The order of events for the water deprotonation and the phosphodiester cleavage process was determined with extensive energy minimizations of the QM/MM system followed by finite temperature string free energy calculations. The geometry was optimized along the Qep RC driving the system from reactant to product. The geometry minimized path was used in the first iteration to determine the minimum free energy pathway in subsequent string free energy calculations. Initially, the first coupled proton transfer and phosphate cleavage step until the formation of the intermediate was probed during minimizations. The reaction coordinate alone was used to drive the phosphate cleavage via the O3' leaving oxygen, the phosphorous atom and the WATnuc oxygen. We did not specify a priori the WATnuc deproto-nation mechanism that facilitates the nucleophilic attack. The QM region included the potential proton acceptors: the 3' downstream pro-Rp oxygen, the Nε of H1126 and the pro-Rp oxygen of the scissile phosphate. We consistently found that the Nε of H1126 acted as a proton acceptor. Additionally, a hydrogen bond was formed between the 3' downstream pro-Rp oxygen and the other WATnuc proton. To determine the effect of the 3' downstream pro-Rp oxygen, H1126 was excluded from the quantum mechanical treatment. In QM/MM minimizations with the smaller QM region, deprotonation was carried out via the 3' downstream pro-Rp oxygen. This is consistent with earlier simulations of Bh RNase H 13 that lacks the histidine residue. To verify that the histidine is the stronger general base the product state of the reaction was modelled by moving the WATnuc proton pointing towards H1126 to the O3' leaving group yielding a product complex with cleaved phosphodiester bond and a monoprotonated phosphate with the hydrogen pointing towards the 3' downstream pro-Rp oxygen. In the subsequent unrestrained QM/MM energy minimization of this system, the WATnuc proton jumped to the Nε of H1126. This clearly demonstrates the higher basicity of H1126 compared to the 3' downstream pro-Rp oxygen. As H1126 was very flexible in the MD simulations, it is likely that H1126 shuttles both protons from WATnuc to the solvent and acts as the general mediator for proton transfers to and from the solvent. It could also play a role in attracting the active site Mg 2+ ions to the active site, similar to the role of E188 in Bh. The pro-Rp oxygen of the scissile phosphate was not considered as a possible proton acceptor, as mutational studies showed that thio substitution of the pro-Rp oxygen does not decrease the catalytic rate. Alla and Nicholson 20 have proposed a dual functional role of H264 in Hs RNase H acting both as a proton acceptor and a facilitator of the product release. The mutation of the catalytic histidine in Hs RNase H (H264A) reduces the kcat 100-fold but has no effect on the Km, strongly supporting the role of histidine as the proton acceptor. A similar effect of the H→A mutation in RNase H can be seen in Ec, where the H124A substitution decreases the kcat 50-fold and increases Km only by 4fold. The H1126F mutation in HIV-1 RNase H renders the enzyme inactive. In addition, Tisdale et al. showed that even the conservative H1126N mutation makes the virus non-infective, corroborating the crucial role of H1126 for RNase H in the virus life cycle. It is likely that the replication of the viral genome is severely impacted by a slower RNase H catalytic rate. The pH profile of the reaction with maximum activity at pH 7.5 and low activity at low pH is also consistent with the role of the histidine which is protonated at low pH, diminishing the catalytic activity.