Received 27 Apr. 2004 Accepted 4 Aug. 2004 Supported by the State Key Basic Research and Developmental Plan of China (001CB1089-06) and the National Natural Science Foundation of China (30270296). * Author for correspondence. E-mail: <jfhuang@ibcas.ac.cn>. http://www.chineseplantscience.com Acta Botanica Sinica 2004, 46 (11): 1331-1337 Crystal Growth and Characterization of Residual Bacterioferritin in Partially Purified Nitrogenase CrFe Protein Solution from a Mutant UW3 of Azotobacter vinelandii ZHAO Jian-Feng 1 , LIU He-Li 2 , ZHOU Hui-Na 1 , WANG Zhi-Ping 3 , ZHAO Ying 1 , BIAN Shao-Min 1 , LI Shu-Xing 2 , BI Ru-Chang 2 , HUANG Ju-Fu 1* (1. Key Laboratory of Photosynthesis and Environmental Molecular Physiology, Institute of Botany, The Chinese Academy of Sciences, Beijing 100093, China; 2. Institute of Biophysics, The Chinese Academy of Sciences, Beijing 100101, China; 3. Institute of Nuclear Agriculture, Zhejiang University, Hangzhou 310029, China) Abstract: 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-containing 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 Å resolution showed that the crystal belonged to space group H3, with unit-cell parameters a = 124.965 Å, b=124.965 Å and c = 287.406 Å. The detailed structural analysis published in the near future has confirmed that the brick red crystal is that of 24-meric bacterioferritin. Key words: crystal growth and characterization; bacterioferritin; partially purified CrFe protein solution; mutant strain UW3 of Azotobacter vinelandii The biological reduction of molecular nitrogen (N 2 ) is one of the most fundamental processes in nature. This pro- cess is catalyzed by nitrogenase which consists of two protein components (components and ). During the separation of nitrogenase from Azotobacter vinelandii, a nitrogen-fixing bacterium, some important proteins includ- ing bacterioferritin had been purified and characterized. Like the bacterioferritins isolated from Escherichia coli and Pseudomonas aeruginosa, etc., bacterioferritin from A. vinelandii (AvBF) was shown to be a ferritin containing haem in addition to the non-heme iron core (Andrews et al., 1991; Wai et al., 1995). It was said that AvBF is a haem- containing multisubunit protein that performs the same functions of iron storage and iron detoxification as animal ferritins being studied more extensively (Stiefel and Watt, 1979). And now it is proposed that the ferritins’dual func- tions of storing iron and detoxification of iron or protec- tion against oxygen are likely to be bacterioferritins’pri- mary function (Carrondo, 2003). As a bacterioferritin, AvBF’s significant difference from other ferritins, such as the presence of 12-heme groups and structurally disor- dered phospho-hydroxy mineral core, made great sense to elucidate how it functions. It is obvious that X-ray diffraction of AvBF crystal would help us to understand its structure-function relationship. However, its crystal structure has not been reported for a long time, though its preliminary X-ray crystallographic studies were described to show that the AvBF crystals belong to the cubic system, space group I432, with cell dimension 230 Å (Zhao et al., 1984).