Electron Transfer in Inorganic, Organic, and ... - ACS Publications

15 Electron Transfer in Inorganic, Organic, and Biological Systems Downloaded from pubs.acs.org by UNIV OF SOUTHERN CALIFORNIA on 06/18/16. For personal use only.

Electron Transfer Across Model Polypeptide and Protein Bridging Ligands Distance Dependence, Pathways, and Protein Conformational States Stephan S. Isied Department of Chemistry, Rutgers University, New Brunswick, NJ 08904

Studies of intramolecular electron transfer across model peptides and metal-modified proteins are reported in this chapter. Intramolecular electron-transfer studies across polyproline spacers have been com pared in the following three different metal donor-acceptor series: [(NH ) Os-iso-(Pro) -Co(NH ) ], [(NH ) Os-iso-(Pro) -Ru(NH ) ] (iso is isonicotinyl-), and [(bpy) Ru-X-(Pro) -Co(NH ) ], (bpy is 2,2'bipyridyl-, X is 4-carboxy-4'-methyl-2,2'-bipyridyl). In these studies the electronic coupling factor β was measured to be ~0.3-0.4 Å-1 for the ruthenium-bipyridine series with n = four to six prolines, in contrast to β ~ 0.9 Å-1 for the other metal-amine complexes with n = one to four prolines. These experiments suggest the possibility of observing rapid rates of electron transfer across 10 proline residues (~40 Å-1 in related ruthenium-bipyridine series. In the rutheniummodified proteins, intramolecular electron-transfer reactions with histidine-33 ruthenium-modified cytochrome c have shown that the rate of intramolecular reduction of the heme of cytochrome c can be changed by more than 5 orders of magnitude for different ruthenium donor complexes. However, oxidation of the heme of cytochrome c by the ruthenium-bipyridine complexes results in a rate that is substantially slower than predicted. Interpretation of these results using a mechanism by which protein conformational change is associated with the rate of electron transfer is proposed. 3 5

n

3 5

3 5

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0065-2393/91 /0228-0229$06.00/0 © 1991 American Chemical Society

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E T IN INORGANIC, ORGANIC, A N D BIOLOGICAL SYSTEMS

D O N O R - A C C E P T O R M O L E C U L E S separated b y synthetic p e p t i d e s a n d p r o teins have c o n t r i b u t e d significantly to o u r u n d e r s t a n d i n g a n d analysis o f i n t r a m o l e c u l a r electron-transfer reactions i n the past decade. T h e different c o n t r i b u t i o n s i n this v o l u m e attest to the v a r i e t y of elegant e x p e r i m e n t s that b o t h d e m o n s t r a t e various aspects of electron-transfer ( E T ) t h e o r y a n d p r o -

Electron Transfer in Inorganic, Organic, and Biological Systems Downloaded from pubs.acs.org by UNIV OF SOUTHERN CALIFORNIA on 06/18/16. For personal use only.

v i d e n e w challenges a n d questions for the theorist to answer i n o r d e r to i n t e r p r e t the n e w e x p e r i m e n t a l results. M y group's w o r k i n this area has c e n t e r e d a r o u n d m e t a l d o n o r - a c c e p t o r complexes

separated b y s y n t h e t i c p e p t i d e s a n d electron-transfer p r o t e i n s

(Structure 1). W e have d e s i g n e d s i m p l e m o d e l systems that e m p h a s i z e c e r tain p r o p e r t i e s of the b r i d g i n g ligands, as w e l l as of the donors a n d acceptors

ET

Structure 1. Binuclear metal donor-acceptor

complexes.

(1-5). A m o n g the most e x c i t i n g findings f r o m o u r c u r r e n t w o r k is the p r e d i c t i o n that long-range e l e c t r o n transfer across p o l y p e p t i d e s s h o u l d b e o b servable over 3 0 - 4 0 A i n reasonably short t i m e scales ( 5 x 10 1.9 x 1 0 1.2 Χ 1 0 "

àG°(eV)

1

9

5

2

NOTE: In all cases, the M - M distance was 9.0 Â.

-0.25 -0.15 +0.4

232


stantial a n d i n some instances exceeds the electronic factor (4). T h e o r y p r e dicts that the rate of i n t r a m o l e c u l a r e l e c t r o n transfer (Jk ) w i l l decrease w i t h distance a c c o r d i n g to the expression k α e" , w h e r e r is the edge-to-edge distance b e t w e e n the donor a n d acceptor at w h i c h the reaction b e c o m e s nonadiabatic (22). T h e e l e c t r o n i c c o u p l i n g factor β is a constant that is c h a r acteristic of the e l e c t r o n i c i n t e r a c t i o n b e t w e e n the d o n o r a n d acceptor across the b r i d g i n g l i g a n d . et


et

Pr

Polyproline Bridging Ligand. Before i n t r o d u c i n g o u r results o n the rate of e l e c t r o n transfer, a short i n t r o d u c t i o n to the p r o p e r t i e s of the p o l y p r o l i n e b r i d g i n g l i g a n d , w h i c h has b e e n the cornerstone of m y group's studies o n the distance d e p e n d e n c e of e l e c t r o n transfer across p o l y p e p t i d e s , w i l l b e

a)

Chart 1. a, b: Structures of t r a n s - and cis-proline polymers, respectively, from fiber X-ray diffraction (29). c: Structures of t r a n s - and cis-proline monomers. Open circles are nitrogen atoms, closed circles are carbon atoms, and closed circles that are connected by one bond to the main chain are carbonyl oxygen atoms.

15.

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Model Polypeptide and Protein Bridging Ligands

233


g i v e n . T h e o l i g o p r o l i n e p e p t i d e s ( C h a r t 1, a a n d b) p r o v e d to b e reasonably r i g i d m o l e c u l e s (23,24) for s t u d y i n g long-range 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 fer as a f u n c t i o n o f distance b e t w e e n a d o n o r a n d acceptor. T h e structural r i g i d i t y of p r o l i n e (Pro) oligomers i n c o m p a r i s o n to o t h e r naturally o c c u r r i n g a m i n o acids is d u e m a i n l y to the c y c l i c structure of the proline ring. The five-membered r i n g of the p r o l i n e side c h a i n restricts rotation a r o u n d the p e p t i d e b o n d , a n d this r e s t r i c t i o n results i n cis-trans conformational i s o m e r i s m (21-32), as s h o w n i n C h a r t 1, c. P o l y p r o l i n e bridges w e r e u s e d as r i g i d c h e m i c a l spacers i n early studies of e n e r g y transfer b e t w e e n organic donors a n d acceptors (23, 24). I n polar solvents the effic i e n c y o f e n e r g y transfer follows the r " distance d e p e n d e n c e for w e a k d i p o l a r e n e r g y transfer (33). T h e results o f these energy-transfer studies s h o w that p o l y p r o l i n e can b e u s e d as a spectroscopic r u l e r i n the 1 0 - 6 0 - A range (23, 24). 6

F i b e r s of p o l y p r o l i n e c r y s t a l l i z e d from aqueous solution possess a n a l l trans conformation ( > 9 5 % trans). W h e n the same p o l y p r o l i n e is c r y s t a l l i z e d from solvents of l o w e r p o l a r i t y , especially aliphatic alcohols, fibers o f the cis i s o m e r are o b t a i n e d instead. I n C h a r t 1, a a n d b show the fiber structures of cis- a n d f r a n s - p o l y p r o l i n e (34, 35). X - r a y diffraction analysis o f poly-Zp r o l i n e fibers shows clear structural differences b e t w e e n the cis a n d trans forms. A s can be seen i n C h a r t 1, b , b o t h p r o l i n e oligomers are h e l i c a l i n structure, w i t h different u n i t c e l l p r o p e r t i e s . f r a n s - P o l y p r o l i n e makes a lefth a n d e d h e l i c a l cycle e v e r y t h r e e residues, w i t h a 3.12-Â translation p e r r e s i d u e along the h e l i c a l axis. I n the cis i s o m e r a r i g h t - h a n d e d h e l i c a l t u r n consists of 3 1/3 p r o l i n e units w i t h a 1.85-Â translation p e r r e s i d u e . O n e of the i n t e r e s t i n g features of the fiber structure of these t w o p r o l i n e isomers ( C h a r t 1, a) is the fact that the trans i s o m e r possesses a n e x t e n d e d structure i n w h i c h p o l a r solvents can h y d r a t e the p e p t i d e b o n d s a n d stabilize the o p e n structure. I n less p o l a r m e d i a the cis conformation is m o r e c o m p a c t a n d is s t a b i l i z e d w h e n the p o l y m e r turns the h y d r o c a r b o n part o f the p r o l i n e r e s i d u e to the w e a k l y p o l a r solvents. T h e c o n v e r s i o n b e t w e e n trans- a n d d s - p o l y p r o l i n e isomers ( C h a r t 1, a) occurs w i t h a half-life o f a p p r o x i m a t e l y 1-2 m i n at r o o m t e m p e r a t u r e (enthalpy change Δ Η * ~ 20 k c a l m o l " ; e n t r o p y change A S * ~ 0) (36). ( F o r h i g h - m o l e c u l a r - w e i g h t o l i g o m e r s , several h o u r s are r e q u i r e d to c o m p l e t e this isomerization.) T h e i n t e r c o n v e r s i o n b e t w e e n the trans a n d cis isomers is k n o w n to be one of the slowest processes c o n t r o l l i n g conformational changes i n peptides a n d proteins (37,38). T h e studies d e s c r i b e d i n this chapter w e r e c a r r i e d out i n aqueous acidic m e d i a , u n d e r 1

conditions i n w h i c h the e x t e n d e d trans conformation of the p r o l i n e o l i g o m e r s is k n o w n to p r e d o m i n a t e (>95%) (39-43). Distance Dependence. W i t h this i n t r o d u c t i o n to the p r o l i n e b r i d g i n g p e p t i d e a n d the sensitivity of inorganic d o n o r - a c c e p t o r systems i n c o n t r o l l i n g rates of electron-transfer reactions, the [ ( N H a ^ O s - L - R ^ N F Q s ] (Os-

234


L - R u ) series [where L is iso(Pro) a n d iso is the i s o n i e o t i n y l group] w i l l b e discussed as a f u n c t i o n of the n u m b e r of p r o l i n e residues separating the d o n o r f r o m the acceptor. T a b l e II shows h o w the rate of i n t r a m o l e c u l a r e l e c t r o n transfer can be changed b y more than 8 orders of m a g n i t u d e as the distance b e t w e e n the donor a n d acceptor is increased b y the i n t r o d u c t i o n of a d d i t i o n a l p r o l i n e residues. I n this w o r k the donors a n d acceptors are k e p t the same a n d therefore this substantial change i n rate m u s t b e a t t r i b u t e d to the distance d e p e n d e n c e of the rate of i n t r a m o l e c u l a r e l e c t r o n transfer.


n

F o r molecules w i t h one, two, or three p r o l i n e s , the t e m p e r a t u r e d e p e n d e n c e of the rate of i n t r a m o l e c u l a r e l e c t r o n transfer has also b e e n s t u d i e d , a n d this i n f o r m a t i o n has b e e n u s e d to separate the d i s t a n c e - d e p e n d e n t c o m p o n e n t f r o m the electronic c o m p o n e n t of the reorganization energy. T h i s separation was done b y u s i n g a m o d i f i e d v e r s i o n of the transition-state expression w h e r e In k + A H V R T is p l o t t e d vs. distance ( F i g u r e 1, O s - L Ru). F r o m these plots the electronic c o u p l i n g factor, β ~ 0 . 6 - 0 . 7 A " , can b e calculated for the O s - L - R u series, w h e r e L is iso(Pro) . W i t h data o n a s i m i l a r series of molecules (the O s - L - C o series, w h e r e L is iso(Pro) ) that 1

n

n

Table II. Intramolecular Rates of Electron Transfer, Activation Parameters, and Distances for the [ ( N ^ O s - L - R i ^ N i y s ] Series η

OS

C^m\\\\\\S\^ RU

NOTE: L is iso(Pro)„.

k (s- ) 1

ΔΗ* AS* (kcal/mol) (calldeg mol)

M-M (A)

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Model

Polypeptide

and Protein

Bridging

235

Ligands

In (k ) + &m/RT et


20

In (k ) et

10

o'

8

• • • . » . . . •

»

12

16

20

Distance, Â Figure 1. Plot of In k vs. distance (lower curve) and In k + A H / R T vs. distance (upper curve) for the [(NH3)50s -L-Ru (NH )5] series; η = 0-4. e t

et

m

n

3

#

4+

w e s y n t h e s i z e d a n d s t u d i e d e a r l i e r , E n d i c o t t (44) o b t a i n e d a slightly h i g h e r β for t h e O s - L - C o series o f m o l e c u l e s . I n t h e O s - L - C o m o l e c u l e s , t h e rate of e l e c t r o n transfer occurs at significantly l o w e r t i m e scales because o f the larger r e o r g a n i z a t i o n e n e r g y o f the Co(III) m e t a l center. T h i s r e o r g a n i z a t i o n e n e r g y m a y b e e x p e c t e d to p r o d u c e m o r e conformational v a r i a b i l i t y o f the b r i d g i n g l i g a n d — e s p e c i a l l y i n t h e longer p r o l i n e b r i d g e s (η ~ 3 o r 4). I f d i r e c t m e t a l - m e t a l overlap o r o v e r l a p t h r o u g h t h e solvent i n these t w o systems is n e g l i g i b l e , t h e n one w o u l d expect similar results to b e o b s e r v e d for β i n b o t h systems. A v e r y i m p o r t a n t a s s u m p t i o n i n v o l v e d i n this analysis is that t h e r e o r ganization e n e r g y is associated w i t h t h e activation e n t h a l p y o f the r e a c t i o n w i t h n o e n t r o p i e c o n t r i b u t i o n s . T h i s a s s u m p t i o n is j u s t i f i e d because t h e d o n o r a n d acceptor are h y d r o p h i l i c a n d s i m i l a r i n n a t u r e . T h e charge o n the p r e c u r s o r a n d successor c o m p l e x i n t h e electron-transfer reaction is also the same [Os(II)Ru(III) - » Os(III)Ru(II)]. F o r a m e t a l - t o - m e t a l distance of 21 Â, a rate o f 50 s

- 1

was o b s e r v e d (5).

T h e d r i v i n g force for these reactions i n T a b l e I I is s m a l l ; r e d u c t i o n p o t e n t i a l £ ° ~ 250 m V . T h i s observation that r a p i d rates of e l e c t r o n transfer c a n b e o b t a i n e d at these l o n g distances (21 A ) e v e n w i t h these s m a l l d r i v i n g forces suggests that r a p i d e l e c t r o n transfer s h o u l d b e observable at m u c h l o n g e r distances w i t h p r o p e r c o n t r o l o f t h e d r i v i n g force a n d t h e r e o r g a n i z a t i o n energy.

236


T o achieve these longer-range

Polyproline Complexes.

electron

transfers, a n e w series of p o l y p r o l i n e complexes (I) was d e s i g n e d a n d s y n t h e s i z e d (bpy is 2 , 2 ' - b i p y r i d i n e ) (6). Ο

II

[(bpy^Ru ^


1

Series 1 T h e s e c o m p l e x e s have b e e n a s s e m b l e d from the reaction o f [ ( b p y ) R u ( b p y 2

n

C O O H ) ] a n d [ ( N H ) C o ( P r o ) O H ] b y u s i n g standard p e p t i d e synthetic t e c h 3

5

n

n i q u e s (45). T h e reason for choosing the r u t h e n i u m b i p y r i d i n e system is the availability of the Ru(I) oxidation state (more accurately d e s c r i b e d as R u L ) , n

e

w h i c h is a v e r y strong reductant; E° — - 1 . 3 V vs. N H E (normal h y d r o g e n electrode). T h e r e d u c t i o n p o t e n t i a l for the R u

I / n

c o u p l e is expected to b e

s i m i l a r to that for [Ru(bpy) ], E° = - 1 . 2 V vs. N H E . (See, for e x a m p l e , ref. 3

46.) W i t h such a strong r e d u c i n g agent, the i n t r a m o l e c u l a r electron-transfer reaction can t h e n take place w i t h m u c h larger d r i v i n g forces t h a n i n the [ ( N H ) O s - i s o ( P r o ) - R u ( N H ) 5 ] series. F u r t h e r m o r e , the Ru(III) b i p y r i d i n e 3

5

n

3

can b e u s e d as an oxidant w h e n o t h e r Ru(II) reductants are u s e d . T h e series of complexes (I) w e r e p u r i f i e d a n d c h a r a c t e r i z e d b y several p h y s i o c h e m i c a l t e c h n i q u e s . T h e c i r c u l a r d i c h r o i c ( C D ) spectra of series I (n — 1-7) are s h o w n i n F i g u r e 2. A s the n u m b e r of p r o l i n e residues increases from 1 to 4, a significant shift to l o w e r energy is o b s e r v e d i n the C D spectra. B e y o n d η = 4, no significant changes are o b s e r v e d . T h i s shift is a t t r i b u t e d to the formation of the p o l y p r o l i n e II left-handed h e l i x (6). S i m i l a r results w e r e o b t a i n e d e a r l i e r o n r e l a t e d molecules b y u s i n g

I 3

C a n d H N M R spec 1

troscopy (5). O t h e r e v i d e n c e for the secondary structure o f the p o l y p r o l i n e h e l i x c a m e f r o m early studies b y S t r y e r a n d H a u g l a n d (23) a n d G a b o r (24) o n s i m i l a r p o l y p r o l i n e p e p t i d e s b r i d g e d b y d o n o r a n d acceptor m o l e c u l e s o f the t y p e , D - ( P r o ) — A , w h e r e η is 1-12 p r o l i n e s , A is the d a n s y l e n e r g y n

acceptor at the a m i n o p r o l i n e t e r m i n a l , a n d D is the n a p h t h y l d o n o r at the c a r b o x y l p r o l i n e t e r m i n a l . T h e i r studies s h o w e d that for energy transfer across p o l y p r o l i n e s (n — 5 - 1 2 ) , the efficiency of e n e r g y transfer decreases w i t h the i n c r e a s i n g n u m b e r of p r o l i n e residues. T h i s decrease indicates a 5 0 % transfer efficiency at 34.6 Â a n d shows the r " d e p e n d e n c e p r e d i c t e d 6

b y Fôrster (33) for weak d i p o l e - d i p o l e c o u p l i n g . F i g u r e 3 shows h o w the rate of e l e c t r o n transfer changes w i t h the n u m b e r of p r o l i n e s separating the r u t h e n i u m a n d the cobalt. I n t r a m o l e c u l a r e l e c t r o n transfer does not decrease as fast as e x p e c t e d i f the n u m b e r of p r o l i n e units b e t w e e n the d o n o r a n d acceptor adopts the secondary h e l i c a l structure ( F i g u r e 3). F u r t h e r m o r e , the t e m p e r a t u r e d e p e n d e n c e o f the r e action ( F i g u r e 4) demonstrates that the distance d e p e n d e n c e is m a i n l y c o n t r o l l e d b y e l e c t r o n i c effects because the change i n t e m p e r a t u r e does not change the slope o f the plot. A n e l e c t r o n i c c o u p l i n g factor, β ~ 0 . 3 - 0 . 4 A " , is calculated for this 1

Figure 2. Circular dichroic (CD) spectra of the proline-bridged electron-transfer series I for η = 1-7 taken in aqueous solution. The CD maximum located at *~200 nm (ττ-ττ*) is characteristic of the trms-polyproline (II) structure. This feature shifts to longer wavelengths as η is increased from 1 to 4.

NANOMETERS


to

E T IN INORGANIC, ORGANIC, AND BIOLOGICAL SYSTEMS


238

In k

Prolines

(n)

Figure 3. A plot of In k + A H / R T vs. the number of proline residues for η = 2-6 prolines. The smaller slope for η = 4-6 corresponds to the stabili zation of the polyproline secondary structure. et

In k

#

et

27

T h r o u g h - b o n d Distance

o

32

(A)

Figure 4. Temperature dependence of the rate of intramolecular electron trans fer for the complexes Ru-iso(Pro) -Co (I), for η = 4-6. n


15.

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Model Polypeptide and Protein Bridging Ligands

series of m o l e c u l e s . T h i s β can b e c o m p a r e d d i r e c t l y w i t h that i n the e a r l i e r systems. T h e m o r e facile i n t r a m o l e c u l a r electron-transfer reaction i n these l o n g e r p r o l i n e s m a y be a t t r i b u t e d to a better m a t c h i n g of the orbitals of the d o n o r w i t h that o f the b r i d g e . T h i s i m p r o v e m e n t m a y reflect changes i n the e l e c t r o n i c structure of the p o l y p r o l i n e b r i d g e after it has a d o p t e d the h e l i c a l structure (Structure 2). Regardless of i n t e r p r e t a t i o n , the o r i g i n a l p o i n t m a d e i n this chapter that e l e c t r o n transfer can be o b s e r v e d at r a p i d rates o v e r 40 Â is n o w f u r t h e r s t r e n g t h e n e d , because the distance b e t w e e n the R u a n d the C o i n these series is approximately 30 Â. E x c h a n g i n g the Co(III) acceptor w i t h the Ru(III) or other acceptors w i t h m u c h l o w e r i n n e r - s p h e r e reorganization energy is one approach to f u r t h e r e x t e n d the distance d e p e n d e n c e of i n t r a m o l e c u l a r e l e c t r o n transfer to —40 A (i.e., 10 p r o l i n e residues separating the d o n o r a n d the acceptor). W e are c u r r e n t l y c a r r y i n g out these experiments (6). E l e c t r o n transfer across r i g i d h y d r o c a r b o n spacers has b e e n elegantly d e m o n s t r a t e d o n a variety of systems, starting w i t h the p i o n e e r i n g w o r k of M i l l e r a n d C l o s s (47). D i f f e r e n t organic h y d r o c a r b o n spacers have b e e n investigated b y C l o s s , D e r v a n , P a d d e n R o w , a n d others; these studies are s u m m a r i z e d i n ref. 47. It is desirable to compare saturated organic h y d r o c a r b o n spacers a n d p e p t i d e spacers. A l t h o u g h the c u r r e n t data is l i m i t e d , a qualitative c o m p a r i s o n indicates that electronic transmission across p e p t i d e s is m o r e facile t h a n across saturated hydrocarbons (a l o w e r β was o b s e r v e d for the p e p t i d e b r i d g i n g groups).

Protein Donor-Acceptor Complexes W e have s h o w n that it is possible to e x t e n d the concept of d o n o r - a c c e p t o r complexes to an electron-transfer p r o t e i n b y covalently attaching a w e l l d e f i n e d transition m e t a l c o m p l e x to a spécifie a m i n o a c i d side c h a i n i n the p r o t e i n (7-9). U s i n g a v a r i e t y of r u t h e n i u m - a m i n e complexes to m o d i f y horse-heart c y t o c h r o m e c, w e isolated r u t h e n i u m - m o d i f i e d proteins i n w h i c h the m o d i f i c a t i o n b y the r u t h e n i u m c o m p l e x occurs at the H i s - 3 3 p o s i t i o n . V a r i a t i o n of the t y p e of Ru(III) c o m p l e x attached to the cyt c can change the p r o t e i n m o i e t y f r o m an acceptor to a donor. W h e n t h e m o d i f i e d p r o t e i n [cyt e - R u ( N H ) ] is p r e p a r e d i n t h e 3

5

>—Co(NH3>

5

Structure 2. Stabilized secondary structure adopted by type I complexes, Ruiso(Pro) -Co, for η = 6. Similar helices are present for η = 4 and 5. n

240


Ru(III)cyt c(III) state a n d t h e n r e d u c e d w i t h a v a r i e t y of radicals generated b y pulse radiolysis t e c h n i q u e s , i n t r a m o l e c u l a r e l e c t r o n transfer from the r u t h e n i u m site to the h e m e site occurs w i t h a rate constant, k = 53 s" , Δ £ ° for cyt c = 0.26 V , a n d àE° for [ ( N H ) R u ( H i s ) ] = 0.10 V . S i m i l a r results have b e e n o b t a i n e d b y flash photolysis t e c h n i q u e s (II). T h e t e m p e r a t u r e d e p e n d e n c e , concentration d e p e n d e n c e , a n d p H d e p e n d e n c e of this electron-transfer reaction w e r e investigated. T h e results of this i n v e s tigation s h o w e d that the rate of e l e c t r o n transfer is i n d e p e n d e n t of c o n c e n tration a n d m o d e r a t e l y sensitive to t e m p e r a t u r e (ΔΗ* ~ 3.5 k c a l M " a n d A S * ~ - 3 9 eu). T h e electron-transfer reaction is i n d e p e n d e n t of p H t h r o u g h p H 5 - 9 ; t h e n it increases b e l o w p H 5 as the native conformation of the cyt c changes (9). 1


3

5

I I / m

1

T h e observation that i n t r a m o l e c u l a r e l e c t r o n transfer b e t w e e n the r u t h e n i u m site a n d the h e m e site occurs at distances of 1 2 - 1 5 Â is e x t r e m e l y significant, because i t represents the first observation of an i n t r a m o l e c u l a r electron-transfer reaction w i t h i n an electron-transfer p r o t e i n . T h e m a g n i t u d e of the rate constant, 53 s" , is s i m i l a r to o t h e r rate constants that are k n o w n to o c c u r w i t h i n the native cyt c m o l e c u l e (8, 9). T h i s finding l e d us to q u e s t i o n w h e t h e r the u n i m o l e c u l a r rate o b s e r v e d is r a t e - l i m i t i n g i n e l e c t r o n transfer (eq 1) o r i n a protein-associated conformational change (eqs 2a a n d 2b). 1

Ru cyt c n

where k

et

i n

Ru cyt c m

11

(1)

is the rate constant for i n t r a m o l e c u l a r e l e c t r o n transfer Ru cyt c n

m

Ru *cyt c n

m

Ru *cyt c n

Ru cyt c m

(2a)

m

11

(fast)

(2b)

a n d k is the rate constant for a p r o t e i n conformational change (8-11). T o answer this q u e s t i o n , a series of r e l a t e d r u t h e n i u m molecules that are m o r e o x i d i z i n g t h a n cyt c w e r e s y n t h e s i z e d a n d c h a r a c t e r i z e d . T h e redox potentials of the Ru(II)/(III) c o u p l e , greater than 0.26 V , a l l o w one to reverse the d i r e c t i o n of e l e c t r o n transfer i n the m o d i f i e d cyt c s u c h that e l e c t r o n flow i n the Ru(III)cyt c(II) is from the h e m e to the r u t h e n i u m . T h u s , one can change the h e m e of cyt c f r o m a n e l e c t r o n acceptor to an e l e c t r o n donor. T h e rationale b e h i n d these e x p e r i m e n t s is rather s i m p l e ; i f the u n i m o l e c u l a r rate o b s e r v e d is r a t e - l i m i t i n g i n e l e c t r o n transfer, t h e n s i m i l a r v a r i a t i o n i n rates of e l e c t r o n transfer s h o u l d b e o b s e r v e d for r e d u c t i o n a n d oxidation œ

(J0). S t r u c t u r e 3 shows the structure of cyt c a n d the positions of the h e m e relative to the r u t h e n i u m - m o d i f i e d sites. T a b l e I I I s u m m a r i z e s the rates of i n t r a m o l e c u l a r e l e c t r o n transfer for the r e d u c t i o n a n d oxidation of cyt c b y a n u m b e r of r u t h e n i u m complexes. T h e rate of r e d u c t i o n of cyt c can be changed b y m o r e than 5 orders of


15.

ISIED

Model Polypeptide and Protein Bridging

Ligands

241

Structure 3. Ruthenium-modified cytochrome c showing the relative of the heme and the ruthenium sites.

position

m a g n i t u d e , d e p e n d i n g o n the redox p o t e n t i a l a n d the reorganization e n e r g y of the r u t h e n i u m - m o d i f i e d species (Table III). T w o types of c o m p l e x e s coo r d i n a t e d to H i s 33 of c y t o c h r o m e c can b e i d e n t i f i e d . I n the first t y p e , e l e c t r o n transfer takes place from (or to) a r u t h e n i u m t o r b i t a l . T h i s c o n d i t i o n is satisfied for the oxidation a n d r e d u c t i o n reactions of c y t o c h r o m e c b y the different r u t h e n i u m complexes (I—II a n d V - V I I i n T a b l e III). T h e other type of reactions are those i n w h i c h the e l e c t r o n is transferred f r o m a r a d i c a l a n i o n l i g a n d attached to the r u t h e n i u m b o u n d at the cis or trans p o s i t i o n to the i m i d a z o l e m o i e t y of the H i s 33. T h i s c o n d i t i o n is satisfied for the r e d u c t i o n of cyt c w i t h reactions i n complexes I I I - I V (Table III). 2 g

T h e r u t h e n i u m - m o d i f i e d proteins constitute an i n t e r e s t i n g series of m o d i f i e d proteins i n w h i c h the distance b e t w e e n the r u t h e n i u m l a b e l a n d t h e h e m e g r o u p is r e l a t i v e l y w e l l - d e f i n e d . T h e variation i n the r e d u c t i o n p o tential of the r u t h e n i u m complexes a l l o w e d us to study, for the first t i m e ,

242


Table III. Rates of Intramolecular Electron Transfer and Reduction Potential of Ruthenium-Cytochrome c Complexes Cyt c

Ruthenium-Modified


3

4

5

U

y

U

Py

Py

P

fc fe

2

(VI) [Ru(bpy) (im)HH/ni) 2

(VII) [Ru(bpy)(terpy)]-(II/III) (VIII) [Ru(bpy) OH ]-(II/III) 2

Intramolecular ET, k (s- )

0.26 -0.01 0.13 -1.3 -1.3 0.92 0.79 0.74 0.65

5 x 10 55 2.8 Χ 10 2.0 Χ 10 40 55 40 40

&

2

Direction of ET

1

fl

Native H H cytochrome c (I) c-[(NH ) Ru(OH)]-(II/III) (II) [(NH ) Ru]-(II/III) (III) [R (bpy)(bp -)( )]-(II/II-) (IV) [R (b )(bpy-)(im)]-(H/II-) (V) [Ru(b y) (py)]-(II/III) 3

E° (V)

—

— Ru —> heme Ru —> heme Ru —> heme Ru —» heme Heme —» R u Heme —> R u Heme —> R u Heme - » R u

2

5

5

Έ° is the reduction potential of the eyt c or substituted cyt c vs. the normal hydrogen electrode (NHE). bpy~ is the bipyridine radical anion; bpy is bipyridine; py is pyridine; im is imidazole; and terpy is terpyridine. è

the r e d u c t i o n a n d oxidation of the cyt c f r o m a remote site, H i s 33, w h i c h is a p p r o x i m a t e l y 15 A away f r o m the h e m e group. T h e s c h e m e for s t u d y i n g the i n t r a m o l e c u l a r electron-transfer step b y u s i n g pulse radiolysis techniques is o u t l i n e d i n S c h e m e I. I n this scheme oxidation of the R u - c y t c species b y C 0 * generates a n o n e q u i l i b r i u m d i s t r i b u t i o n b e t w e e n the R u c y t c a n d R u e y t c . T h e relaxation to e q u i l i b r i u m d i s t r i b u t i o n is t h e n taken as a measure of the rate of i n t r a m o l e c u l a r e l e c t r o n transfer from the r u t h e n i u m site to the h e m e site or vice versa. S i m i l a r reactions can be o b s e r v e d for the r e d u c t i o n of cyt c w i t h C 0 * a n d 3

n

m

m

11

2

C0 * 2

+

Rn cytc m

m

e

+

(*q)

(red) RiAytc

Ru cytc n

m

Hi (red)

Ru cytc

1 0

n

cty

Ru cytc n

K

Ru cytc ni

n

et (ox)

n

Scheme I. CO* and C0 ~ are radicals generated from a chemical precursor by using pulse radiolysis techniques; CO* ~^ k ; C0 ~ —> k . CO* was generated in 0.1 M NaHC0 . C0 ~ was generated in 0.1 M NaHC0 . e was generated in 0.13 M t-BuOH. All experiments were conducted at pH 7-8 in 0.05-0.1 M phosphate buffer. 3

et(red)

2

3

3

et(ox)

3

(aq)

n

15.

ISIED

Model Polypeptide

and Protein Bridging

243

Ligands

( ) (Scheme I). T h e first two entries i n T a b l e I I I clearly show that the rate of r e d u c t i o n of cyt c b y the r u t h e n i u m - a m m i n e complexes decreases w i t h a d e c r e a s e i n d r i v i n g f o r c e . T h u s , i n g o i n g f r o m [ R u ( N H ) O H ] to [ R u ( N H ) ] , the rate changes b y an o r d e r of m a g n i t u d e (10). T h i s change is consistent w i t h a s i m p l e electron-transfer step that follows M a r c u s t h e o r y . F u r t h e r increase i n d r i v i n g force can l e a d to f u r t h e r increase i n rate, as is o b s e r v e d for the [ ( b p y ) R u L ] complexes. (The e l e c t r o n i n these complexes is l o c a l i z e d o n the b p y ligands, a n d therefore these complexes are m o r e c o r r e c t l y f o r m u l a t e d as R u L . ) e

aq

3

3

4

5


2

n

#

A different t y p e of b e h a v i o r is o b s e r v e d for the cyt c oxidation w i t h the r u t h e n i u m - b i p y r i d i n e complexes, w h e r e the d i r e c t i o n of e l e c t r o n transfer is expected to be f r o m the h e m e to the Ru(III) l a b e l . T h r e e r e l a t e d cyt c derivatives i n this series have b e e n c h a r a c t e r i z e d a n d s t u d i e d . T h e m a i n difference b e t w e e n these r u t h e n i u m labels is i n the d r i v i n g force of the reaction (Table III). T h e rates of oxidation of cyt c i n these three complexes are e q u a l w i t h i n e x p e r i m e n t a l error. T h e rate constant for this process is — 4 0 - 5 5 s" . T h i s i n s e n s i t i v i t y of rate to d r i v i n g force for these complexes, as w e l l as the m a g n i t u d e of the o b s e r v e d rate constant, argues against a s i m p l e i n t r a m o l e c u l a r electron-transfer step as the r a t e - l i m i t i n g step i n these reactions. T h e r e f o r e the oxidation of cyt c b y this R u l a b e l does not s e e m to b e l i m i t i n g i n e l e c t r o n transfer. E a r l i e r w e i n t e r p r e t e d this p h e n o m e n o n i n t e r m s of a d i r e c t i o n a l e l e c t r o n transfer (10). H o w e v e r , these data m a y b e a c c o m m o d a t e d b y changes i n the electron-transfer p a t h w a y (i.e., different pathways are operational for r u t h e n i u m - a m m i n e complexes t h a n for the r u t h e n i u m - b i p y r i d i n e complexes). F u r t h e r w o r k is r e q u i r e d to define the m o l e c u l a r a n d electronic events that l e a d to these different rates. 1

1 1 1

I n c o n c l u s i o n , w e have shown that i n s i m p l e d o n o r - a c c e p t o r complexes w h e r e peptides mediate b e t w e e n the d o n o r a n d acceptor, rates of e l e c t r o n transfer can v a r y over m a n y orders of m a g n i t u d e i n a p r e d i c t a b l e way. I n p r o t e i n s , h o w e v e r , electronic a n d conformational states m a y interfere w i t h electron-transfer rates t h r o u g h specific p r o t e i n d y n a m i c a l changes that take c o n t r o l of the electron-transfer process. U n d e r s t a n d i n g the e l e m e n t a r y steps associated w i t h e l e c t r o n transfer w i l l b e one of the future aims that w o u l d h e l p i n u n d e r s t a n d i n g the structure a n d function of electron-transfer p r o teins.

Acknowledgments I thank the graduate students a n d postdoctoral fellows w h o p a r t i c i p a t e d i n this w o r k a n d w h o s e names are m e n t i o n e d i n the p u b l i c a t i o n s , A . Vassilian, M . O g a w a , J . W i s h a r t , R. B e c h t o l d , a n d B . v a n H e m e l r y c k . T h e c y t o c h r o m e c w o r k was investigated b y a n u m b e r of talented students, i n c l u d i n g G . Worosila, M . Gardineer, A . Greway, and M . O . K . C h o . Finally I acknowledge the h e l p a n d c o n t i n u e d support of H a r o l d S c h w a r z of the B r o o k h a v e n

244


N a t i o n a l L a b o r a t o r y , w h o i n t r o d u c e d o u r g r o u p to the p u l s e radiolysis t e c h niques.


References

D.

J. J.

Β.

Β.

1. Isied, S. S. Prog. Inorg. Chem. 1984, 32, 443-517. 2. Isied, S. S.; Vassilian, A . J. Am. Chem. Soc. 1984, 106, 1726. 3. Isied, S. S.; Vassilian, Α.; Magnuson, R.; Schwarz, H . J. Am. Chem. Soc. 1985, 107, 7432-7438. 4. Isied, S. S.; Vassilian, Α.; Wishart, J.; Creutz, C,; Schwarz, H.; Sutin, N . J. Am. Chem. Soc. 1988, 110, 635. 5. Vassilian, Α.; Wishart, J.; van Hemelryck, B.; Schwarz, H.; Isied, S. S. J. Am. Chem. Soc. 1990, 112, 7278. 6. Ogawa, M. Y.; Wishart, J. F; Isied, S. S., unpublished results. 7. Isied, S. S.; Worosila, G . ; Atherton, S. J . J. Am. Chem. Soc. 1982, 104, 7659-7661. 8. Isied, S. S.; Kuehn, C.; Worosila, G . J. Am. Chem. Soc. 1984, 106, 5145. 9. Bechtold, R.; Gardineer, M. B . ; Kazmi, Α.; van Hemelryck, B . ; Isied, S. S. J. Phys. Chem. 1986, 90, 3800. 10. Isied, S. S. In Electron Transfer in Biology and the Solid State: Inorganic Compounds with Unusual Properties; Johnson, M. K.; King, R. B.; Kurtz, M., Jr.; Kutal, C.; Norton, M. L.; Scott, R. Α., Eds.; Advances in Chemistry 226; American Chemical Society: Washington, D C , 1990; pp 91-100. 11. Bechtold, R.; Kuehn, C.; Lepre, C.; Isied, S. S. Nature (London) 1986, 322, 286. 12. Schanze, K . ; Sauer, K . J. Am. Chem. Soc. 1988, 110, 1180. 13. Sisido, M.; Tanaka, R.; Inai, Y.; Imanishi, Y. J. Am. Chem. Soc. 1989, 111, 6790-6796. 14. Farraggi, M.; DeFelippis, M. R.; Klapper, M. H. J. Am. Chem. Soc. 1989, 111, 5141. 15. Therien, M. J.; Bowler, Β. E.; Selman, M. A.; Gray, Η. B.; Chang, I-J.; Winkler, R. In Electron Transfer in Inorganic, Organic, and Biological Systems; Bolton, R.; Mataga, N.; M c L e n d o n , G., Eds.; Advances in Chemistry 228; American Chemical Society: Washington, D C , 1991; Chapter 12. 16. Mayo, S. L.; Ellis, W. R., Jr.; Crutchley, R. J.; Gray, Η. B. Science (Washington, D.C.) 1986, 233, 948-952. 17. Yocum, K. M.; Shelton, J. B.; Schroeder, W. Α.; Worosila, G,; Isied, S. S.; Bordignon, E.; Gray, Η. B. Proc. Natl. Acad. Sci. U.S.A. 1982, 79, 7052. 18. Nocera, D . G ; Winkler, J. R.; Yocum, Κ. M.; Bordignon, E.; Gray, Η. B. J. Am: Chem. Soc. 1984, 106, 5145-5150. 19. Natan, M. J.; Baxter, W. W.; Kuila, D.; Gingrich, D. J.; Martin, G. S.; Hoffman, M. In Electron Transfer in Inorganic, Organic, and Biological Systems; Bolton, J. R.; Mataga, N.; McLendon, G., Eds.; Advances i n Chemistry 228; American Chemical Society: Washington, D C , 1991; Chapter 13. 20. Liang, N.; Mauk, A. G.; Pielak, G J.; Johnson, J. Α.; Smith, M.; Hoffman, M. Science (Washington, D.C.) 1988, 240, 311. 21. M c L e n d o n , G.; Hickey, D.; Berghuis, Α.; Sherman, F.; Brayer, G. In Electron Transfer in Inorganic, Organic, and Biological Systems; Bolton, J. R.; Mataga, N.; M c L e n d o n , G., Eds.; Advances in Chemistry 228; American Chemical So ciety: Washington, D C , 1991; Chapter 11. 22. Sutin, N. In Electron Transfer in Inorganic, Organic, and Biological Systems;

15.


23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36. 37. 38. 39. 40. 41. 42. 43. 44. 45. 46. 47.

ISIED

Model Polypeptide and Protein Bridging

Ligands

245

Bolton, J . R.; Mataga, N.; McLendon, G . , E d s . ; Advances in Chemistry 228; American Chemical Society: Washington, D C , 1991; Chapter 3. Stryer, L.; Haugland, R. P. Proc. Natl. Acad. Sci. U.S.A. 1967, 58, 719. Gabor, G . Biopolymers 1958, 6, 809. Steinberg, I. Z.; Harrington, W. F.; Berger, Α.; Sela, M.; Katchalski, Ε. J. Am. Chem. Soc. 1960, 82, 5263. Engel, J. Biopolymers 1966, 4, 945. Schimmel, P. R.; Flory, P. J. Proc. Natl. Acad. Sci. U.S.A. 1967, 58, 52. Cowan, P. M.; McGavin, S. Nature (London) 1955, 176, 501. Torehia, P. Α.; Bovey, F. A. Macromolecules 1971, 4, 246. Deber, C. M.; Bovey, F. Α.; Carver, J. P.; Blout, E . R. J. Am. Chem. Soc. 1970, 92, 6191. Chao, Y. H.; Bersohn, R. Biopolymers 1978, 17, 2761. C h i u , H. C.; Bersohn, R. Biopolymers 1977, 16, 277. Förster, T. Ann. Phys. (Leipzig) 1948, 2, 55. Traub, W.; Shmueli, U . Nature (London) 1963, 195, 1165. Burge, R. E.; Harrison, P. M.; McGavin, S. Acta Crystallogr. 1962, 15, 914. Cheng, Η. N.; Bovey, F. A. Biopolymers 1977, 16, 1465-1472. L i n , L.; Brandt, J. F. Biochemistry 1983, 22, 553-559. Brandt, J. F.; Halvorson, H. R.; Brennan, M. Biochemistry 1975, 14, 4953. Rothe, M.; Theysohn, R.; Steffer, K. D.; Schneider, H.; Amani, M.; Kostrzewa, M. Angew. Chem. Int. Ed. Engl. 1969, 8, 919. Rothe, M.; Rott, H. Angew. Chem. Int. Ed. Engl. 1970, 15, 770. Grathwohl, C.; Wuthrich, K. Biopolymers 1978, 17, 2761. Grathwohl, C.; Wuthrich, K. Biopolymers 1976, 15, 2025 and 2043. Clark, D. S.; Dechter, J. J.; Mandelkern, L. Macromolecules 1979, 12, 626. Endicott, J. F. Acc. Chem. Res. 1988, 21, 59. Stewart, J. M.; Young, J. D. Solid Phase Peptide Synthesis, 2nd ed.; Pierce Chemical Co.: Rockford, IL, 1984. Juris, Α.; Balzani, V.; Barigelletti, F.; Campagna, S.; Belser, P.; von Zelewsky, A. Coord. Chem. Rev. 1988, 84, 85. Closs, G. L.; Miller, J. R. Science (Washington, D.C.) 1988, 240, 440.

R E C E I V E D for review A p r i l 27, 1990. A C C E P T E D revised manuscript September 27, 1990.

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