Competition between Co(NH₃)₆³⁺ and inner sphere Mg²⁺ ions in the HDV ribozyme

Divalent cations play critical structural and functional roles in many RNAs. While the hepatitis delta virus (HDV) ribozyme can undergo self-cleavage in the presence of molar concentrations of monovalent cations, divalent cations such as Mg²⁺ are required for efficient catalysis under physiological conditions. Moreover, the cleavage reaction can be inhibited with Co(NH ₃)₆³⁺, an analogue of Mg(H₂O) ₆²⁺. Here, the binding of Mg²⁺ and Co(NH ₃)₆³⁺ to the HDV ribozyme is studied by Raman microscopic analysis of crystals. Raman difference spectra acquired at different metal ion conditions reveal changes in the ribozyme. When Mg²⁺ alone is introduced to the ribozyme, inner sphere coordination of Mg(H ₂O)_x²⁺ (x ≤ 5) to nonbridging PO ₂^- oxygen and changes in base stretches and phosphodiester group conformation are observed. In addition, binding of Mg²⁺ induces deprotonation of a cytosine assigned to the general acid C75, consistent with solution studies. When Co(NH₃)₆³⁺ alone is introduced, deprotonation of C75 is again observed, as are distinctive changes in base vibrational ring modes and phosphodiester backbone conformation. In contrast to Mg²⁺ binding, Co(NH₃)₆ ³⁺ binding does not perturb PO₂^- group vibrations, consistent with its ability to make only outer sphere contacts. Surprisingly, competitive binding studies reveal that Co(NH₃) ₆³⁺ ions displace some inner sphere-coordinated magnesium species, including ions coordinated to PO₂^- groups or the N7 of a guanine, likely G1 at the active site. These observations contrast with the tenet that Co(NH₃)₆³⁺ ions displace only outer sphere magnesium ions. Overall, our data support two classes of inner sphere Mg²⁺-PO₂^- binding sites: sites that Co(NH₃)₆³⁺ can displace and others it cannot.