assimilation

By Dr. Sandeep Kumar Department of Botany D.D.U. College University of Delhi Nitrogen is a very important constituent of cellular components. Alkaloids, amides, amino acids, proteins, DNA, RNA, , vitamins, hormones and many other cellular compounds contain nitrogen as one of the elements. It is not exaggerating to say that Nitrogen is the key element for it is the most important constituent of proteins and nucleic acids.

Thus N2 plays a significant role in the formation of the above said compounds which in turn control cellular activities. Without nitrogen, no living organism can survive. Paradoxically all the living organisms are virtually submerged in a sea of atmospheric nitrogen (i.e. 78%), but unfortunately not all organisms are endowed with the potentiality to utilize this abundantly available molecular N2 directly.

Only some organisms like certain , blue green algae and few fungi, have the potentiality to utilize molecular N2 directly and fix it. However, most of the are capable of utilizing other forms of nitrogen with ease and facility. The biological process, where ammonium gets converted into is called as .

Further, when this nitrate is converted or reduced into nitrogen gas, it is called as denitrification.

These steps involve various microorganisms, and it is important biologically as well as economically.

Both the steps are a significant part of the , which is one of the most important cycles for our atmosphere. Around 78% of the atmosphere contains nitrogen, which is even essential biological molecule found in proteins and nucleic acid and thus marking it important part of all living being.

The nitrogen cycle is completed in five simple steps: , nitrification, assimilation, ammonification, and denitrification. In this content, Nitrification

Nitrification is an oxidation process (loss of electrons or gain of the oxidationstate by an atom or compound takes place). This process starts with the which gets oxidized into (NO2-), this action is performed by the bacteria Nitrosomonas sp. Later on, this nitrite (NO2-) gets oxidized into nitrate (NO3-), and this action is performed by the Nitrobacter sp.

The bacteria are autotrophic, and the reaction is performed under aerobic condition. The importance of this step in nitrogen cycle is the conversion of ammonia into nitrate, as nitrate is the primary nitrogen source present in the soil, for the . Though nitrite is toxic to the plants.

The activity of nitrifying bacteria gets slower in acidic solution, and are best at pH between 6.5 to 8.5 and temperature vary from 16 to 35°C.

Denitrification Denitrification is the reduction process, where the nitrate is removed in the form of nitrogen and is converted to nitrogen gas. The action is performed by the bacteria like Bacillus, Aerobacter, Lactobacillus, Spirillum, Pseudomonas.

The bacteria are heterotrophs, and the action is completed under anaerobic condition. Even the small amount of oxygen may hamper the process, but there is a need organic carbon. Denitrification is useful for wastewater treatment, aquatic habitats.

The denitrification is performed best at pH between 7.0 to 8.5 and at the temperature between 26 to 38°C.

Differences between Nitrification and Denitrification

1. The biological process where ammonia (NH4+) is oxidized and converted into the nitrate (NO3-) is called as nitrification, while denitrification is biological process involves the conversion of nitrate (NO3-) into nitrogen gas (N2).

2. The nitrifying bacteria like Nitrobacter, Nitrosomonas are involved in nitrification process, while in denitrification process bacteria such as Spirillum, Lactobacillus, Pseudomonas, Thiobacillus shows their involvement.

3. In nitrification process the microbes are autotrophic, require aerobic condition (presence of oxygen) to grow, and even their growth is slow, on the other hand in denitrification process the microbes are heterotrophic, require anaerobic condition (absence of oxygen), and they show rapid growth as well.

4. The precursor of the nitrification process is ammonia, and the end is nitrate, whereas nitrate is the precursor of the denitrification process and nitrogen is the end product.

5. Nitrification is performed best at pH between 6.5 to 8.5, with temperature varying from 16 to 35 degree C. Though denitrification is best at pH between 7.0 to 8.5 and temperature ranging from 26 to 38 degree C. Nitrogen Assimilation - + Nitrogen enters roots as NO3 or NH4 . Nitrate reduction results in the production of st NH4 called N2 fixation. NH4 is 1 incorporated into amino acids via the + synthetase (GS) reaction. The NH4 is incorporated into amino acids in roots and leaves and the amino acids get integrated into proteins. The main if not sole function of some proteins is to provide a store of amino acids (see below).

Ammonical form of N2 is available in soil in the form of urea or NH4 in free- state. Urea, if present, is first split into NH4 and CO2, and NH4 is then utilized directly by metabolic pathways by higher plants. But recent studies indicate that urea can be directly used up by metabolic pathways in certain plants. It should be remembered here, that free ammonia is the only utilizable form of N2 that can be directly incorporated into amino acids. Whatever may be the source of nitrogen, first it has to be converted to NH3 and fixed into . It can be converted or transferred to other forms by various pathways that operate in living systems. NITRATE REDUCTION

Nitrate reduction to NH4 is not a single step process, but it is a series of reactions in which the first step is performed by . This has been isolated and purified from various sources like Aspergillus, bacteria, chlorella, blue green algae, alfa alfa and other higher plants.

The mol. Wt. of it is about 3.5 x 105 daltons. The enzyme is associated with 2 cofactors i.e. FAD and two molybdenum ions. The enzyme also requires reducing power supplied by NADH+H or NADPH+H. The former is available in non chlorophyllous tissues and the latter is found in chloroplast containing leaves.

NITRATE REDUCTASE IS AN INDUCIBLE ENZYME

In the absence of NO3 the amount of this enzyme present in the tissues is very low. With the addition of NO3 as the , the amount of this enzyme increases many fold. However, the induction requires light without which the enzyme induction is not possible to the fullest extent.

The nitrate induced enzyme synthesis can be inhibited by the inhibitors of transcription and translation like actinomycin D and cycloheximide respectively, which indicates that NO3 acts as an inducer of nitrate reductase gene expression. How light modulates the gene expression is not yet clear. Furthermore, phytohormones, particularly cytokinin also induces nitrate reductase synthesis denovo even in the absence of light and NO3. Cytokinin induced NO3 reductase activity can be inhibited with actinomycin or CHI. The mechanism of denovo synthesis of nitrate reductase, though not clear, it is fully accepted that the nitrate reductase is an inducible enzyme.

NITRITE REDUCTION

In most of the higher plants so far studied, the synthesized in cytoplasm are transported into , where the nitrites are reduced to hyponitrite by an enzyme called .

The enzyme has a mol. Wt. of 60-70KD and it has a special heme component called siroheme detected in soret band. Actually there are two forms of nitrite reductases, of which one form uses NADPH+H as the proton/electron donor in photosynthetic tissues, but root tissues and others including bacteria and fungi use NADH+H as the hydrogen donors. The enzyme nitrite reductase possesses flavin and iron groups.

Added to this, they are inducible enzymes. Strangely, these enzymes are induced by than nitrites. However, nitrite reductase brings about the reduction of nitrite to NH4 in a multistep reaction, where the intermediary products remain attached to the surface of enzyme; only the final product is release from the surface.

 In this process, a total of six electrons and six protons are transferred to nitrite to produce ammonia.

Nonetheless in some cases one of the intermediate products like hydroxylamine has been found to be converted to NH4 by the activity of hydroxylamine reductase.

Such reactions have been observed in mesophyll tissues of higher plants, Neurospora, aspergillus and some bacteria. Whether or not, the enzyme nitrite reductase by itself is capable of converting hydroxylamine to NH4 is not clear.

 Still the overall pathway from NO3 or NO2 to NH4 is catalyzed by a group of enzymes or multienzyme complexes, but the synthesis of NH4 is very essential for amino acid synthesis.

Nitrogen Fixation

Nitrogenase is the metalloenzyme that performs biological nitrogen fixation by catalyzing the reduction of N2 to ammonia. Understanding how the nitrogenase metal (FeMo-cofactor) catalyzes the cleavage of the N2 triple bond has been the focus of intense study for more than 50 years.

Goals have included the determination of where and how substrates interact with the FeMo-cofactor, and the nature of reaction intermediates along the reduction pathway. Progress has included the trapping of intermediates formed during turnover of non-physiological substrates (e.g., alkynes, CS2) providing insights into how these molecules interact with the nitrogenase FeMo-cofactor active site.

More recently, substrate-derived species have been trapped at high concentrations during the reduction of N2, a diazene, and hydrazine, providing the first insights into binding modes and possible mechanisms for N2 reduction.

A comparison of the current state of knowledge of the trapped species arising from non-physiological substrates and nitrogenous substrates is beginning to reveal some of the intricacies of how nitrogenase breaks the N2 triple bond.

Structure and Operation of Nitrogenase . The nitrogenase complex exists in both free living nitrogen fixing organisms as well as in symbiotic nitrogen fixing bacteria. Nitrogenase is a complex of two separately isolated proteins- an iron protein or dinitrogen reductase and a molybdenum-iron protein or dinitrogenase. The proteins have a negative redox potential. The MoFe protein (Iron- Molybdenum protein),is a heterotetramer composed of two alpha subunits and two beta subunits. The protein contains two copies of each of two types of clusters: P clusters and FeMo cofactors. Each P cluster contains 8 iron atoms and 7 sulfides linked to the protein by 6 cysteinate residues. Each FeMo cofactor contains one molybdenum atom, 7 iron atoms, 9 sulfides. This protein is responsible for reducing atmospheric nitrogen to ammonia via a series of electron transfers within the protein to the substrate molecule. The reaction requires the addition of six electrons for each nitrogen molecule that is split into two ammonia molecules. The Fe protein (Nitrogenase Reductase -NR) is a dimer and formed by 2 subunits of polypeptide chains linked by a 4Fe-4S cluster. Each monomer contains an ATP .