Biochemistry of Biological Nitrogen Fixation

Biochemistry of Biological Nitrogen Fixation 

Nitrogen fixing bacteria that occur in association with higher plants, especially with those of family Leguminaceae (fabaceae), are termed as symbionts.

The symbiotic association of nitrogen-fixing bacteria with plants roots generally occurs in multicellular structures called nodules. The best characterized symbiotic association involving nodules is one occurring on roots of leguminous plants. About 200 legume species are capable of nitrogen fixation. Nitrogen fixing root nodules can fix 100 to 200 times more nitrogen than free-living micro-organisms.

Plants other than legumes can also develop symbiotic relationship with nitrogen-fixing bacteria, for example Alnus (alder) involves nodule formation by an actinomycete Frankia. Similarly, grasses can also develop symbiotic relationships with nitrogen fixing organisms, but these associations do not involve the production of root nodules. These bacteria remained anchored to the root surface.

Azolla, an aquatic water fern, contains colonies of blue-green algae that fixes nitrogen. This association is especially important in rice fields. The fixed nitrogen leaks out from the fern plant and supplies the nitrogen needs of the rice plant.

Certain lichens growing on the surface of trunks and branches of forest trees are able to fix nitrogen. Nitrogen fixed by lichens may be leached down to the forest floor by rain. Aging of bark causes lichens to fall on the forest floor, where these are decomposed and releases fixed nitrogen in trees.

Development of Nodule

The nodules in legume are produced by the host plant root upon affected by nitrogen fixing gram-negative bacteria of the genus Rhizobium.

The following steps are involved in development of a nodule:

1. Th root excrete substances that attract bacteria and stimulate them to produce a cell-division.
2. Cells in the root cortex divide o form the primary root nodules meristem.
3. Bacteria attracts themselves to root hairs.
4. The cells in the pericycle near xylem are stimulated to divide and form infected threads.
5. The cells of primary nodule meristem and pericycle continue to divide and two masses of dividing cells fuse into a single clump. The infection threads continue to grow.
6. The nodule elongates, its vascular tissue differentiates and make a connection with the root stele.
7. The bacteria enter the nodule cells. Bacterial cells inside infected host cells multiply rapidly and are transformed into nitrogen fixing organelle called bacteriods. These contain enzymes necessary for nitrogen fixation.

Biochemistry of Biological Nitrogen Fixation

The end product of biological fixation is ammonia. It is incorporated into organic compounds such as glutamine or glutamate and is utilized by the micro-organisms or by the host plant in case of symbiotic association. The overall reaction of fixation of molecular nitrogen into ammonia is:

N₂ + 8e + 16 ATP ———————————- 2NH₃ + H₂ + 16 ATP + 16 Pi

The entire reaction is catalyzed by an enzyme complex, nitrogenase.

Conversion of Ammonia to Nitrate

Some plants directly utilize ammonia under certain conditions, but the principal source of nitrogen that is available to higher plants under normal field conditions is nitrate. In most temperate soils, ammonia is rapidly converted to nitrate through nitrification by bacteria that are absorbed by plants through their roots.

Reduction of nitrate to Ammonium

Inside the body of a plant nitrate is assimilated to organic compounds. Prior to assimilation nitrate is reduced to ammonia in the cytosol.

The first step is reduction of nitrate to nitrite. This reaction carried out by enzyme nitrate reductase. NADH is the electron donor. The overall reaction is:

NO₃^– + NADH + H⁺ + 2e ———————————- NO₂ + NAD⁺ + H₂O

Nitrite produced is rapidly transported to into chloroplasts when, it is reduced to ammonia by the activity of nitrite reductase. Ferredoxin is the electron donor. The overall reaction is:

NO₂ + 6Fd_red + 8H⁺ + 6e ———————————- NH₄⁺ + 6Fd_ox + 2H₂O

Incorporation of Ammonia into Organic Compounds

Ammonia and ammonium, ions do not accumulate in plant cells but are instead rapidly incorporated into organic compounds. The conversion of ammonia into carbon compounds takes place through two possible ways. Both of these possible ways result in same product, glutamate

The first pathway involves the reductive amination of alpha-ketoglutarate to produce glutamate. The reaction is catalyzed by glutamate dehydrogenase.

Alpha-ketoglutarate + NADH + NH₃ ———————————- Glutamate + NAD⁺ + H₂O

The second pathway involves a reaction with glutamate to form its amides, glutamine. This reaction is catalyzed by glutamate dehydrogenase.

Glutamate + NH₃ + ATP ———————————- Glutamine + ADP + P

Glutamine can be converted to glutamate by glutamate synthetase by the following reaction:

Glutamine + alpha-ketoglutarate ———————————- 2 Glutamate

Transamination Reactions

Once assimilated into glutamate, the nitrogen can be further incorporated into other amino acids through transamination reaction by enzymes aminotransferases.

Glutamate + Oxaloacetate ———————————- Aspartate + alpha-ketoglutarate

The two amides glutamate and aspartate are metabolic reservoirs for the temporary storage of excess ammonia.