MgATP-Bound and Nucleotide-Free Structures of a Nitrogenase Protein Complex between the Leu 127Δ-Fe-Protein and the MoFe-Protein

• Published in: Biochemistry (Easton) Vol. 40; no. 3; pp. 641 - 650
• Main Authors: Chiu, Hsiu-Ju, Peters, John W, Lanzilotta, William N, Ryle, Matthew J, Seefeldt, Lance C, Howard, James B, Rees, Douglas C
• Format: Journal Article
• Language: English
• Published: United States American Chemical Society 23.01.2001
• Subjects: Adenosine Triphosphate - chemistry; Adenosine Triphosphate - genetics; Adenosine Triphosphate - metabolism; Azotobacter vinelandii - enzymology; Azotobacter vinelandii - genetics; Binding Sites - genetics; Crystallography, X-Ray; Electron Transport - genetics; Hydrolysis; Leucine - genetics; Leucine - metabolism; Molybdoferredoxin - chemistry; Molybdoferredoxin - metabolism; Mutagenesis, Site-Directed; Nitrogenase - chemistry; Nitrogenase - genetics; Nitrogenase - metabolism; Protein Conformation; Protein Structure, Secondary - genetics; Sequence Deletion
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi001645e

A mutant form of the nitrogenase iron protein with a deletion of residue Leu 127, located in the switch II region of the nucleotide binding site, forms a tight, inactive complex with the nitrogenase molybdenum iron (MoFe) protein in the absence of nucleotide. The structure of this complex generated with proteins from Azotobacter vinelandii (designated the L127Δ-Av2−Av1 complex) has been crystallographically determined in the absence of nucleotide at 2.2 Å resolution and with bound MgATP (introduced by soaking) at 3.0 Å resolution. As observed in the structure of the complex between the wild-type A. vinelandii nitrogenase proteins stabilized with ADP·AlF4 -, the most significant conformational changes in the L127Δ complex occur in the Fe-protein component. While the interactions at the interface between the MoFe-protein and Fe-proteins are conserved in the two complexes, significant differences are evident at the subunit−subunit interface of the dimeric Fe-proteins, with the L127Δ-Av2 structure having a more open conformation than the wild-type Av2 in the complex stabilized by ADP·AlF4 -. Addition of MgATP to the L127Δ-Av2−Av1 complex results in a further increase in the separation between Fe-protein subunits so that the structure more closely resembles that of the wild-type, nucleotide-free, uncomplexed Fe-protein, rather than the Fe-protein conformation in the ADP·AlF4 - complex. The L127Δ mutation precludes key interactions between the Fe-protein and nucleotide, especially, but not exclusively, in the region corresponding to the switch II region of G-proteins, where the deletion constrains Gly 128 and Asp 129 from forming hydrogen bonds to the γ-phosphate and activating water for attack on this group, respectively. These alterations account for the inability of this mutant to support mechanistically productive ATP hydrolysis. The ability of the L127Δ-Av2−Av1 complex to bind MgATP demonstrates that dissociation of the nitrogenase complex is not required for nucleotide binding.