Sequential and Coupled Proton and Electron Transfer Events in the S → S 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 (MnCa) 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 = τ/τ) of the four S transitions in a PSII membrane particle preparation in HO and DO buffers. We found a small KIE (1.2-1.4) for manganese oxidation by ET from MnCa to the tyrosine radical (Y) in the S → S and S → S transitions and for manganese reduction by ET from substrate water to manganese ions in the O-evolving S → S step, but a larger KIE (∼1.8) for manganese oxidation in the S → S step (subscript, number of accumulated oxidizing equivalents; superscript, charge of MnCa). Kinetic lag phases detected in the XAS transients prior to the respective ET steps were assigned to S → S (∼150 μs, HO; ∼380 μs, DO) and S → S (∼25 μs, HO; ∼120 μs, DO) 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 MnCa 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 → S transition, assigned as proton-coupled electron transfer. Observation of a lag phase in the classical S → S transition verifies that the associated PT is a prerequisite for subsequent ET, which completes MnCa oxidation to the all-Mn(IV) level.