Significant cleavage by hammerhead ribozymes requires activation by divalent metal ions. Several models have been proposed to account for the inf luence of metal ions on hammerhead activity. A number of recent papers have presented data that have been interpreted as supporting a one-metal-hydroxide-ion mechanism. In addition, a solvent deuterium isotope effect has been taken as evidence against a proton transfer in the rate-limiting step of the cleavage reaction. We propose that these data are more easily explained by a two-metal-ion mechanism that does not involve a metal hydroxide, but does involve a proton transfer in the rate-limiting step.Hammerhead ribozymes are one of several types of RNA enzymes (1). The term hammerhead refers to a small domain, found in some viral satellite RNAs, that is required for RNA self-cleavage (2). This small domain is necessary for cleavage activity on RNA sequences that form complementary base pairs with the hammerhead (3). Crystal structures have been obtained for hammerhead ribozymes complexed with different noncleavable substrate analogues (4, 5). Because of its small size and simple structure, the hammerhead has been extensively studied as a prototypic RNA enzyme. Fig. 1 depicts a hammerhead ribozyme that is base paired to a 17-nucleotide substrate RNA. This hammerhead, called HH16 (6), has 8 bp in both recognition helices that flank the cleavage site on the substrate. Cleavage occurs on the 3Ј side of a cytosine residue in the substrate strand.Several models have been proposed for the cleavage of RNA phosphodiester bonds by hammerhead ribozymes; most models include a solvated metal hydroxide ion at the catalytic center (4,7,8). In these metal-hydroxide-ion models ( Fig. 2A) a divalent metal hydroxide binds to the pro-R oxygen on the phosphate at the site of cleavage and activates the ribozyme primarily by removing a proton from the 2Ј-OH of the attacking nucleotide residue. The activated 2Ј-O Ϫ species then acts as the nucleophile by attacking the phosphodiester bond.A two-metal-ion model has also been proposed for the hammerhead ribozyme (9). This model is based on mechanisms that have been put forward to explain the activities of alkaline phosphatase and the 3Ј-5Ј exonuclease of DNA polmerase I (10, 11). Similar two-metal-ion models have been proposed for a wide variety of protein and RNA enzymes that mediate phosphoryl transfer reactions (9). In one such model for the hammerhead ribozyme (Fig. 2B), both divalent metal ions bind to the pro-R oxygen on the phosphate at the site of cleavage. The first metal ion (in site A) interacts directly with the 2Ј-oxygen of the attacking ribonucleotide residue, while the second metal ion (in site B) interacts with the 5Ј-oxygen leaving group. In a two-metal-ion mechanism, the metal ion in site A is thought to facilitate the formation of the 2Ј-oxyanion that acts as the nucleophile, and the metal ion in site B is considered to stabilize the oxyanion leaving group (11). Both metal ions may also stabilize the specific geometry ...