Metabolism of Nitrogen

Nitrogen is the most prevalent element in living organisms.

The atmosphere contain near about 78% of N₂ by volume.

Plants compete with microbes for the limited nitrogen that is available in soil.

Thus, it is a limiting nutrient for both natural and agricultural ecosystems.

N₂ cycle can be conveniently discussed under the following steps

(i)   N₂ fixation     (ii) Ammonification    (iii) Nitrification    (iv) Denitrificatio

(I) NITROGEN FIXATION

Nitrogen exists as two nitrogen atoms joined by a very strong triple covalent bond (N º N).

The process of conversion dinitrogen (N₂) to ammonia is termed as nitrogen fixation.

Following are the methods of N₂-fixation:

A. Physico-Chemical method : During thunder, lightening and by using UV rays, atmospheric N2 and oxygen combine to form oxides of nitrogen which form nitrous and nitric acid with water. This may form nitrates of calcium, potassium and ammonium.

B. Industrial N₂ fixation: Industrial combustions, forest fires, automobile exhausts and power generating stations are also sources of atmospheric nitrogen oxides.

C. Biological N₂ fixation : Only certain prokaryotic species are capable of fixing N₂. Biological N₂ fixation may be asymbiotic, symbiotic or through loose symbiosis. Biological N₂ fixation is called diazotrophy and agents of this process are called diazotrophs.

Some important N₂ fixing organisms

(a) Asymbiotic N₂ fixers:

Bacteria

(i)   Aerobic – Azotobacter, Beijerinckia

(ii)  Facultative Aerobic – Klebsiella, Bacillus

(iii) Anaerobic – Clostridium

(iv) Photosynthetic – Chromatium, Rhodospirillum

Blue Green Algae – Anabaena, Aulosira, Nostoc, Scytonema etc. Heterocyst is present in these blue green algae which is responsible for N₂ fixation

(b)    Symbiotic N₂ fixers:

(i) In root nodule of legumes – Rhizobium

(ii) In root nodule of Alnus, Casuarina, Myrica – Frankia

(iii) In leaf nodule of Dioscorea, Pavetta and Psychotria – Klebsiella

(iv) In coralloid root of Cycas – Anabaena cycadae

(v) In fronds of Azolla – Anabaena azollae

(vi) In thallus of Anthoceros – Nostoc

(c) Intermediate: Loose symbiosis with the roots of Sorghum, Zea etc. by Azospirillum.

Rhizobium -Legume Symbiosis

Principal stages of nodule formation are summarised as follows:

1. Rhizobia are Gram negative aerobic rod-shaped bacteria. This genus is responsible for symbiotic N₂ fixation in legumes

2.Legume roots secrete some specific chemicals (e.g., Flavinoids) which attract the bacteria. Rhizobia multiply an colonise-the surroundings of roots and get attached to epidermal root hair cells.

3.The root hairs curl by the action of nod factors secreted by bacteria and the bacteria invade the root hair.

4.An infection thread is produced, carrying the bacteria into the cortex region of root.

5.Cortical cells are stimulated divide rapidly. It is due to auxins secreted by plants and cytokinins secreted by bacteria.

6.Bacteria enters only polyploid cells of cortex. Some of them enlarge and become membrane bound structures called bacteroids. These form the seat of N₂ fixation. These specialised cortical cells now form nodules. Nodules establish a direct vascular connection with the host for exchange of nutrients.

7.The nodules contain a red coloured pigment called leghaemoglobin (LHb). The globin part of leghaemoglobin is formed by host genome, while the heme portion is formed by bacteria.

8.This pigment is O₂ carrier and is also called scavenger of O₂.

9. Nitrogenase enzyme (synthesized by nif genes of bacteria) is required to fix N₂. It is an O₂ sensitive enzyme made up of two unequal sub units. Large component has Fe-Mo moiety, while, small component has only Fe-moiety. Here Mo acts as an acceptor and donor of electrons, when N₂ is reduced to NH₃ LHb maintains anaerobic conditions.

10.N₂ fixation requires energy, so it is an active process.

N₂ + 8e^– + 8H⁺ + l6 ATP —® 2NH₃ + H₂ + 16ADP + 16Pi

11.N₂ fixation occurs under the controJ of plant nod gene and bacterial nod, nif and fix gene cluster.

12.During this process, atmospheric N₂ is reduced by the addition of hydrogen atom.

13.Strong reducing agents e.g., NADPH₂, FMNH₂, Ferredoxin are also required.

14.Donor of electron and H⁺ is generally glucose-6-phosphate; certain cofactors like -TPP, Mg⁺⁺ and CoA are also involved.

15. ATP is provided by the host respiration process.

16.NH₃ so formed is used for the synthesis of amino acids. These acts as building blocks for the synthesis of various types of protein.

(II) AMMONIFICATION

Plants absorb inorganic nitrogen and convert it into proteins

 After the death of organisms and plants, proteins are broken into ammonia by the following two steps:

(a)  Proteolysis: It is breakdown of protein

(b) Deamination : Ammonia is released from the amino acids.

Amino acid + H₂O organic acid + ammonia

It is done by Bacillus ramosus, B. vulgaris, and B. mycoides. This ammonia is converted into nitrate which is absorbed by' the plants.

(III) NITRIFICATION

It is oxidation of ammonia into nitrate, it involves following steps:

   (a)     Conversion of ammonia into nitrate

2NH₃ + 3O₂  2NO₂^– + 2H⁺ + 2H₂O + Energy

(b)     Conversion of nitrite into nitrate.

2NO₂^– + O₂  2NO₃^– + energy

Nitrate assimilation : Nitrate cannot be used by the plant as such. It is first converted into ammonia before being incorporated into organic compounds. Nitrate is reduced in two steps -

The process of nitrate reduction to ammonia is called nitrate assimilation and is accomplished in two steps mediated by two specific enzymes:

(a)     First, the nitrate is reduced to nitrite by an enzyme called nitrate reductase. This enzyme is a flavoprotein and contains molybdenum.

(b)     The nitrite ions are then reduced to ammonia by an enzyme called nitrite reductase. Ferredoxin is the most direct source of electrons for nitrite reduction and hence, it occurs specifically in leaves. Therefore, nitrite ions formed in other parts of the plant are tranported to leaves and further reduced to ammonia. Nitrite reductase does not require molybdenum but contains copper and iron.

Ammonia thus formed, is fixed by the organic acids to produce amino acids which form other nitrogenous compounds.

(IV) DENITRIFICATION

Nitrates are broken down into gaseous nitrogen or nitrolls oxides by some microorganisms.

It is called denitrification e.g., Thiobacillus denitrificans, Micrococcus denitrificans, Pseudomonas denitrificans.

FATE OF AMMONIA

At physiological pH, the ammonia is protonated to form NH₄⁺ ions, which is quite toxic to plants and hence cannot be accumulated by them. This is commonly used up to synthesize ­

(a) Amino acids and or        (b) Amides

(a) Amino Acids

Amino acids are generally the initial products of N₂ assimilation. Mainly, these two processes are used by plants to synthesise amino acid :

(i) Reductive Amination : In this process, NH₃ reacts with a-ketoglutaric acid and forms glutamic acid.

   a-Ketoglutaric acid + NH₄⁺ + NAD(P)H  Glutamic acid + H₂O + NAD (P).

(ii)     Transamination : It involves the transfer of amino group from one amino acid to the keto group of other keto acid.

Glutamic acid is the main amino acid from which other 17 amino acids are formed through transamination. Enzyme required for this reaction is transaminase.

(b) Amides

­Two most important amides present in plants are asparagine and glutamine.

These are formed from two amino acids, glutamic acid and aspartic acid.

For this reaction, glutamine synthetase and asparagine synthetase are required respectively.

Amides contain more N₂ than amino acids and are structural part of most proteins, these are transported through xylem vessels.

Inaddition, along with the transpiration stream the nodules of some plants (e.g., soyabean) export the fixed nitrogen as ureides (allantoin, allantoic acid and citrulline).

Important definitions/ formulae:

Some important N₂ fixing organisms

(a) Asymbiotic N₂ fixers:

Bacteria

(i) Aerobic – Azotobacter, Beijerinckia

(ii) Facultative Aerobic – Klebsiella, Bacillus

(iii) Anaerobic – Clostridium

(iv)      Photosynthetic – Chromatium, Rhodospirillum

Blue Green Algae – Anabaena, Aulosira, Nostoc, Scytonema etc. Heterocyst is present in these blue green algae which is responsible for N₂ fixation.

(b) Symbiotic N₂ fixers:

(i) In root nodule of legumes – Rhizobium

(ii) In root nodule of Alnus, Casuarina, Myrica – Frankia

(iii) In leaf nodule of Dioscorea, Pavetta and Psychotria – Klebsiella

(iv) In coralloid root of Cycas – Anabaena cycadae

(v) In fronds of Azolla – Anabaena azollae

(vi) In thallus of Anthoceros – Nostoc

(c) Intermediate: Loose symbiosis with the roots of Sorghum, Zea etc. by Azospirillum.