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Sub-picosecond C=C bond photo-isomerization:Evidence for the role of excited state mixing

Damianos Agathangelou, Partha Pratim Roy, María del Carmen Marín,Nicolas Ferre, Massimo Olivucci, Tiago Buckup, Jérémie Léonard, Stefan

Haacke

To cite this version:Damianos Agathangelou, Partha Pratim Roy, María del Carmen Marín, Nicolas Ferre, MassimoOlivucci, et al.. Sub-picosecond C=C bond photo-isomerization: Evidence for the role of excitedstate mixing. 2020. �hal-02996783�

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Sub-picosecondC=Cbondphoto-isomerization:1

Evidencefortheroleofexcitedstatemixing23

Photo-isomérisationsub-picosecondedeliaisonsC=C:4

L’importancedumélangedesétatsexcités56DamianosAgathangelou1,*,ParthaPratimRoy2,†,MaríadelCarmenMarín3,NicolasFerré4,7

MassimoOlivucci3,5,TiagoBuckup2,JérémieLéonard1,StefanHaacke18

91UniversitédeStrasbourg−CNRS,InstitutdePhysiqueetChimiedesMatériauxdeStrasbourg,1067034Strasbourg,France112Physikalisch-ChemischesInstitut,Ruprecht-KarlsUniversitätHeidelberg,69120Heidelberg,12Germany133Biotechnology,PharmacyandChemistryDepartment,UniversityofSiena,Siena53100,Italy144Aix-MarseilleUniv.-CNRS,ICR,13397Marseille,France155ChemistryDepartment,BowlingGreenStateUniversity,BowlingGreen,Ohio43403,United16States17

Abstract18

Sub-picosecond photo-isomerization is the major primary process of energy conversion in19retinalproteinsandhasassuchbeeninthefocusofextensivetheoreticalandexperimental20workoverthepastdecades.Inthisreviewarticle,werevisitthelong-standingquestionasto21howtheproteintunesthe isomerizationspeedandquantumyield.Wefocusonourrecent22contributionstothisfield,whichunderscoretheconceptofadelicatemixingofreactiveand23non-reactiveexcitedstates,asaresultofstericpropertiesandelectrostaticinteractionswith24the protein environment. Further avenues and new approaches are outlined which hold25promise foradvancingourunderstandingof these intimatelycoupledchromophore-protein26systems.2728

Résumé29

La photo-isomérisation sub-picoseconde est la principale réaction initiant la conversion30d'énergie dans les protéines de rétinal, si bien qu’elle fait l'objet de travaux théoriques et31expérimentauxapprofondisdepuisplusdetrenteans.Danscetarticlederevue,nousrevisitons32la question toujours ouverte de savoir comment la protéine détermine la vitesse33d'isomérisationetsonrendementquantique.Alalumièredenoscontributionsrécentesence34domaine, nous décrivons le concept d'un mélange d'états excités réactifs et non-réactifs,35délicatement ajusté par les interactions stériques et électrostatiques avec l'environnement36

*Nowat:LUMOSlab,UniversityofMichigan,DepartmentofPhysics,USA†Nowat:UniversityofBerkeley,DepartmentofChemistry,USA

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protéique.Denouvellesperspectivesetapprochesprometteuses sontdécritesquipourront1faireprogresserlacompréhensiondecessystèmeschromophore-protéineintimementcouplés.23Keywords: Photo-isomerisation, retinal proteins, chromophore-protein coupling, non-4adiabaticdynamics,photo-sensoryproteins,chargetransferstate56 7

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1Manyorganisms,frommicrobacteriatovertebrates,usephotosensitiveretinalproteinsfor2photo-chemicalenergyconversionorphoto-sensoryfunctions[1–3].Visible light-absorbing3retinalproteinsrelyontheprotonatedSchiffbaseofretinal(rPSB)aschromophore,covalently4boundtotheinteriorofabarrel-like“opsin”protein.Theirbiologicalactivityistriggeredbya5regioselectiveandstereoselectiveC=CdoublebondphotoisomerizationofrPSB,e.g.11-cisto6all-trans for the visual pigment rhodopsin (Rho), all-trans to 13-cis for the proton pump7bacteriorhodopsin (bR), or both 13-cis to all-trans and all-trans to 13-cis for the bistable,8photochromicAnabaena SensoryRhodopsin (ASR). In all cases, theprotein scaffoldexerts9electrostatic and steric interactions on the chromophore [4–7], which tune the rPSB10photoreaction dynamics, e.g. the excited state lifetime (ESL) and its photoisomerization11quantumyield(IQY),ultimatelydeterminedbythebranchingratiobetweenreactiveandnon-12reactive channels. The pronounced influence of the protein interaction on rPSB photo-13reactivityisevidencedbycomparingwiththephotoreactiondynamicsandIQYofrPSBinother14environments,e.g.insolution[8–14]orinvacuum[15].15

ThephotoisomerizationofrPSBoccursthroughaconicalintersection(CInt),[16,17]whichis16anintersectionseambetweentheexcited(S1)andground(S0)statepotentialenergysurfaces17(PES),wherethemoleculetransientlyadoptsaconfigurationwithanearly90°twistedreacting18doublebond.WhileithasbeenshownthatthereisnocorrelationbetweenESLandIQY[18–1921],manytheoreticalpredictionsagree,thatthePESstopographyandthenuclearmotionsat20theCIntdecideonthebranchingbetweenreactiveandnon-reactivepathways,henceonthe21overallIQY[17,22,23].Thereislargescientificinterestatinvestigatingthephotoreactionsof22retinalproteinsinordertodeciphertheintra-andinter-molecularparameterswhichtune23thephotoreactivityofrPSB,asamodelsystemfornon-adiabaticdynamicsthroughaCInt.In24addition, such a fundamental understanding would be the key to define chemical design25strategies to control the photoreactivity of C=C double bond isomerization in retinal26chromophores or similar compounds. In particular, there is broad interest in producing27rhodopsinmutantswithanenhancedESLand,asaconsequence,fluorescencequantumyield28(FQY) for applications in optogenetics [24,25]. Moreover, understanding the dynamic29structure-propertyrelationshipswillmostlikelyindicatenewavenuesfortailoringmolecular30devices based on ultrafast C=C double isomerization to perform photoswitching or rotary31motion,atthemolecularscale,withoptimumefficiency[26].32

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ChapterIgivesabriefandnecessarilyincompleteaccountofrecentexperimentalstudieson1the effect of the environment of the photophysics of rPSB’s, with an emphasis on the2electrostaticproperties,inparticularthepresenceorprotonationstateofcounterions.3ChaptersIItoIVconcentrateonbothelectrostaticandstericeffectsontheESLandFQYof4rPSBindifferentretinalproteinsandrhodopsin-mimickingphoto-switchesinsolution.Steric5effects,inparticularthedistortionoftherPSBinthegroundstateof11CinRhoasopposed6tothemoreplanarATinbRisheldresponsibleforthefasterphoto-isomerizationreactionin7Rho[27–32],aswillbecorroboratedinthesechapters.InchapterII,wereviewtheconceptof81Bu/2Agmixingbetweenthefirsttwoexcitedstates(Error!Referencesourcenotfound.),and9howittranslatesbothstericandelectrostaticpropertiescomprehensively.Chapter III then10treats the experimental manifestation and quantitative explanation of these effects for11AnabaenaSensoryRhodopsinandsomeselectedmutants.ChapterIVextendsthiscombined12treatment to rhodopsin-mimickingphoto-switches,beforechapterV summarises themain13conceptsandoutlinesfutureavenues.1415I. Anopenquestionstudiedondifferentsystems16

The protonation of the retinal Schiff base in VIS-absorbing retinal proteins makes these17systems distinctively different from their UV-absorbing sisters, which function with a18deprotonatedchromophore[33].Thecharge-transfercharacterofthe lowestexcitedstate19[34,35],S1,isattheheartofthehighsensitivitytothepresenceofcounterionsanddynamic20

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Figure 1: Schematic representation of A: the charge transfer character and bond lengthalternation(BLA)ofthemostrelevantelectronicstatesS0,S1andS2ofrPSBinFranck-Condongeometry,andB:Twoextremecasesofweakandstrongmixingofthe1Buand

2Agcharacterof the first two excited states, presented together with S0 as a function of an effectiveisomerizationcoordinate,limitedtothefirst90°rotationofthereactivedoublebond(seealsofig.11).Notetheconical intersection (CI)betweenS1andS0. Refer tochapter II for amoredetaileddiscussion.

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electrostatic interactions, thereby controlling the excited state dynamics. This will be1highlightedinthefollowingparagraphs.2

3a. IsolatedrPSB’sinsolutionandgasphase4

Since11-cis(11C)andall-trans(AT)isomersarethegroundstateconformationsofthevisual5photo-receptorandofthelargemajorityofmicrobialretinalproteins,respectively,thesewere6studiedquiteextensivelyasisolatedmoleculesinsolutionandtoalesserextentinthegas7phase.Itwasfirstnoticedthattheabsorptionspectrainsolutionaresignificantlyblue-shifted8withrespect to thesituation inproteincavities (referredtoas the“opsinshift”). Itcanbe9quantitatively explained by a stronger polarizability of the protein environment and10interactionoftherPSBwithitscounterioninsolution,reducedthoughforpolarsolvents,and,11forATrPSB,byanenhancedring/chainco-planarizationinproteins[36–39].Insolution,the1211-cis isomerphoto-isomerizationproducesspecificallytheATisomer, like inproteins.The13IQYishoweveronlyabout25%invarioussolventsversus67%inRhodopsin.Conversely,the14photo-isomerizationofATinsolutionproducesavarietyofproductisomers,unlikeinproteins15whereitis100%bond-selective,andoccurswithatotalIQYintherangeof15%[40–42].16Femtosecondtransientabsorptionandfluorescencespectroscopyconsistentlyshowedthat17theexcitedstate lifetimesandthusthe isomerizationtimesaresignificantly longerthan in18proteins,withadominantdecaycomponent in the3-4ps range, forboth the11CandAT19rPSB’s [8,10,12,43–45]. For AT, sub-picosecond fluorescence decay components were20attributedtointernalconversionandthedominantnon-reactivebackreaction[10,21].The21ESLincreaseatlowertemperaturespointstotheexistenceofasmallexcitedstatebarrier[12].22Conversely, in the case of 11C in solution, the sub-ps component was attributed to the23minority, barrier-less reactive channel while the 4-ps component would characterize the24dominantnon-reactivechannel[45].25

Intheabsenceofacounterionoranypolarisableenvironment,i.e.forrPSB’singasphase,26the absorption spectra of AT and 11C rPSB’s are red-shifted with respect to the protein27spectra,duetoarelativedestabilisationofS0[46,47].TheenergiesoftheUV/VISelectronic28transitionsareobtainedbyrecordingthephoto-fragmentationactionspectraofthecationic29chromophoresheldinanionstoragering.Recently,thisapproachwasextendedtotwo-pulse30experiments,whichallowedtorecordtheexcitedstateslifetimesinvacuo[15].Afirstpump31pulseresonantlytunedontotheS0-S1transitionexcitesthemolecules,and,afteradelaytime32t, asecondpumppulseat800nmpromotesthemoleculesfurthertoS3.Thisadditionalexcess33energystoredinthemoleculeleadstoenhancedphoto-fragmentation,ascomparedtothe34pumppulse-onlycase.DuetothelimitedESL,thefragmentationenhancementisdependent35on t, and the delay-dependent signal measures the time-dependent fraction of excited36molecules. A series of comparative experiments, including the effect of temperature37concluded that, in vacuo, 11C rPSBhasanalmostbarrierless fastdecay (≈0.4psat room38temperature),whileATshowsabarrier-controlled>3psdecay.39

Hence,bothsolutionandgasphaseexperimentssuggest,thatinvisualrhodopsintheESLis40inheritedfromthe“intrinsic”propertiesofthe11Cchromophore[15,45],whereasinbacterial41rhodopsins,theproteintunestheexcitedstatepotentialenergysurfaces(PES)ofATinterms42of barrier height and bond selectivity [15,44]. The origin of these environment-induced43modificationsofthePESwillbediscussedindetailinchapterII,onthebasisofcomputational44results involving mixing of the first two excited states. While such modelling allows45rationalizingtheeffectoftheproteinenvironmentontheESL,thequestionabouthowthe46

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proteinstunetheIQYof11CandATchromophoresremainslargelyopen.Sofar,onlyinthe1unique case of rhodopsin – showing the fastest photoisomerization reaction - a direct2connection was established between the specific vibrational motions driven by the PES3topographyand the IQY,pointing to the central roleof the relativephasebetweena few4essentialvibrationalmodesatthemomentofdecayattheCInt[23].56

b. Effects of mutations and pH on excited state dynamics of retinal proteins – a7selection8

9Figure 1: Crystallographic structure of bR (pdb entry 5ZIL), at pH=7, highlighting the retinal chromophore RET (green),10covalentlyboundtoLys216(blue).TheprotonatedSchiffbaselinkingLys216andRET,isconnectedtothecounterion,the11deprotonatedasparticacidD85,viaawatermolecule.Right:ThezoomshowstheH-bondingnetworkinvolvingthreewaters12andkeyresidues.TheireffectsonbR’sESLandIQYwasstudiedbyfemtosecondspectroscopy(seetext).13

The most studied microbial rhodopsin is bR, a light-activated proton pump, found in14Halobacteriumsalinarum[48].Initslight-adaptedform,itbindstherPSBprimarilyintheAT15form.Inwild-typebR,ATà13Cphoto-isomerizationoccurswithin≈0.5ps[49–52],andwith16aquantumyieldof≈65%[53–55].AsshowninFigure1,thecrystallographicstructure,atpH=7,17identifiesthedeprotonatedasparticacidD85asthemaincounterionoftherPSBconnected18viaaH-bondnetworkwithD212andR82viathreewatermolecules[28].Firstexperimental19workbyEl-SayedandLanyi[56,57]reportedthatthereplacementofchargedresiduesinthe20mutantsD85N,D212NandR82Qbyneutralonesincreasestheexcitedstatelifetimeandthus21isomerization timebya factorof5 to20.Replacementsofunchargedresidues inmutants22D115NorY185Fdidnotchangethelifetimessignificantly.Theexactanalysiswascomplicated23bythefactthatmutations,muchlikeacidification[58],induceamixtureofATand13Cisomers24in thegroundstate.Theobservationswereexplainedbyavalencebondresonancemodel25describingtheexcitedstateasalinearcombinationofwavefunctions,theelectronicdensities26ofwhichhavethepositivechargetranslocatedfromtheSchiffbasetodifferentCatomswith27oddnumbersalongthepolyenechain.Thisprovokesforeverygroundstatedoublebonda28

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reactive singlebond character in theexcited state, butdependingon the fractionof local1positivecharge.DuetothenearbynegativelychargedD85andD212,thepositivechargeis2stabilisedbestonC13intheexcitedstateofwild-typebR,providinganintuitiveexplanation3ofthebondselectivityoftheATà13Cisomerizationprocess.Theexcitedstatesinglebond4characterofC13=C14allowsthenforabarrierlessandthereforeultrafastisomerization.These5effects are absent when D85 and D212 are replaced by neutral residues or if they are6protonated(neutral)asinR82QatpH=4[56].TheresultsforD85Nwerelaterconfirmedand7complementedby femtosecondphotonechospectroscopy,whichprovidedevidence fora8largesub-100fsdielectricrelaxationoftheproteinenvironment[59].9

AnothermorerecentexampleforlargeeffectsonESLandIQYrelatedtothechromophore10environment,was recently reported for KR2, fromKrokinobacter eikastus. In 2013, itwas11discovered as the first light-activated Na+ pump [60]. Its crystallographic structure was12resolved [61,62], highlighting particular motifs responsible for Na+ binding at the protein13periphery,andmostimportantlyatransientNa+bindingsite,inthevicinityoftherPSB,made14up of D116, N112 and D251, and, with respect to bR, a modified H-bonding network.15ComparingthecrystalstructuresatneutralandacidicpHallowedtoidentifytheflippingof16theretinalcounterion,theasparticacidD116,asakeyelementforNa+pumping.Atneutral17pH,D116isH-bondedwithretinal’sSchiffbase(rotamer1),butduringtheMintermediateof18KR2’sphotocycle,protonationofD116viatheSchiffbase(SB)occurs.Thiswouldleadtoits19re-orientation(rotamer2)anddisruptionoftheotherwisestrongH-bondingnetwork,thus20enablingNa+translocation[61].TheoverallstructuralchangesassociatedwithNa+pumping21were very recently analyzed by time-resolved serial X-ray crystallography and QM/MM22simulations,coveringthepico-tomilli-secondtimescales[63].However,thechangesinthe23SB-D116distancewereobservedtobemuchsmallerthanexpected,andnoclearevidencefor24theexistenceoftworotamericstateswasfoundinthephotocycleatpH=8.0.25

Regardingultrafastevents,Hontanietal. reportedaspectroscopic investigationof the full26photo-cycle,andinparticulartheprimaryATà13CisomerizationtooccurinKR2within≈20027fs,atpH=8.0[64].ThisissignificantlyfasterthanforothermicrobialrhodopsinsbearingAT28rPSBinthegroundstateandwasattributedtostericeffects,i.e.distorsionoftheATisomer29[65].Taharaetal.investigatedtheeffectofpHontheprimaryphoto-reactionbyfemtosecond30transient absorption spectroscopy (TAS) [66], with the main objective of obtaining new31insights intominority excited state specieswith > 3 ps lifetimes observed at pH=8.0 [65].32ProtonationofD116occursforpH<7,andleadstoasignificantred-shiftoftheabsorption33spectrum[60],duetodestabilizationoftheprotonatedSBgroundstate(S0).KR2atpH<7and34theD116Nmutantshowseveralpsexcitedstatedecaycomponents,inadditiontothe0.1835psESL,whoseamplitudesincreaseforlowerpHvalues.TheoppositeisobservedforpH>7.A36goodcorrelationwiththepH-dependentIQYshowedthatonlythe0.18psdecaychannelis37productiveintermsofisomerization.Amoredetailedanalysisofthedatasuggeststhat,rather38thantheprotonationstateofD116N,thekeyparameterfavoringthefastandefficientexcited39statepathwayistheorientationofthatresidueanditsfractionofpopulationabletoformH-40bondswith retinal’sSchiffbase [66].Whilea similar co-existenceofmultipleexcitedstate41decay components was previously attributed to reactive and non-reactive excited state42pathways[21,44,56,67,68],thisstudypointstothecentralroleofstructuralinhomogeneities43in the ground state, modulated by the protonation of D116N. Similar conclusions were44recentlydrawnbythesameauthorsforthephoto-isomerizationreactivityinproteorhodopsin45[69].Inconclusion,KR2appearstobeaparticularretinalprotein,affordingaflexibleandthus46disordered Schiff base environment, required for the binding of Na+, but at the cost of a47

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relativelylowisomerizationIQY.Nevertheless,intensiveresearchisunderwaytouseKR2as1aplatformforgeneticmodificationsinviewofapplicationsinoptogenetics[3,24,70,71].2Lastbutnotleast,itisworthnotingthattheproteinenvironmentshouldnotbeconsidered3asa rigidgridofpoint charges. Inaddition toultrafastnuclearmotionof the surrounding4residuesandtheirrelatedcharges[72,73],severalexperimentsperformedonbRpointtoa5dynamicelectronicresponse,uponexcitationofrPSB,inparticularforthepolarisablenearby6Trpresidues,suchasTrp86[74–76].Fromacomputationalpointofview,theseeffectswould7requireaquantummechanicaltreatmentofthelarge“rPSB/polarizableresidue”system[73]8andareoutofthescopeofthepresentreview.910II. Theoreticalframework:PotentialenergysurfacesandCInt11

SincetherPSBisaspecialcaseofalinearpolyenechain,theroleofpossibleinteractionsof12thefirstandsecondexcitedstates(S1andS2)hasbeenfrequentlyevokedandexaminedin13thepast[5,77–82].Adoptingthesymmetrynomenclatureofpolyenes,theelectronicground14state is referred to 1Ag, and the first excited states to 1Bu and 2Ag symmetry (cf. Error!15Reference source not found.), even though the chromophore is asymmetric due to the16positive charge on the Schiff base end. Hence, at the FC point (i.e., at the S0 equilibrium17structure),S0has1Agcovalentcharacterwithalternatingsingle/doublebondlengths,anda18positivechargelocatedontheC=NH2frameworkend.Withrespecttothegroundstate,the191Bu state can be described by a bonding p to an anti-bonding p* electronic transition,20delocalizedallovertheretinalbackbone.Suchamodificationtriggersasubstantialstructural21change,characterizedbysingleanddoublebondinversion(cf.Error!Referencesourcenot22found.),hencethebondlengthalternation(BLA,definedastheaveragesinglebondlength-23averagedoublebondlength)changessign.NegativeBLAisnecessaryfortheisomerizationto24occur,i.e.1Buisthereactiveconfiguration.Inaddition,thestatehasapronouncedcharge-25transfer(CT)character,whichimpliesthatthepositivechargeontheC=NH2frameworkend26isreducedandtranslocatedtothepolyenebackbone.Bycontrast,theS2PEShas2Agdiradical27(DIR)characterwithastronglyreducedBLA.Comparedto1Bu,isomerizationishindered,and28thestatehasnon-reactivecharacter.Last,similarto1Ag,thepositivechargeremainslocated29ontheC=NH2Schiffbaseend[83].Thedifferentchargedistributionsinthesethreestateslead30toasituationwhere theirenergydifferences,DE(S1-S0)andDE(S2-S1),areverysensitive to31electrostaticinteractionsandthustostaticordynamicmodificationsoftheenvironment(see32below).33

Fromrecentquantumchemicalcomputations,itisnowwellrecognizedthattheenergiesof34the1Buand2Agstatesarestronglydependentontheisomerizationcoordinate,hydrogen-out-35of-planemotionandC=Cdihedralangles,andthattheymayevencomeintodegeneracy[84–3688]. In suchextremecases, the 1Bu/2Agmixing is complete, and the systemoscillates in S137betweentheCTandDIRcharacter,withafrequencydeterminedbyBLA.Sucharegimewas38veryrecentlyreportedtooccurinbR,duringthefirst100fsafterexcitationandonlyifthe39laserpulsewastunedtoshorterwavelengthsinordertoexplorehighervibronicstatesinS140[87].41In recent years, several papers reported on QM/MM calculations, which addressed the42dynamic1Bu/2Agcouplingalongthereactioncoordinate,inmoregeneralcases.Blendingnon-43reactive2AgcharacterintotheS1state,extendstheexcitedstatelifetime.Itturnsoutthatthe44strengthof1Bu/2AginteractiondependscriticallyontheGSgeometryoftherPSB(e.g.11-cis45vs.all-trans)andontheelectrostaticpropertiesoftheenvironment,nomatterifitisasolvent46ortheproteincavity.Thethencreatedexcitedstatepotentialenergylandscapemaybefully47

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reactive or present barriers (cf. Error! Reference source not found.-B), and is thereby1determiningtheESLandthustheFQYandisomerizationreactionspeed.2Since 13-cis rPSB inmicrobial protein environments have ESL close to the ones of 11-cis3rhodopsin,andbothtypesofchromophoresarefoundtobenon-planar,itwasconcludedthat4apre-twistissynonymousofadisplacedexcitedstatepotential[30,89–91](inparticularalong5hydrogen-out-of-planemotion(HOOP),whichdisplaylargerRamanactivities,cf.sectionIII).6Thiswouldleadtoamoreeffectiveaccelerationoftheexcitedstatewavepacketalongthe7reaction coordinate [81,92]. Recent multi-configurational QM/MM simulations go a step8furtherandrationalisetheeffectoftherPSBgeometryontheexcitedstateenergylandscape9computing the S1 and S2 energies of the full chromophore within CASSCF or CASPT210approximations, and embedded in the respective natural protein environments (bovine11rhodopsin for 11-cis – 11C/Rho; ASR for 13-cis (13C) and all-trans (AT), AMBER force12fields)[86].13

14Figure2:QM/MMtrajectoriesofAT/ASR(A),13C/ASR(B),and11C/Rho(C).S0(red),S1(green),andS2(blue)Themainout-15of-plane(deviationlargerthan±5°)dihedralanglesoftheS0equilibriumstructureoftherPSBsaregivenatthetop,together16withthebicyclepedalmotiondrivingtheisomerizationonS1withreversedbondorder.Theverticaldashedarrowrepresents17weak fluorescence. Labels ASRAT, ASR13C and Rh correspond, respectively, to AT/ASR, 13C/ASR and 11C/Rho in the text.18Reproducedwithpermissionfrom[86].19

Figure2showstheQM/MMtrajectoriesforthethreecasesinthefirst200fsoruntiltheS1/S020CIntisreached.ThecomputedenergiesarefoundtooscillatefollowingtheBLA.For11C/Rho,21S1andS2areseparatedatalltimesandtheypreservetheir1Buand2Agcharacter,respectively22untiltheCIntisreached,i.e.the1Bu/2Agcouplingisnegligible(panelC).S1thereforepreserves23astronglyreactiveCTcharacter,andFigure3showsthatbondlengthinversioniscomplete24(BLA <0) along the C5=C6–C7=C8–C9=C10–C11=C12 fragment of the chromophore. Both25effectsleadinthespecificproteinenvironmenttoabarrierlesspotentialandarapiddown-26hill progression reaching the CIntwithin less than 100 fs, in agreementwith experiments27[31,93–96].2829

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1Figure3:BLAchangesinASRandRho.(A)BLAchangesfortheAT/ASRchromophorealongthefullconjugatedchain(solid2line),alongtheC5=C6–C7=C8-C9=C10–C11=C12–C13=C14fragment(dottedline),andalongthe–C13=C14–C15=Nfragment3(dashedline).(B)Samedataforthe13C/ASRchromophore.(C)TheBLAalongthefullconjugatedchainforthe11C/Rho(solid4line),alongtheC5=C6–C7=C8–C9=C10–C11=C12fragment(dottedline),andalongthe–C11=C12–C13=C14–C15=Nfragment5(dashedline).Reproducedwithpermissionfrom[86].6

In13C/ASR,afteraninitialBLAdisplacement,S1andS2aredegenerateinthe40-80fstime7interval,andthedihedralangles,alongtheC13=C14&C15=Naxes,remainalmostplanar[86].8Duringthesameperiod,Figure3showsthatBLAispositive, i.e.S1 isdominatedbyanon-9reactiveDIRcharacterassociatedwithaverylimitedchargetransferfromthe-C=Nmoiety.10Onlyafter80fs,doesS1displayanegativeBLA,asubstantialchargetransferandareactive111Bucharacter,withadown-hillprogressiontowardstheCIntby≈150fs, inagreementwith12experiments [19,90,97]. ForAT/ASR, S1andS2are found to remaindegenerateduring the13whole simulation intervalof200 fs (Figure2).BLA ispositive,meaninganon-reactiveDIR14characterprevails(Figure3-A),explainingthelongerESLobservedforATthanforthe13CrPSB15inASR[19,90,97].16

The above empirical rule stating that the chromophore’s isomeric state combined with17protein-imposeddeformationswouldcontroltheESLcannowbeexplainedratherintuitively,18in terms of the dominant 2Ag or 1Bu character. In fact, AT/ASRmaintains four conjugated19double bonds (C5=C6–C7=C8– C9=C10–C11=C12–) with a reduced, but still positive BLA,20ratherthanbondlengthinversion(Figure3-A).Suchapolyene-likepatternaccommodatesthe21non-reactiveDIRelectronicstructurebetterthantheCTstructure.Ontheotherhand,dueto22thepre-twistedβ-ionone ringonone sideand the rapidly twistingC11=C12on theother,23

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11

11C/Rhooffersashorterfragmentwithtwoconjugateddoublebonds(C7=C8–C9=C10–C11)1withanegative (inverted)averageBLAvalue (Figure3-C).Asaconsequence,ΔE(S2-S1)will2rapidlydecreasetowardsadegeneracyalongthephotoisomerizationcoordinateinAT/ASR3and13C/ASRbutnotinRho.4

In subsequent publications, the concept of 1Bu/2Ag mixing was further investigated5computationallyfortheenvironment-induceddifferencesof11-cisrPSBinmethanolandin6Rho[84],andfortheeffectsofsubstitutionsoftheall-transrPSBinsolutionwithelectron-7donatingor–withdrawinggroups[84,88].Experimentaldata,regardingtheESL[11,98],where8consistentlyreproducedandexplainedintermsofthebuild-upofanexcitedstatebarrieror9flat,population-stabilisingregionsduetoanincreased2AgcharacterintheS1state,limitingits10reactivity.Insummary,theconcept,whichemergesfromthesetheoreticalresults,isthatboth11stericandelectrostaticeffectsactthroughthemodificationsoftheS1/S2energylandscapein12determiningtheESLofrPSBindifferentproteinenvironments(exemplifiedindetailinchapter13III),andalsoofrhodopsin-mimickingphoto-switchesinsolution(chapterIV).1415III. Effectsofpointmutations:thecaseofAnabaenaSensoryRhodopsin16

17Anabaena Sensory Rhodopsin (ASR) is amembrane protein of 261 residues (26 kDa) found in the18eubacterium Anabaena (Nostoc) sp. PCC7120 [99], and is part of the family of archeal microbial19rhodopsin, asmuch as bR or sensory rhodopsin fromHalobacterium salinarum [1,100]. Themain20structuraldifferenceswithrespecttobRarethereplacementofAsp212inbRbyPro206,anon-polar21residue,inthevicinityoftherPSBandahydrogen-bondingnetworkfromLys210toGlu36[101].22

Regardingtheultrafastphoto-isomerization,itismostnotablethatASRbearsamixtureofall-23trans,15-anti(AT)and13-cis,15-syn(13C)rPSBisomers,therelativecontentofwhichdepends24ontheillumination,so-called 'light-adaptation'conditions.Whenleft inthedark,thedark-25adapted (DA) formadopts 97%AT,which drops to less than 50%under illuminationwith26orangelight,atneutralpH[102].Inthecontextofobtaininginsightintothemechanismsby27whichretinalproteinscatalyzetherPSBphoto-isomerizationreaction,ASRisamodelsystem28allowingtostudy,withinthesameprotein, theprotein–rPSB interactionsfortwodifferent29rPSB conformers at the same time. Interestingly, at pH=7, both isomers displaymarkedly30differentexcitedstatelifetimes(ESL):~0.7psforAT,andapprox.150fsfor13C[19,90,97,103].31Thiswasempiricallyrelatedtothefactthat13Cistwistedinthegroundstateconformation32[101],butdifferentelectrostaticordynamicinteractionswiththeproteinchargedistribution33may also play a role. The different excited state lifetimes were rationalized by quantum34chemistry computations of the excited state potential energy surface (PES) along the35minimumenergypathsinASR[22],andwithexcitedstatetrajectorycalculation[84,86,104].36AsshowninFigure2,itwasfoundthattheS1PESof13Cisbarrierless,whilethatoftheAT37formdisplaysaplateauoraslightbarrierthatcouldaccountforashortrestingperiodofthe38excitedstatepopulation.ThepreciseoriginofthesedifferentshapesoftheS1PESforboth39isomers,andthemechanismoftheirfine-tuningbytheproteininteractionhasmotivatedthe40researcheffortsdescribedbelow.Whilesectionsaandbsummarizeprimarilyexperimental41work on the electronic and vibrational dynamics of AT and 13C/ASR, as a function of42mutations, section c reviews a combined experimental/theoretical study of two cases of43mutationswhichtrackthemolecularoriginofa12-foldenhancementoftheESL.44

45a. Effectofmutationson theESL:Rationalising spectral shiftsandeffectsonexcitedstate46

lifetimes47

12

Our first joint paper on mutation-induced effect in ASR, published in 2018, reported a1combined experimental and computational study of the spectral shifts, the ground state2vibrationalspectraandisomerizationreactiontimesoftheATand13Cisomersinthemutants3L83Q,V112NandW76F[105].4

Effectsofthemodifiedproteinelectrostaticsontheisomer-specificabsorptionspectra.In5L83QandV112N, thenonpolar residuesValineand Leucineare replacedby thepolarbut6neutralGlutamine(Q)andAsparagine(N),respectively,whiletheW76Fmutationreducesthe7electron density and removes a small dipole close to the rPSB backbone (Figure 4). All8mutationsleadtoblue-shiftsoftheDAandLAgroundstateabsorptionspectrawithrespect9towt-ASR[105].Light-adaptationwascarriedouteitherwithanorange(OA,590nm)ora10blue (BA, 460nm) LED, and the light-adaptation with the highest 13C isomer ratio was11retained.Thelatterwashigherthan50%inallcases,asforwt-ASR.V112NandW76Fexhibit12ablue-shiftunderLAaswt-ASRandmanyothermutants.Theabsorptionspectrumofthe13C13isomerofthesemutantsshiftsby2nm(V112N)and8nm(W76F).L83Q,theblue-mostshifted14mutant,surprisinglyshowstheoppositeeffect,withthe13Cisomerbeing6nmred-shifted15withrespecttoAT.1617

18Figure4:CalculatedstructuresoftheATrPSBandthemutatedresiduesL83,W76andV112NinDAASR(A).Thereplacement19ofTrp76byaPhenylalaninereducestheelectrondensityclosetotheATrPSBbackbone(B).IntroductionofthepolarGluat20position83andAspatposition112(C&D)changestheelectrostaticinteractionswithrPSBinbothS0andS1.21

From a purely computational structure of the mutated protein-chromophore complexes,22obtainedby theautomatic retinalproteinmodel (ARM) [106], themutation-inducedshifts23were correctly reproduced, aswell as theamountof LA-inducedblue-shifts forW76Fand24V112N. For L83Q, ARM produced a wrong orientation of the dipoles of Q83, which was25corrected“manually”. Thisproducedabsorptionmaximaconsistentwith theobservations,26including the inverted LA effect. By comparing both the effects of steric and electrostatic27modifications,itwasconcludedthatthelattereffectsaredominant,inparticularforW76F.28Indeed,themutation-inducedchangesoftherPSBconfigurationscause,withrespecttothe29wt-ASR, rather a red-shift and not the experimentally observed blue-shifts. For both the30V112NandW76Fmutants,achangefromValinetoAsparagine,neartheb-iononeringofthe31

13

chromophore,andTryptophantoPhenylalanineproducesadestabilizationofS1andasmaller1stabilizationofS0.Hence,theobservedblue-shiftoftheS0-S1transitionenergyisacombined2effectofS1up-shiftandS0down-shift.3

Effects of themodified protein electrostatics on the reaction kinetics. The excited state4lifetimes and the isomerization reaction times were determined by broadband transient5absorptionspectroscopyinthe340-750nmrange,coveringthecharacteristicbandsofground6statebleach(GSB),excitedstateabsorption (ESA)andstimulatedemission(SE),aswell the7photo-productabsorption intheJandKstatesofbothATand13C isomers.The latterare8obtainedbythestandardproceduresubtractingtheexperimentallydeterminedcontribution9ofATexcitedisomers,fromtheLAdata.ValuesforESLwereextractedfromESAandSEdecay10times. For the AT isomers (DA), the ESA decay appeared to be bi-exponential, while it11displayedmono-exponentialdecaysforthesmallsignalSEregionprobed(700-750nm).When12the SE is studied at longerwavelengths andmaximum signal, allmutants display amulti-13exponentialexcitedstatedecay,as found forwt-ASR [90],and inagreementwith theESA14decays (tobepublished).The fast componentwas found tobe significantly reducedwith15respect to the0.75ps ESLofAT inwt-ASR,with L83Q showinganESL as short as 120 fs.16Whetherthesecondslowercomponent(inthe0.5-0.7psrange,amplitude≤30%)alsoleads17toisomerizationisdifficulttoascertainsincetheriseofthephoto-product(PPA)orJ-state18absorption naturally allows to unambiguously determine only the fastest component. A19potential slower rise appears to be rather compensated by the signal decay to the J-to-K20vibrationalrelaxation(Figure5).Wethereforequotedthe“isomerizationreactiontime”(IRT)21extractedfromacombinedanalysisofGSB,ESA,SEandPPA:L83Q-120fs,V112N–230fs22andW76F340fsfortheATisomers.Thesevaluesarepresentlyreconsideredonthebasisof23thenear-IRSE,whichallow todetermineESLwithhigheraccuracy (higher signalandzero24background,Figure5).Nevertheless,theinitiallyreportedincidentalcorrelationisconfirmed:25thelargerthespectralblue-shiftoftheGSA,thesmallertheIRT.26

27Figure5:TransientabsorptiondatafortheASRmutantL83QinDAconditions(≈100%AT).(A):CombinedUV-VISandnear-IR28probwavelengths,forthedelaytimesindicatedinthelegend.Groundstatebleach(GSB),excitedstateabsorption(ESA),and29stimulatedemission(SE)highlightsignalfromS1.Inthe550-600nmrangeESAturnsinto“hot”groundstateabsorptionby30≈200fs.(B)Kineticsatselectedwavelengths.Thereddashedlineisaguidetotheeyewitha200fsrisetimeforthe“hot”31groundstateabsorption.32

For13C,themutationsdidnotaltertheIRTasmuchasforAT,butthevaluesratherindicate33theoppositetrend:asmallbutsignificantincreaseofESLandIRT.Thisanimportantresult,34which confirms the initial strategy of choosing ASR as a model system: the protein-35chromophoreinteractionsactdifferently,namelyinanoppositefashion,onthetwoisomer36

14

conformations. The molecular basis for this observation is unclear and is the subject on1ongoingworkbothcomputationallyandexperimentally.Acentralquestionistofigureoutin2asmuchLAchangestheentirecoupledprotein-chromophoresysteminwhatwecallthe“13-3cisisomer”.i.e.boththechromophoreandtheproteinlikelyundergoLA-inducedstructural4changes.56

b. Detailsfromfemtosecondvibrationalspectroscopy7

First vibrational spectra obtained by femtosecond non-resonant (pump-)DFWMwere also8reportedinthispaper[105]fortheisomergroundstatesinDAandLA,aswellastheirphoto-9isomerized K states (pump delay 100 ps). The procedure for extracting AT- and 13C-pure10vibrationalspectrawasexplainedindetail,andexploitedmorethoroughlyinasubsequent11publication[91],summarisedanddiscussedinthefollowing.12ThevibrationalspectraofbothATand13C isomers inASRboth inthegroundandexcited13stateswereanalysedindetail[91].Theexperimentaltechniquesusedrelyondegeneratefour-14wave-mixing(DFWM)andimpulsivevibrationalspectroscopy(IVS),whichcanbeperformed15alsointheexcitedstate,duetotheactionofanactinicpumppulseasafunctionofdelaytime16[107–110].17ThegroundstatevibrationalspectraofbothDAandLAformsofASRwereobtainedbynon-18resonant DFWM and IVS, and compared to traditional spontaneous Raman spectra. Non-19resonant schemes used a spectrally broad sub-15fs pulse centered at 650-660 nm, and20revealedverydifferentresonancesinthehigh-frequencyrangeabove1000cm-1and,most21importantly,amuchstrongerRamanactivity for13C in thehydrogen-out-of-plane (HOOP)22regionaround800-900cm-1.Alltheseobservationsarein-linewiththedifferencesbetween23AT and cis rPSB conformations in other retinal proteins, like bR or channel-rhodopsin.24SupportedbyMDsimulationsonthegroundstate,itwassuggestedthatthelargerdistortion25hintedonbytheHOOPactivityislocatedattheC14-C15bond,ratherthanonthereactive26C13=C14bond,inagreementwithX-raydataofASR[101].Asfor11-cisrPSBinRho,thispre-27twistofthe13Cchromophore,asopposedtotheplanarATconfiguration,wasidentifiedas28oneofthereasonsfortheshorterESLof13C.29Thestructural changes in theexcitedstatewere followedbyexcitingeach isomerwithan30actinicpump.ThepurespectraoftheexcitedstatewasobtainedsincetheGSspectraofeach31isomerandthephoto-productsKATandK13Ccouldbemeasuredseparately[91].Inparticular,32thefrequencyoftheC=CstretchandCH3rockingmodesshowedatransientblue-shiftforboth33isomers, consistent with a reduced conjugation length, induced by the torsional motion.34Indeed,ina90°twistedconformation,theC14–C15–NandC5=C13moietiesaredisconnected35electronically.Most importantly thedynamicsof thetransient frequencyshiftsmirrorsthe36excitedstateandreaction lifetimesqualitatively.Theblue-shiftoccursalmost immediately37uponexcitationfor13Canddecayswithahalf-timeof150fs.TheATfrequencyshiftsreach38maximumonlyafter0.2psandadopttheKATvaluesonlyby0.6ps.Thesestudiesalsoshowed39thattheamplitudeoftheseisomerization-inducedchangesintheCH3rockfrequencieswere40differentforATasfor13C,whichistakenasanindicationthatisomerizationcausesalarger41rotationaroundthecentralC13=C14bondforATthanfor13C,inagreementwithprevious42FTIRresultsonthecryo-trappedKintermediates[102].43Inaddition,lowfrequencyvibrations(<400cm-1),relatedtoexcitedstatemodes,identified44previously in transient absorption experiments [97], were shown to appear after a short45inductiontime;shorterfor13CthanforAT.46

15

Effectsofmutationsonthegroundstatevibrationalspectra.ThemutantsL83QandV112N1werestudiedbyfemtosecondDFWMandIVSintheDAandLAstates[111],underexactlythe2sameconditionsastheonesusedintheaboveTAexperiments.Itwasfoundthatmutations3leadtoanenhancedHOOPmodeactivityintheATgroundstateofL83QandV112Nindicating4apre-distorsionofthechromophoreeitheraroundtheactiveC13=C14bondorneighbouring5C14−C15 bond of the ground state compared to the AT-isomer in the wt-ASR [111].6Additionally, thehigh frequencyvibrations related toC-C singlebondorC=Cdoublebond7stretch,aswellasCH3rockmodeschangeconsistentlywitha reducedconjugation length,8inducedbythepre-distortion.Inagreementwithpreviousexperimentalandtheoreticalwork,9thepre-distortionisheldresponsibleforthedrasticreductionoftheexcitedstatelifetimes10forthemutants,ascomparedtoATinwt-ASR.Ontheotherhand,themutationsdonotseem11toenhancethepre-distortionalreadyobservedforthe13Cisomerinwt-ASR,inagreement12withasmallincreaseoftheESLfor13C.13Insummary,time-resolvedvibrationalspectroscopy,hereaccessedviadegenerateFWMand14IVS,providednewinsightsonstructuralchangesduringtheisomerizationprocess.Forwild-15type ASR and its mutants, the observations are perfectly in line with the theoretical16predictionsoutlinedinchapterII(Figure2),underliningheretheroleofapre-twistin13C/ASR17forreducingtheexcitedstatebarrierandleadingtotheshorterESL.1819

c. TowardsarationalpredictionoftheexcitedstatelifetimeofATrPSBinmutants20

Whileadetailedandcomprehensivecomputationalanalysisofthemutation-inducedeffects21on the excited state lifetimes of AT and 13C for the above mutants is still ongoing, we22examined further mutations with the aim of achieving an ASRmutant with an increased23excited state lifetime and thus FQY. This is of particular importance in the area of24optogenetics,combined,ifpossible,withanincreasedabsorptionofthemutantsinthered25ornear-IRregionofthespectrum[24].Werecentlyreportedanalmost10-foldincreaseof26FQY in theASR doublemutantW76S/Y179F, and rationalised the observationswithin the27context of the modified protein electrostatic environment, including the computational28analysisoftheexcitedstateelectronicstructureandlifetimesofwt-ASRandtheL83Qmutant29[112]. The theoretical framework outlined in section II, is central to understanding the30mutation-inducedmodificationsofthe1Bu/2Agmixingalongthereactioncoordinate,which,in31thispaperwastraceddowntotheelectrostaticinteractionsofthemutatedresidues.32

The experiments were performed on the DA forms of wt-ASR and on the L83Q andW76S/Y179F33mutants,togetherwithtwolight-adaptedformsofthelatter.Wefocusedonthemutationeffectson34theATisomers,whichaccumulatesto>95%inDAwt-ASRandL83Q,andishighestinorange-adapted35form of W76S/Y179F (39%). While fluorescence of the non-canonical 7- and 9-cis isomers was36minimisedinthedoublemutant,byexcitationwith>520nmlight,contributionsfrom11-and13-cis37cannotbeexcluded.WefoundthattheFQYwaslowestforwt-ASRandincreasedbyafactorof2.2for38L83Q,andby7.9and6.9fororange-andgreen-adaptedW76S/Y179F,respectively.39

Themutation-induced shifts of the ground state absorption and steady-state emission spectra are40reportedandreproducedwithhighaccuracybytheQM/MMmethod.ThelatterisbasedontheARM41protocol[113],whichemploystheCASSCFmethodwhichisanabinitio(wavefunction-based)multi-42configurational quantum chemical (MCQC)method coupled with the Ambermolecularmechanics43force field to obtain the S0 QM/MMmodels of rhodopsins semi-automatically. Vertical excitation44energiesarethencomputedusingmulticonfigurationalsecond-orderperturbationtheory(CASPT2)to45account for themissing dynamic electron correlations. The level of agreementwith the observed46trends in absorptionand fluorescenceexperiments indicate that thedescriptionsof the steric and47

16

electrostatic effects of the different protein environments on the S0 and S1 relaxed structure are1trustful,andcanbeextendedtodescribingtheS1dynamics.2Thesewereexperimentallyprobedbymeasuring thedecayofstimulatedemission (SE) in the850-31250 nm range, with ≈60 fs time resolution. The SE decays turned out to be almost wavelength-4independent,andbi-phasicforallstudiedASRvariants.WethuscomparedtheaveragevaluesofESL5with the relative increase of FQY, and found that the FQY increases proportionally to ESL for6W76S/Y179F with respect to wt-ASR, according to 𝐹𝑄𝑌 = 𝐸𝑆𝐿×𝑘+ with 𝑘+ the radiative rate,7generallyassumedtobeconstant. Interestingly, L83Qshoweda2.2-times largerFQY thanwt-ASR,8despiteanalmost2-foldreductionoftheESL,indicatingthatL83Qhasahigherradiativeratethanthe9wild-type. This and the effects of the mutations on ESL were explained by calculations of the S110relaxation/reactionpathsandbycomputing200fssemi-classicaltrajectories(Figure6).11

Indeed,relaxedscansoftheS0,S1andS2energiesalongtheC12−C13=C14−C15dihedralangle,12computing minimised energies for fixed dihedral angles, indicate that the S1 profile is13barrierlessforL83Q,whileitisflatteranddisplaysshallowbarriersofca.3and6kcal/molfor14wt-ASR and the doublemutant, respectively. The barriers have amaximum close to 120°15torsionofthereactiveC13=C12doublebond.Theexcitedstatelifetimesarebestsimulated16by trajectories computing the time-dependent energies of the S1 and S2 states and their17crossingwithS0ataconicalintersection(Figure6).Herethekineticenergiesinallvibrational18modesof the chromophore are included, leading to different and time-dependent energy19differences, inparticularbetweenS1andS2.OnlyforL83Qdothecomputationspredictan20ultrafastS1/S0crossingby≈100fs,consistentwiththefastest0.27psdecaytimeobservedin21SE.Ontheotherhand,Figure6showsthatwt-ASRandW76S/Y179FdonotreachtheCIwithin22thesimulationtime,consistentwiththeobservedshortestdecaytimesabove500fsandwith23thecomputedS1barriersintherelaxedenergyscans.24

25

26

hν hν’CIW76S/Y179FS0

S1

S2

EF

ECI

FC

W76S/Y179FAT

5.45.3

0 0

B

D

F

17

Figure6:(A,C,E):CASPT2//CASSCF/AMBERenergyprofilesalongtheS1(greensquares)isomerizationpathofL83Q,wt-ASR,1andW76S/Y179F,respectively,computedintermsofarelaxedscanalongtheC12−C13=C14−C15dihedralangle.Notethe2appearance of an excited state barrier for wt-ASR, andW76S/Y179F. Energy profiles of S0 and S2 are indicated by blue3diamondsandredtriangles,respectively.(B,D,F):ExcitedstateQM/MMFCtrajectoriesofL83Q,wt-ASR,andW76S/Y179F,4computedatthree-rootstate-averaged-CASSCF/AMBERleveloftheoryandcorrectedattheCASPT2level.S0(bluediamonds),5S1(greensquares),andS2(redtriangles)CASPT2/CASSCF/AMBERenergyprofilesalongtheFCtrajectories.Thereactionpath6diagraminpanelEisdifferentfromthecorrespondingpanelinref.112.Infact,thepresentedpathhasbeencomputedusing7a3-rootCASSCFlevel-ratherthan2-rootstateaverage-tobeconsistentwiththeresultspresentedinpanelsAandC.8

9Figure7:Evolutionofthedihedralangles(A)andchargesontheSchiffbaseend(B)alongtheisomerizationpathforL83Q.10TheC12−C13=C14−C15dihedralangleis≈-170°(left)and-120°(left).Adaptedfromref.[112].11

Interestingly, themutationsactthroughdifferentmechanismsonthe1Bu/2Agmixing.ForL83Q,the12reactive character of S1 is induced through electrostatic interactions imposed by the protein13environment,whileinW76S/Y179FthestabilisationofemissiveregionsonS1isduetosubtlechanges14ofthechromophorestructure.ThiswasgivenevidenceforbycomputingtheS1energyprofilesalong15theisomerizationpathsinvacuo,i.e.fortheisolatedrPSBtakenwithitsproteingeometry.Indeed,in16thecaseoftheL83Qchromophorestructure,theenergyprofileappearstobeverysimilartotheone17detected for the rPSB in wt-ASR and W76S/Y179F protein environment, which would lead to a18picosecondexcitedstatelifetimeandanenhancedemission.Sincethesearenotobserved,theshort19ESLinL83Qisaconsequenceoftheproteinenvironment.Thecomputationsindicateareorientation20oftheπ-conjugatedchainalongthereactionpathinawaythatenhancestheinteractionwiththepolar21sidechainsofQ83.Atthesametime,thepositivechargeontheSchiffbaseendisfurtherreduced22uponincreasingtheC12−C13=C14−C15dihedralangle(Figure7).Botheffectsleadtoanelectrostatic23stabilisationofS1alongthereactionpathsandthustoabarrierlessprogressionontheS1PES.24

25Ontheotherhand,theinvacuoanalysisforwt-ASRandW76S/Y179Fshowsnoqualitativechangesin26theenergyprofileswith respect to the respectiveproteinenvironments. This indicates that the S127barrier is not induced but only marginally modulated by electrostatic interaction. Rather, it is an28intrinsicfeatureoftherPSBconformation.The increasedS1barrier inW76S/Y179F isduetosubtle29geometricalchangeswithrespecttotherPSBgeometry inwt-ASR. Indeed,thecomputationsshow30thatitispossibletotransformthewt-ASRS2,S1,andS0energyprofilesintoW76S/Y179F-likeenergy31profilesbysimplychangingthebackbonedihedralanglesalongthewt-ASRreactioncoordinatetothe32correspondingW76S/Y179Fvalues.Thisisremarkablebecausethosearelimitedchangesthatindicate33

18

thefluorescenttuning inrhodopsinsmightbeachievedalsothroughsubtlegeometrical (i.e.steric)1effects.23In conclusion, this combined experimental and computational study outlines two pathways for a4rationaldesignofASRmutantswithenhancedESLandFQY.Itwasshownthatmutationscaninduce5stericeffectsthatinduceapronounced1Bu/2Agmixing,blendingnon-reactivecharacterintoS1through6geometrical effects. Alternatively, itmight be interesting to engineer themutations such that the7dynamic electrostatic interactions with the increasing CT character of the rPSB are of repulsive8charactersoastodestabiliseS1andmaintainasignificant1Bu/2Agmixing.9

10

IV. Retinal-inspired molecular switches: towards controlling photoreactivity by11syntheticchemicaldesign12

13An alternative, complementary strategy to decipher the tuning mechanism of the rPSB14photoreactivity in rhodopsins has been to design amodel chromophore thatmimics – in15solution - theelectronicstructure,PES topologiesandeventuallyphotoreactivityof11-cis16rPSBinthevisualpigmentofRhodospin(Rho)[114,115]. InRho,thephotoisomerizationis17remarkably fast (<200 fs) and efficient (isomerization quantum yield, IQY = 67%) [93,94].18Aboveall,thisphotoreactionisvibrationallycoherent,auniquemechanismenablingtofunnel19theenergyoftheabsorbedphotonspecificallyintotheisomerizationcoordinateonatime20scalefasterthanvibrationaldephasing,andheldresponsiblefortheenhancedIQYinRho[23].21Revealing the chemical design principles underlying such an efficient light to mechanical22energy transductionat themolecular scalewouldbeof central interest in theprospectof23designingefficientmolecularswitchesandmotors[116].24

25Figure8:Chemicalstructureof(A)the11-cisisomerofrPSBinRho,and(B)theZisomeroftheIPcompounds.BothIPand26rPSBhaveasimilarπ-electronsystem(bluemolecularbackbones)withanS1statecharacterizedbyabondlengthalternation27andpartialreductionoftheprotonatedSchiffbasethroughchargetransferalongthecarbonbackbone.Theyalsohavean28analogousphotoreactioncoordinate,whichinvolvesthetorsionaroundthecentralC=Cdoublebond(curvedarrows)aswell29as out-of-planemotions of the purple bonds. Several IP compounds havebeen synthesized and studied theoretically and30experimentally.Theydifferbythesubstitutionsinorange:R1=HorMe,R2=HorMe.31

AsdiscussedaboveinthecaseofASRandduetotheCTcharacteroftheS1state,amajor32parameter influencing the photophysical properties of rPSB is the protein electrostatic33environment and the counterion neutralizing its positive charge [56,117]. In the originally34proposedN-alkylated IP compounds –whichwe nameMeO-NAIP - the electron-donating35MeO group on C6’ stabilizes the S1 state by favoring its CT character, thusmimicking the36stabilizingeffectoftheelectrostaticproteinenvironmentinRho[114].Besidesfine-tuningthe37S1 CT character, another central ingredientof thebiomimetic design is the intramolecular38steric hindrance imposed by the twomethyl groups on C2’ and themethyl group on C5.39AltogethertheyimposetheZconfigurationoftheMeO-NAIPtobethemoststableS0isomer,40in a non-planar - pre-twisted – geometry. Recently, we revealed the effect of the Me41substituentonC5ontheIPphotoreactivitybyinvestigatingthe“demethylated”MeO-NAIP42

(A) (B)

19

compound,nameddMe-MeO-NAIP,whichfeaturesnomethylonC5(R1=HinFigure8).Aswe1will illustrate below, we demonstrated that while the parent MeO-NAIP undergoes a2vibrationallycoherentphotoreactionmimicking thatof rPSB inRho, this isnot thecaseof3dMe-MeO-NAIP.InawaysimilartothecaseofrPSBinretinalproteinsandmutants,wecould4concludethatthisdifferenceinphotoreactivitiesobservedwithinthetwoIPcompounds is5closely related to the S1-S2 energy gap and mixing, itself controlled by electrostatic and6(intramolecular)stericproperties.7UV-Visormid-IRtransientabsorption(TA)spectroscopyaswellasfemtosecondfluorescence8spectroscopywereusedtoinvestigatetheultrafastphotoisomerizationreactionofavariety9ofIPcompoundsinsolution[20,115,118–122].Whileallcompoundsundergoasubpicosecond10photoisomerization, they can be categorized within two photoisomerization scenarios,11correspondingtotwoqualitativelydistinctspectroscopicsignatures,asillustratedinFigure9.12For the so-called “parent”NAIP compound [121] and its zwitterionic derivative [119], the13excitedstateabsorption(ESA)andstimulatedemission(SE)fromtheS1population(Figure914a)areveryshort-lived,theyspectrallyshifttotheblueandtotheredrespectively,andthey15decay impulsively by 250 fs (vertical dashed line across Figure 9 a,c,e). The ground state16photoproduct absorption (PA) band immediately follows, which initially appears after the17decay of the SE in the low-energy spectral range (600-700nm), and rapidly blue shifts to18~430nmwithin~0.5ps.SuchdynamicspectralshiftsoftheS1andearlyS0bandshavebeen19interpretedasthesignaturesofapopulationwhichevolvesballisticallyfromtheFCregionto20thephotoproductgroundstatethroughtheCintintheformofavibrationalwavepacket[119].21Averysimilarred-shiftingSEimpulsivelyfollowedbyablueshiftingPAhasbeenreportedin22Rhodopsintooandinterpretedthesameway[94].Furtherevolutioninthegroundstateis23accompaniedbyapronouncedlow-frequencyoscillation(~80cm-1,correspondingto~500fs24period,Figure9e)interpretedasthesignatureofavibrationalwavepacketoscillatingaround25theS0PESminimumalongtheC=Ctorsioncoordinate[119,121].Importantlythisvibrational26wavepacketwasdemonstratedtobetriggeredbytheS1reactivemotion,meaningthatthis27low-frequencyvibrationalcoherenceispreservedthroughtheCIntandexponentiallydamped28(witha~300fstimeconstant)afterthedecaytoS0[118].29Conversely,the“demethylated”NAIPcompound,whereC5carriesanHatominplaceofa30methylsubstituent,hasaverysimilargroundstatespectrumbutqualitativelydifferentTA31signatures (seeFigure9b,d,f):TheSEsignaturedoesnotextentthis far tothe low-energy32range,itisspectrallynarrowerandlonger-lived.TheSEandESAbandsdecayinconcertwith33theriseofthePAbandwhichappearsgraduallyat430nm,ratherthanimpulsivelyinredpart34oftheprobingwindow.Hencethereisnosimilarindicationofavibrationallycoherentreactive35motionthatwouldimpulsivelydrivethesystemthroughtheCIntasdiscussedabove.More36precisely,uponlightexcitationwithaveryshort(8fs)pumppulse,avibrationallycoherent37motion is detected in S1 along (in particular 232 cm-1mode, see Figure 9 f).However, no38vibrational coherence is transferred to the ground state, and in particular no vibrational39activityisdetectedat80cm-1.4041

20

1Figure9:Transientabsorptionspectroscopy(TAS)oftworepresentativesoftheNAIPcompoundsfamilyobtainedwithan8-2fs,400-nmpumppulse.CompoundZ-1carriesamethylinC5anditsC=Cdoublebondispre-twistedinS0.ItsTASsignatures3(a)arequalitativelydifferentfromthoseofthe“demethylated”,planarcompoundE-2(b)wherethemethylgrouponC5is4replacedbyaHatom.(c,d)Selectionoftransientkineticstracesfromtheabovedatasets,andtheirfits.(e,f)Theresidualsof5the fits are oscillatory signals revealing the vibrational dynamics accompanying the photoreaction.Abbreviations: GSB =6GroundStateBleach;SE=StimulatedEmission;ESA=ExcitedStateAbsorption;PA=PhotoproductAbsorption.Reproduced7withpermissionfrom[118].8

Torationalizewhythesetwocompoundsundergosuchqualitativelydifferentphotoreactions,9wefirstnotice,thatthemoststablegroundstateisomerisZ,witha~15°pretwistedC=Cbond,10for the C5-methylated compound, but E in a planar conformation for the demethylated11compound. These differences are controlled by the intramolecular steric hindrance12introducedwiththemethylsubstituentonC5,whichalsoaffectstheS1electronicstructure13and photoreactivity of the compounds, as rationalized by QM/MM modeling at the14CASSCF/CASSPT2 level [118]. Indeed, in the planar compound, the S1 and S2 states are15computed to be nearly degenerate, resulting in a flat S1 PES, while in the pre-twisted16compoundthedegeneracyisliftedandtheS1PESslopeisverysteep.Consequently,inthe17lattercase,themotionalongthetorsioncoordinateisstronglyacceleratedwhichdrivesthe18systemballisticallytowardstheCInt,suchthatthephotoproductformationoccursfasterthan19vibrationaldecoherenceofthe80cm-1mode,resultinginthepronouncedoscillatorysignal20observedafterthedecaytoS0.Intheformercase,amorediffusiveexplorationoftheS1PES21potential would be responsible for an increased excited state lifetime and vibrational22

21

decoherencewouldbeeffectivealreadybythetimethesystemreachestheCintanddecays1toS0(Figure10).2

3Figure10: Schematic representationsof the twodistinctphotoisomerization scenarios in theNAIP compounds. (a) The4reactivemotionofthepretwistedMeO-NAIPisgovernedbyasteepS1PESdrivingthesystemballisticallytowardstheCInt.5UponimpulsivedecaytoS0,thisS1motioncoherentlypopulatestheS0vibrationalmotionalongthetorsionmodeat80cm-1.6(b)Instead,fortheplanardemethylatedcompound,theS1andS2statesarenearlydegenerate,resultinginaflatPESproviding7amuchweakeraccelerationalongthetorsionmode,andamorediffusivemotiontowardstheCInt.ThecrossingofS1andS28leadsinfacttoanavoidedcrossing.Thecorrespondinglossofsynchronizationthenexplainstheobservedlossofensemble9coherence.Twodecaychannels(“U”=“unsuccessful”,and“S”=”successful”)arerepresented,withtheevolutionsontheS1or10S0PESrepresentedinredandblue,respectively.The20to25%quantumyieldindicatesthatchannelUisdominating.Adapted11from[118].12

BycomparingtheTAsignaturesandphotoreactionscenariosforeachisomerofhalfadozen13ofNAIPderivatives[20],weobservethatallcompoundsunderinvestigationobeyeitherthe14first or the second photoreaction scenario. Moreover, based on DFT modeling of the15compounds S0 equilibrium structures,we establish a perfect correlation between the C=C16doublepre-twistandthephotoreactionscenario:Allcompoundsfeaturingasignificant(>10°)17pre-twistobeythefirstphotoreactionscenario,andallcompoundsfeaturingamoreplanarS018conformation(<10°pre-twist)obeythesecondone.Thisallowsustogeneralizethecentral19roleoftheintramolecularstericeffectandpre-twistatcontrollingthePEStopographyviathe20fine tuning of the S1-S2 statemixing in these retinal-inspired compounds. As reviewed in21chapterII&III,thesameeffectofapre-twistofthechromophoreisoperativein13C/ASRin22reducingtheexcitedstatebarrier[111].23Amajoroutcomeofourlateststudy[20],isthatwhiletheintramolecularsterichindranceand24relatedS1-S2 statemixingcontrols thephotoreactiondynamics,weobservenocorrelation25between the IQY – in the range 15 % to 35 % for all IP compounds investigated – and26observablessuchasexcitedstatelifetime,vibrationalcoherence,absorptionwavelengthsor27degreeofpre-twisting.ThemolecularmechanismcontrollingtheIQYinultrafastC=Cdouble28boundisomerizationthusremainsanopenquestionsofar.2930V. Summaryandconclusions31

32Insummary,forthesub-picosecondisomerizationreactionsofrPSBinaproteincavityandfor33therelatedbio-mimeticswitchesinsolution,modificationsoftheESLcanbeinducedbysteric34effects (ground state pre-twist) and by changes in the electrostatic environment. The35experimentalandtheoreticalstudiesontheATand13CisomersASRanditsmutantshave36identified both effects, and how they are brought about by point mutations. For the37photoswitches,steric[118]andelectronic[20,123]effects,broughtaboutbysubstitutionsof38theNAIPframeworkwereinvestigatedexperimentally.Thesestudiesshowed,inadditionto39

22

thechangeinESL,aclearinfluenceonthesurvivalofvibrationalcoherencesintheground1state. The computational framework shows that both steric and electrostatic parameters2affecttheexcitedstatestructuredueto1Bu/2Agmixing,andhowitdeterminestheCTand3reactivecharacterintermsofBLAofthelowestexcitedstateS1.Acentralpointisthatthe4simulationsallowtopredicthowthismixingchangesdynamicallywhenthechromophores5evolvealongthereactioncoordinate.ShallowexcitedstatebarriersappearwhenS1alternates6betweenareactiveCTandnon-reactiveDIRcharacter,leadingtolongerESLandhigherFQY.7TherecentexperimentalobservationofBLA-mediated1Bu/2Agoscillationsdemonstratesthis8effectclearly,withinthefirst100fsoftheexcitedstatereactioninbR[87].9In order to obtain a more direct experimental insight, and a direct comparison with the10simulations,measuring the time-dependent S1-S2 splittingwould provide new insights for11systemswithreactivevs.non-reactiveexcitedstatedynamics.TheS1-S2transitionisexpected12to lie inthenear-tomid-IRregion(1000-8000cm-1) [84,118],butthehightimeresolution13required(<50fs)makestheenvisagedVIS-pump/IR-probespectroscopychallenging.14LikemutationeffectsdiscussedinchapterIII,pHisknowntoinducesmallshiftsintheASR15absorptionmaximumwavelength[124].Anewcomputationalprotocolhasbeendevisedand16successfullyappliedtodeterminethemolecularoriginofsuchapHeffect[125].Thenextstep,17stillongoing,focusesonthepHeffectontheESL.Preliminaryresultspointtoabi-exponential18decay from S1, modulated by its pH-dependent interaction with S2. Further upcoming19developmentsrelatetocomputingtheexcitedstatevibrationalspectraoftherPSB,inrelation20withtheveryinformativefemtoseconddata[91,111],andapossibleinclusionofthedynamic21polarisationofthenearestneighbourresidues[15,74,75].Unliketheherereportedtransient22spectroscopies,ultrafastX-raydiffractionhasthepotentialofrevealingthedetaileddynamic23structuralandelectronicchangesof the rPSBand itsnearestenvironment,provided these24experimentsbecarriedoutundersinglephotonexcitationconditions[63,72,73,126].25Beyondtheexcitedstatelifetimesandvibrationalcoherences,anothercentralpropertyisthe26reactionquantumyield(IQY),forwhichwestillhavetounderstandandlearnthelessonsthat27theoutstandinghighvaluesfoundinRhoandbRcanteachus,whenthesearecomparedwith28themodestvaluesobtainedfor thebiomimeticswitches insolution [20]or the lowvalues29(<2%)observedforthecrowedalkenesdesignedasmolecularrotarymachine[26].Whileitis30generallyacceptedthatthebranchingintoforwardandbackwardreactiontakesplaceinthe31vicinity of the conical intersection [23], it remains to be rationalised how chemical32substitutionsandeffectsoftheenvironmentcontroltheIQY.Femto-andattosecondphoto-33electronspectroscopyholdsthepromisetomapoutthewavepacketevolutionacrosstheCI34inrealtime,andoneofthemainchallengesforthenextyearswillbetoapplytheseemerging35sophisticatedtoolsforisolatedchromophoresingasphase[127,128]andsolution[129].36

37

VI. Acknowledgements38The authors acknowledge financial support from the French-German ANR-DFG project39“Femto-ASR”, (ANR-14-CE35-0015-01)andANRsupportviagrantNo.ANR-11-JS04-010-0140“IPQCS”, and the “Investissement d’Avenir” program: Labex NIE ANR- 11-LABX-0058_NIE,41LabexCSCANR-10-LABX-0026_CSC,EquipexUNIONANR-10-EQPX-52-01.Otherfundingcame42fromtheUniversitédeStrasbourgforaUSIASFellowship,fromthe“Alsace”region,theNSF43GrantNo.CHE-1710191,andthe ItalianMIURforgrantPRIN2015.M.Oacknowledgesthe44OhioSupercomputerCenter,theMésocentreoftheUniversitédeStrasbourgandCINECAfor45granted computer time. The Centre de Calcul Intensif d’Aix-Marseille is acknowledged for granting46accesstoitshighperformancecomputingresources.47

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Highqualitysamplesforwild-typeASRanditsmutantscamefromH.KandoriandK.Jungand1their teams. The contributions of J. Brazard, A. Cheminal and O. Crégut are kindly2acknowledged,aswellas fromS.Fusi,M.Paolino,L.Barneschi,E.Pieri,V.LedentuandL.3PedrazaGonzález.4 5

24

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