Model Systems for the Primary Photochemical Events of

22

Downloaded by UNIV OF ARIZONA on November 11, 2012 | http://pubs.acs.org Publication Date: June 1, 1982 | doi: 10.1021/ba-1982-0201.ch022

Model Systems for the Primary Photochemical Events of Photosynthesis and Electron Transfer in Bioenergetic Membranes PAUL A. LOACH, JENNIFER A. RUNQUIST, JOSEPHINE L. Y. KONG, THOMAS J. DANNHAUSER, and KENNETH G. SPEARS Northwestern University, Department of Biochemistry and Molecular Biology and Department of Chemistry, Evanston, IL 60201

This chapter describes two model systems that, when coupled together, are designed to effectively reproduce the primary photochemical event of bacterial photosynthesis and subsequent secondary electron transport. In the first model, covalently linked porphyrin-quinone complexes were synthesized and exhibited photochemical charge separation from the excited singlet state. This effect was demonstrated by quenching of fluorescence and formation of a porphyrin (or zinc porphyrin) cation radical and a quinone anion radical in homogeneous solution at room temperature, at 77 K, and in phosphatidylcholine liposomes. The quantum yield was estimated to be near 0.1 for the complex incorporated into liposomes with a radical half-life of 1.5 min. In the second model, for secondary electron transport, metalloporphyrins such as hemin dimethyl ester catalyze electron transport across a phosphatidylcholine lipid bilayer at very high rates, comparable to in vivo electron transport. Catalysis was shown to proceed by an electroneutral diffusion mechanism. These results are discussed from the point of view of future model work and suggest that in vivo electron transport through cytochrome b heme centers may occur in an electroneutral fashion (i.e., by coupled electron and hydrogen ion flow). 0065-2393/82/0201-0515$ 12.75/0 © 1982 A m e r i c a n C h e m i c a l Society In Electrochemical and Spectrochemical Studies of Biological Redox Components; Kadish, K.; Advances in Chemistry; American Chemical Society: Washington, DC, 1982.

516

BIOLOGICAL REDOX COMPONENTS


^ j ^ T e d e v e l o p e d t w o m o d e l s y s t e m s t h a t at first m a y n o t s e e m c l o s e l y related. H o w e v e r , they are each part o f an e v e n t u a l l y more c o m p l i c a t e d s y s t e m . I n t h e m o r e c o m p l i c a t e d m o d e l , t h e first s y s t e m i s e x p e c t e d e v e n t u a l l y t o p r o v i d e c h a r g e s e p a r a t i o n across a l i p i d b i l a y e r as a r e s u l t o f l i g h t a b s o r p t i o n . T h e s e c o n d s y s t e m w i l l b e c a t a l y t i c i n s u b s e q u e n t secondary e l e c t r o n transport. T h u s , w e c a n effectively m o d e l the primary photochemical event a n d c o u p l e d secondary elec t r o n flow o f b a c t e r i a l p h o t o s y n t h e s i s , w h i c h w i l l p r o v i d e i n s i g h t s t o u n d e r s t a n d i n g a variety o f i n v i v o systems. Structurally s i m i l a r por p h y r i n c o m p l e x e s w e r e s y n t h e s i z e d for t h e t w o m o d e l s y s t e m s .

Characteristics of the Primary Photochemical Events in Bacterial Photosynthesis T h e best understood o f the p r i m a r y p h o t o c h e m i c a l events i n photosynthetic systems are those i n photosynthetic bacteria l i k e

Rhodospirillum rubrum a n d Rhodopseudomonas sphaeroides ( 1 - 5 ) . M a j o r properties of this s y s t e m are the f o l l o w i n g . T h e p r i m a r y e l e c t r o n donor consists o f t w o o r m o r e p r o t e i n - b o u n d b a c t e r i o c h l o r o p h y l l m o l e c u l e s ( 6 - 1 7 ) , w h i c h a c c e p t w i t h h i g h e f f i c i e n c y e x c i t e d s i n g l e t state e n e r g y f r o m t h e a n t e n n a c o m p l e x ( e s ) (6, I S , 1 9 ) . A n e l e c t r o n i s d o n a t e d b y t h i s c o m p l e x f r o m i t s e x c i t e d s i n g l e t state t o a n o t h e r m o l e c u l e w i t h i n a t i m e p e r i o d t h a t is less t h a n 10 p s (20-22). T h e e l e c t r o n c o m e s to r e s t for a b o u t 1 0 0 ps o n a n u b i q u i n o n e m o l e c u l e , w h i c h s e r v e s as t h e first s t a b l e e l e c t r o n a c c e p t o r ( 2 3 - 2 7 ) . T h e q u a n t u m y i e l d for t h i s i n i t i a l c h a r g e s e p a r a t i o n i n d i c a t e s a v e r y h i g h e f f i c i e n c y [ ^ 0 . 9 5 ; (28-31)]. T h e approximate redox potential span accomplished b ythe time the e l e c t r o n h a s r e a c h e d t h e first s t a b l e e l e c t r o n a c c e p t o r u b i q u i n o n e is a b o u t 0 . 5 t o 0 . 9 V (2, 3 2 ) . A c y t o c h r o m e , s u c h as c y t o c h r o m e c i n R . rubrum a n d Rps. sphaeroides, serves as t h e s e c o n d a r y e l e c t r o n d o n o r to t h e o x i d i z e d b a c t e r i o c h l o r o p h y l l d o n o r u n i t (28,33) w i t h a n e l e c t r o n t r a n s f e r rate t h a t v a r i e s f r o m a b o u t 0 . 5 /xs t o a f e w m i l l i s e c o n d s d e p e n d i n g o n t h e b a c t e r i a . T h i s c y t o c h r o m e is v e r y t i g h t l y c o u p l e d a n d a l s o o x i d i z e d w i t h a v e r y h i g h q u a n t u m y i e l d (28, 31). F o u r b a c teriochlorophyll molecules, t w o bacteriopheophytin molecules, a n d one to three ubiquinoneio m o l e c u l e s (probably o n e , b u t d e p e n d s o n d e f i n i t i o n ) a r e t i g h t l y b o u n d b y o n e o r t w o p o l y p e p t i d e c o m p o n e n t s as t h e r e a c t i o n c e n t e r o r p h o t o t r a p c o m p l e x (5). T h i s i n t e g r a l c o m p l e x i s m o s t l y c o n t a i n e d w i t h i n t h e p h o t o s y n t h e t i c m e m b r a n e (5, 34-37). 2

C h a r g e s e p a r a t i o n t o t h e first s t a b l e u b i q u i n o n e m o l e c u l e a p p e a r s t o b e e l e c t r o g e n i c a l l y d i s p o s e d s u b s t a n t i a l l y across t h e p h o t o s y n t h e t i c membrane w i t h the bacteriochlorophyll donor unit b e i n g near the o u t e r s u r f a c e o f t h e m e m b r a n e i n t h e i n t a c t c e l l a n d t h e first s t a b l e u b i q u i n o n e n e a r t h e i n n e r s u r f a c e (38, 39). A n i r o n a t o m is f o u n d n e a r

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

22.

LOACH ET AL.

Photosynthesis and Electron Transfer

t h e first s t a b l e u b i q u i n o n e (Q ) A

517

a n d t h e s e c o n d u b i q u i n o n e (QB), b u t is

a p p a r e n t l y n o t c o o r d i n a t e d t o e i t h e r o f t h e m , at l e a s t at l o w t e m p e r a t u r e s (5, 15, 40, 41).

T h i s i r o n is n o t i n a t y p i c a l i r o n s u l f u r

center,

a l t h o u g h c y s t e i n e r e s i d u e s m a y p l a y s o m e r o l e i n its b i n d i n g (5, 43).

42,

I f the electron does not participate i n n o r m a l secondary electron

transfer,, i t m a y r e t u r n d i r e c t l y to t h e p r i m a r y e l e c t r o n d o n o r p r e s u m a b l y b y t u n n e l i n g (44, 45).

unit,

S e c o n d a r y e l e c t r o n flow f r o m t h e

first s t a b l e u b i q u i n o n e m o l e c u l e (QA) to a s e c o n d u b i q u i n o n e m o l e c u l e (QB) o c c u r s i n a b o u t

1 0 0 /xs (27). o - P h e n a n t h r o l i n e b l o c k s t h i s


l a t t e r e l e c t r o n t r a n s f e r s t e p , w h i c h a l s o d o e s n o t o c c u r at l o w t e m p e r a tures. R e c e n t p i c o s e c o n d spectroscopy m e a s u r e m e n t s suggest that sev eral short-lived intermediate

electron acceptors

may be

identified.

O n e o r m o r e o f t h e b a c t e r i o c h l o r o p h y l l m o l e c u l e s r e s p o n s i b l e for t h e 8 0 0 - n m a b s o r b a n c e i n t h e r e a c t i o n c e n t e r m a y s e r v e as a n i n t e r m e d i a t e e l e c t r o n acceptor that d i r e c t l y receives the

e l e c t r o n f r o m the

bac

t e r i o c h l o r o p h y l l o f t h e d o n o r u n i t i n less t h a n a f e w p s (46-48).

This

e l e c t r o n is t h e n t h o u g h t to b e p a s s e d o n to a s i n g l e b a c t e r i o p h e o p h y t i n m o l e c u l e i n a b o u t 5 p s a n d f r o m t h e r e to QA i n a b o u t 2 0 0 p s . I f e l e c t r o n flow to QA is b l o c k e d b y p r i o r r e d u c t i o n , t h e e l e c t r o n m a y r e t u r n f r o m r e d u c e d b a c t e r i o p h e o p h y t i n to t h e o x i d i z e d d o n o r u n i t a n d f o r m a c o m p l e x i n its e x c i t e d t r i p l e t state (49, 50).

M u c h o f the

foregoing

i n f o r m a t i o n is s u m m a r i z e d i n S c h e m e I .

Model System I O n the basis o f the p r e c e d i n g d e s c r i p t i o n o f the i n v i v o r e a c t i o n center, the f o l l o w i n g properties s h o u l d b e a n i n h e r e n t part o f a g o o d m o d e l s y s t e m : (1) p h o t o c h e m i s t r y s h o u l d o r i g i n a t e f r o m t h e

metal

l o p o r p h y r i n (or m o r e e x a c t l y , m a g n e s i u m ( I I ) b a c t e r i o c h l o r i n ) e x c i t e d s i n g l e t state, (2) t h e q u a n t u m

y i e l d for c h a r g e

s e p a r a t i o n to

stable

p r o d u c t s s h o u l d b e n e a r 1.0, (3) t h e m e t a l l o p o r p h y r i n s h o u l d b e

the

e l e c t r o n d o n o r a n d a b e n z o q u i n o n e s h o u l d b e t h e first s t a b l e ( l i f e t i m e l o n g e r t h a n 1 /xs) e l e c t r o n a c c e p t o r , a n d (4) t h e c h a r g e - s e p a r a t e d

prod

u c t s s h o u l d b e s t a b l e for at l e a s t 100 m s at r o o m t e m p e r a t u r e . T h e versatile photochemical activity of porphyrins i n general, a n d c h l o r o p h y l l i n p a r t i c u l a r , has l o n g b e e n k n o w n (51-53).

Photochemi

cal reaction between chlorophyll and benzoquinones i n homogeneous s o l u t i o n has

established

that h i g h concentrations

of

benzoquinone

(e.g., 0.1 M ) q u e n c h t h e e x c i t e d s i n g l e t state w i t h o u t p r o d u c i n g d e t e c t a b l e q u a n t i t i e s o f o x i d i z e d a n d r e d u c e d s p e c i e s (54, 5 5 ) , a l t h o u g h l o w e r concentrations charge

separation

out

of benzoquinone o f the

triplet

(e.g.,

1 m M ) can result

state w i t h

reasonably

in

stable

o x i d i z e d a n d r e d u c e d s p e c i e s (56, 57). S i m i l a r p h o t o c h e m i c a l a c t i v i t y



518

Fel+c teChlfc ]

2

BChl BPh")Q Q

5

QQ0

A

B


| ~ 2 0 0 ps

Scheme I. Simple linear scheme for the primary photochemical events in photosynthetic bacteria as observed in R. r u b r u m and R p s . sphae roides. BChl represents the primary electron donor bacteriochlorophyll complex absorbing at 865 nm, BChl is an additional molecule of bacteriochlorophyll that absorbs at 800 nm and is believed to be a transitory electron acceptor, BPh is a bacteriopheophytin molecule believed to also play a role as an intermediate electron acceptor, Q represents the first stable electron acceptor that is a tightly bound ubiquinone molecule, Q is a secondary electron acceptor also thought to be a ubiquinone molecule, and Fe c represents the ferrous oxidation state of cytochrome c. 865

800

A

B

2+

2

2

w a s a l s o d e m o n s t r a t e d i n f r o z e n s y s t e m s as w e l l as i n h e t e r o g e n e o u s s y s t e m s [ e . g . , f i l m s a n d l i p o s o m e s (58, 59)]. Because selectively controlling t h e reaction path i n this type o f s y s t e m is d i f f i c u l t , w e b e g a n a p r o j e c t o f s y n t h e s i z i n g c o v a l e n t l y l i n k e d porphyrin

dimers

a n d trimers

a n d covalently

linked

porphyrin-

quinone complexes. T h e covalently linked porphyrin dimer system w a s u s e f u l f o r s t u d y i n g t h e t r a n s f e r o f e x c i t e d s i n g l e t state e n e r g y f r o m o n e p o r p h y r i n c e n t e r t o a n o t h e r (60). M a n y c o v a l e n t l y l i n k e d p o r p h y r i n s p e c i e s h a v e b e e n s y n t h e s i z e d (60-81). R e c e n t l y , w e s y n t h e s i z e d (82-86) a s e r i e s o f c o v a l e n t l y l i n k e d p o r p h y r i n - q u i n o n e c o m p l e x e s , w h i c h a p p e a r t o b e e x c e e d i n g l y p r o m i s i n g c o m p l e x e s for s y s t e m a t i c a l l y p r o b i n g p h o t o c h e m i c a l charge separation. W i t h these


22.

LOACH ET AL.

Photosynthesis and Electron Transfer

519

m o d e l c o m p l e x e s w e are i m p o s i n g a c o n d i t i o n w h i c h w a s n o t p o s s i b l e with

the

simple

models.

For example,

i n the

covalently

linked

p o r p h y r i n - q u i n o n e c o m p l e x e s , 1 : 1 q u i n o n e to p o r p h y r i n s y s t e m s c a n b e s t u d i e d i n a " g o o d " s o l v e n t s y s t e m as w e l l as i n l i m i t e d p r o t i c o r l i m i t e d c h a r g e e n v i r o n m e n t s . T h u s , i t s h o u l d b e p o s s i b l e to a p p r o a c h m u c h m o r e s p e c i f i c a l l y c o n d i t i o n s t h a t w e r e p r o b a b l y s e l e c t e d for t h e p r o t e i n b i n d i n g site o f the r e a c t i o n c e n t e r o f p h o t o s y n t h e t i c

systems

over m i l l i o n s o f years o f e v o l u t i o n .


Experimental for Model System I T h e synthesis o f covalently l i n k e d p o r p h y r i n - q u i n o n e complexes was p r e v i o u s l y reported (82,83). T h e c o m p o u n d s s t u d i e d are s h o w n i n Structure I.

M represents a metal such as z i n c or two protons for the free base porphyrins. A l t h o u g h the o x i d i z e d form o f the q u i n o n e is s h o w n , the complexes w e r e also prepared w i t h the q u i n o n e r e d u c e d to a h y d r o q u i n o n e . W h e n M = Z n a n d η = 3, the c o m p o u n d is referred to as Z n P - 3 - Q . I f the p o r p h y r i n is a free base p o r p h y r i n a n d does not contain a metal, the c o m p o u n d is referred to as P - 3 - Q . I f the b e n z o q u i n o n e is r e d u c e d to the h y d r o q u i n o n e , then the corre s p o n d i n g abbreviations are Z n P - 3 - Q H and P - 3 - Q H . Solvents used were a l l o f spectroscopic grade. Fluorescence measure ments w i t h continuous light were made w i t h a P e r k i n - E l m e r M P F - 4 4 A fluorescence spectrometer. E x c i t a t i o n was u s u a l l y at the w a v e l e n g t h maxi m u m o f the Soret b a n d , w h i c h was adjusted to an absorbance o f 0.30 ± 0.02 n m . T h e emission spectra were r e c o r d e d w i t h an excitation b a n d w i d t h o f 3 n m and an emission b a n d w i d t h of 5 n m . O c c a s i o n a l l y , excitation at 590 n m was used for P - 3 - Q . A l l measurements were at room temperature i n air. T h e fluorescence lifetimes were measured b y time-correlated photon c o u n t i n g techniques w i t h excitation b y a m o d e - l o c k e d dye laser p u m p e d b y a m o d e - l o c k e d argon i o n laser. T h e d y e laser pulses were o f ~ 2 ps full w i d t h at h a l f m a x i m u m ( F W H M ) as measured b y autocorrelation methods a n d the 2

2




520

pulse spacing was 10.0 ns. T h e optics a n d methods for time-correlated photon c o u n t i n g w i t h lasers were p r e v i o u s l y d e s c r i b e d (87-89). T h e excitation c o n d i tions for these experiments were λ = 600 n m , 1-10 m j o f average energy i n c i dent on the sample i n ~ l - m m c o l l i m a t e d b e a m . T h e fluorescence c o l l e c t i o n optics were —12 w i t h an intermediate focus to select the m i d d l e 3 - 4 m m o f the 10-m m cuvette. N e u t r a l density a n d color filters a n d an aperture were used to adjust the fluorescence intensity. C o r n i n g 2-58 a n d 2-59 filters, w h i c h w h e n c o u p l e d w i t h the l i m i t e d r e d response o f an A m p e r e x X P 2 0 2 0 p h o t o m u l t i p l i e r tube, effectively gave fluorescence isolation near ~ 6 5 0 n m . E l e c t r o c h e m i c a l measurements were taken using c y c l i c voltammetry. T h e solvents used i n these studies had to be pure and dry. D i s t i l l e d i n glass acetonitrile from B u r d i c k a n d Jackson was used as r e c e i v e d and was stored under nitrogen at a l l times. D i c h l o r o m e t h a n e ( D i s t i l l e d i n glass, B u r d i c k and Jackson) was d r i e d b y s h a k i n g w i t h activated m o l e c u l a r sieves for 24 h on a shaker. T h e supporting electrolyte, tetra-n-butylammonium perchlorate ( T B A P ) (Southwestern A n a l y t i c a l C h e m i c a l s ) was d r i e d under v a c u u m at 100°C for 48 h a n d stored i n a v a c u u m desiccator over phosphorus pentoxide. A l l data were obtained at room temperature u t i l i z i n g methods and apparatus previously d e s c r i b e d (90, 91). C y c l i c voltammetry o f Z n P - 3 - Q H was performed w i t h an "adder" type operational amplifier potentiostat d e s c r i b e d p r e v i o u s l y (92). T h e current flow i n g b e t w e e n the w o r k i n g a n d auxiliary electrode was recorded as a function of the reference signal voltage w i t h a H e w l e t t Packard M o d e l 7001 AX-Y recor der. E P R signals were recorded w i t h a Varian E - 3 spectrometer (Varian Asso ciates). I l l u m i n a t i o n o f the sample was p r o v i d e d b y the output o f a 1000-watt tungsten projection b u l b ( G E M o d e l D F D ) . T h e e x c i t i n g light passed through a 5-cm thick water filter and two C o r n i n g color filters (3-67, 4-94) before b e i n g focused onto the w i n d o w o f the E P R sample cavity. A l l samples were anaerobic u t i l i z i n g procedures p r e v i o u s l y d e s c r i b e d (25, 32). 2

Results and Discussion for Model System I T h e absorbance s p e c t r a o f Z n P - 3 - Q a n d Z n P - 3 - Q H are s h o w n i n F i g u r e 1. T h e v i s i b l e r e g i o n o f t h e s p e c t r a reflects o n l y t h e s p e c t r a l properties o f the Z n p o r p h y r i n part o f the m o l e c u l e , b u t b a n d s i n the U V r e g i o n a r e d u e to b o t h t h e q u i n o n e g r o u p a n d t h e Z n p o r p h y r i n g r o u p . T h e s p e c t r a a b o v e 3 5 0 n m a r e i d e n t i c a l w i t h t h a t o f 5( 4 - c a r b o m e t h o x y p h e n y l ) - 1 0 , 1 5 , 2 0 - t r i t o l y l p o r p h y r i n a t o z i n c i n the same s o l v e n t . A d i f f e r e n c e s p e c t r u m o f t h e U V r e g i o n is p l o t t e d t o s h o w t h e q u i n o n e g r o u p m o r e c l e a r l y a n d t h i s s p e c t r u m is c o m p a r e d w i t h a s i m i l a r d i f f e r e n c e s p e c t r u m for 1 , 4 - b e n z o q u i n o n y l e t h a n o i c a c i d . T h e s e data s h o w that i n this solvent s y s t e m the q u i n o n e g r o u p does not p e r t u r b t h e Z n p o r p h y r i n a b s o r b a n c e s p e c t r u m or v i c e v e r s a . T h e r e fore, t h e r e is n o e v i d e n c e o f c o m p l e x f o r m a t i o n a n d t h e s p e c t r a l p r o p erties a p p e a r to b e m e r e l y the s u m o f the t w o parts o f the m o l e c u l e . 2

P r e v i o u s l y r e p o r t e d N M R d a t a (83) a l s o i n d i c a t e d t h a t i n c h l o r o f o r m t h e r e w a s n o e v i d e n c e for s i g n i f i c a n t i n t e r a c t i o n b e t w e e n



22.

LOACH ET AL.

Photosynthesis

and Electron

521

Transfer

0.4LU Ο

0.30.2-

g co ω

Model Systems for the Primary Photochemical Events of

Recommend Documents