248

Biochimica et Biophysica Acta, 546 (1979) 248--255

© Elsevier/North-Holland Biomedical Press

BBA 47651 MAGNETIC FIELD-INDUCED INCREASE OF THE YIELD OF ( B A C T E R I O ) C H L O R O P H Y L L EMISSION OF SOME PHOTOSYNTHETIC BACTERIA AND OF C H L O R E L L A V U L G A R I S

H. RADEMAKER, A.J. HOFF and L.N.M. DUYSENS Department of Biophysics, Huygens Laboratory of the State University, P.O. Box 9504, Wassenaarseweg 78, 2300 RA Leiden (The Netherlands)

(Received August 16th, 1978) Key words: Magnetic field; Fluorescence yield; Bacteriochlorophyll; Reaction centre; Photosystem H

Summary In photosynthetic bacteria, in which the iron-ubiquinone complex X is prereduced, a magnetic field induces an increase of the emission yield, which is correlated with the decrease in reaction center triplet yield reported previously (Hoff, A.J., Rademaker, H., van Grondelle, R. and Duysens, L.N.M. (1977) Biochim. Biophys. Acta 460, 547--554). Our results support the hypothesis that under these conditions charge recombination of the oxidized primary donor and the reduced primary acceptor predominantly generates the excited singlet state of the reaction center bacteriochlorophyll. In Chlorella vulgaris and spinach chloroplasts, at 120 K, the magnetic field has an effect similar to that found in bacteria, which suggests that an intermediary electron acceptor between P-680 and Q is present in Photosystem II also.

Introduction

Recently it has been found that a static magnetic field decreases the quantum yield of the photoinduced triplet state in photosynthetic bacteria in which X, the secondary acceptor (an iron-ubiquinone complex) was prereduced [1,2]. The p h e n o m e n o n was explained in terms of the so-called radical pair mechanism: charge separation results in the radical pair P*I-, where P is the primary donor (a bacteriochlorophyll dimer) and I the primary acceptor (a bacteriopheophytin; [ 3--8 ]). The spins on P* and I- oscillate between a singlet state (P÷I-) s and a virtual triplet s t a t e (P+I-) w (see Ref. 9 for an introduction to

249

anfenna

energy transfer

B"

BT

k/

reactioncenter uJ

P'I L kh

kh

(t7

kg

'V B

PI

Fig. 1. S c h e m e for e n e r g y t r a n s f e r a n d e l e c t r o n r e d i s t r i b u t i o n r e a c t i o n s o c c u r r i n g in t h e a n t e n n a b a c t e r i o c h l o r o p h y l l a n d t h e r e a c t i o n c e n t e r , w i t h r e d u c e d s e c o n d a r y a c c e p t o r X. kh, k f, kic a n d kis c are the r a t e c o n s t a n t s for e n e r g y t r a n s f e r , f l u o r e s c e n c e , r a d i a t i o n l e s s d e c a y a n d i n t e r s y s t e m crossing to t h e t r i p l e t s t a t e B T o r p T r e s p e c t i v e l y , k c is t h e r a t e c o n s t a n t for c h a r g e s e p a r a t i o n , k t , k s a n d kg axe t h e r a t e cons t a n t s f o r t h e r e c o m b i n a t i o n of P+I- t o t h e t r i p l e t s t a t e , t h e e x c i t e d singlet s t a t e a n d t h e g r o u n d s t a t e , r e s p e c t i v e l y , c~ is t h e a n g u l a r f r e q u e n c y for i n t e r c o n v e r s i o n b e t w e e n t h e singlet a n d t r i p l e t s t a t e o f P+I-.

the theory of the radical pair mechanism). From (P+I-) T the charges will combine to the reaction center triplet state, pW, whereas from (P*I-) s the charges may combine to either the excited singlet state P*I or to the ground state P I. Indications for an efficient back reaction to P*I have recently been obtained by the study of the fluorescence yield as a function of temperature in bacterial cells at low redox potentials [10,11]. The various possible reaction pathways are schematically depicted in Fig. 1. In a magnetic field B the triplet state is composed of three sublevels, TI, To and T-l, corresponding to the values of the magnetic q u a n t u m number ms ; the energy levels are separated by the Zeeman energy g fl B. In a high magnetic field only To is sufficiently close to the singlet level for the interconversion (P+I-) s -~ (P+I-) w to occur, so that the probability to recombine to pTI is reduced by the presence of the field. The decrease of the triplet yield increases the concentration of (P÷I-) s and thus the rate for recombining to the state P ' I ; consequently the concentration of P*I and the emission yield increases. In this communication we examine the effect of a static magnetic field on the emission yield of bacteriochlorophyll of whole cells and chromatophores of the photosynthetic bacteria Rhodopseudomonas sphaeroides (strain 2.4.1 and its carotenoid-less m u t a n t R-26) and Rhodospirillum rubrum (strain S1). We have found that in state PIX- the emission yield is an increasing function of the strength of the magnetic field and is correlated with a decreasing yield of triplet formation. Our results support the hypothesis [ 11], that charge recombination predominantly generates the excited singlet state P* I. In Chlorella vulgaris and spinach chloroplasts at 120 K the curve of the yield of the fluorescence vs. the field strength, measured at 685 or 695 nm, was similar to that found for bacteria. This suggests that also in Photosystem II an intermediary electron acceptor is present between P-680 and Q. Materials and Methods Cells of Rps. sphaeroides, R. rubrum and Cl. vulgaris were grown as described earlier [12,13]. The suspensions of algae and bacteria were taken as

250

magnet L1

1 L2~

~S F2

Fig. 2. S c h e m a t i c r e p r e s e n t a t i o n of t h e a p p a r a t u s u s e d for f l u o r e s c e n c e m e a s u r e m e n t s . W, t u n g s t e n i o d i n e l a m p ; L1 a n d L 2 , lenses; S, s a m p l e cell; F, filters; F 1 , Balzers K 2 a n d S c h o t t B G 1 2 / 4 m m ; F2, S c h o t t K V 5 5 0 ; F 3 , S c h o t t K V 5 5 0 a n d K o d a k W r a t t e n 87C ( f o r b a c t e r i a ) , or S c h o t t K V 5 5 0 + S c h o t t R G 6 6 5 / 3 m m + i n t e r f e r e n c e filter ( f o r algae a n d c h l o r o p l a s t s ) ; L G , 2 - c m d i a m e t e r lucite light guide; PM, p h o t o m u l t i p U e r t u b e ( D u m o n t K M 2 2 9 0 f o r b a c t e r i a , EMI 9 6 5 8 B for algae a n d c h l o r o p l a s t s ) . T h e m e a s u r i n g light i n t e n s i t y f o r b a c t e r i a was ~ 5 n E / c m 2 .

grown and diluted with growth m e di um to give the desired absorbance. C h r o m a t o p h o r e s were prepared by sonication o f cells in growth m edi um and resuspended after differential centrifugation in a buffer containing 50 mM m o r p h o l i n o p r o p a n e sulphonate and 50 mM KC1 at pH 7. Chloroplasts were prepared as described elsewhere [14]. Fluorescence spectroscopy was carried o u t in a s p e c t r o p h o t o m e t e r m o u n t e d in a 9-inch Varian magnet (Fig. 2) [ 2 ] . F o r th e experiments with bacterial cells and c h r o m a t o p h o r e s an $1 t ype p h o t o m u l t i p l i e r ( D u m o n t KM2290) was used. This multiplier showed a small e ff e ct o f the magnetic field on its sensitivity when placed near the magnet gap. With a 90-cm lucite light guide between sample cell and p h o t o m u l t i p l i e r the apparent change in emission at B = 0.6 Tesla (6 kG) was smaller than the accuracy o f the m e a s u r e m e n t (5 • 10 .4 o f the fluorescence), as checked with the fluorescing dye h e x a m e t h y l i n d o t r i c a r b o c y a n i n e iodide. In bacteria appreciable fluorescence increases in a magnetic field occurred only after addition of dithionite. Fractional variations in emission in the u n r e d u c e d controls, presumably in the state P*, were less than 10 -3. This indicates t hat orientation effects are small. T he t e m p e r a t u r e , which was varied by blowing cold nitrogen through a dewar vessel containing a sample cell of 2 m m thickness, was m o n i t o r e d with a Cu-constantan t h e r m o c o u p l e extending into the cell.

Results Fig. 3A shows a typical recording o f the fluorescence F of whole cells of

Rps. sphaeroides at r o o m t e m p e r a t u r e in an external magnetic field, which was linearly increasing with time. In Fig. 3B part of this curve is redrawn as the relative change in fluorescence AF/F induced by the field. Fig. 3C exhibits the e ff e ct for c h r o m a t o p h o r e s o f Rps. sphaeroides at r o o m t em perat ure and at 175 K. It is seen t ha t for bot h types of preparation a well-defined increase of fluorescence occurs with increasing value of B which saturates at B ~ 0.15 T. The value o f B at which half the ef f ect is attained (Bin) is a b o u t 25 mT. The curves appear c o m p l e m e n t a r y to the curves o f the carotenoid triplet yield vs.

251 A

l B o-I

I

'~-

002

B

003

to 2g5K

ooi

ooi 175K IL

I

005

I 010

I

I

I

015

02

025

magnetic

fieldlTI

0 I/----.--"I-"---~F

0

005

010 ~

I

I

I

03

02

025

magnetbc field (TI

Fig. 3. ( A ) U p p e r t r a c e : m a g n e t i c field as a f u n c t i o n o f t i m e . T h e m a g n e t i c field w a s c a l i b r a t e d w i t h an A E G N M R G a u s s m e t e r . L o w e r t r a c e : r e s u l t i n g f l u o r e s c e n c e c h a n g e o f Rps. s p h a e r o i d e s cells, a b s o r b a n c e at 8 5 0 n m 0 . 2 , r e d u c e d b y a d d i n g solid s o d i u m d i t h i o n i t e . (B) S a m e e x p e r i m e n t , r e p l o t t e d as r e l a t i v e c h a n g e o f f l u o r e s c e n c e ( F ( B ) - - F ( B = O))]F(B = 0). (C) R e l a t i v e f l u o r e s c e n c e c h a n g e o f Rps. s p h a e r o i d e s c h r o m a t o p h o r e s , A 8 5 0 = 0.9 in a 5 - m m c u v e t t e as a f u n c t i o n o f m a g n e t i c field. D i t h i o n i t e c o n c e n t r a t i o n 50 m M .

magnetic field previously measured [1,2]. The value of Bl/2 is approximately the same as found for the magnetodependence of the triplet yield *, which indicates that the phenomena are related. The relative change in fluorescence at 0.5 T and B1/2 is plotted as a function of temperature in Fig. 4 for whole cells of Rps. sphaeroides. It is seen that AF/F, the fractional increase in fluorescence, shows a m a x i m u m at about 250 K, and t h a t B~/2 shows a minimum at about the same temperature. Chromatophores of Rps. sphaeroides showed a similar behaviour. The magnetic field effect for cells and chromatophores of the carotenoid-less m u t a n t R-26 was similar to t h a t for the wild-type, viz. AF/F (295 K ) ~ 0.9%, AF/F (100 K) < 0.1%. For whole cells o f R . rubrum at room temperature only a very low magnetic field effect was detected, AF/F ~ 0.2%, but for chromatophores an effect similar to that of Rps. sphaeroides, viz. AF/F ~ 1.5% and B,/2 35 mT, which values decreased with lower temperatures. In Fig. 5 the magnetic field effect for spinach chloroplasts and for Cl. vulgaris is displayed, measured at ~ 1 2 0 K. The curves are similar to those found for bacteria at higher temperatures. At temperatures above 0°C, the fluorescence of chloroplasts as well as of Chlorella showed a rather complex behaviour in a magnetic field with an initial increase and subsequent decrease with time on switching on the field. This complex pattern is probably caused in part by orientation of the pigment molecules by the magnetic field, as earlier observed and studied by Geacintov et al. [15], and was not further studied by US.

* A s is apparent f r o m F i g . 2 A in R e f . 2, t h e v a l u e O f B l / 2 f o r t h e t r i p l e t y i e l d w a s 25 m T , a n d n o t 50 m T as i n a d v e r t e n t l y stated.

252

0.02

x

~"~'~~×-~l~"

0.01 I

l

I

100

150

I

I

,

l

200 250 300 temperature (K)

100

,

150

200

250

300

temperature (K}

Fig. 4. ( A ) R e l a t i v e f l u o r e s c e n c e c h a n g e i n d u c e d b y a m a g n e t i c field o f 0 . 5 T in w h o l e cells o f Rps. w i t h r e d u c e d a c c e p t o r X as a f u n c t i o n o f t e m p e r a t u r e . (B) M a g n e t i c field value at w h i c h h a l f t h e e f f e c t o f 0 . 5 T is r e a c h e d , as a f u n c t i o n o f t e m p e r a t u r e .

sphaeroides

-

15

Q

3

T ,,oo 10

2

05

0 0

01

0.2

0.3

OL.

0.5

~-- mognetic field{T)

b

/ I 01

l 0.2

J 03

l 0 t-

I 0.5

=- magnetic tield (T)

F i g . 5. R e l a t i v e f l u o r e s c e n c e c h a n g e , m e a s u r e d at 6 8 5 n m , as a f u n c t i o n o f e x t e r n a l m a g n e t i c field f o r : (a) CI. vulgaris; a b s o r b a n c e 0 . 0 8 at 6 8 0 n m , t e m p e r a t u r e ~ 1 2 0 K, 10 # M 3 - ( 3 ' , 4 ' - d i c h l o r o p h e n y l ) - l , 1 d i m e t h y l u r e a , a n d 10 m M h y d r o x y l a m i n e a d d e d ; s a m p l e w a s f r o z e n in t h e light. (b) s p i n a c h c h l o r o p l a s t s ; c h l o r o p h y l l c o n c e n t r a t i o n 33 ~tM, t e m p e r a t u r e ~ 1 2 0 K; d i t h i o n i t e a d d e d .

Discussion

The increase in fluorescence yield with a magnetic field of bacterial cells and chromatophores under reducing conditions may arise from a field-induced enhancement of the rate of the back reaction from the state P*I- to the excited singlet state P*I as suggested in the Introduction. Starting with this hypothesis and making certain assumptions we can correlate quantitatively the fractional fluorescence change AF/F with the fractional change in triplet yield ~kPw/P w induced by the magnetic field. We find (see below) that AF/F is proportional to ~kPw/P T with a negative proportionality factor. This is in accordance with our finding that, at a given temperature, the fluorescence increase and the decrease in triplet yield saturate and reach the half-value at about the same field strengths. In principle the above mechanism might be counteracted by another fieldinduced change in fluorescence yield. It has been shown that the probability for the conversion of the state P÷I- to the triplet state pTI is lower in a magnetic field [1,2]. This means that the concentration of reaction centers in the state P I increases when applying a magnetic field and consequently the yield of fluorescence decreases. Hence it is important to assess the steady state concentration of reaction centers in the triplet state. At our measuring light

253

intensity (~ 5 nE/cm ~) and sample absorbance (0.2), ~ 2 5 quanta are absorbed per s per reaction center. The reaction center triplet state is rapidly transferred to the reaction center carotenoid. During the lifetime of this carotenoid triplet, which is ~ 4 ps between 80 and 300 K, the trap is closed [10,16]. At lower temperatures the transfer to the carotenoid becomes inhibited and the lifetime of pTI increases. Under our conditions, and assuming a 100% triplet yield, the fraction of reaction centers in the triplet state never exceeds 10 -4 . Assuming 15% field induced depression of the triplet yield [2], the increase of the fraction of reaction centers in the ground state P I is smaller than 2 • 10 -s, so we can safely neglect its effect on the fluorescence. At low temperature (1.4 K) y e t another magnetic field effect on the fluorescence of photosynthetic bacteria has been reported [17]. This effect originates from field-induced mixing of populating and depopulating probabilities, which affects the concentration of the reaction center triplet state. At temperatures above say 20 K, however, this effect is abolished by rapid spin-lattice relaxation between the triplet sublevels. As mentioned in the previous section, in some instances orientational effects can be important. For chromatophores and whole bacterial cells however, these effects are negligible at room temperature, as demonstrated by the absence of a magnetic field effect in non-reduced samples. We assume that in all preparations at low temperatures, no field-induced orientation will occur. We will now derive the relationship between triplet yield and fluorescence. To this end we first calculate PF, the total probability that an excitation is emitted as fluorescence [ 11]:

PF

=

( 1 - - T)pf + Tp} + (1 -- T)phP'F + lr pp ht , D "f

P ) is the probability for fluorescence after one excitation transfer step, P~ is the probability for fluorescence after returning from the reaction center, Pr and p} are the probabilities for immediate emission of fluorescence for an antenna bacteriochlorophyll and the reaction center, respectively, Ph and p t are the probabilities for energy transfer, T is the fractional concentration of reaction centers. If we assume PF = P'F = P~, we get (1 -- T)pf + Tp} PF = 1 -- (1 -- T)p h -- Tpth

(1)

and its analogue for the total triplet yield (1 PT

= 1 --

(1

T)pisc + T p [ T ) p h - - Tpth

--

(2)

where Pisc is the probability for intersystem crossing in an antenna bacteriochlorophyll and p~ the probability that a reaction center triplet is formed from P*I. Assuming that the rate constants for deexeitation and energy transfer are equal for antenna and reaction center chlorophylls, we can express Pe and PT in terms of these rate constants, the rate constants for charge separation and recombination, and ¢~, the probability that P÷I- will recombine to the excited singlet state P* I:

PF - k f ( 1 - - A T ) kl + k h A T

(3)

254

kisc(1 - - A T ) + kAT(1 -- (1 + ~)0,)/(1 - - 0 s ) k 1 + khAT

PT -

(4)

with A = k c ( 1 - - O s ) / ( k + k c ( 1 - - ~ b . ) ) and a = k g / k s. The rate constants are defined in Fig. 1. kl = k r + kic + kisc is the rate of deexcitation of an antenna bacteriochlorophyll molecule other than via energy transfer, k = k l + kh is the total deexcitation rate. We assume that the rate constants are not altered by a magnetic field. The only parameter that is influenced by the field is 0.. Elimination of 0s yields PT = --BPF + C

(5)

with B = 1 + [kic + a(k I ÷ (k 1 + khT)kc/k)]/k f

(6)

and C

=

1

+

a(1

+

(1

--

V)kc/k)

(7)

From Eqns. 5, 6 and 7 the relationship between the fractional yields of fluorescence and total triplet yield is given by

AF/F = z~kPF/PF = --[PT/(C - - PT) ] [ A P T / P T ]

(8)

PF and PT are the fluorescence yield and triplet yield in zero magnetic field. If we assume kg = 0, the triplet yield PT at 80 K will be close to 1. We now consider changes caused by a saturating magnetic field. At room temperature PT was estimated to be ~0.3 [11], so that PT/(C --PT) ~ 0.3/(1 -- 0.3) ~ 0.43. With ~PT/PT "~---0.05 (Rademaker, H., unpublished), the right hand side of Eqn. 8 is equal to 0.43 . 0.05 = 0.022, in good agreement with our measured value APF/PF ~-- 0.018. Since we have assumed kg = 0, in whole cells of Rps. sphaeroides only a small part of the radical pairs apparently combines to the ground state. In chromatophores we measured ~kPT/P T ~ - ~ - - 0 . 1 2 [2]. If we assume again kg = 0 and PT = 0.3, the right hand side of Eqn. 8 is equal to 0.05, a factor 2 different from our measured value APF/PF = 0.024. Our assumption kg = 0 may not be correct for this case. If k g > 0, a higher value of C is obtained resulting in a lower calculated value of AF/F. Alternatively, PT may have a lower value, e.g. caused by a less efficient energy transfer between the antenna pigments and the reaction center. If we assume that the temperature dependence of APT/P T is similar to that found in reaction centers [1], we can explain the rise in AF/F in the temperature region of 80--250 K by the rise in [APT [/PT, and the decrease at higher temperatures by the decrease of PT/(C--PT), due to the decrease of PT [11]. The curve of B,/2 vs. T can be qualitatively understood with the aid of the model calculations of Werner et al. [18]. These authors predict a decrease in B,/2 with decreasing ks, owing to the corresponding sharpening of the radical pair singlet level (the fuzzier the levels are, the higher a magnetic field one needs to pull T~I out of the region where these levels can still mix with the singlet level). The m i n i m u m in B , / : can be explained by an increase in k t with decreasing T, which below 240 K takes over the effect of decreasing k s. Because of lack of data on PT, it is as y e t n o t possible to apply analogous

255

calculations for the magnetic field effect of the Photosystem II fluorescence of chloroplasts and Chlorella. The similarity between the low field experimental results obtained from these preparations with the results found for the bacterial system suggests that the radical pair mechanism is operative for Photosystem II also. This indicates that an intermediate acceptor is present between the primary donor of Photosystem II, P-680, and the primary acceptor, Q. Recent results obtained with optical difference spectroscopy suggests that such an intermediate might be pheophytin [19]. Note added in proof (Received January 31st, 1979) We have learned that Voznyak et al. [20] have carried out similar experiments in purple bacteria and arrived in a qualitative way at similar conclusions as we did. Acknowledgements We are indebted to Mr. A.H.M. de Wit for expert microbiological assistance, to Dr. J.H. van der Waals for the loan of the magnet and to Dr. R. van Grondelle for many stimulating discussions. This investigation was supported by the Netherlands Foundation for Biophysics, financed by the Netherlands Organization for the Advancement of Pure Research (ZWO}. References 1 Blankenship~ R.E., Schaafsma, T.J. and Parson, W.W. (1977) Biochim. Biophys. Acta 461, 297--305 2 Hoff, A . J , Rademaker~ H., van Grondelle, R. and Duysens, L.N.M. (1977) Biochim. Biophys. Acta 460, 547--554 3 Fajer, J., Brune, D.C., Davis, M.S., Forman, A. and Spaulding, L.D. (1975) Proc. Natl. Acad. Sci. U.S. 72, 4 9 5 6 - - 4 9 6 0 4 Kaufman, K.J., Pe tty, K.M., D u t t o n , P.L. and Rentzepis, P.M. (1976) Biochem. Biophys. Res. Co mmun. 7 0 , 8 3 9 - - 8 4 5 5 Clayton, R.K. and Y a m a m o t o , T. (1976) Biophys. J. 1 6 , 2 2 2 6 Shuvalov, V.A. and Klimov, V.V. (1976) Biochim. Biophys. Acta 440, 587--599 7 Tiede, D.M., Prince, R.C. and Dutton, P.L. (1976) Biochim. Biophys. Acta 449, 447--460 8 Van Grondelle, R., Romiin, J.C. and HoLmes, N.G. (1976) FEBS Lett. 72, 187--192 9 Adrian, F.J. (1977) in Chemically Induced Magnetic Polarisation (Muus, L.T., Atkins, P.W., McLaughlan, K.A. and Pedersen, J.B., eds.), pp. 77--105, Reidel Publ. Co., Dordrecht 10 HoLmes, N.G., van Grondelle, R., Hoff, A.J. and Duysens, L.N.M. (1976) FEBS Lett. 70, 185--190 11 Van Grondelle, R., HoLmes, N.G., Rademaker, H. and Duysens, L.N.M. (1978) Biochim. Biophys. Acta 503, 10--25 12 Slooten, L. (1972) Biochim. Biophys. Acta 256, 452--466 13 Hoo genhout, H. and Amesz, J. (1965) Arch. Mikrobiol. 50, 10--24 14 Amesz, J., Pulles, M.P.J. and Velthuys, B.R. (1973) Bioehim. Biophys. Acta 3 2 5 , 4 7 2 - - 4 8 4 15 Geacintov, N.E., van Nostrand, F., Pope, M. and Tinkel, J.B. (1971) BiochLm. Biophys. Acta 226, 486--491 16 Monger, T.G. and Parson, W.W. (1977) Biochim. Biophys. Acta 4 6 0 , 3 9 3 - - 4 0 7 17 Gorter de Vries, H. and Hoff, A.J. (1978) Chem. Phys. Lett. 55, 395--398 18 Werner, H.-J., Schulten, K. and Weller, A. (1978) BiochLm. Biophys. Acta 5 0 2 , 2 5 5 - - 2 6 8 19 Klimov, V.V., Klevanik, A., Shuvalov, V.A. and Krasnovsky, A.A. (1977) FEBS Lett. 82, 183--186 20 Voznyak, W.M., Jelfimov, J.I. and Proskuryakov, I.I. (1978) Doklady Akad. Nauk 242, 1 2 0 0 - - 1 2 0 3