Function of Quinones and Quinonoids in Green-Plant Photosystem I

10 Function of Quinones and Quinonoids in Green-Plant Photosystem I Downloaded by COLUMBIA UNIV on March 20, 2013 | http://pubs.acs.org Publication Date: May 5, 1991 | doi: 10.1021/ba-1991-0228.ch010

Reaction Center Masayo Iwaki and Shigeru Itoh Division of Bioenergetics, National Institute for Basic Biology, 38 Nishigonaka, Myodaijicyo, Okazaki 444, Japan

In photosystem I photosynthetic reaction centers of green plants (spinach), the constituent phylloquinone (2-methyl-3-phytyl-l ,4naphthoquinone, vitamin K ) that functions as the secondary electron acceptor (A ) was replaced by various quinones and quinonoid compounds. Most of the quinones and quinonoids tested replaced the function of A and suppressed the charge recombination between the reduced primary electron acceptor chlorophyll a (A ) and the oxidized primary donor (P700 ). Redox midpoint potential (E ) values of the semiquinone quinone couple of reconstituted quinones in situ in the photosystem I reaction center were estimated to be about 300 mV more negative than those in dimethylformamide (DMF). This result is different from the situation in the purple bacterial reaction center, in which reconstituted quinones showed E values about 400 mV more positive than those in DMF. The variation indicates that there are different quinone environments in these two types of reaction centers. 1

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G T R E E N P L A N T S H A V E T W O T Y P E S of photosynthetic reaction centers (RC) f u n c t i o n i n g i n series: photosystems I a n d I I . T h e p h o t o s y s t e m (PS) I I R C r e c e n t l y isolated (I) seems to have a structure essentially s i m i l a r to that of p u r p l e bacterial R C , whose tertiary structure has b e e n d e t e r m i n e d b y X ray crystallography (2, 3). I n b o t h P S II a n d p u r p l e bacterial R C s , four to six c h l o r o p h y l l s , two p h e o p h y t i n s , a n d two quinones are e m b e d d e d o n two

0065-2393/91/0228-0163$06.00/0 © 1991 American Chemical Society

In Electron Transfer in Inorganic, Organic, and Biological Systems; Bolton, J., et al.; Advances in Chemistry; American Chemical Society: Washington, DC, 1991.

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E T IN INORGANIC, ORGANIC, A N D BIOLOGICAL SYSTEMS

p o l y p e p t i d e s o f about 3 0 - k D a m o l e c u l a r mass (1-4). A m i n o a c i d sequences of the p o l y p e p t i d e s of P S I I a n d p u r p l e bacterial R C s show o n l y 3 0 % h o m o l o g y to each other, b u t h i g h h o m o l o g y i n essential moieties c o n s t i t u t i n g b i n d i n g sites for the prosthetic groups (2). O n the other h a n d , the core part of the P S I R C c o m p l e x is c o m p o s e d of large p o l y p e p t i d e s of about 8 0 - k D a m o l e c u l a r mass, m o r e t h a n 50 c h l o r o p h y l l a m o l e c u l e s [reaction c e n t e r c h l o r o p h y l l (P700), p r i m a r y e l e c t r o n acceptor c h l o r o p h y l l ( A ) , a n d m o s t l y antenna c h l o r o p h y l l s ; about t w o p h y l loquinone (2-methyl-3-phytyl-l,4-naphthoquinone) molecules; a n d a 4 F e - 4 S c e n t e r ( F , t e r t i a r y e l e c t r o n acceptor)] (4-6). A s m a l l s u b u n i t p o l y p e p t i d e of 9 - k D a m o l e c u l a r mass attached to the large-core p o l y p e p t i d e s containsj o t h e r 4 F e - 4 S centers F a n d F , w h i c h accept electrons f r o m F (7) ( F i g u r e 0

Downloaded by COLUMBIA UNIV on March 20, 2013 | http://pubs.acs.org Publication Date: May 5, 1991 | doi: 10.1021/ba-1991-0228.ch010

x

A

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x

i). T h e a m i n o a c i d sequences of the P S I p o l y p e p t i d e s show almost no h o m o l o g y to those o f P S I I - p u r p l e - b a c t e r i a l - t y p e R C s (5). T h i s , as w e l l as the difference i n prosthetic groups, makes it difficult to consider the e v o l u t i o n a r y r e l a t i o n s h i p b e t w e e n these two types of R C complexes. R e c e n t

-i.sr Ρ700

111 X

-1.0

m Φ

> > Φ

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Ξ

. NADP

"5 >

Ε

UJ 0.0

beck rmectlon 0.5 < -3

-12 time

(log(sec))

Figure 1. Electron-transfer pathway in PS I RC. Key: P700, ground state; P700*, lowest singlet excited state; P700 , triplet state of PS I primary electron donor chlorophyll; Fd, ferredoxin. Inset figure shows structure of phylloqui none (Αι) reproduced after refs. 2 and 3. Reaction times are ob tained from refs. 4, 6, and 31-35. Continued on next page. T


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studies, h o w e v e r , indicate that the quinones f u n c t i o n as the secondary elec t r o n acceptor, b o t h i n P S I (4, 6, 8-20) [some arguments still r e m a i n (21, 22)] a n d P S I I - p u r p l e - b a c t e r i a l - t y p e R C s (23, 24). T h u s , c o m p a r i s o n of q u i none reactions i n these different types of R C s s h o u l d give n e w i n f o r m a t i o n c o n c e r n i n g the electron-transfer m e c h a n i s m a n d the e v o l u t i o n a r y r e l a t i o n ship of p h o t o s y n t h e t i c R C s . T h e energy levels a n d reaction times o f electron-transfer steps i n P S I R C a n d p u r p l e bacterial (Rhodobacter sphaeroides) R C are c o m p a r e d i n F i g u r e 1. E l e c t r o n transfer i n p u r p l e b a c t e r i a l R C is c h a r a c t e r i z e d b y two u b i q u i n o n e s (2,3-dimethoxy-5-methyl-6-(prenyl) - l , 4 - b e n z o q u i n o n e ) f u n c t i o n i n g i n series as the secondary ( Q ) a n d tertiary ( Q ) e l e c t r o n acceptors (23). R o t h of these quinones interact w i t h an F e atom (2, 3). I n some p u r p l e bacteria such as Rhodopseudomonas viridis, Q is m e n a q u i n o n e [ 2 - m e t h y l 3 - ( p r e n y l ) - l , 4 - n a p h t h o q u i n o n e ] . T h i s situation is almost the same i n P S I I R C , w h i c h has plastoquinone [ 2 , 3 - d i m e t h y l - 5 - ( p r e n y l ) - l , 4 - b e n z o q u i n o n e ] as Q a n d Q (4, 24). T h e large energy gap b e t w e e n the p r i m a r y e l e c t r o n acceptor p h e o p h y t i n a n d Q seems to m a t c h the reorganization e n e r g y of 10


A

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— P870 -12 time

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Figure 1. Continued. Electron-transfer pathway in purple bacterial RC. Key: P870, ground state; P870*, lowest singlet excited state; P870 , triplet state of the primary electron donor bacteriochlorophyll aimer; Bph, bacteriopheophytin. Inset figure shows structure of QA site ofRps. viridis RC reproduced after refs. 2 and 3. Reaction times are obtained from refs. 2, 3, 23, and 25. T


166


the reaction (25) a n d to partially c o n t r i b u t e to t h e activationless b e h a v i o r o f the Q reaction i n this type of R C . T h e q u i n o n e - e x t r a c t i o n - r e c o n s t i t u t i o n studies have b e e n extensively c a r r i e d out i n isolated b a c t e r i a l R C s to solve the m e c h a n i s m o f efficient e l e c t r o n transfer i n R C (25-30). O n t h e o t h e r h a n d , t h e P S I R C contains p h y l l o q u i n o n e as t h e secondary e l e c t r o n acceptor ( A ) , w h i c h undergoes a o n e - e l e c t r o n redox step, does not protonate, a n d mediates e l e c t r o n transfer b e t w e e n A a n d F (8-20). R e a c t i o n o f A however, has not b e e n w e l l - c h a r a c t e r i z e d yet. W e first r e p o r t e d t h e extraction a n d r e c o n s t i t u t i o n m e t h o d of this p h y l l o q u i n o n e i n s p i n a c h P S I R C (15) a n d d e m o n s t r a t e d t h e c h e m i c a l i d e n t i t y o f A as p h y l l o q u i n o n e ( I I , 1 5 , 16). T h i s identification was later c o n f i r m e d i n cyanobacterial m e m b r a n e s b y another extraction m e t h o d (9). O n e m o l ecule o f p h y l l o q u i n o n e is r e q u i r e d to regain t h e A f u n c t i o n ( 9 , 1 6 ) , a n d t h e role o f o t h e r w e a k l y b o u n d p h y l l o q u i n o n e is not k n o w n yet. T h e f u n c t i o n a l p h y l l o q u i n o n e b i n d i n g site, designated as t h e Q$ site, also b i n d s h e r b i c i d e s (17, 18) a n d various artificial q u i n o n e s (19, 20). T h e e n e r g y d i a g r a m o f t h e P S I R C is c h a r a c t e r i z e d b y a s m a l l energy gap for each electron-transfer step, 10-fold faster rate for t h e q u i n o n e reactions, a n d e x t r e m e l y l o w redox potentials for c o m p o n e n t s f u n c t i o n i n g o n t h e r e d u c i n g side (31-35). T h e P S I R C thus p r o v i d e s a n e x p e r i m e n t a l system to test a n d refine t h e e l e c t r o n transfer m e c h a n i s m p r o p o s e d i n t h e p u r p l e bacterial R C (25). T h e reason that P S I R C r e q u i r e s p h y l l o q u i n o n e b u t not plastoquinone a n d h o w i t distinguishes t h e f o r m e r f r o m t h e latter, w h i c h exists i n 10 times l a r g e r amounts i n t h e same t h y l a k o i d m e m b r a n e (36), are also o f interest. W e h e r e s u m m a r i z e a n d e x t e n d the q u i n o n e - r e c o n s t i t u t i o n study i n P S I R C . E x t r a c t i o n o f t h e o r i g i n a l p h y l l o q u i n o n e from P S I R C enhances t h e charge r e c o m b i n a t i o n b e t w e e n A ~ a n d P 7 0 0 [ w h i c h p r o d u c e s e i t h e r t h e t r i p l e t P 7 0 0 state (9-11) o r e x c i t e d singlet P 7 0 0 * , w h i c h p r o d u c e s d e l a y e d fluorescence (II)] a n d results i n a decrease i n t h e a m o u n t o f P 7 0 0 detectable i n t h e m i c r o s e c o n d - t o - m i l l i s e c o n d t i m e range. Various q u i n o n e s o r q u i n o n o i d c o m p o u n d s , i n t r o d u c e d i n place o f p h y l l o q u i n o n e i n t o P S I R C , s u p press t h e charge r e c o m b i n a t i o n a n d mediate t h e e l e c t r o n transfer b e t w e e n A ~ a n d i r o n - s u l f u r centers. A

x

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Materials and Methods L y o p h i l i z e d photosystem I particles, w h i c h w e r e o b t a i n e d b y t r e a t i n g s p i n ach chloroplasts w i t h d i g i t o n i n a n d contain almost no photosystem I I , w e r e t w i c e extracted w i t h a 1:1 m i x t u r e of d r i e d a n d water-saturated d i e t h y l e t h e r , f o l l o w e d b y one extraction w i t h d r y d i e t h y l ether; this p r o c e d u r e c o m p l e t e l y r e m o v e d t h e t w o p h y l l o q u i n o n e molecules c o n t a i n e d i n t h e P S I R C (15). T h e p h y l l o q u i n o n e - d e p l e t e d particles w e r e also d e p l e t e d o f about 8 5 % o f the a n t e n n a c h l o r o p h y l l c o m p l e m e n t a n d a l l carotenoids (15-20, 37, 38). H o w e v e r , P 7 0 0 , A , F , F , a n d F w e r e almost unaffected (15-20). T h e 0

x

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extracted particles w e r e d i s p e r s e d i n 50 m M glycine O H buffer ( p H 10) a n d t h e n d i l u t e d i n 50 m M Tris C l [Tris(hydroxymethyl)aminomethane C l ] buffer ( p H 7.5) c o n t a i n i n g 0 . 3 % (ν/ν) T r i t o n X - 1 0 0 (polyethylene g l y c o l p-isooct y l p h e n y l ether). A f t e r 30 m i n of i n c u b a t i o n , grayish u n d i s s o l v e d materials w e r e e l i m i n a t e d b y centrifugation. T h e clear supernatant l i q u i d was d i l u t e d about 50 times w i t h 50 m M Tris C l buffer ( p H 7.5), c o n t a i n i n g 3 0 % (v/v) g l y c e r o l to give a final P 7 0 0 concentration of 0.25 μ Μ , w h i c h was u s e d for r e c o n s t i t u t i o n a n d measurements. A l l extraction p r o c e d u r e steps w e r e d o n e b e l o w 4 °C. T o reconstitute quinones or q u i n o n o i d s , the suspension of the extracted PS I particles was i n c u b a t e d for a day at 0 °C i n the dark w i t h various q u i n o n e s or q u i n o n o i d s d i s s o l v e d i n e t h y l alcohol, h e x y l alcohol, or d i m e t h y l sulfoxide. T h e q u i n o n e - r e c o n s t i t u t e d R C s w e r e stable w h e n stored b e l o w 10 °C. M o r e t h a n 7 0 % o f the P S I R C s w e r e r e c o n s t i t u t e d b y this m e t h o d (15-20). N e x t , 10 m M ascorbate a n d 0.1 m M d i c h l o r o i n d o p h e n o l c o u p l e w e r e a d d e d to the reaction m e d i u m to p r o v i d e seconds-time-scale r e d u c t i o n o f the s m a l l a m o u n t o f P 7 0 0 not r a p i d l y r e d u c e d b y i n t r i n s i c c o m p o n e n t s . Q u i n o n e s a n d q u i n o n o i d s u s e d as candidates to reconstitute A w e r e 2,6d i a m i n o - 9 , 1 0 - a n t h r a q u i n o n e ; fluoren-9-one; anthrone [9(10H)-anthracenone] ( A l d r i c h , M i l w a u k e e ) ; 9,10-anthraquinone; m e n a d i o n e ( 2 - m e t h y l - l , 4 naphthoquinone) (Katayama, O s a k a , Japan); p h y l l o q u i n o n e ( 2 - m e t h y l - 3 - p h y t y l - 1 , 4 - n a p h t h o q u i n o n e ) ; m e n a q u i n o n e - 4 [2-methyl-3-(prenyl) - 1 , 4 - n a p h t h o q u i n o n e ] (Sigma, St. L o u i s ) ; 2 , 3 , 5 , 6 - t e t r a m e t h y l - l , 4 - b e n z o q u i n o n e ; 1,4n a p h t h o q u i n o n e ; l - n i t r o - 9 , 1 0 - a n t h r a q u i n o n e (Tokyokasei, T o k y o , Japan); 1amino-9,10-anthraquinone; 2,3-dichloro-l,4-naphthoquinone; 2-methyl9,10-anthraquinone (Wako, O s a k a , Japan); 2 , 3 - d i m e t h y l - l , 4 - n a p h t h o q u i n o n e ; u b i q u i n o n e - 1 0 ; a n d plastoquinone-9. +

x

4

T h e activity o f the r e c o n s t i t u t e d P S I particles was assayed b y m e a s u r i n g the f l a s h - i n d u c e d absorption change of P 7 0 0 at 695 n m i n a s p l i t - b e a m spectrophotometer (17) at 6 °C. T h e i n t e n s i t y of the actinic flash (532 n m , 10 ns F W H M , 0.7 H z ) from a f r e q u e n c y - d o u b l e d N d - Y A G laser ( Q u a n t a Ray, D C R - 2 - 1 0 ) was attenuated to excite about a q u a r t e r o f the R C s to a v o i d sample damage. Signals w e r e averaged b e t w e e n 32 a n d 128 scans, as r e q u i r e d i n each case.

Results Kinetics of Flash-Induced P700 in PS I RCs Containing Various Quinones and the Redox Properties of Quinones. I n P S I R C s i n +

w h i c h native p h y l l o q u i n o n e is extracted w i t h d i e t h y l e t h e r , o n l y a s m a l l a m o u n t of P 7 0 0 was detected i n the m i c r o s e c o n d - t o - m i l l i s e c o n d t i m e range after excitation w i t h a laser flash (trace of n o additions i n F i g u r e 2). T h i s scarcity is d u e to the r a p i d r e t u r n of an e l e c t r o n o n A ~ to P 7 0 0 (charge r e c o m b i n a t i o n , see F i g u r e 1) w i t h a characteristic t o f 35 ns (9-12). T h i s +

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168


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SHE)

Figure 2. Flash-induced absorption change of P700 in the presence of various naphtho- and anthraquinones in phylloquinone-depleted PS I particles measured at 695 nm. The concentration of added quinone is shown in parentheses. Redox properties of quinones are represented by their E values, referred to the standard hydrogen electrode, measured in DMF (25, 27, 28). NQ and AQ represent 1,4-naphthoquinone and 9,10-anthraquinone, respectively. The figure is redrawn from Figure 1 in ref. 20, with new data added. lf2

reaction produces a s m a l l a m o u n t of t r i p l e t state P 7 0 0 , w h i c h decays w i t h a t o f 80 [this t is l o n g e r t h a n that of a t y p i c a l f e w - m i c r o s e c o n d decay s e e n i n intact P S I R C , because o f the co-extraction o f carotenoids (10)] a n d also is d e t e c t e d at 695 n m ( F i g u r e 2). A s m a l l a m o u n t of P 7 0 0 is p r o d u c e d a n d decays s l o w l y (t = 3 0 - 1 0 0 ms) t h r o u g h r e d u c t i o n e i t h e r b y a n a d d e d a s c o r b a t e - d i c h l o r o i n d o p h e n o l couple or by partially p h o t o r e d u c e d i r o n - s u l f u r centers. T

m

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T h e f l a s h - i n d u c e d P 7 0 0 extent i n the m i c r o s e c o n d - t o - m i l l i s e c o n d range was i n c r e a s e d w h e n various n a p h t h o - a n d a n t h r a q u i n o n e s , i n c l u d i n g those k n o w n as i n h i b i t o r s i n P S II (39), w e r e s u b s t i t u t e d for the i n t r i n s i c p h y l l o +


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q u i n o n e i n t h e P S I R C ( F i g u r e 2). T h i s increase indicates that a l l o f these q u i n o n e s can oxidize A " at a rate r a p i d e n o u g h to c o m p e t e w i t h the charge r e c o m b i n a t i o n w i t h P700 . B e n z o q u i n o n e s w e r e rather p o o r i n this activity except for d u r o q u i n o n e (19). T h e concentration o f each q u i n o n e s h o w n i n F i g u r e 2 was adjusted to p r o v i d e t h e m a x i m u m l e v e l of r e c o n s t i t u t i o n . 0

+

K i n e t i c patterns o f the flash-induced P 7 0 0 after the flash, o n the o t h e r h a n d , s e e m e d to v a r y according to t h e redox p o t e n t i a l o f the r e c o n s t i t u t e d q u i n o n e g i v e n b y the polarographically m e a s u r e d p o t e n t i a l Ε value o f the s e m i q u i n o n e * / q u i n o n e c o u p l e i n d i m e t h y l f o r m a m i d e ( D M F ) . T h i s result suggests that the m i d p o i n t p o t e n t i a l value o f q u i n o n e at the Q site (E i n situ w i t h respect to those o f A a n d F ) is c r u c i a l i n d e t e r m i n i n g the reaction kinetics o f the q u i n o n e as r e c e n t l y shown (19) a n d that t h e E i n situ is r e l a t e d to E in D M F . +

1 / 2

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( j )


0

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A group o f quinones i n d u c e d a h i g h extent o f flash-oxidized P 7 0 0 f o l l o w e d b y a slow decay (t = 3 0 - 1 0 0 ms), as r e p o r t e d for p h y l l o q u i n o n e (16). T h e slow decay t i m e indicates that the P 7 0 0 is n o t r e r e d u c e d b y A ~ or A " , b u t b y ( F / F ) ~ o r b y an external reductant ( F i g u r e 1) (16, 19). P h y l l o q u i n o n e a n d most anthraquinones (except l - n i t r o - 9 , 1 0 - a n t h r a q u i n o n e a n d 2,6-diamino-9,10-anthraquinone) b e l o n g to this g r o u p . T h e s e q u i n o n e s appear to m e d i a t e e l e c t r o n flow f r o m A ~ to F a n d t h e n to F / F because the r e d u c t i o n o f F / F c a n actually b e detected at 4 0 5 n m , w h i c h is a n isosbestic w a v e l e n g t h of P 7 0 0 / P 7 0 0 ( F i g u r e 3) (i.e., t h e y fully r e c o n s t i t u t e d the f u n c t i o n o f A ) . T h e E values o f these quinones i n situ i n the Q j site are, thus, expected to b e i n t e r m e d i a t e b e t w e e n those of A a n d F (19, 20) [i.e., b e t w e e n - 1 0 0 0 (40) a n d - 7 2 0 m V (41)]. +

m

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x

W i t h a q u i n o n e that has a n e x t r e m e l y l o w p o t e n t i a l , 2 , 6 - d i a m i n o - 9 , 1 0 a n t h r a q u i n o n e , b o t h the i n i t i a l extent o f flash-induced P 7 0 0 a n d the extent +

of t h e slow-decay phase w e r e s m a l l e v e n at t h e saturated c o n c e n t r a t i o n (50 μ Μ ) . T h i s q u i n o n e m a y b e i n c o m p l e t e l y r e d u c e d b y A ~ , p r o b a b l y b e cause t h e E o f the q u i n o n e is comparable to o r l o w e r than that o f A . Q u i n o n e s that have h i g h e r potentials [such as 2 , 3 - d i c h l o r o - l , 4 - n a p h t h o q u i n o n e , 1,4-naphthoquinone, 2 - m e t h y l - l , 4 - n a p h t h o q u i n o n e ( m e n a d i one), a n d l - n i t r o - 9 , 1 0 - a n t h r a q u i n o n e ] also p r o m o t e a h i g h i n i t i a l extent o f the flash-induced P 7 0 0 , a result i n d i c a t i n g that t h e y efficiently accept e l e c trons f r o m A " . H o w e v e r , the decay o f P700 was c o m p o s e d o f fast (ti/2 — 0 . 1 - 1 ms) a n d slow (t = 3 0 - 1 0 0 ms) phases. T h e extent o f the latter decreased w i t h the raising o f the E value o f the tested q u i n o n e . T h i s result m a y b e e x p l a i n e d i f these quinones have E values comparable w i t h o r m o r e positive than that o f F so that t h e y cannot fully reduce F a n d therefore, F / F . I n this case, i t is expected that t h e r e m a i n i n g s e m i q u i n o n e * " t h e n w i l l d i r e c t l y r e d u c e P 7 0 0 as the fast phase. T h i s m e c h a n i s m is t y p i f i e d i n the q u i n o n e w i t h the most positive £ value, 2,3-dichloro-l,4-naphthoqui none. It p r o d u c e d a h i g h extent of flash-induced P 7 0 0 , w h i c h decays r a p 0

m

0

+

+

0

m

1 / 2

m

x

A

x

B

+

1 / 2

+



170


E

1

/

2

in D M F

(V

v.s.

SHE)

Figure 3. A: Dependence of the relative extent of the quinone-induced increase of the slow-decay phase of P700 [reduction of P700 + by (F IF )~] on the Ej/2 value of added quinone in DMF. B: Dependence of the relative extent of (FJF )~ measured by the absorption change at 405 nm on the E value of quinone in DMF. Solid lines represent one-electron Nernsfs theoretical curve calculated with -0.4-V E,„ values. The flash-induced kinetics ofP700 or (F / F )~ were measured as in Figure 2 at the optimal concentration of quinones and plotted against E value. Quinones used were 1, 2,3-dichloro-l,4naphthoquinone; 2, l-nitro-9,10-anthraquinone; 3,1,4-naphthoquinone; 4, 2methyl-1,4-naphthoquinone (menadione); 5, 2,3-dimethyl-l,4-naphthoquinone; 6, menaquinone-4; 7, phylloquinone; 8, l-chloro-9,10-anthraquinone; 9, 9,10-anthraquinone; 10, 2-methyl-9,10-anthraquinone; 11, l-amino-9,10-anthraquinone; 12, 2-amino-9,10-anthraquinone; 13, l,2-diamino-9,10-anthraquinone; and 14, 2,6-diamino-9,10-anthraquinone. +

B

A

B

1/2

+

B

m


A

10.

IWAKI & ITOH


171

Quinonoids

i d l y (t = 100 μβ), b u t almost no increase of the slow decay phase ( F i g ure 2). T h e s e m i q u i n o n e * " / q u i n o n e c o u p l e of this q u i n o n e i n the site is e s t i m a t e d to have an £ v a l u e too positive to r e d u c e F a n d so the s e m i quinone*" f o r m e d w i l l r e d u c e P 7 0 0 d i r e c t l y w i t h a fast rate, as o b s e r v e d . m

m

x

+

D e p e n d e n c e o f P 7 0 0 k i n e t i c s o n the redox p r o p e r t y of q u i n o n e s (as s h o w n i n F i g u r e 2) was f u r t h e r analyzed w i t h 14 q u i n o n e s ( F i g u r e 3). T h e i n i t i a l h e i g h t o f the f l a s h - i n d u c e d P 7 0 0 was almost i n d e p e n d e n t of the redox p r o p e r t y o f q u i n o n e s except at e x t r e m e l y negative potentials, as seen i n F i g u r e 2. T h i s i n d e p e n d e n c e indicates that, for R C s w i t h r e c o n s t i t u t e d q u i n o n e s , the efficiency a n d rate of acceptance o f an e l e c t r o n f r o m A ~ is sufficiently h i g h to c o m p e t e against the c h a r g e - r e c o m b i n a t i o n reaction b e t w e e n P 7 0 0 a n d A ~ for most o f q u i n o n e s tested, except for those w i t h the e x t r e m e l y l o w redox potentials. O n the other h a n d , decay kinetics of P 7 0 0 v a r i e d d e p e n d i n g o n q u i n o n e species. F i g u r e 3 A indicates the relative extent of the slow-decay phase w i t h respect to the i n i t i a l extent o f P 7 0 0 f o r m a t i o n . Because the slow phase represents the extent of P 7 0 0 that is not r e r e d u c e d b y the flash-reduced q u i n o n e , this extent represents the a b i l i t y of the q u i n o n e to r e d u c e F . +


0

+

0

+

+

+

x

If the reaction rate b e t w e e n q u i n o n e a n d F is assumed to b e fast e n o u g h to a l l o w e q u i l i b r i u m b e t w e e n t h e m before reaction o f F w i t h F / F or P 7 0 0 , this c u r v e m a y b e u s e d to represent the redox titration o f F b y u s i n g q u i n o n e s o f different redox p o t e n t i a l . T h e c u r v e indicates that the quinone with an Ε value o f - 0 . 4 V i n D M F gives the h a l f m a x i m a l increase o f the slow phase. T h i s increase suggests that q u i n o n e s w i t h this E value show almost the same E v a l u e as F [E = - 0 . 7 2 V (41)] i n the P S I R C p r o t e i n . T h e points w i t h different q u i n o n e s are fitted w i t h the t h e o r e t i c a l o n e - e l e c t r o n N e r n s t i a n c u r v e . T h i s correspondence suggests that £ values o f q u i n o n e s i n situ i n P S I R C are shifted about 0.3 V to the negative side from those i n D M F . F r o m this figure the £ i n situ v a l u e of p h y l l o q u i n o n e (closed circles i n F i g u r e 3) can b e e s t i m a t e d to be 0.1 V m o r e negative t h a n that o f F (i.e., - 0 . 8 2 V ) . x

x

A

+

B

x

m

m

m

x

m

m

m

x

T h e a m o u n t of r e d u c e d F / F was d i r e c t l y m e a s u r e d b y m o n i t o r i n g their flash-induced absorption change at an isosbestic w a v e l e n g t h o f P 7 0 0 + / P 7 0 0 (405 nm). D i f f e r e n c e spectra of F / F o b s e r v e d i n the P S I R C r e c o n s t i t u t e d w i t h l o w - p o t e n t i a l quinones (not shown) w e r e s i m i l a r to that o b s e r v e d i n the p h y l l o q u i n o n e - r e c o n s t i t u t e d P S I R C (16). T h e i n i t i a l a m o u n t of flash-induced ( F / F ) ~ d e p e n d e d o n the £ v a l u e of q u i n o n e ( F i g u r e 3 B ) . T h i s d e p e n d e n c e confirms the i n d i r e c t e s t i m a t i o n f r o m the reaction kinetics o f P 7 0 0 s h o w n i n F i g u r e 3 A . T h e p l o t is also w e l l - f i t t e d w i t h the theoretical c u r v e w i t h a - 0 . 4 - V £ value. T h e l i n e a r r e l a t i o n s h i p b e t w e e n the E value at the Q

+

d

Table I. Dissociation Constant and Binding F r e e Energy Between Q u i n o n e Quinonoid and PS I Quinone Binding Q Site é

Compounds

log[K

0

Quinones" Duroquinone 1,4-Naphthoquinone 2- M ethyl-1,4-naphthoquinone 2,3-Dimethyl-1,4-naphthoquinone Phylloquinone Menaquinone-4 9,10-Anthraquinone Quinonoids Fluoren-9-one Anthrone Inhibitors Atrazine 4,7-Dimethyl-o-phenanthroline Antimycin A Myxothiazole

Function of Quinones and Quinonoids in Green-Plant Photosystem I

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