Optical Properties and Structure of Tetrapyrroles Proceedings of a Symposium held at the University of Konstanz West Germany, August 12-17,1984

Editors Gideon Blauer • Horst Sund

W DE

G

Walter de Gruyter • Berlin • New York 1985

Editors

Gideon Blauer, P h . D . Professor of Biophysical Chemistry Institute of Life Sciences The Hebrew Universi ty Jerusalem, Israel

Horst Sund, Dr. rer. nat. Professor of Biochemistry Fakul tä t fur Biologie Univers i tä t Konstanz D-7750 Konstanz, West Germany

Bayerische Staatsbibliothek

München D L i b r a r y of Congress C a t a l o g i n g i n P u b l i c a t i o n D a t a

M a i n entry under title: Optical properties and structure of tetrapyrroles. Bibliography: p. Includes indexes. 1. Tetrapyrroles-Congresses. 2. Tetrapyrroles-Optical properties-Congresses. I. Blauer, G ideon . II. Sund, Horst, 1926-QD441.067 1985 547'593 84-29791 I S B N 0-89925-009-2 (U.S.)

C I P - K u r z t i t e l a u f n a h m e der Deutschen Bibliothek

Optical properties and structure of tetrapyrroles : proceedings of a symposium held at the Univ . of Konstanz, West Germany, August 12 - 17,1984 / ed. G ideon Blauer ; Horst Sund. -Ber l in ; N e w York : de Gruyter, 1985. (Ber l in . . . ) I S B N 3-11-010054-1 N E : Blauer, G i d e o n [Hrsg.] ; Univers i tä t <Konstanz> ; Tetrapyrroles I S B N 0-89925-009-2 (New York . . . ) Gewebe

3 110100541 Walter de Gruyter • Berl in • N e w York 0-89924-009-2 Walter de Gruyter, Inc., N e w York

Copyright © 1985 by Walter de G r u y t e r & C o . , Berl in 30. A l l rights reserved, including those of translation into foreign languages. N o part of this book may be reproduced in any form - by photoprint, microfilm or any other means nor transmitte d nor translated into a machine language without written permission from the publisher. P r i n t i n g : Ger ike G m b H , Ber l in . B ind ing : Dieter M i k o l a i , Berl in. - Printed in Germany.

CONTENTS

L i s t of Contributors XI

SECTION I. GENERAL C h a i r m a n : S. B e y c h o k

An H i s t o r i c a l Survey of Porphyrins, t h e i r Related Spectra, and t h e i r I n t e r p r e t a t i o n by A.D. Adler and M. Gouterman 3 Discussion 16

Routes to Functional Porphyrin Assemblies (Molecular Complexes, Covalent Redox P a i r s , and Reactive Domains in V e s i c l e s ) by J.-H. F u h r h o p a n d T. L e h m a n n 19 Discussion 40

Organism Dependent Pathways of T e t r a p y r r o l e Biosynthesis by D. Dörnemann a n d H. S e n g e r 43 Discussion 60

SECTION I I . PORPHYRINS AND METALLOPORPHYRINS C h a i r m e n : H. R a p o p o r t , P.-S. S o n g a n d H. Z u b e r

Transient Absorption Spectra and Excited State K i n e t i c s of T r a n s i t i o n Metal Porphyrins by D. Holten and M. Gouterman 63 Discussion 89

Metal Complexes of T e t r a p y r r o l e Ligands. XXXVII. Str u c t u r e and O p t i c a l Spectra of Sandwich-Like Lanthanoid Porphyrins by J.W. B u c h l e r a n d M. K n o f f 91 Discussion 104

Excited State E l e c t r o n Transfer of Carbonyl[mesotetra(p-tolyl) porphyrinato osmium(11)]Complexes. One and Two E l e c t r o n Photo-redox Processes by A . V o g l e r , J . K i s s l i n g e r a n d J.W. B u c h l e r .... 107 Discussion 119

Photophysical P r o p e r t i e s of Hematoporphyrin and i t s D e r i v a t i v e s and t h e i r Use i n Tumor Phototherapy by A. Andreoni and R . cubeddu 121 Discussion 133

O p t i c a l P r o p e r t i e s of Ferriheme-Quinidi ne Complexes by G. B l a u e r 137 Discussion 152

VIII Contents

Resonance Raman Studies of Haemoglobin by K. Nagai 157 Discussion 173

Op t i c a l P r o p e r t i e s of Heme Bound to the Central Exon Fragment of Globins and to Corresponding Fragments i n Cytochromes by S. B e y c h o k 175 Discussion 200

Heme Conf i g u r a t i o n i n Cytochrome c by Y . P . Myer 203 Discussion 225

C o r r e l a t i o n between Near-Infrared, ESR Spectra and Oxida t i o n -Reduction P o t e n t i a l s i n Low-Spin Heme Proteins by A. S e h e j t e r , I . Vi g a n d W.A. E a t o n 227

C i r c u l a r Dichroism C o n t r i b u t i o n s of Heme-Heme and C h l o r o p h y l l -Chlorophyll I n t e r a c t i o n s by R.W. woody 239 Discussion 256

Op t i c a l P r o p e r t i e s of Conjugated Proteins which Transport Haem, Porphyrins and B i l i r u b i n by B. Ketterer 261 Discussion 276

SECTION I I I . BILE PIGMENTS C h a i r m e n : R.P.F. G r e g o r y , //. S u n d a n d R.W. W o o d y

Light Absorption of Bi1 atrienes-abc and 2,3-Dihydrobi1 atrienes-abc by H. F a l k 281 Discussion 293

Mechanism of Phototherapy of Neonatal Jaundice. R e g i o s p e c i f i c Photoisomerization of B i l i r u b i n s by A.F. McDonagh and D.A. L i g h t n e r 297 Discussion 308

E f f e c t s of Environment on Photophysica1 Processes of B i l i r u b i n by A . A . L a m o l a 311 Discussion 3i'7

The Molecular Model of Phytochrome Deduced from Opt i c a l Probes b y P.-S. S o n g 331 Discussion 345

Phytochrome, the Vis u a l Pigment of P l a n t s : Chromophore Structure and Chemistry of Photoconversion by w. Rüdiger, P . Eilfeld a n d F. Thiimmler 349 Discussion 364

Recent Advances i n the Photophysics and Photochemistry of Small, Large, and Native Oat Photochromes by K. Schaffner, S.E. B r a s l a v s k y a n d A.R. H o l z w a r t h 367 Discussion 380

Contents IX

Time-Resolved Fluorescence D e p o l a r i s a t i o n of Phycocyanins i n D i f f e r e n t States of Aggregation by S. Schneider, P. Geiselhart, T. M i n d l , P. H e f f e r l e , F. Dörr, W. J o h n a n d H. S c h e e r 383

The Influence of an External E l e c t r i c a l Charge on the C i r c u l a r Dichroism of Chromopeptides from Phycocyanin by c h . scharnagl, E. Köst-Reyes, S. S c h n e i d e r a n d J . O t t o 397 Discussion 407

B i l i n s and B i 1 i n - P r o t e i n Linkages i n Phycobi1iproteins: S t r u c t u r a l and Spectroscopic Studies by H. Rapoport and A.N. Glazer 411 Discussion 421

S t r u c t u r a l Organization of T e t r a p y r r o l e Pigments i n L i g h t -Harvesting Pigment-Protein Complexes by H . zuber 425 Discussion 442

The C r y s t a l and Molecular S t r u c t u r e of C-Phycocyanin from Mastigo-ciadus laminosus and i t s I m p l i c a t i o n s f o r Function and Evolution by T. S c h i r m e r , W. B o d e , R. H u b e r , W. S i d l e r a n d H. Z u b e r 445 Discussion 449

SECTION IV. CHLOROPHYLLS C h a i r m e n : M. G o u t e r m a n a n d A.A. L a m o l a

O p t i c a l Spectra of Photosynthetic Reaction Centers. T h e o r e t i c a l and Experimental Aspects by A.J. Hoff 453 Discussion 472

C i r c u l a r Dichroism of C h l o r o p h y l l s i n Protein Complexes from Ch l o r o p l a s t s of Higher P l a n t s : A C r i t i c a l Assessment by R.P. F. G r e g o r y 475 Discussion 488

Optical P r o p e r t i e s and St r u c t u r e of Chlorophyll RC I by D. Dörnemann a n d H. S e n g e r 489 Discussion 505

Long-Wavelength Absorbing Forms of Bacteriochlorophyl 1 s. I I . Struc­t u r a l Requirements f o r Formation i n T r i t o n X-100 M i c e l l e s and i n Aqueous Methanol and Acetone by H . scheer, B. Paulke and J . G o t t s t e i n 507 Discussion 521

Concluding Remarks by M. Gouterman 523

Index of Co n t r i b u t o r s 525

Subject Index 527

TIME-RESOLVED FLUORESCENCE DEPOLARISATION OF PHYCOCYANINS IN DIFFERENT STATES OF AGGREGATION

S i e g f r i e d Schneider, Peter G e i s e l h a r t , Toni Mindl, Peter H e f f e r l e and F r i e d r i c h Dörr I n s t i t u t für P h y s i k a l i s c h e und T h e o r e t i s c h e Chemie, Technische Universität München, L i c h t e n b e r g s t r . 4 D 8046 Garching, FRG

Wolfhart John and Hugo Scheer Botanisches I n s t i t u t der Universität Menzingerstr.67, D 8000 München, FRG

I n t r o d u c t i o n

B i l i p r o t e i n s are the main p h o t o s y n t h e t i c l i g h t h a r v e s t i n g pigments i n c y a n o b a c t e r i a , red algae and cryptophytes (1-4). In the former two organisms, they are present as h i g h l y o r­ganized s t r u c t u r e s , the phycobilisomes, which are not an i n t e g r a l p a r t of the p h o t o s y n t h e t i c membrane, but r a t h e r attached to i t s s u r f a c e (2,3,5,6). They c o n t a i n up to s e v e r a l hundred b i l i p r o t e i n s c a r r y i n g up to 2000 chromophores i n a s i n g l e f u n c t i o n a l u n i t , but a l s o a number of so c a l l e d l i n k e r p e p t i d e s which are probably r e s p o n s i b l e f o r the a t ­tachment and i n t e r n a l o r g a n i z a t i o n of the phycobilisomes (7-10). E x c i t a t i o n energy i s captured e f f i c i e n t l y by the phycobilisomes and t r a n s f e r e d with a high quantum y i e l d v i a s e v e r a l i n t e r m e d i a t e acceptors to the c h l o r o p h y l l o u s r e a c t i o n c e n t e r s w i t h i n the p h o t o s y n t h e t i c membrane. This energy t r a n s f e r has been the s u b j e c t of a c t i v e r e s e a r c h over the past decade, i n v o l v i n g both the i n v e s t i g a t i o n of e n t i r e p h y c obilisomes and of fragments thereof (11-19). In view

Optical Properties and Structure of Tetrapyrroles © 1985 by Walter de Gruyter & Co., Berlin • New York

384 S. Schneider et al

o f t h e c o m p l e x s t r u c t u r e o f t h e f o r m e r , we have f o c u s e d m a i n l y on a s i n g l e b i l i p r o t e i n , C - p h y c o c y a n i n ( P C ) , w h i c h i s g e n e r a l l y t h e m a j o r p i g m e n t o f c y a n o b a c t e r i a l p h y c o b i l i ­somes ( 1 7 , 1 9 ) . PC h a s a p r o n o u n c e d t e n d e n c y f o r a g g r e g a t i o n ( 4 ) , w h i c h i s a l s o s t r o n g l y i n f l u e n c e d by t h e d i f f e r e n t l i n k e r p e p t i d e s ( 1 0 ) . These l i n k e r p e p t i d e s a l s o seem t o be i m p o r t a n t i n f i n e - t u n i n g o f t h e a b s o r p t i o n o f b i l i p r o -t e i n s . I n t h i s p a p e r we want t o r e p o r t on t h e p h o t o p h y s i c a l p r o p e r t i e s o f s u c h a c o m p l e x b e t w e e n PC and l i n k e r p e p t i d e s , v i z . PC 636, w h i c h has been i s o l a t e d f r o m g r e e n - l i g h t a d a p t e d c u l t u r e s o f t h e c y a n o b a c t e r i u m M a s t i q o c l a d u s l a m i n o s u s . The t i m e r e s o l v e d f l u o r e s c e n c e i s c o m p a r e d t o t h a t o f common PC 618, w h i c h i s f r e e o f a d d i t i o n a l l i n k e r s .

S a m p l e P r e p a r a t i o n

C u l t u r e s o f M a s t i g o c l a d u s l a m i n o s u s were grown as d e s c r i b e d e a r l i e r w i t h f l u o r e s c e n t t u b e s p r o d u c i n g o n l y s m a l l amounts o f r e d l i g h t . Monomers and t r i m e r s o f PC were p r e p a r e d a s r e p o r t e d e a r l i e r ( 1 9 ) . PC 636 was e l u t e d f r o m t h e DEAE c o l ­umns b e t w e e n p h y c o e r y t h r o c y a n i n , a s e c o n d p i g m e n t f o r m e d i n g r e e n l i g h t , and t h e m a j o r PC. I t was d i a l y s e d a g a i n s t p h o s p h a t e b u f f e r (80 mM, pH 6.0) and u s e d w i t h o u t d e l a y a n d w i t h o u t s t o r a g e i n t h e f r e e z e r .

The c h r o m a t o g r a p h y o f a c r u d e e x t r a c t o f M. l a m i n o s u s on DEAE c e l l u l o s e p r o d u c e s g e n e r a l l y APC I , PC and APC I I i n o r d e r o f i n c r e a s i n g s a l t c o n c e n t r a t i o n . When t h e c e l l s a r e g r o w n u n d e r l i g h t o f r e d u c e d i n t e n s i t y a t ^ ^ 6 0 0 n m , two a d d i ­t i o n a l p i g m e n t s a r e f o r m e d i n s m a l l a m o unts. One i s t h e w e l l known p h y c o e r y t h r o c y a n i n (24) e l u t i n g f i r s t f r o m t h e c o l u m n , w h i c h i s f o l l o w e d by a p h y c o c y a n i n named PC 636 a c c o r d i n g t o i t s e x t r e m l y r e d s h i f t e d a b s o r p t i o n s p e c t r u m . The m a i n a n a l y t i c a l d i f f e r e n c e o f t h i s p i g m e n t seems t o be t h e p r e -

Fluorescence D e p o l a r i z a t i o n 385

s e n c e o f two c o l o r l e s s p e p t i d e s i n a d d i t i o n t o t h e common PC s u b u n i t s . I t has a s l i g h t l y h i g h e r b u o y a n t d e n s i t y on a s u c r o s e g r a ­d i e n t , and d i s s o c i a t e s upon p r o l o n g e d s t a n d i n g o r f r e e z i n g i n b u f f e r o f l o w i o n i c s t r e n g t h . D e t a i l s o f t h e p r e p a r a t i o n and i t s p r o p e r t i e s a r e t o be p u b l i s h e d s e p a r a t e l y .

M e a s u r e m e n t s and d a t a a n a l y s i s

The f l u o r e s c e n c e d e c a y c u r v e s w ere m e a s u r e d u s i n g a s y n c h r o ­n o u s l y pumped m o d e - l o c k e d r i n g dye l a s e r ( r h o d a m i n e 6G, 80 MHz r e p e t i t i o n r a t e , p u l s e w i d t h ss 1 p s ) i n c o n j u n c t i o n w i t h a r e p e t i t i v e l y w o r k i n g s t r e a k c a m e r a ( f o r d e t a i l s s ee e.g. ( 1 6 ) ) . The a p p a r e n t t i m e r e s o l u t i o n o f t h i s s y s t e m i s a p p r o ­x i m a t e l y 25 ps w i t h o u t d e c o n v o l u t i o n p r o c e d u r e ; i t a l l o w s

1 3 m e a s u r e m e n t s w i t h l o w e x c i t a t i o n i n t e n s i t i e s (10 p h o t o n s

2 p e r p u l s e and cm ). The f l u o r e s c e n c e d e c a y c u r v e s m e a s u r e d w i t h t h e a n a l y z e r p a r a l l e l , ( I ( t ) ) , and o r t h o g o n a l , ( I ( t ) ) ,

P s t o t h e p o l a r i z a t i o n o f t h e e x c i t i n g beam a r e t r a n s f e r e d t o a m i n i c o m p u t e r w h e r e , a f t e r p r o p e r c o r r e c t i o n f o r t h e s y s t e m s r e s p o n s e , t h e e x p r e s s i o n s I ( t ) = I p ( t ) + 2 I g ( t ) and D ( t )= I ( t ) - I ( t ) a r e c a l c u l a t e d . I ( t ) m e a s u r e s t h e d e c a y p s o f t h e e x c i t e d s t a t e p o p u l a t i o n ( e l e c t r o n i c l i f e t i m e ) and D ( t ) t h e p r o d u c t o f t h e f o r m e r w i t h t h e c o r r e l a t i o n f u n c ­t i o n o f t h e a b s o r p t i o n a n d e m i s s i o n d i p o l e s ( 1 7 , 2 0 ) . I n c o n t r a s t t o t h e a n i s o t r o p y f u n c t i o n R ( t ) t h e d i f f e r e n c e f u n c t i o n D ( t ) i s a d d i t i v e and c a n be e v a l u a t e d i f more t h a n one e m i t t i n g s p e c i e s i s p r e s e n t . L a c k i n g b e t t e r i n f o r m a t i o n , we a p p r o x i m a t e t h e c o r r e l a t i o n f u n c t i o n by an e x p o n e n t i a l . The b e s t f i t s f o r b o t h f u n c t i o n s ( I and D) a r e d e t e r m i n e d u n d e r t h e a s s u m p t i o n o f a b i e x p o n e n t i a l r e s p o n s e f u n c t i o n (two e m i t t i n g s p e c i e s ) by means o f a M a r q u a r d t a l g o r i t h m . D e p e n d i n g on t h e S/N r a t i o o f t h e r e c o r d e d f l u o r e s c e n c e d e ­c a y c u r v e s and t h e i r r e l a t i v e m a g n i t u d e , t h e f i t p a r a m e t e r s

386 S. Schneider et al

d e r i v e d may be s u b j e c t t o c o n s i d e r a b l e e r r o r . We w i l l , t h e r e ­f o r e , d i s c u s s t h e i r t r e n d s r a t h e r t h a n t h e i r a b s o l u t e m a g n i ­t u d e .

R e s u l t s and D i s c u s s i o n

I t i s f o u n d t h a t i n a l l c a s e s t h e d e c a y c u r v e s c a n be f i t t e d s u f f i c i e n t l y w e l l as c o n v o l u t i o n s o f b i e x p o n e n t i a l s . The f i t p a r a m e t e r s , e.g. t h e d e c a y t i m e s ( T ^ , T ^ i n p s e c ) a n d t h e r e l a t i v e a m p l i t u d e s ( A ^ , i n %) o f t h e s h o r t - and l o n g - l i v e d c o m p o n e n t , r e s p . , a r e g i v e n i n t h e i n s e r t s i n f i g u r e 1. The m e a s u r e m e n t s were p e r f o r m e d a t t h r e e d i f f e r ­e n t t e m p e r a t u r e s , n a m e l y a t 18°C ( A ) , a t 36°C ( B ) , t h e t e m p e r a t u r e t h e a l g a e a r e grown, and a t 52°C (C) where i r r e ­v e r s i b l e t h e r m a l d e n a t u r a t i o n s t a r t s t o become e f f e c t i v e . P a r t i a l d e n a t u r a t i o n a l r e a d y t a k e s p l a c e ' a t l o w e r t e m p e r a ­t u r e s . S t a t i c m e a s u r e m e n t s show a d r a s t i c l o s s i n f l u o r e s ­c e n c e y i e l d (up t o f o u r o r d e r s of m a g n i t u d e ) , w h i c h i s much l a r g e r t h a n t h e d e c r e a s e i n a b s o r p t i o n c o n n e c t e d w i t h a c o n ­f o r m a t i o n a l c h a n g e o f t h e chromophore ( 2 1 ) . The t i m e - i n t e ­g r a t e d f l u o r e s c e n c e i n t e n s i t i e s e x p r e s s e d as A^T^+A^^T^ a l s o c o n f i r m t h e r e d u c t i o n a t h i g h e r t e m p e r a t u r e . I t i s f o u n d a s a g e n e r a l r u l e t h a t t h e d e c r e a s e i s more p r o n o u n c e d i n t h e a l p h a t h a n i n t h e b e t a s u b u n i t and l a r g e r f o r t h e mono­mer t h a n f o r t h e t r i m e r . The n o r m a l i z e d f l u o r e s c e n c e d e c a y c u r v e s a l s o show s m a l l b u t d i s t i n c t v a r i a t i o n s w i t h t e m p e r a ­t u r e . T h e r e f o r e t h e r e s u l t s p r e s e n t e d i n f i g u r e 1 must be t a k e n a s e v i d e n c e f o r an i n t e r m e d i a t e s t a t e b e i n g p r e s e n t d u r i n g t h e p r o c e s s o f t h e r m a l d e n a t u r a t i o n .

The a l p h a s u b u n i t o f PC c o n t a i n s o n l y one c h r o m o p h o r e . I f i t i s s t a b i l i z e d by n o n c o v a l e n t i n t e r a c t i o n w i t h t h e p r o t e i n t o a d o p t o n l y one c o n f o r m a t i o n , a s i n g l e e x p o n e n t i a l d e c a y i s e x p e c t e d w i t h a l i f e t i m e o f 1.5 t o 2.5 ns ( l i f e t i m e o f

Fluorescence D e p o l a r i z a t i o n 387

I. MONOMFR

A) 4 3 3 / 1 8 7 0 ( 5 7 / 4 3 ) 8) 3 6 7 / 2 4 7 8 ( 5 9 / 4 1 ) C) 2 8 9 / 1 7 8 5 ( 6 S / 3 S )

1 . MONOMER

P ) 2 4 8 / 1 8 1 1 ( 5 5 / 4 5 ) B) 2 2 2 / 1 7 6 3 1 5 4 / 4 8 ) C ) 7 0 0 / 1 3 7 8 1 6 1 / 3 9 )

2 1 , T R I M E R

P ) 5 5 8 / 1 3 9 2 ( 8 6 / 1 4 )

B ) 2 9 6 / 1 5 0 7 ( 5 1 / 4 9 )

C ) 2 3 5 / 1 6 3 3 ( 6 1 / 3 9 )

I i TRIMER

n) 62/ 260(77/2il

B) 64/ 856(72/2aJ C ) 95/1138(75/25J

2 1 . RLGE

P ) 1 1 2 / 7 0 5 ( 8 6 / 1 4 )

B) 1 2 7 / 5 6 1 ( 8 3 / 1 7 )

C ) 1 5 5 /

1000 2000 Hps)

1000 2000 Mps)

Fig.1 : Dependence of f l u o r e s ­cence decay of PC 618 on aggregation and temperature (T=18°(A),36°(B),52 Ö(C))

388 S. Schneider et a l

t h e c h r o m o p h o r e i n a n a t i v e e n v i r o n m e n t ) . I n s t e a d , an a d d i ­t i o n a l s h o r t - l i v e d component i s f o u n d , whose l i f e t i m e v a r i e s w i t h t e m p e r a t u r e b e t w e e n 690 and 1060 p s e c . A s i m i l a r be­h a v i o u r was v e r i f i e d f o r t h e a l p h a s u b u n i t o f S p i r u l i n a p l a t e n s i s (17) and A n a baena v a r i a b i l i s ( 2 2 ) . S i n c e a g g r e ­g a t i o n o f t h e s u b u n i t s i s u n l i k e l y , one must assume a t l e a s t two d i f f e r e n t s e t s o f e m i t t i n g s p e c i e s ( c h r o m o p h o r e - p r o t e i n -a r r a n g e m e n t s ) . The l o n g - l i v e d s p e c i e s must be c l o s e t o t h a t i n n a t i v e e n v i r o n m e n t , w h i l s t t h e s h o r t - l i v e d f o r m s h o u l d be c l o s e r t o t h e d e n a t u r e d , l e s s i n t e r a c t i n g s p e c i e s . The f a s t e r d e c a y i n t h e d i f f e r e n c e f u n c t i o n D ( t ) , f u r t h e r m o r e , s i g n a l s t h a t t h e f a s t e r component i s s u b j e c t t o a d e p o l a r i z a ­t i o n m e c hanism w i t h T 1500 p s e c . S i n c e no a c c e p t o r m o l e c u l e s a r e p r e s e n t , t h e d e p o l a r i s a t i o n s h o u l d be due t o o r i e n t a -t i o n a l r e l a x a t i o n o f t h e l e s s r i g i d l y bound c h r o m o p h o r e s .

The b e t a s u b u n i t c o n t a i n s two c h r o m o p h o r e s i n d i f f e r e n t p r o ­t e i n e n v i r o n m e n t . The r e s p e c t i v e a b s o r p t i o n maxima a r e s e p a ­r a t e d by a b o u t 20 nm. The s t a t i o n a r y f l u o r e s c e n c e s p e c t r a o f b o t h s u b u n i t s a r e e s s e n t i a l l y e q u a l , w h i c h i n d i c a t e s an e f f i c i e n t e n e r g y t r a n s f e r f r o m t h e " s e n s i t i z i n g " t o t h e " f l u o r e s c i n g " c h r o m o p h o r e . The e n e r g y t r a n s f e r i s a l s o m a n i ­f e s t e d i n t h e f l u o r e s c e n c e d e c a y c u r v e s . The s h o r t - l i v e d c o mponent (T^ 300ps) i s i n t e r p r e t e d as " l e a k a g e " f l u o r e s ­c e n c e f r o m t h e s - c h r o m o p h o r e , whose l i f e t i m e i s s h o r t e n e d due t o e n e r g y t r a n s f e r t o t h e f - c h r o m o p h o r e i n t h e same s u b -u n i t . The d e p o l a r i s a t i o n t i m e o f t h e f a s t component i s much s h o r t e r t h a n t h a t o f t h e a l p h a s u b u n i t and d e c r e a s e s w i t h i n c r e a s i n g t e m p e r a t u r e f r o m 400 t o 150 p s e c . The l o n g e r l i f e ­t i m e i s c l o s e t o t h e s h o r t e r one i n t h e a l p h a s u b u n i t ; a l i f e t i m e o f 2 n s , w h i c h w o u l d be e x p e c t e d f o r t h e f - c h r o m o -p h o r e i n a n a t i v e e n v i r o n m e n t i s n o t d e t e c t e d , p o s s i b l y f o r e x p e r i m e n t a l r e a s o n s . An u n a m b i g u o u s i n t e r p r e t a t i o n i s p r e ­s e n t l y n o t p o s s i b l e , b e c a u s e d i f f e r e n t s u b s e t s o f chromo-p h o r e - p r o t e i n - a r r a n g e m e n t s c a n n o t be e x c l u d e d i n v i e w o f

Fluorescence D e p o l a r i z a t i o n 389

0̂0 500 600 X/nm m

Fig.2 : A b s o r p t i o n and e m i s s i o n s p e c t r a of t r i m e r s of PC-618 ( ) and PC-636 ( )

the p r e p a r a t i o n sequence, which i n v o l v e s a d e n a t u r a t i o n -r e n a t u r a t i o n sequence.

The i s o t r o p i c decay curves of monomers and t r i m e r s are s i m i ­l a r to each o t h e r . The s h o r t l i f e t i m e i s i n the range of 200-500 psec and r e p r e s e n t s the l i f e t i m e of the s-chromo-phores, which are quenched by energy t r a n s f e r . The longer one between 1600 and 2500 psec c h a r a c t e r i z e s the t e r m i n a l acceptor, i . e . the f-chromophore i n the n a t i v e environement. Chromophores e x c i t e d v i a energy t r a n s f e r r a t h e r than d i r e c t l y by photon a b s o r p t i o n should emit a l e s s p o l a r i z e d f l u o r e s ­cence. Only the s h o r t - l i v e d leakage f l u o r e s c e n c e i s p a r t l y p o l a r i z e d , but the d e p o l a r i z a t i o n times are moderately s h o r t . In c o n t r a s t to S p i r u l i n a p l a t e n s i s (17) we observe f o r PC from the t h e r m o p h i l i c algae no lengthening of the d e p o l a r i ­z a t i o n time with temperature.

390 S. Schneider et al

H

0 4 0 0 8 0 0 1 2 0 0 1 6 0 0 2 0 0 0

TIME (ps)

Fiq.3 : Fluorescence decay of PC-636 e x c i t e d at 580 nm and recorded with i n t e r f e r e n c e f i l t e r s /j. =614 nm (A), 2 • = 635 nm (B) and = 654 nm ( O ^ S o l i d l i n e s are c a l c u l a t e d f i t curves with parameters T<|/T2 i - n P s e c a n ^ A-/A 9 i n % i n p a r e n t h e s i s . (A) 1 72/ 1700 (75/25) (B) 609/ 4200 (66/34) (C) 610/10100 (64/36)

In the i n t a c t a l g a , the energy i s e f f i c i e n t l y t r a n s f e r e d to the n o n f l u o r e s c i n g r e a c t i o n c e n t e r . The observed e m i s s i o n i s only leakage f l u o r e s c e n c e from PC ( f a s t component with l i f e t i m e of approx. 130 psec) and All o p h y c o c y a n i n (slow com­ponent) . Since the f r a c t i o n of emission from d i r e c t l y ex­c i t e d chromophores i s sm a l l , the emission i s e s s e n t i a l l y u n p o l a r i z e d . Furthermore, i t i s found t h a t an i n c r e a s e i n temperature does not r e s u l t i n a l o n g - l i v e d component, which would i n d i c a t e a pr e v e n t i o n of energy t r a n s f e r .

In PC-618, the d i f f e r e n c e i n e x c i t a t i o n energy of s- and

Fluorescence D e p o l a r i z a t i o n 391

0.2

0. 16

0. 12

J / 0.08

0.04

1600 2000 TIME ( p s )

Fig.4 : C a l c u l a t e d d i f f e r e n c e f u n c t i o n s I -I normalized with r e s p e c t to the maximum of the i s o t r o p i c @ecly curve I ( t ) . The f l u o r e s c e n c e was recorded with c u t - o f f f i l t e r s , whose c u t - o f f wavelength i s 20 nm l a r g e r than the e x c i t a t i o n wave­le n g t h . S o l i d l i n e s are c a l c u l a t e d f i t curves w i t h parameters

V (B) 36/1 1 30 ( 86/1 4 ) = 600 nm (C) 42/1 165 ( 85/1 5 ) = 590 nm

[\/T7 i n ps and A 1/A 9 i n % i n p a r e n t h e s i s . [À) 3 5/ 760 ( 8 6 / U r 2 = 610 nm

(D) 53/1290 (69/31) /I exc exc = 580 nm

f-chromphores, res p . , i s small (^20 nm). S e l e c t i v e e x c i t a ­t i o n of both chromophores i s more d i f f i c u l t than i n PC-636, i n which the a b s o r p t i o n of the f-chromophores i s s h i f t e d b a t h o c h r o m i c a l l y due to i n t e r a c t i o n with two c o l o r l e s s pro­t e i n s ( f i g . 2 ) . The f l u o r e s c e n c e decay curves of t r i m e r i c PC-636 prove to be s t r o n g l y dependent on the r e c o r d i n g wave­le n g t h ( f i g . 3 ) . Upon short-wavelength e x c i t a t i o n (580 nm) the longer wavelength emission (both from s- and f-chromo-phores) i s dominated by a f a s t component ( T ^ 600 psec) as i n the case of PC-618. I t s l i f e t i m e c o u l d be r e l a t e d to the energy t r a n s f e r time from s- to f-chromophores.

392 S. Schneider et a l .

I 30000

o

Ü

—• 20000

+

10000

°0 400 800 1200 1600 2000 TIME ( p s )

Fiq.5 : I s o t r o p i c f l u o r e s c e n c e decay of t r i m e r s of PC-636. The f l u o r e s c e n c e was recorded with c u t - o f f f i l t e r s , whose c u t - o f f wavelength was 20 nm l a r g e r than the e x c i t a t i o n wave­le n g t h . S o l i d l i n e s are f i t curves c a l c u l a t e d with parame­t e r s T./T 9 i n ps and A /A i n % i n p a r e n t h e s i s . (A) 61 0/3200 (63/37) ^ ~ v r = 6 1 0 n m

(B) 535/4700 (55/45) J ^ = 580 nm

The l i f e t i m e of the slow component (emission from t e r m i n a l f-chromophores) v a r i e s somewhat with o b s e r v a t i o n wavelength, a f a c t , which could again be i n d i c a t i v e f o r a nonuniform chromophore-protein-arrangement. If the recorded f l u o r e s c e n c e i s r e s t r i c t e d to near-resonant emission (614 nm), a much f a s t e r component dominates the decay curve. We presume t h a t t h i s i s i n part a consequence of resonant energy t r a n s f e r w i t h i n the s-chromophore ma n i f o l d . The l a t t e r can be v e r i f i e d by i n s p e c t i o n of the normalized d i f f e r e n c e curves d i s p l a y e d i n f i g . 4. Upon sh o r t wavelength e x c i t a t i o n (s-chromophore e x c i t a t i o n ) a pronounced f r a c t i o n of the s h o r t - l i v e d emission i s p o l a r i z e d . The decay time of ^40 ps i s r e l a t e d to the energy t r a n s f e r time w i t h i n the s-chromophore manifold (homo-transfer). Such a t r a n s f e r does not change the number

Fluorescence D e p o l a r i z a t i o n 393

of e x c i t e d molecules of t h i s s p e c i e s . A c c o r d i n g l y , i n the i s o t r o p i c decay f u n c t i o n 1=1^+2 I g no such extremely s h o r t ­l i v e d component can be d e t e c t e d ( f i g . 5).

Keeping the r e s u l t s f o r the alp h a - and the beta-subunits i n mind, one c o u l d of course argue t h a t the f a s t component observed i n the decay curves of monomers and t r i m e r s i s due to a second chromophore-protein arrangement, which d i f f e r s s i g n i f i c a n t l y from t h a t i n the n a t i v e s p e c i e s . We b e l i e v e , however, t h a t an a r t e f a c t i a l m i croheterogeneity can be e x c l u ­ded i n the case of PC 636, because i t r e q u i r e s much mi l d e r m a n i p u l a t i o n s than those a p p l i e d i n the p r e p a r a t i o n s of PC-subunits. Any h e t e r o g e n e i t y i s then expected to be i n h e r ­ent to the b i l i p r o t e i n .

From the l a r g e body of measurements denoted to the study of f l u o r e s c e n c e decay and energy t r a n s f e r w i t h i n f u n c t i o n a l l y i n t a c t p h y c o b i l i s o m e s and i t s c o n s t i t u e n t aggregated b i l i -p r o t e i n s one may conclude t h a t the t r a n s f e r times are g r e a t ­l y reduced when the s i z e of the aggregate, i . e . the number of chromophores i s i n c r e a s e d . I t might be that i n a monomeric u n i t the d i s t a n c e between the chromophores i s l a r g e r than between chromophores of a t r i m e r belonging to d i f f e r e n t mono­mers. T h i s c o u l d a l s o imply t h a t i n hexamers (and higher aggregates) the important " v e r t i c a l " energy t r a n s f e r between adjacent t r i m e r s i s f a s t e r than the " h o r i z o n t a l " t r a n s f e r w i t h i n one t r i m e r , which c a r r i e s no energy i n the d i r e c t i o n of the r e a c t i o n c e n t e r (23).

F i n a n c i a l support by the Deutsche Forschungsgemeinschaft i s g r a t e f u l l y acknowledged.

394 S. Schneider et a l .

References

1. Scheer, H.: i n " L i g h t Reaction Path of P h o t o s y n t h e s i s " (F. K. Fong, ed.), p. 7-45, Springer, B e r l i n 1982

2. Gantt, E. : Ann. Rev. P l a n t P h y s i o l . 3_2, 327-347 ( 1 981 ) 3. G l a z e r , A.N.: Ann. Rev. Biochem. 52̂ , 1 25-1 57 ( 1 983) 4. McColl, R.: Photochem. , P h o t o b i o l . 3j>, 899-904 (1 982 ) 5. Mörschel, E., K o l l e r , K., Wehrmeyer, W.: Arch. Micro­

b i o l . 125,43-51 (1980) 6. Wanner, G., Köst, H.-P.: Protoplasma 102,97-109 (1980) 7. G l i c k , R.E., Z i l i n s k a , B.A.": Pl a n t P h y s i o l . 69, 991-997

(1982) 8. R e d l i n g e r , T., Gantt, E.: Proc. N a t l . Acad. S c i . US

79,5542-5546 (1982) 9. K o l l e r , K.P., Wehrmeyer, W., Moerschel, E.: Eur. J . Bio­

chem. 9_1, 57-63 (1 978 ) 10. Yu, M.H., G l a z e r , A.N., W i l l i a m s , R.C.: J . B i o l . Chem.

256,13130-13136 (1981) 11. S e a r l e , G.F.W., Barber, J . , P o r t e r , G.,Tredwell, C.J.:

Biochim. Biophys. Acta 50 1 ,246-256 (1 978 ) 12. Kobayashi, T., Degenkolb, E.O., Behrson, R, Rentzepis,

P.M., M c C o l l , R., Berns, D.S.:Biochemistry 1 8 , 507 3-507 B (1979)

13. Breton, J . , Geacintov, N.E.: Biochim. Biophys. Acta 59_4,1-32 (1 980 )

14. P e l l e g r i n o , F., Wong, D., Alfano, R.R., Z i l i n s k a s , B.: Photochem. P h o t o b i o l . 3_4, 69 1-696 (1 981)

15. Holzwarth, A.R., Wendler, J . , Wehrmeyer, W.: Photochem. P h o t o b i o l . 16,479-487 (1982)

16. H e f f e r l e , P., Nies, M., Wehrmeyer, W., Schneider, S.: Photobiochem. Photobiophys. 5,41-51 (1983),id.325-334

17. H e f f e r l e , P., John, W., Scheer, H., Schneider, S.: Photochem. P h o t o b i o l . 3_9 , 22 1 -232 (1 984)

18. G i l b r o , T., Sundstroem, A., Sundstroem, V., Holzwarth, A.R.: FEBS L e t t . J_6_2,64-68 (1 983)

19. H e f f e r l e , P., G e i s e l h a r t , P., Mindl, T.,Schneider,S.: Z. N a t u r f o r s c h . 39c (1984),in p r i n t

20. Fleming, G.R., M o r r i s , J.M., Robinson, G.W.: J.Chem.Phys. J_7 , 9 1 (1 976 )

21. Scheer, H., Formanek, H., Schneider, S.: Photochem.Photobiol. 36,259 (1982)

Fluorescence D e p o l a r i z a t i o n 395

22. S w i t a l s k i , S.C. and Sauer, K.: Photochem.Photobiol., i n p r i n t

23. Scheer, H.: i n " P h o t o s y n t h e t i c Membranes: Enc y c l o p e d i a of P l a n t P h y s i o l o g y " , ( S t a e h l i n , A., Ar t z e n , C , eds.) S p r i n g e r , H e i d e l b e r g , i n p r i n t

24. Füglistaller, P., Suter, F., Zuber, H.: Hoppe-Seyler's Z. p h y s i o l . Chemie 364,691-7 12 (1983)

Received June 28, 1984

Discussion to t h i s and next l e c t u r e was combined, see page 407-410.