical origins with iron ions on selected physicochemical properties of the resulting modified starch. Native and modified starches were determined for amylose, protein, fat, and phosphorus content as well as water binding capacity and solubility in water. In addition, the number of iron ions was assayed by atomic absorption spectroscopy (AAS). The thermal properties of samples were also examined using a differential scanning calorimeter (DSC). Flow curves were plotted and described using the Ostwald de Waele model, and the degree of paste structure recovery was determined. Samples enriched with iron ions were treated thermally and radical generation in the starch structure was assayed by means of electron paramagnetic resonance (EPR). AAS analysis proved the presence of iron ions in the investigated modified starch preparations. The results achieved established that enriching starch with iron ions affected the rheological and thermal properties of starch preparations. However, the extent and nature of these changes depended on the botanical origin of the starch. This was also a factor in the thermal generation of radicals, a process, which appeared to be more effective in potato starch preparations.
Two kinds of barley genotypes with various water-stress tolerances, tolerant Cam/B1 and sensitive Maresi, were subjected to 10-day soil-drought stress in seedling and flag leaf developmental phases. After this time, both genotypes regardless of the growth stage showed a decrease in quantum yield of PSII photochemistry (ΦPSII) upon stress treatment; however, this effect was stronger in the sensitive plants than in the tolerant ones. The drought stress in the flag leaf stage was associated with an increase in superoxide dismutase (SOD) level in both genotypes, whereas in seedlings, this effect was observed only for Maresi. The activity of other enzymes (catalase and peroxidase) was changed only in small degree. An increase in proline levels and activities of Δ1-pyrroline-5-carboxylate synthetase (P5CS) and ornithine delta-aminotransferase (OAT) were observed independently of genotype and the phase of plant development, whereas the activity pyruvate dehydrogenase (PDH) decreased in tolerant genotype. Moreover, changes in the concentration of monocarbohydrates (glucose and fructose) and dicarbohydrates (saccharose, raffinose and maltose) were found: in seedlings, the amount of all soluble sugars increased, while in flag leaves decreased. The drought treatment resulted in a drop in starch level in the tolerant genotype, but in the sensitive one, the content of this substance increased in both developmental stages. EPR studies allowed the determination of the amount and character of organic radicals present in leaves. In control conditions, the content of these radical species was higher in the sensitive genotype than in tolerant one and decreased upon water stress, with the exception of flag leaves of the sensitive plant. Simulation procedure revealed four types of signals in the EPR spectra. One of them was attributed to a chlorophyll a cation and decreased upon drought. The second, ascribed to semiquinone radicals, reflected the redox balance disturbed by water deficit. The two remaining signals were connected with carbon-centred radicals situated in the carbohydrate matrix. Their number was correlated with starch concentration.
Water oxidation at the oxygen-evolving complex (OEC) of photosystem II (PSII) involves multiple redox states called Sn states (n = 0-4). The S1 → S2 redox transition of the OEC has been studied extensively using various forms of spectroscopy, including electron paramagnetic resonance (EPR) and Fourier transform infrared (FTIR) spectroscopy. In the S2 state, two isomers of the OEC are observed by EPR: a ST = 1/2 form and a ST = 5/2 form. DFT-based structural models of the OEC have been proposed for the two spin isomers in the S2 state, but the factors that determine the stability of one form or the other are not known. Using structural information on the OEC and its surroundings, in conjunction with spectroscopic information available on the S1 → S2 transition for a variety of site-directed mutations, Ca(2+) and Cl(-) substitutions, and small molecule inhibitors, we propose that the hydrogen-bonding network encompassing D1-D61 and the OEC-bound waters plays an important role in stabilizing one spin isomer over the other. In the presence of ammonia, PSII centers can be trapped in either the ST = 5/2 form after a 200 K illumination procedure or an ammonia-altered ST = 1/2 form upon annealing at 273 K. We propose a mechanism for ammonia binding to the OEC in the S2 state that takes into account the hydrogen-binding requirements for ammonia binding and the specificity for binding of ammonia but not methylamine. A discussion regarding the possibility of spin isomers of the OEC in the S1 state, analogous to the spin isomers of the S2 state, is also presented.
Electron paramagnetic resonance (EPR) spectra of the reduced quinone-iron acceptor complex in reaction centers were measured in a variety of environments and compared with spectra calculated from a theoretical model. Spectra were obtained at microwave frequencies of 1, 9, and 35 GHz and at temperatures from 1.4 to 30 K. The spectra are characterized by a broad absorption peak centered at g = 1.8 with wings extending from g approximately equal to 5 to g less than 0.8. The peak is split with the low-field component increasing in amplitude with temperature. The theoretical model is based on a spin Hamiltonian, in which the reduced quinone, Q-, interacts magnetically with Fe2+. In this model the ground manifold of the interacting Q-Fe2+ system has two lowest doublets that are separated by approximately 3 K. Both perturbation analyses and exact numerical calculations were used to show how the observed spectrum arises from these two doublets. The following spin Hamiltonian parameters optimized the agreement between simulated and observed spectra: the electronic g tensor gFe, x = 2.16, gFe, y = 2.27, gFez = 2.04, the crystal field parameters D = 7.60 K and E/D = 0.25, and the antiferromagnetic magnetic interaction tensor, Jx = -0.13 K, Jy = -0.58 K, Jz = -0.58 K. The model accounts well for the g value (1.8) of the broad peak, the observed splitting of the peak, the high and low g value wings, and the observed temperature dependence of the shape of the spectra. The structural implications of the value of the magnetic interaction, J, and the influence of the environment on the spin Hamiltonian parameters are discussed. The similarity of spectra and relaxation times observed from the primary and secondary acceptor complexes Q-AFe2+ and Fe2+Q-B leads to the conclusion that the Fe2+ is approximately equidistant from QA and QB.
Electron paramagnetic resonance (EPR, or ESR for electron spin resonance) spectroscopy is based on the fact that substances containing unpaired electrons are paramagnetic. This paramagnetism may be due to the presence of transition elements which have unfilled shells, or it may be due to the transitory presence of oxidized or reduced substances. In the case of photosynthetic materials, this oxidation and reduction (i.e., the transfer of an electron from one substance to another) is set in motion by light. The basic observation, made in the mid-1950s (Commoner et al., 1956), was that photosynthetic materials become paramagnetic when illuminated. Two prominent light-induced resonances in plants and a single one in bacteria were described in early papers, and numerous speculations on their origin and significance advanced. It is now generally accepted that the light-induced EPR signal at g = 2.002 (signal I) in plants is a direct measure of the oxidation state of the photosystem I reaction center, P-700. However, between the time when the signal was first described and a sense of certainty as to its significance, some 15 years elapsed. We now know that light-induced resonances in photosynthetic organisms or subcellular preparations of them are indeed probes into events essential to the overall process of photosynthesis.
The history of EPR applications to the study of manganese dates to the very first successful resonance experiments by Zavoisky (1945). Since then there have been numerous EPR studies of Mn(II) in diverse materials. The reader may find references to many of these studies in review articles and monographs on EPR (Abragam and Bleaney, 1970; Goodman and Raynor, 1970; König, 1968; Kaiser and Kevan, 1968). Applications of EPR to studies of Mn(II) complexes with proteins began with studies by Malmström et al. (1958) wherein the strong isotropic EPR signal for “unbound” Mn(H2O)
62+was used to determine dissociation constants for Mn(II)-protein complexes. Such analytical applications of the EPR signal for Mn(H2O)
62+(Cohn and Townsend, 1954) for measuring dissociation constants have continued. However, during the last decade the EPR signals for the protein-bound Mn(II) have been measured for enzymes and other proteins, and this latter application is the basis for the present chapter.
This chapter discusses the primary reactions of photosystems I and II of algae and higher plants. In photosynthetic organisms, the “primary reactions” fulfil the objective of converting the energy of light into a primary form of chemical energy which lasts for a time compatible with ordinary biochemical processes—that is, milliseconds. In these reactions, a rather large fraction, approximately 40%, of the photon energy is stored as chemical free energy. The primary reactions can be viewed from two major perspectives. Firstly, from a photochemical point of view: pigment molecules are excited to their lowest excited singlet state, which reacts in an electron transfer reaction, the first step of a process of charge separation. Secondly, from a biochemical point of view the reactions take place in highly organized complexes, the reaction centres, which are made up of several classes of molecules that cooperate in fulfilling complementary roles, such as: architectural support, light absorption, energy transfer and electron transfer. All oxygenic organisms, ranging from cyanobacteria to algae and higher plants, contain photosystem I (PS I) and PS II reaction centres, with only minor variations in spite of their large taxonomic and ecological diversity.
Publisher Summary This chapter discusses electron paramagnetic resonance (EPR) in photosynthesis. Practically, all aspects of EPR spectroscopy come to the fore, individually or in combination, in the various photosynthetic systems of plants and bacteria, in intact cells or in isolated subcellular particles or purified reaction center proteins. EPR has been instrumental in the demonstration that the primary electron donor, P, in bacterial reaction centers (RC) is a bacteriochlorophyll dimer. In normal photosynthesis, in all photosystems the charge on the photoreduced intermediary acceptor is quickly transported to the next primary acceptor. When this acceptor is (photo) chemically pre-reduced or removed by extraction, however, this negative charge cannot be further transported, and recombines with the positive charge on the primary donor. The recombination product is either the singlet ground or excited state, or the triplet excited state of P. The primary donor of photosystem II (P-680) is much more difficult to observe with EPR than that of photosystem PS I, because in normally functioning PS II the photo-oxidized donor is very rapidly (within at most a few hundred ns) reduced by an electron donor called Z. The reduced intermediary acceptor I (BPh) is normally too short-lived to be observable by EPR. However, it can be photoaccumulated at cryogenic temperatures in isolated RCs of, for example, Rb. Sphaeroides, when reduced Cyt c is added, because of slow, irreversible electron donation to P + .
The pathways of electron donation in Photosystem II (PS II) as studied by electron paramagnetic resonance (EPR) and time-resolved optical spectroscopy are discussed. The EPR studies have defined two competing pathways of electron donation in PS II, from cytochrome b559 and from the Mn site of the oxygen-evolving center. The kinetics of re-reduction of the primary electron donor of PS II (P680). as measured by optical spectroscopy, are re-evaluated in light of the EPR results. We propose that the 35-μs kinetic component is due to the reduction of chlorophyll, an alternate electron donor on the cytochrome b559 pathway, rather than to the reduction of P680. The chlorophyll/cytochrome b559 pathway has been proposed to be part of a cyclic electron transfer pathway around PS II; we suggest that photooxidation of chlorophyll is the first step leading to photoinhibition and that cytochrome b559 serves to protect PS II from photoinhibition by rapidly re-reducing the oxidized chlorophyll (Thompson, L.K.; Brudvig, G.W. Biochemistry, 1988, 27: 6653). These results and proposals are summarized in an overall scheme of electron transfer pathways and rates in PS II.
In this review, the main research developments that have led to the current simplified picture of photosystem I are presented. This is followed by a discussion of some conflicting reports and unresolved questions in the literature. The following points are made: (1) the evidence is contradictory on whether P700, the primary donor, is a monomer or dimer of chlorophyll although at this time the balacnce of the evidence points towards a monomeric structure for P700 when in the triplet state; (2) there is little evidence that the iron sulfur centers FA and FB act in series as tertiary acceptors and it is as likely that they act in parallel under physiological conditions; (3) a role for FX, probably another iron sulfur centrer, as an obligatory electron carrier in forward electron transfer has not been proven. Some evidence indicates that its reduction could represent a pathway different to that involving FA and FB; (4) the decay of the acceptor 'A2-' as defined by optical spectroscopy corresponds with 700+ {Mathematical expression} recombination under some circumstances but under other conditions it probably corresponds with P700+ A1- recombination; (5) P700+ A1- recombination as originally observed by optical spectroscopy is probably due to the decay of the P700 triplet state; (6) the acceptor A1- as defined by EPR may be a special semiquinone molecule; (7) A0 is probably a chlorophyll a molecule which acts as the primary acceptor. Recombination of P700+ A0- gives rise to the P700 triplet state. A working model for electron transfer in photosystem I is presented, its general features are discussed and comparisons with other photosystems are made.
A characteristic EPR spectrum is observed from Photosystem I (PS I) particles that are illuminated under reducing conditions. This signal is thought to arise from the secondary electron acceptor of PS I, A1. This EPR spectrum, along with data from other spectroscopic methods, has been taken as evidence that A1 is phylloquinone. In the work reported here, we have used a methionine auxotroph of Anabaena to deuterate specifically the 2-methyl group on the phylloquinone ring. Mass spectrometry of the isolated quinone shows 65% yield in the labeling procedure. The photoaccumulated A1 EPR spectra in protonated and deuterium-labeled cells are indistinguishable, from which we conclude that the photoaccumulated radical is not phylloquinone.
The cytochromes in spinach chloroplasts were studied using EPR spectroscopy. In addition to the low-spin heme signals previously assigned, cytochrome f (gz 3.51), high-potential cytochrome b-559 (gz 3.08) and cytochrome b-559 converted to a low-potential form (gz 2.94), a high-spin heme signal was induced by 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ). However, this signal cannot be due to cytochrome b-563 in its native form. The orientation of the cytochromes in the thylakoid membrane was studied in magnetically oriented chloroplasts. Cytochrome b-559 in the native state and in the low-potential form was found to have its heme plane perpendicular to the membrane plane. The orientation was the same for cytochrome b-559 oxidized by low-temperature illumination, which suggests that also the reduced heme is oriented perpendicular to the membrane.
The reaction of hydroxylamine, a substrate analogue of the water-oxidizing complex (WOC), with spinach photosystem II (PSII) membranes has been further studied by using EPR spectroscopy to monitor the stepwise oxidation of donors and reduction of electron acceptors during successive low-temperature illuminations. In addition to its well-known binding on the donor side of PSII, hydroxylamine binds in the dark with high affinity to a site that structurally interacts with the primary electron acceptor FeQ/sub A/â». Binding in the dark to this acceptor site causes conversion of the normal g = 1.9 EPR signal for FeQ/sub A/) to g = 2.1 on the first turnover. These results indicate that the binding site for NHâOH overlaps with or interacts with the binding site for Q/sub B/. The EPR microwave power saturation of the g = 2.1 signal at 5.5 K is similar to that found for the endogenous ferrosemiquinone acceptors. These results indicate a structural change in the primary acceptor site upon binding NHâOH, with no change in oxidation state of the iron or the semiquinone. In contrast, NHâOH does not bind in the dark to PSII centers exhibiting the other major form of the primary acceptor, which exhibit the g = 1.82 EPR signal, since no change in the EPR signal is observed. The authors also find that the high-affinity binding of NHâOH within the WOC produces no observable EPR-active products in the dark. They conclude that this binding site is closely associated with manganese, since there exists no blockage in the photooxidation of other donors like high-potential cytochrome bâââ or signal II (¹â¶Â°Tyr x Dâ protein).
Electron paramagnetic resonance (EPR) spectroscopy and O2 evolution assays were performed on photosystem II (PSII) membranes which had been treated with 1 M CaCl2 to release the 17, 23 and 33 kilodalton (kDa) extrinsic polypeptides. Manganese was not released from PSII membranes by this treatment as long as a high concentration of chloride was maintained. We have quantitated the EPR signals of the several electron donors and acceptors of PSII that are photooxidized or reduced in a single stable charge separation over the temperature range of 77 to 240 K. The behavior of the samples was qualitatively similar to that observed in samples depleted of only the 17 and 23 kDa polypeptides (de Paula et al. (1986) Biochemistry25, 6487–6494). In both cases, the S2 state multiline EPR signal was observed in high yield and its formation required bound Ca2+. The lineshape of the S2 state multiline EPR signal and the magnetic properties of the manganese site were virtually identical to those of untreated PSII membranes. These results suggest that the structure of the manganese site is unaffected by removal of the 33 kDa polypeptide. Nevertheless, in samples lacking the 33 kDa polypeptide a stable charge separation could only be produced in about one half of the reaction centers below 160 K, in contrast to the result obtained in untreated or 17 and 23 kDa polypeptide-depleted PSII membranes. This suggests that one function of the 33 kDa polypeptide is to stabilize conformations of PSII that are active in secondary electron transfer events.
The reaction of hydroxylamine, a substrate analogue of the water-oxidizing complex (WOC), with spinach photosystem II (PSII) membranes has been further studied by using EPR spectroscopy to monitor the stepwise oxidation of donors and reduction of electron acceptors during successive low-temperature illuminations. In addition to its well-known binding on the donor side of PSII, hydroxylamine binds in the dark with high affinity to a site that structurally interacts with the primary electron acceptor FeQ/sub A//sup -/. Binding in the dark to this acceptor site causes conversion of the normal g = 1.9 EPR signal for FeQ/sub A/) to g = 2.1 on the first turnover. These results indicate that the binding site for NH/sub 2/OH overlaps with or interacts with the binding site for Q/sub B/. The EPR microwave power saturation of the g = 2.1 signal at 5.5 K is similar to that found for the endogenous ferrosemiquinone acceptors. These results indicate a structural change in the primary acceptor site upon binding NH/sub 2/OH, with no change in oxidation state of the iron or the semiquinone. In contrast, NH/sub 2/OH does not bind in the dark to PSII centers exhibiting the other major form of the primary acceptor, whichmore » exhibit the g = 1.82 EPR signal, since no change in the EPR signal is observed. The authors also find that the high-affinity binding of NH/sub 2/OH within the WOC produces no observable EPR-active products in the dark. They conclude that this binding site is closely associated with manganese, since there exists no blockage in the photooxidation of other donors like high-potential cytochrome b/sub 559/ or signal II (/sup 160/Tyr x D/sub 1/ protein).« less
The decay kinetics for the S2 and S3 states of the oxygen-evolving complex in Photosystem II have been measured in the presence of an external electron acceptor. The S2- and S3-states decay monophasically with half-decay times at 18°C of 3–3.5 min and 3.5–4 min, respectively. The results also show that S3 decays via S2 under these circumstances. The temperature dependence of the individual S-state transitions has been measured in single flash experiments in which the multiline EPR signal originating from the S2 state has been used as spectroscopic probe. The half-inhibition temperatures are for S0 to S1 220–225 K, for S1 to S2 135–140 K, for S2 to S3 230 K and for the S3-to-S0 transition 235 K.
Effects of Ca extraction by pH 3.0 treatment on the electron transport on donorside of Photosystem II (PS II) were investigated. The flash O2 yieldwas abolished by the treatment, and was restored to normal by the addition of Ca2+ with almost no change in the oscillation pattern. The light-induced fluorescence increase was lost by the treatment and was well restored by addition of Ca2+. Among the othercations tested, only Sr2+ was effective. The treated membranes exhibited a light-dependent EPR Signal IIfast, superimposed on dark-stable Signal IIslow, but the Signal IIfast was lost on addition of Ca2+, suggesting a Ca-dependent reversible conversion between Signal IIvery fast, and Signal IIfast. The peak temperatures of thermoluminescence B- and Q-bands arising from S2Q−B and S2Q−A charge recombinations, respectively, were elevated to high temperatures after the treatment, and were largely reversed to theirrespective normal temperatures by the addition of Ca2+. When excited by a series of flashes, the amplitude of the modified B-band generated by the 1st flash did not change any more after the 2nd flash. These results were interpreted as indicating that extraction of one of two Ca from higher plant PS II by pH3.0 treatment induces an abnormal S2 state and thereby inhibits S2 to S3 transition.
In Photosystem II preparations at low temperature we were able to generate and trap an intermediate state between the S1 and S2 states of the Kok scheme for photosynthetic oxygen evolution. Illumination of dark-adapted, oxygen-evolving Photosystem II preparations at 140 K produces a 320-G-wide EPR signal centered near g = 4.1 when observed at 10 K. This signal is superimposed on a 5-fold larger and somewhat narrower background signal; hence, it is best observed in difference spectra. Warming of illuminated samples to 190 K in the dark results in the disappearance of the light-induced g = 4.1 feature and the appearance of the multiline EPR signal associated with the S2 state. Low-temperature illumination of samples prepared in the S2 state does not produce the g = 4.1 signal. Inhibition of oxygen evolution by incubation of PS II preparations in 0.8 M NaCl buffer or by the addition of 400 μM NH2OH prevents the formation of the g = 4.1 signal. Samples in which oxygen evolution is inhibited by replacement of Cl− with F− exhibit the g = 4.1 signal when illuminated at 140 K, but subsequent warming to 190 K neither depletes the amplitude of this signal nor produces the multiline signal. The broad signal at g = 4.1 is typical for a spin system in a rhombic environment, suggesting the involvement of non-heme Fe in photosynthetic oxygen evolution.
In spinach photosystem I particles, constituent phylloquinone (2-methyl-3-phytyl-1,4-naphthoquinone; vitamin K1) was replaced by 14 different benzo-, naphtho- and anthraquinones. All of the quinones tested suppressed the nanosecond charge recombination between the reduced electron acceptor chlorophyll a, A−0, and the oxidized primary donor, P700+, suggesting that they replace the function of the electron acceptor A1 and rapidly oxidize A−0. The binding affinity of these quinones for the photosystem I reaction center increased in the order of benzoquinone < naphthoquinone < anthraquinone. The phytyl tail of phylloquinone was also shown to increase the binding affinity. The flash-induced kinetics of P700+ varied independently of the dissociation constants. Only the quinones (including phylloquinone) which are estimated to exhibit an in situ redox midpoint potential (Em) value between those of A0 and the iron-sulfur center Fx fully replaced the function of A1. These results confirm that A1 is phylloquinone and indicate that the phylloquinone-binding site in the photosystem I reaction center gives an environment in which the Em value for the semiquinone-quinone couple is significantly lower than that in the QA site in the reaction center of purple bacteria.Quinone; Phylloquinone; Vitamin K1; Photosystem I; Reaction center; Electron transfer; Photosynthesis
Electron transfer in irreversibly acetate-inhibited PS II preparations from Synechococcus is investigated with EPR. A 1.4 mT wide EPR line with a decay rate consistent with the P+680 Q−A back reaction is tentatively assigned to be a broadened P+680 spectrum. With reversible inhibition of water cleavage by the ADRY reagent FCCP added with acetate electron transfer is studied as well. Under these conditions the spectrum of the immediate donor to P+680 is the same as the spectrum of Z+ in systems with irreversibly inhibited water cleavage (signal II fast) and the same as that of D+ (signal II slow). The consequences are discussed.
The multiline EPR signals arising from manganese in the S2 state of the oxygen-evolving system of spinach and the cyanobacterium Anacystis nidulans have very similar properties and are affected identically by NH3, suggesting that the system is highly conserved. The temperature dependence of the signal amplitude follows Curie behavior down to sub-helium temperatures. This is in contrast to previous reports, which were taken as evidence for a tetrameric manganese cluster. Thus, it seems that it is not yet possible from EPR data alone to distinguish between this model and a dimeric structure.
Oxygen ligands from water are bound to the manganese cluster giving rise to the ‘multiline’ EPR signal from the S2 state of photosystem II, as shown by the observation of hyperfine broadening of the EPR signal in the presence of 17O-enriched water. The binding must occur in the S0, S1 or S2 state. The estimated upper limit for the 17O hyperfine coupling constant, 0.5 mT, excludes Superoxide and hydroxyl radicals in the S2 state, but not peroxide.
Upon addition of hydroxylamine to chloroplasts or photosystem II preparations, the EPR signal of Z⨥ disappears and a new signal is observed. From its shape and g-value this signal is identified with the oxidized reaction center chlorophyll, P680+. The decay of P680+ occurs with a halftime of ⪅ 200 μs and apparently is the result of a back reaction with the reduced form of the primary acceptor, QA. This mode of hydroxylamine inhibition is reversible. These observations indicate that hydroxylamine, in addition to its well known inhibitory action on the oxygen evolving complex, is also able to disrupt physiological electron flow to P680 itself.
In this contribution, the authors provide a more complete interpretation of earlier EPR data and include new experimental data on the magnetic properties of the g = 4.1 Sâ state EPR signal and the Sâ state multiline EPR signal produced by 245 K illumination in active state samples. An interpretation of the temperature-dependence data of the Sâ state EPR signal from NHâCl-treated PSII membranes was presented. These data can be best explained by a model of the Sâ state which consists of a mixed-valence manganese tetramer.
The complex magnetic-resonance spectrum of oxygen gas is observed at 9340 Mc/sec in magnetic fields up to 9000 oersted. Some 40 lines are resolved. A partial analysis of the spectrum is made with the help of Henry's recent Zeeman theory of O2. Other lines are identified by the temperature dependence of the relative intensities. Line widths are measured by several techniques and range from 0.6 to 4 Mc/sec/mm Hg at room temperature. The temperature and pressure dependence of the line widths are investigated.
The authors report the results of a Moessbauer study of the low-potential iron-sulfur cluster F{sub X} in the Photosystem I core protein of Synechococcus 6301. The Moessbauer spectrum of F{sub X} in the oxidized state shows an isomer shift of 0.42 mm/s, which is in good agreement with the 0.43 mm/s isomer shift found in (4Fe-4S) proteins but not with the isomer shift of 0.26 mm/s found in (2Fe-2S) proteins. In the reduced state the spectrum is asymmetrically broadened at 80 K, indicating the presence of two very closely spaced doublets with an average isomer shift of 0.55 mm/s, which is also in agreement with (4Fe-4S) proteins. At 4.2 K, the spectrum exhibits broadening and magnetic splitting similar to what is observed for (4Fe-4S) proteins and quite unlike (2Fe-2S) proteins. Given the assumption that the iron atoms of F{sub X} are tetrahedrally coordinated with sulfur ligands, the data strongly support the assignment of F{sub X} as a (4Fe-4S) cluster.
We have investigated the effects of temperature on the formation and decay of the light-induced multiline EPR signal species associated with photosynthetic oxygen evolution (Dismukes, G.C. and Siderer, Y. (1980) FEBS Lett. 121, 78–80). (1) The decay rate following illumination is temperature dependent: at 295 K the half-time of decay is about 40 s, at 253 K the half-time is approx. 40 min. (2) A single intense flash of light becomes progressively less effective in generating the multiline signal below about 240 K. (3) Continuous illumination is capable of generating the signal down to almost 160 K. (4) Continuous illumination after a preilluminating flash generates less signal above 200 K than at lower temperatures. Our results support the conclusion of Dismukes and Siderer that the S2 state gives rise to this multiline signal; we find that the S1 state can be fully advanced to the S2 state at temperatures as low as 160 K. The S2 state is capable of further advancement at temperatures above about 210 K, but not below that temperature.
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.
The dominant interaction of ${\mathrm{O}}_{2}$ with a magnetic field is through the electronic spin magnetic moment. However, a precise comparison with experiment of the results of calculating the microwave paramagnetic spectrum, assuming only this interaction, shows a systematic discrepancy. This discrepancy is removed by introducing two corrections. The larger (approximately 0.1 percent, or 7 gauss) is a correction for the second-order electronic orbital moment coupled in by the spin-orbit energy. Its magnitude is proportional to the second-order term ${$\mu${}}^{$'${}$'${}}$ in the spin-rotation coupling constant. The smaller (approximately 1 gauss) is a correction for the rotation-induced magnetic moment of the molecule. Since the dependence of this contribution on quantum numbers is quite unique, this coefficient can also be determined by fitting the magnetic spectrum. A total of 120 $X$-band and 78 $S$-band lines were observed. The complete corrections have been made on 26 lines with a mean residual error of roughly 0.5 Mc/sec. This excellent agreement confirms the anomalous electronic moment to 60 parts per million (ppm) and also confirms the validity of the Zeeman-effect theory.
— Using isolated chloroplasts and techniques as described by Joliot and Joliot[6] we studied the evolution of O2 in weak light and light flashes to analyze the interactions between light induced O2 precursors and their decay in darkness. The following observations and conclusions are reported: 1. Light flashes always produce the same number of oxidizing equivalents either as precursor or as O2. 2. The number of unstable precursor equivalents present during steady state photosynthesis is ∼ 1.2 per photochemical trapping center. 3. The cooperation of the four photochemically formed oxidizing equivalents occurs essentially in the individual reaction centers and the final O2 evolution step is a one quantum process. 4. The data are compatible with a linear four step mechanism in which a trapping center, or an associated catalyst, (S) successively accumulates four + charges. The S4+ state produces O2 and returns to the ground state S0. 5. Besides S0 also the first oxidized state S+ is stable in the dark, the two higher states, S2+ and S3+ are not. 6. The relaxation times of some of the photooxidation steps were estimated. The fastest reaction, presumably S*1←S2, has a (first) half time ≤ 200 μsec. The S*2 state and probably also the S*0 state are processed somewhat more slowly (˜ 300–400 μsec).
The positions of powder lines in the electron paramagnetic spectra of high‐spin ferric systems (d5,S = ) have been calculated by solving the spin Hamiltonian = gβB⋅S + ⅓D[3Sz2 − S(S + 1)] + E(Sx2 − Sy2) for a broad range of parameters. Powder lines are obtained for every transition when the magnetic field points along the principal axes of the fine structure tensor. However, it was found that for most transitions extra powder lines are often found when the field lies in any of the principal planes but not along the axes. Particular attention is directed to the transition responsible for the g′ ≈ 4.2 absorption in nearly rhombic (E / D ∼ ⅓) ferric complexes. The calculations show that, depending on the value of the ratio between the microwave quantum and the parameter D, this transition may consist of 3–6 powder lines near g′ = 4.2. The g′ values for all these powder lines were also obtained from a third‐order perturbation calculation which assumes nearly rhombic symmetry and D > gβB. The 9.2‐ and 34‐GHz spectra of Fe(III)–EDTA diluted in the corresponding diamagnetic Co(III) compound and the 2.7‐, 9.2‐, and 34‐GHz spectra of native human serum transferrin have been analyzed by the aid of the calculations. It was determined that for FeEDTA∣ D ∣ = 0.83cm−1 and ∣ E / D ∣ = 0.31, while for transferrin ∣ D ∣ = 0.27cm−1 and ∣ E / D ∣ = 0.31–0.32.
The stability of EPR Signal IIs in oxygen-evolving PS II particles in the different S-states has been studied as a function of storage time at 77 K. Signal IIs in dark-adapted samples retains the same intensity after 1 month storage at 77 K, while that in the S2 state produced by illumination at 195 K decays with a half-life time of about 5 days without decay in the manganese multiline intensity observed at 4.5 K. Signal IIs, once decayed in the S2 state, recovers its original intensity upon dark adaptation of the sample at 210 K accompanied by a decrease in the multiline intensity. A model for the charge recombination process for the S2 state involving Signal IIs is discussed.
Binding of NH3 to the S2 state of the O2-evolving complex of photosystem II (PSII) causes a structural change in the Mn site that is detectable with low-temperature electron paramagnetic resonance (EPR) spectroscopy. Untreated spinach PSII membranes at pH 7.5 produce a S2 state multiline EPR spectrum when illuminated at either 210 K or at 0°C in the presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) having an average hyperfine line spacing of 87.5 G. The temperature dependence of the S2 state multiline EPR signal observed from untreated samples deviates from the Curie law above 5 K, with a maximum signal intensity at 6.9 K as has been previously observed. In contrast, 100 mM NH4Cl-treated PSII membranes at pH 7.5 exhibit a new S2 state EPR spectrum when illuminated at 0°C in the presence of DCMU. The novel S2 state EPR spectrum from NH4Cl-treated PSII membranes has an average hyperfine line spacing of 67.5 G and a temperature dependence obeying the Curie law except for small deviations at low temperature. We assign the new S2 state EPR signal from NH4Cl-treated PSII membranes to a form of the S2 state having one or more NH3 molecules directly coordinated to the Mn site. NH3 does not bind to Mn in the dark-stable S1 state present before illumination, since generation of the S2 state in NH4Cl-treated PSII membranes by illumination at 210 K does not yield the new S2 state EPR spectrum. Since inhibition of O2 evolution activity in the presence of NH4Cl probably occurs through binding of NH3 to the O2-evolving complex in competition with substrate H2O molecules, these results indicate that the EPR-detectable Mn site functions as the substrate-binding site of the O2-evolving complex.
The mechanism of ammonia inhibition of photosynthetic oxygen evolution has been examined by the pulsed EPR technique of electron spin-echo envelop modulation (ESEEM), revealing the direct coordination of an ammonia-derived ligand to the catalytic Mn complex during the S{sub 1} {yields}S{sub 2} transition of the oxygen evolution S-state cycle. ESEEM experiments were performed on the multiline Mn EPR signal observed in photosystem II enriched spinach thylakoid membranes which were treated with either {sup 14}NH{sub 4}Cl or {sup 15}NH{sub 4}Cl (100 mM, pH 7.5). {sup 15}NH{sub 4}Cl treatment produced modulation of the electron spin-echo signal which arises from an I = 1/2 {sup 15}N nucleus with an isotropic hyperfine coupling A({sup 15}N) = 3.22 MHz. {sup 14}NH{sub 4}Cl treatment produced a different ESEEM pattern resulting from an I = 1 {sup 14}N nucleus with A({sup 14}N) = 2.29 MHz, and with electric quadrupole parameters e{sup 2}qQ = 1.61 MHz and {eta} = 0.59. The {sup 14}n electric quadrupole parameters are interpreted with respect to possible chemical structures for the ligand. An amido (NH{sub 2}) bridge between metal ions is proposed as the molecular identity of the ammonia-derived ligand to the catalytic Mn of photosystem II.
The S2-state multiline EPR signal observed in photosynthetic membrane preparations has been previously well characterized at X-band frequencies (9.1-9.5 GHz). These studies have indicated that the signal, centered at g = 2, arises from a multinuclear mixed-valence Mn center of the O2-evolving complex. In the present study, the multiline EPR signal from spinach photosystem II enriched membranes is characterized at an S-band frequency (3.9 GHz). At this lower frequency, the resolution and complexity of the signal increase markedly compared with its appearance in the X-band. While the multiline signal covers similar magnetic field ranges at the two frequencies, the S-band signal has a greater number of lines, narrower line widths, and a different overall appearance. Replacement of Cl- with Br- or 1H2O with 2H2O in the buffer shows that neither exchangeable Cl- nor protons cause superhyperfine structure in the S-band multiline signal. Membrane preparations oriented on mylar sheets show dependence of the S-band signal on the angle between the mylar sheet normal and the magnetic field direction, indicating that the multiplicity of lines is in part due to signal anisotropy. The results, combined with previous work at X-band, indicate that a minimal working model for the species responsible for the multiline signal is a mixed-valence binuclear Mn complex with an anisotropic hyperfine interaction that includes second-order contributions.
The electron spin resonance spectrum of P680+ has been measured in photosystem II membranes at room temperature under repetitive flash excitation by using gated integration techniques. Oxygen evolution was inhibited in the samples used in these experiments, and the lifetime of the radical is extended into the 150-200-μs range. Three different treatments were used that allowed us to determine the spectral characteristics of P680+ when the paramagnetic species YZ+ was also present. These results were compared to the P680+ spectral properties that we measured under conditions in which YZ was in its reduced, diamagnetic form. With Tris-inactivated membranes, where YZ+ but not manganese was present, only a low P680+ signal amplitude could be measured, which precluded an accurate determination of the line width. With NaCl-washed membranes and membranes treated with K3Fe(CN)6, in which YZ+ and manganese were both present during the measurement, the field-modulated P680+ spectrum is 8.9 G wide. This is 1 G wider than the spectrum measured when YZ remains reduced, as happens in membranes inhibited with NH2OH. The broadening of the P680+ spectrum that occurs when its immediate donor is oxidized is attributed to a magnetic dipole-dipole interaction between P680+ and YZ+. The extent of broadening allows us to estimate that the center-to-center distance between the two radicals is 10-15 Å.
Time-resolved ESR has been used to study electron-transfer reactions in oxygen-evolving photosystem II membrane fragments. The exogenous acceptor dichlorobenzoquinone (DCBQ) is reduced by photosystem II; the ESR spectrum of the resulting DCBQ radical overlaps the center but not the wings of the ESR spectra of the endogenous tyrosine radicals YD+ and YZ+. Here YZ+ denotes the species that is involved in electron transfer between the reaction center chlorophyll, P680, and the manganese-containing, oxygen-evolving complex, and YD+ denotes the stable photosystem II radical. By using appropriate magnetic fields, we recorded kinetic transients of YZ+ under repetitive flash conditions with DCBQ present. We also used 1 mM K3Fe(CN)6 as an exogenous acceptor when recording kinetic traces of YZ+, although at concentrations above 5 mm we observe an additional signal that could be due to P680+. The kinetic traces of YZ+ obtained with DCBQ or 1 mM K3Fe(CN)6 are similar and show two phases. The slower phase has a half-time of 1.2 ms and corresponds to the reduction of YZ+ by the S3 state; the faster phase reflects reduction of YZ+ by both S1 and S2. By using flowing, dark-adapted PSII membranes, we resolved the YZ+S1 reaction (t1/2 = 100 μs) on the first flash and found it to be significantly faster than the YZ+S2 reaction (t1/2 = 300 μs) which occurs on the second flash. A high-resolution ESR spectrum of YZ+ in O2-evolving PSII membranes was obtained with gated integration techniques and found to be similar to the spectra of YD+ and of Yz+ in inhibited membranes. Thus, the magnetic interaction between spins on YZ+ and the manganese in the oxygen-evolving complex broadens the YZ+ spectrum negligibly. These results support the idea that a single electron carrier, YZ, operates between P680+ and the manganese ensemble in the oxygen-evolving complex and functions on all four S-state transitions.
The microwave power for half-saturation (P1/2) for the radical in photosystem II giving rise to signal IIslow (SIIs) has been measured by EPR in samples illuminated by a series of flashes. The charge storage state of the oxygen-evolving complex (S0-S4) was monitored by measuring the multiline EPR signal arising from the S2 state. The following results were obtained: (1) SIIs becomes easier to saturate after tris(hydroxymethyl)aminomethane (Tris) washing, a treatment that partially removes the Mn cluster. (2) P1/2 for SIIs oscillates with the flash number. P1/2 is lower in S1 (in dark-adapted material and after four flashes) than in S2, S3, or S0. (3) P1/2(S2) = P1/2(S3). (4) At 8 K P1/2(S2) > P1/2(S2), but at 20 K P1/2(S0) < P1/2(S2). (5) P1/2 for SIIs increases with temperature (8-70 K) in the S1 state. SHs is more difficult to saturate in S2, S3, and S0 than in S1 over the investigated temperature range. In addition, the increase in P1/2 is complex around 20-30 K in S2, S3, and S0. (6) In S0, P1/2 for SIIs decreases with time (decay half-time 30-60 s) to a stable level significantly above the dark level. The data are explained in terms of cross relaxation between the radical giving rise to SIIs and an efficient relaxer, which is suggested to be the Mn cluster. This relaxes more slowly in S1 than in the other S states. Since it is known that a mixed-valence Mn cluster is present in S2, and because P1/2 of SIIs in S3 and S0 is comparable to that in S2, it is suggested that mixed-valence Mn clusters are present in the S3 and S0 states also. Different models with these features can be proposed, the simplest of which is the following: S0 [Mn(H)-Mn(III)], S1 [Mn(III)-Mn(III)], S2 [Mn(III)-Mn(IV)], and S3 [Mn(III)-Mn(IV)].
Photosynthetic organisms are able to oxidize organic or inorganic compounds upon the absorption of light, and they use the extracted electron for the fixation of carbon dioxide. The most important oxidation product is oxygen due to the splitting of water. In eukaryotes these processes occur in photosystem II of chloroplasts. Among prokaryotes photosynthetic oxygen evolution is restricted to cyanobacteria and prochloron-type organisms. How water is split in the oxygen-evolving complex of photosystem II belongs to the most important question to be answered. The primary charge separation occurs in the reaction center of photosystem II. This reaction center is a complex consisting of peripheral and integral membrane proteins, several chlorophyll A molecules, two pheophytin A molecules, two and three plastoquinone molecules, and one non-heme iron atom. The location of the photosystem II reaction center is still a matter of debate. Nakatani et al. (l984) concluded from fluorescence measurements that a protein of apparent molecular weight 47,000 (CP47) is the apoprotein of the photosystem II reaction center. A different view emerged from work with the photosynthetic reaction centers from the purple bacteria. The amino acid sequence of the M subunit of the reaction center from Phodopseudomonas (Rps.) sphaeroides has sequence homologies with the D1 protein from spinach. A substantial amount of structural information can be obtained with the reaction center from Rhodopseudomonas viridis, which can be crystallized. Here the authors discuss the structure of the photosynthetic reaction center from the purple bacterium Rps. viridis and describe the role of those amino acids that are conserved between the bacterial and photosystem II reaction center.
A study of electron paramagnetic resonance (EPR) signals from components on the electron donor side of photosystem II has been performed. By measurement of EPR signal IIslow (D+) it is shown that, after three flashes, D+ decays slowly in the dark at room temperature in the fraction of the centers that was in the S0 state (t1/2 of 20 min in thylakoid membranes and 50 min in photosystem II enriched membranes). This reaction is accompanied by a conversion of S0 to S1. The concentration of S1 was estimated from the amplitude of the S2-state multiline EPR signal that could be generated by illumination at 200 K. These observations indicate that D+ accepts an electron from S0 in a dark reaction in which D and S1 are formed. In addition, the reactions by which D donates an electron to S2 or S3 have been directly measured by monitoring both signal IIslow and the multiline signal. The redox interactions between the D/D+ couple and the S states are explained in terms of a model in which D/D+ has a midpoint potential between those of S0/S1, and S1/S2. In addition, this model provides explanations for a number of previously unrelated phenomena, and the proposal is put forward that the reaction between D+ and Mn2+ is involved in the so-called photoactivation process.
Electron paramagnetic resonance (EPR) signals arising from components in photosystem II have been studied in membranes isolated from spinach chloroplasts. A broad EPR signal at g = 4.1 can be photoinduced by a single laser flash at room temperature. When a series of flashes is given, the amplitude of the g = 4.1 signal oscillates with a period of 4, showing maxima on the first and fifth flashes. Similar oscillations occur in the amplitude of a multiline signal centered at g ≃ 2. Such an oscillation pattern is characteristic of the S2 charge accumulation state in the oxygen-evolving complex. Accordingly, both EPR signals are attributed to the S2 state. Earlier data from which the g = 4.1 signal was attributed to a component different from the S2 state [Zimmermann, J.-L., & Rutherford, A. W. (1984) Biochim. Biophys. Acta 767, 160-167; Casey, J. L., & Sauer, K. (1984) Biochim. Biophys. Acta 767, 21-28] are explained by the effects of cryoprotectants and solvents, which are shown to inhibit the formation of the g = 4.1 signal under some conditions. The g = 4.1 signal is less stable than the multiline signal when both signals are generated together at low temperature. This indicates that the two signals arise from different populations of centers. The differences in structure responsible for the two different EPR signals are probably minor since both kinds of centers are functional in cyclic charge accumulation and seem to be interconvertible. The difference between the two EPR signals, which arise from the same redox state of the same component (a mixed-valence manganese cluster), is proposed to be due to a spin-state change, where the g = 4.1 signal reflects an S = 3/2 state and the multiline signal an S = 1/2 state within the framework of the model of de Paula and Brudvig [de Paula, J. C., & Brudvig, G. W. (1985) J. Am. Chem. Soc. 107, 2643-2648]. The spin-state change induced by cryoprotectants is compared to that seen in the iron protein of nitrogenase.