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  Section: General Biotechnology / Plant Biotechnology
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Biological Nitrogen Fixation

Mechanism of Nitrogen Fixation in Root Nodules
A characteristic feature of the healthy root nodules of leguminoids plants is the presence of a special red pigment like haemoglobin often known as "leghaemoglobin (LHb). LHb has characteristics similar to myoglobin or a variety of haemoglobin found in animal. It is red in color due to presence of iron. For the first time LHb was isolated and crystallized from soybean root nodules.

LHb is found only in healthy nodules. The unhealthy plants or white nodules do not develop LHb; therefore, N2 fixation does not takes place in such nodules. LHb is present outside the bacteroid membrane (peribacteroid space) but in their close contact. Recent studies suggest that the peribacteroid membrane may separate the bacteroids from the oxygen buffering system. LHb regulates O2 concentration as bacteroids are aerobic and consume O2. 

Indirectly LHb promotes O2 utilization in bacteroids and favors nitrogen fixation. The conditions where host plants suffer from O2 deficiency, LHb serves to facilitate the movement of O2 to O2 poor tissues in a proper way. It has been found that at ambient atmospheric concentration (0.2 atm.), O2 becomes a limiting factor for N2 fixation and the reduced O2 diffusion results in prompt inhibition of nitrogenase activity in nodules (Sprent, 1972). 

Oxygen levels above 0.5 atm. inhibit N2 fixation due to inactivation of nitrogenase by excessive oxygen.


Non-Symbiotic N2 fixation




Ecology of diazotrophs


Special features of diazotrophs



Sites of N2 fixation



Nitrogenase and reductants



Presence of hydrogenase



Self regulatory systems


Mechanism of N2 fixation

Symbiotic N2 fixation


Establishment of symbiosis



Host specificity and root hair curling



Infection of root hairs



Nodule development



Nodule development and maintenance


Factors affecting nodule development


Mechanism of N2 fixation in root nodules

Genetics of diazotrophs


Nod genes


Nif genes



Nif gene cloning


Hup genes

LHb combines with O2 to form oxyleghaemoglobin (OLHb) and makes it available at the surface of bacterial membrane where O2 is diffused into it under low concentration of O2 in root nodule. Thus LHb acts as a carrier of O2 and helps in accomplishment of bacterial respiration and consequent provision of ATP for N2 fixation. Bergerson (1971) has described that without LHb, diffusion of O2 through thick nodule tissue would be completely inadequate to meet the necessary ATP requirement. Therefore, the efficiency of the nodules to fix N2 can be examined by estimating the concentration of LHb. 

LHb is found only in root nodules of legumes. It is not found in actinorrhizic nodules i.e. nodules formed by Frankia in roots of non-leguminous plants. Therefore, presence of O2 buffering system has not been reported so far in actinorrhizic nodules. 

LHb facilitates O2 uptake by terminal oxidases and increases ATP production for nitrogenase activity. It also creates an O7 free environment around the active site of nitrogenase which fails to function in the presence of O^ (Fig. 11.7).
  Fixation of nitrogen (N2).
Fig. 11.7. Fixation of nitrogen (N2).
Both the metalloproteins, nitrogenase (Mo-Fe-protein) and nitrogenase reductase (Fe-protein) are essential for nitrogenase activity. Fe-protein interacts with ATP and Mg++, and Mo-Fe-protein catalyses the reduction of N2 to NH3, H+ to H2 and acetylene to ethylene.

The reduced ferredoxin or flavodoxin serves as a source of reductant for electron transfer during N2 fixation (Fig. 11.7).  

From reduced form of ferredoxin (Fd res.) electrons (e-) flow to Fe-protein which reduce to Mo-Fe-protein (in nitrogenase enzyme complex) with subsequent release of inorganic phosphate, Pi. This enzyme complex gets energy from Mg ATP which in turn is produced after bacterial respiration. Finally, Mo-Fe-protein passes on the electron to reducible substrate i.e. N2 (or other substrates). The equation of N7 fixation in nodules of legumes may be written as :

N2 + 16 ATP + 8e- + 10H+2NH+4 + H+2 + 16ADP + 16Pi

It is obvious that ammonia is the first stable product of N2 fixation. But it is not clear whether neutral ammonia or cationic ammonium (NH4+) is formed. Soon after formation, it is transferred through 3 layered bacteroid membrane (two of bacteria and third of host origin) to host cells, where it is enzymatically converted into many products.


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