The role of methionine 156 in cross-subunit nucleotide interactions in the iron protein of nitrogenase.

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.