Crystal Growth and Characterization of Residual Bacterioferritin in Partially Purified Nitrogenase CrFe Protein Solution from a Mutant UW3 of Azotobacter vinelandii

摘要

While attempting to obtain large crystals of nitrogenase CrFe protein, brown crystals and brick red crystals were simultaneously or independently obtained from CrFe protein preparation, which was partially purified from a mutant UW3 of Azotobacter vinelandii Lipmann 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 Av1 (MoFe protein), while the protein of the brick red crystal was composed of ～20 kD subunits. And only the larger subunits rather than the smaller ones were detectable by Western blot to the antibody of Av1. Comparing with the large subunits, the amount of the small subunits in the partially purified CrFe protein solution 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-contain ing proteins with different electro-phoretic 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 A. vinelandii (AvBF). X-ray diffraction to 2.34 A resolution showed that the crystal belonged to space group H3, with unit-cell parameters a = 124.965 A, b=124.965 A and c = 287.406 A. The detailed structural analysis published in the near future has confirmed that the brick red crystal is that of 24-meric bacterioferritin.