Electron-nuclear double resonance spectroscopic evidence for a hydroxo-bridge nucleophile involved in catalysis by a dinuclear hydrolase

Despite the current availability of several crystal structures of purple acid phosphatases, to date there is no direct evidence for solvent-derived ligands occupying terminal positions in the active enzyme. This is of central importance, because catalysis has been shown to proceed through the direct attack on a metal-bound phosphate ester by a metal-activated solvent-derived moiety, which has been proposed to be either (i) a hydroxide ligand terminally bound to the ferric center or (ii) a bridging hydroxide. In this work we use ²H Q-band (35 GHz) pulsed electron-nuclear double resonance (ENDOR) spectroscopy to identify solvent molecules coordinated to the active mixed-valence (Fe³⁺Fe²⁺) form of the dimetal center of uteroferrin (Uf), as well as to its complexes with the anions MoO₄, AsO₄, and PO₄. The solvent-derived coordination of the dinuclear center of Uf as deduced from ENDOR data includes a bridging hydroxide and a terminal water/hydroxide bound to Fe²⁺ but no terminal water/hydroxide bound to Fe³⁺. The terminal water is lost upon anion binding while the hydroxyl bridge remains. These results are not compatible with a hydrolysis mechanism involving a terminal Fe³⁺-bound nucleophile, but they are consistent with a mechanism that relies on the bridging hydroxide as the nucleophile.