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The Diverse Roles of [4Fe-4S] Clusters in Nitrogenase Iron-Sulfur Cluster Assembly and Catalysis

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Nitrogenase catalyzes the enzymatic reduction of dinitrogen (N2) into ammonia (NH3) at ambient temperatures and pressures. N2 reduction by molybdenum (Mo) nitrogenase requires the catalytic molybdenum-iron protein (NifDK) and its reductase, the iron protein (NifH). In addition to its role in N2 reduction, NifH proteins can independently reduce carbon dioxide (CO2) to carbon monoxide (CO) and short-chain hydrocarbons. Nitrogenase proteins contain iron-sulfur cluster cofactors that are crucial to these activities. NifH uses a [Fe4S4] cluster for transferring electrons to NifDK and for independently reducing C1, whereas the site of N2 reduction on NifDK is the complex [MoFe7S9C-homocitrate] M-cluster. M-cluster, however, is synthesized ex-situ by other proteins from [Fe4S4] cluster precursors. The radical S-adenosylmethionine enzyme NifB uses [Fe4S4] cluster units for the biosynthesis of the [Fe8S9C] L-cluster, the immediate precursor to M-cluster. NifH and NifB rely on [Fe4S4] clusters to perform very different functions, mediated by their protein environments in ways that aren’t well understood. The focus of this dissertation is on how the distinct protein environments of NifH and NifB proteins modulate the reactivity of their [Fe4S4] clusters.

Three [Fe4S4] cluster modules, and their amino acid ligands, were identified on NifB by spectroscopic and biochemical characterization of mutant proteins. Interestingly, a histidine residue that serves as a transient nitrogen ligand to a [Fe4S4] precursor cluster was crucial for L-cluster assembly. Mutation of this ligand was found to interfere with the structural transformations of iron-sulfur clusters of NifB.

To understand how the [Fe4S4] cluster at the active site of NifH catalyzes C1 substrate reductions, I solved the structures of two NifH proteins by X-ray crystallography. The first was NifH in the all-ferrous [Fe4S4]0 state from Azotobacter vinelandii (AvNifH). This form, produced by treating the protein with a strong reductant, is the most active in substrate reduction. The structure solved from a crystal in this form revealed two arginine residues that have an asymmetric arrangement that can serve to capture and protonate substrates. Another NifH protein, Methanosarcina acetivorans (MaNifH), can reduce CO2 and CO to short-chain hydrocarbons. MaNifH in the [Fe4S4]1+ state, produced by the presence of dithionite, demonstrates a similar asymmetric arrangement of a pair of arginines. These crystallization solution also contained bicarbonate, an alternative CO2 source. Excitingly, additional density, potentially a captured substrate, was observed near the [Fe4S4] cluster. These structures provided parameters for density functional theory (DFT) calculations provided details that enabled propose mechanisms for CO2 and CO reduction by [Fe4S4] clusters.

Together, these results contribute towards building a mechanistic model of a novel [Fe4S4]-based system for converting greenhouse gases into hydrocarbon fuels, and for understanding how [Fe4S4] clusters are used to synthesize more complex metallocofactors.

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