10394 Biochemistry, Vol. 48, No. 43, 2009 1CSE. If azide remains in the observed site, the His 64 side chain must play a dynamic, mobile role to fulfill its expected charge- relay function, interacting with both anion and Ser 221. Alter- natively, it is possible that, in addition to the imidazole reposi- tioning so as to H-bond to Ser 221, the azide (or other anion) also moves from the observed site, a distance of ∼2 A˚ , to occupy a more Asp 32-like position compatible with a static charge-relay triad as in the wild type. This last possibility is most economical, in spite of the fact that it requires a small steric shift from the observed structure, because it preserves the fundamental roles and interactions of the wild-type mechanism, simply replacing the Asp 32 carboxylate with the triggering anion. In this model, some motion of the His 64 imidazole appears to be required for its effective interaction with Ser 221, but the precise dynamics of His 64 remain unresolved. 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