The Role of Methionine 156 in Cross-subunit Nucleotide Interactions in the Iron Protein of Nitrogenase

• Published in: The Journal of biological chemistry 1998-11, Vol.273 (45), p.29678-29685
• Main Authors: Bursey, Evan H., Burgess, Barbara K.
• Format: Article
• Language: English
• Subjects: ACTIVIDAD ENZIMATICA; ACTIVITE ENZYMATIQUE; Adenosine Triphosphate - metabolism; ADP; ANALYTICAL METHODS; ATP; AZOTOBACTER VINELANDII; Azotobacter vinelandii - enzymology; BINDING; BINDING SITES; CIRCULAR DICHROISM; COENZIMAS; COENZYME; COENZYMES; Electron Spin Resonance Spectroscopy; ENZYMIC ACTIVITY; ESPECTROMETRIA; FER; HIDROLISIS; HIERRO; HYDROLYSE; HYDROLYSIS; IRON; METALLOPROTEINE; METALLOPROTEINS; METALPROTEINAS; METHIONINE; Methionine - metabolism; METIONINA; MG-ADP; MG-ATP; Models, Molecular; MOLECULAR CONFORMATION; MUTANT; MUTANTES; MUTANTS; NITROGENASA; NITROGENASE; Nitrogenase - chemistry; Nitrogenase - metabolism; Oxidoreductases; Protein Binding; Protein Conformation; SPECTRAL DATA; SPECTROMETRIE; SPECTROMETRY; SPECTROSCOPY; Substrate Specificity; TECHNIQUE ANALYTIQUE; TECNICAS ANALITICAS; X RAY DIFFRACTION
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.273.45.29678

A variant Fe protein has been created at the completely conserved residue methionine 156 by changing it to cysteine. The Azotobacter vinelandii strain expressing M156C is unable to grow under nitrogen-fixing conditions, and the purified protein cannot support substrate reduction in vitro. This mutation has an effect on the Fe protein's ability to undergo the MgATP-induced conformational change as evidenced by the fact that M156C is chelated in the presence of MgATP with a lower observed rate than wild-type. While the electron paramagnetic resonance spectra of this protein are similar to those of the wild-type Fe protein, the circular dichroism spectrum is markedly different in the presence of MgATP, showing that the conformation adopted by M156C following nucleotide binding is different from the wild-type conformation. Although competition activity and chelation assays show that this Fe protein can still form a complex with the MoFe protein, this altered conformation only supports MgATP hydrolysis at 1% the rate of wild-type Fe protein. A model based on x-ray crystallographic information is presented to explain the importance of Met-156 in stabilization of the correct conformation of the Fe protein via critical interactions of the residue with Asp-43 and nucleotide in the other subunit.