Protonation status of metal-bound ligands can be determined by quantum refinement.

The protonation status of key residues and bound ligands are often important for the function of a protein. Unfortunately, protons are not discerned in normal protein crystal structures, so their positions have to be determined by more indirect methods. We show that the recently developed quantum refinement method can be used to determine the position of protons in crystal structures. By replacing the molecular-mechanics potential, normally used in crystallographic refinement, by more accurate quantum chemical calculations, we get information about the ideal structure of a certain protonation state. By comparing the refined structures of different protonation states, the one that fits the crystallographic raw data best can be decided using four criteria: the R factors, electron density maps, strain energy, and divergence from the unrestrained quantum chemical structure. We test this method on alcohol dehydrogenase, for which the pK(a) of the zinc-bound solvent molecule is experimentally known. We show that we can predict the correct protonation state for both a deprotonated alcohol and a neutral water molecule.