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 = tau(D2o)/tau(H2O)) 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 (Y-z(center dot+)) in the S-0(n) -> S-1(+) and S-1(n) -> S-2(+) transitions and for manganese reduction by ET from substrate water to manganese ions in the O-2-evolving S-3(n) -> S-0(n) step, but a larger KIE (similar to 1.8) for manganese oxidation in the S-2(n) -> S-3(+) 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 S-3(+) -> S-3(n) (similar to 150 mu s, H2O; similar to 380 mu s, D2O) and S-2(+) -> S-2(n) (similar to 25 mu s, H2O; similar to 120 mu s, D2O) steps and attributed to PT events according to their comparatively large KIE (similar to 2.4, similar to 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 S-2(n) -> S-3(+) transition, assigned as proton-coupled electron transfer. Observation of a lag phase in the classical S-2 -> S-3 transition verifies that the associated PT is a prerequisite for subsequent ET, which completes Mn4Ca oxidation to the all-Mn(IV) level.