Competition between Co(NH) and inner sphere Mg 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 Mg are required for efficient catalysis under physiological conditions. Moreover, the cleavage reaction can be inhibited with Co(NH ), an analogue of Mg(HO) . Here, the binding of Mg and Co(NH ) 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 Mg alone is introduced to the ribozyme, inner sphere coordination of Mg(H O) (x ≤ 5) to nonbridging PO oxygen and changes in base stretches and phosphodiester group conformation are observed. In addition, binding of Mg induces deprotonation of a cytosine assigned to the general acid C75, consistent with solution studies. When Co(NH) 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 Mg binding, Co(NH) binding does not perturb PO group vibrations, consistent with its ability to make only outer sphere contacts. Surprisingly, competitive binding studies reveal that Co(NH) ions displace some inner sphere-coordinated magnesium species, including ions coordinated to PO groups or the N7 of a guanine, likely G1 at the active site. These observations contrast with the tenet that Co(NH) ions displace only outer sphere magnesium ions. Overall, our data support two classes of inner sphere Mg-PO binding sites: sites that Co(NH) can displace and others it cannot. © 2009 American Chemical Society.