Water binding to the Mn4O5Ca cluster of the oxygen-evolving complex (OEC) of Photosystem II (PSII) poised in the S-2 state was studied via (H2O)-O-17- and (H2O)-H-2-labeling and high-field electron paramagnetic resonance (EPR) spectroscopy. Hyperfine couplings of coordinating O-17 (I = 5/2) nuclei were detected using W-band (94 GHz) electron electron double resonance (ELDOR) detected NMR and Davies/Mims electron-nuclear double resonance (ENDOR) techniques. Universal N-15 (I = 1/2) labeling was employed to clearly discriminate the O-17 hyperfine couplings that overlap with N-14 (I = 1) signals from the D1-His332 ligand of the OEC (Stich et al. Biochemistry 2011, 50 (34), 7390-7404). Three classes of 170 nuclei were identified: (i) one mu-oxo bridge; (ii) a terminal Mn-OH/OH2 ligand; and (iii) Mn/Ca-H2O ligand(s). These assignments are based on O-17 model complex data, on comparison to the recent 1.9 angstrom resolution PSII crystal structure (Umena et al. Nature 2011, 473, 55-60), on NH3 perturbation of the O-17 signal envelope and density functional theory calculations. The relative orientation of the putative O-17 mu-oxo bridge hyperfine tensor to the N-14(N-15) hyperfine tensor of the D1-His332 ligand suggests that the exchangeable mu-oxo bridge links the outer Mn to the Mn3O3Ca open-cuboidal unit (O-4 and OS in the Umena et al. structure). Comparison to literature data favors the Ca-linked OS oxygen over the alternative assignment to O4. All O-17 signals were seen even after very short (<= 15 s) incubations in (H2O)-O-17 suggesting that all exchange sites identified could represent bound substrate in the S, state including the p-oxo bridge. H-1/H-2 (I = 1/2, 1) ENDOR data performed at Q- (34 GHz) and W-bands complement the above findings. The relatively small H-1/H-2 couplings observed require that all the mu-oxo bridges of the Mn4O5Ca cluster are deprotonated in the S-2 state. Together, these results further limit the possible substrate water-binding sites and modes within the OEC. This information restricts the number of possible reaction pathways for O-O bond formation, supporting an oxo/oxyl coupling mechanism in S-4.