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

固氮酶类还原酶CFBC/CfbD在辅因子F430的生物合成过程中催化底物六电子还原为六氢氢氯二胺(Ni-H6SHCD)。通过这个反应，酶在底物分子中引入了六个新的立体中心。还原酶cfbc/cfbD与固氮酶在分子结构和部分功能方面是相关的。CFBC/CfbD由两个蛋白质组分组成，一个是还原酶组分，一个是催化组分。还原酶组分是由CFBC构建的，它是一种含有亚基间[4Fe-4S]簇的同源二聚体蛋白。催化组分由CfbD组成，CfbD也是含有亚基间[4Fe-4S]簇的同源二聚体。提出了外给电子体将一个电子转移到CFBC的铁硫团簇上。在与ATP结合后，CFBC被认为经历了构象变化，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CFBC向CFbD的铁硫团簇的电子转移是必不可少的。CfbD的还原团簇最终将电子转移到CfbD内束缚的衬底上。ATP水解和随后的络合物解离完成了第一个电子转移过程。总体而言，这个假设的循环必须进行六次才能实现完全的底物还原。概述的反应周期主要基于对固氮酶的观察。在CFBC/CFBD的情况下，建议的步骤必须进行实验测试，这是本项目的目标。在这个项目中，将研究两个铁硫团簇之间以及CfbD团簇和衬底之间的电子转移机制。为此，将测定各种不同条件下铁-硫团簇的氧化还原电位，以确定哪些条件有利于电子转移的发生。此外，它将被测试，是否发生构象变化，允许电子转移。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，确定CFBC/CFbD的结构是另一个目标。总体而言，该项目旨在阐明CFBC/CfbD和固氮酶之间的共同特征和差异。

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.