Electron−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.

Probing the role of the divalent metal ion in uteroferrin using metal ion replacement and a comparison to isostructural biomimetics

“…Alternatively, pK es1 could be assigned to the deprotonation of a terminal Fe(III)-bound water molecule. However, the absence of this water ligand in resting Uf [13] make this assignment less likely. Also, the significant change of pK es1 upon replacing Fe(II) [40].…”

“…The divalent metal ion is coordinated to the oxygen atom of the bridging aspartate, the nitrogen atoms of two histidine residues and an asparagine oxygen atom. On the basis of recent spectroscopic and crystallographic data, the Fe(III) in the resting state is five-coordinate, including a bridging (hydr)oxo ligand; the M(II) site is likely to be six-coordinate with a terminal water molecule completing its first coordination sphere [13]. The enzyme thus provides an asymmetric binuclear active site with an NO 4 Fe(III) site and an N 2 O 4 divalent metal site (Fig.…”

The Catalytic Mechanisms of Binuclear Metallohydrolases

“…9,79 However, the observation (by ENDOR) that no terminal Fe(III)-bound hydroxide group may be present in the resting state of pig PAP argues against this (although the possibility that substrate binding may promote the binding of a water molecule to Fe(III) cannot be ruled out at present). 86 It could also be shown that fluoride can replace the bridging hydroxide group in pig PAP. 99 Upon the further addition of phosphate, a ternary PAP‚F‚PO 4 complex was formed, where phosphate binds to both metal ions, as observed in the crystal structure of pig PAP.…”

Synthesis and characterization of the tetranuclear iron(iii) complex of a new asymmetric multidentate ligand. A structural model for purple acid phosphatases

“…6). As far as the interdimer interactions (J 23 = J 3 and J 14 = J 2 ) are concerned, these could not be a priori considered equal, due to the proton localization on one of the bridging oxides, and the subsequent asymmetry of the interdimer bridges [Fe(1)-O(16)-Fe(4) = 146.81 (21) • , Fe(2)-O(8)-Fe(3) = 144.00 (22) • ]. Thus, to test whether these are indeed equal, initial fitting attempts considering J 2 = J 3 were carried out.…”