非血红素含铁酶是一类在自然界中广泛存在的酶，一般通过催化活化氧气以实现对底物的氧化，在基因表达、生物代谢等过程中发挥重要作用。但是由于其催化反应过程中中间体的寿命短，光谱特征不强等特点，从实验上研究其催化反应过程存在很多困难和挑战，因此理论计算成为研究非血红素含铁酶性质及机理的重要手段。本论文主要利用密度泛函理论方法研究了人类脱氧羧腐胺赖氨酸羟化酶h-DOHH、N-亚硝基脲合成酶SznF及核糖体加氧酶NO66三种非血红素含铁酶的催化反应机理，得到了以下研究结果：

1、人类脱氧羧腐胺赖氨酸羟化酶（h-DOHH）是翻译起始因子5A（eIF5A）修饰过程中的关键酶。其反应位点由两个铁离子构成，属于非血红素双铁酶，与其它双铁酶活性中心结构不同的是，在h-DOHH的过氧化物中间体中O₂^2-离子以μ-η¹:η¹的方式与Fe^III离子配位，且与底物结合在双铁中心的不同侧。与其他非血红素双铁酶中过氧化物中间体不稳定容易被氧化的反应特点不同的是实验研究表明h-DOHH中过氧化物中间体非常稳定，只有与底物结合才能引发后续反应的进行。针对h-DOHH这些特性，我们使用对称性破缺的密度泛函理论方法（broken-symmetry DFT）对它的催化反应机理进行了研究，计算结果表明h-DOHH的催化过程包含四个连续步骤：（1）底物结合到活性位点并引发O₂^2-从双铁的一侧扭转至双铁之间；（2）O₂^2-结合模式的改变触发O-O键断裂，该步反应为整个催化过程的决速步，活化能垒为21.0kcal/mol；（3）O-O键断裂后，靠近底物一侧的O夺取底物中的H原子并生成羟基自由基；（4）羟基自由基与底物复合完成羟化。研究发现Fe^III/III-peroxo复合物具有特殊稳定性的原因在于μ-η¹:η¹的配位方式使得O-O键断裂需要越过32.5kcal/mol的能垒，而O₂^2-周围的疏水环境使之难以质子化，而与底物结合后，该复合物被活化，促使羟化反应顺利进行，从而揭示了底物结合引发反应进行的机制；同时，研究还确认了在反应过程中，O-O断裂后生成具有闭合结构的[Fe^IV₂(μ-O)₂]⁴⁺中间体，而非实验研究者猜测的开放结构的[Fe^IV-O-Fe^{IV=O]4+中间体。} 

2、非血红素含铁酶 SznF 是负责链脲佐菌素（N ^δ -羟基-N ^ω -甲基-N ^ω -亚硝基-瓜氨酸， SZN）中 N-亚硝基脲药效团合成的关键酶。实验结果表明，SznF 催化的 N-亚硝化反应步 骤是通过分子内氧化重排来完成的，这种仅通过一个金属中心催化完成分子内氧化重排并 实现 N-N 键合成的反应在酶催化和有机合成过程中非常少见。由于 SznF 活性位点的结构 与非血红素 FeII/酮戊二酸依赖加氧酶和外二醇芳环裂解双加氧酶（EDOs）活性中心的配位 方式相似，所以实验研究者根据以上两种酶的反应过程猜测了两种可能的反应机理，但是 我们的研究发现这两种反应机理在能量上并不可行。进一步对 SznF 和其他类似的非血红 素含铁氧化酶进行比较，我们认为在反应过程中可能不会生成高价 Fe^IV -O 中间体，因此提 出了另外两条可能的 SznF 催化反应机理，这两条反应途径均不生成 Fe^IV -O 中间体。研究 结果最终确认，由我们提出的其中一条新颖的反应途径最有可能代表 SznF 催化反应过程。 II 在该机理对应的反应过程中，SznF 通过酶的金属 Fe 中心和底物共同提供电子完成了 O2 的 四电子还原，从而避免了在反应过程中生成高价 Fe^IV=O 中间体，这样的反应过程在非血红 素含铁氧化酶中比较罕见。具体的反应机理为：O₂ 分子首先配位结合到活性中心并进攻底 物的 C ^ε 原子形成过氧精氨酸中间体，该过程中 O₂ 从底物获得两个电子；随后底物的 C ^ε -N ^ω 键断裂并在 C ^ε 位置生成碳正离子，碳正离子极化 O-O 键并使其异裂；对反应机理的研究 表明，在 C ^ε -N ^ω 键断裂后的反应过程非常快速，因此可以避免反应体系之间发生 NO 的交 换，这与实验中检测到的 SznF 催化 N-亚硝化反应中的 N 原子全部来源于同一个精氨酸中 胍基的事实完全相符。 

3、核糖体加氧酶 NO66 是一种非血红素含铁羟化酶，它将肽链中的组氨酸残基转化为 S-3-羟基组氨酸，从而调节细胞的生长和增殖。我们结合分子动力学模拟与 ONIOM 计算 研究了NO66的催化反应机理，同时还探讨了底物周围的二层配体Tyr328及三层配体Ser21 在底物结合及释放方面的作用。研究表明 NO66 的羟化机理同大多数非血红素 FeⅡ /酮戊二 酸依赖加氧酶类似，通过生成的高价的 Fe^IV=O 中间体夺取底物上的 H 原子，然后生成羟 基自由基并结合至底物来完成羟化反应。而动力学模拟计算则表明，NO66 中的 Tyr328 和 Ser421 残基通过氢键作用使 Fe^IV=O 基团和底物保持特定的取向，从而影响反应的立体选 择性。同时，Tyr328 对产物的释放有重要影响。

The non-heme iron-containing enzymes are ubiquitous in nature and perform a wide range of functions involving O₂ activation. However, non-heme iron active sites are much more difficult to study because they do not exhibit the intense spectral features characteristic and the intermediates along the reaction pathway are short-lived. Therefore, theoretical calculations have become an important method for studying the properties and mechanisms of non-heme iron-containing enzymes. This thesis explores the catalytic mechanism of human deoxyhypusine hydroxylase (h-DOHH), N-nitrosourea–producing enzyme SznF and ribosomal oxygenases (NO66).

Human deoxyhypusine hydroxylase (h-DOHH) is a critical enzyme for hypusination of eukaryotic translation initiation factor 5A (eIF5A). The h-DOHH has a nonheme diiron active site that the peroxide moiety binds in the opposite side of the substrate in a μ-η¹:η¹ manner which doesn’t resemble that in other non-heme diiron-containing enzymes. Meanwhile, diiron(III)-peroxo complexed in h-DOHH was found to be extremely stable, in which only the binding of the substrate can trigger the subsequent reactions. In this work, extensive DFT calculations reveal that the catalytic mechanism of h-DOHH consists of four consecutive steps: (1) peroxo isomerization triggered by substrate binding; (2) rate-determining O–O bond cleavage and formation of the [Fe^IV₂(μ-O)₂]⁴⁺ compound; (3) H atom abstraction from the substrate; and (4) OH· rebound to the substrate. This work not only rationalizes the exceptional stability of the diiron(III)-peroxo complex in h-DOHH, but also confirms that h-DOHH uses a diamond shape [Fe^IV₂(μ-O)₂]^4+. core to complete crucial H atom abstraction from the substrate rather than the proposed open core [Fe^IV-O-Fe^{IV=O]4+ .}

The non-heme iron-dependent enzyme SznF catalyzes a critical N-nitrosation step during the N-nitrosourea pharmacophore biosynthesis in Streptozotocin (SZN). The intramolecular oxidative rearrangement process is known to proceed at the Fe^II -containing active site in the cupin domain of SznF, but its mechanism has not been elucidated to date. In this study, based on the density IV functional theory calculations, a unique mechanism was proposed for the N-nitrosation reaction catalyzed by SznF in which a four-electron oxidation process is accomplished through a series of complicated electron transferring between the iron center and substrate to bypass the high-valent Fe^IV=O species. In the catalytic reaction pathway, the O2 binds to the iron center and attacks on the substrate to form the peroxo-bridge intermediate by obtaining two electrons from the substrate exclusively. Then, instead of cleaving the peroxo bridge, the C^€ -N ^ω bond of the substrate is heterolytically cleaved first to form a carbocation intermediate, which polarizes the peroxo bridge and promotes its heterolysis. After O-O bond cleavage, the following reaction steps proceed barrierlessly so that the N-nitrosation is accomplished without NO exchange among reaction species.

Human ribosomal oxygenases (ROXs) NO66 a kind of non-heme iron-containing enzyme which binds the 60S ribosomal protein L8 (RPL8) as a substrate and catalyzes histidine 3S hydroxylation which regulates the growth and proliferation of cells. In this study, the molecular dynamic simulation and ONIOM calculations were performed to study the mechanism of NO66 and the role played by second-shell residue Tyr328 and third-shell residue Ser421 in substrate binding and expelling. The results indicated that the mechanism of NO66 is similar with most of the FeII -and 2-oxoglutarate-dependent oxygenases which the hydroxylation of the substrate is accomplished by the high-valent Fe^IV=O intermediate. The Tyr328 and Ser421 residues are shown to guide the stereoselectivity by holding the substrate and Fe^IV=O group in a specific orientation through hydrogen bonds among them. The results of molecular dynamic simulation also indicate that the Tyr328 residue is involved in expelling the product from the binding pocket after the reaction is complete.