Purification and Characterization of the Molybdenum-Iron Protein Component of Nitrogenase from Soybean Nodule Bacteroids

The Mo-Fe protein from bacteroids of soybean nodules has been purified to a high degree of homogeneity as judged by disc gel electrophoresis and ultracentrifugal analysis. The purification procedure included: polypropylene glycol fractionation, heat treatment, and chromatography on DEAE-cellulose and Sephadex G-200. Specific activities of several preparations of the purified protein ranged between 850 and 1,000 for C2H2 and 260 and 300 for N2. When the Mo-Fe protein was assayed with an optimum level of iron protein, an apparent Km for N2 of 0.068 atm was obtained. The time for 50% inactivation of the protein under 0.2 atm of O2 at 30° is 4.5 min. A molecular weight of 197,600 for the protein was determined by low speed sedimentation equilibrium experiments and a molecular weight of 202,000 was determined by the high speed sedimentation equilibrium method. Sedimentation equilibrium analysis of the protein in 6 m guanidine hydrochloride indicated one size of subunit with a molecular weight of about 50,000. Treatment of the protein with 2-mercaptoethanol and sodium dodecyl sulfate followed by electrophoresis in gels containing sodium dodecyl sulfate also produced one size of subunit. A v of 0.732 ml per g was calculated from the amino acid composition and ultracentrifugal analysis revealed a s020, w value of 9.99. The ultraviolet spectrum of the protein exhibited an absorbance maximum at 279 nm and a molar extinction coefficient at this wave length of 3.69 x 105 cm-1 m-1. A broad general absorbance was observed in the visible spectrum between 350 and 600 nm and no obvious shoulders or peaks were apparent. Analyses of samples of the protein revealed a mean of 1.3 molybdenum, 28.8 iron, and 26.2 acid-labile sulfide atoms per molecule. All the common amino acids were present.