Mechanism of iron-sulfur cluster biosynthesis : Analysis of a framework and the reaction mechanism for the two iron-sulfur cluster assembly systems.

铁硫簇生物合成机制：分析两种铁硫簇组装系统的框架和反应机制。

• 批准号: 15510174
• 负责人: TAKAHASHI Yasuhiro
• 金额: $ 2.18万
• 依托单位: Osaka University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2003
• 资助国家: 日本
• 起止时间: 2003 至 2004
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-15510174/
• 关键词: iron-sulfur cluster; iron-sulfur protein; biosynthesis; ISC machinery; SUF machinery; structure-function; X-ray crystal structure; 分子間相互作用

Iron-sulfur (Fe-S) proteins are present in almost all living organisms and exhibit diverse functions, which include electron transport, redox and non-redox catalysis, and sensing for regulatory processes. They contain clusters of iron and sulfur atoms with variable complexity, such as [2Fe-2S], [3Fe-4S], and [4Fe-3S] clusters. We have recently found two independent systems, called ISC and SUF, essential for the biosynthesis of Fe-S clusters, although still very little is known about the mechanistic details. In this study we have undertaken genetic, biochemical and structural approaches to elucidate the complex mechanism by which the Fe-S clusters are assembled.1. We have isolated and analyzed 14 psuedorevertants generated from a mutant strain lacking HscA and HscB in the ISC machinery, which revealed various suppressor mutations located in IscU. We also found that the IscU homolog from the thermophilic bacterium Aquifex aeolicus exceptionally carries the intermediate Fe-S cluster in the stable form that was further stabilized by introduction of several mutations.2. Purification and biochemical characterization of the components of the SUF machinery have revealed dimeric form of SufD and the SufC_2SufD_2 oligomer as well as the SufB_1SufC_2 SufD_1 complex, suggesting that dynamic alteration of the oligomeric status is involved in the reaction. Essential residues for the SufD function have also been identified by mutagenic studies.3. We have determined the crystal structures of E.coli yfhJ (IscX), SufA, SufC, and SufD. The structure of SufA revealed the active-site architecture comprising of four cysteine residues. We have also identified a salt bridge that appears to trigger the conformational change of SufC. The structure of SufD revealed an unprecedented β-helix dimer. The docking model between SufC and SufD suggests the role of SufC to alter the oligomeric status of SufD.

铁硫蛋白（Fe-S）几乎存在于所有生物体内，并具有多种功能，包括电子传递、氧化还原和非氧化还原催化以及调节过程的传感。它们含有不同复杂程度的铁和硫原子簇，如[2Fe-2S]、[3Fe-4S]和[4Fe-3S]簇。我们最近发现了两个独立的系统，称为ISC和SUF，对Fe-S簇的生物合成至关重要，尽管对其机制细节知之甚少。在这项研究中，我们采用了遗传、生化和结构方法来阐明Fe-S簇组装的复杂机制。我们分离并分析了从ISC机制中缺乏HscA和HscB的突变株中产生的14个伪逆转物，发现IscU中存在各种抑制突变。我们还发现来自嗜热细菌风湿水蛭的IscU同源物异常地以稳定的形式携带中间的Fe-S簇，该簇通过引入几个突变进一步稳定。对SUF机制的组分进行纯化和生化表征，发现SufD、SufC_2SufD_2低聚物以及SufB_1SufC_2 SufD_1配合物的二聚体形式，表明该反应参与了低聚物状态的动态改变。SufD功能的基本残基也已通过诱变研究确定。我们测定了大肠杆菌yfhJ （IscX）、SufA、SufC和SufD的晶体结构。SufA的结构揭示了由四个半胱氨酸残基组成的活性位点结构。我们还发现了一个盐桥，它似乎触发了SufC的构象变化。SufD的结构揭示了一个前所未有的β-螺旋二聚体。SufC与SufD的对接模型表明，SufC可以改变SufD的寡聚状态。

项目成果

Characterization of cluster N5 as a fast-relaxing [4Fe-4S] cluster in the Nqo3 subunit of the proton-translocating NADH-ubiquinone oxidoreductase from Paracoccus denitrificans.

将 N5 簇表征为脱氮副球菌质子转位 NADH-泛醌氧化还原酶 Nqo3 亚基中的快速松弛 [4Fe-4S] 簇。

• DOI: 10.1074/jbc.m212275200
• 发表时间: 2003
• 期刊: The Journal of biological chemistry
• 作者: Yano,Takahiro;Sklar,Joseph;Nakamaru-Ogiso,Eiko;Takahashi,Yasuhiro;Yagi,Takao;Ohnishi,Tomoko
• 通讯作者: Ohnishi,Tomoko

Marco Kriek: "Effect of iron-sulfur cluster assembly proteins on the expression of Escherichia coli lipoic acid synthase"Protein Expr.Purif.. 28・2. 241-245 (2003)

Marco Kriek：“铁硫簇组装蛋白对大肠杆菌硫辛酸合酶表达的影响”Protein Expr.Purif.. 28・245 (2003)。

Takahiro Yano: "Characterization of cluster N5 as a fast-relaxing [4Fe4S] cluster in the Nqo3 subunit of the proton-translocating NADH-ubiquinone oxidoreductase from Paracoccus denitrificans"J.Biol.Chem.. 278・18. 15514-15522 (2003)

Takahiro Yano：“脱氮副球菌质子转位 NADH-泛醌氧化还原酶 Nqo3 亚基中 N5 簇的表征”J.Biol.Chem.. 15514-15522（2003 年） ）

An Intestinal Parasitic Protist Entamoeba histolytica Possesses a Non-redundant NIF-like System for Iron-Sulfur Cluster Assembly under Anaerobic Conditions.

肠道寄生原生生物溶组织内阿米巴拥有非冗余的类 NIF 系统，可在厌氧条件下组装铁硫簇。

• 发表时间: 2004
• 期刊: J.Biol.Chem. 279-16
• 作者: Nemoto;Hisao;Vahab Ali
• 通讯作者: Vahab Ali

Characterization of cluster NS as a fast-relaxing [4Fe-4S] cluster in the Ngo3 subunit of the proton-translocating NADH-ubiquinone oxidoreductase from Paracoccus denitrificans.

NS 簇的表征为脱氮副球菌质子转位 NADH-泛醌氧化还原酶 Ngo3 亚基中快速松弛的 [4Fe-4S] 簇。

• 发表时间: 2003
• 期刊: J.Biol.Chem. 278-18
• 作者: Nemoto;Hisao;Takahiro Yano
• 通讯作者: Takahiro Yano