![Fixation of nitrogen as calcium cyanamide
When N2 gas is passed over heated calcium carbide
at 800-1000oC, a mixture of calcium cyanamide and
carbon is obtained.
CaC2 + N2 [ CaCN2 + C]
Calcium carbide nitrolim
It is used as a fertilizer under the name of nitrolim.
It is added to plants before sowing below the surface
of soil.
It undergoes a series of changes producing
ammonia in the soil.](https://image.slidesharecdn.com/nitrogenase-dr-240330142544-26c15777/85/Nitrogenase-functioning-in-biological-system-9-320.jpg)
![Properties of some representative nitrogenase
Organism Component MW Metal content
Azotobacter vinelandii [MoFe]
[Fe]
234000
64000
2Mo, 34-38Fe, 26-28S
3,4 Fe, 2.8S
Azotobacter chrococum [MoFe]
[Fe]
227000
65400
2Mo, 24Fe, 20S
4Fe, 3.9S
Clostridum pasterurianum [MoFe]
[Fe]
221800
55000
2Mo, 24Fe, 24S
4Fe, 4S
Klebsiella pneumoniae [MoFe]
[Fe]
299000
66800
2Mo, 32Fe, 24S
4Fe, 3.8S
Anabaena cylindrica [MoFe]
[Fe]
223000
60000
2Mo, 20Fe, 20S
Rhodospirillum rubrum [MoFe]
[Fe]
215000
60000
2Mo, 25-30Fe, 19-22S](https://image.slidesharecdn.com/nitrogenase-dr-240330142544-26c15777/85/Nitrogenase-functioning-in-biological-system-19-320.jpg)
Nitrogenase functioning in biological system
- 1. NITROGENASE DR. ALKA GUPTA DEPARTMENT OF CHEMISTRY BRAHMANAND COLLEGE, KANPUR
- 2. NITROGENASE • The third fundamental process in nature that is carried out by living cells in addition to photosynthesis and respiration is nitrogen fixation. • Constituents of the living cell contain nitrogen and act as a reservoir of atmospheric nitrogen are amino acids, nucleic acid, purines, pyrimidines, porphyrins, alkaloids and vitamins. COMPONENTS OF NITROGEN CYCLE Nitrate ion Nitrite ion Hyponitrite ion Nitrogen gas Hydroxyl amine Ammonia NO3 - NO2 - N2O2 2- N2 NH2OH NH3 +5 +3 +1 0 -1 -3 Thus, in nature nitrogen may exist in highly oxidized form (NO3 -) or in highly reduced state (NH3).
- 3. Fig.1 : Nitrogen cycle
- 4. NITROGEN FIXATION The conversion of atmospheric nitrogen into useful nitrogenous compounds by natural or artificial methods is known as fixation of nitrogen. Nitrogen present in various nitrogen compounds is known as combined or fixed nitrogen. Basic requirements of nitrogen fixation Presence of enzyme nitrogenase and hydrogenase. A protective mechanism for the enzyme nitrogenase against O2. A non-heme iron protein-ferredoxin as electron carrier.
- 5. Hydrogen donating system (viz. pyruvate, hydrogen, sucrose, glucose, etc.) A constant supply of ATP Presence of thiamine pyrophosphate (TPP), Coenzyme-A, inorganic phosphates and Mg2+ as cofactors. Presence of cobalt and molybdenum A carbon compound for trapping released ammonia.
- 6. Methods of Nitrogen Fixation (A) Artifical fixation Fixation of nitrogen as nitric acid: N2 and O2 of the air are allowed to combine to form nitric oxide at 3000oC. The equilibrium mixture is suddenly cooled when it combines with more of O2 to form nitrogen dioxide. N2 + O2 2NO – 43.2 K. Cals 2NO + O2 2NO2 It on dissolution in water forms nitric acid which is used for the manufacture of nitrogenous fertilizers. 4NO2 + 2H2O + O2 4HNO3
- 7. Fixation of nitrogen as ammonia and ammonium salts Haber’s Process : A mixture of N2 and H2 in the ratio 1:3 is passed at a pressure of 200-500 atmospheres into a reaction vessel containing finely divided iron as a catalyst and molybdenum as promoter heated to 450-500oC. N2 + 3H2 2 NH3 + 24, K.Cals Conversion of NH3 in to HNO3 by ostwald’s process. The NH3 so obtained can be oxidised to nitric oxide by passing a mixture of NH3 and air in the ratio 1:8 over heated platinum gauze at 800oC. 4 NH3 + 5O2 4 NO + 6H2O
- 8. Then nitric oxide combines with the excess of O2 of the air to form nitrogen dioxide which is absorbed in water in presence of excess of air and gives nitric acid 4 NO + 2O2 4 NO2 4 NO2 + 2H2O + O2 4 HNO3 Now NH3 and HNO3 may be converted into ammonium salts and nitrates which are useful fertilizers.
- 9. Fixation of nitrogen as calcium cyanamide When N2 gas is passed over heated calcium carbide at 800-1000oC, a mixture of calcium cyanamide and carbon is obtained. CaC2 + N2 [ CaCN2 + C] Calcium carbide nitrolim It is used as a fertilizer under the name of nitrolim. It is added to plants before sowing below the surface of soil. It undergoes a series of changes producing ammonia in the soil.
- 10. CaCN2 + H2O + CO2 H2NCN + CaCO3 Calcium cyanamide Cyanamide H2NCN + H2O NH2CONH2 Urea NH2CONH2 + H2O 2NH3 + CO3 Ammonia The ammonia so produced is converted into nitrates (by nitrifying bacteria) which are finally assimilated by plants. Fixation of nitrogen as nitrides When atmospheric nitrogen is passed over red hot oxides of B, Al, Mg or Si and coke, nitrides are obtained.
- 11. B2O3 + 3C + N2 2 BN + 3CO Boron nitride Al2O3 + 3C + N2 2 Al N + 3CO Aluminium nitrid 3MgO + 3C + N2 Mg3 N2 + 3CO Magnesium nitride These nitrides are decomposed by water and NH3 is evolved Al N + 3 H2O Al(OH)3 + NH3 Mg3N2 + 6H2O 3 Mg (OH)2 + 2NH3
- 12. (B) NATURAL FIXATION By Lightning discharges: Under the influence of lightning discharges during thunder clouds N2 and O2 of the air combine together to form nitric oxide N2 + O2 2NO Nitric oxide It is oxidised by excess of O2 to give nitrogen dioxide 2 NO + O2 2 NO2 Nitrogen dioxide
- 13. It reacts with rain water in presence of excess of O2 to produce nitric acid which is washed away by rain and form nitrates with the basic material of the soil and stored in the soil as plant food. H NO2 + 2H2O + O2 4 HNO3 Nitric acid 2HNO3 + CaCO3 Ca(NO3)2 + H2O + CO2
- 14. By Symbiotic bacteria • These bacteria grow in nodules of the plants belonging to the family leguminaced such as peas, beans, gram etc. • These are capable of converting the atmospheric nitrogen into nitrogenous compounds. The enzymes found in bacteroids are called nitrogenase, e.g. Azotobacter, Gleocapsa and Anabaena etc.
- 15. The key enzyme- nitrogenase Recent studies have shown that enzyme nitrogenase consists of two protein components or subunits. They are composed of two metallo proteins which are commonly referred as: Non heme iron protein commonly called Fe- protein or dinitrogen reductase. Iron molybdenum protein called MoFe protein (or dinitrogenase) or MFe (M =Mo, V and Fe) cofactor.
- 16. • The Fe protein component is common to all nitrogenases is a dimer with a single Fe4S4 (ferritins) cluster bound between two equivalent subunits and frequently known as “P cluster” as shown in Fig.2. Fig.2: Model for the structure of the P cluster in nitrogenase.
- 17. The function of Fe-protein components is to react with ATP and reduce MoFe protein, which then reduces N2 to ammonia. •The MoFe protein contains P-cluster and the Fe- Mo co-factor. The structure of Fe-Mo cofactor cluster is shown in Fig.3. Fig.3: Representation of core of Fe-Mo-Cofactor
- 18. The two entities, MoFe3S3 (left) and Fe4S3 (right) are bridged by three sulphur atoms. The Fe-Fe distances between bridged ion sites average 2.5Å, close enough to be considered as metal-metal bonded. Only two protein ligands Cys and His coordinate the cofactor to the protein. Thus nitrogenases react to natural N2 through the metal present in these proteins.
- 19. Properties of some representative nitrogenase Organism Component MW Metal content Azotobacter vinelandii [MoFe] [Fe] 234000 64000 2Mo, 34-38Fe, 26-28S 3,4 Fe, 2.8S Azotobacter chrococum [MoFe] [Fe] 227000 65400 2Mo, 24Fe, 20S 4Fe, 3.9S Clostridum pasterurianum [MoFe] [Fe] 221800 55000 2Mo, 24Fe, 24S 4Fe, 4S Klebsiella pneumoniae [MoFe] [Fe] 299000 66800 2Mo, 32Fe, 24S 4Fe, 3.8S Anabaena cylindrica [MoFe] [Fe] 223000 60000 2Mo, 20Fe, 20S Rhodospirillum rubrum [MoFe] [Fe] 215000 60000 2Mo, 25-30Fe, 19-22S
- 20. Mode of action of nitrogenase The overall reaction is as follows: N2 + 6H+ + 6e- 2NH3 The reduction of nitrogen to ammonia requires at least 6 protons and 6 electrons. Besides, it requires at least 12 molecules of ATP (at least 4 ATP molecules are needed for each pair of electron transferred to N2). Very recently, in view of the fact that N2 fixation is always accompanied by evolution of some H2, the equation is modified as follows: N2 + 8H+ + 8e- 2NH3 + H2 • The enzyme hydrogenase is present in almost all the microbes involved in N2 fixation. The enzyme catalyzes H2 2H+ + 2e–
- 21. BIOLOGICAL NITROGEN FIXATION In contrast to the chemical fixation of nitrogen, biological fixation occurs at one atmosphere pressure and at temperature of living cells in the presence of appropriate enzymes. Biological fixation of nitrogen is accomplished either by non-symbiotic micro-organisms or symbiotic system. Non symbiotic micro-organisms: That can live independently such as aerobic organisms of the soil (e.g. Azotobacter), soil anerobes (e.g. Clostridium sp.) photosynthetic bacteria (e.g. Rhizobium rubium), and cyanobacteria (e.g. Anabaena sp.)
- 22. Symbiotic system: consists of bacteria (Rhizobia) living in symbiosis with members of the Leguminoseae such as clover, alfalfa and soyabeans. Except the legumes about 190 species of shrubs and trees, including the Sierra sweet Bay, Ceanothus and alder are nitrogen fixers. Essential feature of symbiotic fixation The development of nodular tissue on the roots of legumes after infection by a strain of Rhizobia, specific for the given legume. Legume alone is unable to fix nitrogen, free living Rhizobia bacteria can fix N2 only when grown with a limiting supply of organic nitrogen and oxygen.
- 23. Biological nitrogen fixation is carried out by a highly conserved complex of proteins called the nitrogen complex. Two key components of this complex are dinitrogenase reductase and dinitrogenase. Dinitrogenase reductase: It is a dimer of two identical subunits (Mr 60,000. It contains a single Fe4-S4 redox center which can be oxidized and reduced by one electron. It also has two binding sites for ATP. (Mr= relative molecular mass).
- 24. Dinitrogenase: It is a tetramer with two copies of two different sub units (combined Mr 240000). Dinitrogenase contains both iron and molybdenum, and its redox center has a total of 2Mo, 32Fe, and 30S per tetramer. About half of the Fe and S is present as four Fe4-S4 centers and remainder is present as part of a novel iron molybdenum cofactor of unknown structure. Fig.4: Fe4-S4 centers (ferredoxins)
- 25. Process of Nitrogen Fixation • The laguminous plants which have nitrifying bacteria in the nodes of their roots, absorb the N2 from atmosphere • This N2 is reduced to NH3 by employing solar energy in presence of coenzymes mainly ferredoxin e.g. 8Fe-S and Mo/Fe protein known as nitrogenase.
- 26. The conversion of atmospheric nitrogen into its compounds takes place as below: Inhibitors N2 NH3 H2, CO N3 - N2+ NH3 CO N2O N2 + H2O CO Endogenous RCN RCH3 + NH3 CO RNC RNH2, CH4, C2H4, C2H6 CO RCN RCH3 + NH3 CO
- 27. The reduction of N2 is due to endogenous reaction of H2O as 2H2O H3O+ + OH– H3O+ H2O + H+ and at pH 7 N2+ 8H+ + 6e– 2NH4 + + Eo= -280 mV. •The Mo-Fe protein of Azotobacter appears to consists two Mo-containing units. The Fe sites of Mo/Fe protein in which possesses two unique Fe sites, are specific to that protein and to nitrogen fixation. •In oxidised state Fe+n appears to have n = 3 to have high spin configuration. Reduction with HSO3- converts about half the iron to high spin Fe+2 (or Fe+).
- 28. • Mo precipitate in electron transfer process, just as the Fe sites do and it seems that the processes are 2e– in nature at Mo and sulphur remains present in the co-ordination sphere of Mo ( Mo+6 Mo+2, Mo+5 Mo+3) • R.W.F. Hardy et al. proposed that N2 fixation occurs at a Mo-S-Fe site after ATP-4 induced reduction of Mo+n to Mo+(n-2), the possibilities are
- 29. • This scheme amounts for the ATP required reductive diphosphorylation step. The Fe at this site (presumabily Fe+3) is prepared for the entry of N2 by reduction to Fe+. Upon co- ordination of N2 to Fe+, the active site is suggested to be appear as (1a). • (1a) on further reduction by H+ changes into a complex having diazene bridging as (1b).
- 30. • Further one or two electron reduction of this bridged diazene (1b) results in bridging hydrazine (1c) which on further reduction and protonation yields N–N bond and two NH3 molecules as below:
- 31. • In any NO3 or NO2 are formed in plants, these are reduced in presence of nitrate and nitrite reductase enzymes as. NO3 - + NADH +H+ NO2 - + NAD + H2O NO2 + 6e- + 8H+ NH4 ++ 2H2O+ NH3+ H+ The NH3 molecules released during the electron reduction and protonation again form NH4 + which combines with organic acids or ketones present in the plants as.
- 32. • When further NH3 reacts with glutamate or asparte or their acids in presence of ATP, Mg2+ and glutamine synthetase, the amines (e.g. glutamine or aspartagine), which are excellent carries of N2 for protein synthesis are formed as.
- 33. Molybdenum Nitrogenase Nitrogenase is an enzyme involved in the fixation of nitrogen which occurs in bacteria. This enzyme is comprised of two protein chains. (i)The lower molecular weight protein contains on Fe4S4 cluster. (ii)The larger protein, which, itself is tetrameric, involves two molybdenum atoms and large number of iron atoms and sulphide ions. Both proteins are required for activity. Although Fe4S4 clusters are thought to be the redox centers, molybdenum is vitally important.
- 34. It has been shown that bacteria grown in the presence of tungsten VI oxide rather than molybdenum (VI) oxide, can incorporate tungsten but show no nitrogen fixing activity. Thus nitrogen actually coordinates with molybdenum during the fixation process. Fig.5: (a) Proposed arrangement of Mo, S and Fe in the active site of nitrogenase (b) Proposed catalytic cycle of nitrogen fixation and conversion.
- 35. • Thus the nitrogen compounds are of great importance both for constructive and destructive purposes. • Our country is essentially an agricultural one. Hence we require nitrogenous fertilizers whose manufacture is based upon the fixation of nitrogen. • Nitric acid is used in the manufacture of explosives viz. nitro-glycerine, picric acid, dynamite, T.N.T. etc. which can be used both for constructive and destructive purposes.