Identification of dimethylbenzimidazole axial coordination and characterization of N-14 superhyperfine and nuclear quadrupole coupling in Cob(II)alamin bound to ethanolamine deaminase in a catalytically-engaged substrate radical-cobalt(II) biradical state

Cobalt(II)-N-14 superhyperfine and N-14 nuclear quadrupole couplings in cry otrapped free and ethanolamine deaminase-bound cob(II)alamin have been char acterized in the disordered solid state by using X-band electron spin-echo envelope modulation (ESEEM) spectroscopy. Enzyme-bound cob(II)alamin was cr yotrapped after formation by substrate-initiated, thermally activated cleav age of the cobalt-carbon bond of adenosylcobalamin. Free dimethylbenzimidaz ole axial base-on cob(II)alamin was formed by photolysis of the correspondi ng adenosylcobalamin and cryotrapped in glycerol-aqueous glass. Three-pulse ESEEM experiments were performed by using microwave pulse excitation at th e g(perpendicular to) value of Co-II at magnetic field values of 287.0 and 345.0 mT and over a range of tau values from 227 to 1316 ns. Two common set s of N-14 features are distinguished in the ESEEM spectra. One set is assig ned to the remote (N1) nitrogen in the dimethylbenzimidazole alpha-axial li gand by using two independent approaches: (a) comparison of ESEEM from cob( ll)alamin with ESEEM from cob(II)inamide-ligand model compounds and (b) fro m the correspondence between the N1 N-14 nuclear quadrupole parameters deri ved from ESEEM simulations and those computed by using density functional t heory. The second set is assigned to the corrin ring N-14 nuclei. The resul ts identify the coenzyme's on-board dimethylbenzimidazole moiety as the alp ha-axial ligand to cob(II)alamin in ethanolamine deaminase in the substrate radical-Co-II biradical catalytic intermediate state, Thus, Co-II is a pen tacoordinate, alpha-axial liganded complex during turnover. We infer that d imethylbenzimidazole is also the alpha-axial ligand to the intact coenzyme in the resting enzyme. A 14% increase in the isotropic hyperfine coupling o f the remote dimethylbenzimidazole N-14 nucleus in enzyme-bound versus free base-on cob(II)alamin shows an enhanced delocalization of unpaired spin de nsity from Co-II onto the axial ligand, which would contribute to the accel eration of the cobalt-carbon bond cleavage rate in situ.