The assignment of the two substrate water sites of the tetra-manganese penta-oxygen calcium (Mn ₄O ₅Ca) 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 ₄O ₅Ca 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 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.
The electronic properties of the Mn₄OₓCa cluster in the S₂ state of the oxygen-evolving complex (OEC) were studied using X- and Q-band EPR and Q-band ⁵⁵Mn-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 Mnᴵᴵᴵ ion in the cluster is of the same magnitude and geometry for both species types, suggesting that the Mnᴵᴵᴵ 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 ST=½ spin model of the S₂ 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 (MnA) and the other three Mn ions that form the trimeric part of the cluster (MnB, MnC, MnD), 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.
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.
Fluorine electron-nuclear double resonance (
F 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
F ENDOR almost 2-fold. We first demonstrate this on a model fluorine-containing Gd(III) complex with a well-resolved
F 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
F and Gd(III), in which the Gd-F distance is too long to produce a well-resolved
F 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
F ENDOR approach may be applicable to Gd-F distances as large as 20 Å, widening the current ENDOR distance window.
Identifying the two substrate water sites of nature's water-splitting cofactor (Mn
CaO
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 S
state of native Ca-PSII and biosynthetically substituted Sr-PSII employing Time-Resolved Membrane Inlet Mass Spectrometry (TR-MIMS) and a Time-Resolved
O-Electron-electron Double resonance detected NMR (TR-
O-EDNMR) approach. TR-MIMS resolves the kinetics for incorporation of the oxygen-isotope label into the substrate sites after addition of H
O to the medium, while the magnetic resonance technique allows, in principle, the characterization of all exchangeable oxygen ligands of the Mn
CaO
cofactor after mixing with H
O. This unique combination shows i) that the central oxygen bridge (O5) of Ca-PSII core complexes isolated from
has, within experimental conditions, the same rate of exchange as the slowly exchanging substrate water (W
) in the TR-MIMS experiments and ii) that the exchange rates of O5 and W
are both enhanced by Ca
→Sr
substitution in a similar manner. In the context of previous TR-MIMS results, this shows that only O5 fulfills all criteria for being W
. This strongly restricts options for the mechanism of water oxidation.
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•Spin echo signal of spin-correlated P3HT+/PCBM− RPs is treated numerically.•When integration window is centered at moment τ distortions are minimal.•Calculations reveal discrepancy ...between predictions of SCRP model and experiment.•Formation of AE (emission/absorption) polarization is revealed.•Convincing agreement between theory and experiment is achieved.
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.
The electronic properties of the Mn
4O
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
55Mn-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.
► The
S
2 electronic structures of the OECs of
T. elongatus and plant PS II are similar. ► Both can be explained using the same coupling scheme. ► The inferred on-site ZFS of the Mn
III changes for the two OECs. ► Methanol changes the electronic coupling scheme.
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.
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