A minimal QM system of Glu197 and Glu441 side chains with the interacting water molecule was used. Backbone charges of both Glu were averaged to avoid the potential bias of the initial state assignment. Amber2 charge compensation scheme was used. GROMACS pull code was used to put umbrella restraints on the reaction coordinate. 100 000 kJ/mol/nm 2 restraints were used during the initial conjugate gradient minimization and then decreased to 5 000 kJ/mol/nm 2 for umbrella sampling runs in 11 windows with centers in range from -1.5Å to 1.5Å with 0.3Å increments. Each window was sampled for 5 ps with 1 fs time step in 100 replicates. First 0.5 ps were discarded, and subsequent data was grouped in sets of 20 runs per window to yield 5 independent profile reconstructions with the binless WHAM method. Sufficient histogram overlap was achieved for each (Figure ). Position restraints of 100 kJ/mol/nm 2 were active on protein heavy atoms in the MM region only. Velocity-rescale thermostat was used with no pressure control.