搜索
FemtosecondFluorescenceDynamicsofFlavoproteins:ComparativeStudiesonFlavodoxin,ItsSite-DirectedMutants,andRiboflavinBindingProteinRegardingUltrafastElectronTransferinProteinNanospacesNoboruMataga,*HaikChosrowjan,andSeijiTaniguchiInstituteforLaserTechnology,Utsubo-Hommachi1-8-4,Nishiku,Osaka550-0004,JapanFumioTanakaMiePrefecturalCollegeofNursing,Yumegaoka1-1-1,Tsu514-0166,JapanNobuoKidoUnitBiosystems,SchoolofInformaticsandSciences,NagoyaUniVersity,Furo-cho,Chikusaku,Nagoya464-8602,JapanMasayaKitamuraDepartmentofAppliedandBioappliedChemistry,GraduateSchoolofEngineering,OsakaCityUniVersity,Sumiyoshiku,Osaka558-8585,JapanReceiVed:February28,2002;InFinalForm:May24,2002Wehavestudiedthefluorescencedynamicsof“nonfluorescent”flavoproteinsincludingflavodoxin(FD),itsmutantsW60F,Y98F,andW60F/Y98F,andriboflavinbindingprotein(RBP)withthefemtosecondfluorescenceup-conversionmethodandhaveobservedthefluorescencequenchingdynamicsofFDanditsmutantsforthefirsttime.Thestrongfluorescencequenchingintheseflavoproteinsseemstobecausedbyultrafastelectrontransfer(ET)fromaromaticaminoacidresiduestotheexcitedflavinchromophoreinstackedconfigurationaccordingtoprevioustransientabsorptionstudies.Inthepresentwork,wehavemadecomparativestudiesonthedynamicsoffluorescencequenchingduetoETtotheexcitedchromophoreinRBPandFD.WehaveobservedalsofluorescencedynamicsofFDmutantswhereactiveelectrondonorsTrpâNHandTyrâOHarepartially(eitherofthem)orcompletelyreplacedbyinactivephenylalanineanddirectlydemonstratedtheETmechanismoftheultrafastfluorescencequenchinginPNSofFD.IntroductionFlavoproteinswiththeflavinchromophoreareratherubiq-uitousinvariousbiologicalsystems,wheretheyundergoimportantredoxreactions.1Inmostcases,theirreactionsarenotlight-driven;however,considerablestudiesonphotoinducedelectrontransfer(ET)reactionsofflavinsasmodelstofacilitatetheelucidationofthereactionmechanismsinthosebiologicalsystemshavebeenperformed.1Ontheotherhand,althoughexamplesareratherfew,someflavinenzymesseemtoplayalsoimportantrolesinphotobio-logicalreactions.Forinstance,oneexampleistheDNAphotolyasethatphotorepairsacyclobutanepyrimidinedimerproducedbyultravioletlightinDNA.TheDNAphotolyasehastheflavinchromophoreinreducedformandsplitthecyclobu-taneringofthedimerbyphotoinducedreduction.2,3Whentheflavinchromophoreisintheoxidizedform,itcanactasastrongelectronacceptorinthephotoexcitedstate.Therefore,ifaromaticaminoacidresiduestryptophan(TrpâNH)andtyrosine(TyrâOH)areplacedclosetotheflavinchromophoreinproteinnanospace(PNS,proteinenvironmentofafewnanometerscalesurroundingthechromophore),strongquenchingoftheflavinfluorescenceduetotheETfromthearomaticaminoacidresiduescantakeplace.Actually,thebrightfluorescenceoftheflavinchromophore,duetothecommonconjugateð-electronicpartofisoalloxazine(ISO),insolutionisstronglyquenchedinPNSandtherearemany“nonfluores-cent”oronlyveryweaklyfluorescentflavoproteins.Thismaybethemostimportantcharacteristicsofflavoproteinsfortheirfunctionsasphotoreceptors.ExperimentalproofoftheETmechanismofthefluorescencequenchingdynamicsinPNSofflavoproteinswasratherscarce.4WehavepreviouslytriedtodetectthephotoinducedETreactionbymeansofthepicosecond(withexcitinglaserpulseof25psfwhm)time-resolvedtransientabsorptionspectralmeasure-mentsonsomeflavoproteinsandalsosolutionsofflavinchromophoreswithaddedquencherssuchasindoleandphenolcorrespondingtoTrpâNHandTyrâOH,respectively.5,6WehavedetectedthetransientabsorptionspectrathatcanbeascribedtotheradicalionsformedbythephotoinducedETreactionbetweentheexcitedflavinchromophoreandindoleinsolutions.5Wehaveexaminedalsobymeansofthepicosecondtransientabsorptionspectralmeasurementsthe“nonfluorescent”flavo-proteins,flavodoxin(FD)fromDesulfoVibrioVulgaris,strainMiyazakiandriboflavinbindingprotein(RBP).6BothoftheseflavoproteinshavestackedstructureofPNSwheretheISOoftheflavinchromophore,flavinmononucleotide(FMN)forFDandriboflavin(RF)forRBP,issandwichedbetweenTrpâNHandTyrâOHwithveryshortISO-aminoacidinterplanar8917J.Phys.Chem.B2002,106,8917-892010.1021/jp020574lCCC:$22.00©2002AmericanChemicalSocietyPublishedonWeb08/08/2002distancesofca.3.74.3Å.7,8TheobservedtransientabsorptionspectracouldbeascribedmainlytotheradicalionpairstateformedbyETfromthenearbyTrpâNHtotheexcitedflavinchromophore(F*)inPNS,i.e.,F*âââTrpâNHfF-âââTrpâNH+,forFD.6Despitethestackedsandwichstructure,thephotoin-ducedETfromTrpâNHseemstobepredominantduetothefasterETowingtothelargerenergygap,-¢GET,forthereactionF*âââ.TrpâNHfF-âââTrpâNH+.Thechargerecombi-nationdecayoftheproducedionpairstateseemstobemuchslower(decaytimeduetorecombinationinthe100psregime)comparedwiththeveryfastphotoinducedchargeseparation.6Ontheotherhand,theobservedspectraofRBPimmediatelyafterexcitationwereverybroadcomparedwiththoseofFDanditwasdifficulttoconfirmtheultrafastETtotheexcitedriboflavinchromophorefromthearomaticaminoacidresidues.6Contrarytosuchresultsofourprevious10pstransientabsorptionspectralmeasurements,recentfluorescencedynamicsstudiesonRBPbymeansofthefemtosecondfluorescenceup-conversionmethodhaveindicatedultrafastflu