Pulsed polarographic study of the oxygen-evolving system op photosynthesis in chlorella

A C T A U N I V E R S I T A T I S L O D Z I E N S I S FOLIA BIOCHIMICA ET BIOPHYSICA 2 , 1983

P e te r M a r ó ti , Gabor L a c z kó , A ndréa R i n g l e r , L á s z l ó S z a l a y

PULSED POLAROGRAPHIC STUDY

OF THE OXYGEN-EVOLVING SYSTEM OP PHOTOSYNTHESIS IN CHLORELLA

A p u l s e d p o l a r o g r a p h i o d e v io e was b u i l t f o r m ea s u rin g th e amount o f oxyg en e v o lv e d i n p h o t o s y n t h e t i c s y st em s i l l u m i n a t e d w i th l i g h t f l a s h e s . The o x y g e n - y i e l d se q u e n c e o f C h l o r e l l a s u s p e n s i o n s under 30 ¡¿s f l a s h e s a t a r e p e t i t i o n r a t e o f 2 Hz r e v e a l e d t h a t th e e x p e r i m e n ta l o s c i l l a t o r y p a t t e r n o f th e y i e l d v s . number o f f l a s h e s ca n be d e s c r i b e d t h e o r e t i c a l l y w it h th e l i n e a r f o u r - s t e p model (K ok’ s m odel) w it h th e e x c e p t i o n o f th e h e i g h t o f th e y i e l d a f t e r the f i r s t f l a s h . The d e v i a t i o n o f th e or y and e x p e rim e n t ca n be e x p l a i n e d by th e d a rk r e l a x a t i o n p r o c e s s e s i n th e o x i d i z e d s t a t e s ( S - s t a t e s ) o f th e w a t e r - s p l i t t i n g enzyme, s in o e n e a r l y h a l f o f th e r e a c t i o n c e n t e r s can be f ou nd i n S s t a -t e s h a v in g no p o s i t i v e a c c u m u l a te d o h a r g e s i n d a r k - a d a p t e d s y s t e m s . B a s e d upon th e homogeneous h y p o t h e s i s f o r the i n i t i a l d i s t r i b u t i o n o f the t r a n s i t i o n p r o b a b i l i t i e s among th e S - s t a t e s , th e a v e r a g e t r a n s i t i o n p r o b a b i l i t i e s a r e 1 0 , 75 and 15% f o r th e n o n r e a c t i v i t y ( m i s s e s ) , norm al r e a c t i -v i t y and d o u b l e - r e a c t i -v i t y ( d o u b l e - h i t s ) o f th e r e a c t i o n c e n t e r s , r e s p e c t i v e l y . I n t r o d u c t i o n G reen p l a n t p h o t o s y n t h e s i s p r i m a r i l y i m p l i e s o x i d a t i o n - r e - d u c t i o n r e a c t i o n s d r i v e n by two l i g h t r e a o t i o n s i n s e r i e s , i n two d i s t i n c t s y s t e m s , p h o tos y st em I (PS I ) and p h o to sy ste m I I (PS I I ) , r e s p e c t i v e l y ( P i g . 1 ) . The l i g h t i s a b s o r b e d by l i g h t - - h a r v e s t i n g p ig m en ts o f t h e s e s y s t e m s , and th e e x c i t a t i o n energy m i g r a t e s v i a e x o i t o n t r a n s f e r to th e r e a c t i o n c e n t e r s . The e x c i -t e d r e a c -t i o n o e n -t e r s ( s p e c i a l c h l o r o p h y l l - a f o r m s ) a r e a b l e to d o n a te an e l e c t r o n to th e p rimary a c c e p t o r m o le c u le (G o r X ) , r e -s u l t i n g i n th e prim ary c h a r g e -s e p a r a t i o n , i . e . the d i r e c t c o n v

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PS I and PS I I r e s p e c t i v e l y ; M i s th e w a te r s p l i t t i n g enzyme Z sch em a t f o t o s y n t e z y r o ś l i n z i e l o n y c h . S k ł a d n i k i ła ń c u c h a t r a n s -p o r t u e le k tro n ów u ło ż o n e w k o l e j n o ś c i i o h -p o t e n c j a łó w n orm a ln yc h . S t r z a ł k i pionowe w s k a z u ją fo to o h e m ic z n e r o z d z i e l e n i e ła d u nku sp o -wodowane p o c h ło n i ęc ie m kwantu hv w dwu f o t o s y s t e m a c h . N astęp n e

s t r z a ł k i w s k a z u ją p r z e n o s z e n i e p o jed y n c z eg o e l e k t r o n u . P7q0 i Pg80 s ą pierwotnymi d ono ram i, X i Q - pierwotnymi a k c e p to r a m i

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s i o n o f th e l i g h t en erg y i n t o c h e m ic a l e n e rg y ; PS I p r od u c e s a s t r o n g r e d u c i n g a g e n t (NADPH), p er f or m i n g th e f i x a t i o n o f C02 i n t o c a r b o h y d r a t e s and o x i d i z e s th e p oo l o f p l a s t o q u i n o n e mole- o u l e a . PS I I o x i d i z e a w a t e r , th e r eb y e v o l v i n g oxy gen and r e d u -c i n g th e p l a s t o q u i n o n e p o o l.

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to c a r b o h y d r a t e s v i a th e C a l v i n o y o l e h a s been c l e a r l y e l u c i d a t e d , th e pa th o f oxyg en h as t i l l to be e x p l a i n e d . A ltho ug h much e x p e r i m e n t a l e v i d e n c e h a s been accu-r m u la t ed a g a i n s t l i g h t - i n d u o e d w a t e r d e c o m p o s i t io n ( p h o t o l y s i s ) and i n f a v o u r o f th e c h a r g e -a o o u m u l a t i n g s y s te m on th e donor s i d e o f PS I I , th e mechanism o f 02 e v o l u t i o n from HgO i s s t i l l unknown 0

The p r e s e n t work d e a l s w it h the q u e s t i o n o f th e oxyg en l i b e -r a t i o n , and how th e te c h n i q u e o f p u l s e d p o l a ro g ra p h y and mathe-m a t i c a l a n a l y s i s o f th e t r a n s i t i o n p r o b a b i l i t i e s between d if f e re n t o x i d i z e d s t a t e s o f th e o x y g e n - e v o l v i n g enzyme h a s h e l p e d us to g e t a o oh er en t view o f th e p r o c e s s e s . Oxygen p o l a r o g r a p h The p r i n c i p l e o f th e p o l a r o g r a p h i c c e l l ( P i g . 2 ) i s s i m i l a r to t h a t d e s c r i b e d by J o 1 i o t and J o l i o t [ 3 ] . A d ro p o f a l g a l s u s p e n s i o n i s h om ogeneously s p r e a d on a p la tin u m e l e c t r o d e . In o r d e r to a t t a i n a s i n g l e l a y e r o f c e l l s , the C hl o- r e l l a c o n c e n t r a t i o n s h o u ld be a d j u s t e d . In a chamber o f 0 .1 5 mm ~ ” Q 1 t h i c k n e s s w i th an a l g a l c o n c e n t r a t i o n o f 4 . 5 * 1 0 c e l l ml , a m on ola yer i s formed on the e l e c t r o d e a f t e r th e o e l l s s e t t l e to th e b o tt om . T h is c an be ch ec ked v i s u a l l y ( t h e c o l o r o f th e e l e c t r o d e s h o u l d be f a i n t l y g r e e n ) , e l e c t r i c a l l y ( t h e e l e o t r o d e r e s -p on se s h o u l d be s t e a d y w i t h i n s e v e r a l

%

a f t e r 15 min o f s t a b i -l i z a t i o n ) , or m i c r o s c o p i c a -l -l y . The a -l g a -l s u s p e n s i o n i s c o v e re d w i t h a c e l l o p h a n e membrane. B etween t h i s membrane and a p l e x i -g l a s s c o v e r , th e c u l t u r e medium f l o w s . The medium c o n t a i n s 2 M KC1 t o e n s u r e s u f f i c i e n t e l e c t r i c c o n d u c t a n c e . A c o n s t a n t oxygen con-c e n t r a t i o n i n th e con-chamber i s e n s u r e d by an e q u i l i b r i u m between th e oxyg en u p t a k e by th e a l g a e and th e oxygen s u p p l y by th e f l o w in g b u f f e r . The g l a s s window on th e c ov er r e n d e r s p o s s i b l e i l l u -m i n a t io n w it h a Xe f l a s h tu be o f a b ou t 30 p s f l a s h d u r a t i o n and a r e p e t i t i o n r a t e o f 2 Hz .

The w i r in g d ia g r a m o f the a p p a r a t u s c o n s i s t s o f t h r e e main p a r t s ( P i g . 3 ) . The f i r s t p a r t e n s u r e s th e s u i t a b l e v o l t a g e ( - 0 . 7 V) f o r O ^ - d e p o s i t i o n by p o l a r i z i n g th e p la t in u m e l e o t r o d e w i th r e s p e c t t o th e Ag/AgCl e l e c t r o d e . T hi s i s a c h ie v e d w ith a

fla s h unit

F i g . 2 . D ev ic e f o r m e a su ri n g oxyg en ex o h a n g e. E x p la n a t i o n s e e i n t e x t

U r z ą d z e n i e do pomiaru wymiany t l e n o w e j . W y j a ś n i e n ia w t e k ś c i e

y C T p o flc T B O flJIH H 3M e p eH H fl OÓMOHa K HCJIO pOfla. 06T.HC H6HHH B T eKC TO

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F i g . 3- W iring d ia g r a m o f th e s y s t em f o r oxygen m ea sure m ents. E x p l a n a t i o n Se e i n t e x t

Schemat e l e k t r y c z n y u r z ą d z e n i a do pomiarów t l e n u . W y ja śn i e n i a w t e k ś c i e

3 jie K T p im e c K a a cx e w a y c i p o f ic T B a ą jih H3MepeHHił K H C Jiopo^ a.

co m m erc ia l i n t e g r a t e d o p e r a t i o n a l a m p l i f i e r ( F a i r c h i l d , 7 0 9 ) w it h f u l l f e e d - b a c k (1 a m p l i f i c a t i o n ) . From an e l e c t r i o a l p o i n t o f v ie w , th e i l l u m i n a t e d p o l a r o g r a p h i a c e l l oan be c o n s i d e r e d a cur-r e n t g e n e cur-r a t o cur-r . The s m a l l c u cur-r cur-r e n t th cur-roug h the m e a s u r in g d e v i o e i s tr a n s f o r m e d i n t o e l e c t r i o v o l t a g e v i a a c u r r e n t - t o - v o l t a g e c o n v e r t e r , an o p e r a t i o n a l a m p l i f i e r w ith low b i a s c u r r e n t ( F a i r - o h i l d , 7 4 0 , 2 nA ), i n the s ec o n d p a r t o f th e d i a g r a m . In o r d e r t o m easu re only the f l a s h r e s p o n s e o f th e p o l a r o g r a p h , i . e . to a v o i d th e d e t e c t i o n o f the s lo w e r v a r i a t i o n s o f th e oxygen c on-c e n t r a t i o n , a d i f f e r e n t i a l a m p l i f i e r ( F a i r on-c h i l d , 7 4 1 ) i s c onne c-t e d w ic-t h c-the p r e v i o u s s t e p . The s i g n a l i s f e d i n t o a m u l t ic h a n -n e l a -n a l y z e r (K F K I -s y s te m , NTA 5 1 2 ) where i t oan be s t o r e d . The s i g n a l - t o - n o i s e r a t i o oan be im pro ved by r e p e t i t i o n o f th e mea-su r e m e n t .

The u n i c e l l u l a r g r e e n a l g a e ( C h l o r e l l a p y r e n o i d o s a , C h ic k - - s t r a i n Emerson 3 ) were grown and h a r v e s t e d un d er s t a n d a r d c on- d i t i o n s : 4 mW/cm l i g h t f l u x , Tamiya c u l t u r e medium a t room t e m p e r a t u r e .

R e s u l t s

F i g u r e 4 d e m o n s t r a te s a t y p i c a l O ^ - e v o l u t io n p a t t e r n (Yn ) o f d a r k - a d a p t e d C h l o r e l l a c e l l s e x p o s e d to a s e r i e s o f 30 jxs l i g h t f l a s h e s s p a c e d 0 . 5 a a p a r t . S e v e r a l i n t e r e s t i n g o b s e r v a -t i o n s can be made. The most s t r i k i n g f e a t u r e i s th e o s c i l l a t o r y b e h a v i o r o f th e 0 2 y i e l d w i th a p e r i o d i c i t y o f f o u r which d i s a p -p e a r s a f t e r 3 or 4 p e r i o d s due to a s t r o n g d am pi ng . S i m i l a r r e -s u l t -s were o b t a i n e d f i r -s t by J o 1 i o t e t a l . [4 ] and c o r -r o b o -r a t e d l a t e -r by o t h e -r s ( s e e e . g . [ 5 ] ) . A f t e r th e f i r s t and th e sec on d f l a s h p r a c t i c a l l y a n e g l i g i b l e amount o f oxygen c o u l d be d e t e c t e d (Y 1 “ Y^ “ 0 ) , and th e f i r s t maximum a p p e a r s not a f t e r the 3rd f l a s h but a f t e r th e 4 th one. The f u r t h e r maxima oa n be found a f t e r th e 8 th and 1 2th f l a s h e s , and th e minima a f t e r th 6 th and 1 0 th f l a s h e s . The s t e a d y s t a t e 0^ e v o l u t i o n Ygg i s a t t a i n e d a t th e 14th or 1 5 th f l a s h .

In o rd e r t o c h ec k whether th e f l a s h energy i s h ig h enough to s a t u r a t e tho oxyg en e v o l u t i o n , th e ab ove m easu reme nts were c a r r i e d ou t und er th e same e x p e r i m e n t a l c o n d i t i o n s , but w ith a t t e n u

-P i g . 4 . Oxygen e v o l u t i o n i n d ar k a d a p t e d C h l o r e l l a o e l l a i l l u m i -n a t e d by a s e r i e s o f 30 p s s a t u r a t i n g f l a s h e s , a s f u n c t i o n o f f l a s h number. Yss means the s t e a d y s t a t e oxygen e v o l u t i o n und er f l a s h e s w i th d i f f e r e n t i n t e n s i t i e s ( i n a r b i t r a r y u n i t s ) : • -100%,

x -62%, 0-30% and a -141^

W ytwarzanie t l e n u p r z e z komórki C h l o r e l l a a d ap tow a ne do oiemno- ś c i , o ś w i e t l o n e s e r i ą 30 ^sekundowych b łysków n a s y c a j ą c y c h w z a -l e ż n o ś c i od -l i c z b y b ły skó w . Yss o z n a c z a s t a c j o n a r n y poziom wy-t w a r z a n i a wy-t l e n u w od p o w ie d z i na b ł y s k i o r ó ż n e j i n wy-t e n s y w n o ś c i (w

j e d n o s t k a c h umownych): • - 10

O/o,

x - 62$ , o - 30%, a - 14% O ó p a 3 0 B a H H e K H C ^ o p o ^ a b K J i e T K a x C h l o r e l l a n p H c n o c o f i j i e H H u x k T e M - H O T e , o o B e m e H H u x c e p e i i 3 0 Minc Hacuinaiomax B c n u r a e K , b i|)y H K i;H n U H C Ji a

BcnumeK. yss

0Ó03HauaeT cTaunoHapHoe 0<5pa30BaHne KHCJiopo^a Bcnum-

K aM H p a 3 H 0 S H H T eH C H BH O C TH I ip 0 H 3 B 0 J I b H H e eHHHHUH • - 1 0 0 % , X -6 2 % , o - 3 0 % h a - 1 4 %

a t e d f l a s h e n e r g y . The p a t t e r n d o e s n ot ch ang e s i g n i f i c a n t l y : th e p e r i o d i c i t y and th e l o c a t i o n s o f th e maxima and minima a r e p r e s e r v e d - e x c e p t f o r a s m a l l s h i f t o f th e t h i r d maximum i n th e o a s e o f the s m a l l e s t e n e rg y . The l i g h t - s a t u r a t i o n c u r v e o f th e s t e a d y - s t a t e oxygen e v o l u t i o n i n d i c a t e s t h a t th e h i g h e s t f l a s h i n t e n s i t y (100% i n r e l a t i v e u n i t s ) c an be s a t i s f a c t o r i l y c o n s i -d e r e -d a s s a t u r a t i n g .

The O g - y ie l d seq u en oe o f C h l o r e l l a grown und er sy n c h r o n i z e d (1 6 h l i g h t and 8 h d a r k ) and a s y n c h r o n i z e d c o n d i t i o n s was p r a c - t i o a l l y un c ha n g ed .

I t was found t h a t the r a t i o ^4 / ^ 3 was vo!ry s e n s i t i v e to th e d a r k - a d a p t a t i o n o f th e a l g a e . When th e sam ple was k e p t i n d a r k -n e s s f o r 30 s a f t e r s t e a d y f l a s h i n g , Y^ became e q u a l to, or even g r e a t e r tha n Y^ i f th e time f o r d a r k - a d a p t a t i o n was l e s s than 3 0s.

The O g -r es p o n s e d i d n ot f o l l o w the e x c i t i n g f l a s h p rom ptly , b u t had a d e l a y o f a b o u t 15 ms, i n g ood ag reem en t w it h the f i n -d i n g o f Z a n k e 1 [1 0 ] b a s e d on d e l a y e d f l u o r e s c e n c e measu-r e m e n t s .

D i s c u s s i o n

The k i n e t i c p r o p e rt y o f th e damped o s c i l l a t i o n can be s u c c e s s f u l l y e x p l a i n e d by a l i n e a r f o u r - s t e p m od e l, f i r s t i n t r o -d uc e-d by K o k e t a l . [6]. A c c o rd in g to t h i s m od el, each PS I I c e n t r e o c o u p i e s c y c l i c a l l y f i v e s t a t e s , S Q- S4> a s s o c i a t e d w it h th e a c c u m u l a ti o n o f f o u r p o s i t i v e c h a r g e s ; th e s u b s c r i p t s r e f e r t o th e number o f p o s i t i v e o x i d i z i n g e q u i v a l e n t s s t o r e d on the " S " - c o m p l e x . The t r a n s i t i o n from one B t a t e to th e n e x t r e q u i r e s th e a b s o r p t i o n o f one photon :

S i n c e the O g - r es p o n s e f o l l o w s 15 ms a f t e r th e f l a s h , one can a r g u e t h a t th e t r a n s i t i o n from one s t a t e t o an o th er o c c u r s v i a a m eta-s t a b l e eta-s t a t e w it h a r a t e c o n eta-s t a n t k

*

(1 5 ms)- 1 . To e x p l a i n th e damping i n th e o s c i l l a t o r y p a t t e r n , Kok assum ed t h a t even w ith s a t u r a t i n g f l a s h e s , a c e n te r h a s a s m a l l p r o b a b i l i t y o f n ot r e a c -t i n g ( " m i s s e s " ) . Depending on th e f l a s h d u r a t i o n , t h e r e i s a g i v e n p r o b a b i l i t y f o r a c e n t e r to r e a c t tw i c e (" d o u b l e h i t s " ) :

a ^ , (3^ and Yj_ d en ote the p r o b a b i l i t i e s o f m i s s e s , normal r e a c -t i o n s and d ou b le h i t s , r e s p e c t i v e l y . The q u a n t i t a t i v e a n a l y s i s o f Kok’ s model was f i r s t e l a b o r a t e d by D e l r i e u [ l , 2], and l a t e r d e v e l o p e d by L a v o r e l [ 7 ], L a v o r e l a nd l e m a s s o n [8] a nd T h i b a u l t [ 9 ] . I n th e q u a n t i

-t a -t i v e -t r e a -t m e n -t , Kok’ s model i s c o n s i d e r e d a M a r k o f f - p r o c e s s . The e f f e c t o f a f l a s h may be d e s c r i b e d a s th e o p e r a t i o n o f a ma- t r i x K on a s t a t e v e c t o r S'- r e s u l t i n g i n a new s t a t e v e c -t o r S< n + 1 >:

s (n +1) „ g s ( n ) . (1)

The components o f a r e d e f i n e d a s th e r e l a t i v e c o nc en tration s o f c e n t e r s i n s t a t e s

SQ ,

S ^ , S2 and a f t e r n f l a s h e s , and th e e le m en ts o f R a r e th e t r a n s i t i o n p r o b a b i l i t i e s between d i -f -f e r e n t s t a t e s : 0 0 * 2 Æ

3

(

3

0 a , 0 _{r 3} *0 0 l 0 4 0 0 _T

₁

₍

₃

₂ a 3 L e t u s f o c u s our a t t e n t i o n t o th e f o l l o w i n g t h r e e i m p or ta n t q u e s t i o n s . 1. How t o d ete rm in e th e t r a n s i t i o n e l e m e n t s ? 2. I s th e m a t rix -m o d el a p p r o p r i a t e o r n o t ? 3. What c an be s a i d a bo ut th e i n i t i a l S - s t a t e d i s t r i b u t i o n ? Ad 1 and 2 . U n f o r t u n a t e l y , th e d i r e c t d e t e r m i n a t i o n o f th e m a t r i x ele m en ts o f K i n Eq . (1) i s n ot p o s s i b l e . However, f o u r q u a n t i t i e s , w ith l i m i t e d c o n t e n t o f i n f o r m a t i o n , a r e r e a d i l y d e-r i v e d fe-rom th e e x p e e-r i m e n t a l d a t a (Y v a l u e s ) . The g e n e r a l r e c u r -r e n c e law d ed uced f-rom th e e i g e n v a l u e p-roblem o f th e m a t r i x K (Kx =

X x)

i s

V i - d, • V 3 + d 2 • - «3 • V i v - 0 . ( 3 )

where , e»2 , anc* ^4 a r e q u a s i s y m m e tr i c a l f u n c i t o n s o f the t r a n s i t i o n c o e f f i c i e n t s . A c c e p t i n g th e homogeneous h y p o t h e s i s (oijL = a , (3i = ¡3 and =

f),

they c an be e x p r e s s e d a s

d, = ¿ a ,

d2 = 6 « 2 - 2 * 2 ,

^

_ ( 4 )

dj

= 4ot3 - U | 2 + 4 ( 3 2

,

d u

=

a u -

¡34 + y4- 2 c i2y 2+ 4 a ( 32tf.

The d^ ( i = 1, 2 , 3» 4 ) v a l u e s can be d i r e c t l y c a l c u l a t e d from th e e x p e r i m e n t a l seq uen c e Yn : they a r e the s o l u t i o n s o f a s y s t e m o f f o u r l i n e a r e q u a t i o n s , where f i v e c o n s e c u t i v e Y^ v a -l u e s e n te r eac h e q u a t i o n a s c o e f f i c i e n t s . The c a l c u l a t i o n s can be c o n v e n i e n t ly p erform ed on a programmable p o c k e t c a l c u l a t o r (T e x a s I n s t r u m e n t s , SR 5 2) and the r e s u l t s a r e g i v e n i n T a b le 1. The numbers (n ) i n th e f i r s t column d eno te th e s t a r t i n g v a l u e s o f th e t r a i n s o f f l a s h e s a p p l i e d f o r c a l c u l a t i o n o f d^ i n Eq. ( 3 ) . The l a s t column c o n t a i n s the d i “ d 2 " d3 ”

dA

v a l u e s c a_ l c u l a t e d from d i f f e r e n t t r a i n s o f f l a s h e s . T h is e x p r e s s i o n s e r v e s a s a r e l a t i v e l y s im p l e method f o r c h e c k in g the v a l i d i t y o f th e m od el: i t s h o u l d a lw a y s be e q u a l to 1, a s

X

= 1 i s an e ig e n v a -l u e o f the m a t r ix K. A very s u r p r i s i n g o b s e r v a t i o n i s t h a t the f i r s t f l a s h a c t s i n a s p e c i f i c way n ot c o n s i s t e n t w i th th e model. The e f f e c t s o f th e f o l l o w i n g f l a s h e s (up to th e 8t h ) , however, a r e i n r e a s o n a b l y g ood a g reem en t w ith th e m odel. I f n S 8, we a g a i n g e t d e v i a t i o n from th e p r e d i c t i o n s o f Kok’ s m od e l; t h i s d i s c r e -p a n c y , however, ca n be a t t r i b u t e d to th e i n a c c u r a c y o f th e mea-surem en t i n t h i s domain, m a in ly due t o the s t r o n g d amping . The r e a s o n f o r th e d e v i a t i o n o f th e f i r s t f l a s h b e h a v i o r i s unknown. S i m i l a r c o n c l u s i o n s c an be drawn from th e a n a l y s e s o f L a v o- r e 1 [ 7 ] and T h i b a u l t [ 9].

The problem o f c a l c u l a t i o n o f th e t r a n s i t i o n p r o b a b i l i t i e s from the d^ v a l u e s ( E q . ( 4 ) ) a r i s e s from th e n o n - l i n e a r and o v e rd e t e r m i n e d p r o p e r t i e s o f th e e q u a t i o n s (e ve n a n o r m a l i z a -t i o n r e l a -t i o n , a + (3 +

y

= 1, s h o u l d be c o n n e c t ed w ith the s e t o f e q u a t i o n s und er ( E q . ( 4 ) ) .

A

g r a p h i c a l optimum c a l c u l a t i o n was c a r r i e d ou t to o b t a i n th e a v e r a g e t r a n s i t i o n p r o b a b i l i -t i e s :

a

= 0 . 1 0 , ¡3 = 0 . 7 5 and f = 0 . 1 5 . Wot a l l th e q ua nta o f l i g h t a b s o r b e d by th e sy s tem a d van c e th e S - s t a t e s f o r w a r d . There i s an i n e f f i c i e n c y o f a p p r o x i m a t e l y 10$ , which c o r r e s p o n d f a i r l y w e l l t o the v a l u e o b ta i n e d by o t h e r s [7] w ith s i m u l a t i o n methods. Ad 3 . As r e g a r d s the S - s t a t e d i s t r i b u t i o n o f th e d a r k a d a p -t e d c e l l s we r e f e r -t o P i g . 4: no O g -r e sp o n s e s c o u l d be d e t e c t e d

T a b l e 1 V a lu e s o f S d e r i v e d from Eq . ( 3 ) u s i n g t r a i n s

o f f l a s h e s s t a r t i n g w ith th e n - t h f l a s h

W a rto ś c i d^ S otrzym ane z rów n a n ia (3 ) d l a s e r i i błysków p o c z y n a ją c od n - t e g o b ły s k u

BejimiHHbi d^ S nojiyueHHwe o yp aB H em w ( 3 )

Aj ih p a^ a BcnaneK Ha^HHaioinerocH c n-ioii bc id ju ik h

n _dl d2 i

dj

d i _{d1_d2+d3~d4} 2 , 3 , 4 , 5, 0 -4 6 6 - 0 -0 4 7 0 -1 2 8 - 0 - 3 4 7 0 -9 8 8 3 , 4 , 5, 6, 0 -4 2 2 - 0 - 0 8 3 0 -0 97 -0 3 9 0 0 -9 93 4 , 5 , 6, 7 , 0 -3 7 6 - 0 -1 1 7 0 -0 9 4 -0 - 4 1 0 0 -9 9 8 2 , 4 , 6, 7 , 0 -3 9 3 -0 - 0 2 4 0 -2 2 8 - 0 - 3 4 3 0 -9 8 8 © , 2, '3 , 4 , 0 -4 42 -0 -3 6 8 0 -1 8 5 - 0 - 4 4 3 1-437 © , 3, 5 , 7 , 0 -0 8 8 - 0 - 7 4 9 0 -1 1 3 -0 - 2 4 8 1-19 8 © , 4 , 5 , 6, 0 -1 2 7 - 0 - 5 2 2 0 -1 5 0 -0 -0 4 1 0 -8 3 9 5 , 6, 7 , ( 8 ) , 0 -5 47 - 0 - 1 1 5 0 -1 5 9 - 0 -0 9 2 0 -8 93 6, 7 , ® , © , 0-561 - 0 - 1 5 9 0 - 2 4 6 - 0 - 2 4 0 1-205 7 , © , © , © , 0 -4 4 4 - 0 - 0 6 8 0 - 1 5 0 _{- 0 -4 5 7 1-117} 2 , 5 , 7 , © , 0 -3 1 9 - 0 - 1 7 4 0 -2 6 6 - 0 - 1 1 8 0 -8 76 a f t e r the f i r s t and th e s ec o n d f l a s h e s : Y1 = Y2 = 0 , ( 5 ) a d d i t i o n a l l y

* 3

=

(1

- a ) 2 (1 - s £ o ) ) ( 6 ) and Y4 = (1 - a ) 3 S < °> + 3 a( 1 - a ) 2 (1 - s £ ° > ) ( 7 ) may be d e r i v e d from Eq . ( 1 ) u s i n g th e homogeneous h y p o t h e s i s . Frcra

S u b s t i t u t i n g th e e x p e r i m e n t a l d a t a (Y^/Y^ = 0 . 9 ) . we o b t a i n 0 . 4 7 and 0 . 5 3 f o r s £ 0 ' and f o r S ^ ° \ r e s p e c t i v e l y ( s i 0 ^ and

/ _ N O I ^

S^ ' a r e e q u a l t o 0 a c o o r d i n g t o Eq . ( 5 ) ) . T h is would mean t h a t p r a c t i c a l l y h a l f o f th e o e n t e r s a r e i n s t a t e S Q and h a l f i n S^ i n a d a r k - a d a p t e d s a m p le . The r e l a t i v e l y h ig h r a t i o S ()/ S1 we obtained c a n be a t t r i b u t e d e i t h e r to a th o rou g h d a r k - a d a p t a t i o n , or to some s p e c i a l i t y o f th e o x y g e n - e v o l v i n g s ys tem o f our C h l o r e l l a s t r a i n , or more s p e c i f i c a l l y t o th e r a p i d r e l a x a t i o n p r o c e s s e s o f th e h i g h e r S s t a t e s . Acknowled gements The a u t h o r s a r e i n d e b t e d to J . L a v o r e l f o r p r o v i d i n g many u s e f u l s u g g e s t i o n s i n c o n s t r u c t i n g th e p o l a r o g r a p h i o d e v io e . REFERENCES [ 1 ] D e l r i e u M. J . , Photochem. P h o t o b i o l . 2 0 , 357 ( 1 9 7 4 ) . [ 2 ] D e l r i e u M. J . , Photochem. P h o t o b i o l . 2 0 , 441 ( 1 9 7 4 ) . [ 3 ] J o l i o t P. , J o l i o t A ., B i oc h im . B i o p h y s . A cta 1 53 , 625 ( 1 9 6 8 ) . [ 4 ] J o l i o t P. , B a r b i e r i G . , C h a b a u d R . , Photochem. P h o t o b i o l . 1£, 309 ( 1 9 6 9 ) . [ 5 ] F o r b u s h B. , K o k B. , Mc G l ó i n M., P hoto-chem. P h o t o b i o l . 14 , 307 ( 1 9 7 1 ) . [6] K o k B. , F o r b u s h B. , Mc G l o i n M., fh otoohem. P h o t o b i o l . n , 457 ( 1 9 7 0 ) . [ 7 ] L a v 0 r e 1 J . , J . t h e o r . B i o l . 5 7 , 171 ( 1 9 7 6 ) . [8] L a v o r e l J . , L e m a s s o n C . , B ioo h im . B i o p h y s . A c ta 4 3 0 , 501 ( 1 9 7 6 ) . [ 9 ] T h i b a u 1 t P . , J . t h e o r . B i o l . 7 3 , 271 ( 1 9 7 8 ) . [1 0 ] Z a n k e l K. L . , B io c h im . B i o p h y s . A g ta 2 4 5 , 373 ( 1 9 7 1 ) . I n s t i t u t e o f B i o p h y s i c s J d z s e f A t t i l a U n i v e r s i t y S z e g e d , Hungary

P e t e r M a r ó t i , Gabor L a c z kó , A nd res R i n g l e r , L a s z l ó S z a la y BADANIA UKŁADU

FOTOSYNTETYCZNEJ GENERACJI TLENU METODĄ POLAROGRAFII IMPULSOWEJ

Skonatruowano u r z ą d z e n i e do b a d a n ia metodą p o l a r o g r a f i i im-p u l so w e j i l o ś c i t l e n u w ytw arz an ej w u k ła d a c h f o t o s y n t e t y c z n y c h o- s w i e t l a n y o h b ły sk a m i ś w i a t ł a . O k re ś lon o sekw e na ję w y d a jn o ś o i t l e -nowej z a w i e s in C h l o r e l l a o ś w i e t l a n y c h b ły sk a m i 30-psekundowymi

powtarz anymi z c z ę s t o ś c i ą 2 Hz i s t w ie r d z o n o , że se k w e n c ja t a k a może być o p i s a n a t e o r e t y c z n i e z a pomocą l in i o w e g o modelu c z t e r o - eta pow ego (model K o k a ), k t ór y j e d n a k n i e d a j e w ła śo iw y c h p r z e w i-dywań w y d a jn o ś c i t l e n u po pierwszym b ł y s k u . O d c h y len i e t e o r i i od ek sp ery m en tu s p o ł e c z n e g o może być w y j a ś n io n e p r o c e sa m i r e l a k s a c j i oiem nej w s t a n a c h u t l e n i o n y c h ( s t a n a c h S ) enzymu r o z s z c z e p i a j ą c e -go wodę, gdyż s t w i e r d z o n o , że p raw ie połowa centrów r e a k c j i o s t a n a c h S0 w u k ła d a o h ad aptowanych do c ie m n o śo i n i e p o s i a d a na-gromad zonych ładunków d o d a t n i c h . W o p a r o i u o h i p o t e z ę homogennego r o z k ła d u począ tkow ego praw dop od obień stw p r z e j ś ć pomięd zy s ta n a m i S otrzymano n a s t ę p u j ą c e p r z e c i ę t n e p ra wd o po d ob ieństw a p r z e j ś ć po-międ zy c e n tr a m i r e a k o j i : 10% d l a n i e r e a k t y w n o ś c i ( c h y b i e n i a ) , 75 # d l a n orm a ln ej r e a k t y w n o ś c i i 15% d l a podwójnej r e a k ty w n o ś c i (p o -dwójne t r a f i e n i a ) .

FleTep MapoTH, Ta6op JIhuko, AH,npeac PHHrjiep, JIhcjio Cajiaii nyJIbC-n0JIHP0rPAi>H4ECKHE HCCJIEflOBAHMH

<tOTOCHHTETiWECKOil CHCTEMH 0EPA30BAHHH KHCJIOPOHA Y CHLORELLA

I l o c T p o H J iH n y j i b c - n o j i a p o r p a i H M e c K o e y c T p o i ł c T B O , ą ; i a H 3M e p e H H H ko j iH M e c TB a K H c a o p o ^ a o O p a a o B a H H o r o b (JjOTOCHHTeTHHeoKoii c n c T e M e H JIJIIO M H H H p OBaH H oii BCIIŁJIIIKaMH C B e T a . n o c a e .H O B a T e J I b H O O T b Bh IX O A a K U C —

jiopo.ua cacn eH 3HH C h l o r e l l a n p H 30 m m c Bcm jinK ax, n o B T o p a e m i x o M a c T O T o tł 2 T 3 m o k h o on ncaib T e o p e i a u e c K H a p a n o M o m n Ji H H e iłH o ił « ł e m - p e c T y n e H u a T H o M M o ^ e jiH ( M O ,ą ejib Kona) K 0 T 0 p a a , o ^ H a K O , H e o x B a i t i B a e i

B e ^ H ^ H H H B H X O fla n o c j i e n e p B o f t B c n u m K H . O r iu i O H e H H e t e o p H H o t 3 K c n e - p H M e w T a m o * h o o f i i a c H H T b n p o u e c c a u H T e M H O B o ft p e j i H K c a u a H b O K H C Jie H - h u x c o c T o a H n a x (S - c o c T o a H a a ) 3 H 3 n u a p a c n e r m a i o m e r o B O f l y , T a K K a K , i i o m t h n o ji o B H H y i ;e H T p o B p e a K i jH H m o x h o H a ftT H b S - c o c T o a H H a x b c h c - T e M a x a f l a n T H p o B a H H h i x b T e M H O ie 6 e s H a K o r u ie H H h ix n o j i o x i i T e J i b H u x 3 a - p a ^ o B . I l o j i a r a a r o i i o r e H H o e H a H a j i b H o e p a c n p e , n e j i e H H e B e p o a T H O c i e i t n e - pexo,na n o S - c o c T o a H H f l x , n a r a j i n e p e ^ H e e b e p o a T h o c t h n e p e x o , n a : 1096 Ę J ia H 0 p M a J Ib H 0 2 p e a K T H B H O C T H A 15% fl J ia U B o S h o K peaKTH B H O C T H UBOtt— Hue y n a p u u e H T p o B p e a K i y iH .