Characterization of a nitrogenase CrFe protein from a mutant UW3 of Azotobacter vinelandii grown on a Cr-containing medium

Abstract

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, which grew well on the Cr-containing medium. Compared with MoFe protein (OP Av1) from wild-type strain OP, ∼50% protein in the preparation had 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 in 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. The circular dichroism (CD) spectrum in the preparation at ∼450 nm was similar to that of OP Av1, while the relative intensities of three EPR signals appearing at the same g values (g≈4.3, 3.7 and 2.0) were different. The EPR-based calculated results showed that (1) the ratios of Mo to Cr and of Fe to (Cr+Mo) of the CrFe protein preparation were 0.41 and 15, respectively, indicating that in the preparation, a ratio of Cr-containing protein to MoFe protein was about 2.5; (2) the ratios of activity to Fe and to Cr of the preparation were close to the ratios of activity to Fe and to Mo of OP Av1, respectively; (3) the ratios of the EPR signal intensities at the above three g values to Cr of CrFe protein were about ∼83%, 0% and ∼40% of those to Mo of OP Av1, respectively. 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 instead of simple replacement of Mo by Mn in the cofactor.