Photosystem II (PSII) is the water-plastoquinone photo-oxidoreductase central to oxygenic photosynthesis. PSII has been extensively studied for its ability to catalyze light-driven water oxidation at a MnCaO cluster called the oxygen-evolving complex (OEC). Despite these efforts, the complete reaction mechanism for water oxidation by PSII is still heavily debated. Previous mutagenesis studies have investigated the roles of conserved amino acids, but these studies have lacked a direct structural basis that would allow for a more meaningful interpretation. Here, we report a 2.14-Å resolution cryo-EM structure of a PSII complex containing the substitution Asp170Glu on the D1 subunit. This mutation directly perturbs a bridging carboxylate ligand of the OEC, which alters the spectroscopic properties of the OEC without fully abolishing water oxidation. The structure reveals that the mutation shifts the position of the OEC within the active site without markedly distorting the MnCaO cluster metal-metal geometry, instead shifting the OEC as a rigid body. This shift disturbs the hydrogen-bonding network of structured waters near the OEC, causing disorder in the conserved water channels. This mutation-induced disorder appears consistent with previous FTIR spectroscopic data. We further show using quantum mechanics/molecular mechanics methods that the mutation-induced structural changes can affect the magnetic properties of the OEC by altering the axes of the Jahn-Teller distortion of the Mn(III) ion coordinated to D1-170. These results offer new perspectives on the conserved water channels, the rigid body property of the OEC, and the role of D1-Asp170 in the enzymatic water oxidation mechanism.