title: Spectroscopic and computational characterization of the base-off forms of cob(II)alamin.
abstract: The one-electron-reduced form of vitamin B(12), cob(II)alamin (Co(2+)Cbl), is found in several essential human enzymes, including the cobalamin-dependent methionine synthase (MetH). In this work, experimentally validated electronic structure descriptions for two "base-off" Co(2+)Cbl species have been generated using a combined spectroscopic and computational approach, so as to obtain definitive clues as to how these and related enzymes catalyze the thermodynamically challenging reduction of Co(2+)Cbl to cob(I)alamin (Co(1+)Cbl). Specifically, electron paramagnetic resonance (EPR), electronic absorption (Abs), and magnetic circular dichroism (MCD) spectroscopic techniques have been employed as complementary tools to characterize the two distinct forms of base-off Co(2+)Cbl that can be trapped in the H759G variant of MetH, one containing a five-coordinate and the other containing a four-coordinate, square-planar Co(2+) center. Accurate spin Hamiltonian parameters for these low-spin Co(2+) centers have been determined by collecting EPR data using both X- and Q-band microwave frequencies, and Abs and MCD spectroscopic techniques have been employed to probe the corrin-centered pi --> pi* and Co-based d --> d excitations, respectively. By using these spectroscopic data to evaluate electronic structure calculations, we found that density functional theory provides a reasonable electronic structure description for the five-coordinate form of base-off Co(2+)Cbl. However, it was necessary to resort to a multireference ab initio treatment to generate a more realistic description of the electronic structure of the four-coordinate form. Consistent with this finding, our computational data indicate that, in the five-coordinate Co(2+)Cbl species, the unpaired spin density is primarily localized in the Co 3d(z(2))-based molecular orbital, as expected, whereas in the four-coordinate form, extensive Co 3d orbital mixing, configuration interaction, and spin-orbit coupling cause the unpaired electron to delocalize over several Co 3d orbitals. These results provide important clues to the mechanism of enzymatic Co(2+)Cbl --> Co(1+)Cbl reduction.
authors.affiliation: Department of Chemistry, University of Wisconsin-Madison, Madison Wisconsin 53706, USA.
authors.forename: Matthew D
authors.initials: MD
authors.lastname: Liptak
authors.forename: Angela S
authors.initials: AS
authors.lastname: Fleischhacker
authors.forename: Rowena G
authors.initials: RG
authors.lastname: Matthews
authors.forename: Joshua
authors.initials: J
authors.lastname: Telser
authors.forename: Thomas C
authors.initials: TC
authors.lastname: Brunold
mesh_terms: D003198:Computer Simulation; D004578:Electron Spin Resonance Spectroscopy / Q000379:methods; D008956:Models, Chemical; D008968:Molecular Conformation; D014805:Vitamin B 12 / Q000031:analogs & derivatives* / Q000737:chemistry
chemical_list: C097751:cob(II)alamin; D014805:Vitamin B 12