title: Electro-nuclear double resonance spectroscopic evidence for a hydroxo-bridge nucleophile involved in catalysis by a dinuclear hydrolase.
abstract: 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.
authors.affiliation: Department of Chemistry, Northwestern University, Evanston, Illinois 60208, USA.
authors.forename: Stoyan K
authors.initials: SK
authors.lastname: Smoukov
authors.forename: Luca
authors.initials: L
authors.lastname: Quaroni
authors.forename: Xuedong
authors.initials: X
authors.lastname: Wang
authors.forename: Peter E
authors.initials: PE
authors.lastname: Doan
authors.forename: Brian M
authors.initials: BM
authors.lastname: Hoffman
authors.forename: Lawrence
authors.initials: L
authors.lastname: Que
mesh_terms: D000135:Acid Phosphatase; D001149:Arsenates / Q000737:chemistry / Q000378:metabolism; D001665:Binding Sites; D002384:Catalysis; D006867:Hydrolases / Q000737:chemistry* / Q000378:metabolism; D007501:Iron / Q000737:chemistry / Q000378:metabolism; D007527:Isoenzymes; D008667:Metalloproteins / Q000737:chemistry* / Q000378:metabolism; D008982:Molybdenum / Q000737:chemistry / Q000378:metabolism; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods; D010710:Phosphates / Q000737:chemistry / Q000378:metabolism; D012997:Solvents; D000071681:Tartrate-Resistant Acid Phosphatase; D014867:Water / Q000737:chemistry / Q000378:metabolism
chemical_list: D001149:Arsenates; D007527:Isoenzymes; D008667:Metalloproteins; D010710:Phosphates; D012997:Solvents; D014867:Water; C044659:molybdate; D008982:Molybdenum; D007501:Iron; D006867:Hydrolases; D000135:Acid Phosphatase; D000071681:Tartrate-Resistant Acid Phosphatase; C025657:arsenic acid