Purification and Activation In Vitro of MoFe Protein from a nifE Deleted Mutant Strain of Azotobacter vinelandii

The Δnif E MoFe protein (Δnif E Av1) was obtained by a chromatography on DE52, Sephacryl S-300 and Q-Sepharose columns from the unheated crude extract of nifE-deleted mutant strain (DJ35) of Azotobacter vinelandii Lipmann. The analysis by SDS-PAGE showed that the Δnif E Av1 was similar to OP MoFe protein (Av1) of A. vinelandii in the kinds and molecular weights of subunits (α and β subunit). When complemented with nitrogenase Fe protein (Av2), the Δnif E Av1 had hardly any proton-reduction activity, but could be significantly activated by FeMoco extracted from OP Av1. After theΔnif E Av1 was treated with an excess o-phenanthroline (o-phen) and chromatographied on Sephadex G-25 column under atmosphere of Ar, Δnif E Av1© was obtained. In the presence of both Av2 and MgATP regeneration system, the Δnif E Av1©, rather than Δnif E Av1, was significantly activated in vitro by a reconstituent solution containing Mn which composedof KMnO₄, ferric homocitrate, Na₂S, Na₂S₂O₄(DT) and dithiothreitol (DTT). But in the absence of MgATP or Av2, the activation of ΔnifE Av1© did not happen. It indicates that activation ofΔnif E Av1 by RS-Mn requires the pretreatment with o-phen andthe simultaneous presence of Av2 and MgATP.

Keywords
Δnif E Av1; purification; activation and assembly in vitro ; FeMoco and reconstituent solution containing Mn