Electrostatic, Steric, and Reorganizational Control ... - ACS Publications

8 Electrostatic, Steric, and Downloaded by UNIV OF SOUTHERN CALIFORNIA on June 18, 2016 | http://pubs.acs.org Publication Date: May 5, 1989 | doi: 10.1021/ba-1990-0226.ch008

Reorganizational Control of Electron Self-Exchange in Cytochromes Dabney White Dixon and Xiaole Hong Department of Chemistry, Georgia State University, Atlanta, GA 30303

To probe the factors that control biological electron transfer, we analyzed the electron self-exchange reactions of cytochromes c, c , and b . Numerical analysis of the crystal structures gave heme ex posuresand dipole moments of the proteins. Molecular modeling of the electron-transfer complexes allowed calculation of the heme-heme distance. These values, in conjunction with the experimental rate constants as a function of temperature and ionic strength, give the reorganizational energies, λ, which are 0.7, 0.5, and 1.2 eV for cytochromes c, c , and b , respectively. The numerical values of the reorganizational energies are sensitive to the assumptions made about the heme exposure, but the order (i.e., cytochrome c < cytochrome c < cytochrome b ), remains the same for reasonable values of the heme exposure. The parameters derived from this anal ysis were used to analyze studies of the bimolecular electron transfer between cytochromes c and c , the bimolecular electron transfer between cytochromes c and b , and the intracomplex electron transfer between cytochromes c and b . The correlation between the self— 551

5

551

5

551

5

551

5

5

exchange parameters and those obtained from these studies is very good.

ELECTRON TRANSFER IS ONE OF THE MOST BASIC

b i o l o g i c a l reactions, i m portant i n photosynthesis, oxidative p h o s p h o r y l a t i o n , oxidation o f e n d o g e nous a n d exogenous substrates, a n d maintenance o f e n z y m e s i n active states (1-8). T h e rate constant is c o n t r o l l e d b y a v a r i e t y o f factors, i n c l u d i n g t h e d r i v i n g force, distance, a n d reorganizational e n e r g y o f t h e system (9-21). 0065-2393/90/0226-0161$06.00/0 © 1990 American Chemical Society

Johnson et al.; Electron Transfer in Biology and the Solid State Advances in Chemistry; American Chemical Society: Washington, DC, 1989.

162

E L E C T R O N TRANSFER IN BIOLOGY A N D T H E SOLID STATE

T h e nature of the i n t e r v e n i n g residues a n d orientation of the p r o s t h e t i c groups are c o n s i d e r e d i m p o r t a n t . I n a d d i t i o n , conformational change is c o m i n g i n t o focus as a c o n t r o l l i n g factor for b i o l o g i c a l e l e c t r o n transfer (22-25).

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C u r r e n t u n d e r s t a n d i n g of the factors that c o n t r o l e l e c t r o n transfer comes from a variety of types of studies. M e a s u r e m e n t of electron-transfer rate constants b e t w e e n physiological partners is c e r t a i n l y i m p o r t a n t , p a r t i c u l a r l y i n an effort to u n d e r s t a n d h o w electron transfer occurs i n v i v o (1-9). H o w e v e r , u n t i l the advent of s i t e - d i r e c t e d mutagenesis, these studies d i d not a l l o w systematic variation o f the properties of the p r o t e i n s . T h e r e f o r e , i n the last two decades great effort has gone into m e a s u r e m e n t of e l e c t r o n transfer rate constants b e t w e e n n o n p h y s i o l o g i c a l partners (15-21). S u c h studies i n c l u d e b i 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 two proteins o r b e t w e e n a p r o t e i n a n d an inorganic or organometallic c o m p l e x . M o r e r e c e n t l y , these studies have b e e n e x p a n d e d to i n c l u d e 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 two proteins w i t h i n a c o m p l e x or b e t w e e n the p r o t e i n redox site and an organometallic species attached to the p r o t e i n surface (16-18). B e cause diffusional considerations are nonexistent a n d p r o t e i n - p r o t e i n e l e c trostatic i n t e r a c t i o n factors are m i n i m a l i n i n t r a m o l e c u l a r e l e c t r o n transfer, these studies a l l o w particular focus o n the d e p e n d e n c e of e l e c t r o n transfer o n distance, o n the i n t e r v e n i n g residues, o n the reorganizational e n e r g y o f the t w o centers, a n d o n the orientation o f the two redox centers. S t i l l another approach to p r o b i n g the factors that c o n t r o l electron transfer comes from m e a s u r e m e n t o f e l e c t r o n self-exchange, e l e c t r o n transfer b e t w e e n the o x i d i z e d a n d r e d u c e d forms of the same m o l e c u l e (20, 26): c y t + cyt ±¥ c y t + cyt °\ a

b

r e d

a

r e d

o x

b

E l e c t r o n self-exchange rate constants are o f f u n d a m e n t a l i m p o r t a n c e because the t h e r m o d y n a m i c d r i v i n g force ( A G ) is zero i n this r e a c t i o n , a n d h e n c e there is no net d r i v i n g force for the reaction. Differences i n the e l e c t r o n self-exchange rate constants b e t w e e n similar proteins arise from differences i n the electrostatic, geometric, a n d reorganizational character istics of the proteins. Self-exchange rate constants for a variety of b - a n d c-type cytochromes, m e a s u r e d d i r e c t l y v i a N M R spectroscopic t e c h n i q u e s , are g i v e n i n T a b l e I. T h e s e rate constants span at least five orders o f m a g n i t u d e , from 1 0 to 1 0 M " s" . A n u n d e r s t a n d i n g of the factors that d e t e r m i n e this w i d e range is i m p o r t a n t for a clear p i c t u r e of b i o l o g i c a l e l e c t r o n transfer. 0

2

7

1

1

O u r goal i n this chapter is to delineate the factors that c o n t r o l e l e c t r o n transfer i n three h e m e proteins: c y t o c h r o m e c (48, 49), c y t o c h r o m e c ^ (50), a n d c y t o c h r o m e b (51-54). Self-exchange is a b i m o l e c u l a r process a n d as such can b e expressed (20) as 5

-AG * r

fc t e

where k

et

=

SKaV K n

e l

exp

'

(1)

is the o b s e r v e d rate constant for electron transfer; S is the steric


8.

DIXON & H O N G

163

Electron Self-Exchange in Cytochromes

factor, w h i c h reflects the hypothesis that e l e c t r o n transfer occurs p r i m a r i l y at the exposed h e m e edge i n the cytochromes; K is the association constant a

for formation of the p r e c u r s o r state from the two separated electron-transfer partners; v is the nuclear frequency factor; K is the average p r o b a b i l i t y of n

d

passing from the transition state to products; A G * is the free energy of r

activation (the s u m of b o t h i n n e r - s p h e r e a n d outer-sphere reorganizational energies); R is the gas constant; a n d T is the t e m p e r a t u r e . W e m e a s u r e d the self-exchange rate constants of these three proteins as a f u n c t i o n of i o n i c Downloaded by UNIV OF SOUTHERN CALIFORNIA on June 18, 2016 | http://pubs.acs.org Publication Date: May 5, 1989 | doi: 10.1021/ba-1990-0226.ch008

strength a n d t e m p e r a t u r e ; details of these measurements are r e p o r t e d else w h e r e (42, 46, 55). T h e rate constants, i n c o m b i n a t i o n w i t h p r o t e i n d i p o l e m o m e n t s calculated from the crystal structures a n d m o l e c u l a r m o d e l i n g of the electron-transfer complexes, allow us to estimate each of the parameters i n e q 1 a n d thus p r o v i d e a d e t a i l e d p i c t u r e of the factors that c o n t r o l e l e c t r o n transfer i n cytochromes.

Steric Considerations A l l t h r e e of the h e m e proteins discussed h e r e i n have part of the h e m e itself exposed to solvent (56, 57). E a r l y suggestions that e l e c t r o n transfer occurs m a i n l y t h r o u g h the exposed h e m e edge i n b i m o l e c u l a r e l e c t r o n transfer (57) w e r e followed b y various experiments to establish this. M u c h of the w o r k has i n v o l v e d d e r i v a t i z a t i o n of specific residues o n the p r o t e i n surface. I n g e n e r a l , alterations close to the exposed h e m e edge have a larger effect o n e l e c t r o n transfer than do derivatizations r e m o v e d f r o m the h e m e (58). M o r e r e c e n t l y , a n u m e r i c a l analysis of the h e m e exposure, a s s u m i n g e x p o n e n t i a l falloff of the electron-transfer rate constant, i n d i c a t e d that most of the elec t r o n transfer occurs though the exposed h e m e edge (55). E v e n i f e l e c t r o n transfer is largely at o r near the exposed h e m e edge, a n u m b e r of different complexes may b e i n v o l v e d , as has b e e n discussed b y N o r t h r u p et a l . for the c y t o c h r o m e c - c y t o c h r o m e c peroxidase system (59), b y M a u k et a l . for the reaction b e t w e e n the d i m e t h y l ester of c y t o c h r o m e c a n d c y t o c h r o m e b (60), a n d b y Slayton et a l . for reactions of c y t o c h r o m e b w i t h various partners (61). 5

5

I f w e assume that electron transfer occurs largely t h r o u g h the exposed h e m e edge, t h e n i t is necessary to k n o w the fraction of the p r o t e i n surface that is h e m e to analyze the data. T o compare the electron-transfer rate constants of the three proteins discussed h e r e i n , w e calculated this fraction b y u s i n g a p r o b e sphere of 1.5 A a n d the C o n n o l l y a l g o r i t h m (62), as i m p l e m e n t e d i n the B I O G R A F m o l e c u l a r m o d e l i n g p r o g r a m (63). T h e fraction of the surface area of the p r o t e i n that is h e m e , , is 0.007, 0.012, a n d 0.038 for c y t o c h r o m e c [we made appropriate substitutions i n the X - r a y structure of the p r o t e i n from t u n a (48, 49)], c y t o c h r o m e c (50), a n d c y t o c h r o m e b (51), respectively. 5 5 1

5

T h e form of b o v i n e c y t o c h r o m e b s t u d i e d consists of 82 a m i n o a c i d residues a n d differs from the l i p a s e - s o l u b i l i z e d f o r m that has b e e n charac5


Johnson et al.; Electron Transfer in Biology and the Solid State Advances in Chemistry; American Chemical Society: Washington, DC, 1989. 1 10 4 4 7.5

5.5 5.5 -8 7

112 108 112 109 104

104 104 87 86

Horse heart CDNP-Lys-13 Horse heart CDNP-Lys-72 Euglena gracilis C552 Chlorobium thiosulfatophilum C555

2

Rhodospirillum rubrum c Saccharomyces cerevisiae c I Crithidia oncopelti Cm Candida krusei c Horse heart c

-6

134

a

Charge on Fe(lll) Protein

Paracoccus denitrificans C550

Cytochrome

Amino Acids, No.

7.0 7.0 7.0 7.0

7

6.9 7.0 7.3 7 6.6

7.5

b

pD

0.1-1 M salt 0.1 M phosphate + 0.1 M KC1

M-V

35 35 36 37

28 29 30 29, 31 31-34

27

Ref.

8.

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DIXON & H O N G

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CMCMCMCM--H00COCM 00 00 00 00 H 00

o

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165

166


t e r i z e d crystallographically b y r e m o v a l of t w o residues from the a m i n o t e r m i n u s a n d n i n e residues f r o m the carboxyl t e r m i n u s . T h e s e q u e n c e has r e c e n t l y b e e n r e d e t e r m i n e d from the b o v i n e l i v e r c y t o c h r o m e b

5

cDNA

clone (52) a n d f r o m gas-phase sequence analysis of t r y p t i c p e p t i d e s o b t a i n e d from a t r y p t i c hydrolysate of apocytochrome b (46). T h e c o r r e c t e d s e q u e n c e 5

differs from that r e p o r t e d e a r l i e r (53, 54) i n that residue 57 is n o w k n o w n to b e aspargine a n d residues 11 a n d 13 are k n o w n to b e g l u t a m i c a c i d a n d g l u t a m i n e , respectively. Downloaded by UNIV OF SOUTHERN CALIFORNIA on June 18, 2016 | http://pubs.acs.org Publication Date: May 5, 1989 | doi: 10.1021/ba-1990-0226.ch008

Cytochrome b

5

is somewhat different from cytochromes c a n d c ^

in

that one of the h e m e propionates is e x t e n d e d out i n t o the solvent. T o d e t e r m i n e the c o n t r i b u t i o n of this p r o p i o n a t e , w e r e m o v e d

the t e r m i n a l

C H C 0 H atoms from the data set. T h e h e m e fraction decreased to 0.027. 2

2

W e u s e d the larger 0.038 i n the calculations i n this chapter; the s m a l l e r h e m e exposure w o u l d result i n a smaller reorganizational energy. I f electron transfer w e r e to occur o n l y t h r o u g h the exposed h e m e edge, t h e n the steric factor, S, w o u l d b e s i m p l y the fraction o f the surface o f the p r o t e i n that is h e m e squared o r . T h i s is, h o w e v e r , a m i n i m u m v a l u e 2

because, as M a r c u s a n d S u t i n p o i n t e d out (20), it is i n g e n e r a l necessary to integrate over a l l m u t u a l orientations a n d distances of the r e a c t i n g p a i r . M a r c u s a n d S u t i n u s e d a value o f S o f 0.01 i n t h e i r analysis o f the e l e c t r o n self-exchange rate constant o f c y t o c h r o m e c (20). G i v e n that the surface o f c y t o c h r o m e c is 0 . 7 % h e m e , this value o f S assumes that e l e c t r o n transfer is e n h a n c e d b y a d d i t i o n a l factor of 15. W e made d e t a i l e d n u m e r i c a l analyses o f the X - r a y structures o f c y t o chromes c, C55 a n d b , b y u s i n g a m o d e l i n w h i c h e l e c t r o n transfer falls off L5

5

exponentially w i t h distance (55). T h e m o d e l assumed that the o r i e n t a t i o n of the two hemes a n d the nature of the i n t e r v e n i n g residues h a d no effect o n the electron-transfer rate constant a n d that e l e c t r o n transfer o c c u r r e d o n l y at the surface of the p r o t e i n . T h e analysis s h o w e d that e l e c t r o n transfer at the h e m e edge accounts for 4 0 % (cytochrome c) to 8 0 % (cytochrome b ) of 5

the total e l e c t r o n transfer. T h e s e values w o u l d c o r r e s p o n d to e n h a n c e m e n t s of 2.4 for c y t o c h r o m e c a n d 1.3 for c y t o c h r o m e b . F o r this c h a p t e r w e chose 5

an e n h a n c e m e n t factor of 5, b e t w e e n the u p p e r a n d l o w e r estimates. T h e h e m e exposures a n d h e m e - h e m e

distances calculated from the

crystal structures m a y not represent the values f o u n d i n solution. I n a study of the c y t o c h r o m e c - c y t o c h r o m e b

5

i n t e r a c t i o n , W e n d o l o s k i et a l . (64) r a n

picosecond d y n a m i c s a n d f o u n d substantial motions of the residues i n the interface b e t w e e n the two hemes. I n p a r t i c u l a r , t h e y o b s e r v e d that the side c h a i n o f p h e n y l a l a n i n e - 8 2 m o v e d to a b r i d g i n g p o s i t i o n b e t w e e n the t w o h e m e s i n the complex. T h e y also f o u n d that the i n t e r - i r o n distances i n two simulations w e r e 1 . 1 - 2 . 1 A smaller than the 17.8-A distance i n the static model. P r o t e i n motions i n general are u n d e r i n c r e a s i n g study (65-68). i n cytochromes have b e e n m e a s u r e d w i t h N M R spectroscopic

Motions

techniques


8.

167


DIXON & H O N G

(69, 70). Studies of a m i d e N H exchange, i n c l u d i n g w o r k o n the cytochromes (71, 72), r e v e a l that relatively large p r o t e i n motions o c c u r o n a b i o l o g i c a l t i m e scale (73). M y o g l o b i n has also b e e n investigated b y m o l e c u l a r m o d e l i n g (74). Its crystal structure shows no c h a n n e l large e n o u g h to a d m i t to ligands such as carbon monoxide. T h e r e f o r e , p r o t e i n

fluctuations

are necessary to

accommodate the C O b i n d i n g . T h e m o d e l i n g indicates that m o r e t h a n one c h a n n e l from the p r o t e i n surface to the h e m e opens a n d closes d y n a m i c a l l y a n d allows C O b i n d i n g . T h e b i n d i n g of x e n o n to m y o g l o b i n shows s i m i l a r Downloaded by UNIV OF SOUTHERN CALIFORNIA on June 18, 2016 | http://pubs.acs.org Publication Date: May 5, 1989 | doi: 10.1021/ba-1990-0226.ch008

characteristics (75).

The Association Constant, K

a

T h e association constant of the two cytochromes i n the p r o p e r geometry for e l e c t r o n transfer, K , can b e estimated b y calculating the effective v o l u m e a

over w h i c h the reaction occurs along the reaction coordinate m u l t i p l i e d b y an electrostatic w o r k t e r m [exp (-w /RT)] r

K

(20),

—ID

= 4 i r N r * 8 ( r ) exp —

(2)

r

w h e r e N is Avogadro's n u m b e r , r is the s u m of the r a d i i of the two e l e c t r o n transfer p r o t e i n s , 8(r) is the range of i n t e r n u c l e a r separations that c o n t r i b u t e significantly to the reaction rate, a n d w is the w o r k to b r i n g the two proteins into the p r o p e r geometry for e l e c t r o n transfer. T h e value for 8(r) is usually taken to be that at w h i c h the electron-transfer rate constant falls to lie of its value at the distance of closest approach. I n general, e l e c t r o n transfer is thought to fall off exponentially as exp [-p(d - d )] (d is the distance b e t w e e n the two hemes i n the electron-transfer complex); 8(r) may b e e s t i m a t e d as p" . F o r c y t o c h r o m e c, r = 33.2 A (47). W i t h P = 0.9 A"1 a n d h e n c e 8(r) = 1.1 A, w e calculate 4 i r N r 8 ( r ) = 9.3 M . r

0

1

2

1

Values for c y t o c h r o m e e ^ a n d c y t o c h r o m e b are g i v e n i n T a b l e I I . M u l t i p l i c a t i o n of these values b y the appropriate w o r k t e r m s (discussed i n the next section) gives the K values. T h e s e K values are r o u g h a p p r o x i mations because the specifics of the p r o t e i n surface cannot be t a k e n i n t o account. T h e y are not d i r e c t l y comparable w i t h e x p e r i m e n t a l m e a s u r e m e n t of p r o t e i n association, because the latter i n c l u d e s geometries that are not p r o d u c t i v e for e l e c t r o n transfer. O u r N M R spectroscopic studies have s h o w n no e v i d e n c e for d i m e r i z a t i o n of cytochromes c or c at concentrations u p to approximately 10 m M . C y t o c h r o m e b shows small changes i n the c h e m ical shifts a n d l i n e w i d t h s of the h e m i n m e t h y l resonances as a f u n c t i o n of concentration. I f these changes reflect p r o t e i n d i m e r i z a t i o n , t h e n the e q u i l i b r i u m constant is 2 5 - 3 0 M " (55). 5

a

a

5 5 1

5

1


168


Table II. Calculation of Reorganization Energies for k = S K v K exp (-G */RT) et

Factor Heme fraction surface area Steric factor 2

1

1


0

e

SK V K l A

N

n

eI

0.007 0.0012 16.6 9.27 2.7 0.097 8.9 4.9 x i o 5.9 x 10 5.1 x 10 4.2 0.72

Radius (A) 4irr dr Work (kcal mol ) K (M- ) Heme-heme distance K i = exp - d )) a

a

r

Cytochrome c

Cytochrome c si

3

k (exptl.) A G * (kcal mol ) X(eV)

3

et

1

r

5

0.038 0.0361 15.9 8.50 3.1 0.045 7.5 1.7 x i o 2.9 X 10 2.7 x 10 6.85 1.2

3

2

8

8

6

e

Cytochrome b

5

0.012 0.0036 14.4 6.98 0.30 4.2 8.6 6.5 x i o 9.8 x 10 5.1 x 10 3.1 0.54

3

6

NOTE: \L = 0.1 M , 2 5 ° C .

The Work Term and Electrostatic Considerations B i m o l e c u l a r e l e c t r o n transfer i n b i o l o g i c a l systems occurs b e t w e e n

species

that u s u a l l y have a n e t charge as w e l l as a substantially a s y m m e t r i c charge d i s t r i b u t i o n . G i v e n a n appropriate m o d e l , o n e c a n use t h e d e p e n d e n c e o f the electron-transfer rate constant o n i o n i c s t r e n g t h to calculate t w o r e l a t e d parameters: t h e e n e r g y n e e d e d to f o r m t h e electron-transfer c o m p l e x (i.e., the w o r k term) at a g i v e n i o n i c strength a n d t h e rate constant extrapolated to infinite i o n i c s t r e n g t h . O f t h e t h e o r e t i c a l approaches p r e s e n t l y available, w e (55) a n d others (76, 77) find that o f v a n L e e u w e n (78) to b e t h e best at t h e i o n i c strengths u s e d i n N M R e x p e r i m e n t s ( 0 . 1 - 1 . 5 M ) . T h i s f o r m a l i s m treats each p r o t e i n as b o t h m o n o p o l e a n d a d i p o l e . T h e expression i s : In A

= -[Z Z m

rei

+ (ZD)(1 + K r ) + (DD)(1 + K r ) ] ^jL^f( ) 2

K

(3a)

w h e r e k is t h e B o l t z m a n n constant, q is t h e charge o n t h e e l e c t r o n , € is t h e d i e l e c t r i c constant, e is t h e p e r m i t t i v i t y o f v a c u u m , a n d T is t h e t e m p e r 0

ature. ZD

=

Z

Electrostatic, Steric, and Reorganizational Control ... - ACS Publications

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