S2-state Multiline EPR Signal Research Articles

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-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.

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.

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.

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.

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

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

Does histidine 332 of the D1 polypeptide ligate the manganese cluster in photosystem II? An electron spin echo envelope modulation study.

The tetranuclear manganese cluster in photosystem II is ligated by one or more histidine residues, as shown by an electron spin echo envelope modulation (ESEEM) study conducted with [(15)N]histidine-labeled photosystem II particles isolated from the cyanobacterium Synechocystis sp. strain PCC 6803 [Tang, X.-S., Diner, B. A., Larsen, B. S., Gilchrist, M. L., Jr., Lorigan, G. A., and Britt, R. D. (1994) Proc. Natl. Acad. Sci. U.S.A. 91, 704-708]. One of these residues may be His332 of the D1 polypeptide. Photosystem II particles isolated from the Synechocystis mutant D1-H332E exhibit an altered S(2) state multiline EPR signal that has more hyperfine lines and narrower splittings than the corresponding signal in wild-type PSII particles [Debus, R. J., Campbell, K. A., Peloquin, J. M., Pham, D. P., and Britt, R. D. (2000) Biochemistry 39, 470-478]. These D1-H332E PSII particles are also unable to advance beyond an altered S(2)Y(Z)(*) state, and the quantum yield for forming the S(2) state is very low, corresponding to an 8000-fold slowing of the rate of Mn oxidation by Y(Z)(*). These observations are consistent with His332 being close to the Mn cluster and modulating the redox properties of both the Mn cluster and tyrosine Y(Z). To determine if D1-His332 ligates the Mn cluster, we have conducted an ESEEM study of D1-H332E PSII particles. The histidyl nitrogen modulation observed near 5 MHz in ESEEM spectra of the S(2) state multiline EPR signal of wild-type PSII particles is substantially diminished in D1-H332E PSII particles. This result is consistent with ligation of the Mn cluster by D1-His332. However, alternate explanations are possible. These are presented and discussed.

The tetranuclear manganese cluster in photosystem II: location and magnetic properties of the S2 state as determined by saturation-recovery EPR spectroscopy.

The spin-lattice relaxation enhancement of the dark-stable tyrosine radical, YD., by the S2 state of the O2-evolving complex (OEC) of photosystem II (PSII) has been measured by using saturation-recovery EPR spectroscopy. Two forms of the S2 state have been compared: the multiline EPR signal species in untreated PSII and the altered multiline EPR signal species in NH3-treated PSII. Previous work has shown that the non-single-exponential spin-lattice relaxation kinetics of YD. in S2-state PSII result from a dipole-dipole interaction with the Mn4 cluster of the OEC. By taking into account the temperature variation of the effective magnetic moment of the S2-state multiline EPR signal form of the OEC, we provide a quantitative analysis of its temperature-dependent enhancement of the spin-lattice relaxation of YD.. Different spin states of the Mn4 cluster in the S2 state are responsible for the effect at different temperature regimes: for T </= 10 K, it is the ground spin state (S = 1/2); for T >/= 30 K, it is the first excited spin state; and at intermediate temperatures, the contributions of the two spin states are comparable. The relaxation enhancement of YD. is equivalent for both forms of the S2-state multiline EPR signal examined, indicating that the magnetic properties of the Mn4 cluster are very similar in the S2 state for both untreated and NH3-treated PSII. EPR progressive microwave-power saturation has also been used to assess the spin-lattice relaxation properties of the Mn4 cluster giving the altered S2-state multiline EPR signal in the NH3 derivative of PSII. The Orbach mechanism is shown to provide the dominant relaxation pathway; the energy difference between the ground and first excited spin states is estimated to be 30 +/- 2 cm-1, which is very similar to the value found for the S2-state multiline EPR signal species in untreated PSII. Below 4 K, the effectiveness of the S2-state multiline EPR signal species as a spin relaxation enhancer of YD. drops dramatically. This is interpreted to occur because of temperature-dependent 55Mn nuclear spin-lattice relaxation which causes averaging of the effective Larmor frequency of the S2-state multiline EPR signal species during the time scale for spin-lattice relaxation of YD.; because the line shape of the S2-state multiline EPR signal is dominated by isotropic 55Mn nuclear hyperfine splittings, such nuclear relaxation processes allow frequencies in near resonance with that of YD. to be accessed, thereby producing a greater relaxation enhancement. By using a dipolar model that includes the line shapes of both the YD. and S2-state multiline EPR signals, the spin-lattice relaxation enhancement of YD. is analyzed to obtain a lower limit of 22 A for the distance between YD. and the OEC. Together with recent studies showing a close proximity of the Mn4 cluster to YZ., these results provide further support for an asymmetric location of the Mn4 cluster with respect to the two redox-active tyrosines in PSII.

Reversible binding of nitric oxide to tyrosyl radicals in photosystem II. Nitric oxide quenches formation of the S3 EPR signal species in acetate-inhibited photosystem II.

Continuous illumination at temperatures above 250 K of photosystem II samples which have been depleted of calcium or chloride or treated with fluoride, acetate, or ammonia results in production of a broad radical EPR signal centered at g = 2.0. This EPR signal, called the S3 EPR signal, has been attributed to an organic radical interacting with the S2 state of the oxygen-evolving complex to give the species S2X+ (X+ = organic radical). A tyrosine radical has been proposed as the species responsible for the S3 EPR signal. On the basis of experiments demonstrating that nitric oxide binds reversibly to the tyrosyl radical in ribonucleotide reductase, nitric oxide has been used to probe the S3 EPR signal in acetate-treated photosystem II. In experiments using manganese-depleted photosystem II, nitric oxide was found to bind reversibly to both redox-active tyrosines, YD* and YZ*, to form EPR-silent adducts. Next, acetate-treated photosystem II was illuminated to form the S3 EPR signal in the presence of nitric oxide to test whether the S3 EPR signal behaves like YZ*. Under conditions that produce the maximum yield of the S3 EPR signal in acetate-treated photosystem II, no S3 EPR signal was observed in the presence of nitric oxide. Upon removal of nitric oxide, the S3 EPR signal could be induced. Quenching of the S3 EPR signal by nitric oxide yielded an S2-state multiline EPR signal. Its amplitude was 45% of that found for uninhibited photosystem II illuminated at 200 K; this yield is the same as the yield of the S3 EPR signal under equivalent conditions but without nitric oxide. These results suggest that the S3 EPR signal is due to the configuration S2YZ* in which the S2 state of the oxygen-evolving complex gives a broadened multiline EPR signal as a result of exchange and dipolar interactions with YZ*. The binding of nitric oxide to YZ* to form a diamagnetic YZ-NO species uncouples the S2 state from YZ*, yielding a noninteracting S2-state multiline EPR signal species.

Chemical oxidation and reduction of the O2-evolution center in Photosystem II

Treatment of Photosystem II (PS II) with low concentrations of hydroxylamine is known to cause a two-flash delay in the O2-evolution pattern, and in the formation of the S2-state multiline EPR signal, due to the two-electron reduction of the S1-state by hydroxylamine to form the S-1-state. Past work has shown that these delays are not reversed by washing out the hydroxylamine nor by adding DCBQ or ferricyanide to oxidize the residual hydroxylamine, but are reversed by illumination with two saturating flashes followed by a 30-min dark incubation. We have examined the effects of treatments aimed at restoring the normal flash-induced O2-evolution pattern and S2-state multiline EPR signal after treatment of PS II with 40 μM hydroxylamine. In agreement with past work, we find that the two-flash delay in O2 evolution is not reversed when the hydroxylamine is removed by three cycles of centrifugation and resuspension in hydroxylamine-free buffer nor by adding ferricyanide or DCBQ to oxidize the unreacted hydroxylamine. However, the normal flash-induced O2-evolution pattern is restored by illumination with two saturating flashes followed by a 30-min dark incubation (after the sample was first treated with 40 μM hydroxylamine and the unreacted hydroxylamine was removed); illumination with one saturating flash followed by a 30-min dark incubation is only partially effective. These results show that ferricyanide and DCBQ are not effective at oxidizing the S-1-state to the S1-state. In contrast, adding hypochlorite (OCl(-)) after treatment with hydroxylamine restored the normal flash-induced O2-evolution pattern and also restored the formation of the S2-state multiline EPR signal by illumination at 200 K. We conclude that hypochlorite is capable of oxidizing the S-1-state to the S1-state. This is the first example of a chemical treatment that advances the delayed flash-induced O2 evolution pattern.

Formation and flash-dependent oscillation of the S2-state multiline EPR signal in an oxygen-evolving Photosystem-II preparation lacking the three extrinsic proteins in the oxygen-evolving system

In PS-II-enriched membranes lacking the three extrinsic water-soluble proteins in the oxygen-evolving system (18, 24 and 33 kDa), but still evolving oxygen to some extent, the formation of the multiline EPR signal originating from the S2-state is dependent on the concentration of Cl−. In 200 mM Cl− the multiline signal was observed after the first flash and oscillated with the flash number with a period of four. At 20 mM Cl− no signal could be observed in this material. These results suggest that the extrinsic proteins are not necessary for multiline signal formation and that complete advancement through the S-states can occur in their absence when sufficient Cl− is present.