A third divalent ion was also implicated in the mechanism by Samara and Yang. However, as they deduce importance of the third divalent ion from the in crystallo reaction further investigation into the dynamics of the ion recruitment for the real reaction in solution and in the presence of the histidine is still needed. Experimentally, it has been observed that at high Mg 2+ concentrations (>50mM in Bh RNase H) the reaction stalls , which has led to the hypothesis that a third metal ion could bind to the product or disturb the nucleophilic attack even before it was detected in the in crystallo reaction. In their recent work Samara and Yang have shown that the third divalent metal ion coordinates to the scissile phosphate and to the 3' downstream phosphate and that product release can only take place after its departure. Interestingly, H→A mutation in Hs or Ec RNase H removes the attenuation of the reaction at high Mg 2+ concentrations, suggesting that the catalytic histidine plays an important role in the recruitment and displacement of the third metal ion. We therefore investigated the product complex of the cleavage reaction in HIV-1 RT/RNase H at the catalytic (6mM), zero and high (50mM) MgCl2 concentrations with classical MD simulations. In multiple 100 ns long MD simulations we found two prominent binding sites for cations in the product complex. The term "binding site" used in this context refers to the ion sampling a small region of space for some extended period of time close to the active site and shall not be indicative of first shell binding via displacement of one of the first shell water ligands of the solvated K + or Mg 2+ ions. One of the sites (F in Figure , Figure ) was located close to D1085, MgA and MgB and was populated mostly by potassium ions. This binding site was also occupied during the reaction for Bh RNase H crystal structure and was probably populated because of the double negative charge of the scissile phosphate. The second binding site (ion C in Figure , Figure , Figure ) was located between the scissile phosphate and the 3' downstream phosphate, and was populated by Mg 2+ and K + ions alike. C was also found to be occupied in the product state of the in crystallo reaction. The length of the binding events for the ions varied within the replicates from very short binding (~5ns) to quite stable association with the scissile phosphate with retention times of up to 80 ns in binding site C. The relation of potassium vs. magnesium binding is biased towards more potassium ions as they were more abundant in the simulation (150 mM K + vs 50 mM Mg 2+ ). As is expected from the classical description of the ions, we do not observe loss of the first solvation shell during the relatively short simulation time. Instead, we observe coordination to the phosphate oxygens mediated via second shell interactions. The simulations show that the binding of a metal ion in site C causes conformational changes in the RNA strand of the hybrid that disrupt the interaction of the RNA with the DNA strand (Figure ). Complete dissociation could not be observed likely due to the length of the simulation, the length of the hybrid and/or due to the limitations of the nucleic acid force field. Nevertheless, a general trend for destabilization is visible (measured in terms of RMSD of the active site nucleotide and the two nucleotides in the 3' direction of the RNA strand, Figure ). As suggested for the H→A mutation in Hs RNase H , the catalytic histidine H1126 placed on a flexible loop next to the active site seemed to participate in the binding of the third ion. We measured the position of the histidine using its Nα-Cα-Cβ-Cγ dihedral angle, which varied from -45° to 60° for the inwards facing position ready for the catalysis and from -170° to -180° for the outwards position ready for metal ion recruitment and shuttling of the abstracted proton to the solvent (Figure ). When a third metal ion was in the binding site MgC, we found the H1126 to be almost exclusively facing inwards as a second shell ligand of the third ion (Figure ). The metal ion dissociated from the binding site shortly after the histidine changed its conformation from in-to outwards in several of the replicas, further supporting the role of the histidine in recruitment of a third ion similarly to that suggested by Alla and Nicholson experimentally. This effect is most likely electrostatic, as first shell coordination of a Mg 2+ or K + by the histidine is not observed in our simulations. This is congruent with findings of low affinity of nitrogens for earth alkali metals. In the Bh in crystallo reaction, the residue K196 placed at the C-terminus on a flexible loop stabilizes the scissile phosphate. In HIV-1 RNase H a lysine (K1137) is present as well, which, as the Cterminus of the HIV-1 protein extends further, is placed in an alpha helix of HIV-1 RNase H instead of the Cterminal loop of Bh RNase H. K1137 in HIV-1 RNase H points away from the active site and is too far away to stabilize the cleaved phosphate (Figure ). A possible hypothesis for the function of the third divalent ion in the catalysis and product release can be inferred from Aa-RNase III and from the in crystallo study of the Bh RNase H mechanism. Aa-RNase III performs a similar 3'-5' endonucleolytic cleavage like RNase H with the help of two metal ions but the architecture of its active site is slightly different (DDEE motif). In the crystal of the product complex of Aa-RNase III (res. 1.7 Å, PDB: 2NUG), two conformations of the scissile phosphate were observed with different occupancies. The minor conformation with occupancy 0.3 closely resembles the product state of the HIV-1 RNase H reaction from the QM/MM with the scissile phosphate pro-Sp oxygen coordinating both MgA and MgB. However, the major conformation in the crystal with an occupancy of 0.7 has the scissile phosphate out of the active site, bent away from MgA and MgB and closer to MgC (Figure ). The scissile phosphate O is replaced by a water molecule. Gan et al. have proposed that the minor conformation is a model of the complex directly after the reaction took place and the major conformation is the first step towards product release in Aa-RNase III. The product release is facilitated by MgC. In the major conformation, the scissile phosphate is a first shell ligand of MgC. Interestingly, the main binding site for the third ion and the scissile phosphate in the HIV-1 RNase H MD simulations are in the same position as in the minor conformation of the Aa-RNase III crystal, suggesting similarity between the Aa-RNase III and RNase H product release mechanism. The stabilization through the third ion likely has stabilized this state in Aa-RNase III enough in order to be detected by conventional crystallography. Attempts to crystallize the Bh RNase H product complex with 100% occupation of a third Mg 2+ have not been successful and in the in crystallo reaction the third ion was only detectable in 500mM Mn 2+ or in 5mM Mg 2+ / 200mM Li + buffer. It was found that the concentration of Mg 2+ needed for optimal catalysis is higher than the concentration to fully occupy the A and B sites. In analogy to our observations, Samara and Yang found the third ion to bind to the product state. In the crystal it can be seen that with MgC the 5' phosphate product is shifted by 1 Å. The suggested role of the histidine in the recruitment of the third ion is consistent with the observation that for H→A mutation the attenuation of RNase H activity at high Mg 2+ concentration is lost. Bh E188A mutant, which has a lower activity, also displays no attenuation at high Mg 2+ concentrations. Nowotny and Yang have attributed the lower activity of Bh E188A to an abnormally high affinity to the cleavage product which supports the interpretation that a third metal ion is important for the product release. In Hs RNase H264A mutation increases the rate constant for the product release 12-fold which further supports the role of histidine in recruitment of the third ion.