Ely_2011_J.Biol.Inorg.Chem_16_777

The organophosphate-degrading enzyme from Agrobacterium radiobacter (OpdA) is a highly efficient catalyst for the degradation of pesticides and some nerve agents such as sarin. OpdA requires two metal ions for catalytic activity, and hydrolysis is initiated by a nucleophilic hydroxide that is bound to one of these metal ions. The precise location of this nucleophile has been contentious, with both a terminal and a metal-ion-bridging hydroxide as likely candidates. Here, we employed magnetic circular dichroism to probe the electronic and geometric structures of the Co(II)-reconstituted dinuclear metal center in OpdA. In the resting state the metal ion in the more secluded alpha site is five-coordinate, whereas the Co(II) in the solvent-exposed beta site is predominantly six-coordinate with two terminal water ligands. Addition of the slow substrate diethyl 4-methoxyphenyl phosphate does not affect the alpha site greatly but lowers the coordination number of the beta site to five. A reduction in the exchange coupling constant indicates that substrate binding also triggers a shift of the mu-hydroxide into a pseudoterminal position in the coordination sphere of either the alpha or the beta metal ion. Mechanistic implications of these observations are discussed.