Nitrogen cycle

M.Prasad NaiduMSc Medical Biochemistry,

Ph.D.Research Scholar

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.

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.

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

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.

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.

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

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.

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.

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.

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

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.

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.

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.

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.

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

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.

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.

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

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)

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.

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.

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.

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.

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.

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.

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

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.

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

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.

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.

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.

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.

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.

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.

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

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

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

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.