Sequential and Coupled Proton and Electron Transfer Events in the S₂ → S₃ Transition of Photosynthetic Water Oxidation Revealed by Time-Resolved X-ray Absorption Spectroscopy

The choreography of electron transfer (ET) and proton transfer (PT) in the S-state cycle at the manganese-calcium (Mn₄Ca) complex of photosystem II (PSII) is pivotal for the mechanism of photosynthetic water oxidation. Time-resolved room-temperature X-ray absorption spectroscopy (XAS) at the Mn K-edge was employed to determine the kinetic isotope effect (KIE = τD₂O/τH₂O) of the four S transitions in a PSII membrane particle preparation in H₂O and D₂O buffers. We found a small KIE (1.2-1.4) for manganese oxidation by ET from Mn₄Ca to the tyrosine radical (YZ•+) in the S₀n → S₁⁺ and S₁n → S₂⁺ transitions and for manganese reduction by ET from substrate water to manganese ions in the O₂-evolving S₃n → S₀n step, but a larger KIE (∼1.8) for manganese oxidation in the S₂n → S₃⁺ step (subscript, number of accumulated oxidizing equivalents; superscript, charge of Mn₄Ca). Kinetic lag phases detected in the XAS transients prior to the respective ET steps were assigned to S₃⁺ → S₃n (∼150 μs, H₂O; ∼380 μs, D₂O) and S₂⁺ → S₂n (∼25 μs, H₂O; ∼120 μs, D₂O) steps and attributed to PT events according to their comparatively large KIE (∼2.4, ∼4.5). Our results suggest that proton movements and molecular rearrangements within the hydrogen-bonded network involving Mn₄Ca and its bound (substrate) water ligands and the surrounding amino acid/water matrix govern to different extents the rates of all ET steps but affect particularly strongly the S₂n → S₃⁺ transition, assigned as proton-coupled electron transfer. Observation of a lag phase in the classical S₂ → S₃ transition verifies that the associated PT is a prerequisite for subsequent ET, which completes Mn₄Ca oxidation to the all-Mn(IV) level.