J. Heyrovský Institute of Physical Chemistry, Czech Academy of Sciences, Dolejškova 3, 18200 Prague, Czech Republic.
Institute of Organic Chemistry and Biochemistry, Czech Academy of Sciences, Flemingovo nám. 2, 16610 Prague, Czech Republic.
Phys Chem Chem Phys. 2024 Jul 31;26(30):20280-20295. doi: 10.1039/d4cp01725k.
This study investigates the factors modulating the reactivity of 5'-deoxyadenosyl (5'dAdo˙) radical, a potent hydrogen atom abstractor that forms in the active sites of radical SAM enzymes and that otherwise undergoes a rapid self-decay in aqueous solution. Here, we compare hydrogen atom abstraction (HAA) reactions between native substrates of radical SAM enzymes and 5'dAdo˙ in aqueous solution and in two enzymatic microenvironments. With that we reveal that HAA efficiency of 5'dAdo˙ is due to (i) the formation of 5'dAdo˙ in a pre-ordered complex with a substrate, which attenuates the unfavorable effect of substrate:5'dAdo˙ complex formation, and (ii) the prevention of the conformational changes associated with self-decay by a tight active-site cavity. The enzymatic cavity, however, does not have a strong effect on the HAA activity of 5'dAdo˙. Thus, we performed an analysis of in-water HAA performed by 5'dAdo˙ based on a three-component thermodynamic model incorporating the diagonal effect of the free energy of reaction, and the off-diagonal effect of asynchronicity and frustration. To this aim, we took advantage of the straightforward relationship between the off-diagonal thermodynamic effects and the electronic-structure descriptor - the redistribution of charge between the reactants during the reaction. It allows to access HAA-competent redox and acidobasic properties of 5'dAdo˙ that are otherwise unavailable due to its instability upon one-electron reduction and protonation. The results show that all reactions feature a favourable thermodynamic driving force and tunneling, the latter of which lowers systematically barriers by ∼2 kcal mol. In addition, most of the reactions experience a favourable off-diagonal thermodynamic contribution. In HAA reactions, 5'dAdo˙ acts as a weak oxidant as well as a base, also 5'dAdo˙-promoted HAA reactions proceed with a quite low degree of asynchronicity of proton and electron transfer. Finally, the study elucidates the crucial and dual role of asynchronicity. It directly lowers the barrier as a part of the off-diagonal thermodynamic contribution, but also indirectly increases the non-thermodynamic part of the barrier by presumably controlling the adiabatic coupling between proton and electron transfer. The latter signals that the reaction proceeds as a hydrogen atom transfer rather than a proton-coupled electron transfer.
本研究探讨了调节 5'-脱氧腺嘌呤（5'dAdo˙）自由基反应性的因素，5'dAdo˙自由基是一种在自由基 SAM 酶的活性部位形成的强氢原子提取剂，否则会在水溶液中快速自降解。在这里，我们比较了在水溶液中和两种酶微环境中，天然 RADICAL SAM 酶底物和 5'dAdo˙之间的氢原子提取（HAA）反应。由此我们发现，5'dAdo˙的 HAA 效率归因于：（i）5'dAdo˙与底物形成预有序复合物，从而减弱了底物与 5'dAdo˙复合物形成的不利影响，（ii）由紧密的活性腔防止与自降解相关的构象变化。然而，酶腔对 5'dAdo˙的 HAA 活性没有强烈影响。因此，我们基于包含反应自由能对角线效应和异步性和挫折的非对角线效应的三组分热力学模型，对水中 5'dAdo˙进行的 HAA 进行了分析。为此，我们利用了非对角线热力学效应与电子结构描述符之间的直接关系，即在反应过程中反应物之间电荷的再分配。这使得能够获得由于其在单电子还原和质子化后不稳定而无法获得的 5'dAdo˙的 HAA 能力的氧化还原和酸碱性质。结果表明，所有反应都具有有利的热力学驱动力和隧道效应，后者通过将势垒系统地降低约 2 kcal/mol。此外，大多数反应都经历了有利的非对角线热力学贡献。在 HAA 反应中，5'dAdo˙充当弱氧化剂以及碱，而且 5'dAdo˙促进的 HAA 反应具有质子和电子转移相当低的异步性。最后，该研究阐明了异步性的关键和双重作用。它作为非对角线热力学贡献的一部分直接降低了势垒，但也通过控制质子和电子转移之间的绝热耦合间接地增加了非热力学部分的势垒。后者表明该反应作为氢原子转移而不是质子偶联电子转移进行。
