We conclude, then, that the steric obstruction resulting from crystal packing of the dimer has significantly limited the conformational flexibility of His157 and suggest that the likely mobility of this residue in the monomer, which has a solvent exposed active site, may facilitate its protonation and serve a functional role. Taken together with the crystal structure active site occlusion, these factors may explain the single Nε-protonation of His157 observed even at acidic conditions. However, the single protonation of His157 solely at the Nε position in our crystal structures does not rule out formation of the doubly protonated second shell histidine residue under similar acidic operando conditions where the active site would not be barricaded by crystal packing.