Nitrogen fixation

Contents :

Nitrogen fixation

  • Introduction
  • Nitrogen fixation
  • Non – biological nitrogen fixation
  • Biological nitrogen fixation
  • Nitrogenase complex
  • Symbiotic nitrogen fixation
  • Asymbiotic nitrogen fixation
  • Energetics and regulation
  • Conclusion
  • References



  • Nitrogen is an essential constituent of all biomolecules both in plants and in animals
  • Most of the plants obtain nitrogen from the soil in the form of nitrate or ammonium ion, but it is limited
  • The atmosphere consists of 78% of molecular nitrogen but plants unable to convert this molecular nitrogen into a useful form because of the lack the enzyme nitrogenase
  • Only prokaryote species possess this enzyme
  • Nitrogen fixation is the first step of nitrogen cycle where molecular nitrogen is reduced by nitrogen-fixing bacteria to yield ammonia
  • Nitrogen cycle has 3 process

Nitrogen fixation

  • Ammonification
  • Nitrification
  • Denitrification

Nitrogen fixation:


  • The process of reducing dinitrogen to ammonia so that plants can absorb nitrogen is known as nitrogen fixation

Types of nitrogen fixation

Nitrogen fixation


Non – biological nitrogen fixation:


  • Nitrogen may be fixed by the electrical discharge of lightning in the atmosphere

Nitrogen fixation 1

  • The nitrous oxide formed combines with oxygen to form nitric oxide

Nitrogen fixation 2

  • Nitric oxide readily dissolves in water to produce nitric and nitrous acids

Nitrogen fixation 3

  • These acids readily release the hydrogen, forming nitrate and nitrite ions.
  • The nitrate can be readily utilized by plants and microorganisms.

Nitrogen fixation 4

Biological nitrogen fixation :


  • It’s a prokaryote domain because of the presence of nitrogenase enzyme
  • prokaryotes which fix nitrogen – nitrogen fixers
  • It includes both free-living and symbiotic associations with plants
  • Diazatrophs – certain strains of bacteria of genus Rhizobium that shows symbiotic association with legumes via root nodules
  • These species convert molecular nitrogen to ammonia

Nitrogen fixation 5

  • Ammonia thus produced is incorporated either into glutamate by glutamate dehydrogenase or into glutamine by glutamine synthetase

Basic requirements of nitrogen fixation:


  • Basic requirements for Nitrogen fixation are
  • Nitrogenase enzyme complex
  • Protective mechanism against Oxygen – leghaemoglobin
  • Ferredoxin
  • Hydrogen releasing system or electron donor (Pyruvic acid )
  • The constant supply of ATP
  • Coenzymes and cofactors like CoA, inorganic phosphate and Mg+2
  • Cobalt and Molybdenum

Nitrogenase complex :


  • Biological nitrogen fixation is carried out by a highly conserved complex of proteins called as nitrogenase complex
  • Which is mainly consists of 2 important proteins
  • Fe protein (dinitrogenase reductase)
  • Mo-Fe protein (dinitrogenase)
  • Structure
  • Dinitrogenase reductase – is a dimer of 2 identical subunits
  • It contains a single 4Fe – 4S redox center bound between the subunits
  • This can be oxidized and reduced by 1 electron
  • Also, it has 2 binding sites for ATP or ADP
  • Dinitrogenase – is a tetramer with 2 copies of 2 different subunits (𝜶𝟐 − 𝜷𝟐 heterodimer)
  • Contains both iron and molybdenum
  • Its redox centers have 2 MO, 32 Fe and 30 S per tetramer
  • And it has 2 binding sites for reductase
  • About half of the iron and sulfur is present as 2 bridged pairs of 4Fe – 4S centers called  as P cluster
  • P cluster – consists of 2[ 4Fe – 4S] clusters linked through additional sulfide ion

The action of the nitrogenase enzyme :


Nitrogen fixation

  • For reducing nitrogen into ammonia nitrogenase requires 8 electrons
  • At first, dinitrogen is reduced by transferring of electrons to dintrogenase reductase
  • Dinitrogenase has 2 binding sites for reductase
  • The 8 electrons are transferred from reductase to dinitrigenase one at a time :
  • Reduced reductase binds to dinitrogenase and transfers a single electron, oxidized form dissociates in a repeating cycle
  • Each cycle requires the hydrolysis of ATP molecule by dinitrogenase reductase
  • Immediate source of electrons to reduce reductase is reduced ferredoxin
  • Ultimate source of electrons to reduce ferredoxin is pyruvate

Nitrogen fixation

Symbiotic nitrogen fixation :


  • Symbiotic nitrogen fixation occurs in plants that harbor nitrogen-fixing bacteria within their tissues
  • The best-studied example is the symbiotic association between roots of legumes and bacteria of the genus Rhizobium
  • This association results from the root nodules in legumes
  • Root nodules – it is an enlarged multicellular structure on roots

Legume – rhizobium association will fix 25 – 60 kg of molecular nitrogen annually

Root nodule formation :

Nitrogen fixation

Asymbiotic nitrogen fixation:


Nitrogen fixation

Asymbiotic nitrogen fixation:


  • The free-living nitrogen-fixing organisms are called are symbiotic – organisms.
  • It includes Aerobic bacteria, anaerobic bacteria, and blue-green algae
  • Bacteria: types –
  • Free-living aerobic: Azotobacter
  • Free-living anaerobic: Clostridium
  • Blue-green algae: types –
  • Filamentous (in heterocystous) -Oscillatoria
  • Filamentous (heterocystous) – Nostoc, Anabaena

Energetics and regulation :


  • Nitrogen fixation is energetically costly – because it requires

16 ATPs to reduce one molecule of nitrogen


Nitrogen fixation 6

  • Thus to avoid this wastage of energy it must be regulated
  • When soil nitrogen (NO3 or NH4) levels are high, the formation of nodules is inhibited thus regulating nitrogen fixation
  • Some of the inhibitors also regulate nitrogen fixation – hydrogen, nitrous oxide, and nitric oxide
  • Nitrogenase enzyme also regulated at its genetic level
  • It is achieved by transcriptional level modification
  • Transcriptional level regulation
  • Here the Nif genes are mainly regulated
  • If genes – are the genes encode for nitrogenase enzyme
  • Regulation of nif genes transcription is done by the nitrogen sensitive NifA protein
  • When there aren’t enough fixed nitrogen available NtrC triggers NifA expression
  • And NifA activates the rest of the Nif genes
  • If there is a sufficient amount of reduced nitrogen or oxygen is present, another protein is activated: NFL
  • NFL inhibits NifA activity resulting in the inhibition of nitrogenase formation
  • NFL is regulated by the products of glnB and glnK
  • The Nif genes can be found on bacterial chromosomes, but in symbiotic bacteria, they are often found on plasmids
  • Post-translational regulation
  • In free-living diazotrophs, this is the additional level of nitrogenase regulation
  • During energy limiting or nitrogen sufficient condition, the nitrogenase complex is rapidly, reversibly inactivated by ADP – ribosylation of Fe protein
  • It occurs at a specific arginine residue. i.e. Arg 101
  • The presence of ADP ribose group prevents the association of Fe protein with MO – Fe protein
  • Thus it results in regulating the nitrogen fixation

Nitrogen fixation

Conclusion :


  • Nitrogen is a limiting nutrient for plants, even though molecular nitrogen is readily available in the atmosphere
  • Plants do not have the nitrogenase enzyme thus have to depend on prokaryotes to absorb nitrogen
  • Nitrogen fixation is the first step of the nitrogen cycle and it is of two types biological and nonbiological nitrogen fixation
  • Biological nitrogen fixation occurs in two ways i. e symbiotic, where it involves the prokaryotic interaction with plants via root nodules and a symbiotic where free-living microorganisms fix the atmospheric nitrogen into the soil
  • Nonbiological nitrogen fixation involves the lighting process to fix atmospheric nitrogen into the soil
  • Because of the high demand for energy, this nitrogen fixation is tightly regulated by various methods

References :

  • Introduction to plant physiology – Hopkins .W, Hunter. N
  • Lehninger Principles of Biochemistry – Nelson, Cox
  • Biochemistry – Donald Voet, Judith G. Voet

Nitrogen fixation – Wikipedia

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