Our mechanistic investigations evidenced one favorable reaction route, that first requires a small conformational fluctuation from the dominant active site conformation to reach a suitable organization of the active site. This RNA cleavage mechanism differs from that suggested for RNase H in several aspects: the most favorable route for deprotonation of the nucleophilic water molecule is here a Mg 2+ -bound glutamate, and the leaving group is then reprotonated by a water molecule that moves along the reaction path to adopt a bridging position between the two active site Mg 2+ ions, thus taking the place of the scissile phosphate in the reactant state. Note that a water molecule is visible in the X-ray structure of isolated M5 at this specific position, with ill-determined protonation state, so that a hydroxide ion may well bridge the two Mg 2+ ions in the absence of the RNA subtrate. Our product structure could thus be reminiscent of that of the uncomplexed active site. Experimentally testing the nature of the base for the initial deprotonation will prove challenging: mutating Glu96 would presumably prevent the initial proton transfer, but it may also be detrimental for Mg 2+ binding in the active site, and the two effects will be difficult to deconvolute.