Antennas and Reaction Centers of Photosynthetic Bacteria Structure, Interactions, and Dynamics

Proceedings of an International Workshop Feldafing, Bavaria, F.R.G., March 23-25,1985

Editor: M. E. Michel-Beyerle

With 168 Figures

Springer-Verlag Berlin Heidelberg New York Tokyo

Contents

P a r t I Antennas: Structure and Energy Transfer

S t r u c t u r e of A n t e n n a Po l ypep t i d es . B y H . Zube r 2

T h e C r y s t a l a n d M o l e c u l a r S t r u c t u r e o f C - P h y c o c y a n i n B y R . H u b e r 15

C - P h y c o c y a n i n f rom M a s t i g o c l a d u s l a m i n o s u s . I so la t ion and P rope r t i e s of S u b u n i t s and S m a l l Aggregates . B y W. J o h n , R . F i s che r , S. Siebzehnrübl, a n d H . Scheer ( W i t h 9 F igures ) 17

P i cosecond T i m e - R e s o l v e d , P o l a r i z e d F luorescence Decay of Phycob i l i s omes and C o n s t i t u e n t B i l i p r o t e ins Isolated f rom M a s t i g o c l a d u s l a m i n o s u s B y S. Schne ider , P. Ge i s e lha r t , T M i n d l , F. Dörr, W. J o h n , R. F i s che r , and H . Scheer ( W i t h 4 F igures ) 26

F luorescence B e h a v i o u r of C r y s t a l l i z e d C - P h y c o c y a n i n (Tr imer ) f rom M a s t i g o c l a d u s l a m i n o s u s B y S. Schne ider , P. Ge i s e lha r t , C . Scharnag l , T Sch i rmer , W. B o d e , W. S id l e r , a n d H . Z u b e r ( W i t h 4 F igures ) 36

Ene rgy -T rans f e r K i n e t i c s i n Phycob i l i s omes B y A . R . H o l z w a r t h ( W i t h 2 F i gures ) 45

E x c i t o n S ta t e a n d E n e r g y Trans fe r i n B a c t e r i a l M e m b r a n e s : T h e Ro l e of P i g m e n t - P r o t e i n C y c l i c U n i t S t ruc tures B y R . M . P ea r l s t e i n a n d H . Z u b e r 53

Ca ro t eno i d -Bac t e r i o ch l o r ophy l l Interact ions B y R . J . C o g d e l l ( W i t h 1 F i gure ) 62

B a c t e r i o c h l o r o p h y l l a- and c -P ro t e in Comp lexes f rom Ch lorosomes of G r e en Su l fu r B a c t e r i a C o m p a r e d w i t h Bac t e r i o ch l o rophy l l c Aggregates i n C H 2 C 1 2 - H e x a n e . B y J . M . O l s on , P .D . G e r o l a , G . H . v a n B r a k e l , R . F M e i b u r g , a n d H . Vasme l ( W i t h 8 F igures ) . . . 67

Reverse -Phase H i gh -Pe r f o rmance L i q u i d C h r o m a t o g r a p h y of A n t e n n a P i g m e n t - a n d C h l o r o s o m a l P ro t e ins of C h l o r o f l e x u s a u r a n t i a c u s . B y R . Feick ( W i t h 2 F igures ) 74

VII

F luorescence -Detec ted M a g n e t i c Resonance of the A n t e n n a Bac t e r i o ch l o r ophy l l T r ip l e t States of P u r p l e P h o t o s y n t h e t i c B a c t e r i a . B y A . A n g e r h o f er, J . U . von Schütz, a n d H . C . Wo l f ( W i t h 1 F i gure ) 78

H i g h - R e s o l u t i o n 1 H N M R of L i g h t - H a r v e s t i n g C h l o r o p h y l l -P ro t e ins . B y C . D i j k e m a , G .F .W. Searle, and T . J . Schaa f sma 81

C r y s t a l l i z a t i o n and L i n e a r D i c h r o i s m Measurements of the B 8 0 0 -850 A n t e n n a P i g m e n t - P r o t e i n C o m p l e x f r om R h o d o p s e u d o r n o n a s s p h a e r o i d e s 2.4.1 B y J . P . A l l e n , R . The i l e r , a n d G . Feher ( W i t h 2 F igures ) 82

C r y s t a l l i z a t i o n of the B800 -850 -comp l ex f r om R h o d o p s e u d o r n o n a s a c i d o p h i l a S t r a i n 7750 B y R . J . C o g d e l l , K . Wool ley, R . C . Mackenz i e , J . G . L indsay , H . M i c h e l , J . Dob l e r , and W . Z i n t h ( W i t h 6 F igures ) 85

L i n e a r D i c h r o i s m ( L D ) and A b s o r p t i o n S p e c t r a of C r y s t a l s of B 8 0 0 - 8 5 0 L i g h t - H a r v e s t i n g Complexes of R h o d o p s e u d o r n o n a s c a p s u l a t a . B y W. Mäntele, K . Steck, T. Wacker , W. We l te , B . L e vo i r , and J . B r e t o n ( W i t h 5 F igures ) 88

P a r t II Reaction Centers: Structure and Interactions

T h e C r y s t a l S t ruc tu r e of the Pho tosyn the t i c R e a c t i o n Cen t e r f r om ' R h o d o p s e u d o r n o n a s v i r i d i s B y J . Deisenhofer and H . M i c h e l ( W i t h 2 F igures ) 94

S ing le C r y s t a l s f rom Reac t i on Centers of R h o d o p s e u d o r n o n a s v i r i d i s S tud i ed by Po l a r i z ed L i g h t . B y W. Z i n t h , M . Sander , J . D o b l e r , W. K a i s e r , and H . M i c h e l ( W i t h 3 F igures ) 97

O n the A n a l y s i s of O p t i c a l S p e c t r a of R h o d o p s e u d o r n o n a s v i r i d i s R e a c t i o n Centers B y E . W . K n a p p a n d S . F F i scher ( W i t h 3 F igures ) 103

Or i en t a t i on of the C h r o m o p h o r e s in the R e a c t i o n Cen te r of R h o d o p s e u d o r n o n a s v i r i d i s . C o m p a r i s o n of L o w - T e m p e r a t u r e L i n e a r D i c h r o i s m Spec t r a w i t h a M o d e l De r i v ed f rom X - R a y Crys ta l l o g raphy . B y J . B r e t o n ( W i t h 4 F i gures ) 109

C a l c u l a t i o n s of Spect roscop ic Proper t i es of B a c t e r i a l R e a c t i o n Cente rs . B y W.W. P a r s o n , A . Scherz , and A . Warshe l ( W i t h 5 F igures ) 122

O n the Tempera ture -Dependence of the L o n g Wave l eng th F luorescence and A b s o r p t i o n of R h o d o p s e u d o r n o n a s v i r i d i s R e a c t i o n Centers . B y P O . J . Scherer, S.F. F i s che r , J . K . H . Hörber, M . E . M i che l -Beye r l e , a n d H . M i c h e l ( W i t h 3 F igures ) 131

VIII

L o c a l Env i r onments of P i gmen t s in R e a c t i o n Centers of Pho tosyn the t i c B a c t e r i a f rom Resonance R a m a n D a t a B y M . L u t z and B . R o b e r t ( W i t h 4 F igures ) 138

T h e Sp in -Po l a r i z a t i on P a t t e r n of the A m = 1 T r ip l e t E P R Spec t rum of R p s . v i r i d i s R eac t i on Centers B y F . G . H . van W i j k , P G a s t , and T J . Schaa f sma 146

T r i p l e t State Invest igat ion of Cha r g e Separa t i on and S y m m e t r y in Single Crys ta l s of R . v i r i d i s R eac t i on Centers B y J . R . Nor r i s , D . E . B u d i l , H . L . C r e s p i , M . K . B o w m a n , P. G a s t , C . P L i n , C . H . C h a n g , and M . Schiffer 147

Tr ip l e t -minus-S ing l e t A b s o r b a n c e Dif ference Spectroscopy of Pho tosyn the t i c Reac t i on Centers by Abso rbance -De t e c t ed Magne t i c Resonance . B y A . J . Hoff ( W i t h 11 F igures ) 150

E N D O R Studies of the P r i m a r y D o n o r i n B a c t e r i a l R e a c t i o n Centers . B y W. L u b i t z , F. L e n d z i a n , M . P l a t o , K . Möbius, and E . Tränkle ( W i t h 6 F igures )

E N D O R of Semiquinones in R C s f rom R h o d o p s e u d o r n o n a s s p h a e r o i d e s . B y G . Feher, R . A . Isaacson, M . Y . O k a m u r a , and W. L u b i t z ( W i t h 10 F igures )

P h o t o i n d u c e d Charge Separa t i on in B a c t e r i a l Reac t i on Centers Invest igated by Tr ip l e t s and R a d i c a l P a i r s B y J . R . Nor r i s , D . E . B u d i l , S.V. K o l a c z k o w s k i , J . H . Tang , and M . K . B o w m a n ( W i t h 5 F igures )

Sp in D ipo l a r Interact ions of R a d i c a l P a i r s i n Pho tosyn the t i c R e a c t i o n Centers . B y A . O g r o d n i k , W. L e r s ch , M . E . M i che l -Beye r l e , J . Deisenhofer , and H . M i c h e l ( W i t h 4 F igures ) 198

P r o t e i n / L i p i d Interact ion of Reac t i on Cen t e r a n d A n t e n n a P ro t e ins . B y J . R ieg ler , W . M . H e c k l , J . Peschke, M . Lösche, and H . Möhwald ( W i t h 6 F igures ) 207

T h e A r ch i t e c tu r e of Pho t osys t em II i n P l a n t Pho tosynthes i s . W h i c h Pep t ide Subun i t s C a r r y the R e a c t i o n Cente r of P S II? B y A . Trebst and B . D e p k a ( W i t h 3 F igures ) 216

P a r t III Electron-Transfer: Theory and Model Systems

A p p l i c a t i o n of E lec t ron-Trans fe r T h e o r y to Several Systems of B i o l og i ca l Interest. B y R . A . M a r c u s and N . S u t i n 226

Effects of D i s tance , Energy a n d M o l e c u l a r S t ruc tu re on L o n g -D i s t ance E lec t ron-Trans fe r Be tween Mo lecu l es B y J . R . M i l l e r ( W i t h 4 F igures ) 234

IX

164

174

190

Ul t ra f as t E l e c t r o n Transfer i n B i o m i m e t i c Mode l s of P h o t o s y n t h e t i c R e a c t i o n Centers . B y M . R . Was ie l ewsk i , M . P N i e m c z y k , W . A . Svec, and E . B . P ew i t t ( W i t h 5 F igures ) 242

E l e c t r o n Trans fer T h r o u g h A r o m a t i c Spacers i n B r i d g e d E l e c t r o n -D o n o r - A c c e p t o r Mo lecu les . B y H . He i te le a n d M . E . M i che l -Beye r l e 250

E l e c t r o n Trans fer i n R i g i d l y L i n k e d D o n o r - A c c e p t o r Sys tems B y S.F. F i s che r , I. N u s s b a u m , a n d P . O . J . Scherer ( W i t h 3 F igures ) . 256

E l e c t r o n C o n d u c t i o n A l o n g A l i p h a t i c C h a i n s B y R . B i t t l , H . T r eu t l e in , and K . Schu l ten ( W i t h 4 F igures ) 264

P a r t I V Reaction Centers: Structure and Dynamics

K i n e t i c s and Mechan i sms of In i t i a l E l ec t ron -Trans f e r Reac t i ons i n R h o d o p s e u d o r n o n a s s p h a e r o i d e s R e a c t i o n Centers B y W.W. P a r s o n , N . W . T Woodbury , M . Becker , C . K i r m a i e r , a n d D . H o l t e n ( W i t h 3 F igures ) 278

Femtosecond Studies of the Reac t i on Cen te r of R h o d o p s e u d o r n o n a s v i r i d i s : T h e Very F i r s t D y n a m i c s o f the E l ec t ron -T rans f e r Processes . B y W. Z i n t h , M . C . Nuss , M . A . F r a n z , W. K a i s e r , a n d H . M i c h e l ( W i t h 5 F igures ) 286

A n a l y s i s of T ime-reso lved F luorescence of R h o d o p s e u d o r n o n a s v i r i d i s R e a c t i o n Centers B y J . K . H . Hörber, W. Göbel, A . O g r o d n i k , M . E . M i che l -Beye r l e , a n d E .W . K n a p p ( W i t h 3 F igures ) 292

T h e C h a r a c t e r i z a t i o n of the Q A B i n d i n g Si te of the R e a c t i o n Cen t e r of R h o d o p s e u d o r n o n a s s p h a e r o i d e s . B y M . R . G u n n e r , B . S . B r a u n , J . M . B r u c e , and P L . D u t t o n ( W i t h 2 F igures ) 298

P a r t V Model Systems on Structure of Antennas and Reaction Centers

S t r u c t u r e and Energet ics in R e a c t i o n Centers and Semi -synthe t i c C h l o r o p h y l l P r o t e i n Comp lexes . B y S . G . Boxe r ( W i t h 4 F i gures ) .. 306

S m a l l O l i gomers of Bac te r i och lo rophy l l s as i n v i t r o M o d e l s for the P r i m a r y E l e c t r o n Donors and L i gh t -Ha r v e s t i n g P i gmen t s i n P u r p l e P h o t o s y n t h e t i c B a c t e r i a B y A . Scherz , V. Rosenbach , and S. M a l k i n ( W i t h 7 F igures ) 314

E x p e r i m e n t a l , S t r u c t u r a l and Theo r e t i ca l M o d e l s of Bac t e r i o ch l o rophy l l s a, d and g. B y J . Fajer, K . M . B a r k i g i a , E . F u j i t a , D . A . Goff, L . K . H a n s o n , J . D . H e a d , T H o r n i n g , K . M . S m i t h , and M . C . Zerner ( W i t h 6 F igures ) 324

X

E N D O R C h a r a c t e r i z a t i o n of H y d r o g e n - B o n d i n g to Immob i l i z ed Q u i n o n e A n i o n Rad i ca l s . B y P . J . O ' M a l l e y , T . K . Chandrasheka r , a n d G.T . B a b c o c k ( W i t h 3 F i gures ) 339

C o n c l u d i n g R e m a r k s . Some Aspec t s of Ene rgy Transfer i n An t ennas and E l e c t r o n Trans fer i n R e a c t i o n Centers of Pho t o syn the t i c B a c t e r i a . B y J . J o r t n e r and M . E . M iche l -Beye r l e ( W i t h 6 F igures ) 345

Index of Con t r i bu to r s 367

XI

C-Phycocyan in from M a s t i g o c l a d u s l a m i n o s u s . Isolation and Propert ies of Subunits and Smal l Aggregates

W. J o h n , R . F i s c h e r , S. Siebzehnrübl, a n d H. S c h e e r

Ins t i tu t für Bo tan ik der Universität München, Menz inge r S t r . 67 D-8000 München 19, F . R . G .

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

P h o t o s y n t h e t i c organisms cover most of t h e i r energy needs w i t h s u n l i g h t . They have c o n s e q u e n t l y developed a v a r i e t y of a d a p t a t i o n mechanisms t o compete e f f i c i e n t l y f o r i t . In h i g h e r p l a n t s , a dominant mechanism i s the growth towards t h e l i g h t . A q u a t i c and microorganisms adapt commonly by chr o m a t i c a d a p t a t i o n of the p h o t o s y n t h e t i c antenna. The C h l o r o p h y l l s a and b are r a t h e r i n e f f i c i e n t i n c o l l e c t i n g green l i g h t , and s e v e r a l a d d i t i o n a l pigment Systems have ev o l v e d t o f i l l t h i s h ole i n the a c t i o n spectrum.

The p h y c o b i l i p r o t e i n s comprise one such group of antenna pigments. They are used i n c y a n o b a c t e r i a , r ed a l g a and c r y p t o p h y t e s ( 1 ) . In the former two, they are h i g h l y aggregated t o g e t h e r w i t h c o l o r l e s s l i n k e r P o l y p e p t i d e s ( 2 ) , i n t h e p h y c o b i l i s o m e s ( 3 ) . These are m i c r o s c o p i c s t r u c t u r e s s i t u a t e d at the o u t e r s u r f a c e o f the t h y l a k o i d membrane, which t r a n s f e r t h e i r e x c i t a t i o n energy e f f i c i e n t l y (quantum y i e l d £95%) t o 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 membrane. In

Cooperation w i t h the group of S. SCHNEIDER ( G a r c h i n g ) , we have r e c e n t l y begun t o study the f l u o r e s c e n c e p r o p e r t i e s of small aggregates and s u b u n i t s of phycocyanins ( 4 ) . The aim o f t h i s work i s an understanding of t h e energy t r a n s f e r i n th e s e pigments i n r e l a t i o n t o t h e s i z e and s t r u c t u r e of the assembly. T h i s r e p o r t i s concerned w i t h the s t a b i l i t y and the photochemical r e a c t i v i t y o f C- phycocyanin (PC) and i t s s u b u n i t s from M. laminosus. Time- r e s o l v e d p o l a r i z e d f l u o r e s c e n c e data are presented i n the accompanying r e p o r t of the Gar c h i n g group.

2. M a t e r i a l s and Methods

M. laminosus was grown i n Suspension c u l t u r e i n CASTENHOLZ medium T5"). C- PC was i s o l a t e d as r e p o r t e d e a r l i e r ( 4 ) . The s u b u n i t s were i s o l a t e d by p r e p a r a t i v e e l e c t r o f o c u s i n g on Agarose g e l s (Pharmacia, München) i n 8M urea, and r e n a t u r e d w i t h o u t d e l a y on a d e s a l t i n g column ( B i o g e l P6, B i o r a d ) .

A b s o r p t i o n s p e c t r a were re c o r d e d on a model 320 ( P e r k i n Elmer, U e b e r l i n g e n ) spectrophotometer. The c e l l h o l d e r was the r m o s t a t e d , and temperatures measured i n t h e c u v e t t e s w i t h a Pt 100 r e s i s t o r . A b s o r p t i o n d i f f e r e n c e s p e c t r a were measured w i t h a ZWS I I d u a l - wavelength photometer (Sigma, B e r l i n ) i n s p l i t - beam mode. The data were d i g i t i z e d and s t o r e d w i t h model 11+ Computer (Ap p l e , München) u s i n g a s e i f - made program, which was developed f o r o b t a i n i n g high wavelength accuracy.

F l u o r e s c e n c e s p e c t r a were reco r d e d on a OMR 22 ( Z e i s s , Oberkochen) s p e c t r o f 1 u o r i m e t e r . The c e l l h o l d e r s were t h e r m o s t a t e d , and the

17

temperatures recorded i n the c u v e t t e s w i t h a Pt 100 r e s i s t o r . The s p e c t r a are u n c o r r e c t e d f o r the s p e c t r a l response f u n c t i o n o f t h e apparatus. C i r c u l a r d i c h r o i s m s p e c t r a were r e c o r d e d on a model Mark V d i c h r o g r a p h ( J o b i n - Yvon ISA, München) equipped w i t h a s i l e x Computer (Leanord, L i l l e ) and a m o d i f i e d Software. S e d i m e n t a t i o n c o e f f i c i e n t s were o b t a i n e d i n a model E u l t r a c e n t r i f u g e (Beckman, München) at 20 aC w i t h the Scanner wavelength set at 620nm.

Chemicals and s o l v e n t s were reagent grade u n l e s s o t h e r w i s e s t a t e d . Sodium dodecyl s u l f a t e - P o l y a c r y l a m i d e e l e c t r o p h o r e s i s was done by t h e method of LAEMMLI ( 6 ) .

3. R e s u l t s

3.1 O p t i c a ! S p e c t r a

A l l s t u d i e s were done i n potassium phosphate b u f f e r (50 o r lOOmM) at pH7.5. Under th e s e c o n d i t i o n s , PC i s i s o l a t e d as a t r i m e r (heterohexamer, (<*ß ), Sedimentation c o e f f i c i e n t **4.8S). I t can be d i s s o c i a t e d r e v e r s i b l y i n t o the monomer ( h e t e r o d i m e r , («K/3 )* * 2.8S) by i n c u b a t i o n w i t h potassium t h i o c y a n a t e . The a b s o r p t i o n spectrum of t h e monomer i s s l i g h t l y b l u e - s h i f t e d as compared t o the t r i m e r , but o t h e r w i s e very s i m i l a r ( f i g . 1 ) .

The s p e c t r a shown i n f i g . 1 correspond t o p r e p a r a t i o n s , which are f r e e of c o l o r l e s s l i n k e r - p e p t i d e s . I f the l a t t e r are p r e s e n t , t h e s p e c t r a o f monomers ( o b t a i n e d by t h e same d i s s o c i a t i o n method) remain unchanged. The t r i m e r s p e c t r a are more s t r o n g l y r e d - s h i f t e d , however, and a s e r i e s of s l i g h t l y d i f f e r e n t s p e c i e s a b s o r b i n g between 620 and 638nm can be i s o l a t e d . T h e i r Sedimentation c o e f f i c i e n t s are o n l y s l i g h t l y h i g h e r H5-6S) than t h a t o f the t r i m e r . T h i s i s due t o t h e presence o f v a r y i n g amounts of s e v e r a l c o l o r l e s s l i n k e r - p e p t i d e s . The e x a c t s t o i c h i o m e t r y i s s t i l l under i n v e s t i g a t i o n . An i n c r e a s e d chromophore-chromophore c o u p l i n g i n t h e s e r e d - s h i f t e d complexes i s suggested from t h e i r c i r c u l a r d i c h r o i s m (CD) s p e c t r a . S i m u l t a n e o u s l y w i t h the r e d - s h i f t i n t he a b s o r p t i o n maximum, t h e r e develops a pronounced r e d - s h i f t e d f e a t u r e i n the CD- spectrum, which i n c r e a s e s w i t h an i n c r e a s i n g r e d -s h i f t ( f i g . 2 ) . I t has been r a t i o n a l i z e d as an S- shaped e x c i t o n c o u p l e t ( p o s i t i v e at l o n g e r w a v e l e n g t h s ) , which i s superimposed on t h e a b s o r p t i o n - t y p e CD- spectrum o f the monomer.

E x c i t o n c o u p l i n g s are absent i n l i n k e r - p e p t i d e - f r e e p r e p a r a t i o n s o f PC from M. laminosus, but have been d i s c u s s e d e a r l i e r i n PC from S. p r a t e n s i s (7) and i n p a r t i c u l a r i n a l l o p h y c o c y a n i n s ( 8 ) . The i n c l u c t i o n o f e x c i t o n c o u p l i n g would r e q u i r e a changed geometry of t h e chromophores, which can be c o n s i d e r e d on t h e b a s i s of t h e x - r a y s t r u c t u r e o f t r i m e r i c PC from the same organism (9; see a l s o SCHIRMER et a l . , t h i s monograph). The c l o s e s t chromophore c o n t a c t s i n t h e l i n k e r -p e p t i d e - f r e e t r i m e r are between the x - chromophore o f t h e monomeric u n i t and t h e /31- chromophore of t h e a d j a c e n t one. The c e n t e r - t o -c e n t e r d i s t a n c e (22A) i s j u s t t o o l a r g e f o r e x c i t o n i n t e r a c t i o n s . Changes by o n l y a few Angström may, however, be s u f f i c i e n t t o a l l o w f o r s t r o n g c o u p l i n g s , and i t i s p o s s i b l e t h a t such changes are induced by the l i n k e r - p e p t i d e s . Unless t h e r e i s a g r o s s s t r u c t u r a l change i n v o l v e d , t h e r e d - s h i f t e d s p e c i e s are then most l i k e l y due t o a changed geometry i n t h e c o n t a c t r e g i o n of t h e monomeric u n i t s .

18

F i g . 1: Ab s o r p t i o n s p e c t r a o f monomeric (<xß, — ) and t r i m e r i c ((<*/3 )• ; ) PC from PL laminosus (50mM phosphate b u f f e r , pH 7.5)

-?.oooe-4

Li.2

UM v \ | \ 3

1 612

\ 1 2 6 1 8

\ 4 3 6 3 2

V 4 6 3 5

300.0 400.0 SQO.O COO.O 700.0

F i g . 2: C i r c u l a r d i c h r o i s m s p e c t r a o f t r i m e r i c phycocyanin c o n t a i n i n g l i n k e r p e p t i d e s : aggregates w i t h i n c r e a s i n g wavelength of the red a b s o r p t i o n band.Amax see i n s e r t s .

A

40Ö 500 600 nm X

F i g . 3: A b s o r p t i o n s p e c t r a of the i s o l a t e d s u b u n i t s of PC from M. laminosus (oc:—;#:---). The e m i s s i o n maxima are i n d i c a t e d by the l a b e l s i n t h e upper r i g h t .

19

The a b s o r p t i o n s p e c t r a o f the i s o l a t e d s u b u n i t s ( f i g . 3 ) show c o n s i d e r a b l e d i f f e r e n c e s both w i t h r e s p e c t t o t h e p o s i t i o n and t h e shape of the l o n g - wavelength band. The <x - s u b u n i t ( A max= 616nm) has a narrow band s i m i l a r i n shape t o t h a t of i n t e g r a l PC, whereas t h e ß-subunit has a b l u e - s h i f t e d ( Amax= 605nm) and broadened band. The weighed sum of the two a b s o r p t i o n s p e c t r a i s very s i m i l a r t o t h a t o f monomeric PC (not shown). In s p i t e of t h i s b l u e - s h i f t e d a b s o r p t i o n , t h e f l u o r e s c e n c e maxima are almost at the same p o s i t i o n ( f i g . 3 ) . The <*-subunit c a r r i e s one chromophore, the ß - s u b u n i t two chromophores (/31, /32) at d i f f e r e n t b i n d i n g s i t e s of t h e a p o p r o t e i n ( 1 0 ) , t h e broadening should then r e f l e c t a s l i g h t d i f f e r e n c e i n t h e e x c i t a t i o n e n e r g i e s of t h e two chromophores. T h i s i s indeed supported by t h e c i r c u l a r d i c h r o i s m s p e c t r a ( f i g . 4 ) . The a n i s o t r o p y o f t h e ß - s u b u n i t i s o n l y & 6 0 % as compared t o the a - s u b u n i t . The p o s i t i v e extremum i s c o n s i d e r a b l y b l u e - s h i f t e d w i t h r e s p e c t t o t h e a b s o r p t i o n maximum, and t h e r e i s a l o w - i n t e n s i t y red-wing i n t h e CD-spectrum. I t i s suggested t h a t the /2>-subunit c a r r i e s one o p t i c a l l y a c t i v e ( Amax **595nm) and one much l e s s a c t i v e ( p l a n a r ?) chromophore (Amax **615nm).

-s.oooe-4 300.0 400.0 EOO.O 600.0 700.0 MB

F i g . 4: CD of the i s o l a t e d s u b u n i t s shown i n F i g . 2 (<*:—sßr — ) . The s p e c t r a are n o r m a l i z e d t o equal a b s o r p t i o n s i n the l o n g -wavelength maximum.

Th i s i n t e r p r e t a t i o n i s supported by s i m i l a r CD d a t a and a d d i t i o n a l f l u o r e s c e n c e P o l a r i s a t i o n r e s u l t s of MIMURO et a l . ( t h i s volume). An assignment of t h e i n d i v i d u a l chromophores on t h e ß-subunit t o t h e such d e f i n e d a b s o r p t i o n s i s not y e t p o s s i b l e . Based on t h e amino-acid sequence and che x-ray s t r u c t u r e (9,10), ß 1 i s s i m i l a r t o c t . The a b s o r p t i o n maxima would then r e l a t e /31 t o t h e chromophore a b s o r b i n g at <*615nm. S i n c e these chromophores come c l o s e s t i n the t r i m e r , t h e e x c i t o n s p l i t at the red wing o f the main CD-band i n 1 i n k e r - a s s o c i a t e d t r i m e r s ( v i d e supra) would support t h i s assignment. However, t h e CD-data suggest a r a t h e r d i f f e r e n t c o n f o r m a t i o n f o r the ß- chromophore (Amax<*615nm) and the oc- chromophore, and b i o c h e m i c a l l a b e l i n g w i l l be necessary t o d e c i d e t h i s p o i n t c o n c l u s i v e l y .

3.2 S t a b i i i t i e s

The chromophores of PC are h e l d r i g i d l y i n an e n e r g e t i c a l l y u n f a v o r a b l e , extended co n f o r m a t i o n ( 1 , 9 ) . T h i s c o n f o r m a t i o n i s due t o n o n - c o v a l e n t

20

• 1,0-

A A - 1 0 3

0

-1.0

-2,0-

F i g . 5: A b s o r p t i o n (bottom) and CD- s p e c t r a ( t o p ) o f a b i I i p r o t e i n from P i e r i s b r a s s i c a ( 1 3 ) . The pigment c o n t a i n s b i l i v e r d i n IXy^bound non- c o v a l e n t l y t o an a p o p r o t e i n o f MW**25kDa.

protein-chromophore i n t e r a c t i o n s , which are r e v e r s i b l e - l o s t upon u n f o l d i n g of che p r o t e i n , e.g. w i t h urea ( 1 1 ) . T h i s type of i n t e r a c t i o n i s p r o b a b l y c h a r a c t e r i s t i c f o r a l l p h y c o b i 1 i p r o t e i n s and c r u c i a l t o t h e i r f u n c t i o n as antenna pigments, because they i n c r e a s e the o s c i l l a t o r s t r e n g t h of the v i s i b l e band by almost one o r d e r o f magnitude, and decrease the r a d i a t i o n l e s s decay o f e x c i t e d s t a t e s by more than t h r e e o r d e r s of magnitude ( 1 ) . The s p e c i f i c i t y of t h e s e i n t e r a c t i o n s can be seen by comparison w i t h o t h e r b i 1 i p r o t e i n s , e.g. the b i l i v e r d i n I X a - s e r u m albumin complex ( 1 2 ) , or t h e b i l i v e r d i n I X y - p r o t e i n from P i e r i s b r a s s i c a (13, see f i g . 5 ) . In both the l a t t e r pigments i s che c y c l i c - h e l i c a l c o n f o r m a t i o n of t h e chromophores p r e s e r v e d , which i s c h a r a c t e r i s t i c f o r t h e r e s p e c t i v e chromophores i n S o l u t i o n (14,15). They have, a c c o r d i n g l y , low f l u o r e s c e n c e y i e l d s and weak a b s o r p t i o n s i n t h e v i s i b l e ränge. Here, t h e major i n f l u e n c e of the a p o p r o t e i n i s a p r e f e r e n t i a l b i n d i n g of one o f the two h e l i c a l enantiomers, which l e a d s t o i n t e n s e induced C D - s i g n a l s .

Due t o the pronounced chromophore-protein i n t e r a c t i o n s , t h e chromophores o f PC can be used as very s e n s i t i v e probes t o monitor the S t a t e of the p r o t e i n ( 1 , 1 1 ) . Small a g g r e g a t i o n - i n d u c e d changes have been d i s c u s s e d i n the f i r s t p a r t . Much l a r g e r changes o c c u r , i f the p r o t e i n i s p a r t i c a l l y or f u l l y u n f o l d e d , e.g. wich urea o r heat. A f t e r a p r e v i o u s study w i t h PC from S. p l a t e n s i s , t h i s method has now been used t o i n v e s t i g a t e the s t a b i l i t i e s of t h e PC and i t s s u b u n i t s from NL_ laminosus.

The n o r m a l i z e d a b s o r p t i o n changes upon u r e a - d e n a t u r a t i o n are shown i n f i g . 6 (see legend f o r d e t a i l s ) . The g r o s s changes are s i m i l a r f o r the i n t e g r a l pigment ( t r i m e r ) and i t s s u b u n i t s . Small d i f f e r e n c e s i n t h e f i n a l (= 8M urea) a b s o r p t i o n s are i n d i c a t i v e o f s l i g h t l y d i f f e r e n t c o n f o r m a t i o n s o f n a t i v e chromophores, because the chromophores have t h e same m o l e c u l a r s t r u c t u r e and are expected t o have i d e n t i c a l s p e c t r a i n the denatured s t a t e s . The most n o t a b l e d i f f e r e n c e i s the i n c r e a s e d s t a b i l i t y of the i n t e g r a l pigment, as compared t o both s u b u n i t s . At 4M u r e a , the a b s o r p t i o n o f the i n t e g r a l pigment i s decreased by l e s s than 4%, whereas the decrease o f the s u b u n i t s i s 12% (cx) and 15% ( ß). The urea c o n c e n t r a t i o n s n e c e s s a r y t o induce a 50% decrease o f t h e long-wavelength a b s o r p t i o n s are 6.1, 5.6, 5.4M, r e s p e c t i v e l y . In c o n c r a s t t o the r e s u l t s w i t h PC from S. p l a t e n s i s , t h e r e are no i n d i c a t i o n s f o r a s t e p w i s e u n f o l d i n g e v i d e n t from the a b s o r p t i o n s p e c t r a .

21

A620 [%]

F i g . 6: D e n a t u r a t i o n o f t r i m e r i c PC ( — ) and i t s s u b u n i t s («: — ; ß:...) from M. laminosus w i t h u r e a . R e l a t i v e a b s o r p t i o n s at the l o n g - wavelength ( t o p ) and near-UV maxima (bottom), n o r m a l i z e d t o the a b s o r p t i o n s i n t h e absence o f u r e a . A l l s p e c t r a were recorded from s t o c k samples t r e a t e d s e p a r a t e l y b e f o r e the measurement w i t h the a p p r o p r i a t e amount o f u r e a , e i t h e r as 8M S o l u t i o n or as s o l i d . A l l S o l u t i o n s i n lOOmM phosphate b u f f e r , pH 7.5, T= 25°C.

F i g . 7: D e n a t u r a t i o n of PC from M. laminosus and i t s s u b u n i t s w i t h urea. R e l a t i v e f l u o r e s c e n c e i n t e n s i t i e s c o r r e c t e d f o r a b s o r p t i o n . Xmax, exc.= 600nm;Amax, em. *652nm, d e c r e a s i n g w i t h m c r e a s i n g urea c o n c e n t r a t i o n . L a b e l s and d e t a i l s as i n f i g . 5.

The r e l a t i v e f l u o r e s c e n c e y i e l d s d u r i n g the same t i t r a t i o n e xperiment are showi i n f i g . 7. There i s a l r e a d y a l a r g e d i f f e r e n c e between the t h r e e s p e c i e s i n t h e i r n a t i v e s t a t e s . The da t a f o r the i n t e g r a l pigment i n d i c a t e a s t e p w i s e u n f o l d i n g , w i t h the f i r s t s tep being p o s s i b l y r e l a t e d t o t h e d i s a g g r e g a t i o n of t he t r i m e r i n t o monomers. The/3- s u b u n i t i s l e a s t s t a b l e (50% decrease at 3.5M u r e a ) , whereas oc and t h e i n t e g r a l pigment have a 50% d e c r e a s e at 5.3M urea. Although t h e curve s o f t h e l a s t two s p e c i e s c r o s s - over t w i c e i n t h e i n t e r m e d i a t e r e g i o n , t h e sum o f t h e s u b u n i t s i s always l e s s than t h a t of t h e i n t e g r a l pigment.

The d i f f e r e n c e s of the thermal d e n a t u r a t i o n are even more pronounced among the t h r e e s p e c i e s ( f i g . 8 ) . The " m e l t i n g p o i n t s " (50% dec r e a s e o f t h e l o n g -

22

A620 (7.1 100-1

NN \ \ \ \ \ \ \ •

\ 4 \ \ \ •

50-

F i g . 8: Heat d e n a t u r a t i o n o f PC. A l l s p e c t r a were rec o r d e d 5min a f -t e r t he sample had reached the temperature i n d i c a t e d . A l l mea-surements were done w i t h f r e s h samples. Other d e t a i l s as i n F i g . 5.

— i — 20

80 °C

A350l7oJ 100 n

«M.t W.e M . l MC.I I » 4M.» «W.» KM.S HO.f • « • . • m

F i g . 9: D i f f e r e n c e s p e c t r a f o r the p h o t o r e a c t i o n of PC i n the presence of urea (5M). Top: I r r a d i a t i o n w i t h 606nm l i g h t ( i n t e r f e r e n c e f i l t e r ) . P o s i t i v e bands correspond t o b l e a c h i n g . Bottom: I r r a d i a t i o n o f the sample w i t h 525nm l i g h t a f t e r s a t u r i n g p r e - i r r a d i a t i o n a t 606nm. P o s i t i v e peaks correspond t o i n c r e a s e d a b s o r p t i o n s .

23

wavelength a b s o r p t i o n ) i s at 67° (PC), 61° iß) and 58° («). The i n c r e a s e o f thermal s t a b i l i t y by almost 10° i s s i g n i f i c a n t w i t h r e g a r d t o the t h e r m o p h i 1 i c i t y of M. laminosus, which grows w e l l at temperatures as h i g h as 55°C. At t h i s temperature, t h e a b s o r p t i o n o f t h e i / i t e g r a l pigment has decreased by o n l y 10%. The " m e l t i n g p o i n t " f o r PC from t h e m e s o p h i l i c S. p l a t e n s i s i s 52°C ( 1 1 ) , i . e . 15°C below t h a t o f t h e t h e r m o p h i 1 i c pigment.

The s u b u n i t s d i f f e r a l s o by t h e i r photochemical a c t i v i t i e s . PC i s a l i g h t - h a r v e s t i n g pigment and i t s major d e e x c i t a t i o n p r o c e s s i n the i s o l a t e d S t a t e i s f l u o r e s c e n c e . Another b i l i p r o t e i n , e.g. phytochrom, s e r v e s as " r e a c t i o n c e n t e r " pigment f o r photomorphogenesis i n h i g h e r p l a n t s . Here, a s t r u c t u r a l l y c l o s e l y r e l a t e d chromophore i s p h o t o c h e m i c a l l y h i g h l y a c t i v e . S e v e r a l groups have r e p o r t e d r e c e n t l y t h a t PC can undergo photochemical r e a c t i o n s , t o o , i f i t i s p a r t l y denatured by a v a r i e t y of r e a g e n t s (see r e f . 1 f o r l e a d i n g r e f e r e n c e s ) . T h i s process i s of p o t e n t i a l s i g n i f i c a n c e a l s o from a p h y s i o l o g i c a l p o i n t of view, because the r e a c t i o n s are s i m i l a r t o t h o s e o f so- c a l l e d phycochromes, which are thought be sensory p h o t o r e c e p t o r s i n b l u e - green a l g a . We have s t u d i e d the p h o t o c h e m i s t r y o f PC and i t s s u b u n i t s i n t h e presence o f i n c r e a s i n g amounts o f u r e a . The t y p i c a l response o f the sample i s shown i n f i g . 7. Upon i r r a d i a t i o n at 606nm, t h e r e i s a b l e a c h i n g of t h e long-wavelength maximum, which s a t u r a t e s r a p i d l y . T h i s b l e a c h i n g i s p a r t i a l l y r e v e r s e d upon i r r a d i a t i o n at 525nm, but about t h e f i v e - f o l d time i s r e q u i r e d f o r the back r e a c t i o n .

In i n t e g r a l PC, t h i s photochemical r e a c t i o n i s n e g l i g i b l e i n t h e absence o f urea. I t i n c r e a s e s up t o 4.5M of t h e d e n a t u r a n t , and then decreases a g a i n . The b e h a v i o r can be r a t i o n a l i z e d by a model, i n which t h e n a t i v e chromophore i s so t i g h t l y bound t o t h e P o l y p e p t i d e c h a i n , t h a t i t i s unable t o r e a c t p h o t o c h e m i c a l l y . A p a r t i a l d e n a t u r a t i o n o f the P o l y p e p t i d e l o o s e n s t h e s e i n t e r a c t i o n s s u f f i c i e n t l y t o a l l o w f o r photochemical r e a c t i o n s , whereas a complete u n f o l d i n q opens t h e Channel f o r e f f i c i e n t r a d i a t i o n l e s s d e a c t i v a t i o n and p r o h i b i t s p h o t o c h e m i s t r y as i s t y p i c a l f o r f r e e b i l i v e r d i n s . T h e ß - subuni' shows a s i m i l a r , but much more pronounced response, and t h e maximum r e a c t i o n i s again i n the same r e g i o n . Theoc- s u b u n i t i s , by c o n t r a s t , almost i n a c t i v e and shows an i r r e v e r s i b l e b l e a c h i n g at urea c o n c e n t r a t i o n s ^5M. T h i s i n d i c a t e s again a r a t h e r s p e c i f i c environment o f t h e d i f f e r e n t PC chromophores, w i t h t h e photochemical a c t i v i t y b e i n g m o s t l y l o c a t e d on t h e / 3 -s u b u n i t .

The PC System i s w e l l s u i t e d f o r a d e t a i l e d study o f s p e c i f i c non-c o v a l e n t chromophore p r o t e i n i n t e r a c t i o n s , which are c r u c i a l f o r the f u n t i o n o f many chromoproteins. In combination w i t h s e n s i t i v e and s e l e c t i v e s p e c t r o s c o p i c methods, and t h e r e c e n t advances i n s t r u c t u r e a n a l y s i s , a d e t a i l e d p i c t u r e of t h e s e i n t e r a c t i o n s i s expected i n the near f u t u r e .

Acknowledgements: T h i s work was supported by the Deutsche Forschungsgemei n s c h a f t .

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