The product of the first reaction step is a cleaved phosphodiester bond with the O3' leaving oxygen tightly bound to MgB (Figure ). In earlier studies this intermediate has also been observed and found to be highly unstable (Figure ), and the protonation of the leaving oxygen was found to be essential for the stabilization of the cleavage product. However, the source of the proton was not established. As we will show below, we found that protonation via the E1065 is an energetically feasible pathway. Initially, we tested whether the O3' leaving oxygen could be protonated via a file of water molecules in channel C1 passing under the substrate to the bulk solvent (See Figure ). Based on evidence from kinetic experiments with mutated O2', we assumed that the proton could be shuttled to the O3' via the O2' as there is evidence for a role of the O2' in the reaction. To test this hypothesis a hydronium was placed in the solvent channel. Energy minimizations showed that the proton is transferred to the terminal water molecule in the solvent channel close to the O2', where it is coordinated by a conserved serine (S1086) and a conserved glutamine (Q1062) (see Seq. alignment Fig. ). With restrained energy minimizations including the distances between the terminal water proton, the O2', the H2' and the O3' distance and the restraints from the above simulations we tried to obtain a potential energy estimate of this pathway. However, we consistently found that the proton was not directly transferred to the O3' but via E1065, which was not included in the restraints. We therefore concluded that E1065 could temporarily store the proton and then transfer it to the O3'. The restraints in the QM/MM minimizations to generate the initial path for the string calculations were modified to account for this pathway by including only the distances between E1065:H, O3', P, Onuc, Hnuc and H1126: in the RC. With this final reaction coordinate a good starting string was constructed conducing iterative QM/MM energy minimizations along the RC. In the string calculations, a protonated E1065 was modelled as reactant. The potential energy barrier of the protonation of E1065 could be determined in separate restrained energy minimizations to be about 16 kcal mol -1 . (major conformation scissile phosphate in olive sticks, Mg 2+ in dark grey spheres, PDB 2NUG) and MD simulation of HIV-1 RT/RNase H product complex at 50 mM Mg 2+ at 30 ns in replicate 1 (light grey spheres, white sticks). The Mg 2+ ion C bends the scissile phosphate and the pro-Sp oxygen is replaced with a solvent water (sphere in magenta). U is upstream nucleotide, S is the nucleotide directly in the active site containing the scissile phosphate, D is the 3' downstream nucleotide. pro-Sp and pro-Rp oxygen are abbreviated as Sp and Rp. An interactive version with 3D view of the reaction (Panels A-E) together with Figure is available online ().