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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.
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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.
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PNAS|March15,2016|vol.
113|no.
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