93. Mechanism of Biological Nitrogen Fixation

A PREVIOuS report [Wyss & Wilson, 1941] demonstrated that B2 inhibits fixation of N2 by Azotobacter. Because of the apparent uniqueness of this type of inhibition, such a demonstration furnishes strong evidence that in the symbiotic and free-living N2 fixation processes the mechanism of fixation is very similar if not identical. Further studies described in this paper provide additional support for this important conclusion. Methods Details of the methods used were described by Wyss & Wilson [1941]. Briefly, 25 ml. cultures of Azotobacter were grown under different atmospheres in litre bottles provided with a device which maintained the PO2 constant (0.2 atm.) during the experiment. Under these conditions fixation remains logarithmic for 24-48 hr.; hence the specific rate constant of fixation, k= d ln (a + y)/dt, can be readily estimated from the slope of the line obtained by plotting the log of the total N content against time. In this equation a is the initial N content of the cells and y is the increase after t hr. With cultures of Azotobacter vinelandii, k consistently reaches the surprisingly high values of 0-080-0125 corresponding to a total fixation of 20-30 mg./100 ml. in 24 hr. Somewhat lower values were noted with Az. chroococcum. Harvests were made every 10-12 hr. often in duplicate so that each k value is based on 4-6 separate determinations. Respiration measurements were made in the Warburg apparatus by the usual technique, and micro-N determinations by the method of Johnson [1941]. The pN2 function A fundamental property of any enzyme system is the dependence of the rate of reaction on the concentration of substrate. Burk [1930] found that the rate of N2 fixation by Azotobacter was a function of the pN2 between 0.05 and 5 to 10 atm., being directly proportional to the pN2 between 0-05 and 0 5 atm. Apparently these conclusions were based almost entirely on microrespiration experiments in which H2 was used as the diluent gas. It follows then that the pN2 function discussed by Burk [1934] is not the true one but is a complex of the effects of both N2 and H2. Failure to recognize the inhibition by H2 probably explains two characteristics of the pN2 function which Burk thought surprising, viz. that fixation hardly occurred until a pN2 of 0-05 atm. was present, and that