Characterization and reaction mechanism of the nitrogenase-like reductase CfbC/CfbD involved in cofactor F430 biosynthesis

辅因子F430生物合成中固氮酶样还原酶CfbC/CfbD的表征及反应机制

• 批准号: 311061671
• 负责人: Professorin Dr. Gunhild Monika Layer
• 金额: --
• 依托单位: Lehrstuhl für Pharmazeutische Biologie und Biotechnologie
• 依托单位国家: 德国
• 项目类别: Priority Programmes
• 财政年份: 2016
• 资助国家: 德国
• 起止时间: 2015-12-31 至 2022-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/311061671?language=en
• 关键词: Characterization; reaction; mechanism; nitrogenase; like

The nitrogenase-like reductase CfbC/CfbD catalyzes the sophisticated six-electron reduction of the substrate nickel-sirohydrochlorin-diamide (Ni-SHCD) to nickel-hexahydrosirohydrochlorin-diamide (Ni-H6SHCD) during cofactor F430 biosynthesis. With this reaction, the enzyme introduces six new stereocenters into the substrate molecule. The reductase CfbC/CfbD is related to nitrogenase in terms of molecular architecture and, in part, in terms of function. CfbC/CfbD consists of two protein components, a reductase component and a catalytic component. The reductase component is built by CfbC, which is a homodimeric protein containing an intersubunit [4Fe-4S] cluster. The catalytic component consists of CfbD, which is also a homodimer harboring an intersubunit [4Fe-4S] cluster. It is proposed that an external electron donor transfers an electron to the iron-sulfur cluster of CfbC. After ATP-binding, CfbC is believed to undergo a conformational change, which is required for the interaction with CfbD. This protein-protein interaction is essential for the electron transfer from CfbC to the iron-sulfur cluster of CfbD. The reduced cluster of CfbD finally transfers the electron onto the substrate bound within CfbD. ATP-hydrolysis and subsequent complex dissociation complete the first electron transfer process. Overall, this hypothetic cycle has to proceed six times in order to achieve complete substrate reduction. The outlined reaction cycle is mainly based on observations made for nitrogenase. In the case of CfbC/CfbD the proposed steps have to be tested experimentally, which is the objective of this project. Within this project, the mechanism of electron transfers between the two iron-sulfur clusters as well as between the CfbD cluster and the substrate will be investigated. For this purpose, the redox potentials of the iron-sulfur clusters will be determined under a variety of different conditions in order to define those conditions that are favorable for electron transfer to occur. Further, it will be tested, whether conformational changes take place allowing for the electron transfer. Another aim is the detection of potential substrate radicals by performing single electron transfer experiments. Finally, the determination of the structure of CfbC/CfbD is another objective. Overall, this project is designed to shed light on the common features, but also on the differences between CfbC/CfbD and nitrogenase.

在辅因子 F430 生物合成过程中，固氮酶样还原酶 CfbC/CfbD 催化底物镍-西罗氢氯酸二酰胺 (Ni-SHCD) 复杂的六电子还原为镍-六氢西罗氢氯酸-二酰胺 (Ni-H6SHCD)。通过该反应，酶将六个新的立体中心引入底物分子中。还原酶 CfbC/CfbD 在分子结构方面和部分功能方面与固氮酶相关。 CfbC/CfbD 由两种蛋白质成分组成，即还原酶成分和催化成分。还原酶成分由 CfbC 构建，CfbC 是一种含有亚基间 [4Fe-4S] 簇的同二聚体蛋白质。催化组分由 CfbD 组成，它也是带有亚基间 [4Fe-4S] 簇的同二聚体。提出外部电子供体将电子转移到 CfbC 的铁硫簇。 ATP 结合后，CfbC 被认为会发生构象变化，这是与 CfbD 相互作用所必需的。这种蛋白质-蛋白质相互作用对于电子从 CfbC 转移到 CfbD 的铁硫簇至关重要。减少的 CfbD 簇最终将电子转移到结合在 CfbD 内的底物上。 ATP 水解和随后的复合物解离完成了第一个电子转移过程。总的来说，这个假设的循环必须进行六次才能实现底物的完全减少。概述的反应循环主要基于对固氮酶的观察。对于 CfbC/CfbD，必须对提议的步骤进行实验测试，这是该项目的目标。在该项目中，将研究两个铁硫簇之间以及 CfbD 簇与基底之间的电子转移机制。为此，将在各种不同条件下测定铁硫簇的氧化还原电位，以确定有利于电子转移发生的条件。此外，将测试是否发生允许电子转移的构象变化。另一个目标是通过进行单电子转移实验来检测潜在的底物自由基。最后，CfbC/CfbD结构的确定是另一个目标。总体而言，该项目旨在阐明 CfbC/CfbD 和固氮酶之间的共同特征以及差异。