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 N2 only from the soil.
 N2 present in the soil can be ultimately tracked back to
the atmosphere.
 N2 is very important for plants, as it is a constituent of
proteins, nucleic acids and a variety of compounds.
 Mostly plants obtain N2 from 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
 N2 is 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 N2 content of
the soil.
 This is done because legumes fix the atmospheric N2 in
the soil.

4.  N2 Cycle involves a series of events around N2
of the soil and N2 of 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 N2 is 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 N2-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 N2
fixation.

7.  The conversion of molecular N2 of the
atmosphere is accomplished by 2 methods
 1. Lightning or Atmospheric N2-fixation (or)
Non-biological N2 fixation
 2. Biological Nitrogen Fixation

8.  Non-biological N2 fixation
 During lightning N2 will be oxidized to HNO2.
 These oxides are carried to the ground by rain
and deposited as HNO2 or HNO3.
 This method of N2-fixation is very small.

9.  The conversion of N2 to NH3 is 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 NH4 produced by
rhizobium.
 An enzyme nitrogenase is responsible for N2-fixation.
 These 2 methods of BNF are mainly responsible for maintenance
of N2 content 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 N2 is released as NH3. 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 NH3 formed 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 NH3 to nitrate is known as
nitrification.
 Nitrification requires well aerated soil rich in CaCO3, a temp.
below 300C, a neutral PH 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 NH3 is oxidized to nitrite and is carried out by
nitrosomonas.
 In the second step, nitrite is converted into nitrate by the action of
nitrobacter.
 2NH3 + 3O2 --------------→ 2HNO2 + 2H2O + E
 2HNO2 + 2O2 -----------------→ 2HNO3 + energy

12.  Conversion of nitrate to molecular nitrogen is 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 reactive molecule, which
generally requires red-hot Mg for its reduction.
 But under physiological temperature, N2 is 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 environment for their proper growth and
development.

14.  The researchers prepared the extract of these bacteria
and searched for the N2 reducing property of the
extract.
 The extract converts N2 into NH3.
 The researchers also used radio active labelled N15 in
its molecule.
 Since then, Dilworth & Schollhorn et al (1966) have
discovered that the enzyme nitrogenase reduces not
only the N2 into NH3 but 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 N2 reduction.
 Of the two sub-units one is larger and the other is
smaller.
 The larger sub-unit is called Mo-Fe protein and the
smaller sub-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 larger and the
other 2β- are slightly smaller.
 The 2 PP chains of each pair are identical in
structure

17.  It contains 1-2 Mb atoms, 12-32 Fe atoms and equal no.
of S atoms.
 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
linkage and Fe-Mo covalent linkage.
 Mo-Fe Protein subunit participates in the N2 reduction
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.
 3H2+N2----2NH3; ΔG0=-33.39/mol
 CH3NC--------- CH3NHCH3
 CH3NC------- CH3NH2+CH4
 C2H2 + H2--- C2H4
 N2O+H2---- N2+H2O

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 N2 to NH3
 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 Mg2+ to
form Nase reductase MgATP complex (participates in e- transfer)

21.  2 types of 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 (Na2S2O4
-2)
is used as e- donor.

22.  2 groups of inhibitors which inhibit the activity of Nase
 1. Classical inhibitors: include diff kinds of substrates
which compete for the Nase against N2
 Eg: Cyclopropane, HCN, Nitrogen azide,
CO are competitive inhibitors
2. Regulatory inhibitors: O2 and ATP
N itself inhibits the Nase axn.

23.  The addition of NH3 ( 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 NH3 in
the medium
 1. NH3 simply 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 O2 reduces the activity of
Nase enzymes.
 It oxidizes Fe-S clusters of the Nase
 When the enzymes are exposed to air (O2), 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 types of proteins participates in Nase activity namely
legHbs & nodulins.
 1. Leghaemoglobins: Heme protein- facilitates the free
diffusion of O2 from the cytoplasm – it creates
anaerobic environment for the axn of Nase.– 1st
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 NH3 released
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 O2 into cells.
 PSs pretect the Nase against the oxidizing
property of O2.
 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 N2 -----in association with the roots of
higher plants.( symbiotic N2 fixers).
 They fix the N2 either 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 N2 to NH3,
which is converted into some intermediate metabolites
in plant cells.
 These N -containing compounds directly metabolized
from the NH3 are called Urides.
 The microbial cells freely convert the N2 into NH3
which readily diffuses into the plant cells of root
nodules.
 The cells of root nodule consume NH3 in 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 purine undergoes oxidation & hydrolysis to yield allantonic
acid & alantonin which are readily transferred to the xylem sap of
roots.
 The cells synthesize some non protein AAs like 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 18x106 daltons
 It is 24x103 base pairs in its length

37.  The genes K and D encode 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
 M participates 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
 V influences the specificity of Mo-Fe protein subunits
 A and L are the regulatory genes
 A activates the transcription of other genes
 L represses the transcription of other genes
 X & Y are found in the gene map of nif gene cluster, but their
functions are not yet known
 Q participates in the uptake of Mo during the syn of Nase

38.  The genetic regulation of nif-genes was well studied by
introducing a lac A gene 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-L represses 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.