Free energy profile of the reaction: There were two maxima in the one-dimensional energy profile projected onto the Qep RC, corresponding to the two transition states (Figure ). The formation of the phosphorane TS (TS1) was the highest point in the free energy profile with ~18 kcal mol -1 . The intermediate state was ~4 kcal mol -1 lower in energy compared to TS1. To form the product via TS2 another energy barrier with ~3 kcal mol -1 had to be surmounted to protonate the leaving group. From the plot of the free energy surface on the Qe and Qp coordinates it can be seen, that first a proton transfer, then an electron transfer and then again a proton transfer occurs (Figure ). These barrier heights are in good agreement with experimentally determined barriers calculated from kinetic experiments to 16.7 to 20.5 kcal mol -1 in Ec and Hs RNase H. The measured kcat with modified substrate in HIV-1 RNase H is 1.01 to 0.11 min -1 corresponding to a barrier of 19.8 to 21.2 kcal mol -1 . The modified substrate is, however, likely to have negatively impacted the kcat for the measurement of HIV-1 RNase H. The kcat values measured with the human and the Ec RNase H are therefore considered to be closer to the real value in HIV-1. Role of monovalent ions: Samara and Yang 10 implicate monovalent potassium ions in the catalysis based on the in crystallo analysis of the reaction mechanism in Bh RNase H. In Bh RNase H, K + w (site F in Figure ) coordinates the scissile phosphate and K + U (site C in Figure ) together with a transiently bound Mg 2+ C activates the nucleophile. Bh RNase H likely relies on the cation trafficking to activate the nucleophile because it lacks the histidine residue, that is present in other members of the RNase H family. Therefore, HIV-1, Hs and Ec RNase might be less reliant on monovalent ions as the histidine activates the nucleophile and therefore replaces the function of the monovalent ions. This is also related to the slightly different active site architecture with Bh K197 able to point inwards stabilizing the intermediate, which is not possible for HIV-1 K1137 (Figure ).