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International audience; Iron is an essential nutrient in bacteria. Its ferrous form, mostly present in low oxygen and acidic pH environments, can be imported using the specific Ftr-type transport system, which encompasses the conserved FtrABCD system found in pathogenic bacteria such as Bordetella, Brucella and Burkholderia. The nonpathogenicity and versatile metabolism of Rubrivivax gelatinosus make it an ideal model to study the FtrABCD system. Here, we report a new aspect of its regulation and the role of the periplasmic proteins FtrA and FtrB using in vivo and in vitro approaches. We investigated the metal binding mode and redox state of copper and iron to FtrA by crystallography and biophysical methods. An ‘as isolated’ FtrA protein from the bacterial periplasm contained a copper ion (Cu$^+$) identified by electron paramagnetic resonance (EPR). Copper is coordinated by four conserved side chains (His and Met) in the primary metal site. Structural analysis of R. gelatinosus FtrA and FtrA homologues revealed that copper binding induces a rearrangement of the His95 imidazole ring, releasing thereafter space, as well as both Asp45 and Asp92 side chains, for iron binding in the secondary metal site. EPR highlighted that FtrA can oxidize the bound ferrous ion into the ferric form by reducing the bound Cu$^{2+}$ into Cu$^+$, both metal sites being separated by 7 Å. Finally, we showed that FtrB binds iron and not copper. These results provide new insights into the mechanism of ferrous iron utilization by the conserved FtrABCD iron transporter for which we propose a new functional model.

International audience; The Mn4 CaO5 cluster of Photosystem II (PSII) advances sequentially through five oxidation states (S0 to S4 ). Under the enzyme cycle, two water molecules are oxidized, O2 is generated and four protons are released into the lumen. Umena et al. (2011) have proposed that, with other charged amino acids, the R323 residue of the D1 protein could contribute to regulate a proton egress pathway from the Mn4 CaO5 cluster and TyrZ via a proton channel identified from the 3D structure. To test this suggestion, a PsbA3/R323E site-directed mutant has been constructed and the properties of its PSII have been compared to those of the PsbA3-PSII by using EPR spectroscopy, polarography, thermoluminescence and time-resolved UV-visible absorption spectroscopy. Neither the oscillations with a period four nor the kinetics and S-state-dependent stoichiometry of the proton release were affected. However, several differences have been found: i) the P680+ decay in the hundreds of ns time domain was much slower in the mutant, ii) the S2 QA- /DCMU and S3 QA- /DCMU radiative charge recombination occurred at higher temperatures and iii) the S0 TyrZ• , S1 TyrZ• , S2 TyrZ• split EPR signals induced at 4.2 K by visible light from the S0 TyrZ , S1 TyrZ , S2 TyrZ , respectively, and the (S2 TyrZ• )' induced by NIR illumination at 4.2 K of the S3 TyrZ state differed. It is proposed that the R323 residue of the D1 protein interacts with TyrZ likely via the H-bond network previously proposed to be a proton channel. Therefore, rather than participating in the egress of protons to the lumen, this channel could be involved in the relaxations of the H-bonds around TyrZ by interacting with the bulk, thus tuning the driving force required for TyrZ oxidation. This article is protected by copyright. All rights reserved.

In Photosystem II (PSII), the Mn4CaO5-cluster of the active site advances through five sequential oxidation states (S0 to S4) before water is oxidized and O2 is generated. Here, we have studied the transition between the low spin (LS) and high spin (HS) configurations of S2 using EPR spectroscopy, quantum chemical calculations using Density Functional Theory (DFT), and time-resolved UV-visible absorption spectroscopy. The EPR experiments show that the equilibrium between S2LS and S2HS is pH dependent, with a pKa ≈ 8.3 (n ≈ 4) for the native Mn4CaO5 and pKa ≈ 7.5 (n ≈ 1) for Mn4SrO5. The DFT results suggest that exchanging Ca with Sr modifies the electronic structure of several titratable groups within the active site, including groups that are not direct ligands to Ca/Sr, e.g., W1/W2, Asp61, His332 and His337. This is consistent with the complex modification of the pKa upon the Ca/Sr exchange. EPR also showed that NH3 addition reversed the effect of high pH, NH3-S2LS being present at all pH values studied. Absorption spectroscopy indicates that NH3 is no longer bound in the S3TyrZ state, consistent with EPR data showing minor or no NH3-induced modification of S3 and S0. In both Ca-PSII and Sr-PSII, S2HS was capable of advancing to S3 at low temperature (198 K). This is an experimental demonstration that the S2LS is formed first and advances to S3 via the S2HS state without detectable intermediates. We discuss the nature of the changes occurring in the S2LS to S2HS transition which allow the S2HS to S3 transition to occur below 200 K. This work also provides a protocol for generating S3 in concentrated samples without the need for saturating flashes.

International audience; The Mn4CaO5-cluster in Photosystem II advances through five oxidation states, S0 to S4, before water is oxidized and O2 is generated. The S2-state exhibits either a low-spin, S = 1/2 (S2LS), or a high-spin state, S = 5/2 (S2HS). Increasing the pH favors the S2HS configuration and mimics the formation of TyrZ in the S2LS-state at lower pH values (Boussac et al. Biochim. Biophys. Acta 1859 (2018) 342). Here, the temperature dependence of the S2HS to S3 transition was studied by EPR spectroscopy at pH 8.6. The present data strengthened the involvement of S2HS as a transient state in the S2LSTyrZ → S2HSTyrZ → S3TyrZ transition. Depending on the temperature, the S2HS progresses to S3 states exhibiting different EPR properties. One S3-state with a S = 3 signal, supposed to have a structure with the water molecule normally inserted in S2 to S3 transition, can be formed at temperatures as low as 77 K. This suggests that this water molecule is already bound in the S2HS state at pH 8.6. The nature of the EPR invisible S3 state, formed down to 4.2 K from a S2HS state, and that of the EPR detectable S3 state formed down to 77 K are discussed. It is proposed that in the S2LS to S3 transition, at pH \textless 8.6, the proton release (Sugiura et al. Biochim. Biophys. Acta 1859 (2018) 1259), the S2LS to S2HS conversion and the binding of the water molecule are all triggered by the formation of TyrZ.

WOS:000451491000001; In Photosystem II (PSII), the Mn4CaO5-cluster of the active site advances through five sequential oxidation states (S-0 to S-4) before water is oxidized and O-2 is generated. The V185 of the D1 protein has been shown to be an important amino acid in PSII function (Dilbeck et al. Biochemistry 52 (2013) 6824-6833). Here, we have studied its role by making a V185T site-directed mutant in the thermophilic cyanobacterium Thermosynechococcus elongatus. The properties of the V185T-PSII have been compared to those of the WT*3-PSII by using EPR spectroscopy, polarography, thermoluminescence and time-resolved UV-visible absorption spectroscopy. It is shown that the V185 and the chloride binding site very likely interact via the H-bond network linking Tyr(z) and the halide. The V185 contributes to the stabilization of S-2 into the low spin (LS), S = 1/2, configuration. Indeed, in the V185T mutant a high proportion of S-2 exhibits a high spin (HS), S = 5/2, configuration. By using bromocresol purple as a dye, a proton release was detected in the S(1)Tyr(Z)center dot -\textgreater S(2)(LS)Tyr(Z) transition in the V185T mutant in contrast to the WT*3-PSII in which there is no proton release in this transition. Instead, in WT*3-PSII, a proton release kinetically much faster than the S(2)(LS)Tyr(z)center dot -\textgreater S(3)Tyr(Z) transition was observed and we propose that it occurs in the S(2)(LS)Tyr(Z)center dot -\textgreater S(2)(HS)Tyr(Z). intermediate step before the S(2)(HS)Tyr(Z)center dot -\textgreater S(3)Tyr(Z) transition occurs. The dramatic slowdown of the S(3)Tyr(Z)center dot -\textgreater S(0)Tyr(Z) transition in the V185T mutant does not originate from a structural modification of the Mn4CaO5 cluster since the spin S = 3 S-3 EPR signal is not modified in the mutant. More probably, it is indicative of the strong implication of V185 in the tuning of an efficient relaxation processes of the H-bond network and/or of the protein.

International audience; The site for water oxidation in Photosystem II (PSII) goes through five sequential oxidation states (S0 to S4) before O2 is evolved. It consists of a Mn4CaO5-cluster close to a redox-active tyrosine residue (YZ). Cl- is also required for enzyme activity. By using EPR spectroscopy it has been shown that both Ca2+/Sr2+ exchange and Cl-/I- exchange perturb the proportions of centers showing high (S=5/2) and low spin (S=1/2) forms of the S2-state. The S3-state was also found to be heterogeneous with: i) a S=3 form that is detectable by EPR and not sensitive to near-infrared light; and ii) a form that is not EPR visible but in which Mn photochemistry occurs resulting in the formation of a (S2YZ)' split EPR signal upon near-infrared illumination. In Sr/Cl-PSII, the high spin (S=5/2) form of S2 shows a marked heterogeneity with a g=4.3 form generated at low temperature that converts to a relaxed form at g=4.9 at higher temperatures. The high spin g=4.9 form can then progress to the EPR detectable form of S3 at temperatures as low as 180K whereas the low spin (S=1/2) S2-state can only advance to the S3 state at temperatures≥235 K. Both of the two S2 configurations and the two S3 configurations are each shown to be in equilibrium at ≥235 K but not at 198 K. Since both S2 configurations are formed at 198 K, they likely arise from two specific populations of S1. The existence of heterogeneous populations in S1, S2 and S3 states may be related to the structural flexibility associated with the positioning of the oxygen O5 within the cluster highlighted in computational approaches and which has been linked to substrate exchange. These data are discussed in the context of recent in silico studies of the electron transfer pathways between the S2-state(s) and the S3-state(s).

The main cofactors of Photosystem II (PSII) are borne by the D1 and D2 subunits. In the thermophilic cyanobacterium Thermosynechococcus elongatus, three psbA genes encoding D1 are found in the genome. Among the 344 residues constituting the mature form of D1, there are 21 substitutions between PsbA1 and PsbA3, 31 between PsbA1 and PsbA2, and 27 between PsbA2 and PsbA3. In a previous study (Sugiura et al., J. Biol. Chem. 287 (2012), 13336-13347) we found that the oxidation kinetics and spectroscopic properties of TyrZ were altered in PsbA2-PSII when compared to PsbA(1/3)-PSII. The comparison of the different amino acid sequences identified the residues Cys144 and Pro173 found in PsbA1 and PsbA3, as being substituted in PsbA2 by Pro144 and Met173, and thus possible candidates accounting for the changes in the geometry and/or the environment of the TyrZ/His190 phenol/imidizol motif. Indeed, these amino acids are located upstream of the α-helix bearing TyrZ and between the two α-helices bearing TyrZ and its hydrogen-bonded partner, D1/His190. Here, site-directed mutants of PSII, PsbA3/Pro173Met and PsbA2/Met173Pro, were analyzed using X- and W-band EPR and UV-visible time-resolved absorption spectroscopy. The Pro173Met substitution in PsbA2-PSII versus PsbA3-PSII is shown to be the main structural determinant of the previously described functional differences between PsbA2-PSII and PsbA3-PSII. In PsbA2-PSII and PsbA3/Pro173Met-PSII, we found that the oxidation of TyrZ by P680+● was specifically slowed during the transition between S-states associated with proton release. We thus propose that the increase of the electrostatic charge of the Mn4CaO5 cluster in the S2 and S3 states could weaken the strength of the H-bond interaction between TyrZ● and D1/His190 in PsbA2 versus PsbA3 and/or induce structural modification(s) of the water molecules network around TyrZ.

Charge recombination in the light-induced radical pair SnTyrZ(•)QA(-•) in Photosystem II (PSII) from Thermosynechococcus elongatus has been studied at cryogenic temperatures by time-resolved EPR for different configurations of PSII that are expected to affect the driving force of the reaction (oxidation states S0, S1, or S2 of the Mn4CaO5 cluster; PsbA1, PsbA2, or PsbA3 as D1 protein). The kinetics were independent of temperature in the studied range from 4.2 to 50 K and were not affected by exchange of H2O for D2O, consistent with single-step electron tunneling over the distance of ∼32 Å without any repopulation through Boltzmann equilibration of intermediates lying higher in energy. In PsbA1-PSII, the charge recombinations in the radical pairs SnTyrZ(•)QA(-•) (ket = 3.4 × 10(-3) s(-1) for S1) were slower than in PsbA3-PSII despite an expected lower driving force owing to a downshifted Em(QA/QA(-•)) in PsbA1-PSII. Conversely, the reaction was slower in the presence of S2 than in the presence of S1, despite an expected larger driving force due to an upshifted Em(TyrZ(•)/TyrZ) in S2. These observations indicate that the charge recombination occurs in the Marcus inverted region. Assuming that the driving force of the reaction (-ΔG(0) ≈ 1.2 eV at room temperature for S1) does not vary strongly with temperature, the data indicate an optimal electron transfer rate (for a hypothetical -ΔG(0) = λ) substantially faster than would be predicted from extrapolation of room temperature intraprotein ET rates over shorter distances. Possible origins of this deviation are discussed, including a possible enhancement of the electronic coupling of TyrZ(•) and QA(-•) by aromatic cofactors located in between. Observed similar S1TyrZ(•)QA(-•) charge recombinations in PsbA2-PSII and PsbA3-PSII predict that Em(QA/QA(-•)) in PsbA2-PSII is similar to that in PsbA3-PSII.

Water binding to the Mn(4)O(5)Ca cluster of the oxygen-evolving complex (OEC) of Photosystem II (PSII) poised in the S(2) state was studied via H(2)(17)O- and (2)H(2)O-labeling and high-field electron paramagnetic resonance (EPR) spectroscopy. Hyperfine couplings of coordinating (17)O (I = 5/2) nuclei were detected using W-band (94 GHz) electron-electron double resonance (ELDOR) detected NMR and Davies/Mims electron-nuclear double resonance (ENDOR) techniques. Universal (15)N (I = ½) labeling was employed to clearly discriminate the (17)O hyperfine couplings that overlap with (14)N (I = 1) signals from the D1-His332 ligand of the OEC (Stich Biochemistry 2011, 50 (34), 7390-7404). Three classes of (17)O nuclei were identified: (i) one μ-oxo bridge; (ii) a terminal Mn-OH/OH(2) ligand; and (iii) Mn/Ca-H(2)O ligand(s). These assignments are based on (17)O model complex data, on comparison to the recent 1.9 Å resolution PSII crystal structure (Umena Nature 2011, 473, 55-60), on NH(3) perturbation of the (17)O signal envelope and density functional theory calculations. The relative orientation of the putative (17)O μ-oxo bridge hyperfine tensor to the (14)N((15)N) hyperfine tensor of the D1-His332 ligand suggests that the exchangeable μ-oxo bridge links the outer Mn to the Mn(3)O(3)Ca open-cuboidal unit (O4 and O5 in the Umena et al. structure). Comparison to literature data favors the Ca-linked O5 oxygen over the alternative assignment to O4. All (17)O signals were seen even after very short (≤15 s) incubations in H(2)(17)O suggesting that all exchange sites identified could represent bound substrate in the S(1) state including the μ-oxo bridge. (1)H/(2)H (I = ½, 1) ENDOR data performed at Q- (34 GHz) and W-bands complement the above findings. The relatively small (1)H/(2)H couplings observed require that all the μ-oxo bridges of the Mn(4)O(5)Ca cluster are deprotonated in the S(2) state. Together, these results further limit the possible substrate water-binding sites and modes within the OEC. This information restricts the number of possible reaction pathways for O-O bond formation, supporting an oxo/oxyl coupling mechanism in S(4).

The active site for water oxidation in Photosystem II (PSII) goes through five sequential oxidation states (S 0 to S 4 ) before O 2 is evolved. It consists of a Mn 4 CaO 5 cluster and Tyr Z , a redox-active tyrosine residue. Chloride ions have been known for long time to be required for the function of the enzyme. However, X-ray data have shown that they are located about 7 A away from the Mn 4 CaO 5 cluster, a distance that seems too large to be compatible with a direct involvement of chloride in the water splitting chemistry. We have investigated the role of this anion by substituting I − for Cl − in the cyanobacterium Thermosynechococcus elongatus with either Ca 2 + or Sr 2 + biosynthetically assembled into the Mn 4 cluster. The electron transfer steps affected by the exchanges were investigated by time-resolved UV–visible absorption spectroscopy, time-resolved EPR at room temperature and low temperature cw-EPR spectroscopy. In both Ca-PSII and Sr-PSII, the Cl − /I − exchange considerably slowed down the two S 3 Tyr Z • → (S 3 Tyr Z • )′ → S 0 reactions in which the fast phase, S 3 Tyr Z • → (S 3 Tyr Z • )′, reflects the electrostatically triggered expulsion of one proton from the catalytic center caused by the positive charge near/on Tyr Z • and the slow phase corresponds to the S 0 and O 2 formations and to a second proton release. The t 1/2 for S 0 formation increased from 1.1 ms in Ca/Cl-PSII to ≈ 6 ms in Ca/I-PSII and from 4.8 ms in Sr/Cl-PSII to ≈ 45 ms in Sr/I-PSII. In all cases the Tyr Z • reduction was the limiting step. The kinetic effects are interpreted by a model in which the Ca 2 + binding site and the Cl − binding site, although spatially distant, interact. This interaction is likely mediated by the H-bond and/or water molecules network(s) connecting the Cl − and Ca 2 + binding sites by which proton release may be channelled.

This report describes a protocol to incorporate isotopically labelled aromatic amino acids into the proteins of the thermophilic cyanobacterium Thermosynechoccus elongatus. By using the EPR signal of the two redox active tyrosines of Photosystem II, Tyr(D)(*) and Tyr(Z)(*), as spectroscopic probes it is shown that labelled tyrosines can be incorporated with a high yield in this cyanobacterium. The production of a fully (13)C- or (2)H-labelled enzyme is also described.

The Mn4Ca cluster of photosystem II (PSII) goes through five sequential oxidation states (S0-S4) in the water oxidation process that also involves a tyrosine radical intermediate (TyrZ*). An S2TyrZ* state in which the Mn4Ca cluster and TyrZ* are magnetically coupled to each other and which is characterized by a distinct "split-signal" EPR spectrum can be generated in acetate-treated PSII. This state was examined by high-field EPR (HFEPR) in PSII from Thermosynechococcus elongatus isolated from a D2-Tyr160Phe mutant to avoid spectral contributions from TyrD*. In contrast to the same state in plants, both antiferromagnetic and ferromagnetic spin-spin couplings were observed. The intrinsic g values of TyrZ* in the coupled state were directly measured from the microwave frequency dependence of the HFEPR spectrum. The TyrZ* gx value in the antiferromagnetic centers was 2.0083, indicating that the coupled radical was in a less electropositive environment than in Mn-depleted PSII. Two gx values were found in the ferromagnetically coupled centers, 2.0069 and 2.0079. To put these values in perspective, the second redox-active tyrosine, TyrD*, was examined in various electrostatic environments. The TyrD* gx value changed from 2.0076 in the wild type to 2.0095 when the hydrogen bond from histidine 189 to TyrD* was removed using the D2-His189Leu mutant, indicating a change to a significantly less electropositive environment. BLY3P/6-31+G** density functional calculations on the hydrogen-bonded p-ethylphenoxy radical-imidazole supermolecular model complex showed that the entire range of Tyr* gx values, from 2.0065 to 2.0095, could be explained by the combined effects of hydrogen bonding and the dielectric constant of the local protein environment.

The states induced by illumination at 7 K in the oxygen-evolving enzyme (PSII) from Thermosynechococcus elongatus were studied by EPR. In the S(0) and S(1) redox states, two g approximately 2 EPR signals, a split signal and a g = 2.03 signal, respectively, were generated by illumination with visible light. These signals were comparable to those already reported in plant PSII in terms of their g value, shape, and stability at low temperatures. We report that the formation and decay of these signals correlate with EPR signals from the semiquinone of the first quinone electron acceptor, Q(A)(-). The light-induced EPR signals from oxidized side-path electron donors (Cyt b(559), Car, and Chl(Z)) were also measured, and from these and the signals from Q(A)(-), estimates were made of the proportion of centers involved in the formation of the g approximately 2 signals (approximately 50% in S(0) and 40% in S(1)). Comparisons with the signals generated in plant PSII indicated approximately similar yields for the S(0) split signal. A single laser flash at 7 K induced more than 75% of the maximum split and g = 2.03 EPR signal observed by continuous illumination, with no detectable oxidation of side-path donors. The matching electron acceptor side reactions, the high quantum yield, and the relatively large proportion of centers involved support earlier suggestions that the state being monitored is Tyr(Z)(*)Q(A)(-), with the g approximately 2 EPR signals arising from Tyr(Z)(*) interacting magnetically with the Mn complex. The current picture of the photochemical reactions occurring in PSII at low temperatures is reassessed.

The elusive tyrosinate radical has been stabilized in the layers of synthetic laponite on intercalated tyrosinate ester molecules. According to EPR, XRD, and FT-IR data, the tyrosinate ester molecules adopt a horizontal orientation between the clay surfaces, with the aromatic rings almost perpendicular to the clay layers, giving an intersheet separation of 4.8 Angstrom. The radical remains stable for over two months, while it is quickly reduced by nitric oxide. The observed long-term stability of the radical is due to electrostatic interactions with the negative surface of the clay as well as to sequestration phenomena which protect the radical from polymerization. Langmuir

The S2 state of the oxygen-evolving complex of photosystem II, which consists of a Mn4O5Ca cofactor, is EPR-active, typically displaying a multiline signal, which arises from a ground spin state of total spin ST = 1/2. The precise appearance of the signal varies amongst different photosynthetic species, preparation and solvent conditions/compositions. Over the past five years, using the model species Thermosynechococcus elongatus, we have examined modifications that induce changes in the multiline signal, i.e. Ca(2+)/Sr(2+)-substitution and the binding of ammonia, to ascertain how structural perturbations of the cluster are reflected in its magnetic/electronic properties. This refined analysis, which now includes high-field (W-band) data, demonstrates that the electronic structure of the S2 state is essentially invariant to these modifications. This assessment is based on spectroscopies that examine the metal centres themselves (EPR, (55)Mn-ENDOR) and their first coordination sphere ligands ((14)N/(15)N- and (17)O-ESEEM, -HYSCORE and -EDNMR). In addition, extended quantum mechanical models from broken-symmetry DFT now reproduce all EPR, (55)Mn and (14)N experimental magnetic observables, with the inclusion of second coordination sphere ligands being crucial for accurately describing the interaction of NH3 with the Mn tetramer. These results support a mechanism of multiline heterogeneity reported for species differences and the effect of methanol [Biochim. Biophys. Acta, Bioenerg., 2011, 1807, 829], involving small changes in the magnetic connectivity of the solvent accessible outer MnA4 to the cuboidal unit Mn3O3Ca, resulting in predictable changes of the measured effective (55)Mn hyperfine tensors. Sr(2+) and NH3 replacement both affect the observed (17)O-EDNMR signal envelope supporting the assignment of O5 as the exchangeable μ-oxo bridge and it acting as the first site of substrate inclusion.

Two symmetrically positioned redox active tyrosine residues are present in the photosystem II (PSII) reaction center. One of them, TyrZ, is oxidized in the ns–μs time scale by P680+and reduced rapidly (μs to ms) by electrons from the Mn complex. The other one, TyrD, is stable in its oxidized form and seems to play no direct role in enzyme function. Here, we have studied electron donation from these tyrosines to the chlorophyll cation (P680+) in Mn-depleted PSII from plants and cyanobacteria. In particular, a mutant lacking TyrZ was used to investigate electron donation from TyrD. By using EPR and time-resolved absorption spectroscopy, we show that reduced TyrD is capable of donating an electron to P680+with t1/2≈ 190 ns at pH 8.5 in approximately half of the centers. This rate is ≈105times faster than was previously thought and similar to the TyrZ donation rate in Mn-depleted wild-type PSII (pH 8.5). Some earlier arguments put forward to rationalize the supposedly slow electron donation from TyrD (compared with that from TyrZ) can be reassessed. At pH 6.5, TyrZ (t1/2= 2–10 μs) donates much faster to P680+than does TyrD (t1/2> 150 μs). These different rates may reflect the different fates of the proton released from the respective tyrosines upon oxidation. The rapid rate of electron donation from TyrD requires at least partial localization of P680+on the chlorophyll (PD2) that is located on the D2 side of the reaction center.

The Mn(4)-cluster and the cytochrome c(550) in histidine-tagged photosystem II (PSII) from Synechococcus elongatus were studied using electron paramagnetic resonance (EPR) spectroscopy. The EPR signals associated with the S(0)-state (spin = 1/2) and the S(2)-state (spin = 1/2 and IR-induced spin = 5/2 state) were essentially identical to those detected in the non-His-tagged strain. The EPR signals from the S(3)-state, not previously reported in cyanobacteria, were detectable both using perpendicular (at g = 10) and parallel (at g = 14) polarization EPR, and these signals are similar to those found in plant PSII. In the S(3)-state, near-infrared illumination at 50 K induced a 176-G-wide split signal at g = 2 and signals at g = 5.20 and g = 1.51. These signals differ slightly from those reported in plant PSII [Ioannidis, N., and Petrouleas, V. (2000) Biochemistry 39, 5246-5254]. In accordance with the cited work, the split signal presumably reflects a radical interacting with the Mn(4)-cluster in a fraction of centers, while the g = 5.20 and g = 1.51 signals are tentatively attributed to a high-spin state of the Mn(4)-cluster with zero field splitting parameters different from those in plant PSII, reflecting minor changes in the environment of the Mn(4)-cluster. Biochemical modifications (Sr(2+)/Ca(2+) substitution, acetate and NH(3) treatments) were also investigated. In Sr(2+)-reconstituted PSII, in addition to the expected modified S(2) multiline signal, a signal at g = 5.2 was present instead of the g approximately 4 signal seen in plant PSII. In NH(3)-treated samples, in addition to the expected modified S(2)-multiline signal, a g approximately 4 signal was detected in a small proportion of the reaction centers. This is of note since g approximately 4 spectra arising from the Mn(4)-cluster in the S(2) state have not yet been published in cyanobacterial PSII. The detection of modified S(3)-signals in both perpendicular (at g = 7.5) and parallel (at g = 12) polarization EPR from NH(3)-treated PSII indicate that NH(3) is still bound in the S(3)-state. The acetate-treated PSII behaves essentially as in plant PSII. A study using oriented samples indicated that the heme plane of the oxidized low spin Cytc(550) was perpendicular to the plane of the membrane.

Nonheme Fe(III)-hydroperoxo and Fe(III)-peroxo complexes with aminopyridyl-type ligands have been prepared and characterized by UV/Vis, EPR, mass and Resonance Raman (RR) spectroscopy. The Fe(III)(OOH) species are low-spin and exhibit a deep purple color due to the ligand-to-metal charge transfer (LMCT) hand centered at ca. 550 nm. The RR spectra of the Fe(III)(OOH) complexes display two bands at ca. 620 and 800 cm-1 that are assigned to the respective Fe-O and O-O stretching modes on the basis of the characteristic H/D and 16O/18O frequency shifts. Upon deprotonation, Fe(III)(O2) species are obtained which possess a high-spin configuration of nearly axial symmetry and a LMCT transition in the near infrared (ca. 750 nm). The frequencies of the Fe-O and O-O stretching modes at ca. 465 and 820 cm-1, as well as their respective 16O/18O shifts of -16 and -45 cm-1, indicate an ?2 coordination geometry for the Fe(III)(O2) complex.

The Mn(4) complex which is involved in water oxidation in photosystem II is known to exhibit three types of EPR signals in the S(2) state, one of the five redox states of the enzyme cycle: a multiline signal (spin 1/2), signals at g5 (spin 5/2) and a signal at g=4.1 (or g=4.25). The g=4.1 signal could be generated under two distinct sets of conditions: either by illumination at room temperature or at 200 K in certain experimental conditions (g4(S) signal) or by near-infrared illumination between approximately 77 and approximately 160 K of the S(2)-multiline state (g4(IR) signal). The two g=4.1 signals arise from states which have quite different stability in terms of temperature. In the present work we have compared these two signals in order to test if they originate from the same or from different chemical origins. The microwave power saturation properties of the two signals measured at 4.2 K were found to be virtually identical. Their temperature dependencies measured at non-saturating powers were also identical. The presence of Curie law behavior for the g4(S) and g4(IR) signals indicates that the states responsible for both signals are ground states. The orientation dependence, anisotropy and resolved hyperfine structure of the two g4 signals were also found to be virtually indistinguishable. We have been unable to confirm the behavior reported earlier indicating that the g4(S) signal is an excited state, nor were we able to confirm the presence of signal from a higher excited state in samples containing the g4(S), nor a radical signal in samples containing the g4(IR). These findings are best interpreted assuming that the two signals have a common origin i.e. a spin 5/2 ground state arising from a magnetically coupled Mn-cluster of 4 Mn ions.

The light-driven oxidation of water to dioxygen is catalyzed by the enzyme photosystem II. A four-manganese ion cluster and a tyrosine, YZ, are present in the catalytic site. In preparations inhibited by addition of acetate or removal of the calcium cofactor, it is possible to trap the tyrosyl radical in interaction with the metal cluster. The coupled species is characterized by a broad split EPR signal at 9 GHz. In this work, high-field EPR has been used for further characterization of the coupling. The 285, 190 and 95 GHz EPR spectra of the interacting system are reported. Analysis of these spectra yielded exchange and dipolar couplings of the same magnitude as those found with 9 GHz EPR. However, the high-field spectra show that the coupling between the radical and the manganese cluster has opposite sign in acetate-treated compared to calcium-depleted samples. The sign difference indicates differences in the electronic structure of the radical−metal center pair. Comparisons are made between photosystem...

The Mn(4)-cluster of photosystem II (PSII) from Synechococcus elongatus was studied by electron paramagnetic resonance (EPR) spectroscopy after a series of saturating laser flashes given in the presence of either methanol or ethanol. Results were compared to those obtained in similar experiments done on PSII isolated from plants. The flash-dependent changes in amplitude of the EPR multiline signals were virtually identical in all samples. In agreement with earlier work [Messinger, J., Nugent, J. H. A., and Evans, M. C. W. (1997) Biochemistry 36, 11055-11060; Ahrling, K. A., Peterson, S., and Styring, S. (1997) Biochemistry 36, 13148-13152], detection of an EPR multiline signal from the S(0) state in PSII from plants was only possible with methanol present. In PSII from S. elongatus, it is shown that the S(0) state exhibits an EPR multiline signal in the absence of methanol (however, ethanol was present as a solvent for the artificial electron acceptor). The hyperfine lines are better resolved when methanol is present. The S(0) multiline signals detected in plant PSII and in S. elongatus were similar but not identical. Unlike the situation seen in plant PSII, the S(2) state in S. elongatus is not affected by the addition of methanol in that (i) the S(2) multiline EPR signal is not modified by methanol and (ii) the spin state of the S(2) state is affected by infrared light when methanol is present. It is also shown that the magnetic relaxation properties of an oxidized low-spin heme, attributed to cytochrome c(550), vary with the S states. This heme then is in the magnetic environment of the Mn(4) cluster.

The Mn4 complex which is involved in water oxidation in photosystem II is known to exhibit three types of EPR signals in the S2 state, one of the five redox states of the enzyme cycle: a multiline signal (spin 1/2), signals at g > 5 (spin 5/2), and a signal at g = 4.1 (spin value 3/2 or 5/2). The multiline and g = 4.1 signals are those the most readily observed. The relative proportions of the g = 4.1 signal and of the multiline signal are affected by many biochemical treatments including the substitution of Ca2+and Cl- which are two essential cofactors for O2 evolution. The state responsible for the multiline signal can also be converted, reversibly, to that responsible for the g = 4.1 signal upon the absorption of near-IR light at around 150 K. These infrared-induced effects are confined to the Mn4 cluster, and no other redox change occurs in the enzyme. Here, we have used the IR-induced photochemistry of the Mn4 cluster to measure the changes in magnetization occurring upon interconversion of the stat...

The synthesis and structural characterization of the novel [MnIII,IV2O2(N,Nbispicen)2](ClO4)3·CH3CN complex employing the tetradentate ligand N,Nbispicen = N,N-bis(2-pyridylmethyl)-1,2-diaminoethane are reported. Magnetic properties were determined and show that the ground state is a spin doublet. This can be quantitatively interpreted by antiferromagnetic coupling between a Mn(III) high spin and a Mn(IV) (J = −316 cm−1). The 16 line solution EPR spectrum exhibits an unusual splitting in the low field resonances. The following rhombic tensors were needed to simulate the EPR spectrum: |A1x|= 160 10−4 cm−1, |A1y| = 144 10−4 cm−1, |A1z|= 109 10−4 cm−1, |A2x|= 69 10−4 cm−1, |A2y| = 72 10−4 cm−1, |A2z| = 75 10−4 cm−1, gx = 2.001, gy = 1.996, gz = 1.984. The classical ligand field theory of local [gIII] and [gIV] tensors implemented with the first order perturbation theory to describe the properties of the pair does not result in a satisfying description of the [g1/2] tensor unless a large reduction in the spin-orbit constant is invoked. A simplified version of second-order perturbation theory leads to effects in qualitative agreement with experiment but weak as expected from the large |J| value. The magnitude of these effects depends, however, on the anisotropy effects on each Mn ion. It is suggested that determination of the anisotropy of the magnetic properties of the monomeric Mn(III) and Mn(IV) moieties would be a valuable goal for a future study of these mixed valent dimanganese-di-μ-oxo complexes. The complex exhibits two quasi-reversible waves in the cyclic voltammogram, one at E1/2 = 0.18 V vs SCE for the III/IV █ III/III couple and the other at E1/2 = 0.98 V vs SCE for the III/IV █ IV/IV couple. The UV-Vis spectra of the three redox states have been recorded spectroelectrochemically.

The manganese complex (Mn4) which is responsible for water oxidation in photosystem II is EPR detectable in the S2-state, one of the five redox states of the enzyme cycle. The S2-state is observable at 10 K either as an EPR multiline signal (spin S = 1/2) or as a signal at g = 4.1 (spin S = 3/2 or 5/2). It has recently been shown that the state responsible for the multiline signal is converted to that responsible for the g = 4.1 signal upon the absorption of near-infrared light [Boussac A, Girerd J-J, Rutherford AW (1996) Biochemistry 35 : 6984–6989]. It is shown here that the yield of the spin interconversion may be variable and depends on the photosystem II (PSII) preparations. The EPR multiline signal detected after near-infrared illumination, and which originates from PSII centers not susceptible to the near-infrared light, is shown to be different from that which originates from infrared-susceptible PSII centers. The total S2-multiline signal results from the superposition of the two multiline signals which originate from these two PSII populations. One S2 population gives rise to a "narrow" multiline signal characterized by strong central lines and weak outer lines. The second population gives rise to a "broad" multiline signal in which the intensity of the outer lines, at low and high field, are proportionally larger than those in the narrow multiline signal. The larger the relative amplitude of the outer lines at low and high field, the higher is the proportion of the near-infrared-susceptible PSII centers and the yield of the multiline to g = 4.1 signal conversion. This inhomogeneity of the EPR multiline signal is briefly discussed in terms of the structural properties of the Mn4 complex.

gamma-Ray irradiation at liquid nitrogen temperature of a dimethylformamide solution of the tetranuclear complex [(Mn4O6)-O-IV(bipy)(6)](4+) (bipy = 2,2'-bipyridine) allowed the generation of the first mixed-valence tetranuclear system containing Mn-III and Mn-IV ions and exhibiting a S = 1/2 ground state. The X-band EPR spectrum of this tetranuclear system has been obtained. Simulations have been undertaken and the Mn hyperfine coupling tensors determined clearly show a (MnMn3IV)-Mn-III, composition for the EPR active species. A general approach for the analysis of the isotropic components of the Mn hyperfine tensors is presented in detail. This allowed the determination of the spin projection value for each Mn site. A three J coupling scheme assuming that the linear topology of the starting compound remains is able to reproduce these spin projection values if and only if the Mn-III ion is located at a terminal position in a N4O2 environment. The EPR signal of this [Mn4O6(bipy)(6)](3+) species is compared with the multiline signal observed in the S-2 state of the photosynthetic Oxygen Evolving Complex.

The assignment of the two substrate water sites of the tetra-manganese penta-oxygen calcium (Mn 4 O 5 Ca) cluster of photosystem II is essential for the elucidation of the mechanism of biological O-O bond formation and the subsequent design of bio-inspired water-splitting catalysts. We recently demonstrated using pulsed EPR spectroscopy that one of the five oxygen bridges (μ-oxo) exchanges unusually rapidly with bulk water and is thus a likely candidate for one of the substrates. Ammonia, a water analog, was previously shown to bind to the Mn 4 O 5 Ca cluster, potentially displacing a water/substrate ligand [Britt RD, et al. (1989) J Am Chem Soc 111(10):3522–3532]. Here we show by a combination of EPR and time-resolved membrane inlet mass spectrometry that the binding of ammonia perturbs the exchangeable μ-oxo bridge without drastically altering the binding/exchange kinetics of the two substrates. In combination with broken-symmetry density functional theory, our results show that ( i ) the exchangable μ-oxo bridge is O5 {using the labeling of the current crystal structure [Umena Y, et al. (2011) Nature 473(7345):55–60]}; ( ii ) ammonia displaces a water ligand to the outer manganese (Mn A4 -W1); and ( iii ) as W1 is trans to O5, ammonia binding elongates the Mn A4 -O5 bond, leading to the perturbation of the μ-oxo bridge resonance and to a small change in the water exchange rates. These experimental results support O-O bond formation between O5 and possibly an oxyl radical as proposed by Siegbahn and exclude W1 as the second substrate water.

The whereabouts of the Ca2+ site in Photosystem II (PSII) was investigated by experiments in which Mn2+ was substituted for Ca2+. When stoichiometric amounts of Mn2+ ions were added to Ca2+-depleted PSII, the Mn2+ was not detected by EPR. The titration of Ca2+ back into Ca2+-depleted/Mn2+-containing PSII resulted in the simultaneous release of the Mn2+ and the loss of the two EPR signals which are characteristic of the Ca2+-depleted enzyme (i.e., the stable, modified S2 multiline signal arising from the intrinsic Mn cluster and the split S3 signal from an organic radical interacting with the Mn cluster). These results indicate that the Mn2+ occupies the functional Ca2+ site. The S2 and S3 EPR signal characteristic of this kind of Ca2+-depleted preparation were unaffected by the binding of the Mn2+ Since, from earlier results, it seems likely that the modification and stability of S2 multiline signal in these PSII preparations is due to binding of chelator to or close to the Mn cluster, the present results indicate that the Ca2+ site (at least when occupied by Mn2+) does not overlap with the chelator binding site. Since Mn2+ binding does not effect the S2 EPR signal from the Mn cluster, it can be concluded that the Mn2+ is not involved in detectable magnetic interactions with the cluster. This result indicates that the Mn2+-occupied Ca2+ binding site is outside the first co-ordination sphere of the Mn cluster. The relaxation properties of TyrD. were enhanced by the presence of the Mn2+ when the Mn cluster was in the S1 state.

The compound [Mn2III,IVO2(bisimMe2en)2](ClO4)3·H2O (bisimMe2en = N,N‘-dimethyl-N,N‘-bis(imidazol-4-ylmethyl)ethane-1,2-diamine) was synthesized. It crystallizes in the monoclinic space group C2/c with a = 18.139(8) A, b = 12.694(5) A, c = 17.694(8) A, β = 107.5(6)°, V = 3885(6) A3, and Z = 4. The cation [Mn2III,IVO2(bisimMe2en)2]3+ contains a di-manganese di-μ-oxo unit. The Mn−Mn axis is a C2 axis. The Mn−imidazole distances for MnIII or MnIV are distinct: respectively, 2.208(9) and 2.004(8) A. The ground state is a |SA2,SB3/2,S1/2〉 state separated by 420 cm-1 from the S = 3/2 state. The EPR spectrum has been simulated with |A1x,y| = 160.10-4 cm-1, |A1z| = 136.10-4 cm-1, |A2x,y| = 72.10-4 cm-1, |A2z| = 71.10-4 cm-1, gxy = 1.997, gz = 1.993. The ESEEM spectrum is reported. The 2H nuclei corresponding to the exchangeable hydrogen atoms of imidazole groups in D2O have been detected at 2.4 MHz at 3480 G. In cyclic voltammetry, [Mn2III,IVO2(bisimMe2en)2]3+ presents in oxidation two reversible waves at 1.04 an...

The tyrosine EPR signal(s) have been investigated at room temperature in Ca2+-depleted Photosystem II. It was found that at low, non-saturating microwave powers, the tyrosine signal present after flash illumination decayed monophasically in about 40% of the centers with a t12 of 780 s. This decay is attributed to TyrD° reduction. The radical formed as the formal S3 state and detected as at split EPR signal at helium temperature, decayed monophasically at room temperature with a t12 of 110 s. When the tyrosine EPR signals were monitored at room temperature using a high, saturating microwave power, in addition to the 780 s phase, a faster phase of 110 s was observed. This additional phase is attributed to TyrD°, the amplitude of which is enhanced by dipolar coupling to the fast relaxing S3 state. Its decay corresponds to the loss of the fast relaxing S3 species. Similar effects reported earlier and attributed to the formation of TyrZ° are reinterpreted as a relaxation enhancement of TyrD°. No evidence was found from this approach to attribute the split S3 EPR signal to a TyrZ° signal which is detectable at room temperature.

International audience; FTIR difference and EPR spectroscopies were used to identify the organic radical species formed during the Sz to S3 transition in Ca2+-depleted, EGTA-treated, and polypeptide-reconstituted photosystem II membranes (denoted Sz' and 83', respectively). Ferricyanide was added to the samples to act as an exogenous electron acceptor. Using EPR spectroscopy, it was shown that, under the experimental conditions used, only the species oxidized in the S3' state was detected during the time required for theacquisition of the FTIR difference spectra. No contributions from the electron acceptor side were observed. The corresponding Ss'/Sz' FTIR difference spectra were recorded at 10 °C in HzO, DzO, and with 15Nlabeled photosystem II membranes. Spectra were compared with radical-minus-neutral FTIR difference spectra of amino acid model compounds generated by UV irradiation at low temperature. Under our experimental conditions, we did not observe FTIR difference signals consistent with tyrosine oxidation in the S2' to S3' transition. The infrared signals characteristic of radical formation with 4-methylimidazole and histidine obtained by UV irradiation of 4-methylimidazolium at pH 6 and of a His-Tyr dipeptide at pH 7 are presented. The analogy found between these spectra and the Ss'/Sz' spectmm obtained in situ supports the oxidation of a histidinium in the Sz' to S3' transition.

The oxidized radicals of aromatic amino acids, 4-methylimidazole and phenol were generated by UV irradiation at low temperature. The radicals were monitored by EPR spectroscopy. For the first time, infrared (IR) modes characteristic of the tyrosine, tryptophan, phenylalanine, and histidine radicals were obtained by FTIR difference spectroscopy between the ground state and the radical state. The effects of D- (on Tyr and phenol) or 15N- (on His) labeling on the IR modes of the radicals were studied, as were the influence of hydrogen bonding and of pH. These parameters were studied to model the possible radical structures and environments in proteins. The radicals obtained with tyrosine, phenol, and 4-ethylphenol present six main IR modes: a combination mode at ∼ 2110–2106 cm−1; the v8b(CC) mode at 1550–1556 cm−1, the v7a(CO) and v19b(CC) modes both at 1515–1500 cm−1, which are distinctly affected by D-labeling of the phenol ring; the 14(vCC + δCH) mode at 1290–1288 cm−1, which is strongly modified when the radical is hydrogen bonded; and the 9a(CC) mode at 1163–1159 cm−1. These IR modes partly confirm the assignments made by resonance Raman (RR) spectroscopy and should help to obtain precise structure and force field calculations for the radicals. The deprotonated 4-methylimidazole radical (4-MeIm·) is obtained at pH 12. It has characteristic IR modes at 1593 v(CC), 1425 δCH3, 1376 δCH3, 1315 cm−1, 1213 cm−1 and 1098 cm−1 δ(CH). The methyl modes seem strongly downshifted upon radical formation, while the ring modes appear less affected. In particular, the C4C5 double-bond character is conserved. The protonated 4-methylimidazole radical formed at pH ≤ 6 is characterized by signals at 1433 cm−1, 1380 cm−1, 1310 cm−1, 1227 cm−1, and 1172 cm−1. The histidine and tyrosine radicals present similar IR modes as the corresponding model of their sidechain. For all the amino acids, the vas(COO−) and vs(COO−) modes of the terminal carboxylate were respectively up- and downshifted by ∼ 20 cm−1 upon the radical formation. This effect suggests that, in a protein, the amide bond of the amino acid could also be influenced by the radical formation. © 1995 John Wiley & Sons, Inc.

The main cofactors that determine the photosystem II (PSII) oxygen evolution activity are borne by the D1 and D2 subunits. In the cyanobacterium Thermosynechococcus elongatus, there are three psbA genes coding for D1. Among the 344 residues constituting D1, there are 21 substitutions between PsbA1 and PsbA3, 31 between PsbA1 and PsbA2, and 27 between PsbA2 and PsbA3. Here, we present the first study of PsbA2-PSII. Using EPR and UV-visible time-resolved absorption spectroscopy, we show that: (i) the time-resolved EPR spectrum of Tyr(Z)(•) in the (S(3)Tyr(Z)(•))' is slightly modified; (ii) the split EPR signal arising from Tyr(Z)(•) in the (S(2)Tyr(Z)(•))' state induced by near-infrared illumination at 4.2 K of the S(3)Tyr(Z) state is significantly modified; and (iii) the slow phases of P(680)(+) reduction by Tyr(Z) are slowed down from the hundreds of μs time range to the ms time range, whereas both the S(1)Tyr(Z)(•) → S(2)Tyr(Z) and the S(3)Tyr(Z)(•) → S(0)Tyr(Z) + O(2) transition kinetics remained similar to those in PsbA(1/3)-PSII. These results show that the geometry of the Tyr(Z) phenol and its environment, likely the Tyr-O···H···Ne-His bonding, are modified in PsbA2-PSII when compared with PsbA(1/3)-PSII. They also point to the dynamics of the proton-coupled electron transfer processes associated with the oxidation of Tyr(Z) being affected. From sequence comparison, we propose that the C144P and P173M substitutions in PsbA2-PSII versus PsbA(1/3)-PSII, respectively located upstream of the α-helix bearing Tyr(Z) and between the two α-helices bearing Tyr(Z) and its hydrogen-bonded partner, His-190, are responsible for these changes.

We have used thermoluminescence (TL) and EPR measurements of Photosystem II (PS II) from spinach in order to identify charge pairs responsible for TL bands in the region of 40–50°C including the ‘C-band’ (peak V) and the TL band from PS II depleted of calcium. In intact PS II membrane preparations, in the presence of DCMU, a TL band at 50°C is induced following illumination at 77 K. This band decays, at 30°C, with a half-time of 10 min. This decay corresponds to the disappearance of the EPR signal arising from QA− and an accelerated decay of the organic free radical Tyr D+. It is concluded that recombination of this charge pair is probably responsible for the thermoluminescence emission. In PS II preparations that have been depleted of calcium using a salt/EGTA wash followed by rebinding of the extrinsic polypeptides, a TL band is produced at around 45–50°C following 198 K illumination. In such samples a stable S2 state of the water-splitting complex is present, giving rise to a modified form of the EPR multiline signal. During incubation at 30°C in the dark this signal decays with a half-time around 20–25 min. This decay is not accelerated by the presence of QA− induced by low-temperature illumination of the sample. In contrast, low-temperature illumination does result in an acceleration in the decay of Tyr D+, indicating that Tyr D+/Q−A recombination is again the dominant origin of thermoluminescence. In PS II depleted of calcium by incubation at pH 4.0, the possibility that TL emission temperature is determined by a change in the mid-point redox potential of QA (Krieger, A. and Weis, E. (1992) Photosynthetica 27, 89–98) was investigated by comparing TL from equivalent samples of control and Ca2+-depleted PS II. It was shown that the emission temperature of the high temperature TL band induced by illumination at 77 K did not differ significantly between control and treated samples, suggesting that, under the conditions used, the potential of QA does not change significantly.

The quinone-iron complex of the electron acceptor complex of Photosystem II was studied by EPR spectroscopy in Thermosynechococcus elongatus. New g ∼ 2 features belonging to the EPR signal of the semiquinone forms of the primary and secondary quinone, i.e., Q(A)(•-)Fe(2+) and Q(B)(•-)Fe(2+), respectively, are reported. In previous studies, these signals were missed because they were obscured by the EPR signal arising from the stable tyrosyl radical, TyrD(•). When the TyrD(•) signal was removed, either by chemical reduction or by the use of a mutant lacking TyrD, the new signals dominated the spectrum. For Q(A)(•-)Fe(2+), the signal was formed by illumination at 77 K or by sodium dithionite reduction in the dark. For Q(B)(•-)Fe(2+), the signal showed the characteristic period-of-two variations in its intensity when generated by a series of laser flashes. The new features showed relaxation characteristics comparable to those of the well-known features of the semiquinone-iron complexes and showed a temperature dependence consistent with an assignment to the low-field edge of the ground state doublet of the spin system. Spectral simulations are consistent with this assignment and with the current model of the spin system. The signal was also present in Q(B)(•-)Fe(2+) in plant Photosystem II, but in plants, the signal was not detected in the Q(A)(•-)Fe(2+) 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.

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.

The synthesis and structural characterization of the novel [MnIII,IV2O2(N,Nbispicen)2](ClO4)3·CH3CN complex employing the tetradentate ligand N,Nbispicen = N,N-bis(2-pyridylmethyl)-1,2-diaminoethane are reported. Magnetic properties were determined and show that the ground state is a spin doublet. This can be quantitatively interpreted by antiferromagnetic coupling between a Mn(III) high spin and a Mn(IV) (J = −316 cm−1). The 16 line solution EPR spectrum exhibits an unusual splitting in the low field resonances. The following rhombic tensors were needed to simulate the EPR spectrum: |A1x|= 160 10−4 cm−1, |A1y| = 144 10−4 cm−1, |A1z|= 109 10−4 cm−1, |A2x|= 69 10−4 cm−1, |A2y| = 72 10−4 cm−1, |A2z| = 75 10−4 cm−1, gx = 2.001, gy = 1.996, gz = 1.984. The classical ligand field theory of local [gIII] and [gIV] tensors implemented with the first order perturbation theory to describe the properties of the pair does not result in a satisfying description of the [g1/2] tensor unless a large reduction in the spin-orbit constant is invoked. A simplified version of second-order perturbation theory leads to effects in qualitative agreement with experiment but weak as expected from the large |J| value. The magnitude of these effects depends, however, on the anisotropy effects on each Mn ion. It is suggested that determination of the anisotropy of the magnetic properties of the monomeric Mn(III) and Mn(IV) moieties would be a valuable goal for a future study of these mixed valent dimanganese-di-μ-oxo complexes. The complex exhibits two quasi-reversible waves in the cyclic voltammogram, one at E1/2 = 0.18 V vs SCE for the III/IV █ III/III couple and the other at E1/2 = 0.98 V vs SCE for the III/IV █ IV/IV couple. The UV-Vis spectra of the three redox states have been recorded spectroelectrochemically.

The radical formed as the formal S3 charge storage state in Ca(2+)-depleted photosystem II and detected as a split EPR signal was previously assigned to an oxidized histidine radical on the basis of its UV spectrum. In a recent paper [Hallahan, B. J., Nugent, J. H. A., Warden, J. T., & Evans, M. C. W. (1992) Biochemistry 31, 4562-4573], this assignment was challenged, and it was suggested that the signal arises instead from the well-known tyrosine radical Tyrz., the electron carrier between the photooxidized chlorophyll and the Mn cluster. Here, we provide evidence that the measurements of the Tyr., on which the new interpretation was based, are artifactual due to the use of saturating microwave powers. Other than a relaxation-enhancement effect, the formation of the split S3 signal is accompanied by no change in the Tyr. signal. Although essentially unrelated to the origin of the S3 radical, several other experimental and interpretational problems in the work of Hallahan et al. (1992) are pointed out and rationalized. For example, the inability of Hallahan et al. (1992) to observe the split S3 signal in samples containing DCMU or without a chelator, in contrast to our observations, is attributed to a number of technical problems including the incomplete inhibition of the enzyme. We thus conclude that the assignment of the split S3 signal as His., although not proven, remains the most reasonable on the basis of current data.

Electronic absorption, electron paramagnetic resonance (EPR), and Soret-excited resonance Raman (RR) spectra are reported for bis(N-alkylimidazole) complexes of various iron(III)-"basket-handle" (Fe(III)BHP(+)) and "picket-fence" (Fe(III)PFP(+)) porphyrins in methylene chloride. The Fe(III)BHP(+) derivatives consist of four cross-trans (CT) and two adjacent-cis (AC) -linked in which the composition and the length of the handles are variable (CT Fe(III)[(C(11)Im)(2)(+)], CT and AC Fe(III)[((C(4))(2)phi)(2)](+), CT Fe(III)[((C(3))(2)phi)(C(12))](+), CT and AC Fe(III)[((C(3))(2)phi)(2)](+)). The meso-alphaalpha betabeta and meso-alphabeta alphabeta atropisomers of Fe(III)-tetrakis(o-pivalamidophenyl)-porphyrins represents the Fe(III)PFP(+) derivatives (Fe(III)alphaalpha betabeta-T(piv)PP(+) and Fe(III)alphabeta alphabeta-T(piv)PP(+), respectively). The absorption and RR data obtained for these ferric compounds were compared to those previously published for the homologous ferrous complexes (Picaud, T., Le Moigne, C., Loock, B., Momenteau, M. and Desbois, A. J. Am. Chem. Soc. 2003, 125, 11616 and Le Moigne, C., Picaud, T., Boussac, A., Loock, B., Momenteau, M. and Desbois, A. Inorg. Chem. 2003, 42, 6081). The Soret band position of the eight investigated ferric compounds is observed between 417 and 424 nm, indicating that none of the complexes possesses a planar heme. The EPR spectra show that most of the Fe(III)BHP(+) complexes and all the Fe(III)PFP(+) complexes are rhombic B-type hemichromes (g(max) = 2.86-2.96). Notable exceptions concern the bis(N-methylimidazole) complexes of two CT Fe(III)BHP(+). The Fe(III)BHP(+) with the shortest handles (Fe(III)[((C(3))(2)phi)(2)](+)) exhibits a g value at 2.80. When the handles are lengthened by two methylene units (Fe(III)[((C(3))(2)phi)(2)](+)), the EPR spectrum corresponds to a mixture of two "highly anisotropic low-spin " or "large g(max)" type I EPR signals, a major species at g = 3.17 and a minor species at g = 3.77. All these EPR data were converted in terms of dihedral angle formed by the rings of the axial ligands. The RR spectra of the Fe(III)BHP(+) and Fe(III)PFP(+) complexes exhibited variable frequencies for the structure-sensitive nu(2) and nu(8) lines (1558-1563 cm(-1) and 386-401 cm(-1), respectively). In considering the ability of the different superstructures to stabilize particular out-of-plane distortions, this vibrational information was analyzed in terms of heme structure through changes in core size and Fe-N(pyrrole) bond length, in relation to changes in coordination geometry. The bis(N-methylimidazole) complex of Fe(III)[((C(3))(2)phi)(2)](+) was found to be the most distorted with a strongly ruffled tetrapyrrole. Because of a handle asymmetry, the heme conformation of the bis(N-methylimidazole) complex of Fe(III)[((C(3))(2)phi)(C(12))](+) was deduced to be a composition of ruffled and domed structures. The heme structure of the other complexes is a mixture of ruffled and saddled or ruffled and waved conformations. Taking into account our previous data on the ferrous series, this investigation provides information about the reorganization of the heme structure upon iron oxidation. The general trend is a decrease of either the core-size, or the Fe-N(pyrrole) bond length, or both. However, we demonstrated that the heme superstructures precisely control the nature and the extent of the tetrapyrrole reshaping. These results point out similar possible effect in the heme proteins, considering both an analogy between porphyrin superstructures and amino acids forming the heme sites and the diversity of the heme environments in the proteins.

THE evolution of oxygen as a result of light-driven water oxidation occurs in plants and is catalysed by photosystem-II (PS-II). A manganese-cluster probably acts both as the active site and as a charge-accumulating device (for a review, see ref. 1). The enzyme cycle involves five redox states which are denoted as S0–S4, depend-ing on the number of positive equivalents stored2. Oxygen is released after formation of the transient S4 state. Ca2+ is an obligatory cofactor in this process and its depletion inhibits the enzyme cycle at the step before water oxidation, that is, after formation of the S3 state3. In chelator-treated, Ca2+-depleted PS-II, a new electron paramagnetic resonance (EPR) signal arising from a formal S3 state has been reported4. It was suggested that the S3 EPR signal could originate from the oxidation of an amino acid, interacting magnetically with the manganese cluster4. There are only a few examples of amino-acid oxidation in enzyme chemistry and these are limited to tyrosine5 and tryptophan6. Here we report evidence that the S2 to S3 transition occurring in Ca2+-depleted PS-II corresponds to the oxidation of histidine.

The effects of NH3 on the oxygen evolving enzyme have been investigated with EPR and steady-state O2 evolution. The following results were obtained. At low light intensity O2 evolution occurs in all centers even though ammonia is bound. This binding occurs in the S2 state and results in a modification of the multiline signal as reported earlier. However, the oscillations with flash number of the amplitude of the EPR signal are virtually unaffected, indicating that NH3 binding does not prevent S-state advancement. Inhibition of O2 evolution by NH3 measured at light intensities that are nearly saturating for untreated photosystem II is interpreted as being due to a slow down in the rate of S-state cycling. At very high light intensities NH3 is not able to inhibit oxygen evolution presumably because NH3 binding is S state dependent and the susceptible S state (S2) is turned over too quickly. NH3 binding resulting in the modified multiline signal does not occur in S1. When S1 is formed from fully NH3 modified S2 by deactivation or by three further flashes, the S1 state does not have NH3 bound. NH3 thus dissociates easily from S1. Earlier reports of NH3 binding in S1 may be explained by the observation that NH3 binding can occur upon incubation of samples in S2 at temperatures as low as 198 K. Evidence is obtained for an NH3 binding occurring slowly (30 s) in S3. This binding results in a block in S-state advancement as suggested earlier [Velthuys, B. R. (1975) Thesis, University of Leiden]. The results are interpreted in two possible models: (1) NH3 binding in S2 occurs in a substrate site, but it is rapidly exchanged by water upon S4 formation. (2) NH3 binding in S2 is not in a substrate site but instead in a structural site and remains bound while water is oxidized. Inherent in this model is that other NH3 binding sites, i.e., the Cl- site, and the slow NH3 binding site in S3 could be the true substrate sites. Some mechanistic implications are discussed.

The active site for water oxidation in photosystem II (PSII) consists of a Mn 4 Ca cluster close to a redox-active tyrosine residue (TyrZ). The enzyme cycles through five sequential oxidation states (S 0 to S 4 ) in the water oxidation process. Earlier electron paramagnetic resonance (EPR) work showed that metalloradical states, probably arising from the Mn 4 cluster interacting with TyrZ, can be trapped by illumination of the S 0 , S 1 and S 2 states at cryogenic temperatures. The EPR signals reported were attributed to S 0 TyrZ, S 1 TyrZ and S 2 TyrZ, respectively. The equivalent states were examined here by EPR in PSII isolated from Thermosynechococcus elongatus with either Sr or Ca associated with the Mn 4 cluster. In order to avoid spectral contributions from the second tyrosyl radical, TyrD, PSII was used in which Tyr160 of D2 was replaced by phenylalanine. We report that the metalloradical signals attributed to TyrZ interacting with the Mn cluster in S 0 , S 1 , S 2 and also probably the S 3 states are all affected by the presence of Sr. Ca/Sr exchange also affects the non-haem iron which is situated approximately 44 Å units away from the Ca site. This could relate to the earlier reported modulation of the potential of Q A by the occupancy of the Ca site. It is also shown that in the S 3 state both visible and near-infrared light are able to induce a similar Mn photochemistry.

The Mn4Ca complex that is involved in water oxidation in PSII is affected by near-infrared (NIR) light in certain redox states and these phenomena can be monitored by electron paramagnetic resonance (EPR) at low temperature. Here we report the action spectra of the NIR effects in the S2 and S3 states in PSII from plants and the thermophilic cyanobacterium Thermosynechococcus elongatus. The action spectra obtained are very similar in both S states, indicating the presence of the same photoactive form of the Mn4Ca complex in both states. Since the chemical nature of the photoactive species is not known, an unequivocal interpretation of this result cannot be made; however, it appears to be more easily reconciled with the view that the redox state of the Mn4Ca cluster does not change from the S2 to the S3 transition, at least in those centers sensitive to NIR light. The temperature dependence of the NIR effect and the action spectra for S2 indicate the presence of structural heterogeneity in the Mn4Ca cluster.

The thermophilic cyanobacterium, Thermosynechococcus elongatus, has been grown in the presence of Sr2+ instead of Ca2+ with the aim of biosynthetically replacing the Ca2+ of the oxygen-evolving enzyme with Sr2+. Not only were the cells able to grow normally with Sr2+, they actively accumulated the ion to levels higher than those of Ca2+ in the normal cultures. A protocol was developed to purify a fully active Sr(2+)-containing photosystem II (PSII). The modified enzyme contained a normal polypeptide profile and 1 strontium/4 manganese, indicating that the normal enzyme contains 1 calcium/4 manganese. The Sr(2+)- and Ca(2+)-containing enzymes were compared using EPR spectroscopy, UV-visible absorption spectroscopy, and O2 polarography. The Ca2+/Sr2+ exchange resulted in the modification of the EPR spectrum of the manganese cluster and a slower turnover of the redox cycle (the so-called S-state cycle), resulting in diminished O2 evolution activity under continuous saturating light: all features reported previously by biochemical Ca2+/Sr2+ exchange in plant PSII. This allays doubts that these changes could be because of secondary effects induced by the biochemical treatments themselves. In addition, the Sr(2+)-containing PSII has other kinetics modifications: 1) it has an increased stability of the S3 redox state; 2) it shows an increase in the rate of electron donation from TyrD, the redox-active tyrosine of the D2 protein, to the oxygen-evolving complex in the S3-state forming S2; 3) the rate of oxidation of the S0-state to the S1-state by TyrD* is increased; and 4) the release of O2 is slowed down to an extent similar to that seen for the slowdown of the S3TyrZ* to S0TyrZ transition, consistent with the latter constituting the limiting step of the water oxidation mechanism in Sr(2+)-substituted enzyme as well as in the normal enzyme. The replacement of Ca2+ by Sr2+ appears to have multiple effects on kinetics properties of the enzyme that may be explained by S-state-dependent shifts in the redox properties of both the manganese complex and TyrZ as well as structural effects.

First, the crystal structure of cytochrome c-550 (the psbV1 gene product) from the thermophilic cyanobacterium Thermosynechococcus elongatus has been determined to a resolution of 1.8 A. A comparison of the T. elongatus cytochrome c-550 structure to its counterparts from mesophilic organisms, Synechocystis 6803 and Arthrospira maxima, suggests that increased numbers of hydrogen bonds may play a role in the structural basis of thermostability. The cytochrome c-550 in T. elongatus also differs from that in Synechocystis 6803 and Arthrospira maxima in its lack of dimerization and the presence of a trigonal planar molecule, possibly bicarbonate, tightly bound to the heme propionate oxygen atoms. Cytochromes c-550 from T. elongatus, Synechocystis 6803 and Arthrospira maxima exhibit different EPR spectra. A correlation has been done between the heme-axial ligands geometries and the rhombicity calculated from the EPR spectra. This correlation indicates that binding of cytochrome c-550 to Photosystem II is accompanied by structural changes in the heme vicinity. Second, the psbV2 gene product has been found and purified. The UV-visible, EPR and Raman spectra are reported. From the spectroscopic data and from a theoretical structural model based on the cytochrome c-550 structure it is proposed that the 6th ligand of the heme-iron is the Tyr86.

The iron-containing superoxide dismutase (FeSOD) from the thermophilic cyanobacterium Thermosynechococcus elongatus has been isolated. The protein crystallizes readily and we have determined the structure to 1.6 A resolution. This is the first structural characterization of an FeSOD isolated from a cyanobacterium and one of the highest resolution FeSOD structures determined to date. The activity of the T. elongatus FeSOD has been measured both at 25 degrees C and 50 degrees C and it has been spectroscopically characterized. The T. elongatus FeSOD EPR spectra at pH 5.1, 7.5 and 10.0 are similar. This indicates that no change in the geometry of the Fe(III) site occurs over a wide range of pH. This is in contrast to the other FeSODs described in the literature.

Despite crystallographic structures now available and intensive work in the past decades, little is known about the higher redox states of the catalytic cycle of Photosystem II, the enzyme responsible for the presence of O(2) on Earth and at the beginning of the process that has produced both the biomass and the fossil fuels. In one of the highest oxidation states, the S(3)-state, only signals at g-values higher than 4 have been detected so far at the X-band. In this work, we report for the first time the complete X-band EPR spectrum for the S(3)-state of Photosystem II. Simulations show that, for a spin state S = 1, as was previously suggested for S(3), it is not possible to account for all the features observed. A satisfactory simulated spectrum was obtained for a spin state S = 3 with zero-field splitting parameters D = 0.175 cm(-1) and E/D = 0.275. The detection of the full EPR signal for S(3) opens the door for new investigations and a better understanding of the catalytic cycle of Photosystem II.

The Mn4 complex which is involved in water oxidation in photosystem II (PSII) is known to exhibit two types of EPR signals in the S2 state, one of the five redox states of the enzyme cycle: either a multiline signal (S = 1/2) or a signal at g = 4.1 (S = 3/2 or S= 5/2). The S = 1/2 state can be converted to that responsible for the g = 4.1 signal upon the absorption of near-infrared (IR) light [Boussac, A., Girerd, J.-J., and Rutherford, A.W. (1996) Biochemistry 35, 6984-6989]. It is shown here that a third state gives rise to signals at g = 10 and 6. This state is formed by IR illumination of the S = 1/2 state at 65 K, a temperature where IR illumination leads to the loss of the S = 1/2 signal but to no formation of the g = 4.1 state. On the basis of the corresponding decrease of the S = 1/2 state, the new state can be trapped in approximately 40% of the PSII centers. Warming of the sample above 65 K, in the dark, leads to the loss of the g = 10 and 6 resonances with the corresponding appearance of the g = 4.1 signal. It is suggested that the IR-induced conversion of the S = 1/2 state into the g = 4.1 state at 150 K involves the transient formation of the new state. The new state is attributed to a S = 5/2 state of the Mn4 complex (although a S value5/2 is also a possibility). Spectral simulations indicate an E/D ratio of -0.05 with D/= 1 cm-1. The resonances at g = 10 and 6 correspond to the gz of the +/-5/2 and +/-3/2 transition, respectively. The temperature-dependent conversion of this S = 5/2 state into the g = 4.1 state is proposed to be due to relaxation of the ligand environment around the Mn4 cluster that leads to a change in the zero field splitting parameters, assuming an S = 5/2 value for the g = 4.1 state. The new form of the S2 state reported here may explain some earlier data where the S2 state was present and yet not detectable as either a S = 1/2 or a g = 4.1 EPR signal.

The evolution of oxygen as a result of light-driven water oxidation is catalyzed by photosystem II (PS2) in which a cluster of four manganese ions acts both as a charge accumulating device and as the active site. During the enzyme cycle, the oxidizing side of PS2 goes through five different redox states that are denoted Sn, n varying from 0 to 4. Oxygen is released during the S3 to S0 transition in which S4 is a transient state (1,2). The Mn4 is known to exhibit in the S2-state a multiline EPR signal (spin 1/2) (1–7, and references therein), a signal at g = 4.1 or g = 4.25 (1–7) (spin = 5/2, (8–12)) and signals at g 5 (spin 5/2) (8–12). A different multiline signal has also been observed in the S0-state (13-15). The detection of an S0 signal by cw-EPR required the presence of methanol in the sample (13–15). In the present study we report experiments on the nature of the interaction of methanol with the Mn4-cluster, on the number of spins contributing in the S0-multiline signal and on the effects of infrared light.