Excitation energy transfer between chlorophyll a and chlorophyll b obtained from fluorescence meaeurment's

0, Szurkowskl, S, Motchondanl, R. M. Leblanc D. Fr«ckowiak

EXCITATION ENERGY TRANSFER BETWEEN CHLOROPHYLL A AND CHLOROPHYLL B OBTAINED

FROM FLUORESCENCE MEASURMENTS

In this study, th* fluorescence lifetime of chlorophyll a, chlorophyll b and their mixtures, and the possibility of migration of energy absorbed by chlorophyll a to chlorophyll b was examined.

With an aim to evaluate the yield of excitation energy tran-sfer between ordered chlorophyll a and chlorophyll b molecules the fluid oriented medium-nematic liquid crystal (LC) mixture MB9A (p-methoxybenzylldene p-butylanlline) and EBBA (p-ethoxy- benzylidene p-butylaniline) la used. LC is oriented by deposi-tion of thin layer of sample between stretched polyvinyl alcohol films [1] or between silicon oxide orienting layers; pigment ¡eo- lacules are oriented similarly by strong interaction with LC mo-lecules [2]. Chlorophylls in LC are monomeric except the highest

-2 concentration used (10 M/1).

Tha results of photoacoustic spectra suggest the excitation energy transfer from chi a to chi b in investigated system C3]. In order to check this unexpected result the mean lifeti-mes of chi a, chi b and their mixture were measured. Lifetilifeti-mes ware measured for chi a and chi b solutions and for their mi“ xtura in the same solvent. Table 1 gathers the results of life-times measurements for chi a chi b and their mixture in the sa-me LC. Every one results from Tab. 1 is obtained by averaging of three or four Independent measurements. As can be seen from

T a b 1 e 1 Fluorescence lifetime of chi a, chi b

and chi a + cfil b mixture in MB8A_ + EBBA (Ca - 1.7 • 10"*tVl. C b - 3.6 • 10"3 M/1)

Czas życia flgorescencji chi a, chi b i mieszaniny chl a+ chi b w MBBA ♦ EBBA CCa - 1,7- 10" mole/1, Cb « 3,6 • 10*3 mola/1)

Sample Warelength Lifetime [nm] of excitation [nm] of fluorescence [nm] | j Chl a 405 > 620 5.56*0.03 405 > 695 5.53-0.02 436 > 620 5.59-0.02 1 466 > 620 5.35*0.02 ] 491 > 620 5.17-0.02 Chl b 405 > 620 6.61-0.03 405 > 695 8.91-0.03 436 > 620 8.81*0.03 466 > 620 8.75*0.03 491 > 620 8.46*0.03 Chl a -» chl b 405 > 620 7.82-0,03 405 > 695 8.01*0.02 435 > 620 7.99*0.03 436 > 695 8.13-0.02 466 > 620 7.65*0.03 466 > 695 7.72*0.02 491 > 620 7.49*0.05 491 > 695 7.70*0.04

Tab. 1 for chi a + chi b mixture ia much clo8er to that of chi b alone, then for the chi a solution in LC, evan at higher chi a than chi b concentration. Measurement were done using two regions of fluorescence spectra:

1) using filter transmitting wavelenghta \ > 620 nm e.g. in-cluding whole chi a and chi b emission bands;

2) trough the filter transmitting >- > 695 nw .<hich giv* mo-re chi *, then chi b fluomo-rescence.

Comparing «11 reiu.lt® gather ? < T«d J *r»e ;o C ;.J-<ie, thar bacauea tff •trong o u r yp ir,fc nf e*»iesioi' - -c.tr.> of chi a chi b and their aggregate 1 <c " not po»> x'ol v ",#'j»ure

H»ton«jJnr to every ©n© i»' * .« •■i.gne-t* locate : gr».«nc »t* xturo. TVi rt -lit ©* T for pigme «> t .> re* related with two ef fecta i

1) the indepen'. nt excitation of eve / one pigment follov^sci by the overlapping of their cmiaeioni' r. harectarizad by different

2) the change In tha fractions of emitting pigments by the excitation energy trarafer.

Firet effect ia trivial. To check if second affect pays m~ oortant role in our eysten we analyaed firat lifetimes of pig.-aft ¡»Sxture u*ving Pearlstein, Turner»an and Sordrin formula [4j

(l ♦ tan2^2^ i^ i t,n * (* * tan2Sj) f2$0 tan 5j>

tan 3 ® ^ w" ”■ )

(l ♦ tanz62 )fJ'i>1 ♦ (1 ♦ tan S1)f2«i2 wheret 6 • obeerved phase ehift for pigment mixture;

6^, fi2 - phase ahifta for the pigment» in separated sam- plaa i

f^, fg - fractions of light absorbed by chi t and chi b respectively;

$2 - the yialda of fluorescence of chi a and chi b 7 — 1

tan 6 » - tax; u « 7.36 • 10 a

us is 2IIv frequency of light nodulotion usetl vr> phase fluorone» ter. It was found that % obtained from exf>*rs Biant ia different fro fc^se ut'ted fro« formula (i). For example at " * ¿Si r*r \? ^« al * ”0,41; whereas the experimental value is

t8n 5exp " ’°*56

It v* ari evidence itrsut in our soaole the emission if at >st partiably excited by ET.

e:.ci-tation energy transfer between Investigated plgmente the formula proposed by K n o x [4] is usedj (,2 * A^ fl)w 5# t • arc tan 1 2

(

)

wherei A ^ A2 »re natural radiative rate of chi b and chi a (A^ •tJi* *2 * T o S )!

Kl # K2 " totfll decay constant* for uncouplad *y*t*mej F - rate excitation energy tranefer«. It la auppaaad

in calculation that energy la transfarad fro« chi b to chi aj

5fl'f2 ' Phas® shift predicted when absorbed fraction

are f^ and f2#

Data used is f1# fg calculation for two wavelength* of axel- tating ligth are gathered in Tab. 2.

and A2 are obtained fro« red absorption banda of chi b and chi a respectively using formulas

A - — • 3.10" 9 V2 A\> E «ax To

*

>1 where t 3 - position of band maxiau« In ca I

A 3 - half bend width (in cm”1 )»

E - excitation at «axlauw of abaorptlon fT^c«** . ASX

K1 'icfil b *nd K2 " xcRI a arB <>bt*lnwd trom «ti-me a of separated pigments, f^, f2 and w ara daflnad aa In fo-rmula (1).

Data used in calculations and reaulta ara gatharad In Tab. 2. It was found that In all cases 5 ~ »Chl a “ r,t* ^ from chi b to chi a is negative. It mean* that rata of ET fro« chi a to chi b predominates In Investigated ayatea. Therefore the yield of ET from chi a to chi b is calculated aa $ ebl # chl • - -Kchl b - chi a • ''chl a* The accuracy of calculation la better for not very low every one f^ and f2 , therefore such re-sult« are shown in Tab. 2.

Valu es u s e d I n t h e ca lculation o f t h e ef fi cien cy o f t h e exc it atio n en er gy tra n s fe r on t h e b a s i s o f f or m ul a ( 2) W ar to ści u ż y t a d o ob lic zeń wyd a jnoś ci tr ans f e ru ai gr a c ji en erg i i w e d ł u g f o rm uł y ( 2 ) Wa v el en gt h o f ex ci ta-t i o n (n» ) P i g m e n t A ( a “ 1 ) X (s' 1 ) f ( n s ) t a n 5 V z K c h l b * c h l 6 ^c hl a-c hi b 46 6 c hi a ( 2 ) C h i b U ) 2 . 76 x 1 0 7 2.8 3 x 1 0 7 1 8 .7 x i O 7 1 1 .4 x 10 7 0.5 7 0. 4 3 5 . 3 5 8. 75 -0 . 56 3 0 a* -2. 2 x IO7 0.1 2 m 4 9 1 c h i a ( 2 ) c h i b < 1 ) 2. 78 x 1 0 7 2.8 3 X 107 1 9.3 x 1 0 7 1 1 , 8 x 1 0 7 0 , 7 1 0. 29 5 . 17 8 . 46 -0 .55 13 m -5 .0 x IO 7 0.2 6

„2 7-lg. l. Fluorescence spectra of chi » (Ca ■ 2.3 10 M) ?.»xc» 47C np curve 1 '■ j ?>. » 430 na (curve 2>; chi b (cb -* 5,2 - 10'-* M ) X 470 nn~ (.curve 5)} chi a chi b mixture (CL » 1.7 • 10“ Mj C b « 3,6 • 10'3 M'l X e* » 430 n« (curve

3,»; X * 480 na (curv« T )

6aG m

Widma f lr:or rucenęji chi a (Ca » 2,3 * 10' mola 5

A.e<0 * 470 n» • .< '• <30 nm (krzywe 2); chi b (Cb * » 5 , 2 • 10"5*©l»/Ai9xcn 47v no (krsywe 5:, chi o «• chi b ri.fiazanine (Ca * 1,7 • 10 “«ula, J*, * 3,6 • 10 mole)

-All used excitation wavelength« are located In a region of chlorophylls Soret band«. The difference a _ CH1 b ob' tained for varic< s are related with different fractions

exc

of monomer* and aggregate« of pigment absorbing at various exci-tations.

One can conclude, thet similarly as from PAS results, analy-sis of lifetimes of fluorescence of pigment mixture suggest» some way of migration of energy obeerved by chi a molecules to chi b. The lifetime of aggregate has to be longer than that of chi a, closer to t of chi b.

The thermal deactivation of excitation energy in aggregate has to ba higher than thet of chi a.

In fluorescence spectrum of pigment mixture additional maxi-mum located at 695 nm was found Fig. 1. This maximaxi-mum is not o o curing in spectra of separate pigments, therefore it is probably conneetea with mixed aggregates of both and LC molecules.

Acknowledgement

We would like to acknowledge the financial support from The Natural Sciences and Engineering Research Council of Canada and Polish Academy of Sciences (grant MR. II.7.1.1.). We would alao like to thank dr H. Manlkowski for helping us with lifetime surements. One of us (O.F.) wish to th»nk the Université du Qu-ébec » Trois Rivierés for visiting pro!ostorship in the Centre de Recherche an Photobiophysique.

[ l] * o y a <b a H . V a *■ a * a k 1 Y. , M a t * u u r

N ., M a d a H., K o l a y a s h i S., Mol. Cryst. Llq. Cryst. 72. 127 (1980.

f’2] P r 9 c k o w i a k 0., B e a m a n 0., S t i l l -m a n M. 0., Biochim. Blophy**. Acta 681, 274 (1982 V [3] F r « c k o w i a k 0,, H a t c h s n . d a n 1 S., L

e-b l a n c Rt M., S z u r k o w s k i O,. Photoacoustlc spectra cf chlorophyll a and chlorophyll b In nematic li-quid crystal (to bo published)*

[4] K n o x R, S., Excitation energy transfer and migration! theoretical considerations In Bioenergética of Photosynthe-sis, Now York (1S75).

Institute of Phyelca Poznan Technical University Centre de Recherche an Photoblophyslque

Université du Québec a Trola Rivléréa Québec, Canada

D. Szurkowski, S. Hotchendani, R. M. Leblanc D, Frąckowiak

PRZEKAZYWANIE ENERGII POBUDZENIA POMIĘDZY CHLOROFILEM A 1 CHLOROFILEM B

-.V ŚWIETLE POMIARÓW FLUORESCENCJI

Badania czasu Zycla fluorescencji chlorofilu a 1 chlorofilu b oraz rieszanin chlorofili tugeruje m o ż l i w o ś ć migracji ener-gii absorbowanej przez chlorofil a 00 chlorofilu b„