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Inhibition by sulfonamides of the photoreductive step in spinach chloroplast photosynthesis
BIOCHIMIE, 1972, 54, 1205-1211.
Inhibition by sulfonamides of the photoreductive step in spinach chloroplast photosynthesis. C. FRANqOIS. Laboratoire de B i o c h i m i e , Institut L~,)n Fredericq, Universitd de Liege, 17 Place Delconr, 4000 Liege, Belgiqae. (23/5/1972). Summary. - - Spinach chloroplasts, their grana fraction and their photosystem I and II fractions have been prepared. The photoreduetion of water by the non-cyclic photosystem preparations has been shown to be inhibited by several sulfonamides ; the sensitivity of the photoreduction has no relation with the inhibition of the carbonic anhydrase activity in lysed chloroplasts. A sulfonamide-sensitive unit thus exists in the electron transport sequence of the lamellae and seems to be different from the DCMU-sensitive site. Spinach ferredoxin, ferredoxin-NADP reductase and plastocyanin were prepared and used to reconstitute the non-cyclic electr6n transport sequence. This system proved to be sensitive to other sulfonamides and arguments in favor of this second sensitive site being the ferredoxin - - ferredoxin-NADP reduetase - - membrane complex are discussed. It is concluded that carbonic anbydrase may not be involved in the sensitivity of photosynthesis to sulfonamides.
it was r e c e n t l y r e p o r t e d [1, 2] that Diamox, a w i d e l y - u s e d d i u r e t i c d r u g and a p o w e r f u l i n h i b i tor in animals, of the e n z y m e c a r b o n i c a n h y d r a s e (carbonate h y d r o - l y a s e , E.N. 4.2.1.1), was also an i n h i b i t o r of p h o t o s y n t h e s i s in isolated s p i n a c h chloroplasts ; o t h e r s u l f o n a m i d e s w e r e c o n s i d e r e d as i n a c t i v e ; azide and nitrite are also p o w e r f u l i n h i b i t o r s . E v e r s o n and Slack (3) h a v e f u r t h e r rep o r t e d that c a r b o n i c a n h y d r a s e is l o c a t e d in the chloroplasts of the Calvin-cycle plants and is i n h i bited by azide, nitrite, Diamox, the i n h i b i t i o n of the latter t w o b e i n g r e l i e v e d by 5 mM H,CO-.~. Judging f r o m the effect of these 3 molecules on both c a r b o n i c a n h y d r a s e a c t i v i t y and on p h o t o s y n t h e sis, E v e r s o n [41, and H a t c h and Slack [5] have p r o p o s e d that c a r b o n i c a n h y d r a s e is a key unit in the s e q u e n c e m a k i n g up the p h o t o s y n t h e t i c Abbreviations : DCMU : 3-(3',4'-dichlorophenyl)-l,l-dimethylurea (Dupont). DPIP : 2,6-diehlorophenol-indophenol (Merck). DPC : 1,5-diphenylcarbazide (BDH). Diamox : acetazolamide : 2-acetylamino-l,3,4-thiadiazole-5-snlfonamide (Lederle). Navidrex : 3-eyclopentylmethyl-6-chloro-7-sulfamyl3,4-dihydro-l,2,4-benzothiadiazine (Ciba). C1 13850 : NS-t-butyl-2-acetylamino-l,3,4-thiadiazole-5snlfonamide (Lederle). C1 11366 : 2-benzenesulfonamide-l,3,4-thiadiazole-5sulfonamide (Lederle). Furosemide : 4-chloro-N (2-furylmethyl)-5-snlfamylanthranilie acid (Hoechst). Esmarin : 3-dichlormethyl-3,4-dihydro-6-chloro-7-sulfa myl-2-H- 1,2,4-benzothiadiazine-l,l-dioxide (Merck). Methazolamide : 2-acetylimino-2,3-dihydro-3-methyl1,3,4-thiadiazole-5- sulfonamide (Lederle).
m a c h i n e r y and that it is likely to be l o c a t e d w h e r e CO 2 or HCO-~ is d e l i v e r e d to the e n z y m e u n i t c a r r y i n g the c a r b o x y l a t i o n step in the d a r k reactions of the Calvin- and the C4-dicarboxylie a c i d p a t h w a y s . The latter r e l a t i o n s h i p recalls the relationship, generally a d m i t t e d to be p r e s e n t in animals, of the i n h i b i t i o n of c a r b o n i c a n h y d r a s e by D i a m o x and s i m i l a r m o l e c u l e s on the one h a n d , w i t h the i n t e r f e r e n c e of these m o l e c u l e s on the ion t r a n s l o c a t i o n s (in the k i d n e y , stomach, c i l i a r y processes, salt gland in birds, fish gills) on the o t h e r hand. T h e latter r e l a t i o n s h i p w a s r e c e n t l y quest i o n e d by us. We h a d s h o w n that s u l f o n a m i d e s depress the r e s p i r a t i o n in w h o l e tissue t h r o u g h a d i r e c t i n h i b i t i o n of the o x y g e n c o n s u m p t i o n in m i t o c h o n d r i a isolated f r o m several tissues of the dog a n d the r a t [6], suggesting that a n o t h e r sulfon a m i d e - s e n s i t i v e site is p r e s e n t in the energyp r o d u c i n g m a c h i n e r y . Consequently w e w e r e led to cast doubts on the m e c h a n i s m offered by E v e r son [41 on h o w s u l f o n a m i d e s i n h i b i t p h o t o s y n t h e sis, and to investigate w h e t h e r , in analogy w i t h t h e i r action on the o x i d a t i v e m e t a b o l i s m of mitoc h o n d r i a , s u l f o n a m i d e s m i g h t i n t e r f e r e w i t h the r e d u c t i v e , or light r e a c t i o n s of the chloroplasts. This r e p o r t p r e s e n t s e v i d e n c e that some sulfonamides i n h i b i t the n o n c y c l i c e l e c t r o n t r a n s p o r t in s p i n a c h c h l o r o p l a s t s and c h l o r o p l a s t f r a g m e n t s at a site situated at the r e d u c i n g side of cytoc h r o m e b559, w h i l e o t h e r s u l f o n a m i d e m o l e c u l e s i n t e r f e r e w i t h i n the f e r r e d o x i n - NADP sequence.
C. Franfois.
1206 MATERIALS AND METHODS.
Sucrose ( b i o c h e m i c a l grade) and D.PI,P w e r e p u r c h a s e d f r o m Merck, NADP and NADPH f r o m Sigma, DPC f r o m BDH and DCMU f r o m Dupont. The sulfonamides, listed u n d e r Abbreviations, w e r e all k i n d l y p r o v i d e d to us by the C o m p a n i e s mentioned. S p i n a c h chloroplasts w e r e p r e p a r e d f r o m comulercially available fresh leaves by the m e t h o d of W a l k e r and Hill [7]. A c r u d e f e r r e d o x i n e x t r a c t was p r e p a r e d a c c o r d i n g to San P i e t r o [8] ; the 75 p. c e n t a c e t o n e p r e c i p i t a t e w a s r e d i s s o l v e d for e x t r a c t i o n and c e n t r i f u g e d ; the s u p e r n a t a n t was dialysed, c o n c e n t r a t e d in the dialysis bag w i t h A q u a c i d e (Calbiochem) a n d k e p t frozen. P l a s t o c y a n i n w a s o b t a i n e d f r o m fresh s p i n a c h leaves a c c o r d i n g to Katoh [9~. 02 p r o d u c t i o n w a s m e a s u r e d p o l a r o g r a p h i c a l l y w i t h the Clark e l e c t r o d e of the Gilson O x y g r a p h in a 1.6 ml cell. I l l u m i n a t i o n was o b t a i n e d f r o m a w h i t e light 150 W tungsten bulb p l a c e d at a dist a n c e of 6 cm f r o m the r e a c t i o n vessel. The incubation m e d i u m w a s the f o l l o w i n g : sucrose 0.6 M ; Na2HPO 4 5 m M ; MgCI2 5 m M ; MnCI 2 10~ m M ; EDTA 10 m M ; KHC03 4 m M ; Tris 7.5 r a M ; p H 7.50. It was first d e o x y g e n a t e d in the m e a s u r i n g vessel by b u b b l i n g Ne until 0 2 was r e d u c e d to a l o w level. Chloroplasts, substrates a n d i n h i b i t o r s w e r e then added. An 02 c o n c e n t r a t i o n of a r o u n d 10 p. cent air s a t u r a t i o n was o b t a i n e d and used at the start of each i n u m i n a t i o n p e r i o d . The r e a c t i o n was c a r r i e d out u n t i l an 02 c o n c e n t r a t i o n of a r o u n d 30 p. cent air saturation w a s r e a c h e d . All r e a c t i o n s e v o l v i n g O2 w e r e r u n w i t h i n such limited 02 c o n c e n t r a t i o n s . The o x y g r a p h was standar-
dized against a i r - s a t u r a t e d w a t e r (0.270 mM at 22"). The Is0 value ( c o n c e n t r a t i o n of i n h i b i t o r n e c e s s a r y for a 50 p. cent i n h i b i t i o n of the i n i t i a l rate) w a s estimated by i n t e r p o l a t i o n bet~veen values d i f f e r i n g f r o m the I50 by less t h a n ± 20 p. cent. Total c h l o r o p h y l l and c h l o r o p h y l l a and b w e r e e s t i m a t e d on an 80 p. cent acetone extract, a c c o r d i n g to Strain et al. [10]. RESULTS. T h e effect of several s u l f o n a m i d e s w e r e tested on separate p r e p a r a t i o n s of s p i n a c h chloroplasts, u s i n g NADP + ( + f e r r e d o x i n ) , the natural e l e c t r o n acceptor, or a r t i f i c i a l acceptors. W i t h both k i n d s of acceptors, the p h o t o c h e m i c a l o x i d a t i o n of water, reflected in the 02 evolution, is r e d u c e d by D i a m o x and C1 1385,0, but not by the o t h e r sulfona m i d e N a v i d r e x . This is s h o w n in table I w h i c h lists the c o n c e n t r a t i o n s of each drug (I~0) necessary to i n h i b i t 50 p. cent of the 02 e v o l u t i o n in s p i n a c h chloroplasts. T h e values v a r y s o m e w h a t for different e l e c t r o n a c c e p t o r s w i t h the same i n h i b i t o r and this m a y be due to the fact that m e m b r a n e p e r m e a b i l i t i e s differ a c c o r d i n g to the steady-state e n e r g y level of the chloroplasts, or to d i f f e r e n c e in b i n d i n g to i n e r t proteins. The i n h i b i tion of the 0 2 p r o d u c t i o n w i t h NADP ÷ as the elect r o n acceptor, supports the o b s e r v a t i o n by E v e r son [4] that 1 mM D i a m o x i n h i b i t s 50 p. cent of the C'Oe fixation in isolated s p i n a c h chloroplasts. H o w e v e r , we s h o w h e r e that the Hill r e a c t i o n w i t h K3Fe(CN) 6 or DPIP, an e l e c t r o n t r a n s f e r p r o d u c i n g no NADPH and unable to s u p p o r t CO 2 fixation, is as well 50 p. cent i n h i b i t e d by D i a m o x in the same range of c o n c e n t r a t i o n , and by o t h e r s i m i l a r m o l e c u l e s (see below).
TABLE I.
Sulfonamide concentration (15o) necessary to inhibit 50 p. cent of the photochemical water oxidation b.q isolated spinach chloroplasts in the presence of 3 different electron acceplors at 15°C. Sulfona mide Diamox (raM)
13 13.850 (mM)
Navidrex
2,5 0.13 0.60
0.1 0.30 0.20
inactive (*) inactive (') inactive C)
Acceptor NADP + DPIP Ferri~yanide
0.7 mM 0.3 mM 8 mM
Oz estimation is described under Methods. The initial rate of 02 evolution (~mole O~.min-1 per mg chlorophyll) at 15°C in the control amounted respectively to 1.15 with ferricyanide and DPIP, and to 0.252 with NADP. (*) A 5 mM Navidrex concentration elicited no measurable inhibition.
BIOCHIMIE, 1072, 54, n" 9.
Inhibition of photosynthesis by sulfonamides. T h e s e r e s u l t s s u g g e s t t h a t t h e i n h i b i t i o n of t h e CO 2 f i x a t i o n is n o t to b e a t t r i b u t e d o n l y to a n interference with a carbonic anhydrase activity l i n k e d to t h e s e q u e n c e of t h e d a r k r e a c t i o n s , as h a s b e e n d e d u c e d b y E v e r s o n [4] f r o m t h e n a r r o w s p e c i f i c i t y of t h e s e c o m p o u n d s f o r c a r b o . n i c a n h y d r a s e , b u t t h a t o n e s i t e of a c t i o n of t h e s u l f o n a m i d e s c a n b e v i s u a l i z e d as r e s i d i n g i n t h e p h o t o chemical reaction sequence. In order to confirm the existence in the chlorop l a s t of a s u l f o n a m i d e - s e n s i t i v e s i t e d i f f e r e n t f r o m the carbonic anhydrase, seven different molecules of t h a t c l a s s of c o m p o u n d s w e r e t e s t e d i n l h e i r e f f e c t o n t h e Hill r e a c t i o n i n a g r a n a p r e p a r a t i o n , on the one hand, and in their inhibitory capacity o n t h e c a r b o n i c a n h y d r a s e a c t i v i t y of a p r e p a r a t i o n of l y s e d c h l o r o p l a s t s , o n t h e o t h e r h a n d . As figure 1 clearly shows for the 7 molecules tested, t h e s e n s i t i v i t y of t h e p h o t o c h e m i c a l reaction s e q u e n c e is n o t r e l a t e d w h a t s o e v e r to t h e s e n s i t i -
tN
1207
v i t y of c a r b o n i c a n h y d r a s e . As t h e p h o t o r e a c t i o n was tested on the grana preparation, when the carbonic anhydrase activity was tested on lysed chloroplasts, the difference in the responses are not likely to be attributed to a selective compartm e n t a t i o n of t h e t a r g e t s a n d of t h e d r u g s , b u t n m s t l i k e l y to a s p e c i f i c a f f i n i t y of t h e d r u g s t o w a r d t h e s e n s i t i v e sites. A Icm
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0.7:
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0.25 .=
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~f
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"D
oM
o
-6
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4',o
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i f
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Fro. 2. - - A b s o r p t i o n spectra at 25°C of subchlorop l a s t p h o t o s y s t e m I (broken line) a n d p h o t o s y s t e m II (solid line) f r a c t i o n s o b t a i n e d b y the m e t h o d of Vern o n and Shaw (1971).
/ J
_I
oT
/
-4-
-0
-'5
-~
Log I s o ( m o l o r )
-'3 for corbonic
22 anhydros~
Fro. 1. - - Lack of r e l a t i o n s h i p between I50 (molar c o n c e n t r a t i o n of s u l f o n a m i d e causing 50 p. cent i n h i bition) for the Hill r e a c t i o n in a c h l o r o p l a s t g r a n a p r e p a r a t i o n (with KaFe(CN)6) a t 15°C, a n d I.~0 for the carbonic a n h y d r a s e activity in lysed chloroplasts at 0°C. Equal i n h i b i t i o n for b o t h reactions should correspond w i t h the b r o k e n line. D, Diamox ; L, Furosem~de ; M, Methazolamide ; N, Navidrex ; T, C1 13850 ; O, C1 11366 ; E, E s m a r i n . The g r a n a f r a c t i o n was p r e p a r e d according to P a r k a n d P o n (1961). Carbonic a n h y d r a s e activity was carried out 61ectrom e t r i c a l l y at 0°C b y a m o d i f i c a t i o n [3] of the m e t h o d of R a n g h t o n a n d Booth. I n h i b i t o r s were added to the e n z y m e a n d buffer at 0°C one rain before the r e a c t i o n w a s started by a d m i x i n g the substrate. Carbonic a n h y d r a s e activity in the c h l o r o p l a s t lysate a m o u n t e d to 23 U per mg chlorophyll. The Hill r e a c t i o n m e a s u r e m e n t is described u n d e r Methods a n d 8 mM Fe(CN)6K3 is p r e s e n t as the electron aeceptor. The control r a t e a m o u n t e d to 1.15 ~mole O2.min-1 p e r mg c h l o r o p h y l l at 15°C. F o r N a n d E, i n h i b i t i o n of the Hill r e a c t i o n was a b s e n t at 10-2 M. BIOCHIMIE, 1'972, 54, n ° 9.
T h e l a t t e r a b s e n c e of r e l a t i o n s h i p , t o g e t h e r w i t h t h e o b s e r v a t i o n t h a t t h e g r a n a f r a c t i o n is d e v o i d of c a r b o n i c a n h y d r a s e a c t i v i t y , s u g g e s t s t h e e x i s t e n c e of a s e c o n d s u l f o n a m i d e - s e n s i t i v e site, i n d e p e n d e n t of t h e e n z y m e c a r b o n i c a n h y d r a s e ; a recent demonstration that the same compounds i n h i b i t t h e r e s p i r a t i o n of a n i m a l m i t o c h o n d r i a h a s l e d u s to c o n c l u d e t h a t a s e c o n d s u l f o n a m i d e - s e n s i t i v e s i t e is p r e s e n t i n t h e a n i m a l c e l l s [6]. T h e l o c a t i o n of t h e s e n s i t i v e s i t e w i t h i n grana fraction was investigated.
the
Several spinach chloroplast preparations were f r a c t i o n a t e d i n t h e p r e s e n c e o f T r i t o n X-100 f o r s e p a r a t i o n of t h e t w o p h o t o s y s t e m s , a c c o r d i n g to V e r n o n a n d S h a w [11]. E a c h f r a c t i o n so o b t a i n e d displayed a characteristic absorption spectrum (fig. 2), a n d a g o o d e n r i c h m e n t i n t h e c o r r e s p o n d ing chlorophyll species : the chlorophyll a/chlorop h y l l b r a t i o r e a c h e d v a l u e s h i g h e r t h a n 6.5 i n t h e p h o t o s y s t e m I p r e p a r a t i o n s . I t w a s 1.27, 2.09 82
C. Francois.
1208
and t,79 respectively in three photosystem II fractions each obtained from a separate chloroplast preparation. Some of the sulfonamide molec u l e s s h o w n a b o v e to b e a c t i v e o n w h o l e m e m branes were tested on each photosystem. As shown i n t a b l e II, p h o t o s y s t e m I is insensitive to these molecules (but is sensitive to other molecules, see D i s c u s s i o n ) , a s w e l l a s t o D , ~ M U . O n t h e co, n t r a r y , when water alone, or water plus DPC, serves as the electron donor, and DrPIP as the electron acceptor, photosystem II displays a sensitivity at the concentrations active on the whole chloroplasts.
A) T h e p r e s e n t o b s e r v a t i o n s seem to indicate that the sulfonamides tested act within the electron/hydrogen transfer sequence somewhere between water (or DPC) and the unit reducing DPI,P (or ferricyanide) : the latter unit is localized down the phosphorylation site [12] and is likely C y t b~ 9 [13~. According to A r n o n [14] p h o t o s y s t e m II includes a second photoreduction step whereby reduced plastoeyanin i s u s e d to r e d u c e t h e o x i d i z e d chlorophyll and the photoact reduces a bound
TABLE II.
Rate (in p. cent of the control) of substrate reduction by purified photosgstem I and photosgstem II in the presence of DCMU or snlfonamides. B
+
+ crude ferredoxin extract
]
Electron pathway
PhotosyMem 11
Photosyslcm II
Plmtosystem 1
no addition
D PI P It._, to N A I) P
no addition
H~O to DPIP [lt~O and I mM iDPC lo DI}IP '
[erredoxill
+ ferl;e,h}xia - - NADP reductase + I}lastocyanine Ascorbatn to NADP
luhihitors DCMU
1 X 3 X
10-'~M . . . . . . 10-6M . . . . . .
100; 100
i
1; 0
18; 33 100
0,6 mM ..... 2,0 mM ..... 10,0 mM .....
100 100
58 30
60 31
100 100 100
C1 13 850
0,2 mM ..... 0,5 mM ..... 1,3 mM .....
100 92
53 27
62 28
10o 100 100
C1 11 366
2,0 mM .....
100
45
46
50
Diamox
S p i n a c h c h l o r o p l a s t s p r e p a r e d as d e s c r i b e d b y W a l k e r a n d Hill (1967). C h l o r o p l a s t s f r a c t i o n a t i o n a c c o r d i n g to V e r n o n a n d S h a w (1971) w i t h 40 m g c h l o rophyll per g detergent. a) N A D P + r e d u c t i o n b y p h o t o s y s t e m s I ( w i t h a s c o r b a t e + D P I P ) w a s m e a s u r e d a c c o r d i n g to t h e s a m e a u t h o r s , e x c e p t t h a t p l a s t o c y a n i n a n d e x c e s s T r i t o n X-100 w e r e o m i t t e d ; c r u d e f e r r e d o x i n p r e p a r a t i o n , o b t a i n e d as r e p o r t e d u n d e r Methods, was added in saturating amount. Initial rate in controls amounted to 33 a n d 64 ~ m o l e s . h - 1 N A D P + r e d u c e d p e r m g c h l o r o p h y l l a t 25°C i n t w o separate photosystem I preparations. b) D P I P p h o t o r e d u c t i o n w a s c a r r i e d o u t a c c o r d i n g to V e r n o n a n d S h a w (1971). D P C (BDH) w a s r e c r y s t a l l i z e d f r o m h o t e t h a n o l . T h e i n i t i a l c o n t r o l r a t e f o r 2 s e p a r a t e p r e p a r a t i o n s w a s 109 a n d 215 rrmole.h-1 D P I P r e d u c e d p e r m g c h l o r o p h y l l a t 25°C w i t h w a t e r as t h e sole e l e c t r o n d o n o r ; ' w i t h D P C p r e s e n t at 0.5 raM, t h e r e d u c t i o n r a t e a m o u n t e d to 170 f o r o n e p r e p a r a t i o n a n d w i t h 1 m M D P C p r e s e n t , to 471 ;~mole-1 D P I P p e r m g c h l o r o p h y l l i n a n o t h e r p r e p a r a t i o n . T h e l i g h t e n e r g y W a s o b t a i n e d f r o m a 100 W w h i t e l i g h t t u n g s t e n b u l b p l a c e d at l 0 cn~' f r o m t h e p h o t o m e t e r c u v e t t e a n d t h e l a t t e r r e m o v e d e v e r y 30 sec l o r spectrophotometric absorbance measurements in a thermostated Zeiss spcctrophotometer. c) N A D P r e d u c t i o n b y a s c o r b a t e w a s m e a s u r e d w i t h t h e r e c o n s t i t u t e d s y s t e m of M a l k i n (197l) ; see t e x t .
BIOCHIMIE, 1972, 54, n ° 9.
Inhibition of photosynthesis by sulfonamides. ferredoxin. It has been r e p o r t e d by Malkin [15] that, a c c o r d i n g to this schema, photosystem II p r e p a r a t i o n s can c a r r y out electron t r a n s f e r from ascorbate to NADP ÷ p r o v i d e d p l a s t o c y a n i n , soluble f e r r e d o x i n and ferredoxin-NADP reductase are added. Using a crude f e r r e d o x i n extract o b t a i n e d by the method of San Pietro [8] as a source of the latter two factors, a n d p l a s t o c y a n i n p r e p a r e d a c c o r d i n g to Katoh [9], one photosystem II fraction r e d u c e d NADP + at the rate of 26.3 ~mole.h-* per mg c h l o r o p h y l l at 26°C ( i l l u m i n a t i o n as i n e x p e r i m e n t s of table II). The rate was u n c h a n g e d by the p r e s e n c e of 3.6 × 10-6 M D~CMU, or i n c r e a s ing c o n c e n t r a t i o n s of Diamox up to 10 raM, C1 13850 up to 1.3.5 mM a n d Methazolamide up to 3.3 raM. It m a y thus be c o n c l u d e d that the latter sulfonamide molecules are not i n t e r f e r i n g on the sequence from p l a s t o c y a n i n to NADP i n photosystem II, as it is conceived by A r n o n [14] ; this is i n agreement w i t h the results of table II, a n d the i n t e r f e r e n c e of these molecules at a step b e t w e e n water a n d c y t o c h r o m e b559 accounts for their effect on the water p h o t o r e d u c t i o n (table I). B) However, other thiadiazole sulfonamides, w h e r e an aromatic nucleus has been added on the 2-amino group in the thiadiazole ring, p r o v e d to i n h i b i t the electron t r a n s p o r t from p l a s t o c y a n i n to N A D P ; one of these molecules is C1 113,06, w h i c h i n h i b i t s 50 p. cent at 2 raM. We were thus led to suspect that a n o t h e r sulfonamide-sensitive site is p r e s e n t w i t h i n the latter sequence of elect r o n - h y d r o g e n transfer. I n order to c o n f i r m this observation, we decided to test the molecules on a r e c o n s i i t u t e d f e r r e d o x i n - - f e r r e d o x i n - N A D P reductase system fed electrons w i t h purified photosystems I or II. S p i n a c h f e r r e d o x i n was p u r i f i e d to an A420/A276 of 0.31, a c c o r d i n g to B u c h a n a n a n d A r n o n [16] a n d ferredoxin-NADP reductase (E.N.1.6.99.4) e n r i c h e d to an A456/A275 of 0.048 b y the p r o c e d u r e of Shin [17]. The w a s h e d chloroplast p r e p a r a t i o n utilized in the test described i n [17] was replaced by a photosystem I or a photosystem II fraction p r e p a r e d as reported i n table H. I l l u m i n a t i o n was o b t a i n e d as described i n table II. As expected, there was no electron t r a n s f e r from DPIP-ascorbate to NADP with photosystem II. Photosystem I p r o d u c e d 5,0 ~moles NADPH.h-1 per mg c h l o r o p h y l l at 25°C and this activity was i n h i bited by two of the h y d r o p h o b i c molecules (but not by C1 11366) r e p o r t e d above to act b e t w e e n p l a s t o c y a n i n a n d NADP (with ascorbate a n d photosystem II). The two photosystems differ as to the specific m e m b r a n e p h o t o r e d u c t i o n step, but
BIOCHIMIE, 1972, 54, u ° 9.
1209
they share in vitro the c o m m o n h y d r o g e n t r a n s f e r sequence from f e r r e d o x i n to NADP. The latter results seem to i n d i c a t e that the c o m m o n h y d r o g e n t r a n s f e r sequence, c o m p r i s i n g a m e m b r a n e - l i n k e d p h o t o r e d u c e d center, f e r r e d o x i n and f e r r e d o x i n NADP reductase, r e p r e s e n t s a second s u l f o n a m i d e sensitive site i n the chloroplast. Some specificity of the m e m b r a n e appears i n the different b e h a v i o r of C1 113,66 w h e n photosystem I or photosystem II is used to f u r n i s h the r e d u c i n g equivalents (table I I ) ; m o r e o v e r w h e n the m e m b r a n e p h o t o u n i t s were omitted a n d the f e r r e d o x i n .... ferredoxin-NADP reductase complex tested in the artificial sequence from NADPH to c y t o c h r o m e c (Shin [17]), n o n e of the molecules, s h o w n above to be active on the whole system, was i n h i b i t o r y . It m a y thus be c o n c l u d e d that some s t r u c t u r a l r e q u i r e m e n t s confers activity of s u l f o n a m i d e s t o w a r d s a site located at the m e m b r a n e - f e r r e doxin-flavoprotein level.
DISCUSSION. Several molecules s h o w n here (A) to interfere w i t h the p h o t o r e d u c t i o n sequence b e t w e e n w a t e r a n d c y t o c h r o m e b~.~9 in photosystem H (fig. 1) belong to s u l f o n a m i d e s b e a r i n g a thiadiazole nucleus, but one, F u r o s e m i d e , possesses a c h l o r i n a t e d a n t h r a n i l i c n u c l e u s ; several other s u l f o n a m i d e s are inactive, a n d one w h i c h i n h i b i t e d the NADP p r o d u c t i o n p r o v e d to 'activate the Hill r e a c t i o n (with f e r r i c y a n i d e ) by 100 p. cent. The activity on the ferredoxin-NADP sequence (B), on the other h a n d , seems to be a p r o p e r t y of some very h y d r o p h o b i c thiadiazole molecules, like CI 11366. Accordingly, it seems reasonable to admit that the s u l f o n a m i d e group c o m m o n to all the active molecules studied is the group i n t e r f e r i n g at specific sites, but that an a d d i t i o n a l s t r u c t u r a l c h a r a c t e r is r e q u i r e d for activity. A s i m i l a r v a r i a t i o n i n the i n h i b i t o r y p o w e r of a series of s u l f o n a m i d e s was r e p o r t e d in studies on c a r b o n i c a n h y d r a s e [18], m i t o e h o n d r i a l r e s p i r a t i o n [6] and x a n t h i n e oxidase [19], as may be a n t i c i p a t e d for the i n t e r a c tion b e t w e e n drugs a n d m e m b r a n e components. The s u l f o n a m i d e s described u n d e r A) do act on chloroplast fractions w h i c h are also sensitive to DCMU. F r o m the following c o n s i d e r a t i o n s , it seems that the latter sulfonamide-sensitive site is different from the DCMU-sensitive site : I)CMU raises the variable f u o r e s c e n c e of the s h o r t - w a v e l e n g t h photocenter, by i n h i b i t i n g its 83
I210
C. F r a n f o i s .
r e o x i d a t i o n [13]. P r e l i m i n a r y e x p e r i m e n t s s h o w e d that the sulfonamides do not raise the variable f l u o r e s c e n c e of t h e i s o l a t e d p h o t o s y s t e m I I f r a c t i o n . B e s i d e s , w e h a v e o b s e r v e d ( t a b l e II), t h a y b y r a i s i n g to 1 m M t h e c o n c e n t r a t i o n of D P C a d d e d as a n e l e c t r o n d o n o r to i s o l a t e d p h o t o s y s t e m II, t h e e l e c t r o n f l o w c o m p l e t e l y i n h i b i t e d b y DCMU, is r e e s t a b l i s h e d at 33 p. c e n t of t h e c o n t r o l v a l u e . T h i s is i n a c c o r d a n c e w i t h v a l u e s r e p o r t e d b y V e r n o n a n d S h a w [20], S h n e y o . u r a n d A r n o n [2I] and Malkin [15]. On the contrary, the inhibition by the sulfonamides was not relieved when DPC was added. If t h e s u l f o n a m i d e - s e n s i t i v e s i t e i n t h e n o n cyclic photosystem differs from the DCMU-sensit i v e site, w e feel t h a t t h e p r e s e n t i n v e s t i g a t i o n does not bring arguments capable of locating the sulfonamide:sensitive u n i t i n r e l a t i o n to t h e DCMU-sensitive unit, Although only some sulfonamides are active on t h e f e r r e d o x i n - N A D P s e q u e n c e ( e x p e r i m e n t s B), t h e s i m p l i c i t y of t h i s s y s t e m m a y r a i s e a n i n t e r e s t i n g s u g g e s t i o n . W e h a v e r e p o r d e d [61_ t h a t t h e m i t o c h o n d r i a l r e s p i r a t i o n is s u l f o n a m i d e - s e n s i t i v e . A d d i t i o n a l e x p e r i m e n t s (to b e p u b l i s h e d ) h a v e i n d i c a t e d t h a t t h e s e n s i t i v e s i t e is p r e s e n t i n t h e electron transport chain and the smallest sensitive f r a g m e n t i s o l a t e d is t h e p a r t i c u l a t e N A D H d e h y d r o g e n a s e , a c o m p l e x of f l a v o p r o t e i n w i t h a n o n h e i n e i r o n p r o t e i n . W e h a v e also r e c e n t l y e s t a b l i s h e d [19] t h a t s o m e e l e c t r o n - t r a n s p o r t p a t h w a y s i n x a n t h i n e o x i d o r e d u c t a s e s a r e s e n s i t i v e to s u l f o n amides: these enzymes are soluble models for complex electron transport chains and possess a flavine and a non-heme iron, besides persulfide group and molybdenum (Massey and Edmondson [22]). W e feel t h a t t h e l a t t e r s e n s i t i v i t y to s u l f o n a m i d e s of t h e c l o s e l y r e l a t e d n o n - h e m e i r o n flavoproteins points to the very similar ferredoxin-f e r r e d o x i n - N A D P r e d u c t a s e c o m p l e x as o n e t a r g e t for these inhibitors. No suggestion will be put forward about the n a t u r e of t h e o t h e r s e n s i t i v e site, b e c a u s e t h e s e n s i t i v e s e q u e n c e , f r o m w a t e r to c y t o c h r o m e b is m u c h t o o c o m p l e x ; to o u r k n o w l e d g e , n o flavop r o t e i n h a s e v e r b e e n d e m o n s t r a t e d to b e p a r t o f t h a t s e q u e n c e , a l t h o u g h M c K e n n a a n d B i s h o p [23] have suggested that an endogenous flavin may be i n v o l v e d i n t h e p h o t o o x i d a t i o n of Mn2+.
A cJcn~wledgments. We are g r a t e f u l to Prof. E. Schoffeniels for c o n s t a n t e n c o u r a g e m e n t a n d to Dr. J.-M. Michel for c a r r y i n g out the fluorescence tests. Diamox was given by Lederle (Brussels) ; C1 13850, C1 11366 a n d Methazola m i d e by the A m e r i c a n C y a n a m i d C y ; E s m a r i n by
BIOCHIMIE, 1'972, 54, n ° 9.
Merck ; F u r o s e m i d e b y Hoechst a n d Navidrex b y Ciba. This work was supported by g r a n t n ° 790 f r o m the Fonds de la Recherche fondamentale collective to Prof. E. Schoffeniels. RI~.SUM~. La p h o t o r d d u c t i o n de l ' e a u p a r des p r 6 p a r a t i o n s de ehloroplastes d'6pinard, leurs g r a n a ou leur photosyst~me non-cyclique isol6 est inhib6e p a r p l u s i e u r s sulf o n a m i d e s ; cette sensibilit6 n ' e s t pas en r e l a t i o n avec le pouvoir d ' i n h i b i t i o n de ces moI~cules sur F a n h y drase c a r b o n i q u e des chloroplastes ; il existe done u n site sensible, different de l ' a n h y d r a s e , d a n s la e h a l n e de t r a n s p o r t d'61ectrons du systbme p h o t o s y n t h 6 t i q u e non-cyclique. La sensibilit6 de la p h o t o r 6 d u c t i o n du COa aux s u l f o n a m i d e s ne d6pend done pas, ou pas uniquen~ent, d'une i n h i b i t i o n de l ' a n h y d r a s e carbonique. La sensihilit6 du systbme non-cyelique aux s u l f o n a mides semble diff~rente de sa sensibilit6 au DCMU. La s6quence de t r a n s p o r t ferredoxine-NADP r6ductase, ferredoxine, lamelles du systbme eyelique o u uon-cyclique, p l a s t o e y a n i n e a 6t6 reeonstitude h p a r t i r des unit6s isol6es. Elle est sensible h certaines mol6cules de sulfonamides, et l'identit6 de ce second site sensible avec la ferredoxine-NADP r~duetase est discut6e. ZUSAMMENFASSUNG.
Die P h o t o r e d u k t i o n des Wassers d u r c h Prfiparation e n yon C h l o r o p l a s t e n des Spinats, i h r e r G r a n a f r a k t i o n e n oder ihres isolierten n i c h t - zyklischen P h o t o sytems wird yon m e h r e r e n S u l f o n a m i d e n i n h i b i e r t ; diese Sensibilit~t ist nicht m i t dem I n h i b i t i o n s v e r m 6 gen dieser Molekfile a u f die C a r b o a n h y d r a s e d e r Chlor o p l a s t e n in E i n k l a n g zu bringen. Also existiert eine sensible Etappe, die sich yon der A n h y d r a s e u n t e r scheidet, in der T r a n s p o r t k e t t e d e r E t e k t r o n e n des n i e h t - z y k l i s c h e n p h o t o s y n t h e t i s c h e n Systems. Die Sensibilit~it der P h o t o r e d u k t i o n des CO~ gegeniiber Sulfona m i d e n hiingt also nicht, oder n i e h t allein, yon einer I n h i b i t i o n der C a r b o a n h y d r a s e ab. Die Sensihilit$it des n i c h t - z y k l i s c h e n Systems gegeniiber S u l f o n a m i d e n seheint yon der bet DCMU g e f u n d e n e n untersehiedlich. Die T r a n s p o r t s e q u e n z Ferredoxin-NADP-Reduktase, Ferredoxin, M e m b r a n des zyklisehen oder nieht-zyklisehen Systems, P l a s t o z y a n i n , w u r d e a u s g e h e n d aus den isolierten E l e m e n t e n r e k o n s t r u i e r t . Sic ist einigen Sulf o n a m i d m o l e k f i l e n gegenfiber empfindlich, u n d die !dentlt~it dieser zweiten sensiblen Etappe m i t der F e r r e d o x i n - N A D P - R e d u k t a s e w i r d h i e r er6rtet. REFERENCES. 1. Ikemori, M. :¢ Nishida, K. (1968) Physiol. Plantarum, 21, 292. 2. Everson, R. G. (1969) Nature, 222, 876. 3. Everson, R. G. ,~ Slack, C. R. (1968) Phytochemistry,, 7, 581-584. 4. Everson, R. G. (1970) Phytochemistry, 9, 25-32. 5. Hatch, M. D. a Slack, C. R. (1970) Proff. Phytochem., 2, 35-106. 6. Franqois, C. & Deprez, Ch. (1971) Arch. internat. Physiol. Biochim., 79, 993-1007. 7. Walker, D. A. a Hill, R. (1967) Biochim. Biophys. Acla, 131, 330-338. 8. San Pietro, A. (1963) In Meth. in Enzymology, Colo~vick, S. P. a Kaptan, N. O., eds, 6, 439-445. 9. Katoh, S. (1971) In Meth. in Enzymology, San Pietro, A., ed., 23, 277-289. 10. Strain, H. H., Cope, B. T. a Svec, W. A. (1971) In Meth. in Enzymology, San P i e t r 0 , A., ed., 23, 452-476.
Inhibilion of p h o l o s y n l h e s i s 11. Vernon, L. P. ~ Shaw, E. R. (197I) In Melh. il~ Enzymology, San Pietro, A., ed., 23, 277-289. 12. Avron, M. (1967) In Current Topics in Bioenergetics, Sanadi, D. R., ed., 2, 1-19. 13. B o a r d m a n , N. K. (1968) In Advances in Et~zymology, Nord, F. F., ed., 30, 2-79. 14. Arnon, D. I. (1971) Proc. Natl Acad. Sci., U.S.A., 68, 2883-2892. 15. Malkin, R. (1971) Biochim. Biophys. Acta, 253, 421-427. 16. B u c h a n a n , B. B. ~ Arnon, D. I. (1971) In Meth. in Enzymology, San Pietro, A., ed., 23, 413-440.
BIOCHIMIE, 1972, 54, n ° 9.
by s n l [ o n a m i d e s .
(1971) In Meth. in Enzymology, San A., ed., 23, 440-447. H. (1967) Physiol. Bey., 47, 595. C. (1972) Arch. internat. Physiol. Biochim., 80, 600-601. Vernon, L. P. • Shaw, E. R. (1969) Biochem. Biophys. Res. Commun., 36, 878-884. S h n e y o u r , A. ~ Avron, M. (1971) Biochim. Biophys. Acta, 253, 412-420. Massey, V. t~ E d m o n d s o n , D. (1970) J. Biol. Chem., 245, 6595-6598. McKenna, J. M. ,~ Bishop, N. I. (1967) Biochim. Biophys. Acta, 131, 339-349.
17. Shin, M. Pietro, 18. Maren, T. 19. Franqois, 20. 21. 22. 23.
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Inhibition by sulfonamides of the photoreductive step in spinach chloroplast photosynthesis. C. FRANqOIS. Laboratoire de B i o c h i m i e , Institut L~,)n Fredericq, Universitd de Liege, 17 Place Delconr, 4000 Liege, Belgiqae. (23/5/1972). Summary. - - Spinach chloroplasts, their grana fraction and their photosystem I and II fractions have been prepared. The photoreduetion of water by the non-cyclic photosystem preparations has been shown to be inhibited by several sulfonamides ; the sensitivity of the photoreduction has no relation with the inhibition of the carbonic anhydrase activity in lysed chloroplasts. A sulfonamide-sensitive unit thus exists in the electron transport sequence of the lamellae and seems to be different from the DCMU-sensitive site. Spinach ferredoxin, ferredoxin-NADP reductase and plastocyanin were prepared and used to reconstitute the non-cyclic electr6n transport sequence. This system proved to be sensitive to other sulfonamides and arguments in favor of this second sensitive site being the ferredoxin - - ferredoxin-NADP reduetase - - membrane complex are discussed. It is concluded that carbonic anbydrase may not be involved in the sensitivity of photosynthesis to sulfonamides.
it was r e c e n t l y r e p o r t e d [1, 2] that Diamox, a w i d e l y - u s e d d i u r e t i c d r u g and a p o w e r f u l i n h i b i tor in animals, of the e n z y m e c a r b o n i c a n h y d r a s e (carbonate h y d r o - l y a s e , E.N. 4.2.1.1), was also an i n h i b i t o r of p h o t o s y n t h e s i s in isolated s p i n a c h chloroplasts ; o t h e r s u l f o n a m i d e s w e r e c o n s i d e r e d as i n a c t i v e ; azide and nitrite are also p o w e r f u l i n h i b i t o r s . E v e r s o n and Slack (3) h a v e f u r t h e r rep o r t e d that c a r b o n i c a n h y d r a s e is l o c a t e d in the chloroplasts of the Calvin-cycle plants and is i n h i bited by azide, nitrite, Diamox, the i n h i b i t i o n of the latter t w o b e i n g r e l i e v e d by 5 mM H,CO-.~. Judging f r o m the effect of these 3 molecules on both c a r b o n i c a n h y d r a s e a c t i v i t y and on p h o t o s y n t h e sis, E v e r s o n [41, and H a t c h and Slack [5] have p r o p o s e d that c a r b o n i c a n h y d r a s e is a key unit in the s e q u e n c e m a k i n g up the p h o t o s y n t h e t i c Abbreviations : DCMU : 3-(3',4'-dichlorophenyl)-l,l-dimethylurea (Dupont). DPIP : 2,6-diehlorophenol-indophenol (Merck). DPC : 1,5-diphenylcarbazide (BDH). Diamox : acetazolamide : 2-acetylamino-l,3,4-thiadiazole-5-snlfonamide (Lederle). Navidrex : 3-eyclopentylmethyl-6-chloro-7-sulfamyl3,4-dihydro-l,2,4-benzothiadiazine (Ciba). C1 13850 : NS-t-butyl-2-acetylamino-l,3,4-thiadiazole-5snlfonamide (Lederle). C1 11366 : 2-benzenesulfonamide-l,3,4-thiadiazole-5sulfonamide (Lederle). Furosemide : 4-chloro-N (2-furylmethyl)-5-snlfamylanthranilie acid (Hoechst). Esmarin : 3-dichlormethyl-3,4-dihydro-6-chloro-7-sulfa myl-2-H- 1,2,4-benzothiadiazine-l,l-dioxide (Merck). Methazolamide : 2-acetylimino-2,3-dihydro-3-methyl1,3,4-thiadiazole-5- sulfonamide (Lederle).
m a c h i n e r y and that it is likely to be l o c a t e d w h e r e CO 2 or HCO-~ is d e l i v e r e d to the e n z y m e u n i t c a r r y i n g the c a r b o x y l a t i o n step in the d a r k reactions of the Calvin- and the C4-dicarboxylie a c i d p a t h w a y s . The latter r e l a t i o n s h i p recalls the relationship, generally a d m i t t e d to be p r e s e n t in animals, of the i n h i b i t i o n of c a r b o n i c a n h y d r a s e by D i a m o x and s i m i l a r m o l e c u l e s on the one h a n d , w i t h the i n t e r f e r e n c e of these m o l e c u l e s on the ion t r a n s l o c a t i o n s (in the k i d n e y , stomach, c i l i a r y processes, salt gland in birds, fish gills) on the o t h e r hand. T h e latter r e l a t i o n s h i p w a s r e c e n t l y quest i o n e d by us. We h a d s h o w n that s u l f o n a m i d e s depress the r e s p i r a t i o n in w h o l e tissue t h r o u g h a d i r e c t i n h i b i t i o n of the o x y g e n c o n s u m p t i o n in m i t o c h o n d r i a isolated f r o m several tissues of the dog a n d the r a t [6], suggesting that a n o t h e r sulfon a m i d e - s e n s i t i v e site is p r e s e n t in the energyp r o d u c i n g m a c h i n e r y . Consequently w e w e r e led to cast doubts on the m e c h a n i s m offered by E v e r son [41 on h o w s u l f o n a m i d e s i n h i b i t p h o t o s y n t h e sis, and to investigate w h e t h e r , in analogy w i t h t h e i r action on the o x i d a t i v e m e t a b o l i s m of mitoc h o n d r i a , s u l f o n a m i d e s m i g h t i n t e r f e r e w i t h the r e d u c t i v e , or light r e a c t i o n s of the chloroplasts. This r e p o r t p r e s e n t s e v i d e n c e that some sulfonamides i n h i b i t the n o n c y c l i c e l e c t r o n t r a n s p o r t in s p i n a c h c h l o r o p l a s t s and c h l o r o p l a s t f r a g m e n t s at a site situated at the r e d u c i n g side of cytoc h r o m e b559, w h i l e o t h e r s u l f o n a m i d e m o l e c u l e s i n t e r f e r e w i t h i n the f e r r e d o x i n - NADP sequence.
C. Franfois.
1206 MATERIALS AND METHODS.
Sucrose ( b i o c h e m i c a l grade) and D.PI,P w e r e p u r c h a s e d f r o m Merck, NADP and NADPH f r o m Sigma, DPC f r o m BDH and DCMU f r o m Dupont. The sulfonamides, listed u n d e r Abbreviations, w e r e all k i n d l y p r o v i d e d to us by the C o m p a n i e s mentioned. S p i n a c h chloroplasts w e r e p r e p a r e d f r o m comulercially available fresh leaves by the m e t h o d of W a l k e r and Hill [7]. A c r u d e f e r r e d o x i n e x t r a c t was p r e p a r e d a c c o r d i n g to San P i e t r o [8] ; the 75 p. c e n t a c e t o n e p r e c i p i t a t e w a s r e d i s s o l v e d for e x t r a c t i o n and c e n t r i f u g e d ; the s u p e r n a t a n t was dialysed, c o n c e n t r a t e d in the dialysis bag w i t h A q u a c i d e (Calbiochem) a n d k e p t frozen. P l a s t o c y a n i n w a s o b t a i n e d f r o m fresh s p i n a c h leaves a c c o r d i n g to Katoh [9~. 02 p r o d u c t i o n w a s m e a s u r e d p o l a r o g r a p h i c a l l y w i t h the Clark e l e c t r o d e of the Gilson O x y g r a p h in a 1.6 ml cell. I l l u m i n a t i o n was o b t a i n e d f r o m a w h i t e light 150 W tungsten bulb p l a c e d at a dist a n c e of 6 cm f r o m the r e a c t i o n vessel. The incubation m e d i u m w a s the f o l l o w i n g : sucrose 0.6 M ; Na2HPO 4 5 m M ; MgCI2 5 m M ; MnCI 2 10~ m M ; EDTA 10 m M ; KHC03 4 m M ; Tris 7.5 r a M ; p H 7.50. It was first d e o x y g e n a t e d in the m e a s u r i n g vessel by b u b b l i n g Ne until 0 2 was r e d u c e d to a l o w level. Chloroplasts, substrates a n d i n h i b i t o r s w e r e then added. An 02 c o n c e n t r a t i o n of a r o u n d 10 p. cent air s a t u r a t i o n was o b t a i n e d and used at the start of each i n u m i n a t i o n p e r i o d . The r e a c t i o n was c a r r i e d out u n t i l an 02 c o n c e n t r a t i o n of a r o u n d 30 p. cent air saturation w a s r e a c h e d . All r e a c t i o n s e v o l v i n g O2 w e r e r u n w i t h i n such limited 02 c o n c e n t r a t i o n s . The o x y g r a p h was standar-
dized against a i r - s a t u r a t e d w a t e r (0.270 mM at 22"). The Is0 value ( c o n c e n t r a t i o n of i n h i b i t o r n e c e s s a r y for a 50 p. cent i n h i b i t i o n of the i n i t i a l rate) w a s estimated by i n t e r p o l a t i o n bet~veen values d i f f e r i n g f r o m the I50 by less t h a n ± 20 p. cent. Total c h l o r o p h y l l and c h l o r o p h y l l a and b w e r e e s t i m a t e d on an 80 p. cent acetone extract, a c c o r d i n g to Strain et al. [10]. RESULTS. T h e effect of several s u l f o n a m i d e s w e r e tested on separate p r e p a r a t i o n s of s p i n a c h chloroplasts, u s i n g NADP + ( + f e r r e d o x i n ) , the natural e l e c t r o n acceptor, or a r t i f i c i a l acceptors. W i t h both k i n d s of acceptors, the p h o t o c h e m i c a l o x i d a t i o n of water, reflected in the 02 evolution, is r e d u c e d by D i a m o x and C1 1385,0, but not by the o t h e r sulfona m i d e N a v i d r e x . This is s h o w n in table I w h i c h lists the c o n c e n t r a t i o n s of each drug (I~0) necessary to i n h i b i t 50 p. cent of the 02 e v o l u t i o n in s p i n a c h chloroplasts. T h e values v a r y s o m e w h a t for different e l e c t r o n a c c e p t o r s w i t h the same i n h i b i t o r and this m a y be due to the fact that m e m b r a n e p e r m e a b i l i t i e s differ a c c o r d i n g to the steady-state e n e r g y level of the chloroplasts, or to d i f f e r e n c e in b i n d i n g to i n e r t proteins. The i n h i b i tion of the 0 2 p r o d u c t i o n w i t h NADP ÷ as the elect r o n acceptor, supports the o b s e r v a t i o n by E v e r son [4] that 1 mM D i a m o x i n h i b i t s 50 p. cent of the C'Oe fixation in isolated s p i n a c h chloroplasts. H o w e v e r , we s h o w h e r e that the Hill r e a c t i o n w i t h K3Fe(CN) 6 or DPIP, an e l e c t r o n t r a n s f e r p r o d u c i n g no NADPH and unable to s u p p o r t CO 2 fixation, is as well 50 p. cent i n h i b i t e d by D i a m o x in the same range of c o n c e n t r a t i o n , and by o t h e r s i m i l a r m o l e c u l e s (see below).
TABLE I.
Sulfonamide concentration (15o) necessary to inhibit 50 p. cent of the photochemical water oxidation b.q isolated spinach chloroplasts in the presence of 3 different electron acceplors at 15°C. Sulfona mide Diamox (raM)
13 13.850 (mM)
Navidrex
2,5 0.13 0.60
0.1 0.30 0.20
inactive (*) inactive (') inactive C)
Acceptor NADP + DPIP Ferri~yanide
0.7 mM 0.3 mM 8 mM
Oz estimation is described under Methods. The initial rate of 02 evolution (~mole O~.min-1 per mg chlorophyll) at 15°C in the control amounted respectively to 1.15 with ferricyanide and DPIP, and to 0.252 with NADP. (*) A 5 mM Navidrex concentration elicited no measurable inhibition.
BIOCHIMIE, 1072, 54, n" 9.
Inhibition of photosynthesis by sulfonamides. T h e s e r e s u l t s s u g g e s t t h a t t h e i n h i b i t i o n of t h e CO 2 f i x a t i o n is n o t to b e a t t r i b u t e d o n l y to a n interference with a carbonic anhydrase activity l i n k e d to t h e s e q u e n c e of t h e d a r k r e a c t i o n s , as h a s b e e n d e d u c e d b y E v e r s o n [4] f r o m t h e n a r r o w s p e c i f i c i t y of t h e s e c o m p o u n d s f o r c a r b o . n i c a n h y d r a s e , b u t t h a t o n e s i t e of a c t i o n of t h e s u l f o n a m i d e s c a n b e v i s u a l i z e d as r e s i d i n g i n t h e p h o t o chemical reaction sequence. In order to confirm the existence in the chlorop l a s t of a s u l f o n a m i d e - s e n s i t i v e s i t e d i f f e r e n t f r o m the carbonic anhydrase, seven different molecules of t h a t c l a s s of c o m p o u n d s w e r e t e s t e d i n l h e i r e f f e c t o n t h e Hill r e a c t i o n i n a g r a n a p r e p a r a t i o n , on the one hand, and in their inhibitory capacity o n t h e c a r b o n i c a n h y d r a s e a c t i v i t y of a p r e p a r a t i o n of l y s e d c h l o r o p l a s t s , o n t h e o t h e r h a n d . As figure 1 clearly shows for the 7 molecules tested, t h e s e n s i t i v i t y of t h e p h o t o c h e m i c a l reaction s e q u e n c e is n o t r e l a t e d w h a t s o e v e r to t h e s e n s i t i -
tN
1207
v i t y of c a r b o n i c a n h y d r a s e . As t h e p h o t o r e a c t i o n was tested on the grana preparation, when the carbonic anhydrase activity was tested on lysed chloroplasts, the difference in the responses are not likely to be attributed to a selective compartm e n t a t i o n of t h e t a r g e t s a n d of t h e d r u g s , b u t n m s t l i k e l y to a s p e c i f i c a f f i n i t y of t h e d r u g s t o w a r d t h e s e n s i t i v e sites. A Icm
1.0-
i
0.7:
0.S(
tE
0.25 .=
-i-
~f
~-2-
eL
/ ff
"D
oM
o
-6
J
4',o
5~o
' ~o
'
tiff
/
E
i f
oO oD
Fro. 2. - - A b s o r p t i o n spectra at 25°C of subchlorop l a s t p h o t o s y s t e m I (broken line) a n d p h o t o s y s t e m II (solid line) f r a c t i o n s o b t a i n e d b y the m e t h o d of Vern o n and Shaw (1971).
/ J
_I
oT
/
-4-
-0
-'5
-~
Log I s o ( m o l o r )
-'3 for corbonic
22 anhydros~
Fro. 1. - - Lack of r e l a t i o n s h i p between I50 (molar c o n c e n t r a t i o n of s u l f o n a m i d e causing 50 p. cent i n h i bition) for the Hill r e a c t i o n in a c h l o r o p l a s t g r a n a p r e p a r a t i o n (with KaFe(CN)6) a t 15°C, a n d I.~0 for the carbonic a n h y d r a s e activity in lysed chloroplasts at 0°C. Equal i n h i b i t i o n for b o t h reactions should correspond w i t h the b r o k e n line. D, Diamox ; L, Furosem~de ; M, Methazolamide ; N, Navidrex ; T, C1 13850 ; O, C1 11366 ; E, E s m a r i n . The g r a n a f r a c t i o n was p r e p a r e d according to P a r k a n d P o n (1961). Carbonic a n h y d r a s e activity was carried out 61ectrom e t r i c a l l y at 0°C b y a m o d i f i c a t i o n [3] of the m e t h o d of R a n g h t o n a n d Booth. I n h i b i t o r s were added to the e n z y m e a n d buffer at 0°C one rain before the r e a c t i o n w a s started by a d m i x i n g the substrate. Carbonic a n h y d r a s e activity in the c h l o r o p l a s t lysate a m o u n t e d to 23 U per mg chlorophyll. The Hill r e a c t i o n m e a s u r e m e n t is described u n d e r Methods a n d 8 mM Fe(CN)6K3 is p r e s e n t as the electron aeceptor. The control r a t e a m o u n t e d to 1.15 ~mole O2.min-1 p e r mg c h l o r o p h y l l at 15°C. F o r N a n d E, i n h i b i t i o n of the Hill r e a c t i o n was a b s e n t at 10-2 M. BIOCHIMIE, 1'972, 54, n ° 9.
T h e l a t t e r a b s e n c e of r e l a t i o n s h i p , t o g e t h e r w i t h t h e o b s e r v a t i o n t h a t t h e g r a n a f r a c t i o n is d e v o i d of c a r b o n i c a n h y d r a s e a c t i v i t y , s u g g e s t s t h e e x i s t e n c e of a s e c o n d s u l f o n a m i d e - s e n s i t i v e site, i n d e p e n d e n t of t h e e n z y m e c a r b o n i c a n h y d r a s e ; a recent demonstration that the same compounds i n h i b i t t h e r e s p i r a t i o n of a n i m a l m i t o c h o n d r i a h a s l e d u s to c o n c l u d e t h a t a s e c o n d s u l f o n a m i d e - s e n s i t i v e s i t e is p r e s e n t i n t h e a n i m a l c e l l s [6]. T h e l o c a t i o n of t h e s e n s i t i v e s i t e w i t h i n grana fraction was investigated.
the
Several spinach chloroplast preparations were f r a c t i o n a t e d i n t h e p r e s e n c e o f T r i t o n X-100 f o r s e p a r a t i o n of t h e t w o p h o t o s y s t e m s , a c c o r d i n g to V e r n o n a n d S h a w [11]. E a c h f r a c t i o n so o b t a i n e d displayed a characteristic absorption spectrum (fig. 2), a n d a g o o d e n r i c h m e n t i n t h e c o r r e s p o n d ing chlorophyll species : the chlorophyll a/chlorop h y l l b r a t i o r e a c h e d v a l u e s h i g h e r t h a n 6.5 i n t h e p h o t o s y s t e m I p r e p a r a t i o n s . I t w a s 1.27, 2.09 82
C. Francois.
1208
and t,79 respectively in three photosystem II fractions each obtained from a separate chloroplast preparation. Some of the sulfonamide molec u l e s s h o w n a b o v e to b e a c t i v e o n w h o l e m e m branes were tested on each photosystem. As shown i n t a b l e II, p h o t o s y s t e m I is insensitive to these molecules (but is sensitive to other molecules, see D i s c u s s i o n ) , a s w e l l a s t o D , ~ M U . O n t h e co, n t r a r y , when water alone, or water plus DPC, serves as the electron donor, and DrPIP as the electron acceptor, photosystem II displays a sensitivity at the concentrations active on the whole chloroplasts.
A) T h e p r e s e n t o b s e r v a t i o n s seem to indicate that the sulfonamides tested act within the electron/hydrogen transfer sequence somewhere between water (or DPC) and the unit reducing DPI,P (or ferricyanide) : the latter unit is localized down the phosphorylation site [12] and is likely C y t b~ 9 [13~. According to A r n o n [14] p h o t o s y s t e m II includes a second photoreduction step whereby reduced plastoeyanin i s u s e d to r e d u c e t h e o x i d i z e d chlorophyll and the photoact reduces a bound
TABLE II.
Rate (in p. cent of the control) of substrate reduction by purified photosgstem I and photosgstem II in the presence of DCMU or snlfonamides. B
+
+ crude ferredoxin extract
]
Electron pathway
PhotosyMem 11
Photosyslcm II
Plmtosystem 1
no addition
D PI P It._, to N A I) P
no addition
H~O to DPIP [lt~O and I mM iDPC lo DI}IP '
[erredoxill
+ ferl;e,h}xia - - NADP reductase + I}lastocyanine Ascorbatn to NADP
luhihitors DCMU
1 X 3 X
10-'~M . . . . . . 10-6M . . . . . .
100; 100
i
1; 0
18; 33 100
0,6 mM ..... 2,0 mM ..... 10,0 mM .....
100 100
58 30
60 31
100 100 100
C1 13 850
0,2 mM ..... 0,5 mM ..... 1,3 mM .....
100 92
53 27
62 28
10o 100 100
C1 11 366
2,0 mM .....
100
45
46
50
Diamox
S p i n a c h c h l o r o p l a s t s p r e p a r e d as d e s c r i b e d b y W a l k e r a n d Hill (1967). C h l o r o p l a s t s f r a c t i o n a t i o n a c c o r d i n g to V e r n o n a n d S h a w (1971) w i t h 40 m g c h l o rophyll per g detergent. a) N A D P + r e d u c t i o n b y p h o t o s y s t e m s I ( w i t h a s c o r b a t e + D P I P ) w a s m e a s u r e d a c c o r d i n g to t h e s a m e a u t h o r s , e x c e p t t h a t p l a s t o c y a n i n a n d e x c e s s T r i t o n X-100 w e r e o m i t t e d ; c r u d e f e r r e d o x i n p r e p a r a t i o n , o b t a i n e d as r e p o r t e d u n d e r Methods, was added in saturating amount. Initial rate in controls amounted to 33 a n d 64 ~ m o l e s . h - 1 N A D P + r e d u c e d p e r m g c h l o r o p h y l l a t 25°C i n t w o separate photosystem I preparations. b) D P I P p h o t o r e d u c t i o n w a s c a r r i e d o u t a c c o r d i n g to V e r n o n a n d S h a w (1971). D P C (BDH) w a s r e c r y s t a l l i z e d f r o m h o t e t h a n o l . T h e i n i t i a l c o n t r o l r a t e f o r 2 s e p a r a t e p r e p a r a t i o n s w a s 109 a n d 215 rrmole.h-1 D P I P r e d u c e d p e r m g c h l o r o p h y l l a t 25°C w i t h w a t e r as t h e sole e l e c t r o n d o n o r ; ' w i t h D P C p r e s e n t at 0.5 raM, t h e r e d u c t i o n r a t e a m o u n t e d to 170 f o r o n e p r e p a r a t i o n a n d w i t h 1 m M D P C p r e s e n t , to 471 ;~mole-1 D P I P p e r m g c h l o r o p h y l l i n a n o t h e r p r e p a r a t i o n . T h e l i g h t e n e r g y W a s o b t a i n e d f r o m a 100 W w h i t e l i g h t t u n g s t e n b u l b p l a c e d at l 0 cn~' f r o m t h e p h o t o m e t e r c u v e t t e a n d t h e l a t t e r r e m o v e d e v e r y 30 sec l o r spectrophotometric absorbance measurements in a thermostated Zeiss spcctrophotometer. c) N A D P r e d u c t i o n b y a s c o r b a t e w a s m e a s u r e d w i t h t h e r e c o n s t i t u t e d s y s t e m of M a l k i n (197l) ; see t e x t .
BIOCHIMIE, 1972, 54, n ° 9.
Inhibition of photosynthesis by sulfonamides. ferredoxin. It has been r e p o r t e d by Malkin [15] that, a c c o r d i n g to this schema, photosystem II p r e p a r a t i o n s can c a r r y out electron t r a n s f e r from ascorbate to NADP ÷ p r o v i d e d p l a s t o c y a n i n , soluble f e r r e d o x i n and ferredoxin-NADP reductase are added. Using a crude f e r r e d o x i n extract o b t a i n e d by the method of San Pietro [8] as a source of the latter two factors, a n d p l a s t o c y a n i n p r e p a r e d a c c o r d i n g to Katoh [9], one photosystem II fraction r e d u c e d NADP + at the rate of 26.3 ~mole.h-* per mg c h l o r o p h y l l at 26°C ( i l l u m i n a t i o n as i n e x p e r i m e n t s of table II). The rate was u n c h a n g e d by the p r e s e n c e of 3.6 × 10-6 M D~CMU, or i n c r e a s ing c o n c e n t r a t i o n s of Diamox up to 10 raM, C1 13850 up to 1.3.5 mM a n d Methazolamide up to 3.3 raM. It m a y thus be c o n c l u d e d that the latter sulfonamide molecules are not i n t e r f e r i n g on the sequence from p l a s t o c y a n i n to NADP i n photosystem II, as it is conceived by A r n o n [14] ; this is i n agreement w i t h the results of table II, a n d the i n t e r f e r e n c e of these molecules at a step b e t w e e n water a n d c y t o c h r o m e b559 accounts for their effect on the water p h o t o r e d u c t i o n (table I). B) However, other thiadiazole sulfonamides, w h e r e an aromatic nucleus has been added on the 2-amino group in the thiadiazole ring, p r o v e d to i n h i b i t the electron t r a n s p o r t from p l a s t o c y a n i n to N A D P ; one of these molecules is C1 113,06, w h i c h i n h i b i t s 50 p. cent at 2 raM. We were thus led to suspect that a n o t h e r sulfonamide-sensitive site is p r e s e n t w i t h i n the latter sequence of elect r o n - h y d r o g e n transfer. I n order to c o n f i r m this observation, we decided to test the molecules on a r e c o n s i i t u t e d f e r r e d o x i n - - f e r r e d o x i n - N A D P reductase system fed electrons w i t h purified photosystems I or II. S p i n a c h f e r r e d o x i n was p u r i f i e d to an A420/A276 of 0.31, a c c o r d i n g to B u c h a n a n a n d A r n o n [16] a n d ferredoxin-NADP reductase (E.N.1.6.99.4) e n r i c h e d to an A456/A275 of 0.048 b y the p r o c e d u r e of Shin [17]. The w a s h e d chloroplast p r e p a r a t i o n utilized in the test described i n [17] was replaced by a photosystem I or a photosystem II fraction p r e p a r e d as reported i n table H. I l l u m i n a t i o n was o b t a i n e d as described i n table II. As expected, there was no electron t r a n s f e r from DPIP-ascorbate to NADP with photosystem II. Photosystem I p r o d u c e d 5,0 ~moles NADPH.h-1 per mg c h l o r o p h y l l at 25°C and this activity was i n h i bited by two of the h y d r o p h o b i c molecules (but not by C1 11366) r e p o r t e d above to act b e t w e e n p l a s t o c y a n i n a n d NADP (with ascorbate a n d photosystem II). The two photosystems differ as to the specific m e m b r a n e p h o t o r e d u c t i o n step, but
BIOCHIMIE, 1972, 54, u ° 9.
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they share in vitro the c o m m o n h y d r o g e n t r a n s f e r sequence from f e r r e d o x i n to NADP. The latter results seem to i n d i c a t e that the c o m m o n h y d r o g e n t r a n s f e r sequence, c o m p r i s i n g a m e m b r a n e - l i n k e d p h o t o r e d u c e d center, f e r r e d o x i n and f e r r e d o x i n NADP reductase, r e p r e s e n t s a second s u l f o n a m i d e sensitive site i n the chloroplast. Some specificity of the m e m b r a n e appears i n the different b e h a v i o r of C1 113,66 w h e n photosystem I or photosystem II is used to f u r n i s h the r e d u c i n g equivalents (table I I ) ; m o r e o v e r w h e n the m e m b r a n e p h o t o u n i t s were omitted a n d the f e r r e d o x i n .... ferredoxin-NADP reductase complex tested in the artificial sequence from NADPH to c y t o c h r o m e c (Shin [17]), n o n e of the molecules, s h o w n above to be active on the whole system, was i n h i b i t o r y . It m a y thus be c o n c l u d e d that some s t r u c t u r a l r e q u i r e m e n t s confers activity of s u l f o n a m i d e s t o w a r d s a site located at the m e m b r a n e - f e r r e doxin-flavoprotein level.
DISCUSSION. Several molecules s h o w n here (A) to interfere w i t h the p h o t o r e d u c t i o n sequence b e t w e e n w a t e r a n d c y t o c h r o m e b~.~9 in photosystem H (fig. 1) belong to s u l f o n a m i d e s b e a r i n g a thiadiazole nucleus, but one, F u r o s e m i d e , possesses a c h l o r i n a t e d a n t h r a n i l i c n u c l e u s ; several other s u l f o n a m i d e s are inactive, a n d one w h i c h i n h i b i t e d the NADP p r o d u c t i o n p r o v e d to 'activate the Hill r e a c t i o n (with f e r r i c y a n i d e ) by 100 p. cent. The activity on the ferredoxin-NADP sequence (B), on the other h a n d , seems to be a p r o p e r t y of some very h y d r o p h o b i c thiadiazole molecules, like CI 11366. Accordingly, it seems reasonable to admit that the s u l f o n a m i d e group c o m m o n to all the active molecules studied is the group i n t e r f e r i n g at specific sites, but that an a d d i t i o n a l s t r u c t u r a l c h a r a c t e r is r e q u i r e d for activity. A s i m i l a r v a r i a t i o n i n the i n h i b i t o r y p o w e r of a series of s u l f o n a m i d e s was r e p o r t e d in studies on c a r b o n i c a n h y d r a s e [18], m i t o e h o n d r i a l r e s p i r a t i o n [6] and x a n t h i n e oxidase [19], as may be a n t i c i p a t e d for the i n t e r a c tion b e t w e e n drugs a n d m e m b r a n e components. The s u l f o n a m i d e s described u n d e r A) do act on chloroplast fractions w h i c h are also sensitive to DCMU. F r o m the following c o n s i d e r a t i o n s , it seems that the latter sulfonamide-sensitive site is different from the DCMU-sensitive site : I)CMU raises the variable f u o r e s c e n c e of the s h o r t - w a v e l e n g t h photocenter, by i n h i b i t i n g its 83
I210
C. F r a n f o i s .
r e o x i d a t i o n [13]. P r e l i m i n a r y e x p e r i m e n t s s h o w e d that the sulfonamides do not raise the variable f l u o r e s c e n c e of t h e i s o l a t e d p h o t o s y s t e m I I f r a c t i o n . B e s i d e s , w e h a v e o b s e r v e d ( t a b l e II), t h a y b y r a i s i n g to 1 m M t h e c o n c e n t r a t i o n of D P C a d d e d as a n e l e c t r o n d o n o r to i s o l a t e d p h o t o s y s t e m II, t h e e l e c t r o n f l o w c o m p l e t e l y i n h i b i t e d b y DCMU, is r e e s t a b l i s h e d at 33 p. c e n t of t h e c o n t r o l v a l u e . T h i s is i n a c c o r d a n c e w i t h v a l u e s r e p o r t e d b y V e r n o n a n d S h a w [20], S h n e y o . u r a n d A r n o n [2I] and Malkin [15]. On the contrary, the inhibition by the sulfonamides was not relieved when DPC was added. If t h e s u l f o n a m i d e - s e n s i t i v e s i t e i n t h e n o n cyclic photosystem differs from the DCMU-sensit i v e site, w e feel t h a t t h e p r e s e n t i n v e s t i g a t i o n does not bring arguments capable of locating the sulfonamide:sensitive u n i t i n r e l a t i o n to t h e DCMU-sensitive unit, Although only some sulfonamides are active on t h e f e r r e d o x i n - N A D P s e q u e n c e ( e x p e r i m e n t s B), t h e s i m p l i c i t y of t h i s s y s t e m m a y r a i s e a n i n t e r e s t i n g s u g g e s t i o n . W e h a v e r e p o r d e d [61_ t h a t t h e m i t o c h o n d r i a l r e s p i r a t i o n is s u l f o n a m i d e - s e n s i t i v e . A d d i t i o n a l e x p e r i m e n t s (to b e p u b l i s h e d ) h a v e i n d i c a t e d t h a t t h e s e n s i t i v e s i t e is p r e s e n t i n t h e electron transport chain and the smallest sensitive f r a g m e n t i s o l a t e d is t h e p a r t i c u l a t e N A D H d e h y d r o g e n a s e , a c o m p l e x of f l a v o p r o t e i n w i t h a n o n h e i n e i r o n p r o t e i n . W e h a v e also r e c e n t l y e s t a b l i s h e d [19] t h a t s o m e e l e c t r o n - t r a n s p o r t p a t h w a y s i n x a n t h i n e o x i d o r e d u c t a s e s a r e s e n s i t i v e to s u l f o n amides: these enzymes are soluble models for complex electron transport chains and possess a flavine and a non-heme iron, besides persulfide group and molybdenum (Massey and Edmondson [22]). W e feel t h a t t h e l a t t e r s e n s i t i v i t y to s u l f o n a m i d e s of t h e c l o s e l y r e l a t e d n o n - h e m e i r o n flavoproteins points to the very similar ferredoxin-f e r r e d o x i n - N A D P r e d u c t a s e c o m p l e x as o n e t a r g e t for these inhibitors. No suggestion will be put forward about the n a t u r e of t h e o t h e r s e n s i t i v e site, b e c a u s e t h e s e n s i t i v e s e q u e n c e , f r o m w a t e r to c y t o c h r o m e b is m u c h t o o c o m p l e x ; to o u r k n o w l e d g e , n o flavop r o t e i n h a s e v e r b e e n d e m o n s t r a t e d to b e p a r t o f t h a t s e q u e n c e , a l t h o u g h M c K e n n a a n d B i s h o p [23] have suggested that an endogenous flavin may be i n v o l v e d i n t h e p h o t o o x i d a t i o n of Mn2+.
A cJcn~wledgments. We are g r a t e f u l to Prof. E. Schoffeniels for c o n s t a n t e n c o u r a g e m e n t a n d to Dr. J.-M. Michel for c a r r y i n g out the fluorescence tests. Diamox was given by Lederle (Brussels) ; C1 13850, C1 11366 a n d Methazola m i d e by the A m e r i c a n C y a n a m i d C y ; E s m a r i n by
BIOCHIMIE, 1'972, 54, n ° 9.
Merck ; F u r o s e m i d e b y Hoechst a n d Navidrex b y Ciba. This work was supported by g r a n t n ° 790 f r o m the Fonds de la Recherche fondamentale collective to Prof. E. Schoffeniels. RI~.SUM~. La p h o t o r d d u c t i o n de l ' e a u p a r des p r 6 p a r a t i o n s de ehloroplastes d'6pinard, leurs g r a n a ou leur photosyst~me non-cyclique isol6 est inhib6e p a r p l u s i e u r s sulf o n a m i d e s ; cette sensibilit6 n ' e s t pas en r e l a t i o n avec le pouvoir d ' i n h i b i t i o n de ces moI~cules sur F a n h y drase c a r b o n i q u e des chloroplastes ; il existe done u n site sensible, different de l ' a n h y d r a s e , d a n s la e h a l n e de t r a n s p o r t d'61ectrons du systbme p h o t o s y n t h 6 t i q u e non-cyclique. La sensibilit6 de la p h o t o r 6 d u c t i o n du COa aux s u l f o n a m i d e s ne d6pend done pas, ou pas uniquen~ent, d'une i n h i b i t i o n de l ' a n h y d r a s e carbonique. La sensihilit6 du systbme non-cyelique aux s u l f o n a mides semble diff~rente de sa sensibilit6 au DCMU. La s6quence de t r a n s p o r t ferredoxine-NADP r6ductase, ferredoxine, lamelles du systbme eyelique o u uon-cyclique, p l a s t o e y a n i n e a 6t6 reeonstitude h p a r t i r des unit6s isol6es. Elle est sensible h certaines mol6cules de sulfonamides, et l'identit6 de ce second site sensible avec la ferredoxine-NADP r~duetase est discut6e. ZUSAMMENFASSUNG.
Die P h o t o r e d u k t i o n des Wassers d u r c h Prfiparation e n yon C h l o r o p l a s t e n des Spinats, i h r e r G r a n a f r a k t i o n e n oder ihres isolierten n i c h t - zyklischen P h o t o sytems wird yon m e h r e r e n S u l f o n a m i d e n i n h i b i e r t ; diese Sensibilit~t ist nicht m i t dem I n h i b i t i o n s v e r m 6 gen dieser Molekfile a u f die C a r b o a n h y d r a s e d e r Chlor o p l a s t e n in E i n k l a n g zu bringen. Also existiert eine sensible Etappe, die sich yon der A n h y d r a s e u n t e r scheidet, in der T r a n s p o r t k e t t e d e r E t e k t r o n e n des n i e h t - z y k l i s c h e n p h o t o s y n t h e t i s c h e n Systems. Die Sensibilit~it der P h o t o r e d u k t i o n des CO~ gegeniiber Sulfona m i d e n hiingt also nicht, oder n i e h t allein, yon einer I n h i b i t i o n der C a r b o a n h y d r a s e ab. Die Sensihilit$it des n i c h t - z y k l i s c h e n Systems gegeniiber S u l f o n a m i d e n seheint yon der bet DCMU g e f u n d e n e n untersehiedlich. Die T r a n s p o r t s e q u e n z Ferredoxin-NADP-Reduktase, Ferredoxin, M e m b r a n des zyklisehen oder nieht-zyklisehen Systems, P l a s t o z y a n i n , w u r d e a u s g e h e n d aus den isolierten E l e m e n t e n r e k o n s t r u i e r t . Sic ist einigen Sulf o n a m i d m o l e k f i l e n gegenfiber empfindlich, u n d die !dentlt~it dieser zweiten sensiblen Etappe m i t der F e r r e d o x i n - N A D P - R e d u k t a s e w i r d h i e r er6rtet. REFERENCES. 1. Ikemori, M. :¢ Nishida, K. (1968) Physiol. Plantarum, 21, 292. 2. Everson, R. G. (1969) Nature, 222, 876. 3. Everson, R. G. ,~ Slack, C. R. (1968) Phytochemistry,, 7, 581-584. 4. Everson, R. G. (1970) Phytochemistry, 9, 25-32. 5. Hatch, M. D. a Slack, C. R. (1970) Proff. Phytochem., 2, 35-106. 6. Franqois, C. & Deprez, Ch. (1971) Arch. internat. Physiol. Biochim., 79, 993-1007. 7. Walker, D. A. a Hill, R. (1967) Biochim. Biophys. Acla, 131, 330-338. 8. San Pietro, A. (1963) In Meth. in Enzymology, Colo~vick, S. P. a Kaptan, N. O., eds, 6, 439-445. 9. Katoh, S. (1971) In Meth. in Enzymology, San Pietro, A., ed., 23, 277-289. 10. Strain, H. H., Cope, B. T. a Svec, W. A. (1971) In Meth. in Enzymology, San P i e t r 0 , A., ed., 23, 452-476.
Inhibilion of p h o l o s y n l h e s i s 11. Vernon, L. P. ~ Shaw, E. R. (197I) In Melh. il~ Enzymology, San Pietro, A., ed., 23, 277-289. 12. Avron, M. (1967) In Current Topics in Bioenergetics, Sanadi, D. R., ed., 2, 1-19. 13. B o a r d m a n , N. K. (1968) In Advances in Et~zymology, Nord, F. F., ed., 30, 2-79. 14. Arnon, D. I. (1971) Proc. Natl Acad. Sci., U.S.A., 68, 2883-2892. 15. Malkin, R. (1971) Biochim. Biophys. Acta, 253, 421-427. 16. B u c h a n a n , B. B. ~ Arnon, D. I. (1971) In Meth. in Enzymology, San Pietro, A., ed., 23, 413-440.
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(1971) In Meth. in Enzymology, San A., ed., 23, 440-447. H. (1967) Physiol. Bey., 47, 595. C. (1972) Arch. internat. Physiol. Biochim., 80, 600-601. Vernon, L. P. • Shaw, E. R. (1969) Biochem. Biophys. Res. Commun., 36, 878-884. S h n e y o u r , A. ~ Avron, M. (1971) Biochim. Biophys. Acta, 253, 412-420. Massey, V. t~ E d m o n d s o n , D. (1970) J. Biol. Chem., 245, 6595-6598. McKenna, J. M. ,~ Bishop, N. I. (1967) Biochim. Biophys. Acta, 131, 339-349.
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