Secondly, arginine is always considered to be in a protonated state for a variety of protein environments. Asp292 was kept unprotonated because the negatively charged Asp292 can form a stronger salt bridge with positively charged Arg490 than when Asp292 is uncharged. The reasons for keeping Lys353 in a protonated state are given in the Supplementary Material. QM/MM geometry optimizations were carried out on input structures 1-23 with the 8 residue, QM Region I. With QM Region I, optimization of 3-11 leads to structures in which there is no transfer of hydrogen, and the protonation state of the surrounding amino acid residues in the input structure (as shown in Figure ) remains the same in the optimized structures. However, geometry optimization of 1-2 results in structures in which a hydrogen (H22) transfers from N19 of Arg360 to equatorial O1 of vanadate. This transfer does not occur for any of the other structures, 3-23. Considering that the pKa of Arg360 is more than 11, one can argue that the monoprotonated 1 or 2 is not stable.