| Nitrogenase represents a class of prokaryotic metalloenzymes that catalyze the reduction of dinitrogen (N2) to ammonia (NH 3) under ambient conditions. The most active and best-understood member of these binary systems is the molybdenum nitrogenase, which is composed of iron (Fe) protein and the molybdenum-iron (MoFe) protein. The biosynthesis of the iron-molybdenum cofactor (FeMoco) of the MoFe protein occurs on NifEN, a scaffold protein that has significant homology to the MoFe protein. NifEN hosts the maturation of a precursor to the mature FeMoco before it is delivered to the MoFe protein. Recently, the Ribbe lab identified that the FeMoco precursor and mature FeMoco on NifEN are structurally similar to the FeMoco on the MoFe protein. The goals of this thesis were to investigate NifEN's role in catalysis, the "ex situ" assembly of FeMoco on NifEN, how the FeMoco is transferred from NifEN to the MoFe protein, and if NifEN could act as an assembly scaffold for heterometals of other known alternative nitrogenases (vanadium and iron-only nitrogenase). The functions of NifEN in catalysis and assembly were investigated via activity assays, EPR spectroscopy, and XAS/EXAFS analysis. These experiments determined that NifEN, which contains the FeMoco-precursor, has the ability to reduce some substrates of the MoFe protein (C2H2 and N3-), although at a lower rate. FeMoco maturation on NifEN was determined to be dependent on dithionite concentration, suggesting an essential role of redox chemistry in this process. Homocitrate, which is required during FeMoco maturation, has an impact on the redox properties of FeMoco and is essential for the transfer of FeMoco between NifEN and the MoFe protein. Vanadium and iron can be heterologously inserted into the NifEN-associated precursor in a redox-dependent process, albeit at much reduced efficiencies. This observation suggests that the selective heterometal insertion into the molybdenum, vanadium and iron-only nitrogenases is likely accomplished through different redox potentials of the homologous Fe proteins and their respective scaffold proteins depending on the nitrogenase system. |
