Biological nitrogen fixation is catalyzed by the complex metal containing enzyme termed nitrogenase. This enzyme is composed of two separable metal-containing enzyme components that interact with each other to catalyze MgATP hydrolysis, intermolecular electron transfer and the reduction of dinitrogen gas to ammonia. The crystal structure of a nitrogenase Fe protein single site deletion variant reveals a distinctly new conformation of the Fe protein and indicates that upon binding of MgATP, the Fe protein undergoes a dramatic conformational change that is largely manifested in the rigid body reorientation of the homodomeric Fe protein subunits with respect to one another. The observed conformational state allows the rationalization of a model of structurally and chemically complementary interactions that occur upon initial complex formation with the MoFe protein component that are distinct from the protein-protein interactions that have been characterized previously for stabilized nitrogenase complexes. The crystallographic results, in combination with complementary UV-visible absorption, EPR and resonance Raman spectroscopic data, indicate that the [4Fe-4S] cluster of both the Fe protein deletion variant and the native Fe protein in the presence of MgATP can reversibly cycle between a regular cubane-type [4Fe-4S] cluster in the reduced state and cleaved form involving two [2Fe-2S] fragments in the oxidized state. Resonance Raman studies indicate that this novel cluster conversion is induced by glycerol and the crystallographic data suggest that glycerol is bound as a bridging bidentate ligand to both [2Fe-2S] clusters fragments in the oxidized state.