Competition between Co(NH(3)(6)3+ 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 Mg(2+) are required for efficient catalysis under physiological conditions. Moreover, the cleavage reaction can be inhibited with Co(NH(3))(6)(3+), an analogue of Mg(H(2)O)(6)(2+). Here, the binding of Mg(2+) and Co(NH(3))(6)(3+) 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(2+) alone is introduced to the ribozyme, inner sphere coordination of Mg(H(2)O)(x)(2+) (x </= 5) to nonbridging PO(2)(-) oxygen and changes in base stretches and phosphodiester group conformation are observed. In addition, binding of Mg(2+) induces deprotonation of a cytosine assigned to the general acid C75, consistent with solution studies. When Co(NH(3))(6)(3+) 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(2+) binding, Co(NH(3))(6)(3+) binding does not perturb PO(2)(-) group vibrations, consistent with its ability to make only outer sphere contacts. Surprisingly, competitive binding studies reveal that Co(NH(3))(6)(3+) ions displace some inner sphere-coordinated magnesium species, including ions coordinated to PO(2)(-) groups or the N7 of a guanine, likely G1 at the active site. These observations contrast with the tenet that Co(NH(3))(6)(3+) ions displace only outer sphere magnesium ions. Overall, our data support two classes of inner sphere Mg(2+)-PO(2)(-) binding sites: sites that Co(NH(3))(6)(3+) can displace and others it cannot.