Study On Nitrogenase Protein In Azotobacter Vinelandii Mutant DJ194 And UW3

A DEAE column absorbing the crude extract of DJ194 was developed by stepwise elution with 0.26 mol/L NaCl. The protein in the 0.26 mol/L fraction were well separated by a chromatography on the Q-Sepharose column developed with NaCl gradient (0.15—0.40 mol/L) and further purified by a chromatography on a Sephacryl S-200 column. The purified protein were designated tentatively asΔnifZAv2_Q2. The relative mobility of the main band ofΔnifZAv2_Q2 on SDS gel was somewhat higher than that of one of bands with the molecular weight of 32 kDa for a partially purified OP Av2. The protein was identified to be one of Fe proteins and had similar the Fe content and the ability to complement with OP Av1. But the protein had a higher relative mobility on native PAGE gel and much higher C₂H₂-reduction activity (6150 nmol C₂H₄·min^-1·mg protein^-1) than those of OP Av2. The C₂H₂-, H⁺ and N₂ (reflected by the difference of H₂ evolution)-reduction activities of OP Av1 complemented withΔnifZAv2_Q2 were∽2.2 times of those when completed with OP Av2. Thus, it is indicated that the protein is one of Av2 with somewhat different characteristics of the structure and function. Perhaps, it is the special characteristics ofΔnifZAv2_Q2 that make it not only decrease the NaCl concentration for its elution from the DEAE column but also have the supernormal activity, purity and mobility on native PAGE gel.Through the anoxic chromatography on the columns of DEAE-52, Q-Sepharose and Sephacryl S-200, a CrFe protein preparation was obtained from UW3, a mutant of Azotobacter vinelandii. Compared with MoFe protein (OP Av1) from wild-type strain OP,～50% protein in the preparation had the similar subunits composition, and had immune reaction with antibody of OP Av1. The preparation had～40% of C₂H₂-, H⁺-and N₂ (expressed by the difference of H⁺- reduction activity between under Ar and under N₂) -reduction activity of OP Av1 and similar electron pairs to those of OP Av1. And metal analysis showed that the preparation contained Fe, Cr and Mo. Compared with OP Av1, the circular dichroism (CD) spectrum of the preparation at～450 nm was similar, while the relative intensities of three EPR signals appeared at the same g values (g≈4.3, 3.7 and 2.0) were different. The results indicate further that CrFe protein might be a new nitrogenase component I protein with a similar function and structure including metallocluster to those of MoFe protein except that Cr replaces Mo in the cofactor.