Studies On The Purification, Characterization And Crystallization Of FeMoco-deficient Nitrogenase MoFe Proteins From Mutants (UW₄₅,DJ₅₄ And DJ₃₅) Of Azotobacter Vinelandii

1. Purification and crystallization of nitrogenase MoFe protein ofAzotobacter vinelandi i mutants UW45 DJ54 and DJ35Both the partly purified nifB MoFe protein and Fe protein were obtained by two DEAE 52 columns from the crude extract of a nifB mutated strain UW45 of Azotobacter vinelandii Lipmann. The partially purified nifB' MoFe protein was further purified by chromatogaphy on Sephacryl S-300 column and DEAE 52 column,and the obtained nifB' MoFe protein was almost homogeneous as determined by commassie staining of SDS-PAGE. The analysis of SDS-PAGE showed that nifB' MoFe protein was similar to OP MoFe protein in the kinds of subunits (a and P subunit). The partially purified nifB Fe protein was similar in a substrate-reducing activity to OP Fe protein purified from wild-type strain Azotobacter vinelandii when complemented with OP MoFe protein. When complemented with Fe protein,the nifB MoFe protein had hardly any FT-reducing activity,but could be significantly reactivated by FeMoco extracted from OP MoFe protein to a high proton-reducing activity,which was very close to the highest value of C2H2-reducing activity reported previously in other laboratorys. Under suitable conditions,dark brown short rhombohedron crystals could be obtained from nifB MoFe protein. Both of the longest sides of the biggest crystal were O.lmm. The possibility and time of the formation of crystals,and number,size,quality,and shape of crystals obviouslydepended not only on the kinds and concentrations of the components in the crystalline solution,but also on the methods for crystallization and technical bias,etc. The preliminary results showed that the crystals could be formed from the nifB' MoFe protein.By using the similar separation and purification methods,A nifH and A nifE MoFe protein were obtained from Azotobacter vinelandii mutants DJ54 and DJ35. The results of the complement activity experiments showed that A nifH MoFe could be activated by FeMoco. Like the crystal of nifB' MoFe protein,the crystals of both A nifH protein and A nifE MoFe protein were obtained at the first in the world when the first experiment on the crystallization was done. The preliminary studies showed that they were A nifH or A nifE MoFe protein crystals. 2. Proceedings of the reseaches on the novel nitrogenase MnFe proteinand CrFe proteinSeveral new batches of MnFe protein and CrFe protein were prepared. Their substrate reduction activities have been determined by the complementation with UW45 Fe protein. Several relatively good MnFe and CrFe protein crystals have been obtained under suitable conditions,while the studies on optimization of conditions of crystal growth is continued in order to obtain better ones.For the last space experiments,the rate of crystallization of MnFe protein was one hundred percent,and the crystals obtained were thicker than the ones obtained on the earth. We are continuing to do the match experiments on the earth in order to obtain the suitable conditions for the crystallization on the next spacecraft.