Multiline EPR Signal Research Articles

The Two Alternative Oxidation State Assignments of Manganese Ions: What S2 CW-EPR Multiline (ML) Signal Simulations Reveal?

Characterizing the photosystem II (PSII) sample, continuous wave electron paramagnetic resonance (CW-EPR) simulations of the S2 ML signal at X-band frequencies was our focus. This can help increase our understanding of how the manganese (Mn) atoms in the catalytic site of the PSII magnetically interact using ML signals. It can also be used to further the understanding of possible water-splitting mechanisms in the oxygen-evolving complex (OEC). The question that remains is how much does each manganese (Mn) ion contribute to the ML signal through its hyperfine interactions in the OEC? Currently, there are two proposals for the average oxidation states of the Mn ions, denoted the ‘high’ oxidation paradigm (HOP) and the ‘low’ oxidation paradigm (LOP). Majority of PSII researchers favour the HOP. Various experiments have been conducted to investigate the two alternative oxidation states, including EPR (Jin et al. in Phys Chem Chem Phys 16(17):7799–7812, https://doi.org/10.1039/c3cp55189j, 2014; Baituti in Hyperfine Interact 238(1), https://doi.org/10.1007/s10751-017-1440-8, 2017; Ioannidis et al. in Biochemistry, https://doi.org/10.1021/bi060520s, 2006). The S2 ML EPR signal simulation using the 55Mn hyperfine coupling constants, with one very large, one medium, and two small hyperfine values, fits the experimental data. The Mn1 has a large hyperfine coupling, which agrees well with earlier data by Jin et al. [19]. Three large fractional anisotropy observed on three Mn centers (Mn1,3,4), suggests the presence of three MnIII ions, and Mn2 center is likely to be MnIV ion, hence favouring the LOP (MnIII MnIV MnIII MnIII).

Electronic structure of the oxygen evolving complex in photosystem II, as revealed by55Mn Davies ENDOR studies at 2.5 K

We report the first (55)Mn pulsed ENDOR studies on the S2 state multiline spin ½ centre of the oxygen evolving complex (OEC) in Photosystem II (PS II), at temperatures below 4.2 K. These were performed on highly active samples of spinach PS II core complexes, developed previously in our laboratories for photosystem spectroscopic use, at temperatures down to 2.5 K. Under these conditions, relaxation effects which have previously hindered observation of most of the manganese ENDOR resonances from the OEC coupled Mn cluster are suppressed. (55)Mn ENDOR hyperfine couplings ranging from ∼50 to ∼680 MHz are now seen on the S2 state multiline EPR signal. These, together with complementary high resolution X-band CW EPR measurements and detailed simulations, reveal that at least two and probably three Mn hyperfine couplings with large anisotropy are seen, indicating that three Mn(III) ions are likely present in the functional S2 state of the enzyme. This suggests a low oxidation state paradigm for the OEC (mean Mn oxidation level 3.0 in the S1 state) and unexpected Mn exchange coupling in the S2 state, with two Mn ions nearly magnetically silent. Our results rationalize a number of previous ligand ESEEM/ENDOR studies and labelled water exchange experiments on the S2 state of the photosystem, in a common picture which is closely consistent with recent photo-assembly (Kolling et al., Biophys. J. 2012, 103, 313-322) and large scale computational studies on the OEC (Gatt et al., Angew. Chem., Int. Ed. 2012, 51, 12025-12028, Kurashige et al. Nat. Chem. 2013, 5, 660-666).

Participation of Glutamate-333 of the D1 Polypeptide in the Ligation of the Mn4CaO5 Cluster in Photosystem II

In the 1.9 Å structural model of photosystem II (PDB: 3ARC), the amino acid residue Glu333 of the D1 polypeptide coordinates to the oxygen-evolving Mn₄CaO₅ cluster. This residue appears to be highly significant in that it bridges the two Mn ions (Mn(B3) and the "dangling" Mn(A4)) that are also bridged by the oxygen atom O5. This oxygen atom has been proposed to be derived from one of two substrate water molecules and to become incorporated into the product dioxygen molecule during the final step in the catalytic cycle. In addition, the backbone nitrogen of D1-Glu333 interacts directly with a nearby Cl⁻ atom. To further explore the influence of this structurally unique residue on the properties of the Mn₄CaO₅ cluster, the D1-E333Q mutant of the cyanobacterium Synechocystis sp. PCC 6803 was characterized with a variety of biophysical and spectroscopic methods, including polarography, EPR, X-ray absorption, and FTIR difference spectroscopy. The kinetics of oxygen release in the mutant were essentially unchanged from those in wild-type. In addition, the oxygen flash yields exhibited normal period-four oscillations having normal S state parameters, although the yields were lower, indicative of the mutant's lower steady-state dioxygen evolution rate of approximately 30% compared to that of the wild-type. The S₁ state Mn-XANES and Mn-EXAFS and S₂ state multiline EPR signals of purified D1-E333Q PSII core complexes closely resembled those of wild-type, aside from having lower amplitudes. The S(n+1)-minus-S(n) FTIR difference spectra showed only minor alterations to the carbonyl, amide, and carboxylate stretching regions. However, the mutation eliminated a negative peak at 3663 cm⁻¹ in the weakly H-bonding O-H stretching region of the S₂-minus-S₁ FTIR difference spectrum and caused an approximately 9 cm⁻¹ downshift of the negative feature in this region of the S₁-minus-S₀ FTIR difference spectrum. We conclude that fully functional Mn₄CaO₅ clusters assemble in the presence of the D1-E333Q mutation but that the mutation decreases the yield of assembled clusters and alters the H-bonding properties of one or more water molecules or hydroxide groups that are located on or near the Mn₄CaO₅ cluster and that either deprotonate or form stronger hydrogen bonds during the S₀ to S₁ and S₁ to S₂ transitions.

Stability of the S3 and S2 State Intermediates in Photosystem II Directly Probed by EPR Spectroscopy

The stability of the S(3) and S(2) states of the oxygen evolving complex in photosystem II (PSII) was directly probed by EPR spectroscopy in PSII membrane preparations from spinach in the presence of the exogenous electron acceptor PpBQ at 1, 10, and 20 °C. The decay of the S(3) state was followed in samples exposed to two flashes by measuring the split S(3) EPR signal induced by near-infrared illumination at 5 K. The decay of the S(2) state was followed in samples exposed to one flash by measuring the S(2) state multiline EPR signal. During the decay of the S(3) state, the S(2) state multiline EPR signal first increased and then decreased in amplitude. This shows that the decay of the S(3) state to the S(1) state occurs via the S(2) state. The decay of the S(3) state was biexponential with a fast kinetic phase with a few seconds decay half-time. This occurred in 10-20% of the PSII centers. The slow kinetic phase ranged from a decay half-time of 700 s (at 1 °C) to ~100 s (at 20 °C) in the remaining 80-90% of the centers. The decay of the S(2) state was also biphasic and showed quite similar kinetics to the decay of the S(3) state. Our experiments show that the auxiliary electron donor Y(D) was oxidized during the entire experiment. Thus, the reduced form of Y(D) does not participate to the fast decay of the S(2) and S(3) states we describe here. Instead, we suggest that the decay of the S(3) and S(2) states reflects electron transfer from the acceptor side of PSII to the donor side of PSII starting in the corresponding S state. It is proposed that this exists in equilibrium with Y(Z) according to S(3)Y(Z) ⇔ S(2)Y(Z)(•) in the case of the S(3) state decay and S(2)Y(Z) ⇔ S(1)Y(Z)(•) in the case of the S(2) state decay. Two kinetic models are discussed, both developed with the assumption that the slow decay of the S(3) and S(2) states occurs in PSII centers where Y(Z) is also a fast donor to P(680)(+) working in the nanosecond time regime and that the fast decay of the S(3) and S(2) states occurs in centers where Y(Z) reduces P(680)(+) with slower microsecond kinetics. Our measurements also demonstrate that the split S(3) EPR signal can be used as a direct probe to the S(3) state and that it can provide important information about the redox properties of the S(3) state.

Effect of Ca2+/Sr2+ Substitution on the Electronic Structure of the Oxygen-Evolving Complex of Photosystem II: A Combined Multifrequency EPR, 55Mn-ENDOR, and DFT Study of the S2 State

The electronic structures of the native Mn(4)O(x)Ca cluster and the biosynthetically substituted Mn(4)O(x)Sr cluster of the oxygen evolving complex (OEC) of photosystem II (PSII) core complexes isolated from Thermosynechococcus elongatus, poised in the S(2) state, were studied by X- and Q-band CW-EPR and by pulsed Q-band (55)Mn-ENDOR spectroscopy. Both wild type and tyrosine D less mutants grown photoautotrophically in either CaCl(2) or SrCl(2) containing media were measured. The obtained CW-EPR spectra of the S(2) state displayed the characteristic, clearly noticeable differences in the hyperfine pattern of the multiline EPR signal [Boussac et al. J. Biol. Chem.2004, 279, 22809-22819]. In sharp contrast, the manganese ((55)Mn) ENDOR spectra of the Ca and Sr forms of the OEC were remarkably similar. Multifrequency simulations of the X- and Q-band CW-EPR and (55)Mn-pulsed ENDOR spectra using the Spin Hamiltonian formalism were performed to investigate this surprising result. It is shown that (i) all four manganese ions contribute to the (55)Mn-ENDOR spectra; (ii) only small changes are seen in the fitted isotropic hyperfine values for the Ca(2+) and Sr(2+) containing OEC, suggesting that there is no change in the overall spin distribution (electronic coupling scheme) upon Ca(2+)/Sr(2+) substitution; (iii) the changes in the CW-EPR hyperfine pattern can be explained by a small decrease in the anisotropy of at least two hyperfine tensors. It is proposed that modifications at the Ca(2+) site may modulate the fine structure tensor of the Mn(III) ion. DFT calculations support the above conclusions. Our data analysis also provides strong support for the notion that in the S(2) state the coordination of the Mn(III) ion is square-pyramidal (5-coordinate) or octahedral (6-coordinate) with tetragonal elongation. In addition, it is shown that only one of the currently published OEC models, the Siegbahn structure [Siegbahn, P. E. M. Acc. Chem. Res.2009, 42, 1871-1880, Pantazis, D. A. et al. Phys. Chem. Chem. Phys.2009, 11, 6788-6798], is consistent with all data presented here. These results provide important information for the structure of the OEC and the water-splitting mechanism. In particular, the 5-coordinate Mn(III) is a potential site for substrate 'water' (H(2)O, OH(-)) binding. Its location within the cuboidal structural unit, as opposed to the external 'dangler' position, may have important consequences for the mechanism of O-O bond formation.

Participation of Glutamate-354 of the CP43 Polypeptide in the Ligation of Manganese and the Binding of Substrate Water in Photosystem II

In the current X-ray crystallographic structural models of photosystem II, Glu354 of the CP43 polypeptide is the only amino acid ligand of the oxygen-evolving Mn(4)Ca cluster that is not provided by the D1 polypeptide. To further explore the influence of this structurally unique residue on the properties of the Mn(4)Ca cluster, the CP43-E354Q mutant of the cyanobacterium Synechocystis sp. PCC 6803 was characterized with a variety of biophysical and spectroscopic methods, including polarography, EPR, X-ray absorption, FTIR, and mass spectrometry. The kinetics of oxygen release in the mutant were essentially unchanged from those in wild type. In addition, the oxygen flash yields exhibited normal period four oscillations having normal S state parameters, although the yields were lower, correlating with the mutant's lower steady-state rate (approximately 20% compared to wild type). Experiments conducted with H(2)(18)O showed that the fast and slow phases of substrate water exchange in CP43-E354Q thylakoid membranes were accelerated 8.5- and 1.8-fold, respectively, in the S(3) state compared to wild type. Purified oxygen-evolving CP43-E354Q PSII core complexes exhibited a slightly altered S(1) state Mn-EXAFS spectrum, a slightly altered S(2) state multiline EPR signal, a substantially altered S(2)-minus-S(1) FTIR difference spectrum, and an unusually long lifetime for the S(2) state (>10 h) in a substantial fraction of reaction centers. In contrast, the S(2) state Mn-EXAFS spectrum was nearly indistinguishable from that of wild type. The S(2)-minus-S(1) FTIR difference spectrum showed alterations throughout the amide and carboxylate stretching regions. Global labeling with (15)N and specific labeling with l-[1-(13)C]alanine revealed that the mutation perturbs both amide II and carboxylate stretching modes and shifts the symmetric carboxylate stretching modes of the α-COO(-) group of D1-Ala344 (the C-terminus of the D1 polypeptide) to higher frequencies by 3-4 cm(-1) in both the S(1) and S(2) states. The EPR and FTIR data implied that 76-82% of CP43-E354Q PSII centers can achieve the S(2) state and that most of these can achieve the S(3) state, but no evidence for advancement beyond the S(3) state was observed in the FTIR data, at least not in a majority of PSII centers. Although the X-ray absorption and EPR data showed that the CP43-E354Q mutation only subtly perturbs the structure and spin state of the Mn(4)Ca cluster in the S(2) state, the FTIR and H(2)(18)O exchange data show that the mutation strongly influences other properties of the Mn(4)Ca cluster, altering the response of numerous carboxylate and amide groups to the increased positive charge that develops on the cluster during the S(1) to S(2) transition and weakening the binding of both substrate water molecules (or water-derived ligands), especially the one that exchanges rapidly in the S(3) state. The FTIR data provide evidence that CP43-Glu354 coordinates to the Mn(4)Ca cluster in the S(1) state as a bridging ligand between two metal ions but provide no compelling evidence that this residue changes its coordination mode during the S(1) to S(2) transition. The H(2)(18)O exchange data provide evidence that CP43-Glu354 interacts with the Mn ion that ligates the substrate water molecule (or water-derived ligand) that is in rapid exchange in the S(3) state.

An Alternative Method for Calcium Depletion of the Oxygen Evolving Complex of Photosystem II As Revealed by the Dark-Stable Multiline EPR Signal

The dark-stable multiline EPR signal of photosystem II (PSII) is associated with a slow-decaying S(2) state that is due to Ca(2+) loss from the oxygen evolving complex. Formation of the signal was observed in intact PSII in the presence of 100-250 mM NaCl at pH 5.5. Both moderately high NaCl concentration and decreased pH were required for its appearance in intact PSII. It was estimated that only a portion of oxygen evolving complexes was responsible for the signal (about 20% in 250 mM NaCl), based on the loss of the normal S(2)-state multiline signal. The formation of the dark-stable multiline signal in intact PSII at pH 5.5 could be reversed by addition of 15 mM Ca(2+) in the presence of moderately high NaCl, confirming that it was the absence of Ca(2+) that led to its appearance. Formation of the dark-stable multiline signal in NaCl-washed PSII, which lacks the PsbP (23 kDa) and PsbQ (17 kDa) subunits, was observed in about 80% of the sample in the presence of 150 mM NaCl at pH 5.5, but some signal was also observed under normal buffer conditions. In both intact and NaCl-washed PSII, the S(2)Y(Z). signal, which is also characteristic of Ca(2+) depletion, appeared upon subsequent illumination. Formation of the dark-stable multiline signal took place in the absence of Ca(2+) chelator or polycarboxylic acids, indicating that the signal did not require their direct binding as has been proposed previously. The conditions used here were milder than those used to produce the signal in previous studies and included a preillumination protocol to maximize the dark-stable S(2) state. Based on these conditions, it is suggested that Ca(2+) release occurred through protonation of key residues that coordinate Ca(2+) at low pH, followed by displacement of Ca(2+) with Na(+) by mass action at the moderately high NaCl concentration.

Substitution of chloride by bromide modifies the low-temperature tyrosine Z oxidation in active photosystem II

Chloride is an essential cofactor for photosynthetic water oxidation. However, its location and functional roles in active photosystem II are still a matter of debate. We have investigated this issue by studying the effects of Cl − replacement by Br − in active PSII. In Br − substituted samples, Cl − is effectively replaced by Br − in the presence of 1.2 M NaBr under room light with protection of anaerobic atmosphere followed by dialysis. The following results have been obtained. i) The oxygen-evolving activities of the Br −-PSII samples are significantly lower than that of the Cl −-PSII samples; ii) The same S 2 multiline EPR signals are observed in both Br − and Cl −-PSII samples; iii) The amplitudes of the visible light induced S 1Tyr Z • and S 2Tyr Z • EPR signals are significantly decreased after Br − substitution; the S 1Tyr Z • EPR signal is up-shifted about 8 G, whereas the S 2Tyr Z • signal is down-shifted about 12 G after Br − substitution. These results imply that the redox properties of Tyr Z and spin interactions between Tyr Z • and Mn-cluster could be significantly modified due to Br − substitution. It is suggested that Cl −/Br − probably coordinates to the Ca 2+ ion of the Mn-cluster in active photosystem II.

Characteristics of the Dark-Stable Multiline EPR Signal of Ca2+-Depleted Photosystem II

Photosystem II (PSII), which produces molecular oxygen using energy from light absorption, requires Ca2+ and Cl- ions as inorganic cofactors. PSII shows two electron paramagnetic resonance signals that have been associated with the depletion or disruption of Ca2+ at the catalytic Mn4Ca cluster: a dark-stable multiline signal from an S2 state that decays very slowly, and a broad metalloradical signal from an S2YZ state that is unable to proceed to higher oxidation states. The conditions for their formation were explored to help clarify how they are correlated. The dark-stable multiline signal was found to form in PSII prepared at pH 5.5 using itaconic acid buffer, a relative of citrate. The signal was very similar to the previously reported signal that is observed after EDTA treatment of PSII lacking the PsbP and PsbQ subunits. Both of these treatments, which employ Ca2+ chelators, also resulted in formation of the S2YZ signal when PSII was illuminated in the presence of an electron acceptor. Treatment of intact PSII with fluoride, which is a competitor of Cl- activation, resulted in formation of the S2YZ signal, but not the dark-stable multiline signal. Fluoride may also interfere with Ca2+ function as a result of the high stability of the CaF2 complex. These findings are examined in relation to the requirements of PSII for Ca2+ and Cl-. (Supported by UNCG Office of Research).

Elucidating the site of action of oxalate in photosynthetic electron transport chain in spinach thylakoid membranes

The effects of oxalate on PS II and PS I photochemistry were studied. The results suggested that in chloride-deficient thylakoid membranes, oxalate inhibited activity of PS II as well as PS I. To our knowledge, this is the only anion so far known which inhibits both the photosystems. Measurements of fluorescence induction kinetics, YZ* decay, and S2 state multiline EPR signal suggested that oxalate inhibited PS II at the donor side most likely on the oxygen evolving complex. Measurements of re-reduction of P700+ signal in isolated PS I particles in oxalate-treated samples suggested a binding site of oxalate on the donor, as well as the acceptor side of PS I.

Direct quantification of the four individual S states in Photosystem II using EPR spectroscopy

EPR spectroscopy is very useful in studies of the oxygen evolving cycle in Photosystem II and EPR signals from the CaMn 4 cluster are known in all S states except S 4. Many signals are insufficiently understood and the S 0, S 1, and S 3 states have not yet been quantifiable through their EPR signals. Recently, split EPR signals, induced by illumination at liquid helium temperatures, have been reported in the S 0, S 1, and S 3 states. These split signals provide new spectral probes to the S state chemistry. We have studied the flash power dependence of the S state turnover in Photosystem II membranes by monitoring the split S 0, split S 1, split S 3 and S 2 state multiline EPR signals. We demonstrate that quantification of the S 1, S 3 and S 0 states, using the split EPR signals, is indeed possible in samples with mixed S state composition. The amplitudes of all three split EPR signals are linearly correlated to the concentration of the respective S state. We also show that the S 1 → S 2 transition proceeds without misses following a saturating flash at 1 °C, whilst substantial misses occur in the S 2 → S 3 transition following the second flash.

Formation Spectra of the EPR Split Signals from the S0, S1, and S3 States in Photosystem II Induced by Monochromatic Light at 5 K

The interaction EPR split signals from photosystem II (PSII) have been reported from the S0, S1, and S3 states. The signals are induced by illumination at cryogenic temperatures and are proposed to reflect the magnetic interaction between YZ* and the Mn4Ca cluster. We have investigated the formation spectra of these split EPR signals induced in PSII enriched membranes at 5 K using monochromatic laser light from 400 to 900 nm. We found that the formation spectra of the split S0, split S1, and split S3 EPR signals were quite similar, but not identical, between 400 and 690 nm, with maximum formation at 550 nm. The major deviations were found between 440 and 480 nm and between 580 and 680 nm. In the regions around 460 and 680 nm the amplitudes of the formation spectra were 25-50% of that at 550 nm. A similar formation spectrum was found for the S2-state multiline EPR signal induced at 0 degrees C. In general, the formation spectra of these signals in the visible region resemble the reciprocal of the absorption spectra of our PSII membranes. This reflects the high chlorophyll concentration necessary for the EPR measurements which mask the spectral properties of other absorbing species. No split signal formation was found by the application of infrared laser illumination between 730 and 900 nm from PSII in the S0 and S1 states. However, when such illumination was applied to PSII membranes poised in the S3 state, formation of the split S3 EPR signal was observed with maximum formation at 740 nm. The quantum yield was much less than in the visible region, but the application of intensive illumination at 830 nm resulted in accumulation of the signal to an amplitude comparable to that obtained with illumination with visible light. The split S3 EPR signal induced by NIR light was much more stable at 5 K (no observable decay within 60 min) than the split S3 signal induced by visible light (50% of the signal decayed within 30 min). The split S3 signals induced by each of these light regimes showed the same EPR spectral features and microwave power saturation properties, indicating that illumination of PSII in the S3 state by visible light or by NIR light produces a similar configuration of YZ* and the Mn4Ca cluster.

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

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

Enhancement of Y D• spin relaxation by the CaMn 4 cluster in photosystem II detected at room temperature: A new probe for the S-cycle

The long-lived, light-induced radical Y D • of the Tyr161 residue in the D2 protein of Photosystem II (PSII) is known to magnetically interact with the CaMn 4 cluster, situated ∼ 30 Å away. In this study we report a transient step-change increase in Y D • EPR intensity upon the application of a single laser flash to S 1 state-synchronised PSII-enriched membranes from spinach. This transient effect was observed at room temperature and high applied microwave power (100 mW) in samples containing PpBQ, as well as those containing DCMU. The subsequent decay lifetimes were found to differ depending on the additive used. We propose that this flash-induced signal increase was caused by enhanced spin relaxation of Y D • by the OEC in the S 2 state, as a consequence of the single laser flash turnover. The post-flash decay reflected S 2 → S 1 back-turnover, as confirmed by their correlations with independent measurements of S 2 multiline EPR signal and flash-induced variable fluorescence decay kinetics under corresponding experimental conditions. This flash-induced effect opens up the possibility to study the kinetic behaviour of S-state transitions at room temperature using Y D • as a probe.

Evidence That Azide Occupies the Chloride Binding Site near the Manganese Cluster in Photosystem II

The effect of adding azide to photosystem II (PS II) membrane samples (BBY preparation), with or without chloride, has been investigated using continuous wave (CW) and pulsed EPR spectroscopy. In the BBY samples with 25 mM chloride, we observed that the inhibition induced by azide is partly recovered by the addition of bicarbonate. Electron spin-echo envelope modulation (ESEEM) was used to search for spin transitions of 15N nuclei magnetically coupled to the S2 state Mn cluster (multiline EPR signal form) in 15N (single terminal label) azide-treated samples with negative results. However, an 15N ESEEM peak was observed in parallel chloride-depleted PS II samples when the 15N-labeled azide is added. However, this peak is absent in chloride-depleted samples incubated in buffer containing both chloride and [15N]azide. Thus these results demonstrate an azide binding site in the immediate vicinity of the Mn cluster, and since this site appears to be competitive with chloride, these results provide further evidence that chloride is bound proximal to the Mn cluster as well. Discussion on the possible interplay between azide, chloride, and bicarbonate is provided.

Effects of Ethylene Glycol and Methanol on Ammonia-Induced Structural Changes of the Oxygen-Evolving Complex in Photosystem II

Ammonia is an inhibitor of water oxidation and a structural analogue for substrate water, making it a valuable probe for the structural properties of the possible substrate-binding site on the oxygen-evolving complex (OEC) in photosystem II (PSII). By using the NH(3)-induced upshift of the 1365 cm(-)(1) IR mode in the S(2)Q(A)(-)/S(1)Q(A) spectrum and the NH(3)-modified S(2) state EPR signals of PSII as spectral probes, we found that ethylene glycol has clear effects on the binding properties of the NH(3)-specific site on the OEC. Our results show that in PSII samples containing 30% (v/v) ethylene glycol, the affinity of the NH(3)-specific binding site on the OEC is estimated to be more than 10 times lower than that in PSII samples containing 0.4 M sucrose. In addition, our results show that the NH(3)-induced upshift of the 1365 cm(-)(1) IR mode in the S(2)Q(A)(-)/S(1)Q(A) spectrum is dependent on the concentration of ethylene glycol, but not dependent on the concentration of sucrose (up to 1.5 M) or methanol (up to 5.4 M). By comparing the concentration dependence of sucrose and ethylene glycol on NH(3)-induced spectral change and also by comparing the sucrose and ethylene glycol data at similar concentrations ( approximately 1 M), we conclude that ethylene glycol has a clear effect on the NH(3)-induced spectral changes. Furthermore, our results also show that ethylene glycol alters the steric requirement of the amine effect on the upshift of the 1365 cm(-)(1) mode in the S(2)Q(A)(-)/S(1)Q(A) spectrum. In PSII samples containing 30% (v/v) ethylene glycol, only NH(3), not other bulkier amines (e.g., Tris, AEPD, and CH(3)NH(2)), has a clear effect on the upshift of the 1365 cm(-)(1) mode in the S(2)Q(A)(-)/S(1)Q(A) spectrum; in contrast, in PSII samples containing 0.4 M sucrose, both NH(3) and CH(3)NH(2) have a clear effect. On the basis of the results mentioned above, we propose that ethylene glycol acts directly or indirectly to decrease the affinity or limit the accessibility of NH(3) and CH(3)NH(2) to the NH(3)-specific binding site on the OEC in PSII. Finally, we also applied the same approach to test whether methanol is able to compete with ammonia on its binding site on the OEC. We found that 4% (v/v) methanol does not have any significant effect on the NH(3)-induced upshift of the 1365 cm(-)(1) mode in the S(2)Q(A)(-)/S(1)Q(A) spectrum and the NH(3)-modified S(2) state g = 2 multiline EPR signal. Our results suggest that methanol is unable to compete with NH(3) upon binding to the Mn site of the OEC that gives rise to the altered S(2) state g = 2 multiline EPR signal.

Specific loss of the extrinsic 18 KDa protein from Photosystem II upon heating to 47 °C causes inactivation of oxygen evolution likely due to Ca release from the Mn-complex

Exposure of Photosystem II (PS II) membrane particles from spinach to a temperature of 47 degrees C caused the rapid release of the 18 kDa protein in parallel to inactivation of oxygen evolution. Previously, it has been suggested that the first heat-jump response involves rapid Ca release from the Mn complex of O2-evolution, followed by the slower release of (2 + 2) MnII ions [Pospisil P et al. (2003) Biophys J 84: 1370-1386]. Here, the predicted biphasic MnII release to the bulk was verified by atomic absorption spectroscopy (AAS). Analysis of laser flash-induced delayed fluorescence transients suggests that the loss of the essential Ca ion from the Mn4Ca complex in the dark is due to the loss of the 18 kDa protein. The S2-state multiline EPR signal of the Mn complex was still generated in heat-treated PS II presumably lacking Ca, but retaining four Mn ions.

Interactions of Chloride and Formate at the Donor and the Acceptor Side of Photosystem II

Chloride is required for the maximum activity of the oxygen evolving complex (OEC) while formate inhibits the function of OEC. On the basis of the measurements of oxygen evolution rates and the S(2) state multiline EPR signal, an interaction between the action of chloride and formate at the donor side of PS II has been suggested. Moreover, the Fe(2)+Q-A EPR signals were measured to investigate a common binding site of both these anions at the PS II acceptor side. Other monovalent anions like bromide, nitrate etc. could influence the effects of formate to a small extent at the donor side of PS II, but not significantly at the acceptor side of PS II. The results presented in this paper clearly suggest a competitive binding of formate and chloride at the PS II acceptor side.

Molecular interference of Cd 2+ with Photosystem II

Many heavy metals inhibit electron transfer reactions in Photosystem II (PSII). Cd 2+ is known to exchange, with high affinity in a slow reaction, for the Ca 2+ cofactor in the Ca/Mn cluster that constitutes the oxygen-evolving center. This results in inhibition of photosynthetic oxygen evolution. There are also indications that Cd 2+ binds to other sites in PSII, potentially to proton channels in analogy to heavy metal binding in photosynthetic reaction centers from purple bacteria. In search for the effects of Cd 2+-binding to those sites, we have studied how Cd 2+ affects electron transfer reactions in PSII after short incubation times and in sites, which interact with Cd 2+ with low affinity. Overall electron transfer and partial electron transfer were studied by a combination of EPR spectroscopy of individual redox components, flash-induced variable fluorescence and steady state oxygen evolution measurements. Several effects of Cd 2+ were observed: (i) the amplitude of the flash-induced variable fluorescence was lost indicating that electron transfer from Y Z to P 680 + was inhibited; (ii) Q A − to Q B electron transfer was slowed down; (iii) the S 2 state multiline EPR signal was not observable; (iv) steady state oxygen evolution was inhibited in both a high-affinity and a low-affinity site; (v) the spectral shape of the EPR signal from Q A −Fe 2+ was modified but its amplitude was not sensitive to the presence of Cd 2+. In addition, the presence of both Ca 2+ and DCMU abolished Cd 2+-induced effects partially and in different sites. The number of sites for Cd 2+ binding and the possible nature of these sites are discussed.

Ammonia-induced structural changes of the oxygen-evolving complex in photosystem II as revealed by light-induced FTIR difference spectroscopy.

Light-induced Fourier transform infrared difference spectroscopy has been applied to studies of ammonia effects on the oxygen-evolving complex (OEC) of photosystem II (PSII). We found that NH(3) induced characteristic spectral changes in the region of the symmetric carboxylate stretching modes (1450-1300 cm(-1)) of the S(2)Q(A)(-)/S(1)Q(A) FTIR difference spectra of PSII. The S(2) state carboxylate mode at 1365 cm(-1) in the S(2)Q(A)(-)/S(1)Q(A) spectrum of the controlled samples was very likely upshifted to 1379 cm(-1) in that of NH(3)-treated samples; however, the frequency of the corresponding S(1) carboxylate mode at 1402 cm(-1) in the same spectrum was not significantly affected. These two carboxylate modes have been assigned to a Mn-ligating carboxylate whose coordination mode changes from bridging or chelating to unidentate ligation during the S(1) to S(2) transition [Noguchi, T., Ono, T., and Inoue, Y. (1995) Biochim. Biophys. Acta 1228, 189-200; Kimura, Y., and Ono, T.-A. (2001) Biochemistry 40, 14061-14068]. Therefore, our results show that NH(3) induced significant structural changes of the OEC in the S(2) state. In addition, our results also indicated that the NH(3)-induced spectral changes of the S(2)Q(A)(-)/S(1)Q(A) spectrum of PSII are dependent on the temperature of the FTIR measurement. Among the temperatures we measured, the strongest effect was seen at 250 K, a lesser effect was seen at 225 K, and little or no effect was seen at 200 K. Furthermore, our results also showed that the NH(3) effects on the S(2)Q(A)(-)/S(1)Q(A) spectrum of PSII are dependent on the concentrations of NH(4)Cl. The NH(3)-induced upshift of the 1365 cm(-1) mode is apparent at 5 mM NH(4)Cl and is completely saturated at 100 mM NH(4)Cl concentration. Finally, we found that CH(3)NH(2) has a small but clear effect on the spectral change of the S(2)Q(A)(-)/S(1)Q(A) FTIR difference spectrum of PSII. The effects of amines on the S(2)Q(A)(-)/S(1)Q(A) FTIR difference spectra (NH(3) > CH(3)NH(2) > AEPD and Tris) are inverse proportional to their size (Tris approximately AEPD > CH(3)NH(2) > NH(3)). Therefore, our results showed that the effects of amines on the S(2)Q(A)(-)/S(1)Q(A) spectrum of PSII are sterically selective for small amines. On the basis of the correlations between the conditions (dependences on the excitation temperature and NH(3) concentration and the steric requirement for the amine effects) that give rise to the NH(3)-induced upshift of the 1365 cm(-)(1) mode in the S(2)Q(A)(-)/S(1)Q(A) spectrum of PSII and the conditions that give rise to the altered S(2) state multiline EPR signal, we propose that the NH(3)-induced upshift of the 1365 cm(-1) mode is caused by the binding of NH(3) to the site on the Mn cluster that gives rise to the altered S(2) state multiline EPR signal. In addition, we found no significant NH(3)-induced change in the S(2)Q(A)(-)/S(1)Q(A) FTIR difference spectrum at 200 K. Under this condition, the OEC gives rise to the NH(3)-stabilized g = 4.1 EPR signal and a suppressed g = 2 multiline EPR signal. Our results suggest that the structural difference of the OEC between the normal g = 2 multiline form and the NH(3)-stabilized g = 4.1 form is small.