Inverse thio effects in the hepatitis delta virus ribozyme reveal that the reaction pathway is controlled by metal ion charge density

The hepatitis delta virus (HDV) ribozyme self-cleaves in the presence of a wide range of monovalent and divalent ions. Prior theoretical studies provided evidence that self-cleavage proceeds via a concerted or stepwise pathway, with the outcome dictated by the valency of the metal ion. In the present study, we measure stereospecific thio effects at the nonbridging oxygens of the scissile phosphate under a wide range of experimental conditions, including varying concentrations of diverse monovalent and divalent ions, and combine these with quantum mechanical/molecular mechanical (QM/MM) free energy simulations on the stereospecific thio substrates. The R_P substrate gives large normal thio effects in the presence of all monovalent ions. The S_P substrate also gives normal or no thio effects, but only for smaller monovalent and divalent cations, such as Li⁺, Mg²⁺, Ca²⁺, and Sr²⁺; in contrast, sizable inverse thio effects are found for larger monovalent and divalent cations, including Na⁺, K⁺, NH₄⁺, and Ba²⁺. Proton inventories are found to be unity in the presence of the larger monovalent and divalent ions, but two in the presence of Mg²⁺. Additionally, rate-pH profiles are inverted for the low charge density ions, and only imidazole plus ammonium ions rescue an inactive C75Δ variant in the absence of Mg²⁺. Results from the thio effect experiments, rate-pH profiles, proton inventories, and ammonium/imidazole rescue experiments, combined with QM/MM free energy simulations, support a change in the mechanism of HDV ribozyme self-cleavage from concerted and metal ion-stabilized to stepwise and proton transfer-stabilized as the charge density of the metal ion decreases.