Characterizing multiple metal ion binding sites within a ribozyme by cadmium-induced EPR silencing.

In ribozyme catalysis, metal ions are generally known to make structural andor mechanistic contributions. The catalytic activity of a previously described Diels-Alderase ribozyme was found to depend on the concentration of divalent metal ions, and crystallographic data revealed multiple binding sites. Here, we elucidate the interactions of this ribozyme with divalent metal ions in solution using electron paramagnetic resonance (EPR) spectroscopy. Manganese ion titrations revealed five high-affinity Mn(2+) binding sites with an upper K(d) of 0.6+/-0.2 muM. In order to characterize each binding site individually, EPR-silent Cd(2+) ions were used to saturate the other binding sites. This cadmium-induced EPR silencing showed that the Mn(2+) binding sites possess different affinities. In addition, these binding sites could be assigned to three different types, including innersphere, outersphere, and a Mn(2+) dimer. Based on simulations, the Mn(2+)-Mn(2+) distance within the dimer was found to be approximately 6 A, which is in good agreement with crystallographic data. The EPR-spectroscopic characterization reveals no structural changes upon addition of a Diels-Alder product, supporting the concept of a preorganized catalytic pocket in the Diels-Alder ribozyme and the structural role of these ions.