Nitrogen Metabolism

 

Carbon, hydrogen, oxygen and nitrogen are the major constitutes of body of all the living organisms. These elements contribute to organize various biomolecules present in a cell. In the atmosphere molecular nitrogen is 78% by volume. Nitrogen is the second most important constituent next to carbon in living organisms. It is an important component of amino acids, proteins, enzymes, vitamins, alkaloids and some growth hormones. Proteins present in living organisms contain about 16% nitrogen. Nitrogen is present in the atmosphere as dinitrogen or nitrogen gas. Molecular Nitrogen or diatomic nitrogen (N2) is highly stable as it is triple bonded (N≡N). Because of this stability, molecular nitrogen as such is not very reactive in the atmosphere under normal conditions.

Nitrogen is part of nitrogenous bases and amino acids, which are the building blocks of nucleic acids and proteins respectively. DNA transfers genetic information to subsequent generations of organisms. About 78% of the atmosphere is made of nitrogen, but molecular nitrogen is not directly available to plants and animals, the nitrogen cycle makes this nitrogen available to plants. Nitrogen cycle converts this nitrogen into a usable form. Lightning fixes Nitrogen to NH3 and nitrogen fixing bacteria like Rhizobium also convert N2 into NH3. Most plants absorb nitrates from soil and reduce it to NH3 in the cells for further metabolic reactions. Dead organisms and their excreta like urea are decomposed by bacteria into NH3 and by a different set of bacteria into nitrates. These are left in the soil for use by plants. In this way Nitrogen cycle is self regulated.

Nitrogen Fixation

The conversion of molecular nitrogen into compounds of nitrogen especially ammonia is called nitrogen fixation. This nitrogen fixation may take place by two different methods – abiological and biological. In abiological nitrogen fixation the nitrogen is reduced to ammonia without involving any living cell. Abiological fixation can be of two types: industrial and natural.

Industrial Nitrogen Fixation

Burning fossil fuels, using synthetic nitrogen fertilizers and cultivation of legumes are all examples of human actions that produce fix nitrogen. Industrial nitrogen fixation through the Haber-Bosch process is a deliberate attempt of humans to produce ammonia. This process was invented in the early 1900s by German scientist Fritz Haber. It requires high pressure (200 atmospheres) and temperature (400°C), and the resulting ammonia is used as a synthetic nitrogen fertilizer to enrich land for crop growth. The Haber process has become very popular and - with help from new crop varieties- has led to a great increase in agricultural productivity, necessary for the growing global population. However, negative effects to the environment have also accompanied this process, as the large increase in fixed nitrogen is contaminating the surface water, groundwater, and the atmosphere - producing dangerous consequences.

Natural nitrogen fixation

In natural process nitrogen can be fixed especially during electrical discharges in the atmosphere. It may occur during lightning storms when nitrogen in the atmosphere can combine with oxygen to form oxides of nitrogen. These oxides of nitrogen may be hydrated and trickle down to earth as combined nitrite and nitrate.

Biological nitrogen fixation

Biological nitrogen fixation is reduction of molecular nitrogen to ammonia by a living cell in the presence of enzymes called nitrogenases. This process of nitrogen fixation is primarily confined to microbial cells like bacteria and cyanobacteria. These microorganisms may be independent and free living.

Mechanism of Biological Fixation of Nitrogen

Nitrogen fixation requires

(i)   the molecular nitrogen

(ii)   a strong reducing power to reduce nitrogen like reduced FAD (Flavin adenine dinucleotide) and reduced NAD (Nicotinamide Adenine Dinucleotide)

(iii)   a source of energy (ATP) to transfer hydrogen atoms from NADH2 or FADH2 to dinitrogen and

(iv)  enzyme nitrogenase

(v)  compound for trapping the ammonia formed since it is toxic to cells.

(vi)  The reducing agent (NADH2 and FADH2) and ATP are provided by photosynthesis and respiration. 

The overall biochemical process involves stepwise reduction of nitrogen to ammonia. The enzyme nitrogenase is a Mo-Fe containing protein and binds with molecule of nitrogen (N2) at its binding site. This molecule of nitrogen is then acted upon by hydrogen (from the reduced coenzymes) and reduced in a stepwise manner. It first produces diamide (N2H2) then hydrazime (N2H4) and finally ammonia (2NH3). NH3 is not liberated by the nitrogen fixers. It is toxic to the cells and therefore these fixers combine NH3 with organic acids in the cell and form amino acids. In legumes, nitrogen fixation occurs in specialized bodies called root nodules. Nitrogen fixation is coupled to the hydrolysis of 16 equivalents of ATP and is accompanied by the co-formation of one equivalent of H2. The conversion of N2 into ammonia occurs at a metel cluster called FeMoco, an abbreviation for the iron-molybdenum cofactor. 

•  The mechanism proceeds via a series of protonation and reduction steps wherein the FeMoco active site hydrogenates the N2 substrate.
• In free-living diazotrophs, nitrogenase-generated ammonia is assimilated into glutamate through the glutamine synthetase /glutamate synthase pathway.
• The microbial nif genes required for nitrogen fixation are widely distributed in diverse environments.
• Nitrogenase is rapidly degraded by oxygen. For this reason, many bacteria cease production of the enzyme in the presence of oxygen.
• Many nitrogen-fixing organisms exist only in anaerobic conditions, respiring to draw down oxygen levels, or binding the oxygen with a protein such as leghaemoglobin.

Biological Nitrogen fixation may be categorized into following types:

1.  Non- Symbiotic/ asymbiotic Biological Nitrogen Fixation.
2. Associative Biological Nitrogen Fixation.
3. Symbiotic Biological Nitrogen Fixation.

Non- Symbiotic/ asymbiotic Biological Nitrogen Fixation:

Soil contains a number of free living nitrogen fixing organisms. These include a number of aerobic and anaerobic bacteria and blue green algae. Biological nitrogen fixation by microorganisms living freely or staying out of plant cell is called non-symbiotic Biological Nitrogen Fixation.

The asymbiotic nitrogen fixers can be classified as follows:

Free living aerobic nitrogen fixing bacteria: 

1. Photosynthetic: Chlorobium, Chromatium
2. Non-Photosynthetic: Azotobacter, Azomonas, Derxia, Beijerinckia 

    Free living anaerobic nitrogen fixing bacteria:

        1. Photosynthetic: Rhodospirillum

        2. Non-Photosynthetic: Clostridium

    Free living chemosynthetic bacteria:  

1. Heterotrophic: Desulfovibro
2. Cyanobacteria or Blue green algae: Heterocyst bearing: Nostoc, Anabaena, Rivularia, Calothrix.
3. Non-Heterocyst bearing: Oscillatoria, Gloeocapsa, Lyngbya, Plectonema.
4. Free living Fungi:  Yeasts and Pullularia

The asymbiotic free living nitrogen fixers are quite primitive. These organisms fix nitrogen more actively under poor aeration, provided no hydrogen gas is being produced. Certain bacteria, living in close contact with the roots of cereal and grasses, fix nitrogen. This association is a loose mutualism, called associative Symbiosis. The bacteria reside in the transition zone between soil and root (the rhizosphere) and sometimes enter the roots. Some of the fixed nitrogen is absorbed by the roots and in turn the bacteria get nourishment from the carbohydrates released by the roots. Some of the examples are: 1. Azospirillum brasilense in association with cereal roots. 2. Beijerinckia in association with the roots of Sugarcane. 3. Azotobacter paspali in association with roots of tropical grass- Paspalum notatum. Symbiotic nitrogen fixation is part of a mutualistic relationship in which plants provide a niche and fixed carbon to bacteria in exchange for fixed nitrogen. Such an association between bacteria and host is ecological, long term and mutually beneficial to both, microbial partner fixes atmospheric nitrogen. 

The various examples of Symbiotic biological nitrogen fixation can be grouped under the following three categories:

1.  Nitrogen Fixation through nodule formation in leguminous plants: Symbiotic nitrogen fixers in large number of legume plants include genus Rhizobium mainly. They established themselves inside specialized structures on the roots called root nodules. The bacteria fix nitrogen only when they are present inside the nodules. The association is regarded as symbiotic because the host plant supplies the nodule bacteria the required organic carbon (carbohydrates). In return micro-organism supply fixed nitrogen to the host plant. Bradyrhizobium japonicum is a slow growing symbiont of Soybeans. Azorhizobium caulinodans is a stem nodule forming symbiont in Sesbania species.

2.Nitrogen Fixation through nodule formation in non-leguminous plants: Many plants belonging to families other than Leguminosae are known to produce root nodules. The important among them are primarily trees and shrubs. The important examples of non-leguminous plants that produce root nodules and fix nitrogen are:

1. Genus Frankia forms root nodules in association with Alnus sp., Casuarina equisetifolia, Myrica gale, etc.

2. Rhizobium also root nodules in genus Parasponia.

3. Leaf nodules are formed by bacteria Klebsiella in genus Psychotria and by bacteria Burkholderia in genus Pavetta zimermanniana.

4. Nitrogen Fixation through Non-nodulation: In some plants symbiotic nitrogen fixation occurs but nodules are not formed. Such associations are Pseudo symbiotic (Pseudo symbiosis). Some of the examples are:

• Lichens, an association with fungi and algae(cyanobacteria or green algae
• Anthoceros, a Bryophyte, associated with Nostoc.
• Azolla, a fern, in association withAnabaena.
• Cycas, a Gymnosperm, in association with Anabaena or Nostoc, blue green algae in its coralloid roots.
• Gunnera macrophylla, an angiospermin association with Nostoc in its stem.
• Roots of Digitaria, Sorgham and Maize associated with Spirillum.
• Symbiotic diazotrophs are more advanced and efficient (100-200 times) than the asymbiotic diazotrophs. 

Regulation of nitrogen fixation

Several bacteria have the amazing capacity to fix atmospheric nitrogen to ammonia under ambient conditions. The ability of microorganisms to use nitrogen gas as the sole nitrogen source and engage in symbioses with host plants confers many ecological advantages, but also incurs physiological penalties because the process is oxygen sensitive and energy dependent. Consequently, biological nitrogen fixation is highly regulated process by sophisticated regulatory networks that respond to multiple environmental cues. Nitrogen fixation is regulated at the transcriptional level in response to environmental oxygen and ammonium levels. Because the nitrogenase components are oxygen labile, it is advantageous for bacteria to repress transcription when oxygen levels are high. It is also advantageous to repress the expression of the metabolically expensive nitrogenase system when the cellular level of fixed nitrogen is sufficiently high. The degree to which each stimulus affects transcription is characteristic of the particular diazotroph. Expression of nitrogenase in symbiotic diazotrophs is fairly insensitive to ammonium because export of ammonium to their symbiont suppresses ammonium levels. The expression of nif genes in free-living diazotrophs is more sensitive to cellular ammonium levels. The oxygen sensitivity of nitrogenase and the energetic requirements for nitrogen fixation impose physiological constraints on diazotrophs, necessitating tight regulation of nitrogen fixation (nif) genes in response to the levels of fixed nitrogen, carbon, energy and the external oxygen concentration. Common regulatory components and similar regulatory networks are used to control nitrogen fixation, but there is considerably plasticity in the regulatory networks, which differ from species to species, dependent on host physiology. In the Proteobacteria, most nif genes are activated by the enhancer-binding protein NifA together with the RNA polymerase sigma factor σ⁵⁴. The expression of NifA and, in many cases its activity, is controlled by regulatory cascades that are responsive to different environmental cues. 

Four proteins regulate nitrogen fixation in response to oxygen or redox signals. In symbiotic bacteria, FixL (a haemoprotein sensor histidine kinase) and NifA provide a hierarchical response to the oxygen concentration. In other diazotrophs, NifA is not directly responsive to oxygen but its activity is regulated by a partner flavoprotein, NifL, that senses the redox status. The histidine protein kinase RegB and its homologues respond to redox through an active cysteine and might sense the electron flux through a high affinity cbb₃-type oxidase. The nitrogen status is communicated to target regulatory proteins by the PII signal-transduction proteins that are covalently modified by uridylylation under nitrogen-limiting conditions. Many bacteria contain more than one homologue of PII, enabling hierarchical regulation in response to the level of fixed nitrogen. In free-living diazotrophic bacteria that have the NifL–NifA regulatory system, the PII-like protein GlnK regulates NifA activity by completely different mechanisms, illustrating the plasticity of protein–protein interactions in these systems. 

In K. pneumoniae, control of nif gene expression focuses on NifA (the nifA gene product), a ς⁵⁴ (rpoN gene product)-dependent transcriptional activator, responsible for control of all major nif gene cluster transcription. Transcription of nifA is under the control of the ntrBC gene products, which comprise a global two-component transcriptional activator system, responsible for cellular nitrogen regulation. In the paradigm system, K. pneumoniae, the nifA gene is cotranscribed with nifL, which encodes a redox- and nitrogen-responsive regulatory flavoprotein (NifL). NifL acts as a negative regulator of NifA, effectively adding another level of regulation in response to oxygen and fixed nitrogen. Oxidized NifL is also sensitive to the presence of nucleotides in vitro, with increased inhibition especially in response to ADP. 

In A. vinelandii and Rhodospirillum rubrum, expression of nifA is not under the control of the ntrBC gene products, and it remains unclear whether nifA expression is under nitrogen control. In Rhizobium meliloti, redox-dependent control of nifA expression occurs in response to fixL and fixJ, which encode a two-component regulatory system responsive to oxygen. This system apparently replaces the ntrBC control found in K. pneumoniae. R. meliloti also lacks NifL, but NifA still appears to be inhibited by oxygen stimulus. Similarly, there is no evidence for NifL in Rhodobacter capsulatus. R. capsulatus contains nif-related genes analogous to ntrBC, but the expression of an rpoN-like gene is found to be sensitive to oxygen and fixed nitrogen status. 

To prevent unproductive nitrogen fixation during energy-limiting or nitrogen-sufficient conditions, the nitrogenase complex is rapidly, reversibly inactivated by ADP-ribosylation of Fe protein. The ADP-ribosylation system has been identified in R. rubrum and R. capsulatus (purple, nonsulfur photosynthetic bacteria), Azospirillum brasilense and Azospirillum lipoferum (microaerophilic, associative bacteria), and Chromatium vinosum (a purple sulfur bacterium). In nitrogenase, the presence of the ADP-ribose group apparently prevents association of Fe protein with MoFe protein, rather than blocking electron transfer between complexed Fe protein and MoFe protein. ADP-ribosylated Fe protein differs from unmodified Fe protein in only a few characteristics. The two subunits of the inactive Fe protein dimer are not equivalent because ADP-ribosylation occurs on only one subunit. Modified Fe protein retains the native [Fe₄S₄] cluster, which can be chemically oxidized and reduced. It also retains the oxygen lability of the active Fe protein. ADP-ribosylated Fe protein has the ability to bind MgATP and to undergo the conformational change that gives access of the [Fe₄S₄] cluster to chelators.