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
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
