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Eur J Biochem. 1975 Dec 15;60(2):467-76.

Nitrogenase from Azotobacter chroococcum. Purification and properties of the component proteins.

Abstract

1. A large-scale purification of the nitrogenase components from Azotobacter chroococcum yielded two non-haem iron proteins, both of which were necessary for nitrogenase activity and each had a specific activity of approximately 2000 +/- 300 nmol of acetylene reduced/mg protein per min in the presence of sautrating amounts of the other. This procedure freed the Mo-Fe protein from a protein contaminant which had an electron paramagnetic resonance signal at g = 1.94. 2. Both proteins were purified to homogeneity as determined by disc gel electrophoresis and ultracentrifugal analysis. Both proteins were oxygen-sensitive but not cold-labile. Ultracentrifugal analysis indicated that both proteins dissociated to a slight degree at concentrations below 2 mg/ml. 3. The larger of the two proteins had a molecular weight of 227 000 and contained 1.9 +/- 0.3 atoms of Mo, 23 +/- 2 atoms of Fe, 20 +/- 2 acid-labile sulphide and 47 tryptophan residues/mol. The protein consists of 4 subunits of mol. wt 60 000 (approx.). The reduced protein showed electron paramagmetic resonance signals at g = 4.29, 3.65 and 2.013 but not in the area of g = 5 to 6. Upon oxidation abosrbance increased throughout the visible region of the ultraviolet visible spectrum, with a maximum difference between oxidised and reduced protein occurring at 430 nm. 4. The smaller protein had a molecular weight of 64 000 and contained 4 g-atoms of Fe and 4 acid-labile sulphide groups/mol but no tryptophan. It had two subunits of mol. wt 30 800. The reduced protein showed electron paramagnetic resonance signhe protein retained almost full activity after oxidation with phenazine methosulphate. The ultraviolet visible spectrum of oxidised protein was clearly different from that of the oxygen-inactivated protein: it had a sharp peak at 269 nm and a broad absorbance between 340 and 470 nm with a maximum difference between oxidised and reduced forms at 430 nm. Oxygen-inactivated protein showed a sharp peak at 277.5 nm and broad peaks from 305 to 360, 400 to 425 and 435 to 475 nm. 5. Amino acid analyses of both proteins showed that most common amino acids were present with a preponderance of acidic residues. Analyses of compositional relatedness showed that the nitrogenase proteins from A. chroococcum were most closely related to those from A. vinelandii and least so to those from Clostridium pasteurianum.

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