Spectroscopic and Computational Characterization of the Base-off Forms of Cob(II)alamin

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• 作者：Matthew D. Liptak ; Angela S. Fleischhacker ; Rowena G. Matthews ; Joshua Telser ; Thomas C. Brunold
• 刊名：Journal of Physical Chemistry B
• 出版年：2009
• 出版时间：April 16, 2009
• 年：2009
• 卷：113
• 期：15
• 页码：5245-5254
• 全文大小：263K
• 年卷期：v.113,no.15(April 16, 2009)
• ISSN：1520-5207

文摘

The one-electron-reduced form of vitamin B₁₂, cob(II)alamin (Co²⁺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” Co²⁺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 Co²⁺Cbl to cob(I)alamin (Co¹⁺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 Co²⁺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 Co²⁺ center. Accurate spin Hamiltonian parameters for these low-spin Co²⁺ 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 π → π* 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 Co²⁺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 Co²⁺Cbl species, the unpaired spin density is primarily localized in the Co 3d_z²-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 Co²⁺Cbl → Co¹⁺Cbl reduction.