Activation In Vitro of Mutant MoFe Proteins from Azotobacter vinelandii by Reconstituent Solutions

nifB-MoFe protein (nifB-Av1), △nifE MoFe protein (△nifE Av1) and △nifZ MoFe protein (△nifZ Av1) were obtained by chromatography on DE52, Sephacryl S-300 and Q-Sepharose columns from nifB point-mutated,nifE deleted and nifZ deleted mutant stains (UW45, DJ35 and DJ194) of Azotobacter vinelandii Lipmann,respectively. When complemented with nitrogenase Fe protein (Av2), △nifZAv1 had partial activity and both nifB- Av1 and △nifE Av1 had hardly any activity, but could be obviously activated by FeMoco extracted from wild-type MoFe protein (OP Av1) or △nifZAv1. After being incubated with excess O-phenanthroline (O-phen)for 150 min at 30 C and subjected to chromatography on a Sephadex G-25 column in an Ar atmosphere, nifB-Av1(C), △nifE Av1(C) and △nifZAv1(C) were obtained, respectively. Based on a calculation of Fe atoms in the Ophen-Fe compound with ε 512nm = 11 100, lost Fe atoms of nifB-Av1, AnifEAv1 and △nifZAv1 were estimated to be 1.35, 2.89 and 8.44 per molecule of protein, respectively. As a result of the Fe loss,△nifZAv1 loses its original activity. In the presence of both MgATP and Av2, these Fe-losing proteins, but not the original proteins untreated with O-phen, could be significantly activated by reconstituent solution (RS) composed of dithiothreitol, ferric homocitrate, Na2S and Na2MoO4, or K2CrO4, or KMnO4. But in the absence of MgATP or Av2, the activation did not occur, with the exception that △nifZAv1(C) was partially activated, and the activity was only 17%. These findings indicate that: (i) △nifZ Av1 with half P-cluster content is somewhat different from FeMoco-deficient nifB- Av1 and△nifE Av1 with respect to protein conformation either before or after treatment with O-phen; (ii) full activation of these proteins with RS requires pretreatment with O-phen and the simultaneous presence of MgATP and Av2.