The Calvin Cycle (AKA Light-Independent Reactions)

The Calvin cycle is the second stage in photosynthesis, where solar energy is used to fix carbon dioxide and synthesize carbohydrates. PhotosynthesisandRespiration,Science TheCalvinCycle(AKALight-IndependentReactions) ArpapornSutipatanasomboon 04/30/2022 TheCalvin(-Benson-Bassham)(CBB)cycle,alsonamedreductivepentosephosphatepathwayordarkreactions,isagroupofbiochemicalreactionsinphotoautotrophs. Thesereactionsformthelight-independentstageofphotosynthesis,wheretheenergyconvertedfromlightisusedtoassimilatecarbondioxidefromtheatmosphere. Thefixedcarbonmoleculesareincorporatedintocarbohydrates,whichareconsumedbytheheterotrophs.ThecarbohydrateintermediatesproducedduringtheCalvincyclecanalsobeconvertedtoprecursorsofproteinsandlipidsthatalsofeedconsumersinthefoodchain. TheCalvinCycleandPhotosynthesis Photosynthesisineukaryoticorganismssuchascyanobacteria,greenalgae,andplantsoccursinchloroplastsandcanbedividedintolight-dependentandindependentstages.Thelight-dependentreactionsbeginwhenphotoreceptorsinthethylakoidmembranecapturephotons,energyfromsunlight.ThesolarenergyexciteselectronsinphotoreceptorsclusteredintophotosystemsIandII(PSIandII).Theexcitedelectronsgothroughtheelectrontransportchain,reducingnicotinamideadeninedinucleotidephosphate(NADP+)intoNADPHandoxidizingwaterintooxygen.   Duringthetransferofelectrons,protongradientsaregeneratedacrossthethylakoidmembrane,leadingtoadenosinetriphosphate(ATP)synthesisinthestroma;thisprocessiscalledphotophosphorylation. Figure1:Thetwostagesofphotosynthesis:light-dependentreactionsandtheCalvincycle.(Source:ModifiedfromBoyer,2006andHeldt,2005).[1,2]ThesecondstageofphotosynthesisoccursinarepeatedsetofbiochemicalreactionscalledtheCalvin-Benson-BasshamorCalvincycle,darkreactions,orthereductivepentosephosphatepathway(namedafterthecycle’sfirstsubstrate,apentosephosphate).Thecyclestartsbycapturingatmosphericcarbondioxide(CO2),incorporatingitintoafive-carbonsubstrate,thusresultinginasix-carbonintermediate.Subsequently,ATPandNADPHgeneratedduringthelight-dependentreactionsareconsumedtotransformthesix-carbonproductintotwomoleculesofathree-carbonphosphatesugar(triosephosphate).Afractionofthetriosephosphateistransportedtothecytoplasmforcarbohydratesynthesis–thefinalproductofphotosynthesis.MostofthetriosephosphatemoleculesremaininthechloroplastsothattheyareusedtoregeneratethefirstsubstrateoftheCalvincycle–theyleavethechloroplastonlyafterthey’vebeenconvertedtoDHAP.NB:ADPandNADP+fromenergy-consumingreactionsarerecycledtothelight-dependentreactionssothattheycanparticipateintheelectrontransferprocess.[1,2] Figure2:OverviewoftheCalvincycle(Source:MikeJonesUser:Adenosine,CCBY-SA2.5,viaWikimediaCommons).     ReactionsintheCalvinCycle Despitethenamedarkreactions,theCalvincycledoesnotnecessarilyoccurinthedark.Infact,theCalvincycleiscoupledwithwateroxidation,thelastelectrontransferreactioninPSIIthatoccursafterthecaptureofsunlightenergy.ElectrontransferhelpsgenerateNADPHandATP,whichareusedintheCalvincycle.Therefore,theCalvincycleismorelikelytohappenduringdaylightafterthelight-dependentstagehassufficientlyproducedNADPHandATP.ThebiochemicalreactionsintheCalvincyclecanbegroupedintothreephasesbasedontheirtasks: 1. CarbonFixation Alsoknownasthecarboxylationphase,thefirststepintheCalvincyclecomprisesonlyoneirreversiblereactioncatalyzedbytheenzymeribulose-1,5-bisphosphatecarboxylase/oxygenase(RubisCO).Readhowenzymesareabletoactasbiocatalysts.Here,atmosphericcarbondioxide(CO2)isfixedandincorporatedintoafive-carbonpentosephosphate,ribulose-1,5-bisphosphate(RuBP).Thereactionproducesahighlyreactivesix-carbonketoacidintermediate,2-carboxy3-ketoarabinitol1,5-bisphosphate.Theintermediateishydrolyzedandsplitinhalftoyieldtwo3-phosphoglycerate(3PGA)moleculesultimately.EachisthensubjectedtosuccessivereactionsintheCalvincycleandtransformedintophosphatesugars.RubisCoisthemostabundantenzymeonEarthandtheonlyonecapableoffixingCO2fromtheatmosphere.Thus,therateofcarbonfixationisconsiderablyslow,whichisthoughttobethereasonbehinditslargequantityinphotosynthetictissue.Hence,RubisCo-catalyzedcarboxylationactsasboththerate-limitingandcommittedstepoftheCalvincycle.[2,3] HowdoesphotorespirationaccompanyRubisCo-catalyzedcarbonfixation? ApartfromCO2,RubisCoalsocapturesatmosphericoxygen(O2)andassimilatesittoRuBPduringphotorespiration.Insteadoftwo3PGAmolecules,RubisCo-catalyzedoxygencondensationproducesone3PGAandanothertwo-carbonmolecule,2–phosphoglycolate(2PGC). The3PGAmoleculeproducedfromphotorespirationcansupplytheCalvincycleinasimilarmannertothoseproducedfromcarbonfixation.However,thetwo-carbonproduct,2PGC,mustbetranslocatedandmodifiedbeforere-enteringthechloroplastandconvertedto3PGAbeforethefixedoxygenmoleculecanparticipateintheCalvincycle.[2] 2. ReductionPhase Each3PGAgeneratedfromcarbonfixationundergoessuccessivereductionreactionssothatitistransformedintoatriosephosphate(C3H7O6P).Thethree-carbonsugarphosphatecanserveasaprecursorinthebiosynthesisofcarbohydratesandtheregenerationofRuBPinthelastphaseoftheCBBcycle. Thetransformationof3PGAtotriosephosphateoccursintwosteps: 2.1. Thephosphorylationof3PGA Thisfirststepin3PGAtransformationiscatalyzedbytheenzyme3-phosphoglyceratekinase.ThereactiondephosphorylatesATPandproduces 1,3-bisphosphoglycerate(BPG),ADPandinorganicphosphate. 2.2. Thegenerationofglyceraldehyde3-phosphate(G3P) Thissecondstepin3PGAtransformationiscatalyzedbyNADP-glyceraldehyde3-phosphatedehydrogenase.Thecarboxylicacidphospho-anhydrideportionoftheBPGreactswiththethiolgroupintheactivecenteroftheenzyme,resultingintheformationofathioesterbond.Subsequently,thethioesterbondishydrolyzedtoformtheproductglyceraldehyde3-phosphate(G3P).ThioesterbondhydrolysisrequiresasubstantialamountofenergyandiscoupledwiththeoxidationofNADPHtoNAPD+.Thus,thegenerationofG3PfromBPGreductionisirreversible.Thus,thereductionphaseofthecyclecanbesummarizedinto:[2]3PGA+ATP+NADPH—>Triosephosphate(G3P⇌DHAP)Combinedwiththecarbonfixation,bothstagescanbesummarizedinto:RuBP+CO2+ATP+NADPH —>Triosephosphate(G3P⇌DHAP) G3Pisthefirstsynthesizedtriosephosphateanditisconvertedtoitsisomerbytheenzymetriosephosphateisomeraseintodihydroxyacetonephosphate(DHAP).BothG3PandDHAPcontainthree-carbonatoms,andDHAPismorefavorableintheequilibriumthanG3P.[2]        Isafractionoftriosephosphateusedtosynthesizesugarandstarch? One-sixthofthesynthesizedtriosephosphate(G3PandDHAP)isusedasprecursorsinthebiosynthesisofsugarandstarch.Whentwotriosephosphatemoleculesareavailable,theycondense,formingasix-carbonmolecule,fructose-1,6-bisphosphate(FBP).Itissubsequentlyhydrolyzedintofructose-6-phosphate,anisomerofglucose-6-phosphatethatservesasoneoftheprecursorsinsugar,starch,andcellulosebiosynthesis.[2]Generally,starchissynthesizedfromtriosephosphatethatremainsinthestromaduringtheday.Sugarandcellulosearesynthesizedaftertriosephosphateistransportedtothecytosolviaaspecifictransporter.[3]Inadditiontosugarandstarch,G3PandDHAPcanbeconvertedintoaminoacidsandfattyacids,whicharebuildingblocksforproteinsandlipids.  3. RegenerationPhase Themajorityofthetriosephosphateremainsinthestromaandundergoesseveralchemicalrearrangementreactions.Attheendoftheregenerationphase,thecarbon-fixingRuBPisregeneratedandavailabletorestartthecycle.Inthisphase,thereactionsconsistofaseriesofaldolcondensation,dephosphorylation,andtransketolasereactionsthatleadtothegenerationofafive-carbonsugar-phosphate–ribulose-5-phosphate(Ru5P).Thestepsinclude:[2,3]  3.1. Theformationoffructose-6-phosphate(F6P) Thethree-carbontriosephosphateistransformedintosix-carbonfructose-6-phosphate(F6P)intwosteps:ThecondensationofG3PandDHAPbytheenzymealdolase.Thisreactionresultsinasix-carbonmolecule,fructose-1,6-bisphosphate(FBP).ThehydrolysisofFBPbytheenzymefructose-1,6-bisphosphataseirreversiblydephosphorylatesFBP,convertingitintoF6P. 3.2. Thegenerationofribulose-5-phosphate(Ru5P) Inthisstep,F6BcanexittheCBBcycletosupplyothermetabolicpathwaysaspercellularneeds.  F6BwhichremainsintheCBBcycleparticipatesinseveraltransfersofcarbonatomssothatitistransformedintoafive-carbonpentosephosphatemolecule,ribulose-5-phosphate(Ru5P).Theenzymestransketolaseoraldolasecancatalyzethetransferofcarbonatoms.Transketolasemovesthecarbonatomsfromtheketoneportionofthedonormoleculeandaddsthemtothealdehydegroupoftheacceptingmolecules.Aldolasecatalyzesthecondensationbetweenketoneandaldehydemolecules.Thefollowingarethemetabolitesandthenumberoftheircarbonatomsgeneratedfromtransketolase-catalyzedcarbontransfer:3.2.1.  Erythrose-4-phosphate(E4P)Erythrose-4-phosphate(E4P)isafour-carbonmetaboliteresultingfromtheremovaloftwocarbonatomsfromF6B,facilitatedbytheenzymetransketolase.3.2.2.  Xylulose-5-phosphate(X5P)Xylulose-5-phosphate(X5P)isafive-carbonmetabolitegeneratedfromtwotransketolase-dependenttransfers.ThefirstX5PissynthesizedfromthetransferoftwocarbonatomsfromF6Btothethree-carbontriosephosphate,G3P.Lateron,anotherX5Pisgeneratedinasimilarmanner.However,thetwocarbonatomsarefromtheketonegroupofaseven-carbonmetabolite,sedoheptulose-7-phosphate(S7P).X5PisaRu5PepimerandcanbeconvertedtoRu5Pbytheenzymeribulosephosphateepimerase.3.2.3.  The3-carbonsugarsSedoheptulose-1,7-bisphosphate(SBP)andsedoheptulose-7-phosphate(S7P)areseven-carbonmetabolitesinthelight-independentstageofphotosynthesis.SBPisgeneratedfromthetransferofDHAP,thethree-carbonmetabolite,tothefour-carbonmetabolite,E4P,whichiscatalyzedbytransaldolase.  S7PissubsequentlygeneratedfromtheirreversibledephosphorylationofSBP,catalyzedbytheenzymesedoheptulose-1,7-bisphosphatase.3.2.4.  Ribose-5-phosphate(R5P)Ribose-5-phosphate(R5P)isafive-carbonmetabolitethatistheresidualfromthetransferoftwocarbonatomsfromS7PtoG3PtoformX5P.R5PisaRu5PisomerandcanbeconvertedtoRu5Pbytheenzymeribosephosphateisomerase.   Thus,theprocessesinthegenerationofRu5Pcanbesummarizedinto:[2]FBP+2G3P+DHAP+2H2O —>2X5P+R5P+2PiThetwowatermoleculesareusedinthehydrolysisofFBPintoF6PandSBPintoS7P,whichalsoproducestwoinorganicphosphates.X5PandR5PareenzymaticallyconvertedintoRu5P.Thus,threeRu5P(3×5carbonatoms)aregeneratedfromfivemoleculesoftriosephosphate(5×3carbonatoms),twoofwhicharecondensedintoFBPinthepreviousstepoftheregenerationphase. 3. Phosphorylationofribulose-5-phosphate(Ru5P) Inthelaststepoftheregenerationphase,Ru5Pisirreversiblyphosphorylatedbytheenzymeribulosephosphatekinase.ThereactionconsumesATPandtransformsRu5Pintoribulose-1,5-bisphosphate(RuBP). RuBPsynthesizedattheendoftheregenerationphasereplenishestheRuBPinCO2fixation.IfATPandNADPHareavailable,theregeneratedRuBPwillbeconsumedinthenextroundofthecycle.Theregenerationphasecanbesummarizedintothefollowingequation:[3]5Triosephosphate+3ATP+2H2O—>3RuBP+3ADP+3PiBasedonthesummarizedequation,threeRuBPmoleculesareregeneratedfromfivemoleculesoftriosephosphate,which,basedonthepreviousstagesarefromthreeroundsofCO2fixation. Rubisco-catalyzedCO2fixationandassimilationintoRuBPresultsintwo3PGA.EachconsumesoneATPandNADPHandtransformsintoatriosephosphate(C3H7O6P)molecule.One-sixthofthetriosephosphatesproducedbythecycleisusedincarbohydratesynthesispathways,whiletherestremainsandenterstheregenerationphase. Thus,theoverallCalvincyclecanbesummarizedinto:[3]3CO2+5H20+9ATP+6NADPH—>C3H7O6P+9ADP+9Pi+6NADP+SinceADP,inorganicphosphates,andNADP+eventuallyresupplythelightreactions,itcanbeconcludedthatthemanybiochemicalreactionsinthedarkreactioncontributeonlyoneproducttophotosynthesis,athree-carbontriosephosphate,glyceraldehyde3-phosphate(G3P).TwoormoreoftheG3Pmoleculesaresubsequentlyusedtosynthesizestarchandsugar. InConclusion TheCalvincyclestartswithRubisCo-fixationofatmosphericCO2andassimilationintoafive-carbonribulose-1,6-bisphosphate(RuBP),followedbyareductionphase,andtheregenerationofitscarbon-acceptingsubstrate,RuBP.Alongtheway,afractionofthethree-carbonproduct,glyceraldehyde3-phosphate(G3P),leavesthecycleandservesasprecursorsinthebiosynthesisofsugarandstarch.ItmarksthesecondstageofphotosynthesiswhenATPandNADPHproducedduringthefirststageareconsumedtosetthestageforcarbohydratesynthesis.Andofcourse,italsoenablesphotosynthesistoprovidefoodandrawmaterialstoheterotrophsintheecosystem.   References: BoyerR,ConceptsinBiochemistry,3rdedition.NewJersey:JohnWiley&Sons;2006.HeldtH-WandPiechullaB,PlantBiochemistry.4thedition.SanDiago,California:AcademicPress;2011.BhatlaSCandLalMA,PlantPhysiology,Development,andMetabolism.Singapore:SpringerNatureSingapore;2018 ArpapornSutipatanasomboon ArpaSutipatanasomboonisaresearchscientistbasedintheBangkokMetropolitanRegion.ShebeganherscientificjourneyatMahidolUniversityinBangkok,Thailand,wheresheobtainedherBachelor’sinPlantSciences.Followingherpassionforresearch,ArpamovedtoGermanytopursuehergraduatestudiesattheUniversityofCologne.ShesubsequentlyearnedherMaster’sinBiologicalSciencesandcompletedherPh.D.workingontheintersectionbetweencelldeathandproteostasisinArabidopsisthaliana.Apartfromresearch,shehasdevelopedinterestsintechnologytransfer,intellectualproperty,andIPmanagement.Hergoalistouseherresearchandexpertisetoassistplantbreedersandtomakescienceandscientificknowledgeaccessibletoeveryone. 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