Thermal and Photoinduced Long Distance Electron Transfer in

Downloaded by UNIV OF CALIFORNIA SANTA BARBARA on April 1, 2018 | https://pubs.acs.org Publication Date: April 30, 1986 | doi: 10.1021/bk-1986-0307.ch011

11 Thermal and Photoinduced Long Distance Electron Transfer in Proteins and in Model Systems 2

George McLendon, John R. Miller1, Ken Simolo, Karen Taylor, A. Grant Mauk , and Ann M . English 3

Department of Chemistry, University of Rochester, Rochester, NY 14627

All biological energy (and, thus, all fossil energy) is ultimately derived from a series of basic electron transfer reactions, starting with the primary charge separation in photosynthesis. The subsequent energy flow proceeds through a series of subsequent redox reactions, largely involving metallo-proteins in which the energy of reduction is coupled to proton transport and manufacture of ATP for biosyntheses. (Fig 1).

Figure 1. Mitochondral electron transport chain. Despite the obvious importance of such redox reactions, until recently such reactions remained rather poorly characterised, and poorly understood. 1

Current address: Chemistry Division, Argonne National Labs, Argonne, IL 60438 Current address: Department of Biochemistry, School of Medicine, University of British Columbia, Vancouver, British Columbia, V6T 1Y6 Canada Current address: Enzymology Research Group, Departments of Chemistry and Biology, Concordia University, Montreal, Quebec, H3G 1M8 Canada

2

3

0097-6156/ 86/ 0307-0150S06.00/ 0 © 1986 American Chemical Society

Lever; Excited States and Reactive Intermediates ACS Symposium Series; American Chemical Society: Washington, DC, 1986.


11.

McLENDON ET AL.

151

Long-Distance Electron Transfer

Within the past few years, however, rapid advances have occurred in several key areas, including: 1) electron transfer theory1-3 2) experiments on model reactions (eg: electron transfer at long, fixed distance),4-6 3) experimental techniques for monitoring rapid biolo gical electron transfer,7-10 and 4) structural characterization of the redox proteins themselves,11-15 including detailed models for the protein-protein complexes within which electron transfer occurs. As a result of these advances, rapid experimental progress in protein redox chemistry is occurring. In this paper we focus on two prototypic protein redox couples, cytochrome (c)c/cytochrome b5 (b5) and c/cytochrome c peroxidase (ccp) which have been the subjects of detailed studies in our labs. Theories of Protein-Protein Electron Transfer. The sketch presented here is necessarily brief Details of the theory can be found in several recent reviews.1-3 Like any reaction, the rate constant for electron transfer can be written as the product of a prefactor times an activation barrier: -AG /k T k = A exp An unusual feature of biological electron transfer reactions is that the reactive prosthetic groups are held at fixed distances within a protein matrix and these distances are greater than the collision distance. Thus, the prefactor for biological g

electron t r a n s f e r i s not r e l a t e d t o a s i m p l e c o l l i s i o n f r e q u e n c y . I n s t e a d , A i s governed by the o v e r l a p o f donor and a c c e p t o r wavef u n c t i o n s , ( F i g 2 ) , s i n c e such o v e r l a p d e t e r m i n e s the p r o b a b i l i t y o f e l e c t r o n exchange between the donor and a c c e p t o r . When ttjiis o v e r l a p i s l a r g e ( i e : e l e c t r o n i c i n t e r a c t i o n energy > 100 cm ) when r e a c t a n t s a t t a i n the c o r r e c t t r a n s i t i o n s t a t e n u c l e a r c o n f i g u r a t i o n w i l l p r o c e e d to p r o d u c t s , w i t h a p r o b a b i l i t y κ=1. Such r e a c t i o n s a r e called adiabatic. I f the o v e r l a p i s s m a l l , t h e n the appropriate n u c l e a r c o n f i g u r a t i o n can be r e a c h e d many times w i t h o u t net r e a c t i o n , and the r e a c t i o n i s c a l l e d n o n a d i a b a t i c . As d e t a i l e d e l s e w h e r e , the o v e r l a p w i l l g e n e r a l l y d e c r e a s e e x p o n e n t i a l l y w i t h i n c r e a s i n g donoracceptor distances: Α « exp - (aR) where the parameter α may depend on the n a t u r e of the donor and a c c e p t o r , the donor i o n i z a t i o n p o t e n t i a l , as w e l l as the n a t u r e o f the " s t u f f " (eg: p r o t e i n m a t r i x ) i n between the donor and a c c e p t o r s . For,many r e a c t i o n s , α has been e x p e r i m e n t a l l y found t o be α = 1 . 2 ± 0 . 2 ' A We now t u r n t o the. a c t i v a t i o n energy. The most w i d e l y used approach t o r e l a t e AG t o s t r u c t u r a l parameters of the r e a c t a n t s i s due t o Marcus ( f i g 3 ) . In e s s e n c e , Marcus t h e o r y s t a t e s t h a t the a c t i v a t i o n f r e e energy AG , i s d e t e r m i n e d by the b a l a n c e between the r e o r g a n i z a t i o n energy, λ, and the f r e e energy o f r e a c t i o n , AG. The r e o r g a n i z a t i o n energy λ can be u n d e r s t o o d as the energy r e q u i r e d f o r a v e r t i c a l t r a n s i t i o n between the energy minimum of the r e a c t a n t s u r f a c e and the p r o d u c t s u r f a c e a t the same n u c l e a r c o o r d i n a t e . In such a p i c t u r e , λ e s s e n t i a l l y d e f i n e s the F r a n c k Condon f a c t o r f o r v i b r a t i o n a l o v e r l a p o f r e a c t a n t and p r o d u c t s t a t e s . The t o t a l λ i s made up o f i n t e r n a l (λ.) and medium (λ ) d i s p l a c e m e n t s : λ = + \ . An energy λ. i s r e q u i r e d s i n c e , i n g e n e r a l , bond l e n g t h s (and a n g l e s ) w i l l change between an o x i d i z e d and r e d u c e d m o l e c u l e . Thus, Ai can be modeled by a harmonic o s c i l l a t o r t r e a t m e n t . I f ηω >> kT, then r e a c t i o n a l o n g t h i s v i b r a t i o n a l c o o r d i n a t e r e q u i r e s n u c l e a r tunne ling. S e m i c l a s s i c a l and quantum m e c h a n i c a l t r e a t m e n t s have been Q



EXCITED STATES AND REACTIVE INTERMEDIATES

Nucleor Configuration

Nucleor Configuration

F i g u r e 2. Top: Note t h a t f o r simple ( S l a t e r ) w a v e f u n c t i o n s , the o v e r l a p between donor (D) and a c c e p t o r (A) d e c r e a s e s e x p o n e n t i a l l y as d i s t a n c e (R) i n c r e a s e s . Bottom: t h i s o v e r l a p c a n e q u i v a l e n t l y be viewed as an i n t e r a c t i o n energy, Η , between r e a c t a n t and p r o d u c t s u r f a c e s , l e a d i n g t o an a v o i d e d c r o s s i n g , ( a ) When is l a r g e (>100 cm ) t h e r e a c t i o n remains on t h e lower s u r f a c e , and t h e r e a c t i o n i s " a d i a b a t i c " . (b) When Η i s s m a l l , some t r a j e c t o r i e s may c r o s s t o t h e upper "R" s u r f a c e and r e t u r n t o the r e a c t a n t w e l l w i t h o u t making p r o d u c t s . Α β

Α

β


11.

MCLENDON ET AL.

153


d e v e l o p e d f o r such c a s e s , and have been a p p l i e d t o low temperature data f o r the photosynthetic r e a c t i o n centers. A second i m p o r t a n t energy, Xs, a r i s e s from r e p o l a r i z a t i o n o f t h e medium around a developing charge. A c l a s s i c a l continuum t r e a t m e n t s u g g e s t s 2 1 1 1 As = Ae ( ~ - - ) (-) ( f o r r >> c o l l i s i o n a l ) op s D

where D and D a r e t h e o p t i c a l and s t a t i c d i e l e c t r i c c o n s t a n t s and r i s t h e d i s t a n c e between donop agd a c c e p t o r . The continuum treatment has been q u e s t i o n e d r e c e n t l y . ' F o r r e a c t i o n s i n polymers ( l i k e p r o t e i n s ) whether t h e s t a t i c d i e l e c t i c c o n s t a n t i s t h e a p p r o p r i a t e parameter i s a l s o q u e s t i o n a b l e . Recent d a t a f o r e l e g t r o n t r a n s f e r i n d r y l e x a n f i l m s ( D ~ 2.4) s u g g e s t λ - 1.0 V, w h i l e c l a s s i c a l c a l c u l a t i o n s r e q u i r e λ < 0.3 V. Thus, i n o r d e r t o u n d e r s t a n d b i o l o g i c a l e l e c t r o n t r a n s f e r i n a t h e o r e t i c a l c o n t e x t , we wish t o c h a r a c t e r i z e t h e parameters which c o n t r o l t h e p r e f a c t o r , A, t h r o u g h t h e d i s t a n c e dependence (the " a " parameter) and c h a r a c t e r i z e t h e r e o r g a n i z a t i o n energy, λ.


p

s

S

3

The Cytochrome c/cytochrome b5 Complex: S t r u c t u r a l F e a t u r e s . One i m p o r t a n t f e a t u r e o f t h e c/b5 system i s t h e d e t a i l e d s t r u c t u r a l i n f o r m a t i o n which i s a v a i l a b l e f o r t h e s e p r o t e i n s . The s t r u c t u r e o f c y t c i s ^ n o w n a t - 1.5Â r e s o l u t i o n i n b o t h t h e o x i d i z e d and reduced states. EXAFS s t u d i e s have a l s o been r e p o r t e d , which show no o b s e r v a b l e changes i ^ m e t a l c o o r d i n a t i o n geometry on oxidation/reduction. The cytochrome b5 s ^ g u c t u r e has a l s o been s o l v e d a t h i g h r e s o l u t i o n by Matthews e t a l . Based on t h e s e s t u d i e s and t h e known c h e m i c a l p r o p e r t i e s o f t h e s e c y t o c h r o m e s , i n 1976 Salemme proposed a n o v e l model f o r t h e s t r o n g n o n c o v a l e n t c/b5 complex. T h i s model i s g r a p h i c a l l y shown i n f i g . 4. Key f e a t u r e s i n c l u d e an e l e c t r o s t a t i c b i n d i n g r e g i o n i n which s e v e r a l l y s i n e r e s i d u e s on c y t c a l i g n w i t h a p p r o p r i a t e a c i d i c amino a c i d s on cytochrome b5 t o form s t r o n g " s a l t - l i n k s " . In t h e p r o p o s e d complex s t r u c t u r e , t h e hemes a r e s e p a r a t e d by a n e a r e s t c o n t a c t d i s t a n c e o f - 8 A, (16 A Fe-Fe d i s t a n c e ) and a r e p r e d i c t e d t o be i n p a r a l l e l p l a n e s . A v a r i e t y o f subsequent s t u d i e s have examined s e v e r a l f e a t u r e s o f t h i s model, and g e n e r a l l y s u p p o r t i t s p r e d i c t i o n s . F o r example, t h e c ( I I I ) / b 5 ( I I I ) binding constant, Κ , i s quite s e n s i t i v e t o . i o n i c s t r e n g t h , d e c r e a s i n g from Κ ~ 3 Χ 1 θ ' M a t μ = 0 M t o K ~10 M at μ = 10 mM, c o n f i r m i n g t h e importance o f i o n i c i n t e r a c t i o n s i n s t a b i l i z i n g t h e complex. Some s p e c i f i c r e s i d u e s i n v o l v e d iûgthis i n t e r a c t i o n have been i d e n t i f i e d by t h e NMR s t u d i e s o f Moore. In agreement w i t h t h e model, r e s o n a n c e s f o r L y s 13, L y s 72, & Phe 82 a r e s e l e c t i v e l y broadened. ^ Energy t r a n s f e r measurements by McLendon e t a l suggest a Zn-Fe d i s t a n c e o f c a . 17 A, i n good agreement w i t h Salemme's p r e d i c t i o n . Thus t h e g e n e r a l s t r u c t u r a l f e a t u r e s o f t h e model appear t o be w e l l founded. Μ

M

E l e c t r o n T r a n s f e r K i n e t i c s i n t h e c y t c / c y t b5 Complex. Pulse r a d i o l y s i s t e c h n i q u e s have been used t o i n v e s t i g a t e t h e r e a c t i o n sequence




Nuclear Configuration

F i g u r e 3. Schematic o f Marcus t h e o r y ( z e r o i n t e r a c t i o n ) . Key: (a) a c t i v a t e d p r o c e s s , AG < λ; (b) a c t i v a t i o n l e s s , AG = λ ; ( c ) i n v e r t e d , AG > λ

F i g u r e 4. Tom P o u l o s ,

Computer model o f the c y t c / c y t b^ complex

(courtesy

Genex).


11.


McLENDON ET AL.

Fe(III) b5/Fe(III)

Fe(II) b5/Fe

I ] [ I

c

e (aq) ^> k~1CTM

c

155

F e ( I I ) b5 \ F e ( I I I ) c s

> Fe(III) b5/Ee(II)c k = 1.6 ± . il χ 1 0 s 5


-ο

A t low i o n i c s t r e n g t h , (μ ύ 10 M) where t h e complex i s f u l l y formed, t h e t r a n s f e r r a t e i s f i r s t _ o r d e r and i s independent o f t h e c o n c e n t r a t i o n s o f [b ] , [ c ] o r [e a q ] . As shown i n f i g 5, t h e decay o f i n i t i a l l y produced F e ( I I ) b5 — > F e ( I I I ) b 5 measured a t ^28 nm i s k i n e t i c a l l V g C O u p l e d t o t h e c o n v e r s i o n F e ( I I I ) c — > F e ( I I ) c measured at 416 nm. A t h i g h i o n i c s t r e n g t h (μ=100 mM P h o s p h a t e ) , where t h e complex i s broken up, we o b s e r v e a second o r d e r r a t e which i n c r e a s e s l i n e a r l y w i t h i n c r e a s i n g [ c ] . The second o r d e r r a t e c o n s t a n t s o obtained i s k = 4X10 M s which a g r e e s w e l l w i t h jjggependent stopped f l o w measurements under i d e n t i c a l c o n d i t i o n s . fei

S p e c i e s Dependence. We have found t h a t t h i s i n t r a m o l e c u l a r r a t e i s rather s e n s i t i v e t o small perturbations. F o r example, when t h e p r i m a r y sequence o f c y t c i s a l t e r e d , t h e r a t e c o n s t a n t c a n change by o v e r a f a c t o r o f 4: k . . , = 4000s" k = 6000s . chicken cow « k

horse

"

l

8

0

0

s

k

"

tuna

"

2

H

0

0

s

k

~

yeast

=

1

2

0

0

s

"

S i m i l a r examples o f t h e dependence o f i n t r a m o l e c u l a r t r a n s f e r r a t e s on p r o t e i n p r i m a r y ^ g t r u c t u r e a r e found i n r e a c t i o n s i n t h e c y t c / c y t c p e r o x i d a s e system. Meâl S u b s t i t u t i o n : P h o t o i n d u c e d E l e c t r o n T r a n s f e r . The d a t a f o r t h e Fe b5/Fe c r e a c t i o n , by t h e m s e l v e s , a r e i n s u f f i c i e n t t o e s t a b l i s h e i t h e r t h e r e o r g a n i z a t i o n energy λ, o r t h e exchange energy Η , f o r the p r o t e i n c o u p l e . As one approach t o t h i s problem, we p r e p a r e d and c h a r a c t e r i z e d d e r i v a t i v e s o f cytochrome c i n which F e i s r e p l a c e d by m e t a l s w i t h v e r y d i f f e r e n t r e a c t i v i t y (eg: Z n ( I I ) ) . S i n c e Znc, and the a n a l o g o u s f r e e base, Η porphc, have f i l l e d d s h e l l s , they have r e l a t i v e l y l o n g l i v e d e x c i t e d s t a t e s , which can s e r v e as s t r o n g reducing agents: E ° 3 * ' = 0.8 V s 10 msec. Α β

(

E

3

Z

+

S

n

c

0

/

Λ

(

V

Z

n

c

T

)

S

2

m

s

e

c

)

°( porphc*)/porphc) ' porphc We, and o t h e r s , have shown t h a t t h e s e d e r i v a t i v e s m a i n t a i n t h e same s t r u c t u r e as n a t i v e cytochrome c, as j u d g e d by c i r c u l a r d i c h r o ism, h i g h r e s o l u t i o n magnetic resonance,^gnd by b i n d i n g t o F e ( I I I ) c y t b5, o r o t h e r p h y s i o l o g i c a l p a r t n e r s . Thus, by combining p u l s e - r a d i o l y s i s s t u d i e s o f Fec/Feb5 o r porphc" /Feb5 w i t h p h o t o l y s i s s t u d i e s o f metal s u b s t i t u t e d cytochromes i t i s p o s s i b l e t o vary t h e r e a c t i o n e x o t h e r m i c i t y from AG = 0.2 eV t o AG = 1.1eV. The r e s u l t s of f l a s h p h o t o l y s i s s t u d i e s w i t h porphc/b5 and Znc/b5 a r e shown i n f i g 6. Data from a l l d e r i v a t i v e s a r e summarised i n f i g u r e 7, as a plot of k v s . AG. C o n s i d e r i n g t h e u n c e r t a i n t i e s i n r a t e s which may r e s u l t from s m a l l c o n f o r m a t i o n a l d i f f e r e n c e s t h e d a t a can be a d e q u a t e l y d e s c r i b e d u s i n g s i m p l e Marcus t h e o r y ( s o l i d l i n e ) . This r e s u l t s u g g e s t s a t o t a l r e o r g a n i z a t i o n energy f o r r e a c t i o n o f λ = 0.7 eV. I t remains unknown how such r e o r g a n i z a t i o n energy might be p a r t i t i o n e d between A i and As. P r e l i m i n a r y measurements o f t h e temperature dependence o f t h e r e a c t i o n Fe b5/Fe c suggest Eact - 4 e t


156



.02·

1

3

5

F i g u r e 5. I n t r a m o l e c u l a r e l e c t r o n t r a n s f e r k i n e t i c s i n the c/b complex. Key: t o p , decay of f e ( I I ) b . (428 nm); bottom, growth of F e ( I I ) c (416 nm). t

Time F i g u r e 6. Top: quenching of (porph c y t c ) by F e ( I I I ) c y t b jilO μιη each, pH 7, ImMpi) (460 nm) ; bottom: quenching of (Zn c y t c ) - by F e ( I I I ) c y t b (460 nm).


II.

McLENDON ET AL.

157



k c a l / M o l ( f i g 8) which i s somewhat h i g h e r than the v a l u e o f 3.6 k c a l / M o l c a l c u l a t e d from Marcus t h e o r y assuming λ = 0.7 eV as i n f i g 7. However, the o b s e r v e d v a l u e might c o n t a i n some c o n t r i b u t i o n from c o n f o r m a t i o n a l changes which a f f e c t o r b i t a l o v e r l a p , and s t u d i e s a r e ongoing. The Marcus type f i t shown i n f i g u r e 7 i m p l i c i t l y assumes t h a t the p r i m a r y e f f e c t o f metal s u b s t i t u t i o n i s t o change the e x o t h e r m i c i t y , w h i l e h o l d i n g c o n s t a n t b o t h the r e o r g a n i z a t i o n energy and the donor acceptor e l e c t r o n i c coupling. We have a l r e a d y n o t e d t h a t e x t e n s i v e c o n f o r m a t i o n a l s t u d i e s s u g g e s t t h a t the metal s u b s t i t u t e d cytochromes c (eg: Z n c y t c , p o r p h c y t c ) a r e e s s e n t i a l l y i s o s t r u c t u r a l w i t h F e c y t c . Thus we expect t h a t f o r the Zn, Fe, and p o r p h y r i n c y t o chromes m e t a l - p o r p h y r i n dependent v a r i a t i o n s i n s t r u c t u r e w i l l not g r e a t l y a f f e c t the g e n e r a l t r e n d o b s e r v e d f o r the dependence o f r a t e on AG. The q u e s t i o n of metal dependent e f f e c t s on e l e c t r o n i c c o u p l i n g requires d e t a i l e d examination. There a r e two p o s s i b l e s o u r c e s f o r such e f f e c t s ^ F i r s t , as the donor b i n d i n g energy d e c r e a s e s from Fe t o Zn , the e l e c t r o n i c m i x i n g , e x p r e s s e d i n the dependence o f r a t e on d i s t a n c e k « exp-(aR) s h o u l d change. In tljie^simplest b a r r i e r I P

IP

F

o

r

t

n

i

s

t n e o r v

t u n n e l i n g t h e o r y α = ^ d n o r ~" medium^ > then, α depends s t r o g g l v ^ o n IP 88n8r. I n m o r ^ d e t a i l e d t h e o r i e s , based on superexchange, ' α depends weakly ( r o u g h l y l o g a r i t h m i c a l l y ) on IP donor. The a v a i l a b l e e x p e r i m e n t a l d a t a f o r the dependence o f α on IP s u p p o r t the superexchange model : α depends weakly on IP. Thus, we expect the shape o f the r a t e v s . AG p l o t w i l l be m i n i m a l l y a f f e c t e d by changes i n t h e " a " parameter among the d i f f e r e n t p o r p h y r i n s . A second t y p e of v a r i a t i o n i n e l e c t r o n i c c o u p l i n g c o u l d o c c u r . I t i s p o s s i b l e t h a t i n the Fe system, the donor w a v e f u n c t i o n i s h i g h y l o c a l i z e d a t the i r o n , whereas i n the e x c i t e d s t a t e Zn p o r p h y r i n , the e l e c t r o n i s c l e a r l y w i d e l y d e l o c a l i z e d around the r i n g I f t h i s were t r u e , then the " e f f e c t i v e / d i s t a n c e f o r the Fe r e a c t i o n would be c a : 4 A l o n g e r than f o r the Zn r e a c t i o n , c o r r e s p o n d i n g t o a hundred fold rate difference. However, the a v a i l a b l e e v i d e n c e s t r o n g l y s u g g e s t s t h a t e x t e n s i v e d e r e a l i z a t i o n o c c u r s between the Fe c e n t e r and the p o r p h y r i n π system. F o r example, combined ΝMR s t u d i e s and t h e o r e t i c a l c a l c u l a t i o n s o f F e ( I I I ) p o r p h y r i n s s u g g e s t the s p i n i s e x t e n s i v e l y d e l o c a l i z e d i n t o the π system. (φ = 0.7Φ+ 0.3Φ )· F u r t h e r m o r e , e x c e s s s p i n d e n s i t y , and a s s o c i a t e d e l e B S r B n d e n s i t y , i n cytochrome c i s d i r e c t e d a t the heme edge from which e l e c t r o n t r a n s f e r would o c c u r , r e f l e c t i n g an a n i s o t r o p i c i n t e r a c t i o n o f the d x z , d yz o r b i t a l s w i t h the a x i a l m e t h i o n i n e . ' I f we t a k e the c o n s e r v a t i v e e s t i m a t e s o f 10$ e l e c t r o n d e n s i t y a t the r e a c t i v e edge o f the Fe system, and -25$ e l e c t r o n d e n s i t y a t the edge f o r the more d e l o c a l i z e d Zn system, then a maximum " n o r m a l i z a t i o n f a c t o r " o f about two f o l d i n the e l e c t r o n i c f r e q u e n c y f a c t o r can be e s t i m a t e d t o c o r r e c t f o r d i f f e r e n t i a l w a v e f u n c t i o n o v e r l a p i n the F e c y t v s . Zncyt system. 0

m

1

Μ 0

ρ

η

We c o n c l u d e t h a t the b a s i c t r e n d o f i n c r e a s i n g r a t e w i t h i n c r e a s i n g AG i n the c/b5 system p r i m a r i l y r e f l e c t s a F r a n c k Condon term r a t h e r than an e l e c t r o n i c term. However, s i n c e s m a l l r a t e d i f f e r e n c e s may be p h y s i o l o g i c a l l y s i g n i f i c a n t , " t u n i n g " of the e l e c t r o n i c f a c t o r i s c e r t a i n l y worthy o f f u r t h e r s t u d y .



158


«Η 0

1

1

1

05

1

15

-AG (eV) F i g u r e 7. P l o t of In ( i n t r a c o m p l e x ) e l e c t r o n t r a n s f e r r a t e (10 μπι each, pH 7, ImM phosphate) vs AG.

F i g u r e 8.

Arrhenius plot

f o r data

i n F i g u r e 5.



11. MCLENDON ET AL.

159

A Second Example: Cytochrome c P e r o x i d a s e / c y t o c h r o m e c . Cytochrome c p e r o x i d a s e ( c e p ) c a t a l y z e s r e d u c t i o n o f HÔ^ i n y e a s t , w i t h cytochrome c p r o v i d i n g t h e r e d u c i n g equivalents: Fe


ES

III

ccp + H 0 2

IV °+ Fe cep (ES) + H 0

2

2

+ (2) F e ( I I ) c y t c -> (2) F e ( I I I )

cytc + Fe(III)ccp

D e t a i l e d c r y s t a l l o g r a p h i c s t r u c t u r e s a r e a v a i l a b l e f o r c c p , and c , and a d e t a i l e d s t r u c t u r a l p r o p o s a l e x i s t s f o r t h e n o n c o v a l e n t c c p / c y t c complex, as shown i n f i g u r e 9. I n t h i s complex, t h e hemes are r o u g h l y p a r a l l e l w i t h a c l o s e s t approach d i s t a n c e o f c a 16 A, (24 A Fe-Fe). A wide v a r i e t y o f p h y s i c a l e v i d e n c e , i n c l u d i n g c h e m i c a l c r o s s l i n k i n g s t u d i e s , s u p p o r t s t h i s proposed s t r u c t u r e . Thus, t h e i n t e r e s t i n g i n i t i a l q u e s t i o n s f o r r e l a t i n g s t r u c t u r e t o a c t i v i t y a r e : what a r e t h e r a t e s o f r e a c t i o n , and how do t h e s e r a t e s depend on r e a c t i o n e x o t h e r m i c i t y , metal s i t e s t r u c t u r e , and p r o t e i n structure. The c/ccp system i s w e l l s u i t e d f o r a d d r e s s i n g such questions. Fe c c p can be produced i n a v a r i e t y o f r e a c t i v e s t a t e s i n c l u d i n g F e ( I I ) ( h i g h s p i n ) , F e ( I I ) (low s p i n ) , F e ( I I I ) ( h i g h s p i n ) , F e ( I I I ) (low s p i n ) , and F e ( I V ) , and metal s u b s t i t u t i o n t o i n t r o d u c e Zn, Mn and m e t a l s i s f a c i l e . S i m i l a r l y , a v a r i e t y o f m e t a l l o c y t o c h r o m e c d e r i v a t i v e s can be produce^, e g : Ç ç p D c y t c , F e ( I I I ) c y t c , Z n c y t c , porph c y t c . F e

c c

P

/ F e

m

HI

n

The r e a c t i o n F e ccp/Fe cytc Fe ccp/Fe c y t c proceeds w i t h ΔΕ = 0.4V. The r e a c t i o n haç been monjôred both by p u l s e r a d i o l y s i s , and by s i m p l e m i x i n g o f Fe c c p + Fe c y t c , with equivalent r e s u l t s : k = 0.25 ± 0.07 s ( f i g u r e 10) I t i s i n t e r e s t i n g t h a t a dependence of r a t e on t h e p r i m a r y s t r u c t u r e o f t h e p r o t e i n i s ^ o b s e r v e d : ( a t c o n s t a n t AG) f o r h o r s e c y t c / c c p ( y e a s t ) k = 0.25 s but f o r yeasty cytc/(yeast) ccp k = 4 s and f o r tuna c y t c / y e a s t c c p k = 0.1 s , even though the g e n e r a l t h r e e d i m e n s i o n a l s t r u c t u r e s a r e e s s e n t i a l l y i d e n t i c a l f o r h o r s e , tuna and y e a s t cytochrome^ c . These d e t e r m i n a t i o n s d i s p r o v e an e a r l i e r s u g g e s t i o n based on modulated e x c i t a t i o n s p e c t r o s c o p y , t h a t k ~ 10 s . C l e a r l y t h e r a t e i s slow, but does t h i s slow r a t e r e f l e c t λ o r Η ? ^ The c o m p a r a t i v e ^ r a t e s t u d y o f porpnc /Fe ccp -> porphc/Fe c c p (ΔΕ - 0.9V k=100 s ) s u g g e s t s a h i g h r e o r g a n i z a t i o n energy (X-2V) for t h i s couple. I t i s l i k e l y t h a t much o f t h i s r e o r g a n i z a t i o n energy i s an i n n e r s p h e r e r e o r g a n i z a t i o n , r e f l e c t i n g t h e c o o r d i n a t i o n change between t h e h i g h s p i n 6 c o o r d i n a t e F e ( I I I ) / 5 c o o r d i n a t e Fe(II) couple. As a l r e a d y n o t e d , i t i s p o s s i b l e t o compare t h e r e a c t i v i t y o f another o x i d a t i o n s t a t e of ccp v i a the r e a c t i o n Fe(IV) ccp + cytc(red) Fe(III) ccp + c y t c (ox). For F e ( I I ) c y t c ΔΕ - IV and k Ξ 10^ s while f o r Z n ( I I ) c y t c , ΔΕ . . =°θ]^ k = 2 ± 0.4 s and f o r H porph c y t c reaction ' 2 ΔΕ^ ,. = 0.1V k = 4±0.4x10 s . We a g a i n see a s t r o n g reaction dependence o f r a t e i n d r i v i n g f o r c e , c o n s i s t e n t w i t h λ ~ 1.4V. Γβ

t

0




160

F i g u r e 9. Computer model of c y t c / c c p complex, ( c o u r t e s y Tom P o u l o s ) .

0.6.

ο 0

0.2 0.4

0.6 0.8

1

t (s) F i g u r e 10. R e a c t i o n of horse c y t c F e ( I I I ) / F e ( I I ) c c p top 436 nm (ccp). R e a c t i o n of horse c y t c F e ( I I I ) / F e ( I I ) c c p m i d d l e 416 nm ( c y t c ) . R e a c t i o n of y e a s t c y t c F e ( I I I ) / F e ( I I ) c c p bottom 436 nm.


11.

MCLENDON ET AL.


161


Thus, the p i c t u r e o b t a i n e d from the c y t c / c c p system i s q u i t e s i m i l a r t o t h a t i n the c y t c / c y t b 5 / b system: λ f o r p r o t e i n - p r o t e i n e l e c t r o n t r a n s f e r appears t o be l a r g e . Comparisons With Other I n t r a m o l e c u l a r P r o t e i n Redox R e a c t i o n s . A l t h o u g h work r e m a i n s l i m i t e d , p r o g r e s s i n t h i s a r e a i s i n d e e d r a p i d , and many new r e s u l t s have been r e p o r t e d w i t h i n the l a s t y e a r . E x p e r i m e n t s can be d i v i d e d i n t o two b a s i c c a t e g o r i e s : t r a n s f e r between p h y s i o l o g i c a l p a r t n e r s (eg: c y t b5/cytC.; c y t c / c y t c p e r o x i d a s e ( c c p ) , c y t b5/Hb), and t r a n s f e r between two groups i n the same g p r o t e i n r a n g i n g from Hoffman's s t u d i e s of α Znporph/gFeporph Hb, to s t u d i e s of Ru s u b s t i t u t e d p r o t e i n s ( c y t c , a z u r i n o r m y o g l o b i n ) by Gray and I s e i d . C u r r e n t l y a v a i l a b l e r e s u l t s a r e summarized i n T a b l e I. S e v e r a l p o i n t s a r i s e from t h i s c o m p i l a t i o n . F i r s t of a l l , f o r the p r o t e i n - p r o t e i n redox c o u p l e s , the dependences of r a t e on AG, and/or t e m p e r a t u r e , a r e g e n e r a l l y c o n s i s t e n t w i t h m o d e r a t e l y l a r g e r e o r g a n i z a t i o n e n e r g i e s (0.8V < λ < 2.0 V ) . Thus the m o d e r a t e l y l a r g e v a l u e of λ i n f e r r e d f o r the c/b5 c o u p l e appears l i k e l y t o be a common phenomenon r a t h e r than an anomaly. Recent model r e a c t i o n s (not p r o t e i n ) ^ i n low d i e l e c t r i c media l i k e i s o o c t a n e or polycarbonate show r e l a t i v e l y l a r g e e x p e r i m e n t a l s o l v e n t r e o r g a n i z a t i o n e n e r g i e s , r a n g i n g from λ = 0.6 - 1.0V w h i l e c l a s s i c a l d i e l e c t r i c continuum t h e o r y p r e d i c t s λ = 0 - 0.3V. The r e a s o n s f o r the f a i l u r e of continuum t h e o r y a r e not u n d e r s t o o d , but may r e f l e c t the h i g h l o c a l e l e c t r i c f i e l d s which o c c u r w i t h i n s e v e r a l angstroms of an i n j e c t e d c h a r g e . In t h i s c o n t e x t , v a l u e s o f λ £ 1V f o r p r o t e i n s do not seem anomalous, but a r e q u i t e i n l i n e w i t h o b s e r v a t i o n s o f s i m p l e redox r e a c t i o n s i n low d i e l e c t r i c media. We a l s o note t h a t the r a t e s o b s e r v e d f o r the p r o t e i n complexes a t o p t i m a l e x o t h e r m i c i t y a r e s i m i l a r t o t h o s e ^ o b s e r v e d a t o p t i m a l AG and s i m i l a r d i s t a n c e s i n small molecules. A second p o i n t of i n t e r e s t i s t h a t the λ v a l u e s i n f e r r e d f o r h i g h s p i n F e ( I I I ) complexes (eg: Hb) a r e much l a r g e r t h a n t h o s e seen f o r low s p i n systems (eg: c y t c ) . The slow r e a c t i o n s seen f o r h i g h s p i n Fe heme p r o t e i n s l i k e l y r e f l e c t a l a r g e i n t e r n a l r e o r g a n i z a t i o n energy a s s o c i a t e d w i t h the r e a c t i o n from 5 c o o r d i n a t e F e ( I I ) t o 6 coordinate F e ( I I I ) . A f i n a l p o i n t i s t h a t e l e c t r o n t r a n s f e r r e a c t i o n s of the Ru m o d i f i e d p r o t e i n s a r e g e n e r a l l y slow when compared w i t h the p r o t e i n p r o t e i n r e a c t i o n s or w i t h s i m p l e model systems. The r e a s o n s f o r t h i s d i s c r e p a n c y a r e f a r from c l e a r . L i k e l y e x p l a n a t i o n s i n c l u d e an underestimate of λ (the date of I s e i d , and some d a t a o f W i n k l e r e t al. s u g g e s t λ £ 0.8V), and an u n d e r e s t i m a t e o f the a p p r o p r i a t e d i s t a n c e f o r e l e c t r o n t r a n s f e r . I f t r a n s f e r p r o c e e d s from the heme d i r e c t l y t o the Ru atom, r a t h e r than v i a the i m i d a z o l e l i g a n d , then the l a r g e r d i s t a n c e l i s t e d i n the T a b l e i s a p p r o p r i a t e . Some e v i d e n c e f o r t h i s p o s s i b i l i t y comes from s t u d i e s o f a model r u t h e n i u m - p o r p h y r i n system.25 At h i g h e x o t h e r m i c i t y , a r a t e c o n s t a n t o f k ύ 10 s i s o b s e r v e d f o r e l e c t r o n t r a n s f e r from the p o r p h y r i n t o Ru. W h i l e r a p i d , t h i s r a t e i s f a r s l o w e r than e x p e c t e d f o r an a d i a b a t i c system (k > 10 s ). We i n f e r , t h e r e f o r e , t h a t a d i a b a t i c i t y i n t h i s system i s p r e c l u d e d by the poor o v e r l a p o f the donor and a c c e p t o r o r b i t a l s r e f l e c t i n g i n p a r t the h i g h l y metal


162


Table I .

Some R a t e s o f I n t r a m o l e c u l a r B i o l o g i c a l E l e c t r o n T r a n s f e r Reactions

System

I I

I I I

Fe cytb /Fe cytc λ * III Z n c y t c /Fe b 3 * III H p o r f c /Fe b III Zncytb_/Fe cytc - I l l H p o r f c /Fe b J


û

0

c

2

F e

i l

R(A)

k(s

0.2

8 (l6)

1.5X10

0.8

8

0.4

8

3X1 0 4

1.1

8

1.1

8

0.05

8(16)

0.01

0.9

8

8X1 0

3

d

1.0

8

2X10

3

c

0.9

20

100

0.9

16(24)

2X1 0

0.4

16

2

a

m

b

/Fe Hb III ZnHb/Fe b_ III ZnbJFe Hb III α Zn β Fe Hb

ref

3

b

5

1X10

5

m

')

4X1 0

3

c

5

5

5

I V

Fe ccp(ES)/Fe

I ] :

cytc I I

ccp(ES)/Zn cytc ccp(ES)/H porfcytc 2

I I

I

I I

Fe ccp/Fe" ' cytc Fe

I I I

ccp/porfcytc

c c p E S / p r o t e i n r a d i c a l (TRP) 3 * III Znccp /Fe c -.11 . , I l l

Thermal and Photoinduced Long Distance Electron Transfer in

Recommend Documents