Spectroscopic and Computational Studies of Cobalamin Species with Variable Lower Axial Ligation: Implications for the Mechanism of Co-C Bond Activation by Class I Cobalamin-Dependent Isomerases
Spectroscopic and Computational Studies of Cobalamin Species with Variable Lower Axial Ligation: Implications for the Mechanism of Co-C Bond Activation by Class I Cobalamin-Dependent Isomerases
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DOI：
10.1021/ic502665x
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发表时间：
2015-04-20
影响因子：
4.6
通讯作者：
Brunold, Thomas C.
中科院分区：
文献类型：
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作者：
Conrad, Karen S.;Jordan, Christopher D.;Brunold, Thomas C.
5 '-脱氧腺苷钴胺素（辅酶B-12，辅酶Cbl）作为几种酶的辅因子，在发酵和催化中起重要作用。所有这些酶通过促进辅因子Co-C键的均裂来启动催化，以响应底物结合到它们的活性位点。尽管有相当多的研究工作，在促进Co-C键均裂较低的轴向配体的作用仍然不完全理解。在本研究中，我们的特点是几个衍生物的C2 H3Cbl和它的单电子还原形式，钴（II）Cbl，通过使用电子吸收和磁性圆二色光谱。为了补充我们的实验数据，我们对这些物种进行了计算，以及额外的Co（II）Cbl类似物。采用量子力学/分子力学方法优化了所有物种的几何构型，并将优化后的几何构型用于含时密度泛函理论计算吸收光谱。总的来说，我们的研究结果表明，在较低的轴向配体的碱性降低导致的变化，在Co（II）的状态，复制的影响后，看到的Co（II）Cbl的I类异构酶，取代较低的轴向二甲基苯并咪唑配体的CBL CBL与蛋白质衍生的组氨酸（His）残基结合的辅因子的电子结构。酶活性位点中His配体碱性的这种降低可以通过在Co-C键均裂过程中保守残基催化三联体DXHXGXK吸收质子来实现。
5'-deoxyadenosylcobalamin (coenzyme B-12, AdoCbl) serves as the cofactor for several enzymes that play important roles in fermentation and catabolism. All of these enzymes initiate catalysis by promoting homolytic cleavage of the cofactors Co-C bond in response to substrate binding to their active sites. Despite considerable research efforts, the role of the lower axial ligand in facilitating Co-C bond homolysis remains incompletely understood. In the present study, we characterized several derivatives of AdoCbl and its one-electron reduced form, Co(II)Cbl, by using electronic absorption and magnetic circular dichroism spectroscopies. To complement our experimental data, we performed computations on these species, as well as additional Co(II)Cbl analogues. The geometries of all species investigated were optimized using a quantum mechanics/molecular mechanics method, and the optimized geometries were used to compute absorption spectra with time-dependent density functional theory. Collectively, our results indicate that a reduction in the basicity of the lower axial ligand causes changes to the cofactors electronic structure in the Co(II) state that replicate the effects seen upon binding of Co(II)Cbl to Class I isomerases, which replace the lower axial dimethylbenzimidazole ligand of AdoCbl with a protein-derived histidine (His) residue. Such a reduction of the basicity of the His ligand in the enzyme active site may be achieved through proton uptake by the catalytic triad of conserved residues, DXHXGXK, during Co-C bond homolysis.