Department of Biochemistry, Instituto de Química, Universidade de São Paulo, Av. Prof. Lineu Prestes 748, 05508-900 São Paulo, São Paulo, Brazil.
J Chem Inf Model. 2023 Jun 12;63(11):3510-3520. doi: 10.1021/acs.jcim.3c00292. Epub 2023 May 17.
The interconversion between fumarate and succinate is fundamental to the energy metabolism of nearly all organisms. This redox reaction is catalyzed by a large family of enzymes, fumarate reductases and succinate dehydrogenases, using hydride and proton transfers from a flavin cofactor and a conserved Arg side-chain. These flavoenzymes also have substantial biomedical and biotechnological importance. Therefore, a detailed understanding of their catalytic mechanisms is valuable. Here, calibrated electronic structure calculations in a cluster model of the active site of the Fcc fumarate reductase were employed to investigate various reaction pathways and possible intermediates in the enzymatic environment and to dissect interactions that contribute to catalysis of fumarate reduction. Carbanion, covalent adduct, carbocation, and radical intermediates were examined. Significantly lower barriers were obtained for mechanisms via carbanion intermediates, with similar activation energies for hydride and proton transfers. Interestingly, the carbanion bound to the active site is best described as an enolate. Hydride transfer is stabilized by a preorganized charge dipole in the active site and by the restriction of the C1-C2 bond in a twisted conformation of the otherwise planar fumarate dianion. But, protonation of a fumarate carboxylate and quantum tunneling effects are not critical for catalysis of the hydride transfer. Calculations also suggest that the driving force for enzyme turnover is provided by regeneration of the catalytic Arg, either coupled with flavin reduction and decomposition of a proposed transient state or directly from the solvent. The detailed mechanistic description of enzymatic reduction of fumarate provided here clarifies previous contradictory views and provides new insights into catalysis by essential flavoenzyme reductases and dehydrogenases.
延胡索酸盐和琥珀酸盐之间的相互转化是几乎所有生物体能量代谢的基础。该氧化还原反应由一个大型酶家族，延胡索酸还原酶和琥珀酸脱氢酶，利用黄素辅因子和保守的Arg 侧链的氢化物和质子转移来催化。这些黄素酶在生物医学和生物技术方面也具有重要意义。因此，详细了解它们的催化机制是有价值的。在这里，采用活性位点的簇模型对 Fcc 延胡索酸还原酶的电子结构进行了校准计算，以研究酶环境中的各种反应途径和可能的中间产物，并剖析有助于延胡索酸还原催化的相互作用。研究了碳负离子、共价加合物、碳正离子和自由基中间体。通过碳负离子中间体的机制得到了明显更低的势垒，氢化物和质子转移的活化能相似。有趣的是，与活性位点结合的碳负离子最好被描述为烯醇化物。氢化物转移通过活性位点中的预组织电荷偶极和在 otherwise planar 的富马酸二阴离子的扭曲构象中限制 C1-C2 键来稳定。但是，富马酸羧酸盐的质子化和量子隧穿效应对于氢化物转移的催化并不是关键的。计算还表明，酶周转的驱动力是由催化 Arg 的再生提供的，该再生要么与黄素还原和所提出的瞬态的分解偶联，要么直接来自溶剂。这里提供的富马酸酶还原的详细机制描述澄清了先前的矛盾观点，并为必需的黄素酶还原酶和脱氢酶的催化提供了新的见解。
