Studies On The Nitrogenase Component Ⅰ From DJ194, DJ35 And UW3 Mutants Of Azotobacter Vinelandii

The paper based chiefly on the study about disassembly and assembly in vitro of dinitrogenases which were deleted in metalloclusters .The feasibility of disassembly and assembly in vitro of dinitrogenase mutant was demonstrated once more.Moreover, the possibility of existance of novel nitrogenases,such as CrFe protein and MnFe protien ,with high purity, containing no FeMoco, was comformed by disassembly and assembly in vitro.The results provided new evidence for the existance of novel nitrogenase.Otherwise,CrFe protein and MnFe protein were purified and the characteristics of them have been studied preliminary,at the same time,the growth of Δ nifZ Av1 crystals have been studying.Thereby,the study not only had breadth and depth,but also had innovation.Through the anerobic chromatography on the anion-exchange column DEAE-52 and Q-Sepharose,and on the gel filtration column Sephacryl S-200 or S-300,four different Azotobacter vinelandii Lipmann mutants nitrogenase were obtained. Δ nifZ MoFe protein (ΔnifZ Av1) preparation,purity over 90%, was obtained from the nifZ-deleted strain DJ194.Purer ΔnifE Av1 preparation,a FeMoco-dificient MoFe protein, was purified from nifE gene deleted mutant designated DJ35.Futher, two new nitrogenase components I, designated CrFe protein and MnFe protein,were puriied from UW3/Cr and UW3/Mn,which were cultured in Mo- and NH₄⁺-free modified Burk's media containing Na₂CrO₄ and MnSO₄.The optimun conditions of crystal growth for high purified ΔnifZ Av1 preparation were carried out again.In the lab I studied, The conclusion,ΔnifZ Av1 preparation obtained from DJ194 was a MoFe protein,containing two intact FeMoco metalloclusters,but just one intact P-cluster metallocluster,with half activity,was demonstrated first time.After being incubated with excess O-phenanthroline in an anoxic condition,The Fe atoms coming from the P-cluster metallocluster of Δ nifZ MoFe protein were chelated by O-phenanthroline.By calculation, Fe atoms of ΔnifZ Av1 lost were estimated to be about 8 per molecule of ΔnifZ Av1 preparation,namely all 8 Fe atoms to form one intact P-cluster. After chromatography on a Sephadex G-25 column in an Ar atmosphere ,Fe...