Incorporation of Ammonia into Organic Compounds

In this article we will discuss about the incorporation of ammonia into organic compounds.

Nitrogen is an essential element for all living organisms and also viruses, coming next in importance to carbon. Nitrogen forms about 14% of the dry weight of the living matter, where it is primarily present as a constituent of proteins and nucleic acids.

Although molecular nitrogen abounds in the earth’s atmosphere, the biochemical mechanism for its utilization as a source of nitrogen is restricted to a small number of prokaryotic species, both photosynthetic and non-photosynthetic.

All other living organisms — plants, animals and microorganisms of different kinds — are solely dependent on combined nitrogen. Whereas plants and most bacteria and fungi can use inorganic nitrogenous salts, like ammonium or nitrate, animal organisms can utilize only organic nitrogenous compounds as source of nitrogen.

Plants and microorganism which utilize nitrate as nitrogen source have to reduce it to the level of ammonia before incorporation into various organic compounds of their cells, because in their cellular constituents nitrogen is present in a reduced state.

The major ports of entry of inorganic nitrogen into organic compounds are shown in Fig. 8.67:

Reduction of nitrate to the level of ammonia involves conversion of nitrogen from its highest oxidized state (+5) to the most reduced state (-3), requiring transfer of 8 electrons. This conversion is supposed to take place in 4 steps, each step consisting of a two-electron transfer reaction.

The probable steps and intermediates are as follows, though the components are not well-established:

The above sequence is known as assimilatory nitrate reduction pathway which is distinguished from a dissimilatory pathway operating in nitrate respiration (also known as de-nitrification). The first step in the above pathway involves reduction of nitrate to nitrite.

The reaction is catalysed by nitrate reductase. In microorganisms, plants and fungi, nitrate reductase is a soluble cytoplasmic enzyme. In Neurospora, the enzyme is a molybdenum containing flavo-protein. The probable pathway of electron flow to nitrate is NADPH₂ —> FAD Mo —> NO₃^–.

Molybdenum undergoes valency change from Mo⁵⁺ to Mo⁶⁺ during electron transfer for reduction of nitrate to nitrite. Reduction of nitrite is catalysed by nitrite reductase. Further electron transport for production of NH₃ requires a highly electronegative reductant. In green plants, reduced ferredoxine produced by the light-reaction of photosynthesis probably acts as the terminal reductant of nitrite to ammonia. The probable path for electron flow to nitrite is ferredoxine (reduced) —> NADP —> FAD —> NO₂^–.

The final product of nitrate reduction, ammonia, is then incorporated into organic compounds by the several alternative routes described below:

Incorporation of Ammonia into Organic Compounds:

The different ports of entry of ammonia have been shown in Fig. 8.67. One of the most important routes of incorporations of ammonia is reductive amination of α-ketoglutaric acid in which NADPH₂ acts as H-donor. The enzyme catalyzing the reaction is glutamic acid dehydrogenase.

Glutamic acid, produced by the reaction, can transfer the amino group to other keto acids by transamination:

Glutamic acid can accept another molecule of NH₃ to produce an amide called glutamine.

The reaction is catalysed by the enzyme glutamine synthetase which requires ATP:

Glutamine may transfer its amido group to aspartic acid producing another amide, asparagine and glutamic acid:

Asparagine may also be produced by direct amination of aspartic acid.

The reaction also requires ATP:

Although glutamic acid dehydrogenase reaction provides the main port of entry of ammonia into organic compounds, a few other reactions operate in specific organisms, by which ammonia is incorporated. For example, the members of the genus Bacillus lack the enzyme glutamic acid dehydrogenase and they employ a-alanine dehydrogenase reaction for amination of pyruvate.

The enzyme is NAD-linked:

Another enzyme, aspartase, may incorporate ammonia into fumaric acid producing aspartic acid, though the enzyme is probably involved more in deamination of aspartic acid, rather than its formation.

A further route of entry of ammonia is catalysed by the enzyme carbamyI phosphate synthetase. This enzyme uses CO2, NH3 and ATP as substrates to form carbamyl phosphate which is an important intermediate in the synthesis of ornithine, arginine and pyrimidine bases of nucleic acid.

Transformations of Nitrogen and Nitrogenous Compounds in Nature- the Nitrogen Cycle:

Atmospheric nitrogen and various inorganic as well as organic nitrogenous compounds present in the biosphere undergo transformation from one form to another through biological and non-biological agencies. These transformations constitute the nitrogen cycle of nature which is of profound significance for the sustenance of life in this planet. It is important, therefore, to consider the nitrogen cycle in some detail.

A schematic representation of the nitrogen cycle is given in Fig. 8.68:

The atmospheric molecular nitrogen is converted to ammonia by biological means, or to nitrate and ammonia by non-biological means, like lightning and man-made technology. The process is known as nitrogen fixation.

Plants and microorganisms can take up ammonium salts as nitrogen source to synthesize proteins and other nitrogenous compounds. Ammonia is oxidized to nitrate in nature through the process of nitrification by the nitrifying bacteria. Nitrate, so produced is taken up by plants and metabolized through the assimilatory nitrate reduction pathway for syntheses of proteins and other nitrogenous metabolites.

Nitrate can also be converted back to molecular nitrogen by the process of de-nitrification. The process is carried out by microorganisms under anaerobic condition through dissimilatory nitrate reduction pathway.

Animal organisms take up nitrogen in the form of proteins and other organic nitrogenous compounds from plants or animals which feed on plants. The nitrogenous compounds of dead remains of all living organisms — animals, plants and microorganisms — are decomposed in nature by microbial activity leading to liberation of nitrogen in the form of ammonia.

The process is known as ammonification which is another important component of the nitrogen cycle. In addition, most terrestrial vertebrates excrete urea as an end-product of nitrogen metabolism, while birds and reptiles generally excrete uric acid. These are also converted to ammonia through microbial activities. The nitrifying bacteria can quickly oxidize ammonia into nitrate.

A part of the incompletely degraded residues of the dead organisms, specially of plants, remains in soil as humus. The nitrogenous materials of humus are slowly degraded in the soil and used for nourishment of plants and microorganisms.

It can be seen from Fig. 8.68 that ammonia occupies a central position in the nitrogen cycle. The bulk of ammonia originates through the process of ammonification, while a considerable amount comes from nitrogen fixation.

Incorporation of ammonia into amino acids initiates its journey into the living system. Plants and microorganisms are the primary users of ammonia, while the animal world depends on them for supply of nitrogen. So they can be considered as secondary users.

The two other important microbiological process, viz. ammonification and nitrogen fixation, are discussed below: 

1. Ammonification:

Ammonification includes the microbial degradation of proteins and other nitrogenous compounds resulting in release of ammonia.

The process may be divided into two parts:

i. Degradation of proteins to the stage of amino acids, and

ii. Deamination of amino acids to release ammonia.

Proteolysis involves enzymatic hydrolysis of proteins into amino acids. The enzymes belong to the class hydrolases and they are variously known as proteinases, proteases or peptidases. Although protein hydrolysis is a normal process of digestion in animals and some specialized plants, like the insectivorous plants, in this section only protein decomposition in natural habitats by microorganisms will be treated.

Many naturally occurring microorganisms including bacteria, actinomycetes, fungi etc. produce extracellular proteinases i.e. the enzymes are secreted in the environment where they attack proteins. These enzymes are distinguished into two main types, exopeptidases and endo-peptidases, both of which are capable of hydrolyzing the peptide bonds which bind the amino acids together to form the long polypeptides.

The exopeptidases hydrolyse the polypeptide chain from one end releasing each time the last amino acid, while the endo-peptidases cause hydrolytic cleavage of peptide bonds in the middle of the molecule, so that a long molecule is cleaved into several shorter chains. Some of the endo-peptidases attack at specific sites e.g. the peptide bond between two specific amino acids, like lysine and arginine.

These shorter peptide chains are further hydrolysed by exopeptidases to produce amino acids:

Among the actively proteolytic bacteria are members of the genera Proteus, Bacillus, Clostridium, Pseudomonas etc. The end-products of proteolysis are the amino acids making up the protein. The amino acids so released in the environment may be taken up by resident microorganisms for protein synthesis. The uptake of amino acids requires specific transport systems in the cell membrane. Alternatively, amino acids produced by proteolysis may be further degraded by microorganisms.

When the amino acids are not absorbed by microorganisms as nutrients, they are either decarboxylated to yield primary amines, or may be deaminated to release ammonia. The latter process is known as ammonification.

Decarboxylation of amino acids mostly occurs when the conditions are anaerobic and acidic, leading to formation of CO₂ and primary amines. Some of the common primary amines are cadaverin, a decarboxylation product of lysine, agmatin of arginine, histamine of histidine, ethanolamine of serine, cysteamine of cysteine etc.

The obnoxious odour emitted by decomposing animal dead bodies is due to some of these primary amines:

The alternative fate of amino acids produced by proteolysis is oxidative deamination resulting in formation of ammonia and a keto acid.

A probable intermediate in the process is an imino acid:

Oxidative deamination of glutamic acid, for example, produces α-ketoglutaric acid and HN₃, NADP acting as H-acceptor:

Ammonia is also obtained from urea which is excreted in the urine of mammals as the end-product of nitrogen metabolism. Moreover, urea is applied to soil as a major nitrogenous fertilizer. Urea is hydrolysed by many microorganisms to ammonia and CO₂ with the help of urease. Ammonia in the form of ammonium ion can be taken up by plants, or, alternatively, can be converted to nitrate by nitrifying bacteria and then taken up by plants.

Many microorganisms inhabiting in soil and aquatic environments are capable of urea hydrolysis. Some of the notable bacteria are Proteus vulgaris, P. mirabilis, Bacillus pasteurii and Sporosarcina ureae.

2. Di-Nitrogen Fixation:

Reduction of molecular nitrogen to ammonia and its incorporation into organic compounds is commonly known as nitrogen fixation or, more precisely, di-nitrogen fixation, because nitrogen molecule contains two atoms of nitrogen bound by three very stable bonds (N=N). Although the total reserve of nitrogen in the earth’s atmosphere is about 3.9 x 10¹⁵ tons, yet it is the most common limiting factor in soil affecting crop production.

This is because plants by themselves, or animals, are unable to utilize molecular nitrogen as nitrogen source. The ability of nitrogen fixation is restricted only to some specific prokaryotic organisms — the cyanobacteria, some eubacteria and a few archaebacteria. Total amount of nitrogen fixed by these organisms amounts to about 175 x 10⁶ tons per year.

Besides biological fixation, nitrogen is converted to compounds like nitric oxide by abiotic natural phenomena, like lightning. Such compounds are washed down to the earth’s surface by rain and thereby they enrich the soil and water bodies with nitrogenous compounds.

The amount of nitrogen fixed in this way is nearly 70 x 10⁶ tons per year. Apart from these natural processes, molecular nitrogen is used for production of nitrogenous fertilizers like urea, ammonium sulfate and other compounds by man-made technology.

Such technology generally involves reduction of N₂ by H₂ at high temperature and pressure in presence of a catalyst yielding ammonia. Biological nitrogen fixers also perform the same reaction, but at the atmospheric temperature and pressure catalysed by an enzyme complex — nitrogenase.

The ability of living organisms to reduce N₂ to NH₃ and to grow at the cost of the fixed nitrogen is known as diazotrophy. The diazotrophs can be ecologically divided into two broad groups. One group includes organisms capable of nitrogen fixation under free-living conditions and the other group comprising those which fix nitrogen in association with other organisms.

When the nature of such association is mutually beneficial, it is called symbiosis. Some nitrogen-fixers form a more lose type of association with hosts. These are commonly known as associative diazotrophs. The difference between symbiotic fixers and the associative fixers is that the symbiotic fixers like rhizobia are unable to fix nitrogen when growing outside the symbiotic partner. However, this distinction is not always rigorous.

The nitrogen-fixing ability of an organism can be detected by several means. A straightforward approach is to grow the organism in a culture medium from which combined nitrogen, either inorganic or organic, is withdrawn. If the organism is capable of growth in such a nitrogen-free medium, it is assumed that the organisms are capable of using atmospheric nitrogen.

A more sophisticated approach is to provide the organism with heavy nitrogen isotope (¹⁵N₂) in the gaseous atmosphere. If the heavy isotope is incorporated into the cell protein of the organism, it can be detected by mass-spectrometry. A third — rather indirect — approach of testing nitrogen-fixing ability is the acetylene reduction test which is based on the fact that the enzyme-complex nitrogenase cannot only reduce N₂ to NH3, but also several other compounds including acetylene (HC=CH) to ethylene (H₂C=CH₂).

The method involves exposure of the organism to acetylene in the gas phase for some time and to detect the presence of ethylene by gas chromatography. This method is quick and reliable. It has greatly facilitated detection of nitrogen-fixing capacity and has significantly expanded the list of di-nitrogen-fixers. Thus, besides the well-known free-living and symbiotic nitrogen-fixers, many new organisms have been now known to possess this capability and many more may be discovered in future.

A. Classification of Diazotrophic Organisms:

A classification of diazotrophic organisms based on their metabolic characteristics and oxygen relation is given below together with few representative genera:

The taxonomic arrangements of the genera of major nitrogen-fixing bacteria have undergone sweeping changes in the recent past. The systematic positions according to the Bergey’s Manual of Systematic Bacteriology (Second Edition, 2001) are presented in Table 8.5. In the table the cyanobacteria have not been included.

B. The Diazotrophs:

From the chart shown above, it can be seen that diazotrophic organisms are of diverse metabolic pattern. They are also morphologically different and belong to different taxonomic groups. Ecologically they may be differentiated into two broad groups; the free-living, and symbiotic, including the “associative” forms.

(i) Free-living diazotrophs:

The acetylene-reduction assay of nitrogenase has proved the presence of the enzyme nitrogenase in a large group of morphologically as well as physiologically diverse group of prokaryotic organisms.

Free-living N₂-fixers include both heterotrophic bacteria like Azotobacter, Beijerinckia, Clostridium — all of which are residents of soil or aquatic habitats, as well as autotrophic forms. Among the autotrophs are the anoxygenic photosynthesizes, like Rhodospirillum, Rhodobacter, Chlorobium, Chloroflexus etc. and oxygenic photosynthetic cyanobacteria.

Although the heterotrophic N₂-fixers are very common in natural habitats, their contribution to nitrogen addition to soil and aquatic bodies may not be very significant. This is because N₂-fixation demands high energy supply. The heterotrophic fixers have to depend on the organic substrates for deriving this energy either by respiration or fermentation. But such organic substrates are generally in short supply in the habitats where they grow.

Many of these heterotrophic bacteria concentrate in the vicinity of the root systems of higher plants (the rhizosphere) where organic substances are excreted. These bacteria utilize these substances for their sustenance and N₂-fixation. In contrast, the cyanobacteria make a much larger contribution to global N₂-fixation, because they derive energy by oxygenic photosynthesis.

Some 125 species of more than 30 genera of cyanobacteria are known to be N₂-fixers at an average rate of 3 to 11 kg N₂/hectare/year. Much higher rate of N₂-fixation has been reported in the rice-fields of India amounting to up to 80 kg N₂/hectare/year. Among the well-known genera of N₂-fixers are Nostoc, Anabaena, Spirulina, Synechococcus, Pleurococcus, Oscillatoria, Scytonema etc. Some of the cyanobacteria can also enter into symbiotic association with fungi forming the composite organisms called lichens, while others with different plant groups.

(ii) Associative diazotrophs:

Many associative interactions between diazotrophs and higher plants have been reported in the last few decades. Such association may occur on the leaf-surface (phyllosphere) leading to phyllocoenoses or on the root surface (rhizocoenoses) or in the soil surrounding the root-system (rhizosphere).

Some of the well-established associations are shown in Table 8.6:

(iii) Symbiotic diazotrophs:

The symbiotic diazotrophs belong to three groups of prokaryotes. These are the cyanobacteria, the rhizobia and the organisms belonging to the actinorhizal genus Frankia. Among these, the rhizobial symbiosis with leguminous plants has been the best studied.

Moreover, when they grow in soil or common bacteriological culture media, rhizobia are unable to fix molecular nitrogen. They do so only in symbiotic association with appropriate host plants. In contrast, the cyanobacteria and members of Frankia can fix nitrogen also when they grow independently.

Cyanobacteria can grow as a micro-symbiont in association with several fungi and plants of various groups, starting from bryophytes to angiosperms. Cyanobacterial genera, like Nostoc, Anabaena, Tolypothrix etc. form symbiotic association with several ascomycetes and basidiomycetes to produce a composite thallus, known as lichens.

Among bryophytes, the gametophytic thalli of Blasia, Calycularia, Anthoceros and Notothylas are known to house Nostoc colonies where they presumably fix N₂ and help the hosts by supplying fixation products. The water- fern Azolla contains Anabaena azollae in their leaf cavities.

Azolla has been used as a nitrogenous fertilizer for enrichment of rice-paddies in different countries. The gymnosperms, Cycas and Zamia produce morphologically specialized roots, called coralloid roots in which Nostoc colonies grow as micro-symbionts. Among the angiosperms, Gunnera is well known for having pockets of Nostoc- containing cells in the roots.

Members of the genus Frankia are mycelium-forming heterotrophic bacteria belonging to the actinomycetes. They are microaerophilic and extremely slow growing organisms which enter into symbiotic association with non-leguminous trees and shrubs and produce actively N₂-fixing root nodules. The root-nodules are known as rhizothamnia. The quantity of N₂ fixed by some members, e.g. in Alnus rugosa may be as high as 150 kg/hectare/year. Frankia can fix N₂ also in culture.

The last and the most well-known group of symbiotic N₂-fixers comprises the rod-shaped, Gram- negative, non-sporing bacteria, commonly known as rhizobia. All rhizobia were previously included in a single genus designated as Rhizobium having several species. But later it has been split into six genera.

The recognized species of these genera are shown in Table 8.7:

Rhizobia enter into symbiotic association with leguminous plants (Family Fabaceae) of all kinds, trees, shrubs and herbs. Outside this family of plants, at least one plant, Parasponia sp., belonging to the Family Ulmaceae, is known to form symbiotic association with rhizobia.

In general, rhizobia form effective root-nodules in the appropriate host plants. Within these nodules, the bacteria exist as nitrogen-fixing bacteroids. Besides roots, rhizobia like Azorhizobium caulinodans can form nodules also on the stem in Sesbania rostrata and Aeschynomene aspera. The bacteria causing leaf-nodules or galls in some species of Rubiaceae and Myrsineae are also believed to be related to rhizobia.

The great majority of rhizobial bacteria, however, infect different plants of the Family Fabaceae producing root-nodules. The leguminous plants distributed in 16,000 to 19,000 species are of great agricultural importance, because they not only serve as a very important source of vegetable protein to humans, but also provide fodder to animals. More importantly, the agricultural leguminous crops enrich the soil with fixed nitrogen which benefits a non-leguminous crop in the crop-rotation system.

Rhizobia show a high degree of host specificity. This means that all rhizobia do not form symbiotic association with all leguminous host. A particular host species can form effective N₂-fixing root nodules with a few strains, or even a single strain of rhizobia.

Similarly, a single strain of the bacteria can infect only a few species, or in some cases only a single species of host plant. A successful symbiotic association is indicated by the formation of effective nodules which have a flesh-coloured interior due to the presence of a kind of haemoglobin, called leg-haemoglobin (leguminous haemoglobin).

Atmospheric nitrogen can be fixed only by such effective nodules and be made available to the host plant. The bacteria, in turn, receive photosynthetic as energy source from the host plant. This kind of ‘give and take’ is the basis of symbiosis.

Although rhizobia actively fix N₂ within the specialized environment of the root nodule, they are unable to utilize N₂ in the soil or in nitrogen-free culture media, because they do not synthesise the enzyme nitrogenase under such conditions. Under these conditions, they grow as ordinary heterotrophic organisms using nitrogenous compounds. There is an exception to this general rule. Azorhizobium caulinodans which form stem-nodules in Sesbania rostrata has been reported to fix N₂ in culture media and to utilize the fixed nitrogen for growth.

The rhizobia are distinguished into two types, depending on their growth rate — the fast-growing and the slow-growing species. The fast-growing species belong to the genera Rhizobium and Sinorhizobium.

The fast-growing and the slow-growing rhizobia also differ in several other characteristics as shown in Table 8.8:

(a) Host-micro-symbiont interactions in leguminous plants:

Legume-rhizobial symbiosis comprises often very complex interactions between the two partners. A successful symbiosis resulting in the formation of actively N₂-fixing effective root or rarely stem nodules is established only when an appropriate strain of the micro-symbiont infects the specific host. Otherwise, meeting of rhizobia with the host either results in no infection, or, even if infection occurs, only ineffective nodules are formed which are without leg-haemoglobin. Such nodules do not fix N₂.

Rhizobia are soil-inhabiting, free-living heterotrophic bacteria which show locomotion with the help of peritrichous or sub-polar flagella. Interaction between an appropriate host and a proper strain of the micro-symbiont starts by secretion of a chemical substance by the host which attracts the swimming bacteria towards its root zone.

Thus, the chemical substance acts as a signal which is different in case of different hosts and the attraction of the bacteria is also specific. These signals have been identified as flavonoids. Different flavonoids attract different strains of rhizobia. This explains at least partly the basis of host-rhizobial specificity. Two of these flavonoid attractants have been identified as luteolin and genistein.

The structures of these compounds are shown:

Attracted by these flavonoids, specific rhizobia swim close to the root system of the respective host plants. Then the bacteria respond by synthesizing certain proteins, like Nod D. These proteins in conjunction with the plant-secreted flavonoids induce the expression of other nod genes of the bacteria. These genes specify the synthesis and excretion of certain polysaccharides, called Nod-factors. The Nod-factors initiate nodulation in the host plants by acting in several ways.

They induce root-hair curling initiate cell division of the root-cortex and transcription of nodulin genes of the host genome. One of these Nod-factors produced by Sinorhizobium meliloti has been isolated and purified. The compound has been found to be a sulfated tetraglucosamino glycolipid.

Its structure is shown:

The events leading to establishment of legume-rhizobial symbiosis can be diagrammatically represented as shown in Fig. 8.69:

Nod-genes of bacteria are several in number. It has been shown that nod genes A, B and C are common to all rhizobia and these gene specify the oligosaccharide backbone of the different Nod- factors. There are also additional nod genes which specify the side groups of Nod-factors to generate host-specific lipopolysaccharides. The nodulin genes of host plants which are induced by the host-specific Nod-factors produce noduline proteins which function in nodule organogenesis.

(b) Formation of root nodules in leguminous plants:

The physical interaction between rhizobia and host begins with attachment of the bacterial cells to the surface of root hairs. This attachment is controlled by two factors, one produced by the micro-symbiont and the other by the host. The bacterial factor is a calcium-binding protein, known as rhicadhesin, and the host factor is a lectin produced on the root surface.

Due to attachment of the bacteria, the root-hair tip is curled by the Nod-factor. The attached bacteria do not breach the root hair wall, but they pass into an infection thread. This thread is a trans-cellular tunnel produced by the invagination of the root-hair cell envelope and it consists of the root hair cell wall, externally surrounded by the cell membrane. The rhizobia enters through the tunnel and continue cell division being embedded in a matrix of glycoprotein. Thus the bacteria are not in direct contact with the cytoplasm of the root-hair cell.

An infection thread is shown diagrammatically in Fig. 8.70:

The infection thread continues growth through the epidermal cell and the other cell layers until it reaches the root cortex. The thread may form branches and the ends of the branches appear in different cells of the cortex.

In these cells rhizobia are released from the terminal un-walled portions of the infection thread in the form of small droplets by the process of endocytosis. Each droplet containing one or few rhizobia remains enclosed by the plant cell membrane.

The rhizobia within these droplets are then transformed generally into irregular-shaped bacteroids. These bacteroid-containing bodies are known as symbiosomes and the surrounding membrane as peri-bacterial membrane (PBM). After rhizobia are transformed into bacteroids, they do not further multiply by cell division. Each symbiosome may contain one to few bacteroids.

The transformation of rhizobia into bacteroids initiates activation of several genes which are not expressed in bacteria. One of such genes is that coding for the heme moiety of leghaemoglobin. The product of this gene i.e. the heme is then trans-located across the PBM into the cytoplasm of the cortical cells.

The globin part of leghaemoglobin is synthesized by the host cell and the two moieties are combined to form the complete molecule of leghaemoglobin that remains localized in the cortical cells of the host containing the symbiosomes. Another gene-complex that is activated in the bacteroids is that coding for the nitrogen-fixing enzyme complex — nitrogenase.

Side by side, the release of rhizobia from the infection thread in the host cell triggers changes in these cells also. The cortical cells begin to divide at a rapid rate under the influence of the Nod-factor produced by the rhizobia and growth hormones.

This leads to swelling of the part of the root to form a nodule. According to some workers, only tetraploid cortical cells can act as primordial of nodules. The tetraploid cortical cells containing bacteroids initiate cell division and the surrounding cells also start dividing resulting eventually into bulging of the root portion to form a nodule.

Pattern of nodulation varies depending on the host species. In some plants, nodules are mainly restricted on the tap-root, while, in others, mainly on the root branches. In still others, mixed type nodule distribution may occur.

The shape and size of individual nodules are also variable. Again, in some host species, many nodules remain clustered, while in other species nodules may be isolated. But in all cases, the effective nodules have a flesh-colour inside due to the presence of leg-haemoglobin, while ineffective nodules are white.

The morphological features associated with root nodule formation in leguminous plants are diagrammatically described in Fig. 8.71:

In the root nodules, the host feeds the bacteroids with TCA cycle intermediates like malate and succinate. The bacteroids use them as substrate to make NADH₂ and ATP via an electron transport system. NADH₂ and ATP are utilized by the bacteroids for reduction of molecular nitrogen to ammonia.

Ammonia is then trans-located across the symbiosome membrane into the cytoplasm of the host cortical cells and is converted to glutamic acid and glutamine. From glutamine, the amido amino group is transferred to aspartic acid producing asparagine which is trans-located to other parts of the host plant through its vascular system.

These biochemical exchanges are shown diagrammatically in Fig. 8.72:

The most abundant protein in the root nodules is leghaemoglobin, reaching up to 20% of the total protein. It is present in the cytoplasm of the host cortical cells having symbiosomes, but not within the symbiosomes. The protein part of leghaemoglobin is synthesized by the host cell, while the heme prosthetic group is produced by the bacteroids.

The function of leghaemoglobin is to transport oxygen to the bacteroids, just as haemoglobin does in mammals. Due to high oxygen demand of the bacteroids, a hypoxic (low oxygen pressure) condition prevails in the cytoplasm of the host cortical cells containing bacteroids. Leghaemoglobin is supposed to play the role of maintaining a controlled oxygen supply to the symbiotic system.

Such controlled oxygen supply is of great importance, because the enzyme-complex nitrogenase is highly sensitive to oxygen. The problem of supplying oxygen to the highly aerobic bacteroids without harming the oxygen-labile nitrogenase is excellently managed by leghaemoglobin. It binds oxygen and carries it to the electron transport system of bacteroids located in their membrane where oxygen is used as the terminal electron acceptor producing water. Thus, oxygen is never set free to react with the oxygen-sensitive nitrogenase which is located within the symbiosomes (see Fig. 8.72).

The question how is the nitrogenase protected in cyanobacteria from oxygen is interesting, because these organisms are not only aerobic, but they also carry out photosynthesis with oxygen evolution. The N₂-fixing property is found mainly in filamentous cyanobacteria, though some unicellular forms like Gloeothece also have the property.

In the filamentous genera, the ability to fix N₂ is not present in all the cells of the trichome, but is restricted to some specialized cells, called heterocyst’s. These cells are thick-walled. And they are devoid of photosystem II which is involved in photosynthetic oxygen evolution. The nitrogenase-complex present in these cells is, therefore, not exposed to oxygen produced through photosynthesis.

The thick wall of the heterocyst’s probably resists diffusion of oxygen from the surrounding medium. Most of the unicellular cyanobacteria are characterized by production of a thick glycocalyx which forms a several-layered capsule around single cells as well as groups of cells. These polysaccharide coverings probably protect the nitrogenase in case of unicellular N₂-fixing cyanobacteria.

(c) Biochemical mechanism of di-nitrogen fixation:

The triple-bonded nitrogen molecule is highly stable and its reduction to ammonia requires a strongly electronegative reductant. The first indication of the presence of such a reductant was obtained in the cell-free extract of the anaerobic N₂-fixer, Clostridium pasteurianum, and the compound was identified as ferredoxin which has a redox potential of -0.43 volts and which also acts as an electron- acceptor in photosynthesis.

Ferredoxin is an iron-containing non-heme protein. Later, another reductant was found to play a similar role in other N₂-fixers. This compound was named flavodoxin. Depending on the species, one of these two reductants is responsible for supply of electrons for reduction of the nitrogen molecule.

Reduction of N₂ is a high energy-demanding process. This energy is supplied by hydrolysis of ATP. Rhizobia which carry out aerobic respiration produce ATP by oxidative phosphorylation. The anaerobic N₂-fixers like Clostridia have to depend on fermentation for this ATP generation, while the photosynthetic cyanobacteria and the anoxygenic photosynthetic bacteria can use photophosphorylation for providing ATP necessary for N₂-fixation. It has been calculated that reduction of one molecule of nitrogen (N₂) to two molecules of ammonia (NH₃) requires a total of at least 8 electrons and hydrolysis of at least 16 ATP molecules producing ADP+Pi;

N₂+ 8H⁺+ 8e^–+ 16ATP 2NH₃ + H₂ + 16 ADP + 16Pi

The enzyme-complex catalyzing the above reaction is nitrogenase. The nitrogenase complex is composed of two proteins, neither of which can individually catalyse the reduction reaction. Both proteins are highly sensitive to oxygen and they lose catalytic property in presence of free oxygen. One of these two proteins is called Mo-Fe protein because it contains molybdenum and iron.

It is also called di-nitrogenase. It has a molecular weight of 2.2 – 2.5 x 10⁶ Daltons. It is a tetramer i.e. consisting of 4 subunits (polypeptides) which may be identical or of two types depending on species. The di-nitrogenase molecule contains two atoms of Mo, 24-32 non-heme iron atoms and 26-28 sulfur atoms which are acid-labile.

The second component of the nitrogen complex is known as the Fe-S protein because it contains non-heme iron and acid-labile sulfur, but no molybdenum. This protein is called di-nitrogenase reductase. It has a molecular weight of 54 – 60 x 10³ Daltons and consists of two identical subunits. The molecule contains 4 non-heme Fe and 4 acid-labile S (sulfide). In an active nitrogenase complex, the two component proteins are present in the ratio of 1 to 2 FeS protein to 1 Mo-Fe protein.

The mechanism of reduction of N₂ to NH₃ involves a transfer of an electron from the reduced electron donor — ferredoxin or flavodoxin — to the Fe atom of an oxidized form of the Fe-S protein which is thereby reduced. The reduced Fe-S protein next reacts with Mg salt of ATP and the protein undergoes a conformational change. Meanwhile, the Mo-Fe protein combines with a nitrogen molecule through Mo atoms.

The MO-Fe protein with its attached N₂ then reacts with Mg-ATP-Fe-S complex to produce a 1: 1 nitrogenase complex. Within this complex, an electron is transferred from the Fe-atom of Fe-S protein to a Fe atom of the Mo-Fe protein with simultaneous hydrolysis of ATP to form ADP + Pi. The electron accepted by Mo-Fe protein is used for reduction of N=N to -N=N- Thus, one cycle of electron transfer from the donor (ferredoxin, fd or flavodoxin, fid) to N=N is completed via Fe-S and MoFe components of nitrogenase. This reaction-cycle is repeated six times to effect complete reduction of N₂ to NH₃.

The electron transport chain can be represented in a simplified manner as shown:

In this diagrammatic representation the involvement of ATP has not been included.

A more detailed representation for giving an idea of the N₂-reduction process is shown in Fig. 8.73 in which one cycle of electron-flow from donor to N₂ molecule has been included:

In addition to di-nitrogen, the enzyme nitrogenase can also reduce acetylene, azide, cyanide, nitrous oxide, etc. The ability to reduce acetylene to ethylene has been used for developing an easy and quick method of assaying nitrogenase.