In the oxygen-evolving complex (OEC) of photosystem II(PSII) molecular oxygen is formed from two substrate water molecules that are ligated to mu-oxo bridged cluster containing four Mn ions and one Ca ion (Mn4OxCa cluster; O-x symbolizes the unknown number of mu-oxo bridges; x >= 5). There is a long-standing enigma as to when, where, and how the two substrate water molecules bind to the Mn4OxCa cluster during the cyclic water-splitting reaction, which involves five distinct redox intermediates (S-i-states; i = 0,...,4). To address this question we employed hyperfine sublevel correlation (HYSCORE) spectroscopy on (H2O)-O-17-enriched PSII samples poised in the paramagnetic S-2 state. This approach allowed us to resolve the magnetic interaction between one solvent exchangeable O-17 that is directly ligated to one or more Mn ions of the Mn4OxCa cluster in the S-2 state of PSII. Direct coordination of O-17 to Mn is supported by the strong (A approximate to 10 MHz) hyperfine coupling. Because these are properties expected from a substrate water molecule, this spectroscopic signature holds the potential for gaining long-sought information about the binding mode and site of one of the two substrate water molecules in the S-2 state of PSII.