NMR Studies on Chloroplast Membranes

38 N M R Studies on Chloroplast Membranes T. WYDRZYNSKI and GOVINDJEE

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Department of Physiology and Biophysics, University of Illinois, Urbana, Ill. 61801 †

N. ZUMBULYADIS, P. G. SCHMIDT, and H. S. GUTOWSKY Department of Chemistry, University of Illinois, Urbana, Ill. 61801

In green plant photosynthesis the mechanism by which water is photo-oxidized and oxygen is produced still remains largely unsolved (see review 1). However, it is known that manganese is directly involved (2). Inasmuch as the unpaired electron spin of Mn(II) can lead to large increases in magnetic relaxation rates of nuclei bound near the ion, it appeared to us that manganese would be a natural paramagnetic probe and that proton magnetic relaxation could be used to study the oxygen evolving mechanism. In this communication we present our initial findings, some of which have been reported earlier (3, 4). The results indicate that a significant contribution to proton relaxation rates of chloroplast membrane suspensions does arise from interactions with membrane-bound manganese. Furthermore light-induced changes in the relaxation rates suggest that proton relaxation is monitoring the oxygen evolving system. Materials and Methods Chloroplast Preparation. Chloroplast thylakoid membranes were isolated either from commercial spinach (Spinacea oleracea) or green house grown peas (Pisum sativa) in a medium consisting of 50 mM N-2-hydroxyethylpiperazine-N -2-ethanesulfonic acid (HEPES) buffer adjusted to pH 7.5 with NaOH, 400 mM sucrose and 10 mM NaCl. The chloroplasts were given an osmotic shock in a similar medium containing 100 mM sucrose and finally resuspended in the original isolation medium. Chlorophyll concentration was adjusted to 3 mg Chl/ml in all samples. 1

Nuclear Relaxation Measurements. The inversion recovery method (180° - τ - 90° sequence) was used to determine the spin lattice relaxation rate (T ). The spin-spin relaxation rate (T ) was measured from the exponential decay of the echo am plitudes in a Carr-Purcell (Meiboom-Gill modification) (CPMG) train of rf pulses. The experimental uncertainties in T and T data are within +5%. †Present Address : Research Laboratories, Eastman-Kodak Company, Rochester, New York 14650 -1

1

-1

2

-1

1

-1

2

471

Resing and Wade; Magnetic Resonance in Colloid and Interface Science ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

472

MAGNETIC

RESONANCE

In order to measure 1i ght-î nduced changes the nmr probe was designed to provide the best o p t i c a l geometry w h i l e s t i l l maint a i n i n g a good s i g n a l - t o - n o i s e r a t i o . A t i g h t f i t t i n g P l e x i g l a s plug was i nserted into the bottom of a 12 mm nmr tube to support a t h i η l a y e r of sample (~ 100 μΐ t o t a l volume) i n the region of the nmr c o i l s . I l 1umi nat ion was f rom the top. Thi s arrangement a 11 owed f o r a large surface area and hence maximum a b s o r p t i o n of l i g h t by the whole sample. In the f l a s h i ng 1 ight experiments T2*" was measured a f t e r a sequence of l i g h t f l a s h e s . The CPMG t rain was i ni tîated s imultaneously w i t h the l a s t 1ight f l a s h of the sequence. The time i n t e r v a l between success i ve f l a s h e s i n a sequence was 2 sec. A dark adaptât ion per iod of 7 mi η was allowed between each sequence of l i g h t f l a s h e s . Although t h i s procedure i s somewhat m o d i f i e d from the one u s u a l l y employed to measure oxygen, we found that i t d i d not a f f e c t the oxygen y i e l d p a t t e r n . Li ght f l a s h e s were obta i ned from a strobe l i g h t (Strobotac type 1538-A, General Radio Co.) and were of short durât ion (2.4 Msec a t h a l f height w i t h an extended t a i 1 up to 10 Msec). 1

ResuIts and P i s c u s s i o n Paramaqnet i c Contribut ions to the Water Proton Relaxât ion Rates of C h l o r o p l a s t Membrane Suspensions. Suspens ions of da rkadapted c h l o r o p l a s t membranes have a large e f f e c t on the water proton relaxât ion r a t e s . Upon wash i ng the memb ranes twi ce i n b u f f e r medium Τ," decreases i n general by about 50$. Simple wash i ng u s u a l l y has 1i t t l e e f f e c t on c h l o r o p l a s t a c t i v i t y but undoubtedly serves to remove l o o s e l y bound paramagnetic ions. Howeve r, not al1 ions are removed ; f o r example, i t has been r e ported that about 35$ of the manganese i s l o s t upon repeated washings ( 5 ) . Washed c h l o r o p l a s t s represent the c o n t r o l i n the f o l l o w i ng experiments. In washed c h l o r o p l a s t s any paramagnetic c o n t r i b u t i o n t o water proton relaxât ion wi11 depend on the access ib i 1 i t y of water to the t i g h t l y bound metal ions i n the membrane. When EDTA i s added to washed c h l o r o p l a s t s the relaxât ion rates decrease. For examp1e, at 26 MHz and 26°C 1 mM EDTA reduces Τ,~ to about one f o u r t h of the c o n t r o l value (TABLE I ) . As shown i n TABLE I the magnitude of the e f f e c t of EDTA depends to a large degree on temperature and nmr f requency. I t appears f rom these resu1ts that the t i g h t l y bound paramagnet i c ions do have a major i n f l u ence on the relaxât ion rates i n washed c h l o r o p l a s t s . For a system such as c h l o r o p l a s t membranes the measu red relaxation rate, T ~ ) ^ (or T 2 " ) bs) cons i de red as the sum of c o n t r i b u t i o n s from a l 1 s i tes i n the membrane access i b l e t o the s o l v e n t water, plus the r e l a x a t i o n rate of f ree water: 1

1

1

t

c a n

1

Q

S

D e

0

P.

Τ," ) obs = Τ? Τ 7 + i »I 1

T V ' ) f ree


(D


Conditions

c

Chloroplasts

0. 18

0.20 0.18

0.56 0.16

38°

0. 19

0. 75 0. 15

25°

2.80 2.45 2.30

0.21

26°

0.78 0.40

8°

2.24

2.94 2.89

2

T -'

8°

a

2.29

2.69 2.34

38°

2.27

3.02 2.42

24°

1

(sec- ) 26 MHz

2.39

3. 34 2.64

9. 5°

R a t e s o f Pea C h l o r o p l a s t s

16 MHz

Relaxation

C h l o r o p l a s t s w e r e t r e a t e d w i t h t rî s - a c e t o n e med i urn a c c o r d i ng t o p r o c e d u re o f Yamash i t a and Tomi t a (9) and t h e n w a s h e d o n c e wi t h b u f f e r m e d i a .

' A f t e r î s o l a t i o n c h l o r o p l a s t s w e r e washed t w i c e i n b u f f e r medium and r e s u s p e n d e d t o a concentrât i o n o f 3 mg c h l o r o p h y l 1 / m l .

Relaxât i o n r a t e s c o r r e c t e d by s u b t r a c t i ng r a t e s o f b u f f e r medium f rom o b s e r v e d r a t e s o f c h l o r o p l a s t suspens i o n s .

T r i s - A c e t o n e Washed

EDTA

8°

1

T,"' ( s e c " ) 26 MHz

0. 44 0. 22

a

26°

16 MHz

Washing on Water Proton

0.50 0.14

E f f e c t o f EDTA a n d T r i s - A c e t o n e

^Washed C h l o r o p l a s t s Washed C h l o r o p l a s t s + 1 mM

TABLE I .

474

MAGNETIC

RESONANCE

where P\ i s the f r a c t i o n of water i n s i t e i . Most water i s f r e e (Pf » 1) and T, " ) f e ' taken as the r e l a x a t i o n rate i n the b u f f e r medium without c h l o r o p l a s t s . The q u a n t i t y "Π " ) T, ) f ee • t h e r e f o r e the r e l a x a t i o n c o n t r i b u t i o n due t o the mem branes and i s denoted simply T, " ( o r T 2 " ) i n t h i s communication. In macromolecular systems T j " o f H 0 i s u s u a l l y influenced most s t r o n g l y by paramagnetic s i t e s . The r e l a x a t i o n rate T, " o f water a t such a s i t e i s u s u a l l y dominated by e l e c t r o n nuclear dipole-dipole interactions: 1

s

r e e

r e

1

O

D

S

s

r

1

1

1

2

1

m

2

,

2

Y 9 S(S+l)p

2

3T

2

I

Ρ

ï^"Ï5

7T

C

c

+

(2)

where Y j i s the nuclear magnetogyric r a t i o , S i s the t o t a l e l e c t r o n s p i n , g i s the e l e c t r o n i c g - f a c t o r , β i s the Bohr magnetron, r i s the distance between the nucleus and the paramagnetic i o n , uuj and uu are the nuclear and e l e c t r o n i c Larmor frequencies r e s p e c t i v e l y , and T i s the c o r r e l a t i o n time. The d i p o l e - d i p o l e i n t e r a c t i o n may be modulated by any o f several time-dependent processes such t h a t : s

c

J_ T

=

_L J _ +

T

c

s

T

R

J_

+ T

M

where T s i s the e l e c t r o n i c r e l a x a t i o n time, i s the r o t a t i o n a l c o r r e l a t i o n time and τ i s the exchange l i f e t i m e . The s h o r t e s t of these c o r r e l a t i o n times dominates. An expression s i m i l a r t o (2) can be obtained f o r the s p i n s p i n r e l a x a t i o n r a t e , T " , but contains a d d i t i o n a l terms asso c i a t e d with s c a l a r c o u p l i n g not u s u a l l y important f o r T," . Μ

1

2 m

1

, γ VS(S+1)0 T

2 i m

15

r

6

2

v

3T c

13T

l+uuj

S e where A i s the e l e c t r o n - n u c l e a r hyperf i ne c o u p l i n g constant and T i s the c o r r e l a t i o n time f o r the s c a l a r i n t e r a c t i o n . e

C h l o r o p l a s t Manganese and Water Proton R e l a x a t i o n , Several treatments are known t o a f f e c t c h l o r o p l a s t managnese (e.g. see r e f . 6 ) . For example, washing c h l o r o p l a s t s w i t h 0.8 M t r i s (hydroxymethyl) aminomethane ( t r i s ) b u f f e r a t pH >8 a l t e r s the en vironment o f manganese such that a Mn(Il) e s r s i g n a l appears ( 7 ) . The current hypothesis i s that some o f the manganese i s released to the i n s i d e o f the membrane v e s i c l e ( 6 ) , but i s not removed


38.

Chforoplast Membranes

wYDRZYNSKi ET AL.

475

from the c h l o r o p l a s t s . However, t h e amount o f manganese a f f e c t e d is s t i l l a matter of controversy (see r e f . 6 ) . Nevertheless, t r i s w a s h i n g i n a c t i v a t e s t h e o x y g e n e v o l v i n g mechanism, b u t leaves t h e r e s t o f t h e e l e c t r o n t r a n s p o r t c h a i n i n t a c t and f u n c t i o n a l t o t h e e x t e n t t h a t p h o t o r e d u c t i o n o f NADP+ can be r e s t o r e d by a d d i n g exogenous e l e c t r o n d o n o r s ( 8 ) . T h i s s u g g e s t s t h a t t r i s w a s h i n g does not a f f e c t o t h e r p a r a m a g n e t i c c e n t e r s i n t h e e l e c t r o n t r a n s p o r t c h a i n up t o t h e p r i m a r y a c c e p t o r o f P h o t o s y s t e m I . We f i n d t h a t t r i s w a s h i n g g e n e r a l l y c h a n g e s T," of c h l o r o p l a s t s , but t h a t t h e m a g n i t u d e and d i r e c t i o n o f t h e change v a r y w i t h t h e source of the p l a n t m a t e r i a l . Some e x a m p l e s a r e shown i n TABLE II. 1

TABLE I I .

E f f e c t o f T e t r a p h e n y l b o r o n and T r i s - W a s h i n g on P r o t o n T|" of Spinach C h l o r o p l a s t s

Water

1

a

Conditions 1 Washed C h l o r o p l a s t s Washed C h l o r o p l a s t s + 5 mM °Tris Washed C h l o r o p l a s t s T r i s Washed C h l o r o p l a s t s +5 mM TPB"

TPB"

T,-' (sec) Sample No. 2 3

4

0.86 1.64

0.90 1.70

1.04

1.03

0.82 0.88

-

1.36

0.41

R a t e s c o r r e c t e d by s u b t r a c t i n g r a t e s o f b u f f e r medium f r o m obs e r v e d r a t e s o f c h l o r o p l a s t s u s p e n s i o n s . Measurements were made a t 26 MHz, 24°C. b

As

C

Trî s wash î ng a c c o r d i ng t o p r o c e d u re o f Yamash i t a and

i n TABLE I . Butler

(8).

R e c e n t l y Y a m a s h i t a and Tomi t a ( 9 ) have f o u n d t h a t a more c o m p l e t e e x t r a c t i o n o f manganese f r o m t h e membrane i s o b t a i n e d when 20$ a c e t o n e i s i n c l u d e d d u r i ng t r i s w a s h i ng. Aga i η p h o t o r e d u c t i o n o f NADP+ can be r e s t o r e d wi t h added e l e c t r o n d o n o r s . When c h l o r o p l a s t s a r e t r e a t e d i n t h i s way T j ~ ' and T ""' i s cons i d e r a b l y r e d u c e d (TABLE I ) . I n t e r e s t i n g l y , t h e r a t e s do n o t show e i t h e r a marked f r e q u e n c y o r t e m p e r a t u r e d e p e n d e n c y . A l t h o u g h t h e s e r e s u l t s i n d i c a t e t h a t bound manganese does i n f l u e n c e t h e p r o t o n relaxât i o n , c o n t r i b u t i o n s f r o m o t h e r p a r a m a g n e t i c c e n t e r s c a n n o t be r u l e d o u t ; however, t h e y p r o b a b i y do not have a domi n a t i ng e f f e c t . F o r e x a m p l e , t h e c o p p e r bound i n p l a s t o c y a n i n, a component o f t h e e l e c t ron t r a n s p o r t c h a i n, i s n o t a c c e s s i b l e t o t h e b u l k w a t e r and has 1i t t l e e f f e c t on o b s e r v e d water proton r e l a x a t i o n rates (JO). Wi t h r e s p e c t t o i r o n , h i g h s p i n F e ( I I ) and h i g h and low s p i n F e ( I I I ) have much f a s t e r e l e c t r o n i c relaxât i o n r a t e s t h a n M n ( I I ) and a re l e s s e f f i c i e n t i n relaxât ion by c o m p a r i s o n ( 1 1 ) . 2


476

MAGNETIC RESONANCE

W a t e r P r o t o n R e l a x a t i o n as a M o n i t o r o f Manganese O x i d a t i o n States. I t i s n o t known what o x i d a t i o n s t a t e s o f manganese e x i s t i n c h l o r o p l a s t membranes. The e l e c t r o n i c r e l a x a t i o n r a t e , how e v e r , i s s t r o n g l y dependent on the o x i d a t i o n s t a t e . For example, t h e v a l u e s o f T f o r M n ( I I ) a r e g e n e r a l l y IO" - IO" s e c , depend i n g o n t h e nmr f r e q u e n c y a n d c h e m i c a l e n v i r o n m e n t ( J J _ ) . Mn(III), on t h e o t h e r h a n d , h a s a much s h o r t e r e l e c t r o n s p i n r e l a x a t i o n t i m e . A r e c e n t s t u d y by V i 1 l a f r a n c a e t . a l . (12) y i e l d e d a v a l u e o f T ¥ 3 χ 10"' s e c f o r M n ( I I I ) bound t o a s u p e r o x i d e d i m u t a s e f r o m JE. c o l i . This d i f f e r e n c e i n T i s s u f f i c i e n t t o account f o r a much g r e a t e r r e l a x a t i o n e f f e c t by M n ( I I ) t h a n M n ( l l l ) . I f the e l e c t r o n i c r e l a x a t i o n o f metal ions i s dominating the p r o t o n r e l a x a t i o n i n c h l o r o p l a s t membranes, t h e n c h a n g e s i n o x i d a t i o n s t a t e w i l l be r e f l e c t e d i n t h e r e l a x a t i o n r a t e s . O x i d a t i o n s t a t e s o f bound i o n s c a n be s h i f t e d by a d d i n g redox r e a g e n t s . B u t many r e d o x r e a g e n t s upon o x i d a t i o n o r r e duction give r i s e t o f r e e r a d i c a l intermediates which could i n t e r f e r e wi t h t h e p r o t o n r e l a x a t i o n r a t e s . One r e d u c t a n t w h i c h does n o t a p p e a r t o f o r m f r e e r a d i c a l i n t e r m e d i a t e s i s t h e t e t r a phenylboron anion (TPB"). The o x i d a t i o n o f TPB" i s a two e l e c tron transfer (13): 8

9

s

1

s

s

B(C H )r ^-^iCôHsh + B(C H ) 5

6

6

B(C H ) 6

5

2

+

+ HOH

5

+

» ( C H ) B0H + H 6

5

+ 2e"

2

+

2

TPB i s known t o a c t a s a r e d u c t a n t i n t h e o x y g e n e v o l v i n g system o f c h l o r o p l a s t s (14, 15). When TPB" i s added t o t h e c h l o r o p l a s t s u s p e n s i o n , T," i n c r e a s e s (TABLE I I ) . F i g u r e 1 shows T|" a s a f u n c t i o n o f TPB" c o n c e n t r a t i o n i n unwashed chloroplasts. The t i t r a t i o n c u r v e shows s e v e r a l p l a t e a u s w h i c h may be i n d i c a t i v e o f s e v e r a l f r a c t i o n s o f i o n s b e i n g s u c c e s s i v e l y r e d u c e d by TPB". TPB" i t s e l f h a s no e f f e c t on t h e b u f f e r medium. I n t e r e s t i n g l y , TPB" a l s o h a s no e f f e c t i n t r i s - w a s h e d chlorop l a s t s (TABLE I I ) s u g g e s t i n g t h a t i t i s a c t i n g o n manganese i n volved in 0 evolution. I n a number o f c a s e s i t has been f o u n d ( 1 1 ) t h a t T o f M n ( I l ) and o t h e r p a r a m a g n e t i c i o n s depends on t h e s t r e n g t h o f t h e a p p l i e d m a g n e t i c f i e l d . The v a l u e o f T IS d e t e r m i n e d by c r y s t a l l a t t i c e f i e l d f l u c t u a t i o n s having a c o r r e l a t i o n t i m e , T , such that: 1

1

2

s

S

v

s

s

ν

s

ν

where Β i s a c o n s t a n t c o n t a i n i n g t h e v a l u e o f t h e r e s u l t a n t e l e c t r o n i c s p i n and the z e r o f i e l d s p l i t t i n g parameters. A t low m a g n e t i d f i e l d s T i s o f t e n t h e s h o r t e s t c o r r e l a t i o n s


38.

Chtoroplast Membranes

wYDRZYNSKi ET AL.

477

t i m e and domi n a t e s t h e r e l a x a t i o n o f n u c l e i bound t o t h e p a r a m a g n e t i c s i t e s ( E q . 3 and 4 ) . A t h i g h e r f i e l d s t r e n g t h s T can i n c r e a s e and TR o r may t h e n become t h e d o m i n a n t c o r r e l a t i o n time. T|" r e a c h e s a maximum as t h e f i e l d i n c r e a s e s and t h e n declines. On t h e o t h e r h a n d , T ~ has terms d e p e n d i n g d i r e c t l y on T ( E q . 3 ) . F o r t h e c a s e o f a f i e l d d e p e n d e n t T J -I \ f o u n d t o i n c r e a s e t o a p l a t e a u as t h e magnet i c f i e l d i n c r e a s e s . F i g u r e 2 shows t h e f r e q u e n c y dependence f o r T," and T ~' f o r a normal c h l o r o p l a s t s u s p e n s i o n and f o r one c o n t a i n i n g 5 mM TPB". The T j " f o r normal c h l o r o p l a s t s shows a b r o a d maximum and t h e n a s l o w d e c l i ne as t h e nmr f r e q u e n c y i s i n c r e a s e d . How ever, the T " increases s i g n i f i c a n t l y at the h igher f requencies. Thi s b e h a v i o r does s u g g e s t t h a t e l e c t r o n i c r e l a x a t i o n domi n a t e s t h e p r o t o n r e l a x a t i o n i n normal c h l o r o p l a s t s . However, t h e l a c k of a d i s t i n c t peak i n T|" is peculiar. T h i s may i n d i c a t e t h e e x i s t e n c e o f a d i s t r i b u t i o n o f c o r r e l a t i o n t i m e s . On t h e o t h e r hand when TPB" i s added t o t h e c h l o r o p l a s t s , T,~ and T " show a f r e q u e n c y dependence d i s t i n c t l y c h a r a c t e r i s t i c o f e l e c t r o n i c domination of proton r e l a x a t i o n . The c o r r e l a t i o n t i m e c a l c u l a t e d a t t h e peak i n T " w i t h TPB" i s a p p r o x i m a t e l y 6 χ 1 0 " s e c a t 24 MHz, w h i c h i s w i t h i n t h e e x p e c t e d range o f T f o r M n ( I I ) . T h i s r e s u 1 1 i s c o n s i s t e n t wi t h t h e i dea t h a t TPB" r e d u c e s a f r a c t i o n o f manganese i n a h i g h e r o x i d a t i o n s t a t e t o a l o w e r o x i d a t i o n s t a t e wh i c h i s more e f f i c i e n t i n ρ r o t o n relaxât i o n . s

1

1

2

S >

c

2

S

1

2

1

1

2

1

1

1

2

1

9

t

s

L i g h t E f f e c t s on Water P r o t o n Relaxât i o n R a t e s o f C h l o r o p l a s t Membranes: R e l a t i o n s h i p t o t h e Oxygen E v o l v i n g Mechanism. In a s e r i e s o f m i c r o s e c o n d 1 i g h t f l a s h e s t h e y i e l d o f o x y g e n e v o l v e d f r o m i s o l a t e d c h l o r o p l a s t s o r w h o l e a l g a l e e l 1 s shows a dampled o s e i 1 l a t o r y p a t t e r n , h a v i ng a p e r i o d o f f o u r wi t h peaks a f t e r t h e 3 r d , 7 t h , and 11th f l a s h e s ( ] _ ) . Based on t h i s u n i q u e p a t t e r n Kok and c o - w o r k e r s ( 1 6 ) have p r o p o s e d a f o u r s t e p model i n wh i c h some c h e m i c a l i n t e r m e d i a t e a c c u m u l a t e d up t o f o u r o x i d i z i ng e q u i v a l e n t s upon s u c c e s s i v e p h o t o a c t i v a t i o n s o f the o x y g e n evolving centers:

So

-i^U s,

s

s

2

3

J h L

-^

s I

4

t 4H+

+ 0 *^"

^ 2

2

() 5

HOH

Here S i n d i c a t e s t h e o x i d a t i o n s t a t e o f t h e i n t e r m e d i a t e ; S r e p r e s e n t s t h e most o x i d i z e d s t a t e . The ρ r i ma r y p h o t o r e a c t i o n of t h e o x y g e n e v o l v i ng s y s t e m i s t h e e x c i t a t i o n o f t h e r e a c t i o n center chlorophyl1 molecule P , w h i c h i s o x i d i z e d upon r e d u c t i o n o f t h e p r i m a r y e l e c t r o n a c c e p t o r Q; P o t h e n r e c e i v e s an e l e c t r o n from the S i n t e r m e d i a t e , perhaps v i a another i n t e r m e d i a t e l a b e l e d Ζ ( f o r d e t a i l s see r e v i e w , Γ7). When f o u r o x i d i z i n g e q u i v a l e n t s have a c c u m u l a t e d and t h e S 4 s t a t e i s f o r m e d , two w a t e r m o l e c u l e s r e a c t t o p r o d u c e o x y g e n and t h e o r i g i n a l So 4

6 8 0

+

6 8


478

MAGNETIC RESONANCE

state. The i d e n t i t y of the charge accumulating intermediate i s unknown, although i t has been suggested t o i n v o l v e manganese (-2, 18-20). However, there has been no d i r e c t experimental evidence t o show that c h l o r o p l a s t manganese undergoes changes in o x i d a t i o n s t a t e during photosynthesis. Data from previous s e c t i o n s i n d i c a t e that proton r e l a x a t i o n monitors membrane-bound manganese and suggest that the r e l a x a t i o n rates are s e n s i t i v e t o changes in o x i d a t i o n s t a t e s . To determine whether proton re l a x a t i o n could be r e l a t e d t o the oxygen e v o l v i n g mechanism we measured the s p i n - s p i n r e l a x a t i o n rate i n b r i e f f l a s h e s o f l i g h t . Figure 3 shows T2*" o f c h l o r o p l a s t membranes as a f u n c t i o n of f l a s h number ( 4 ) . S i m i l a r data have been obtained from seven other preparations o f spinach and l e t t u c e c h l o r o p l a s t s . The o s c i l l a t o r y p a t t e r n f o r T 2 " shows some s t r i k i n g s i m i l a r i t i e s t o the oxygen y i e l d p a t t e r n . As i n oxygen measurements, maxima occur a f t e r the 3rd, 7th, 11th and 15th f l a s h e s . A l s o , the T "' o s c i l l a t i o n s damp out a f t e r the 17th f l a s h , corresponding t o a s i m i l a r damping o f the o s c i l l a t i o n s i n the oxygen y i e l d . These important p a r a l l e l s i n the two types o f data s t r o n g l y imply that proton r e l a x a t i o n i s monitoring the oxygen-evolving mechanism. However, there are some s i g n i f i c a n t d i f f e r e n c e s . A f t e r the f i r s t f l a - h where no oxygen i s evolved, the r e l a x a t i o n rate shows a large decrease which has no subsequent counterpart. Minima i n the r e l a x a t i o n rates then occur a f t e r the 4 t h , 8 t h , 12th, e t c . f l a s h e s . Minima i n the oxygen y i e l d , on the o t h e r hand, occur a f t e r the 6 t h , 10th, 14th, e t c . f l a s h e s . The r e l a x a t i o n rates s t e a d i l y increase from the 4th t o the 7th f l a s h , from the 8th t o the 11th f l a s h and so on, w h i l e the trend i s the opposite f o r oxygen e v o l u t i o n , the y i e l d s t e a d i l y dropping from the 3rd t o the 6th f l a s h and from the 7th t o the 10th f l a s h . These d i f f e r ences i n T "' and oxygen y i e l d patterns may be explained on the basis that the r e l a x a t i o n rates d i f f e r f o r each o f the S s t a t e s whereas oxygen e v o l u t i o n only takes place during the S 4 t o S trans i t ion. The time s c a l e s f o r the formation ( t , / ~ 600 Msec) and l i f e times ( t , / ~ 10-30 sec) o f the i n d i v i d u a l S s t a t e s are s u f f i c i e n t l y d i f f e r e n t (X) from the s p i n - s p i n r e l a x a t i o n times ( T ~ 100 msec) so as not t o introduce a complex behavior i n the T data. The s p i n echo amplitudes always y i e l d a s i n g l e exponential decay. The changes i n T ~* are not caused by the oxygen produced i n photosynthesis. We estimated that the amount o f oxygen produced a f t e r the t h i r d f l a s h i s less than 4$ of the t o t a l oxygen present in the sample when e q u i l i b r a t e d as i t i s w i t h the a i r . The amount was c a l c u l a t e d t o have l e s s than 1$ e f f e c t on T ~' whereas the maximum l i g h t - i n d u c e d changes are about 20$. Figure 4 shows T 2 " as a f u n c t i o n of f l a s h number f o r t r i s washed c h l o r o p l a s t s . There i s an i n i t i a l l i g h t - i n d u c e d decrease in Τ "' , but the o s c i l l a t i o n s are absent. As pointed out e a r l i e r , 1

1

2

2

0

2

2

2

2

2

2

1

2


Chloroplast

38. wYDRZYNSKi ET AL. 1

~i

τ

ι

1

5

6

Membranes 1

1

1

479

1

1

Γ

ι

1

r

4.0 h

S* 3.0 h

2,0

4

7

8

10

9

II

12

Λ-

75

50

100

Tetraphenylboron Concentration, mM

Figure 1. T,' measured as a function of tetraphenylboron (TPB) concentration in unwashed spinach chloroplasts. Measurements were made at 26.9 MHz, 24°C. 1

I

τ

i

1

I

1

) With T P B

.

-

•

5

O

1

-

• v.

Control

4

. 3

_

/ o

if

-

1 With T P B

1 1

1

10

20

1

30

I

40

I

50

I

oT, Control

!

60

70

80

90

v/MHz Figure 2. Frequency dependence of T/ and TV for a spinach chloro plast suspension and for one containing 5mM tetraphenylboron (TPB); 26°C. J

1


480

MAGNETIC RESONANCE

I

1

I

I

I

1

MI

-, I

I

2

3

4

5

6

7

I

8

1

9

I

I

10 II

I

I

1

1

1

«

«

1

1

1—

12 13 14 15 16 1? 18 19 20 21

Flash Number Biochimica Biophysica Acta

Figure 3. Τ / measured as a function of number of light flashes in unwashed spinach chloroplasts. The procedure is given in "Materials and Methods." Measurements were made at 26.9 MHz, 24°C (5). 1

τ

r

1

1

1

1

1

r

Flash Number Figure 4. TV measured as a function of number of light flashes in washed and tris-washed lettuce chloroplasts. Measurements were made at 26.9 MHz, 24°C. 1


38.

w Y D R Z Y N S K i ET AL.

481

Chloroplast Membranes

t r i s wash î ng î n a c t î v a t e s t h e o x y g e n - e v o l v î ng a p p a r a t u s , b u t l e a v e s t h e r e s t o f t h e e l e c t r o n t r a n s p o r t c h a i n I n t a c t and f u n c tional . A l t h o u g h s t e p w i s e c h a n g e s In manganese o x l d a t i o n s t a t e s and c o n s e q u e n t c h a n g e s In e l e c t r o n l c r e l a x â t i o n t i m e c o u l d p r o v i de a s i m p i e q u a i i t a t Ive e x p l a n a t i o n o f t h e f l a s h i n g 1 i g h t r e s u l t s , o t h e r mechanisms c o u l d l e a d t o o s c i 1 l a t i o n s i n T ~ . Such p o s s i b i 1 i t i e s i n c l u d e d i f f e r e n c e s In t h e a c c e s s o f w a t e r t o t h e bound paramagnet i c i o n s and m o d i f i c a t i o n s In c h e m i c a l e x c h a n g e r a t e s as an i n d i r e c t r e s u 1 1 o f change a c c u m u l a t i o n . We hope t h a t f u r t h e r e x p e r i m e n t s w i l l c l a r i f y t h e mechani sm i n v o l v e d . 1

2

C o n c l u d i ng Remarks C h l o r o p l a s t membranes r e p r e s e n t an e x t r e m e l y comp1 ex s y s t e m f o r p h y s i c a l chemi c a l s t u d i e s . U n f o r t u n a t e l y , e x p e r i m e n t s on t h e o x y g e n e v o l v i ng mechanIsm a r e c o n f i ned t o t h e use o f i n t a c t mem b r a n e s as t h e o x y g e n e v o l v i ng c a p a c i t y i s r a p i d l y l o s t i n a t tempts t o i so l a t e submemb rane ρ r o t e i η f r a g m e n t s . NMR r e l a x â t i o n measurements o f w a t e r i n c h l o r o p l a s t s u s p e n s i o n s In p a r t r e f l e c t t h e s y s t e m ' s comp1 e x i t y . On t h e o t h e r h a n d , some s i m p l I f i c a t i o n Is a c h i e v e d I η t h a t t h e m a j ο r c o n t r I but Ion t o r e l a x â t i o n enhancement a p p e a r s t o be membrane bound manganese i o n s . Most i m p o r t a n t 1 y t h e p a t t e r n o f r e l a x â t i o n r a t e ( T ~ ) i n f l a s h i ng l i g h t b e a r s c l o s e s i m i l a r i t i e s t o t h e o x y g e n y i e l d d e m o n s t r a t i n g t h a t nmr c a n s e r v e as a p r o b e o f t h e o x y g e n e v o l v i ng mechani sm. The deta11 s u n d e r l y i n g t h i s o b s e r v â t i o n a re t h e s u b j e c t o f o u r cu r r e n t w o r k . 1

2

Acknowledgements We t h a n k t h e Nat i o n a l S c i e n c e Foundat i o n f o r f i n a n c i a l s u p p o r t t o G (GM 36751) and t o HSG (MPS 7 3 - 0 4 9 8 4 ) , t h e N a t i o n a l I n s t i t u t e s o f H e a l t h t o PGS (GM 18038) and t h e O f f i c e o f Naval R e s e a r c h t o HSG (NR 0 5 6 - 5 4 7 ) . TW was s u p p o r t e d by HEW PHS GM 7283-1 (Sub P r o j . 604) t r a i n i n g g r a n t i n C e l l u l a r and M o l e c u l a r Β iology.

Literature Cited 1. 2. 3. 4. 5.

Joliot, P. and Kok, B. in "Bioenergetics of Photosynthesis" (Govindjee, ed.) pp. 387-412, Academic Press, New York (1975). Cheniae, G. Ann. Rev. Plant Physiol. (1970) 21, 467-498. Wydrzynski, T., Zumbulyadis, Ν., Schmidt, P. G. and Govindjee Biochim. Biophys. Acta (1975) 408, 349-354. Wydrzynski, T., Zumbulyadis, Ν., Schmidt, P. G., Gutowsky, H. S. and Govindjee Proc. Natl. Acad. Sci., U.S.A. (1976) 73, 1196-1198. Blankenship, R. Ε., Babcock, G. T. and Sauer, K. Biochim.


482

MAGNETIC RESONANCE

Biophys. Acta (1975) 387, 165-175. Blankenship, R. E. and Sauer, K. Biochim. Biophys. Acta (1974) 357, 252-266. 7. Lozier, R., Baginsky, M. and Butler, W. L. Photochem. Photobiol. (1971) 14, 323-328. 8. Yamashita, T. and Butler, W. L. Plant Physiol. (1968) 43, 1978-1986. 9. Yamashita, T. and Tomita, G. Plant Cell Physiol. (1974) 15, 252-266. 10. Blumberg, W. E. and Peisach, J. Biochim. Biophys. Acta (1966) 126, 269-273. 11. Dwek, R. A. "Nuclear Magnetic Resonance (NMR) in Biochem istry: Application to Enzyme Systems" Claredon Press, Oxford (1973). 12. Villafranca, J. J., Yost, F. J. and Fridovich, I. J. Biol. Chem. (1974) 249, 3532-3536. 13. Geske, D. H. J. Chem. Phys. (1959) 63, 1062-1070. 14. Homann, P. Biochim. Biophys. Acta (1972) 256, 336-344. 15. Erixon, K. and Renger, G. Biochim. Biophys. Acta (1974) 333, 95-106. 16. Kok, B., Forbush, B. and McGloin, M. Photochem. Photobiol. (1970) 11, 457-475. 17. Govindjee and Govindjee, R. in 'Bioenergetics of Photo synthesis" (Govindjee, ed.) pp. 1-50, Academic Press, New York (1975). 18. Olson, J. M. Science (1970) 168, 438-446. 19. Renger, G. Z. Naturforsch. (1970) 25b, 966-971. 20. Earley, J. E. Inorg. Nucl. Chem. Lett. (1973) 9, 487-490.

6.


NMR Studies on Chloroplast Membranes

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