Biochemical and structural characterization of the cross-linked complex of nitrogenase: comparison to the ADP-AlF4(-)-stabilized structure.
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.
Schmid
Benedikt
B
Division of Chemistry and Chemical Engineering 114-96, California Institute of Technology, Pasadena, CA 91125, USA.
Einsle
Oliver
O
Chiu
Hsiu-Ju
HJ
Willing
Andreas
A
Yoshida
Mika
M
Howard
James B
JB
Rees
Douglas C
DC
D000244:Adenosine Diphosphate / Q000737:chemistry; D017607:Aluminum Compounds / Q000737:chemistry; D016948:Azotobacter vinelandii / Q000201:enzymology*; D003432:Cross-Linking Reagents / Q000737:chemistry*; D018360:Crystallography, X-Ray; D004795:Enzyme Stability; D005022:Ethyldimethylaminopropyl Carbodiimide / Q000737:chemistry; D005459:Fluorides / Q000737:chemistry; D005998:Glycine / Q000031:analogs & derivatives* / Q000737:chemistry; D008983:Molybdoferredoxin / Q000737:chemistry; D009097:Multienzyme Complexes / Q000737:chemistry*; D009591:Nitrogenase / Q000737:chemistry*; D019159:Nonheme Iron Proteins / Q000737:chemistry; D011485:Protein Binding; D055672:Static Electricity
D017607:Aluminum Compounds; D003432:Cross-Linking Reagents; D008983:Molybdoferredoxin; D009097:Multienzyme Complexes; D019159:Nonheme Iron Proteins; C050992:tetrafluoroaluminate; C018556:glycine amide; D000244:Adenosine Diphosphate; D009591:Nitrogenase; D005459:Fluorides; D005022:Ethyldimethylaminopropyl Carbodiimide; D005998:Glycine