HOW DO MICROBES "FIX" NITROGEN FROM THE AIR? — NICHOLAS 453 



two isotopes, apart from ordinary nitrogen-14, are available: the 

 stable heavy isotope nitrogen-15 and the more recently developed radio- 

 active nitrogen-13. The use of the stable isotope is time consuming 

 and needs expensive mass spectrometers. Nitrogen-13, on the other 

 hand, has to be made continuously in a cyclotron and experiments have 

 to be done within about 2 hours because of its vei-y short half-life of 

 only 10.05 minutes. Nevertheless, the tracer has been effectively used 

 in some recent experiments with Azotohacter. 



An important advance has been made with the development of 

 methods for preparing cell-free extracts of microorganisms that fix 

 atmospheric nitrogen. This discovery occurred, as is often the case, 

 in a number of laboratories almost simultaneously and independently. 

 In 1960, the Du Pont organization in the U.S.A. succeeded with 

 Clostridium pasteurianum, the Long Ashton workers with Azotohacter 

 vinelandii, the Wisconsin laboratory with Rhodospirillum rubrum and 

 blue-green algae, and a California group with the sulphur bacterium 

 Ghromatium. In all cases, fixation depended on special methods of 

 cell disruption and on supplies of special substrates. For the first 

 time, it became possible to study the enzymes concerned in nitrogen 

 fixation outside the living cell. 



The Du Pont group found that sodium pyruvate was required for 

 nitrogen fixation in extracts of C. pasteurianum. No other compound 

 will initiate the activation of the gas. Since about 100 parts of 

 pyruvate have to be added to the extracts to fix 1 part of nitrogen 

 gas as ammonia it is clear that some intermediate product in the 

 metabolism of pyruvate, rather than pyruvate itself, is required for 

 nitrogen fixation. This intermediate factor has not been identified. 

 However, it has been possible to separate two enzyme complexes from 

 the extracts. The first is the reducing system which produces hydrogen 

 from pyruvate and the second is the nitrogen-fixing component. 

 Neither will, on its own, activate nitrogen gas, but when they are 

 recombined active fixation takes place. An unexpected result was 

 the identification of a new reducing protein, containing iron, which 

 is required for the hydrogen production from pyruvate by this bac- 

 terium. The substance has now been isolated, purified, and named 

 ferredoxin. It has a molecular weight of about 15,000, contains about 

 5 atoms of iron per molecule of protein, and is a most important elec- 

 tron carrier, especially in bacteria that live without oxygen. It is also 

 found in green plants. 



The enzymes that fix nitrogen in C. pasteurianum are present in the 

 soluble parts of the cell ; in Azotohacter vinelandii, on the other hand, 

 they are located in particles which are components of the cell mem- 

 branes. Another difference is that sodium pyruvate has no effect on 

 fixation in Azotohacter — fixation is stimulated instead by unidentified 

 reducing factors excreted into the culture medium during early growth. 



