Nitrogen cycle

nitrogen

1. M.Prasad Naidu
MSc Medical Biochemistry,
Ph.D.Research Scholar

2. Nitrogen is abundantly present (78%) in the
atmosphere.
But green plants can not utilize the atmospheric N2
directly.
Plants can take up N
2only from the soil.
N
2present in the soil can be ultimately tracked back to
the atmosphere.
N
2is very important for plants, as it is a constituent of
proteins, nucleic acids and a variety of compounds.
Mostly plants obtain N
2from the soil as nitrates and
ammonium salts.
As plants continuously absorb nitrate and ammonium
salts, the soil gets depleted of fixed nitrogen.

3. Besides this the leaching effect of rain and denitrifying
action of some bacteria lower the nitrogen content of
the soil.
This loss is compensated by the processes of lightning
and nitrogen fixation
N
2is supplied in the form of fertilizers to agricultural
crops.
The crop rotation with cereals and legumes has been
practiced for a long time to increase the N
2content of
the soil.
This is done because legumes fix the atmospheric N
2in
the soil.

4. N
2Cycle involves a series of events around N
2
of the soil and N
2of atmosphere. These events
include
1. Nitrogen fixation
2. Ammonification and
3. Nitrification

5. Wilfrath and Hellreigal first discovered the fact
that legumes fix the atmospheric nitrogen in
the soil.
The fixed N
2is directly consumed by cereals
during crop-rotation.
Beijerinck in 1922 first isolated the bacteria
from the root nodules of leguminous plants
and named it Rhizobium leguminosarum.

6. Later a large number of organisms were
reported for their N
2-fixing capacity.
The research workers of the Central Research
Laboratory in the USA first isolated an enzyme
nitrogenase from the bacteria Closteridium
pasieurianum in the year 1960.
Later, in 1966 Dilworth and Schollhorn
discovered the activities of nitrogenase in N
2
fixation.

7. The conversion of molecular N
2of the
atmosphere is accomplished by 2 methods
1. Lightning or Atmospheric N
2-fixation(or)
Non-biological N
2fixation
2. Biological Nitrogen Fixation

8. Non-biological N
2fixation
During lightning N
2will be oxidized to HNO
2.
These oxides are carried to the ground by rain
and deposited as HNO
2or HNO
3.
This method of N
2-fixation is very small.

9. The conversion of N
2to NH
3is called BNF.( brought about by
asymbiotic and symbiotic micro organisms.
Asymbiotic micro organisms are free living bacteria and
Cyanobacteria (blue green algae )
Symbiotic bacteria namely Rhizobium are associated with root
nodules of leguminous plants.
Legumes are capable of utilizing the NH
4produced by
rhizobium.
An enzyme nitrogenase is responsible for N
2-fixation.
These 2 methods of BNF are mainly responsible for maintenance
of N
2content in the soil.

10. Plants synthesize organic nitrogenous compounds with the help of
ammonium or nitrate.
After the death of plants and animals, the nitrogenous compounds are
broken down into a number of simpler substances.
In this process most of the N
2is released as NH
3. This process is called
ammonification.
It is due to the activity of bacteria(Bacillus ramosus, B.vulgaris,
B.mycoides), actinomycetes and fungi(Penicillium.sp., Aspergillus sp.,).
The quantity of NH
3formed depends on these factors:
1. The type of ammonifying organism involved,
2. Soil acidity, soil aeration and moisture content,
3. The chemical composition of the nitrogenous material and
4. The supply of carbohydrates.

11. The process of oxidation of NH
3to nitrateis known as
nitrification.
Nitrification requires well aerated soil rich in CaCO
3, a temp.
below 300C, a neutral P
H
and absence of organic matter.
The bacteria involved in this process are called nitrifying bacteria.
Nitrification is carried out in 2 steps.
In the first step NH
3is oxidized to nitrite and is carried out by
nitrosomonas.
In the second step, nitrite is converted into nitrate by the action of
nitrobacter.
2NH
3+ 3O
2--------------→ 2HNO
2+ 2H
2O + E
2HNO
2+ 2O
2-----------------→ 2HNO
3+ energy

12. Conversion of nitrate to molecular nitrogenis called
denitrification. This is the reverse process of
nitrification. i.e.,
Nitrate is reduced to nitrites and then to nitrogen gas.
This process occurs in waterlogged soils but not in well
aerated cultivated soils.
Anaerobic bacteria. Eg. Pseudomonas denitrificans,
Thiobacillus denitrificans.

13. Nitrogen is a highly un reactivemolecule, which
generally requires red-hot Mg for its reduction.
But under physiological temperature, N
2is made into
its reactive form by an enzyme catalyst, nitrogenase.
The research workers of Central Research Laboratory
first isolated the enzyme from the bacteria C.
pasieurianum.
They are the bacteria inhabiting the soil; they prefer
anerobic environmentfor their proper growth and
development.

14. The researchers prepared the extractof these bacteria
and searched for the N
2reducing property of the
extract.
The extract converts N
2into NH
3.
The researchers also used radio active labelled N
15
in
its molecule.
Since then, Dilworth& Schollhornet al (1966) have
discovered that the enzyme nitrogenase reduces not
only the N
2into NH
3but also acetylene into ethylene.
The ethylene is measured by using gas
chromatographic methods.

15. The isolated & purified Nitrogenase enzyme is made of 2
protein units.
The absence of any one of these protein units from the
nitrogenase causes the failure of N
2reduction.
Of the two sub-units one is largerand the other is
smaller.
The largersub-unit is called Mo-Fe proteinand the
smallersub-unit is called ferrus protein.

16. The larger sub-unit consists of 4 PP chains,
(Mol.Wt.200,000 to 245,000 dts)
Of the 4 PP chains 2α-chains are largerand the
other 2β-are slightly smaller.
The 2 PP chains of each pair are identical in
structure

17. It contains 1-2 Mbatoms, 12-32 Featoms and equalno.
of Satoms.
Some of the ferrous & Sulfur atoms are arranged in 4+4
clusters, while the others have different arrangements
such as Fe-Fe covalent linkage, 2Fe-Mo covalent
linkageand Fe-Mo covalent linkage.
Mo-Fe Protein subunit participates in the N
2reduction
hence the name nitrogenase.
It also contains Fe-Mo co-factor which consists of 7
ferrous atoms per Mo atom.

18. Transfers e-from Ferridoxin / Flavodoxin to
nitrogenase
Consists of 2 smaller PP chains.
Mol.wt 60,000 to 60,700 dts
2 PP chains are more or less identical
Each PP contains 4 iron & 4 Sulfurs.
It catalyses the binding of Mg-ATP with the protein.
The nitrogenase is a binary enzyme.
The nitrogenase differs from one source to the other in
size, structure and activities.

19. Besides the N reduction, Nase also reduces
acetylene, hydrozen azides, nitrous oxides,
cyclopropane, etc.
3H
2+N
2----2NH
3; ΔG
0
=-33.39/mol
CH
3NC---------CH
3NHCH
3
CH
3NC-------CH
3NH
2+CH
4
C
2H
2+ H
2---C
2H
4
N
2O+H
2----N
2+H
2O

20. Nase needs ATP for activation (the rate of Nase axn increases with
the conc of ATP in the cells)
ATP is hydrolysed to yield E which is used in N reduction
Under invitro conditions, Nase needs 12-15 ATPs to reduce one
molecule of N
2to NH
3
The e-released from ATP molecules move from nitrogenase
reductase to nitrogenase and the subunits readily dissociates from
each other.
ATP does not react directly with Nase alone, it reacts with Mg
2+
to
form Nase reductase MgATP complex(participates in e-transfer)

21. 2 typesof e-donors or reductants are found in N-fixing
organisms.
1.Ferridoxins 2. Flavodoxins
They serve as e-donors to activate Nase during the N
reduction
Ferridoxins(5600-24000)
Flavodoxins(14000-22800)dts
In azotobacter & Blue green algae NADPH serves as an
e-donor.
Under invitro conditions, Sodiumdithionite (Na
2S
2O
4
-2
)
is used as e-donor.

22. 2groupsofinhibitorswhichinhibittheactivityofNase
1.Classicalinhibitors:includediffkindsofsubstrates
whichcompetefortheNaseagainstN
2
Eg:Cyclopropane,HCN,Nitrogenazide,
COarecompetitiveinhibitors
2.Regulatoryinhibitors:O
2andATP
NitselfinhibitstheNaseaxn.

23. The addition of NH
3( in the form of ammonium salts)
induces rapid growth of N fixing micro organisms,
while it reduces the rate of N fixation.
The Nase has the following responses towards NH
3in
the medium
1. NH
3simply switches off the Nase activity
2. It inhibits the production of Nase enzyme
3. It may reduce both Nase production and Nase
action.

24. The high conc of O
2reduces the activity of
Nase enzymes.
It oxidizes Fe-S clusters of the Nase
When the enzymes are exposed to air (O
2), it
induces the denaturation of the enzyme within
10 min or even within a min.

25. The increased conc of H in the cell inhibits the
activity of Nase enzyme.
The enzyme directly starts to reduce the
Hydrogen ions into Hydrogen
During this reduction some amt of E is released
This E inhibits the Nase activity.

26. Nase also requires some globular pro for its normal N
reducing activity.
2 typesof proteins participates in Nase activity namely
legHbs & nodulins.
1. Leghaemoglobins: Heme protein-facilitates the free
diffusion of O
2from the cytoplasm –it creates
anaerobic environment for the axn of Nase.–1
st
isolated from the root nodules of legumes.

27. Another globular protein found in the root nodules of
plants infected with Rhizobium.
It is produced before the root nodule starts to fix the N
from the atmosphere.
Facilitates the proper utilization of NH
3released
during N fixation.
Induces activation of a no of enzymes like uricase,
glutamine synthetase, ribokinase

28. The aerobic mos produce carbohydrates
especially polysaccharides.
PSs hinder the free diffusion of O
2into cells.
PSs pretect the Nase against the oxidizing
property of O
2.
Thus the PS permit the Nase activity in aerobic
micro organisms.
The aerobic mos also have some adaptations
for the protection of Nase against the
damaging agencies in the cell.

29. Enzyme protein association
Rapid respiratory metabolism
Association with rapid oxygen consumers
Association with acid lovers
Time specific Nase activity
Protection through colonization of bacteria
Special separation of the N2 fixing system

30. Anaerobic microbes actively reduce N into
NH3
This NH3 is widely used in the metabolism of
plants.
In general, Nase is denatured when it is
exposed to the O2 present in the atmosphere
But the Nase of Closteridium shows high rate
of tolerance of O2.
So the organisms like Closteridium fix N2 even
under aerobic condition.

31. Microbes ---fix N
2-----in association with the roots of
higher plants.( symbiotic N
2fixers).
They fix the N
2either under aerobic / anerobic
Eg: Rhizobium leguminosarum, R. japonicum,
R.trifolli, etc,
They invade the roots of leguminous plants and non-
leguminous plants like Frankia, Casurina etc, for their
growth & multiplication
After the establishment of symbiotic association, they
start to fix the atmosphere N in the soil.

32. 1. Soil moisture:-moderate( ↑ and ↓ moisture of the
soil reduce the rate of N fixation in soil)
2. Effect of Drought:-the increased water deficiency
causes decrease in the conc of legHb in the root
nodules. (↓N fixation)
3. Oxygen tension:-↑ O2 tension in the soil causes ↓ in
the rate of N fixation by microbes.
4. Effect of the pH of the soil solution:-
An ↑ in the soil salinity ↓ the rate of N fixation.
5. Light intensity:-In photosynthetic microbes, light
induces a high rate of Photosynthesis resulting in high
rate of N fixation.

33. During N fixation, the microbes reduce the N
2to NH
3,
which is converted into some intermediate metabolites
in plant cells.
These N -containing compounds directly metabolized
from the NH
3are called Urides.
The microbial cells freely convert the N
2into NH
3
which readily diffuses into the plant cells of root
nodules.
The cells of root nodule consume NH
3in the form of
Urea.
They contain a no.of enzymes (glutamine synthetase,
glutamate synthetase, aspartate amino transferase )
which participate in the synthesis of glu, gln, & asp.

34. These compounds may either participate in the synthesis of
nucleic acids / some non protein AAs / AAs like Arg, Gln & Asp.
The purineundergoes oxidation & hydrolysis to yield allantonic
acid & alantoninwhich are readily transferred to the xylem sap of
roots.
The cells synthesize some non protein AAslike homoserine, y-
methylene glutamine, citrulline, canavanine etc which are
transferred to the ….
The glutamate produced is converted to Arg & ….
Gln & Asp are converted to Asn & …..
All the various substances are transported to the various parts of
the plants which utilize them for their cellular metabolism.

35. N-fixation is expressed by the activity of a
group of genes called nif-genes.
Nif-genes are isolated from diff species of
micro organisms ( Klebsiella penumoniae,
Phodopsedomonas, Rhizobium, Azatobacter
vinelandii, Closteridium )
The structure of nif-genes of Klebsiella
pneumoniae was best studied.

36. Stericher et al 1971 isolated
Structurally it is a cluster of genes located in
chromosomal DNA
It consists of 17 genes located in 7 operons.
Mol wt is 18x10
6
daltons
It is 24x10
3
base pairs in its length

37. The genes K and Dencode for the syn of MoFe protein & H
encodes for the syn of Fe protein.
F & J participate in the transfer of e-to the Nase subunit of the
enzyme ( nitrogenase)
N,E & B participate in the syn & processing of Fe-Mo Cofactor
Mparticipates in the processing of Fe-Protein subunits which are
the produts of gene H
S & V are involved in the processing of Mo-Fe protein subunits
Vinfluences the specificity of Mo-Fe protein subunits
A and L are the regulatory genes
Aactivates the transcription of other genes
Lrepresses the transcription of other genes
X & Y are found in the gene map of nif gene cluster, but their
functions are not yet known
Qparticipates in the uptake of Mo during the syn of Nase

38. The genetic regulation of nif-genes was well studied by
introducing a lac Agene into the diff individual
operons of nif genes
Only 2 genes were involved in the expression of nif-
genes viz nif-A and nif-L
The product of nif-A acts as an activator for the
regulation of nif genes
The product of nif-Lrepresses the regulation of nif
genes
They possibly regulate all operons of the nif gene
cluster

39. Besides these 2 regulator genes, some other
genes also participate in the expression of nif-
genes
The gene narD participates in the processing of
Mo during the regulation of nif genes and in
the synthesis of Nase
The unc gene influences the ATP supply for the
regulation & syn of Nase.