Probing the functional role of Ca²⁺ in the oxygen-evolving complex of photosystem II by metal ion inhibition

Photosynthetic oxygen evolution in photosystem II (PSII) takes place in the oxygen-evolving complex (OEC) that is comprised of a tetranuclear manganese cluster (Mn₄), a redox-active tyrosine residue (Y_z), and Ca²⁺ and Cl^- cofactors. The OEC is successively oxidized by the absorption of 4 quanta of light that results in the oxidation of water and the release of O₂. Ca²⁺ is an essential cofactor in the water-oxidation reaction, as its depletion causes the loss of the oxygen-evolution activity in PSII. In recent X-ray crystal structures, Ca ²⁺ has been revealed to be associated with the Mn₄ cluster of PSII. Although several mechanisms have been proposed for the water-oxidation reaction of PSII, the role of Ca²⁺ in oxygen evolution remains unclear. In this study, we probe the role of Ca²⁺ in oxygen evolution by monitoring the S₁ to S₂ state transition in PSII membranes and PSII core complexes upon inhibition of oxygen evolution by Dy ³⁺, Cu²⁺, and Cd²⁺ ions. By using a cation-exchange procedure in which Ca²⁺ is not removed prior to addition of the studied cations, we achieve a high degree of reversible inhibition of PSII membranes and PSII core complexes by Dy³⁺, Cu ²⁺, and Cd²⁺ ions. EPR spectroscopy is used to quantitate the number of bound Dy³⁺ and Cu²⁺ ions per PSII center and to determine the proximity of Dy³⁺ to other paramagnetic centers in PSII. We observe, for the first time, the S₂ state multiline electron paramagnetic resonance (EPR) signal in Dy³⁺- and Cd ²⁺-inhibited PSII and conclude that the Ca²⁺ cofactor is not specifically required for the S₁ to S₂ state transition of PSII. This observation provides direct support for the proposal that Ca²⁺ plays a structural role in the early S-state transitions, which can be fulfilled by other cations of similar ionic radius, and that the functional role of Ca²⁺ to activate water in the O-O bond-forming reaction that occurs in the final step of the S state cycle can only be fulfilled by Ca²⁺ and Sr²⁺, which have similar Lewis acidities.