MECHANISM OF NITROGEN FIXATION AND NITRATE ASSIMILATION
The molecular nitrogen is reduced into ammonia as the end product before it enters into the metabolic system of plants. Nitrogen fixation requires:
- A reducing power like NDPH, FMNH2.
- A source of energy like ATP.
- Enzyme nitrogenase.
- Compounds for trapping ammonia formed by the reduction of dinitrogen.
The enzyme nitrogenase has iron and molybdenum which take part in attachment of a molecule of nitrogen. Bonds between the two atoms of nitrogen become weakened by their attachment to the metallic component. The weakened molecule of nitrogen is acted upon by hydrogen from a reduced coenzyme. It produces diamide, hydrazine and ammonia. Ammonia is not liberated. It is toxic even in small quantities. The nitrogen fixing microorganisms protect themselves from it by providing organic acids. Ammonia and organic acids react to give rise to amino acids. A part of the fixed nitrogen is handed over to the host by the symbiotic nitrogen fixing microorganisms. Free living nitrogen fixers do not immediately enrich the soil, it is only after the death that the fixed nitrogen enters the cycling pool. It occurs in two steps, ammonification and nitrification.
Ammonification: Plants absorb inorganic nitrogen and convert it into organic compounds, that is protein. After the death and decay of organic plants, the protein again comes back to the soil. In the soil, the proteins are broken into ammonia by active putrefying bacteria (bacteria of decay). This is done in following two steps:
- Proteolysis. In this step, proteins break down into their constituent amino acids. Protein breaks into peptide in the presence enzyme, proteases. The peptides break down into amino acid in the presence of enzyme, peptidases. Bacteria like Clostridium, Proteus, Pseudomonas, Bacillus etc., take part in the above-decomposition of protein.
- Deamination: In this step, amino acids are transferred into ammonia. The process involves removal of amino group from amino acid which is then transferred into ammonia. The process is facilitated by many species of bacillus such as Bacillus ramosus, Bacillus vulgaris, and Bacillus mycoides. Ammonia thus formed accumulates in the soil and some of it also diffuses into the atmosphere. Ammonia does remain in gaseous state in the soil but is changed to ionic form. Ammonia can be used by plants directly provided the pH of soil is more than 6 and the plant contain abundant organic acid. Unlike nitrates, very few plants can store ammonium ions, for example (Begonia, Oxalis). The ammonia is readily converted into nitrate which is absorbed by plants.
Nitrification: Oxidation of ammonia into nitrate is known as nitrification. Many autotrophic bacteria utilize this oxidation process to derive energy for their metabolic activities. Nitrification involves following two steps:
- In the first step, ammonia is oxidised into nitrite. This oxidation process is facilitated by Nitrosomonas.
- In the second step, nitrite is further oxidised into nitrate by Nitrobacter.
Most of the bacteria performing nitrification (for example, Nitrosococcus, Nitrosomonas and Nitrobacter) are chemoautotrophs. They use the energy liberted during nitrification in the synthesis of organic substances from carbon dioxide and a hydrogen donor. They are thus autotrophs which do not use solar energy for the synthesis of food.
Nitrates may also be broken down into gaseous nitrogen or nitrous oxide by some microorganism. This is called denitrification. Under anaerobic condition (for example, water-logging, oxygen depletion) some microorganism use nitrate and other oxidized ions as a source of oxygen. In the process, nitrates are reduced to gaseous compounds of nitrogen. As a result of denitrification, the soil becomes deficient in available nitrogen (nitrites). However, at the same time, a certain amount of elemental nitrogen diffuses into the atmosphere from the soil. This balances the amount of nitrogen removed from the atmosphere by biological nitrogen fixation. Denitrification of soil not only depletes the soil of an important nutrient but also causes acidification which is equally harmful in solubilisation of harmful metals. Common bacteria causing denitrification of soil are Pseudomonas denitrificans, Thiobacillus denitrificans, Micrococcus denitrificans, Serratia, Achromobacter. Denitrification can be represented by the following equation:
NITRATE ASSIMILATION: Nitrate is the most important source of nitrogen to the plants. It accumulates in the cell sap of several plants and takes part in reducing osmotic potential. Nitrate is first reduced to ammonia and then it is used by the plants. Reduction of nitrate occurs in two steps.
- Reduction of nitrate to nitrite. It is carried out by the agency of an inducible enzyme called nitrate reductase. The enzyme is a molybdoflavoprotein. Nitrate reductase is found attached loosely to cell membrane (Butz and Jackson, 1977). It requires a reduced coenzyme (NADH or NADPH) for its activity. The reduced coezyme is brought in contact with nitrate by FAD or FMN.
- Reduction of nitrite: Nitrite is reduced in the presence of an enzyme called, nitrite reductase. The enzyme is a metalloflavoprotein. It contains copper and iron. It occurs inside chloroplast in the leaf cells and leucoplasts of other cells. Nitrite reductase requires reducing power. It is NADPH in illumininated cells and NADH in others. The process of reduction also requires ferredoxin, which occurs in higher plants mostly in green tissue. The product of nitrite reduction is ammonia.
2NO2+7 NAD(P)H+7 H+→2NH3+4H2O+7 NAD(P)+
Ammonia is not liberated. It combines with some organic acid to produce amino acid. Amino acid then forms various types of nitrogenous compounds.
SYNTHESIS OF AMINO ACID
The first organic compounds of nitrogen assimilation are amino acids. They are synthesized by following three methods:
- Reductive amination: Ammonia combines with a keto organic acid like α-ketoglutaric acid and oxaloacetic acid in the presence of enzyme dehydrogenase (for example, glutamate dehydrogenase, aspartate dehydrogenase),and a reduced coenzyme NADH or NAD(P)H to form amino acid.
Other amino acids such as aspartic acid and alanine can also be synthesized by direct incorporation of ammonia into oxaloacetic acid and pyruvic acid respectively.
Oxaloacetic acid+NH3→aspartic acid
2 Catalytic amidation: Ammonia combines with catalytic amounts of glutamic acid in the presence of an enzyme, glutamine synthetase and ATP to produce an amide, called glutamine. Amides are amino acid derivatives in which -OH component carboxylic group (-COOH) is replaced by another amino group (-NH2). Amides are, therefore, double aminated keto acids. The two most amides are glutamine and asparagine. They are formed by amidation of glutamic acid and aspartic acid respectively.
Glutamine reacts with α-ketoglutaric acid in the presence of enzyme glutamate synthetase to form two molecules of glutamate. Reduced coenzyme, (NADH or NADPH) is required.
Glutamine+ α-ketoglutaric acid+NAD(P)H→2 Glutamate+NAD(P)
- Transamination: This reaction involves transfer of amino group of one amino acid to the keto group of keto acid. The enzyme required is transaminase or aminotransferase. Glutamic acid is the primary amino acid involved in transfer of amino group. As many as 17 different amino acids can be synthesized.
Glutamic acid+Oxaloacetic acid→ α-ketoglutaric acid+ Aspartic acid.