The activation of cobalamin requires a reduction from cobalamin(III) to cobalamin(II). The reduction by glutathione and dithiothreitol was followed using visible spectroscopy and electron paramagnetic resonance. In addition the oxidation of glutathione was monitored. Glutathione first reacts with oxidized cobalamin(III). The binding of a second glutathione required for the reduction to cobalamin(II) is presumably located in the dimethyl benzimidazole ribonucleotide ligand cavity. The reduction of cobalamin(III) by dithiothreitol, which contains two thiols, is much faster even though no stable cobalamin(III) complex is formed. The reduction, by both thiol reagents, results in the formation of thiyl radicals, some of which are released to form oxidized thiol products and some of which remain associated with the reduced cobalamin. In the reduced state the intrinsic lower affinity for the benzimidazole base, coupled with a trans effect from the initial glutathione bound to the β-axial site and a possible lowering of the pH results in an equilibrium between base-on and base-off complexes. The dissociation of the base facilitates a closer approach of the thiyl radical to the cobalamin(II) α-axial site resulting in a complex with ferromagnetic exchange coupling between the metal ion and the thiyl radical. This is a unique example of "internal spin trapping" of a thiyl radical formed during reduction. The finding that the reduction involves a peripheral site and that thiyl radicals produced during the reduction remain associated with the reduced cobalamin provide important new insights into our understanding of the formation and function of cobalamin enzymes.

中文翻译：

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钴胺素与谷胱甘肽和二硫三醇的络合和还原过程中硫自由基的内自旋捕获

钴胺素的激活需要从钴胺素（III）减少到钴胺素（II）。使用可见光谱和电子顺磁共振跟踪谷胱甘肽和二硫苏糖醇的还原。此外监测谷胱甘肽的氧化。谷胱甘肽首先与氧化的钴胺素（III）反应。还原为钴胺素（II）所需的第二种谷胱甘肽的结合可能位于二甲基苯并咪唑核糖核苷酸配体腔中。即使没有形成稳定的钴胺素 (III) 复合物，含有两个硫醇的二硫苏糖醇还原钴胺素 (III) 的速度也快得多。两种硫醇试剂的还原导致硫自由基的形成，其中一些被释放以形成氧化的硫醇产物，而其中一些仍然与还原的钴胺素结合。在还原状态下，对苯并咪唑碱的固有亲和力较低，加上初始谷胱甘肽与 β 轴位点结合的反式效应和可能降低的 pH 值导致碱基和碱基复合物之间的平衡。碱基的解离有利于硫基更接近钴胺素(II) α-轴位点，导致金属离子和硫基之间具有铁磁交换耦合的络合物。这是还原过程中形成的硫基自由基的“内部自旋俘获”的独特例子。还原涉及外围位点以及还原过程中产生的硫基自由基仍然与还原的钴胺素相关的发现为我们理解钴胺素酶的形成和功能提供了重要的新见解。