Thio effects and an unconventional metal ion rescue in the genomic hepatitis delta virus ribozyme

Metal ion and nucleobase catalysis are important for ribozyme mechanism, but the extent to which they cooperate is unclear. A crystal structure of the hepatitis delta virus (HDV) ribozyme suggested that the pro-R_P oxygen at the scissile phosphate directly coordinates a catalytic Mg²⁺ ion and is within hydrogen bonding distance of the amine of the general acid C75. Prior studies of the genomic HDV ribozyme, however, showed neither a thio effect nor metal ion rescue using Mn²⁺. Here, we combine experiment and theory to explore phosphorothioate substitutions at the scissile phosphate. We report significant thio effects at the scissile phosphate and metal ion rescue with Cd²⁺. Reaction profiles with an S_P-phosphorothioate substitution are indistinguishable from those of the unmodified substrate in the presence of Mg²⁺ or Cd²⁺, supporting the idea that the pro-S_P oxygen does not coordinate metal ions. The R _P-phosphorothioate substitution, however, exhibits biphasic kinetics, with the fast-reacting phase displaying a thio effect of up to 5-fold and the slow-reacting phase displaying a thio effect of ∼1000-fold. Moreover, the fast- and slow-reacting phases give metal ion rescues in Cd²⁺ of up to 10- and 330-fold, respectively. The metal ion rescues are unconventional in that they arise from Cd²⁺ inhibiting the oxo substrate but not the R_P substrate. This metal ion rescue suggests a direct interaction of the catalytic metal ion with the pro-R_P oxygen, in line with experiments with the antigenomic HDV ribozyme. Experiments without divalent ions, with a double mutant that interferes with Mg²⁺ binding, or with C75 deleted suggest that the pro-R_P oxygen plays at most a redundant role in positioning C75. Quantum mechanical/molecular mechanical (QM/MM) studies indicate that the metal ion contributes to catalysis by interacting with both the pro-R_P oxygen and the nucleophilic 2′-hydroxyl, supporting the experimental findings.