Institute of Organic Chemistry and Biochemistry, Gilead Sciences & IOCB Research Center, Academy of Sciences of the Czech Republic, Flemingovo náměstí 2, 166 10 Praha 6, Czech Republic.
Inorg Chem. 2012 Mar 5;51(5):2806-20. doi: 10.1021/ic2018067. Epub 2012 Feb 14.
Large-scale quantum and molecular mechanical methods (QM/MM) and QM calculations were carried out on the soluble Δ(9) desaturase (Δ(9)D) to investigate various structural models of the spectroscopically defined peroxodiferric (P) intermediate. This allowed us to formulate a consistent mechanistic picture for the initial stages of the reaction mechanism of Δ(9)D, an important diferrous nonheme iron enzyme that cleaves the C-H bonds in alkane chains resulting in the highly specific insertion of double bonds. The methods (density functional theory (DFT), time-dependent DFT (TD-DFT), QM(DFT)/MM, and TD-DFT with electrostatic embedding) were benchmarked by demonstrating that the known spectroscopic effects and structural perturbation caused by substrate binding to diferrous Δ(9)D can be qualitatively reproduced. We show that structural models whose spectroscopic (absorption, circular dichroism (CD), vibrational and Mössbauer) characteristics correlate best with experimental data for the P intermediate correspond to the μ-1,2-O(2)(2-) binding mode. Coordination of Glu196 to one of the iron centers (Fe(B)) is demonstrated to be flexible, with the monodentate binding providing better agreement with spectroscopic data, and the bidentate structure being slightly favored energetically (1-10 kJ mol(-1)). Further possible structures, containing an additional proton or water molecule are also evaluated in connection with the possible activation of the P intermediate. Specifically, we suggest that protonation of the peroxide moiety, possibly preceded by water binding in the Fe(A) coordination sphere, could be responsible for the conversion of the P intermediate in Δ(9)D into a form capable of hydrogen abstraction. Finally, results are compared with recent findings on the related ribonucleotide reductase and toluene/methane monooxygenase enzymes.
采用大规模量子力学和分子力学方法（QM/MM）和量子力学计算对可溶的Δ(9)去饱和酶（Δ(9)D）进行了研究，以探讨光谱定义的过氧二铁（P）中间物的各种结构模型。这使我们能够为Δ(9)D 的反应机制的初始阶段制定一个一致的机制图像，Δ(9)D 是一种重要的二价非血红素铁酶，它能在烷链中切割 C-H 键，导致双键的高度特异性插入。通过证明可以定性地再现已知的光谱效应和底物结合对二价 Δ(9)D 引起的结构扰动，对方法（密度泛函理论（DFT）、含时密度泛函理论（TD-DFT）、QM（DFT）/MM 和静电嵌入的 TD-DFT）进行了基准测试。我们表明，与 P 中间物的实验数据相关性最佳的结构模型与 μ-1,2-O(2)(2-) 结合模式相对应。证明 Glu196 与一个铁中心（Fe(B)）的配位是灵活的，单齿配位提供了与光谱数据更好的一致性，而双齿结构在能量上略受青睐（1-10 kJ mol(-1)）。还评估了与 P 中间物的可能活化有关的其他可能结构，包括额外的质子或水分子。具体来说，我们建议过氧部分的质子化，可能在 Fe(A)配位球中先结合水分子，可能是导致 P 中间物在 Δ(9)D 中转化为能够进行氢提取的形式的原因。最后，将结果与最近关于相关核苷酸还原酶和甲苯/甲烷单加氧酶的研究结果进行了比较。
