Histidine is among the most versatile amino acids by virtue of its imidazole ring, which is capable of shuttling protons and binding metals at each of its two nitrogen atoms, Nπ (Nδ) and Nτ (Nε). Despite having similar basicities and a tautomeric relationship, the nitrogen atoms used in copper enzymes are differentiated, with near exclusive Nτ-ligation associated with substrate activation sites and exclusive Nπ-ligation with electron-transfer sites.1 A conclusive demonstration of innate thermodynamic preferences between these ligation modes would clarify how the protein matrix distinguishes structure and function in biological copper sites. To this end, ligand competition experiments were performed at -145 °C using methylated monodentate imidazoles and histidines bonded to synthetic µ-η2:η2-peroxodicopper(II) cores—faithful models of the oxygenated binuclear copper sites in tyrosinase enzymes. These experiments show an intrinsic preference for Nτ-ligation at Cu(II) centers, achievable with a histidine-derived ligand. This preference is ascribed enthalpically to the greater basicity of Nτ and entropically to the greater molecular volume of the resulting metal complex compared to the Nπ-ligated isomer. These results support that the Nτ-ligation observed in copper enzymatic sites is the intrinsic thermodynamic form, while the Nπ-ligation observed in electron-transfer sites appears, by corollary, to be entatic in origin—requiring evolved protein structural influences. This structural distinction provides a robust indicator of biological copper-site function and offers insight into the evolutionary interplay between structure and reactivity.