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

Abstract

The electronic structures of the native Mn₄O_xCa cluster and the biosynthetically substituted Mn₄O_xSr 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 Mn^III 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 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’ (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.