Multiline Electron Paramagnetic Resonance Signal Research Articles

Insights into the protonation state and spin structure for the g = 2 multiline electron paramagnetic resonance signal of the oxygen-evolving complex.

In photosystem II (PSII), one-electron oxidation of the most stable oxidation state of the Mn4CaO5 cluster (S1) leads to formation of two distinct states, the open-cubane S2 conformation [Mn1(III)Mn2(IV)Mn3(IV)Mn4(IV)] with low spin and the closed-cubane S2 conformation [Mn1(IV)Mn2(IV)Mn3(IV)Mn4(III)] with high spin. In electron paramagnetic resonance (EPR) spectroscopy, the open-cubane S2 conformation exhibits a g = 2 multiline signal. However, its protonation state remains unclear. Here, we investigated the protonation state of the open-cubane S2 conformation by calculating exchange couplings in the presence of the PSII protein environment and simulating the pulsed electron-electron double resonance (PELDOR). When a ligand water molecule, which forms an H-bond with D1-Asp61 (W1), is deprotonated at dangling Mn4(IV), the first-exited energy (34 cm-1) in manifold spin excited states aligns with the observed value in temperature-dependent pulsed EPR analyses, and the PELDOR signal is best reproduced. Consequently, the g = 2 multiline signal observed in EPR corresponds to the open-cubane S2 conformation with the deprotonated W1 (OH-).

Alteration of the O2-Producing Mn4Ca Cluster in Photosystem II by the Mutation of a Metal Ligand.

The O2-evolving Mn4Ca cluster in photosystem II (PSII) is arranged as a distorted Mn3Ca cube that is linked to a fourth Mn ion (denoted as Mn4) by two oxo bridges. The Mn4 and Ca ions are bridged by residue D1-D170. This is also the only residue known to participate in the high-affinity Mn(II) site that participates in the light-driven assembly of the Mn4Ca cluster. In this study, we use Fourier transform infrared difference spectroscopy to characterize the impact of the D1-D170E mutation. On the basis of analyses of carboxylate and carbonyl stretching modes and the O-H stretching modes of hydrogen-bonded water molecules, we show that this mutation alters the extensive network of hydrogen bonds that surrounds the Mn4Ca cluster in the same manner as that of many other mutations. It also alters the equilibrium between conformers of the Mn4Ca cluster in the dark-stable S1 state so that a high-spin form of the S2 state is produced during the S1-to-S2 transition instead of the low-spin form that gives rise to the S2 state multiline electron paramagnetic resonance signal. The mutation may also change the coordination mode of the carboxylate group at position 170 to unidentate ligation of Mn4. This is the first mutation of a metal ligand in PSII that substantially impacts the spectroscopic signatures of the Mn4Ca cluster without substantially eliminating O2 evolution. The results have significant implications for our understanding of the roles of alternate active/inactive conformers of the Mn4Ca cluster in the mechanism of O2 formation.

Reconciling Structural and Spectroscopic Fingerprints of the Oxygen-Evolving Complex of Photosystem II: A Computational Study of the S2 State.

The catalytic cycle of photosynthetic water oxidation occurs at the Mn4CaO5 oxygen-evolving complex (OEC) of photosystem II. Extensive spectroscopic data have been collected on the intermediates, especially the S2 (Kok) state, although the proton and electron inventories (Mn oxidation states) are still uncertain. The "high oxidation" paradigm assigns S2 Mn oxidation level (III, IV, IV, IV) or (IV, IV, IV, III), whereas a "low oxidation" paradigm posits two additional electrons. Here, we investigate the geometric (X-ray diffraction, extended X-ray absorption fine structure) and spectroscopic (electron paramagnetic resonance (EPR), electron nuclear double resonance (ENDOR)) properties of the S2 state using quantum chemical density functional theory calculations, focusing on the neglected low paradigm. Two interconvertible electronic spin configurations are predicted as ground states, producing multiline ( S = 1/2) and broad ( S = 5/2) EPR signals in the low paradigm oxidation state (III, IV, III, III) and with W2 as OH- and O5 as OH-. They have "open" ( S = 5/2) and "closed" ( S = 1/2) Mn3CaO4-cubane geometries. Other energetically accessible isomers with ground spin states 1/2, 7/2, 9/2, or 11/2 can be obtained through perturbations of hydrogen-bonding networks (e.g., H+ from His337 to O3 or W2), consistent with experimental observations. Conformers with the low oxidation state configuration (III, IV, IV, II) also become energetically accessible when the protonation states are O5 (OH-), W2 (H2O), and neutral His337. The configuration with (III, IV, III, III) agrees well with earlier low-temperature EPR and ENDOR interpretations, whereas the MnII-containing configuration agrees partially with recent ENDOR data. However, the low oxidation paradigm does not yield isotropic ligand hyperfine interactions in good agreement with observed values. We conclude that the low Mn oxidation state proposal for the OEC can closely fit most of the available structural and electronic data for S2 at accessible energies.

Electron paramagnetic resonance study of the electron transfer reactions in photosystem II membrane preparations from Arabidopsis thaliana

Arabidopsis thaliana is widely used as a model organism in plant biology as its genome has been sequenced and transformation is known to be efficient. A large number of mutant lines and genomic resources are available for Arabidopsis. All this makes Arabidopsis a useful tool for studies of photosynthetic reactions in higher plants. In this study, photosystem II (PSII) enriched membranes were successfully isolated from thylakoids of Arabidopsis plants and for the first time the electron transfer cofactors in PSII were systematically studied using electron paramagnetic resonance (EPR) spectroscopy. EPR signals from both of the donor and acceptor sides of PSII, as well as from auxiliary electron donors were recorded. From the acceptor side of PSII, EPR signals from Q Aˉ Fe 2+ and Pheˉ Q Aˉ Fe 2+ as well as from the free Pheˉ radical were observed. The multiline EPR signals from the S 0- and S 2-states of CaMn 4O x -cluster in the water oxidation complex were characterized. Moreover, split EPR signals, the interaction signals from Y Z and CaMn 4O x -cluster in the S 0-, S 1-, S 2-, and the S 3-state were induced by illumination of the PSII membranes at 5 K and characterized. In addition, EPR signals from auxiliary donors Y D , Chl + and cytochrome b 559 were observed. In total, we were able to detect about 20 different EPR signals covering all electron transfer components in PSII. Use of this spectroscopic platform opens a possibility to study PSII reactions in the library of mutants available in Arabidopsis.

The reduced S<sub>-1</sub> and S<sub>-2</sub> oxidation states of the O<sup>2</sup>-evolving complex of photosystem II: An EPR microwave power saturation study

Progressive microwave power saturation (P1/2) measurements have been performed on the tyrosine D radical (YD •) of photosystem II (PSII) in order to examine its relaxation enhancement by the oxygen-evolving complex (OEC) poised to the reduced S−1 and S−2 oxidation states by NO treatment. Analysis of the power saturation curves showed that the S−1 oxidation state of the OEC does not enhance the relaxation of YD •: it therefore possesses a diamagnetic ground state. In contrast, the Mn(II)-Mn(III) multiline electron paramagnetic resonance (EPR) signal characteristic of the S−2 oxidation state of the OEC was shown to provide a relaxation enhancement pathway for YD •, however less efficient relative to the one provided by the S2-state multiline EPR signal. We also examined the YD • relaxation enhancement characteristics of the EPR-silent oxidation state produced after brief (1–5 min) dark incubation at 0°C of a PSII sample poised to the EPRactive S−2 state. This EPR-silent oxidation state denoted as “0°C incubation” state was shown to possess remarkably similar P1/2 values with the EPR-active S−2 state in the overall examined temperature range (6–20 K). In addition, these values remained unchanged after successive cycles of the OEC between the EPR-active S−2 state and the “0°C incubation” state. The data presented in this work point to the conclusion that the “0°C incubation” state is indeed an S−2 oxidation state with half-integer spin.

EPR Characterization of Photosystem II from Different Domains of the Thylakoid Membrane

We report electron paramagnetic resonance (EPR) studies on photosystem II (PSII) from higher plants in five different domains of the thylakoid membrane prepared by sonication and two-phase partitioning. The domains studied were the grana core, the entire grana stack, the grana margins, the stroma lamellae and the purified stromal fraction, Y100. The electron transport properties of both donor and acceptor sides of PSII such as oxygen evolution, cofactors Y D, Q A, the CaMn 4-cluster, and Cytb 559 were investigated. The PSII content was estimated on the basis of oxidized Y D and Q A (-) Fe (2+) signal from the acceptor side vs Chl content (100% in the grana core fraction). It was found to be about 82% in the grana, 59% in the margins, 35% in the stroma and 15% in the Y100 fraction. The most active PSII centers were found in the granal fractions as was estimated from the rates of electron transfer and the S 2 state multiline EPR signal. In the margin and stroma fractions the multiline signal was smaller (40 and 33%, respectively). The S 2 state multiline could not be induced in the Y100 fraction. In addition, the oxidized LP Cytb 559 prevailed in the stromal fractions while the HP form dominated in the grana core. The margins and entire grana fractions have Cytb 559 in both potential forms. These data together with previous analyses indicate that the sequence of activation of the PSII properties can be represented as: PSII content > oxygen evolution > reduced Cytb 559 > dimerization of PSII centers in all fractions of the thylakoid membrane with the gradual increase from stromal fractions via margin to the grana core fraction. The results further support the existence of a PSII activity gradient which reflects lateral movement and photoactivation of PSII centers in the thylakoid membrane. The possible role of the PSII redox components in this process is discussed.

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

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

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

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

Analysis of Dipolar and Exchange Interactions between Manganese and Tyrosine Z in the S2YZ• State of Acetate-Inhibited Photosystem II via EPR Spectral Simulations at X- and Q-Bands

Upon room-temperature illumination, acetate-inhibited photosystem II membranes are known to exhibit a 240 G wide X-band (∼9.5 GHz) electron paramagnetic resonance (EPR) signal at 10 K. This EPR signal arises from an interaction between the S = 1/2 multiline S2 state of the tetranuclear manganese cluster and an oxidized tyrosine residue, YZ•. In the present study, the exchange and dipolar interactions between the two paramagnetic species are simulated at X- and Q-band (∼33 GHz) frequencies utilizing second-order perturbation theory. The positions and relative intensities of the hyperfine lines in the S = 1/2 S2 state multiline EPR signal of the noninteracting Mn4 cluster are accurately simulated by including g anisotropy and four sets of axially symmetric 55Mn hyperfine tensors. These parameters are then used to simulate the dipolar and exchange interactions giving rise to the interacting S2YZ• (formerly referred to as S3) EPR signal. Relative intensities of components of the S2YZ• EPR spectrum, at both X-...

Characterization of the interaction between manganese and tyrosine Z in acetate-inhibited photosystem II.

When acetate-inhibited photosystem II (PSII) membranes are illuminated at temperatures above 250 K and quickly cooled to 77 K, a 240 G-wide electron paramagnetic resonance (EPR) signal is observed at 10 K. This EPR signal arises from a reciprocal interaction between the spin 1/2 ground state of the S2 state of the Mn4 cluster, for which a multiline EPR signal with shifted 55Mn hyperfine peaks is observed, and the oxidized tyrosine residue, YZ*, for which a broadened YZ* EPR spectrum is observed. The S2YZ* EPR signal in acetate-inhibited PSII is the first in which characteristic spectral features from both paramagnets can be observed. The observation of distinct EPR signals from each of the paramagnets together with the lack of a half-field EPR transition indicates that the exchange and dipolar couplings are weak. Below 20 K, the S2YZ* EPR signal in acetate-inhibited PSII is in the static limit. Above 20 K, the line width narrows dramatically as the broad low-temperature S2YZ* EPR signal is converted to a narrow YZ* EPR signal at room temperature. The line width narrowing is interpreted to be due to averaging of the exchange and dipolar interactions between YZ* and the S2 state of the Mn4 cluster by rapid spin-lattice relaxation of the Mn4 cluster as the temperature is increased. Decay of the S2YZ* intermediate at 200 K shows that the g = 4.1 form of the S2 state is formed and that a noninteracting S2-state multiline EPR signal is not observed as an intermediate in the decay. This result shows that a change in the redox state of YZ induces a spin-state change in the Mn4 cluster in acetate-inhibited PSII. The interconversion between spin states of the Mn4 cluster in acetate-inhibited PSII supports the idea that YZ oxidation or YZ* reduction is communicated to the Mn4 cluster through a direct hydrogen-bonding pathway, possibly involving a ligand bound to the Mn4 cluster.

A divalent metal site in the small subunit of the manganese-dependent ribonucleotide reductase of Corynebacterium ammoniagenes.

Based on its metallo-cofactor, the manganese-dependent ribonucleotide reductase (Mn-RRase) responsible for delivery of DNA precursors in the Mn-requiring Gram-positive bacterium Corynebacterium (formerly Brevibacterium) ammoniagenes ATCC 6872 is no longer considered as a simple analogue of the aerobic Fe-RRase of Escherichia coli but as the prototype of the class IV enzymes (1). Deliberate dissociation of the Mn-RRase holoenzyme and an improved sample preparation of the dimeric CA2 protein allowed further characterization of the inherent metallo-cofactor by Q-band electron paramagnetic resonance (EPR) spectroscopy. At 40 K, a distinct hyperfine sextet (I = 5/2,55Mn) pattern with a weak zero-field splitting was detected in the CA2 protein prepared from manganese-sufficient cells displaying high RRase activity as expected. This Q-band Mn(II) signal was absent in the apo-CA2 protein obtained from manganese-depleted cells devoid of this enzymatic activity. The presence of a mixed valence manganese cluster in the C. ammoniagenes RRase is excluded since no complex multiline EPR signals were detected in the CA2 protein even at very low (8 K) temperature. The observed Mn(II) spectrum indicates a protein-bound manganese which was modified in the presence of 5.7 mM p-methoxyphenol, but is insensitive toward 10 mM EDTA. Thus, the manganese appeared to be either strictly bound or buried within a hydrophobic pocket of the CA2 protein, inaccessible for EDTA.

Detection of an EPR multiline signal for the S0* state in photosystem II.

The S0* state was generated by incubation of dark-adapted (S1 state) photosystem II membranes either with the exogenous two electron reductant hydrazine and subsequent 273 K illumination in the presence of DCMU or by dark incubation with low amounts of the one electron reductant hydroxylamine. In agreement with earlier reports, the S1 and S-1 states were found to be electron paramagnetic resonance (EPR) silent. However, in the presence of 0.5-1.5% methanol, a weak EPR multiline signal centered around g = 2.0 was observed at 7 K for the S0* states generated by both procedures. This signal has a similar average line splitting to the well-characterized S2 state multiline EPR signal, but can be clearly distinguished from that and other modified S2 multiline signals by differences in line position and intensities. In addition, at 4 K it can be seen that the S0* multiline has a greater spectral breadth than the S2 multilines and is composed of up to 26 peaks. The S0* signal is not seen in the absence of methanol and is not affected by 1 mM EDTA in the buffer medium. We assign the S0* multiline signal to the manganese cluster of the oxygen evolving complex in a mixed valence state of the form MnIIMnIIIMnIIIMnIII,MnIIMnIIIMnIVMnIV, or MnIIIMnIIIMnIIIMnIV. Addition of methanol may be helpful in future to find an EPR signal originating form the natural S0 state.

Analysis of the interaction of water with the manganese cluster of photosystem II using isotopically labeled water.

The association of water with the Mn of the water oxidizing complex was investigated using H2(17)O- and 2H2O-reconstituted lyophilized photosystem II particles. The pulsed electron paramagnetic resonance (EPR) technique of electron spin echo envelope modulation (ESEEM) was used to investigate the interaction of the magnetic 2H and 17O nuclei with the paramagnetic S2 state of the Mn complex and other photosystem II components. ESEEM offers a much more specific and sensitive detection of this type of interaction than continuous wave (CW) EPR. Unlike earlier reports using CW EPR, these experiments did not detect any interaction of water with the multiline EPR signal from the S2 state of the Mn complex. No signals indicating specific interaction of either H or O with the multiline signal were detected. Signals due to 2H and 17O were detected only at the Larmour frequency, indicating nonspecific "distant ENDOR" effects. A weak interaction with 17O was detected both in S1, when the Mn is EPR silent, and in S2, but only on the high-field side of g = 2. This interaction may be with the Rieske iron-sulfur center in the cytochrome b6f complex. The results were the same whether the multiline signal was generated by 200 K illumination of dark-frozen samples, or by room temperature illumination in the presence of the inhibitor 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU). Illumination at room temperature in the presence of an electron acceptor to allow multiple turnovers of the system with cycling of the S states did not result in the appearance of any new interactions. These results appear to exclude close (less than 6 A) binding of water to the Mn center giving rise to the multiline signal, and also to exclude mechanisms in which water oxidation involves the breaking and re-formation of the mu-oxo bridges of the Mn complex. They cannot, however, exclude models in which water binding to the manganese complex and direct oxidation by the manganese complex occur in the higher S states, or are catalyzed by one bis(mu-oxo) Mn dimer while oxidizing equivalents are accumulated in the S2 state by a second bis(mu-oxo) Mn dimer.

Properties of the chloride-depleted oxygen-evolving complex of photosystem II studied by electron paramagnetic resonance.

The effects of different Cl- depletion treatments in photosystem II (PS-II)-enriched membranes have been investigated by electron paramagnetic resonance (EPR) spectroscopy and by measurements of oxygen-evolving activity. The results indicated that the oxygen-evolving complex of PS-II exhibits two distinct Cl(-)-dependent properties. (1) After Cl(-)-free washes at pH 6.3, a reversibly altered distribution of structural states of PS-II was observed, manifested as the appearance of a g = 4 EPR signal from the S2 state in a significant fraction of centers (20-40%) at the expense of the S2 multiline signal. In addition, small but significant changes in the shape of the S2 multiline EPR signal were observed. Reconstitution of Cl- to Cl(-)-free washed PS-II rapidly reversed the observed effects of the Cl(-)-free washing. The anions, SO4(2-) and F-, which are often used during Cl- depletion treatments, had no effect on the S2 EPR properties of PS-II under these conditions in the absence or presence of Cl-. Flash experiments and measurements of oxygen evolution versus light intensity indicated that the two structural states observed after the removal of Cl- at pH 6.3 originated from oxygen-evolving centers exhibiting a lowered quantum yield of water oxidation. (2) Depletion of Cl- in PS-II by pH 10 treatment reversibly inhibited the oxygen-evolving activity to approximately 15%. The pH 10 treatment depleted the Cl- from a site which is considered to be equivalent to that studied in most earlier work on Cl(-)-depleted PS-II. The S2 state in pH 10/Cl(-)-depleted PS-II was reversibly modified to a state from which no S2 multiline EPR signal was generated and which exhibited an intense S2 g = 4 EPR signal corresponding to at least 40% of the centers but possibly to a much larger fraction of centers. The state responsible for the intense S2 g = 4 signal generated under these conditions is unlike that observed after removal of Cl- from PS-II at pH 6.3, in that this state was more stable in the dark, showing a half-decay time of approximately 1.5 h at 0 degrees C, and was unable to undergo further charge accumulation. Nevertheless, a fraction of centers, probably different from those exhibiting the S2 g = 4 signal, was able to advance to the formal S3 state, giving rise to a narrow EPR signal around g = 2. Addition of the anions SO4(2-) or F- to pH 10/Cl(-)-depleted PS-II affected the properties of PS-II, resulting in EPR properties of the S2 state similar to those reported earlier following Cl- depletion treatment of PS-II in the presence of these anions. Surprisingly, after addition of F-, the g = 4 EPR signal showed a damped flash-dependent oscillation. In addition, a narrow signal around g = 2, corresponding to the formal S3 state, also showed a damped flash-dependent oscillation pattern. The presence of oscillating EPR signals (albeit damped) in F(-)-treated pH 10/Cl(-)-depleted PS-II indicates functional enzyme turnover. This was confirmed by measurements of the oxygen-evolving activity versus light intensity which indicated that in approximately 45% of oxygen-evolving centers the enzyme turnover was slowed by a factor of 2. The distinct Cl- depletion effects in PS-II observed under the two different Cl- depletion treatments are considered to reflect the presence of two distinct Cl(-)-binding sites in PS-II.

Simulation of the S2 state multiline electron paramagnetic resonance signal of photosystem II: a multifrequency approach

The S2 state electron paramagnetic resonance (EPR) multiline signal of Photosystem II has been simulated at Q-band (35 Ghz), X-band (9 GHz) and S-band (4 GHz) frequencies. The model used for the simulation assumes that the signal arises from an essentially magnetically isolated MnIII-MnIV dimer, with a ground state electronic spin ST = 1/2. The spectra are generated from exact numerical solution of a general spin Hamiltonian containing anisotropic hyperfine and quadrupolar interactions at both Mn nuclei. The features that distinguish the multiline from the EPR spectra of model manganese dimer complexes (additional width of the spectrum (195 mT), additional peaks (22), internal "superhyperfine" structure) are plausibly explained assuming an unusual ligand geometry at both Mn nuclei, giving rise to normally forbidden transitions from quadrupole interactions as well as hyperfine anisotropy. The fitted parameters indicate that the hyperfine and quadrupole interactions arise from Mn ions in low symmetry environments, corresponding approximately to the removal of one ligand from an octahedral geometry in both cases. For a quadrupole interaction of the magnitude indicated here to be present, the MnIII ion must be 5-coordinate and the MnIV 5-coordinate or possibly have a sixth, weakly bound ligand. The hyperfine parameters indicate a quasi-axial anisotropy at MnIII, which while consistent with Jahn-Teller distortion as expected for a d4 ion, corresponds here to the unpaired spin being in the ligand deficient, z direction of the molecular reference axis. The fitted parameters for MnIV are very unusual, showing a high degree of anisotropy not expected in a d3 ion. This degree of anisotropy could be qualitatively accounted for by a histidine ligand providing pi backbonding into the metal dxy orbital, together with a weakly bound or absent ligand in the x direction.

The manganese center of oxygen-evolving and calcium-depleted photosystem II: a pulsed EPR spectroscopy study

The environment of the multi-manganese center in the O2-evolving complex (OEC) of plant photosystem II (PS II) under conditions of Ca2+ depletion has been probed using pulsed electron paramagnetic resonance (EPR) spectroscopy, and the following results are reported: (1) In Ca(2+)-depleted PS II membranes treated with the chelator [ethylenebis(oxyethylenenitrilo)]tetraacetic acid (EGTA), the modified Mn EPR signal arising from the OEC in the S2 state and the split EPR signal from the S3 state could be detected in the absorption mode by recording the amplitude of a two-pulse echo as a function of the external magnetic field. The formation of the S3 signal (g approximately 2.004; delta Hpp = 164 G) is not accompanied by the disappearance of the Mn EPR signal, although the signal becomes difficult to detect in CW EPR. This result supports the previous interpretation of the split S3 EPR signal as arising from the interaction of an organic radical with the Mn cluster [Boussac, A., Zimmermann, J. L., Rutherford, A. W., & Lavergne, J. (1990) Nature 347, 303-306]. (2) The two-pulse electron spin echo envelope modulation (ESEEM) spectra of the S2 state formed in Ca(2+)-depleted PS II membranes obtained from 14N- and 15N-labeled material are different. This indicates that nitrogen nuclei from nitrogen-containing protein residues are coupled to the Mn center in the S2 state of the inhibited enzyme. In addition, comparison with the two-pulse ESEEM data obtained for the S2 state in the untreated enzyme suggests that the coupling may be altered by the Ca2+ depletion and/or EGTA treatment. (3) The treatment of Ca(2+)-depleted PS II membranes with sodium pyrophosphate also induced a stable S2 state characterized by a modified multiline EPR signal that is similar to that obtained in EGTA-treated PS II membranes. Comparison of the ESEEM data obtained for the pyrophosphate and 14N and 15N samples treated with EGTA suggests that the modification induced by the EGTA treatment is accompanied by the binding of (an) EGTA molecule(s) to or near the Mn center. (4) ESEEM data obtained for the S3 state formed in the pyrophosphate or EGTA-treated enzyme are quite similar to those obtained for the corresponding S2 state. The data are also compared with ESEEM data obtained on oxidized 4(5)-methylimidazole obtained by UV irradiation. These results are discussed with respect to the current assignment of the S3 radical as arising from oxidation of a histidine residue.

Spectroscopic characterization of photoinhibited Photosystem II and kinetic resolution of the triggering of the D reaction center protein for degradation

Photoinhibition of isolated thylakoids results in inactivation of Photosystem II electron transport and proteolysis of the D 1 reaction center protein. At low, non-freezing temperatures the mechanism of inactivation for Photosystem II electron transport can be experimentally studied without interference of secondary damaging effects, since the degradation of the D 1 -protein does not occur. Here we have applied electron paramagnetic resonance (EPR) spectroscopy to characterize the sequential events leading to inhibition of PS II electron transport and triggering of the D 1 -protein for degradation at 2 C. Two principle kinetics of inactivation and damage were observed: (i) inactivation with a half-time of 1 to 1.5 h in case of steady-state electron transport through Photosystem II, the induction of the S 2 -state multiline EPR signal, the EPR signal from Q A -Fe 2− and lowering of the F v / F m fluorescence ratio. This is explained by over-reduction of the first quinone acceptor (Q A ) leading to impairment of its function. (ii) Inhibition with a half-time of 3–4 h in case of EPR Signal II slow ; inhibition of the primary charge separation reaction and release of manganese from its site in the oxygen evolving system. We were also able, for the first time, to follow the kinetics for the triggering of the D 1 -protein for degradation. This triggering followed the slower kinetic phase and is likely to be the result of conformational changes in the protein induced by the highly reactive singlet oxygen. Additionally, a dark-stable cationic radical with g = 2.0031 and 10–11 G wide was progressively induced during the inhibition and was tentatively attributed to a carotenoid cation.

Spectroscopic characterization of photoinhibited Photosystem II and kinetic resolution of the triggering of the D 1 reaction center protein for degradation

Photoinhibition of isolated thylakoids results in inactivation of Photosystem II electron transport and proteolysis of the D 1 reaction center protein. At low, non-freezing temperatures the mechanism of inactivation for Photosystem II electron transport can be experimentally studied without interference of secondary damaging effects, since the degradation of the D 1-protein does not occur. Here we have applied electron paramagnetic resonance (EPR) spectroscopy to characterize the sequential events leading to inhibition of PS II electron transport and triggering of the D 1-protein for degradation at 2 C. Two principle kinetics of inactivation and damage were observed: (i) inactivation with a half-time of 1 to 1.5 h in case of steady-state electron transport through Photosystem II, the induction of the S 2-state multiline EPR signal, the EPR signal from Q A-Fe 2− and lowering of the F v/ F m fluorescence ratio. This is explained by over-reduction of the first quinone acceptor (Q A) leading to impairment of its function. (ii) Inhibition with a half-time of 3–4 h in case of EPR Signal II slow; inhibition of the primary charge separation reaction and release of manganese from its site in the oxygen evolving system. We were also able, for the first time, to follow the kinetics for the triggering of the D 1-protein for degradation. This triggering followed the slower kinetic phase and is likely to be the result of conformational changes in the protein induced by the highly reactive singlet oxygen. Additionally, a dark-stable cationic radical with g = 2.0031 and 10–11 G wide was progressively induced during the inhibition and was tentatively attributed to a carotenoid cation.

Nitrogen ligation to manganese in the photosynthetic oxygen-evolving complex: continuous-wave and pulsed EPR studies of photosystem II particles containing nitrogen-14 or nitrogen-15

The possibility of nitrogen ligation to the Mn in the oxygen-evolving complex from photosystem II was investigated with electron paramagnetic resonance (EPR) and electron spin echo envelope modulation (ESEEM) spectroscopies using 14N- and 15N-labeled preparations. Oxygen-evolving preparations were isolated from a thermophilic cyanobacterium, Synechococcus sp., grown on a medium containing either 14NO3- or 15NO3- as the sole source of nitrogen. the substructure on the "multiline" EPR signal, which arises from Mn in the S2 state of the enzyme, was measured with continuous-wave EPR. No changes were detected in the substructure peak positions upon substitution of 15N for 14N, indicating that this substructure is not due to superhyperfine coupling from nitrogen ligands. To detect potential nitrogen ligands with superhyperfine couplings of lesser magnitude than could be observed with conventional EPR methods, electron spin-echo envelope modulation experiments were also performed on the multiline EPR signal. The Fourier transform of the light-minus-dark time domain ESEEM data shows a peak at 4.8 MHz in 14N samples which is absent upon substitution with 15N. This gives unambiguous evidence for weak hyperfine coupling of nitrogen to the Mn of the oxygen-evolving complex. Possible origins of this nitrogen interaction are discussed.

Strong light photoinhibition of electrontransport in Photosystem II. Impairment of the function of the first quinone acceptor, Q A

Electron paramagnetic resonance (EPR) spectroscopy has been applied in an investigation on the mechanism for photoinhibition of the electron transport in Photosystem II. The experiments were performed in vitro in thylakoid membranes and preparations of Photosystem-II-enriched membranes. Photoinhibition resulted in inhibition of the oxygen evolution and EPR measurements of the S 2 state multiline EPR signal show that its induction by illumination at 198 K was decreased with the same kinetics as the oxygen evolution. Further EPR measurements show that the reduction of Q A was inhibited with the same kinetics as the oxygen evolution. The amount of photoreducible pheophytin was estimated from photoaccumulation experiments under reducing conditions and the results show that the primary charge separation reaction was inhibited much slower than the oxygen evolution or the reduction of Q A. These results indicate that photoinhibition inhibits the electron transfer between pheophytin and Q A probably by impairment of the function of Q A. In the inhibited centers the primary charge separation reaction is still operational. It is suggested that the event leading to photoinhibition of the electron transport is the double reduction of Q A which then leaves its site. Photoinhibition also results in rapid oxidation of cytochrome b-559 and a change of cytochrome b-559 from its high potential form to its low potential form. The reaction is quantitative and proceeds with the same kinetics as the inhibition of oxygen evolution. The potential shift of cytochrome b-559 suggests that photoinhibition induces early conformational changes in Photosystem II.