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Metal–Dithiolene Bonding Contributions to Pyranopterin Molybdenum Enzyme Reactivity

Authors:
Jing Yang at University of New Mexico
Jing Yang
John Enemark at The University of Arizona
John Enemark
Martin L. Kirk
Martin L. Kirk
Martin L. Kirk
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Abstract and Figures

Here we highlight past work on metal–dithiolene interactions and how the unique electronic structure of the metal–dithiolene unit contributes to both the oxidative and reductive half reactions in pyranopterin molybdenum and tungsten enzymes. The metallodithiolene electronic structures detailed here were interrogated using multiple ground and excited state spectroscopic probes on the enzymes and their small molecule analogs. The spectroscopic results have been interpreted in the context of bonding and spectroscopic calculations, and the pseudo-Jahn–Teller effect. The dithiolene is a unique ligand with respect to its redox active nature, electronic synergy with the pyranopterin component of the molybdenum cofactor, and the ability to undergo chelate ring distortions that control covalency, reduction potential, and reactivity in pyranopterin molybdenum and tungsten enzymes.
Fold angle distortions as a function of redox orbital electron occupancy in a series of Cp2M IV (bdt) complexes. (Adapted with permission from J. Am. Chem. Soc. 2018, 140, 14777-14788. Copyright (2018) American Chemical Society).
Fold angle distortions as a function of redox orbital electron occupancy in a series of Cp2M IV (bdt) complexes. (Adapted with permission from J. Am. Chem. Soc. 2018, 140, 14777-14788. Copyright (2018) American Chemical Society).
… 
Pictorial description of how the ligand fold angle modulates the degree of mixing between the dithiolene out-of-plane S orbitals (Sπ + ) and the in-plane Mo(xy) redox orbital. The chelate ring fold
Pictorial description of how the ligand fold angle modulates the degree of mixing between the dithiolene out-of-plane S orbitals (Sπ + ) and the in-plane Mo(xy) redox orbital. The chelate ring fold
… 
(A) Solid state resonance Raman profiles and 5K mull electronic absorption spectrum for Tp*Mo V O(bdt). (B) Resonance Raman spectrum for Tp*Mo V O(bdt) (293K) using 514.5 nm excitation (75 mW). (C) General molecular orbital diagram for Tp*Mo V O(dithiolene) complexes. The z-axis is oriented along the Mo≡O bond and the energies of the molecular orbitals are not drawn to scale. Transitions are described in the text. (Adapted with permission from Inorganic Chemistry, 1999, 38, 1401. Copyright (1999) American Chemical Society).
(A) Solid state resonance Raman profiles and 5K mull electronic absorption spectrum for Tp*Mo V O(bdt). (B) Resonance Raman spectrum for Tp*Mo V O(bdt) (293K) using 514.5 nm excitation (75 mW). (C) General molecular orbital diagram for Tp*Mo V O(dithiolene) complexes. The z-axis is oriented along the Mo≡O bond and the energies of the molecular orbitals are not drawn to scale. Transitions are described in the text. (Adapted with permission from Inorganic Chemistry, 1999, 38, 1401. Copyright (1999) American Chemical Society).
… 
Frontier orbital energies as a function of fold angle in Mo IV O(SPh)2( i Pr2Dt 0 ), which possesses a dithione π-acceptor ligand. (Adapted with permission from Inorganic Chemistry, 2016, 55, 785-793. Copyright (2016) American Chemical Society).
Frontier orbital energies as a function of fold angle in Mo IV O(SPh)2( i Pr2Dt 0 ), which possesses a dithione π-acceptor ligand. (Adapted with permission from Inorganic Chemistry, 2016, 55, 785-793. Copyright (2016) American Chemical Society).
… 
Low-frequency rR spectra for wt, Q102G, and Q197A XDH, Mo IV −4-TV (a) and Mo IV −2,4-TV (b). Raman spectra were collected on resonance with the Mo(IV) → P MLCT band using 780 nm laser excitation (Adapted with permission from Inorganic Chemistry, 2017, 56, 6830-6837. Copyright (2017) American Chemical Society).
+1
Low-frequency rR spectra for wt, Q102G, and Q197A XDH, Mo IV −4-TV (a) and Mo IV −2,4-TV (b). Raman spectra were collected on resonance with the Mo(IV) → P MLCT band using 780 nm laser excitation (Adapted with permission from Inorganic Chemistry, 2017, 56, 6830-6837. Copyright (2017) American Chemical Society).
… 
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Inorganics2020,8,19;doi:10.3390/inorganics8030019www.mdpi.com/journal/inorganics
Review
Metal–DithioleneBondingContributionsto
PyranopterinMolybdenumEnzymeReactivity
JingYang
1
,JohnH.Enemark
2
andMartinL.Kirk
1,
*
1
DepartmentofChemistryandChemicalBiology,TheUniversityofNewMexico,MSC032060,
Albuquerque,NM87131‐0001,USA;yangjing@unm.edu
2
DepartmentofChemistryBiochemistry,UniversityofArizona,Tucson,AZ85721,USA;
jenemark@email.arizona.edu
*Correspondence:mkirk@unm.edu;Tel.:+1‐505‐277‐5992
Received:2February2020;Accepted:2March2020;Published:5March2020
Abstract:Herewehighlightpastworkonmetal–dithioleneinteractionsandhowtheunique
electronicstructureofthemetal–dithioleneunitcontributestoboththeoxidativeandreductivehalf
reactionsinpyranopterinmolybdenumandtungstenenzymes.Themetallodithioleneelectronic
structuresdetailedherewereinterrogatedusingmultiplegroundandexcitedstatespectroscopic
probesontheenzymesandtheirsmallmoleculeanalogs.Thespectroscopicresultshavebeen
interpretedinthecontextofbondingandspectroscopiccalculations,andthepseudo‐Jahn–Teller
effect.Thedithioleneisauniqueligandwithrespecttoitsredoxactivenature,electronicsynergy
withthepyranopterincomponentofthemolybdenumcofactor,andtheabilitytoundergochelate
ringdistortionsthatcontrolcovalency,reductionpotential,andreactivityinpyranopterin
molybdenumandtungstenenzymes.
Keywords:metal–dithiolene;pyranopterinmolybdenumenzymes;fold‐angle;tungstenenzymes;
electronicstructure;pseudo‐Jahn–Tellereffect;thione;molybdenumcofactor;Moco
1.Introduction
Itisnowwell‐establishedthatallknownmolybdenum‐containingenzymes[1–3],withthesole
exceptionofnitrogenase,containacommonpyranopterindithiolene(PDT)(Figure1)organic
cofactor(originallycalledmolybdopterin(MPT)),whichcoordinatestotheMocenteroftheenzymes
throughthesulfuratomsofthedithiolenefragment.Todate,thePDTcomponent[4]ofthe
molybdenumcofactor(Moco)istheonlyknownoccurrenceofdithioleneligationinbiological
systems.Thiscofactorisalsofoundinanaerobictungstenenzymes,anditmaybeoneofthemost
ancientcofactorsinbiology[5].Thestudyofmetal–dithiolenecompounds(metallodithiolenes)has
undergonearecentrenaissance,withtheirsynthesis,geometricstructure,spectroscopy,bonding,
andelectronicstructurehavingbeenrecentlyhighlighted[4,6–20].Here,webrieflyreviewthe
discoveryofmetallodithiolenecompounds[13,21].Thishistoryisfollowedbyamoreextensive
discussionofkeyinvestigationsintothemyriadrolesofthedithioleneligandsinthestructure,
bondingandreactivityofmetalcompounds,usingmultiplespectroscopictechniques,aswellas
theoreticalcalculations.Throughoutthisreview,thekeyimplicationsoftheseresultsforMoandW
enzymesarediscussed.
Inorganics2020,8,192of14
Figure1.Thereducedtetrahydroformofthepyranopterindithiolene(PDT)coordinatedtoMointhe
molybdenumcofactor(Moco).Intheenzymes,theMoioncanredoxcyclebetweentheMoIV,MoV,
andMoVIoxidationstates.
Intheearly1960s,severalresearchgroupsreportedintenselycoloredsquareplanarmetal
complexeswithchelatingsulfur‐donorligandsthatcouldstabilizemetalcompoundsinarangeof
formaloxidationstatesrelatedbyone‐electronoxidation‐reduction(i.e.,redox)reactions(Figure2)
[22–24].McClevertygavethesenovelligandsthegeneralname“dithiolene”inordertoemphasize
theirdelocalizedelectronicstructures[25].Theseligandsarealsodescribedasbeing“non‐innocent”
duetotheparticipationofthedithioleneligandsinthemultipleone‐electronreactionsoftheirmetal
complexesandtheinabilitytoassignaspecificoxidationstatetothemetalionorthedithiolene
ligands[11].
Figure2.Squareplanermetallodithiolenecomplexes.R=CN,CH3,Ph,CF3.
Importantly,thesenon‐innocentdithioleneligandscanmodulatethenatureofthecovalent
bondingwithtransitionmetalionsviathevariousredoxstatesaccessibletothedithiolene(Figure3)
[13].Theene‐1,2‐dithiolateisthereducedformoftheligandandpossessessixπ‐electrons.Thisligand
formisbothaσ‐donorandπ‐donorthatusuallyformsstrongcovalentbondswithanoxidized
transitionmetalion,asisobservedintheactivesitesofmostpyranopterinMoandWenzymes(e.g.,
Mo(V)/Mo(VI)‐dithiolenebonds).Theradicalanionformwithfiveπ‐electronsisusuallyfoundin
moleculeschelatedbymultipledithioleneligands,whereextendeddelocalizationoftheπ‐electrons
andmixed‐valencyassistsinthestabilizationofthemetal–ligandbonds.Thefullyoxidized1,2‐
dithioneformoftheligandpossessesonlyfourπ‐electronsandcanbedescribedbytworesonance
structures(e.g.,the1,2‐dithioneand1,2‐dithiete).Thelow‐lyingemptyπ*orbitalsoftheS=Cbonds
inthedithionecanacceptπ‐electrondensityfromelectron‐richlow‐valenttransitionmetals[16,17],
therebystabilizingsuchcompounds.However,dithione‐containinglow‐valentmetalcomplexesare
encounteredmuchlessfrequentlythanhigh‐valenttransitionmetalionscoordinatedbyreduced
formsofdithioleneligands.
Figure3.Dithioleneredoxstatesandresonancestructuresfortheoxidizeddithione/dithieteforms.
(AdaptedwithpermissionfromInorganicChemistry,2016,55,785–793.Copyright(2016)American
ChemicalSociety).
Inorganics2020,8,193of14
In1982,JohnsonandRajagopalanproposedthatMococonsistedoftheMoioncoordinatedby
thedithiolenefragmentofthePDT(Figure1),fromtheresultsofanelegantseriesofdegradative,
analyticalandspectroscopicstudiesofsulfiteoxidase[26].Thisproposedstructurewassubsequently
confirmedbyX‐raycrystallography[27,28],andnumerousexamplesarenowknown[29].
Molybdenumandtungstenenzymesaretheonlyknownexamplesofdithiolenecoordinationin
biology,andgiventhe“non‐innocent”behaviorofdithioleneligandsinsimplemetalcompounds,
onemayaskwhatroledoesdithiolenecoordinationplayinmolybdenumenzymes?Throughaseries
ofexamplesinvolvingsmallmoleculesandenzymes,wewilladdressthisimportantquestionand
howitrelatestocontrolofmetal–ligandcovalency,reductionpotentials,andreactivityin
pyranopterinMoandWenzymes.
2.Mo–DithioleneBonding
2.1.EarlyDescriptionsofMo–DithioleneBonding
Someinsightintotheroleofdithiolenecoordinationinenzymesisprovidedbythe
organometalliccompoundsofthegeneralformulaCp2M(bdt),whereCpisC5H5−,andMiseitherMo,
VorTi.Thefoldangleofthedithioleneliganddependsontheformald‐electroncountofthemetal,
andthisanglerangesfromnearlyplanar(9°)forMo(d2),to35°forV(d1),and46°forTi(d0)(Figure
4).LauherandHoffman[30]relatedthisincreaseinthefoldanglewithdecreasedd‐electroncount
todonationfromthefilledout‐of‐planeSπ+orbitaltothein‐planemetald‐orbital(Figure5).Forthe
molybdenumenzymes,thesemodelcompoundresultsimplythattheMo–dithiolenefoldanglein
MococouldberelatedtotheformaloxidationstateoftheMoatom,withMo(VI)(d0)sitespossessing
arelativelylargefoldangleandMo(V)(d1)andMo(IV)(d0)sitespossessingsmallerfoldangles.
Accuratefoldanglesaredifficulttodetermineforlargeproteinmolecules,butvaluesrangingfrom
6–33°havebeencalculatedforvariousmolybdenumenzymes[31].Thebindingofsubstrateor
inhibitors,and/ordynamicconformationalchangesintheprotein,areexpectedtomodulatethe
activesitechelatefoldangleandtherebyaffectenzymereactivity[4,32].
Figure4.Foldangledistortionsasafunctionofredoxorbitalelectronoccupancyinaseriesof
Cp2MIV(bdt)complexes.(AdaptedwithpermissionfromJ.Am.Chem.Soc.2018,140,14777–14788.
Copyright(2018)AmericanChemicalSociety).
Figure5.Pictorialdescriptionofhowtheligandfoldanglemodulatesthedegreeofmixingbetween
thedithioleneout‐of‐planeSorbitals(Sπ+)andthein‐planeMo(xy)redoxorbital.Thechelateringfold
Inorganics2020,8,194of14
isalongthedithioleneS–Svector.(AdaptedwithpermissionfromProc.Natl.Acad.Sci.USA.2003,
100,3719–3724.Copyright(2003) NationalAcademyof Sciences.
2.2.SpectroscopicInvestigationsofMo–DithioleneBonding
2.2.1.ElectronParamagneticResonance(EPR)Spectroscopy
Animportantspectroscopicsignatureofmolybdenumenzymes,suchasxanthineoxidaseand
sulfiteoxidase,isauniqueMo(V)electronparamagneticresonance(EPR)spectrum.TheEPRspectra
oftheenzymesdisplayarelativelylargeaverageg‐value(gave=1.97)andrelativelysmall95,97Mo
hyperfineinteractions(hfi)comparedtotheEPRspin‐Hamiltonianparametersfromtypicalinorganic
Mo(V)complexesthatpossesshardN,O,andCldonorligands.TheuniqueEPRparametersfor
molybdenumenzymeshavebeenascribedtocovalentdelocalizationofelectrondensitybetweenthe
Mo(V)centerandthesulfuratomsofthecoordinatedpyranopterindithioleneunit[33].Theoxo‐
Mo(V)modelcompoundTp*MoO(bdt)(Figure6,whereTp*ishydrotris‐(3,5‐dimethyl‐1‐
pyrazolyl)borateandbdtis1,2‐benzenedithiolate))displaysMo(V)EPRspin‐Hamiltonian
parametersthatareverysimilartothoseobservedintheenzymes.Thissupportstheproposalof
dithiolenecoordinationinMoenzymes[34],whichhasbeenconfirmedbyX‐raycrystalstructures
[2].RecentmultidimensionalvariablefrequencypulsedEPRstudiesofsulfiteoxidase,wherethe
sulfuratomsofMocohavebeenisotopicallylabeledwith33S(I=3/2),haveprovideddirect
experimentalevidencefordelocalizationofMo(V)spindensityontotheSatomsofthedithiolene
fragmentofMoco[35,36].Densityfunctionaltheory(DFT)computationsshowspinpolarization
effectsandstrongcovalentintermixingbetweenthein‐planemetaldxyorbitalandout‐of‐planepz
orbitalsofthePDTdithioleneSatoms,whichprovideamechanismfortheobservationofasignificant
33Shyperfineinteraction[12,36].
Figure6.TheTp*MoVO(bdt)model.Notethattheapicaloxoligandcanbechangedtoaterminal
sulfidoornitrosyltoprobetheelectronicstructureoftheMo–dithioleneunit.Thebdtligandcanalso
beconvenientlyinterchangedwithalargevarietyofotherdithiolenes.
2.2.2.ElectronicAbsorptionandResonanceRamanSpectroscopies
ExperimentalinvestigationoftheelectronicstructuresoftheMocentersofenzymesisdifficult
becauseoftheintenseabsorptionsfromotherchromophores(e.g.,theb‐typehemeinsulfiteoxidase
andironsulfurcentersandFADinxanthineoxidase)[37–41].However,theeffectsofdithiolene
coordinationonelectronicstructurehavebeeninvestigatedformodeloxo‐Mo(V)compounds(Figure
6)byelectronicabsorption,XAS,magneticcirculardichroism(MCD),and resonanceRaman(rR)
spectroscopies[12,14–17,32,33,42–51].ForTp*MoO(bdt),theelectronicabsorptionsat19,400cm−1
(Band4)and22,100cm−1(Band5)areassignedtoS➝Mochargetransferbands(Figure7A)[12].
TheseassignmentshavebeenconfirmedbyrRspectroscopy(Figure7A,B),whichshowsthree
resonantlyenhancedvibrationsat362.0,393.0,and931.0cm−1.Thelowerfrequencyvibrations(ν1and
ν6)canbeassignedtosymmetricS–Mo–Sstretchingandbendingvibrations,andthe931.0cm−1
frequency(ν3)isprimarilytheMo≡Ostretch.Figure7Cshowsamolecularorbitaldiagramthatis
consistentwiththespectroscopicdataofFigures7A,B.Band5ofFigure7Aisassignedas𝜓opa”➝
𝜓xza”,𝜓yza’(bluearrow,Figure7C),atransitionwhichformallyresultsinthepromotionofanelectron
fromanout‐of‐planedithiolenemolecularorbitaltothenearlydegenerateModxz,yz‐basedorbitals,
whicharestronglyantibondingwithrespecttotheapicalMo≡Obond.Thisbandassignmentis
Inorganics2020,8,195of14
supportedbytherRenhancementofν
3
(squares)withexcitationintoBand5(Figures7A,B).The
preferentialenhancementofvibrationsν
1
(diamonds)andν
6
(circles)uponexcitationat514.5nm
(Figures7A,B)supportsassignmentofBand4astheelectronictransitionψ
ipa”
➝ψ
xya’
(redarrow,
Figure7C),
whichpromotesanelectronfromtheantisymmetricin‐planedithioleneorbital(ψ
ipa”
)to
thehalf‐filledin‐planeMod
xy
(ψ
xya’
)orbital.Theintensityofthiselectronictransitionillustratesthe
covalencyofin‐planemetal–dithiolenebondingandsuggeststhatsuchapseudo‐σ‐mediatedprocess
couldplayaroleinone‐electrontransferstepsofenzymecatalysis.
Figure7.(A)SolidstateresonanceRamanprofilesand5Kmullelectronicabsorptionspectrumfor
Tp*Mo
V
O(bdt).(B)ResonanceRamanspectrumforTp*Mo
V
O(bdt)(293K)using514.5nmexcitation
(75mW).(C)GeneralmolecularorbitaldiagramforTp*Mo
V
O(dithiolene)complexes.Thez‐axisis
orientedalongtheMo≡Obondandtheenergiesofthemolecularorbitalsarenotdrawntoscale.
Transitionsaredescribedinthetext.(AdaptedwithpermissionfromInorganicChemistry,1999,38,
1401.Copyright(1999)AmericanChemicalSociety).
3.SynergisticInteractionsbetweentheDithioleneandPterinComponentsofthePDT
ElectroniccouplingbetweenthedithioleneandthepterincomponentsofthePDTismost
prevalentinthedihydropyranopterinformofthePDT[4,15,20,29,52].Thiscouplingisdramatically
reducedinatetrahydropyranopterinduetothelossofextendedπ‐conjugationinthesesystems.
Two‐electronoxidationofthetetrahydropyranopterincomponentofthePDTcanresultinan
unusualasymmetricdithioleneknownasthe“thiol–thione”formthatleadstobondandelectronic
asymmetryinthemetal–dithiolenecore[4,15,52].AsdepictedinFigure8,thetwo‐electronoxidized
10,10a‐dihydropyranopterincanundergoaninducedinternalredoxreactionuponprotonationatthe
N‐5positionthatinvolvesasubsequentchargetransferbetweenthedithiolenechelateandthe
piperazineringofthepterin.Thisprotonationresultsinadominantmonoanionicthiol–thionechelate
formoftheligandwhenboundtoMoorW.Thisthiol–thionecharactercanalsooccurintheabsence
ofprotonationbytheconceptofresonance,whichmayalsobedescribedasconfigurationalmixing
betweenthethiol–thioneanddithiolstates.Thistypeofthiol–thionechelatehasbeenobservedand
studiedinasmallmoleculeMo(IV)systems[4,15,20,52].Inthesesystems,excitedstatethiol–thione
characterwasshowntobeadmixedintothegroundstateconfigurationusingavarietyof
spectroscopicandcomputationalprobesoftheelectronicstructure.Theanalysisofthedataindicates
Inorganics2020,8,196of14
thatatwo‐electronoxidizedpterinisinherentlyelectronwithdrawing,allowingforalow‐lying
dithiolene→ pterinintraligandchargetransfer(ILCT)statetomixwiththegroundstatetoprovide
avariabledegreeofthiol–thionecharacterintheelectronicgroundstate.
(a)(b)
Figure8.OxidizedPDTligands:dihydropyranopterin(a)andprotonateddihydropyranopterin(b)
yieldingthethiol/thione.
Definitivespectroscopicsignaturesareassociatedwiththepresenceofadihydropterinformof
thePDTligand.Itisobservedthatthedithiolene→ pterinintraligandchargetransfer(ILCT)bandis
intense(E=20,000–27,500cm−1;ε~10,000–16,000M−1cm−1)[52],andthereisconsiderableresonance
enhancementofnumerousRamanvibrationsthatcanbeassignedasoriginatingfrompterinand
dithioleneC=CandC=Nvibrations.Keyresonanceenhancedvibrationalmodesthatcanbeused
tocharacterizethepresenceofdihydropterinthiol–thionecharacterintheenzymesincludethe1508
cm−1and1549cm−1pyranopterin–dithiolenestretchingfrequenciesthatwereobservedinthisMo(IV)
cyclizedpyranopterindithiolenemodelcompound.ThisoxidizedpyranringclosedformofthePDT
hasyettobedefinitivelyobservedinanypyranopterinMoenzyme,butitspresencewouldhave
profoundimplicationsontheelectronicstructureoftheMosite.Namely,thechangeinligandcharge
from−2to−1leadstoanasymmetricreductioninthechargedonatedbythemonoanionicligand
comparedtothedianionicdithiolene.Chargeeffectsonoxygenatomtransfercatalysishaverecently
beenexploredinmodelcompoundsshowingdramaticrateenhancementsintheoxidativehalf
reactionthatleadstosubstratereduction[53].Thisreactivitycorrelateswithalargeshiftinthe
Mo(VI/V)reductionpotentialbetweencationic[Tpm*MoO2Cl]+(−660mVvs.Fc+/Fc)andcharge
neutralTp*MoO2Cl(−1010mVvs.Fc+/Fc)[53].Thesameeffectonredoxpotentialandreactivity
wouldbeexpectedinenzymesthatcouldadoptanoxidizedPDTwithathiol–thioneconfiguration.
Thepresenceofathiol–thioneformofthePDTinanenzymewouldalsohaveaconsiderableimpact
ontheactivesiteelectronicstructure,andenablethepyranopterintoplayamoresignificantrolein
catalysisbyfine‐tuningtheMoredoxpotentialandprovidingaπ‐pathwayforelectrontransfer
regenerationoftheactivesite[52].Additionally,theasymmetryinthedithiolene(thiol/thione)charge
donationwouldbeexpectedtoresultinasignificanttranseffectortransinfluenceonoxoorsulfido
ligandsthatarecoordinatedtotheMoorWionandorientedtranstothethionesulfur.
4.TheElectronicBufferEffectandFoldAngleDistortions
4.1.PhotoelectronSpectroscopy(PES)Studies
AcommonstructuralfeatureofthelargegroupofpyranopterinMoenzymesthatcatalyzea
widerangeofoxidation/reductionreactionsincarbon,sulfur,andnitrogenmetabolismis
coordinationbythesulfuratomsofone(ortwo)uniquedithiolenegroupsderivedfromtheside
chainofanovelsubstitutedpterin(PDT,Figure1).Giventheelectroniclabilityofthedithiolene,a
possibleroleofdithiolenecoordinationinmolybdoenzymesistobuffertheinfluenceofotherligands
andchangesintheformaloxidationstateofthemetal.Gas‐phasephotoelectronspectroscopy(PES)
isapowerfultoolforprobingmetal–ligandcovalencyinisolatedmolecules.Gas‐phaseultraviolet
PESofthemolybdenummodelcomplexeswiththegeneralformulaTp*MoE(tdt)(Figure6,whereE
=O,S,orNO,andtdt=3,4‐toluenedithiolate),exhibitnearlyidenticalfirstionizationenergies(6.88–
6.95eV)eventhoughthereisadramaticdifferenceintheelectronicstructurepropertiesoftheaxial
ligand.Collectively,theseresultshaveprovideddirectexperimentalevidenceforthe“electronic
buffer”effectofdithioleneligands[54].
Additionalevidencefortheelectronicbuffereffectofdithioleneligandshasbeenprovidedby
gas‐phasecoreandvalenceelectronionizationenergymeasurementsoftheseriesofmolecules
Inorganics2020,8,197of14
Cp2M(bdt)(Figure4,Cp=η
5‐cyclopentadienyl,M=Ti,V,Mo,andbdt=benzene‐1,2‐dithiolate).
Comparisonofthegas‐phasecoreandvalenceionizationenergyshiftsprovidesaunique
quantitativeenergymeasureofvalenceorbitaloverlapinteractionsbetweenthemetalandthesulfur
orbitalsthatisseparatedfromtheeffectsofchargeredistribution.Theresultsexplainthelarge
amountofsulfurcharacterintheredox‐activeorbitalsandtheelectronicbufferingofoxidationstate
changesinmetal–dithiolenesystems.Theexperimentallydeterminedorbitalinteractionenergiesalso
revealapreviouslyunidentifiedoverlapinteractionofthepredominantlysulfurHOMOofthebdt
ligandwiththefilledπorbitalsoftheCpligands,suggestingthatdirectdithioleneinteractionswith
otherligandsboundtothemetalcouldbesignificantforothermetallodithiolenesystemsinchemistry
andbiology[55].
4.2.ALargeFoldAngleDistortioninaMo(IV)–DithioneComplex
Mo(IV)–dithionecomplexesaremuchrarerthanMo(V)/Mo(VI)‐dithiolenecomplexes.Recently,
adetailedspectroscopicandcomputationalstudywasperformedonanovelMo(IV)–dithione
complex,MoO(SPh)2(iPr2Dt0)(whereiPr2Dt0=N,N′‐isopropylpiperazine‐2,3‐dithione)[17].The
structureofthisunusualmoleculewasdeterminedbyx‐raycrystallographyanddisplaysa
remarkablylargedithiolenefoldangle(η=70°).Thislargefoldanglewascomparedtothatobserved
inmorethan75othermetallodithiolenecomplexesfoundintheCambridgecrystallographic
database,wherefoldangleswerefoundtorangefrom0.3°to37.3°withanaveragevalueforηof
12.5°[17].ThelargefoldangledistortioninthemetallodithioleneringofMoO(SPh)2(iPr2Dt0)is
reflectedinitsunusualelectronabsorptionspectrum.ThecombinationofanelectronrichMo(IV)
centerandelectrondonatingthiolate(SPh)ligandsresultsinthepresenceoflow‐energyMo(IV)d(x2‐
y2)➝dithioneMLCTandthiolate➝dithioneLL’CTtransitionsasaresultofthestrongπ‐acceptor
characterofthedithioneligand.ThesespectralassignmentsaresupportedbyresonanceRaman
profilesconstructedforthe378cm−1S–Mo–Ssymmetricstretchandthe945cm−1MoOstretchin
additiontotheresultsofTDDFTcomputations.Thedonor–acceptornatureofthecomplexwas
revealedinamolecularorbitalfragmentsanalysisusingadonorfragment,[(PhS)2Mo(IV)]2+(F1)and
anacceptorfragment,[iPr2Dt0](F2).Theanalysisshowedthat21%oftheF1HOMOwasmixedinto
theF2fragmentLUMOata70ofoldangle.Incontrast,only5%oftheF1HOMOwasmixedintoF2
fragmentLUMOinaplanerconfiguration(η=0°),correlatingtheeffectiveπ‐acceptorabilityofthe
dithionewiththeligandfoldangle.TheeffectsofthisHOMO‐LUMOmixingalsoaffectstheHOMO‐
LUMOgap,withtheHOMO‐LUMOgapincreasingatlargerfoldangles(Figure9).Theincreased
covalencythatresultsfromthefoldangledistortionrepresentsanexampleofastrongpseudo‐Jahn–
Tellereffect,videinfra,involvingvibroniccouplingbetweenthegroundstateandalow‐energy
excitedstateinthenon‐distorted(η=0°)geometryofthismolecule.Ascanofthepotentialenergy
surfaceasafunctionofthisfoldangledistortioncoordinateresultsinanasymmetricdoublewell
potential(Figure10),withtheglobalminimumrepresentingagroundstategeometrywiththe
dithioneligandfolddistortedtowardtheapicaloxoligand.Thus,anoxidizeddithioneformofthe
PDTpresentinanenzymeactivesitewouldbeexpectedtopossessaverylargeligandfoldangle,
unlessthepolypeptideenforcesamoreplanerfoldanglegeometry.
Inorganics2020,8,198of14
Figure9.FrontierorbitalenergiesasafunctionoffoldangleinMoIVO(SPh)2(iPr2Dt0),whichpossesses
adithioneπ‐acceptorligand.(AdaptedwithpermissionfromInorganicChemistry,2016,55,785–793.
Copyright(2016)AmericanChemicalSociety).
Figure10.AdoublewellinthegroundstatepotentialenergysurfaceofMoIVO(SPh)2(iPr2Dt0)asa
functionoftheligandfoldangle.(AdaptedwithpermissionfromInorganicChemistry,2016,55,785–
793.Copyright(2016)AmericanChemicalSociety).
4.3.Low‐FrequencyPyranopterinDithioleneVibrationalModesinXanthineOxidase/Dehydrogenase
Low‐frequencydithiolenedistortionsthatarecoupledtolargeelectrondensitychangesatthe
Moionrepresentanexampleoftheelectronicbuffereffect[54],andhavebeenprobedinbovine
xanthineoxidase(XO)andR.capsulatusxanthinedehydrogenase(XDH)usingresonanceRaman
spectroscopy[40].Computationshaveshownthatexcitingintoalow‐energyMo(IV)→product
metal‐to‐ligandchargetransfer(MLCT)bandresultsinalargedegreeofchangetransferfromthe
Mo(IV)HOMOtotheproductLUMO,resultinginanexcitedstatewithsignificantMo(V)hole
character(e.g.,Mo(IV)–P0→Mo(V)–P∙).Thus,theopticalchargetransferprocessmimicsthe
instantaneousone‐electronoxidationoftheMoion,whichisencounteredintheelectrontransfer
reactionsoftheenzymes.
TheMo(IV)→2,4‐TVandMo(IV)→4‐TV(2,4‐TV=2,4‐thioviolapterin;4‐TV=4‐thioviolapterin)
MLCTbandsarered‐shiftedrelativetotheMo(IV)→violapterinMLCTband[39,40,56–59].Thered‐
shiftoftheMoIV–2,4‐TVandMoIV–4‐TVMLCTbandseliminatesspectraloverlapwiththeabsorption
envelopeofthe2Fe–2SspinachferredoxinclustersandFAD.TheeliminationoftheFADfluorescence
backgroundandspurioussignalsderivingfrom2Fe–2SvibrationscontributingtotheRaman
spectrumallowfortheacquisitionofveryhigh‐qualityresonanceRamandata.Multiplelow‐
frequency(200–400cm−1)Ramanvibrationsareobservedtobeenhancedwhenusinglaserexcitation
onresonancewiththeMo(IV)→productMLCTband[40],andthesehavebeenassignedasa
vibrationalmodeinvolvingdithiolenefolding,Mo≡Orocking,andpyranopterinmotions(BandA:
MoIV–4‐TV=234cm−1;MoIV–2,4‐TV=236cm−1),aringdistortionvibrationthatpossessesbothMo–
SHandpyranopterinmotions(BandB:MoIV–4‐TV=290cm−1;MoIV–2,4‐TV=286cm−1),thesymmetric
S–Mo–Sdithiolenecorestretchingvibration(BandC:MoIV–4‐TV=326cm−1;MoIV–2,4‐TV=326cm−1),
andthecorrespondingasymmetricS–Mo–Sdithiolenestretch(BandD:MoIV–4‐TV=351cm−1;MoIV–
2,4‐TV=351cm−1)(Figure11).Thus,theinstantaneousgenerationofaholeontheMocenter(Mo(IV)–
P0→Mo(V)–P∙)byphotoexcitationisfeltbythedithiolenechelateandextendsallthewaytothe
aminoterminusofthePDT.ThemostresonantlyenhancedmodeinthisspectralregionisBandC,
thesymmetricS–Mo–Sdithiolenecorestretching,andthefrequencyofthismodeandBandDare
similartothoseobservedinTp*MoO(bdt)[12,32],whichwereassignedasthechelateringsymmetric
S–Mo–Sstretchingandbendingvibrations,respectively.BandAissignificant,sinceitpossesses
dithioleneringfoldingcharacterindicatingthatelectrondensitychangesatMoarebufferedbya
distortionalongthislow‐frequencycoordinate,ashasbeenobservedinthevariousmodelsystems
describedinthisreview.TheseobservationsstronglysupportanelectrontransferroleforthePDTin
catalysis,withthedithiolenecontributingtotheMo–Scovalencynecessaryforincreasingthe
Inorganics2020,8,199of14
electroniccouplingmatrixelementforelectrontransfer(H
DA
)andtoaffecttheMoreductionpotential
viathecovalencyintheMo–S
dithiolene
bonds.
(a)(b)
Figure11.Low‐frequencyrRspectraforwt,Q102G,andQ197AXDH,Mo
IV
−4‐TV(a)andMo
IV
−2,4‐
TV(b).RamanspectrawerecollectedonresonancewiththeMo(IV)→ PMLCTbandusing780nm
laserexcitation(AdaptedwithpermissionfromInorganicChemistry,2017,56,6830–6837.Copyright
(2017)AmericanChemicalSociety).
5.VibrationalControlofCovalency
AcombinationofMCD,electronicabsorption,electronparamagneticresonance,resonance
Raman,andphotoelectronspectroscopieshasbeenusedinconjunctionwiththeorytoreveal
vibrationalcontrolofmetal–ligandcovalencyinaseriesofCp
2
M(bdt)complexes(M=Ti,V,Mo;Cp
=η
5
‐C
5
H
5
)[60](Figure4).Theworkisimportantbecauseithasallowedforadetailedunderstanding
ofhowredoxorbitalelectronoccupancy(Ti(IV)=d
0
,V(IV)=d
1
,Mo(IV)=d
2
,)affectsthenatureofthe
M–dithiolenebondingschemeatparityoftheligandsetandatparityofcharge.Inthisseriesof
complexes,largechangesinthemetallodithiolenefoldangleandelectronicstructureareobservedas
electronsaresuccessivelyremovedfromtheredoxorbital(Figure4).Theseelectronoccupancyeffects
onthefoldangledistortionarenowunderstoodintermsofthepseudo‐Jahn–Tellereffect(PJT).PJT‐
derivedmoleculardistortionsoriginatefromthemixingoftheelectronicgroundstate(Ψ
0
)with
specificexcitedstates(Ψ
i
)[61,62].Thegroundstate–excitedstateenergygap(2Δ),thematrixelements
(F
0i
)ofthevibroniccontributiontotheforceconstant(F),andtheprimarynon‐vibronicforceconstant
(K
0
)allgovernthedegreeoftheligandfolddistortionaccordingto:
𝐹

𝛹
󰇻𝜕𝐻 𝜕𝑄
󰇻𝛹
(1)
𝐹
∆∙𝐾
(2)
AtthecriticalthresholddefinedbyEquation(2),themetallodithiolenecentersofCp
2
M(bdt)can
distortalongthedithiolenefoldanglecoordinatetoyieldadoublewellpotentialenergysurface
(Figure12),andthemagnitudeofthePJTdistortionismaximizedbyalargeF,asmallΔ,andasmall
K
0
.Thus,thePJTdistortionintheseCp
2
M(bdt)complexeseffectivelycouplessoftfoldanglebending
modesintheM‐dithiolenechelateringtotheinherentelectronicstructureofthesystemviathed‐
electroncount.Importantly,themixingoflow‐energychargetransferstatesintothegroundstateby
thePJTeffectcontrolsthecovalencyoftheM–Sbonds.
OneoftheuniqueaspectsofMo–SandW–Sbondingisthesmallenergygapbetweenfilled
dithiolene‐basedorbitalsandthelowestenergymetal‐basedorbital,whichnaturallyleadstolow‐
energychargetransferstatesthatcanmixwiththegroundstate.Modesofteningalongthedithiolene
foldcoordinateisimportantinpyranopterinMoandWenzymessincethisleadstoapotentialenergy
surfacewherealargerangeofdithiolenefoldanglesmaybesampledwithoutpayingaprohibitive
energypenalty.ThiseffectismaximizedwhenF
2
≅Δ∙K
0
.Thus,alow‐energypathwayisoperative
thatcanminimizeenergeticallyunfavorablereorganizationalenergycontributionsalongthereaction
coordinate,whichaccompanyredoxchangesatthemetalion.Asmentionedpreviously,thesefold
Inorganics2020,8,1910of14
angledistortionshavebeenshowntobekinematicallycoupledtolowfrequencypyranopterinmodes
inXOandcontributetolow‐energybarriersforelectrontransferregenerationoftheactivesite.
However,intheenzymestheremaybeeitheracompetingoradditiverelationshipbetweenactive
sitedistortionsthataredrivenviathed‐electroncountofthemetalionanddistortionsthatare
imposedbytheprotein.Vibroniccouplingeffectsthatderivefromdifferentoccupancynumbersfor
theredox‐activeorbitalwillfunctiontomodulatetheenzymereductionpotentialintheoxidative
andreductivehalfreactionsofpyranopterinMoandWenzymes,andthisoccursbymodulatingthe
degreeofmetal–ligandcovalencyvialow‐frequencydistortionsattheactivesite.
Figure12.Theexcitedstate(black)andgroundstate(red)potentialenergysurfacesassociatedwith
varyingvaluesofF2(Dotted:F2=0,solid:F2=Δ∙K0,dashed:F2=2Δ∙K0)WhentheconditionF2>Δ∙K0
ismetoneobservesthatthesingle‐wellgroundstatepotentialenergysurfacedistortsintoadouble‐
wellpotential.TheF2>Δ∙K0criteriadescribeastrongPJTeffect.(AdaptedwithpermissionfromJ.Am.
Chem.Soc.2018,140,14777–14788.Copyright(2018)AmericanChemicalSociety).
6.Conclusions
Thisreviewfocusesontheelectronicstructures,molecularstructures,andspectroscopic
propertiesofwell‐characterizedmetallodithiolenecompoundsinordertoprovidedeepinsightinto
therole(s)ofmetal–dithiolenebondinginpyranopterindithiolenecontainingenzymes(Figure1).
Thediscovery,intheearly1960s,thattransitionmetaldithiolenecompoundsundergoaseriesofone‐
electronoxidation‐reductionreactions(Figure2),providedthefirstevidenceforthe“non‐innocence”
ofdithioleneligandsandthehighlycovalentnatureofmetal–dithiolenebonding.Additionallinks
betweenmetal–dithiolenecovalencyandelectronicandmolecularstructurewerepositedfrom
theoreticalstudiesofbentmetallocene−dithiolenecompounds(Figure4)byLauherandHoffmanin
1976[30],whorelatedmetal−dithiolenechelatering“folding”withthemetaliond‐electron
configuration.InvestigationsofModithiolenecompoundsbyelectronicabsorption,resonance
Raman,andEPRspectroscopiesshowedthatS➝Mochargetransferbandsdominatethevisible
spectrumandthatthereissubstantialdelocalizationofspindensityontotheSatomsofthedithiolene.
Recentcomprehensivestudiesofbentmetallocene−dithiolenecompoundshaveshownthatlow‐
energyligandfolddistortionsarisefromapseudo‐Jahn−Teller(PJT)effect,whichinvolvesvibronic
couplingoftheelectronicgroundstatewithelectronicexcitedstatestocontrolmetal−ligand
covalency[60](Section5).Thisvibroniccouplingprocessmayplaycriticalrolesinthecatalyticcycles
ofpyranopterinMoandWenzymesbydynamicand/orstaticmodulationofredoxpotentialsand
providingasuperexchangepathwayforelectrontransferthroughthePDTframework.However,
greaterunderstandingofhowgeometricandelectronicstructurecontrolreactivity,anddefine
functioninMoandWenzymes,willrequirelinkingtheconceptsthathavebeendevelopedfor
metallodithiolenestotheemergingresultsfromstudiesofwell‐characterizedcompoundsthatmimic
thepterincomponentofPDT(Section3).Exploringthesynergisticinteractionsbetweenthe
dithioleneandpterincomponentsofthePDTandthemetalionwillbechallenging,butsuchresearch
promisestoprovideimportantinsightsintothesecriticallyimportantenzymes.
AuthorContributions:J.H.E.,J.Y.,andM.L.K.collectivelyconceivedanddraftedthisarticle.Allauthorshave
readandagreedtothepublishedversionofthemanuscript.
Inorganics2020,8,1911of14
Funding:M.L.K.’sresearchcontributionstothisarticlewerefundedbytheNationalInstitutesofHealth(R01‐
GM‐057378).
ConflictsofInterest:Theauthorsdeclarenoconflictofinterest.
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... The relationship between these enzyme families and the PDT is underscored by the observation that outof-plane PDT distortions appear to be related to enzyme function [16]. Determining how the geometric structure of the PDT correlates with the Mo oxidation state [18,19], and how the PDT contributes to the unique active site electronic structures of the enzyme active sites remains a very Abbreviations: rR, resonance Raman; SO, sulfite oxidase; DMSOR, dimethyl sulfoxide reductase; XO, xanthine oxidase; PDT, pyranopterin dithiolene; MPT, molybdopterin; EXAFS, extended X-ray absorption fine structure; AO, aldehyde oxidase; E-P, enzyme-product; XDH, xanthine dehydrogenase; DFT, density functional theory; NBO, natural bond orbital; CODH, carbon monoxide dehydrogenase; Q k , normal coordinate vibration along mode q; bdt, 1,2-benzenedithiolate; tdt, 3,4toluenedithiolate; qdt, quinoxaline dithiolate; Tp*, hydrotris-(3,5-dimethyl-1-pyrazolyl)borate; LMCT, ligand-to-metal charge transfer; LUMO, lowest unoccupied molecular orbital; SERR, surface enhanced resonance Raman; SAM, self-assembled monolayer; MCD, magnetic circular dichroism; EPR, electron paramagnetic resonance; BSOR, biotin sulfoxide reductase; DMS, dimethyl sulfide; NIR, near infrared; BSO, biotin sulfoxide; FAD, flavin adenine dinucleotide; MLCT, metal-toligand charge transfer; Moco, molybdenum cofactor; XAS, X-ray absorption spectroscopy. enzymes that comprise the DMSO reductase family are very diverse in nature and catalyze a wide range of chemical transformations. ...
... Perhaps the most characterized benchmark oxomolybdenum dithiolene complexes are Tp*MoO(bdt) [88], Tp*MoO(tdt) [88], and Tp*MoO(qdt) [89] (Tp* = hydrotris-(3,5-dimethyl-1-pyrazolyl)borate; bdt = 1,2-benzenedithiolate; tdt = 3,4-toluenedithiolate; qdt = quinoxaline dithiolate). These paramagnetic d 1 Mo(V) complexes are important for understanding the electronic structure of molybdoenzymes that possess a single terminal Mo ---O bond oriented cis to a dithiolene chelate, since this coordination geometry is encountered in many pyranopterin containing Mo enzyme forms [1][2][3]6,17,19,88,90]. The [MoO(dithiolene)] 1+ unit in these molecules possesses idealized C s symmetry, with the mirror plane bisecting the two S atoms of the dithiolene and containing the Mo ---O unit. ...
... Similarly, the resonance Raman spectra of the DMSOR family enzyme arsenite oxidase was interpreted in the context of one PDT dithiolene possessing a dithiolate structure with the second having more π-delocalization [112]. Model studies of oxo-Mo(IV) dithiolene complexes display evidence of thiol/thione character in a single dithiolene [12,13], complicating the issue of ν(C-S) and ν(C=C) vibrational assignments and highlighting aspects of the non-innocent nature of the dithiolene when bound to Mo [1,10,[12][13][14]19,90,107]. ...
Resonance Raman spectroscopy of pyranopterin molybdenum enzymes
Article
  • Jun 2022
  • J INORG BIOCHEM
Resonance Raman spectroscopy (rR) is a powerful spectroscopic probe that is widely used for studying the geometric and electronic structure of metalloproteins. In this focused review, we detail how resonance Raman spectroscopy has contributed to a greater understanding of electronic structure, geometric structure, and the reaction mechanisms of pyranopterin molybdenum enzymes. The review focuses on the enzymes sulfite oxidase (SO), dimethyl sulfoxide reductase (DMSOR), xanthine oxidase (XO), and carbon monoxide dehydrogenase. Specifically, we highlight how Mo-Ooxo, Mo-Ssulfido, Mo-Sdithiolene, and dithiolene CC vibrational modes, isotope and heavy atom perturbations, resonance enhancement, and associated Raman studies of small molecule analogs have provided detailed insight into the nature of these metalloenzyme active sites.
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... HCOO − → CO2 + 2e − + H + (8) With the single (as far as is presently known) exception of nitrogenase [9][10][11][12], molybdenum is found coordinated by the cis-dithiolene group (-S-C=C-S-) of one or two molecules of a pyranopterin cofactor (Figure 1). In a parallel situation to the haem ring, this unique cofactor is not an "innocent scaffold" and it is considered to be co-responsible to modulate the active site reactivity, besides acting as a "wire" to conduct the electrons to, or from, the other redox-active centres of the enzyme (intramolecular electron transfer, when this is the case) [13][14][15][16][17][18][19][20]. In addition to the pyranopterin cofactor, the molybdenum ion is coordinated by oxygen and/or sulfur and/or selenium terminal atoms and/or by enzyme-derived amino acid residues ( Figure 1), which are also expected to have key roles in catalysis (although their individual roles are not yet understood in many molybdoenzymes). ...
... With the single (as far as is presently known) exception of nitrogenase [9][10][11][12], molybdenum is found coordinated by the cis-dithiolene group (-S-C=C-S-) of one or two molecules of a pyranopterin cofactor (Figure 1). In a parallel situation to the haem ring, this unique cofactor is not an "innocent scaffold" and it is considered to be co-responsible to modulate the active site reactivity, besides acting as a "wire" to conduct the electrons to, or from, the other redox-active centres of the enzyme (intramolecular electron transfer, when this is the case) [13][14][15][16][17][18][19][20]. In addition to the pyranopterin cofactor, the molybdenum ion is coordinated by oxygen and/or sulfur and/or selenium terminal atoms and/or by enzyme-derived amino acid residues (Figure 1), which are also expected to have key roles in catalysis (although their individual roles are not yet understood in many molybdoenzymes). ...
Bringing Nitric Oxide to the Molybdenum World—A Personal Perspective
Article
Full-text available
  • Aug 2023
  • MOLECULES
Molybdenum-containing enzymes of the xanthine oxidase (XO) family are well known to catalyse oxygen atom transfer reactions, with the great majority of the characterised enzymes catalysing the insertion of an oxygen atom into the substrate. Although some family members are known to catalyse the “reverse” reaction, the capability to abstract an oxygen atom from the substrate molecule is not generally recognised for these enzymes. Hence, it was with surprise and scepticism that the “molybdenum community” noticed the reports on the mammalian XO capability to catalyse the oxygen atom abstraction of nitrite to form nitric oxide (NO). The lack of precedent for a molybdenum- (or tungsten) containing nitrite reductase on the nitrogen biogeochemical cycle contributed also to the scepticism. It took several kinetic, spectroscopic and mechanistic studies on enzymes of the XO family and also of sulfite oxidase and DMSO reductase families to finally have wide recognition of the molybdoenzymes’ ability to form NO from nitrite. Herein, integrated in a collection of “personal views” edited by Professor Ralf Mendel, is an overview of my personal journey on the XO and aldehyde oxidase-catalysed nitrite reduction to NO. The main research findings and the path followed to establish XO and AO as competent nitrite reductases are reviewed. The evidence suggesting that these enzymes are probable players of the mammalian NO metabolism is also discussed.
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Advancing Our Understanding of Pyranopterin-Dithiolene Contributions to Moco Enzyme Catalysis
Article
Full-text available
  • Nov 2023
  • MOLECULES
The pyranopterin dithiolene ligand is remarkable in terms of its geometric and electronic structure and is uniquely found in mononuclear molybdenum and tungsten enzymes. The pyranopterin dithiolene is found coordinated to the metal ion, deeply buried within the protein, and non-covalently attached to the protein via an extensive hydrogen bonding network that is enzyme-specific. However, the function of pyranopterin dithiolene in enzymatic catalysis has been difficult to determine. This focused account aims to provide an overview of what has been learned from the study of pyranopterin dithiolene model complexes of molybdenum and how these results relate to the enzyme systems. This work begins with a summary of what is known about the pyranopterin dithiolene ligand in the enzymes. We then introduce the development of inorganic small molecule complexes that model aspects of a coordinated pyranopterin dithiolene and discuss the results of detailed physical studies of the models by electronic absorption, resonance Raman, X-ray absorption and NMR spectroscopies, cyclic voltammetry, X-ray crystallography, and chemical reactivity.
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A bacterial tungsten-containing aldehyde oxidoreductase forms an enzymatic decorated protein nanowire
Article
Full-text available
  • Jun 2023
Aldehyde oxidoreductases (AORs) are tungsten enzymes catalyzing the oxidation of many different aldehydes to the corresponding carboxylic acids. In contrast to other known AORs, the enzyme from the denitrifying betaproteobacterium Aromatoleum aromaticum (AORAa) consists of three different subunits (AorABC) and uses nicotinamide adenine dinucleotide (NAD) as an electron acceptor. Here, we reveal that the enzyme forms filaments of repeating AorAB protomers that are capped by a single NAD-binding AorC subunit, based on solving its structure via cryo-electron microscopy. The polyferredoxin-like subunit AorA oligomerizes to an electron-conducting nanowire that is decorated with enzymatically active and W-cofactor (W-co) containing AorB subunits. Our structure further reveals the binding mode of the native substrate benzoate in the AorB active site. This, together with quantum mechanics:molecular mechanics (QM:MM)-based modeling for the coordination of the W-co, enables formulation of a hypothetical catalytic mechanism that paves the way to further engineering for applications in synthetic biology and biotechnology.
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Fine‐Tuning Redox Properties of Heteroleptic Molybdenum Complexes through Ligand‐Ligand‐Cooperativity
Article
  • Apr 2023
Heteroleptische Molybdänkomplexe mit 1,5‐Diaza‐3,7‐Diphosphacyclooctan (P 2 N 2 ) und non‐innocent (nicht‐unschuldigen) Dithiolenliganden wurden synthetisiert und elektrochemisch charakterisiert. Dabei wurde festgestellt, dass die Reduktionspotentiale der Komplexe durch einen synergistischen Effekt moduliert werden, welcher durch DFT‐Berechnungen als Ligand‐Ligand‐Kooperativität über nicht‐kovalente Wechselwirkungen identifiziert werden konnte. Dieses Ergebnis konnte durch elektrochemische Studien in Kombination mit UV/Vis‐Spektroskopie und temperaturabhängiger NMR‐Spektroskopie bestätigt werden. Das beobachtete Verhalten erinnert an eine enzymatische Redoxmodulation durch Effekte basierend auf Interaktionen in der zweiten Ligandensphäre.
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Fine‐Tuning Redox Properties of Heteroleptic Molybdenum Complexes through Ligand‐Ligand‐Cooperativity
Article
Full-text available
Heteroleptic molybdenum complexes bearing 1,5‐diaza‐3,7‐diphosphacyclooctane (P2N2) and non‐innocent dithiolene ligands were synthesized and electrochemically characterized. The reduction potentials of the complexes were found to be fine‐tuned by a synergistic effect identified by DFT calculations as ligand‐ligand cooperativity via non‐covalent interactions. This finding is supported by electrochemical studies combined with UV/Vis spectroscopy and temperature‐dependent NMR spectroscopy. The observed behavior is reminiscent of enzymatic redox modulation using second ligand sphere effects.
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A bacterial tungsten-containing aldehyde oxidoreductase forms an enzymatic decorated protein nanowire
Preprint
Full-text available
  • Jan 2023
Aldehyde oxidoreductases (AOR) are tungsten enzymes catalysing the oxidation of many different aldehydes to the corresponding carboxylic acids. In contrast to other known AORs, the enzyme from the denitrifying betaproteobacterium Aromatoleum aromaticum (AOR Aa ) consists of three different subunits (AorABC) and utilizes NAD as electron acceptor. Here we reveal that the enzyme forms filaments of repeating AorAB protomers which are capped by a single NAD-binding AorC subunit, based on solving its structure via cryo-electron microscopy. The polyferredoxin-like subunit AorA oligomerizes to an electron-conducting nanowire that is decorated with enzymatically active and W-cofactor (W-co) containing AorB subunits. Our structure further reveals the binding mode of the native substrate benzoate in the AorB active site. This, together with QM:MM-based modelling for the coordination of the W-co, enables formulation of catalytic mechanism hypothesis that paves the way for further engineering of AOR for applications in synthetic biology and biotechnology.
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Protonation and Non-Innocent Ligand Behavior in Pyranopterin Dithiolene Molybdenum Complexes
Article
  • Aug 2022
The complex [TEA][Tp*MoIV(O)(S2BMOPP)] (1) [TEA = tetraethylammonium, Tp* = tris(3,5-dimethylpyrazolyl)hydroborate, and BMOPP = 6-(3-butynyl-2-methyl-2-ol)-2-pivaloyl pterin] is a structural analogue of the molybdenum cofactor common to all pyranopterin molybdenum enzymes because it possesses a pyranopterin-ene-1,2-dithiolate ligand (S2BMOPP) that exists primarily in the ring-closed pyrano structure as a resonance hybrid of ene-dithiolate and thione-thiolate forms. Compound 1, the protonated [Tp*MoIV(O)(S2BMOPP-H)] (1-H) and one-electron-oxidized [Tp*MoV(O)(S2BMOPP)] [1-Mo(5+)] species have been studied using a combination of electrochemistry, electronic absorption, and electron paramagnetic resonance (EPR) spectroscopy. Additional insight into the nature of these molecules has been derived from electronic structure computations. Differences in dithiolene C-S bond lengths correlate with relative contributions from both ene-dithiolate and thione-thiolate resonance structures. Upon protonation of 1 to form 1-H, large spectroscopic changes are observed with transitions assigned as Mo(xy) → pyranopterin metal-to-ligand charge transfer and dithiolene → pyranopterin intraligand charge transfer, respectively, and this underscores a dramatic change in electronic structure between 1 and 1-H. The changes in electronic structure that occur upon protonation of 1 are also reflected in a large >300 mV increase in the Mo(V/IV) redox potential for 1-H, resulting from the greater thione-thiolate resonance contribution and decreased charge donation that stabilize the Mo(IV) state in 1-H with respect to one-electron oxidation. EPR spin Hamiltonian parameters for one-electron-oxidized 1-Mo(5+) and uncyclized [Tp*MoV(O)(S2BDMPP)] [3-Mo(5+)] [BDMPP = 6-(3-butynyl-2,2-dimethyl)-2-pivaloyl pterin] are very similar to each other and to those of [Tp*MoVO(bdt)] (bdt = 1,2-ene-dithiolate). This indicates that the dithiolate form of the ligand dominates at the Mo(V) level, consistent with the demand for greater S → Mo charge donation and a corresponding increase in Mo-S covalency as the oxidation state of the metal is increased. Protonation of 1 represents a simple reaction that models how the transfer of a proton from neighboring acidic amino acid residues to the Mo cofactor at a nitrogen atom within the pyranopterin dithiolene (PDT) ligand in pyranopterin molybdenum enzymes can impact the electronic structure of the Mo-PDT unit. This work also illustrates how pyran ring-chain tautomerization drives changes in resonance contributions to the dithiolene chelate and may adjust the reduction potential of the Mo ion.
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The Role of the Pyranopterin Dithiolene Component of Moco in Molybdoenzyme Catalysis
Chapter
  • Jun 2019
An overview of the pyranopterin dithiolene (MPT) component of the molybdenum cofactor (Moco) and how MPT may contribute to enzymatic catalysis is presented. The chapter begins with a brief review of MPT and Moco biosynthesis and continues to explore the nature of what is arguably the most electronically complex ligand in biology. To explore this complexity, we have dissected MPT into its relevant molecular components. These include the redox-active ene-1,2-dithiolate (dithiolene) and pterin moieties, which are bridged by a pyran that may be found in ring-opened or ring-closed configurations. The various redox possibilities of MPT bound to Mo are presented, along with the electronic structure of the redox components. MPTs are found to display a remarkable conformational variance in pyranopterin Mo enzymes. This is discussed in terms of a relationship to enzyme function and the potential for the observed non-planer distortions to reflect different MPT oxidation and tautomeric states. The chapter ends with a series of case studies featuring model compounds that highlight how biomimetic small molecule studies have contributed to furthering our understanding of the roles this remarkable ligand plays in the catalytic cycles of the enzymes.
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Remote Charge Effects on the Oxygen-Atom-Transfer Reactivity and Their Relationship to Molybdenum Enzymes
Article
  • Jan 2019
We report the syntheses, crystal structures, and characterization of the novel cis-dioxomolybdenum(VI) complexes [Tpm∗MoVIO2Cl](MoO2Cl3) (1) and [Tpm∗MoVIO2Cl](ClO4) (2), which are supported by the charge-neutral tris(3,5-dimethyl-1-pyrazolyl)methane (Tpm∗) ligand. A comparison between isostructural [Tpm∗MoVIO2Cl]⁺ and Tp∗MoVIO2Cl [Tp∗ = hydrotris(3,5-dimethyl-1-pyrazolyl)borate] reveals the effects of one unit of overall charge difference on their spectroscopic and electrochemical properties, geometric and electronic structures, and O-atom-transfer (OAT) reactivities, providing new insight into pyranopterin molybdoenzyme OAT reactivity. Computational studies of these molecules indicate that the delocalized positive charge lowers the lowest unoccupied molecular orbital (LUMO) energy of cationic [Tpm∗MoO2Cl]⁺ relative to Tp∗MoO2Cl. Despite their virtually identical geometric structures revealed by crystal structures, the MoVI/MoV redox potential of 2 is increased by 350 mV relative to that of Tp∗MoVIO2Cl. This LUMO stabilization also contributes to an increased effective electrophilicity of [Tpm∗MoO2Cl]⁺ relative to that of Tp∗MoO2Cl, resulting in a more favorable resonant interaction between the molydenum complex LUMO and the highest occupied molecular orbital (HOMO) of the PPh3 substrate. This leads to a greater thermodynamic driving force, an earlier transition state, and a lowered activation barrier for the orbitally controlled first step of the OAT reaction in the Tpm∗ system relative to the Tp∗ system. An Eyring plot analysis shows that this initial step yields an O-MoIV - OPPh3 intermediate via an associative transition state, and the reaction is ∼500-fold faster for 2 than for Tp∗MoO2Cl. The second step of the OAT reaction entails solvolysis of the O-MoIV - OPPh3 intermediate to afford the solvent-substituted MoIV product and is 750-fold faster for the Tpm∗ system at -15 °C compared to the Tp∗ system. The observed rate enhancement for the second step is ascribed to a switch of the reaction mechanism from a dissociative pathway for the Tp∗ system to an alternative associative pathway for the Tpm∗ system. This is due to a more Lewis acidic MoIV center in the Tpm∗ system.
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Ground State Nuclear Magnetic Resonance Chemical Shifts Predict Charge-Separated Excited State Lifetimes
Article
  • Nov 2018
  • INORG CHEM
Dichalcogenolene platinum(II) diimine complexes, (LE,E′)Pt(bpy), are characterized by charge-separated dichalcogenolene donor (LE,E′) → diimine acceptor (bpy) ligand-to-ligand charge transfer (LL′CT) excited states that lead to their interesting photophysics and potential use in solar energy conversion applications. Despite the intense interest in these complexes, the chalcogen dependence on the lifetime of the triplet LL′CT excited state remains unexplained. Three new (LE,E′)Pt(bpy) complexes with mixed chalcogen donors exhibit decay rates that are dominated by a spin−orbit mediated nonradiative pathway, the magnitude of which is proportional to the anisotropic covalency provided by the mixed-chalcogen donor ligand environment. This anisotropic covalency is dramatically revealed in the 13C NMR chemical shifts of the donor carbons that bear the chalcogens and is further probed by S K-edge XAS. Remarkably, the NMR chemical shift differences also correlate with the spin−orbit matrix element that connects the triplet excited state with the ground state. Consequently, triplet LL′CT excited state lifetimes are proportional to both functions, demonstrating that specific ground state NMR chemical shifts can be used to evaluate spin−orbit coupling contributions to excited state lifetimes.
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Implications of Pyran Cyclization and Pterin Conformation on Oxidized Forms of the Molybdenum Cofactor
Article
  • Sep 2018
The large family of mononuclear molybdenum and tungsten enzymes all possess the special ligand molybdopterin (MPT), which consists of a metal-binding dithiolene chelate covalently bound to a pyranopterin group. MPT pyran cyclization/scission processes have been proposed to modulate the reactivity of the metal center during catalysis. We have designed several small molecule models for the Mo-MPT cofactor that allow detailed investigation into how pyran cyclization modulates electronic communication between the dithiolene and pterin moieties, and how this cyclization alters the electronic environment of the molybdenum catalytic site. Using a combination of cyclic voltammetry (CV), vibrational spectroscopy (FT-IR and rR), electronic absorption spectroscopy, and x-ray absorption spectroscopy (XAS), distinct changes in the MoO stretching frequency, Mo(V/IV) reduction potential, and electronic structure across the pterin-dithiolene ligand are observed as a function of pyran ring closure. The results are significant for they reveal that a dihydropyranopterin is electronically coupled into the Mo-dithiolene group due to a coplanar conformation of the pterin and dithiolene units, providing a mechanism for the electron-deficient pterin to modulate the Mo environment. A spectroscopic signature identified for the pyranodihydropterin-dithiolene ligand on Mo is a strong dithiolenepterin charge transfer transition. In the absence of a pyran group bridge between pterin and dithiolene, the pterin rotates out of plane, largely decoupling the system. The results support a hypothesis that pyran cyclization/scission processes in MPT may function as a molecular switch to electronically couple and decouple the pterin and dithiolene to adjust the redox properties in certain pyranopterin molybdenum enzymes.
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Xanthine oxidase–product complexes probe the importance of substrate/product orientation along the reaction coordinate
Article
  • Jul 2017
A combination of reaction coordinate computations, resonance Raman spectroscopy, spectroscopic computations, and hydrogen bonding investigations have been used to understand the importance of substrate orientation along the xanthine oxidase reaction coordinate. Specifically, 4-thiolumazine and 2,4-dithiolumazine have been used as reducing substrates for xanthine oxidase to form stable enzyme-product charge transfer complexes suitable for spectroscopic study. Laser excitation into the near-infrared molybdenum-to-product charge transfer band produces rR enhancement patterns in the high frequency in-plane stretching region that directly probe the nature of this MLCT transition and provide insight into the effects of electron redistribution along the reaction coordinate between the transition state and the stable enzyme-product intermediate, including the role of the covalent Mo–O–C linkage in facilitating this process. The results clearly show that specific Mo-substrate orientations allow for enhanced electronic coupling and facilitate strong hydrogen bonding interactions with amino acid residues in the substrate binding pocket.
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Consensus structures of the Mo(V) sites of sulfite-oxidizing enzymes derived from variable frequency pulsed EPR spectroscopy, isotopic labelling and DFT calculations
Article
  • Jun 2017
Sulfite-oxidizing enzymes from eukaryotes and prokaryotes have five-coordinate distorted square-pyramidal coordination about the molybdenum atom. The paramagnetic Mo(V) state is easily generated, and over the years four distinct CW EPR spectra have been identified, depending upon enzyme source and the reaction conditions, namely high and low pH (hpH and lpH), phosphate inhibited (Pi) and sulfite (or blocked). Extensive studies of these paramagnetic forms of sulfite-oxidizing enzymes using variable frequency pulsed electron spin echo (ESE) spectroscopy, isotopic labeling and density functional theory (DFT) calculations have led to the consensus structures that are described here. Errors in some of the previously proposed structures are corrected.
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Vibrational Probes of Molybdenum Cofactor–Protein Interactions in Xanthine Dehydrogenase
Article
  • Jun 2017
The pyranopterin dithiolene (PDT) ligand is an integral component of the molybdenum cofactor (Moco) found in all molybdoenzymes with the sole exception of nitrogenase. However, the roles of the PDT in catalysis are still unknown. The PDT is believed to be bound to the proteins by an extensive hydrogen-bonding network, and it has been suggested that these interactions may function to fine-tune Moco for electron- and atom-transfer reactivity in catalysis. Here, we use resonance Raman (rR) spectroscopy to probe Moco-protein interactions using heavy-atom congeners of lumazine, molecules that bind tightly to both wild-type xanthine dehydrogenase (wt-XDH) and its Q102G and Q197A variants following enzymatic hydroxylation to the corresponding violapterin product molecules. The resulting enzyme-product complexes possess intense near-IR absorption, allowing high-quality rR spectra to be collected on wt-XDH and the Q102G and Q197A variants. Small negative frequency shifts relative to wt-XDH are observed for the low-frequency Moco vibrations. These results are interpreted in the context of weak hydrogen-bonding and/or electrostatic interactions between Q102 and the -NH2 terminus of the PDT, and between Q197 and the terminal oxo of the Mo≡O group. The Q102A, Q102G, Q197A, and Q197E variants do not appreciably affect the kinetic parameters kred and kred/KD, indicating that a primary role for these glutamine residues is to stabilize and coordinate Moco in the active site of XO family enzymes but to not directly affect the catalytic throughput. Raman frequency shifts between wt-XDH and its Q102G variant suggest that the changes in the electron density at the Mo ion that accompany Mo oxidation during electron-transfer regeneration of the catalytically competent active site are manifest in distortions at the distant PDT amino terminus. This implies a primary role for the PDT as a conduit for facilitating enzymatic electron-transfer reactivity in xanthine oxidase family enzymes.
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Spectroscopic and electronic structure studies of Mo model compounds and enzymes
Chapter
  • Jan 2017
Electronic structure contribution to reactivity in pyranopterin Mo enzymes is presented and discussed. This is accomplished by relating spectroscopic studies of relevant model compounds with those of the enzymes. This has contributed to a greater understanding of how the pyranopterin dithiolene functions in catalysis, and has led to an enhanced understanding of how electronic structure controls reactivity in sulfite oxidase, xanthine oxidase, CO dehydrogenase, DMSO reductase and MOSC family proteins.
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