Purification, Characterization And Crystallization Of Novel Nitrogenase Containing Cr Or Mn

A Nif-Azotobacter vinelandii Lipmann mutant UW3 was cultured in Mo-and NH4+-free modified Burk's media containing MnSO4 or Na₂CrO₄. It was found that within a certain range, the existence of Mn or Cr could stimulate the growth of UW₃. Judged from the growth curves and C2H2-reduction activity curves, the stimulation effect might be acquired through Mn or Cr taking part in the constitution of FeMco instead of Mo, which could affect the nitrogen fixation activity of nitrogenase. Two nitrogenase components â… (designated MnFe protein and CrFe protein) were purified from UW3/Mn and UW3/Cr by anion-exchange and gel filtration column chromatography, and the characteristics of the two proteins were studied. It was shown by anaerobic native-and SDS-PAGE that the two proteins were both tetramers composed of two kinds of subunits, whose molecular weights were a bit less than those of α and β subunits in OP MoFe protein, respectively. The subunits also had immune reaction with OP MoFe protein antibody. The C₂H₂-reducion, H₂-evolving under Ar, and nitrogen-fixation activities of CrFe protein were respectively 36%, 38% and 43% of those of OP MoFe protein, and the general activity of MnFe protein was about 50% of that of OP MoFe protein. Besides, based on the H+-reduction activities under N2, the electron utilizing ratios of nitrogen fixation of MnFe protein and CrFe protein were calculated to be similar to that of OP MoFe protein. It was proved by the data of metal contents analysis that Mn and Cr respectively existed in the two proteins, but there was still some contaminating Mo. Comparing with that of OP MoFe protein, the visible circular dichroism molar ellipticity ([θ]) of the two protein significantly decreased except for [θ]450nm. CrFe protein and MnFe protein had EPR singals at g≈4.3,3.7 and 2.0, which is the same as DT-reduced OP MoFe protein. However, their relative signal intensities at each position were different. When the signal possibly induced by contaminating Mo was eliminated, the three signal intensities of CrFe protein were 20%, 0% and 10% of those of DT-reduced MoFe protein, and MnFe protein 112%, 49% and 65%, respectively. The results indicated that the main difference between the metalloclusters of CrFe protein, MnFe protein and MoFe protein was the M in FeMco (M=Cr, Mn or Mo) instead of P-cluster. Using vapor diffusion in hanging drop method, the crystallization conditions of MnFe protein and CrFe protein were screened and primarily optimized, and the precipitants in which Tris/Hepes, NaCl, MgCl2 and PEG 8000 were principal variables were chosen for further studies on the optimal concentrations of each components. Based on the results, the conditions of vapor diffusion in sitting drop method and liquid-liquid diffusion method were optimized. Using liquid-liquid diffusion method, large single crystals of two proteins were respectively obtained under certain condition. SDS-PAGE analysis of the crystals showed that they were composed of two kinds of subunits similar to those of OP MoFe protein, respectively. The effect of micro-gravity on the crystallization of anaerobic protein were studied by Space Shuttle "Shenzhou 3" hitching experiment. The results indicated that micro-gravity was helpful to avoid the formation of twin crystals, and possess the potential to obtain large single crystals after long-term growing. It is significant and feasible to develop the nitrogenase structure and function research with the assistance of space science.