Biochemical and Structural Characterization of the Cross-Linked Complex of Nitrogenase:  Comparison to the ADP-AlF4 --Stabilized Structure

• Published in: Biochemistry (Easton) 2002-12, Vol.41 (52), p.15557-15565
• Main Authors: Schmid, Benedikt, Einsle, Oliver, Chiu, Hsiu-Ju, Willing, Andreas, Yoshida, Mika, Howard, James B, Rees, Douglas C
• Format: Article
• Language: English
• Subjects: Adenosine Diphosphate - chemistry; Aluminum Compounds - chemistry; Azotobacter vinelandii - enzymology; Cross-Linking Reagents - chemistry; Crystallography, X-Ray; Enzyme Stability; Ethyldimethylaminopropyl Carbodiimide - chemistry; Fluorides - chemistry; Glycine - analogs & derivatives; Glycine - chemistry; Molybdoferredoxin - chemistry; Multienzyme Complexes - chemistry; Nitrogenase - chemistry; Nonheme Iron Proteins - chemistry; Protein Binding; Static Electricity
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi026642b

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 β400 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 Å 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-AlF4 -. 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-AlF4 - structure.