SpectroscopicAnalysisandChemicalModificationoftheH134CMutantoftheThermusthermophius Rieske Protein

VictorRodríguezandLauraM.Hunsicker-Wang

TrinityUniversity,SanAntonio,TX

AbstractTheRieske proteinisfoundinthebc1complex(complexIII)andplaysanimportantroleintransportingelectronsandprotonsthroughtheelectrontransportchain.TheRieskeproteincontainsa[2Fe-2S]clusterligatedbya2-cystinesand2- histidines.ThereductionpotentialofthisclusterispH-dependentandvariesacrossspecies.AnH134CmutantoftheThermus thermophilus Rieske substitutesoneoftheligatinghistidines foracysteine,changingtheligationstructureoftheclusterfroma2Cys-2Histoa3Cys-1Hisenvironment.Tostudytheeffectsofthismutation,theproteinissubjectedtomodificationwithdiethylpyrocarbonate(DEPC)andtopHchanges.Thebehaviorofthisproteiniscomparedtothewildtypeproteinasobservedthroughcirculardichroism andUVvisiblespectroscopy.Becauseofthesimilarityintheiron-sulfurclusterligands,H134Cwillalsobecomparedtoanothermitochondrialprotein,mitoNEET,whichcontainsa3Cys-1Hisenvironment.

Rieske Proteins

• TheRieske proteinisfoundincomplexIII(cytochromebc1 complex)aspartoftheelectrontransportchain.

• Thiscomplexoxidizesubiquinol toubiquinoneandtransportstheelectronsthroughthecomplexintotheFe-SclusterinReiske.

http://www.scienceprofonline.org/metabolism/electron-transport-chain-cellular-respiration.htmlhttp://en.wikipedia.org/wiki/Coenzyme_Q_%E2%80%93_cytochrome_c_reductase

H134Cmutant&MitoNEET

• TheRieske proteinFe-Sclusterhasa2-His2-Cysligationenvironment.

• H134Cmutant- theligationstructureisalteredtohaveaonehistidine andthreecysteines.

• ThisligationenvironmentissimilartothatofMitoNEET,aknowndiabetesdrugactivator.

• ResultsfromtheseexperimentswillbeusedtocomparetothatofMitoneet tobetterunderstandthedifferenceswiththisligationenvironment.

Rieske

MitoNEET

PDBID3REE

pH-DependentUV-Visiblespectra

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

300 400 500 600 700 800

Absorbance(A)

Wavelength(nm)

4.39

6.63

7.54

8.65

9.61

10.41

10.97

11.99

• PreviousstudiesshowthatH134ChasalargerangeofpHstability

• H134ChasapKa of9.86.DatacollectedbyAbhishek Chhetri

H134C

H134CCrystals• CrystalsforH134CRieskemutantwereisolatedunderthefollowingconditions:30%PEG4000,0.2MMgCl2, 0.1MTris-HCl pH8.5or20%PEG2000MME,0.01MNiCl2·6H2O,0.1MTris pH8.5.

• Bestdatacollectedto1.66Åresolution!

• SpacegroupP2221Preliminaryelectrondensitymap(2Fo-Fc)showing3Cys,1His[2Fe2S]cluster

His154Cys 134

Cys 132 Cys 151

DEPCmodification

• Deprotonatedhistidines reactwithDEPC.• Intruncatedwildtype,notonlywasmodificationobserved,but

alsoreduction.

DEPC

DEPC=diethylpyrocarbonateKonkleetal.2010Biochemistry49,7272-7281

ReactionwithDEPC- UV-Vis

pH6

pH7

pH8

pH6

pH7

pH8

Konkleetal.2010Biochemistry49,7272-7281

truncTtRp H120Q/H162Q

• truncTtRpandH120Q/H162QaremodifiedbyDEPC• HigherpHcausesfastermodificationandmoremodificationintruncTtRp

EffectofpHonDEPCreaction- CD

pH=6 pH=7

pH=8 pH=9

H120Q/H162Q

Konkleetal.2010Biochemistry49,7272-7281• MorespectralchangeswithhigherpH• Proteinsbecomereducedaftermodification

H134CReactionwithDEPC- UV

-0.05

0.05

0.15

0.25

0.35

0.45

0.55

200 300 400 500 600 700 800

Difference

Wavelength(nm)

pH6.0

-0.05

0.05

0.15

0.25

0.35

0.45

0.55

200 300 400 500 600 700 800

Difference

Wavelength(nm)

pH7.6

• H134CwasreactedwithDEPC• differenceUV-Visspectraover40minutesatdifferentpHvalues.

-0.05

0.05

0.15

0.25

0.35

0.45

0.55

200 300 400 500 600 700 800

Difference

Wavelelngth(nm)

pH8.2

H134CismodifiedbyDEPC,buttheLMCTbandsareminimallyaffected

-0.1

0

0.1

0.2

0.3

0.4

0.5

200 300 400 500 600 700 800

Difference

Wavelength(nm)

pH9.0

pH-dependenceofmodification

• ReactionrateincreaseswithpH,buttheextentofmodificationdecreasesabovepH7.6

• TheprofilediffersfromtruncTtRp (seepreviousslide)

ΔAb

s.240nm

Time(min)

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

0.4

0.45

0.5

0 5 10 15 20 25

pH6.0

pH7.6

pH8.2

pH9.0

• H134CreactedwithDEPCover90minutes(upperfigure).

• AchangeinsignalisobservedovertimeandfollowasimilarpattertowhatwasobservedforreductioninwildtypeRieske.

• Tofullyunderstandthechangesofthesignals,themutantproteinwasreducedandoxidize(lowerfigure)

CDspectrachangebuttheproteindoesnotappeartobecomereduced

H134CReactionwithDEPC- CD

24hourreactionwithDEPC

• TheCDsignalat450nmincreasesuntil3hours,andthenreturnstotheoriginallevel.

• OneinterpretationisthattheDEPCmodificationreversesovertime,indicatingalabileadduct.

-13

-11

-9

-7

-5

-3

-1

1

3

5

230 280 330 380 430 480 530 580

mde

g

Wavelength(nm)

H134CatpH8.2reacetdwithDEPCfor24hours

unreacted

0hours

3hours

10hours

23hours

27hours

0

2

4

0 3 6 9 12 15 18 21 24 27Absorbtion

Time(hours)

H134CreactedwithDEPCat450nm

Conclusions

• H134CpKa ofproteinis9.86.• Crystalstructureconfirmsthe3Cys1HisStructure

• ProteinismodifiedbyDEPCandnotreducedwithina90minutereaction.

• DEPCmodificationmayreversesoverlongertimes,indicatingalabileadductinthis3Cys,1Hisligationenvironment.

Acknowledgements

• McNairScholarProgram• NationalScienceFoundation• P.JohnHartLabattheUniversityofTexasHealthScienceCenterSanAntonioforcrystals