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

Abstract

The Fe protein of nitrogenase plays multiple roles in substrate reduction and cluster maturation via its redox-active [Fe 4 S 4 ] cluster. Here we report the synthesis and characterization of a water-soluble [Fe 4 Se 4 ] cluster that is used to substitute the [Fe 4 S 4 ] cluster of the Azotobacter vinelandii Fe protein (Av-NifH). Biochemical, EPR and XAS/EXAFS analyses demonstrate the ability of the [Fe 4 Se 4 ] cluster to adopt the super-reduced, all-ferrous state upon its incorporation into AvNifH. Moreover, these studies reveal that the [Fe 4 Se 4 ] cluster in AvNifH already assumes a partial all-ferrous state ([Fe 4 Se 4 ] 0 ) in the presence of dithionite, where its [Fe 4 S 4 ] counterpart in AvNifH exists solely in the reduced state ([Fe 4 S 4 ] 1 + ). Such a discrepancy in the redox properties of the AvNifH-associated [Fe 4 Se 4 ] and [Fe 4 S 4 ] 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.

Selenocyanate Derived Se-Incorporation into the Nitrogenase Fe Protein Cluster

“…reported that an Fe protein with an Fe4Se4 cluster is reduced to the all ferrous state in the presence of dithionite, rendering it EPR silent in perpendicular mode EPR. 35 In this context, the signal observed in Figure 2-figure supplement 7 may correspond to the [Fe4S4] 1+ state while the [Fe4Se4] 0 state is not observed. Our results cannot distinguish between these two possible interpretations.…”

“…The exchange of selenium into an Fe 4 S 4 cluster through reaction with KSeCN represents a new approach for generating Fe 4 Se 4 -containing Fe proteins from native Fe 4 S 4 -containing proteins and a selenium source that complements existing reconstitution approaches using apoproteins (proteins deficient in the native Fe 4 S 4 cluster) and a (i) selenium source, iron source, and reductant or (ii) with synthetic clusters. 34,35 The capability of preparing chalcogenide substituted clusters opens new avenues for exploring the multiple roles of the Fe protein in biological nitrogen fixation.…”

Nitrogenase Fe Protein: A Multi-Tasking Player in Substrate Reduction and Metallocluster Assembly

“…Progress has been made, however, toward establishing the potential physiological relevance of the all-ferrous state of the Fe protein. A recent study demonstrated that contrary to its native counterpart, a variant of the A. vinelandii NifH protein containing an [Fe 4 Se 4 ] cluster in place of the [Fe 4 S 4 ] cluster readily displayed the all-ferrous specific, g = 16.4 EPR signal in the presence of dithionite ( E 1/2 = −0.47 at 2 mM), a solution potential where the native [Fe 4 S 4 ] cluster was only capable of adopting the reduced [Fe 4 S 4 ] 1+ state [ 47 ]. This observation implies that the Fe protein could potentially access the all-ferrous state under certain physiological conditions.…”

“…However, there are clear distinctions between the functionality of NifH in catalysis and assembly. Apart from the distinctive nature of substrate reduction and cluster assembly that underlies the differences between these processes, a recent study has demonstrated the requirement of a more negative reduction potential for NifH to fulfill its function in nitrogenase catalysis than those for the same protein to carry out its functions in nitrogenase assembly [ 47 ]. Moreover, while the formation of the P-cluster involves interactions between NifH and an NifDK variant that are analogous to those between NifH and NifDK during catalysis, maturation of the M-cluster occurs on a biosynthetic scaffold that is homologous to, but clearly distinct from NifDK, and involves more chemical transformations than those occurring during the process of P-cluster maturation.…”

“…The differential C 1 substrate reactivities of Ma NifH and Ma VnfH have been shown to be associated with the strengths (i.e., reduction potentials) of their respective all-ferrous states in C 1 substrate reduction, with Ma VnfH having a weaker all-ferrous state at a more positive reduction potential than the stronger all-ferrous state adopted by its Ma NifH counterpart at a more negative reduction potential [ 19 ]. Consistent with this observation, a variant of Av NifH containing an [Fe 4 Se 4 ] cluster in place of the [Fe 4 S 4 ] cluster was shown to adopt the all-ferrous state at a more positive potential than its native Av NifH counterpart and, consequently, reduce CO 2 to CO at a reduced efficiency than the latter in the presence of Eu II -DTPA, where both Fe proteins existed fully in the super reduced, all ferrous state [ 47 ].…”