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Identification of Dimethylbenzimidazole Axial Coordination and Characterization of 14 N Superhyperfine and Nuclear Quadrupole Coupling in Cob(II)alamin Bound to Ethanolamine Deaminase in a Catalytically-Engaged Substrate Radical−Cobalt(II) Biradical State

1999; American Chemical Society; Volume: 38; Issue: 39 Linguagem: Inglês

10.1021/bi983067w

1943-295X

Shyue‐Chu Ke, Maricel Torrent, Djamaladdin G. Museav, Keiji Morokuma, Kurt Warncke,

Heme Oxygenase-1 and Carbon Monoxide

Cobalt(II)−14N superhyperfine and 14N nuclear quadrupole couplings in cryotrapped free and ethanolamine deaminase-bound cob(II)alamin have been characterized in the disordered solid state by using X-band electron spin−echo envelope modulation (ESEEM) spectroscopy. Enzyme-bound cob(II)alamin was cryotrapped after formation by substrate-initiated, thermally activated cleavage of the cobalt−carbon bond of adenosylcobalamin. Free dimethylbenzimidazole axial base-on cob(II)alamin was formed by photolysis of the corresponding adenosylcobalamin and cryotrapped in glycerol−aqueous glass. Three-pulse ESEEM experiments were performed by using microwave pulse excitation at the g⊥ value of CoII at magnetic field values of 287.0 and 345.0 mT and over a range of τ values from 227 to 1316 ns. Two common sets of 14N features are distinguished in the ESEEM spectra. One set is assigned to the remote (N1) nitrogen in the dimethylbenzimidazole α-axial ligand by using two independent approaches: (a) comparison of ESEEM from cob(II)alamin with ESEEM from cob(II)inamide−ligand model compounds and (b) from the correspondence between the N1 14N nuclear quadrupole parameters derived from ESEEM simulations and those computed by using density functional theory. The second set is assigned to the corrin ring 14N nuclei. The results identify the coenzyme's on-board dimethylbenzimidazole moiety as the α-axial ligand to cob(II)alamin in ethanolamine deaminase in the substrate radical−CoII biradical catalytic intermediate state. Thus, CoII is a pentacoordinate, α-axial liganded complex during turnover. We infer that dimethylbenzimidazole is also the α-axial ligand to the intact coenzyme in the resting enzyme. A 14% increase in the isotropic hyperfine coupling of the remote dimethylbenzimidazole 14N nucleus in enzyme-bound versus free base-on cob(II)alamin shows an enhanced delocalization of unpaired spin density from CoII onto the axial ligand, which would contribute to the acceleration of the cobalt−carbon bond cleavage rate in situ.