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 2 only from the soil.  N 2 present in the soil can be ultimately tracked back to the atmosphere.  N 2 is very important for plants, as it is a constituent of proteins, nucleic acids and a variety of compounds.  Mostly plants obtain N 2 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  N 2 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 N 2 content of the soil.  This is done because legumes fix the atmospheric N 2 in the soil.

4.  N 2 Cycle involves a series of events around N 2 of the soil and N 2 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 N 2 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 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 2 of the atmosphere is accomplished by 2 methods  1. Lightning or Atmospheric N 2 -fixation(or) Non-biological N 2 fixation  2. Biological Nitrogen Fixation

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

9.  The conversion of N 2 to NH 3 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 NH 4 produced by rhizobium.  An enzyme nitrogenase is responsible for N 2 -fixation.  These 2 methods of BNF are mainly responsible for maintenance of N 2 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 N 2 is 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 3 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 NH 3 to nitrate is 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 3 is 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 2 O + E  2HNO 2 + 2O 2 -----------------→ 2HNO 3 + 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, N 2 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 N 2 reducing property of the extract.  The extract converts N 2 into NH 3.  The researchers also used radio active labelled N 15 in its molecule.  Since then, Dilworth & Schollhorn et al (1966) have discovered that the enzyme nitrogenase reduces not only the N 2 into NH 3 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 N 2 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 2 α - chains are larger and the other 2 β - 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 N 2 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.  3H 2 +N 2 ----  2NH 3 ; Δ G 0 =-33.39/mol  CH 3 NC---------  CH 3 NHCH 3  CH 3 NC-------  CH 3 NH 2 +CH 4  C 2 H 2 + H 2 ---  C 2 H 4  N 2 O+H 2 ----  N 2 +H 2 O

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 2 to 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 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 ( Na 2 S 2 O 4 -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 N 2  Eg: Cyclopropane, HCN, Nitrogen azide, CO are competitive inhibitors 2. Regulatory inhibitors: O 2 and ATP N itself inhibits the Nase axn.

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 3 in the medium  1. NH 3 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 O 2 reduces 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 types of proteins participates in Nase activity namely legHbs & nodulins.  1. Leghaemoglobins : Heme protein- facilitates the free diffusion of O 2 from 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 3 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 O 2 into 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 2 fixers).  They fix the N 2 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 N 2 to NH 3, which is converted into some intermediate metabolites in plant cells.  These N -containing compounds directly metabolized from the NH 3 are called Urides.  The microbial cells freely convert the N 2 into NH 3 which readily diffuses into the plant cells of root nodules.  The cells of root nodule consume NH 3 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 18x10 6 daltons  It is 24x10 3 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.