The Fe protein of nitrogenase reduces two C1 substrates, CO₂ and CO, under ambient conditions when its [Fe₄S₄] cluster adopts the all-ferrous [Fe₄S₄]⁰ state. Here, we show disparate reactivities of the nifH- and vnf-encoded Fe proteins from Methanosarcina acetivorans (designated MaNifH and MaVnfH) toward C1 substrates in the all-ferrous state, with the former capable of reducing both CO₂ and CO to hydrocarbons, and the latter only capable of reducing CO to hydrocarbons at substantially reduced yields. EPR experiments conducted at varying solution potentials reveal that MaVnfH adopts the all-ferrous state at a more positive reduction potential than MaNifH, which could account for the weaker reactivity of the MaVnfH toward C1 substrates than MaNifH. More importantly, MaVnfH already displays the g = 16.4 parallel-mode EPR signal that is characteristic of the all-ferrous [Fe₄S₄]⁰ cluster at a reduction potential of -0.44 V, and the signal reaches 50% maximum intensity at a reduction potential of -0.59 V, suggesting the possibility of this Fe protein to access the all-ferrous [Fe₄S₄]⁰ state under physiological conditions. These results bear significant relevance to the long-lasting debate of whether the Fe protein can utilize the [Fe₄S₄]^0/2+ redox couple to support a two-electron transfer during substrate turnover which, therefore, is crucial for expanding our knowledge of the reaction mechanism of nitrogenase and the cellular energetics of nitrogenase-based processes.

The Fe protein of nitrogenase plays multiple roles in substrate reduction and metallocluster assembly. Best known for its function to transfer electrons to its catalytic partner during nitrogenase catalysis, the Fe protein is also a key player in the biosynthesis of the complex metalloclusters of nitrogenase. In addition, it can function as a reductase on its own and affect the ambient reduction of CO₂ or CO to hydrocarbons. This review will provide an overview of the properties and functions of the Fe protein, highlighting the relevance of this unique FeS enzyme to areas related to the catalysis, biosynthesis, and applications of the fascinating nitrogenase system.

Nitrogenase is a versatile metalloenzyme that reduces N₂, CO and CO₂ at its cofactor site. Designated the M-cluster, this complex cofactor has a composition of [(R-homocitrate)MoFe₇S₉C], and it is assembled through the generation of a unique [Fe₈S₉C] core prior to the insertion of Mo and homocitrate. NifB is a radical S-adenosyl-L-methionine (SAM) enzyme that is essential for nitrogenase cofactor assembly. This review focuses on the recent work that sheds light on the role of NifB in the formation of the [Fe₈S₉C] core of the nitrogenase cofactor, highlighting the structure, function and mechanism of this unique radical SAM methyltransferase.

The Fe protein of nitrogenase plays multiple roles in substrate reduction and cluster maturation via its redox-active [Fe₄ S₄ ] cluster. Here we report the synthesis and characterization of a water-soluble [Fe₄ Se₄ ] cluster that is used to substitute the [Fe₄ S₄ ] cluster of the Azotobacter vinelandii Fe protein (AvNifH). Biochemical, EPR and XAS/EXAFS analyses demonstrate the ability of the [Fe₄ Se₄ ] cluster to adopt the super-reduced, all-ferrous state upon its incorporation into AvNifH. Moreover, these studies reveal that the [Fe₄ Se₄ ] cluster in AvNifH already assumes a partial all-ferrous state ([Fe₄ Se₄ ]⁰ ) in the presence of dithionite, where its [Fe₄ S₄ ] counterpart in AvNifH exists solely in the reduced state ([Fe₄ S₄ ]¹⁺ ). Such a discrepancy in the redox properties of the AvNifH-associated [Fe₄ Se₄ ] and [Fe₄ S₄ ] clusters can be used to distinguish the differential redox requirements for the substrate reduction and cluster maturation of nitrogenase, pointing to the utility of chalcogen-substituted FeS clusters in future mechanistic studies of nitrogenase catalysis and assembly.