The catalytic activity of the binuclear glyoxalase II (GlxII) enzyme is closely linked to the type and charge of metal ions in its active site. Using hybrid quantum mechanics/molecular mechanics (QM/MM) calculations, we investigated the reaction mechanism of human GlxII, which features two Zn(II) ions in its active site. By systematically replacing these Zn(II) ions with Fe(II), Fe(III), or Co(II), we evaluated the impact of metal substitutions on reaction energetics and active-site geometry. Our results reveal that the type and position of the metal ions are critical to the catalytic activity of GlxII. Substitution of the Zn(II) ion in the three-histidine site with Fe(II), Fe(III), or Co(II) significantly increased the activation barrier, indicating that these configurations are less favorable. In contrast, substituting Zn(II) in the two-histidine site with either Fe(II) or Co(II) resulted in a reduced activation barrier and produced geometries closely resembling those observed when both metal sites are occupied by Zn(II). Additionally, moving the metal ions from the QM to the MM region inhibited the reaction, highlighting their direct chemical involvement in catalysis beyond electrostatic stabilization. These results underscore that the metal ions chemically participate in the catalytic process beyond their electrostatic contributions. Collectively, our results provide insights into the structural and electronic factors governing GlxII catalysis, offering a theoretical framework to complement and refine experimental studies.