Metabolism of Nitrogen

Nitrogen Cycle:

Apart from carbon, hydrogen, and oxygen, nitrogen is the most prevalent element in living organisms. Nitrogen is a constituent of amino acids, proteins, hormones, chlorophylls and many of the vitamins. Plants compete with microbes for the limited nitrogen that is available in the soil. Thus, nitrogen is a limiting nutrient for both natural and agricultural eco-systems. Nitrogen exists as two nitrogen atoms joined by a very strong triple covalent bond (N º N). The process of conversion of nitrogen (N₂) to ammonia is termed as nitrogen fixation.

In nature, lightning and ultraviolet radiation provide enough energy to convert nitrogen to nitrogen oxides (NO, NO₂, N₂O). Industrial combustions, forest fires, automobile exhausts and power-generating stations are also sources of atmospheric nitrogen oxides. Decomposition of organic nitrogen of dead plants and animals into ammonia is called ammonification. Some of this ammonia volatilises and re-enters the atmosphere but most of it is converted into nitrate by soil bacteria in the following steps:

2NH³ + 3O₂ → 2N + 2H⁺ + 2H₂O ------ (i)

2N + O₂ → 2N ------ (ii)

Ammonia is first oxidised to nitrite by the bacteria Nitrosomonas and/or Nitrococcus. The nitrite is further oxidised to nitrate with the help of the bacterium Nitrobacter. These steps are called nitrification. These nitrifying bacteria are chemoautotrophs. Ammonia is transported in three forms by ammonia transporters.

The nitrogen cycle showing relationship between the three main nitrogen pools – atmospheric soil, and biomass

The nitrate thus formed is absorbed by plants and is transported to the leaves. In leaves, it is reduced to form ammonia that finally forms the amine group of amino acids. The medium around plant roots turns alkaline on nitrate uptake. Therefore, nitrate uptake is always accompanied by cation uptake or anion removal to maintain ionic balance. Nitrate present in the soil is also reduced to nitrogen by the process of denitrification. It is carried by bacteria Pseudomonas and Thiobacillus.

Biological Nitrogen Fixation:

Very few living organisms can utilise the nitrogen in the form N₂, available abundantly in the air. Only certain prokaryotic species are capable of fixing nitrogen. Reduction of nitrogen to ammonia by living organisms is called biological nitrogen fixation. The enzyme, nitrogenase which is capable of nitrogen reduction is present exclusively in prokaryotes. Such microbes are called N₂- fixers.

N º N NH₃

The nitrogen-fixing microbes could be free-living or symbiotic. Examples of free-living nitrogen-fixing aerobic microbes are Azotobacter and Beijernickia while Rhodospirillum is anaerobic and Bacillus free-living. In addition, a number of cyanobacteria such as Anabaena and Nostoc are also free-living nitrogen-fixers. Anabaena is a blue-green alga composed of barrel-shapped cells held in a gelatinous matrix. So, it can fix atmospheric nitrogen.

Symbiotic Biological Nitrogen Fixation:

Several types of symbiotic biological nitrogen fixing associations are known. The most prominent among them is the legume-bacteria relationship. Species of rod-shaped Rhizobium has such relationship with the roots of several legumes such as alfalfa, sweet clover, sweet pea, lentils, garden pea, broad bean, clover beans, etc. The most common association on roots is as nodules. These nodules are small outgrowths on the roots. The microbe, Frankia, also produces nitrogen-fixing nodules on the roots of non-leguminous plants (e.g., Alnus). Both Rhizobium and Frankia are free-living organisms present in the soil, but as symbionts can fix atmospheric nitrogen.

Uproot any one plant of a common pulse, just before flowering. There will be near-spherical outgrowths on the roots. These are nodules. If you cut through them you will notice that the central portion is red or pink. The nodules are in pink due to the presence of leguminous haemoglobin or leg-haemoglobin.

Nodule Formation:

Nodule formation involves a sequence of multiple interactions between rhizobium and roots of the host plant. Principal stages in the nodule formation are summarised as follows:

Rhizobia multiply and colonise the surroundings of roots and get attached to epidermal and root hair cells. The root-hairs curl and the bacteria invade the root-hair. An infection thread is produced carrying the bacteria into the cortex of the root, where they initiate the nodule formation in the cortex of the root. Then the bacteria are released from the thread into the cells which leads to the differentiation of specialised nitrogen fixing cells. The nodule thus formed, establishes a direct vascular connection with the host for the exchange of nutrients. These events are depicted in below figure.

Development of root nodules in soyabean : (1) Rhizobium bacteria contact a susceptible root hair, divide near it, (2) Successful infection of the root hair causes it to curl, (3) Infected thread carries the bacteria to the inner cortex. The bacteria get modified into rod-shaped bacteroids and cause inner cortical and pericycle cells to divide. Division and growth of cortical and pericycle cells lead to nodule formation, (4) A mature nodule is complete with vascular tissues continuous with those of the root.

The nodule contains all the necessary biochemical components, such as the enzyme nitrogenase and leghaemoglobin. Leghaemoglobin is the haemoglobin like red pigments found in the root nodules of legumes and reported to function as an oxygen-carrying pigment in symbiotic nitrogen fixation. The enzyme nitrogenase is a Mo-Fe protein and catalyses the conversion of atmospheric nitrogen to ammonia, the first stable product of nitrogen fixation.

Steps of conversion of atmospheric nitrogen to ammonia by nitrogenase enzyme complex found in nitrogen-fixing bacteria

The reaction is as follows:

N₂ + 8e⁻ + 8H⁺ + 16ATP → 2NH₃ + H₂ + 16ADP + 16P_i

The enzyme nitrogenase is highly sensitive to the molecular oxygen; it requires anaerobic conditions. The nodules have adaptations that ensure that the enzyme is protected from oxygen. To protect these enzymes, the nodule contains an oxygen scavenger called leg-haemoglobin. It is interesting to note that these microbes live as aerobes under free-living conditions (where nitrogenase is not operational), but during nitrogen-fixing events, they become anaerobic (thus protecting the nitrogenase enzyme). From above reaction that the ammonia synthesis by nitrogenease requires a very high input of energy (8 ATP for each NH₃ produced). The energy required, thus, is obtained from the respiration of the host cells.

The fate of Ammonia:

At physiological pH, the ammonia is protonated to form N (ammonium) ion. While most of the plants can assimilate nitrate as well as ammonium ions, the latter is quite toxic to plants and hence cannot accumulate in them. There are two main ways in which the N is used to synthesise amino acids in plants:

i. Reductive Amination: In these processes, ammonia reacts with a-ketoglutaric acid and forms glutamic acid as indicated in the equation given below :

ii. Transamination: It involves the transfer of an amino group from one amino acid to the keto group of a keto acid. Glutamic acid is the main amino acid from which the transfer of NH₂, the amino group takes place and other amino acids are formed through transamination. The enzyme transaminase catalyses all such reactions. For example,

The two most important amides – asparagine and glutamine – found in plants are a structural part of proteins. They are formed from two amino acids, namely aspartic acid and glutamic acid, respectively, by addition of another amino group to each. The hydroxyl part of the acid is replaced by another NH₂ – radicle. Since amides contain more nitrogen than the amino acids, they are transported to other parts of the plant via xylem vessels. In addition, along with the transpiration stream the nodules of some plants (e.g., soyabean) export the fixed nitrogen as ureides. These compounds also have a particularly high nitrogen to carbon ratio.