The QM region comprised the ribose phosphate backbone of A117 and C116, the two active site Mg 2+ ions, the water molecules in their first hydration shell as well as protein residue side chains involved in Mg 2+ inner-sphere coordination (Glu96, Glu10, Asp56), for a total of 94 atoms (see Figure ). Starting structures with active site geometries appropriate for each mechanism were selected from the above described MD simulations and further equilibrated for ∼ 60 ps at the QM(DFTB3)/MM-MD 48 level after conversion of the input and parameter files from Gromacs to Amber format using the Parmed tool. The masses of the hydrogens involved in proton transfer were adjusted to 2 amu to increase the weight of the proton transfer events in the overall path mass-weighted reaction coordinate s. A 12.0 Å cutoff radius was used for electostatic and Lennard-Jones QM/MM interactions in B3LYP calculations while DFTB3 simulations employed Ewald for long-range electrostatic interactions. The RNase M5 reaction free energy landscape was explored using a dynamic path sampling approach, the adaptive string method (ASM), developed by one of us.