HOW DO MICROBES "FIX" NITROGEN FROM THE AIR? — NICHOLAS 455 



This means either that intermediates between nitrogen and ammonia 

 are tightly held by enzyme surfaces or that a labeled enzyme is formed, 

 or both. When fractions were exposed to the tracer for brief periods, 

 the isotope which was tightly bound to one protein fraction after the 

 shortest exposure became dispersed into three protein fractions after 

 a longer one. This agam supports the theory that enzyme-bound 

 intermediates are formed before ammonia, but no one has yet isolated 

 or identified them. 



It has been known for some time that trace metals including molyb- 

 denum, iron, copper, and boron are essential for nitrogen fixation, and, 

 more recently, cobalt has been added to the list. Their precise func- 

 tions in the process are not known. The teclinique of electron para- 

 magnetic resonance (EPR) has been used in attempts to identify sig- 

 nals associated with free radicals, particular states of transition metals 

 (iron, molybdenum, copper, manganese, etc.) and other systems con- 

 taining unpaired electrons in bacterial extracts that fix nitrogen. 

 Cell-particles that fix nitrogen prepared from Azotohacter were found 

 to give signals for flavin semiquinone, for molybdenum in a highly 



Azotobacter particles 



grown on N2 grown on NH4 



9 = 1-97 g=200 



g=|-94g=2 00 g=(94 



Gas with 



H2 



+m€diurn 



Gas with 



N' 



■AA^ 



H 



Figure 4. — Electron paramagnetic resonance spectra (EPR) of particles prepared from 

 Azotobacter vinelandii (OP). Signals recorded in gauss (g). Left column, particles 

 actively fixing nitrogen. Right column, particles that do not fix nitrogen, from cells 

 grown with an ammonium salt as nitrogen source. Upper row, spectra of particles as 

 isolated. In the nitrogen fixing particles there is a signal at g= 1.97 probably associated 

 with molybdenum valency 5 + . Middle row, after adding 10^1 of culture medium in 

 which the bacterium had been growing and gassing with hydrogen. Lower curve, 

 gassing with nitrogen after the hydrogen and medium treatment. 



