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 S0 n → S1 + and S1 n → S2 + transitions and for manganese reduction by ET from substrate water to manganese ions in the O2-evolving S3 n → S0 n step, but a larger KIE (∼1.8) for manganese oxidation in the S2 n → 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 + → S3 n (∼150 μs, H2O; ∼380 μs, D2O) and S2 + → S2 n (∼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 S2 n → 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.