Biochemistry-II - Nitrogen Fixation, Assimilation, Elimination

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Study GuideBiochemistry-IINitrogen Fixation, Assimilation,Elimination1.Bacterial Nitrogenase and Nitrogen FixationNitrogen is essential for life, but most organisms cannot useatmospheric nitrogen (N)directly.Nitrogen fixationthe conversion of Ninto ammonia (NH)is carried outonly by certain bacteria.These nitrogen-fixing bacteria exist in two forms:Free-living bacteriain soil or waterSymbiotic 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-intensive16 ATP moleculesare required to reduceone molecule of NHydrogen gas (H)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 (N)oReduces Nto ammonia (NH)Figure 1Mechanism of Nitrogen FixationThe process occurs step by step:Reduced ferredoxin donates electrons tonitrogenase reductaseNitrogenase reductase transfers electrons tonitrogenase, using ATPNitrogenase bindsNElectrons and protons are added graduallyTwo molecules of ammonia (NH)are releasedHydrogen gas (H)is released as an unavoidable by-product

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Study GuideRole of HydrogenaseThe release of Hrepresents aloss of energy. To reduce this loss:Many nitrogen-fixing organisms containhydrogenaseHydrogenase captures electrons from HThese electrons are transferred back toferredoxinThis 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 plantsLower agricultural yield compared to non-fixing plantsFor example:Maize (corn)produces more biomass per fieldSoybeans, which fix nitrogen, have lower overall energy yieldOxygen Sensitivity of NitrogenaseNitrogenase isextremely sensitive to oxygen. Even small amounts of Ocan inactivate theenzyme.To protect nitrogenase:Root nodules containleghemoglobinLeghemoglobin binds oxygen tightlyThis 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 (NHNO):Providesreduced nitrogen (NH₄⁺)directlyAlso suppliesnitrate (NO₃⁻), which plants can reduceStep 1: Nitrate ReductionNitrate is reduced to nitrite bynitrate reductase:NADH or NADPH acts as theelectron donorThe exact donor depends on the organismStep 2: Nitrite ReductionNitrite is then reduced to ammonia bynitrite reductase:Important intermediates:Nitric oxide (NO)Hydroxylamine (NHOH)These intermediates:Remain bound to the enzymeDo not dissociateinto the cell

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Study GuideSummary of Nitrogen MetabolismNitrogen fixation convertsN→ NHRequiresnitrogenase, ATP, and reducing powerExtremely sensitive to oxygenLeghemoglobin protects nitrogenaseNitrate and nitrite can also be reduced to ammoniaAmmonia is theusable form of nitrogenfor metabolismKey TakeawaysOnly bacteria fix atmospheric nitrogenNitrogenase requires16 ATP per NHrelease is unavoidableNitrogen fixation limits plant growthNitrate and nitrite reduction provide alternative nitrogen sourcesAmmonia is central to nitrogen metabolism2.Ammonium UtilizationAlthoughammonia (NH)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:GlutamateGlutamineCarbamoyl 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 DehydrogenaseUsesα-ketoglutarateas the carbon skeletonIncorporatesNH₄⁺Requiresreducing power (NADPH or NADH)A key feature of this enzyme is that it has ahigh Kfor ammonia.This means:It works best whenammonia concentration is highIt 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 SynthetaseUsesglutamateas the nitrogen acceptorIncorporatesNH₄⁺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 nitrogenα-ketoglutarate is converted intoglutamateReducing 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 (GSGOGAT) pathway2.2Regulation of Ammonium AssimilationWhich pathway is used depends onnitrogen availability.When Ammonia Is AbundantGlutamate dehydrogenase is more activeItshigh Kallows it to function efficientlyCommon in bacteria growing inammonium-rich mediaWhen Ammonia Is ScarceGSGOGAT pathway dominatesUsesATPto trap ammonia efficientlyPrevents loss of ammonia from the cellCommon when nitrogen comes fromnitrogen fixationThis regulation ensures that:Nitrogen is used efficientlyToxic ammonia does not accumulateNitrogen is conserved under limiting conditionsImportance of Glutamate and GlutamineOnce formed:Glutamateacts as the main amino-group donor in transamination reactionsGlutaminedonates nitrogen for:
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