The hammerhead ribozyme crystal structure identified a specific metal ion binding site referred to as the P9/G10.1 site. Although this metal ion binding site is approximately 20 A away from the cleavage site, its disruption is highly deleterious for catalysis. Additional published results have suggested that the pro-R(P) oxygen at the cleavage site is coordinated by a metal ion in the reaction's transition state. Herein, we report a study on Cd(2+) rescue of the deleterious phosphorothioate substitution at the cleavage site. Under all conditions, the Cd(2+) concentration dependence can be accounted for by binding of a single rescuing metal ion. The affinity of the rescuing Cd(2+) is sensitive to perturbations at the P9/G10.1 site but not at the cleavage site or other sites in the conserved core. These observations led to a model in which a metal ion bound at the P9/G10.1 site in the ground state acquires an additional interaction with the cleavage site prior to and in the transition state. A titration experiment ruled out the possibility that a second tight-binding metal ion (< 10 microM) is involved in the rescue, further supporting the single metal ion model. Additionally, weakening Cd(2+) binding at the P9/G10.1 site did not result in the biphasic binding curve predicted from other models involving two metal ions. The large stereospecific thio-effects at the P9/G10.1 and the cleavage site suggest that there are interactions with these oxygen atoms in the normal reaction that are compromised by replacement of oxygen with sulfur. The simplest interpretation of the substantial rescue by Cd(2+) is that these atoms interact with a common metal ion in the normal reaction. Furthermore, base deletions and functional group modifications have similar energetic effects on the transition state in the Cd(2+)-rescued phosphorothioate reaction and the wild-type reaction, further supporting the model that a metal ion bridges the P9/G10.1 and the cleavage site in the normal reaction (i.e., with phosphate linkages rather than phosphorothioate linkages). These results suggest that the hammerhead undergoes a substantial conformational rearrangement to attain its catalytic conformation. Such rearrangements appear to be general features of small functional RNAs, presumably reflecting their structural limitations.