Biochemistry,  Biology


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All the living organisms are basically composed of carbon, hydrogen, oxygen, nitrogen and many other forms of chemical elements. These elements contribute to finally organize various biomolecules present in a cell. Nitrogen is next to carbon in importance in living organisms. In a living cell, nitrogen is an important constituent of amino acids, proteins, enzymes, vitamins, alkaloids and some growth hormones. Therefore, the study of nitrogen metabolism is absolutely essential because the entire life process is dependent on these nitrogen-containing molecules.

Molecular Nitrogen

Nitrogen is primarily present in the atmosphere freely as dinitrogen or nitrogen gas. It is present in the combined form as Chile saltpetre or sodium nitrate and Chile in South America is the major source of this nitrate nitrogen.

Molecular Nitrogen or diatomic nitrogen (N2) is highly stable as it is triple bonded (N≡N). Because of this stability, molecular nitrogen as such is not very reactive in the atmosphere under normal conditions. In the atmosphere, molecular nitrogen is 78.03% by volume and it has a very low boiling point (-195.8oC) which is even lower than that of oxygen. Proteins present in living organisms contain about 16% nitrogen.

Nitrogen Cycle

Nitrogen is an essential constituent of living beings. Nitrogenous bases are part of nucleic acids and proteins are made up of amino acids of which Nitrogen is an important constituent. You already know about the importance of these two biomolecules.

Air has 78% N2 but most of the living beings cannot utilize this atmospheric Nitrogen. Nitrogen cycle converts this nitrogen into a usable form. Lightning fixes Nitrogen to NH3, and nitrogen-fixing bacteria like Rhizobium (which live in roots of leguminous plants like pea, beans, pulses etc.) also convert N2 into NH3. Most plants absorb nitrates from soil and reduce it to NH3 in the cells for further metabolic reactions. Dead organisms and their excreta like urea are decomposed by bacteria into NH3 and by a different set of bacteria into nitrates. These are left in the soil for use by plants. In this way, Nitrogen cycle is self-regulated but human activities have caused steady loss of soil Nitrogen.

Nitrogen Fixation (Biological and Abiological)

The conversion of molecular nitrogen into compounds of nitrogen especially ammonia is called nitrogen fixation. Nitrogen fixation is a reductive process i.e., nitrogen fixation will stop if there is no reducing condition or if oxygen is present. This nitrogen fixation may take place by two different methods – abiological and biological.

Abiological Nitrogen Fixation

In abiological nitrogen fixation, the nitrogen is reduced to ammonia without involving any living cell. Abiological fixation can be of two types: industrial and natural. For example, in Haber’s process, synthetic ammonia is produced by passing a mixture of nitrogen and hydrogen through a bed of catalyst (iron oxides) at a very high temperature and pressure.

This is industrial fixation wherein nitrogen gets reduced to ammonia.

In natural process, nitrogen can be fixed especially during electrical discharges in the atmosphere. It may occur during lightning storms when nitrogen in the atmosphere can combine with oxygen to form oxides of nitrogen

These oxides of nitrogen may be hydrated and trickle down to earth as combined with nitrite and nitrate.

Biological Nitrogen Fixation

Chemically, this process is same as abiological. Biological nitrogen fixation is reduction of molecular nitrogen to ammonia by a living cell in the presence of enzymes called nitrogenases.

Nitrogen fixation by free-living organisms and symbiotic nitrogen fixation

Nitrogen fixation is a distinctive property possessed by a select group of organisms, because of the presence of the enzyme nitrogenase in them.

The process of nitrogen fixation is primarily confined to microbial cells like bacteria and cyanobacteria. These microorganisms may be independent and free living.

ClostridiumAnaerobic bacteria (Non-photosynthetic)
KlebsiellaFacultative bacteria (Non-photosynthetic)
AzotobacterAerobic bacteria (Non-photosynthetic)
RhodospirillumPurple, non-sulphur bacteria (Photosynthetic)
AnabaenaCyanobacteria (Photosynthetic)

Some microbes may become associated with other organisms and fix nitrogen. The host organism may be a lower plant or higher plant. The host organism and the nitrogen-fixing microbes establish a special relationship called symbiosis and this
results in symbiotic nitrogen fixation

LichensCyanobacteria and Fungus
BryophyteCyanobacteria and Anthoceros
PteridophyteCyanobacteria and Azolla.
GymnospermCyanobacteria and Cycas
AngiospermsLegumes and Rhizobium.
AngiospermsNon-leguminous plants and actinomycete (Such as Alnus, Myrica, Purshia).
AngiospermBrazilian grass (Digitaria), Corn and Azospirillum.

Mechanism of biological fixation of nitrogen

Nitrogen fixation requires

(i) the molecular nitrogen

(ii) a strong reducing power to reduce nitrogen like reduced FAD (Flavin adenine dinucleotide) and reduced NAD (Nicotinamide Adenine Dinucleotide)

(iii) a source of energy (ATP) to transfer hydrogen atoms from NADH2 or FADH2 to dinitrogen and

(iv) enzyme nitrogenase

(v) compound for trapping the ammonia formed since it is toxic to cells.

The reducing agent (NADH2 and FADH2) and ATP are provided by photosynthesis and respiration.

The overall biochemical process involves stepwise reduction of nitrogen to ammonia. The enzyme nitrogenase is a Mo-Fe containing protein and binds with a molecule of nitrogen (N2) at its binding site. This molecule of nitrogen is then acted upon by hydrogen (from the reduced coenzymes) and reduced in a stepwise manner. It first produces diamide (N2H2) then hydrazime (N2H4) and finally ammonia (2NH3).

NH3 is not liberated by the nitrogen fixers. It is toxic to the cells and therefore these fixers combine NH3 with organic acids in the cell and form amino acids. The general equation for nitrogen fixation may be described as follows:

Molecular nitrogen is a very stable molecule. Therefore, sufficient amount of cell energy in the form of ATP is required for stepwise reduction of nitrogen to ammonia.

In legumes, nitrogen fixation occurs in specialized bodies called root nodules. The nodules develop due to interaction between the bacteria Rhizobium and the legume roots. The biochemical steps for nitrogen fixation are same. However, legume nodules possess special protein called LEGHEMOGLOBIN. The synthesis of leghemoglobin is the result of symbiosis because neither bacteria alone nor legume plant alone possesses the protein. Recently it has been shown that a number of host genes are involved to achieve this. In addition to leghemoglobin, a group of proteins called nodulins are also synthesized which help in establishing symbiosis and maintaining nodule functioning.

Leghemoglobin is produced as a result of interaction between the bacterium and legume roots. Apparently, Rhizobium gene codes for Heme part and legume root cell gene codes for Globin moiety. Both the coded products together consitute the final protein leghemoglobin. During N2-fixaion, function of Leghemoglobin is to act as Oxygen-scavenger so that the enzymes, Nitrogenages then, convert N2 to NH3 under anaerobic condition.

Leghemoglobin is considered to lower down the partial pressure of oxygen and helps in nitrogen fixation. However, this function is specific for legumes only because free-living microbes do not possess nitrogen-fixing leghemoglobin. Moreover, it has also not been found in cyanobacterial symbiosis with other plants, which fix N2 under aerobic condition.

Nitrate and ammonia assimilation by plants

As pointed out in the previous section, nitrogen fixation is confined to selected microbes and plants. But all plants require nitrogen because it has a role to play in the general metabolism. Therefore, plants which do not fix nitrogen, use other combined nitrogen sources such as nitrate and ammonia for carrying on metabolic activity.

Nitrate is absorbed by most plants and reduced to ammonia with the help of two different enzymes. The first step conversion of nitrate to nitrite is catalyzed by an enzyme called nitrate reductase. This enzyme has several other important constituents including FAD, cytochrome, NADPH or NADH and molybdenum.

The overall process of nitrate reduction takes place in the cytosol and is an energy-dependent reaction.

The enzyme nitrate reductase has been studied in many plants and it is observed that the enzyme is continuously synthesized and degraded. The enzyme nitrate reductase is inducible. This means that increase in nitrate concentration in the cytosol induces more of nitrate reductase to be synthesized. However, when excess NH4+ is produced then it has a negative effect on the synthesis of nitrate reductase. In plants, it has also been observed that light also increases nitrate reductase when nitrate is available.

In the second step, the nitrite so formed is further reduced to ammonia and this is catalyzed by the enzyme nitrite reductase. Nitrite present in the cytosol is transported into chloroplast or plastids where it is reduced to ammonia.

The enzyme nitrite reductase is able to accept electrons from sources such as NADH, NADPH or FADH2. Besides, reduced ferredoxin has also been shown to provide electrons to nitrite reductase for reducing nitrite to ammonia. Ammonia so formed has to be utilized quickly by plants because accumulation of ammonia has a toxic effect. Some plants including algae leach out excess ammonia which can further be oxidized to nitrite and nitrate by microorganisms in the soil or water.

Amino acid synthesis by plants

As you have noticed that ammonia formation is achieved by plants either by (i.) nitrogen fixation or (ii) by reduction of nitrate to nitrite. Ammonium (NH4+) is the most reduced form of inorganic combined nitrogen. This ammonium now becomes the major source for the production of amino acids, which are the building blocks of enzymes and proteins. Amino acids have two important chemical groups. (i) amino group (NH2) and (ii) carboxy1 group (-COOH).

Ammonium so produced is the major source of amino group. However, the carboxyl group has to be provided by other organic molecule synthesized by the plants. There are two major reactions for amino acid biosynthesis in plants:

Reductive amination reaction

In this reaction, ammonia combines with a keto acid. The most important keto acid is the alpha-ketoglutaric acid produced during the operation of Krebs cycle. The keto acid then undergoes enzymatic reductive amination to produce an amino acid.

It has been noted that reductive amination respresents the major ‘port of entry’ for ammonia into the metabolic stream in plants. This initiates synthesis of glutamic acid followed by other amino acids.

Transamination reaction

This is another very important reaction for amino acid biosynthesis. The reaction involves transfer of amino group, from already synthesized amino acid to the keto acid.

In the above reaction, aspartic acid has transferred its amino group (NH2) to the α-ketoglutaric acid to synthesize glutamic acid and release keto acid. The reaction is catalyzed by enzymes called transaminases. A large number of amino acids are synthesized by this transamination reaction. Amino acids are organic molecules containing nitrogen. The incorporation of amino group, from ammonium, into keto acids represents the major step for synthesis of nitrogenous organic biomolecules.

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