The identity of a key intermediate in the S to S transition of nature's water-oxidizing complex (WOC) in Photosystem 2 is presented. Broken-symmetry density functional theory (BS-DFT) calculations and Heisenberg-Dirac-van Vleck (HDvV) spin ladder calculations show that an S state open cubane model of the WOC containing a μ-hydroxo O4 changes from an = / form to an = /, form upon deprotonation of W1. Combined with X-band electron paramagnetic resonance (EPR) spectral analysis, this indicates that the = 4.1 EPR signal corresponds to an = / form of the WOC with W1 present as a water ligand to Mn, while the = 4.8/4.9 form observed at high pH values corresponds to an = / form, with W1 as a hydroxo ligand. The latter is also likely to represent the form needed to progress to S in the functioning enzyme.