Cobalt hexammine inhibition of the hammerhead ribozyme

The effects of Co(NH3)(6)(3+) On the hammerhead ribozyme are analyzed using several techniques, including activity measurements, electron paramagnetic resonance (EPR), and circular dichroism (CD) spectroscopies and thermal de naturation studies. Co(NH3)(6)(3+) efficiently displaces Mn2+ bound to the ribozyme with an apparent dissociation constant of K-d app = 22 +/- 4.2 mu M in 500 mu M Mn2+ (0.1 M NaCl). Displacement of Mn2+ coincides with Co(NH3 )(6)(3+) inhibition of hammerhead activity in 500 mu M Mn2+, reducing the a ctivity of the WT hammerhead by similar to 15-fold with an inhibition const ant of K-i = 30.9 +/- 2.3 mu M. A residual 'slow' activity is observed in t he presence of Co(NH3)(6)(3+) and low concentrations of Mn2+. Under these c onditions, a single Mn2+ ion remains bound and has a low-temperature EPR sp ectrum identical to that observed previously for the highest affinity Mn2site in the hammerhead ribozyme in 1 M NaCl, tentatively attributed to the A9/G10.1 site [Morrissey, S. R., Horton, T. E., and DeRose, V. J. (2000) J. Am. Chem. Soc. 122, 3473-3481]. Circular dichroism and thermal denaturatio n experiments also reveal structural effects that accompany the observed in hibition of cleavage and Mn2+ displacement induced by addition of Co(NH3)(6 )(3+). Taken together, the data indicate that a high-affinity CO(NH3)(6)(3) site is responsible for significant inhibition accompanied by structural changes in the hammerhead ribozyme. In addition, the results support a mode l in which at least two types of metal sites, one of which requires inner-s phere coordination, support hammerhead activity.