Histidine N_τ-Imidazole Ligation to Copper in Proteins: Innate or Entatic?

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, N_π (N_δ) and N_τ (N_ε). Despite having a tautomeric relationship and similar basicities, the nitrogen atoms used in copper enzymes are differentiated functionally, with near exclusive N_τ-ligation associated with substrate activation sites and exclusive N_π-ligation with electron-transfer sites. The results herein show that the innate thermodynamic preference is N_τ-ligation to Cu(II) centers, a conclusion drawn from ligand competition experiments at -145 °C between histidine imidazoles within synthetic μ-η²:η²-peroxodicopper(II) cores. The findings from these faithful models of oxygenated binuclear copper sites in tyrosinase enzymes are informed by spectroscopic data from a series of related complexes with monodentate methylated imidazoles. This preference of histidine N_τ-ligation is ascribed enthalpically to greater basicity and entropically to the greater molecular volume of the resulting metal complex compared to its N_π-ligated isomer. These results support that the N_τ-ligation observed in copper enzymatic sites is the innate thermodynamic form, independent of the protein matrix, while the N_π-ligation observed in electron transfer sites is presumably entatic in origin─requiring evolved protein structural influences. This structural distinction provides a powerful indicator of function among the biological copper sites.