| Abstract
| - Despite the current availability of several crystal structures of purple acid phosphatases, to datethere 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 ona metal-bound phosphate ester by a metal-activated solvent-derived moiety, which has been proposed tobe either (i) a hydroxide ligand terminally bound to the ferric center or (ii) a bridging hydroxide. In this workwe use 2H Q-band (35 GHz) pulsed electron−nuclear double resonance (ENDOR) spectroscopy to identifysolvent 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 coordinationof the dinuclear center of Uf as deduced from ENDOR data includes a bridging hydroxide and a terminalwater/hydroxide bound to Fe2+ but no terminal water/hydroxide bound to Fe3+. The terminal water is lostupon anion binding while the hydroxyl bridge remains. These results are not compatible with a hydrolysismechanism involving a terminal Fe3+-bound nucleophile, but they are consistent with a mechanism thatrelies on the bridging hydroxide as the nucleophile.
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