1m34
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Nitrogenase Complex From Azotobacter Vinelandii Stabilized By ADP-Tetrafluoroaluminate
OverviewOverview
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.
About this StructureAbout this Structure
1M34 is a Protein complex structure of sequences from Azotobacter vinelandii with CA, MG, ALF, HCA, CFM, CLF, SF4 and ADP as ligands. Active as Nitrogenase, with EC number 1.18.6.1 Full crystallographic information is available from OCA.
ReferenceReference
Biochemical and structural characterization of the cross-linked complex of nitrogenase: comparison to the ADP-AlF4(-)-stabilized structure., Schmid B, Einsle O, Chiu HJ, Willing A, Yoshida M, Howard JB, Rees DC, Biochemistry. 2002 Dec 31;41(52):15557-65. PMID:12501184
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