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NifSdirectedassemblyofatransient2Fe2SclusterwithintheNifUproteinPram


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Document on Subject : "NifSdirectedassemblyofatransient2Fe2SclusterwithintheNifUproteinPram"— Transcript:

1 NifS-directedassemblyofatransient[2Fe-2S
NifS-directedassemblyofatransient[2Fe-2S]clusterwithintheNifUproteinPramvadeeYuvaniyama*,JeffreyN.Agar,ValerieL.Cash*,MichaelK.Johnson,andDennisR.Dean**DepartmentofBiochemistry,VirginiaTech,Blacksburg,VA24061-0346;andDepartmentofChemistryandCenterforMetalloenzymeStudies,UniversityofGeorgia,Athens,GA30602EditedbyRichardH.Holm,HarvardUniversity,Cambridge,MA,andapprovedOctober26,1999(receivedforreviewSeptember28,1999)TheNifSandNifUproteinsfromAzotobactervinelandiiarere-quiredforthefullactivationofnitrogenase.NifSisahomodimericcysteinedesulfurasethatsuppliestheinorganicsul®denecessaryforformationoftheFe-Sclusterscontainedwithinthenitrogenasecomponentproteins.NifUhasbeensuggestedtocomplementNifSeitherbymobilizingtheFenecessaryfornitrogenaseFe-SclusterformationorbyprovidinganintermediateFe-Sclusterassemblysite.Asisolated,thehomodimericNifUproteincontainsoneone21,1clusterpersubunit,whichisreferredtoastheper-manentcluster.Inthisreport,weshowthatNifUisabletointeractwithNifSandthatasecond,transient[2Fe-2S]clustercanbeassembledwithinNifUinvitrowhenincubatedinthepresenceofferricion,-cysteine,andcatalyticamountsofNifS.Approximatelyonetransient[2Fe-2S]clusterisassembledperhomodimer.Thetransient[2Fe-2S]clusterspeciesislabileandrapidlyreleasedonreduction.Weproposethattransient[2Fe-2S]clusterunitsareformedonNifUandthenreleasedtosupplytheinorganicironandsulfurnecessaryformaturationofthenitrogenasecomponentproteins.Theroleofthepermanent[2Fe-2S]clusterscontainedwithinNifUisnotyetknown,buttheycouldhavearedoxfunctioninvolvingeithertheformationorreleaseoftransient[2Fe-2S]clusterunitsassembledonNifU.BecausehomologstobothNifUandNifS,respectivelydesignatedIscUandIscS,arefoundinnon-nitrogen®xingorganisms,itispossiblethatthefunctionofNifUproposedherecouldrepresentageneralmechanismforthematurationofFe-Scluster-containingproteins.ron-sulfurclustersarefoundinnumerousproteinsthathaveimportantredox,catalytic,orregulatoryproperties(forarecentreview,seeref.1).Moreover,Fe-Sclustersareintimatelyinvolvedintherespectivefunctionsoftheseproteins.Forexample,Fe-Sclustersareknowntoactaselectroncarriersorenvironmentalsensorsortobeinvolvedinsubstratebindingandactivation.Advancesinourunderstandingofthestructures,organization,andreactivityofcertainbiologicallyrelevantFe-Sclustershaveinvolveddeterminationofthespectroscopicandelectronicpropertiesofprotein-boundFe-Sclusters,character-izationofclusterschemicallyextrudedfromtheirpolypeptidematrices,andpreparationofsyntheticFe-Sclusters.Untilre-cently,however,thebiologicalmechanismbywhichFe-Sclustersareformedhasreceivedscantattention.ItwasshownmanyyearsagothatFe-Sclusterscouldbespontaneouslyincorporatedintoapo-formsofcertainferredoxinsbysimplyincubatingtheminasolutionthatcontainsironandsulfide(2).However,consideringthetoxicityoffreeironandsulfide,itisunlikelythatprotein-boundFe-Sclustersarespontaneouslyformedinvivofromfreeironandsulfide.ItismorelikelythattheironandsulfurnecessaryforFe-Sclusterformationaredeliveredtotheclusterassemblysitebyintermediatecarrierproteins.PreviousworkhasledtotheproposalthattheNifUandNifSnitrogenfixation-specificgeneproductsareinvolvedintheacquisitionofironandsulfurnecessaryforthematurationofthetwonitrogenasecomponentproteins,bothofwhichcontainFe-Sclusters(3).ItwasshownthatNifSisapyridoxalphosphate--cysteinedesulfuraseandthatanenzyme-boundpersulfideisanintermediateinthatreaction(4,5).Thus,NifShasbeentargetedasthesourceofinorganicsulfidenecessaryfornitrogenaseFe-Sclusterformation.WehaverecentlyfoundthatNifUisamodularproteinwithtwodistincttypesofiron-bindingsites(seeFig.1).OneofthesebindingsitetypesislocatedwithinthecentralthirdoftheNifUprimarysequence(6,7)andbindsa[2Fe-2S]cluster(oneclusterbindingsitepersubunit).Thesecondtypeofsiteisalabilemononucleariron-bindingsite(7)locatedwithintheN-terminalthirdoftheNifUprimaryse-quence(onemononuclearsitepersubunit).Becausethethe21,1clusterspresentinisolatedNifUaretightlyboundtotheprotein(6),thelabilemononuclearsiteisthelikelysourceofironfornitrogenaseFe-Sclusterassembly.IfNifSandNifUdohavecomplementaryfunctionsinthemobilizationofsulfurandironfornitrogenaseFe-Sclusterassembly,severaldifferentpathwaysforthisprocesscanbeconsidered.Forexample,NifSandNifUcouldeitheroperateindependentlyduringFe-Sclusterformationortheycouldfunctiontogether.IfNifUandNifSfunctiontogether,thenironandsulfurcouldbeseparatelyreleasedfromeachofthemduringclusterassemblyoranFe-Sclusterprecursorcouldbepreformedandthenreleased.InthecurrentworkwehaveaddressedtheseissuesbyaskingwhetherNifUandNifSareabletoformamacromolecularcomplexandwhetheralabileFe-SclusterspeciescanbeformedonNifU.ExperimentalProceduresPlasmidsandStrains.ConstructionofplasmidsusedfortheheterologousexpressionofalteredformsofNifUinEscherichiahasbeendescribed(7).PlasmidpDB822wasusedtoex-pressafull-lengthversionofNifUforwhichtheCysresidueissubstitutedbyAla.ThisformofNifUisdesignatedAla).PlasmidpDB1041wasusedtoexpressafull-lengthversionofNifUforwhichtheAspresiduewassubsti-tutedbyAla[designatedNifU(AspAla)].PlasmidpDB937wasusedtoexpressatruncatedformofNifUthatincludesthefirst131residuesofNifU.ThistruncatedformofNifUisdesignatedNifU-1.PlasmidpDB1044wasobtainedbytheoligonucleotide-directedmutagenesisofpDB937.ThisplasmidexpressesaformofNifU-1thathastheAspresiduesubstitutedbyAlaandisdesignatedNifU-1(AspAla).Fig.1isaschematicrepresenta-tionofNifUanddifferentformsofNifUusedinthecurrentBiochemicalManipulations.ThepurificationofNifSandalteredformsofNifUwasperformedaspreviouslydescribed(4,7).TheinvitroFe-Sclusterbiosyn

2 theticsystemincludesthefollowing:0.05mMN
theticsystemincludesthefollowing:0.05mMNifU-1dimer(orotherformofNifU-1or0.1mMferricammoniumcitrate5mM1mMscap]l-cysteineMNifS0.1MNaClina25mMHCl(pH7.4)buffer.Allvolumeswere1.0ml,andreactions Thispaperwassubmitteddirectly(TrackII)tothePNASof®ce.Towhomreprintrequestsshouldbeaddressed.E-mail:deandr@vt.eduorjohnson@Thepublicationcostsofthisarticleweredefrayedinpartbypagechargepayment.Thisarticlemustthereforebeherebymarkedªºinaccordancewith18U.S.C.§1734solelytoindicatethisfact.January18,2000vol.97no.2599±604 werecarriedoutanoxicallyinaseptum-sealedcuvetteunderanAratmosphere.ProteinsampleswerepurifiedunderAr,andbuffersusedwereextensivelydegassedandspargedwithArbeforeuse.AnoxicconditionsweremaintainedwithaSchlenkapparatusandorananaerobicglovebox.Reactionswereini-tiatedbytheadditionof-cysteineandweremonitoredbyUV-visiblespectroscopyasdescribedbelow.NifUandNifScomplexformationwasmonitoredbygelexclusionHPLC(BeckmanSystemGold)columnchromatog-raphywithaZorbaxGF-250column.Volumesof100containing6nmolofNifUorNifS(or6nmolofNifUplus6nmolofNifS)ina25mMTrisHCl(pH7.4)buffercontaining20mM1.0mMDTTwereinjectedontothecolumn.SamplesthatcontainedamixtureofNifUandNifSwerepreincubatedatroomtemperaturefor8minbeforeloadingthecolumn.ForsamplemixturesinwhichamolarexcessofNifUorNifSwasused,thesamplecontainedapproximately6nmolofoneproteinand12nmoloftheother.Elutionoftheproteinsampleswasmonitoredbyvisibleat405nm.ResultsobtainedbyusingtheBeckmanSystemGoldHPLCwerealsoindependentlyconfirmedwithanAmersham-PharmaciaFPLCchromatographysystemfittedwithaSuperose12column.SpectroscopicMethods.AllsampleconcentrationsarebasedonproteindeterminationsandareexpressedperNifUorNifU-1monomer.UV-visibleabsorptionspectrawererecordedunderanoxicconditionsinseptum-sealed1-mmand1-cmcuvettes,byusingaShimadzu3101PCscanningspectrophotometeroraCarydiodearrayspectrophotometer.X-band(9.6GHz)EPRspec-trawererecordedbyusingaBrukerESP-300EEPRspectrom-eterequippedwithadual-modeER-4116cavityandanOxfordInstrumentsESR-9flowcryostat.ResonanceRamanspectrawererecordedbyusinganInstru-mentsSA(Edison,NJ)RamanorU1000spectrometerfittedwithacooledRCA31034photomultipliertubewith90ÉscatteringgeometryandlinesfromaCoherentRadiation(PaloAlto,CA)Sabre10-Wargonionlaser.Spectrawererecordeddigitallywithphoton-countingelectronics,andthesignalnoiseratiowasimprovedbysignalaveragingmultiplescans.BandpositionswerecalibratedbyusingtheexcitationfrequencyandCCl,andthesepositionsareaccurateto1cm.Samplesconsistedofldropletsofconcentratedprotein(2±4mM)thatwereplacedinacustomdesignedsamplecellattachedtothecoldfingerofanAirProductsDisplexmodelCSA-202Eclosed-cyclerefrigerator.Thesampletemperaturewasmaintainedat18Kduringscanningtominimizelaser-inducedsampledegradation.Bandscausedbythefrozenbuffersolutionshavebeensub-tractedfromallofthespectrashowninthisworkafternormal-izationoflatticemodesoficecenteredat229cmNifUandNifSComplexFormation.ThatNifUandNifSdonotformatightcomplexwasdeterminedintwodifferentways.First,NifUwasnotfoundtocopurifywithNifSwhenNifSwasisolatedfromcrudeextractspreparedfromnitrogen-fixingAzotobactervine-cells.Second,specificimmunoprecipitationofeitherNifUorNifSfromA.vinelandiicrudeextractsdidnotresultinthecoprecipitationofthecomplementaryprotein.However,NifUandNifScanformatransientcomplexbecauseanequimolarmixtureofNifUandNifSresultsintheappearanceofanewpeakduringsize-exclusioncolumnchromatographywhencomparedwithindividuallychromatographedsamplesofeitherNifUorNifS(Fig.2).CalibrationofthecolumnanddenaturinggelelectrophoresisofthepeakfractionoftheNifU-NifScomplexindicateformationofaheterotetramericcomplex.WhenatwofoldmolarexcessofNifUwasmixedwithNifS,apeakcorrespondingtotheNifU-NifScomplexandapeakcorrespond-ingtouncomplexedNifUcouldberesolvedbygelexclusionchromatography.Theconverseexperimentinvolvingtheaddi-tionofatwofoldmolarexcessofNifSalsoresultedintheappearanceoftwopeaksduringchromatographyofthesample,onecorrespondingtoNifSandtheothercorrespondingtotheNifU-NifScomplex.TheshoulderrecognizedintheNifU-NifScomplexfractionshowninFig.2isreproducibleandprobablyindicatesthatdissociationofthecomplexoccursduringchro-matography.ExperimentalRationale.InpreviousworkwefoundthatsevencysteinescontainedwithinNifUarerequiredforitsfullinvivofunction(7).Fourofthesecysteines(Cys,Cys,Cys,and)arecontainedwithinacentralsegmentofNifUandprovidetheligandsforthe[2Fe-2S]clusterpresentineachmonomerofthedimericNifUproteinasisolated.Becausethe[2Fe-2S]clusterscontainedwithinisolatedNifUaretightlyboundandcannotbereleasedbychelatingreagents,werefertothemasthepermanentclustersandconsideritunlikelythattheyrepresentprecursorsdestinedforassemblyofthenitrogenasemetalloclusters.Wethereforebecameinterestedinwhetherasecond,morelabileclustermightbeassembledelsewhereonNifU.Thethreeothercysteines(Cys,Cys,andCys Fig.1.SchematicrepresentationofNifUanddifferentformsofNifU.Theuppermostlinerepresentsfull-lengthNifU.Abovethelineareindicatedthecysteine(C)andaspartate(D)residuesrelevanttothepresentwork.Thenumericalresiduepositionsareindicatedbelowtheline.SubstitutingresiduespresentinalteredformsofNifUorNifU-1areindicatedbyabold,underlinedletterattheappropriateposition.Dashedbracketsatthetopofthe®gureindicatetherespectivemononuclearirontransient[2Fe-2S]clusterdomainandpermanent[2Fe-2S]clusterdomainwithintheNifUprimarysequence. Fig.2.ComplexformationbetweenNifUandNifS.The®gureshowstheelutionpro®lesofNifS,NifU,oranequimolarmixtureofNifUandNifS,byusingsizeexclusionchromatography.ConditionsusedaredescribedinterialsandMethodsetal. required

3 forfullinvivoNifUfunctionweretargetedast
forfullinvivoNifUfunctionweretargetedasthemostlikelyresiduestoparticipateinprovidingsuchanassemblysiteforformationofatransientcluster.ThepossibilityforassemblyofasecondFe-SclusteronNifUwasalsoconsideredbecausewerecentlyobtainedevidenceforthepresenceofonemononuclearFe-bindingsitewithineachsubunitoftheNifUproteinasisolated(7).Thus,weconsideredthepossibilitythatbindingofFeatthemononuclearsitecouldrepresentanintermediatestageintheassemblyofatransientFe-SclusteronNifU.BindingofFeatthemononuclearsite(s)requiresresiduesCys,Cys,andOurabilitytotestwhetheratransientFe-SclustercouldbeassembledonNifUwascomplicatedbythepresenceofonepermanent[2Fe-2S]clusterineachmonomerofintactisolatedNifU.Thisproblemwascircumventedintwodifferentways.First,aportionofNifUwasrecombinantlyexpressedandthenisolatedthatcorrespondsonlytotheN-terminalthirdoftheNifUcodingsequence.ThistruncatedformofNifUisreferredtoasNifU-1,anditdoesnotincludethosecysteinesthatprovidethecoordinatingligandstothepermanent[2Fe-2S]clustercontainedwithinfull-lengthNifU.Thus,theisolatedformofNifU-1,whichisahomodimer,doesnotcontainthepermanent[2Fe-2S]clusterscontainedwithinfull-lengthNifU(seeFig.1).Second,afull-lengthformofalteredNifUthatcarriesanalaninesubstitutionforoneofthe2Fe-2Scluster-coordinatingresidues(residueCys)wasalsorecombinantlyproducedandisolated.ThisalteredNifUproteinisreferredtoasNifU(CysAla)andalsodoesnotcontainthepermanent[2Fe-2S]clusterinitsisolatedform(Fig.1).PurifiedsamplesofNifU-1andAla)werethenusedinexperimentsdescribedbe-low,whichdemonstratethattheycanserveasscaffoldsforNifS-catalyzedformationof[2Fe-2S]NifU-1andNifU(Ala)CanServeasScaffoldsforAssemblyofaa21Cluster.ItwaspossibletodevelopanoptimizedinvitroFe-Sclusterbiosyntheticsystemthatincludesa0.05mMNifU-1dimerorNifU(CysAla)0.1mMferricammoniumcitrate1mM-cysteineMNifS.Inthisbiosyntheticsystem,NifSispresentatonlyverylowlevelswhencomparedwithNifU-1orNifU(CysAla).Onereasonforperformingthebiosyntheticassayinthiswaywastoensurethatthepyridoxal-phosphatechromophorepresentinNifSwouldnotinterferewiththeabilitytodetectFe-SclusterformationbyUV-visibleabsorptionandresonanceRamanspectroscopies.Also,byusingonlycatalyticamountsofNifS,itwaspossibletomonitorthetime-dependentformationofFe-Sclusters.AtypicalexperimentinwhichNifU-1wasusedasascaffoldforFe-SclusterassemblyisshowninFig.3.Thesedatashowthatthereisatime-dependentassemblyofachromophoricspeciesinNifU-1thatexhibitsabsorbanceinflectionsat325,420,465,and550nm(Fig.3).ThisUV-visibleabsorptionspectrumischaracteristicof[2Fe-2S]cluster-containingproteins(8),althoughtheabsorptionpeaksarelesswelldefinedinoursamplewhencomparedwithsimilarspectrafromtypical[2Fe-2S]cluster-containingproteins.Themostlikelyexplanationfortherelativelyfeaturelessnatureoftheabsorbtionspectrumisthat,onceformed,the[2Fe-2S]clusterisrelativelyunstableatambienttemperature.Thisinstabilityismanifestedbyacon-comitantaccumulationofacolloidalprecipitateofironsulfidethatisresponsibleforthespectralinflectionsbecomingincreas-inglylesswelldefineduponincubationofthesamplebeyondabout200min.Acolloidaliron-sulfideprecipitatealsoaccumu-lateswhenNifU-1isomittedfromthereactionmixture,butthisoccursatamuchslowerratethantherateobservedforFe-Sclusterbiosynthesisinthecompletesystem.Fig.3(curvesaandb)showsthetimedependenceof[2Fe-2S]clusterassemblyontheNifU-1scaffold.ThesedataalsoshowthatclusterformationisdependentontheconcentrationofNifS,withanapproximatedoublingintherateofclusterformationwhentheamountofNifSinthereactionmixtureisdoubled.TheresultsofcontrolexperimentspresentedinFig.3alsoshowthatanalteredformofNifS,forwhichtheactivesiteresiduewassubstitutedbyalanine(5),isnotactiveinclusterformation.AlteredformsofNifU-1,inwhichanyoneofthethreecysteineresidues(Cys,Cys,andCys)hasbeensubstitutedbyalanine,werealsoineffectiveinclusterassembly.Finally,no[2Fe-2S]clusterformationoccurredifanyoftheabove-mentionedcomponentsofthebiosyntheticmixturewasomitted.WhenNifU(CysAla)wassubstitutedforNifU-1intheassemblymixture,aspectrumhavingaverysimilarlineshapeandintensityandthesameabsorbancemaximawasobserved(datanotshown). Fig.3.invitroFe-Sclusterassembly.()UV-visiblespectrumofNifU-1beforeinvitroFe-Sclusterassembly[before-cysteinewasaddedtoinitiateassembly(lowerspectrum)andafter140minofinvitroclusterassembly(upperspectrum)].Thepostassemblyspectrumshowninisthemaximumthatcouldbeobtained.()UV-visiblespectrumofNifU-1(AspAla)beforeinvitroclusterassembly(lowerspectrum)andafter80minofinvitroFe-Sclusterassembly(upperspectrum).Thepostassemblyspectrumshowninrepresentsapproximately60%ofthemaximumthatcouldbeobtained.()TimedependenceofFe-Sclusterassemblyasmonitoredbythechangeinextinctioncoef®cientat465nmvs.timeafterinitiationoftheFe-Sclusterassemblyreaction.Thetimedependenceforclusterassemblyshowninlineaofpanelcorrespondstothesamesampleshowninpanel.Linebofpanelshowsclusterassemblyunderthesameconditionsasforlinea,exceptthathalfoftheamountofNifSwasaddedtotheassemblycocktail.Datashowninthelineslabeledcarecontrols.OnedatasetcorrespondstoconditionsthatarethesameasforlineexceptthatanalteredformofNifShavingtheactive-siteCysresiduesubstitutedbyalaninewasused.Theotherdatasetincorrespondstoconditionsthatarethesameasusedforlinea,exceptthatanalteredformofNifU-1havingtheCysresiduesubstitutedforbyalaninewasused.ConditionsforFe-SclusterassemblyaredescribedinMaterialsandMethodsetal.January18,2000vol.97no.2 IsolationofanNifU-1VariantThatContainsaStabilized[2Fe-2S]The[2Fe-2S]clusterassembledontoNifU-1orAla)islabileinvitro.Forexample,asjudgedbyratios,morethan50%of

4 theclusterwaslostwhengelfiltrationwasuse
theclusterwaslostwhengelfiltrationwasusedtoremoveexcessreagents.Suchlability(alsoseebelow)wasnotunexpectedconsideringthattheproposedphysiologicalfunctionoftheclusteristosupplytheironandsulfurnecessaryfornitrogenasemetalloclusterassembly.Inotherwords,thetransient2Fe-2SclustermusthaveamechanismtoescapefromtheNifUscaffoldduringthematurationofthenitrogenasecomponentproteinsandthereforeshouldnotbetightlyboundtoNifU.DuringthecourseofourstudieswithalteredformsofNifU-1asanapproachtoexaminingthenatureofthemononucleariron-bindingsite,wefortuitouslyidentifiedanalteredformofNifU-1thatcontainssome[2Fe-2S]initsisolatedstate(Fig.3).ThisalteredformofNifU-1hastheresiduesubstitutedbyalanineandisreferredtoasAla).TheisolatedformofNifU-1(AspAla)contained0.15Fepermonomer.Moreover,the[2Fe-2S]clustercontainedwithinisolatedNifU-1(AspAla)isstablewithnolossorchangeintheUV-visiblespectrumevenafterincubationatambienttemperaturefor12h.StabilizationofanFe-SclusterresultingfromanaminoacidsubstitutionhasprecedencefortheFNRproteinfromE.coli.Inthiscaseavariantformwasidentifiedthatcontainsamorestable4Fe-4Sclusterandisaffectedinsignaltransductioneventsdependentonclusterassemblyanddisassembly(9).WhenNifU-1(AspAla)wasusedasascaffoldforclusterassembly(Fig.3),athreefoldincreaseinvisibleabsorptionintensityover200minwasobserved.Theresultingspectrumischaracteristicofabiological[2Fe-2S]centerand,inagreementwiththeFeanalysesoftheisolatedsample,theextinctioncoefficients(e.g.,3.0mM)areindicativeofapproximately0.5clusterspermonomeror1.0clusterperhomodimer.Thisconclusionisbasedontherangeofextinctioncoefficientsfortypical[2Fe-2S]proteins[6±11mM(8)].Analogousbiosyntheticclusterreconstitutionexper-imentswerealsocarriedoutwithafull-lengthformofNifUforwhichtheAspresidueissubstitutedbyalanine[designatedAla)].TheresultingUV-visibleabsorptionspectrumwasindistinguishablefromthatofthestartingspectrumorigi-natingfromthepermanent[2Fe-2S]clusters,exceptforauniform30%increaseinabsorptionintensityinthe300-to800-nmregion.Thisresultisconsistentwiththeformationofaboutoneadditional[2Fe-2S]clusterperhomodimer(datanotshown).Itshouldbenotedthat,inthebiosyntheticsystemdescribedhere,onlyoneferricionisaddedpereachproteinmonomer.Consequently,nomorethanone[2Fe-2S]couldbeformedperhomodimer.Nevertheless,duringthedevelopmentofthebiosyntheticsystem,wefoundthattheadditionofhigherlevelsofferricirondidnotincreasetheamountoftransientclusteraccumulatedwhenanyofthevariousformsofNifUwasusedintheassemblycocktail.Themainconsequenceofatwofoldorfivefoldincreaseintheferricammoniumcitrateconcentrationwastodecreasethequalityoftheabsorptiondataowingtoanincreaseintheaccumulationofcolloidalironsulfide.ResonanceRamanEvidenceforAssemblyoftheTransient[2Fe-2S]ResonanceRamanspectroscopywasusedtoidentifyandfurthercharacterizethetransient[2Fe-2S]clusterspresentinthevariousformsofNifUproteinsinvestigatedinthiswork.Acomparisonofthelow-temperatureresonanceRamanspectraofthepermanent[2Fe-2S]clustersinNifUandofthetransienttransient21assembledinNifU-1byusing488-nmexcitationisshowninFig.4.Inbothspectra,thepatternandfrequencyofbandsintheFe-Sstretchingregionareuniquelyindicativeof[2Fe-2S]clusters(6,10±12).Therelativeinten-sitiesofequivalentbandsareremarkablysimilarforbothclusters,butthefrequenciesareallup-shiftedby6±8cmthetransient[2Fe-2S]clusterinNifU-1whencomparedwiththepermanentclustersinNifU.Hence,thevibrationalassign-mentsmadeforthepermanentclustersinNifU(6)canbetransferreddirectlytothetransientcluster.Also,therelativeintensitiesandfrequenciesofthebandsinNifU-1arealmostidenticaltothoseofthe[2Fe-2S]clusterinhumanferroche-latase(ref.12;Fig.4),whichhasrecentlybeenshowntohavecompletecysteinylligationbyaminoacidsubstitution(13)andcrystallographicstudies(H.A.Dailey,personalcommunica-tion).ThisresultstronglysuggeststhattheclusterassembledonNifU-1hascompletecysteinylligation.Becausethereareonlythreecysteinesavailableineachmonomer,thissituationindi-catesthatthetransientclusterismostlikelybridgedbetweenthesubunits.ThehigherFe-Sstretchingfrequenciesforthetran-sientclusterinNifU-1comparedwiththepermanentclustersinNifUindicatestrongerFe-Sbondsforthetransientstructure.Thus,thelabilityofthetransientclusterismorelikelytobeaconsequenceofenhancedsolventaccessibilityresultingfromitslocationatthesubunitinterface,ratherthanfromanintrinsicthermodynamicinstability.Althoughthelabilityofthetransient[2Fe-2S]clusterhasthusfarimpededourattemptstoobtainresonanceRamanspectrafromreconstitutedfull-lengthNifU,thedecreasedla-bilityinNifU(AspAla)hasprovidedanopportunitytoassesstheresonanceRamanspectrumoftheclusterinafull-lengthversionofNifU(Fig.5).ThespectrumofthereconstitutedformofNifU(AspAla)(Fig.5)isclearlydominatedbyheperma-nentclustersofas-isolatedNifU(Fig.5).Nevertheless,thedifferencebetweenthereconstitutedandas-isolateddatasets(Fig.5)revealsaspectrumverysimilartothatoftherecon-stituted[2Fe-2S]clusterinNifU-1(AspAla)(Fig.5)andhavingapproximatelyhalftheintensityofthepermanentclus-ters.BecauseFe-Sstretchingfrequenciesareverysensitiveto Fig.4.Comparisonofthelow-temperatureresonanceRamanspectraofthethe21clustersinas-isolatedNifU(),reconstitutedNifU-1(),andhumanferrochelatase().Allsamples(2±4mMin100mMTrisHCl,pH7.8,buffer)wereintheformofconcentratedfrozendropletsmaintainedat18K.Thespectrawererecordedbyusing488-nmexcitationwith70-mWoflaserpoweratthesampleandarethesumof19,90,and80scansfor,andrespectively.Eachscaninvolvedadvancingthespectrometerin1cmments(0.5cm)andphotoncountingfor1spointwith6-cmetal. minorperturbationsintheclusterenvironme

5 nt,thisresultindicatesanegligiblechangei
nt,thisresultindicatesanegligiblechangeinthetransientclusterenvironmentonremovaloftheC-terminaldomain.Moreover,thereisaclosesimilarityintheresonanceRamanspectraofthetransientclustersreconstitutedinNifU-1(Fig.4),NifU-1(AspAla)(Fig.5),andNifU(AspAla)(Fig.5).ThemaindifferencesamongthesespecieslieinthefrequenciesofthetotalsymmetricpredominantlyFe-S(Cys)modesassignedat349,337,and340inNifU-1,NifU-1(AspAla),andNifU(AspAla),re-spectively.Thisvariabilityislikelytooriginatefromdifferencesbetweenthewild-typeandAspAlavariantintermsofcysteinylFe-S-C-Cdihedralanglesandorhydrogenbondinginteractionsinvolvingcysteinyl-Satoms.AnattractivepossibilityisthatsubstitutionoftheAspresiduebyalaninereducesclusterlabilitybydecreasingsolventaccessibilityandthatthisfeatureisreflectedintheresonanceRamanasaresultofperturbedhydrogen-bondinginteractionsinvolvingtheligatingcysteineSatoms.Insummary,alloftheresonanceRamanspectraclearlydemonstrateNifS-mediatedassemblyofasimilartransient[2Fe-2S]clusterinavarietyofdifferentformsofNifU.Releaseofthe[2Fe-2S]ClusteronItsReduction.UV-visiblespectraorresonanceRamanspectraoftheFe-Sclustersformedinthebiosyntheticsystemdescribedhereprovidesevidenceforthecatalyticformationofalabile[2Fe-2S]clusterwithintheNifUprotein.Complementaryspectroscopicevidenceforthiscon-clusioncouldnotbeobtainedbyusingeitherelectronparamag-neticresonanceorvariable-temperaturemagneticcirculardi-chroismspectroscopy,becauseoftheextremereductivelabilityofthecluster.Forexample,dithionite-mediatedreductionofthetransient[2Fe-2S]clusterresultedinanimmediate,complete,andirreversiblebleachingofthevisiblespectrum.Also,noparamagneticformofthetransientclustercouldbetrappedevenbyfreezingthesamplewithin10safteradditionofatwofoldexcessofdithionite.Thedithionite-reducedsamplesexhibited2EPRsignalsoverthetemperaturerange10±100Kandnotemperature-dependentmagneticcirculardichroismbands,suggestingthatthe[2Fe-2S]formisunstableanddegradedimmediatelyonreduction.Thelabilityofthetransient[2Fe-2S]clusteronreductionwasalsoshownbyquantitativecaptureoftheFeionreleasedduringdithionitereductionbyusingtheFe-chelatingreagent-dipyridyl(datanotNifUcontainstwodistincttypesofiron-bindingsites.Inas-isolatedNifU,oneofthesetypesofsitesisoccupiedbyaa21,1clusterthatwerefertoasthepermanentcluster(6).Theothertypeofiron-bindingsiteisamononuclearsitethatispredominantlyunoccupiedintheas-isolatedproteinbutcanbeinvitrobytheadditionofferricion(7).Inthecurrentwork,weshowthat-cysteineandcatalyticamountsofNifScanbeusedtoassembleanadditionallabile[2Fe-2S]clusterwithinavarietyofdifferentformsoftheNifUprotein.Thelabilityofthisclusterandthepresenceofthepermanentclustershavesofarpreventeddefinitiveidentificationofthistransientclusterinfull-lengthwild-typeNifU.However,acombinationofUV-visibleabsorptionandresonanceRamanstudieshasprovidedabundantevidencefortheinvitroassemblyofthisclusterinsamplesofNifU-1andtheNifU(CysAla)variant,neitherofwhichcontainsthepermanentclusters.OtherevidencehasbeenobtainedwiththeNifU-1(AspAla)andNifU(AspAla)vari-ants,inwhichthetransientclusterislesslabile.ThesamethreecysteineresiduesnecessaryforironbindingatthemononuclearsitearealsorequiredforNifS-directedassemblyofthetransient[2Fe-2S]cluster(Fig.1).Althoughwecannotyetruleoutthepossibilitythatasetofconditionsexistswherebyonetransienttransient21clustercanbeassembledwithineachsubunit,theavailabledataareconsistentwithamodelinwhichferricionsboundattheindividualmononuclearsitesarerearrangedondonationofsulfurbyNifS,toforma[2Fe-2S]clusterthatisbridgedbetweenthetwoNifUsubunits.Evidencesupportingthismodeofbindingincludes()thelabilityofthetransient[2Fe-2S]cluster;()theiron-bindingstoichiometryatthemono-nuclearsites;()amaximalUV-visibleabsorptionintensityconsistentwithnomorethanonetransient[2Fe-2S]clusterperNifUhomodimer,and()aresonanceRamanspectrumthatisinterpretedintermsoffourcysteineligands,owingtoitsclosecorrespondencetothespectrumofhumanferrochelatase.ThelabilityofthetransientclusteranditsreleasefromthepolypeptidematrixonreductionisconsistentwiththehypothesisthatthefunctionofthetransientclusteristoprovideironandsulfidenecessaryfortheformationoftheFe-Scoresofthenitrogenasemetalloclusters.Theroleofthepermanent[2Fe-2S]clusterscontainedwithinNifUisnotyetknown.However,theobservedreleaseofthetransientclusteronreductionindicatesthattheroleofthepermanentclusterscouldbetoprovidereducingequivalentsforthatprocess.InthiscontextwenotethatbothironscontainedintheNifU-boundtransientclusterareintheferricoxidationstateandthatpreviousworkhasshownthatchemicalreconstitutionofFe-Scluster-containingproteinsismosteffectivewhenferrousionsareusedinthereconstitutionsystem(2).Thus,reductionofthetransientclustermightbeimportantnotonlyforitsreleasebutalsoforplacingironsdestinedfornitrogenasemetalloclustercoreformationintheappropriateoxidationstate.Itisalsopossiblethattheperma-nent[2Fe-2S]clusterscouldhavearedoxfunctionintheacquisitionofironforinitialbindingatthemononuclearsites.Thereisnoapriorireasonwhythepermanentclusterscouldnotparticipateinallofthesefunctions. Fig.5.Low-temperatureresonanceRamanspectraoftransient[2Fe-2S]clusters.()NifU(AspAla)aftertreatmentbytheclusterbiosyntheticsystem;)as-isolatedNifU(AspAla);()differencespectrum(spectrum);()NifU-1(AspAla)aftertreatmentwiththeclusterbiosyntheticsystem.TheconditionsusedarethesameasthosedescribedinFig.4,andeachspectrumisthesumof33scans.etal.January18,2000vol.97no.2 GenesencodinghomologstoNifUandNifSarealsolocatedwithinthegenomesofawidevarietyofnon-nitrogenfixingorganisms(14).Wehavedesignatedt

6 heseasgenestoindicatetheproposedroleofth
heseasgenestoindicatetheproposedroleoftheirproductsinthehousekeepingfunctionofgeneralFe-Sclusterassembly.InlinewiththisproposalthegeneshavebeenfoundtobeessentialforA.vinelandiiviability(14).ThereisalsomountingbiochemicalandgeneticevidencefromotherlaboratoriesthatthegeneproductsareinvolvedinFe-Sclusterassemblyinbothprokaryotic(15)andeukaryotic(16±19)organisms.AcomparisonoftheorganizationoftheNifUproteinanditsproposedhousekeepingcounterpart,designatedIscU,isrelevanttotheworkdescribedhere.Forexample,theIscUproteinisconsiderablytruncatedwhencom-paredwithNifU,bearingsequenceidentityonlytotheN-terminalthirdofNifU.ThisportionofNifUcorrespondstotheNifU-1fragmentdescribedinthepresentwork.TheNifUCys,andCysresiduescontainedwithinthissegmentarealsostrictlyconservedinallgeneproductsidentifiedsofar.Infact,theIscUprimarysequenceisamongthemostconservedsequencemotifsinnature(20).TheIscUproteindoesnotcontainasequencecorrespondingtothepermanent[2Fe-cluster-bindingdomainpresentinNifU.However,thereisanothergenecontainedwithinthegeneclusterwhoseproductdoesharborweakprimarysequenceidentitywhencomparedwiththe[2Fe-2S]cluster-bindingregionofNifU.Thissmallferredoxinhasbeenpurifiedandshowntocontainaa21,1clusterthatisnearlyidenticalinitsspectroscopicandelectronicpropertieswhencomparedwiththe[2Fe-2S]clusterscontainedwithinas-isolatedNifU(21,22).Thus,afunctionanalogoustothatprovidedbytheNifUpermanentclustermightalsobeduplicatedbythisferredoxin.Itisinter-estingthatabacterialferritin-associated[2Fe-2S]ferredoxinhavingthesamespatialarrangementofcluster-coordinatingcysteines,aswellasthesamespectroscopicandelectronicpropertiesasthe-specificferredoxin,hasalsobeenidentifiedE.coli(23,24).Thisobservationhasledtospeculationthatafunctionofthebacterialferritin-associatedferredoxincouldinvolvethereleaseofironfromferritinforFe-Sclusterassembly,asuggestionalsoinlinewithapossibleroleforthepermanentclusterscontainedwithinNifU.Althoughitisnotyetknownwhethera[2Fe-2S]clustercanbeassembledonIscU,bothIscUandIscSfromA.vinelandiibeenrecombinantlyproducedandisolated.IscSexhibitsan-cysteinedesulfuraseactivity(14)similartothatdemonstratedforNifS(4).Also,inpreliminarywork,IscSandIscUhavebeenfoundtoformamacromolecularcomplexsimilartothatde-scribedforNifSandNifU(ourunpublishedresults).DespitethesesimilaritiestheassemblyofFe-SclusterscatalyzedbytheIscsystemmightbeconsiderablymorecomplexthanwehavefoundsofarfortheNifsystem.Forexample,thereareheat-shock-cognate(Hsc)proteinsencodedwithinbacterialgenomesthathavebeensuggestedtohavechaperonefunctionsinvolvingeithertheformationofFe-Sclustersortheirinsertionintovarioustargetproteins(13).Also,inSaccharomycescerevisiaecertainHscproteinshavealreadybeenimplicatedinthephys-iologicalassemblyofFe-Sclusters(16,18,25).Incontrast,therearenoknown-specificgeneproductshomologoustotheHscfamilyofproteins.Finally,thereremainanumberofimportantgapsinourunderstandingofthemobilizationoftheironandsulfurrequiredformaturationofFe-Scluster-containingproteins.AlthoughthecurrentworkdemonstratestheinvitroabilityofNifUtoassem-bleatransient[2Fe-2S]clusterinthepresenceofNifS,whetherthissystemfunctionsbydirectlydonatinga2Fe-2Sunitforinvivoclusterassemblystillneedstobedetermined.IfFe-Sclustersareassembledinthisway,thentherearefundamentalissuescon-cerninghowthetransientclusterisdeliveredtothetargetproteinandhowthe2Fe-2Sunitsmightbeassembledintohigher-orderclusters.Inaddition,itisnotyetknownhowtheNifS-boundpersulfideisphysiologicallyreleasedforassemblyofthetransientcluster.WebelievethattheNifsystemwillcontinuetoprovideamodelforthebiochemical-geneticapproachtoaddresstheseissues.ThisworkwassupportedbygrantsfromtheNationalScienceFounda-tion(MCB9630127toD.R.D.)andtheNationalInstitutesofHealth(GM51962toM.K.J.).1.Beinert,H.,Holm,R.H.&Munck,E.(1997)653±659.2.Malkin,R.&Rabinowitz,J.(1966)Biochem.Biophys.Res.Commun.822±827.3.Dean,D.R.,Bolin,J.T.&Zheng,L.(1993)J.Bacteriol.6737±6744.4.Zheng,L.,White,R.,Cash,V.L.,Jack,R.F.&Dean,D.R.(1993)Proc.Natl.Acad.Sci.USA2754±2758.5.Zheng,L.,White,R.H.&Dean,D.R.(1994)Biochemistry4714±4720.6.Fu,W.,Jack,R.F.,Morgan,T.V.,Dean,D.R.&Johnson,M.K.(1994)Biochemistry7.Agar,J.N.,Yuvaniyama,P.,Jack,R.F.,Cash,V.L.,Smith,A.D.,Dean,D.R.&Johnson,M.K.(2000)J.Biol.Inorg.Chem.,inpress.8.Dailey,H.A.,Finnegan,M.G.&Johnson,M.K.(1994)Biochemistry303±407.9.Khoroshilova,N.,Beinert,H.&Kiley,P.J.(1995)Proc.Natl.Acad.USA2499±2503.10.Han,S.,Czernuszewicz,R.S.&Spiro,T.G.(1989)J.Am.Chem.Soc.3496±3504.11.Han,S.,Czernuszewicz,R.S.,Kimura,T.,Adams,M.W.W.&Spiro,T.G.J.Am.Chem.Soc.12.Crouse,B.R.,Sellers,V.M.,Finnegan,M.G.,Dailey,H.A.&Johnson,M.K.Biochemistry13.Sellers,V.M.,Wang,K.-F.,Johnson,M.K.&Dailey,H.A.(1998)J.Biol.14.Zheng,L.,Cash,V.L.,Flint,D.H.&Dean,D.R.(1998)J.Biol.Chem.13264±13272.15.Nakamura,M.,Saeki,K.&Takahashi,Y.(1999)J.Biochem.10±18.16.Kispal,G.,Csere,P.,Prohl,C.&Lill,R.(1999)EMBOJ.17.Schilke,B.,Voisine,C.,Beinert,H.&Craig,E.(1999)Proc.Natl.Acad.Sci.10206±10211.18.Strain,J.,Lorenz,C.R.,Bode,J.,Garland,S.,Smolen,G.A.,Ta,D.T.,Vickery,L.E.&Culotta,V.C.(1998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