Rajić Martina, Prah Alja, Stare Jernej
Theory Department, Laboratory for Computational Biochemistry and Drug Design, National Institute of Chemistry, Hajdrihova 19, Ljubljana SI-1000, Slovenia.
ACS Omega. 2024 Oct 10;9(42):43046-43057. doi: 10.1021/acsomega.4c06575. eCollection 2024 Oct 22.
The complete two-step hydride transfer mechanism of amine oxidation involved in the metabolism of monoamine neurotransmitters was scrutinized by DFT calculations. In living organisms, this process is catalyzed by monoamine oxidase enzymes. Herein, we focus on some intriguing aspects of the reaction that may have been previously noticed but have not been clarified to date. The first step of the reaction includes the C-H bond cleavage on the methylene group vicinal to the amino group of the monoamine substrate and the subsequent transfer of hydrogen to the N5 atom of the flavin prosthetic group of the enzyme. We confirmed the nature of this step to be hydride transfer by evaluation of the pertinent HOMO-LUMO gap together with analysis of orbital contours alongside the intrinsic reaction coordinate profile. Next, we investigated the rather peculiar intermediate adduct that may form between the amine substrate and the flavin molecule, featuring an unusually long C-N bond of ∼1.62 Å. Although this bond is quite stable in the gas phase, the presence of just a few explicit water molecules facilitates its dissociation almost without energy input so that the amine-flavin intermediate can form an ionic pair instead. We attribute the existence of the unusual C-N bond to a fragile balance between opposing electronic structure effects, as evaluated by the natural bond orbital analysis. In line with this, the intermediate in the solution or in the enzyme active site can exist in two energetically almost equivalent forms, namely, as a covalently bound complex or as an ion pair, as suggested by previous studies. Finally, we characterized the transformation of the intermediate to the fully reduced flavin and imine products via proton transfer from the amino group to the flavin N1 atom, completing the reductive part of the catalytic cycle. Although we found that explicit solvation substantially boosts the kinetics of this step, the corresponding barrier is significantly lower than that in the hydride transfer step, confirming hydrogen abstraction as the rate-limiting step of amine oxidation and validating the two-step hydride transfer mechanism of monoamine oxidases.
通过密度泛函理论（DFT）计算，对单胺神经递质代谢过程中涉及的胺氧化的完整两步氢化物转移机制进行了详细研究。在活生物体中，该过程由单胺氧化酶催化。在此，我们关注该反应中一些有趣的方面，这些方面可能之前已被注意到，但迄今为止尚未得到阐明。反应的第一步包括单胺底物氨基邻位亚甲基上的C-H键断裂，以及随后氢向酶的黄素辅基的N5原子转移。我们通过评估相关的最高占据分子轨道（HOMO）-最低未占据分子轨道（LUMO）能隙，并结合沿本征反应坐标剖面图的轨道轮廓分析，证实了该步骤的本质是氢化物转移。接下来，我们研究了胺底物与黄素分子之间可能形成的相当特殊的中间加合物，其特征是具有约1.62 Å的异常长的C-N键。尽管该键在气相中相当稳定，但仅存在几个明确的水分子就能几乎无需能量输入地促进其解离，从而使胺-黄素中间体能够形成离子对。通过自然键轨道分析评估，我们将异常C-N键的存在归因于相反电子结构效应之间的脆弱平衡。与此一致的是，溶液中或酶活性位点的中间体可以以两种能量几乎相等的形式存在，即作为共价结合的复合物或作为离子对，正如先前研究所表明的那样。最后，我们通过质子从氨基转移至黄素N1原子，表征了中间体向完全还原的黄素和亚胺产物的转化，从而完成了催化循环的还原部分。尽管我们发现明确的溶剂化作用显著提高了该步骤的动力学，但相应的势垒明显低于氢化物转移步骤中的势垒，这证实了氢提取是胺氧化的限速步骤，并验证了单胺氧化酶的两步氢化物转移机制。
