Antennas and Reaction Centers of Photosynthetic …Antennas and Reaction Centers of Photosynthetic...

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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