Metal-ion stoichiometry of the HIV-1 RT ribonuclease H domain: evidence for two mutually exclusive sites leads to new mechanistic insights on metal-mediated hydrolysis in nucleic acid biochemistry

Crystallographic studies of the Mn2+-doped RNase H domain of human immunodeficiency virus type 1 reverse transcriptase (HIV-1 RT) [1] have revealed two bound Mn2+ separated by approximately 4 Angstrom and surrounded by a cluster of four conserved carboxylates. Escherichia coli RNase H is structurally similar to the RNase H domain of HIV-1 RT, but requires one divalent metal cation for its activity [2, 3], implying either that the HIV-1 RT RNase H domain contrasts in its ability to bind two divalent metal ions, or that the crystallographic data reflect specific use of Mn2+ and/or the doping technique employed. Metal binding stoichiometry has been determined for Mn2+ and the biologically more relevant Mg2+ cation by solution calorimetric studies of native and recombinant p66/p51 HIV-1 RT. Three Mn2+ ions bind to HIV-1 RT ape-enzyme: one at the DNA polymerase and two at the RNase H catalytic center, the latter being consistent with crystallographic results. However, only one Mg2+ ion is bound in the RNase H catalytic center. Several mechanistic implications arise from these results, including the possibility of mutually exclusive Mg2+ binding sites that might be occupied according to the specific reaction being catalyzed by the multifunctional RNase H domain. The occurrence of distinct binding stoichiometries for Mg2+ and Mn2+ to multifunctional enzymes has previously been reported [4].