454 ANNUAL REPORT SMITHSONIAN INSTITUTION, 19 64 



Oxidation /reductton 

 state of. nitrogen atom 



HoO 



+ 1 



-I 



-2 



-3 



(nitrous oxfde) 



1^0/ 



NEN 



^HON-NOH(hyponltrlte) 



or 



H_N-NO (nitramide) 



"A 



2H* 



2H2O 



a keto acids 



HN=NH, 

 (diimide) 



V |» 2NH2OH 



a, keto acids 



N2H-N2H 



(hydrazine) 



(hydroxyldmine) 



oxtm$s 



(ammonia) 

 ammo acids 



♦ 



profein and other cell nitrogen constituents 



Figure 3. — Possible pathways of nitrogen fixation. 



Inhibitors of fixation accumulate in cell-free extracts of Azotobacter 

 cells during growth ; these inhibitors may be destroyed by putting the 

 cells in liquid air. 



The various ways in which nitrogen gas might be converted to 

 ammonia are given in figure 3. The uppermost route, via nitrous 

 oxide, is not now thought to be likely ; in fact, nitrous oxide is a potent 

 inhibitor of fixation. Hydroxylamine and hydrazine have both been 

 suggested as possible intermediates but the evidence for either is slim. 

 Eecent work with cell-free extracts of C. fosteurianum by the Wis- 

 consin workers, using nitrogen-15, and by the Long Ashton group 

 with extracts of Azotobacter using radioactive nitrogen-13, showed 

 impressive labeling of nitrogen in ammonia but there was no detectable 

 trace of hydroxylamine or hydrazine. The results suggest that free 

 hydroxylamine or hydrazine are not intermediates in the reaction 

 but do not rule out the possibility that they and other compounds may 

 occur tightly bound to the enzyme protein. In fact evidence for 

 enzyme-bound intermediates has come from experiments with Azo- 

 tobacter^ in which fractions exposed to radioactive nitrogen-13 incor- 

 porated more of the tracer than appeared in the form of ammonia. 



