J. Heyrovský Institute of Physical Chemistry, The Czech Academy of Sciences, Dolejškova 3, Prague 8 182 23, Czech Republic.
Institute of Organic Chemistry and Biochemistry, The Czech Academy of Sciences, Flemingovo nám. 2, Prague 6 166 10, Czech Republic.
J Am Chem Soc. 2020 Jun 10;142(23):10412-10423. doi: 10.1021/jacs.0c01786. Epub 2020 May 29.
A full understanding of the catalytic action of non-heme iron (NHFe) and non-heme diiron (NHFe) enzymes is still beyond the grasp of contemporary computational and experimental techniques. Many of these enzymes exhibit fascinating chemo-, regio-, and stereoselectivity, in spite of employing highly reactive intermediates which are necessary for activations of most stable chemical bonds. Herein, we study in detail one intriguing representative of the NHFe family of enzymes: soluble Δ desaturase (ΔD), which desaturates rather than performing the thermodynamically favorable hydroxylation of substrate. Its catalytic mechanism has been explored in great detail by using QM(DFT)/MM and multireference wave function methods. Starting from the spectroscopically observed 1,2-μ-peroxo diferric intermediate, the proton-electron uptake by is the favored mechanism for catalytic activation, since it allows a significant reduction of the barrier of the initial (and rate-determining) H-atom abstraction from the stearoyl substrate as compared to the "proton-only activated" pathway. Also, we ruled out that a -like intermediate (high-valent diamond-core bis-μ-oxo-[Fe] unit) is involved in the reaction mechanism. Our mechanistic picture is consistent with the experimental data available for ΔD and satisfies fairly stringent conditions required by Nature: the chemo-, stereo-, and regioselectivity of the desaturation of stearic acid. Finally, the mechanisms evaluated are placed into a broader context of NHFe chemistry, provided by an amino acid sequence analysis through the families of the NHFe enzymes. Our study thus represents an important contribution toward understanding the catalytic action of the NHFe enzymes and may inspire further work in NHFe biomimetic chemistry.
目前，计算和实验技术还远不能完全理解非血红素铁（NHFe）和非血红素二铁（NHFe）酶的催化作用。尽管这些酶采用了高反应性的中间产物，这些中间产物对于激活大多数稳定的化学键是必要的，但它们仍表现出令人着迷的化学、区域和立体选择性。在此，我们详细研究了 NHFe 酶家族的一个有趣代表：可溶性 Δ 去饱和酶（ΔD），它不是进行热力学有利的底物羟化，而是使底物去饱和。已经使用 QM（DFT）/MM 和多参考波函数方法详细研究了其催化机制。从光谱观察到的 1,2-μ-过氧双铁中间产物开始， 通过质子-电子摄取的途径是催化激活的首选机制，因为与“仅质子激活”途径相比，它允许从硬脂酰底物中初始（和速率决定）H 原子的提取的势垒显著降低。此外，我们排除了涉及反应机制的 -样中间产物（高价金刚石核双-μ-氧-[Fe]单元）。我们的机理图与 ΔD 可用的实验数据一致，并满足自然界的相当严格的条件：硬脂酸去饱和的化学、立体和区域选择性。最后，通过对 NHFe 酶家族的氨基酸序列分析，将评估的机制置于更广泛的 NHFe 化学背景中。我们的研究因此为理解 NHFe 酶的催化作用做出了重要贡献，并可能激发 NHFe 仿生化学的进一步研究。
