The Deltanif E MoFe protein (Deltanif 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 Delta nif EAv1 was similar to OP MoFe protein (Av1) of A. vinelandii in the kinds and molecular weights of subunits (alpha and beta subunit). When complemented with nitrogenase Fe protein (M), the A nif EAv1 had hardly any proton-reduction activity, but could be significantly activated by FeMoco extracted from OP Av1. After the Delta nif E Av1 was treated with an excess o-phenanthroline (o-phen) and chromatographied on Sephadex G-25 column under atmosphere of Ar, Delta nif E Av1(C) was obtained. In the presence of both Av2 and MgATP regeneration system, the Delta nif EAv1(C), rather than A nif EAv1, was significantly activated in vitro by a reconstituent solution containing Mn which composed of KMnO4, ferric homocitrate, Na2S, Na2S2O4 (DT) and dithiothreitol (DTT). But in the absence of MgATP or Av2, the activation of Delta nifE Av1(C) did not happen. It indicates that activation of Delta nif EAv1 by RS-Mn requires the pretreatment with o-phen and the simultaneous presence of Av2 and MgATP.
Six hundred and 28 mg of NifB(-) Av1 was obtained by a chromatography twice on DE 52 columns and Sephacryl S-300 column from the crude extract (37 677 mg) of a nifB mutated strain UW45 of Azotobacter vinelandii Lipmann. The protein was almost homogeneous as determined by Coomassie staining of SDS gels. The analysis by SDS-PAGE showed that NifB(-)Av1 was similar to Av1 from wild-type strain of A. vinelandii (OP) in the kinds of subunits (alpha and beta subunit). When complemented with Av2, NifB(-)Av1 had hardly any H-reducing activity, but could be significantly activated by FeMoco extracted from Av1. Under a suitable condition for crystallization, short dark-brown rhombohedral crystals could be obtained from NifB(-)Av1. Both of the longest sides of the biggest crystal were 0.1 mm. The time of the formation of crystals and number, size, quality and shape of crystals obviously depended not only on the kinds and concentrations of the components in the precipitant solution, but also on the methods for crystallization and technical bias, etc. The preliminary results showed that the crystal seemed to be formed from NifB(-)Av1.
Under a given condition of crystallization, dark brown short rhombohedron crystals could be obtained from Δ nifZ MoFe protein purified from a nifZ deleted mutant strain of Azotobacter vinelandii Lipmann. Systematic studies on the effect of concentrations of PEG 8000,MgCl 2, NaCl,Tris and buffer pH on the crystallization and crystal growth of the protein showed that the protein could not be crystallized in lower concentrations of the chemicals and lower buffer pH. A large amount of smaller crystals of the protein appeared in a week with gradual increasing in the chemical concentrations and pH≥8.0. When the chemical concentrations were further increased, the time for crystallization was increased and a few high grade crystals of larger size were formed. If the concentrations of the chemicals were continuously increased, many crystals with smaller size, and, sometimes of poor quality appeared again and eventually ceased to produce any crystals. The optimal concentration for each of the above mentioned chemicals varies with other variable factors. Only one bigger crystal (both of the longest two sides: 0.16 mm) could be obtained in a hanging drop of protein sample when the concentrations of PEG 8000, MgCl 2, NaCl,Tris and protein were kept at 1.86%, 300 mmol/L, 400 mmol/L, 53 mmol/L and 4.64 g/L , respectively, with Tris buffer pH 8.2.
