A.H.C.M.Schapendonk
Centre for Agrobiological Research (CABO), Wageningen
Department of Plant Physiological Research, Agricultural
University, Wageningen
Electrical events
associated with primaryphotosynthetic reactions
inchloroplast membranes
Centre for Agricultural
Wageningen - 1980
Publishing and Documentation
ThisworkwassupportedbytheFoundationforBiophysicswith financialaidfromthe
NetherlandsOrganizationfortheAdvancementofPureResearch (Z.W.O.).
ISBN9022007561
Theauthorgraduatedon10December 1980asDoctorindeLandbouwwetenschappenatthe
AgriculturalUniversity,Wageningen,theNetherlands,onathesiswiththesametitle
andcontents.
ThispublicationwillalsobeissuedasPublication 175oftheCentreforAgrobiological
Research (CABO),Wageningen.
(c)CentreforAgriculturalPublishingandDocumentation,Wageningen,1980.
Nopartofthisbookmaybereproducedorpublished inanyform,byprint,photoprint,
microfilmoranyothermeanswithoutwrittenpermissionfromthepublishers.
Abstract
Schapendonk,A.H.C.M., 1980.Electrical events associated with primary photosynthetic
reactions inchloroplast membranes.Agric.Res.Rep. (Versl.landbouwk.Onderz.)905,
ISBN 902200756 1, (viii)+95p.,47 figs,3tables,224refs,Eng.and Dutch summaries.
Also:Doctoral thesis,Wageningen.
A studywasmadeof electricphenomena inisolated photosynthetically-active chloroplast
membranes upon energization.Energization of chloroplast thylakoid membranes givesrise to
a charge separation across themembrane and anassociated transmembrane electric field.
Thekinetics of this fieldweremonitored as potential changes by amicro-electrode inserted ina single chloroplast and theywere compared with theabsorbance change of an
intrinsic pigment (F515)proposed tobeareflection of a transmembrane electric field.
Theobserved discrepancy between the flash-induced P515response and theelectric response
was caused by conformational changes inthemembranal core thatbrought about absorbance
changes ofF515 inaddition to itsreponsiveness to the transmembrane potential.Most of
theconclusions drawnhitherto fromP515kinetics,both after flash excitation and during
prolonged illumination, have tobe regarded with caution because they commonly arebased
onassumptions that are invalid under normalphysiological conditions.Under certain conditions, ifappropriate corrections aremade, theP515kinetics are indeed a reflection
of the transmembrane potential. Inthat case theresults appear tobe ingood agreement
with results obtained with theelectrophysiological approach.
The component of theP515absorbance change, that isascribed to conformational
changes inthemembranal core,may provide auseful tool for studies of fast structural
changes inrelation toenergy coupling,without affecting themembrane integrity.
Freedescriptors: electric potential;P515; 515nm absorbance change; conformational
changes; structural changes;light scattering; thylakoid membrane; photosynthesis; electron
transport; surface potential;ATP synthesis.
Contents
Abbreviations
1. Introduction
1.1 Higherplantphotosynthesis
1.2 Thylakoidstructure
1.3 Generationofanelectricalpotentialacrossenergizedthylakoidmembranes
1.4 Outlineofstudy
1
1
3
4
5
2. Material and methode
2.1 Isolationandcharacterizationofchloroplasts
2.2 Flash-inducedabsorbancedifferencemeasurementsofP515
2.3 AbsorbancechangesofP515and90°-scatteringchangesduringprolonged
illumination
2.4 Salt-inducedabsorbancechangesofP515
2.5 Potentialandresistancemeasurementsbymeansofmicro-capillaryglass
electrodes
2.6 Particleelectrophoresisofchloroplasts
2.7 Chemicalanalyses
7
7
7
9
9
11
13
14
3. Evidence for a light-induced blue bandshift of part of the P515pigment pool
in intact chloroplast membranes
3.1 Introduction
3.2 Material
3.3 Flash-inducedabsorbancedifferencespectra
3.4 Simulationofbandshifts
3.5 Discussion
4. Salt-induced absorbance changes of PS15 in broken chloroplasts
4.1 Introduction
4.2 Materialandmethod
4.3 Salt-inducedabsorbancechanges
4.4 DataforavoltagecalibrationoftheP515absorbancechange
4.5 Discussion
5. Analyses of the flash-induced
5.1 Introduction
5.2 Materialandmethod
PS1S absorbance changes in isolated chloroplasts
15
15
16
17
18
18
21
21
21
22
24
26
28
28
28
5.3 EvidencefortheinductionofafastandaslowP515responseupon saturating
lightflashesinintact chloroplasts
5.4 AnalysesoftheP515responseinbrokenchloroplasts
5.5- Kineticsofthe light-generated transmembranepotential
5.6 Effectofvalinomycin
5.7 ActivationofReactionIIbyPhotosystem1
5.8 Discussion
6. Réaction kinetics of transmembrane •potential changes in relation to ion
permeability
6.1 Introduction
6.2 Effectofelectrolyte concentrationonthedarkkineticsofReactionI
6.3 Decay of Reaction I after pre-illunination
6.4 Measurementoftheelectrical resistanceofthylakoidmembranes
6.5 Discussion
29
32
35
36
38
40
45
45
46
7. Light-dependent conformational changes in the thylakoid membrane
7.1 Introduction
7.2 Material
7.3 Someelectrokineticdataofdifferent chloroplast preparations
7.4 Theresponsivenessofelectrophoreticmobility andReactionIIuponchemical
treatment
7.5 ReactionIIasapossible intrinsicmarkerfortheconformational stateof
thecoupling factor
7.6 Discussion
8. The PS1S response and the photo-electrical
8.1
8.2
8.3
8.4
8.5
8.6
49
50
52
55
55
56
56
57
59
62
response during prolonged illumination
Introduction
Material
P515absorbancechangesandchangesinlightscattering
Protontranslocation
EffectofMg onlight-induced90°-scattering changes
Discussion
65
65
65
66
67
68
72
Summary
75
Samenvatting
78
References
81
Abbreviations
ACMA
9-amino-6-chloro-2-methoxyacrldine
ADP
Adenosine-5'-diphosphate
ATP
Adenosine-S'-triphosphate
CCCP
Carbonylcyanide-m-chloro-phenylhydrazone
DCCD
Dicyclohexylcarbodiimide
DCMU
3-(3,4,dichlorophenyl)-1,1-dimethylurea
DCPIP
Dichlorophenolindophenol
Diquat
N,N'-ethylene-2,2'-dipyridiliumdibromide
EDTA
Ethylenediaminetetraacetate
HEPES
N-2-hydroxyethylpiperazine-N'-2-ethanesulphonicacid
MES
2(N-morpholino)ethanesulphonicacid
NADP
Nicotinamide-adeninedinucleotidephosphate
PMS
N-methylphenazoniummethosulphate
S1,
S-chloro,3-t-butyl,2'-chloro,4'-nitro-salicylanilide
SDS
Sodiumdodecylsulphate
Tricine
N-tris(hydroxymethyl)methyl-glycine
Tris
tris(hydroxymethyl)aminomethane
1 Introduction
1.1 HIGHERPLANTPHOTOSYNTHESIS
Theoverallprocessofphotosynthesis,definedasthelight-drivenCO,reductionwith
concomitant0,evolution,hasbeenknownformorethantwocenturiesbutitwasnotbefore1943thatthehypothesisofRuben (1943)arosethatphotosyntheticCO,reductionrequiredtheelectrochemicalgenerationoftwoenergy-richproducts:adenosinetriphosphate
(ATP)andreducedpyridinenucleotides (NADPH).Thishypothesisgotstrongsupportinthe
earlyfifties (Arnon,1951;Arnonetal., 1954).
Theconceptoftwolightreactions,asproposedbyHill&Bendall (1960),andDuysens
etal. (1961),hasbeenanimportantlandmarkinthesearchforthemechanismbywhich
photosyntheticenergyconversionleadstoNADP reductionandATPproduction.Duysens&
Amesz (1962)proposedthatthetwolightreactionswereoperatinginseries:along-wavelengthPhotosystem1(PS1)andashort-wavelengthPhotosystem2(PS2).Thisconceptcan
bepresentedbytheso-calledZscheme (Fig.1).Lightenergyabsorbedbytheantenna
pigmentmoleculesistransferredtothereactioncenters.InthereactioncentersofPS1
andPS2,theexcitedchlorophyllmolecules (P700andP680,respectively)becomeoxidized
withaconcomitantreductionoftheprimaryelectronacceptors'(P430andC550).Thetwo
lightreactionsareconnectedbyachainofcompoundsundergoingreversibleoxido-reduc-
20ns
«P_
Fig. 1.Simplified visualization ofphotosynthetic electron flow through thevectorially
oriented electron transport chainandcoupled proton translocation across thethylakoid
membrane.Theproton-binding andproton-releasing sitesattheoutsideandtheinsideof
themembrane, respectively areindicated. Reaction sitesofcytb-559andcytb-563are
not indicated; P680andP700:trapping centersofPS2andPS1,respectively; C550andP430:
primary acceptorsofPS2andPS1,respectively;PQ:plastoquinone;cytf:C554 (cytochrome
f); PC:plastocyanin; FD: ferredoxin; FP:flavoprotein;Ma:mangane-containing water splitting protein; S1-S4:different functional statesofthewater splitting enzyme.
tionreactions.ThestrongoxidantthatexistsafterchargetransferinPS2iscapableof
oxidizingH-0.Thelight-dependentuphillelectronandhydrogentransportfromH,0to
NADP servesthepurposetocreatereducingpowerforC0 2reduction.Inaddition,followingthesuggestionofMitchell (1961,1966,1968,1977),avectorialorientationofelectronandhydrogencarriersinthemembranewillleadtotheestablishmentofaproton
motiveforce (pmf).Thepmf,whichconsistsofanelectricalandachemicalcomponent,is
suggestedtobethedrivingforceforphotophosphorylation.Manyexperimentaldatahave
supportedthevalidityofthischemiosmotictheoryforenergycouplinginchloroplastmembranes (Witt,1971;Trebst,1974).Neumann&Jagendorf (1964)recognizedthatprotonuptakeinchloroplastsistightlycoupledwithelectrontransport.Afewyearslater,itwas
suggestedthatthylakoidmembraneswereabletouseàpHgradienttodriveATPsynthesis
(Jagendorf&Uribe,1966;Jagendorf,1967,1975).TheabilityofpHgradientstomimicthe
effectsoflightindeedisrathergoodandextendsoveragreatareaoflight-inducedeffectssuchasPi-ATPexchangereactions (Bachofen&Specht-Jurgensen,1967)andconformationalchangesinCF.,(Ryrie&Jagendorf,1972).Recentlyreversedelectronflowcaused
byapHgradienthasbeenobserved (Schreiber&Avron,1977).
IfthetransmembraneelectrochemicalprotongradientistheenergysourceforATP
synthesisitwouldbeexpectedthatphotophosphorylationstartsafteralagperiod,to
allowtheestablishmentofapmf,sufficientlyhightosupplytheenergyrequiredforATP
synthesis.Suchalagperiod,however,hasbeenobservedtobeverysmallorabsentand
ATPformationwasfoundtoproceedduringthefirstfewmillisecondsofilluminationor
eveninsinglelightflashes,whenthereiseithernooranegligibleprotongradient
formed (Ortetal.,1976;Ort&Dilley,1976;Vinkleretal.,1978).Avron (1978)suggestedthat,inthisearlytimerange,thetransmembraneelectricfieldwasthemajordriving
force.Althoughthelattersuggestionmightexplaintheobservedflash-inducedATPsynthesis,noconvincingevidenceforsuchamechanismisavailable.Furthermore,itseems
unlikelythatthetransmembraneelectricalpotentialishighenoughtospanthephosphate
potential (Vredenbergetal.,1973).WithinthisframeworkIwouldliketocastaglance
attwoalternativemodelsandavarietyofintermediateversions,thatmayprovideinsight
intothemechanismofcoupledelectrontransport (Jagendorf,1975).
Thechemicalhypothesis (Slater,1953;Chance&Williams,1956)proposestheformationofdirectchemicalbondsbetweenelectroncarriersandenzymesorsubstratesofthe
phosphorylationreaction.Oxidationofthecomplexthenraisestheligandbondtoahigh
energylevelandthisenergydrivestheanhydro-bondformationofATP.Thebasicideabehindthismechanismledtotheproposalthatenergycouplingmayinvolveconformational
changes (Boyer,1965).Animportantfeatureofthishypothesis,recentlyrevisedbyBoyer
(1974)andSlater (1974),isthereleaseoftightlyboundATPviaanenergy-linkedconformationalchangeatthecatalyticsiteofthecouplingATPase (CF*)whereastheATPformationitselfrequiresnoenergyinput.Accordingly,severalobservationspointtothe
occurrenceofconformationalchangesinthe (CF..)(Ryrie&Jagendorf,1971;Nelsonetal.,
1972;McCartyetal.,1972;Kraayenhof&Slater,1975)butnosatisfactoryexplanationas
tohowtheenergyistransmittedhasbeengivenasyet.
ThehypothesisproposedbyWilliams (1962)emphasizestheimportanceofelectrontransportmediatedaccumulationofprotonsinthehydrophylicpocketsofthemembrane
itself.ThismaycausealoweringofH 2 0activityduetohydronium formationandfacilitatethedehydrationreactionofpolyphosphate formation.Recently,DelValle-Tasconet al.
(1978)gavesupport forsuchamechanism inchromatophores of Rhodoepirillum
rubrum. In
addition,localpHchangeswithinthemembranehavebeenproposedtoaccount foran importantroleofconformational changesofmembraneproteins (Williams,1972).Seealso
Izawaetal. (1975).
Althoughallthesehypotheses canexplain theobservedformationofATP intheabsenceofeitheratransmembraneproton gradient oratransmembranepotential (Ortet al.,
1976),noconvincing evidence fortheproposedmechanism isavailableuntilnow.Thechemiosmotichypothesis sofarhasprovided themost coherent framework tounderstand andpredictmany facetsofthecouplingmechanism.However,someofthesepredictionshavenot
beenverifiedbyexperimental findings,especially thoseconcerning thenumber ofATPmoleculesformedperpairofelectronsmoving fromH-0tothereducingsiteofPS1.A full
understandingofthismechanismmightrequest theaccomodationofmembrane-locatedevents
(Harris&Crofts, 1978).
1.2 THYLAKOIDSTRUCTURE
Theabilityofthedirect conversionoflightenergy intochemicalenergy istheunique
propertyofspecializedorganelles inphotosynthesizing organisms.Tomeet the requirements
setbythiscomplex task,thestructuralframeworkoftheseorganelles isexpected tobe
highlydifferentiatedand tobeprovidedwithappropriate toolswhichenableastrict
functioning ofthesystem. Itisnowwellestablished thatthe innerchloroplast membranes,havingafolded structureofcloselypackedmembrane stacks (granumlamellae)and
interconnectingmembranes,called stromalamellae,separateaninnerthylakoidphase
fromthestromaphase (Menke, 1961).
Thethylakoidmembrane containsallthechlorophylls and accessorypigments,thecomponentsoftheelectrontransportchainsand theenzymes forphosphorylationofADP.The
matrixofthisstructureconsistsofglycolipids,sulpholipids andfewphospholipids.It
containsavarietyofdifferentproteins (Menke&Ruppel, 1971),eitherasan intrinsic
part,stabilizedbyhydrophobic interactions and ionpairformation (Gitler,1972), or
loselyboundatthesurfacebysaltbridge interaction (Avron, 1963).Chlorophyll aandb,
carotenoids (Thornber, 1975)andcytochromes (Bendallétal., 1971;Lemberg &Barrett,
1973)formcomplexeswithproteins inacooperative andprotective interaction.Freeze
fracturing,deep etchingandserologicaltechniquesrevealed themorphological andfunctionalasymmetry ofthethylakoidmembrane (Trebst, 1974;Anderson, 1975).
The compartmentalized threedimensional structureofthechloroplast andthespecializedcomposition ofthethylakoidmembranes implicitelypoint toahighaccessibilityof
theprimaryphotosyntheticprocesses toregulatory activities suchas energy-dependent
redistribution ofionsbetweenthethylakoid innerspaceand thestromaor structural
changesofthemembrane.A considerationofthisaspect inphotosynthesis research emerged
inrecentyears,andmanyobservations confirmed theoccurrenceofsuchcontrolmechanisms
(Horton&Cramer, 1974;Butler &Kitajima,1975;Kraayenhof&Slater, 1975).Foramore
detailed treatiseonthissubject Iwould liketorefertorecentreviews (Barber,1976;
Kraayenhof,1977a,1977b).
Anextensiveresearchonthebasicoriginoftheintrinsicachievementsofthe
thylakoidmembraneandthecriticalimportanceofthemutualcoherenceofitscomponents,
willleadtoamorecompleteunderstandingofprimaryphotosyntheticprocesses.
1.3 GENERATIONOFANELECTRICALPOTENTIALACROSSENERGIZEDTHYLAKOIDMEMBRANES
Energycapturedbythereactioncentersisusedforelectrontransferfromadonortoan
acceptormolecule.Photo-oxidationoftheelectrondonorsOila TdimerP700andChia T T
P680(Fig.1)andvectorialelectronejectiontowardstheexternallyorientatedelectron
acceptors,P430andC550,respectively,willgiverisetoatransmembraneelectricfield
(Junge&Witt,1968).Whenbothreactioncentersareequallyactivatedinasingle-turnover
lightflash,eachofthemgenerateshalfoftheelectricfield (Schliephakeetal.,1968;
Renger&Wolff,1975;Schapendonk&Vredenberg,1979).Allfurtherreactionsinphotosynthesisaresubsequenttothisprocessandthereforeconsiderableattentionhasbeenpaid
tounderstanditsoriginandfunction.Itmayprovidefurtherknowledgeaboutthecoupling
betweenelectrontransportandphotophosphorylation.Adetailedreviewonthismatterhas
beengivenrecentlybyWitt (1979).
Themagnitudeoftheelectricalpotentialinasingle-turnoverlightflashmeasured
withdifferenttechniques,wasreportedtobe10-20mV (Vredenberg&Tonk,1975),15-35
mV (Schapendonk&Vredenberg,1977),50mV (Schliephakeetal.,1968)and135mV(Zickler
etal.,1976),althoughrecentlyoneoftheauthors (Witt,1979)suggestedthatthelatter
valuemightbeanoverestimation.Exeptfortheelectrodemeasurements (Vredenberg&Tonk,
1975),allavailableinformationonflash-inducedelectricfieldkineticsisobtainedfrom
estimatesmakinguseoftheabsorbanceresponseofanintrinsicprobe,theP515pigment
complex (Witt,1971),orofanexternalpotentialprobe (Bashfordetal.,1978;Schuurmans
etal., 1978).
Junge&Witt (1968)suggestedthatthefield-dependentelectrochromicabsorbanceshift
ofP515wasalinearindicatoroftheelectricfieldacrossthethylakoidmembraneand
consequentlywouldfollowasingle-exponentialdarkdecaywitharateconstant,determined
bythemembranecapacitanceandthemembraneconductance (Bulychev&Vredenberg,1976a).
Accordingly,asingle-exponentialdecayoftheP51Sabsorbancechangeinisotonically
suspendedchloroplastshasbeenreportedtooccurundernon-phosphorylatingconditions
(Witt,1971;Junge&Witt,1968;Boeck&Witt,1972).Thedecayrate,however,seemstobe
dependentonthemembraneintegrityratherthanonthemembraneconductance.Thisisdemonstratedbythescatterinthereportedhalf-lifetimesoftheP515changes,usingdifferentmembraneisolationprocedures (Schmidetal.,1976;Schapendonketal.,1979).
Theoccurrenceofbiphasicdecaykineticsunderphosphorylatingconditions(Rumberg
&Siggel,1968;Jungeetal.,1970;Girault&Galmiche,1978)hasbeeninterpretedtobe
duetoanincreasedprotonconductancethroughtheATPsynthesizingenzymecomplexabove
acriticalpotential (Rumberg&Siggel,1968;Jungeetal.,1970).Ontheotherhand
Nelson (1972)andNeumannetal. (1970)observedthatthedecaykineticsofP515wereindependentontheoccurrenceofATPsynthesis.Moreover,Girault&Galmiche(1976)demon-
stratedthattheaccelerationofthePS15decaycouldeasilybeobtainedbyadditionof
ATP. Theseandotherobservationshavegivenrisetofundamentalcriticismoftheproposed
hypothesis (Rumberg&Siggel,1968)andtendtotheconclusionthattheobservedeffects
somehowreflectaprocesswhichmaybeassociatedwithnucleotidebindingonthemembrane
ormorespecificallyontheCF«.
Supportwasgivenforthesuggestionthatatleastpartofthecarotenoidabsorbance
changesmaybecausedbyconformationalchangesinthemembraneclosetotheregionwhere
thecarotenoidsareattached (Fleischmann&Clayton,1968;Baltscheffskyetal., 1971).
Quantitativeestimatesofthesteady-statetransmembranepotentialacrossthethylakoidduringcontinuousilluminationarealsoambiguousasyet.Dependentonthemethodologicalapproach,valuesof75-10SmV (Barber,1972a),30mV (Strichartz&Chance,1972),
10mV (Schröderetal.,1971)havebeenreportedforthetransmembranepotentialinchloroplasts.Gräber&Witt (1974)concludedfromP515measurementsthatthesteady-state
transmembranepotentialreachedavalueofabout 100mVinintact Chlorella cells.Adirectmeasurementwithmicro-electrodesinintact PeperotrAa chloroplastsshowedasteadystatepotentialinthelightoflessthan10mV.Moreover,thekineticsofthelatterare
quitedifferentfromthoseofthe515nmabsorbancechanges.Consideringthedifferent
methodsused,theapparentlyconflictingresultsinfactarenotnecessarilycontradictorybecausetheintrinsicP515pigmentsmayreacttochangesinlocalelectricfields
(Fleischmann&Clayton,1968)andtochangesinsurfacepotential (Rumberg&Muhle, 1976).
Theselocalizedeventsarenotdetectedwithmicro-electrodes,thataresituatedinthe
bulkphase.Moreover,partoftheP515absorbancechangesiscausedbyslowersecondary
events,reflectedbyscatteringchanges,thatareknowntooccuruponenergization
(Deameretal.,1967;Thome etal., 1975).
1.4 OUTLINEOFSTUDY
Thisstudydealswithelectricaleventsinthylakoidmembranes,associatedwithlightactivitationofthephotosyntheticapparatus.Theseeventswillbediscussedinrelationto
thedynamicfunctionofthemembraneinenergytransductionandenergycoupling.Thekineticpatternofthetransmembranepotentialwillbecharacterizedanddiscussedinview
oftheseeminglyconflictingresultsobtainedwithspectroscopicandelectrophysiological
methods,respectively.Someadditionalinformationwillbepresentedaboutstructural
changesinthemembranecoreinducedbyelectrogenicmechanisms,andtheirpossiblefunctioninphotophosphorylationwillbediscussed.
Experimentshavebeencarriedoutwithintact (ClassI) andbroken(ClassII)chloroplasts.Chapter3dealswiththeinhomogeneousdistributionofthefield-indicating
carotenoid-chlorophyllbpigmentcomplex(P515)inthemembrane,asconcludedfromapparentdifferencesinthespectrum,characteristicfortheelectrochromicresponseofthe
complex.AvoltagecalibrationoftheP515absorbancechangescausedbyasaturating
single-turnoverlightflashisgiveninChapter4.AkineticanalysisoftheP515responseinlightflashesisgiveninChapter5.
Thekineticsofthetransmembranepotentialchanges,reflectedbyafastP515component (resolvedinChapter5 ) ,arestudiedinrelationtoionconductivityandcompared
withthetransmembranepotentialchangesasmeasuredwithmicro-capillary glass electrodes
(Chapter 6). Thecharacteristics ofaslowP515componentarediscussed inrelationto
conformationalchangesinthemembrane (Chapter 7). Finally,anexperimentalapproachis
given,todetectP515absorbancechangesduringprolonged illuminationafter correction
forscattering artifacts (Chapter8 ) .
Partoftheworkpresented inthisbookhasbeenpublished (Schapendonk&Vredenberg,
1977; 1979;Vredenberg &Schapendonk,1978;Tonketal.,1979;Schapendonketal.,1979;
1980).
2 Material and methods
2.1 ISOLATIONANDCHARACTERIZATIONOFCHLOROPLASTS
Forallexperiments,except thosedescribedinChapter6,freshly-grown spinach (Spinaeia
oleraaea, "Amsterdams Reuzenblad"wasculturedonhydroponicsinthelaboratory green
houseaccordingtothemethodofSteiner (1968).Foroptimum growthinwinter theroot
temperaturewasmaintainedat20°Candduringthatperiodtheplantswere illuminated
withhigh-pressuremercury lampsfor8hours.Intactchloroplasts routinelywere isolated
accordingtoamodifiedmethodofNakatani&Barber (1977),except forpreparationsused
inChapter 4.Washed leaves (25g)werecutintosmallpieces(%0.5cm)andgroundfor
2s,usinganUltraTurrax 18/10cavitationdisperserprovidedwith 18Nshaft,in50ml
ofamedium containing 0.33mol/1sorbitol,0.2mmol/1MgCl,,20mmol/1MESand5mgegg
albumen,adjustedtopH6.5withtris.After filtrationthrough8layersofperlonnet
(porediameter40jjm),thefiltratewas centrifugedintwotubesfor40sat815 gina
M5EChillSpincentrifuge.Thepelletswere resuspendedbygentlehand-shakingin10ml
of 0.33mol/1sorbitol,adjustedtopH7.5withtris.However,inthecourseoftheinvestigation,itwasdiscovered thatreplacementoftrisbytricine-NaOHgavebetterCO,"
dependent0,evolution,seealsoCheniae&Martin (1978).Asubsequent centrifugationfor
30-40sat815 gyieldedapreparationof90-100% intactchloroplastsasdeterminedby
ferricyanidereduction (Heber&Santarius, 1970).Theassaymedium consistedof0.33 mol/1
sorbitol,0.5nmol/1K2HP0 4 , 10mmol/1NaHCOj,2mmol/1HEPES,pH7.5,unless otherwi;.u
indicatedinthetext.TheCO^-dependent0,evolutionrateusuallywasintherangebetween
60and80ymol0,permgchlorophyllperhour.
The isolationproceduredidnot takemore than4-5min.Brokenchloroplastswereobtainedbya30sosmoticshockineitherH,0, 10nmol/1MgCl,or10mmol/1 KClandsubsequent additionofdouble strengthassaymedium,containing 0.66nmol/1sorbitol,4mmol/1
HEPES,pH7.5andeither 10mmol/1 MgCl,or10nmol/1KCl,unlessotherwise indicatedin
the text.
2.2 FLASH-INDUCEDABSORBANCE DIFFERENCEMEASUREMENTSOFF51S
Flash-induced absorbance changesweremeasuredinamodifiedAminco-Chance absorption difference spectrophotometer (Fig.2a).Theapparatus operatedinthedcsingle-beammode.A
0.3x0.3cmsample cellwas inserted intoaspecialholder thatcouldbethermostated.
Flashillumination (half-time8us)withaGeneralElectric FT-230flashtubeoraXenon
457flashtubeofwavelengths above665
ranreachedthesampleviaaflexible light guide.
Thephotomultiplier wasshielded fromstray lightbyaWratten 58,BG38andBG18filter
combination.Singleflashesoragroupof2-6flasheswithtime intervalsof100mswere
TRIGGER
SOURCE
- J I—
AMPL.
[FLASH
AVERAGER
CH
RECORDER
/ EJJ
MA
CUV
Fig. 2a.Schematicdiagramoftheequipmenttomeasureflash-inducedabsorbancechangesin
chloroplastsuspensions.Thechopperblade (CH)isatafixedpositionallowingonemeasuringbeamofacertainwavelengthtopassthecuvetteviaanadjustablemirrorassembly
(MA).Transmittedlightisdetectedbyaphotomultiplier (PM1).Aftercompensationwitha
dcvoltageandsubsequentamplification,thesignalcausedbyaflash-inducedchangein
transmission,isfedintoaDatalab.102Asignal-averager.Severalflashresponsesare
averagedandthesignalismonitoredonarecorderoranosciloscope.Apulsegenerating
system,TektronixRate/Rampgenerator26G1triggeringaRampgenerator26G2andtwopulse
generators 26G3,controlsthetimeofflashfiringandprovidestriggerpulsesforthe
averagerandtheosciloscope (Tektronix 7313).
Fig. 2b.Operationmodeofequipmenttomeasureabsorbancechangesand90-scattering
changessimultaneously.Chopperrotationcausesa300Hzmodulatedpassageofthemeasuringbeamofafixedwavelengththroughthecuvette,whichallowsanabsorbance (andscattering)measurementatthiswavelengthagainstadarkreference.Light,scatteredbythe
chloroplastsuspensionisdetectedbyasecondphotomultiplier (PM2).Furtherdetailsare
inthetext.
Fig. 2c.Equipment tomeasure absorbance changes caused by addition of substances toone
of thecuvette compartments. Chopper rotation directsmeasuring light of a selected wavelength alternatively through themeasuring and reference compartment of thecuvette.The
ac signal is fed intoaLock-in amplifier and theresulting dc signal is recorded.
fired.Therepetitionrateusuallywas0.05Hz.Anumberofeither32or64absorbance
responsesweresampledandaveragedusingaDataLab102Asignal-averager.Insomeexperiments38or70flasheswerefiredatarepetitionrateof3-4Hz.Inthiscase6flashes
wereusedforpre-conditioningthesample.
2.3 ABSORBANCECHANGESOFP515AND90°-SCATTERINGCHANGESDURINGPROLONGED ILLUMINATION
Absorbancechangesand90°-scatteringchangesweremeasuredsimultaneouslyinthesameapparatus (Fig.2b)nowoperatinginthechoppedsingle-beammode (monochromaticmeasuring
beamalternatingagainstdark).Scatteringchangesweremonitoredbyasecondphotomultiplierviaaflexiblelightguide,placedagainstthesidewallofthecuvette.ThephotomultiplierswereshieldedfromstraylightbyaWratten58,BG18andBG38filtercombination.Thesamplewasilluminatedbylightfroma250Wtungstenlampplacedinamodifiedlamphouseassemblyofalightprojector.Theexcitationlightwasofwavelengthsabove
665nm.Thetemperatureofthesamplewaskeptat2°C.Beammodulation (300Hz)wasachievedbyarotatingchopperblade.Thesignalofalight-emittingdetectionsystem (Data
TechnologyAD2010E)wasusedasthereferenceinputforaBrookdealLock-inAmplifier
Type401AandaBrookdealPhasesensitiveDetector9412,thatservedtoconvertthemodulatedresponseofthephotomultiplierintoaproportionaldcsignal.Thissignalwasamplifiedandrecorded.
2.4 SALT-INDUCEDABSORBANCECHANGESOFP515
Absorbancechangescausedbytheadditionofsaltsatcharacteristicwavelengthsinthe
regionbetween460and700nmweremeasuredinthesplit-beammodeofoperation(Fig. 2c).
Themonochromaticbeamofmeasuringwavelengthsalternativelypassessampleandreference
compartmentofaspecialcuvette (Fig.3 ) ,seealsoTonketal. (1979).Themotor-driven
pinions (aandb)coupledbythestring (e),rotatesynchronouslyintwosmallcylindrical
channels(5mmdiam.)whichhaveopenconnectionswiththeupperandlowerpartsofsepa-
Fig.3.Cuvette,designedtomeasureabsorbancechangesinchloroplastsuspensionscaused
byadditionofsubstancesthroughinletskandjinoneofthecompartments (g),withreferencetocompartmentf.
ratedreference (£)andmeasuring (g)compartment (34x8x 10mm).Thecentersofthelowerholes (candd)areoff-linewiththeshafts(handi)oftherotatingpinions.Clockwise-directedrotationthereforecausesasuctionintotheupperholesandanupwarddirectedpressureinthecompartments,whichleadstoacounter-clockwisecirculationofthe
suspensionsinbothcompartments.Becauseoftheparallelandsynchronizedflowofparticles,split-beamabsorbancedifferencemeasurementsaremuchlesslimitedinsensitivity
andaccuracybywhirlingeffects.Themixingtimeafterthepulsedinjection(throughinletsjork)ofasmallvolumeofaconcentratedchemicaleffectorinthemeasuringcompartmentwasfoundtobelessthan1s.Thedouble-beammode (monochromaticbeamsofmeasuringandreferencewavelengthalternativelypassingthroughasinglecompartmentof
cuvette)wasusedtomeasureaccuratelyabsorbancedifferencespectraofKCl-induced
changesinthepresenceof3umol/1valinomycinand2mmol/1MgCl-.Saltswereaddedas
concentratedsolutions (1mol/1viamicro-syringes).
10
2.5 POTENTIALANDRESISTANCEMEASUREMENTSBYMEANSOFMICRO-CAPILLARYGLASSELECTRODES
MeasurementswereperformedonchloroplastsofPeperomia metalliaa inathinleafsection
(Fig.4 ) . Growthconditionsofplantsandexperimentalprocedureswereessentiallythe
sameasdescribedbyVredenbergetal. (1973)andBulychevetal. (1973).Fig.5showsthe
diagramoftheexperimentalarrangement.Potentialsweremeasuredbymeansofamicro-capillaryglasselectrode (Fig.6)filledwith3mol/1KClincontactwithaAg-AgClwire,
viaanagarKClbridge (Fig. 7).Thepotential (change)oftheelectrode,insertedintoa
singlechloroplast,wasmeasuredwithreferencetoanexternalelectrode.Theelectrode
signalwasfedintoahigh-impedanceunitygainamplifier (ILPICO-metricAmplifier181),andrecordedonanoscilloscopeoragalvanometricrecorderwithatimeresolution
of0.5ms.Photoreactionswereinducedeitherbyprolongedilluminationfromamodified
lamphouseassemblyofalightprojector (250W ) ,orbyflasheswithhalf-lifetimesof8
ysand80ususingaXenonoraRolleiflashdevice,respectively.Thelightreachedthe
sampleviaaflexiblelightguide(1mmdiameter).Thepositionofthelightguidewasat
adistanceof1to4mmfromtheleafsection.Thepositionoftheelectrodewasstabilized,quenchingenvironmentalvibrationsbyaspeciallyconstructedtable (W.Tonk,unpublished).Photoresponsescouldbeobservedduringaperiodof5-15minafterinsertion
oftheelectrode.
ThemembraneresistancewasrecordedwithswitchSinposition1(Fig. 5).Withthe
q
10 ilresistorinthecurrentcircuit,thecurrentpulses,eitherhyperpolarizingor
depolarizingthemembrane,wereoflowamplitude (lessthan1nA).Thecurrentpulses
Fig.4.Light-microscopepictureofaleafsectionof Peperomia metalliaa withonecell,
containingfourchloroplasts.Theglasselectrodeispositionedneartothecellwall.
Thebarcorrespondstoalengthof20pm.
11
Fig.5.Scheme
Thepotentialis
inserted intoa
tion.Thesignal
toredonanosci
Apulsegenerato
allowedcurrent
plastmembranes
0.
felectricpotentialmeasurements inaleafsectionof Peperomia
metallioa.
measuredacrosschloroplastmembranesbyamicro-capillary glasselectrode
singlechloroplastwithreferencetoanelectrodeinthesurroundingsoluisfedintoahigh-impedanceunitygainamplifierandsubsequentlymoniloscopeorrecorder.
r (switchSinpositionl)Qorafunctiongenerator (switchSinposition2)
injection,limitedbya10 Sresistorinthecircuit,acrossthechloroLight-inducedpotentialchangesweremeasuredwiththeswitchinposition
passedacrossthemembranethroughtheelectrodetowardstheexternalreferenceelectrode.
Thecapacitanceoftheelectrodewascompensatedbyanegative-capacitancecouplingcircuitofthePICO-metricamplifier.
Current-voltagecharacteristicsofthethylakoidmembraneweremeasuredbymeansofa
current-scanningtechnique,basicallysimilartotheonedescribedbyothers(Coster,
1965;Vredenberg&Tonk,1974b).SwitchSisputinposition2,connectingthecurrent
circuitwiththeoutputofafunctiongenerator,operatinginasingle-sweepmode,which
o
generatesavoltagethatchangesatauniformerate.Thecurrentlimitedbythe10 a
loadresistorchangedfrom-1 (inwardcurrent)to+1nA (outwardcurrent)atarateof
0.66nA/s.SpecialattentionwaspaidtothelinearityoftheI-Vcharacteristicofthe
measuringelectrode.
Fig.6.Scanningelectronmicrographoftheopenelectrodetip.Thebarcorrespondstoa
lengthof 1urn.ThemicrographwasmadebytheTechnicalandPhysicalEngineeringResearch
Service,ElectronMicroscopySectionatWageningen.
12
Fig.7.Perspexholder formicro-capillary glasselectrode.TheAg-AgClwire (a)isin
contactwiththeKCl-filledglasselectrode (c)viaaKCl-agarbridge(b).
Insameexperimentschloroplastswereisolatedinamediumcontaining 0.25mol/1
sucrose,25g/1Ficoll,100mg/1bovineserumalbumen,12mnol/1NaCl,20mmol/1HEPES-NaCH,pH 7.6.Onesinglechloroplastwasimmobilizedbysuctiononafire-polished tipof
a glasspipette (tipdiameter 10-12 ym),andtheelectrodecouldbeinserted.Furtherprocedureswereasdescribed inthissectionandappeared tobeapplicabletothissystem
without furtheradaptations.
2.6 PARTICLEELECTROPHORESIS OF CHLOROPLASTS
Theelectrophoreticmobility (u)ofindividual chloroplastswasdetermined inathermostated (25°C)laterallyorientatedrectangularquartzchamber (depth0.7mm,height 14mm)
connected toreversibleCu-CuSO.electrodecompartments.Theinstrument,basically similar
totheonedescribedbyFuhrmannetal.(1964),wasequippedwithphasecontrastoptics
(Leitz,largeworkingdistance)andafibre-opticsactinic illuminatorprovidingred light
(650nmcut-offfilter)withanintensityof220W/m attheelectrophoresis chamber.Measurementswereperformedatconstantcurrents (1-5mA)resultinginelectricfield strengths
between5and35V/cm.Theobservationlightwasfilteredbyagreen (540nm)interference
filterandkeptatthelowestpossibleintensity inordertominimizeactiniceffects.The
objectivewasfocussedononeofthetwopre-determined stationaryphases.Theelectrophoreticmigrationwastimedforbothforwardandbackward (reversedfield)runsovera
knowndistance (37.5or75 ym).Usually 10consecutiverunsofdifferent chloroplasts
ofthesametypewereregisteredandthemeanvaluesof u aregivenintablesandfigures;
withinthesamechloroplastpreparationthestandarddeviationsweremostly 1to3%,neverexceeding 8%.Theelectricalconductance andviscosityofthedifferentmedia,includingchloroplastsweremeasuredwithconductivitybridges (PhilipsPR9501 andRadiometer
CDM3)andanOstwald-typeviscosimeter,respectively.
Glutaraldehyde-treatedchloroplastswereobtainedbyincubatingbrokenchloroplasts
for2minin2.5%glutaraldehyde,followedbytwowashing steps.
ElectrophoreticmeasurementsweredoneinclosecollaborationwithDr.A.Theuvenet,
Dept.ofChemicalCytologyoftheUniversityatNijmegenandDr.R.Kraayenhofandworkers,Dept.ofBiologyoftheFreeuniversityatAmsterdam.
13
2.7 CHEMICAL ANALYSES
-Chlorophyll concentrationsweredeterminedbythemethod ofBruinsma (1961).
- Proteinwasdetermined bytheBiuretreactionofGornall &Bardawill (1949).
- Light-dependentJ^g fluxacrossthylakoidmembraneswasdetectedbyaMg -selective
electrodeplaced inacylindricalperspex cuvette (W.Tonk,unpublished).Stirringofthe
suspensionwas achievedbyrotationofthecuvettecausedbyaconstant flowofairagainstapaddlewheel.Theadvantageofthisstirringmethodabovethemorecommonlyused
magnetic stirrer isasuppresseddeteriorationofthechloroplasts andanimprovement of
thesignaltonoiseratio.Thesuspensionwas illuminated bylight froma 250W tungsten
lampplaced inamodified lamphouseassemblyofalightprojector (Leitz).
- Lightdependentoxygenevolutionwasmonitoredpolarographicallywitheither a separate
oxygenelectrode (RankBrothers)oraGilsonOxygraph.The temperaturewaskept constant
at20°C.
- Phosphorylation activityofchloroplastpreparationswasmeasured byATP consuming
luciferine/luciferasemediated lightemission.Thechloroplast suspensionwas illuminated
underphosphorylating conditionswithlightofwavelengths above 665nm.Immediately after
cessationoftheillumination,thequantumyieldoftheATPhydrolyzing luciferine/
luciferasereactionwasdetectedbyaphotomultiplier.Calibrationwascarried outby
titratingknownamounts ofATP.
-A CF..preparationfromspinachchloroplastswas isolatedbyachloroform-water extractionprocedure offragmented thylakoidmembranes andsubsequent ammoniumsulphateprecipitationoftheproteinfractioninthewaterphase.FurtherpurificationbySepharose 6B
chromatography yielded ahighlypurified CF. fraction.Antibodies againstCF.,elicited
inrabbit,wereadonationofDr.J.VerheyenoftheBiochemistryDepartment,University
ofAmsterdam. Intheexperiments dealingwiththeseantibodypreparations,theserum from
thesameanimalbefore immunizationwasused asacontrol.
14
3 Evidence for a light-induced bluebandshift of part of theP515
pigment poolin intact chloroplast membranes
3.1 INTRODUCTION
Thelight-induceddifference spectruminthe460-S40
ranwavelengthregion,measuredin
algaeandchloroplasts,ischaracterizedbyamaximum around 518nm,aminimumatabout
480
ranandaninflectioninthe490-500
ranwavelengthregion (Duysens,'1954;Roux&
Faludi-Daniel,1977;Bouges-Bocquet, 1977;Conjeaudetal., 1976).Evidencehasbeengiven
thatthisspectrumistheresultofanelectrochromicresponseofchlorophyll b andcarotenoidpigmentstotheelectricfield,generatedbytheprimarylight-inducedchargeseparationinthemembrane (Junge&Witt,1968;Reichetal., 1976).
Reichetal. (1976)showed thatthefrequencyshiftoftheabsorptionofamolecule
wasrelatedtothestrengthoftheelectricfield.Therelationisgivenby:
hAv=-|-ye-yg|?-J(<*e-ag)? 2 ,
inwhichAvisthefrequencyshiftoftheabsorption,duetotheelectrical fieldF*,yg
andyepresent thepermanentdipolemomentofthemoleculeinitsgroundstateandexcited
state,respectively;agandaepresent thepolarizability ofthegroundstateandthe
excitedstaterespectively;histheconstantofPlanck.Becausecarotenoids lackapermanentdipolemomenty,onlythequadraticpartoftherelationwouldbeexpectedtocontributetotheshift.Experiments,however,showedalinearrelationshipbetweentheelectricfieldandtheabsorbanceshiftatafixedwavelength (Jackson&Crofts,1969a;Amesz
&DeGrooth, 1976;Chapter 4).Thus,ithasbeensuggested thatthecarotenoidsareexposedtoahighpermanent field (fint),duetoasymmetrical complex formationwithpolar
molecules (Sewe&Reich, 1977a; 1977b;Reichetal., 1976;Reich&Scheerer, 1976).Inthe
caseofcarotenoidssuchaligand formationhasbeenshowntoinduceadipolemomentrelatedtothestrengthofthepermanent field (Reichetal., 1976)with:
Ay = Aa//-Fint,
inwhich àa// isthepolarizabilityofthecarotenoidmoleculeparallelwiththelongaxis
ofthemolecule.Consequently:
hAv = -|Aa//'fint|f-i(«e-ag)? 2
Sewe&Reich (1977b)estimated that themagnitudeofthepermanent field,causedby
asymmetrical complexformationisoftheorderof1.7x10 V-cnf .Forchromatophoresan
evenhigherfieldstrengthhasbeenreported (Amesz&DeGrooth, 1976),thatmayexceed
15
7 x 1 0 V-cnf .Incomparison,thephoto-electricfieldacrossathylakoidmembranehas
beenapproximatedtobeabout1.2x10 V-cm" (Reich&Scheerer,1976).Therefore,the
quadratictermintheequationcanbeneglected.Witt (1979)inferredthattheabsorbancechangeAA(X)isrelatedtoageneratedelectricfieldofmoderatestrengthaccording
to:
AA(X)=c( 6A / 5x ) |f|
Theconstantcisproportionaltothescalarproductofthemagnitudeoftheinduced
dipolemomentandthecosineoftheanglebetweenthemolecularaxisandthefielddirection.Thismeansthat:
-AA(X)islinearlydependentonthefieldstrength
-theshapeandwavelengthlocationofthedifferencespectrumAA(X)isinvariableand
6A
proportionaltothefirstderivative ( /.)oftheabsorbancespectrum.
DeGrooth&Amesz(1977)suggestedthat,atleastinbacterialchromatophores,the
poolofcarotenoidsshowingelectrochromismmaynotbecompletelyhomogeneousandmayconsistofmoleculeswithslightlydifferentpeakwavelengthsandbandshiftsdependingonthe
strengthandorientationoflocalelectricfields.
Inthischapterwediscussaneffectofcationsonthelight-induceddifferencespectrumofP515inchloroplasts.Itisshownthatinintactchloroplasts,aswellasinbroken
chloroplastsinthepresenceofcations,partofthepigmentsinthepoolrespondina
waythat,uponflashillumination,theirabsorptionbandisshiftedtowardsashorter
wavelength.Thisblueshift,whichgivesrisetotheinflectionaround500runintheoveralldifferencespectrum,isinterpretedintermsofacation-dependentchangeinthe
orientationofthepigmentcomplexwithrespecttoaninducedphoto-electricfield,orof
acation-inducedmigrationoftheprimaryacceptorofPhotosystem2intothehydrophobic
regionofthemembrane.Thus,inthelattercase,partofthepigmentsmightbecomeexposedinthelighttoanoppositelyorientatedfieldbetweentheprimaryacceptorandthe
outersurfaceofthemembrane.
3.2 1IATERIAL
IntactchloroplastsfromfreshspinachleaveswereisolatedasdescribedinChapter
2exceptforthefollowingmodifications.Theincubationmediumofintactchloroplasts
contained330mmol/1sorbitoland30mmol/1tricinepH8.0.Brokenchloroplastswereobtainedbybriefosmoticshockeitherinwater,orsaltsolutioncontaining10mmol/1MgCl,
and15mmol/1KClandsubsequentwashinginincubationmediumcontaining,inadditionto
330mmol/1sorbitoland30mmol/1tricine,nosaltor10mmol/1MgCl?and15mmol/1KCl,
respectively.Stocksuspensionsofchloroplastswerediluted6-10foldinassaymedium
whichwassimilartotheincubationmedium.Insuspensionsofbrokenchloroplasts,20
ymol/1diquatwasaddedaselectronacceptor.Finalchloroplastconcentrationswereequivalenttoapprox100yg/mlchlorophyll.
16
3.3 FLASH-INDUCEDABSORBANCE DIFFERENCE SPECTRA
Fig. 8a showsthespectraofflash-inducedabsorbance changesinintactandbrokenchloroplastsinthepresenceandabsenceofions,respectively.Thetwospectraaredistinctly
different.Theratiooftheabsorbancechangesatthemaximum 515andtheminimum480nm
ishigherinintactchloroplaststhaninbrokenchloroplasts.Inaddition,thespectrum
oftheintactchloroplasts showsaninflectioninthe490-500nmregion.Thespectrumof
brokenchloroplasts,isolatedandincubatedinasalt-containingmediumwasfoundtobesimilartothespectrumofintactchloroplasts (notshown).Thus,thespectraldifferences
AA
-033Fig.8.a)Spectraof flash-induced absorbance changes inintact andbrokenchloroplasts,
(curves2and 1,respectively).
Intact chloroplasts suspensions contained inaddition to sorbitoland tricine, 10mmol/1
MgCl2and 30mmol/1KCl.Broken chloroplastswere prepared and incubated intheabsence
of cations.Theabsorbance changes aregiven inrelativeunits,b)Spectrum obtained by
subtraction (curve 2-1)of the two spectra inFig.8a.
17
appearedtobeduetoioniceffects.
Inductionandrecoveryoftheflash-inducedabsorbancechangesforeachpreparation
hadthesamekineticsatallwavelengthsstudied,butnoattemptwasmadetodistinguish
thekineticsofAA515indifferentchloroplastsuspensions.AdetailedstudyofthesekineticsisgiveninChapter5.ThemagnitudesofAA515werefoundtovarylinearlywith
chlorophyllconcentration.
AsshowninFig.8b,subtractionofthetwospectraofFig.8agivesaspectrumwith
aminimumaround505nm,amaximumat483nmandzeropointsat460,495and535nm.Thus,
itappearsthatthedifferencespectrumofintactchloroplastsiscausedbytwodifferent
bandshifts,onesimilartotheredbandshiftobservedinbrokenchloroplasts (Fig-8a,see
alsoChapter4)andone (Fig.8b),indicativeforabluebandshift.
3.4 SIMULATIONOFBANDSHIFTS
Fig. 9 (curve"a+b")showsacomputeddifferencespectrumcomposedofa1nmredbandshiftofasingleGaussianbandA (maximumat497nm,halfwidth44nm)anda1nmblue
bandshiftofaGaussianbandB (B=0.18A,maximumat495.5nm,halfwidth25nm).For
comparisonthespectraofbandshiftsA (curvea)andB (curveb)aregiven.Thecomputed
differencespectraaanda+bappeartobeareasonablygoodsimulationofthespectra1
and2measuredinthechloroplasts (Fig.8).Obviously,thewidthofthenegativebandof
theexperimentaldifferencespectraissmallerthanthatofthecomputedspectra.Moreover,
thelatterwouldneedasmallbaselineshift (Fig.9)tomatchtheasymmetryobserved
intheexperimentalspectra.
3.5 DISCUSSION
Incubationofchloroplastsinanion-freeenvironmentwillleadtounstacking (Gross&Hess,
1974;Telferetal.,1976),changesinthemembranesurfacecharge (potential)(Barber&
Mills,1976),andtostructuralchangesinthemembrane(Gross&Hess,1974;Murakamiet
al.,1975).Thestructuralchangeswillaffectthemutualorientationofpigments.
Thus,inthepresenceofhighconcentrationsofMg
andK ,whichforinstanceexist
intheintactchloroplasts (Nobel,1969;Vredenberg,1977),thecarotenoidsinthemembrane
mightbecomeexposedtoelectricfieldswithadifferentorientationandmagnitudethan
inanion-freeenvironment.Ifso,ourresultscanbeinterpretedintermsoftwoalternativemodels:(i)Instackedchloroplasts,inthepresenceofmono-anddivalentcations,
partoftheelectrochromicallyrespondingcarotenoidshaveadipoleorientationwhich,
withrespecttothelight-generatedelectricfield,isdifferentfromtheoneofthebulk
ofthecarotenoids.Butintheabsenceofionsallfield-indicatingpigmentsareorientated
inthesamedirection,(ii)Allcarotenoidshavethesamedipoleorientation,bothinthe
absenceandpresenceofions,butasmallpartofthem,notablythoselocatedinthevicinityoftheprimaryacceptorsofthereactioncentersattheoutercoreofthemembrane,
becomeexposedtoanoppositelyorientatedlight-generatedelectricfieldbetweenthe
chargedprimaryacceptorsandtheoutermembranesurface.Thiscouldperhapsoccurifthe
acceptorhasmigratedmoreintothehydrophobicregionofthemembraneunderhigh-salt
AA
540nm
Fig.9. Computed difference spectrum (curvea+b)composed of a 1nmredbandshift (curve
a)of a singleGaussianbandA and a 1nmblue shift (curveb)of aGaussianband B (B•
0.18 A ) .Thebrokenhorizontal line istheoriginal zero lineof the spectra.The spectra
areplotted inrelative absorbanceunits.
conditions.Thepostulatedmigrationwouldbecomereversedintheabsenceofions.AccordingtoaninterpretativemodelproposedbyRenger (1975)th emigrationoftheprimaryacceptorQofPhotosystem 2includingitsproteinaceous shieldingcomplex,couldtakeplace
inthepresenceofsurfactants.Furthermore,afield-drivenmovementofQintothemembranalplanehasbeensuggested (Joliot&Delosme, 1974).
AlthoughIamnotinthepositiontodiscriminatebetweenthetwoalternatives,I
considerthefirstoneasthemostplausibleone.Thefractionofpigmentsthatshowsa
bluebandshift cannotbeestimatedwithprecisionasyet.AnalysesofthedataofFigs8
and9indicate thatwhenthebluebandshift isassumedtobeequaltotheoneofthered
bandshiftofthebulkpigments,theformeroneiscausedby181ofthebulkpigments.The
19
absorptionbandoftheblue-shifting fractionpresumablyhasasomewhatsmallerhalf-width
(25nm)thanthatofthebulk (44run)andislocatedataslightly lowerwavelength.These
differencessuggestadifferentchemicalenvironmentwhichwouldbeinharmonywiththe
modelsdiscussed.
20
4 Salt-induced absorbance changesof P515inbroken chloroplasts
4.1 INTRODUCTION
Voltagecalibrationofthe515nmabsorbancechangeusuallyismadewithreferencetothe
absorbancechangeinresponsetoasingle-turnoversaturatinglightflash.Accordingto
approximationsaboutthenumberofchargesloadingthemembranecapacitance,asingle
flashwouldgenerateatransmembranepotentialofapprox.50mV(Junge&Witt,1968;
Emrichetal.,1969;Witt&Zickler,1973;Witt,1971).Potentialmeasurementswithmicro-capillaryglasselectrodesinsertedinasinglegranumstackofchloroplastsof
Peperomia metalliaa
haveindicatedthatasingle-turnoverlightflashgeneratesapotentialacrossthethylakoidmembraneintherangebetween10and50mV(Bulychevetal.,
1972;Vredenbergetal.,1973;Vredenberg&Tonk,1975;Vredenberg, 1976).
Accordingtotheresultsofstudiesontheeffectofthevoltage-dependentionophore
alamethicinonthekineticsofthe515nmabsorbancechange,ithasbeenconcludedthat
thepotentialgeneratedbyasingleflashisoftheorderof100-200mV(Zickleretal.,
1976; Boheim&Benz, 1978).Thisvalueisatvariancewiththepotentialconcludedfrom
themicro-electrodeapproach.Thediscrepancyemphasizestheneedforanotherindependent
voltagecalibrationmethodoftheelectrogenicchargingofthethylakoidmembrane.
Attemptshavebeenmadetomeasureelectrochromicbandshifts,broughtaboutbya
transmembrane (diffusion)potential,generatedbysaltadditions (Strichartz&Chance,
1972; Gross&Libbey,1972).Theseexperimentsweresuccessfulinbacterialchromatophores
(Croftsetal.,1972;Jackson&Crofts,1969a)andhavebeenusedasacalibrationmethod
fordeterminingthemagnitudeofthetransmembranepotential,associatedwithphotosyntheticenergyconservation,inbacteria.However,thismethodcouldnotbeappliedto
chloroplast (thylakoid)membranesbecausesubstantialchangesinlightscatteringoccur
uponsaltadditions,whichpreventedaccuratemeasurementsofthe515nmbandshift
(Strichartz&Chance,1972;Gross&Libbey, 1972).
ThischapterreportsonexperimentsinwhichKCl-inducedabsorbancechangesaremeasuredinthe460-540nmwavelengthregionunderproperconditionsatwhichscattering
changesareminimized.Theresultsindicatethata515nmabsorbancechangeof1.1x10
correspondswithapotentialacrossthethylakoidmembraneof1mVandthattheelectrogenicpotentialgeneratedbyasingle-turnoversaturatinglightflashisintherange
between15and35mV.
4.2 MATERIALSANDMETHOD
Chloroplastswere'isolatedfromfreshleavesofspinach,growninthelaboratoryglass
house.Washedleaveswerehomogenizedinanisolationmediumcontaining0.33mol/1sor21
bitoland2nmol/1HEPES,adjustedtopH7.1withNaOH.Thehomogenatewasfiltered through
fourlayersofperIonnet (porediameter40ymol/1),andcentrifugedfor2minat2000 g.
Thesedimentedchloroplastsweresubjectedtoanosmoticshockresuspending themin10ml
distilledwater.After1min,10mlofdoublestrengthisolationmediumwasaddedtothe
suspension.Thestocksuspensionofbrokenchloroplastswasdiluted10to15foldinisolationmediumgivingafinalconcentration,equivalentto15-25yg/mlchlorophyll.Chloroplastswereusedwithin1hafterpreparation.
Absorbance changes inducedbysaltadditionorbysingle-turnover lightflasheswere
measuredasdescribedinChapter 2.Theactinicflashwasofwavelengths above665nm.The
absorbancechangesweremeasuredatvariouswavelengths,selectedbyinterferencefilters,
intheregion460-540nm.Anumberofflasheswerefiredatdarkintervalsof0.3s.Usually thesignalsof64flashesweresampledandaveraged.
4.3 SALT-INDUCEDABSORBANCECHANGES
Thecharacteristicsoftheabsorbancechangesat515,540,460and475nmupon successive
additionsof10mmol/1KCland2mmol/1MgCl-,orviae versa, toasuspensionofchloroplastsareshowninFig. 10.Additionof10mmol/1KClcausesanabsorbancedecreaseat
thesewavelengths.At515and540nmthedecreaseisprecededbyatransient increase.It
appearsthatthedecreaseinabsorbanceiscomposedofafastandaslowcomponent.The
relativemagnitudeofthesecomponentsisdifferentatthewavelengthsused.Subsequent
additionof2mmol/1MgCl,causesanincreaseinabsorbanceat515and540nm,whichapproximately equalstheprecedingdecreasecausedbytheadditionofKCl (Figs.10aand10b).
Ascanbeseen,thekineticsoftheMgCl2-inducedabsorbanceincreasearemultiphasic.
Theincreaseat475and460 (Figs.10cand10d)causedbyMgCl,inthepresenceofKClis
relativelysmallascomparedtotheincreaseat515and540nm.Additionof2mMMgCl-to
a suspensionofchloroplastsintheabsenceofKClcausesanincreaseinabsorbanceat515
and540nmandadecreaseat475and460nm.Theabsorbancechanges causedbyMgCl 2at
thesewavelengths appeartobecomposedofaslowandafastcomponent.Subsequent addition
of10mmol/1KClcausesasmallandreversibleabsorbance increaseat515and540nm.Absorbancechangesofthiskindat475and460nmcouldnotberesolvedwithsufficientaccuracy fromtheresponsecausedbythedilutioneffect.Theabsorbance changesbrought
aboutbyMgCl ?additionintheabsenceandpresenceofKCl,respectively,werefoundtobe
saturatedataMgCl?concentrationof1-2mmol/1 (not shown).
ThecharacteristicsoftheabsorbancechangesuponKCladditioninthepresenceof
MgCl,,asobservedat515 (and 540nm) (Figs. 10aand10b),werestudiedinmoredetail.
Theaccuracyofthemeasurementswas improvedbyperformingtheminthe dual-wavelength
operationmodeofthespectrophotometer.Inordertosuppressdisturbances causedbythe
dilutioneffect,measurementsweredonewithreferencewavelengthsinthe570-680nmwavelengthregion,atwhichtheabsorbanceofthechloroplast suspensionwasequaltotheone
atthemeasuringwavelengthsinthe460-540nmregion.Forexample,570and642nmwere
usedasreferencewavelengths formeasurementsat540and515nm,respectively. Itwas
verified thattheabsorbancechangesatthereferencewavelengthsuponadditionofKClin
thepresenceofMgCl-andvalinomycin,werenegligibly smallascomparedtotheonesatthe
22
Fig.10.Kineticsofsalt-inducedabsorbancechangesat540(a),515(b),475(c)and
460ran(d)inacation-freesuspensionofbrokenchloroplasts(15ug/mlchlorophyll).FinalconcentrationsofKClandMgCl2were 10and2mmol/1respectively.AdditionofMgCl2,
KClandH2Oareindicatedbylong,shortandthinupwardpointingarrows,respectively.
TheresponseuponadditionsofH2Oatthesameamountasthesaltsolutionindicatesthe
dilutioneffect.Thespectrophotometerwasoperatinginthesplit-beammode.Anupward
inflectionmeansadecreaseinabsorbance.
KCl
KCl
Fig.11.Absorbancechangesat515nmupontwosubsequentadditionsof10mmol/1KCl(b)
orof10mmol/1NaCland 10mmol/1KCl (a)toachloroplastsuspension (25ug/mlchlorophyll)inthepresenceof2mmol/1MgCl2and2ymol/1valinomycin.Adownward inflection
meansanincreaseinabsorbance.Thespectrophotometerwasoperatinginthedual-wavelengthmode.Thereferencewavelengthwas642nm.Thesmallinitialtransientsignalwas
causedbytheadditionofthesaltsolution.
23
measuringwavelengths.
Fig.11bshowstheKCl-inducedabsorbancechangeat515
ran
inthepresenceof2mmol/1
MgCl2and3umol/1valinomycin.Afastincreaseisfollowedbyaslowdecreasetotheinitiallevel.Asecondadditionof10mmol/1KClcausesasimilarbutsmallerabsorbance
change.Asmalleffectisalsoobserveduponaprioradditionof10mmol/1NaCl(Fig.11a).
Asubsequentadditionof10mmol/1KCl,inthiscase,givesrisetoanabsorbancechange
thatisaboutequaltotheoneobservedintheabsenceofNaClandKClinthesuspending
medium.
4.4 DATAFORAVOLTAGECALIBRATIONOFTHEP515ABSORBANCECHANGE
InFig.12theabsorbancechangeat515
ran
isplottedasafunctionoftheKCladdedto
thechloroplastsuspension.Itappearsthattheincreaseinabsorbanceisproportional
tothelogarithmoftheKClconcentrationgradient,initiallyinducedacrossthethylakoid
membrane,atleastforaconcentrationbelow50mmol/1.Thissuggeststhattheabsorbance
changeisproportionaltothemembranediffusionpotential,setbythesaltaddition.KCl
concentrationsupto50mmol/1didnotaffectthemagnitudeofthelight-inducedabsorbance
changesat515nm.AtaKClconcentrationabove50mmol/1theabsorbanceincreasewas
higherthanwouldbepredictedfromtherelationshipatthelowerconcentrations (not
shown).
Thetimecourseof'thelight-inducedabsorbancechangeat515nminthepresenceand
absenceofMgCl,,respectively,isshowninFig.13.Themagnitudeofthefastinitial
30.0mmol/lKCl
Fig. 12.ThemagnitudeoftheKCl-inducedabsorbancechangesat515nm,measured inthe
presenceof2mmol/1M g C ^ and3umol/1valinomycin,plottedasafunctionofKClconcentration.Chlorophyllconcentrationwas25ug/ml.Otherconditionswereasdescribed inthe
text.
24
Fig. 13.Absorbance changeat 515nmupon single-turnover saturating light flashes inthe
presence (a)and theabsence (b)of 2mmol/1MgCl^, respectively.Chlorophyll concentrationwas 23ug/ml.Other conditionswere asdescribed inthe text.
Fig. 14.Spectrum of absorbance changes induced by additionof 10mmol/1 KCl (opencircles)orby a single-turnover light flash (closed circles), measured inthepresenceof
2mmol/1 MgCl,and 3umol/1valinomycin.Theabsorbance scaleof the spectrum isplotted
inrelativeabsorbanceunits.
increaseinabsorbancecausedbyasingle-turnoverlightflash,isindependentonthepresenceof2mmol/1MgCl,.MgCl,atconcentrationsabove10mmol/1wasfoundtosuppressthe
magnitudeofthisresponsetoacertainextent.Thespectrumofthereversibleabsorbance
changescausedbyadditionof10mmol/1KClandthespectrumofthechangescausedby
single-turnoversaturatinglightflashesappeartobesimilar(Fig. 14).
25
4.5 DISCUSSION
Thespectrumofthereversibleabsorbancechangeinthe460-540nmwavelengthregionupon
additionofKCltoachloroplastsuspensioncontainingMgCl-andvalinomycin(Fig.14)
suggeststhatthechangeiscloselyassociatedwithareactionofP515.
Light-inducedabsorbancechangesofP515havebeenarguedtobeduetoanelectrochromicbandshiftofthesepigments,inresponsetothephoto-electricalfield(potential)
acrossthemembrane (Junge&Witt,1968;Emrichetal.,1969;Witt&Zickler,1973;Witt,
1971).Thespectrumoftheabsorbancechangecausedbyasingle-turnoversaturatinglight
flashisidenticalwiththedifferencespectrumofthesalt-inducedabsorbancechange
(Fig. 14).Asimilarcorrespondencehasbeendemonstratedforbacterialchromatophores
withrespecttothecarotenoidbandshifts (Croftsetal.,1972;Jackson&Crofts,1969a).
Instrictanalogywiththeseexperiments,thedataofFigs.11and12confirmthat
theabsorbancechangesinducedbyKCladditioninthepresenceofMgCl,occurinresponse
toamembrane (diffusion)potential,(i)Theabsorbancechangeuponadditionof10mmol/1
KClinthepresenceofvalinomycinismuchhigherthanuponadditionof10mmol/1NaCl,as
wouldbeexpectedinthepresenceofthisionophoreatwhichthemembranepermeabilityto
K ishighascomparedtothatofNaCl. (ii)ThepresenceofNaClinthesuspendingmedium
doesnotinfluencetheextentoftheabsorbancechangeuponKCladdition,whereastheresponseuponKCladditioninthepresenceofKClissignificantlysmaller,(iii)Theextent
ofthe515nmabsorbancechangeisproportionaltothelogarithmoftheconcentrationof
addedKCl (Fig.12).Moreover,thedecaykineticsoftheabsorbancechangescloselyresemblethekineticsoftheKCl-inducedchangesindelayedlightemissioninbrokenchloroplasts (Barber,1972a;1972b).Thesechangesindeedhavebeeninterpretedintermsofthe
decayofthemembranediffusionpotential,duetotheredistributionofK .Therateof
thisredistributionhasbeenevidencedtobecontrolledbytherateofentryofCI across
themembraneintothethylakoidinnerspace (Barber,1972a).Itwasfoundthatthemagnitudeofthe515nmabsorbancechange(Fig.11),uponadditionofafixedamountofKCl(in
thepresenceofMgCl,andvalinomycin),isproportionaltothechlorophyllconcentration
inthechloroplastsuspension,forconcentrationslowerthan30yg/mlchlorophyll.The
samewasfoundtobetrueforthemagnitudeofthelight-inducedabsorbancechange.
Fig.12showsthata10-fold increaseinK concentrationcorrespondswitha515nm
absorbancechangeofapprox.4.9x10".Intheabsenceofpermeatingionsthisincrease
wouldhavecorrespondedwithachangeinthemembranediffusionpotentialof58.5mV(at
20C),accordingtotheNernstequation.CorrectingforthepresenceofCl~inthemedium
(2mMMgCl2)andsubstitutinganestimated (Barber,1972a)permeabilityratiobetweenCI
andK of0.04inthepresenceofvalinomycin,theGoldmanconstantfieldequationyields
+
that,attheconcentrationsused,a10-foldincreaseinK concentrationcorrespondswith
achangeinthemembranediffusionpotentialofabout35mV.Thustheproportionalityfactorbetween515nmabsorbancechangeandmembranepotentialisabout1.4x10 permV.
ThedataofFig.12mayneedacorrectionfactorforasmallabsorbancechangethatcould
haveoccurredatthereferencewavelength (642nm)used.Accordingtospectraldataof
Reichetal. (1976)thiscorrectionfactorisabout0.8.Thiswouldmeanthattheabsor-4
bancechangeat515nmhasbeen1.1x10 permVatachlorophyllconcentrationof25
26
-4
yg/ml.Atthesameconcentrationasingle-turnoversaturatinglightflash (e.g.Fig.13)
_3
wouldcauseanabsorbancechangeof2.2x 10 ,whichcorrespondswithamembranepotentialof20mV.Similarexperiments,donewithvarioussamples,haveindicatedthatasingle-tumoversaturatinglightflashcausesthegenerationofatransmembranepotential (E)
intherangebetween15and35mVwithanaveragevalueofabout25mV.ThisvalueisaboutequaltotheestimatedvalueofE associatedwiththechargingofthemembrane
capacitance,duetoonechargeseparationinallreactioncenterspresentinthethylakoid
membrane (Junge&Witt,1968;Witt,1971;Vredenberg,1976),andfallswithintherangeof
flash-generatedpotentialsmeasuredwithmicro-electrodes inchloroplastsof P.
metallioa
(Vredenberg&Tonk,1975;Vredenberg, 1976).Valuesofabout 100mVhaverecentlybeenconcludedfromanalysesofthe515nmabsorptionchanges inchloroplastsinthepresenceof
thevoltage-dependentionophorealamethicin (Zickleretal., 1976).Thereasonforthe
apparentdiscrepancyisunknownasyet.Itmightbethat,contrarytotheassumptions
made (Zickleretal., 1976),theproportionalityfactorbetweenconcentrationandthecharacteristicpotentialoftheionophoreisdifferent inartificiallipidbilayersandthylakoidmembranes.Thepresentcalibrationmethodhastheimportantadvantagethatthelightandsalt-induced515nmabsorbancechangesaremeasuredinidenticalsamplesunderequal
conditions.
AscanbeconcludedfromthedataofFig.10,theapproachofsettingK diffusion
potentialsacrossthethylakoidmembraneinordertomeasureitsassociated electrochromic
shiftscouldonlybeappliediflowconcentrationsofMgCl-,orhighconcentrationsof
NaCl (notshown),werepresentinthesuspendingmedium.Inaccordancewiththeresultsof
others (Gross&Libbey,1972),thetimecourseoftheirreversibleabsorbancechangesin
the450-550nmwavelengthregionuponadditionofKClintheabsenceofMgCl,(Fig.10),
indicatesachangeinlightscattering,probablyduetostructuralchangesinthemembrane,
associatedwithcationbindingatfixednegativecharges.SubsequentadditionofMgCl,
resultsinanabsorbanceincreaseatwavelengthsabove500nm.Thiseffecthasbeennoticedbyothers (Gross&Prasher,1974).Itprobablyreflectsthesubstitutionofmonovalentcationsbydivalentcationsatthefixedcharges,causedbychangesofionicconcentrationsneartheelectricaldoublelayer (Barberetal.,1977;Chapter8 ) .
27
5 Analyses of theflash-inducedP515 absorbance changes in isolated
chloroplasts
5.1 INTRODUCTION
Recentlyithasbeenreportedthattheflash-inducedAA515inintact(ClassI)chloroplasts (Horvâthetal.,1978),likeinintactalgalcells (Joliot&Delosme,1974),occurs
withcomplexmulti-phasicriseanddecaykinetics.However,riseanddecayoftheflashinducedpotentialchangesincoupledintactchloroplasts,measuredwithmicro-electrodes,
havebeenshowntofollowsinglefirst-orderreactionkinetics (Bulychevetal.,1972;
Vredenberg&Tonk,1974a;Bulychev&Vredenberg,1976a).Thedecayrateofthepotential
changeafterasingleflashwasfoundtobedependentontheionicpermeabilityofthe
thylakoidmembraneonly (Bulychev&Vredenberg,1976a).Apparentdiscrepanciesonthemagnitudeandkineticsofthelight-inducedtransmembranepotentialchanges,asmeasuredwith
AA515andmicro-electrodes,respectively,havebeenamatterofdiscussion (Junge,1977b;
Rumberg,1977;Vredenberg,1976;Vredenberg&Tonk,1975;Schapendonk&Vredenberg,1977).
Thischapterdealswithanalysesofthekineticsofflash-inducedPS15absorbance
changesandofthetransmembranepotential,studiedonthylakoidmembranesofdark-adapted
spinachand Peperomia metallica
chloroplasts,respectively.Theanalysessuggestthatthe
AA515inintactandbrokenchloroplastsisthecompositeresultofatleasttwodifferent
processes,whicharecalledReactionIandReactionII.ThekineticsoftheAA515caused
byReactionI,characterizedbyarisetime<0.5msandadecayrateof8.6-17.3s~,
showsimilaritieswiththekineticsofthetransmembranepotential,measuredbymeansof
micro-capillaryglasselectrodes.TheabsorbancechangeofP515,associatedwithReaction
IIandcharacterizedbyarisetimeof100-150msandadecayrateof1.1-1.7s
ispre-
sumedtobeinducedbyalocalintramembranalelectricfieldassociatedwithchargeinteractionbetweenacompoundatthereducingsiteofPhotosystem1andcytochromeforplastocyanin.
5.2 MATERIALANDMETHOD
IntactandbrokenchloroplastswereisolatedfromfreshlygrownspinachleavesandpreparedbythemethoddescribedinChapter2,exceptforthefollowingmodificationswith
respecttotheincubationandassaymedium.Intactchloroplastswereincubatedandmeasuredinamediumwhichcontained330mmol/1sorbitol,0.5mmol/1K-HPO.,10mmol/1NaHCCu,
2mmol/1HEPES (pH7.5)and10mmol/1MgCl~.Brokenchloroplastswereobtainedbyosmotic
shockinH,0containingeither10mmol/1MgCl,or10mmol/1KCl.Theassaymediumcontained
330mmol/1sorbitol,30mmol/1tricine(pH7.7),20pmol/ldiquatandeither10mmol/1
MgCl-or17.5mmol/1KCl.Chloroplastconcentrationusuallywasequivalenttoabout50
yg/mlchlorophyll.Flash-inducedelectricpotentialchangesweremeasuredonsinglechlo28
roplastsinintactleavesof Peperonria metalliaa. Experimentalconditionsandmethods
wereasdescribedinChapter2.InhibitionoftheelectrontransportbetweenPhotosystem
2andPhotosystem 1wasachievedbypre-incubationofthesampleinthepresenceofKCN,
bythemethoddescribedelsewhere (Ouitrakul&Izawa,1973),orbyadditionofD O U .
5.3 EVIDENCEFORTHEINDUCTIONOFAFASTANDASLOWP515RESPONSEUPONSATURATINGLIGHT
FLASHESININTACTCHLOROPLASTS
ArepresentativeexampleofthetimecourseoftheÄA515uponasaturatinglightflashin
intactspinachchloroplastsisshowninFigs 15aand15b.Analysesofthesemi-logarithmic
plotsofthesechangesrevealatleast5differentphasesinthekinetics.Aftertheinitialfastabsorbanceincrease,calledphaseaandcompletedwithinatimeshorterthanthe
resolutiontimeofthemeasuringsystem (0.5m s ) , arelativelyslowincreaseinabsorbance
occursinthefirst10-20msuptoanabsorbancelevelwhichismoreorlessconstantfor
thenext20-80ms.Accordingtotheanalysis,thesubsequentdecreaseinabsorbanceappearstobecomposedofthreedifferentphases.Amajorphasecwithahalf-lifetimeof
420msisprecededbyarelativelyfastphasec'withahalf-lifetimeof75ms,andfollowedbyasmallslowphaseddecayingwithahalf-lifetimeof1.5s.Phasesc',andd
arenotresolvedduringthefirst70-100msaftertheflashduetotheslowabsorbance
riseorevenconstantlevelinthistimeperiod.However,extrapolationofthedecayline
ofphasec'totimezero (Fig.15b)resultsinaninitiallevelwhichcoincidesreasonablywellwiththeonereachedinthefastphasearise.Thiscertainlyisnottruefor
phasec.
ThedataofFig.15bsuggestthatthephasec'decayisassociatedwiththephasea
rise.Thissuggestionissubstantiatedbytheanalyzeddataoftheabsorbanceresponsein
adoubleflash (Fig.15c).TheseshowthattheAA515uponasecondflash,characterizedby
afastriseandasingledecayphaseequaltothec'componentanalyzedinthefirstflash,
issuperimposedupontheAA515inducedbythefirstflash.Thebreakbetweenthefast (c')
andtheslow (c)phaseafterthesecondflashisattheabsorbanceleveldeterminedby
phasecanddofthefirstflash.Thekineticsoftheresponseuponthirdandfollowing
flashes,firedattimeintervalsintherangeof10-100ms,werefoundtobesimilarto
thoseinducedbythesecondflash.Thus,wearriveattheconclusionthattherapiddecay
component (phasec')isassociatedwiththefastrise (phase a).Thesecomponentshavebeen
ascribedtoareaction,calledReactionI.
ThedeconvolutionoftheoverallP515responseintoReactionIandIIisshowninFig.
16.TherisekineticsofReactionIIshowaslowabsorbanceincreasewithin140ms.This
iscalledphaseb.ThedecayofReactionIIaftertheflashisbiphasicwithrateconstants
determinedbythecharacteristicrelaxationtimesofphasescandd.Foralargevariety
ofpreparations,thehalf-lifetimeofphasec',canddwasfoundtobeintherangeof
40-80ms,400-600msand1.5-3 s,respectively.Fig.16,representativeforintactspinach
chloroplasts,showsthatthephaseaandb,associatedwithReactionIand II,respectively,areaboutequalinmagnitude.Thespectraofthesechangeswerefoundtobeidentical
withthecharacteristicdifferencespectrumofP515 (e.g.Chapter 4 ) .Thereforewesuggest
thatReactions IandIIareduetoelectrochromicresponsesoftheP515pigmentcomplex.
29
AA(a.u.)
1001
5s
100
10-
V^JuVÔ_
/UW «
H
•
0.5
1
«
02s
100
©rv
02s
Fig. 15.Absorbance changes at 515ranindark-adapted intact spinach chloroplasts, in
duced by single (a,b)or double (c)flashes,recorded and displayed on 5 s (a)and 1s
time scales (b,c). Average of 32 single or double flashes, fired at a rate of 0.05 Hz.
The arrows mark themoments at which the flashes were fired. On the right the corresponding semi-logarithmic plots of the experimental curves are shown and the curve analyses
into exponential phase d (open squares), c (closed stars) and c' (closed circles), respectively, according to an adopted method (Atkins, 1969). (AA is given in arbitrary units
(a.u.)). Extrapolation of phase d and subtraction of the absorbance of phase d from the
experimental absorbance at corresponding times,gives the AA in the absence of phase d.
This absorbance curve yields themeasuring points of phase c.Analogously, phase c' is
resolved (see text). The dotted lineparallel with phase c' (lower right-hand figure) represents the fast phase c' induced by the first flash (middle right-hand figure). Other
dotted lines represent the extrapolated parts of the respective phases.
/N.
5.6x10r
• •
b,
.
-—d
-d
Fig. 16.Resolution (bottompart of figure)of theAA515 induced by asingleflashanda
seriesof twoflashes (upperpart of figure)into twocomponents,Reaction 1 (solid curves)
and Reaction II (brokencurves).Thecurveswere computed according tothedataobtained
from thecurveanalysisdepicted inFig. 15.Further explanations are inthetext.
Thespectrumoftherelatively smallphasedwas found tobepartially dissimilar
tothatofP515.Phased,which isnegligibly smallinbrokenchloroplasts (seebelow),
might represent thedecayofareactiondifferent fromReaction Iand II.Therefore,Reaction IIinintactchloroplastsmightneedasmallcorrection fortheAA515associatedwith
phase d.
According tothedataofFigs 15and 16onewould expect forintactchloroplasts that,
afteronepre-illuminatingflash,singleflashes firedatarepetitionrateof4-S Hz
cause theAA515ofReaction Ionly,i.e.AA515withafastrise (phasea)andasingleexponentialdecay (phasec')witharateconstant ofabout 9s~ .Thisresponse issuperimposeduponaconstant absorbance leveldeterminedbyReaction IIinducedinthepre-illuminating flash(es).Experiments ofthiskindwillbeillustrated forbrokenchloroplasts in
Section5.4.
According totheanalyses (not shown),theeffectofanon-saturating secondflash
(Fig.17b)isidenticaltothatofasaturatingsecondflash (Fig. 17a),except forthe
magnitudeofphasec'.Fig. 17cshowstheresponseuponafirstnon-saturating andasuccessivesaturatingflash.Theabsorbance increase inducedby thesecondflashisenhanced
ascompared totheresponseafterasaturating firstflash.Itsrisekinetics suggest a
contributionofphaseb inthiscase (notresolvedhere).Thedecaykineticsafterthe
second flashappeartobe identicaltothoseofasecondflashobserved aftera saturating
first flash (compareFigs.17band 17c).Theanalysesofthesetimecourses indicatethat
thesecondflashcauses anabsorbance increasethatdecaysexponentially inaphasec',
characterizedbyanintensity-independenthalf-lifetimeof60-80ms.Aftercompletion of
31
©
©
©
-V
Fig. 17.Timecoursesofthe515nmabsorbancechangeinintactchloroplasts,inducedby
twosuccessiveflashes,bothsaturating (a),thesecondnonsaturating (b)andthefirst
nonsaturating (c). Thebrokenlinespresentthekineticsofasinglesaturating(first)
flash.
4
AA 5J6XKT
200ms
Fig. 18.Timecourseofthe515nmabsorbancechangesinducedbytwosuccessiveflashes.
Thetimeintervalbetweenbothflasheswasvariedasindicatedbythearrows.
phasec',the slowphasescanddareobservedthataresolelyduetotheeffectofthe
firstflash(if saturating).
Theeffectofthesecondflashwasfoundtobeinvariablewhenthedark timebetween
theflasheswasvaried from 1to100ms.Atdarktimesabove 100msagradual increasein
thephasescandd,inducedbythesecond flash,wasobserved.Representativecurvesare
showninFig. 18.Atdarktimesshorterthan1msthe absorbancechangeuponthesecond
flashdecreasedsignificantly (withoutaneffect,uponthehalf-timeofphasec'andwas
foundtobecome zeroatdarktimesbelow400to500 ps (notshown).
5.4 ANALYSESOFTHEP515RESPONSEINBROKENCHLOROPLASTS
Experimentsonbrokenspinachchloroplastswereperformedtostudythe effectofvarious
additionsontheP515kineticsintheabsenceofthe"barrier function"exertedbythe
chloroplastenvelopeandstromaphase.Fig.19illustratesthattheP515responseinfresh
brokenchloroplastsalsoiscomposedofaReactionI-andReactionII-type.Ingeneralthe
32
AA(a.u)
50
Fig. 19.Time courses of the 515nm absorbance changes inbroken spinach chloroplasts (in
thepresence of 10mmol/1 MgCl 2 ), induced by repetitive single flashes fired at a repetition rate of 0.05 Hz (aand c)and 4Hz (b).Curve bwasmeasured immediately after pre-illuminationwith 6flashes, fired at arate of 4Hz.On theright in aand b: semi-logarithmic plot of the absorbance change against time.Note thedifferent time scales
inaand b.Thebroken curve (I)in c is identical to the response measured inb, i.e.
characterized by an exponential decay phase c'; curve II is thedifference between the
experimental curve (identical to the one shown ina)and curve I.
occurrencesinbrokenchloroplastswerefoundtobesimilartothoseinintactchloroplasts
providedthatagentleisolationprocedurewasapplied,includingosmoticshockinginthe
presenceofcations.
Fig.19ashowstheP515responseindark-adaptedbrokenchloroplasts,suspendedina
Mg -containingmedium,illuminatedbyflashes,firedatarepetitionrateof0.05Hz.The
semi-logarithmicplotshowsthatthedecaydoesnotfollowfirst-orderreactionkinetics.
Aslowandafastphasecanberesolvedwhich,inanalogywiththesituationinintact
chloroplasts,arecalledphasecandphasec',respectively.Phasec'canberesolvedaftersaturationofReactionIIwithsevenpre-illuminatingflashes(seebelow).Subsequent
flashes,firedatarepetitionrateof4HzcauseaP51Sresponsewhichexhibitsasingle-exponentialdecaywitharateconstantof12s" (Fig.19b).Becauseoftheclearsimilaritieswiththeoccurrencesinintactchloroplasts,thisresponseisattributedtoReaction
I.SubtractionofReactionIfromtheoverallresponsethengivestheresponseofReaction
II,asillustratedinFig.19c.TheincreaseinabsorbancecausedbyReactionIIisabout
501oftheabsorbanceincreaseassociatedwithReactionI.
TherelativemagnitudeofReactionIIinbrokenchloroplastsappearstobedependent
33
ontheioniccompositionofthemedium.Figs 20cand20dshowthatthemagnitudeofReactionIIinchloroplasts,suspendedinaMg -containingmedium,islowerthanthatof
ReactionIIinchloroplastssuspendedinK-containingmedium.Ascanbeconcludedfrom
thesemi-logarithmicplots (insertsFigs20aand 20b),therateconstantofthedecayof
AA(a.u.)
50
•
10
X.
X
1
100
•
200ms
200ms
•
Fig.20.Timecoursesofthe515nmabsorbancechangesinbrokenchloroplastsinducedby
repetitivesingleflashesfiredatarepetitionrateof0.05Hzinthepresenceof10
mmol/1KCl (a)or 10mmol/1MgCl2 (b).Resolution (bottompartoffigure)oftheAA515
responsesintotwocomponents:ReactionIandReactionII.CurvesI (candd)weremeasuredbyfiringrepetitivesingleflashesatarepetitionrateof4Hz,afterpre-illuminationwith6flashes.CurvesII(candd;brokenlines)wereobtainedbysubtracting
curvesIfromtheoverallAA515response (aand b). Theinsertspresentsemi-logarithmic
plotsoftheoverallresponses (aandb)andoftheReactionIdecaykinetics (candd),
respectively.
34
AA1.2X1Ö 3
*
•• c
^
O (7th flash)
2s
Fig.21.Timecourseoftheabsorbance changesat515nminbrokenchloroplasts,inthe
presenceof10mmol/1 KCl,inducedbyaseriesof7flashes,firedat100mstime intervals.ReactionI(phasec',closed circles),wasobtained after extrapolatingtheslow
phasec(closed stars)tothetimeatwhichtheseventh flashwas fired (semi-logarithmic
plot),andsubsequent subtracting fromtheoverall decay.
ReactionIIisindependentontheioniccompositionofthemedium.Thesemi-logarithmic
plotsofReactionI(insertsFig. 20cand20d),indicate thatthedecayofReactionIis
about 25%fasterinthepresenceof10mmol/1Mg ,incomparisonwith itsdecayinthe
presenceof10mmol/1K .MoredatawithrespecttothisioniceffectareinSection6.2.
Inbrokenchloroplasts,agroupofseveralflashes causesacumulative increaseof
theabsorbanceuptoasaturationlevelwhichisreached after4-6flashes,firedattime
intervalsof100ms(Fig.21).Theincreaseoftheabsorbancewhichappearstobedueto
anincreaseofReactionIIisabout 1.*timestheextentofReactionIIafteroneflash
(compareFig.20andFig.21).BecauseReactionIIreachesasaturation levelafterfive
successive flashes,thedecayaftertheseventh flashcanbeanalyzedbyextrapolatingthe
slowphase,associatedwithReaction II,tothetimeatwhichthe7thflashhadbeen fired.
Subtracting theextrapolatedpart fromtheexperimental curvegivesrisetothesingleexponentialdecayofReactionIwithahalf-life timethatequals thehalf-lifetimeof
ReactionIafterasingleflashi.e.approximately 100ms.
5.5 KINETICSOFTHELIGHT-GENERATED TRANSMEMBRANE POTENTIAL
Fig.22showsthesingleanddoubleflashresponseofthepotential acrossthechloroplast
(thylakoid)membraneofa Peperomia chloroplast in situ measuredwithamicro-electrode.
Theanalyses indicateasimilarsingle-exponential potentialdecaybothafterthefirst
andthesecondflashwithahalf-life timeof35ms.Inafewinstancestherelaxationaf-
35
lOmV/div
20ms/div
mV
lOOr
j10
100
200ms
Fig.22.Responseandsemi-logarithmicplotofthephoto-electricpotentialofa Feperomia
metallioa chloroplasttoasaturatinglightflashortoaseriesoftwosaturatinglight
flashesseparatedbyadarktimeof20ms.Measurementsweredoneinacrosssectionofa
Peperomia metallioa leafwithamicro-capillary glasselectrodeinsertedintoasingle
chloroplast.
terthesecondflashwasslightly(% 1(H)fasterthanafterthefirstflash.Thehalf-life
timeofthepotentialdecayforalargenumberofdifferentchloroplastswas30-60ms.The
potentialincreasemeasuredinthefirstflashwasintherangeof15-40mV;theratio
betweenthepotentialchangeinthesecondandfirstflashwas0.6-0.8.Thesimilarities
betweenthekineticsofReactionI(Fig.16)andthekineticsofthetransmembranepotentialchanges,asmeasuredwithmicro-capillaryglasselectrodes (Fig.22), suggestthat
theAA515associatedwithReactionI,isduetothetransmembraneelectricfield.Amore
detailedstudyontheReactionIkineticswillbegiveninChapter6.
5.6 EFFECTOFVALINOMYCIN
Itseemsunlikely,thattheslowdecaykineticsofReactionII,whichmainlydeterminethe
decayoftheoverallP515responseafteraflashinthe0.1-1 stimeperiod (cf.Figs15
and 16),areassociatedwiththedecayofadelocalizedelectricfield.Thissuggestionis
substantiatedbytheresultsdepictedinFig.23,whichshowthehalf-lifetimesofthedecayofReactionIandthemagnitudeofReactionII,afterasingleflash,asafunctionof
thevalinomycinconcentration.ThemagnitudeofReactionIandthedecayrateofReaction
IIwerefoundtobeindependentonthevalinomycinconcentration (notshown).Themagnitude
ofReactionIIappearstobeextremelysensitivetolowconcentrationoftheionophore.
ReactionIIiscompletelyinhibitedatavalinomycinconcentration,equalorlessthan250
36
tv2(ms)
AA(au.)
100
100
tV 2 (ms)
100r
AAla.u.)
100
jJO
250
soo
nmol/l
Fig.23.ThemagnitudeofReactionIIinbrokenspinachchloroplasts(openstars,right
hand scale)andthehalf-lifetimeofReactionI(closedcircles,lefthandscale)plotted
asafunctionoftheamountofvalinomycininthesuspendingmedium,inthepresenceof10
mmol/1MgCl,and 1mmol/1KCl (a),10nmol/lMgCl2and6mmol/1KCl (b),10mmol/1MgCl,
and 10mmol/1KCl (c),or6mmol/1KCl(d).
nmol/l.A501inhibitionofReactionII,measuredinthepresenceofMg + + ,isobtainedat
140nmol/land60nmol/lvalinomycininthepresenceof1mmol/1and10mmol/1KCl (Fig.
23aand 23c),respectively.ReactionIisonlyacceleratedwhenReactionIIhasbeencompletelyinhibited.InthepresenceofMg atavalinomycinconcentrationbelow200nmol/l,
thehalf-lifetimeofthedecayofReactionIishardlydependentontheK +concentration.
ThisindicatesthatMg isamorepermeantionthanK,eveninthepresenceofalow
amountvalinomycin.Thisissubstantiatedbytheobservationthat,ReactionI,inthepresenceof6mmol/1K,isequallyacceleratedby450nmol/lvalinomycin (Fig.23d)and10
mmol/1MgCl 2 (Fig.23b).SeealsoSection6.2.
TheobservedaccelerationoftheoveralldecayofP515inthepresenceoftheionophore
apparentlyisnotduetoanenhancedrateconstantofthedecayofthetransmembraneelectricfieldbuttoadecreaseofthemagnitudeofReactionII.Ingeneral,themagnitude
ofReactionIIinbrokenchloroplastsunlikethatofReactionIwasfoundtobestrongly
dependentonthephysiologicalconditionofthechloroplastsandtobesubstantiallysuppressedbymembrane-modifying substances (Fig.23),divalentcations (Fig.20),ageing
(Fig.24)andfreezing (notshown).SeeChapter7foramoredetaileddiscussion.
37
5.6x10"
200m s
V^Mr
Fig.24.Timecoursesofthe515nmabsorbancechangesinanagedpreparationofion-free
preparedbrokenchloroplastsupondouble-flashexcitation.Repetitionrateofseparate
flashsequenceswas0.05Hz.
5.7 ACTIVATIONOFREACTIONIIBYPHOTOSYSTEM1
Figs 25aand2SbshowrepresentativeexamplesoftheAA515inintactchloroplastsupon
saturatingsingle-turnoverlightflashes,whichexcitebothphotosystemswithexcitation
lightofwavelengthsabove665nm(Fig.25a),orpreferentiallyPhotosystem 1bylightof
anarrowwavelengthbandaround717nm (Fig.25b),respectively.Accordingtotheanalyses,
themagnitudesofReactionIandofReactionIIin717nmlightareabout50$andabout
75%,respectively,ofthemagnitudecausedbyactivatingbothphotosystems.Figs26aand
26bshowtheAA515uponilluminationwith3successivelightflashesofwavelengthsabove
665nmandof717nm,respectively,inintactchloroplasts.Thetimeintervalsbetweenthe
Fig.25.Timecoursesofthe515nmabsorbancechangesinintactchloroplastsuponilluminationwithsingle-turnover lightflashesofwavelengthsabove665nm (a)andofanarrowwavelengthbandaround717nm (b).Flashfrequencywas0.05Hz (noisycurves)or3Hz
(solidcurves).Incaseofthelatter,3pre-illuminatingflasheswerefired (seetext).
ThedottedlinesrepresenttheresultantkineticpatternofReactionIIobtainedafter
subtractionofthesolidcurves (ReactionI)fromtheoverallresponses.
38
•••
Fig.26.Timecoursesofthe515runabsorbancechangesinintactchloroplastsuponilluminationwithtripleflashesofwavelengthsabove665nm (a)andofanarrowwavelength
bandaround717nm (b).Timespanbetweenseparateflasheswas80ms.Seriesof3flashes
werefiredatafrequencyof0.05Hz.Thedottedlinesrepresenttheresponsestosingle
flashesfiredat0.05Hz.
flasheswas80ms.ThedottedcurvesrepresentthePS15absorbanceresponsesuponasingle
flash.Astheabsorbancedecayinthedark,300msafteraflash,isexclusivelydueto
thatofReactionII(cf. Fig.25),itappears,accordingtoFig.26a,andconclusivewith
resultsobtainedwithdoubleflashexperiments,thatReactionIIissaturatedbyasingle
lightflashofwavelengthsabove665nm.Thisapparentlyisnotthecasewhenlightof717
nmwavelengthisused.RepetitiveexcitationofPhotosystem1givesrisetoafurtherincreaseofReactionIIinthesecondandthirdflash (Fig.26b).Itshouldbementioned that,
with717nmlight,thethirdandthesecondflashwerenotcompletelysaturating.Therefore,itmightbethatthesaturationlevelofReactionIIcanbereachedaftertwo successivesaturatingexcitationswithPhotosystem1light.
Fig. 27showstheresponsesinbrokenchloroplasts,intheabsenceandpresenceof
theelectrontransportinhibitorsDCMJandKCN.InthepresenceofD O W (b), theextent
oftheP515responseisabout 50%oftheoneassociatedwithReactionIintheabsenceof
DCMJ (a).TheAA515inthepresenceofDCMUcannotberesolved intoits componentsbecauseReactionIcanonlybemeasuredaccuratelyinfast-repetitiveflashexperiments.However,aswouldbeexpectedinthepresenceofDCMJ,nosignalisobservedunderthatcondition (seealsoRenger&Wolff,1975).
AdditionofDCPIP/ascorbatecreatesaconditioninwhichatotalrecoveryoftheP515
responseismeasured (Fig.27c).Asimilarresponsehasbeenmeasuredafteradditionof
70umol/1PMStoDCMJ-poisoned chloroplasts (notshown).Theresponseafterpre-incubation
ofthechloroplastsinthepresenceof30mmol/1KCNisshowninFig.27d.Themagnitude
ofthesignalisabout40%ofthatoftheReactionIresponseinthecontrolexperiment.
ThedecayoftheresponseinthepresenceofKCNappearstobefasterthanthedecayof
thesignalinDCMJ-poisoned chloroplastsandsuggeststhecompleteabsenceofReactionII
39
®
AA 1-KT3
©
200ms
"•»^J v< Wt*<*tif**Jl4V'+*u*<
Fig.27.Timecourseofthe515nmabsorbancechangesuponilluminationwithsingle-turnoverlightflashesofwavelengthsabove665nmfiredatafrequencyof0.05Hzinbroken
chloroplasts.Chloroplastsweresuspended in330mmol/1sorbitol,30mmol/1tricine(pH
7.6)andeither 17.5mmol/1KCl (a,bandc)or 17.5mmol/1KCN (dand e). a)Controlexperiment;b)inthepresenceof2pmol/1DCMUafterpre-illuminationwith4flashes;c)
inthepresenceof2pmol/1DCMU,3.3mmol/1ascorbateand48pmol/1DCPIP;d)after
pre-incubationin30mmol/1KCN;e)afterpre-incubationinKCNandsubsequentaddition
of3.3mmol/1ascorbateand48pmol/1DCPIP.
andasomewhatfasterdecayrateofReactionI.AdditionofDCPIP/ascorbate(Fig.27e)or
70pmol/1PMS (notshown),inthiscase,createsaconditioninwhichtheflash-induced
AA515equalstheReactionIresponseofthecontrol,exceptforthesomewhatfasterdecay.
ThedarkkineticssuggesttheabsenceofReactionIIinKCN-inhibitedchloroplastsinthe
presenceofDCPIP/ascorbate.
Apparently,reducedDCPIPandPMScanreactwithoxydizedP700inadirectway,independentofplastocyanin.Ouitrakul&Izawa(1975)reportedarecoveryof18%and201of
theelectrontransportrateinKCN-treatedchloroplastsby50pmol/1DCPIPand100umol/1
PMS,respectively.Thus,adirectreactionofthesemediatorswithP700seemsplausible.
A201recoveryoftheelectrontransportrateissufficienttoreduceP700 completelyin
thedarkperiodsbetweentherepetitiveflashes.
5.8 DISCUSSION
Themagnitudeoftheabsorbanceincreaseat515nmhasbeenusedasalinearmeasureofthe
electrictransmembranepotentialofthethylakoids (Witt,1971;Junge,1977a;Gräber&
40
Witt, 1975).A single-exponentialdecayofthe515nmresponseafterasingle-turnover
lightflashisusuallyobservedinaged,oruncoupledion-freepreparedbrokenchloroplasts (Fig.24).Thiswouldindicateavoltage-independentmembraneconductancewhich
determinestherateconstantoffield-dissipatingpassiveionmovements (seealsoChapter
6). Biphasicdecaykineticshavebeenshowntooccurinbrokenchloroplastunderphosphorylatingconditions.Thesearebelievedtobeduetochangesintheprotonconductancethrough
theATPaseaboveacriticalpotential (Junge&Witt,1968;Boeck&Witt,1972;Schliephake
etal., 1968;Rumberg&Siggel,1968;Jungeetal.,1970;Schmidetal.,1976;Girault&
Galmiche,1978).ThishypothesishasledtoconclusionsaboutacorrelationbetweenATP
synthesisandP515kinetics.However,itmaybedoubtedifprotonscontributesignificantly
tothemembraneconductance,whetherunderphosphorylatingconditionsornot (Section6.3).
Moreover,theexperimentswithbrokenchloroplastsdescribedinthischapterwerecarried
outundernon-phosphorylatingconditionsandthereforenomulti-phasicdecaykinetics
wouldbeexpectedtooccurinviewofthishypothesis.Furthermore,ascanbeconcluded
fromFig.21andFig.20a,thetransitionfromafasttoaslowdecayphaseafterthelast
flashofaseriesof7flashes,occursatahigherlevelthanthetransitionafterasingle
flash.Itisdifficulttoreconcilewiththisobservationintermsoftheexistenceofa
distinctcriticalpotentialfortheactivationoftheATP-synthesizingcomplex.
OnthebasisofresultsdepictedinFigs 15-21,wearriveattheconclusionthatthe
flash-induced515nmabsorbancechangeindark-adaptedintactchloroplastsistheconsequenceofatleasttwodifferentprocesses.Oneprocess (ReactionI)ischaracterizedbya
fastrise (phasea)andadecay (phasec')withahalf-lifetimeof60-100ms,theother
(ReactionII)ischaracterizedbyarelativelyslowriseandadecay (phasescandd)with
ahalf-lifetimesof400-800msand 1-3s,respectively.
ThespectrumoftheslowdecayofphasedwasfoundtobedifferentfromthatofP515.
ThisphasemightrepresentthedecayofareactiondifferentfromReactionIandIIand
probablycorrespondstochangesinscatteringlevel (seealsoChapter 8).Theestimated
magnitudeofphasedinasingleflashisabout 101ofthetotalP515response.
TheobservedsimilaritybetweenReactionIkineticsandthekineticsofthetransmembranepotentialchangesasmeasuredwithmicro-capillaryglasselectrodes,providesreasonablegoodevidencefortheconclusionthatthe515nmabsorbancechangeassociatedwith
ReactionIisduetothetransmembraneelectricfield.Thisfield,measuredwithmicroelectrodesisequallyactivatedbybothpigmentsystems (Vredenberg,1974).Accordingto
thedataofFig.25,itappearsthatthemagnitudeofthefastriseofReactionIinPhotosystem1(717nm)lightisabout501ofthemagnitudemeasuredinlightabsorbedbyboth
photosysterns.Moreover,themagnitudeoftheP515responsemeasuredinDCMU-orKCNtreatedchloroplasts (Fig.27band27d,respectively)uponflashlightabsorbedbyboth
systemsisabout50%oftheReactionIresponseintheabsenceoftheinhibitors (Fig.27a).
IlluminationofDCMU-poisonedbrokenchloroplastswith717nmlightflashesunderthese
conditionsdidnotresultinanabsorbanceresponseintheabsenceofelectrondonorsfor
Photosystem 1.Wethereforeconclude,inagreementwiththeinterpretationofRenger&
Wolff (1975),thattheresponsemeasuredinthepresenceoftheseelectron-transportinhibitorsisbroughtaboutbychargeseparationinPhotosystem 2only.AccordingtoFigs
27a,27cand 27e,ReactionIcanberestoredcompletelyinDCMU-orKCN-poisonedchloro41
plastsbytheadditionofelectrondonorsforPhotosystem 1.Thus,itseemsreasonableto
concludethatReactionIisequallyactivatedbyPhotosystem 1andPhotosystem2.
ThedecayoftheoverallP515responseafteraflashinthe0.1-1stimeperiod (cf.
Figs 15-21),whichismainlyduetoReactionII,isdifficulttointerpretintermsof
thedecayofthetransmembraneelectricfield.Thisisnicelyillustratedbytheinhibiting
effectoflowconcentrationsofvalinomycinonthemagnitudeofReactionII.Inthisrespectitisofinteresttorecallthe200to600foldincreaseinP515decayrate,usually
observedinintactchloroplastsinthepresenceof1pmol/1valinomycin.Ifweassumethat
intheabsenceofvalinomycintheK concentrationinthechloroplaststromaisatleast
30mmol/1(Nobel,1969;Gimmleretal.,1974;Vredenberg,1976)andwilldecreaseconsiderablyinthepresenceoftheionophoreatalowoutsideconcentrationofK (Bulychev&
Vredenberg,1976a),andfurtherassumethattheK permeabilitycoefficientofthethylakoidmembraneisincreasedbyafactorof10inthepresenceofvalinomycin (Barber,1972a),
onewouldnotexpectthatthemembraneconductancechangestosuchalargeextent.Ithas
beenreported (Bulychev&Vredenberg,1976a)thatthedecayofthetransmembranepotential
undersimilarbutnotidenticalconditionsisacceleratedbyafactorofnomorethan5.
ReactionIIdependsquitedifferentlyonPhotosystem1andPhotosystem2illumination.
WefoundforavarietyofpreparationsthatexcitationofonlyPhotosystem1,bysingle
lightflashes,causedanactivationofReactionII,thatappearedtobe60-80$oftheresponseafterexcitationofbothphotosystems.AsFig.26shows,ReactionIIissaturated
afterdoubleortripleexcitationofPhotosystem1.Thissuggeststhattheredoxpoiseat
thereducingsideofPhotosystem2partlycontrolsReactionII,possiblybyitsinfluence
ontheredoxstateofcytochromef(Mitchell,1975).Theobservation,thatReactionIIis
mainlydependentonaprocessactivatedbyPhotosystem1excitation,isinagreementwith
theobservation (Fig.22)thatReactionIIisrelativelysmall,orevenabsentinDCMU(Fig. 27b)andKCN-treated (Fig.27d)chloroplasts,respectively,andcanbecompletely
restoredinthepresenceofDCMJafteradditionofelectrondonorsforPhotosystem1.
ThedecayoftheP515responseinthepresenceofD C U (Fig.27b)suggestsacontributionofReactionII,whichinthiscaseisabout20%ofthemaximalresponseunderconditionsatwhichelectrontransferthroughPhotosystem1ispossible (Figs 27aand27c).
WhetherthissmallcontributionofReactionIIhastobeattributedtoPhotosystem1activitycannotbesaidwithcertainty.Photosystem1excitationby717nmlightunderthese
conditionswasfoundnottoresultinaP515response.Butitispossiblethat,uponexcitationofbothPhotosystems,thereducedacceptorofPhotosystem2isoxidizedinthe
darkbyacomponentwhichinitsreducedformservesasa(weak)donorforoxidizedcarriersatthedonorsideofPhotosystem1.Thus,uponexcitation,someelectrontransport
throughPhotosystem1wouldbepossiblewiththeconsequentappearanceofasmallReactionII(andReactionI).Inintactchloroplasts (inthepresenceofDCMU),POmightbe
comere-reducedviathepathway:ferredoxin •*cytb564 •*PQ(Millsetal.,1979).Subsequently,thereducedplastoquinonemightreducetheprimarydonorofPhotosystem2.Such
amechanism,however,isnottobeexpectedinbrokenchloroplasts,whicheasilyhavereleasedferredoxin.
Ourresultsdonotpermitdefiniteconclusionsabouttheprocesswhichisresponsible
fortheappearanceofReactionII.Aslowflash-inducedAA515increasehasbeenreported
42
tooccurinpre-illuminatedchloroplasts (Velthuys,1978),whichhasbeensuggested tobe
causedbyaprotontranslocationattheoxidizingsideoftheplastoquinonepool.Theresultsofourexperiments,however,stillshowaslowAA515associatedwithReactionII,
underconditionsatwhichplastoquinone isintheoxidizedform (Photosystem 1illumination).However,arelationbetweenelectrogeniccyclicelectrontransport andtheslowP515
response (Arnon&Chain,1975;Crowtheretal.,1979;Millsetal.,1979)cannotbeexcludedasyet,provided thatthereactionrateoftheelectrogeniccyclicelectrontransportturnsouttobecompatiblewithReaction IIriseanddecaykinetics.This suggestion
isdifficult toreconcilewiththefact (Fig.23)thatthedecayofReactionIIisunalteredwhereas itsmagnitude issuppressed inthepresenceoflowconcentrations ofvalinomycin.
ThekineticsandspectralcharacteristicsofReaction IIsuggestthatitsmanifestationisareflectionof physico-chemical processes inthemembraneneartheP515pigment
complex.Severalworkers showedthatthedecayofthePS15responseinbroken chloroplasts
hasrelativelyrapidsinglefirst-orderkinetics (seealsoFig.13). Thissuggests that
Reaction IIinthesepreparations doesnotresult inanelectrochromicresponseofthe
P515pigmentcomplex,oralternativelydoesnotoccuratall.Thereasonforthisisunknownasyet.Iassumethatthestructuralorganizationofmembraneconstituents andspecifically thesensitivity of (partof)theP515pigments totrans-andinnermembraneelectricalfieldsareaffectedbysome,asyetunknown,critical stepsintheprocedure of
chloroplastpreparation.
TheabsenceofReactionIIinKCN-poisonedchloroplasts,inthepresenceofelectron
donorsforPhotosystem1(Fig.27e)wouldsuggestafunctional roleofplastocyanin or
cytochrome finthereactionprocess.Thissuggestion isinagreementwiththeobservation
(Malkin,1978)thatadistinctcomponentoftheP515response isdependentontheredox
conditionofacomponentwithamidpointpotentialof+385mV,whichisaboutequaltothe
midpointpotentialsofcytochrome fandplastocyanine.
Thereasonfortheobserveddifference insaturationofReaction IIinintactand
brokenchloroplasts isnotclearasyetbut itshouldberecalled thatdifferences inthe
P515spectrumofbroken-andintactchloroplastweresuggested tobeduetocation-dependentchanges inthepositionofelectroncarriersandotherpigments inthemembrane (Chapter 3). Ithasbeenobserved (Haehnel,1977),thatoxidizedP700canbereduced completely
afteronesaturating flash,whereasonlypartofPCisoxidized.Successive flashesincreasetheoxidationdegreeofthePCpool.Similarresultswithrespecttocytochrome f
wereobtainedbyKoikeetal. (1977).Anelectric interactionbetweenoxidizedPCandP700,
proposed tobepartofonecomplex (Ke,1975),mightexplaintheobservedincreaseof
Reaction IIinsuccessive flashesinbrokenchloroplasts.ThereforeitmightbeinterestingtostudytheoxidationofPCandthereductionofP700inintactchloroplasts.This,
accordingtotheobserved saturationofReaction IIinasingleflash,shouldhaveaone-to-onestoichiometryundertheseconditions.
Itisunlikely thatsome,relatively fast,electrogenicprocess,mediatedbyeither
oxidizedcytochrome forplastocyanin,mayaccount inadirectwayfortheslowriseand
decaykinetics ofReactionII.However,suchanelectrogenicprocessmaywellcauseconformational changes inthemembrane.A chargeseparationbetweeneitheroxidizedplasto43
cyaninorcytochrome£andacatalistatthereducingsideofPhotosystem1mayinduce
changesinthestructuralassemblyoflipidandproteinmolecules.Suchconformational
changesaresupposedtoalterthemutualorientationoffixedchargedgroupsofthemolecularstructureswithinthemembrane.Weproposethatthismayresultin(achangeof)the
intrinsicelectricfield,associatedwiththesechargedisplacements.Asaconsequence
thismayresultinAA515oftheReactionIItype.Itisinterestingtonotethattherise
anddecaykineticsoftheP515absorbancechangesassociatedwithReactionIIcloselyresembletheopticalresponseofthepotentialprobeoxonolVI (Schuurmans,1978;Bashford,
1978).Themagnitudeoftheoxonolabsorbanceshifthasbeenshowntobeinhibitedbyvalinomycin(Schuurmansetal.,1978).Thisinhibitionappearstobesimilartotheinhibition
ofReactionII.Theactionofvalinomycincanbeunderstoodbydiscerningthechaotropic
effectofthisionophore.Adirecteffectofvalinomycinonthephosphorylatingsystemhas
beenreported (Trebst&Reimer,1977)andthechangesinlipidaggregationcausedbythe
antibiotic (Walz,1977)mighthampertheconformationalchangeoftheactivatedcoupling
factor.
Ingeneral,itappearsthattheriseofReactionIIoccursinthesametimerangeas
theflash-inducedresponseofexternalprobeswhichareindicatorsofeitherchangesin
themembranesurfacepotential (Kraayenhof&Arents,1977),orofconformationalchanges
ofthechloroplastATPase (Kraayenhof&Slater,1975).Ithasbeendiscussed (Ortetal.,
1976;Vinkleretal.,1978)thatsomemodeofmembraneenergizationorconformationis
neededfortheonsetofATPsynthesisinthelight.Thisactivationstepappearstobe
completedinthesametimerangeastheriseofReactionII(seealsoChapter7).
44
6 Reaction kineticsof transmembrane potential changesin relation to
ion permeability
6.1 INTRODUCTION
Therelationshipbetweenelectrontransportandprotonpumping intothethylakoid inner
spacehasbeenthesubjectofseveralstudies.NetuptakeofprotonswasobservedbyNeumann
&Jagendorf (1964)inbrokenchloroplastsandbyHeldtetal. (1973)inintactchloroplasts.UsingphenolredasapHindicatororthefluorescencequenchingoffluorescent
amines,thesteady-stateratesofprotontranslocation (Chow&Hope,1977)andthekineticsofonsetanddecayofApH (Chow&Hope,1976)havebeenstudied extensively.
Tomaintainelectricalneutralityinthebulkphases,theH uptakeisbalancedby
eitheranioninfluxorcationefflux.Unfortunately,theknowledgeofpassiveionfluxes
•acrossthethylakoidmembraneanditsconductanceisratherunsatisfactory.Sofar,often
conflicting resultshavebeenreported.TheeffluxofK andMg hasbeenreportedtobe
themainchargebalancing ionmovement (Dilley&Vernon,1965).Bulychev&Vredenberg
(1976b)havegivensupportforthissuggestionandhaveemphasized theconsequenceofthe
relativelyhighconcentrationofbothionsavailableintheintactchloroplast.Deamer&
Packer (1969)reportedCl~tobeamajorco-ionforprotonpumping.TheimportanceofCl~
inthisrespectwasalsodiscernedbyHindetal. (1974).
Nakatanietal. (1978)emphasized theimportanceofsurfacepotentialsofthylakoid
membranesintermsofiondistributionbetweenthemembraneboundaryandthebulkphase
andtheeffectsthereofonthemembraneconductance.Theseresults,confirmedbylater
findings (Nakatanietal., 1979),havegivensupporttotheproposalthatMg mayactas
themaincounterionforprotontransport.Alternatively,Gräber&Witt (1975,1976)concludedthattheestablishmentofaprotongradientcausedanenhancementofthespecific
protonconductance,tosuchanextent,thattheactiveprotonuptakeunder steady-state
conditionsisbalancedbothbypassiveproton leakageandprotoneffluxthroughtheATP-synthesizing complex.Junge (1977a)andHarris&Crofts (1978)discussedanelectrically
gatedATP-synthesizing complexthatwouldenlargethemembraneconductanceduetoahigher
protonpermeabilitycoefficient (seehoweverChapter5and7 ) .
Thesearchforaneffectofthemembranepotentialontheionconductivity remainsa
challengeforabetterunderstandingofion-controlledprocesses,includingATPsynthesis.
Reversiblechangesofthemembraneconductancecausedbylight-dependentprocesses have
beenobservedbyVredenberg&Tonk (1973)attheplasmalemmaofNitella
tranaluoens.
Inthischapter,thedecayrateofReactionI(cf.Chapter 5),proposedtobeproportionaltothethylakoidmembraneconductance,isstudiedinrelationtotheionicenvironmentofthylakoidmembranesofspinachchloroplasts.Fromthisstudyconclusionsare
drawnwithrespecttotherelativecontributionofdifferentionstothepassivemembrane
conductance.AnalysisofReactionIkineticsinducedbyaflash,firedimmediatelyafter
45
thepre-illuminatingperiods,leadstotheconclusionthattheprotonconductanceofthe
thylakoidmembraneisrelativelylowandsuggestsevidencefortheexistenceofaMg -H
exchangemechanism.Additionalinformationontheeffectofenergizationandassociated
changesoftheelectricalresistanceofthemembrane,isderivedfromelectricalstimulationofthylakoidmembranesof Peperomia metalliaa
bymeansofmicro-capillaryglass
electrodes.
6.2 EFFECTOFELECTROLYTECONCENTRATIONONTHEDARKKINETICSOFREACTIONI
Fig.28ashowsthereciprokehalf-lifetimeofReactionIplottedasafunctionofthe
amountofKClpresentintheassaymediumatdifferentpHvalues.AtpH7and8,thedecayappearstobehardlydependentontheconcentrationofKCl,indicativeforalowmembranepermeabilityofbothK andCI undertheseconditions.SimilarresultswereobtainedbySchuldiner&Avron (1971)andYakovleva&Molotkovskii (1979)whomeasureddark
swellingofthylakoidsuponadditionofKCl.LoweringthepHoftheassaymediumapparentlyresultsinanincreaseinthemembraneconductanceasmaybeconcludedfromthepHdependentdecayrateofReactionIintheabsenceofions (Fig.28b).Moreover,atlower
pHthedecayrateofReactionIbecomesmoredependentonsaltconcentration,according
tothealteredslopesinFig.28a.IfCI isreplacedbyaspartate,noappreciableeffect
©
s-1
120r
r 'i
.
8pH
mmol/l KAsp.
*=50
i
O =35
I-*
o
. =10
. =20
*= 5
*= 0
\
o.
•
*
\*\•
X
if
1
6
Fig.28.a)Reciprokeofthehalf-lifetimeoftheReactionIdecayafterasingle-turnover
flash,measuredatdifferentpHvaluesinbrokenspinachchloroplastsplottedasafunctionofKClconcentrationinthesuspendingmedium:pH8,openstars;pH7,closedcrescents;pH6,closedstars.
b)EffectofpHonthereciprokehalf-lifetimeofReactionIinbrokenchloroplasts,suspendedinanion-freeassaymedium,c)EffectofpHonthereciprokehalf-lifetimeof
ReactionIinbrokenchloroplasts,suspended inamediumcontaining5mmol/1KClandin
additionvaryingamountsofpotassiumaspartate (seeinsert).
46
8pH
ofsaltconcentrationonthe-ReactionIdecay kineticswasfound atdifferentpH.The
pH-dependent changesofthedecayratearesimilartothoseintheabsenceofions (compareFigs28band28c).Thus,Iconclude thatthethylakoidmembraneisimpermeableto
bothaspartateandK.Fromtheseresultsitmaybederived that,betweenpH8and6, the
membraneconductanceisdeterminedbytheamountofCl".ThetransportofCl~apparently
isfacilitatedbylowpHascanbeconcluded from itseffectontheslopeoftheconcentrationdependent ReactionIdecayrate (Fig.28a).
Fig.29showsthehalf-life timeofReactionIasafunctionofCl~concentrationat
differentpH.Cl~wasaddedeitheraspotassiumsaltorasmagnesium salt.BecauseK*is
ratherimpermeant (Fig. 28c),theapparent differencesindecay rateatequal Cl"concentrationsaresuggestive foracontributionofaMg fluxtothemembraneconductance.The
reason fortheinitial inclinationinsomeofthecurvesisnotknownasyetbutitmight
reflect secondaryeffectsofMg additionassociatedwith ionredistribution processes
intheelectrical double layeradjacenttotheplaneofshear (c.f.Chapter7and8). Conclusionsaboutthepassivemembrane conductance,however,canbederived fromthe linear
partofthecurves.Fromthedifferencesintheslopesoftheconcentration lines,anestimationcanbemadeoftheMg -andtheCl~-conductivity,respectively.Themembrane
conductanceatpH7,inthepresenceof40mmol/1Cl"addedasaMgsalt,exceedstheone
inthepresenceofthesameamountofCl~addedasK saltbyafactorofabout4.Atthis
pH,theMg conductivity apparentlyisfourtimestheoneofCl".Thisfactorwasabout
2bothatpH6andpH8.The Cl"conductivityhasnopHoptimumbutrather increasesconstantlyupon lowering thepHtoavalueofS(not shown).
Fig.30showstheanion-dependent half-lifetimesofReaction I.Allsaltswereadded
asMg salts.Therefore,theobserveddifferencesinconcentration curvesmaybeattributedsolelytotheanioneffectonthemembrane conductance.Theplots showamoreor
©
8-1
100
100
50-
20
4 0 6 0 0
20
40
600
20
40
60mmol/ICI
Fig.29.Reciprokeofthehalf-life timeofReactionIdecay,measuredatpH8(a),pH7
(b)andpH6(c),plottedasafunctionofCl concentration.Cl wasaddedasMgCl2
(closed stars)orasKCl (closed circles), respectively.
47
©
s-1
100r
100
50 -
50 -
20
40
60
20
40
60
•Cl"
Fig.30.Reciprokeofthehalf-lifetimeofReactionIatpH8(a), pH7(b)andpH6(c)
plottedasafunctionofnegativechargeequivalents:CI (closed stars),SO^ (open
stars)andNO-j"(closedcrescents).AllanionswereaddedasMg-salts.
30mmol/l
Fig.31.Reciprokeofthehalf-lifetimeofthedecayofReactionI,measuredbyfastrepetitive(3Hz)flashfiring,afterpre-illuminationwith6flashes,plottedasafunction
oftheMgCl2concentrationinthesuspendingmedium.Closedcirclespresenttheresults
ofexperimentsperformedonbrokenchloroplastswithoutfurthermanipulation;openstars:
brokenchloroplastsafterCF]extractionbymildEDTAtreatment.
lesssigmoidalconcentrationdependenceindicativeforamorecomplexmechanismthanonly
apassiveiondiffusion.ThepermeabilitycoefficientofNO,"andCl~appeartobeequal
exceptatpH7,asmaybeconcludedfromthemorepronouncedeffectofNO,"additionat
thatpH.TheresultsofSO. additionshow,inaccordancewithresultsreportedbyYakovleva&Molotkovskii (1979),thatthisanionisrelativelyimpermeant.
48
6 0mmol/l
ExtractionofCF.byEDTAtreatmenthasbeenreportedtocreateaconductancechannel
throughCF 0 thatretainsitsselectivityforprotons (O'Keefe&Dilley,1977).Results.
depictedinFig.31areinconflictwiththisinterpretation.ExtractionofCF1bymild
EDTAtreatment (chloroplastswereincubatedfor1minin0.5mmol/1EDTA,2mmol/1tricine
pH 8.0),resultsina101increaseintheReactionIdecayrateintheabsenceofsalts
(notshown).ToinvestigatewetherthissmallaccelerationwascausedbyanincreasedprotonconductancethroughCF Q ,thereciprokeofthehalf-lifetimesofReactionIincontrol-andextractedchloroplastswereplottedasafunctionofMgCl,concentration(Fig.
31).Thedifferencebetweentheslopesofthelines,depictedinthisfigure,indicates
thattheCl~andMg conductivityinextractedchloroplastsisenhanced.Theacceleration
ofReactionIdecaycouldbereversedbyDCCDsubstitution (notshown).DCCDisachemical
effectorthatblockstheionconductingchannelthroughCF Q .Thus,Iconcludethatthe
relativehighmembraneconductanceinextractedchloroplastsisnotexclusivelycausedby
aselectiveincreaseoftheprotonpermeability.
6.3 DECAYOFREACTIONIAFTERPRE-ILLUMINATION
Table1showsthehalf-lifetimesofthedecayofReactionIasafunctionofpre-illumination.-Ifpre-illuminationcausesaprotonaccumulationinthethylakoidinnerspacethis
willleadtoanincreaseofthespecificprotonconductanceofthemembrane.Shortpreilluminationof200to500msindeedresultsinanenhancementoftheReactionIdecay
kineticsbutextensionofthispre-illuminationperiodupto32sdoesnotresultina
furtheraccelerationofthedecayofReactionI.Ithasbeenestablishedthatamaximum
ApHacrossthethylakoidmembraneisachievedafter5-30sofillumination (Hindetal.,
1974;Karlish&Avron,1971).ThusitappearsthatthemaximumaccelerationofReactionI
decayafter0.25sofpre-illuminationisnotdirectlycausedbyanincreasedprotonconductanceduetolight-dependentprotonaccumulationinthethylakoidinnerspace.
Ausländer&Junge (1974)andFowler&Kok(1974a)suggestedthattheremightexist
localizedprotonreservoirsonbothsidesofthethylakoidmembrane.Itisplausiblethat
suchreservoirs,seperatedfromthebulkphase,mayleadtoafastriseofprotonconcentrationinthelight,causingasignificant increaseinprotonconductivitywithinatime
spanobservedabove.AdditionofDCCD,thatisexpectedtodecreasethespecificproton
permeabilitythroughCF« (underphosphorylatingconditions),however,didnotresultina
differenteffectofpre-illuminationcomparedwiththesituationintheabsenceofDCCD.
Table 1.Effectofpre-illuminationonthehalf-lifetimeofReactionI.
Pre-illuminationtime(s)
0.1
0.25
0.5
1.0
2.0
4.0
32
Half-lifetime(ms)
67
39
42
43
41
50
48
49
FurthermoreIobservedthattheeffectofpre-illuminationwasaboutsimilarunderboth
phosphorylatingconditionsandnon-phosphorylatingconditionswhereastheprotonpermeabilityisexpectedtobedistinctlydifferent.Thereforeitseemsunlikelythattheobservedeffectsofpre-illuminationareduetoaprotongradientwhetherlocalizedornot.
IsuggestthattheenhancementofReactionIdecayaftershortpre-illuminationisdueto
afastcompetitivereleaseofcationsfromH bindingsitesattheinsidethylakoidsurface.AlocalincreaseoffreeMg concentrationatthemembraneboundarymaycausethe
observedincreaseofmembraneconductance.Accordingly,IobservedthattheeffectofpreilluminationonReactionIkineticsisconsiderablylessinthepresenceofMgCl,concentrationsabove4mmol/1(notshown).Inthiscase,themembraneconductanceislessaffectedbyproton-inducedcationreleasefromthemembranebecauseoftheexistenceofa
highconcentrationoffreemovingionsattheboundaryofthemembrane (seealsoSection
8.6).
6.4 MEASUREMENTOFTHEELECTRICALRESISTANCEOFTHYLAKOIDMEMBRANES
Thesingle-electrodepulsemethod (Lettvinetal.,1958)opensperspectivestoobtaininformationonthemagnitudeoftheresistanceofthethylakoidmembrane.However,themethodhassomeshortcomingsmainlyduetothefactthattherearenomeansforatestofthe
electrodequalityafterimpalement.Thereforethemethodgivesonlyqualitativeinformationontheresistanceofthethylakoidmembrane.Buthitherto,itistheonlydirectmethodavailable.Afterimpalementoftheelectrode,thetotalresistanceofthecircuit,detectedbythepotentialresponseupona400mshyperpolarizingcurrentinjectionof1nA,
wasfoundtobe50-60Mfi.Theresponseuponsuchacurrentpulseisbiphasic(Fig.32b).The
fastphaseisattributedtothepotentialdifferenceacrosstheelectrodetipandisdetermined
bytheelectroderesistance.Theslowphaseisareflectionofthetransmembranepotentialdifferencegeneratedbythechargingcurrentpassingacrossthemembrane.Extrapolationof
thesemi-logarithmicplot (Fig.32b)oftheslowphasetotimezeroshowsthatthepotentialacrossthemembraneisabout2mVataloadingcurrentof1nAindicatingamembrane
resistanceof2Mf!.Similarly,theresponseupondepolarizingpulsescouldbeanalyzed
(notshown).Therelaxationtimeofthecurrent-inducedpotentialchange (Fig.32b)agrees
fairlywellwiththatofthepotentialdecayafterasingle-turnoverflash(Fig.32a).The
membraneresistancederivedfromsuchexperimentsingeneralvariedbetween2and40Mft,
dependingonthemagnitudeofthepulseandthestateofthechloroplast.Iobserveda
maximumvalueof60MŒatadepolarizingpulseof0.25nA(notshown).Highcurrentinjectionabove1-1.5nAgaverisetoapotentialovershootindicativeforthe"punchthrough"effect(Coster,1965)thatiscausedbyasharpdeclineofthemembraneresistanceaboveacertaincriticalpotential.Thevalidityofthepulsemethodissupported
bytheobserveddisappearanceoftheslowpotentialkineticsinthepresenceoftheionophorevalinomycin(Fig.32c)thatisknowntoincreasethespecificmembraneconductance
forK .ItshouldbenotedthattheamountofK inthechloroplastmayincreasedueto
KClleakageoutoftheelectrodetip.
Themembraneconductanceisdeterminedbythepermeabilityofionsandtheamount
ofionsavailableforchargetransportacrossthemembrane.Theelectrodemeasurements
50
Fig.32.a)Time courseof a flash-induced electricpotential and the semi-logarithmic
plot of itsdecaykinetics.The arrow indicates themoment atwhich theflashwas fired.
b,c)Electrical responsesof theelectrodeand the thylakoidmembraneupon ahyperpolarizingcurrent injection intheabsence (b)and thepresence (c)of 30ymol/1valinomycin.
Thearrows indicate themoment ofpulse injection.Extrapolationof the slowphase inthe
semi-logarithmic plot to time zerogives thevoltage,generated across thethylakoid membrane.
arenotsuitableforaquantitative studyoftheselectiveconductanceofthethylakoid
membranebecausevariationofionconcentrationintheinsideofthechloroplastishamperedbytheenvelopebarrier.Thereforethissortofexperimentswereperformedonbroken
chloroplastsusingtheReactionIkineticsofPS15asthedeterminantprobe (Section6.2).
Fig.33givestheI-Vcharacteristicofthethylakoidmembranemeasuredwithasingle
electrodewithreferencetoanexternalelectrode.Before insertingtheelectrode,its
current-voltage linearitywaschecked.Thischeckisimportant topreventafalsifiedinterpretationofresultsduetoartifactscausedbyanon-linearcurrent-voltagerelationshipoftheelectrode.Itisdiscerned,however,that thiscontrolisnotadefiniteprecautionagainstpossibleartifactsbecausetheconditionoftheelectrodetipmaybedifferentbeforeandafterimpalement.Thedifferent ioniccompositionofthethylakoidinteriorandcontaminating substancesclingingtotheelectrodetipmaycausechangesin
electroderesistance.However,thetypicalbendedshapeofthemembrane current-voltage
relation,depictedinFig.33,wasonlyobservedifreproducable light-dependent potential
responsescouldbeevoked.Ifthelatterwererelativelysmall,orevenabsent,thecur-
51
+1J1A
Fig.33.Current-voltagecharacteristicofthethylakoidmembrane.Thecurvesweremeasuredbyapplyingacurrent,from-1nAto+1nAchangingatauniformrateof0.66nAs
Thebrokenlinerepresentsthecurrent-voltagecharacteristicoftheelectrodebeforeimpalement.
rent-voltagecharacteristicusuallywasstraightalthoughaconsiderableincreaseofresistancewasobservedafterelectrodeimpalement.
Afteraratherconstantvalue,adepolarizingcurrentacrossthethylakoidmembrane
wasfoundtocauseanincreaseoftheapparentresistanceuptoacertainlevel.Beyond
thisleveltheresistancewasfoundtodeclineandmightevenbecomezero.Atthishigh
currentflowspontaneousfiringofactionpotentialswasobservedindicatingadramatic
changeofthefunctionalconditionofthemembrane.
6.5 DISCUSSION
ThedatadepictedinFig.33showthattheelectricalresistanceofthethylakoidmembraneisnotaconstantbutratherdependsonthedirectionandmagnitudeofapolarizing
currentfluxthroughthemembrane.Non-linearcurrent-voltagecharacteristicshavebeen
observedintheplasmamembraneof mitella
tvanslucens
(Vredenberg&Tonk,1973)andalso
inthylakoidmembranesof Peperomia chloroplasts (Bulychevetal.,1976).Althoughthe
latterauthorsdonotspecifytherectifyingpropertyoftheelectrode,theyconcludeaboutthesameresistanceforthethylakoidmembraneatzerocurrent (i.e.20-30Nto).
Assumingaspecificconductanceofthethylakoidmembraneof30yS.cm whichcanbe
calculatedfromthedatainFig.32a(seealsoVredenberg,1976)andatotalresistance
of20MS2(Fig.33)itcanbecalculatedthatthemembranesurfaceinvolvedisabout
4 2
2
1 5 x 1 0 vim.Thesurfaceareaofonethylakoidisabout3.5urn.Thereforeitseemsthat
4
about4.3x10 thylakoidsareinterconnected,correspondingtoaninternalvolumeofa3
3
bout110urn.Thetotalvolumeofa Peperomia chloroplastisabout700ym.Theamountof
thylakoidsbeinginterconnectedmightbeevenhigherbecauseinmycalculationthewhole
thylakoidsurfacewasassumedtoparticipateintheionexchangemechanism.However,only
partofthethylakoidsurface (i.e.themargin)isexposedtothestroma;themajorpart
issituatedinthepartitionzone.Theresistancemeasurementssupporttheevidencethat
granumstacksarenoseperateentitiesbutratherformaunity,interconnectedbystroma
lamellae.Paollilo (1970)pointstotheexistenceofaspiralfretwork,interconnecting
individualgranaandgranumstacks.Evenseveralparallelfretsmaybesimilarlyassoci-
52
atedwithasinglegranun.ForareviewseeCoombs&Greenwood (1976).
Theobservednon-linearcurrent-voltagerelationshipofthethylakoidmembranesuggestsevidenceforanasymmetricaldouble-latticemodelofthethylakoidmembrane.Sucha
modelhasbeendescribedforotherbiologicalmembranes (Coster,1965).Inanasymmetrical
fixed-charge latticemodel,anabrupttransitionoftheelectrostatical potential exists
attheso-calleddepletion zone.Theconcentrationofmobileionsinthevicinityofthis
zoneisdependent onthesignandthemagnitudeoftheappliedvoltage.Fluctuationof
themobileionconcentration atthedepletion zonebyanappliedexternal fieldwill
causeconcentrationgradients intheneighbouring fixedchargeregionsand consequently
adiffusionofionsdowntheconcentrationgradientwilltakeplace.Thecurrent
densityduetoiondiffusiondecreases ifthejunctionpotentialismagnifiedby theexternalappliedvoltage.Alternatively,thecurrentdensity increases iftheappliedvoltageisoppositetothejunctionpotential.Asmaybeconcluded fromFig.33,thecurrent
densitythroughthethylakoidmembrane increases ifthesignoftheappliedvoltageis
negative.Thiswouldsuggestthatthefixednegativecharge-density attheinner thylakoid
surface islessthanattheoutersurface.
Analysesoftherelativecontributionofdifferent ionstothethylakoidmembrane
conductance (Figs.28-32)pertaintofourconclusions:
1.TheprotonconductanceofthethylakoidmembraneatpH5-8isnegligibleascomparedtoitsconductance tootherions,bothafteradarkperiodandapre-illumination
period,longenoughtoestablishamaximumprotongradientacrossthemembrane.Thesmall
acceleration ofReaction Iafterashortperiodoflightapparently isnotduetoadirect
effectofprotonaccumulation.Iproposethatthisacceleration istheresultofanincrease inthepermeability ofotherionsoranincreaseofthecationconcentration in
themembraneboundaryduetoexpulsionofcationsfromthemembraneorinnermembrane
double layerbyproton-binding.A competitiveMg extrusioninthedarkuponH addition
hasbeenobservedbyBose&Hoch (1978)..
2.A similarconclusionisdrawnfromtheobservationthatloweringpH apparently
resultsinanincreasedmembraneconductancewhichisnotduetothehigheramountof
availableprotons.Themechanismoftheproton-inducedpermeability increase isnotknown.
Fromtheeffectofsurface-charge neutralisation ontheiondistributionatlowpH,the
Mg concentrationattheboundarymaybeexpectedtodecreasewhichconsequentlymay
causeadecreaseoftheMg conductivity.However,thechanges insurfacechargedensityinthetrajectfrompH6topH 8areonlyminor (cf. Chapter 7).Apossiblemechanis++
ticexplanationmaybegivenbypH-dependent openingandclosingofspecificMg -conductancechannelsoralternatively aH -Mg antiportmechanism (Karlish&Avron,1968)
facilitatedbylowpH (seeSection 8.6).TheobservedcompetitionofMg andH forbindingsites (Bose&Hoch,1978)makestheantiportmechanismacceptable.Theexistence of
specificion-conductance channelshowever,maynotbeexcluded.Suchchannelsmay fulfill
animportantregulatingrole,expeciallywhenthefunctionofthatspecific ionisdeterminedbyitsdistributionoverdifferentcompartments.Therefore,aselectiveprotonchannelthroughtheATP-synthesizing complexisindispensable,buteventhenthe selectivity
apparently isnotdeterminedbythemembranecomponent (CF0)but itisratheraconsequenceoftheCFjbindingattheoutsideofthechannel (Fig.31).
53
3.Inthisrespecttheobserved increaseofselectiveCI conductivityupon lowering
pHfrom8to5mightbecausedbyadecreaseofnegativesurfacechargesfavouring anincreaseofCl~ionsatthemembraneboundary.
4.Theobserveddifferences inpermeability ofanumberofanionsagreewithobservationsofothersusinganindependentmethod (Schuldiner &Avron, 1971).
54
7 Light-dependentconformational changesinthethylakoid membrane
7.1 INTRODUCTION
Basedonavailablekineticdata,atleast twocategoriesoflight-dependent structural
changes canbedistinguished:
-Ratherslowconformational changesoccurringinthesecondtominute timerangedueto
changesintheionicmicro-environmentanddehydrationofcomponentswithinthemembranal
corethatareprobably correlatedwithionfluxesandwatermovementduring illumination
(Packer,1963;Dilley&Vernon, 1965;Murakami&Packer, 1970;Torres-Pereiraetal.,
1974).
-Relatively fastconformational changeswithahalf-life timeof20-100ms(Kraayenhof&
Slater, 1975;Kraayenhof,1975)thatwere foundtobecorrelatedwithanenergydependent
exposureofnegativesitestowards theoutsidethylakoidmembrane surfaceoranocclusion
ofpositivesitesontheATPase (Giaquintaetal., 1973,1974;Kraayenhof, 1975).
Boyer (1965),proposedamechanismofsequentional conformational-energy transduction
inwhichelectron-transportingproteinsareinvolved.Theseconformational changes,which
areprobablyaccompaniedbychangesinpK ofproteingroupsonthe oxidation-reduction
components (Papa,1976)arelikelytoresultinanalteredchargedensity.
Theoccurrenceofconformational changesintheATPsynthetizing complexwas first
observedbyRyrie&Jagendorf (1972)andtheirfindingswere confirmedbyothers (McCarty
& Fagan, 1973;Lutzetal., 1974;Kraayenhof&Slater, 1975;Harris&Slater, 1975).
Isuggested (Chapter 5)thattheslowcomponentoftheflash-induced absorbance
changeat515nm,wasareflectionofanaltered conformationofproteinsorlipidsinthe
membrane,inducedbyanelectrogenic effectnearPhotosystem 1.Thiswasproposedtolead
toachangeinstrengthandorientationofinternalelectric fieldsneartheP515pigment
complex.ThereforeIinvestigatedwhethertherewassomecommonmechanisticbasisforfast
conformational changesoftheATP-synthetizing complex,ReactionIIandtheproposed
changesinelectricalchargedensityofthemembrane,inthecourseofphotosyntheticenergy transduction.
Mostbiologicalmembranes includingchloroplastmembranes (Dilley&Rothstein,1967;
Gross&Hess,1974;Bergetal., 1974;Gitier, 1971)bearanetnegativesurfacecharge
inthephysiologicalpH-domain.Thecorrespondingnegativeelectricsurfacepotentialmay
beconsideredasanentitythatcontrolstheioniccompositioninthediffusedouble layer
adjacenttothemembrane surfaceandthereby intrinsicmetabolicandstructuralmembrane
properties,includingthetranslocationofions (Gross&Prasher, 1974;Barberetal.,
1977; Theuvenet&Borst-Pauwels,1976a, 1976b).Anumberofsurface-relatedphenomena,such
astheelectrostatic absorptionofdyemolecules (Montai&Gitler,1973;Searleetal.,
1977)anduncouplers (Bakkeretal., 1975)seemtobeingoodharmonywiththe theoryof
55
thediffusedoublelayerdevelopedbyGouyandChapman (Davies&Rideal,1963;Aveyard&
Haydon,1973).McLaughlin (1977)hasreviewedgeneralaspectsofthistheorywithspecial
emphasistoelectrostaticphenomenaofsurfacesofbiologicalmembranes.
A 15?,increaseofthe (negative)surfacechargehasbeenobserveduponillumination
ofintactspinachchloroplasts (Nobel&Mel,1966).Inarecentelectrophoreticstudyon
membranesofintactchloroplastsandofdivalentcation-depletedthylakoidsmembranes,
Nakatanietal. (1978)didnotobserveanylight-inducedincreaseofsurfacechargedensity.
Inthischapteracharacterizationoftheelectrokineticbehaviorofchloroplastsin
responsetoenergizationbylightwillbegiven.Theresultsarediscussedinrelationto
theprocessesthatgiverisetotheReactionIIresponseofP515.SeealsoSchapendonket
al. (1980).
7.2 MATERIAL
Absorbancechangesweremeasuredinchloroplasts,preparedasdescribedinChapter2.All
othermeasurementswerecarriedoutwithchloroplastspreparedasdescribedbelow."Intact"
(outermembrane-containing)chloroplastsweremadefromfreshlygrownormarketspinachby
homogenizationofpre-cutleaves (10g)withacavitationdisperser(IKAUltra-Turrax,
equipedwith18Nshaft)for2x1satmaximumspeed,followedbyfiltrationthrough8
layersofperlonnet (40 \mmeshwidth)and1mincentrifugationat2000 g. Theisolation
andresuspensionmedium (usually50mland2ml,respectively)contained330mmol/1sorbitoland2mmol/1tricine,pH7.2and,whereindicated,5mmol/1MgCl7.Brokenchloroplasts
wereobtainedbysuspendingthepelletsin1ml ti?0 or10mmol/1MgCl,for1min,after
which1mlofdouble-strengthmediumwasadded.Theelectrophoresismediawereasindicatedinthelegendstothetablesandfigure.Themethodformeasuringelectrophoretic
mobilityofchloroplastparticlesisdescribedinChapter2.
7.3 SOMEELECTROKINETICDATAOFDIFFERENTCHLOROPLASTPREPARATIONS
Theelectrophoreticmobility (u)ofchloroplastsandtheeffectofilluminationwerefound
todependonchloroplastintegrity.Brokenchloroplastsshowedaslightlylargerdarkmobilityandthelight-stimulation (àu^. )wasfoundtoberoughly2to5timesthatof
intactones.
Thescreeningofnegativechargesonthethylakoidmembranesbythechloroplastenvelopeisprobablythereasonfortherelativelylowincreaseinmobilityinintactchloroplasts.TheeffectofMgCl2onthemobilityofbrokenchloroplastswasinvestigatedand
theresultsaresummarizedinTable2.Chloroplastsisolatedandbrokenintheabsenceof
MgCl,»butwithoutdrasticdepletionofbivalentcations (Nakatanietal.,1978),showa
decreaseofelectrophoreticmobilitiesandoflight-stimulationuponincreasingtheMgCl,
concentrationintheelectrophoresismedium (Table 2).Theobviousadvantageofusing
brokenchloroplastsisolatedintheabsenceofcations,isthattheyprovidealesscomplicatedmembranesystemmoresuitableforcomparingthecationeffectswiththeoretical
modelsaspredictedbytheGouy-Chapmantheory.Theresultsofthesetestsindeedarefound
56
Table2.EffectofMgCl2ontheelectrophoreticmobilityofbrokenchloroplasts
prepared intheabsenceorpresenceofMgC^ 3 .
Experiment
MgCl2 (mmol/1)
0
0.05
1.0
0.05b
1.0
-K-105 (cm2-V_1-s-1)
A
(%)
")
VL
dark
light
19.9
19.0
13.0
28.5
24.5
14.6
43
29
J2
13.8
13.6
26.9
18.2
95
34
a.Thebrokenchloroplastswereisolated intheabsence (expt.A)orpresence (expt.B)
of5mmol/1MgCl?*Inbothexperimentselectrophoresiswascarriedoutinisolationmedium
plus10iimol/1diquat.Chlorophyllcontentwas 10ug/ml.
b.ThisconcentrationofMgCl2wasintroduced intothemediumwiththechloroplastsuspension.
tobeingoodharmonywiththetheory (Searleetal.,1977;Nakatanietal., 1978).If
chloroplastmembranesarekeptunderamorephysiological conditionbyisolationinthe
presenceo£Mg ions,theyshowasmallerelectrophoreticmobilityinthedark,butthe
stimulatory effectoflightatlowMg concentrationisconsiderably increased.Further
experimentstobereportedhereareperformedwithbrokenchloroplastsonly.
7.4 THERESPONSIVENESSOFELECTROPHORETICMOBILITYANDREACTIONIIUPONCHEMICALTREATMENT
ThepH-dependenceofthedark-andlight-mobilitiesofbrokenchloroplastsisshownin
Fig.34.BothmobilitycurvessharplydeclinebelowpH6andindicateachargeneutralizationpointatpH4.8,roughlyconfirmingtheresultsofNakatanietal. (1978)onnon-
-uK^Ccm2^-1^-!)
40
8 pH
Fig.34.Theelectrophoreticmobilityofbrokenchloroplastsindark (closedcircles)and
light (opencircles)asafunctionofpH,measured intheabsenceofMgCl2-Experimental
conditionswereasinTable2A.
57
energizedchloroplastmembranes.ItappearsthatilluminationcausesexposureofadditionalnegativegroupswithidenticalpK asthegroupsalreadyexposedinthedark.
Fig.35showssingle-flashinducedabsorbancechanges,measuredatpH6.7andpH5.2.
TheslowdecaycomponentrepresentativeforReactionII,whichisclearlyobservedatpH
6.7appearstobeabsentatpH5.2.AtthispHonlyReactionIisobserved.Aplotofthe
ReactionII/ReactionIratio,asafunctionofpH,isdepictedinFig.35.Thereappears
tobeamaximumvalueforthisratioatpH6.7andasharpdeclinebetweenpH6andpH
5.2.ThemagnitudesofReactionIandReactionIIwereanalyzedasdescribedinChapter5.
Table3liststheeffectsofchemicalandphysicaltreatmentsontheelectrophoretic
mobilityofbrokenchloroplasts.TheelectrontransferinhibitorDCMUdoesnotaffectthe
®
b*nj
©
H
Q5
0-
-*S\
6
8
pH
Fig.35.Upperpart:ResponseofP515toflashesfiredatarepetitionrateof0.05Hz,
atpH6.7 (a)andpH5.2 (b),respectively.Lowerpart:theratioReactionII/ReactionI,
plottedasafunctionofpH(c).
58
Table3.Effectsofelectrontransferinhibitor,uncouplersandphysicaltreatmentson
theelectrophoreticmobilityofbrokenchloroplasts3.
Additionortreatment
None
DCMU (0.5ymol/1)
DCMU (0.5ymol/1)+PMS (6ymol/1)-diquat
CCCP (5ymol/1)
S-13(lymol/1)
ACMA (15ymol/1)
1.5min60°C
2min2.5%glutaraldehyde
valinomycin (1ymol/1)
valinomycin(5ymol/1)
-u-105 (cm2-V_1-s-1)
AM
dark
light
14.0
14.0
14.9
17.4
13.5
12.1
5.3
6.7
12.6
12.1
27.0
15.0
27.1
14.8
14.1
14.0
5.1
6.9
20.7
12.5
^
n-»i
93
7
82
-15
4
16
- 4
3
64
3
a.Thebrokenchloroplastswerepreparedinthepresenceof5mmol/1MgCl2.Theelectrophoresismediumcontained330nmol/1sorbitol,10mmol/1KCl,2mmol/1tricinebuffer,
pH7.2,10ymol/1diquat (0.05mmol/1MgCl2introducedwithchloroplast suspension).Chlorophyllcontent 10yg/ml.
dark-mobilitybutpreventsthelightstimulation.AdditionofPMScompletelyrestoresthe
lighteffect.AsimilarobservationhasbeendescribedinChapter5forReactionII.
Theeffectofvalinomycinwastestedunderavarietyofconditions.Wefoundthat
thisantibioticinvariablyeliminatesthelight-stimulationbothinthepresence (Table3)
andabsence (notshown)ofaddedK ions,whichisincompleteagreementwiththeobservedinhibitionofReactionIIbytheionophore (cf.Chapter 5), exeptforthefactthat
ReactionIIappearstobeinhibitedatsomewhatlowerconcentrations.Thisdiscrepancy
mightbeduetothedifferenceintheisolationproceduresused.Treatmentswhichinhibit
ReactionII,arealsofoundtodiminishthelight-inducedincreaseofnegativesurface
charges:Ashortheat-treatment (1.5minat60°C)ofachloroplastsuspensiondrastically
lowersthedark-mobilityandabolishesthelight-effect.Ageingofthechloroplastsfor3
hat25°Cdidnotaffectthedark-mobilitysignificantlybutgraduallydiminishedthe
light-stimulation (notshown).Treatmentofthechloroplastsfor2minwith2.5%glutaraldehyderesultsinadecreaseddark-mobilityandaninhibitionofthelight-stimulation.
West&Packer (1970)reportedthatinthelattercaseATPsynthesisisimpaired,whereas
electrontransferandH uptakestillproceeds.
7.5 REACTIONIIASAPOSSIBLEINTRINSICMARKERFORTHECONFORMATIONALSTATEOFTHECOUPLINGFACTOR
Fig.36showsthesemi-logarithmicplotofthe515
ranabsorbancedecayafteraseriesof
7flashesintheabsenceandthepresenceofMgCl,(Fig.36aand36b,respectively).The
absorbancelevelinthe0.4-1 stimedomainwhichisduetoReactionII(cf.Chapter5)
wasfoundtobeappreciablylowerinthepresencethanintheabsenceofADPandK^HPO.,
bothintheabsence (Fig.36a)andthepresence (Fig.36b)ofMg + + ..Theionicstrengthof
theassaymediumintheabsenceofK-HPO.wasadjustedtoanequivalentmolaritywithKCl.
ThedecaykineticsofReactionIIappeartobesimilar,althoughintheabsenceofMg + + ,
59
AA(a.u.)
40 r
A.
^-n—
10
-*-
05
1s
40
\
,v
®
10
*-*.
0
Q5
1s
Fig.36.Semi-logarithmicplotsoftheabsorbancedecayat515ranafteraseriesof7
flashesfiredat100mstimeintervalsinthepresenceofeither 10mmol/1KCl (a)or
10mmol/1MgCl2(b).TheP515decayunderphosphorylating-(+0.8mmol/1ADPand 1mmol/1
KH2PO4)andnon-phosphorylatingconditionsisindicatedbyopenstarsandclosedcircles,
respectively.Thelogarithmsoftheabsorbancechangesaregiveninarbitraryunits (a.u.).
thedecayisslightlyfasterinthepresenceofADPandK-HPO..ThedecayofReactionI
wasfoundtobeunaffectedbyADPandK2HPO.(Section6.3).Conclusivewithresultsof
Girault&Galmiche(1976)wefoundthatadditionofATPhadqualitativelythesameeffect
onReactionIIasADPandK2HPO..Accordingly,theapparentaccelerationoftheoverall
responseinthepresenceofADPorATPmustbeduetoadecreasedcontributionofReaction
11totheoverallresponse.Thesedatasuggestthattheinductionofchangesinthelocal
electricfield (Reaction II),ispartlyaffectedbyanalteredconformationofamembrane
component,possiblytheATPasecomplex,duetobindingofADPorATP.
Fig.37aand37bshowtheanalysesoftheP51Sresponseintheabsenceandthepresenceof4pmol/1DCCDpermgchlorophyll.InthepresenceofDCCD,ReactionIIappearsto
becompletelyinhibited.TheslowprocessthatisresponsiblefortheDCCDinsensitivepart
oftheabsorbancechange (Fig.37b)isnotyetknownbutitsslowriseanddecaykinetics
whicharedifferentfromthekineticsofReactionII,leadtothesuggestionthatsome
otherasyetunknowneffectmightbeinvolved.Asimilarabsorbancecomponentwhichwe
calledphased(cf.Chapter 5),wasobservedintheabsenceofDCCD.Wedidnotcorrect
60
DCCD(«mol)
-V*
Fig. 37.Timecourseof theP515 absorbance changes inbroken chloroplasts intheabsence
(a)and thepresence (b)of 4umolDCCDpermg chlorophyll,respectively, induced by repetitive single-turnover flashes,fired at arepetition rateof 0.05 Hz.Reaction Ikinetics
(solid curves)weremeasured immediately after pre-illuminationwith 6flashes,firedat
arepetitionrateof 3Hz.In (c)therelative contribution ofReaction I (open squares)
and Reaction II (closed circles) isplotted as a functionof theadded amount ofDCCDper
mgchlorophyll.
forthisphasebecauseofitsrelativelysmallcontribution.Fig.37cshowsthemaximum
extentofReactionIandReaction II,respectively,asafunctionoftheamountofDCCD.
ReactionIIiscompletely inhibitedataDCCDconcentrationatwhichthemagnitudeof
ReactionIcorrespondsto75%ofthecontrol.Theobserved decreaseofReactionIupon
additionofDCCDprobablyiscausedbyasecondaryeffectduetoinhibitionofelectron
transport (Uribe,1971).Thiseffectmightalsoberesponsibleforapartial inhibition
ofReactionII,becausetheonsetoftheconformational changeassociatedwithReactionII
issuggestedtobeprimarily causedbyanelectrogenicmechanism locatedatthePhotosystem1site (Section 5.7). However,thedifferentconcentration levelsofDCCDatwhich
ReactionIandReactionIIareinhibited (Fig.37c),indicate thatReactionIIinhibition
cannotexclusivelybecausedbyinhibitionofelectrontransport,evenwhenDCCDwould
haveapredominanteffectonelectrontransportactivatedbyPhotosystem 1only.Moreover
ithasbeenobservedthatrestorationofelectrontransportinDCCDpoisoned chloroplasts
byadditionof6pmol/1PMSresultsinacompleterecoveryofReactionIbutwithouta
concomitant recoveryofReactionII(notshown).
ExtractionofCF 1bymildEDTAtreatment (chloroplastswereincubatedin0.5mmol/1
EDTAand2nrnol/1tricine (pH8)resultedinapartialinhibitionofReactionII(Fig. 38).
Theabilityofchloroplaststophosphorylateortocarryoutlight-dependentH uptake
isknowntobelostundertheseconditions (Avron,1963;McCarty&Racker,1966).ThisagaingivesanindicationthattheprocessesreflectedbyReactionIIareconnectedwith
61
WV
-wv
Fig.38.TimecourseoftheP515absorbancechangeinbrokenchloroplastsbefore (a)and
afterEDTAtreatment (b),inducedbyrepetitivesingle-turnoverflashesfiredatarepetitionrateof0.05Hz.Theresolvedresponsesareobtainedaccordingtotheanalysis
discussed inChapter5,Fig.15.
thefunctionalintegrityoftheATP-synthesizingsystem.AnattempttoinhibittheconformationalchangesinCF..andconcomitantlyReactionIImorespecificallybytreatmentwith
anti-serumagainstCF,,wasunsuccesfull.TheisolatedCF..fractionwasfoundtobehighly
pureaccordingtotheelectrophoreticbandpatternina SDS-polyacrylamidegel,butthe
antibodiesinhibitedneithertheCa -dependentATPase (J.Verheyen,personalcommunication),northelight-dependentATPsynthesis,northeelectrontransportrate.Although
theantibodiesdidprecipitatewithantiserumandchloroplastagglutinationwasobserved,
noinhibitionwasfoundtooccur,evenwhentheantibodywastitratedoverawideconcentrationrange.Theseresultsareinagreementwiththoseofothers (Nelsonetal.,1973;
Shoshanetal.,1975)whoshowedthatantibodyfunctioninginrelationtoATPsynthesis
isconfinedtoveryspecificpartsoftheCF..ratherthentoanindispensableunityofthe
totalCF
7.6 DISCUSSION
Thepresentexperimentsshowthatilluminationofchloroplastsinthepresenceofsuitable
electrontransfermediatorscausesamarkedenhancementofparticlemobility,theeffect
beingmostpronouncedinbroken(envelope-free)chloroplasts.Thesmallerlight-stimulation(15-301)in"intact"chloroplastswouldconfirmearlierobservationsofothers
(Nobel&Mel,1966).Obviously,amoreorlessintactoutermembraneshieldsthethylakoid
surfacechargeeffects (andchangesthereof).Ontheotherhand,thestructuralandfunctionalintegrityofthethylakoidmembranesthemselvesisaprerequisiteforthelighteffect.Extensivedepletionofdivalentcationsandtris-washingpriortotheelectrophoresisexperiments (Nakatanietal.,1978)leadstoamodificationofgrosschloroplast
structure (granastacking)aswellasofthemolecularstructureofthethylakoidmembrane;
thelipidbilayerisexpectedtobecomelessorderedandmembrane-boundproteins,including
theATPase,areremoved.Earlierworkondivalentcationbinding (Grossetal.,1969;
62
Gross, 1972)showsthatdivalent cationdepletionleadstouncouplingof energy-linked
functions.Thismayexplainthelackoflight-stimulation ofelectrophoreticmobility in
the thylakoidpreparationsusedbyNakatani etal. (1978)and thelowextent orevenabsenceofReaction IIinchloroplast thatarebrokenandincubated inabsenceofsalts.
Incontrast totheobservations ofNobel&Mel (1966)thelight-inducedincreaseof
thenegativemembrane surfacechargedensity, liketheoccurrenceofReaction II,isshown
tobeuncoupler-sensitive (Table3)andseemstogohandinhandwiththelight-induced
conformational changesdetectedasanelectrostatic interactionofchloroplastswith
cationicprobes,suchasaminoacridines (Kraayenhof, 1973),ethylred (Heath, 1973)and
PMS (Homaim,1976).However,itshouldberemembered thattheReactionIIkineticswere
studiedinflashilluminationand theelectrophoretic experimentswerestudied incontinuous illumination.Withthisinmind,oneis leftwiththenotion thatadditionofvalinomycin,acidification,additionofDCMJandPMSandvariousphysical treatments,have
qualitatively thesameeffect onboth theextent ofReaction IIandthe light-stimulated
increase insurfacechargedensity.
Themagnitudeofthechargedensitycorresponds toa zêta (ç)potential of-18mV in
thedarkand-37mV inthelight (Schapendonk etal., 1980).Itseemsveryunlikelythat
thelight-inducedincrease ofnegative surfacecharges isadirect resultofchargereorientationatthephotosyntheticreactioncenters,sincethesechargechangesareexpected
tobe toosmalltobedetectedbyelectrophoresis (Nakatanietal., 1978).From chemical
modification studies,Nakatani etal. (1978)deduced thatthethylakoidsurfacechargeis
mainly determinedbycarboxylgroupsofasparticandglumaticacidwhichsuggests that
membraneproteinsaremost likelyinvolvedinthealteredchargedensityupon illumination,
describedhere.
TheobvioussimilaritybetweentheeffectofpHontheextentofReaction II (Fig.
35)andonthedecreaseofthelight-induced changeofthenegativechargedensity (Fig.
34)respectively,expeciallythenarrowtransitionphasebetweenpH6andpH 5,isin
goodagreementwiththesuggestionthatbothReaction IIandtheobservednegative charge
exposureareanexpressionofareorientationoffixednegative chargesassociatedwith
conformational changes inthemembranalcore.
The factthatbothReactionIIandthelight-stimulated generationoffixednegative
chargescanbecatalysedbyPhotosystem 1only,alsosuggeststheexistenceofa common
basicprinciple (compareTable3andFig.27).IfReaction IIandalsoaminoacridinebindingarebelieved tooriginate fromthesameelectrogenicprocessasthechanges insurfacechargedensity,thenthekineticsoftheformerprocesses showthatthe generation
ofsurfacecharges is much faster thantheenergy-dependent increaseofchloroplast lightscatteringandbulkiontranslocation.ThegenerationofnegativechargesandtherisetimeofReaction IIappeartocoincidewiththelight-induced conformationalchange in
themembrane-boundATPase,monitoredwithacovalent fluorescent label (Kraayenhof, 1977b).
Iwant topaysomeattentiontothefunctional aspectsofbothReaction IIandthe
electrophoretically detectedchargeexposure inthelight,inparticularwithrespectto
theactivityoftheATP-synthesizing complex.Itshouldbenoticed thatthe innersideof
thethylakoidmembrane alsohas anexcess offixednegativecharges,whichwill decrease
uponillumination inconjunctionwithprotonuptake (Witt&Zickler, 1973;Fowler&Kok,
63
1974b).Theresultanteffectmaybealocalizedelectricfieldofsignificantstrength,
whichmightcontributetotheprotonmotiveforce(Rumberg,1977)andwhichmightbedetectedbytheP515pigmentsystem,resultingintheappearanceofReactionII.
Alternativelythereisnodirectevidencethattheproposedchangeinmembraneconformation,reflectedbytheincreaseinsurfacechargedensityandtheappearanceof
ReactionII,isameasureoftheenergizedstateofthethylakoidmembrane,although,the
observedinhibitoryeffectsofuncouplerslikeS13andCCCPontheseprocesses (notshown
forReactionII)wouldconfirmsuchaninterpretation.IfthemagnitudeofReactionII
wouldreflectsuchenergizedstate,orevenwouldbequantitativelylinkedwiththeintermediaryhigh-energystateoftheATP-synthesizingcomplex,thenitsdecreaseinthepresenceofaddedATPcannotbeeasilyunderstood.Moreover,thedecayrateofReactionII
wouldbeenhancedunderphosphorylatingconditions.Accordingtotheresultsshownin
Fig. 36,thisappearsnottobethecase.However,itmightbethattheregulationofthe
capacityoftheATP-synthesizingcomplex,governedbyachangeinitsconformation,goes
handinhandwithmembraneconformationalchanges,thatfindtheirexpressionintheappearanceofReactionIIandthechangeinsurfacechargedensity.TheinhibitionofReactionIIbyEDTAextractionofCF.,orbyadditionofDCCDeveninclinetotheideathat
ReactionIIisassociatedwithaconformationalchangeofthecouplingfactoritself.An
attempttofixtheconformationoftheATPasemorespecificallybyantibodycomplexation
unfortunatelywasunsuccessful.
Harris&Crofts (1978)suggestedthattheactivationofATPproductioniscontrolled
byadistortionofalocalelectricfieldinthevicinityoftheATPasecomplex.ThecharacteristicsofReactionIIsuggestthatundersomeconditionstheP515complexresponds
similarly,tothesedistortedfields.However,modificationofeithertheATPaseorthe
P515complexmightresultinanalteredandmutuallydifferentresponsiveness.Nevertheless,themanifestationofthesedynamicmembranephenomenapointstothefunctionalimportanceofamechanismthatishighlyaccessibletoinfluencesofpHandionmovements
andprovidesapossibilityforregulationoftheATP-synthesizingcapacity.Inthiscontext
itisofinteresttorecall (Chapter5)thatReactionIIisactivatedbyPhotosystem1and
asmaybeconcludedfromdataofCrowtheretal. (1979)itsrateofappearanceissubstantiallyenhancedunderconditionsatwhichcyclicelectrontransportoccurs.Thiswould
mean,ifourinterpretationiscorrect,thatthelight-activationoftheATPaseismainly,
ifnotexclusively,dependenton (cyclic)electrontransportaroundPhotosystem1.Inagreementwiththishypothesis,electron-microscopystudiesofthylakoidmembranesrevealed
thatthemargins,stromalamellaeandendmembranescontaintheATP-synthesizingcomplex
andonlyPhotosystem1,whereasthepartitionregionsofthegranacontainbothPhotosystem1andPhotosystem2butnoATPasecomplex(Saneetal.,1970;McCartyetal., 1971).
FurtherexperimentshavetobedesignedtoclarifytheverydetailsofthemembraneprocessesthatcontroltheefficiencyoftheenergytransductionreactionsthatleadtoATP
productionandiondisplacements.
64
8 TheP515responseandthephoto-electricalresponseduringprolonged
illumination
8.1 INTRODUCTION
Thekineticsandmagnitudeoftheelectricalcomponentoftheelectrochemicalprotongradientincontinuous light,resulting fromcapacitivemembranecharging,followedbyH
uptake andfielddissipatingionfluxes,arestillamatterofdiscussion.Fromchanges
indelayed lightemissioninchloroplastmembranes,Barber (1972b)concluded thatthe
steady-statepotentialinthelightis75-105mV.Fromthedistributionofpermeant ions,
equilibratinginthelightaccordingtoaNernstpotentialequaltothesteady-statepotential,avalueofabout 10mVhasbeendeduced (Rottenbergetal., 1972).Thisvalue
was foundtobesimilartotheoneobtainedbydirectmeasurementswithamicro-electrode
inserted intoastackofthylakoids (Vredenbergetal., 1973).
Thekineticsoftheabsorbancechangesat515nmincontinuous lighthavebeenproposedtoreflectchangesofthetransmembraneelectric field.Thekineticsandmagnitude
oftheseopticalchangesincontinuous light,aswellasinflashlight (Chapter5)were
foundtobedifferentfromthetransmembranepotentialasmeasuredbymicro-electrodes
(Larkum&Bonner,1972;Vredenbergetal.,1973;Thorneetal., 1975;Krendelevaetal.,
1977).Opticalmeasurements,however,havebeencriticizedbecauseoftheoccurrenceof
scatteringchangesthatmaycontributetotheobservedP515changes (Thorneetal., 1975;
Deameretal., 1967).Totesttheeffectofscattering changes,absorbanceandscattering
changesinthelightwererecordedsimultaneouslyat515nmundervariousconditionsand
anattemptwasmadetocorrecttheapparentabsorbancechangesforthescatteringchanges.
Theresultsarepresentedinthischapterandcorrectedopticalchangesarecomparedwith
thepotentialresponsesmeasuredbymicro-electrodes.
8.2 MATERIAL
ChloroplastswerepreparedaccordingtothemethoddescribedinChapter2exceptforthe
followingmodifications.AfterhomogenizationinaOmniMixerfor5-10s,thechloroplasts
werecentrifugedat1650 gfor1min.Itshouldbementioned thatundertheseconditions
thechloroplastenvelopemightbebrokenpriortoosmoticshocking.Themeasuringsystems,
todetectabsorbancechanges,scatteringchangesandlight-dependentMg effluxaredescribedinChapter 2.Althoughthechloroplasts,usedinelectrodeandabsorbance (difference)measurements,areofdifferentorigin,theP515responsesofintactspinachchloroplastswerefoundtobequalitatively similartothoseinintactleavesofPeperomia and
spinach. Peperomia chloroplastsweredifficulttoisolate,thereforespinach chloroplasts
havebeenusedintheP515measurements.
65
8.3 P515ABSORBANCECHANGESANDCHANGESINLIGHTSCATTERING
Thetimecoursesoftheelectricalpotentialinthelight,measuredin Peperomia chloroplastswithmicro-electrodes (Fig.39b),isobviouslydifferentfromthetimecourseof
theabsorbancechangeat515nm (Fig.39a),measuredinisolatedspinachchloroplasts.The
mostprominentdetailistheobservedhighsteady-stateabsorbancelevelreachedinthe
latter.Inordertoinvestigatetowhatextentthishighabsorbancelevelwascausedby
scatteringchanges,aphenomenonthatiswellestablishedinthylakoidpreparations(Itoh
etal.,1963;Packeretal.,1965;Croftsetal.,1967),bothabsorbancechanges (AA)and
90-scatteringchanges (ASc)wererecordedsimultaneously (Fig.40a).90-Scattering
changesofthylakoidmembranes,measuredat515nm,aremostlikelyamanifestationof
selectivedispersionduetoachangeoftherefractiveindexneartheabsorptionpeak
(Latimer&Rabinowitch,1956,1957),leadingtoscatteringmaximaatthelong-wavelength
sidesoftheabsorptionmaxima.Iftherefractiveindexofthemediumisincreasedtowards
thatofthethylakoidmembranes,byadditionof30*0bovineserumalbumen,thescattering
maximumshiftstowardstheabsorbancemaximumandthis,asexpected,resultsinadecrease
ofthelight-induced90-scatteringchanges (compareFigs40aand40b).Thus,incontinuous
light,usingalowchloroplastdensity,asignificantpartoftheapparentabsorbance
changesat515nmmaybecausedby90°-scattering.
Ifscatteringchangesaresuppressed (Fig.40b)thekineticsoftheabsorbancechanges
arequalitativelysimilartothepotentialresponseasprobedwithanelectrode (e.g.compareFig.39band40b).
®
10mV
Fig. 39.Timecourseofthelight-inducedabsorbancechangesat515nmofbrokenspinach
chloroplasts (a)andthetimecourseoftheelectricpotentialacrossthethylakoidsofa
Peperomia metatlioa chloroplastmeasuredbymicro-electrodes (b).Upwardanddownwardpointingarrowsmarktheonsetandendoftheillumination,respectively.
66
AA1-10-3
ASd2510"2
i
1
0.5min
i
0L5
1
min
Fig.40.Simultaneous recordings ofthelight-induced 515tunabsorbance changes (upper
part)and90°-scattering changes (lower part). Chloroplasts aresuspended in0.33mol/1
sorbitol.5mmol/1MgCl2> 5mmol/1 HEPES,pH6.5,intheabsence (a)andthepresenceof
30% bovine serumalbumen (b).Chlorophyll concentrationwasequivalent to25ug/ml chlorophyll (optical path 1cm).Measurements wereperformed inthedouble-beammodewitha
dark reference.Theupward anddownward pointing arrowsmark thestart andtheendofan
illumination period, respectively.
8.4 PROTON TRANSLOCATION
Convincing argumentshavebeengivenbyothers (Deameretal., 1967;Thomeetal., 1975),
favouringtheideathatthe90°-scattering changesreflect small alterationsoftheindex
ofrefractionduetothylakoid loadingofprotonsandconformational changesinthethylakoidmembrane.Fig.41ashowstheeffectofpH,bothonthe515nmabsorbanceandonthe
90-scattering.AloweringofpHresultsinanincreaseofboth90-scatteringandabsorbance.Thelinearrelationbetweentheabsorbance increaseandthescattering increase
indicates thattheformerisadirectreflectionofthelatterwhichinturnmaybeassociatedwithprotonationofthethylakoidmembrane.Thereisadeviationfrom linearityat
highscattering changes (belowpH4.5).Thereasonforthisisunknownasyet.TheAA/ASc
ratio (inthelinearregion)wasfoundtodependonthechloroplast density.
Figs42aand42bshowthetimecoursesoflight-induced absorbanceandscattering
changesinathylakoid suspensionisolatedinthepresenceofMgCl-andincubatedina
lowsalt (0.5mmol/1MgCl,)assaymedium.TheAA/ASC ratiounder theseconditionswasdeterminedaccordingtothemethoddepictedinFig.41.Assumingthatthelight-dependent
processes,andtheloweringofthepHinthedark,giverisetothesameAA/ASc ratio,a
67
2
K) 2
ASc-102 A A•10
0-
AA-102
(ï)
\.
•3
25-
jT
(b)
3-
V AS
\
\.
S.
2/
/
\
*
/
; AA
50- -1
\ \
/
1-
/
/
/
l_^
6.0
I
5.5
•
•
5.0
4.5
4.0pH
25
Fig.41.Simultaneousrecordingsoftheabsorbance (closedcircles)and90-scattering
changes (closedstars)measuredat515
ran,
plottedasafunctionofpH (a).Anupward
inflectionoftheabsorbancecurveandadownwardinflectionofthescatteringcurveindicateanabsorbanceincreaseandascatteringincrease,respectively,b)PlotofAAasa
functionofASc,derivedfromtherecordinginwhichthechloroplastconcentrationwas
equivalentto130ug/mlchlorophyll (opticalpath1 cm).
correctionfortheapparentabsorbanceincreaseinthelightcanbemadebysubtracting
thelight-inducedscatteringchange,multipliedbytheestimatedAA/AScratio(Fig. 42c).
Theresultantcorrectedkineticpattern (Fig.42d)showsatimecourseoftheabsorbance
changemuchmore,ifnotcompletely,compatiblewiththetimecourseoftheelectrode-detectedelectricpotential (Fig. 39b).
Differentandcomplicatedresultswereobtainedinchloroplaststhatwereisolated
andbrokeninion-freemedium.Thesechloroplastsshowedalight-inducedscatteringdecreasethatappearedtobemostpronouncedatpH5.9-6.3andtobenegligibleatpH8
(Fig.43).InthelattersituationthekineticpatternoftheP51Sresponsewasqualitativelysimilartotheresponsemeasuredinthepresenceofions,butcorrectedforscatteringchanges (cf.Fig.42d).ThecorrectionmethodcouldnotbeappliedforthescatteringdecreaseatlowpH.Inthiscase,thecontributionofanincreaseofforwardscattering,isprobablyappreciable,duetoshrinkageoftheunstackedthylakoids.Therefore,
adifferentanalysisandcorrectionprocedureshouldbeappliedinthiscase.
8.5 EFFECTOFMg + +ONLIGHT-INDUCED90°-SCATTERINGCHANGES
Mg
extrusioninion-freepreparedbrokenchloroplastsinthelightwasfoundtobe60-
80nmol/1Mg permgchlorophyllin20s(Fig.44).Iassumethat,inthelight,Mg is
notreleasedfrombindingsitesattheouterthylakoidsurfacebutitisextrudedeither
fromthethylakoidinteriorvolumeorfromtheinnerthylakoidsurface,dissipatingthe
electrochemicalpotentialgradient.Withachloroplastvolumeof26ul/mgchlorophyll
68
50 ASc-10 2
AA-10'
©
y
8-ASc-IO2
AAM O 3
30s
30s
Fig.42.Simultaneous recordingsof light-induced absorbance changes at 515nm (a)and
scattering changes (b)ina suspension ofbroken chloroplasts containing 0.33 mol/1 sorbitol, 2mmol/1MES,pH 6.3, and inaddition 0.5mmol/1MgCl2.Therelationbetween the
absorbance and scattering increase (c)isdetermined according to themethod depicted in
Fig.41.The resolved light-induced absorbance change isdepicted ind. Chloroplast concentrationwas equivalent to 25ug/mlchlorophyll (opticalpath 1cm).Furtherdetails
are inthe text.
(Heldtetal., 1973),extrusionfromthethylakoidinteriorvolumeduringillumination
wouldgiveacalculateddecreaseinMg concentrationof2-5nmol/1.However,only0.24
mmol/1wereavailableatthestartoftheexperimentindicatingthatthemajorpartofMg
ismembrane-bound.ThereleaseofboundMg inthelightmayaccountforthescattering
decreasedepictedinFig.43.Theamountoflight-dependentMg effluxacrossthethylakoidmembranehasbeenreportedtobehighatlowpHandtobenegligibleatpH8.6(Ben-Hayim,1978;Portis&Heldt,1976).ThepHdependenceofthe90°-scatteringdecreaseis
fullycompatiblewiththesefindings(Fig. 43).
Fig. 45showsthekineticsoflight-inducedabsorbanceand90°-scatteringchangesat
pH6.3ineithertheabsenceorpresenceof5mmol/1MgCl,.ThepresenceofthisrelativelyhighMgCl 2concentrationapparentlycreatesaconditionthatfavoursthelight-stimulated90-scatteringincrease (compareFigs.45aand45b).Thisstronglysuggeststhat
light-drivenMg movementandconsequentchangesinMg bindingatthethylakoidsurface
cause90-scatteringchangessimilartoH-induced90°-scatteringchanges.UndercircumstancesthatsufficientMg ispresentinthediffusedoublelayernexttotheinner
thylakoidsurfacetomeetthedemandforcounterions,thecompetitionbetweenthetightly-
69
0.5min
0.5min
0.5min
Fig. 43.Simultaneousrecordingsoflight-induced 515iraiabsorbancechanges(upperpart)
and90-scatteringchanges (lowerpart)atdifferentpH:6(a);7(b);8(c).Chloroplasts
wereisolatedandsuspendedin0.33mol/1sorbitoland2mmol/1HEPES.Chloroplastconcentrationwasequivalentto15yg/mlchlorophyll (opticalpath1 cm).
20nmol
30s
Fig. 44.Light-inducedMg extrusionfromchloroplastmembranesincubated inthepressenceof0.24mmol/1MgCl2measuredwithaMg-sensitiveelectrode.Thebarcorresponds
with20nmolMg + + permgchlorophyll.Arrowsmarkthestartandendofillumination.
boundMg
andH maystopandconsequentlythescatteringdecrease (Fig.43a)willbein-
hibited.Thereasonfortherelativelyhighlight-induced90-scatteringincreaseinthe
presenceof5mmol/1MgCl2 (Fig.45b)ascomparedtotheoneinthepresenceof0.5mmol/1
MgCl2 (Fig.42b)isnotclearasyet.Recentstudies (Krause,1974;Ben-Hayim,1978),
however,supporttheideathatthereleasedMg
isnotdilutedinthemediumbutremains
inthediffusedoublelayernexttothemembrane.ThismayleadtoahighlocalconcentrationofMg
Mg
70
andanassociatedshiftoftheequilibriumtowardscomplexformationof
andfixednegativechargesattheoutsidesurfaceofthethylakoidmembrane.Subse-
©
©
AAKMÖ3
ASC
0.5min
0.5min
Fig.45.Simultaneous recordings of 515umabsorbance changes (upperpart)and 90°-scattering changes (lower part)inthelight.Chloroplastswereprepared and incubated, either
intheabsence (a)or inthepresence (b)of 5mmol/1MgCl 2 - Thechloroplast concentration
was equivalent to27pg/mlchlorophyll (opticalpath 1cm).
quentdehydrationofmembranalcomponentscouldthenleadtoanincreaseof90°-scattering.
OurresultssuggestthatthiseffectoccursatratherhighconcentrationsoffreeMg
C>0.5mmol/1),whichisseeminglyconflictingwithadissociationconstantof51pmol/1
forcomplexformationofMg andnegativechargesonthethylakoidsurfaceashasbeen
proposedbyProchaska&Gross (1975).However,itisdifficulttopredicttheactualion
concentrationsinthediffusedoublelayer,whichareknowntodifferfromthosepresent
inthebulkphase.Moreover,thenetincreaseoftheoutsidesurfacechargedensity(Section7.3)uponilluminationwillshifttheequilibriumtowardscomplexformation.
Unfortunately,wewerenotabletocorrectforMg -inducedscatteringchangesinthe
samewayaswasdoneforH-induced90°-scatteringchanges.Themainreasonforthisisthe
effectofMg onotherprocessesthenthoseundersurveyhere,whichinadditionto90scatteringgiverisetochangesinforwardscattering.Furthermoretheeffectofaddition
ofMg on90°-scatteringand515nmabsorbancechangeswasfoundtobepH-dependent(Fig.
46).BecausethepHofthethylakoidinteriorisknowntodecreaseuponenergization,associatedMg movementsandbindingprocesseswillcause90°-scatteringchangesthatare
dependentontheprevailingpHconditions.ThisinfactcreatesarathercomplicatedsituationthatcannotbeanalyzedbythemethoddepictedinFig.42.
71
AA
60s
ASc610" 2
60s
~"\w
Fig. 46.Effectofadditionof 10mmol/1MgCl2 (indicatedbythearrows)onthe515iunabsorbance (a)andthe90-scattering (b)ofachloroplastsuspensionatpH7.6 (left-hand
part)andpH4.2 (right-hand part),respectively.Chloroplastconcentrationwasequivalent
to80ug/mlchlorophyll.Anupwardinflectionmeansanincreaseinabsorbanceandscattering,respectively.
8.6 DISCUSSION
Theresultspresentedinthischapterindicatethatthe515nmabsorbancechangesincontinuouslight,conclusivelywiththeinterpretationofothers (Thomeetal.,1975;Deamer
etal., 1967),maybeattributedatleastpartly,tolight-induced90°-scatteringchanges.
Inthepast,severalauthorsdidnotdiscerntheinfluenceofscatteringphenomenaonthe
515nmabsorbanceinthelightandconsequentlyconsideredthekineticdataofthese
changesasadirectreflectionofelectrogenicprocessesmonitoredbytheP515pigment
system (Larkum&Bonner,1972;Larkum&Boardman,1974;Krendelevaetal.,1977;Satoh&
Katoh,1977,1979;Yamamoto&Nishimura,1977).Itisnotmyintentiontodiscusstheir
resultsindetailhere.However,'asmaybeconcludedfromtheoverallresponsesofthe515
nmabsorbancechangessuchaninterpretationwillleadtoanoverestimationofthemembranepotential (c.f.Figs 39,42and45).Theobserved90°-scatteringchangesareproposedtobetheresultofalterationsinrefractiveindexofthethylakoidmembranesdue
tobothH-andMg -bindingeffectsonthehydrationofthethylakoidmembranes,andconformationalchangesinthemembranecausedbyMg -binding.
Fig. 47presentsapossiblemechanismforlight-induced90-scatteringchangesinthe
absence (a)andpresence (b)ofMgCl,.IntheabsenceofMg
inthebulkphase (Fig. 47a),
++
stillasmallamountofMg isretainedatspecialbindingsites.Light-drivenprotonaccumulationinthethylakoidinteriorisproposedtoresultinadissociationandsubsequent
extrusionofboundMg inordertoreachasteady-statecondition.Theobservedlight-dependentMg extrusionfromthylakoidmembranesthatwerepreparedintheabsenceofMg
(Fig. 44),supportsthismechanism.Inaddition,partoftheprotonspumpedintothethy72
O
\
» &
#
idböö^
o
o
o
o
QQOQQ' » 0 *
Mfi* ß
Q&WS&2'»
Q<JX»q$
••**
Fig. 47.Schematicvisualisationoflight-inducedchangesofbindingpropertiesofH
(closedovalsymbols)andMg (closedcircles)inchloroplastmembranes,intheabsence
(A)andpresence (B)ofMg inthebulkphase.Negativechargesatthemembraneareindicatedbysmalldashes.Dark-andlight-conditionsarepresented intheleft-andright-handpartofthefigure,respectively.
lakoidbindattheinnersideofthemembrane.Theresultanteffectapparentlyisadecreaseof90°-scatteringmainlycausedbyconformationalchangesassociatedwithMg dissociation.InthepresenceofrelativelyhighMg
concentration(Fig.47b)theamountof
++
freeMg intheboundarylayerisproposedtobesufficientforcounteriontransportand
thereforenoboundMg + +willbeexchangedtobalanceinwardprotonpumping.Protonationof
thethylakoidinteriorwillleadtodehydration,increaseinhydrophobicityandanincrease
in90°-scattering.UndertheseconditionstheextrudedMg
maybindattheoutersurface
(Ben-Hayim,1978),drivenbytheincreasednegativechargedensityattheoutersurface
73
(Section7.3).Thisprocessmayleadtoanadditionalincreaseof90-scattering.Itis
assumedthatbindingofMg
attheoutersurfaceisnegligiblysmallataMg concentra-
tionequalorlessthan0.5mmol/1inthebulkphase.
Thecontributionofthe90-scatteringchangestothe515nmabsorbancechangeis
clearlydemonstratedinFigs40and45.However,aneliminationofallartifactsinsuch
acomplicatedsystemasathylakoidmembranesuspensioncannotbeattainedduetothe
heterogenityoftheabsorbingcompounds,andthegreatnumberofprocessesthattakeplace
uponillumination,probablyaffectingtheP515absorbancechanges.Multiplescatteringand
consequentlyalterationsoftheeffectivepathlengthofthemeasuringlightcausedbymembraneparticlescannotbeexcluded,neitherthedistortionofabsorbancechangesbymutual
shadingofpigmentsassociatedwithanalteredionicenvironmentofthemembraneduring
illumination (Duysens,1956),seealsoChapter4.Osmoticswellingorshrinkageislikely
tooccurinenergizedion-freepreparedchloroplasts (Fig.43)butmaybesmallinchloroplastssuspendedinthepresenceofsaltswithapermeableanion(Deamer&Packer,1969;
Thorne,1975).
ThisleadsmetotheconclusionthattheP515absorbancechangeinchloroplastscannotbetakenasameasureforthetransmembranepotentialincontinuouslight.Aquantitativecorrectionforotherprocessesthataffecttherealelectrochromicabsorbance
changecanonlybemadeunderspecialconditions.
74
Summary
Themainsubjectofthispublicationisacomparativestudyofelectricalphenomenaand
associatedprocesses inchloroplastsmeasuredbymeansofspectroscopicandelectrophysiologicaltechniques.Theprocessesundersurveyaredirectlylinkedwiththeprimary
photo-acts andtheassociatedconversionoflightenergyintochemicalenergy.
A generalintroductiontothesubjectoftheenergy-conserving systeminphotosyntheticmembranes isgiveninChapter1.Themethodsandcomplementary techniquesusedto
studylight-dependent reactionsinchloroplastsaredescribed inChapter2.Aspectroscopicmethod tocharacterizeelectricphenomenainchloroplast (thylakoid)membranes,is
basedonanabsorbancebandshiftofanintrinsicpigmentcomplex (P515)duetolight-inducedchangesinthemembraneelectricfield.Flash-induced absorbancedifference spectra
areshowntobedifferent,dependentonthetypeofchloroplastsused (Chapter 3). The
differencespectrum,characteristic forthebandshiftofintactchloroplasts inthe460S40nmwavelengthregion,couldnot,incontrasttotheoneofion-freepreparedbroken
chloroplasts,beexplainedbyasinglebandshift.Computersimulationofacombinedred
bandshiftofasinglegaussianbandat497nmandabluebandshiftofasinglegaussian
bandat495.5nm,showedagoodsimilaritywiththeobserveddifferencespectruminintact
chloroplasts.Thisledtotheconclusionthatthefield-indicatingpigmentsinintact
chloroplasts arenothomogeneously distributedinthemembranecore.Alternatively,the
bluebandshiftmightbecausedbyanenergy-dependent exposureofpartofthepigments
toanoppositelyorientatedelectric field.
AmethodforthevoltagecalibrationoftheP515absorbancechangescausedbysingleturnoversaturating lightflashesisdiscussed inChapter4.Absorbancechanges,caused
byaddingKCltoasuspensionofbrokenchloroplasts inthepresenceofvalinomycinand
a lowconcentrationofMgCl,,weremeasured inthewavelengthregion460-540nm.ThemagnitudeoftheKCl-inducedabsorbancechangeswasshowntobeproportional tothelogarithm
oftheKClconcentration.Thespectrumoftheseabsorbancechangeswasfoundtobeidenticalwiththedifferencespectrumofthelight-inducedabsorbancechanges,whichwas
attributed totheelectrochromic absorbancebandshiftofP515.Thiswasinterpreted as
evidencethatundertheseconditionsabsorbancechangesofPS15respondexclusively toa
membranediffusionpotentialinducedbysaltaddition.Theresultsindicate thattheelectricpotentialacross thethylakoidmembrane,generatedbyasingle-turnover lightflash,
isintherangebetween 15and35mV.
Analysesofthekineticsofflash-inducedabsorbancechangesofP515andofthetransmembranepotential,asmeasuredbyamicro-electrode insertedinasinglechloroplastof
Peperomia metalliaa (Chapter 5), suggestedevidencethattheP515absorbancechangesare
thecompositeresultofatleasttwoprocesses:ReactionIandReactionII.Absorbance
changes,accompanyingReactionI,areproposedtoberelatedwithachangeofthetrans75
membranepotential.Theyshowedakineticpattern,similartothephoto-electricresponse
measuredwithmicro-electrodes.BothPhotosystem1andPhotosystem2werefoundtocontributeequallytotheflash-inducedabsorbancechangeofP515associatedwithReactionI.
AbsorbancechangesaccompanyingReactionIIareattributedtochangesininnermembrane
electricfieldsduetoalterationsintheconformationofmembraneconstituents.Experimentscarriedoutbothwithbrokenandintactchloroplastsshowedthattheinductionof
ReactionIIisexclusivelydependentonPhotosystem1activity.Thedatasuggestevidence
thattheoxidation-reductionstateofcytochromeforplastocyaninisadeterminantfactor
fortheinductionoftheconformationalchangesinthemembrane.ThesechangesaresuggestedtoresultinanalteredexposureofnegativefixedchargestotheP515pigmentcomplex.
SomeelectricalcharacteristicsofthethylakoidmembraneareconsideredinChapter
6.Thecurrent-voltagerelationofthethylakoidmembranewasfoundtobenon-linear.It
isconcludedthattheresistanceoftheinnermembranesystemisabout20 Mil,whichincreasesbyadepolarizingcurrentinjectiontoavalueof60MJianddecreasestoavalue
of2Mßbyahyperpolarizingcurrentinjection.
Thehalf-lifetimeofthedecayofReactionIafterasingleflashwastakenasa
measureforthepassivemembraneconductance.Itisconcludedthatprotonshardlyattributetothemembraneconductance.AsmallaccelerationofReactionIafterashortpreilluminationperiodcouldnotbeattributedtoadirecteffectofanincreasedproton
conductance,associatedwithprotonaccumulationinthethylakoidvolume.Theselective
conductanceofthethylakoidmembranetoavarietyofionswasfoundtobepH-dependent,
exeptforK,whichwasfoundtoberatherimpermeantinthepHrangefrom8to6.The
conductivityofMg appearedtobeofsignificantimportanceatpH7.Allanionstested,
werefoundtocrossthethylakoidmembraneatincreasingrates,whenthepHwaslowered
from8to6.Itisarguedthatthiseffectisduetothereductionofthefixednegative-chargedensity,favouringahighanionconcentrationinthediffusedouble-layeradjacent
tothemembrane.
Chapter7dealswithacloserstudyofprocessesthataffectReactionII.Theextent
ofReactionIIwasfoundtobehighlysensitivetophysicalandchemicaltreatmentsof
thechloroplastsandtotheionicenvironmentofthethylakoidmembrane.Light-induced
exposureofnegativechargesatthethylakoidoutersurface,detectedbyparticleelectrophoresis,showedasimilarsensitivitytovariousphysicalandchemicaltreatmentsas
ReactionII.Thisobservationledtotheconclusionthatbothphenomenamightbeareflectionofthesameprocess.TherisetimeofReactionIIabsorbancechangesofP515coincideswiththeactivationtimefortheonsetofATPsynthesis,•whichledtoasearchfor
apossibleparticipationofCF.,structuralchangesintheprocessthatunderliesReaction
II.
ItwasfoundindeedthatReactionIIisinhibitedbytreatmentsthatmodifythestructureandoperationoftheATP-synthesizingcomplexsuchasextractionofCF..,additionof
adeninenucleotidesandDCCDbindingonCF„.Itissuggested,however,thattheconformationalchanges,asreflectedbyReactionII,areprobablynotthedrivingforceforphotophosphorylationbuttheyrathercreateaconditioninwhichphotophosphorylationcan
proceedefficiently.However,furtheracquisitionofinsightinthemechanismbywhich
structuralchangesinthemembranemightbecorrelatedwithconformationalchangesofthe
76
couplingfactorwillneedmoredetailed experimentation.
Resultsofacomparitiveexaminationofthekineticsofthetransmembranepotential
changeandofthePS15ahsorbancechangeduringprolongedilluminationaregiveninChapter8.ItisshownthatpartofthePS15absorbancechangesincontinuous lightaredueto
simultaneously occurringscatteringchanges.Itissuggested thatH accumulationandsubsequentH bindingontheinsideofthethylakoidmembranecausethesescatteringchanges.
Aproportionality factor,relatingthescatteringchangetoanassociatedabsorbancechange,
wasobtainedbyacidtitrationofthesechangesinachloroplast suspension.Thisfactor
wastakentocorrectlight-inducedPS15changesforthesimultaneously recorded scattering
changes.Itisshownthatundercertainconditions,theresultanttimecourseofPS15is
qualitatively similartothetimecourseoftheelectricalpotential,measuredwithmicroelectrodes.Thelimitationsofthemethodarediscussed,inparticularinviewoftheoccurrenceofconformationalchangeswhicharecausedbyarathercomplicatedmechanismof
Mg -H exchange,andofMg bindingontheoutsideofthethylakoidsurfaceafterlightinducedextrusionfromthethylakoidinterior.
77
Samenvatting
Dezepublikatiegeefthetresultaatvaneenvergelijkendonderzoekinchloroplastennaar
elektrischeverschijnselenendaarmeegeassocieerdeprocessen,gemetenmetspectroscopischeenelektrofysiologischetechnieken.Deonderzochteprocessenzijndirektverbonden
metdeomzettingvanlichtenergieinchemischeenergie.
Hoofdstuk1geefteenalgemeneinleidingoverhetenergieconserverendesysteem.De
methodesdiebijhetonderzoeknaarliehtafhankelijkereactiesinchloroplastengebruikt
zijn,alsmedeverscheideneaanvullendetechnieken,wordenbeschreveninhoofdstuk2.Een
spectroscopischemethodeomelektrischeverschijnseleninthylakoïdmembranentekarakteriserenisgebaseerdopeenabsorptiebandverschuivingvaneenintrinsiekpigmentcomplex
(P515),tengevolgevandoorlichtgeïnduceerdeveranderingenvanhetelektrischeveld.Verschilspectravanflits-geïnduceerdeabsorptieveranderingenblijkenafhankelijktezijnvan
deintactheidvandechloroplasten (hoofdstuk 3). Integenstellingtothetverschilspectrum,waargenomeningebrokenchloroplasteninhetgolflengtegebiedtussen460en540nm,
kanhetverschilspectruminintactechloroplastenindatgebiednietwordenverklaardmet
deverschuivingvanéénenkelabsorptieband.Computersimulatievaneengecombineerderoodverschuivingvaneenbandbij497nmeneenverschuivingnaarblauwvaneenbandbij495,5
nm,geeftweleenverschilspectrumovereenstemmendmetdatvanintactechloroplasten.Dit
leidttotdeconclusiedatdepigmenten,diedegroottevanhetveldaangeven,inintacte
chloroplastenniethomogeenoverdemembraanverdeeldzijn.Anderzijdszoudeblauwverschuivingveroorzaaktkunnenzijndoorenergie-afhankelijkeblootstellingvaneendeel
vandepigmentenaaneentegenovergesteldgeoriënteerdelektrischveld.
EenkalibratievandeP515-absorptieveranderingen,alsafspiegelingvandekinetiek
vandeelektrischetransmembraanpotentiaal,isbeschreveninhoofdstuk4.AbsorptieveranderingenveroorzaaktdoorKCl-toevoegingaaneensuspensievangebrokenchloroplasten,in
aanwezigheidvanvalinomycineeneenlageMgCl~concentratie,werdengemeteninhetgolflengtegebiedtussen460en540nm.AangetoondwordtdatdedoorKClgeïnduceerdeabsorptieveranderingenevenredigzijnmetdelogaritmevandeKCl-gradiëntoverdemembraan.Het
verschilspectrumvandezeabsorptieveranderingenisidentiekaanhetverschilspectrumvan
dedoorlichtgeïnduceerdeabsorptieveranderingen.Ditwordtgeïnterpreteerdalseenbewijs
dat,onderdieomstandigheden,deoptredendeP515-absorptieveranderingenhetgevolgzijn
vaneendiffusiepotentiaaloverdemembraan.Deresultatentonenaandatdooreenenkele
lichtflitseenpotentiaaloverhetmembraangegenereerdwordt,dieligttussen15and35mV.
Analysesvandekinetiekvandeflits-geïnduceerdeP515-absorptieveranderingenende
kinetiekvandetransmembraanpotentiaal,gemetenmeteenmicro-elektrodeinéénenkele
chloroplastvan Pepevomia metalliea
(hoofdstuk 5),tonenaandatdeP515-absorptieveran-
deringhetsamengestelderesultaatisvantenminstetweeprocessen:reactieIenreactie
IIgenaamd.AbsorptieveranderingengerelateerdaanreactieI,zijnhetgevolgvande
78
transmembraanpotentiaal enhebbeneenvergelijkbaarkinetischpatroonalsdefoto-elektrischeresponsgemetenmetmicro-elektrodes.Fotosysteem 1en 2dragenindezelfdemate
bij totdeinitiële,met reactie Igeassocieerde,absorptieverandering.DeabsorptieveranderingendieaanreactieIIgerelateerd zijnwordentoegeschrevenaanveranderingenvan
lokaleelektrischeveldenindemembraan tengevolgevanconformatieveranderingenvan
bepaaldemembraanstructuren.Experimentenmet zowelgebrokenalsintactechloroplastentonenaan,datreactieIIuitsluitendafhankelijkisvandeaktiviteitvanFotosysteem 1.
Demate,waarinplastocyanineofcytochroomfgeoxydeerd zijn,isbepalendvoordeaanzet
vanconformatieveranderingen indemembraaneneenveranderdeblootstellingvanhetPS15pigmentcomplexaangeladengroepen.
Hoofdstuk6handeltoverenkeleelektrischeeigenschappenvande thylakoïdmembraan.
Derelatietussendegroottevaneenkunstmatig-geïnduceerdestroom IendegemetenspanningV isniet lineair.DaaruitkanwordengeconcludeerddatdeweerstandRvandethylakoïdmembraan afhankelijkisvandegrootteenderichtingvandestroom I.InafwezigheidvaneenstroomI,isRongeveer 20Mn.Eendepolarizerendestroomveroorzaakteen
stijgingvanRtoteenmaximumwaardevan60MR eneenhyperpolarizerendestroomveroorzaakteendalingvanRtoteenwaardevan 2Mn.
Dehalfwaardetijdvanhetvervalvanreactie Inaeenflitswordtalsmaat genomen
voordepassievemembraangeleiding.Hetblijktdatprotonennauwelijksbijdragentotdeze
geleiding.Dekleineversnellingvanreactie Inavoorbelichtingkannietwordenverklaard
met eenverhoogdeprotongeleiding tengevolgevanprotonopname indethylakoïden.DeselektievegeleidingvandethylakoïdmembraanvoorandereionenispHafhankelijk,behalvevoor
K ,dattamelijkslechtdemembraanpasseert inhettrajecttussenpH 8enpH6.DepermeabiliteitvoorMg daarentegenishoogmeteenmaximumbijpH 7.Depermeatiesnelheid
vanalleanionen diewerdengetest,neemt toebijeenpH-dalingvan8naar6.Ditis
waarschijnlijkhetgevolgvaneenverhoogdeanionconcentratie indeelektrischedubbellaaggrenzendaanhetmembraanoppervlak,tengevolgevanneutralisatievandenegatiefgeladenmembraangroependoorprotonen.
Hoofdstuk7behandeltdebasisverschijnselendietengrondslag liggenaanhetoptredenvanreactieII.Demate,waarinreactieIIoptreedtblijkt zeergevoeligte zijnvoor
fysisch-chemischingrijpenindestructuurvande thylakoïdmembraan envoordeionensamenstellingvanhetsuspensiemedium.ToevoegingvanMg enH heeft eensterkremmend
effectopreactie II.Vergrotingvandenegatieveoppervlakteladingvande thylakoïd (buitenzijde)inhetlicht,zoalsdiewordtgemetenineenelektroforese-experiment,heefteenzelfdegevoeligheidvoorverscheidene fysisch-chemischebehandelingenalsreactie II.Daarnaast isdevergrotingvandenegatieveoppervlaktelading,evenalsreactie II,alleenafhankelijkvandeaktiviteitvanFotosysteem 1.Dezeobservaties leidentotdeconclusie,
datbeideverschijnseleneenafspiegeling zijnvanhetzelfdeproces.Destijgtijdvanabsorptieveranderingen tengevolgevanreactie II,isvergelijkbaarmetdetijd,diewordt
gemetenalvorensATP-syntheseeenaanvangneemt.Derhalveisgezochtnaareenmogelijke
betrokkenheidvanreactieIIbijconformatieveranderingeninhetATP-synthetiserendeenzymcomplex.
Overeenkomstigdezesuggestieblijkt reactie IIgeremdtewordendoorstructurele
veranderingenvanditenzym,veroorzaaktdoor:extractievanCF..,toevoegingvanadenine79
nucleotidenenDCCD-bindingaanCF„;maareendirectbewijsvooreencausaalverband tussen degroottevanreactieIIenhetATP-syntheseprocesisnietgevonden(hoofdstuk7).
OpbasisvandekinetiekvanreactieIIwordtgeconcludeerddatdeprocessen dieaan
reactieIItengrondslagliggen,geendrijvendekrachtvormenvoordefotofosforylering,
maardatzijmogelijkerwijsweleenvoorwaardezijnvooreenefficiëntefotofosforyleringsactiviteit.Erisechtermeeronderzoekvereistominzichtteverkrijgeninhetmechanisme,
waardoorstructureleveranderingenindemembraan (reactieII)zijngekoppeldaanconformatieveranderingenvanhetATP-synthetiserendenzym.
DeresultatenvaneenvergelijkendonderzoeknaardekinetiekvandetransmembraanpotentiaalendeP515-absorptieveranderingentijdenslangerebelichtingsperiodenzijnvervatinhoofdstuk8.EendeelvandeP515-absorptieveranderingenincontinuebelichting
wordtveroorzaaktdoorgelijktijdigeveranderingeninstrooiing.Verondersteldwordtdat
dezestrooiingsveranderingenhetgevolgzijnvanprotonopnameenprotonbindingaandebinnenzijdevandethylakoïdmembraan.Deevenredigheidsfactortussenstrooiings-enabsorptieveranderingenwordtverkregendoorzuurtitratievanbeideveranderingen.Destrooiingsveranderingenwordengemetenondereenhoekvan90 methetmeetlicht.Dewaargenomenverhoudingtussenabsorptie-enstrooiingsveranderingwordtgebruiktomdoorlicht-geïnduceerdeP51S-absorptieveranderingentecorrigerenvoorgelijktijdigoptredendestrooiingsveranderingen.HetresulterendetijdsverloopvandeP515-absorptieveranderingblijktdan,
onderbepaaldeomstandigheden,vergelijkbaartezijnmetdetransmembraanpotentiaal,gemetenmetmicro-elektrodes.Demethodeheeftechtereengeringetoepasbaarheid.De
strooiingsveranderingen,tengevolgevanprotonbindingaandebinnenzijdevandethylakoïdmembraanlijkennamelijkvergezeldtegaanvannoganderestructureleveranderingen,die
wordenveroorzaaktdooreenuitwisselingvanMg
tegenprotonen.Demate,waarinditin
strooiingsveranderingentotuitdrukkingkomt,lijktafhankelijkvanderelatievehoeveelheidMg dieaanwezigisindeoplossingendeelektrischedubbellaag,endehoeveelheid
Mg ,diegebondenisaangefixeerdeladingenophetmembraan.
80
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