1M1Y

Chemical Crosslink of Nitrogenase MoFe Protein and Fe Protein

Summary for 1M1Y

• Entry DOI: 10.2210/pdb1m1y/pdb
• Related: 1n2c 3min
• Descriptor: Nitrogenase molybdenum-iron protein alpha chain, Nitrogenase molybdenum-iron protein beta chain, nitrogenase IRON protein 1, ... (8 entities in total)
• Functional Keywords: nitrogenase, nitrogen fixation, chemical crosslink, protein interaction, oxidoreductase
• Biological source: Azotobacter vinelandii; More
• Total number of polymer chains: 16
• Total formula weight: 718060.58

Authors

Schmid, B.,Einsle, O.,Chiu, H.J.,Willing, A.,Yoshida, M.,Howard, J.B.,Rees, D.C. (deposition date: 2002-06-20, release date: 2003-02-11, Last modification date: 2024-02-14)

Primary citation

Schmid, B.,Einsle, O.,Chiu, H.J.,Willing, A.,Yoshida, M.,Howard, J.B.,Rees, D.C.
Biochemical and Structural Characterization of the Crosslinked Complex of Nitrogenase: Comparison to the ADP-AlF4- Stabilized Structure
Biochemistry, 41:15557-15565, 2002

PubMed Abstract: The transient formation of a complex between the component Fe- and MoFe-proteins of nitrogenase represents a central event in the substrate reduction mechanism of this enzyme. Previously, we have isolated an N-[3-(dimethylamino)propyl]-N'-ethylcarbodiimide (EDC) cross-linked complex of these proteins stabilized by a covalent isopeptide linkage between Glu 112 and Lys beta400 of the Fe-protein and MoFe-protein, respectively [Willing, A., et al. (1989) J. Biol. Chem. 264, 8499-8503; Willing, A., and Howard, J. B. (1990) J. Biol. Chem. 265, 6596-6599]. We report here the biochemical and structural characterization of the cross-linked complex to assess the mechanistic relevance of this species. Glycinamide inhibits the cross-linking reaction, and is found to be specifically incorporated into Glu 112 of the Fe-protein, without detectable modification of either of the neighboring residues (Glu 110 and Glu 111). This modified protein is still competent for substrate reduction, demonstrating that formation of the cross-linked complex is responsible for the enzymatic inactivation, and not the EDC reaction or the modification of the Fe-protein. Crystallographic analysis of the EDC-cross-linked complex at 3.2 A resolution confirms the site of the isopeptide linkage. The nature of the protein surfaces around the cross-linking site suggests there is a strong electrostatic component to the formation of the complex, although the interface area between the component proteins is small. The binding footprints between proteins in the cross-linked complex are adjacent, but with little overlap, to those observed in the complex of the nitrogenase proteins stabilized by ADP-AlF(4)(-). The results of these studies suggest that EDC cross-linking traps a nucleotide-independent precomplex of the nitrogenase proteins driven by complementary electrostatic interactions that subsequently rearranges in a nucleotide-dependent fashion to the electron transfer competent state observed in the ADP-AlF(4)(-) structure.

PubMed: 12501184
DOI: 10.1021/bi026642b

Experimental method

X-RAY DIFFRACTION (3.2 Å)