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Biochemistry-II - Nitrogen Fixation, Assimilation, Elimination

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    Study Guide Biochemistry - II – Nitrogen Fixation, Assimilation, Elimination 1. Bacterial Nitrogenase and Nitrogen Fixation Nitrogen is essential for life, but most organisms cannot use atmospheric nitrogen (N ₂ ) directly. Nitrogen fixation — the conversion of N ₂ into ammonia (NH ₃ ) — is carried out only by certain bacteria . These nitrogen - fixing bacteria exist in two forms: • Free - living bacteria in soil or water • Symbiotic bacteria living in the root nodules of leguminous plants such as soybeans, peas, and clover Nitrogen fixation requires a large input of ATP and reducing power and is catalyzed by a specialized enzyme system called nitrogenase . 1.1 Overall Reaction of Nitrogen Fixation The complete reaction for nitrogen fixation is: This equation highlights that: • Nitrogen fixation is energy - intensive • 16 ATP molecules are required to reduce one molecule of N ₂ • Hydrogen gas (H ₂ ) is always released as a by - product Structure of the Nitrogenase Enzyme Nitrogenase is a two - protein complex : 1. Nitrogenase Reductase (NR)

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    Study Guide o Also called the Fe protein o Contains iron o Accepts electrons from reduced ferredoxin o Uses ATP to transfer electrons 2. Nitrogenase (MoFe protein) o Contains molybdenum and iron o Binds molecular nitrogen (N ₂ ) o Reduces N ₂ to ammonia (NH ₃ ) Figure 1 Mechanism of Nitrogen Fixation The process occurs step by step: • Reduced ferredoxin donates electrons to nitrogenase reductase • Nitrogenase reductase transfers electrons to nitrogenase , using ATP • Nitrogenase binds N ₂ • Electrons and protons are added gradually • Two molecules of ammonia (NH ₃ ) are released • Hydrogen gas (H ₂ ) is released as an unavoidable by - product

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    Study Guide Role of Hydrogenase The release of H ₂ represents a loss of energy . To reduce this loss: • Many nitrogen - fixing organisms contain hydrogenase • Hydrogenase captures electrons from H ₂ • These electrons are transferred back to ferredoxin • This helps recover some of the energy lost during nitrogen fixation 1.2 Energy Cost and Plant Growth Nitrogen fixation consumes a large fraction of the energy produced by photosynthesis in nodulated plants. Consequences include: • Slower growth of nitrogen - fixing plants • Lower agricultural yield compared to non - fixing plants For example: • Maize (corn) produces more biomass per field • Soybeans , which fix nitrogen, have lower overall energy yield Oxygen Sensitivity of Nitrogenase Nitrogenase is extremely sensitive to oxygen . Even small amounts of O ₂ can inactivate the enzyme. To protect nitrogenase: • Root nodules contain leghemoglobin • Leghemoglobin binds oxygen tightly • This maintains a low - oxygen environment while still allowing respiration

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    Study Guide Nitrate and Nitrite Reduction In addition to nitrogen fixation, plants and soil bacteria can obtain nitrogen by reducing nitrate (NO ₃⁻ ) and nitrite (NO ₂⁻ ) to ammonia. These compounds are often produced by the oxidation of ammonia in soil. Agricultural Importance A common fertilizer, ammonium nitrate (NH ₄ NO ₃ ) : • Provides reduced nitrogen (NH ₄⁺ ) directly • Also supplies nitrate (NO ₃⁻ ) , which plants can reduce Step 1: Nitrate Reduction Nitrate is reduced to nitrite by nitrate reductase : • NADH or NADPH acts as the electron donor • The exact donor depends on the organism Step 2: Nitrite Reduction Nitrite is then reduced to ammonia by nitrite reductase : Important intermediates: • Nitric oxide (NO ⁻ ) • Hydroxylamine (NH ₂ OH) These intermediates: • Remain bound to the enzyme • Do not dissociate into the cell

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    Study Guide Summary of Nitrogen Metabolism • Nitrogen fixation converts N ₂ → NH ₃ • Requires nitrogenase , ATP, and reducing power • Extremely sensitive to oxygen • Leghemoglobin protects nitrogenase • Nitrate and nitrite can also be reduced to ammonia • Ammonia is the usable form of nitrogen for metabolism Key Takeaways • Only bacteria fix atmospheric nitrogen • Nitrogenase requires 16 ATP per N ₂ • H ₂ release is unavoidable • Nitrogen fixation limits plant growth • Nitrate and nitrite reduction provide alternative nitrogen sources • Ammonia is central to nitrogen metabolism 2. Ammonium Utilization Although ammonia (NH ₃ ) and ammonium ion (NH ₄⁺ ) are essential nitrogen sources, free ammonia is toxic at high concentrations. Therefore, cells must quickly incorporate ammonium into stable organic molecules . These organic forms can then safely store, transport, and distribute nitrogen for biosynthesis. The most important intermediates of nitrogen assimilation are: • Glutamate • Glutamine • Carbamoyl phosphate These compounds serve as nitrogen donors for the synthesis of amino acids, nucleotides, and other nitrogen - containing molecules .

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    Study Guide Formation of Glutamate One direct way to incorporate ammonium is through the enzyme glutamate dehydrogenase . Role of Glutamate Dehydrogenase • Uses α - ketoglutarate as the carbon skeleton • Incorporates NH ₄⁺ • Requires reducing power (NADPH or NADH) A key feature of this enzyme is that it has a high K ₘ for ammonia . This means: • It works best when ammonia concentration is high • It is less effective when ammonia is scarce Therefore, this pathway is mainly used when ammonium is abundant in the environment. Formation of Glutamine When ammonia levels are low, cells use a more efficient but energy - requiring pathway . Role of Glutamine Synthetase • Uses glutamate as the nitrogen acceptor • Incorporates NH ₄⁺ • Requires ATP

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    Study Guide This reaction produces glutamine , which contains two nitrogen atoms and is a safe storage and transport form of nitrogen . 2.1 Formation of Glutamate from Glutamine (GOGAT Pathway) Glutamine can donate its nitrogen to form glutamate through the enzyme glutamate synthase , also called GOGAT (glutamine:2 - oxoglutarate aminotransferase). In this reaction: • Glutamine transfers its amide nitrogen • α - ketoglutarate is converted into glutamate • Reducing power is required

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    Study Guide Two Pathways for Making Glutamate From the reactions above, cells have two ways to synthesize glutamate : 1. Glutamate dehydrogenase pathway 2. Glutamine synthetase + glutamate synthase (GS – GOGAT) pathway 2.2 Regulation of Ammonium Assimilation Which pathway is used depends on nitrogen availability . When Ammonia Is Abundant • Glutamate dehydrogenase is more active • Its high K ₘ allows it to function efficiently • Common in bacteria growing in ammonium - rich media When Ammonia Is Scarce • GS – GOGAT pathway dominates • Uses ATP to trap ammonia efficiently • Prevents loss of ammonia from the cell • Common when nitrogen comes from nitrogen fixation This regulation ensures that: • Nitrogen is used efficiently • Toxic ammonia does not accumulate • Nitrogen is conserved under limiting conditions Importance of Glutamate and Glutamine Once formed: • Glutamate acts as the main amino - group donor in transamination reactions • Glutamine donates nitrogen for:

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