To support the possible role of E1065 as a proton transfer, we calculated its pKa using PROPKA 3.1 obtaining a value of 7.80, significantly larger than its model pKa of 4.5, which suggests that E1065 could accept a proton throughout the catalytic cycle similar to our finding in the QM/MM energy minimizations (Table ). In addition, various experimental evidence supports the role of E1065 in the proton transfer. Firstly, the homologous Ec E48D mutant is substantially less active with a 95% decrease in kcat. This decrease is most likely connected with suboptimal positioning and change in pKa. Secondly, it was found that Ec E48 is less important for Mg 2+ binding compared to the three other aspartates as the Mg 2+ dissociation constant for the Ec E48Q mutant is 1.1 mM compared with 0.71 mM for the wild type but 18.8 mM for Ec D70N. As a result of the latter, Kanaya et al. proposed that the glutamate of the DEDD motif should play an additional role in the catalysis. Our results support that hypothesis, showing the role of E1065 as a mediator in the proton-transfer reaction needed for the stabilization of the leaving group and competition of the cleavage reaction. In the in crystallo reaction in Bh RNase H it can also be observed that if there is only potassium present, Bh E109 and Bh D71 are disordered. Finally, Uchiyama et al. have measured kinetics of Ec RNase H with modified substrate at the O2' position of the nucleotide immediately upstream of the scissile bond which also support the pathway via E1065. Substitution of the O2' into an amino, fluoro or methoxy group or removal of the O2' did not affect the affinity for the substrate but greatly reduced the reaction rate. E1065 interacts with the O2' through a hydrogen bond both in the crystal and in simulations. Altogether this supports a dual role of E1065 in recognizing the RNA strand in the DNA/RNA hybrid via the hydrogen bond and then by shuttling the proton to the leaving O3'. Also consistent with this hypothesis is the 50% decrease in catalytic activity in Ec by mutation of a serine close to the active site (Ec S71A, HIV-1 S1086). This serine is conserved in Bh, Hs, Ec and HIV-1 RNase H (Figure ) and, in our simulations, the proton transfer originates from the terminal water molecule bound to S1086. The binding site for this terminal water is favorable as can be inferred from its presence in multiple crystal structures (e.g. 1ZBL, 2G8I, and 2R7Y, 6DOG) of Bh RNase H (Figure ). Protonation of D1085, which was previously considered as the most likely protonation pathway for Bh RNase H , was not attempted as the pathway via E1065 is in very good agreement with the experimental kinetics. In addition, our pKa calculations show that E1065 (pKa 7.80) is more likely to get protonated than D1085 (pKa 2.71). Thus, E1065 can better stabilize the proton ultimately donated to the O3' alkoxide after the intermediate has formed because of its higher pKa value. Another contributing factor is that the energetic cost of rotating the D1085 necessary for protonation is not necessary if protonation occurs via the pre-positioned E1065. The protonation pathway via the pro-Rp oxygen of the scissile phosphate is not consistent with available experimental findings wherefore it was not considered as a potential proton mediator in our calculations. Uchiyama et al. demonstrated that sulfur substitution of the pro-Rp oxygen of the scissile phosphate does not decrease activity. A decrease would however be expected for sulfur substitution of the pro-Rp oxygen because the phosphorothioate is a weaker base. It can therefore be concluded that protonation via E1065 is the most likely route for the protonation of the leaving group in HIV-1 RNase H and possibly in the other members of the family (Bh E109, Ec E48, Hs E186) in contrast to earlier findings.