The Dual Role of Histidine as General Base and Recruiter of a Third Metal Ion in HIV-1 RNase H

RNase H is a prototypical example for two metal ion catalysis in enzymes. An RNase H activity is present in the HIV-1 reverse transcriptase but also in many other nucleases such as Homo sapiens (Hs) or Escherichia coli (Ec) RNase H. The mechanism of the reaction has already been extensively studied based on the Bacillus halodurans (Bh) RNase H crystal structures, most recently using time-resolved X-Ray crystallography. However, kinetic and mutation experiments with HIV-1, Hs and Ec RNase H implicate a catalytic histidine in the reaction that is not present in Bh RNase H, and the protonation of the leaving group also remains poorly understood. We use quantum mechanics/molecular mechanics (QM/MM) calculations combining Hamiltonian replica exchange with a finite-temperature string method to study the cleavage of the ribonucleic acid (RNA) backbone of a DNA/RNA hybrid catalyzed by the HIV-1 RNase H with a focus on the proton transfer pathway and the role of the histidine. The reported pathway is consistent with kinetic data obtained with mutant HIV-1, Hs and Ec RNase H, the calculated pKa values of the DEDD residues and crystallographic studies. The overall reaction barrier of ∼18 kcal mol-1, encountered in the first step, matches the slow experimental rate of ∼1-100 min-1. Using Molecular dynamics (MD) calculations we are able to sample the recently identified binding site for a third transient divalent metal ion in the vicinity of the scissile phosphate in the product complex. Our results account for the experimental observation of a third metal ion facilitating product release in an Aquifex aeolicus RNase III crystal structure and the Bh RNase H in crystallo reaction. Based on our data we are able to show that the third ion and the histidine are key to product release as had been hypothesized.