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

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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.