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Nitrogen Cycle

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Title: Nitrogen Cycle


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

2
INTRODUCTION
  • 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
INTRODUCTION
  • 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
NITROGEN CYCLE
  • 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
DISCOVERY OF N2 FIXATION
  • 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
DISCOVERY OF N2 FIXATION
  • 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
NITROGEN FIXATION
  • 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
Lightning/Atmospheric N2 fixation
  • 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
Biological N2-fixation
  • 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
AMMONIFICATION
  • 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
NITRIFICATION
  • 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
DENITRIFICATION
  • 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
NITROGENASE COMPLEX
  • 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
NITROGENASE COMPLEX
  • 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
Structure of Nitrogenase Complex
  • 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
Structure of Nitrogenase Complex
  • The larger sub-unit consists of 4 PP chains,
    (Mol.Wt.200,000 to 245,000 dts)
  • Of the 4 PP chains 2a- chains are larger and the
    other 2ß- are slightly smaller.
  • The 2 PP chains of each pair are identical in
    structure

17
Mo-Fe Protein (component I / Nitrogenase)
  • 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 44 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
Smaller subunit ( Coponent II / Nitrogenase
reductase / Fe protein)
  • 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
Substrates for the axn of Nase
  • Besides the N reduction, Nase also reduces
    acetylene, hydrozen azides, nitrous oxides,
    cyclopropane, etc.
  • 3H2N2----?2NH3 ?G0-33.39/mol
  • CH3NC---------? CH3NHCH3
  • CH3NC-------? CH3NH2CH4
  • C2H2 H2---? C2H4
  • N2OH2----? N2H2O

20
Energy supply for Nase axn
  • 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
e- donors for the axn of Nase
  • 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
Role of inhibitors in Nase axn
  • 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
N substrates their effects on Nase axn
  • 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
Effect of O2 conc on Nase axn
  • 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
Effect of H conc on Nase axn
  • 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
Role of proteins in Nase activity
  • 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
Nodulins
  • 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
Aerobic N fixation
  • 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
The important adaptations
  • 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 N2 fixation
  • 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
Symbiotic N fixation
  • 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
Effect of field factors on N fixation
  • 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
Uride metabolism
  • 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
Uride metabolism
  • 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
Genetics of N- fixing genes
  • 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
Structure of Nif gene cluster(Klebsiella
pneumoniae)
  • 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
Functions of different Nif- genes
  • 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
Regulation of Nif genes
  • 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
Regulation of Nif genes
  • 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.
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