In the course of our coupling analysis, we found that histidine pK a values are predicted significantly higher with Amber14SB than with CHARMM36m. We previously observed lower accuracy for the prediction of lysine pK a s with the Amber14SB forcefield: this was traced to the partial charge difference of the backbone between the charged and uncharged lysine species. We hypothesized that this may also play a role for histidine, as here too the backbone partial charges differ between the doubly (denoted HIP) and singly protonated histidine residues (denoted HID and HIE). To further investigate, we computed 22 histidine pK a values that were taken from a full data set previously probed using equilibrium free energy calculations. These calculations were performed using both plain Amber14SB and a modified version, here called Amber14SB-H, where the partial charges of the protonated histidine backbone are those previously reported by Best et al. To account for the fact that two neutral histidine tautomers can exist with the proton present on Nδ (HID) or Nϵ (HIE), we perform two sets of free energy calculations: HIP → HID and HIP → HIE, which yield two relative free energies of deprotonation: ∆∆G δ and ∆∆G ϵ . Taking their difference gives the relative free energy of tautomer interconversion: free energy of deprotonation (Figure ):