A Conformational Mimic of the MgATP-Bound “On State” of the Nitrogenase Iron Protein^,

Abstract

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 homodimeric 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 a 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] cluster fragments in the oxidized state.

Insights into the role of nucleotide-dependent conformational change in nitrogenase catalysis: Structural characterization of the nitrogenase Fe protein Leu127 deletion variant with bound MgATP

“…Although Av2-L127D does not support nitrogen reduction and catalyzes very low rates of MgATP hydrolysis, it is capable of forming a stable complex with the MoFe protein in the absence of added nucleotides that closely resembles the Fe protein:MoFe protein complex stabilized in the presence of MgADP and tetrafluoroaluminate (Av1:Av2 -MgADP AE AlF 4 ) [19,20,26,27]. The structural characterization of the nucleotide-free Av2-L127D revealed a conformation that was strikingly different from that of the previously reported structures of Av2 [23]. In the current work, we have extended these studies to the characterization of the MgATP-bound state of the Av2-L127D.…”

“…Recently, we have been able to structurally characterize a site-directed variant of the Fe protein with unique properties [23]. This site-directed variant has a single amino acid deletion in a nucleotide-dependent ''Switch'' region directly linking the nucleotide binding site to the [4Fe-4S] cluster.…”

A Proposed Role for the Azotobacter vinelandii NfuA Protein as an Intermediate Iron-Sulfur Cluster Carrier

“…Analytical data, coupled with the UV-visible absorption spectrum and extinction coefficients (Fig. 2) 2ϩ cluster in the MgATP-bound nitrogenase iron protein (34). This feature is illustrated in Fig.…”

Probing the MgATP-Bound Conformation of the Nitrogenase Fe Protein by Solution Small-Angle X-ray Scattering

“…Previous SAXS experiments probing the nucleotide-bound states of the Fe protein reported R g values for the nucleotide-bound conformations very similar to those observed for the nucleotide-free native Fe protein (47) (48). In the current study, the effect of nucleotide binding to both the native and L127Δ Fe protein are examined in parallel to directly assess whether the L127Δ Fe protein is a mimic of the MgATP state as suggested previously (33).…”

“…Radii of gyration (R g ) values were obtained from the Guinier plots, using the first 20-22 intensity points beyond the beam stop in the Q range ~ 0.02 -0.06 Å -1 . Theoretical curves were generated with CRYSOL (42) from the crystal structure of the nucleotide-free native Fe protein (2NIP) (30), and the crystal structure of the nucleotide-free L127Δ Fe protein (1RW4) (33). The electron pair distance distribution function, P(r), was calculated using GNOM (43).…”