Intersystem exciton transfer in isolated chloroplasts

Bloehtmtca et Btophyszca Acta, 325 (1973) 530 538

C~Elsevter Scientific Pubhshmg Company, Amsterdam - Printed m The Netherlands BBA 46639

I N T E R S Y S T E M E X C I T O N T R A N S F E R IN I S O L A T E D C H L O R O P L A S T S

J.-M. BRIANTA|S, C. VERNOTTE and I. MOYA Laboratolre de Photosynthkse du C.N R . S , 91190 Gif-sur-Yrette (France)

(Received June 8th, 1973)

SUMMARY The following arguments in favor of exclton transfer between the two photosystems are presented: (1) MgCI/ (1-10 mM range) decreases the mtersystem transfer but does not modify the partition of absorbed photons between the photosystems. MgC12 addition causes a simultaneous increase of excitation life time (~) and of fluorescence intensity (F). The same linear relationship is obtained with or without added Mg 2+ (2) The deactivation of Photosystem II by the Photosystem II to Photosystem I transfer increases with the level of reduced Photosystem II traps. When all Photosystem I[ traps are closed, half of Photosystem II excltons are deactivated by transfer to Photosystem I (3) From the relative values of the 685-nm fluorescence yield and System II electron transport rate in limiting light, measured with and without MgCl z, the values of rate constants of Photosystem II deactivation were calculated. (4) The mtersystem transfer determines a 715-nm variable fluorescence, which is lowered by MgCI 2 addition. When this transfer lS decreased by MgCI2 the efficiency of the transfer between Photosystem II-connected units is enhanced, and a more slgmoldal fluorescence rise is obtained A double-layer model of the thylakoid membrane where each photosystem is restricted to one leaflet is proposed to explain the decrease of the intersystem transfer after adding cations. It is suggested that MgCI 2 decreases the thickness of the Photosystem I polar region, increasing the distance between the pigments of the two photosystems.

INTRODUCTION The addition of cations to chloroplast suspensions reduces changes of the partttJon of absorbed energy between the two photosystems 1-5. These changes could result either from changing the partition of the sensltizers related to each photosystem (Hypothesis I) or by changing an lntersystem exciton transfer (Hypothesis II, sugAbbrewanons: DCMU, 3-(3,4-dlchlorophenyl)-l.l-dlmethylurea; F, fluorescence intensity, T, excitation hfet~me

INTERSYSTEM EXCITON TRANSFER

531

gested by Muratal-3). After considering the extensive analysis done by Malkm 6 and more recently by Sun and Sauer 7'8 on "spdl-over" we concluded that additional experiments were needed an order to choose between these two hypotheses. The results reported here, measuring fluorescence and electron transport in the presence or absence of MgCl2, argue in favor of Hypothesis II; i.e. there is a Photosystem l! --, Photosystem I exclton transfer which is inhibited by Mg 2+. EXPERIMENTAL Chloroplasts used in these experiments were isolated from lettuce leaves, according to the method of Nelson et al. 9.

Excitation hfettme fluorescence mtensiO' relationship Mg 2+ induce an increase of the Photosystem l[ fluorescence antenslty of chloroplasts. The effect is complete after 10 mtn m the dark or light. This phenomenon could result either from an increase of the quantum ymld of fluorescence or from an increase of the sensitization of Photosystem 1I. In order to resolve this ambigmty, we measured fluorescence intensity (F) and lifetime (~) simultaneously during Photosystem II reduction, in the presence or absence of MgCI2, using a phase fluonmeter previously described a0,~l The Photosystem II fluorescence anductlon was followed using the method of Lavorel az. The yaeld of Photosystem lI variable fluorescence pr is: kf

pf =

(1)

kf + kp O + k c + ktl where kf, kp, ka and kc are the rate constants of deactivatmn, respectively, by fluorescence, reductmn of Q, transfer to Photosystem I and other non-radmtwe processes. Q is the concentratmn of the oxidized form of Duysens' quencher. The excitatmn hfettme is. 1

r =

(2)

kf + kp Q + k¢ + ]~a SO~ z =

1

/q

-pe'r

(3)

Therefore, the intensity of variable fluorescence may be written as followed:

F = I'a'kf

(4)

where I = incident intensity absorbed and a - fractmn of photons absorbed by Photosystem 1I Fig. 1 shows that the same linear relationship between r and F is obtained either in presence or m absence of MgC12. This result means that the product (1 - a) of Eqn 4, which determines the slope (z, F) has not been changed upon MgCI 2 additmn. Therefore, this experiment points out that MgCI 2 reduces a real increase of Photosystem ii fluorescence yMd (i.e. of pf) instead of an increase of excitation

532

J.M. BRIANTAIS

15

o

et al

DQ

o Q o

Cp • Cp+ MgCi2

tI[J

I

3

2

4

FLUORESCENCE

Fig 1 E x citation lifetime (r)-fluorescence intensity (F) relationship duri ng P hot os ys t e m II reduction. A phase fluorlmeter gave s i m u l t a n e o u s m e a s u r e m e n t s of F a nd r The frequency of the m o d u l a t e d incident beam was 7 25 M H z The chloroplast suspension, 2-ram thEck, c ont a i ne d 30 Hg of c h l o r o p h y l l per ml o f a 10 - z M Trls NaC1 buffer (pH 7 8) and 0.4 M sucrose The i nduc t i on was developed m a static way or by flowing By the latter mode, an actinic p r e d l u m l n a t l o n , used at various mtensEt]es is a d d e d 2 ms before fluorescence analysis. ThEs gave different energy states c o r r e s p o n d i n g to varEous l n t e r m e d m t e levels between m a x i m u m and m i n i m u m fluorescence yields Exc i t a t i on beam for both analysis and actlnEC light was blue hght ( C o r n m g 4-96 filter). Fluorescence emitted is detected t h r o u g h a C o r n l n g 2-64 filter MgCI2, ( 3 - 0 , + M g C I 2 (7 5 10 -3 M), Q - O .

c o m i n g to P h o t o s y s t e m II centers. The increase of Pr m u s t result f r o m a change o f at least one o f the various ways o f P h o t o s y s t e m II deacttvation. The following experiments were designed to d e t e r m i n e which o f the deactivation processes are affected.

Effect of MgCI2 on Photosystem H and Photosystem I photochemical rates MgCl 2 was f o u n d to decrease the rates o f P h o t o s y s t e m I - m e d m t e d M e h l e r reactions but increase the rate o f P h o t o s y s t e m I [ - m e d i a t e d O2 evolution in ezther TABLE 1 E F F E C T O F MgCI2 A D D I T I O N ON S Y S T E M I A N D S Y S T E M II P H O T O R E A C T I O N S S t a u o n a r y rates were m e a s u r e d m h m l t l n g light intensity at 2. -- 647 rim; (half b a n d w i d t h 12.5 nm) and at 2. > 695 nm (Shott RG 695) The rate values are given m / t m o l e s / m g c h l o r o p h y l l per h C h l o r o p l a s t s at 40Hg c h l o r o p h y l l / m l were suspended in 0 01 M Tris N a C I buffer (pH 7 8) which c o n t a i n e d 0 4 M sucrose. W h e n added, MgCI2 c o n c e n t r a t l o n was 7 5 10 -3 M. The System 1 phot oreaction m i x t u r e c o n t a i n e d D C M U , 10 - 4 M, s o d i u m ascorbate, 10 -z M, me t hyl vl ol oge n, 10-'* M, and s o d i u m azlde, 10 - 4 M. Light induced 0 2 u p t a k e was m e a s u r e d The System I1 reaction mlxturc contained 10- "~ M d l c h l o r o p h e n o h n d o p h e n o l , 0 2 evolution was de t e rmi ne d A C l a r k - t y p e electrode (Gllson Co.I was used to m e a s u r e 02 exchanges The m a g n i t u d e of the error is ~ 3 % The temperature was m a i n t a i n e d at 20 °C

14"avelenqth3 ofexcttatton ( nm

647 695

Oz et,olutton -FMgCIz --MqClz

)

17 7 16 1

12.5 11.9

~ MgCI2/ -- Mg C12 1 42 I 35

02 uptahe ~MgcIz

h,JgCI2 -

104 252

155 296

-~ M qCI-2/ ,~4q Cl2

0 67 0 85

I N T E R S ~ STEM EXCITON T R A N S F E R

533

Photosystem ll or Photosystem I hght (Table l). This is in agreement with earlier reports of photochemical actlwtles using Photosystem lI illumination ~'13. It should be noted, however, that the MgCl 2 effect on Photosystem I is greatest when Photosystem II sensltizers are preferentmlly excited. In addmon, the ratio of ~ Mg2+/ - M g 2+ rates is comparatively greater for Photosystem I at long wavelength than at short wavelengths. In contrast, the ratio of ~ Mg z + / - Mg 2 + rates for Photosystem II vary httle with the two different lights used.

Effect oJ MgC/2 on the photochemical rise of Photoo'stem H fluorescence As shown in Fig. 2, when all Photosystem II traps are open (F0 level), a MgC12 effect occurs but its magnitude is very much smaller than when all Photosystem II traps are closed (F~ level) Although the fluorescence yield Fm is two times larger in the presence of MgC12, the half-rise of the fluorescence intensity is only slightly changed (27 ms without MgCl2, 24 ms with MgCI2). The half-time of Q reduction (determined by the integral of (FM-FE) versus time (according to Malkmt4)) is 43.5 ms in the absence of MgCI 2 and 30.0 ms in the presence of this salt. In addition, Fig. 2 shows that the shape of the photochemical fluorescence rise

w

•

•

FM

p

/ / // !

°

.....

F 0

6o

,0

m zec

o

50

100

I50 MILLISECDND

Fig. 2. Fluorescence reduction of a c h l o r o p l a s t suspenmon. The reaction mi xt ure c o n t a i n e d chlorophyll (20/+g/ml), 5 l0 -5 M D C M U , 0 4 M sucrose and 0.0l M T r i s - N a C l (pH 7.8) in the presence ( 0 - 0 ) o f 5 - 10-3 M MgCI2, or absence ( O - Q ) o f MgCl2. The suspenmon was 1.5-mm thzck. The excitatio n h g h t (480 nm, ~1). = 15 nm) was both analytic and acmlc, its intensity c o r r e s p o n d s to 23 p h o t o n s / c e n t e r per second The fluorescence (Ft) emitte d at 685 nm (_1). -- 15 nm) was me a s ure d. Fo, initial fluorescence yield; FM, m a x i m u m yield o b t a i n e d at the end of the p h o t o c h e m i c a l phase of the reduction. The reset corresponds to a log (F,,t Ft ) plot, calculated from the reduction curves.

J.M. BRIANTAIS et al

534

is clearly sigmoidal m the presence of MgCl 2 a n d nearly exponential xn its absence (see the inset).

Effect of MgCl2 on the relationship between 715 and 685 nm variable fluorescence Lavorel t 5 f o u n d that the emission spectra of variable fluorescence m Chlorella shows a 715-nm band, the kinetics of which c a n n o t be differentmted from the fluorescence changes which occur at 685 n m As Fig. 3 shows, MgCl 2 a d d m o n o n chloroplasts decreases the slope of the linear relationship which correlates 715- a n d 685n m emission d u r i n g the i n d u c t i o n period. Notice that the two curves, with a n d without MgC12, intercept the 715-nm axis at the same point.

I

~LdDRESCENC[ l~5

~6

/ "~

•

Cp+MgLI~

o~

D

I

~

3

4

FLUORESEEN~E G85

Fig 3 Effect of MgCI2 on the relaUonshlp between variable fluorescences measured at 715 and 685 nm. O O, in absence of MgClz; Q - O . m presence of MgCI2 (7.5 • 10-3 M). The fluorescence was recorded at 715 nm and 685 nm (A)~-- 6.4 nm). The Fo level was measured by flowing the suspension in a capillary tubing 1.5-mm m diameter The fluorescence rise curve was obtained by stopping the flow Exc~tation beam 480 nm (A;t = l0 nm) The chloroplast suspension was 0.4 M sucrose, 0.01 M Tns-NaCl buffer (pH 7.8) containing 20/tg chlorophyll/ml.

Variation of MgCl 2 effect depending upon the chloroplast preparations A great variability of the m a g m t u d e of the m a g n e s i u m effect has been observed along the p r e p a r a t i o n s tested. I n preparations which show the "classical" m a g n e s m m effect, the amplitude of the p h e n o m e n o n vanes. F o r example, representative data from three separate experiments are shown in Table II. It seems hkely that at least two i n d e p e n d e n t factors determine the amplitude of the MgCI 2 effect on Photosystem II fluorescence: (1) the n u m b e r of active Photosystem II centers (approximated f r o m the F~/Fo ratio) whlch regulate the effect of Mg z÷ on the Fo level a n d (2) a m e m b r a n e state which regulates the Mg z+ influence on the F M level. Th~s may relate to our observations that some chloroplast preparaUons, including isolated g r a n a a n d stroma lamellae separated after F r e n c h press disruption 16, do n o t show a M g C I / e f f e c t o n fluorescence. I n some cases, these u n u s u a l preparations show a n opposite effect of MgCl2; i.e. a decrease in the 685-nm emission a n d a correlative increase in the Photosystem I Mehler reaction rate, using 650- or 670-nm lights at a limiting intensity.

1NTERSYSTEM EXCITON TRANSFER

535

T A B L E 11 C O M P A R I S O N OF T H E V A R I A B I L I T Y OF MgCI_, E F F E C T IN T H R E E C H L O R O P L A S T PREPARATIONS 685-nm fluorescence t_l). = 6 nm) was measured. Fo was de t e rmi ne d by flowing the s u s p e n s i o n , F~I c o r r e s p o n d s to the steady state reached during c o n s t a n t dluminat~on Other c o n d i t i o n s are the same as m F~g 4

Prepn 1

Prepn 2

Prepn 3

Fo(SMgCI2) Fo( -- M g C I , )

I 17

1.4

1 07

FM( ' MgCIz) FM( -- M gCl2)

1 75

2 05

2 10

F~( ~ MgCI2) Fo( + MgCI2)

3.70

3 30

5 55

DISCUSSION

W e have a t t e m p t e d to gather evidence for the m o d e of action of M g 2+ i n ehcitlng a change m the p a t t e r n o f exciton distribution between Photosystems I and II It was first shown (Fig. I ) t h a t ~ a n d F change m a parallel relatzonshlp with and without MgC12. A l t h o u g h the d a t a does n o t show a rigorous h n e a r relationship a n d e x t r a p o l a t e to a positive ~ value at a zero F value, we can conclude that the observed F increase m the presence o f d w a l e n t cat,on ~s a real increase in the fluorescence yield. A change m the p a r t l t l o m n g o f a b s o r b e d p h o t o n s between the two p h o t o systems or an i n t r o d u c t i o n o f P h o t o s y s t e m I to P h o t o s y s t e m II exclton transfer m the presence o f MgC12 would have caused an increase m F without a change m ~. This was not observed W e have discussed a b o v e the fact that the increase m pf must be due to a reduction in the rate o f some d e a c t w a t l o n process. The rate constants for these processes were defined m Eqn 1. Each c o m p o n e n t of the equation wdl now be discussed w~th respect to o u r findings. The rate c o n s t a n t for fluorescence, k f , we assume does n o t change upon add m o n o f MgClz since the slope o f F v e r s u s r (Eqn 4) was not affected by salt a d d m o n . W e can also conclude that kp • Q is not the factor regulating the salt-reduced change m the fluorescence yield since k v . Q a p p r o a c h e s zero m the presence o f 3-(3,4-d~c h l o r o p h e n y l ) - l , l - d ~ m e t h y l u r e a ( D C M U ) (when the M g 2+ effect is m a x i m a l ) . A decrease o f k¢ by MgCl2 a d d l t l o n w o u l d gwe an increase of the yields o f System II p h o t o c h e m i s t r y and of l n t r a s y s t e m l I transfer, as we have observed. Such a change m kc would not explain the relative decrease o f the 715-nm vartable fluorescence n o r the inhibition o f System ! p h o t o c h e m i s t r y which ~s preferentmlly s e n s m z e d at 647 nm The final f a c t o r included m Eqn 1 which might be affected by MgCI2 is kt~ (excitation transfer f r o m P h o t o s y s t e m I1 to P h o t o s y s t e m I). R e a s o n s for b e h e v m g that o u r d a t a are most consistent w~th a decrease in th~s c o m p o n e n t are as follows. The d a t a o f Table I indicate that there ~s a greater inhibition by MgC12 o f P h o t o s y s t e m I m short wavelength hght, but a u m f o r m effect on P h o t o s y s t e m II

536

J.M. BRIANTAIS e t

al.

m either 647- or long wavelength light. This suggests that the spillover from Photosystem I[ to Photosystem I, but not the reverse, is the pmmary s~te of excitation distribution regulation. Another argument mdzcatmg that ktT is decreased by MgCl 2 addition comes from our finding that the z - F slope does not change upon addition of the salt. This result means that MgCl2 addition does not change the arrival of excltons to Photosystem II so the salt must not increase a Photosystem I to Photosystem II transfer. Our data also suggest that there is increased Intrasystem II exciton transfer. The fluorescence induction was qmte exponentml in the absence of MgCl 2 but became slgmoldal when the salt Js added (Fig. 2); according to kavorel and Joliot 17, the sigmo~dal kinetics are the result of connections between Photosystem II umts. A decrease in Photosystem II ~ Photosystem I excition transfer may explain the ongm of a 715-nm band m the variable fluorescence, since this band may correspond to a Photosystem I emission produced by the transfer. It would determine that the kinetics of reduction of the 715-nm band would follow the 685-nm fluorescence. The decrease of the slope of this linear relationship by MgCI2 would result from the decrease of kt~. A mechamsm by which MgC12 could ehclt a change in k,i must somehow be related to the structural organization of Photosystems I and II within the membrane If the mtersystem transfer occurs according to a F6rster process, ~t will occur mainly m the direction Photosystem lI to Photosystem I because of the overlap between absorption and emission spectra of the two photosystems. Taking Photosystem I and Photosystem lI enriched particles is as representative of each photosystem, the transfer Photosystem II to Photosystem I is five t~mes more probable than Photosystem 1 to Photosystem II transfer on the overlap basis. No change of absorption spectra nor of the posltlon of emission bands was detected on adding MgCI2. therefore, we have to assume that the decrease of the transfer induced by MgCI2 is the result of an increase of the distance between Photosystem I and Photosystem II p~gments Cation addition reduces structural changes of chloroplasts19-22; glutaraldehyde fixation of chloroplasts suppresses the MgCIz-mduced increase in the 685-nm fluorescence 22. Because many results on chloroplast structures suggest an asymmetrical drstmbutlon of the two photosystems in a bilayer membrane 2a, we propose that Mg 2+ increases the distance between the two polar parts of the membrane to which, tetrapyrole rings are hnked. The fact that Mg z+ may cause a structural change in the membrane may be related to the finding that EDTA-washed chloroplasts show a depressed MgCI2 effect 22. Mg 2+ is a cofactor of phosphorylatlon at the concentration which reduces Photosystem II fluorescence y~eld increase The couphng factor (CF1) is locahzed on the outside face of the chloroplast lamellae 2a. Thus we may suggest the following speculative scheme' M g 2+ would determine a conformatlonal change of CF~ and this m turn causes a shrinkage of the polar part of Photosystem I layer and increasing the d~stance between the two polar regions of Photosystem I and Photosystem II. It has prewously been shown that Mg 2+ reduces chloroplast membrane thlckness 21. This may not be inconsistent with our ideas, however, since salts are also known to reduce conformatlonal changes (substructural alteration) within the membrane itself 24.

INTERSYSTEM EXCITON TRANSFER

537

APPENDIX

Magmtude of the vartous pathways of Photosystem H deactivation', rate constant values The ratio of fluorescence yields (Pr) with and without magnesium, for instance when all the traps are closed (blue hght, D C M U added) is: Pr(+ Mg) _ 2 pf(-Mg)

(average value from several experiments).

The ratio of the photochemical yxelds (pp) with and without magnesmm, for instance when all traps are open ( C o r n m g 4-96 blue filter, h m m n g intensity) is: pp(+ Mg) _ 1.2 pv(-Mg)

(average value from several experiments)

We assume, m a d d m o n , that MgCI 2 (10 m M ) abohsh the transfer Photosystem II --. Photosystem I (because such a concentraUon saturates the effect). In presence of D C M U , kp. Q 0 (Q = 0) so pr(+Mg) = kr+k~+/,,, pf( -- Mg) kf + k c

= 2,

so

k,i = k r + / %

(5)

No~' when Q is completely oxidized.

kP'Q+kf+kc+k"kp - Q + k t + k ¢

Pv(+Mg)p p ( - Mg)

1.2

so

ktl = 0.2 ( k p . Q + k r + k c )

(6)

combining (5) and (6) /,p "O -

1-02

(kr+k¢)

0.2

kp • Q = 4k,l

(Q completely oxidized)

(7)

from such values, one calculates pp ( + Mg) 0.80, pp ( - Mg) = 0.66. The Importance of the transfer to Photosystem I, PtJ, will vary, depending upon Q~¢d '--* Qo, equlhbrmm: When all Q is reduced" P,I -

ktl

= 0.5

because of (5)

ktl + kr + k~ When all Q is oxidized. P,i =

ktl

= 0.166

because o f ( 5 ) and (7)

kti + kr + k~ + k p • Q The values of rate constants can be calculated assuming the intrinsic lifetime r, 15 ns and a fluorescence yield = 0.027 when all the traps are open z5"26 and 0.108 when both D C M U a n d MgClz are added from z , = 1 5 n s like, k r = 6 . 7 107s - ~ l s deduced. If Pr ( + M g ÷ D C M U ) = 0.108 = kr,/ke+kc then k~ = 5 53 • 108 s -1 f r o m

J M. B R I A N T A I S et al.

538

relation (5) ktl = 6.2 • 10 s s -~ and f r o m relation (7) kp • Q = 2.48 • 109 S - 1 " M are reduced.

Value of kill Let us consider again the d e a c t i v a t i o n in one P h o t o s y s t e m II unit. A c c o r d i n g to

the computer calculation of J. Lavorel using Monte-Carlo method, to determine the kinetics of disappearance o f open traps in a connected model of Photosystem I! units 18, the efficiency of the transfer between two adjacent Photosystem II units Js 0.7 in Chlorella cells, when all Q is reduced. So, depending on the importance of kti m Chlorella, (from 0-6.2- l0 s s-~), ktl I w o u l d vary f r o m 14, 5-29- 108 s - 1 (values d e d u c e d from P,H = k t l l / k t l l - k r ~- k~- ktl 0.7). ACKNOWLEDGEMENTS

W e w a n t to t h a n k Mrs M. Picaud for her help in the experimental p a r t o f this work. W e are grateful to Dr C J. A r n t z e n for suggestions he gave us d u r i n g the p r o o f writing. This w o r k was s u p p o r t e d in part by D . G R.S.T., G r a n t No. 7270174.

REFERENCES Murata, N. (1969) Btochmt. Blophys Acta 189, 171-18l Murata, N , Tashlro, H. and Takamlya, H (19703 Btochtm. Btophy,s Acta 197, 250-256 Murata, N. (19713 Blochml. Btophys. Acta 245, 365 372 H o m a n n , P. H (19693 Plant Phystol. 44, 932-936 Gross, E L. (1972) Btophys Soc. Meet ( A b s t r ) , 64a Malkm, S (1967) Btophy.s.J. 7, 629-649 Sun, A S K and Sauer, K. (1971) Btochm~ Biophys' Acta 234, 399 414 Sun, A S K and Sauer, K. (1972) Btochtm Btophy.s. Acta 256, 409-427 Nelson, N , Dreshler, Z. and Neumann, J. (1970) J Btol. Chem. 245, 143 151 Moya, 1 (1973)Thesis, Umverslty ParJs-Sud (Orsay) France Vernotte, C and Moya, I. (19733 Photochem. Photobtol. 17, 245 254 Lavorel, J. (1965) Photochem Photobtol. 4. 819-828 Ben Hayylm, G and Avron, M. (1971) Photoehem Photobtol. 14, 389 396 Malkin, S. (1966) Bloehun Btophys Acta 126, 433-442 Lavorel, J (19623 Btochlm Btoph)'~ Acta 60, 510-523 Park, R B and Sane, P. V (19713 Annu Ret, Plant Phv~tol 22, 395 430 Lavorel, J and Johot, P (19723 Biophys J. 12, 815-831 Brlantats, J M (1969) Phyatol. Veq 7, 135 180 lzawa, S and Good, N E (1966) Plant Phy~tol 41, 533 543 Izawa, S and Good, N E (19663 PlantPhystol 41, 544-552 Murakaml, S and Packer, L (19711 Arch Bzochem Btoph)s. 146, 337-347 Mohanty, P , Braun, B Z. and Gowndjee (19733 Btochtm Btophys A~ta 292, 459-476 Arntzen, C J and Brmntals, J. M (1973) in Btoener,qettcs of Photosynthests (Govmdjee, ed ), Academic Press, New York 24 Wang, A Y. 1 and Packer, L. (19733 Btochtm Blophya. Acta 305,488-492 25 Latimer, P , Banntster, T T. and Rabmowltch, E. (19563 Sciences 124, 585-586 26 Teale, F (1960) Btochtm Btoph3's Acta 42, 69-75

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