NITROGEN FIXATION 449 



reaction. If root nodule bacteria from nodules of the pea are grown on arti- 

 ficial media, they lose both their ability to fix nitrogen and their hydrogenase, 

 although this was not always found to be the case. The nitrogen fixation by 

 Rhiznbium trifolii and Azotobacter was found to be inhibited by hydrogen and 

 also by carbon dioxide, both of which react with hydrogenase. Carbon 

 monoxide inhibits the nitrogen fixation in the root nodules {174-o) as well as 

 in Azotobacter (1740). 



The nitrogenase and hydrogenase activities of Azotobacter differ in their 

 sensitivity to carbon monoxide and in the effect on them of pH. Thus 

 nitrogenase is much more effectively inhibited by carbon monoxide than 

 hydrogenase. Nitrogenase was inactive at ;;H 6, at which hydrogenase still 

 possessed 70% of its optimal activity; the optimal pH was the same for both 

 (about 7.5). The activities of hydrogenase measured by the "Knallgas" 

 reaction and by methylene blue reduction are, however, also differently 

 affected by pH {2651). Since nitrogenase action may require the formation 

 of a complex with another enzyme activating the nitrogen, these differences 

 cannot be taken as proof of the existence of two independent enzymes.* 



Hemoglobin in the root nodules. An interesting correlation between the 

 content of hemoglobin in the root nodules (r/. Chapter VII) and their ability 

 to fix nitrogen has been described recently by Virtanen (2890,2891). The 

 root nodules which are effective for nitrogen fixation are red and contain 

 hemoglobin in the nodule, while those which are inactive are green. The 

 latter contain a bile pigment hemoglobin, from which hydrochloric acid 

 splits off iron, the green substance being thus closely related to or identical 

 with choleglobin (Chapter X). Red nodules are transformed into green 

 nodules by keeping the plants in the dark. This transformation is irreversible 

 and accompanied by an irreversible loss of nitrogen-fixing power. In addition 

 to red and green nodules, brown nodules were observed which contain hemi- 

 globin. Fluoride shifts the absorption band in the brown nodules to 610 m/x, 

 the position of the band of hemoglobin fluoride. 



The equilibrium between hemoglobin and hem/globin is said to depend 

 on light intensity. On bright days the oxaloacetic acid content of the nodules 

 is highest and the influence of oxaloacetic acid on the reduction of hem/globin 

 to hemoglobin is explained as a shift of the equilibrium between hem?globin 

 (Hi) + nitrogen and hemoglobin (Hb) -|- hydroxylamine 



Nj + Hi ;:± NH2OH + Hb 



Oxaloacetic 

 acid 



i 



i-Aspartic acid Oxime of oxaloacetic acid 



CO2H H2 CO2H 



HC — NH2 C=NOH 



CH2 CH2 



CO2H CO2H 



* Cf. the recent review of Burris and Wilson (384a). 



