Electro-nuclear double resonance spectroscopic evidence for a hydroxo-bridge nucleophile involved in catalysis by a dinuclear hydrolase. Academic Article uri icon


  • 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 (2)H Q-band (35 GHz) pulsed electron-nuclear double resonance (ENDOR) spectroscopy to identify solvent molecules coordinated to the active mixed-valence (Fe(3+)Fe(2+)) form of the dimetal center of uteroferrin (Uf), as well as to its complexes with the anions MoO(4), AsO(4), and PO(4). 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 Fe(2+) but no terminal water/hydroxide bound to Fe(3+). 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 Fe(3+)-bound nucleophile, but they are consistent with a mechanism that relies on the bridging hydroxide as the nucleophile.

published proceedings

  • J Am Chem Soc

altmetric score

  • 1

author list (cited authors)

  • Smoukov, S. K., Quaroni, L., Wang, X., Doan, P. E., Hoffman, B. M., & Que, L

citation count

  • 63

complete list of authors

  • Smoukov, Stoyan K||Quaroni, Luca||Wang, Xuedong||Doan, Peter E||Hoffman, Brian M||Que, Lawrence

publication date

  • March 2002