Mechanistic characterization of the HDV genomic ribozyme: Solvent isotope effects and proton inventories in the absence of divalent metal ions support C75 as the general acid

The hepatitis delta virus (HDV) ribozyme uses the nucleobase C75 and a hydrated Mg²⁺ ion as the general acid-base catalysts in phosphodiester bond cleavage at physiological salt. A mechanistic framework has been advanced that involves one Mg²⁺-independent and two Mg ²⁺-dependent channels. The rate-pH profile for wild-type (WT) ribozyme in the Mg²⁺-free channel is inverted relative to the fully Mg²⁺-dependent channel, with each having a near-neutral pK _a. Inversion of the rate-pH profile was used as the crux of a mechanistic argument that C75 serves as general acid both in the presence and absence of Mg²⁺. However, subsequent studies on a double mutant (DM) ribozyme suggested that the pK_a observed for WT in the absence of Mg²⁺ arises from ionization of C41, a structural nucleobase. To investigate this further, we acquired rate-pH/pD profiles and proton inventories for WT and DM in the absence of Mg²⁺. Corrections were made for effects of ionic strength on hydrogen ion activity and pH meter readings. Results are accommodated by a model wherein the Mg²⁺-free pK _a observed for WT arises from ionization of C75, and DM reactivity is compromised by protonation of C41. The Brønsted base appears to be water or hydroxide ion depending on pH. The observed pK_a's are related to salt-dependent pH titrations of a model oligonucleotide, as well as electrostatic calculations, which support the local environment for C75 in the absence of Mg²⁺ being similar to that in the presence of Mg ²⁺ and impervious to bulk ions. Accordingly, the catalytic role of C75 as the general acid does not appear to depend on divalent ions or the identity of the Brønsted base.