These findings suggest that while Mg²⁺ provides a more stable catalytic environment, Mn²⁺ enhances catalytic efficiency by reducing energetic barriers and favoring the progression of the reaction by enhanced stabilization of the TS and product states via polarization of the active site. Individual residue interaction analysis is consistent with previously reported experimental mutagenesis, and several of the residues observed as catalytically relevant are also sites involved in pathogenic mutations. This analysis also uncovered additional catalytically relevant amino-acids that could be targets of subsequent experimental mutagenesis studies. Collectively, this study provides critical insights into the mechanistic role of metal ions in polymerase gamma function, with potential implications for understanding metal ion regulation in mitochondrial DNA replication.