NITROGEN CYCLE, METABOLISM AND ASSIMILATION

NITROGEN CYCLE

Nitrogen cycle includes the process of uptake of nitrogen from the environment, its usage by various plants, and returning it back to the environment. In living organisms, is also known as nitrogen metabolism.

First of all, this nitrogen is converted to ammonia. This process is known as ammonification.

N₂—–> NH₃

Further ammonia is converted to nitrites and nitrates, so that plant roots can absorb them. This process is called as nitrification.

NH₃——> NO₂– —–> NO₃^–

These nitrates, nitrates and ammonia are converted back to nitrogen to complete the nitrogen cycle, and this process is known as denitrification.

Ammonification, nitrification and denitrification are collectively known as nitrogen fixation. Nitrogen fixation is always an anaerobic process. It occurs in presence of an enzyme nitrogenase, which is active only under anaerobic conditions. Nitrogenase enzyme is sensitive to oxygen.

Nitrogen fixation can be of two types:

Physical nitrogen fixation –

Only 10% nitrogen fixation occurs via physical process.

This type of nitrogen fixation occurs via a physical method like lightning, thunder, photochemical reaction etc. In this, Nitrogen from atmosphere combines with atmospheric oxygen to form nitric oxide.

N₂ + O₂—->2NO

Then this NO further combines with water vapor of atmosphere to form nitric acid.

NO + H₂O ——-> HNO₃

The nitric acid combines with various salts present in the soil to form nitrates and nitrates.

Biological nitrogen fixation

90% nitrogen is fixed via this method. In this nitrogen fixation occurs with the help of a biological agent/diazotrophs like bacteria, strictly under anaerobic conditions, using the enzyme nitrogenase.

N₂—— (8e^– +8H⁺) —————-> 2NH₃ + H₂ + 16ATP

Qs. Name the enzyme required for biological nitrogen fixation. Describe it.

As. Nitrogenase.

Nitrogenase enzyme is a complex of two subunits: Dinitrogenase reductase and dinitrogenase.

Dinitrogenase reductase	Dinitrogenase.
This is a dimer (of two 30kDa subunits)	This is a tetramer
Two subunits are there joined together by Fe4S4.	Two copies of two subunits are there.
Also called as Fe protein.	Also called as MoFe subunit.

Qs. Name the genes involved in the synthesis of nitrogenase.

As. Nif genes. They also carry out the process of nitrogen fixation.

Qs. Name the genes required for function and regulation of nitrogenase in aerobic nitrogen fixing bacteria.

As. Fix genes.

Qs. What is the site of nitrogen fixation.

As. MoFe cofactor

Qs. What biological agents can fix nitrogen.

As. The biological agents which fix nitrogen are all prokaryotes. These can be bacteria, cyanobacteria, archaea. The bacteria which fix nitrogen are classified into two classes-

Symbiotic nitrogen fixers- these live in symbiotic association with plant, and constitute aerobic prokaryotes. They form nodules in plant. Inside the nodule, environment is anaerobic, which is the compulsory requirement for nitrogen fixation. Nodules can be formed with

Roots of leguminous plants like pea, alfalfa, bean, clover, soybean etc.
Stem of leguminous plants like Sesbania; (By bacteria like e.g. Rhizobium, Bradyrhizobium and Azorhizobium).
Non-leguminous plants like Alnus, Myrica, Casuarina (AMC); by bacteria like Frankia.

Please note that all symbiotic nitrogen fixing bacteria are aerobic; that is why, they form nodules.

Free living nitrogen fixers – these are free living in the soil, and continuously fix nitrogen. These are of two types
Aerobic-

Which form heterocysts like Nostoc, Anaebaena. Heterocysts are single membrane compartments, which are required to maintain anaerobic conditions in aerobic bacteria; they lack Photosystem II, the system which is required to produce oxygen.
Which don’t form heterocysts like Azospirillium, Azotobacter, Pseudomonas(bacteria); Thiobacillus (Archaea). In Azospirillium, nitrogenase is switched off by ammonium ion; this protects nitrogenase from anaerobic environment. In Azotobacter, “significant intensification of metabolism” results in reducing oxygen. It also has a nitrogenase protective system, which protects nitrogenase from oxygen.

Anaerobic

Photosynthetic (also called phototrophs)– which grow due to sunlight e.g. Rhodospirillium, Chlorobium, Chromatium.
Non-photosynthetic (also called chemotrophs) – which grow due to chemical agent e.g. Clostridium, Desulfovibrio, Methanococcus (Archaea)

Qs. How are nodules formed in Rhizobium?

As. Plants secrete chemicals like flavonoids and homoserine. Bacteria in soil are attracted towards these chemicals. Rhicaderin is a plant surface protein which helps in attachment of bacteria and plant.

Nod genes are activated in bacteria as a result of attachment and chemicals.

First of all, rhizobia secrete inactive NodD protein; this protein recognizes flavonoids and is attracted by it. Now NodD protein is activated and it returns back to bacteria; there it induces transcription of Nod A, B and C genes.

Nod genes are of two types:

Common nod genes: include Nod A, B, C. these are present in all Rhizobia strains. These synthesize nod factor, lipo-chito-oligo-saccharides (chitin derivative). These three genes induce root hair curling.

NodA is N-acetyl transferase.

NodB is chitin oligosaccharide deacetylase

Nod C is chitin oligosaccharide synthase.

Host specific nod genes: include Nod P, Q, H /F, E, L. They differ among rhizobia and determine host range.

Bacteria attach to root. From here it forms invagination of its plasma membrane—–> goes into cortex—–> there it enlarge and divide to forms bacteriods (contain rhizobia) —–> which are separated from cytoplasm of the cell and have anaerobic environment—–> nitrogen fixation occurs in bacteroids.

Important: Leghaemoglobin is a haemoglobin derivative which binds to oxygen, and helps in creation of anaerobic environment. Globin of plant and heme of bacterial/rhizobial cell combine to form leghaemoglobin. Leghaemoglobin is present inside cytosol and not inside bacteriods. It gives the nodules a pink color.

Nitrogen assimilation:

Plants absorb nitrogen in the form of ammonium ion, nitrites and nitrates. This nitrogen is further converted in amino acids and finally proteins by active process using H⁺-ATPase pump; this process is known as nitrogen assimilation.

Two enzymes are required for nitrogen assimilation:

Nitrate reductase – converts nitrate to nitrite, in cytosol. In this process NADPH is required.

NO₃— ——->NO₂

This enzyme has two subunits. Each subunit has FAD, Heme (cyt-b557) and Pterin (Mo containing organic molecule) as prosthetic group.

Nitrite reductase – converts nitrite to ammonium ion.

NO₂——-> NH₄⁺

This enzyme has 2 prosthetic groups: one Fe₄S₄, and one Siroheme. This reaction occurs in plastids.

Further it is necessary to convert NH4+ to amino acids to avoid ammonia toxicity. This conversion occurs by two enzymes, glutamine synthetase and glutamate synthetase (also k/a GOGAT).

NH4 ion + glutamate + ATP —— (glutamine synthetase) —-à glutamine + alpha-ketoglutarate + NADH —— (GOGAT) ——–à glutamate

Two types of GOGAT are there:

Accepts electrons from NADH
Accepts electrons from ferredoxin

Further aminotransferases convert glutamate and glutamine into other amino acids.

Reductive amination: one more method of nitrogen assimilation. In this method,

Alpha-ketoglutarate ——–> (glutamate dehydrogenase) ———> glutamate

Glutamate dehydrogenase can use NADH (in mitochondria) or NADPH (in chloroplast).

Qs. Which pump drives the uptake of nitrate into root cells.

Hydrogen pump.