SOME PROPERTIES OF PURIFIED NITROGENASE OF AZOTOBACTER CHROOCOCCUM

The nitrogenase of Azotobacter chroococcum was fractionated into two essential components by anaerobic chromatography and elution with MgCl2. Fraction 1, which contained iron and molybdenum, was not damaged by exposure to air for 30 min at 20° but Fraciton 2, which also contained iron but only traces of molybdenum, was completed inactivated by this treatment. The ratio of these fractions, giving maximum rate of nitrogen fixation, acetylene reduction or ATP-dependent hydrogen evolution was about 2:1 (mg protein of Fraction 1/mg protein Fraction 2) but for isocyanide or cyanide reduction the optimum ratio was about 6:1. Na2S2O4-dependent ATPase activity catalysed by the fractions was not always proportional to the amount of acetylene or isocyanide reduced or hydrogen evolved and the amount of ATP hydrolysed per 2 electrons used for reduction varied. The relative amounts of methane, ethylene and ethane formed from methyl-, vinyl- or ethylisocyanide were different and were affected by isocyanide concentration and by the ratio of the two fractioins. The amounts of products formed from methyl isocyanide were linear with time after a short lag and CO inhibited methane and ethane formation to about equal extents; ethylene production was only slightly inhibited. The observed difference in the optimum ratio for acetylene reduction compared with that for isocyanide is explained by assuming Fraction 2 contains the substrate complexing site, Fraction 1 provides electrons and isocyanide partially inhibits the reaction of Fraction 1 with 2. © 1969.