BIOCHEMISTRY

LESSON 3: NITROGEN FIXATION

Dear Students in this lesson I will explain you the conversion of nitrogen into ammonia to nitrate /nitrite and again in to nitrogen . The role of enzyme into their conversion and utility of nitrogen . other N compounds, or it may be converted by nitrifying bacteria to NO2 and NO3. In turn, NO3 may enter metabolism through reduction to NH4 and subsequent assimilation to amino acids by bacteria, fungi, and plants or can serve as electron acceptor for denitrifying bacteria when oxygen is limiting. Losses from the nitrogen pool occur physically, when nitrogen (especially nitrate) is leached into inaccessible domains in the soil, and chemically, when denitrification releases N2. Plants bhiosynthesize both the suit of nitrogenous compounds that are present in all living organisms and the may chemical species unique to plants (Fig. 1.6). The most important step in assimilation of inorganic N into organic metabolites is the reaction, catalysed by glutamine synthetase, a high-flux process subject to extensive regulation in all plants. The largest requirement for N is the synthesis of amono acids ,which function as the building blocks of proteins as well as precursors to may other compounds. Nitrogen is also

Introduction
The elements nitrogen and sulfur are acquired by plants primarily through interaction with the soil solution. As with other mineral nutrients, the acquisition of nitrogen- and sulfurcontaining ions is mediated by highly evolved morphological, physiological, and bio-chemical mechanisms. Unlike other mineral nutrients, however, the inorganic forms of nitrogen and sulfur are often present in soil in oxidized forms, which must be reduced for the element to be used in metabolism. These conversions take place in highly reducing environments (characterized by low Eo values) and link nitrogen and sulfur assimilation with pathways that generate reducing potential. Nitrate and sulfate reduction are compartmentalized and regulated to facilitate integration with other cellular metabolism. A combination of biochemical and molecular-genetic approaches is further elucidating these pathways. Although we know more about nitrogen metabolism in plants than we do about sulfur metabolism, our understanding of sulfur is increasing impressively with the recent renewed interest in the subject and the advent of new tools with which to study it.

Overview of Nitrogen in the Biosphere and in Plants

Nitrogen is the fourth most abundant element in living organism. It constitutes less than 0.1% of the earths curst but makes up about 80% of the atmosphere, mostly as dinitrogen (N2). Despite the apparent disparity in content, the large mass of the earths curst means that the number of N atoms below ground is about 50 times greater than in the entire atmosphere. Most of this mineral nitrogen is in igneous rocks, but their weathering is not a significant source of entry for inorganic nitrogen into the living world. The cycle of transformation of mineral and organic nitrogen is shown in figure 1.5 The cycle of N between geochemical and biochemical states is quite complex, given the many oxidation states the N atom can occupy in inorganic and organic compounds (Table 1.1). Most of the nitrogen taken up by organism is recycled from a pool of nitrogen compounds that have previously been used by other organisms. New inputs to this pools are generated during chemical reactions that result from natural events (e.g., fire and lightning) or from human activity (e.g., use of internal combustion engines and application of chemical fertilizers). The main input to the pool of accessible in-organic N in natural ecosystems, however, is from atmospheric and dissolved N2 through the biological process of nitrogen fixation, a process that converts N2 to NH3 (Table 1.2) Nitrogen fixation is carried out only by pro-karyotic species. The NH3 produced by N fixation may be assimilated into amino acids and thence to protein and
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Figure 1.5 The nitrogen cycle, Organic nitrogen compounds, which are constituents of all living organisms, are released into the environment by death and decay and are excreated as waste by some animals. Microorganisms deaminate organic nitrogen, utilizing the carbon as a food source and liberating ammonium in the process. Plants and microorganisms can take up nitrate and reduce it to ammonium for subsequent assimilation into organic nitrogen-containing compounds. Many biological process that change the oxidation state of nitrogen are catalysed exclusively by prokaryotes. These include nitrification (in which ammonium or nitrite is oxidized and the energy released is used to fix inorganic carbon), denitrification (in which nitrogen serves as terminal electron acceptor and is reduced during anaerobic respiratation), and nitrogen fixation (In which dinitrogen gas is reduced to ammonium).

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An essential component of nucleic acids , cofactors, and other common metabolities and is a major component of chlorophyll . The characteristic yellow color of nitrogen-taarved plants (chlorosis) freflects their inability to synthesize, adequate amounts of green chlorophyll under N-limited conditions (Fig. 1.7). In addition, several plant hormones contain N or are derived from nitrogenous precursors . Plants synthesize diverse nitrogenous secondary compounds, most prominently the alkaloids Although flavonoids and other plant phenolics do not contain N, their derivation from phenylalanine means their synthesis is linked with amino acid metabolism. Plants may acquire N though NH4 uptake and incorporation into organic compounds; NO3 uptake and reduction to NH4 , or, in the case of plant hosts for nitrogen-fixing bacteria, acquisition of fixed N from bacterial endoymbionts (from the Greek: living together within). Plants display quite diverse strategies in acquiring nitrogen, as is evident both in comparisons of different species with one another and in comparison of individusls of a given species that are grown in different environments. For example, nitrate takern up by roots may be reduced and assimilated in the root) NO3 NH4 Glutamine), Or It May Be Transported as NO3 to shoot, NO3 may be also be stored Table 1.1 Compound Oxidation state of N N2 NH3 NH4 N2O NO NO2 NO2_ NO3 0 -3 -3 +1 +2 +3 +4 +5 Name Dinitrogen (nitrogen gas) Ammonia Ammonia ion Nitrous oxide Nitric oxide Nitrite Nitrogen dioxide Nitrate Figure 1.6 Selected nitrogenous compounds of biological importance. Some (e.g., amino acids, nucleoside bases) are found in all living organism, whereas others are unique to plants.

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Figure 1.7 Nitrogen deficiency phenotype in leaves of sugar beet. Nitrogen deficiency is often associated with uniform yellowing (chlorosis) of older leaves. This feature is obvious for the plants grown in nitrogen deficient soil (left side of photo relative to counterparts grown with sufficient nitrogen (right side of photo) In vacuoles in cells of the root or the shoot. Plants able to establish symbioses exert developmental and physiological control over the formation and function of symbiotic structures, according to their nutritional environment: Legume plants grown in the presence of NO3 , for example, will use this as a source of N nutrition and will not from symbiotic nodules or allow Rhizobium invasion (Fig. 1.8) Acquisition of nitrogen occurs in a context that is physiological, developmental, and environmental. A comprehensive investiga2.503 Copy Right: Rai University 15

tion of nitrogen assimilation requires the study of genes and gene expression, of protein structure and activity, and of root development and physiology. As photoautotrophs, plants are typically limited in their growth by availability of nutrients other than carbon, and nitrogen is frequently the limiting factor for plant productivity. Widespread application of nitrogenous fertilizer has been a key factor in improving agricultural yields during the 19th and 20th centuries). As the human population grows and the demand for agricultural products increases, it becomes all the more important to understand the mechanisms of nitrogen acquisition for plants.

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Overview of Nitrogen Fixation

Nitrogen fixation reduces nitrogen gas to ammonia, at the cost of ATP and reducing equivalents. Dinitrogen constitutes about 80% of the earths atmosphere, but the stability of its triple bond renders it inert and metabolically inaccessible to most organism. Eukaryotes cannot utilize dinitrogen, but some prokaryotes are able to catalyse the enzymatic reduction of this compound to ammonia (Rx. 16.1). Unlike the Haber-Bosch process (see Box 16.1O, the biological nitrogen fixation catalysed by the enzyme nitrogenase occurs at ambient temperatures and atmospheric pressure. Table1.2 Rate of natural and anthropogenic nitrogen fixation Source Amount of N fixeda Lightining Biological N- Fixation in terrestrial systems Biological N-Fixation in marine Systemsb N- fertilizer Synthesis Fossil Fuel Combustion <10 Tg/year 90-140Tg/year 30-300 Tg / year c 80 Tg/year >20 Tg /Year

Figure 1.8 Overview of n uptake by a nonnodulated plant (left), and by a nodulated plant with N-fixing symbionts (right). There is considerable variation in the details of nitrogen assimilation in different plants. Plant roots can import nitrate, ammonium, and other nitrogenous compounds from the soil. For use in synthesis of amines and amides, nitrate must be reduced to nitrite and then to ammonium. Nitrate reduction in the cytosol and storage in the vacuoles are processes that can occur in either the rots or the leaves. Nodulated plants are able to take up fixed nitrogen from the soil (not shown) but, through the action of symbiotic bacteria, can generate ammonium also by reducing N 2. The ammonium from nitrogen fixation is assimilated into amino acids and ultimately incorporated into amide amino acids (glutamine or asparagines) or ureides for export to the leaves. Enzymatic nitrogen fixation is limited to prokaryotes. This trait is associated with members of many eubacterial phylogenies (Fig. 1.9) as well as some methanogenic archaea. A few nitrogenfixing bacteria from diverse taxa (i.e., cyanobacteria, actinomycetes, and the a-roteobacteria) are able to establish symbiotic associations with plants. In such symbioses, nitrogen fixed by bacteria is exchanged for carbon fixed by the plant. Nitrogen fixation by symbiotic bacteria can be highly productive because interaction with the plant allows fixation to occur under optimised physiological conditions, over coming constraints that often limit nitrogen fixation by nonsymbiotic bacteria. Much of what we know about the genetics and biochemistry of nitrogen fixation has been determined in studies of free-living dubacteria, such as Clostridium, Klebsiella, Azotobacter, and Anabaena. Results from these investigations have provided detailed information on factors that constrain all nitrogen-fixing systems, including symbiotic bacteria.

a- The standard unit of measeure is teragram (Tg) a10 11 g equal to 106 metric tons b- This estimate includes both natural ecosystems and agricultural nitrogen fixation c- Estimates differ because of variable data Reaction 1.1: Nitrogenase N2 + 16 ATP + 8e + 8H 2NH3 + H2 + 16ADP + 16Pi

Nitrogen Fixation is Sensitive to Oxygen

Nitrogen fixation is a unique biochemical reaction that consumes energy-rich compounds while requiring strong biological reductants. Because nitrogenase and some of the proteins that supply it with reductant are sensitive to oxygen, may nitrogenfixing bacteria are anaerobes. Neither fermentation nor anaerobic
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resporation oxidizes reduced carbon compounds as efficiently as aerobic respiration, so anaerobic bacteria must process large quantities of substrate to genrrate the ATP required for dinitrogen fixation. In contrast, aerobes have the advantage of high ATP production from aerobic metabolism but must contend with the oxygen sensitivity of nitrogenase. In some cases, free-living nitrogen-fixing organisms use mechanical or biochemical barriers to keep oxygen away from the biological catalysts of nitrogen fixation. In other cases, the nitrogen fixation machinery is segregated spatially in specialized structures. For example, some filamentous cyanobaceria generate heterocysts, thick walled cells that fix nitrogen but cannot complete all the reactions of oxygenic photosynthesis. Heterocysts produce the ATP needed for nitrogen fixation by way to cyclic photophosphorylatin, a light-dependent process that does not create oxygen gas. Some nonfilamentous cyanobacteria segregate photosynthesis from nitrogen fixation temporally, performing oxygenic photosynthesis in the light and nitrogen fixation in the dark.

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Figure 1.10A (A) Schematia diagram of the nitrogenase complex, showing the flow of reducing power and substrates in enzymatic nitrogen fixation. The Fe-protein, encoded by nifH, accepts electrons from a carrier,, e.g., ferredoxin, flavodoxin, or the carrier varies, depending on the biological system involved. The Fe-protein, accompanied by net hydrolysis of ATP. The MoFe-protein, and a 2 B2 heterotetramer of

Figure 1.9 Phylogenetic distribution of nitrogen-fixing eubacteria. A simplified taxonomy of these bacteria shows that, although many groups contain nitrogen-fixing species (highlighted in yellow), nitrogen fixation is not careied out by every representative of these groups.

Enzymology of Nitrogen Fixation

The biological reduction of dinitrogen to ammonia is carried out by two enzymes, dinitrogenase and dinitrogenase reductase. Together, these enzymes are often referred to as nitrogenase (Fig.1.10) Dinitrogenase, a 240-kDa heterotetramer, binds N2 and holds it while it is being reduced. Dinitrogenase reductase, a 64-kDa homodimer, provides dinitrogenase with high-energy electrons; The midpoint reduction potential of the reaction catalysed by dinitrogenase reductasse is about 400 mV when the enzyme is binding ATP. Both proteing have been purified from bacterial species and characterized extensively, and the three-dimensional structures of representative proteins have been determind by X-ray crystallography. For nitrogenase to function, additional proteins are needed to synthesize its unique metal-containing cofactors and to transfer low-potential electrons.
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Figure 1.10B Subunits encoded by nifD and nifK, accepts electrons and binds H + ions and N2 gas in a stepwise cycle, ultimately leading to the production of H2 and ammonia. (B) Docking of the nitrogenase FE protein dimmer (yellow) with half of the nitrogenase MoFe protein (red, nifD; purple, nifH). A 4Fe4S cluster is associated with the Fe protein. The P cluster is near the nifD / nifH interface. FeMoCo (green) is mostly associated with nifD.

Reference
Principles of Biochemistry, Lehninger, Nelson and Cox Worth Publishers, ISBN : 033394657-X

Problems
Q.1 Q.2 Q.3 Q.4 Q.5 Explain nitrogen cycle. Describe nitrogenous compounds of biological importance. Explain the reduction of nitrogen gas to ammonia Is nitrogen fixation sensitive to oxygen ? Explain. Describe the enzymology of nitrogen fixation.
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