Volcmc 31. number 1 C’IlE~lICAL PHYSICS LET-I’ERS IS raru3ry 1975

ESR OF TRIPLET STATES OF CHLOROPHYLLS a, b, cl, c2 AND BACTERIOCHLOROPHYLL a. APPLICATIONS OF TFS AND ELEdrkON SPIN POLARIZATION TO PHOTOSYNTHESiS*.

J.R. NORRIS, R.A. UPHAUS and J.I. KATZ CXemirtry Division, A&onne.hbtioml Loborotory, Argonne, Illinois 60439. USA

Received I? September 1974

ZFS parameters for the title chlorophylls in both ordi‘nary and fully dcuterated form have been d&mined under es- perimental conditions that auow the aggregation state of rhe chlorophylls to be specified. The triplet state spectra are polarized. The electron spin polarization (ESP) con be analyzed by a simple scheme, and is found to bc sensitive to the agrcgotion state of the chlorophyll. Comparis0.n of in vivo and in vitro bacteriochlorop_hyll spectra supports the chIoro- phyll special pair proposal fo; the structure of in viva photo-reactive chlorophyll.

1. Introduction aggregation behavior characteristic of “model’! por- phyrins whose triplet state energy levels have been

We report here ESR triplet state spectra for all of more frequently studied. the major, well-characterized chlorophylls (Chl’s) oc- It has become evident in recent times that only

curring in nature. Although such studies have been aggregated chlorophylls play an active role in photo-

carried out on-porphyrins [ 1,2], which are generally synthesis, as the optical and ESR properties of both supposed to be models for chlorophyll, very little light-harvesting (antenna) and photo-renctiva chloco- was kn,own about the ESR of any Chl triplet [3] _ In phyll can only be explained in terms of cooperative fact, complete zero field splitting parameters had - phenomena involving two or more chlorophyll mole- been reported only for Chl b [4], and some incom- cules [ 111. Although it is clearly necessary to Yrirst plete data was available for Chl a [5] _ To our knowl- understand in vitro monon!eric ch[orophyII systems

edge no data of any kind existed for in vitro Chl cl, (and this includes model porphyrin systems) ~ it is Ch! cz [6] and bacteriochl&ophyll a (Bchl) systems. essential that the effect of aggregation on the photo- Highly important for the application of in vitro induced triplet states of the naturally occurring chlorophyll triplet data to photosynthesis is the recog- chlorophyll be established in order for triplet state nirion that the chlorophylls exhibit a remarkable “pa- ESR data to be nppknble to the interprctatior. of in

city to undergo selfiaggregation to form. dimers and vivo photosynthesis. In vitro triplet data on chloro- oligomers [7] and intkct with mono-functional phyla systenls in which the nature of the chloroph~Il nucleophiles (Lewis bases, L) to form monomeric species present have r.ot or cannot be specified have

chlorophyll species, and with I%functional nucleo- only limited utility insofar as applications to in viva philes to form poiymeric chloiophyll species by coor- photosynthesis is con’cerned. Thus, we have undertaken dination to Mg [8,9]. In brief, the state of chloro- an ESRstudy of in &o monomeric and of certain phyll is extra-ordtijrily solvent, temperature, and’ particularly important aggregated species of chloro- concentration dependent. [lo]. The coordination phyll. As some uncertainties remain in our under- interactidns of the’chlorophylls are quite unlike any standing df the state of chlor,ophyll in the glassy state

at ver$ low temperetures, we have been careful to

*Work performed under the auspices of the U.S. Atomic specify the experimental conditions we have used, Energy .Commission. particularly the solvent system and the chlorophyll

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ESR Qectra were recorded on a.Varian E-9 spec- iroineter u&g ! 0 kHz field modulation of 20 ‘to 40 G. amplitude in combination with’light modulation pro-

: vided by a PAR rotating chopper, essentially accord- ,’ ‘. ,ing to the procedures of Brinen [12] and Levanon and Weissman [13]. Typical microwave power-was

0.2 mW. An Ithaca lock-in amplifier was used to de- -‘te$t the signal produced by a 50 W Eimac xenon lamp’ fil.tQxnd tb.rmdA q. cm: 0.f y+p&~. act” ;.._,_:._.,,,. >-d.rlt-Cl* . .._ c_m_._ _..* .,_ ~bzht ~.m3Ad~.+. i,-e.

‘from 0.03. Hz to_ 3000 Hz was employed. Chlorophylls weie prepar’ed by the method of

Strain and’ Svec [ 141, As chlqrophyll so prepared in- vrlriably contains water, all samples were‘prepared

.from chlorophylls that were rendered strictly an-’ h)drous bn a vacuum line by codistillati6n with de- gassed CC14, Samples were sealed off ii-~ 4 mm o.d. thin-walled quartz tubing with the appropriate de- gassed solvents. ChI concentrationwas maintained =1 X:1O-3 M. Sp. ectra were collected in a Xicolet.

iO74 computer on line.to the demistry Division’s

Sigma 5 central computer. Temperatures from 415 to

,85 K were maintained with an Air Products LDT-3 Helit& system using a chrome1 versus gold 0.07 qt. % iron thermocouple for temperature measurement. ZFS were determined by measurement of peak separa- ‘tions and nbt by simulations.

3. Results ‘,

Fig. 1 illustrates some typical ESR triplet spectra

., of the monomeiic chlorophylls. We have, in addition, extijned, the effects of metal insertion i;, the corn-

pounds zinc tetraphenyl porphyrin (ZnTPP) and zinc pheophytin a (zinc chlorophyll). The ZFS data are .summa&ed ti._tible l_ In all instances the chlorophyll TFS aie relatively small, indicating efficient spin de- localization in the triplet state; OurZFS parameters z,e:significantly lower but probably agree within ex- perimental JintiTlits with .the results of others. Based on

the number of reductid.pyrrole rings in,thc.chloro- -phyfis, we expe_cted the sequence

-&_&z aid not observe this trend we assume the de-

Fig. 1. Triplet spectra‘of in,vitro rrknomeric chlorophik. (a) -:.Bchl; (b) Chl a; (c) Chl b; (d) Chl61; (e) Chlc2. AU chloro-

Iocalization of spins in th’e tiiplet states of the chloro- &ylk’werc of nor&I isotopic composition. Spectra were re-

phyilg iS fai more &mpi@aJed than that based on the corded in ethanol at-20$l J-Iz light modulatiqn, except for.(e), where 80 Hi Ilght modulatibn x&s used. ,‘.

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Table i ZFS p=amctcrs of chlorophylls

CIIEMICAL PIiYSICS LE-ITEKS 1s I’cbru~r~ 1975

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Solvent State of chlorophyll ZFS 0 .;-.-- _--_.--- _- ._--

Q!lorophyll s-ystem”) aggxgationb) speciesc.d,e) IDI (cm-‘) IEI (an-~~ .-.

‘HChla A hl ‘H-ihl 3. L’,_z 0.0274 t 0.0005 0.0032 I c.ccc5

‘HChl a B hI 2HEhl a- L;I_-2 il.0277 5 0.0005 0.0039 c 0.0005

‘H.Chl a C D (‘HChl aI2 0.02ai * o.aocs 0.0032 i 0.0005

*H-Chl ;1 D 0 I(*HChl &I ,I 0.0273 c 0.0005 0.0035 c c.ooo~

:‘HGii‘ 6 A si 5TT P.. . . . I-lClll u-L’,__2 fjJjQs> t ~&qG)s D,>.CQ.S$ _c g.,g,QQ.~.

‘HChl b A M ‘HChl b-L’,_? 0.0290 t 0.0004 0.00~3 c o.cac4

2HChl b B hl *HChl b-L;_?, 0.0289 c o.oco5 0.0050 i 0.0005

*HChl b D 0 [(‘HClll blal,,’ 0.0277 + 0.0005 0.0032 i 0.0005

’ HChI c 1 A hi ‘HChl L’I_-2 cl. 0.0269 0.0005 f 0.0055 E 0.0005

‘HChl Q A M ’ -HChl - L;_* cl 0.0276 L 0.0005 0.00;s c_ 0.0005

‘H-Bchl E M 1 H-Bchl. L’& 0.0226 r O.CflOS 0.0059 5 0.0005

‘H-Bchl B M I H-B chl * L’i __2 0.0224 2 0.0005 0.@0.54 I 0.0005

’ H-Bchl B b! ‘H-Bchl. L;-2 0.0221 2 0.0005 0.0033 : 0.0003

‘H-ZnTPP F hl(?) 7 0,0303 z 0~0001 0

‘H-ZnTPP E hl 7 0.0299 2 0.0005 0.001 I 5 0.cJ006

‘H-ZnChl a F hl ? 0.0296 _c 0.0002 0.0032 I c.aoo?.

a) A. ‘H-ethanol; B, *H-toluene + 10% ‘H-pyridine; C, ‘H-tolucne: D, ‘H-methylcyclohcssne; E, ‘H-mcthyltct-ah~.drofurnne; F, embedded in poly(methylmethacrylate).

b) hi, monomer; D, dimer; 0, otigomer. c) L’, ‘H-ethanol; L”, ‘H-pyridine; L”‘, ‘H-methyltctrahydrofurane, d) Based on room temperature species. The piincipal species at RT has coordination number 5 [21], i.;., L = 1. In neat pohr

solvents, under forcing conditions, or probably at low temperature, hexacoordinste species may also exist to an estcnt deter- mined by the polarity of the medium, i.e., L approaches 2.

e) We ignore the presence of diastereomeric a’ and b’ [22] as probably having little or no effect on ZFS. f) ZFS values were temperature independent within experimental error in the range 5 -50 K.

above simple expectations. The complicated nature of in viva-chloroplasts (to be published) we are now

the spin delocalization is best illustrated by the E val- aware that Chl a and Chl b in their various aggrega-

ucs. Only the conjugated system of an unreduced por- tion states in vivo cannot be distinguished on the basis

phyrin can possess axial symmetry and thus exhibit of zero field splittings alone. The erectron spin polari- zero E value. As expected, Bchl, Chl a and Chl b pos- z&ion (ESP) evident in the tripkt spectra, ito~ever:

sess relatively large E values, but we did not expect the large c’ value observed for ChJ cl aild c2. Thu~,~

appears’to be sensitive,to the aggregation state of the chlqrophyll:

on the basis of E values in tabiF 1, the delocalization All of the spectra we have recorded in chlorophyll

of the tiiplet state in Chl cl and Chl c2 is much more systems exhibit some ESP (fig. I). Spin polarization

like that of Bchl: has b-een prk-iously observed in metal free porphyrins Our results indicate small changes in the ZFS of [ 151 and in photosynthetic bacteria. [16] - As ex-

Chl a and +I b with @_gregakon state that may oi petted, the effect of deuterium, cannot be detected in may not tiZ outside the range of experimental err&. .thk zero field splittings (tabIe I). Howe!ver, the-extent Moreover, becausk we have observed ~hl~triplets in of electTon spin pol&izatio~ IS decreased in 2H-CM’s,

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which is consistent witha longer triplet lifetiine, . . .which iti turn allbws more efficient SJQ-I lattice re- _,

1s February 1975

‘. Based on ,qualitativk considerations of the hi& field, steady state populations with negligible spin lat- tice relaxatib’ri effects we can interpret.the cidorophyll triplet electron spin polarizations in a simple manner. TX ;T,, and Tz refer to the triplet spin substates in ; zero external magnetic~fie1d.x andy are assumed’to Ii: in the plane of the macrocycle and E is perpendic- ular to that plane. We make the reasonable assump-

‘tion that D > 0. Then, by using simple first order ki- netics, we predict polarization schemes based on rela- tive rates of population and depopulation of the T,, ‘T,, and Tz spin substates .m the presence of a high magnetic field. We consider in ihiS comqunicatiorr only limiting conditions such as singlet-tripiet cros- sing via one or two spin s&states. This.method indi- .tites that momeric Chl a, Chl b, Chl cl arid Chl c2 qre polarized via two dominant directions in, the plane of the macrocycle, and that Bcllis polarized via on- ly one main direction in the macrocycle plane.ZnClrl 2 is polarized \v;th prim,arily an in-plane’and out-of- plane spin-orbit interaction and ZnTPP with prima- rily an out-of-plane interaction. As we believe these systems to be monomeric, the out-of-plane mechanism of polarization in the zmc compounds probably in- volves the heavy metal effect of zinc.

More important, the aggregated Chl a and Chl b oligomer species [(Chl a)2] n and [(Chl b)3] ,~ ex- hibit very different polarization patterns which may be associated with in-plane and outof-plane polariza- tions that reflect the keto C=O...Mg and aldehyde ,C=O...Mg interactions involved in oligomer formation. Kinetic:data witl be necessary to establish the details of the polarization. However, it is important to em- phasize, only kinetic data on well-characterized chloro- phyll systems will be useful for this purpose. Our

fmdings strongly indicate that the polarization Infor- matiorrin the spectra is very lke<y to be of much more use. than the ZFS in the study of chlorophyll aggregation in vitro, tid for the study of the role of

chloroplryll~triplet states in photosynthesis. : Finally, we compare our in vitro Bchl triplet re--

.&Its with the in vivo triplet signal attributed to .B,chl-by L&h and Dutton [16-181. Leighand Dutton ’ compared the in vivo ZFS value of 0.0188 cm-l with : the =a_!23 &-n-r, value of .C.hl a, Chl b, and a number

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(?f porphyrins, and conclude; that two Bchl mob cules we-e involved in reducing the mtignitilde of th,e in vivo tliple’t ZFS. This interprgtation was in agree-

‘ment with the special pair concept of-photo-reactive. ,chl’orophyll iri photosynthesis [ 11,19,20]. Although our results indicate that the ZFS of monomeric’Bch1 is much smaller than the value used by Leigh and Dutton, and thus tends to weaken the argument based on a comparison of the ZFS parameters, the best interpretation of photosynthetic bacteria in vivo

trip!et spectra still demands two molecules on the basis of :he polarization in the triplet spectra. Where-

as we can interpret the polarization scheme of in’vitro Bchl in terms of&e in-plane axis, the in vivo triplet polarization cannot be interpreted in terms of a single molecule of Bchl. Thus, both the ZFS and the ESP reasonat ly require the interaction of more-than one molecule of Bchl to account for the triplet signal’ob- served in viva. The chlorophyLl special pair concept for photo-reactive chlorophyll in bacteria thus re-

ceives support’from the doubIet ESR linewidth [I I], from the ENDOR of the doublet state [20], from the ZFS of the triplet state [17], and now from a corn-. parison of the in vitro and in Give ESP of the triplet states.

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Because spm polarization of the triplet spectra ‘may be ;I sensitive function of the state of aggrega- tion and the relative orientations of the chlorophyll molecuks in aggregated chlorophyll, we expect the Fo!ariza;ion information implicit in these triplet spectra I:O be of great value in the study of chloro- phyll function in photosynthesis.

Acknowle.dgement ..

We thank Dr.‘TJ. Schaafsma for pointing out.to. us that the ESP of.in vivo bacteriochlorophyll cannot be accounted for in terms of a single mo!ecule. We are indebted to Dr. Marion Trifunac for helpful dis- cussions and for contributions to the data collection.

References . .

[ 11 $‘.G. van Do& ‘T.J. Schaafsma, M. Soma and J.H. : van der WzaLs, Chem: Phys. Letters 21 (1973) 221.

[21 1.1.. Ghan, W.G. van.Dorp, TJ. Schaafsma and J.H. van dir Waals, Mol. Phys. 22 (1971) 74!, 752.

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CHEMICAL PHYSICS LEtiERS 1.5 February L975

131 D.L. van der Rleulen and Govindjce, J. Sci. Ind. Res. 32

[4] J.M. Lhoste, Compt..Rend. Aad. 51% (Paris) 266D (1965) 1059.

[S] G.T. Ri!&ireva, L.A. Sibel’dina, 2.P. Ribova, L.P. Ksyushin and A.& Krasnovskii, Dokl. Biophysids 181 (1968) 103 (English trnnsl.).

161 H_H. Strain, B.T. Cope, G.N. McDonald, W.A. Svec and

J-J. Katz, Phytochemistry 10 (1971) 1109. (71 K. Ballschmiter, K. Truesdell and J.J. Katz, Biochim.

Biophys. Acta 184 (1969) 604. [S] J.J. Katz, in: Inorganic biochemistry, Vol: 2, ed. G.L.

Eichhorn (Elsebier, Amsterdam, 1973) ch. 29, pp. 1022-1066.

[VI TM. Cotton! A.D. Trifunac, K. BaUschmiter and J.J. ICat?, Biocl:bn. Bibphys. Acta, tb be published.

[IO] K. Ballschmiter and J.J. Katz, J. Am. Chem. Sot. 91 (1969) 2661.

[I I] J.J. Katz and J.R. Norris, in: Current topics in bio- energetics, Vol. 5,‘cds. D.R. Sanadi and L. Pnckcr

(Academic Press, New York, 1973) pp. 41-75. [I?,] J.S. Brinen, J. Chem. Phys. 49 (1968) 586.

[13] II. Levanon and S. Weisrman, Israeli. Clem. LO (1972) 1.

[14] H.H. Strain aicI WA. Svcc, in: The cI;lcrophyLL+. cd+.

LP. Vernon and G.R. Seely (Academic Press, New York, 1966) pp. X-66.

(I.51 H. Levanon and A: Wolberg, Ciiem. Phys. Letters 24 (1974) 96.

[16] P.L. Dutttin. J.S.-Leigh and XI. Seibert. Biochem. Biophys. Res. Commun. 46 (1972) 406.

[17] J.S. Leigh and P.L. Dutton, Biochim. Biophsx. .kcta 357 (1974) 67.

[IS] P.L. Dutton, J.S. Leigh and D.W. Reed, Biochim. Bio- phys. Acta 292 (1973) 654.

[19] J.R. Norris, R.A. Uphaus. H.L. Crespi and J.J. Katz. Proc. Natl. Aud. Sci. US 68 (1971) 625.

[20] J.K. Norris, H. Scheer and J.J. Katz, Arm. NY Acad. Sci., to be published.

[21] J.J. Katz, H.H. Strain, D.L. Leussingand R.C. Doughc~ J. Am. Chem. Sot. 90 (1968) 764.

[22] J.J. Katz, G.D. Norman, W.A. Svec and H.H. Strain, J. Am. Chem. Sot. 90 (1968) 6841.

‘LY,