PHOTOCHEMISTRY AND PHOTOBIOLOGY

Proceedings of the International Conference held at the University

of Alexandria, Egypt, January 5-10, 1983

Volume 2

Edited by Ahmed H. Zewail

C a l i f o r n i a I n s t i t u t e of T e c h n o l o g y

p h r ri> t m \ m Y)

Changes with rays of light

harwood academic publishers chur • london • paris

Utrecht • new york

Volume 2 Contents

Dedication v

Acknowledgments vii

Volume 1 Contents xvii

Foreword xxv

Preface xxvii

Chairman's Introductory Remarks xxix

Director's Opening Address xxxi

Closing Remarks xli

Organizing Committee xliii

Session Chairpersons xlv

C H A P T E R 7 E N E R G Y S T O R A G E , C O L L E C T I O N A N D C O N V E R S I O N

G . P O R T E R

The Photolysis of Water and Photochemical Energy Storage (Acknowledgement) 742

T E R R Y C O L E and A H M E D H. Z E W A I L

Luminescent Solar Concentrators (LSC): Their Physics and Chemistry 743

R A F I K O. L O U T F Y and A H - M E E H Ö R

Sensitization of n-Type Semiconductors by Phthalocyanines 759

J O H N O. W I L L I A M S

Photo-Electrochemical Characterization of rc-ZnSe Epitaxial Layers Grown on n-GaAs Substrates 775

I X

X V O L U M E 2 CONTENTS

C H A P T E R 8 P H O T O B I O L O G Y — O P T I C A L T E C H N I Q U E S

E R I C R. H E N R Y , J A M E S H O F R I C H T E R , J O S E P H H .

S O M M E R and W I L L I A M A . E A T O N

Photolysis of Carbonmonoxyhemoglobin and the Cooperativity Problem 791

L . L I N D Q V I S T , M . P. F O N T A I N E , E . B R E H E R E T , B. A L P E R T

and J. C . A N D R E

Photodissociation of Oxyhemoglobin by Nanosecond Laser Excitation: Study of the Geminate Recombination Kinetics 811

T H O M A S G . SPIRO and J A M E S T E R N E R

Structure and Dynamics in the Photolysis of C O - and 0 2 -Hemoglobin , Monitored by Time-Resolved Resonance Raman Spectroscopy 817

M . O T T O L E N G HI, V. R O S E N B A C H , M . S H E V E S , T. B A A S O V

and N. F R I E D M A N

On the Molecular Mechanism of the Photocycle of Bacteriorhodopsin 829

R O B E R T R. B I R G E and B R I A N M . P I E R C E

The Nature of the Primary Photochemical Events in Bacteriorhodopsin and Rhodopsin 841

V. M A L A T E S T A , V. S O L A N and P. A . H A C K E T T

EfTicient Photochemical Synthesis of Vitamin D Metabolites: 250 H-26 2 H 2 -Cholecalciferol 857

B. R. W A R E and F R E D E R I C K L A N N I

Polymerization and Crosslinking of Actin i n V i t r o and i n V i v o Studied by Fluorescence Photobleaching Recovery 863

J. F Ü N F S C H I L L I N G and I. Z S C H O K K E - G R Ä N A C H E R

Chlorophyll-B in Vesicles Studied by Site-Selection Spectroscopy 879

A . M . A T T A L L A H

Laser Application in Immunology: Detection of Kil ler Cell Activity with Ultramicro Techniques 889

V O L U M E 2 CONTENTS xi

H A S S A N M . Y O U N I S

4,4-Bis(l-Anilinonaphthalene 8-Sulfonate)(Bis-ANS): A Probe of the Conformational Changes in Chloroplast Coupling Factor 1 and the Mechanism of Photophosphorylation in Chloroplasts 899

H A S S A N M . Y O U N I S and S H A F I K A A . E L - K A S S A B A N Y

Involvement of the Energy-Coupling Adenosine Triphosphatase Complex of Chloroplasts in the Mode of Action of Herbicides 917

P I L L - S O O N S O N G

Proton Transfer Processes in Plant and Protozoan Photoreceptors 931

K L A A S A . Z A C H A R I A S S E , B O L E S L A W K O Z A N K I E W I C Z

and W O L F G A N G K Ü H N L E

Micelles and Biological Membranes Studied by Probe Molecules 941

A . H . E L - S E B A E , A . S. E L - B A K A R Y , J. L E P A T O U R E L ,

E . K A D O U S and M . F. M A C K L A D

Effect of Photoperiodism on Fish Susceptibility to Insecticides 961

C H A P T E R 9 P H O T O B I O L O G Y — M A G N E T I C R E S O N A N C E T E C H N I Q U E S

G . L . C L O S S , R. J. M I L L E R and J. R. N O R R I S

The Influence of Electron-Nuclear Hyperfine Interactions on Kinetics in Photochemical Reactions 969

W. L E R S C H , A . O G R O D N I K and M . E . M I C H E L - B E Y E R L E

Comparison of Magnetic Field Experiments on Recombination of Radical Ion Pairs in Reaction Centers of Photosynthetic Bacteria 987

J. B E C K , J. U . V O N S C H Ü T Z , and H . C. W O L F

Fluorescence Detected Magnetic Resonance on Pigment Complexes of Photosynthetic Bacteria 995

xiv V O L U M E 2 CONTENTS

C H A P T E R 11 P H O T O C H E M I S T R Y — L I N E A R A N D N O N L I N E A R

J E A N - C L A U D E L E H M A N N

Recent Developments of Chemical Photophysics in France 1261

E . V . K H O R O S H I L O V A , N. P. K U Z M I N A , V. S. L E T O K H O V

and Y U . A . M A T V E E T Z

Nonlinear Photochemical Synthesis of Biomolecules by Powerful U V Picosecond Pulses 1267

J E F F R E Y I. Z I N K

Excited State Distortions and Transition Metal Photochemistry 1277

A L L A N L . S M I T H , A H A R O N G E D A N K E N and D A Y O

A L U G B I N

Some Aspects of the Photochemistry of the Xenon Fluorides 1299

E . K A S S A B , E . M . E V L E T H , G . C H A M B A U D and B. L E V Y

A Theoretical Investigation of the Electronic Quenching of the Vaiyous Excited States of H 2 by Ground State H 2 1307

S A U L G . C O H E N and J A M A L D. K H A N

Catalysis by Thiol and the Ani l ine-Thiol Pair in Photoreduction of Aromatic Ketones by Amines 1317

M . S. A . A B D E L - M O T T A L E B , M . H . A B D E L - K A D E R and

S. T. E L - A R A B Y

Correlations of the Quantum Yield of Photochemical t r a n s / c i s Isomerization of Some DyestufTs with Their Electronic Structure and with Solvent Polarity Scales 1329

A B D E L - G H A N Y E L - K O U R A S H Y , M . E . S O L I M A N and

M . A . E L - M A N G O U C H

Spectroscopic Studies of the Effect of Radiation Energy with Time on the Rate of Ion-Pair Interaction of Phosphorus(V) 1337

V O L U M E 2 CONTENTS xv

A . C. A L B R E C H T

Multiphoton Studies of Highly Excited States in the Condensed Phase 1345

E L - Z E I N Y M . E B E I D , A B D E L - F A T T A H M . H A B I B and

S A L A H E . M O R S I

Photoreactivity of l,4-Bis(jB-pyridyl-2-vinyl) Benzene in Solution 1355

H E I K E S C H A R P I N G and C O R N E L I U S Z E T Z S C H

U V Photoabsorption Cross Sections of Low Volatile Substances in the Gas Phase Using a Gas Saturation Technique: Benzene, Monochlorobenzene, o-, m-, and p-Dichlorobenzene 1371 J . c. M I C H E A U

Photochemically Induced Dissipative Structures: A Useful Concept in Photochemistry and Photobiology 1381

M . H . A B D E L - K A D E R

Proton Translocation and c i s / t r a n s Photoiosomerization in a Simple Merocyanine Dye: A Chemical Model and its Biological Implications 1397

Electron-Nuclear Multiple Resonance Studies on Primary Products of Bacterial Photosynthesis

W. LUBITZj F. LENDZIAN,t H. SCHEER,§ M. PLATOt and K. MÖBIUS*

f I n s t i t u t für O r g a n i s c h e C h e m i e , F r e i e Universität B e r l i n , T a k u s t r . 3 , D - 1 0 0 0 B e r l i n 3 3 , t I n s t i t u t für Molekülphysik, F r e i e Universität B e r l i n , A r n i m a l l e e 14, D - 1 0 0 0 B e r l i n 3 3 , § B o t a n i s c h e s I n s t i t u t , Universität München, M e n z i n g e r S t r . 6 7 , D - 8 0 0 0 München 19, W e s t G e r m a n y

E N D O R and TRIPLE resonance techniques are applied to bacterial photosynthetic pigment anion and cation radicals in Solution, yielding an almost complete set of isotropic H - and 14N-hyperfine couplings including signs. Assignments to molecular positions are attempted on the basis of the experiments including specific D-labeling. The results from the bacteriochlorophyll a cation radical are compared with those of the in situ light-induced primary donor cation P 870 + (protonated and partially deuter-ated) in the bacterium Rhodopseudomonas sphaeroides R-26. The latter is also studied by liquid State E N D O R / T R I P L E under physiological conditions (25°C, water). This comparison shows that P 870 + is a bacteriochlorophyll a dimer. The different shifts of the individual hyperfine couplings contain information about the structure of this dimer. On the basis of the experiments and MO calculations a specific dimer model is proposed.

INTRODUCTION

In recent years the mechanism of light-induced Charge Separation in photosynthesis has roused the interest of scientists from different fields.1 In such investigations photosynthetic bacteria 2 are easier to study than plants since they have only one photosystem, and the reaction centers (RC's)—in which the primary events take place— have been isolated and well characterized. 3 In the non-sulfur purple bacteria Rhodopseudomonas (Rp.) sphaeroides R-26 & n d Rhodospiril-lum (R.) rubrum G-9, which are studied in this paper, the RC ' s

1057

1058 W. LUBITZ et a l .

contain three protein subunits, four bacteriochlorophyll (BChl) a and two bacteriopheophytin (BPh) a molecules, one or two ubiquinones (UQ), and one non-heme iron (3 + ). 3 The structure of B C h l a is shown in Figure 1.

In the R C the light energy is trapped by singlet excitation of a pigment absorbing at 870 nm (P 870), which donates an electron to an electron transport chain in a few picoseconds. P 870 is currently believed to be a "special pair" of B C h l a molecules; the series of electron acceptors involves probably monomeric B C h l a, BPh a, and iron ubiquinone complexes. 4 Düring the Charge Separation process cation (P 870) and anion (BChl a , B P h a, U Q ) radicals of the various pigments are formed. Much of our present knowledge about these species and their interactions in the R C has therefore evolved from the application of paramagnetic resonance methods5—used in con-junction with optical techniques. The dimeric nature of P 870^ was originally proposed by Norris et a l . 6 on the basis of E P R data. Further support for this model came from electron-nuclear double resonance ( E N D O R ) studies. 7" 1 1 Similar investigations were performed in order to identify and characterize the anion radicals of the primary electron

o 12-17 acceptors. '

FIGURE 1 Molecular structure of BChl a and numbering scheme; R = phytyl, p (in R p . s p h a e r o i d e s ) , R = geranylgeranyl, gg (in R. r u b r u m ) . In BPh a the central Mg is replaced by two hydrogens bound to N { and N I n .

B A C T E R I A L PHOTOSYNTHESIS 1059

In all these studies E N D O R proved to be superior to E P R for the determination of the electron nuclear hyperfine coupling constants (hfc's) due to its much higher spectral resolution. 1 8 Most of the i n v i v o E N D O R work has been carried out in frozen matrices, 7 - 1 1 where dipolar broadening masks all interactions except those belonging to nuclei with relatively small hfs anisotropy, e.g., rotating methyl groups. 1 9 It was only recently that the first detection of liquid State E N D O R on a bacterial R C ( H 2 0 , 25°C) was reported. 2 0 In these experiments the additional application of electron-nuclear-nuclear T R I P L E resonance 1 8 proved to be very helpful for increasing the E N D O R sensitivity and resolution ("Special T R I P L E " ) and for deter-mining the relative signs of the hfc's ("General T R I P L E " ) .

In the present paper it is demonstrated that an almost com-plete set of isotropic 1 4 N - and H-hfc's can be obtained by E N D O R / T R I P L E - i n - s o l u t i o n for the i n v i t r o prepared monomeric pigment radical cations (see also Refs. 21, 22) and anions (see also Refs. 16, 23). These Systems are important as model Compounds for the i n v i v o occurring radical ions. The data for B C h l a + are compared to those of P 870 + in bacterial RC ' s , allowing conclusions about the electronic and geometric structure of the primary donor cation which is of importance for the understanding of the first events in photosyn­thesis.

EXPERIMENTAL

R p . s p h a e r o i d e s R-26 and R. r u b r u m G-9 were grown in Hutners medium. 2 4 R p . s p h a e r o i d e s R-26 R C ' s were isolated by the method of Clayton and Wang 2 5 and further purified on D E A E cellulose. Lauryldimethylaminoxid ( L D A O ) was exchanged against Triton X -100 by chemical reduction on a D E A E cellulose column. Ubiquinone was removed by the method of Okamura et a l . 2 6 RC ' s of R. r u b r u m G-9 were prepared according to Snozzi and Bachofen. 2 7 The R C samples were concentrated by membrane filtration. They were filled under Argon in the sample tubes, which were then sealed. The R C concentration was c. 2-3 x 10~ 4 M .

The bacteriochlorophylls were isolated by the procedure of Strain and Svec. 2 8 BPh a was obtained from B C h l a by treatment with hydrochloric acid . 2 3 The cation radical of B C h l a was prepared by

1060 W. LUBITZ e t a t .

iodine oxidation in C H 2 C l 2 / C H 3 O H (c. 6 :1 , by vol.) under high vacuum condit ions. 2 1 , 2 2 The anion radicals of B C h l a and B P h a were generated by potentiostatically controlled electrolysis in 1,2-dimethoxyethane ( D M E ) , using tetra-«-butylammonium Perchlorate (TBAP) as supporting electrolyte. 2 3 Optimum E N D O R signals 2 9 were obtained at c. 3 x 10~ 4 M for all three radicals.

The measurements were performed with a self-built computer-controlled spectrometer which allows the registration of E P R , E N D O R and General/Special T R I P L E resonance spectra. The experimental details have already been described elsewhere. 1 8 , 3 0

RESULTS AND DISCUSSION

ENDOR and TRIPLE resonance on isolated pigment radicals

A s a representative example the B C h l <Zp is chosen. The E P R spec-trum (g = 2.0026) is completely unresolved. The ENDOR-in-so lu t ion spectrum, however, reveals eleven H-hfc's (Figure 2, bottom); addi-tionally, all four 1 4 N-hfc's could be observed at lower frequencies. For B C h l ßg g the same H - and 1 4 N-hfc's are obtained within experi­mental error (^10 kHz), clearly showing that the hydrophobic side chain (see Figure 1) has no influence on the spin density distribution of the isolated cation radicals. The hfc's including their signs (from General T R I P L E ) are collected in Table I.

Following the method of Katz et a l . 3 1 partially deuterated B C h l a was extracted from biosynthetically labeled bacteria. In such prepar-ations large abundances of protons are only found at positions l a , 5a, 2b and more than two bonds away from the 7r-system; the methine and ß-proton (rings II, IV) positions are highly deuterated. Such D-labeling and further D-exchange experiments were used for assigning the H-hfc's to specific groups of protons in the radical (see Table I).

Our results are in agreement with those of Borg et a l . 2 1 who could resolve seven proton couplings in Solution. Earlier frozen Solution E N D O R experiments on B C h l a + gave less detailed results. 7 - 1 1

The H - and 1 4 N - E N D O R and T R I P L E resonance spectra of the anion radicals of B C h l a and BPh a in Solution have been analyzed in a similar way. 2 3 The isotropic hfc's including their signs and the assignments to molecular positions are also given in Table I. Although

B A C T E R I A L PHOTOSYNTHESIS 1061

I I I I I I I I I I 5 7 9 11 13 15 17 19 21 23 MHz

F I G U R E 2 H-ENDOR-in-solution spectra of P 870 + in RC's and BChl al (both from R p . sphaeroides R-26) at the given temperatures, the signs and assignments of the six largest hfc's (see Table I and II) are indicated. Experimental conditions, BChl a + : mw power 20 mW, r.f. power 100 W (11 G, rot. frame), lOkHzfm of r.f. field, ±50 kHz deviation, no field modulation, time constant 1 s, 4scans; P870 + : in situ illumination (450-900 nm, 500 W Xe high pressure lamp), mw power 80 mW, ±70 kHz fm deviation, 9 scans, rest as above.

the E P R spectra of B C h l a ~ and B P h a ~ are very similar, a distinction of these species is clearly possible by their E N D O R and T R I P L E spectra. 1 8 For all three radical ions discussed in this paper, Pariser-Parr -Pople 1 ( 5 , 3 2 and molecular fragment ab i n i t i o 3 3 calculations are available which support the above assignments (see Ref. 30).

A comparison between the cation and anion radicals (Table I) on the basis of known relationships between hfc's and 7r-spin densit ies 2 3 , 3 0 yields the following result: In the c a t i o n there is little—or even negative—spin density at the four nitrogens and the methine carbons. High values are found for all carbons in the pyrrole rings I

1062 W. LUBITZ et a l

T A B L E I Comparison of isotropic hfc's a (MHz) of BChl a + , BChl a ~ , and BPh a

Position0 Type BChla + BChl a ~ B P h a "

la 5a

methyl protons

methine protons

+4.85 +9.50 ±0.15 + 1.30 +2.30

+7.63 +9.19 -6.23 -6.91 -9.65

+7.10 +8.32 -6.94 -8.02 -8.50

3,4,7,8 ß-protons + 11.61 + 13.00 + 13.59 + 16.43 <

+0.50 +0.95 -1.60

r - i . 5 o \ -1.84 1-2.63

other protons -0.50 -1.65

+0.48 +0.98

Rings II, IV, I, III nitrogens -3.17 -3.05

-2.35 ±0.45

+6.52 +5.86

H l - 1 8 H0 .52

+7.19 +6.16

(-H.20 (-)0.60

a B C h l a + : (I2, C H 2 C l 2 / C H 3 O H ) , H-hfc's: 255 K, 1 4N-hfc's 270 K; BChl a", BPh a ~ : (electrolysis, D M E , TB AP), H-hfc's: 255 K: BChl a ~ , 270 K: BPh a ~ , 1 4N-hfc's: 280 K. Hfs data from E N D O R and Special TRIPLE, signs from General TRIPLE; for details see Refs. 23 and 30.

b For numbering of positions see Figure 1. Except for methyl protons (see Ref. 9, 10, 41), individual assignments in the various types of nuclei cannot be given.

to IV (from 0-proton and methyl hfc's). In both a n i o n s of B C h l a and B P h a the nitrogens in rings II and IV have large and the other two have small spin densities. A t the methine carbons and at carbons 1 and 5 the spin density is large. It has been argued by Fajer et a l . 3 4 that the similarity of the primary Chlorophyll donor and the intermediate Chlorophyll and/or pheophytin acceptors favour electron transfer on a picosecond timescale. The striking differences between the gener-ated cation and anion radicals, however, minimize orbital overlap and thereby inhibit wasteful back reactions.

ENDOR and TRIPLE resonance on pigment radicals in RC's

Illumination of RC ' s from R p . s p h a e r o i d e s R-26 results in a reversible bleaching of the optical band at 870 nm. Concomitantly a narrow Gaussian E P R signal at g = 2.0025 appears which is attributed to the

B A C T E R I A L PHOTOSYNTHESIS 1063

cation radical of P 870. I n s i t u I l luminat ion in the E N D O R cavity allows the detection of the E N D O R spectrum (25°C, water) depicted in Figure 2 (top).

It has been shown 2 0 that in P 870 + all proton nuclei are still in the fast tumbling limit in spite of the long rotational correlation time r R

of 30 ns (molecular weight of R C c. 100 000 daltons, water, 25°C). Therefore, a liquid Solution E N D O R spectrum is obtained. However, the t r value is far from Optimum E N D O R detection conditions 2 9

( T R 1 C . 1 ns), implying lower intensities and fairly large linewidths as compared to the B C h l a + spectrum (Figure 2).

Seven H-hfc's can be deduced in P 870 + which are all positive and belong to one species according to a General T R I P L E experiment. Highest resolution is achieved by use of the Special T R I P L E technique (Figure 3, top). The same spectra are observed when the R C ' s are chemically oxidized by K 3 [Fe (CN) 6 ] . On thermal denaturation of the protein, E P R and E N D O R signals disappear.

In order to assign the E N D O R lines to specific groups of protons in the radical we have also studied deuterated RC ' s , v i d e s u p r a . In Figure 3 the Special T R I P L E spectra of P 870 + in protonated and partially deuterated RC ' s are compared. Since in the latter case only lines belonging to the methyl groups in positions l a and 5a show up (the acetyl and y-proton hfc's are very small) an assignment as given in Figure 3 is straightforward. The additional line in the P 870 + spectra (smallest hfc) might be assigned to the acetyl group (2b) or to y-protons (3a, 4a, 7a, 8a) or to protons from the protein. It is remarkable that there are only two hfc's for the methyl groups in positions l a and 5a (Figures 2 and 3). Assuming a bacteriochlorophyll dimer for P 870 + , this finding already indicates a symmetrical spin distribution over both halves.

Since an assignment of the measured hfc's to molecular positions was achieved in both B C h l a ¥ and P 870~\ the data can be directly compared (Table II). It is obvious that in P 870 + all hfc's are reduced in magnitude but not by a constant factor of 2 as proposed by the simple "special pair" m o d e l . 6 - 1 1

It is interesting that an average reduction factor defined as the ratio of average hfc's in each species is very close to two (1.95). The procedure of comparing hfc's is, however, questionable (unknown changes in the dihedral angles of the ß-protons).32 A better approach to confirm the dimeric nature of P 870 + is to simulate the E P R

1064 W. LUBITZ et a l .

H - Spec ia l T R I P L E 5 a

1a A

R C R p . s p h a e r o i d e s R - 2 6

2 0 ° C i l l u m i n a t e d

0 1 2 3 U 5 6 MHz

FIGURE 3 H-Special TRIPLE spectra of light-induced P 870 + in Solution; bacteria grown with protonated succinate in H 2 0 (top), and in 99.9% D 2 0 (bottom). Experi-mental conditions: total r.f. power 200 W, 4 scans, ±50kHzfm deviation, rest as for E N D O R (Figure 2). The Shoulders on lines la and 5a (bottom) are due to non-averaged hfs anisotropy of the methyl groups and not due to additional couplings. (The deuterated RC's were coagulated resulting in a considerable increased r R value and solid State ENDOR line shapes.)

spectrum by using the E N D O R hfc's. The result is shown in Figure 4, in trace a a dimer and in trace b a monomer was chosen for the Simulation. It is obvious that P 870 + in Rp. sphaeroides is a dimer of B C h l a molecules, which is also in accordance with recent E P R results from Wasielewski et al.35

The reduction factors presented in Table II are in contradiction to earlier solid State E N D O R studies on P 8 7 0 \ where a halving of all observed hfc's was found. 7 " 1 1 Our temperature dependent E N D O R studies showed that this result was due to an incorrect assignment of the observed broad resonances in the frozen State.3 0

B A C T E R I A L PHOTOSYNTHESIS 1065

T A B L E II Comparison of isotropic hfc's a (MHz) of BChl Ö p

+ (255 K) and P 870 +

(298 K) from R p . sphaeroides R-26 (ENDOR/TRIPLE data)

Type (Position) BChl a +

p P 870 + R F a

C H 3 (la) +4.85 +4.00 1.21 C H , (5a) +9.50 +5.60 1.70 0-H] + 11.61 +3.30 3.52 ^ ( 3 , 4 , 7 , 8 ) + 13.00 +4.45 2.92 ^ ( 3 , 4 , 7 , 8 ) + 13.59 +8.60 1.58 ß-W + 16.43 +9.50 1.73

a RF: reduction factor of hfc's a (BChl a ")/a (P 870"). The ß-proton hfc's are not individually assigned, they are compared according to their ordering in magnitude. The smallest hfc of P 870 + (+1.4 MHz) is not considered since no assignment was made.

The comparison between the hfs data obtained for P 87CT in R p . s p h a e r o i d e s R-26 and in another bacterium ( R . r u b r u m G - 9 ) 3 6 shows that the couplings are different. This is true not only for the 0-proton hfc's which are strongly dependent on the geometry of rings II and IV, but also for the methyl couplings. No such differences of the hfc's of isolated B C h l a * and B C h l a * g (different side chains) could be found. These observations point to a possible geometrical distortion of the dimer by the protein environment.

Structural proposal for P870 +

Beyond the dimeric nature of P 870 + the hfs data contain information about its structure. By adopting the E S E results of Bowman and Nor r i s 3 7 P 870 + can be described by a supermolecule, in which the unpaired electron is delocalized in a super M O extending over both B C h l a halves. This requires an interplanar distance ^3.5 Ä (van der Waals distance of aromatic 7r-systems). The E N D O R results have shown that the halves are equivalent, i.e., they must be related to each other by a C 2-symmetry axis. A model which fulfills this requirement has already been proposed . 3 8 - 4 0 In order to show if our experimental observations can be explained in the framework of such a model, we have performed a series of simple H M O calculations on a dimer constructed from the 7r-skeletons of the two B C h l a molecules, including the keto groups at rings I and III. The best

1066 W. LUBITZ e t a l .

b

F I G U R E 4 Experimental and simulated EPR spectra of P 870 + in Solution. For the Simulation the E N D O R couplings and assignments from Table II were used; trace a: Simulation assuming a dimer (twice the number of protons belonging^to each hfc, linewidth 2.3 G obtained from the residual small hfc in P 870 + and the 4 N couplings in B C h l a + ) ; trace b: Simulation assuming a monomer (linewidth 2 1 / 2 x 2.3 = 3.2 G).

agreement with the experiments was obtained for a dimer geometry with the following characteristics (Figure 5): (i) interplanar distance 3-3.5 Ä (ii) maximum overlap in the region of rings III and V , (iii) two parallel planes (shifted against each other by 7-8 Ä), a-face of the first B C h l a partially overlapping the ß-face of the second B C h l a .

This proposed dimer geometry must, however, be considered as preliminary since the theoretical approach—so far—has been very simple (enforced by Computer limitations). More sophisticated calcu-lations (Extended H M O and SCF methods) are already in progress.

B A C T E R I A L PHOTOSYNTHESIS 1067

1 3Ä 7 A

F IGURE 5 Horizontal and perpendicular view of the dimer model. Only the TT-skeletons and the hydrated rings (broken lines) of the two monomeric halves are shown. Standard geometries with X-Ray modifications for the keto group at rings III were used for the Hückel M O calculations (CNDO/2 parametrization).

Furthermore we are currently trying to extend our E N D O R / T R I P L E studies to the primary donor and acceptors in other bacteria and in plant Systems.

Note added i n proof: After completion of this work, a manuscript by O'Malley and Babcock 4 2 has been brought to our notice in which P 870 + is proposed to consist of a monomeric B C h l a+ with altered spin distribution (see also ref. 36).

Acknowledgements

This work was supported by the Deutsche Forschungsgemeinschaft (Sfb 143 and Sfb 161). The skillful assistance of C. Bubenzer (Universität München) in the RC preparations is gratefully acknowledged. H.S. thanks Dr. R. K. Clayton (Ithaca) for providing a stab culture of R p . s p h a e r o i d e s R-26.

1068

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