Wednesday, July 26, 2023 - 3:00pm


Nitrogen fixation, the reduction of N2 to NH3, is a key step in the global nitrogen cycle. Biological nitrogen fixation (BNF) is carried out by diazotrophic microbes, which utilize the metalloenzyme nitrogenase to perform this chemical transformation. BNF by Mo-nitrogenase requires both the catalytic component, NifDK, and the reductase component, NifH. NifDK is the site of N2 reduction and contains two of the most complex metallocofactors known to nature: the [F8S7] P-cluster and the [MoFe7S9C-homocitrate] M-cluster. NifH is a homodimer that contains a single [Fe4S4] cluster and plays a crucial role in the transfer of electrons to NifDK and the assembly of NifDK’s metallocofactors and catalyzes the reduction of C1 substrates. Section 1 of this dissertation focuses on a pair of studies that probe the reactivity of different NifH homologs. Section 2 of this dissertation details progress toward the heterologous expression of functional nitrogenase and related proteins.

NifH is a multipurpose actor that plays an essential role in nitrogen fixation and may have its own industrially relevant applications independent of NifDK. However, the relationship between NifH’s reactivity and redox properties has not been thoroughly studied. Section 1 details a pair of studies that employ novel methods, which can be applied broadly to the study of [Fe4S4] containing proteins, to probe the reactivity of NifH and the redox properties of its metallocofactor. In study 1, the disparate reactivities of two homologs of NifH toward the reduction of C1 substrates in the all-ferrous state are explored and related to differences in the redox potential of the all-ferrous state. In study 2, a synthetic [Fe4Se4] compound is incorporated into NifH, showing redox properties distinct from its [Fe4S4] counterpart that can be used to distinguish the differential redox requirements of nitrogenase catalysis and assembly.

Endowing cereals with nitrogen fixation activity is considered a holy grail. However, previous attempts in the literature fail to demonstrate the expression of functional nitrogenase and related proteins in a heterologous host. Section 2 details work done toward heterologously expressing functional nitrogenase and related proteins in the model organism Escherichia coli.


Joseph Solomon


McGaugh 1246