Photosynthetic Energy Transduction - Advances in Chemistry (ACS

21 Photosynthetic Energy Transduction Spectral and Redox Characteristics of Chlorophyll Radicals in Vitro and in Vivo 1

Downloaded by CORNELL UNIV on May 18, 2017 | http://pubs.acs.org Publication Date: June 1, 1982 | doi: 10.1021/ba-1982-0201.ch021

J. FAJER , I. FUJITA, M. S. DAVIS, A. FORMAN, and L. K. HANSON Brookhaven National Laboratory, Department of Energy and Environment, Upton, NY 11973 Κ. M. SMITH University of California, Department of Chemistry, Davis, CA 95616

Optical, redox, and paramagnetic resonance results, as well as theoretical calculations, are presented for cation and anion radicals of magnesium tetraphenylchlorin, and serve as guides to the properties of chlorophyll ions. Electrochemical cells designed to generate the radicals are described. The spectral signatures of chlorophyll (and pheophytin) radicals in vitro are compared with those of the primary donors and acceptors of plant photosyn thesis to identify the transients observed in vivo. The model studies and the theoretical calculations suggest that the protein environment of the chlorophylls in the reaction center can modulate their properties signifi cantly, and may impose specific orientations as well as hydrogen bonding on the substituent groups of the chromophores.

"Dlant

photosynthesis

functions

v i a t w o photosystems

( P S ) that

c o o p e r a t i v e l y fix c a r b o n d i o x i d e ( P S I) a n d e v o l v e o x y g e n ( P S I I ) . A c o m b i n a t i o n o f optical a n d paramagnetic resonance spectroscopy re cently resulted i n a generalized m e c h a n i s m b y w h i c h green

plants

(and algae) transduce a n i n c i d e n t photon into the oxidants a n d r e d u c tants t h a t d r i v e t h e b i o c h e m i s t r y o f t h e o r g a n i s m s . T h e l i g h t h a r v e s t e d 1

Author to whom correspondence should be addressed. 0065-2393/82/0201-0489$07.25/0 © 1982 A m e r i c a n C h e m i c a l Society Kadish; Electrochemical and Spectrochemical Studies of Biological Redox Components Advances in Chemistry; American Chemical Society: Washington, DC, 1982.

BIOLOGICAL REDOX COMPONENTS

490

b y a n t e n n a p i g m e n t s is f u n n e l e d to a reaction center w h e r e a c h l o r o p h y l l (or c h l o r o p h y l l s ) , P, i s r a i s e d t o its e x c i t e d state, P * , a n d transfers a n e l e c t r o n t o a n e a r b y a c c e p t o r , A ( l a b e l e d I i n P S I I ) , w i t h i n a few picoseconds. T h i s p r i m a r y charge separation is then s t a b i l i z e d b y t h e r a p i d translocation o f the e l e c t r o n to a secondary a c c e p t o r , A , for a n o v e r a l l r e a c t i o n ( 1 - 2 7 ) : x

2

PA A X

2

^

Ρ*ΑχΑ

+

2

+

P A -A x

P AA -

2

t

2

A c c e p t o r A is a n i r o n - s u l f u r p r o t e i n i n P S I (13, 27) a n d a n i r o n plastoquinone ( F e - Q ) c o m p l e x i n P S I I (25, 26). T h e p r i m a r y donors, P , are b e l i e v e d t o b e o x i d i z e d c h l o r o p h y l l s ( C h i ) , P - 7 0 0 i n P S I a n d P - 6 8 0 i n P S I I ( I - 1 0 ) . E l e c t r o n s p i n r e s o n a n c e ( E S R ) (6), e l e c t r o n n u c l e a r d o u b l e resonance ( E N D O R ) (28, 29), a n d optical a n d c i r c u l a r d i c h r o i s m ( 3 0 ) r e s u l t s l e d t o t h e p r o p o s a l t h a t P - 7 0 0 i s a d i m e r (or " s p e c i a l pair") o f chlorophylls, although a m o n o m e r i c enol form o f C h i r e c e n t l y w a s p o s t u l a t e d (31 ). 2


+

+

+

T h e nature o f P - 6 8 0 i n P S I I is c o n s i d e r a b l y m o r e a m b i g u o u s : l i n e w i d t h s o f E S R signals attributed to P - 6 8 0 range from 7 to 9 G (32-37) (vs. 7 G for P - 7 0 0 ) , r e s u l t i n g i n p r o p o s a l s o f m o n o m e r ( 3 3 ) , d i m e r (34-36) a n d e v e n t r i m e r ( 3 2 ) c o n f i g u r a t i o n s . E v e n m o r e p u z z l i n g are t h e large differences i n o x i d a t i o n potentials o f P - 7 0 0 a n d P-680. Titrations o f P-700 y i e l d a m i d p o i n t potential (1-5) ranging be t w e e n + 0 . 4 a n d + 0 . 5 V against the normal h y d r o g e n electrode, w h e r e a s t h e m i n i m u m p o t e n t i a l n e e d e d to o x i d i z e w a t e r to o x y g e n a t t h e p h y s i o l o g i c a l p H sets a l o w e r l i m i t o f + 0 . 8 V (4, 5 ) for P - 6 8 0 . +

+

W e describe here i n vitro magnetic, redox, a n d optical properties o f c h l o r o p h y l l (Structure I) a n d m a g n e s i u m tetraphenylchlorin ( M g T P C , S t r u c t u r e I I ) , a m o d e l c o m p o u n d , a n d t h e i r 7r-cation r a d i c a l s , w h i c h i l l u s t r a t e t h e e n v i r o n m e n t a l factors c a p a b l e o f a l t e r i n g t h e properties o f C h i i n v i v o . T h e oxidation potentials a n d E S R charac teristics o f the c h l o r i n s are sensitive functions o f s o l v e n t a n d c o u n t e r ion. T h e s e changes m a y b e e x p l a i n e d b y theoretical calculations that p r e d i c t t h e e x i s t e n c e o f a n e a r l y d e g e n e r a t e e x c i t e d state w h o s e d e gree o f interaction is i n f l u e n c e d b y t h e perturbations caused b y the a x i a l l i g a n d s , t h a t is, t h e i m m e d i a t e e n v i r o n m e n t o f t h e C h i . C o m p a r i son o f the o p t i c a l , redox, a n d m a g n e t i c data o f the c h l o r i n s i n v i t r o w i t h t h o s e a t t r i b u t e d to P - 6 8 0 s u g g e s t s t h a t m a n y o f t h e p r o p e r t i e s o f P - 6 8 0 can b e rationalized i n terms o f a C h i m o n o m e r ligated b y n e i g h b o r i n g ( p r o t e i n ? ) m o l e c u l e s (38). W e also r e v i e w i n v i t r o a n d i n v i v o o p t i c a l a n d p a r a m a g n e t i c reso n a n c e r e s u l t s t h a t h a v e l e d t o t h e i d e n t i f i c a t i o n o f C h i as t h e p r i m a r y a c c e p t o r , A o f P S I ( 1 3 , 1 4 , 1 7 , 1 8 , 21, 23) a n d o f p h e o p h y t i n ( P h e o , a m e t a l - f r e e C h i ) as t h e a c c e p t o r , I, o f P S I I (17-20, 24, 26). H e r e a g a i n , u

Kadish; Electrochemical and Spectrochemical Studies of Biological Redox Components Advances in Chemistry; American Chemical Society: Washington, DC, 1982.

FAJER ET AL.

Photosynthetic Energy Transduction


21.

491

Structure I

Structure II

m o d e l studies

a n d theoretical calculations suggest that the

e n v i r o n m e n t o f the c h l o r o p h y l l s i n the r e a c t i o n c e n t e r c a n

protein

modulate

t h e i r p r o p e r t i e s a n d m a y i m p o s e s p e c i f i c o r i e n t a t i o n s as w e l l as h y d r o g e n b o n d i n g o n t h e s u b s t i t u e n t g r o u p s o f t h e c h r o m o p h o r e s (18,

39a).

Experimental T h e E S R a n d E N D O R techniques were d e s c r i b e d p r e v i o u s l y (40). T h e radicals were generated e l e c t r o c h e m i c a l l y at p l a t i n u m electrodes i n rigor ously d r i e d a n d degassed solvents u s i n g tetrapropylammonium or tetrab u t y l a m m o n i u m perchlorate as carrier electrolyte. T h r e e e l e c t r o c h e m i c a l c e l l



492

configurations were used. For cation radicals, w h i c h are not sensitive to traces o f oxygen, a c e l l ( F i g u r e 1) was constructed to a l l o w simultaneous c y c l i c voltammetry, c o n t r o l l e d potential electrolysis, a n d optical measurements. T h e w o r k i n g electrode consisted o f a Pt basket w i t h a m e s h bottom into w h i c h was fitted a tube e n d i n g i n a fritted glass disk. T h i s tube contained a m e s h P t basket as a counterelectrode. T h i s configuration offered large effective surface areas for b o t h electrodes a n d a l l o w e d the flow of several m i l l i a m p e r e s i n a solvent o f l o w d i e l e c t r i c constant such as dichloromethane ( e = 9) c o n t a i n i n g 10" M reactant a n d 0.1 M ( C H ) N C 1 0 4 as electrolyte. A t h i n reference elec trode fit d i r e c t l y into the top o f the c e l l . T h e w h o l e assembly was p u r g e d w i t h dry nitrogen or argon a n d the flow o f gas served both to stir the solution a n d to e x c l u d e oxygen a n d water. T h e stirred solution m o v e d past the optical c e l l and a l l o w e d spectra to be recorded. (The entire c e l l assembly r e a d i l y fit into the s a m p l i n g compartments o f C a r y 17 or 219 spectrophotometers, a n d was small enough to be inserted into an o p t i c a l d e w a r w i t h i n the s a m p l i n g compartments for low-temperature measurements.) A t the e n d o f the electrolysis, c y c l i c voltammetry c o u l d be performed on the product v i a the t w o Pt leads, w h i c h e n d i n beads, inserted b e l o w the w o r k i n g electrode. T h e r e v e r s i b i l i t y o f the reaction c o u l d also be c h e c k e d b y regeneration of the parent c o m p o u n d s i m p l y b y reversing the polarity of the w o r k i n g a n d counterelectrodes. 3


3

7

4

For anion radicals, w h i c h require rigorous exclusion o f water a n d oxygen, solutions c o n t a i n i n g the solvent, reactant, a n d carrier electrolyte were con tacted w i t h m o l e c u l a r sieves, A l 0 , a n d degassed on a v a c u u m l i n e i n a side arm o f the c e l l s h o w n i n F i g u r e 2. T h e c e l l assembly was then sealed off a n d the solution was p o u r e d into the c e l l . A Pt w i r e served as quasi-reference electrode. A magnetic stirrer, rotated b y an outside magnet m o u n t e d on the flexible shaft o f a stirring motor, p u m p e d the solution over the w o r k i n g elec trode a n d through an optical c e l l . For E S R measurements, the assembly s h o w n i n F i g u r e 3 was used. Outgassed solutions, prepared as just d e s c r i b e d , w e r e p o u r e d into the c e l l to cover the electrodes, a n d enough current was passed through the solution to convert 8 0 - 9 0 % o f the p o r p h y r i n to radical. (A more elaborate version e m p l o y e d a quasi-reference electrode.) After the c e l l was sealed off from the v a c u u m l i n e , E S R a n d E N D O R measurements were ob tained i n the t h i n side arm, a n d the identity of the product was verified spectrophotometrically i n the attached optical c e l l . C h l o r o p h y l l a n d p h e o p h y t i n were prepared b y standard techniques (38). T h e syntheses o f N - t e t r a p h e n y l p o r p h y r i n (95% N ) , a n d of magnesium tetr a p h e n y l c h l o r i n were reported (38, 41). M e t h y l p y r o p h e o p h o r b i d e - α , deuterated at the 5-, 10-, a n d δ-positions (pyropheo-de, see Structure III) was prepared as follows: m e t h y l pheophorb i d e - a (200 mg) was refluxed u n d e r nitrogen i n 50 m L o f d r y c o l l i d i n e con t a i n i n g 4 m L o f D 0 for 16 h . T h e solvents were r e m o v e d under v a c u u m a n d the residue was c r y s t a l l i z e d from m e t h a n o l - m e t h y l e n e c h l o r i d e to g i v e 196 m g (93%) of c o m p o u n d , partially deuterated at positions 5 and 10. To exchange the δ-position, this material was heated for 4 h at 110°C i n 12 m L of deuteroacetic a c i d a n d 3 m L o f dioxane, under nitrogen. T h e solution was d i l u t e d w i t h m e t h y l e n e c h l o r i d e ; w a s h e d w i t h water, then aqueous s o d i u m bicarbonate; d r i e d over anhydrous s o d i u m sulfate; a n d evaporated to dryness. After crystallization from m e t h y l e n e c h l o r i d e - m e t h a n o l , a 9 6 % recovery o f m e t h y l p y r o p h e o p h o r b i d e - α was obtained. N M R spectroscopy i n d i c a t e d that the δ-proton was > 7 5 % exchanged, that the 10-methylene was totally ex changed, but that the 5-methyl was only about 5 0 % exchanged (labeled 2

15

3

1 5

2


21.

FAJER E T A L .

Photosynthetic

Energy

493

Transduction

reference electrode

inert


gas i n l e t

l e a d for c o u n t e r e l e c t r o d e

f

Jf^Î^

- — c o u n t e r e l e c t r o d e (Pt mesh) w o r k i n g e l e c t r o d e (Pt foil) p o r o u s g l a s s frit

cyclic voltammetry electrodes

2-mm

optical cross s e c t i o n o f the electrodes

working

counterelectrode

electrode glass frit Figure

1.

Electro optical cell for controlled potential cyclic voltammetry, and optical measurements.

electrolysis,



494

p y r o p h e o - d i n F i g u r e 9). T h e partially deuterated material was therefore re treated as follows: 46.5 m g i n 20 m L o f dry p y r i d i n e a n d 2 m L o f D 0 was refluxed for 92 h under nitrogen. After evaporation to dryness, use o f toluene as a solvent chaser, a n d crystallization from m e t h y l e n e c h l o r i d e - m e t h a n o l , 39.5 m g (85% recovery) of the r e q u i r e d pyropheo-de was obtained. N M R spectros copy s h o w e d that the peak assigned to the 5-methyl was totally absent (i.e., > 9 5 % deuteration). T r i ton-treated subchloroplast particles e n r i c h e d i n P S I (one P S I reaction center/30 C h i molecules) or i n P S II (one P S II reaction center/30-40 C h l s ) were prepared a n d the p r i m a r y acceptors were trapped as described i n Refer ences 18, 20, a n d 26. 4


2

to v a c u u m a n d r e a g e n t reservoir

quasi-reference electrode

2-mm

optical

>

Photosynthetic Energy Transduction - Advances in Chemistry (ACS

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