Spin polarization in the lowest triplet state of chlorophyll

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1 Novembbr 1974

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Received’ Ib April li74 Revised manuscript received 21 July.1974

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Chlorophyll-b in glassy solution has a spin-polarized lowest triplet state at and above 7? K. Ths magnitude of the effect is different for MTHF and ethanol as soIvents, in contrast to what is found for the porphin free base. Chlorophyla-a does not exhibit spa-polar~ation under identical conditions as for.c~lorophyli-b. Zero-fidld parameter: are found’to be: chlcrophyll-a @THF) D = (281+ 6) x 104’cni-‘;‘E = (39 + 3) x IO4 “R~; chlorophyll-b (MTHF) D 7 (289 f 4) x 113~ cm-‘;‘E = (49 f 3) X IO4 cm-l, From ESR signal kinetics it follows that for chlorophyl~b, population and depoptilation .mtiy involve the spin level ly), describing ;t spin moving 61 a plane perpendiculv to tithemolecular plane;

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py>Px>Pz; :..

:

A-; t 240 = 40 s-‘;

k y = qa

2 120 s-1;

kz c 75 s -1,

.,

where Pi anri ki denote popuI.ating and decay rates Thus, the kinetic scheme for t$e chIorophyl1 trip&t is different from that of porphyrins with heavier metal ions, but very similar t6 that of the por$i.n free box. The $n-Iattice relaxation the is found to be anisotropic and, Shorter than Ute~decay rates of j~di~du~ spin levels. Nevertheless, spiq polarization can be observed, es~nt~lly.beca~se the ESR signal amp~tudc depends on population differences;

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.’ Upon illuminatik of photosynthetic pigments both ip vitro and in viva, significant steady-state levels of triplets &I be formed [l-3]. Fur~e~ore, it has “been Found that triplet state &inetics:affect the populatiogs of the ground.and excited singlet states of chlorophyll [4] &d of the porphin free base IS]. On the Other hand,.& triplet lifetime df chlorophyll at iooti temperature is &Ithe miliisecond region [I], thus exceeding the pP,riod whi& is significant fo; the ‘photosynthetic &ergy .&version. Therefore, it is ‘, still a matter bi debate as to‘whether the lotiast trip. let state of chlorophyll is part,of.the energy transfer ‘p&.hway & phti~osyn*e~s [3,6]; and more kinetic studies are needed to settle this qu~stibri. AlSo from t+e mo[ecujar point of’view,.the’triplet .’ state of chJoroph.yll is tif interest, ]since.it belongs to ’ tl-k class.‘of porphykis, far which well-developed theories:on.efectronic’stnrcture [7) 3s w&as a large ‘. ”_,.

;.,

amourtt of spectial data [ 1,S,9] are avail&le. Even if the tdplet state is not an essential intermediate in,biological energy cunversion;~t provides an attractive probe to study primary processes in photosynthetic model compounds and in the in vivo unit. For example, Duttoti

and l&h

[3] have recently

._

demonstrated

that the presence’ of trip]& is closely connected to the primary photoche~st~ ir, the photosy~~etic reaction centre of rh~dopseudo~onas spheroides., In :tis letter we present kinetic data tin the lowest triplet stake of chlorophyll-a and b, together with that : of Ihe po@in free base, obttied from the steadystate and transient behaviour of their IO& temperature ESR s3ectra. .,’ :

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Volume 29, number 1

CHEMICAL PHYSICS LETTERS.

tetrahydrofuran

(MTHF) and ethanol were takenwith Varian E-6 spectrometer, equipped with a low power bridge. Porphin free base (Calbioa slightly motied

them) chlorophyll+ and b (Fluka) were checked for purity by TLC and visible spectrum [I, S.,lo]. All operations were carried out in evacuated, grease-free glassware. Samples were irradiated both continuously and chopped with filtered light from a 200 W mercury arc (Osram HBO-200).

ESR signals were amplified and averaged by using a boxcar integrator or PSD (Brookdeal) and a HP 5480 signal analyzer, which also served for background sub traction.

tudes S for e&h of .tie six &m - +I tra&itions at 6
for the popu!ationsNO and IV,,. Since spin relaxation rates m!y be anisctropic, we use different rates LW’~, ldZ and \d3 for ti a!ong any of the molecular axes i = X, y or _z. Fo_IIo+g Sixl and Schwoerer [13], we assume \dI = ldZ =*d and K_l(f) = (1 + 26)NI(f) resulting in the elimination of I$ from the rate equations. Noting tiat for i =x, y the s@al amplitudes ST and St7 at resonance fieId values fl andHi_ are proportional to the population differences No -N, and N-l- No, respectively, the steady state spin polarization

3. Spin polarization

with coittinuous

illumination

In zero magnetic field,,the lowest electronic triplet state of chlorophyll, assumed to have ii~~”character in polar solvents [l 11, is split into three non-degenerate spinlevels Ix>, Iv>, Iz>;the z axis is taken perpendicular to the molecular plane, defined by x and y. As usuai for Ian* states, E,, Ey > E,. Fig. 1 defines the populating rates P,-,+I, the total decay rates k~,+~and spin-lattice

relaxation

rates \v for the spin levels 10)

and 1+1)[12-141. Boltzmann factors exp[(E, - E,,t)/kT], with 1t1,m’ = 0, +I, arc approximated by{1 +6(m-m’)} where 6 3 hv/kT and y i: the microwave.frequency. Steady state ESR signal ampli-

i November -1974

Ri = (SF-

as defined

by a ~oiarsiafim

ratio

q)/($M:)

(0

can be expressed as Pokl - P,k,-, - Plk,,6 R,= I

WqPo+ 2Pl) + P$c()

(2) . I

(ForHIz, $ and SF are interchanged.) Cnderiving eq. terms in 62 have been neglected i&h respect to 6, sinceat77K,6=5X 10s3_ The experimental ESR spectrum provides three values of Ri, one for each pair of transitions at $ and 6 (i = X,y, z). In the absence of relaxation ~vi = 0 and Ri + m, whereas with w + 03 no spin polarization is observed. With ld B kg,kl and S 4 1, (2) is further simplified to

(21,

Ri = ($P,, - k@,YJfi

,

(3)

w-here fraCtional population rates pg and pi are de‘fined by po,+ I E Po,=l/(Po f 2Pi). Note that poIarization is obs-erved.if xC1po- k,pl iS of the same order of magnitude as ~‘6, even if tv’ S Q, kl_ As will be shown below, spin polarization

in the porphin free base and chlorophyti-b arises because both the populating and decay rates are different for the three zeio-field spin states Ii), resulting in p~fpl (F p-1) and ko + kI (= /c_~) for Hli x, y, or-z. Since one may neglect the ra’di$ive comribution fo the total decay rates, the kinetics of the high-field spin componenjs are entirely governed by radiationless processes. Then, the pppulating and decay mechanism . are rather similar in nature, as has been shown by the. Fig. 1. Kinetic s&me for triplet mapetic sublevels 10) and l*l) it high field. For definition of symbols, see text.

‘ory and,experim&ts [lS]-on aromatic and aia-aromatic molecules. Quantitatively, this means that the dif. .

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29, number 1

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‘. .” @ii. 2. Triplet ESR &e&urn of’the porpbiafree base iri MTHF’(4 X 10” hl) at 77 K indiking the presence of spin polarization. : Microwave power 0.05 mW, modulation amplitude 16 G, mod&xion frequency 100 kHz. Spectrum recorded with contiuous illumination. :. ,,

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.. ‘,,‘, ,’ ‘. &ctra of chlordph$-b in X&THF (2 X’104 M) at twp.dkferent, ~e,rnpe~~t”~e~-~~~~d~ k&h conti&oEs &ni-’ : mtion;the hihal ndic;l iignal.is qmitted. Microwave power is: 0.5 mW (a) and 5 mW.(b), microivove frequency-Y&MHz, m’&:’ dulation ampli!udb 16 G, modulation frequency iO0 kHz:In this region the.&ape or the obsemed spectrum is indepe?ddnt of mi- ., .’ ,. . &o,wa~e power. -:. :,

Fig 3: Tripl$~SR

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CHEMICAL

~Volum: 29, number 1

PHYSICS LETTERS

ference between po/pI and k,/k, is rather small for each of the culonica! field directions. Therefore, the ‘difference k,po - kopl can only have.appreciable magniitide with respect to wiS for,short-lived tripIets, ‘i.e., when-k0 a_nd ki are large. In m&it solvents at - 77 K the spin system is only partially polarized due to fast relaxation and one observes no emissive transitions, but an asymmetric intensity distribution as in figs. 2 and 3. The component out of a pair of transitions (e-g., -c and H;;) with lowesr intensity will appea; as emissive if relaxation is sufficiently slowed down, The spectra of figs. 2 and 3 give the following ROlarization ratios: pofphin free base (77 K, MTHF) Ax = +C.30(+0.95), chlorophyll-b

R, = -0.50(+0.06)

, Ry = -0.59(+-0.06)

, RZ = 0 .

Chlorophyll-a d.oes not show spLn polarization under the same conditions as chlorophykb. Applying eq. (3), these results imply that both for the porphin free base and chlorophyll-b, transitions at H;; and I$ are emissive and those at 3 and Hu are absorptive in the absence of relaxation. Anticipating further results from transient ESR responses, following below, we find pY > pX > pz: Combining this result ivith the steady state behaviour of transitions at -c and q, we conclude that the spin-component I_Y) decays faster than !_Y)and/or 1~).This is very different from the kinetic scheme observed for Zn-porphin, [16], where Iz) is the dominant level for populating and decay. It follows from (3) that for isotropic re-

laxation (wx = :vY = wz) R/R;-RZ=O.

(4)

Spin relaxation mu& be anisotropic, since eq. (4) ddes not hold &perimentaUy. Table 1 summarizes the zero-field parameten for the three compounds of interest dissolved in MTHF and ethanol:Results agree with existing data obtained ‘from hi&field h = 2 ESR spectra [ 11,17,18] and with zero-field ttisitions observed for the porphin free.base [S]. N&e that D and E of the porphin free : base are insensitive to a ch-Age in solvent, whereas chlorophyll-b changes its E value by =X30%. Chloror phyll-a’hu

~(1owe.r E value than its partner

Table 1 Zero-field puameters of the porphin Gee bax and chIor@ phyll-a and b. D values were &xhted tram the scparatiort of the outer peaks;E values ffom the weragc of the sepacation between x and y positions in the low and high field POICons bf the spectrum. All measurements wxe curied out at 77 K, escept for chlorophyll-a, where T= 95 K. The vtiations in D and E in this temperature rzmge ztc v,ithin the mwuring erro:

Compound

Solvent.

D (IO-‘! cm-‘)

E(104cm-L)

porphin

ethanol MTHF ethanol

437 A 7 43626 287 A 5

652 3 36 5 4

hlTHF

289 f 4

49 L 3

MT’HF

28156

39c 3

chlorophyll-b chlorophyll-i

66 + 5

. R, = 0 ;

(94 K, MTHF)

R, = +0.26(+0.03)

1 Now.mber 1974

-6, in

agreement with previous conckions from A?77= 2 spectra [17]. The arndunt of spin polarization R, remains constant upon a,change of solvenUn the caxe of the parphin free.base, in contrast with chlorophyil-k~, where Ri is !ower in ethanol than in MTHF. Although more data are needed to substantiate such a conclusion, it is conceivable that the lowering of both E andRi upon

a change in solvent have a common or&n, if it is assumed that the amount of inequiva!ence of themolecular x and y axes, reflected by the magnitude of E, also affects spin relax$ion, and thus Ri. A f&t spin relaxation, accompanied by a low E~vaIue, could also explain the absence of spin polarization in chlorophyll-a, whichis surprising in view of its triplet lifetime being shorter than that of the -b compound. A change in solvent viscosity has a different effect on Rj and E, as is shown by fig. 3: a small temperature increase does noi affer;t D or E, but spin polarization is nearly wiped out. Even if no spin polari~ation is observed, i.e., the Am = 1 ESR spectrum has a symmetric intensity’distributicn around the center field, anisotropic spin relax&ion can affect the reiative intensities of the three pairs of transitions in a different way as is apparent in triplet ESR spectra of some metalloporphyrins [19].

4. Transient Spin polarization

With standard ESR detectioni’quantitative mat&n on the !dnetic scheme of fig. L cannot

inforbe ob-

CHEMICAL PHYSICS LETTERS :

Volume 29, numb-r.1

tained in a simple manner. Therefore, folloG-rg Lhoste [20] and Levanon -arid Weissmann [2 1,221, we have employed,a.chopped light-source and phase: .,_ sensitive detection with reference to the chopping frequency. It appears to be useful to study both the time dependence of each of the Am = *l transitions, as welI as the shape of the spectrum after a certain time has elapsed after the exciting b&has been switched on or off, using a boxcar integrator [23]; such,a spectrum is called a gated.spectrum.-Treatment of our results goes along the same lines as in previous studies on slower systems by Six1 and Schwoerer [ 13; 141 and Clarke [24]. Solving the differential equations leads to a sum of exponent& for the time dependence of the ‘various signal amplitudes. If the relaxation rates are of comparable magnitude or larger than the decay rates, signals ST and S,: have unequal amplitude [13]. ForWI\x, y and z.this is what we observe. Fig. C represents the observed time dependence for the signal amplitudes Sz, SG and St; with a time reso-

,: ..

1 Novcmbe; 1974

lution which is probably limited by the.0.3 ms RCtime of the’spectrometer. After switching tiffthe exciting light, the kl. When switching on the exciting light, signal Sit initially grows proportiOIld t0 Pi - i(Pj +Pk) (i, j, k = X, y or Z). ThUS Lit -

0’

repl’esents a spectrum in statu nascendi with nonnalizetl signal amplitudes reflecting the difference in the populating rates PO and pcl. ii convenient experimental method to obtain a spectrum in statu nascendi is .to measure a gated spectrum with the gate-open-period of the boxcar integrator directly after the switching on of the exciting light. For chlorophyll-b such a gated spectrum is shown in fig. 5. Note the large amplitude of the S; signal resulting from F, > pX, pZ . By evaluating the initial normalized slopes of the transients SC(t), S,f(r) and S,‘(t), a first estimate of the populating rates can be obtained: P, : Py : Pz = 3 : >6 : ,< 2 .. The opposite behaviour of a transition with continuous or pulsed excitation can now be understood. In the fonmer case the Sz transition, for instance, would be emissive in the absence of relaxation, even when the 0nset ofthe same transition is obsarptive. This is due to the‘fact that, although p. > pl, po/ko
,5wCTr
for Sf a negative &gal is absorption, for s’ and Si a p,crtitive ~&nt$is~bsorption

:.

: --

‘. ,x . ,:

,.

I.,

Using phase-sensitive detection \vith reference to the chopping frequency, generally leads to distorted,ESR spec ira, because transients of different shape are integrated. for Hlh, y, z! Some transitions~may even be ab.

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Volume 29, number 1

1 November 1974

CHEhiICaiLPHYSICi LETTERS Gated

ESR

3256 G

160~~

I

+

Spectrum

CHLOROPHYLL-B

.

..

tight

of

in HTHi

mdd

.‘

-H

Fig_ 5. Gated ESR spectrum of chlorophyll-b in MTHF. Response time ESR spectrometer Ct.3 rns; gatewfdth: I ns; deIay w
sent from the spectrum, as is observed for the chlorophyll-b $ and S; transitions at 160 Hz choppingfrequency. Finally, kinetic constants pi, ki and wi can be obtamed by electronic simulation of the ESR transients. As \vi 9 ki,pi, all transients decay to equilibrium with exponentials containing the averages izi Pi and fxi ki [ 131, and in this respect do not provide data on the individual spin components. However, the signs and relative amplitudes of Sf(f) and SIT(t) both for continuous illumination and at a short time afrer the leading and trailing edges of the applied light pulse are determined by ki, pi, and wi. Fig. 6, represents a superposition of the experimental and sjmulated transient S;(r). Using the same values of ki and pi for the five remaining transients leads to close agreement with experimental results, if \u is taken to be different for each of the. three pairs of transitions Li$, SG and $. We fmd that P, : P,, : Pz = 0.3O(iO.O2):0.60(+0.04):0.09(~0.04); : ki : kr : kz =3.1(‘0.5)

: 7.8(?1.5) :.< 1.

The value of ~Ciki, determined from the~simulation experiment, is 320 + 50 s-lin agreement with the

Fig. 6. Superpodtion of simulated znd obser&d tznsient Sof chlorophyll-b in MTHF at a chopping frequency of 15 & ESR response time 0.3 ms Accumulation of = 1000 trznsients Time base: 5 msjdiv.; A = zbsorption;E = emission.

ESR.value of 300 s-l. We combined the Iast number with the ratio k, : ky : k, from the simu!ation experiment, yielding .I kx = 246 I40 s- , ky = 600 t 120 smL, k, < 75 s-r, in agreement with previous conclusions from the steady state S$II polarization. In actual simulation, the error limits are smaller than quoted above, since the choice of kj and pi rhgt be consistent with the 121

.,’

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Voiu&

CHEMICAL PHYSICS IiETl-ERS~

29, number 1

.. Referenies

] magtjtude of the steady state po!arization. Spinlattice rei,axati& rates-are found to be of the order of lti s,-l.$urthermore tvX k buy > wz, in &litative agieement with the results.obtained from (3) using experimental values fo; ki and pi. Probably, the absence of sp& p’ol&ization fdr Hli z is die to the very sm& value of klpO - kOpl iti (3) and not to fast relaxation. Simulatiqn was carried out with a fictitious’Boltzmann difference 6 = 0.1. Since the shape of the transient is largely determined by ~~6, if lvi s ki,pi, this -does not dffect the,results, as long as iviE .is kept unchanged with respect to the actual ESR experiment, and6<1. 0~; results show gopd agreemect with data recent’ ly obtained by Clarke [4], using optically detected magnetic resonande in zero magnetic field, and are qualitatively similar to experimental results on the FO@n free base under cotiditions of siow relaxation

ii] d.P. Gurinovich;,A.N. Scvchenko and K.N. Solovyov, 1

of solvent

affect

Biological Molecules; iisbon

(1974), Contribution

nr.

(Academic Press, New York, 1966). [9] J.E. Falk, Porphyrins and metatloporphyrins

(Elsevier. Amsterdam, 1964). [lo] iJ. Eisner and R.P. Linstead, J. Chem. Sot. (1955) 3749. ill] J.%l. Lhoste, Compt. Rend. Acad. Sci. (Paris) D (1968)

1CS9. [12j J.zI van dar Waals and M.S. de Groot, in:.The triplet st;ite, ed. A.B. Zahlan (Cambiidge Univ. Press, London, 15’67) p. 125. [ 131 H. Six1 and hi. Schwoerer, 2. Niturforsch. 24a (1969) 952; 25a (1970) 1383. [14] M. Schwoerer, in: Proc XVII Congess Ampbre. cd. V.

the zero-.

Hovi O\lorth-Ho:land,Amsterdam,

1973) p. 143.

[15] D. Antheunis, Thesis, Leyden (1974). [16] I.‘!. Ghan, W.G. van Dorp, T.J. Schaafsma and J.H. van der Waals, Mol. Phys. 22 (1971) 741, 753. [ 171 G.T. Rikhireva, L.A. Sibel’dinn, Z.P. Gribova, B.S. hlxinov, L.P. Kayushin and A.A..Krasnovskii, DokL Al;ad. Nauk USSR (Biophysicql Section) 181 (1968) lC:3. [ 181 G.T. Rikhireva, Z.P. Gribova, L.P. Kayushin, A.V. Ulnrikhina and A.A. Krasnokkii, D&l. Akad. Nauk USSR 159 (1964) 196. [19] J.ld. Lhostc,‘C HdjBne and hf. Ptak, in: The triplet state, ed. A.B; Zahlan (Cambridge Univ. Press, London,

‘.

Acknowledgement

-We.tiish t’o‘thank Dr. J&I: van der Waals for per-_ mission to refer, to les&s obtained in his group prior ,’ to publication Onk of us (T.J;S.) is seatly-indebted to Drs. J.H. van der Waals and M. Gouterinan for inspiring disctis+ns on the electronid structure of pori. fihyrins. &Ir: PiA. de Jager has provided us with vahiable’technical asskttance. I “jj~‘.:~‘.‘; ; ..,. ..’ -. :.. ,. .-: .: ; ._. . ..‘.‘. ., :

related comp&nds

[5] W.G. van Doti, T.J.‘Schaafsma, hi. Soma and J.H. van der Waals, Chem. Phys. Letters 21 (1973) 221. [6] AK. Chibisov; Dokl. Aknd. Nauk USSR 205 (1972) 14-2. [7] M. Gouterman, in: Excited stales of matter, ed. C.W. Shoppee, Grad. Studies Texas Tech. Univ. 2:1-174, 1573, and references therein. [S] L.F. Vernon and G.R. Seely, eds., The chlorophylls

field parameter J?, as well ai the observed amount of spin-polarization, which also depends on the direction of the magnetic field with respect to the molecular iti&, due to anisotropic spin-lattice relax&ion rates.

.. .

of chlorophyfl.and

a;.

Summarizing, we coriclude that the kinetic behaviour of the lowest triplet state of chlorophyll-b, as derived from steady state’and transient spin-polarization, is characterized by the presence of a fast.decaying irrplane spin component~which~also has the largest populating rate. In this respect, chlorophyll-b resembles ee porphin frek base rather than porphyrin wit?? heavier metal ions. Both the presence of a different side group as in znd a change

Spectroscdpy

(Science and Engineering Publish&g House, Minsk, 1568) [English transl. Natl. Techn..Inform. Service, Springfield] ch. 7. [3] kT. Gradyushko’, .4N. Sevch&ko; K-N. Solovyov and bI.P..Tsvirko, Photochem. Photobiol. 11 (1968) 387. [ 31 P.L. Dutt
[25].

chlorophyll-a

l-November 1974

1S67) p. 487; J.l’. Kleibeuker and T. J. Schaafsma, unpublished

:

results. [20] J.lil. Lhoste, Stud. Biophys. Ber!in 12(1’968) 135. -, [21] H: Levanon qd S.I. Weissman, J. Am. Chem. Sot. 93 (1371) 4309.

[22] H. Levanon and S-1. Weis_&an, Israel J. Chem. 10 (1372: 1. [23] hf. Plato and K. Mtibiul;hiesstechnik

8 (1972) 22p.

[24] R.H. Chke, Chkm. Phys. Letters 6 (1970) 413. [25] ni. Soma, prisate,communication.

_‘. ,:

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