QM/MM calculations are known to be sensitive to the employed QM region. We employed two different QM region sizes in this study, shown in Figure . The small QM region consisted of the Cu ion, HIS37 and HIS144 (constituting the histidine brace), HIS208 (used as proton donor), TYR213, GLN211 and ASP140. Additionally, GLY38 (bound to HIS37), ALA142 and SER143 (bound to HIS144), and six water molecules were included in the QM region. Two of the water molecules (W419 and W492) were included in the crystal structure, the remaining water molecules are from the solvation model and are labeled W2 and W4-W6. Since we observed large changes in MM energies of up to 30 kJ/mol with the smaller QM region for some protontransfer reactions (particularly 3 --→ 4 and 3 red --→ 4 red ), we mainly show the results for the QM region including two additional water molecules (W1 and W3) and ASP90. In cases where we show results obtained with the small QM region, they are explicitly denoted with the subscript "small". We used the hydrogen link atom approach, and capped TYR213, GLN211, ASP90 and ASP140 at the C α atom, replacing it with a hydrogen atom. HIS37 and SER143 were fully included in the QM region. ALA142 was capped at the backbone N atom and HID144 at the backbone C atom, replacing them with hydrogen link atoms.