Assignment of the slowly exchanging substrate water of nature's water-splitting cofactor.

Identifying the two substrate water sites of nature's water-splitting cofactor (Mn4CaO5 cluster) provides important information toward resolving the mechanism of O-O bond formation in Photosystem II (PSII). To this end, we have performed parallel substrate water exchange experiments in the S1 state of native Ca-PSII and biosynthetically substituted Sr-PSII employing Time-Resolved Membrane Inlet Mass Spectrometry (TR-MIMS) and a Time-Resolved 17O-Electron-electron Double resonance detected NMR (TR-17O-EDNMR) approach. TR-MIMS resolves the kinetics for incorporation of the oxygen-isotope label into the substrate sites after addition of H218O to the medium, while the magnetic resonance technique allows, in principle, the characterization of all exchangeable oxygen ligands of the Mn4CaO5 cofactor after mixing with H217O. This unique combination shows i) that the central oxygen bridge (O5) of Ca-PSII core complexes isolated from Thermosynechococcus vestitus has, within experimental conditions, the same rate of exchange as the slowly exchanging substrate water (WS) in the TR-MIMS experiments and ii) that the exchange rates of O5 and WS are both enhanced by Ca2+→Sr2+ substitution in a similar manner. In the context of previous TR-MIMS results, this shows that only O5 fulfills all criteria for being WS. This strongly restricts options for the mechanism of water oxidation.

Extending the Range of Distances Accessible by 19F Electron-Nuclear Double Resonance in Proteins Using High-Spin Gd(III) Labels.

Fluorine electron-nuclear double resonance (19F ENDOR) has recently emerged as a valuable tool in structural biology for distance determination between F atoms and a paramagnetic center, either intrinsic or conjugated to a biomolecule via spin labeling. Such measurements allow access to distances too short to be measured by double electron-electron resonance (DEER). To further extend the accessible distance range, we exploit the high-spin properties of Gd(III) and focus on transitions other than the central transition (|-1/2⟩ ↔ |+1/2⟩), that become more populated at high magnetic fields and low temperatures. This increases the spectral resolution up to ca. 7 times, thus raising the long-distance limit of 19F ENDOR almost 2-fold. We first demonstrate this on a model fluorine-containing Gd(III) complex with a well-resolved 19F spectrum in conventional central transition measurements and show quantitative agreement between the experimental spectra and theoretical predictions. We then validate our approach on two proteins labeled with 19F and Gd(III), in which the Gd-F distance is too long to produce a well-resolved 19F ENDOR doublet when measured at the central transition. By focusing on the |-5/2⟩ ↔ |-3/2⟩ and |-7/2⟩ ↔ |-5/2⟩ EPR transitions, a resolution enhancement of 4.5- and 7-fold was obtained, respectively. We also present data analysis strategies to handle contributions of different electron spin manifolds to the ENDOR spectrum. Our new extended 19F ENDOR approach may be applicable to Gd-F distances as large as 20 Å, widening the current ENDOR distance window.

Time-domain shape of electron spin echo signal of spin-correlated radical pairs in polymer/fullerene blends.

Temporal shape of electron spin echo (ESE) signal of photoinduced spin-correlated radical pairs (SCRP) in composite of conductive polymer P3HT and substituted fullerene PCBM is studied in details. ESE signals of radical pairs (RP) P3HT+/PCBM- are calculated in realistic model, taking into account finite microwave pulse length. Inhomogeneous broadening of resonant lines and interradical distance distribution are included. Experimentally observed ESE time-domain shape was found to contradict predictions of conventional SCRP theory, which would be valid in the case of very fast electron transfer. Thus, instantaneous formation of singlet SCRP is not the case for P3HT+/PCBM- pair, and spin system has enough time to evolve coherently during sequential electron transfer. While it is impossible to reproduce experimental data within simple singlet SCRP model, assumption of presence of additional - with respect to what is predicted by singlet SCRP theory - AE (absorption/emission) spin polarization gives convincing accordance with the experiment. Density matrix of RP P3HT+/PCBM- is a superposition of two contributions, namely the parts reflecting (i) antiphase polarization of original singlet-born SCRP and (ii) additional AE-polarization which is generated during initial stage of charge separation. AE-polarization affects experimental ESEEM (electron spin echo envelope modulation) traces, as well as ESE shape, making impossible their interpretation via simple singlet SCRP model. However, this effect can be eliminated by averaging of ESEEM traces over EPR spectral positions. Finally, choosing the optimal gate for ESE time-domain integration and proper microwave detection phase tuning are considered.

Characterization of Oxygen Bridged Manganese Model Complexes Using Multifrequency (17)O-Hyperfine EPR Spectroscopies and Density Functional Theory.

Multifrequency pulsed EPR data are reported for a series of oxygen bridged (µ-oxo/µ-hydroxo) bimetallic manganese complexes where the oxygen is labeled with the magnetically active isotope (17)O (I = 5/2). Two synthetic complexes and two biological metallocofactors are examined: a planar bis-µ-oxo bridged complex and a bent, bis-µ-oxo-µ-carboxylato bridge complex; the dimanganese catalase, which catalyzes the dismutation of H2O2 to H2O and O2, and the recently identified manganese/iron cofactor of the R2lox protein, a homologue of the small subunit of the ribonuclotide reductase enzyme (class 1c). High field (W-band) hyperfine EPR spectroscopies are demonstrated to be ideal methods to characterize the (17)O magnetic interactions, allowing a magnetic fingerprint for the bridging oxygen ligand to be developed. It is shown that the µ-oxo bridge motif displays a small positive isotropic hyperfine coupling constant of about +5 to +7 MHz and an anisotropic/dipolar coupling of -9 MHz. In addition, protonation of the bridge is correlated with an increase of the hyperfine coupling constant. Broken symmetry density functional theory is evaluated as a predictive tool for estimating hyperfine coupling of bridging species. Experimental and theoretical results provide a framework for the characterization of the oxygen bridge in Mn metallocofactor systems, including the water oxidizing cofactor of photosystem II, allowing the substrate/solvent interface to be examined throughout its catalytic cycle.

Structure, ligands and substrate coordination of the oxygen-evolving complex of photosystem II in the S2 state: a combined EPR and DFT study.

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.

Ammonia binding to the oxygen-evolving complex of photosystem II identifies the solvent-exchangeable oxygen bridge (µ-oxo) of the manganese tetramer.

The assignment of the two substrate water sites of the tetra-manganese penta-oxygen calcium (Mn4O5Ca) 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 Mn4O5Ca 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 (MnA4-W1); and (iii) as W1 is trans to O5, ammonia binding elongates the MnA4-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.

Detection of the water-binding sites of the oxygen-evolving complex of Photosystem II using W-band 17O electron-electron double resonance-detected NMR spectroscopy.

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).

Electronic structure of a weakly antiferromagnetically coupled Mn(II)Mn(III) model relevant to manganese proteins: a combined EPR, 55Mn-ENDOR, and DFT study.

An analysis of the electronic structure of the [Mn(II)Mn(III)(µ-OH)-(µ-piv)(2)(Me(3)tacn)(2)](ClO(4))(2) (PivOH) complex is reported. It displays features that include: (i) a ground 1/2 spin state; (ii) a small exchange (J) coupling between the two Mn ions; (iii) a mono-µ-hydroxo bridge, bis-µ-carboxylato motif; and (iv) a strongly coupled, terminally bound N ligand to the Mn(III). All of these features are observed in structural models of the oxygen evolving complex (OEC). Multifrequency electron paramagnetic resonance (EPR) and electron nuclear double resonance (ENDOR) measurements were performed on this complex, and the resultant spectra simulated using the Spin Hamiltonian formalism. The strong field dependence of the (55)Mn-ENDOR constrains the (55)Mn hyperfine tensors such that a unique solution for the electronic structure can be deduced. Large hyperfine anisotropy is required to reproduce the EPR/ENDOR spectra for both the Mn(II) and Mn(III) ions. The large effective hyperfine tensor anisotropy of the Mn(II), a d(5) ion which usually exhibits small anisotropy, is interpreted within a formalism in which the fine structure tensor of the Mn(III) ion strongly perturbs the zero-field energy levels of the Mn(II)Mn(III) complex. An estimate of the fine structure parameter (d) for the Mn(III) of -4 cm(-1) was made, by assuming the intrinsic anisotropy of the Mn(II) ion is small. The magnitude of the fine structure and intrinsic (onsite) hyperfine tensor of the Mn(III) is consistent with the known coordination environment of the Mn(III) ion as seen from its crystal structure. Broken symmetry density functional theory (DFT) calculations were performed on the crystal structure geometry. DFT values for both the isotropic and the anisotropic components of the onsite (intrinsic) hyperfine tensors match those inferred from the EPR/ENDOR simulations described above, to within 5%. This study demonstrates that DFT calculations provide reliable estimates for spectroscopic observables of mixed valence Mn complexes, even in the limit where the description of a well isolated S = 1/2 ground state begins to break down.

The electronic structures of the S(2) states of the oxygen-evolving complexes of photosystem II in plants and cyanobacteria in the presence and absence of methanol.

The electronic properties of the Mn(4)O(x)Ca cluster in the S(2) state of the oxygen-evolving complex (OEC) were studied using X- and Q-band EPR and Q-band (55)Mn-ENDOR using photosystem II preparations isolated from the thermophilic cyanobacterium T. elongatus and higher plants (spinach). The data presented here show that there is very little difference between the two species. Specifically it is shown that: (i) only small changes are seen in the fitted isotropic hyperfine values, suggesting that there is no significant difference in the overall spin distribution (electronic coupling scheme) between the two species; (ii) the inferred fine-structure tensor of the only Mn(III) ion in the cluster is of the same magnitude and geometry for both species types, suggesting that the Mn(III) ion has the same coordination sphere in both sample preparations; and (iii) the data from both species are consistent with only one structural model available in the literature, namely the Siegbahn structure [Siegbahn, P. E. M. Accounts Chem. Res.2009, 42, 1871-1880, Pantazis, D. A. et al., Phys. Chem. Chem. Phys.2009, 11, 6788-6798]. These measurements were made in the presence of methanol because it confers favorable magnetic relaxation properties to the cluster that facilitate pulse-EPR techniques. In the absence of methanol the separation of the ground state and the first excited state of the spin system is smaller. For cyanobacteria this effect is minor but in plant PS II it leads to a break-down of the S(T)=½ spin model of the S(2) state. This suggests that the methanol-OEC interaction is species dependent. It is proposed that the effect of small organic solvents on the electronic structure of the cluster is to change the coupling between the outer Mn (Mn(A)) and the other three Mn ions that form the trimeric part of the cluster (Mn(B), Mn(C), Mn(D)), by perturbing the linking bis-µ-oxo bridge. The flexibility of this bridging unit is discussed with regard to the mechanism of O-O bond formation.

Effect of Ca2+/Sr2+ substitution on the electronic structure of the oxygen-evolving complex of photosystem II: a combined multifrequency EPR, 55Mn-ENDOR, and DFT study of the S2 state.

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