The Basic Properties of the Electronic Structure of the Oxygen-evolving Complex of Photosystem II Are Not Perturbed by Ca2+ Removal

Ca2+ is an integral component of the Mn4O5Ca cluster of the oxygen-evolving complex in photosystem II (PS II). Its removal leads to the loss of the water oxidizing functionality. The S-2' state of the Ca2+-depleted cluster from spinach is examined by X-and Q-band EPR and Mn-55 electron nuclear double resonance (ENDOR) spectroscopy. Spectral simulations demonstrate that upon Ca2+ removal, its electronic structure remains essentially unaltered, i.e. that of a manganese tetramer. No redistribution of the manganese valence states and only minor perturbation of the exchange interactions between the manganese ions were found. Interestingly, the S-2' state in spinach PS II is very similar to the native S-2 state of Thermosynechococcus elongatus in terms of spin state energies and insensitivity to methanol addition. These results assign the Ca2+ a functional as opposed to a structural role in water splitting catalysis, such as (i) being essential for efficient proton-coupled electron transfer between Y-Z and the manganese cluster and/or (ii) providing an initial binding site for substrate water. Additionally, a novel Mn-55(2+) signal, detected by Q-band pulse EPR and ENDOR, was observed in Ca2+-depleted PS II. Mn2+ titration, monitored by Mn-55 ENDOR, revealed a specific Mn2+ binding site with a submicromolar K-D. Ca2+ titration of Mn2+-loaded, Ca2+-depleted PS II demonstrated that the site is reversibly made accessible to Mn2+ by Ca2+ depletion and reconstitution. Mn2+ is proposed to bind at one of the extrinsic subunits. This process is possibly relevant for the formation of the Mn4O5Ca cluster during photoassembly and/or D1 repair.