CHARACTERIZATION OF A PHOTOSYSTEM II REACTION CENTER

483

Photosynthesis Research 10." 4 8 3 - 4 8 8 (1986) © Martinus N i j h o f f Publishers, Dordrecht - Printed in the Netherlands

C H A R A C T E R I Z A T I O N OF A P H O T O S Y S T E M II R E A C T I O N C E N T E R C O M P L E X I S O L A T E D BY E X P O S U R E OF PSII M E M B R A N E S TOIA N O N - I O N I C D E T E R G E N T AND HIGH C O N C E N T R A T I O N S OF NaCI DEMETRIOS F. GHANOTAKIS and CHARLES F. YOCUM D i v i s i o n of B i o l o g i c a l sciences, Ann Arbor, MI 4 8 1 0 9 - 1 0 4 8 USA

The U n i v e r s i t y

of M i c h i g a n ,

ABSTRACT A h i g h l y r e s o l v e d PSII r e a c t i o n center complex has been p r e p a r e d by e x p o s u r e of PSII m e m b r a n e s to the d e t e r g e n t octylglucopyranoside at e l e v a t e d ionic strengths; o x y g e n e v o l u t i o n a c t i v i t y is about i,OOO ~ m o l e s O g / h r / m g Ch] in the p r e s e n c e of CaCl 9. A Mn q u a n t i t a t i o n and ~ k i n e t i c study of Z, the donor to P'8-' r e v e a l s that on a Chl basis this new p r e p a r a t i o n s h o w s b a ~ a l m o s t f o u r - f o l d e n r i c h m e n t in Mn and the e l e c t r o n t r a n s p o r t c o m p o n e n t s of PSII. i.

INTRODUCTION

R e s e a r c h in several l a b o r a t o r i e s (3, 9, iO, 13) has p r o d u c e d h i g h l y r e f i n e d p r e p a r a t i o n s of a PSII "core" c o m p l e x which, a l t h o u g h p h o t o c h e m i c a l l y active, does not p o s s e s s the c a p a c i t y to e v o l v e oxygen. M o r e recent work (6, ii, 12) has p r o d u c e d isolated o x y g e n e v o l v i n g r e a c t i o n center c o m p l e x e s from PSII m e m b r a n e s by d i s s o c i a t i n g these m e m b r a n e s w i t h nonionic d e t e r g e n t s such as d i g i t o n i n or o c t y l g l u c o p y r a n o s i d e . The p r o d u c t of these p r o c e d u r e s is a p u r i f i e d PSII r e a c t i o n center c o m p l e x d e p l e t e d of the water s o l u b l e 17 and 23 kDa p o l y p e p t i d e s , and these p r e p a r a t i o n s t h e r e f o r e r e q u i r e the _ p r e s e n c e of n o n - p h y s i o l o g i c a l c o n c e n t r a t i o n s of Caand C1 for o p t i m u m o x y g e n e v o l u t i o n a c t i v i t y (5, 6, ii, 12). In this c o m m u n i c a t i o n we r e p o r t results from a new m e t h o d (5) for i s o l a t i o n of a PSII r e a c t i o n center complex. A manganese q u a n t i t a t i o n and a s p e c t r o s c o p i c study of Z, the p r i m a r y donor to P6B-' r e v e a l s a f o u r - f o l d e n r i c h m e n t , on a Chl basis, in £n~ M n - c o n t e n t as well as the e l e c t r o n t r a n s p o r t c o m p o n e n t s of PSII. i D e d i c a t e d to the m e m o r y of W a r r e n Butler, w h o s e r e s e a r c h p r o v i d e d new insights and ideas about the s t r u c t u r e and f u n c t i o n of PSII. Abbreviations: BZ, benzidine; Chl, chlorophyll; DCBQ, 2,5dichloro-p-benzoquinone; EPR, e l e c t r o n p a r a m a g n e t i c resonance; LHC, light h a r v e s t i n g complex; OGP, l - O - n - o c t y l - b D-glucopyranoside; PSII, P h o t o s y s t e m II; R.C.C, r e a c t i o n c e n t e r complex; Tris, 2 - a m i n o - 2 - ( h y d r o x y m e t h y l ) - l - 3 propanediol.

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484 2.

MATERIALS

AND M E T H O D S

PSII m e m b r a n e s , p r e p a r e d as in (4), w e r e t r e a t e d as in (5) to p r o d u c e the h i g h l y a c t i v e o x y g e n e v o l v i n g PSII r e a c t i o n c e n t e r complex. Tris t r e a t e d s y s t e m s w e r e p r e p a r e d by e x p o s u r e of PSII p r e p a r a t i o n s to 0 . 8 M Tris plus imM EDTA. O x y g e n e v o l u t i o n a c t i v i t y was m e a s u r e d w i t h a C l a r k - t y p e oxygen electrode. Gel e l e c t r o p h o r e s i s was c a r r i e d out as in (2) w i t h the m o d i f i c a t i o n s d e s c r i b e d in the figure c a p t i o n s . E P R s p e c t r o s c o p y was c a r r i e d out on a B r u k e r E R - 2 O O D s p e c t r o m e t e r o p e r a t e d at X - b a n d and i n t e r f a c e d to a N i c o l e t 1180 c o m p u t e r . I n s t r u m e n t m o d i f i c a t i o n s , as well as the f l a s h lamp c i r c u i t r y and the p r o t o c o l for signal averaged, f l a s h i n g - l i g h t k i n e t i c e x p e r i m e n t s , are d e s c r i b e d in ref. 15. 3.

RESULTS

Fig. i, lane 2 shows that the new PSII R e a c t i o n C e n t e r C o m p l e x (R.C.C.) c o n s i s t s of the h y d r o p h o b i c p o l y p e p t i d e s o b s e r v e d in "core" c o m p l e x p r e p a r a t i o n s from PSII, along with two other h y d r o p h o b i c p o l y p e p t i d e s (with m o l e c u l a r w e i g h t s w h i c h we e s t i m a t e to be about 20 and 28 kDa), and the hydrop h i l i c 33 kDa species; the c o m p l e x has b e e n d e p l e t e d of the w a t e r - s o l u b l e 17 and 23 kDa p o l y p e p t i d e s and thus the+pres e n c e of n o n - p h y s i o l o g i c a l c o n c e n t r a t i o n s of both Ca and C1 are r e q u i r e d for o x y g e n e v o l u t i o n a c t i v i t y (see ref. 5). In c o n t r a s t to the p r e p a r a t i o n d e s c r i b e d in (6), the PSII r e a c t i o n c e n t e r c o m p l e x i s o l a t e d by our p r o c e d u r e is very 3a c t i v e w h e n DCBQ is p r e s e n t as an e l e c t r o n acceptor; Fe(CN) 6 a p p e a r s to be a m o r e e f f e c t i v e a c c e p t o r in this new p r e p a r a tion w h e n c o m p a r e d to c o n t r o l PSII m e m b r a n e s , but is not as e f f e c t i v e as DCBQ (data not shown). As s h o w n in T a b l e I, an a n a l y s i s of the m a n g a n e s e cont e n t of this new p r e p a r a t i o n r e v e a l s an a l m o s t f o u r - f o l d enr i c h m e n t of m a n g a n e s e on a Chl basis. Since a series of exp e r i m e n t s c a r r i e d out by M a t s u d a and B u t l e r (8), has c l e a r l y d e m o n s t r a t e d that high p o t e n t i a l Cyt b ~ q r e v e a l s the structural i n t e g r i t y of the p h o t o s y n t h e t i c ~ b r a n e and that disr u p t i o n of that i n t e g r i t y c a u s e s C y t ~ 5 ~ to be m o d i f i e d to lower p o t e n t i a l forms, we s t u d i e d th~ ~ t a t e of C y t K ~ 9 in this n e w p r e p a r a t i o n by use of EPR s p e c t r o s c o p y . We hag~ found t h a t a s i g n i f i c a n t a m o u n t of high p o t e n t i a l C y t ~ q has shifted to lower p o t e n t i a l form(s) in the Complex; eg~fi though add i t i o n of CaCI. r e c o n s t i t u t e d o x y g e n e v o l u t i o n a c t i v i t y , it d i d not r e s t o r ~ C y t 5 5 9 to its h i g h p o t e n t i a l form(s) (data not shown). W h e n a h i g h e r ionic s t r e n g t h (IM NaCI) was used d u r i n g e x p o s u r e of PSII m e m b r a n e s to OGP, the PSII r e a c t i o n center c o m p l e x w h i c h r e s u l t e d was also d e p l e t e d of the w a t e r s o l u b l e 33 kDa p o l y p e p t i d e (Fig. 2) as well as m o s t of the m a n g a n e s e (Table I). This p r e p a r a t i o n s h o w s v e r y l i t t l e o x y g e n evolution a c t i v i t y (data not shown). This m o d i f i e d i s o l a t i o n p r o c e d u r e (35 m M OGP + IM NaCI) was also a p p l i e d in Trist r e a t e d PSII m e m b r a n e s ; the p o l y p e p t i d e c o n t e n t of the rea c t i o n c e n t e r c o m p l e x w h i c h r e s u l t s from such a t r e a t m e n t

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485

is s h o w n in Fig. 2. The k i n e t i c b e h a v i o r of Z + in this TrisPSII R.C.C. was ~ t u d i e d by EPR and was c o m p a r e d to the kinetic b e h a v i o r of Z o b s e r v e d in T r i s - P S l l m e m b r a n e s . As shown Fig. i. Gel e l e c t r o p h o r e s i s patterns of (i) u n t r e a t e d PSII m e m branes; (2) O~ e v o l v i n g r e a c t i o n center c o m p l e x p ~ e p a r e d by e x p o s u r e of PSII m e m b r a n e s to 35 m M OGP + 0.5 M NaCI. A 15% a c r y l a m i d e r e s o l v i n g gel was used and 6M urea was p r e s e n t in the gel. (*) d e n o t e s w a t e r soluble extrinsic polypeptides.

Table

II:

Preparation

Mn C o n t e n t

of v a r i o u s

PSII

Preparations

Mn-Content

PSII M e m b r a n e s PSII R e a c t i o n C e n t e r C o m p l e x (35:1.O) T r i s - P S II M e m b r a n e s T r i s - P S l I R e a c t i o n C e n t e r C o m p l e x (35:~.0) PS II R e a c t i o n C e n t e r C o m p l e x (35:0.5)a. b.

(atoms/250

Chl)

4a 6.0 0.6 0 14.8

See refs. 1 and 4 (x:y); x = c o n c e n t r a t i o n of OGP (mM), y = c o n c e n t r a t i o n of NaCl (M) used for the i s o l a t i o n of the R . C . C .

in Fig. 3B, Z + d e c a y s r e l a t i v e l y s l o w l y in T r i s - t r e a t e d PSII R . C . C . , b u t its d e c a y is d r a m a t i c a l l y a c c e l e r a t e d u p o n a d d i tion of an e x o g e n o u s d o n o r such as b e n z i d i n e . A calculation of the s e c o n d order rate cons~ant, f o r . b e n z i d i n e from the d a t a of Fig. 3B g i v e s k = 6.0 X I0- M - £ . s - l ; this rate c o n s t a n t is h i g h e r c ~ m p a ~ e d ~o that o b s e r v e d in Tris PSII m e m b r a n e s (k = i.O x i0 v M- .s- ).+ A c o m p a r i s o n of the a m p l i t u d e of the k i n e t i c t r a c e s of Z in the T r i s - P S I I r e a c t i o n c e n t e r c o m p l e x (Fig. 3B) w i t h that o b s e r v e d in T r i s - P S i I m e m b r a n e s (Fig.~ 3A) r e v e a l s that, on a Chl basis, an e n r i c h m e n t of Z a p p r o a c h i n g f o u r - f o l d has o c c u r r e d in the T r i s - P S I i R.C.C.

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2A.

2A.

2B.

2B.

Gel e l e c t r o p h o r e s i s p a t t e r n s of (1) u n t r e a t e d PSII m e m b ranes; (2) the r e a c t i o n c e n t e r c o m p l e x p r e p a r e d by e x p o s u r e of PSII m e m b r a n e s to 35mM 0GP plus IM NaCI ( F r a c t i o n B); (3) F r a c t i o n A o b t a i n e d f r o m t r e a t m e n t in 2; (4) the r e a c t i o n c e n t e r c o m p l e x p r e p a r e d by e x p o s u r e of T r i s - t r e a t e d m e m b r a n e s to 35mM OGP p l u s IM NaCI ( F r a c t i o n A); (5) F r a c t i o n A o b t a i n e d f r o m t r e a t m e n t in 4. A c o m b i n a t i o n of a 12% (upper part) and 18% (lower part) a c r y l a m i d e r e s o l v i n g gel was used and 6M urea was p r e s e n t in the gel. Gel e l e c t r o p h o r e s i s p a t t e r n s of (i) the r e a c t i o n c e n t e r c o m p l e x p r e p a r e d by e x p o s u r e of T r i s - P S I I (2) F r a c t i o n A o b t a i n e d from t r e a t m e n t in i; (3) T r i s - t r e a t e d PSII membranes; (4) u n t r e a t e d PSII m e m b r a n e s . Conditions as in Fig. i.

487

[341]

A.Tris PSII

B.Tris

PSII

R.C. Complex

ii]5pM BZ

iii]lOpM BZ

1 !

200 ms

3.

K i n e t i c t r a n s i e n t s of Z + at room t e m p e r a t u r e in A) Trist r e a t e d PSII m e m b r a n e s (4.16 mg Chl/ml) and B) the PSII r e a c t i o n c e n t e r c o m p l e x p r e p a r e d from Tris t r e a t e d PSII m e m b r a n e s (0.56 mg Chl/ml); i) 0 ~ M Bz, ii) 5 ~ M Bz and iii) i0 ~ M BZ. I n s t r u m e n t conditions: time c o n s t a n t 1 ~s, flash rate 0.25 Hz, m o d u l a t i o n 4 Gpp, g a i n i0 x i0 and n u m b e r of scans a v e r a g e d 190 (for A) and 200. (for B). A m i x t u r e of 3 mM Fe(CN) 6 - and 3 mM s e r v e d as an a r t i f i c i a l a c c e p t o r system.

Fe(CN)2-

4.

DISCUSSION

A l t h o u g h the i s o l a t i o n of P h o t o s y s t e m II m e m b r a n e s (i, 7, 14) has a d v a n c e d k n o w l e d g e of the p o l y p e p t i d e c o m p o s i t i o n of PSII, further r e s o l u t i o n of the PSII s y s t e m is n e c e s s a r y for e f f e c t i v e s p e c t r o s c o p i c studies of both the p r i m a r y r e a c t i o n s and the water c l e a v a g e process. Recently, a series of t e c h n i q u e s have b e e n r e p o r t e d for isolation of o x y g e n e v o l v i n g r e a c t i o n c e n t e r c o m p l e x e s (5, 6, ii, 12). As shown in Table I, the a c t i v e PSII r e a c t i o n center c o m p l e x is e n r i c h e d in Mn by a factor of four, and it is t h e r e f o r e a v e r y a t t r a c t i v e s y s t e m for a s p e c t r o s c o p i c c h a r a c t e r i z a t i o n of the M n - c o m p l e x . Low t e m p e r a t u r e ESR and EXAFS e x p e r i m e n t s , which r e q u i r e very c o n c e n t r a t e d samples, wi~l b e n e f i t by use of this new system. The kinetic study of Z shown in Fig. 3 also d e m o n s t r a t e s a 3 . 7 - f o l d e n r i c h m e n t in a c o m p o n e n t of the PSII e l e c t r o n t r a n s p o r t system. In a d d i t i o n to its use in s p e c t r o s c o p i c studies, the reduced number of polyp e p t i d e s p r e s e n t in the PSII r e a c t i o n center c o m p l e x will f a c i l i t a t e further e l u c i d a t i o n of the structural role of the v a r i o u s p o l y p e p t i d e s as well as their r e l a t i o n s h i p to sites of c a t a l y t i c a c t i v i t y .

488 Acknowledgment: This r e s e a r c h was s u p p o r t e d by g r a n t s the N a t i o n a l S c i e n c e F o u n d a t i o n (PCM82-14240) and the C o m p e t i t i v e R e s e a r c h O f f i c e of USDA (G-82-I127).

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