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.