Biochemistry-II - Nitrogen Fixation, Assimilation, Elimination - Quick Guides

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Study GuideBiochemistry-II–Nitrogen Fixation, Assimilation,Elimination1.Bacterial Nitrogenase and Nitrogen FixationNitrogen is essential for life, but most organisms cannot useatmospheric nitrogen (N₂)directly.Nitrogen fixation—the conversion of N₂into ammonia (NH₃)—is carried outonly by certain bacteria.These nitrogen-fixing bacteria exist in two forms:•Free-living bacteriain soil or water•Symbiotic bacterialiving in the root nodules ofleguminous plantssuch as soybeans, peas,and cloverNitrogen fixation requires alarge input of ATPandreducing powerand is catalyzed by aspecialized enzyme system callednitrogenase.1.1Overall Reaction of Nitrogen FixationThe complete reaction for nitrogen fixation is:This equation highlights that:•Nitrogen fixation isenergy-intensive•16 ATP moleculesare required to reduceone molecule of N₂•Hydrogen gas (H₂)is always released as a by-productStructure of the Nitrogenase EnzymeNitrogenase is atwo-protein complex:1.Nitrogenase Reductase (NR)

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Study GuideoAlso called theFe proteinoContains ironoAccepts electrons fromreduced ferredoxinoUses ATP to transfer electrons2.Nitrogenase (MoFe protein)oContainsmolybdenum and ironoBinds molecular nitrogen (N₂)oReduces N₂to ammonia (NH₃)Figure 1Mechanism of Nitrogen FixationThe process occurs step by step:•Reduced ferredoxin donates electrons tonitrogenase reductase•Nitrogenase reductase transfers electrons tonitrogenase, using ATP•Nitrogenase bindsN₂•Electrons and protons are added gradually•Two molecules of ammonia (NH₃)are released•Hydrogen gas (H₂)is released as an unavoidable by-product

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Study GuideRole of HydrogenaseThe release of H₂represents aloss of energy. To reduce this loss:•Many nitrogen-fixing organisms containhydrogenase•Hydrogenase captures electrons from H₂•These electrons are transferred back toferredoxin•This helpsrecover some of the energylost during nitrogen fixation1.2Energy Cost and Plant GrowthNitrogen fixation consumes alarge fraction of the energy produced by photosynthesisinnodulated plants.Consequences include:•Slower growth of nitrogen-fixing plants•Lower agricultural yield compared to non-fixing plantsFor example:•Maize (corn)produces more biomass per field•Soybeans, which fix nitrogen, have lower overall energy yieldOxygen Sensitivity of NitrogenaseNitrogenase isextremely sensitive to oxygen. Even small amounts of O₂can inactivate theenzyme.To protect nitrogenase:•Root nodules containleghemoglobin•Leghemoglobin binds oxygen tightly•This maintains alow-oxygen environmentwhile still allowing respiration

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Study GuideNitrate and Nitrite ReductionIn addition to nitrogen fixation, plants and soil bacteria can obtain nitrogen by reducingnitrate (NO₃⁻)andnitrite (NO₂⁻)to ammonia.These compounds are often produced by theoxidation of ammoniain soil.Agricultural ImportanceA common fertilizer,ammonium nitrate (NH₄NO₃):•Providesreduced nitrogen (NH₄⁺)directly•Also suppliesnitrate (NO₃⁻), which plants can reduceStep 1: Nitrate ReductionNitrate is reduced to nitrite bynitrate reductase:•NADH or NADPH acts as theelectron donor•The exact donor depends on the organismStep 2: Nitrite ReductionNitrite is then reduced to ammonia bynitrite reductase:Important intermediates:•Nitric oxide (NO⁻)•Hydroxylamine (NH₂OH)These intermediates:•Remain bound to the enzyme•Do not dissociateinto the cell

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Study GuideSummary of Nitrogen Metabolism•Nitrogen fixation convertsN₂→ NH₃•Requiresnitrogenase, ATP, and reducing power•Extremely sensitive to oxygen•Leghemoglobin protects nitrogenase•Nitrate and nitrite can also be reduced to ammonia•Ammonia is theusable form of nitrogenfor metabolismKey Takeaways•Only bacteria fix atmospheric nitrogen•Nitrogenase requires16 ATP per N₂•H₂release is unavoidable•Nitrogen fixation limits plant growth•Nitrate and nitrite reduction provide alternative nitrogen sources•Ammonia is central to nitrogen metabolism2.Ammonium UtilizationAlthoughammonia (NH₃)andammonium ion (NH₄⁺)are essential nitrogen sources,free ammoniais toxicat high concentrations. Therefore, cells must quicklyincorporate ammonium into stableorganic molecules. These organic forms can then safely store, transport, and distribute nitrogen forbiosynthesis.The most important intermediates of nitrogen assimilation are:•Glutamate•Glutamine•Carbamoyl phosphateThese compounds serve as nitrogen donors for the synthesis ofamino acids, nucleotides, andother nitrogen-containing molecules.

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Study GuideFormation of GlutamateOne direct way to incorporate ammonium is through the enzymeglutamate dehydrogenase.Role of Glutamate Dehydrogenase•Usesα-ketoglutarateas the carbon skeleton•IncorporatesNH₄⁺•Requiresreducing power (NADPH or NADH)A key feature of this enzyme is that it has ahigh Kₘfor ammonia.This means:•It works best whenammonia concentration is high•It is less effective when ammonia is scarceTherefore, this pathway is mainly used whenammonium is abundantin the environment.Formation of GlutamineWhen ammonia levels are low, cells use amore efficient but energy-requiring pathway.Role of Glutamine Synthetase•Usesglutamateas the nitrogen acceptor•IncorporatesNH₄⁺•RequiresATP

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Study GuideThis reaction producesglutamine, which contains two nitrogen atoms and is asafe storage andtransport form of nitrogen.2.1Formation of Glutamate from Glutamine (GOGAT Pathway)Glutamine can donate its nitrogen to form glutamate through the enzymeglutamate synthase, alsocalledGOGAT(glutamine:2-oxoglutarate aminotransferase).In this reaction:•Glutamine transfers its amide nitrogen•α-ketoglutarate is converted intoglutamate•Reducing power is required

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Study GuideTwo Pathways for Making GlutamateFrom the reactions above, cells havetwo ways to synthesize glutamate:1.Glutamate dehydrogenase pathway2.Glutamine synthetase + glutamate synthase (GS–GOGAT) pathway2.2Regulation of Ammonium AssimilationWhich pathway is used depends onnitrogen availability.When Ammonia Is Abundant•Glutamate dehydrogenase is more active•Itshigh Kₘallows it to function efficiently•Common in bacteria growing inammonium-rich mediaWhen Ammonia Is Scarce•GS–GOGAT pathway dominates•UsesATPto trap ammonia efficiently•Prevents loss of ammonia from the cell•Common when nitrogen comes fromnitrogen fixationThis regulation ensures that:•Nitrogen is used efficiently•Toxic ammonia does not accumulate•Nitrogen is conserved under limiting conditionsImportance of Glutamate and GlutamineOnce formed:•Glutamateacts as the main amino-group donor in transamination reactions•Glutaminedonates nitrogen for:

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