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 environmentfor 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 2α- chains are larger and the other 2β- 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 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. 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. • 3H2+N2----2NH3; ΔG0=-33.39/mol • CH3NC--------- CH3NHCH3 • CH3NC------- CH3NH2+CH4 • C2H2 + H2--- C2H4 • N2O+H2---- N2+H2O

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 allantonicacid & 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-Lrepresses 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.