To carry out the QM/MM energy minimizations, the system was trimmed to a sphere of 20 Å centered on the scissile phosphate group. Atoms further than 15 Å away from that group were kept frozen throughout the simulations. The region treated with molecular mechanics was 4164 atoms in size. The QM region contained 116 atoms including two Mg 2+ ions, the RNA backbone of the active site adenosine, the guanosine in 3' direction of the RNA strand and the side chains of residues Q1062, D1030, E1065, D1085, D1136 and H1126. The hydrogen link atoms were placed between the Cβ and Cα bonds for the aspartates, glutamine and histidine or the Cγ and Cβ bonds for the glutamates. Link hydrogens were restrained between the original bond partners and their bond length was scaled by a factor of 0.7261. Twelve water molecules were included in the QM region of which three coordinate MgA. To probe for the different proton transfers proposed in the literature , we also included all water molecules that make hydrogen bonds to the scissile phosphate, to the Bh D132 equivalent D1085, to the O2' and the Q1062 amide oxygen. The charge of this QM region was -2. To probe for a possible new protonation pathway, a water molecule in the solvent channel egressing from the O2'/Q1062 into the bulk solvent was protonated. The total number of atoms therefore was 117 and total charge of the QM region was -1 for the string calculation. All calculations were performed with the CHARMM 27 force field 31 and charmm40b2 as the MD engine interfaced with Q-Chem 4.4 using full electrostatic embedding of the MM region. All calculations were carried out with the 6-31+G(d) basis set. Density functional theory calculations and the B3LYP exchange-correlation functional were used. The reaction coordinate (RC) was defined by the bond distances between reactive atoms. The RC included the proton transfer (Qp) and electron transfer (Qe). Qep is taken as = + . The Qe coordinate includes r2-r1 (WATnuc:OH2 -RNA:P -RNA:O3', Figure ) . The Qp coordinate includes r4-r3 (H1126:NE2 -WATnuc:H -WATnuc:O, and r6-r5 (RNA:O3' -RNA:H2' -E1065:OE1, Figure ).