Many proteins use Asx and Glx (x = n, p, or u) side chains as key functional groups in enzymatic catalysis and molecular recognition. In this study, NMR spin relaxation experiments and molecular dynamics simulations are used to measure the dynamics of the side chain amide and carboxyl groups, (13)C(γ/δ), in Escherichia coli ribonuclease HI (RNase H). Model-free analysis shows that the catalytic residues in RNase H are preorganized on ps-ns time scales via a network of electrostatic interactions. However, chemical exchange line broadening shows that these residues display significant conformational dynamics on μs-ms time scales upon binding of Mg(2+) ions. Two groups of catalytic residues exhibit differential line broadening, implicating distinct reorganizational processes upon binding of metal ions. These results support the "mobile metal ion" hypothesis, which was inferred from structural studies of RNase H.