Characterization of a Nitrogenase Iron Protein Substituted with a Synthetic [Fe Se ] Cluster.

The Fe protein of nitrogenase plays multiple roles in substrate reduction and cluster maturation via its redox-active [Fe S ] cluster. Here we report the synthesis and characterization of a water-soluble [Fe Se ] cluster that is used to substitute the [Fe S ] cluster of the Azotobacter vinelandii Fe protein (AvNifH). Biochemical, EPR and XAS/EXAFS analyses demonstrate the ability of the [Fe Se ] cluster to adopt the super-reduced, all-ferrous state upon its incorporation into AvNifH. Moreover, these studies reveal that the [Fe Se ] cluster in AvNifH already assumes a partial all-ferrous state ([Fe Se ] ) in the presence of dithionite, where its [Fe S ] counterpart in AvNifH exists solely in the reduced state ([Fe S ] ). Such a discrepancy in the redox properties of the AvNifH-associated [Fe Se ] and [Fe S ] 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.