Characterization of a Nitrogenase Iron Protein Substituted with a Synthetic [Fe4Se4] Cluster

Abstract

AbstractThe Fe protein of nitrogenase plays multiple roles in substrate reduction and cluster maturation via its redox‐active [Fe4S4] cluster. Here we report the synthesis and characterization of a water‐soluble [Fe4Se4] cluster that is used to substitute the [Fe4S4] cluster of the Azotobacter vinelandii Fe protein (AvNifH). Biochemical, EPR and XAS/EXAFS analyses demonstrate the ability of the [Fe4Se4] cluster to adopt the super‐reduced, all‐ferrous state upon its incorporation into AvNifH. Moreover, these studies reveal that the [Fe4Se4] cluster in AvNifH already assumes a partial all‐ferrous state ([Fe4Se4]0) in the presence of dithionite, where its [Fe4S4] counterpart in AvNifH exists solely in the reduced state ([Fe4S4]1+). Such a discrepancy in the redox properties of the AvNifH‐associated [Fe4Se4] and [Fe4S4] 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.