CHARACTERIZATION OF A VARIANT IRON PROTEIN OF NITROGENASE THAT IS IMPAIRED IS ITS ABILITY TO ADOPT THE MGATP-INDUCED CONFORMATIONAL CHANGE

An Azotobacter vinelandii nitrogenase iron protein mutant has been cre ated which contains an alanine to glycine substitution at amino acid 1 57, The strain expressing this mutant Fe protein is able to grow under nitrogen-fixing conditions. This contrasts with an A. vinelandii stra in described previously which is unable to grow under nitrogen-fixing conditions and which expresses an Fe protein variant that has an alani ne 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 t he A157G strain grows at 55% of the rate of the wild-type strain, puri fied A157G; Fe protein is only able to support substrate reduction in in vitro assays at a rate that is approximately 20% of the rate suppor ted by the wildtype Fe protein. Electron paramagnetic resonance, circu lar dichroism spectroscopies, and enzymatic activity data indicate tha t the A157G Fe protein adopts the correct conformation upon the bindin g of MgATP, However, kinetic studies using chelation show that this pr otein undergoes the conformational change more slowly than the wild-ty pe protein. Thus, this mutant has lower activity because of an impaire d ability to undergo this conformational change. Comparison of two ava ilable xray crystal structures of the native Fe protein alone and comp lexed with the MoFe protein has provided us with a model to explain th e change in activity in alanine 157 mutants. Steric interactions with the side chain of residue 157 influence the protein's ability to under go 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 MgAT P binding.