Purification, Characterization, Crystal Growth And Identification Of Nitrogenase MoFe Protein From Mutant DJ35 And Bacterioferritin From UW3 Of Azotobacter Vinelandii

1. Purification, reconstitution in vitro and crystallization of nitrogenase MoFe protein from Azotobacter vinelandii mutant DJ35The △nifE MoFe protein (△nifE Avl) was obtained by a chromatography on DEAE Cellulose 52, Sephacryl S-300 and Q-Sepharose Fast Flow columns from the unheated crude extract of nifE-deleted mutant strain (DJ35) of Azotobacter vinelandii Lipmann. The analysis by SDS-PAGE and Western blotting shows that the △ nifE Av1 was similar to OP MoFe protein (Avl) of A. vinelandii in the kinds and numbers {α2β2). When complemented with nitrogenase Fe protein (Av2), the AnifE Avl had hardly any proton-reduction activity, but could be significantly activated by FeMoco extracted from OP Av1.After the AnifE Avl was anaerobically treated with excess o-phenanthroline (o-phen) and chromatographied on Sephadex G-25 column under argon, A nifE Avl(?) was obtained. In the presence of both Av2 and MgATP regeneration system, the partial Fe-deficient △nifE Avl(?), rather than AnifE Av1, was significantly activated in vitro by a reconstituent solution containing Mn/Cr composed of KMnO4/Na2CrO4, ferric homocitrate, Na2S, Na2S2O4 (DT) and dithiothreitol (DTT). But in the absence of MgATP or Av2, the activation of △nifE Av1(?) did not happen. It indicates that the activation of △nifE Avl by RS-Mn/RS-Cr requires the pretreatment with o-phen and the simultaneous presence of Av2 and MgATP. △nifZ Avl and NifB" Avl, which were purified from nifZ-deleted mutant strain (DJ194) and nifB point mutated strain (UW45) respectively, were anaerobically treated with excess o-phen and chromatographied on Sephadex G-25 column under argon, then partial Fe-deficient △nifZ Avl(?) and NifB- Avl(?) were obtained. Like AnifE Avl(?), △nifZ Avl(?) and NifB" Av1(?) were significantly activated by a reconstituent solution containing Cr/Mo in the presence of Av2 and MgATP.To obtain large single crystal of △nifE Avl suitable for X-ray diffraction, a body of studies were performed on the crystallization conditions such as kinds and concentrations of precipitants, pH value of buffer, diffusion method and protein batch.And under a suitable condition, dark brown short rhombohedron crystals of △nifE Av1 were obtained and identified for the first time.2. Crystal growth and identification of residual bacterioferritin in partially purified nitrogenase CrFe proteinWhile attempting to obtain large crystals of nitrogenase CrFe protein, brown crystals and brick red crystals were simultaneously or independently formed from CrFe protein preparation, which was partially purified from a mutant UW3 of A. vinelandii grown on Mo-, ammonia-free but Cr-containing medium. SDS-PAGE and anoxic native-PAGE analysis consistently showed that the protein of the brown crystal was mainly composed of subunits (~60 kD) similar to those of OP Avl, while the protein of the brick red crystal was composed of ~20 kD subunits. And only the larger subunits other than the smaller ones were detectable by Western blotting to the antibody of OP Avl. Comparing with the large subunits, the amount of the small subunits in the partially purified CrFe protein was much smaller, indicating that the protein composed of the smaller subunits was one of contamination proteins for CrFe protein. Detection by 3, 5-diaminobenzoic acid of native-PAGE gels showed that the proteins forming the brick red crystal and the brown crystal were two kinds of iron-containing proteins with different electrophoretic mobility on the gel. The analysis of matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) proved that the protein forming the brick red crystal was bacterioferritin of Azotobacter vinelandii (AvBF). X-ray diffraction to 2.34 A resolution showed that the crystal belonged to space group H3, with unit-cell parameters a=l24.965 A, b=124.965 A and c=287.406 A. The first detailed structural analysis has also confirmed that the brick red crystal is that of 24-meric bacterioferritin.