laseradman
adman 时间:2021-01-03 阅读:()
CorrectionBIOCHEMISTRYCorrectionfor"Redox-coupledprotontransfermechanisminni-tritereductaserevealedbyfemtosecondcrystallography,"byYohtaFukuda,KaManTse,TakanoriNakane,ToruNakatsu,MamoruSuzuki,MichihiroSugahara,ShigeyukiInoue,TetsuyaMasuda,FumiakiYumoto,NaohiroMatsugaki,ErikoNango,KensukeTono,YasumasaJoti,TakashiKameshima,ChangyongSong,TakakiHatsui,MakinaYabashi,OsamuNureki,MichaelE.
P.
Murphy,TsuyoshiInoue,SoIwata,andEiichiMizohata,whichappearedinissue11,March15,2016,ofProcNatlAcadSciUSA(113:2928–2933;firstpublishedFebruary29,2016;10.
1073/pnas.
1517770113).
TheauthorsnotethatFig.
4appearedincorrectly.
Thecor-rectedfigureanditslegendappearbelow.
www.
pnas.
org/cgi/doi/10.
1073/pnas.
1604061113Fig.
4.
Updatedreactionmechanismofnitritereduction.
DashedlinesrepresentH-bonds.
StrongandweakH-bondsinvolvedinPCETarecoloredasinFig.
2B.
Chainlinesmeansterichindrancebetweenthenearface-onsubstrateandHis255.
www.
pnas.
orgPNAS|April12,2016|vol.
113|no.
15|E2207CORRECTIONDownloadedbyguestonNovember21,2020DownloadedbyguestonNovember21,2020DownloadedbyguestonNovember21,2020DownloadedbyguestonNovember21,2020DownloadedbyguestonNovember21,2020DownloadedbyguestonNovember21,2020DownloadedbyguestonNovember21,2020DownloadedbyguestonNovember21,2020Redox-coupledprotontransfermechanisminnitritereductaserevealedbyfemtosecondcrystallographyYohtaFukudaa,b,1,KaManTsea,1,TakanoriNakane(中根崇智)c,1,ToruNakatsud,e,MamoruSuzukie,f,MichihiroSugaharae,ShigeyukiInouee,g,TetsuyaMasudae,h,FumiakiYumotoi,NaohiroMatsugakii,ErikoNangoe,KensukeTonoj,YasumasaJotij,TakashiKameshimaj,ChangyongSonge,k,TakakiHatsuie,MakinaYabashie,OsamuNurekic,l,MichaelE.
P.
Murphym,TsuyoshiInouea,2,SoIwatae,n,andEiichiMizohata(溝端栄一)a,2aDepartmentofAppliedChemistry,GraduateSchoolofEngineering,OsakaUniversity,2-1Yamadaoka,Suita,Osaka565-0871,Japan;bDepartmentofBiochemistryandMolecularBiophysics,ColumbiaUniversity,NewYork,NY10032;cDepartmentofBiologicalSciences,GraduateSchoolofScience,TheUniversityofTokyo,7-3-1Hongo,Bunkyo-ku,Tokyo113-0033,Japan;dDepartmentofStructuralBiology,GraduateSchoolofPharmaceuticalSciences,KyotoUniversity,Sakyo,Kyoto606-8501,Japan;eRIKENSPring-8Center,1-1-1Kouto,Sayo-cho,Sayo-gun,Hyogo679-5148,Japan;fInstituteforProteinResearch,OsakaUniversity,3-2Yamadaoka,Suita,Osaka565-0871,Japan;gDepartmentofCellBiologyandAnatomy,GraduateSchoolofMedicine,TheUniversityofTokyo,7-3-1Hongo,Bunkyo-ku,Tokyo113-0033,Japan;hDivisionofFoodScienceandBiotechnology,GraduateSchoolofAgriculture,KyotoUniversity,Gokasho,Uji,Kyoto611-0011,Japan;iStructuralBiologyResearchCenter,KEKHighEnergyAcceleratorResearchOrganization,Tsukuba,Ibaraki305-0801,Japan;jJapanSynchrotronRadiationResearchInstitute,1-1-1Kouto,Sayo-cho,Sayo-gun,Hyogo679-5198,Japan;kDepartmentofPhysics,PohangUniversityofScienceandTechnology,Pohang790-784,Korea;lGlobalResearchCluster,RIKEN,2-1Hirosawa,Wako-shi,Saitama351-0198,Japan;mDepartmentofMicrobiologyandImmunology,UniversityofBritishColumbia,Vancouver,BC,CanadaV6T1Z3;andnDepartmentofCellBiology,GraduateSchoolofMedicine,KyotoUniversity,Yoshidakonoe-cho,Sakyo-ku,Kyoto,606-8501,JapanEditedbyEdwardI.
Solomon,StanfordUniversity,Stanford,CA,andapprovedFebruary2,2016(receivedforreviewSeptember9,2015)Proton-coupledelectrontransfer(PCET),aubiquitousphenome-noninbiologicalsystems,playsanessentialroleincoppernitritereductase(CuNiR),thekeymetalloenzymeinmicrobialdenitrifica-tionoftheglobalnitrogencycle.
AnalysesofthenitritereductionmechanisminCuNiRwithconventionalsynchrotronradiationcrystallography(SRX)havebeenfacedwithdifficulties,becauseX-rayphotoreductionchangesthenativestructuresofmetalcentersandtheenzyme–substratecomplex.
Usingserialfemtosecondcrys-tallography(SFX),wedeterminedtheintactstructuresofCuNiRintherestingstateandthenitritecomplex(NC)stateat2.
03-and1.
60-resolution,respectively.
Furthermore,theSRXNCstructurerepre-sentingatransientstateinthecatalyticcyclewasdeterminedat1.
30-resolution.
ComparisonbetweenSRXandSFXstructuresrevealedthatphotoreductionchangesthecoordinationmannerofthesubstrateandthatcatalyticallyimportantHis255canswitchhydrogenbondpartnersbetweenthebackbonecarbonyloxygenofnearbyGlu279andtheside-chainhydroxylgroupofThr280.
Thesefindings,whichSRXhasfailedtouncover,proposearedox-coupledprotonswitchforPCET.
Thisconceptcanexplainhowpro-tontransfertothesubstrateisinvolvedinintramolecularelectrontransferandwhysubstratebindingacceleratesPCET.
OurstudydemonstratesthepotentialofSFXasapowerfultooltostudyredoxprocessesinmetalloenzymes.
copper|bioinorganicchemistry|freeelectronlaser|SADphasing|damage-freestructureSincetheinventionoftheHaber–Boschprocess,theamountoffixednitrogeninsoilsandwatershasbeenincreasing,andthistrendhassignificantimpactontheglobalenvironment(1,2).
Fixednitrogenisoxidizedtonitrite(NO2)ornitrate(NO3)bynitrificationandthenconvertedtogaseousdinitrogen(N2)bymicrobialdenitrification,whichclosesthenitrogencycle.
Micro-organismsinvolvedindenitrificationcoupletheirrespiratorysystemstostepwisereductionofnitrogenoxidestoN2(NO3→NO2→NO→N2O→N2)(3,4).
ThereductionofNO2totoxicnitricoxide(NO2+2H++e→NO+H2O)isreferredtoasthekeystepindenitrificationandcatalyzedbyeithercd1-hemenitritereductase(cd1NiR)orcoppernitritereductase(CuNiR)(3,4).
Althoughthecatalyticmechanismofcd1NiRiswellunderstood(5,6),thatofCuNiRiscontroversial(7).
CuNiRisahomotrimericproteincontainingtwodistinctCusitespermonomer(SIAppendix,Fig.
S1).
Type1Cu(T1Cu)withaCys–Met–His2ligandsetisanelectronacceptorincorporatednearthemolecularsurface,whereastype2Cu(T2Cu)withaHis3ligandsetisacatalyticcenter,whichis12distantfromthemolecularsurfaceandlocatedbetweentwoadjacentmonomers(7,8).
Spaced12.
5apart,thetwoCusitesarelinkedbyaCys–Hisbridgeandasensorloop.
WhereastheCys–Hisbridgeisanelectronpathway,thesensorloopisthoughttocontrolelectrondistributionbetweenT1CuandT2Cu(9).
Twoconservedresidues,Asp98andHis255(Alcaligenesfaecalisnumbering),arelocatedabovetheT2Cusiteandbridgedbyawatermoleculecalledbridgingwater(SIAppendix,Fig.
S1).
TheyareessentialtotheCuNiRactivitybecausetheyassistprotonSignificanceCoppernitritereductase(CuNiR)isinvolvedindenitrificationofthenitrogencycle.
SynchrotronX-raysrapidlyreducecoppersitesanddecomposethesubstratecomplexstructure,whichhasmadecrystallographicstudiesofCuNiRdifficult.
UsingfemtosecondX-rayfreeelectronlasers,wedeterminedintactstructuresofCuNiRwithandwithoutnitrite.
Basedontheobtainedstructures,weproposedaredox-coupledprotonswitchmodel,whichprovidesanexplanationforproton-coupledelectrontransfer(PCET)inCuNiR.
PCETiswidelydistributedthroughbiogenicprocessesinclud-ingrespiratoryandphotosyntheticsystemsandishighlyexpectedtobeincorporatedintobioinspiredmoleculardevices.
OurstudyalsoestablishesthefoundationforfuturestudiesonPCETinothersystems.
Authorcontributions:Y.
F.
andE.
M.
designedresearch;Y.
F.
,K.
M.
T.
,T.
Nakane,T.
Nakatsu,M.
Suzuki,M.
Sugahara,S.
Inoue,T.
M.
,F.
Y.
,N.
M.
,E.
N.
,K.
T.
,Y.
J.
,T.
K.
,C.
S.
,T.
H.
,M.
Y.
,O.
N.
,M.
E.
P.
M.
,S.
Iwata,andE.
M.
performedresearch;E.
N.
,K.
T.
,Y.
J.
,T.
K.
,C.
S.
,T.
H.
,andM.
Y.
contributednewreagents/analytictools;K.
M.
T.
purifiedandcrystallizedproteinsandper-formedtheassay;T.
Nakaneprocessedserialfemtosecondcrystallography(SFX)dataandperformedsingle-wavelengthanomalousdiffractionphasing;T.
Nakatsu,M.
Suzuki,M.
Sugahara,S.
Inoue,T.
M.
,F.
Y.
,andN.
M.
collectedSFXdata;E.
N.
,K.
T.
,Y.
J.
,T.
K.
,C.
S.
,T.
H.
,andM.
Y.
contributedtheSFXsystems;S.
IwatasupervisedtheSPring-8AngstromCompactFree-ElectronLaserSFXProject;E.
M.
collectedSFXdataandcollectedandprocessedsynchrotronradiationcrystallographydata;Y.
F.
,K.
M.
T.
,T.
Nakane,andE.
M.
ana-lyzeddata;andY.
F.
,K.
M.
T.
,T.
Nakane,M.
E.
P.
M.
,T.
I.
,andE.
M.
wrotethepaper.
Theauthorsdeclarenoconflictofinterest.
ThisarticleisaPNASDirectSubmission.
FreelyavailableonlinethroughthePNASopenaccessoption.
Datadeposition:Crystallography,atomiccoordinates,andstructurefactorshavebeendepositedintheProteinDataBank,www.
pdb.
org[PDBIDcodes4YSC(SFXRS),4YSE(SRXRS),5D4H(SRXNC),5D4I(SFXNC),5D4J(SRXRSCL),5F7B(SRXRSRT),5F7A(SRXNCRT);andCoherentX-rayImagingDataBankID:34].
1Y.
F.
,K.
M.
T.
,andT.
Nakanecontributedequallytothiswork.
2Towhomcorrespondencemaybeaddressed.
Email:inouet@chem.
eng.
osaka-u.
ac.
jpormizohata@chem.
eng.
osaka-u.
ac.
jp.
Thisarticlecontainssupportinginformationonlineatwww.
pnas.
org/lookup/suppl/doi:10.
1073/pnas.
1517770113/-/DCSupplemental.
2928–2933|PNAS|March15,2016|vol.
113|no.
11www.
pnas.
org/cgi/doi/10.
1073/pnas.
1517770113transfer(PT)tothesubstrate(10–12).
Althoughintramolecularelectrontransfer(ET)fromT1CutoT2Cucanoccurintherestingstate(RS)(13,14),thedifferencesintheredoxpotentialsofT2CuminusT1CuaresmallandsometimesnegativeintheabsenceofNO2,meaningthatintramolecularETbeforeNO2bindingisnotenergeticallyfavorable(15,16).
Bycontrast,intramolecularETisdramaticallyacceleratedinthepresenceofNO2(15,17).
Anex-planationforthisgating-likephenomenonisthatsubstratebindingraisestheredoxpotentialofT2CuandshiftstheequilibriumoftheETreaction(16).
However,pHdependenceofintramolecularETinthepresenceofNO2cannotbeexplainedbysuchachangeofredoxpotentials(15).
Instead,Kobayashietal.
(15)proposedthatreduction-inducedstructuralchangeofHis255isresponsibleforthegating-likemechanism.
BecauseithasbeenrecentlyproventhatintramolecularETinCuNiRisaccompaniedbyPTandhenceproton-coupledET(PCET)(17,18),onecanreadilyspeculatethatintramolecularETcontributesPTtoNO2andthatthestructuralchangeofHis255isinvolvedinPCET.
CrystalstructuresofCuNiRfromRhodobactersphaeroides(RhsNiR)impliesthispossibilitybe-causeHis287inRhsNiR,whichcorrespondstoHis255,seemstoshowpH-andredox-dependentconformationalchanges(19,20).
However,presumablybecauseofX-rayradiationdamagesimpliedbyrerefinementofRhsNiRstructures(21),electrondensityaroundHis287wassounusualthatinterpretationofitisdifficult(SIAp-pendix,Fig.
S2).
Crystalstructuresdeterminedbysynchrotronradiationcrystal-lography(SRX)haveprovidedinsightsintotheenzymaticmecha-nismofCuNiR(22–25),andthesestudiesaresummarizedelsewhere(7).
High-resolutionnitritecomplex(NC)structuresrevealedanO-coordinationofNO2showinganearface-onbindingmode(22,23),whereasCu(II)-NO2modelcomplexesshowaverticalbindingmode(7,26–29).
Thenearface-oncoordinationmanneristhoughttofacilitateitsconversiontoside-onNO,whichwasobservedinthecrystalstructuresofCuNiRexposedtoNO(22,23,25).
Skepticaleyeshave,however,beencastontheseCuNiRstructuresbecauseSRXdatamightbeaffectedbysomeproblemsconnectedtothehighradiationdosedeliveredonthecrystals.
First,strongsynchro-tronX-rayscausenotonlyradiationdamagestoaminoacidresiduesbutalsophotoreductionofmetalloproteins(30,31).
Althoughacomparisonbetweenoxidizedandreducedstatesisnecessarytocloselyinvestigateredoxreactions,completelyoxidizedstructuresarealmostimpossibletodeterminebySRX.
Indeed,theCucentersinCuNiRarerapidlyreducedbyexposuretosynchrotronX-rays(21,32).
Second,followingthephotoreductionofT2Cu,NO2iseasilyreducedandproducesNOandwaterinSRX(21).
Conse-quently,electrondensityatthecatalyticsiteofanNCstructureisderivedfromthemixtureofbothsubstrateandproduct,makinginterpretationofdatacomplicatedandunreliable.
Third,cryogenicmanipulationsforreducingradiationdamagesinSRXhavealsobeenfocusedasafactorthatchangesthepopulationofaminoacidresidues(33,34)andenzyme–substratecomplexes(35).
Crystallo-graphic(36),computational(37),andspectroscopic(38–40)studiesactuallyshowthatbindingmodesofNO2andNOinCuNiRcrystalstructurescandifferfromthoseinphysiologicalenvironments.
WehereventuredtousephotoreductioninSRXtoinitiateachemicalreactionandtotrapanenzymaticallyproducedin-termediarystate(30,31).
Furthermore,tovisualizeintactCuNiRstructuresintherestingandNCstates,weappliedserialfemtosec-ondcrystallography(SFX)withX-rayfreeelectronlasers(XFELs)(41),whichenablesdamage-freestructuraldeterminationofmetal-loproteins(42,43)andevaluationofthenativeconformationalpopulationatroomtemperature(RT)(44).
BycomparingSRXandSFXdata,wediscussPCETandnitritereductioninCuNiR.
ResultsandDiscussionRSStructuresDeterminedbySFXandSRX.
TheSFXandcryogenicSRXstructuresofCuNiRfromA.
faecalis(AfNiR)(45,46)inRSwererefinedto2.
03-and1.
20-resolution,respectively(SFXRSandSRXRS,SIAppendix,TablesS1andS2).
Wealsocol-lectedSRXdataat293K,whichisthetemperatureintheSFXexperiment,andthestructurewasdeterminedat1.
56-resolution(SRXRSRT,SIAppendix,TableS2).
AlthoughtheT1CusiteisrapidlyreducedbysynchrotronX-rays(21,32),thereisnosig-nificantdifferenceinthegeometrybetweentheSRXandSFXstructures(SIAppendix,TableS3).
BecausethetypicaldifferencesoftheT1CugeometriesbetweenthereducedandoxidizedstatesareAdmanET(1997)Structureofnitriteboundtocopper-con-tainingnitritereductasefromAlcaligenesfaecalis.
Mechanisticimplications.
JBiolChem272(45):28455–28460.
47.
SolomonEI,SzilagyiRK,DeBeerGeorgeS,BasumallickL(2004)Electronicstructuresofmetalsitesinproteinsandmodels:Contributionstofunctioninbluecopperpro-teins.
ChemRev104(2):419–458.
48.
NakaneT,etal.
(2015)Nativesulfur/chlorineSADphasingforserialfemtosecondcrystallography.
ActaCrystallogrDBiolCrystallogr71(Pt12):2519–2525.
49.
GhoshS,DeyA,SunY,ScholesCP,SolomonEI(2009)Spectroscopicandcomputa-tionalstudiesofnitritereductase:Protoninducedelectrontransferandbackbondingcontributionstoreactivity.
JAmChemSoc131(1):277–288.
50.
HalfenJA,TolmanWB(1994)Syntheticmodelofthesubstrateadducttothereducedactivesiteofcoppernitritereductase.
JAmChemSoc116:5475–5476.
51.
HalfenJA,etal.
(1996)Syntheticmodelingofnitritebindingandactivationbyre-ducedcopperproteins.
Characterizationofcopper(I)-nitritecomplexesthatevolvenitricoxide.
JAmChemSoc118:763–776.
52.
KujimeM,IzumiC,TomuraM,HadaM,FujiiH(2008)Effectofatridentateligandonthestructure,electronicstructure,andreactivityofthecopper(I)nitritecomplex:Roleoftheconservedthree-histidineligandenvironmentofthetype-2coppersiteincopper-containingnitritereductases.
JAmChemSoc130(19):6088–6098.
53.
LiY,HodakM,BernholcJ(2015)Enzymaticmechanismofcopper-containingnitritereductase.
Biochemistry54(5):1233–1242.
54.
DouzouP,HoaGHB,PetskoGA(1975)Proteincrystallographyatsub-zerotemperatures:Lysozyme-substratecomplexesincooledmixedsolvents.
JMolBiol96(3):367–380.
55.
FukudaY,etal.
(2016)Redox-coupledstructuralchangesinnitritereductaserevealedbyserialfemtosecondandmicrofocuscrystallography.
JBiochemmvv133.
56.
BoulangerMJ,MurphyMEP(2002)Crystalstructureofthesolubledomainofthemajoranaerobicallyinducedoutermembraneprotein(AniA)frompathogenicNeisseria:Anewclassofcopper-containingnitritereductases.
JMolBiol315(5):1111–1127.
57.
LawtonTJ,BowenKE,Sayavedra-SotoLA,ArpDJ,RosenzweigAC(2013)Characterizationofanitritereductaseinvolvedinnitrifierdenitrification.
JBiolChem288(35):25575–25583.
58.
ZhangH,BoulangerMJ,MaukAG,MurphyMEP(2000)Carbonmonoxidebindingtocopper-containingnitritereductasefromAlcaligenesfaecalis.
JPhysChemB104:10738–10742.
59.
FukudaY,etal.
(2014)Structuralinsightsintothefunctionofathermostablecopper-containingnitritereductase.
JBiochem155(2):123–135.
60.
TenboerJ,etal.
(2014)Time-resolvedserialcrystallographycaptureshigh-resolutionintermediatesofphotoactiveyellowprotein.
Science346(6214):1242–1246.
61.
KupitzC,etal.
(2014)Serialtime-resolvedcrystallographyofphotosystemIIusingafemtosecondX-raylaser.
Nature513(7517):261–265.
62.
OtwinowskiZ,MinorW(1997)ProcessingofX-raydiffractiondatacollectedinos-cillationmode.
MethodsEnzymol276:307–326.
63.
McCoyAJ,etal.
(2007)Phasercrystallographicsoftware.
JApplCryst40(Pt4):658–674.
64.
EmsleyP,LohkampB,ScottWG,CowtanK(2010)FeaturesanddevelopmentofCoot.
ActaCrystallogrDBiolCrystallogr66(Pt4):486–501.
65.
MurshudovGN,etal.
(2011)REFMAC5fortherefinementofmacromolecularcrystalstructures.
ActaCrystallogrDBiolCrystallogr67(Pt4):355–367.
66.
WinnMD,etal.
(2011)OverviewoftheCCP4suiteandcurrentdevelopments.
ActaCrystallogrDBiolCrystallogr67(Pt4):235–242.
67.
ChenVB,etal.
(2010)MolProbity:All-atomstructurevalidationformacromolecularcrystallography.
ActaCrystallogrDBiolCrystallogr66(Pt1):12–21.
68.
SugaharaM,etal.
(2015)Greasematrixasaversatilecarrierofproteinsforserialcrystallography.
NatMethods12(1):61–63.
69.
TonoK,etal.
(2013)Beamline,experimentalstationsandphotonbeamdiagnosticsforthehardx-rayfreeelectronlaserofSACLA.
NewJPhys15(8):083035.
70.
WhiteTA,etal.
(2012)CrystFEL:Asoftwaresuiteforsnapshotserialcrystallography.
JApplCryst45(2):335–341.
71.
DuisenbergAJM(1992)Indexinginsingle-crystaldiffractometrywithanobstinatelistofreflections.
JApplCryst25:92–96.
72.
SheldrickGM(2010)ExperimentalphasingwithSHELXC/D/E:Combiningchaintracingwithdensitymodification.
ActaCrystallogrDBiolCrystallogr66(Pt4):479–485.
Fukudaetal.
PNAS|March15,2016|vol.
113|no.
11|2933BIOCHEMISTRY
P.
Murphy,TsuyoshiInoue,SoIwata,andEiichiMizohata,whichappearedinissue11,March15,2016,ofProcNatlAcadSciUSA(113:2928–2933;firstpublishedFebruary29,2016;10.
1073/pnas.
1517770113).
TheauthorsnotethatFig.
4appearedincorrectly.
Thecor-rectedfigureanditslegendappearbelow.
www.
pnas.
org/cgi/doi/10.
1073/pnas.
1604061113Fig.
4.
Updatedreactionmechanismofnitritereduction.
DashedlinesrepresentH-bonds.
StrongandweakH-bondsinvolvedinPCETarecoloredasinFig.
2B.
Chainlinesmeansterichindrancebetweenthenearface-onsubstrateandHis255.
www.
pnas.
orgPNAS|April12,2016|vol.
113|no.
15|E2207CORRECTIONDownloadedbyguestonNovember21,2020DownloadedbyguestonNovember21,2020DownloadedbyguestonNovember21,2020DownloadedbyguestonNovember21,2020DownloadedbyguestonNovember21,2020DownloadedbyguestonNovember21,2020DownloadedbyguestonNovember21,2020DownloadedbyguestonNovember21,2020Redox-coupledprotontransfermechanisminnitritereductaserevealedbyfemtosecondcrystallographyYohtaFukudaa,b,1,KaManTsea,1,TakanoriNakane(中根崇智)c,1,ToruNakatsud,e,MamoruSuzukie,f,MichihiroSugaharae,ShigeyukiInouee,g,TetsuyaMasudae,h,FumiakiYumotoi,NaohiroMatsugakii,ErikoNangoe,KensukeTonoj,YasumasaJotij,TakashiKameshimaj,ChangyongSonge,k,TakakiHatsuie,MakinaYabashie,OsamuNurekic,l,MichaelE.
P.
Murphym,TsuyoshiInouea,2,SoIwatae,n,andEiichiMizohata(溝端栄一)a,2aDepartmentofAppliedChemistry,GraduateSchoolofEngineering,OsakaUniversity,2-1Yamadaoka,Suita,Osaka565-0871,Japan;bDepartmentofBiochemistryandMolecularBiophysics,ColumbiaUniversity,NewYork,NY10032;cDepartmentofBiologicalSciences,GraduateSchoolofScience,TheUniversityofTokyo,7-3-1Hongo,Bunkyo-ku,Tokyo113-0033,Japan;dDepartmentofStructuralBiology,GraduateSchoolofPharmaceuticalSciences,KyotoUniversity,Sakyo,Kyoto606-8501,Japan;eRIKENSPring-8Center,1-1-1Kouto,Sayo-cho,Sayo-gun,Hyogo679-5148,Japan;fInstituteforProteinResearch,OsakaUniversity,3-2Yamadaoka,Suita,Osaka565-0871,Japan;gDepartmentofCellBiologyandAnatomy,GraduateSchoolofMedicine,TheUniversityofTokyo,7-3-1Hongo,Bunkyo-ku,Tokyo113-0033,Japan;hDivisionofFoodScienceandBiotechnology,GraduateSchoolofAgriculture,KyotoUniversity,Gokasho,Uji,Kyoto611-0011,Japan;iStructuralBiologyResearchCenter,KEKHighEnergyAcceleratorResearchOrganization,Tsukuba,Ibaraki305-0801,Japan;jJapanSynchrotronRadiationResearchInstitute,1-1-1Kouto,Sayo-cho,Sayo-gun,Hyogo679-5198,Japan;kDepartmentofPhysics,PohangUniversityofScienceandTechnology,Pohang790-784,Korea;lGlobalResearchCluster,RIKEN,2-1Hirosawa,Wako-shi,Saitama351-0198,Japan;mDepartmentofMicrobiologyandImmunology,UniversityofBritishColumbia,Vancouver,BC,CanadaV6T1Z3;andnDepartmentofCellBiology,GraduateSchoolofMedicine,KyotoUniversity,Yoshidakonoe-cho,Sakyo-ku,Kyoto,606-8501,JapanEditedbyEdwardI.
Solomon,StanfordUniversity,Stanford,CA,andapprovedFebruary2,2016(receivedforreviewSeptember9,2015)Proton-coupledelectrontransfer(PCET),aubiquitousphenome-noninbiologicalsystems,playsanessentialroleincoppernitritereductase(CuNiR),thekeymetalloenzymeinmicrobialdenitrifica-tionoftheglobalnitrogencycle.
AnalysesofthenitritereductionmechanisminCuNiRwithconventionalsynchrotronradiationcrystallography(SRX)havebeenfacedwithdifficulties,becauseX-rayphotoreductionchangesthenativestructuresofmetalcentersandtheenzyme–substratecomplex.
Usingserialfemtosecondcrys-tallography(SFX),wedeterminedtheintactstructuresofCuNiRintherestingstateandthenitritecomplex(NC)stateat2.
03-and1.
60-resolution,respectively.
Furthermore,theSRXNCstructurerepre-sentingatransientstateinthecatalyticcyclewasdeterminedat1.
30-resolution.
ComparisonbetweenSRXandSFXstructuresrevealedthatphotoreductionchangesthecoordinationmannerofthesubstrateandthatcatalyticallyimportantHis255canswitchhydrogenbondpartnersbetweenthebackbonecarbonyloxygenofnearbyGlu279andtheside-chainhydroxylgroupofThr280.
Thesefindings,whichSRXhasfailedtouncover,proposearedox-coupledprotonswitchforPCET.
Thisconceptcanexplainhowpro-tontransfertothesubstrateisinvolvedinintramolecularelectrontransferandwhysubstratebindingacceleratesPCET.
OurstudydemonstratesthepotentialofSFXasapowerfultooltostudyredoxprocessesinmetalloenzymes.
copper|bioinorganicchemistry|freeelectronlaser|SADphasing|damage-freestructureSincetheinventionoftheHaber–Boschprocess,theamountoffixednitrogeninsoilsandwatershasbeenincreasing,andthistrendhassignificantimpactontheglobalenvironment(1,2).
Fixednitrogenisoxidizedtonitrite(NO2)ornitrate(NO3)bynitrificationandthenconvertedtogaseousdinitrogen(N2)bymicrobialdenitrification,whichclosesthenitrogencycle.
Micro-organismsinvolvedindenitrificationcoupletheirrespiratorysystemstostepwisereductionofnitrogenoxidestoN2(NO3→NO2→NO→N2O→N2)(3,4).
ThereductionofNO2totoxicnitricoxide(NO2+2H++e→NO+H2O)isreferredtoasthekeystepindenitrificationandcatalyzedbyeithercd1-hemenitritereductase(cd1NiR)orcoppernitritereductase(CuNiR)(3,4).
Althoughthecatalyticmechanismofcd1NiRiswellunderstood(5,6),thatofCuNiRiscontroversial(7).
CuNiRisahomotrimericproteincontainingtwodistinctCusitespermonomer(SIAppendix,Fig.
S1).
Type1Cu(T1Cu)withaCys–Met–His2ligandsetisanelectronacceptorincorporatednearthemolecularsurface,whereastype2Cu(T2Cu)withaHis3ligandsetisacatalyticcenter,whichis12distantfromthemolecularsurfaceandlocatedbetweentwoadjacentmonomers(7,8).
Spaced12.
5apart,thetwoCusitesarelinkedbyaCys–Hisbridgeandasensorloop.
WhereastheCys–Hisbridgeisanelectronpathway,thesensorloopisthoughttocontrolelectrondistributionbetweenT1CuandT2Cu(9).
Twoconservedresidues,Asp98andHis255(Alcaligenesfaecalisnumbering),arelocatedabovetheT2Cusiteandbridgedbyawatermoleculecalledbridgingwater(SIAppendix,Fig.
S1).
TheyareessentialtotheCuNiRactivitybecausetheyassistprotonSignificanceCoppernitritereductase(CuNiR)isinvolvedindenitrificationofthenitrogencycle.
SynchrotronX-raysrapidlyreducecoppersitesanddecomposethesubstratecomplexstructure,whichhasmadecrystallographicstudiesofCuNiRdifficult.
UsingfemtosecondX-rayfreeelectronlasers,wedeterminedintactstructuresofCuNiRwithandwithoutnitrite.
Basedontheobtainedstructures,weproposedaredox-coupledprotonswitchmodel,whichprovidesanexplanationforproton-coupledelectrontransfer(PCET)inCuNiR.
PCETiswidelydistributedthroughbiogenicprocessesinclud-ingrespiratoryandphotosyntheticsystemsandishighlyexpectedtobeincorporatedintobioinspiredmoleculardevices.
OurstudyalsoestablishesthefoundationforfuturestudiesonPCETinothersystems.
Authorcontributions:Y.
F.
andE.
M.
designedresearch;Y.
F.
,K.
M.
T.
,T.
Nakane,T.
Nakatsu,M.
Suzuki,M.
Sugahara,S.
Inoue,T.
M.
,F.
Y.
,N.
M.
,E.
N.
,K.
T.
,Y.
J.
,T.
K.
,C.
S.
,T.
H.
,M.
Y.
,O.
N.
,M.
E.
P.
M.
,S.
Iwata,andE.
M.
performedresearch;E.
N.
,K.
T.
,Y.
J.
,T.
K.
,C.
S.
,T.
H.
,andM.
Y.
contributednewreagents/analytictools;K.
M.
T.
purifiedandcrystallizedproteinsandper-formedtheassay;T.
Nakaneprocessedserialfemtosecondcrystallography(SFX)dataandperformedsingle-wavelengthanomalousdiffractionphasing;T.
Nakatsu,M.
Suzuki,M.
Sugahara,S.
Inoue,T.
M.
,F.
Y.
,andN.
M.
collectedSFXdata;E.
N.
,K.
T.
,Y.
J.
,T.
K.
,C.
S.
,T.
H.
,andM.
Y.
contributedtheSFXsystems;S.
IwatasupervisedtheSPring-8AngstromCompactFree-ElectronLaserSFXProject;E.
M.
collectedSFXdataandcollectedandprocessedsynchrotronradiationcrystallographydata;Y.
F.
,K.
M.
T.
,T.
Nakane,andE.
M.
ana-lyzeddata;andY.
F.
,K.
M.
T.
,T.
Nakane,M.
E.
P.
M.
,T.
I.
,andE.
M.
wrotethepaper.
Theauthorsdeclarenoconflictofinterest.
ThisarticleisaPNASDirectSubmission.
FreelyavailableonlinethroughthePNASopenaccessoption.
Datadeposition:Crystallography,atomiccoordinates,andstructurefactorshavebeendepositedintheProteinDataBank,www.
pdb.
org[PDBIDcodes4YSC(SFXRS),4YSE(SRXRS),5D4H(SRXNC),5D4I(SFXNC),5D4J(SRXRSCL),5F7B(SRXRSRT),5F7A(SRXNCRT);andCoherentX-rayImagingDataBankID:34].
1Y.
F.
,K.
M.
T.
,andT.
Nakanecontributedequallytothiswork.
2Towhomcorrespondencemaybeaddressed.
Email:inouet@chem.
eng.
osaka-u.
ac.
jpormizohata@chem.
eng.
osaka-u.
ac.
jp.
Thisarticlecontainssupportinginformationonlineatwww.
pnas.
org/lookup/suppl/doi:10.
1073/pnas.
1517770113/-/DCSupplemental.
2928–2933|PNAS|March15,2016|vol.
113|no.
11www.
pnas.
org/cgi/doi/10.
1073/pnas.
1517770113transfer(PT)tothesubstrate(10–12).
Althoughintramolecularelectrontransfer(ET)fromT1CutoT2Cucanoccurintherestingstate(RS)(13,14),thedifferencesintheredoxpotentialsofT2CuminusT1CuaresmallandsometimesnegativeintheabsenceofNO2,meaningthatintramolecularETbeforeNO2bindingisnotenergeticallyfavorable(15,16).
Bycontrast,intramolecularETisdramaticallyacceleratedinthepresenceofNO2(15,17).
Anex-planationforthisgating-likephenomenonisthatsubstratebindingraisestheredoxpotentialofT2CuandshiftstheequilibriumoftheETreaction(16).
However,pHdependenceofintramolecularETinthepresenceofNO2cannotbeexplainedbysuchachangeofredoxpotentials(15).
Instead,Kobayashietal.
(15)proposedthatreduction-inducedstructuralchangeofHis255isresponsibleforthegating-likemechanism.
BecauseithasbeenrecentlyproventhatintramolecularETinCuNiRisaccompaniedbyPTandhenceproton-coupledET(PCET)(17,18),onecanreadilyspeculatethatintramolecularETcontributesPTtoNO2andthatthestructuralchangeofHis255isinvolvedinPCET.
CrystalstructuresofCuNiRfromRhodobactersphaeroides(RhsNiR)impliesthispossibilitybe-causeHis287inRhsNiR,whichcorrespondstoHis255,seemstoshowpH-andredox-dependentconformationalchanges(19,20).
However,presumablybecauseofX-rayradiationdamagesimpliedbyrerefinementofRhsNiRstructures(21),electrondensityaroundHis287wassounusualthatinterpretationofitisdifficult(SIAp-pendix,Fig.
S2).
Crystalstructuresdeterminedbysynchrotronradiationcrystal-lography(SRX)haveprovidedinsightsintotheenzymaticmecha-nismofCuNiR(22–25),andthesestudiesaresummarizedelsewhere(7).
High-resolutionnitritecomplex(NC)structuresrevealedanO-coordinationofNO2showinganearface-onbindingmode(22,23),whereasCu(II)-NO2modelcomplexesshowaverticalbindingmode(7,26–29).
Thenearface-oncoordinationmanneristhoughttofacilitateitsconversiontoside-onNO,whichwasobservedinthecrystalstructuresofCuNiRexposedtoNO(22,23,25).
Skepticaleyeshave,however,beencastontheseCuNiRstructuresbecauseSRXdatamightbeaffectedbysomeproblemsconnectedtothehighradiationdosedeliveredonthecrystals.
First,strongsynchro-tronX-rayscausenotonlyradiationdamagestoaminoacidresiduesbutalsophotoreductionofmetalloproteins(30,31).
Althoughacomparisonbetweenoxidizedandreducedstatesisnecessarytocloselyinvestigateredoxreactions,completelyoxidizedstructuresarealmostimpossibletodeterminebySRX.
Indeed,theCucentersinCuNiRarerapidlyreducedbyexposuretosynchrotronX-rays(21,32).
Second,followingthephotoreductionofT2Cu,NO2iseasilyreducedandproducesNOandwaterinSRX(21).
Conse-quently,electrondensityatthecatalyticsiteofanNCstructureisderivedfromthemixtureofbothsubstrateandproduct,makinginterpretationofdatacomplicatedandunreliable.
Third,cryogenicmanipulationsforreducingradiationdamagesinSRXhavealsobeenfocusedasafactorthatchangesthepopulationofaminoacidresidues(33,34)andenzyme–substratecomplexes(35).
Crystallo-graphic(36),computational(37),andspectroscopic(38–40)studiesactuallyshowthatbindingmodesofNO2andNOinCuNiRcrystalstructurescandifferfromthoseinphysiologicalenvironments.
WehereventuredtousephotoreductioninSRXtoinitiateachemicalreactionandtotrapanenzymaticallyproducedin-termediarystate(30,31).
Furthermore,tovisualizeintactCuNiRstructuresintherestingandNCstates,weappliedserialfemtosec-ondcrystallography(SFX)withX-rayfreeelectronlasers(XFELs)(41),whichenablesdamage-freestructuraldeterminationofmetal-loproteins(42,43)andevaluationofthenativeconformationalpopulationatroomtemperature(RT)(44).
BycomparingSRXandSFXdata,wediscussPCETandnitritereductioninCuNiR.
ResultsandDiscussionRSStructuresDeterminedbySFXandSRX.
TheSFXandcryogenicSRXstructuresofCuNiRfromA.
faecalis(AfNiR)(45,46)inRSwererefinedto2.
03-and1.
20-resolution,respectively(SFXRSandSRXRS,SIAppendix,TablesS1andS2).
Wealsocol-lectedSRXdataat293K,whichisthetemperatureintheSFXexperiment,andthestructurewasdeterminedat1.
56-resolution(SRXRSRT,SIAppendix,TableS2).
AlthoughtheT1CusiteisrapidlyreducedbysynchrotronX-rays(21,32),thereisnosig-nificantdifferenceinthegeometrybetweentheSRXandSFXstructures(SIAppendix,TableS3).
BecausethetypicaldifferencesoftheT1CugeometriesbetweenthereducedandoxidizedstatesareAdmanET(1997)Structureofnitriteboundtocopper-con-tainingnitritereductasefromAlcaligenesfaecalis.
Mechanisticimplications.
JBiolChem272(45):28455–28460.
47.
SolomonEI,SzilagyiRK,DeBeerGeorgeS,BasumallickL(2004)Electronicstructuresofmetalsitesinproteinsandmodels:Contributionstofunctioninbluecopperpro-teins.
ChemRev104(2):419–458.
48.
NakaneT,etal.
(2015)Nativesulfur/chlorineSADphasingforserialfemtosecondcrystallography.
ActaCrystallogrDBiolCrystallogr71(Pt12):2519–2525.
49.
GhoshS,DeyA,SunY,ScholesCP,SolomonEI(2009)Spectroscopicandcomputa-tionalstudiesofnitritereductase:Protoninducedelectrontransferandbackbondingcontributionstoreactivity.
JAmChemSoc131(1):277–288.
50.
HalfenJA,TolmanWB(1994)Syntheticmodelofthesubstrateadducttothereducedactivesiteofcoppernitritereductase.
JAmChemSoc116:5475–5476.
51.
HalfenJA,etal.
(1996)Syntheticmodelingofnitritebindingandactivationbyre-ducedcopperproteins.
Characterizationofcopper(I)-nitritecomplexesthatevolvenitricoxide.
JAmChemSoc118:763–776.
52.
KujimeM,IzumiC,TomuraM,HadaM,FujiiH(2008)Effectofatridentateligandonthestructure,electronicstructure,andreactivityofthecopper(I)nitritecomplex:Roleoftheconservedthree-histidineligandenvironmentofthetype-2coppersiteincopper-containingnitritereductases.
JAmChemSoc130(19):6088–6098.
53.
LiY,HodakM,BernholcJ(2015)Enzymaticmechanismofcopper-containingnitritereductase.
Biochemistry54(5):1233–1242.
54.
DouzouP,HoaGHB,PetskoGA(1975)Proteincrystallographyatsub-zerotemperatures:Lysozyme-substratecomplexesincooledmixedsolvents.
JMolBiol96(3):367–380.
55.
FukudaY,etal.
(2016)Redox-coupledstructuralchangesinnitritereductaserevealedbyserialfemtosecondandmicrofocuscrystallography.
JBiochemmvv133.
56.
BoulangerMJ,MurphyMEP(2002)Crystalstructureofthesolubledomainofthemajoranaerobicallyinducedoutermembraneprotein(AniA)frompathogenicNeisseria:Anewclassofcopper-containingnitritereductases.
JMolBiol315(5):1111–1127.
57.
LawtonTJ,BowenKE,Sayavedra-SotoLA,ArpDJ,RosenzweigAC(2013)Characterizationofanitritereductaseinvolvedinnitrifierdenitrification.
JBiolChem288(35):25575–25583.
58.
ZhangH,BoulangerMJ,MaukAG,MurphyMEP(2000)Carbonmonoxidebindingtocopper-containingnitritereductasefromAlcaligenesfaecalis.
JPhysChemB104:10738–10742.
59.
FukudaY,etal.
(2014)Structuralinsightsintothefunctionofathermostablecopper-containingnitritereductase.
JBiochem155(2):123–135.
60.
TenboerJ,etal.
(2014)Time-resolvedserialcrystallographycaptureshigh-resolutionintermediatesofphotoactiveyellowprotein.
Science346(6214):1242–1246.
61.
KupitzC,etal.
(2014)Serialtime-resolvedcrystallographyofphotosystemIIusingafemtosecondX-raylaser.
Nature513(7517):261–265.
62.
OtwinowskiZ,MinorW(1997)ProcessingofX-raydiffractiondatacollectedinos-cillationmode.
MethodsEnzymol276:307–326.
63.
McCoyAJ,etal.
(2007)Phasercrystallographicsoftware.
JApplCryst40(Pt4):658–674.
64.
EmsleyP,LohkampB,ScottWG,CowtanK(2010)FeaturesanddevelopmentofCoot.
ActaCrystallogrDBiolCrystallogr66(Pt4):486–501.
65.
MurshudovGN,etal.
(2011)REFMAC5fortherefinementofmacromolecularcrystalstructures.
ActaCrystallogrDBiolCrystallogr67(Pt4):355–367.
66.
WinnMD,etal.
(2011)OverviewoftheCCP4suiteandcurrentdevelopments.
ActaCrystallogrDBiolCrystallogr67(Pt4):235–242.
67.
ChenVB,etal.
(2010)MolProbity:All-atomstructurevalidationformacromolecularcrystallography.
ActaCrystallogrDBiolCrystallogr66(Pt1):12–21.
68.
SugaharaM,etal.
(2015)Greasematrixasaversatilecarrierofproteinsforserialcrystallography.
NatMethods12(1):61–63.
69.
TonoK,etal.
(2013)Beamline,experimentalstationsandphotonbeamdiagnosticsforthehardx-rayfreeelectronlaserofSACLA.
NewJPhys15(8):083035.
70.
WhiteTA,etal.
(2012)CrystFEL:Asoftwaresuiteforsnapshotserialcrystallography.
JApplCryst45(2):335–341.
71.
DuisenbergAJM(1992)Indexinginsingle-crystaldiffractometrywithanobstinatelistofreflections.
JApplCryst25:92–96.
72.
SheldrickGM(2010)ExperimentalphasingwithSHELXC/D/E:Combiningchaintracingwithdensitymodification.
ActaCrystallogrDBiolCrystallogr66(Pt4):479–485.
Fukudaetal.
PNAS|March15,2016|vol.
113|no.
11|2933BIOCHEMISTRY
- laseradman相关文档
- 本公司adman
- 风险投资adman
- utvecklingenadman
- 知ADMAN解决方案
- 广告二十四,php改写wordpress广告插件adman
- adman成都格志科技有限公司怎么样？
华圣云 HuaSaint-阿里云国际站一级分销商，只需一个邮箱即可注册国际账号，可代充值
简介华圣云 HuaSaint是阿里云国际版一级分销商（诚招募二级代理）,专业为全球企业客户与个人开发者提供阿里云国际版开户注册、认证、充值等服务，通过HuaSaint开通阿里云国际版只需要一个邮箱，不需要PayPal信用卡，不需要买海外电话卡，绝对的零门槛，零风险官方网站：www.huasaint.com企业名：huaSaint Tech Limited阿里云国际版都有什么优势?阿里云国际版的产品...
VirMach（8元/月）KVM VPS，北美、欧洲
VirMach，成立于2014年的美国IDC商家，知名的低价便宜VPS销售商，支持支付宝、微信、PayPal等方式付款购买，主打美国、欧洲暑假中心产品，拥有包括洛杉矶、西雅图、圣何塞、凤凰城在内的11个数据中心可以选择，可以自由搭配1Gbps、2Gbps、10Gbps带宽端口，有Voxility DDoS高防IP可以选择（500Gbps以上的防御能力），并且支持在控制面板付费切换机房和更换IP（带...
HostKvm四月优惠：VPS主机全场八折,香港/美国洛杉矶机房$5.2/月起
HostKvm是一家成立于2013年的国外主机服务商，主要提供基于KVM架构的VPS主机，可选数据中心包括日本、新加坡、韩国、美国、中国香港等多个地区机房，均为国内直连或优化线路，延迟较低，适合建站或者远程办公等。本月商家针对全场VPS主机提供8折优惠码，优惠后美国洛杉矶VPS月付5.2美元起。下面列出几款不同机房VPS主机产品配置信息。套餐：美国US-Plan0CPU：1cores内存：1GB硬...
adman为你推荐
-
美国主机租用美国服务器租用整的这么便宜 啊域名空间代理哪里的域名空间商比较好?vps主机什么是vps主机asp主机空间有ASP虚拟主机空间，还需要另外买Access数据库么？vps试用请问有什么网站可以提供免费vps试用的？想用它来刷一下外国pt站空间域名服务器和空间域名什么意思网站空间购买在哪里购买网站空间手机网站空间谁有上手机网站刷空间人气的网址100m虚拟主机100M虚拟主机有多大，能放多少东西虚拟主机mysql在虚拟主机如何打开数据库？