In this article we will discuss about Processes of Nitrogen Metabolism.
Processes of Nitrogen Metabolism:
Like carbon, hydrogen and oxygen, nitrogen is also one of the most prevalent essential macro-elements which regulates plant growth, especially in agricultural systems. The main source of nitrogen for the construction of nitrogenous organic compounds is the atmosphere.
It occurs in such essential biomolecules as nucleic acids, proteins, some of the phytohormones and in many of the vitamins. So, nitrogen as component of these biomolecules and many other compounds is involved in most of the biochemical reactions contributing to life activities.
Plants require higher amounts of nitrogen as it is important in their structure and metabolism. Nearly, 80 per cent of the earth’s atmosphere is composed of nitrogen, bathing the entire plant world, but unfortunately most plants cannot utilize it in its elementary form. For its supply they have to depend on the soil and from there they acquire nitrogen in inorganic form either as ammonium compounds or as nitrate.
Only certain prokaryotic systems can use the atmospheric nitrogen directly.
In a stable ecological system, the atmospheric nitrogen is converted into a metabolically useful form by a few prokaryotic life styles for supply to higher plants and animals. Ammonia, the most useful form, is initially converted to glutamate and glutamine and then to other nitrogen-containing compounds required for the growth and maintenance of the life of plants.
The other sources of nitrogen in the soil are nitrate (NO3), ammonium ions (NH4+) or organic nitrogen, which are obtained from the nitrogen cycle (Fig. 10.1) and farm manure. Nitrate is converted to ammonia prior to metabolism. Ammonium ions and organic nitrogen in the form of amino acids mobilized from the proteins or partially destroyed proteins, can be absorbed directly by the roots of the plants.
With death and decay of organic systems the amino acids, purines and pyrimidine’s are cycled back to ammonia, some of which is lost from the nitrogen cycle by the processes which convert it to NO2 – and then to nitrogen gas or to NO3 –. So, nitrogen metabolism includes both anabolic and catabolic processes.
The anabolic processes are:
(i) Nitrogen fixation
(ii) Amino acid synthesis
(iii) Protein synthesis
The catabolic processes are:
(i) proteolysis and amino acid destruction
Therefore, it is evident that nitrogen is available in several forms for the plant system, like nitrate, ammonia, organic nitrogen and molecular nitrogen in the environment. The continuous inter-conversion of these forms to maintain the constancy of the amount of nitrogen in atmosphere, by physical and biological processes constitute the nitrogen cycle (Fig. 10.1).
It essentially involves:
(i) Ammonification and nitrification
(iii) Nitrogen fixation
(i) Ammonification and Nitrification:
Conversion of organic nitrogen to NH+ by soil microbes is called ammonification. The organic nitrogen in the soil comes from the animal excreta as well as from the decaying plant and animal remains.
In warm (30°-35°C) and moist soils at about neutral pH, ammonia is further oxidized first to produce nitrite and then to nitrate within a few days of its formation. This oxidation is known as nitrification. Certain microorganisms are responsible for carrying out both these processes.
The ammonifying saprophytic bacteria are Bacillus mycoides, B. vulgaris and B. ramosus. Certain fungi and actinomycetes can also release ammonia from the natural organic compounds. Two genera of chemoautotrophic bacteria, Nitrosomonas and Nitrobacter, are responsible for nitrification.
Nitrosomonas converts ammonia only into nitrite which is then further converted to nitrate by Nitrobacter as follows:
Both the reactions are exergonic; the energy released through the oxidation of ammonia or of nitrite is utilized by the bacteria in their chemosynthesis.
(a) Nitrate Assimilation:
Nitrate which is the most abundant form of nitrogen in the soil, and the root systems of higher plants absorb it in the form of NO3–. Since it cannot be directly used by the plants, it must first be reduced to the NH3 level before incorporation into the nitrogenous compounds.
For the first time, nitrate is reduced to nitrite by the enzyme nitrate reductase, requiring respiratory energy according to the following reaction:
The nitrate reductases have been purified from soybean leaves, Neurospora and bacteria like E. coli, Pseudomonas aeruginosa, etc. This enzyme is a complex metalloenzyme that form homodimers. It has binding sites for NAD (P) H and for nitrate.
Three cofactors —FAD, heme-Fe and molybdenum cofactor (MoCo) — form the redox centers that facilitate the chain of electron transfer reactions as follows:
MoCo is a molybdenum ion complexed with an organic molecule pterin, which acts as a metal chelator. Each nitrogen reductase (NR) subunit is 1000 amino acid long and contains all three cofactors.
Most plant NR use NADH, but some use either NADH or NADPH. The genes for nitrate reductase from several higher plants have been cloned. The process is repeated in the further reduction of nitrite through the intermediates, hyponitrite and hydroxylamine, to form NH3.
Each step involves the addition of two electrons by reduced NAD+ (NADP +). This reduction process of NO3 – to NH3 and its incorporation into the cellular proteins by aerobic microorganism and higher plants, is referred to as nitrate assimilation.
Nitrite reductase (NiR) transfers electrons from ferredoxin to nitrite as follows:
NO2 + 6Fdred + 8H+ + 6e → NH4 + 6Fdox + 2H2O
The source of electrons is reduced ferredoxin (Fdred), produced in chloroplasts by photosynthetic noncyclic electron transfer. In non-photosynthetic tissues nitrite reduction also utilizes Fdred in plastids.
Where NADPH produced from oxidative pentose phosphate pathway reduces ferredoxin by an enzyme Fd-NADP+ reductase. The enzyme NiR consists of a single polypeptide containing two prosthetic groups: an iron sulfur center (Fe4S4) and a specialized heme (siroheme).
The enzyme is a nuclear-encoded protein with an N-terminal transit peptide that is cleaved from the mature enzyme. The precursor peptide is targeted to the plastids by the transit peptide. NiR is a monomer of 60 to 70 kDa molecular mass having functional domains and cofactors that shuttle electrons from Fdrcd to nitrite.
The two functional domains are bridged by a sulfur ligand. NiR is regulated transcriptionally in coordination with NR. As nitrite is toxic, cells must contain enough NiR to reduce all the nitrite produced by NR.
The conversion of nitrate and nitrite into ammonia, nitrogen gas and nitrous oxide (N 2O) is called de-nitrification. The process ending in the release of gaseous nitrogen into the atmosphere, completes the nitrogen cycle.
In this process through a series of reactions nitrates are reduced to ammonia and free nitrogen by the anaerobic bacteria like Pseudomonas denitrificans, Bacillus subtilis. Thiobacillus denitrificans.Micrococcus, Azotobacter, Clostridium, etc. These bacteria use NO3 – as an electron acceptor during respiration instead of O2, thus obtaining energy for survival.
(iii) Nitrogen Fixation:
Nitrogen fixation is defined as the conversion of elementary di-nitrogen (N2O) into organic form to make it available for plants. Rotation of leguminous and non-leguminous field crops in the ancient agricultural practice was based on the observation that this process gave a better yield if the non- leguminous crops grew following the leguminous crops.
Nobody, however, realized that the benefit was related to fixation of air nitrogen until Baussingault in 1838 established that the soil fertility increased due to some bacteria found in root nodules of leguminous crops as well as in soil and that these bacteria were capable of fixing atmospheric nitrogen.
Nitrogen fixation is of two types:
Non-biological nitrogen fixation and biological nitrogen fixation.
(a) Non-Biological or Physical Nitrogen Fixation:
Nitrogen is an extremely stable molecule. It exists in di-nitrogen form (N2) having a triple bond (N = N) between two nitrogen atoms. The nitrogen bond has the shortest length of 1.095 Å, the highest ionization potential (15.58 eV) and the highest stretching frequency. For this reason it is highly resistant to chemical attack.
To break this triple bond about 225 kcal of energy is required but it is very difficult to achieve. In the fertilizer industry nitrogen is reduced to ammonia at very high temperature and pressure over an iron catalyst.
Nitrogen may also be fixed through the electrical discharges that occur during lightning. In this process, the atmospheric nitrogen combines with oxygen to produce oxides of nitrogen which are subsequently hydrated by water vapor and carried to earth as nitrites and nitrates by rain.
(b) Biological Nitrogen Fixation:
During the process of evolution some bacterial species have acquired the capacity to reduce nitrogen to ammonia, a process governed by a set of genes called the nitrogen fixation (nlf) genes These species are termed ‘nitrogen-fixing’ organisms. Such organisms include either non-symbiotic microorganisms that can live independently or certain bacteria living in symbiosis with higher plants.
The former group encompasses certain species of heterotrophic bacteria, both aerobic (Azotobacter sp.) and anaerobic (Clostridium sp.); photosynthetic bacteria (Rhodospirillum sp.) and several blue-green algae (Cyanophyta).
The symbiotic system consists of bacteria of the genus Rhizobium together with many members of the family Leguminosae, such as peas, beans, clovers, soybean etc., to form an important nitrogen-fixing cooperative. An essential feature of the symbiotic fixation is the development of nodules on the roots of the plants.