Characterization of a variant iron protein of nitrogenase that is impaired in its ability to adopt the MgATP-induced conformational change.
An Azotobacter vinelandii nitrogenase iron protein mutant has been created which contains an alanine to glycine substitution at amino acid 157. The strain expressing this mutant Fe protein is able to grow under nitrogen-fixing conditions. This contrasts with an A. vinelandii strain described previously which is unable to grow under nitrogen-fixing conditions and which expresses an Fe protein variant that has an alanine to serine mutation at position 157. The A157S Fe protein was unable to support substrate reduction by nitrogenase because of an inability to undergo a required MgATP-induced conformational change. Although the A157G strain grows at 55% of the rate of the wild-type strain, purified A157G Fe protein is only able to support substrate reduction in in vitro assays at a rate that is approximately 20% of the rate supported by the wild-type Fe protein. Electron paramagnetic resonance, circular dichroism spectroscopies, and enzymatic activity data indicate that the A157G Fe protein adopts the correct conformation upon the binding of MgATP. However, kinetic studies using chelation show that this protein undergoes the conformational change more slowly than the wild-type protein. Thus, this mutant has lower activity because of an impaired ability to undergo this conformational change. Comparison of two available x-ray crystal structures of the native Fe protein alone and complexed with the MoFe protein has provided us with a model to explain the change in activity in alanine 157 mutants. Steric interactions with the side chain of residue 157 influence the protein's ability to undergo the initial MgATP-induced conformational change. In the case of the A157G mutant, however, once the correct conformation is attained, the protein can participate in all subsequent reactions including complex formation, electron transfer, and MgATP hydrolysis. Thus, the role of alanine 157 is to stabilize the proper initial conformation upon MgATP binding.
Bursey
E H
EH
Department of Molecular Biology and Biochemistry, University of California, Irvine, California 92697-3900, USA.
Burgess
B K
BK
D000255:Adenosine Triphosphate / Q000737:chemistry*; D016948:Azotobacter vinelandii / Q000201:enzymology* / Q000235:genetics; D002942:Circular Dichroism; D004578:Electron Spin Resonance Spectroscopy; D004579:Electron Transport; D006868:Hydrolysis; D009591:Nitrogenase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D010088:Oxidoreductases; D011485:Protein Binding; D011487:Protein Conformation
D000255:Adenosine Triphosphate; D010088:Oxidoreductases; D009591:Nitrogenase; C029130:nitrogenase reductase