All QM:MM calculations were conducted using the Gaussian16 C.01 program .The QM:MM models obtained from MD simulations were stripped from sodium ions and most of the water molecules, leaving only H2O molecules penetrating a 20 Å radius from Cys493. The positions of all residues and water molecules above the 15 Å radius from Cys493 were frozen in geometry optimization. The overall charge of the QM:MM models were -7 and -3 for holo-and apo-BSS models, respectively. The fumarate was modeled in the monoprotonated state according to previous docking studies . Two sizes of high layer (HL) were used in the study a small one (S-HL, Fig. ) used for geometry optimization and vibrational analysis and a big one (B-HL Fig. ), used for single point correction of the energy.