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

Abstract

nifB− MoFe protein (nifB− Av1), ΔnifE MoFe protein (ΔnifE Av1) and ΔnifZ MoFe protein (ΔnifZ 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), ΔnifZ Av1 had partial activity and both nifB− Av1 and ΔnifE Av1 had hardly any activity, but could be obviously activated by FeMoco extracted from wild-type MoFe protein (OP Av1) or ΔnifZ Av1. 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©, 4n/fE Av1© and ΔnifZ Av1© were obtained, respectively. Based on a calculation of Fe atoms in the O-phen-Fe compound with ɛ512nm= 11 100, lost Fe atoms of nifB− Av1, ΔnifEAv1 and ΔnifZAv1 were estimated to be 1.35, 2.89 and 8.44 per molecule of protein, respectively. As a result of the Fe loss, ΔnifZ Av1 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, Na2 and Na2MoO4, or K2CrO4, or KMnO4. But in the absence of MgATP or Av2, the activation did not occur, with the exception that ΔnifZ Av1© was partially activated, and the activity was only 17%. These findings indicate that: (i) ΔnifZ Av1 with half P-cluster content is somewhat different from FeMoco-deficient nifB− Av1 and ΔnifE 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. (Managing editor: Wei Wang)