The MnCaO cluster, featuring four ligand water molecules (W1 to W4), serves as the water-splitting site in photosystem II (PSII). X-ray free electron laser (XFEL) structures exhibit an additional oxygen site (O6) adjacent to the O5 site in the fourth lowest oxidation state, S, forming MnCaO. Here, we investigate the mechanism of the second water ligand molecule at the dangling Mn (W2) as a potential incorporating species, using a quantum mechanical/molecular mechanical (QM/MM) approach. Previous QM/MM calculations demonstrated that W1 releases two protons through a low-barrier H-bond toward D1-Asp61 and subsequently releases an electron during the S to S transition, resulting in O at W1 and protonated D1-Asp61. During the process of MnCaO formation, O, rather than HO or OH, best reproduced the O5···O6 distance. Although the catalytic cluster with O at O6 is more stable than that with O at W1 in S, it does not occur spontaneously due to the significantly uphill deprotonation process. Assuming O at W2 incorporates into the O6 site, an exergonic conversion from Mn1(III)Mn2(IV)Mn3(IV)Mn4(IV) (equivalent to the open-cubane S valence state) to Mn1(IV)Mn2(IV)Mn3(IV)Mn4(III) (equivalent to the closed-cubane S valence state) occurs. These findings provide energetic insights into the deprotonation and structural conversion events required for the MnCaO formation.