Electron spin echo envelope modulation (ESEEM) spectroscopy reveals several interactions that serve to weaken the Co–C5′ bond, through stabilization of the cleaved state, thereby facilitating the 5′-deoxyadenosyl (5′-dAdo^•) radical formation in unlabeled and ¹⁵N-labeled lysine 5,6-aminomutase. In the first place, we show that the Co^II–His133β interaction enhances the spin delocalization from Co^II to the α-axial ligand, reducing the 5′-dAdo^•–Co^II recombination probability in situ and allowing nascent 5′-dAdo^• to migrate toward the substrate. Next, using [5′-²H]-deoxyadenosylcobalamin, we show that the C5′ methyl group of 5′-dAdoH is at closest contact with the substrate radical. In the case of 5′-dAdo^•, this would allow the 5′-methylene radical to be rapidly quenched by H-transfer from the substrate. Finally, we show that the spin density is transferred noncovalently from the substrate radical to the adenine N7 of 5′-dAdoH. In the one-radical substrate–5′-dAdo^•–product H-exchange triad, which is central in the mechanism of action of B₁₂ enzymes, such transfer would promote the forward H-transfer reaction by dynamically lowering the free energy of the substrate radical, thereby facilitating its formation and quenching of 5′-dAdo^•. In the reverse H-transfer reaction following the 1,2-rearrangement, the reversibility of the noncovalent spin transfer allows the product radical to restore full radical character to reform 5′-dAdo^• and a diamagnetic product. The role of dynamical and reversible spin partitioning as a mechanism for modulating the radical stability is likely common in B₁₂ enzymes. This study leads to a better comprehension about how an enzyme controls the reaction trajectory of 5′-dAdo^• exquisitely with specificity. The ESEEM results support the positioning of the interacting partners in the catalytic form of lysine 5,6-aminomutase obtained by modeling.

中文翻译：

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电子自旋回波包络调制光谱揭示腺苷钴胺素依赖的赖氨酸 5,6-氨基变位酶如何定位自由基对中间体并调节其稳定性以实现高效催化

电子自旋回波包络调制（ESEEM）光谱揭示了几个相互作用投放到削弱联合C5'键，通过切割的状态的稳定化，从而有利于5'-脱氧腺苷（5'-墙裙^•）在未标记的和自由基形成¹⁵ N-标记的赖氨酸 5,6-氨基变位酶。首先，我们表明 Co ^II -His133β 相互作用增强了从 Co ^II到 α 轴配体的自旋离域，降低了 5'-dAdo ^• -Co ^II原位重组概率并允许新生的 5'-dAdo ^•向基板迁移。接下来，使用 [5′- ²H]-脱氧腺苷钴胺素，我们表明 5'-dAdoH 的 C5' 甲基与底物自由基最接近。在 5'-dAdo ^•的情况下，这将允许 5'-亚甲基自由基被底物的 H-转移迅速淬灭。最后，我们表明自旋密度从底物自由基非共价转移到 5'-dAdoH 的腺嘌呤 N7。在单自由基底物 5′-dAdo ^•产物H 交换三联体中，这是 B ₁₂酶作用机制的核心，这种转移将通过动态降低自由基的自由能来促进正向 H 转移反应。底物自由基，从而促进其形成和淬灭 5'-dAdo ^•. 在 1,2-重排后的反向 H-转移反应中，非共价自旋转移的可逆性允许产物自由基恢复完整的自由基特性，以重新形成 5'-dAdo ^•和抗磁性产物。动态和可逆自旋分配作为调节自由基稳定性的机制的作用可能在 B ₁₂酶中很常见。这项研究有助于更好地理解酶如何精确地、特异性地控制 5'-dAdo ^•的反应轨迹。ESEEM 结果支持通过建模获得的赖氨酸 5,6-氨基变位酶催化形式的相互作用伙伴的定位。