THE AZOTOBACTER-VINELANDII NIFEN COMPLEX CONTAINS 2 IDENTICAL [4FE-4S] CLUSTERS

The nifE and nifN gene products from Azotobacter vinelandii form an al pha(2)/beta(2) tetramer (NifEN complex) that is required for the biosy nthesis of the nitrogenase FeMo cofactor. In the current model for Nif EN complex organization and function, the complex is structurally anal ogous to the nitrogenase MoFe protein and provides an assembly site fo r a portion of FeMo cofactor biosynthesis. In this work, gene fusion a nd immobilized metal-affinity chromatography strategies were used to e levate the in vivo production of the NifEN complex and to facilitate i ts rapid and efficient purification. The NifEN complex produced and pu rified in this way exhibits an FeMo cofactor biosynthetic activity sim ilar to that previously described for the NifEN complex purified by tr aditional chromatography methods. UV-visible, EPR, variable-temperatur e magnetic circular dichroism, and resonance Raman spectroscopies were used to show that the NifEN complex contains two identical [4Fe-4S](2 +) clusters. These clusters have a predominantly S = 1/2 ground state in the reduced form, exhibit a reduction potential of -350 mV, and are likely to be coordinated entirely by cysteinyl residues on the basis of spectroscopic properties and sequence comparisons. A model is propo sed where each NifEN complex [4Fe-4S] cluster is bridged between a Nif E-NifN subunit interface at a position analogous to that occupied by t he P clusters in the nitrogenase MoFe protein. In contrast to the MoFe protein P clusters, the NifEN complex [4Fe-4S] clusters are proposed to be asymmetrically coordinated to the NifEN complex where NifE cyste ines-37, -62, and -124 and NifN cysteine-44 are the coordinating Ligan ds. On the basis of a homology model of the three-dimensional structur e of the NifEN complex, the [4Fe-4S] cluster sites are likely to be re mote from the proposed FeMo cofactor assembly site and are unlikely to become incorporated into the FeMo cofactor during its assembly.