The document discusses nitrogen fixation and the nitrogen cycle. It notes that while nitrogen gas makes up 78% of the atmosphere, plants cannot use it directly and must obtain nitrogen from the soil in the forms of nitrates and ammonium salts. Nitrogen fixation is carried out through both biological and non-biological processes, with biological nitrogen fixation being the primary means of fixing atmospheric nitrogen in the soil through symbiotic bacteria like Rhizobium that form nodules on legume roots. The nitrogenase enzyme is responsible for this nitrogen fixation through an energy-intensive process requiring ATP.

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.