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 Mn4OxCa cluster and the biosynthetically substituted Mn4OxSr cluster of the oxygen evolving complex (OEC) of photosystem II (PSII) core complexes isolated from Thermosynechococcus elongatus, poised in the S2 state, were studied by X- and Q-band CW-EPR and by pulsed Q-band 55Mn-ENDOR spectroscopy. Both wild type and tyrosine D less mutants grown photoautotrophically in either CaCl2 or SrCl2 containing media were measured. The obtained CW-EPR spectra of the S2 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 (55Mn) 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 55Mn-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 55Mn-ENDOR spectra; (ii) only small changes are seen in the fitted isotropic hyperfine values for the Ca2+ and Sr2+ containing OEC, suggesting that there is no change in the overall spin distribution (electronic coupling scheme) upon Ca2+/Sr2+ 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 Ca2+ site may modulate the fine structure tensor of the MnIII ion. DFT calculations support the above conclusions. Our data analysis also provides strong support for the notion that in the S2 state the coordination of the MnIII 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 MnIII is a potential site for substrate 'water' (H2O, 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.