Nitrogen fixation is the process by which nitrogen is converted from its stable dinitrogen form in the atmosphere into ammonia. This process is essential because plants cannot use atmospheric nitrogen. It is carried out by nitrogen-fixing bacteria that contain the nitrogenase enzyme complex. There are two types of biological nitrogen fixation - symbiotic fixation occurs through root nodules in legumes formed via their association with Rhizobia bacteria, and asymbiotic fixation by free-living bacteria and cyanobacteria in soil. Nitrogen fixation requires a large amount of energy, so it is tightly regulated by various mechanisms at the genetic level and through feedback inhibition when fixed nitrogen is abundant.

1. Nitrogen fixation
BY
MANDARA V M
II YR. III SEM MSC BIOCHEMISTRY
M S C W

2. Contents :
• Introduction
• Nitrogen fixation
• Non – biological nitrogen fixation
• Biological nitrogen fixation
• Nitrogenase complex
• Symbiotic nitrogen fixation
• Asymbiotic nitrogen fixation
• Energitics and regulation
• Conclusion
• References

3. Introduction:
• Nitrogen is an essential constituent of all biomolecules both
in plants and in animals
• Most of the plants obtain nitrogen from soil in the form of
nitrate or ammonium ion, but it is limited
• Atmosphere consists 78% of molecular nitrogen but plants
unable to convert this molecular nitrogen into a useful form
because the lack the enzyme nitrogenase
• Only prokaryote species posses this enzyme

4. • 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
• Ammonification
• Nitrification
• Denitrification

5. Nitrogen fixation:
• The process of reducing dinitrogen to ammonia so that
plants can absorb nitrogen is known as nitrogen
fixation
• Types of nitrogen fixation

6. Non – biological nitrogen fixation:
• Nitrogen may be fixed by the electrical discharge of lightning in the
atmosphere
• The nitrous oxide formed combines with oxygen to form nitric oxide
• Nitric oxide readily dissolves in water to produce nitric and nitrous acids
• These acids readily release the hydrogen, forming nitrate and nitrite ions.
• The nitrate can be readily utilized by plants and microorganisms.

7. Biological nitrogen fixation :
• It’s a prokaryote domain because of the presence 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
• Ammonia thus produced is incorporated either into glutamate by
glutamate dehydrogenase or into glutamine by glutamine
synthetase

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

9. 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 protiens
• 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 centre 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

10. • Its redox centres has 2 MO, 32 Fe and 30 S per tetramer
• And it has 2 binding site for reductase
• About half of the iron and sulphur is present as 2 bridged pairs of 4Fe
– 4S centres called as P cluster
• P cluster – consists 2[ 4Fe – 4S] clusters linked through additional
sulphide ion

11. Action of nitrogenase enzyme :
• For reducing nitrogen into ammonia nitrogenase
requires 8 electrons
• At first dinitrogense is reduced by transferring of
electrons to dintrogenase reductase
• Dinitrogenase has a 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 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

12. Symbiotic nitrogen fixation :
• Symbiotic nitrogen fixation occurs in plants that harbour 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 form the root nodules in legumes
• Root nodules – it is a enlarged multicellular structures on roots
• Legume – rhizobium association will fix 25 – 60 kg of molecular
nitrogen annually

13. Root nodule formation :

14. Asymbiotic nitrogen fixation:
• The free living nitrogen fixing organisms are called are
asymbiotic – 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 (non heterocystous) -Oscillatoria
• Filamentous (heterocystous) – Nostoc, Anabaena

15. Energitics and regulation :
• Nitrogen fixation is energetically costly – because it requires
16 ATPs to reduce one molecule of nitrogen
• 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

16. • Transcriptional level regulation
• Here the Nif genes are mainly regulated
• Nif genes - are the genes encode for nitrogenase enzyme
• Regulation of nif genes transcription is done by the nitrogen
sensitive NifA protein
• When there isn'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: NifL
• NifL inhibits NifA activity resulting in the inhibition of
nitrogenase formation
• NifL 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

17. • 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 association of Fe
protein with MO – Fe protein
• Thus it results in regulating the nitrogen fixation

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

19. References :
• Introduction to plant physiology – Hopkins .W, Hunter. N
• Lehninger principles of Biochemistry – Neslon, Cox
• Biochemistry – Donald Voet, Judith G. Voet
• Nitrogen fixation – Wikipedia