Cross-linking site in Azotobacter vinelandii complex.
The Fe-protein and the MoFe-protein of the Azotobacter vinelandii nitrogenase complex can be chemically cross-linked by 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (Willing, A., Georgiadis, M.M., Rees, D. C., and Howard, J. B. (1989) J. Biol. Chem. 264, 8499-8503). In this reaction, one of the identical subunits of the Fe-protein dimer is linked by an isopeptide bond to each beta-subunit of the MoFe-protein tetramer. The reaction has been found to be highly specific with greater than 85% of amino acid residues Glu-112 (Fe-protein) and Lys-399 (MoFe-protein) cross-linked to each other. Although Glu-112 is located in a highly conserved amino acid sequence, it is found in only half of the known Fe-protein sequences. Likewise, Lys-399 is not a conserved residue in the MoFe-protein. Glu-112 appears to be part of an anionic cluster of nine carboxylic acids which is located between the proposed thiol ligands for the Fe:S center. In contrast, the basic residue cluster which includes Lys-399 has been found in only in the Azotobacter MoFe-protein. Thus, this crosslinking reaction either is unique to Azotobacter nitrogenase or must involve other residues in the MoFe-protein of other species. Because Lys-399 and Glu-112 form a specific cross-link, it is probable that they are part of the interaction site leading to productive complex formation. This information should be useful for the model building of the complex from the crystallographic structures of the individual components.
Willing
A
A
Department of Biochemistry, University of Minnesota, Minneapolis 55455.
Howard
J B
JB
D000595:Amino Acid Sequence; D001395:Azotobacter / Q000201:enzymology*; D001665:Binding Sites; D003432:Cross-Linking Reagents; D005288:Ferredoxins / Q000302:isolation & purification*; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D008983:Molybdoferredoxin / Q000302:isolation & purification* / Q000378:metabolism; D009591:Nitrogenase / Q000302:isolation & purification* / Q000378:metabolism; D010088:Oxidoreductases; D010449:Peptide Mapping
D003432:Cross-Linking Reagents; D005288:Ferredoxins; D046911:Macromolecular Substances; D008983:Molybdoferredoxin; D010088:Oxidoreductases; D009591:Nitrogenase; C029130:nitrogenase reductase