Chiral Recognition by Metal-Ion Complexes in Electron-Transfer

Downloaded by UNIV OF CALIFORNIA SANTA BARBARA on September 3, 2015 | http://pubs.acs.org Publication Date: May 5, 1989 | doi: 10.1021/ba-1990-0226.ch013

13 Chiral Recognition by Metal-Ion Complexes in Electron-Transfer Reactions 1

Rosemary A. Marusak, Thomas P. Shields, and A. Graham Lappin

Department of Chemistry, University of Notre Dame, Notre Dame, IN 46556

Chiral induction in the reaction between horse cytochrome c(II) and [Co(ox) ] (ox is oxalate(2-)) was investigated. The usefulness of [Λ-Co(ox) ] as a stereoselective probe is established in the oxidation of [Co(en) ] (en is 1,2-diaminoethane), in which two products are formed: [Co(en) ] by an outer-sphere pathway with an enantio meric excess of8%Δ and [Co(en) (ox)] by an inner-sphere pathway with an enantiomeric excess of 1.5% Λ. Stereoselectivity in the re action with cytochrome c(II) averages 9%, with a preference for the Λ form of the oxidant. Equilibrium dialysis experiments with cyto chrome c(III) indicate that [Λ-Co(ox) ] binds preferentially to the protein and that this ion pair is a good model for an active electron 3-

2-

3

3-

3

2+

3

3+

3

+

2

3-

3

-transfer precursor complex. The mechanism is discussed in terms of known anion binding sites on the protein surface.

REPORTS OF CHIRAL INDUCTION IN ELECTRON-TRANSFER REACTIONS in volving metal-ion complexes have been published over the past decade (1-5). The induction is a direct measure of the relative reactivities of an optically active reagent, Δ - Α (ox is oxidized) with the enantiomeric forms of the electron-transfer reaction partners A - B and A - B (red is reduced), expressed as k Jk± , eqs 1 and 2. οχ

red

à

1

r e d

x

Δ-Α

ο χ

Δ-Α

ο χ

reà

+ à-B

+ A-B

A-A r

e

d

- ^ à-A

reà

r e d

+ Δ-Β

ο χ

(1)

+ Δ-Β

ο χ

(2)

Address correspondence to this author. 0065~2393/90/0226-0237$06.00/0 © 1990 American Chemical Society

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

238

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

T h e reaction b e t w e e n [Co(edta)]" [ e d t a tetraacetate(4-)] a n d [ C o ( e n ) ] 3

2 +

4-

is

1,2-diaminoethane-N,Ν,Ν',Ν'-

(en is 1,2-diaminoethane) is perhaps the

most extensively s t u d i e d system from the p o i n t of v i e w o f stereoselectivity (4, 5). It is a n outer-sphere process, a n d w h e n [â-Co(edta)]~ is u s e d , t h e p r o d u c t , [ C o ( e n ) ] , shows a 1 0 % e n a n t i o m e r i c excess o f the Λ i s o m e r , a 3

3 +

Δ Λ process w h e r e k /k àA

àA

is 1.22. T h e s e stereoselectivity studies are pos


sible because o f the substitution inertness o f the cobalt(III) complexes a n d the s l o w e l e c t r o n i c self-exchange b e t w e e n cobalt(III) a n d cobalt(II), w h i c h p r e v e n t s r a c e m i z a t i o n b y a self-exchange m e c h a n i s m . C o m p a r i s o n s w i t h i o n - p a i r i n g stereoselectivities for i n e r t isostructural analogues (5, 6) suggest a d o m i n a n t role for p r e c u r s o r c o m p l e x

stereoselec

t i v i t y i n the c h i r a l i n d u c t i o n . A m o d e l o f t h e p r e c u r s o r c o m p l e x has b e e n d e r i v e d from studies w i t h structurally r e l a t e d d e r i v a t i v e s . I n the m o d e l , the orientation o f the [Co(edta)]" oxidant is w e l l d e f i n e d ,

hydrogen-bonding

t h r o u g h its carboxylate face to the a m i n e protons o f the [ C o ( e n ) ] 3

2 +

reduc-

tant. T h i s latter reagent is i n d i s c r i m i n a t e i n its interactions because o f its relatively h i g h s y m m e t r y a n d its conformational l a b i l i t y . T h e i m p o r t a n c e o f h y d r o g e n b o n d i n g t h r o u g h the carboxylate face of the oxidant i n this reaction has p r o m p t e d studies w i t h the r e l a t e d c o m p l e x [ C o i o x ^ ] " , i n w h i c h carboxylate faces are also available, as oxidant (7). T h e 3

c o m p l e x is r e a d i l y r e s o l v e d (8); i t has a r e d u c t i o n potential of 0.57 V (vs. the n o r m a l h y d r o g e n electrode) (9) a n d a l o w self-exchange rate (10), w h i c h are i d e a l for stereoselectivity studies. H o w e v e r , it is heat- a n d l i g h t - s e n s i t i v e a n d is p r o n e to r a p i d r a c e m i z a t i o n , e v e n i n the s o l i d state. O n e reason for investigating the use o f [ C o i o x ^ ] " as a stereoselective 3

oxidant lies i n its value as a p r o b e o f the mechanisms o f e l e c t r o n transfer w i t h metalloproteins b e l o w t h e i r isoelectric points. C h i r a l r e c o g n i t i o n i n reactions o f metalloproteins has p r o v e d to b e rather difficult to detect, d e spite the obvious chirality of the reagents i n v o l v e d . E a r l y w o r k e r s i n the field failed to detect rate differences i n the r e d u c t i o n of Δ - a n d A - [ C o ( e n ) ] 3

3 +

b y parsley f e r r e d o x i n ( I I ) a n d the r e d u c t i o n o f horse c y t o c h r o m e c b y Δ and A-[Co(sep)]

2 +

(sep is s e p u l c h r a t e ,

1,3,6,8,10,13,16,19-octaazabicy-

clo[6,6,6]eicosane) (12). H o w e v e r , rate measurements are rather i n s e n s i t i v e a n d cannot d i s t i n g u i s h stereoselectivities b e l o w the o r d e r o f 1 0 % or so. M o r e r e c e n t l y , B e r n a u e r a n d Sauvain (13) r e p o r t e d stereoselectivity i n the reaction of c h i r a l iron(II) complexes w i t h plastocyanin. T h e i n t i m a t e m e c h a n i s m i n v o l v e d is not k n o w n , as the i r o n reagents have b e e n s h o w n to favor i n n e r sphere mechanisms (14). Stereoselectivity i n the r e d u c t i o n o f [ C o i o x ^ ] " b y cobalt(II) i n 1,23

d i a m i n o e t h a n e solutions is r e p o r t e d i n this chapter to demonstrate the use fulness o f this reagent as a stereoselective oxidant. Because of the u t i l i t y o f this reagent, [ C o i o x ) ^ " is e m p l o y e d as a c h i r a l p r o b e i n the oxidation o f 3

horse cytochrome c(II). T h i s particular reaction is chosen for several reasons. F i r s t , the p r o t e i n carries a + 6 charge at p H 7; the - 3 charge o n the oxidant


13.

239

Chiral Recognition by Metal-Ion Complexes

MARUSAK E T AL.

enhances the electrostatic attraction b e t w e e n the reagents, a factor i m p o r t a n t i n i n f l u e n c i n g the stereoselectivity. S e c o n d , the reaction is r e l a t i v e l y w e l l characterized, a n d two p r e v i o u s k i n e t i c studies are r e p o r t e d (15,16). F i n a l l y , e l e c t r o n transfer at cytochrome c takes place t h r o u g h the exposed h e m e edge, a r o u n d w h i c h there are w e l l - d e f i n e d a n i o n - b i n d i n g sites. N M R spec troscopic studies (17, 18) w i t h the paramagnetic analogue [ C r f a x ^ ] " have p i n p o i n t e d three areas o n the p r o t e i n surface at w h i c h b i n d i n g takes place, a w e a k site distant from the h e m e edge a n d two stronger b i n d i n g sites i n the v i c i n i t y of the reaction center. T h e role of these b i n d i n g sites i n the electron-transfer process w i l l b e discussed.


3

Experimental Details The preparation of K [Co(ox) ] · 3 . 5 H 0 (molar absorptivity, €βο5 = 165 M cm ) and resolution of the complex were carried out by literature methods (7, 8). The absolute configuration is taken as [ A - H s a r C o i o x ^ ] " (A€ 22 = 3.80 M cm" ) (19). The complex is light-sensitive, and all manipulations were carried out i n the dark. Racemization of the complex amounted to 10-12% over a 2-h period; this value was applied as a correction factor i n the studies. The reaction stoichiometry and products were determined by high-performance liquid chromatography ( H P L C ) with an ionexchange column (Waters Protein Pak S P - 5 P W Sephadex). Typical reaction conditions involved addition of [ C o ^ x ^ ] (ΙΟ" M ) to a solution containing cobalt(II) ( Ι Ο M ) i n excess 1,2-diaminoethane (2.0 x 10 —10 M ) and appropriate supporting electrolyte, with rapid stirring and under an atmosphere of argon to prevent aerial oxidation of the cobalt(II) complex. In some experiments, oxalate ion and l,2- C-oxalate ion (3 x ΙΟ M ) were added to the cobalt(II) solution. After completion of the reaction, the mixture was cooled and ice-cold 6 M HC1 added so that the resultant p H was less than 1. Aliquots of this mixture were then subject to analysis. The two products [Co(en) ] and [Co(en) (ox)J were isolated on a 1- χ 10-cm column (Sephadex SP C-25) and a 1- X 20-cm column (Dowex 5 0 X 2 400), washed with water and dilute acid, and eluted with 1.0 M H C 1 and 0.01 M HC1, respectively. The electron-transfer stereoselectivity was determined by examining the optical purity of the reaction products. Absolute configurations are [A-( + )-Co(en) (ox)] (€500 = 103 M " cm" , A € = 2.65 M cm" ) (20) and [A-( + )-Co(en) ] (€467 = 88 M c m , & € = 1 9 0 M cm ) (21). Kinetic measurements were made anaerobically at 25.0 °C and 0.1 M ionic strength (NaC10 ), under pseudo-first-order conditions with an excess of reductant and with an excess of the 1,2-diaminoethane ligand as a buffer. The decomposition of [ C o ^ x J J - was monitored at 605 n m . Horse cytochrome c (Sigma, Type VI) was used without further purification. Samples of the oxidized protein were dialyzed i n appropriate bufier solutions for at least 2 h before use. The reduced protein was obtained by the addition of a few crystals of sodium dithionite to the oxidized protein, followed by dialysis with argonsaturated buffer under an atmosphere of argon. In a typical equilibrium dialysis experiment, 5 m L of ΙΟ M cytochrome c(III) (10 M Tris buffer, 0.1 M ionic strength (KC1)) was dialyzed against 5 m L of 3 x 1 0 M racemic [Co(ox);}] - in the same buffer for 1 h at 0 °C. The [ C o i o x J J solution was then examined for optical activity. Electron-transfer stereoselectivities were determined by mixing 2 m L of 5 X 10 M cytochrome c(II) in appropriate buffer at 0.1 M ionic strength (KC1) with 3

3

3

1

1

6

3-

3

2

2

13

1

-3

3

3+

2

+

+

2

1

1

m

1

1

1

2

1

520

1

3

3+

1

-1

1

4

3

-3

-2

-3

3-

4


3

240


equal volumes containing a two- to fivefold excess of [Co(ox) ] ~, also in buffer at 23 °C. After the reaction proceeded to completion, the protein was removed on an ionexchange column (Sephadex SP C-25) and the optical activity in the resulting [Coiox^] " was determined. Kinetic measurements were made at 550 nm under pseudo-first-order conditions, with an excess of [Coiox^] " in 5 X 10 M buffer and ionic strength 0.1 M (KC1). 3

3

3


3

3

Results and Discussion Oxidation o f Cobalt(II) i n 1 , 2 - D i a m i n o e t h a n e Solutions b y [ C o (ox^] ". T h e s t o i c h i o m e t r i c r e d u c t i o n o f [ C o ( o x ) ] ~ b y cobalt(II) i n aqueous solutions o f 1,2-diaminoethane (en) results i n the formation o f t w o products that c a n b e separated b y H P L C o r c o n v e n t i o n a l c h r o m a t o g r a p h y ( F i g u r e 1). T h e products are i d e n t i f i e d spectroscopically as [ C o ( e n ) ] a n d [ C o ( e n ) ( o x ) ] , a n d the relative a m o u n t o f each is d e p e n d e n t o n t h e e n concentration. A t l o w [en], t h e d o m i n a n t p r o d u c t is [ C o ( e n ) ( o x ) ] . T h e p r o p o r t i o n o f [ C o ( e n ) ] increases w i t h i n c r e a s i n g [en] ( F i g u r e 2), a result 3

3

3

3 +

3

2

+

2

3

+

3 +

[Co(en) (ox)]+ 2

Figure 1. HPLC analysis (SP-5PW Sephadex) of the products of the reaction of [Co(ox) ] ~ with cobalt(H) in 1,2-diaminoethane solutions, (a), [en] = 0.122 M; (b), [en] = 0.183 M. The wavelength is 346 nm, where excess [cobalt(II)] has little absorbance. There is an initial Schlieren gradient caused by the passage of excess 1,2-diaminoethane, but the cation peahs are readily identified. 3

3



13.

MARUSAK ET AL.

Chiral Recognition by Metal-Ion Complexei

241

20-

0 0 — • 0.0

1

1

•

1

0.1

1

0.2

1

1

0.3

0.4

[en], (M) Figure 2. Plot of percent [Co(en) ] product as a function of [en] for the oxidation of [cobalt(U)] in 1,2-diaminoethane solutions by [Co(ox) ] ~ at 25.0 °C and 0.10 M ionic strength. 3

3+

3

3

suggesting that the reaction involves two p a r a l l e l pathways differing i n t h e i r d e p e n d e n c e o n [en]. T h e kinetics of the reaction are also consistent w i t h this observation. T h e reaction is first-order i n [ C o ( o x ) ] " a n d [Co(II)] concentrations. T h e second-order rate constant, k , shows a strong d e p e n d e n c e o n [en] ( F i g u r e 3), w h i c h is e x p l a i n e d b y the m e c h a n i s m i n eqs 3 - 5 , w i t h the r e s u l t i n g rate l a w , e q 6. Best-fit parameters are k = 390 ± 20 M s a n d k = 3300 ± 300 M s" , w i t h a value for K = 2000 M " (22). T h e s e parameters show reasonable agreement w i t h values for k lk estimated f r o m the stoiehiometry results. 3

3

so

1

0

1

1

3

{

[Co(en) ] 2

[Co(en) ] 2

[Co(en) ] 3

2+

2+

+

2+

3

K

2+

3

[Co(ox) ] -

[Co(en) (ox)]

[Co(ox) ] -

[Co(en) ]

3

{

Q

+ en < = ± [Co(en) ] 3

1

1

3

2

3

3

b +

3+

+

= 2000 M "

+ "[Co(ox) ] -" 2

2

+ lCo(ox) ] -" 3

4

1

(3) (4) (5)

K k [en] 3

0

1 + K [en] 3


W

242


w h e r e fc is the o b s e r v e d rate constant. P a t h w a y k , w h i c h leads to formation of [ C o ( e n ) l , is most l i k e l y outer-sphere i n nature because intermediates are o b s e r v e d i n the reaction. P a t h w a y k w h i c h leads [ C o ( e n ) ( o x ) ] , is s h o w n from experiments r u n i n the p r e s e n c e of free Q

obs

3

3 +

i9

+


2

1 3

0.00

0.02

0.04 [en]

0.06

0.08

the no to C-

0.10

(M)

Figure 3. Plot of the second-order rate constant, k , as a function of [en] for the oxidation of [cobalt(II)] in 1,2-diaminoethane solutions by [Co(ox) ]^ at 25.0 °C and 0.10 M ionic strength. so

3

4

-1 400

1

1

'

500

1

(00

wavelength (nm) Figure 4. Circular dichroism spectra of (a), [Co(ox)3]^. Continued on next page. In Electron Transfer in Biology and the Solid State; Johnson, M., et al.; Advances in Chemistry; American Chemical Society: Washington, DC, 1989.

13.

MARUSAK E T AL.


243

l a b e l e d oxalate i o n to incorporate none of the a d d e d l a b e l . H e n c e , t h e oxalate i n [ C o ( e n ) ( o x ) ] is d e r i v e d f r o m the [ C o i o x ^ ] " as a result of an i n n e r - s p h e r e 2

+

3

e l e c t r o n transfer i n w h i c h a d o u b l y b r i d g e d oxalate i o n is transferred. Stereoselectivity i n this reaction c a n b e investigated b y t h e use o f o p tically active [ A - C k ^ o x ^ ] " as oxidant. T h e reaction products are o p t i c a l l y 3

stable a n d n o t p r o n e to racemization b y a self-exchange m e c h a n i s m . T h e Downloaded by UNIV OF CALIFORNIA SANTA BARBARA on September 3, 2015 | http://pubs.acs.org Publication Date: May 5, 1989 | doi: 10.1021/ba-1990-0226.ch013

c i r c u l a r d i c h r o i s m spectra o f t h e isolated products u n d e r w e l l - d e f i n e d c o n ditions are shown i n F i g u r e 4. T h e s e results indicate that for t h e [ C o ( e n ) ] 3

3 +

t h e r e is a n 8 % e n a n t i o m e r i c excess o f t h e Δ i s o m e r ; for t h e [ C o ( e n ) ( o x ) ] 2

+

there is a 1.5% e n a n t i o m e r i c excess of the Λ i s o m e r . T h e s e stereoselectivities

c

i

-0.1-

Figure 4. (b), [Co(en) ] produced in the reaction of [Co(ox) ] ~ with [cobalt(II)] in 1,2-diaminoethane solutions; and (c), [Co(en) (ox)] produced in the reaction of [Co(ox) ]^ with [cobalt(II)] in 1,2-diaminoethane solutions. 3

3+

3

2

3

+

3


244


are modest b u t nevertheless demonstrate the u t i l i t y of [ C o ( o x ) ] " as a stereoselective oxidant. S o m e w h a t s u r p r i s i n g l y , c h i r a l i n d u c t i o n i n the o u t e r sphere reaction is greater than that i n the i n n e r - s p h e r e reaction. T h e d o u b l y b r i d g e d i n t e r m e d i a t e i n the i n n e r - s p h e r e reaction holds the c h i r a l centers a r o u n d 5 A apart ( F i g u r e 5), so that there is little intimate contact. H e n c e , transfer of c h i r a l i t y is difficult. O n the other h a n d , for the outer-sphere reaction, i n t i m a t e contact b e t w e e n the coordination spheres is possible, a n d indeed likely.


3

3

sA

Figure 5. Representation of the oxalate-bridged inner-sphere intermediate proposed in the reaction of [Co(ox) ] ~ with [Co(en) ] , showing the separation between the chiral centers. 3

3

2

2+

A s w i t h the c o r r e s p o n d i n g oxidation b y [ C o ( e d t a ) ] , o u t e r - s p h e r e stereoselectivity reflects the h i g h s y m m e t r y a n d conformational flexibility of the [ C o ( e n ) ] reductant. W h e r e the reductant is m o r e r i g i d a n d sterically d e m a n d i n g , as w i t h [ C o ( ( ± ) - c h x n ) ] ((±)-chxn is r a c - l , 2 - d i a m i n o cyclohexane), stereoselectivities can b e m u c h larger, a p p r o a c h i n g 7 0 % e n a n t i o m e r i c excess (23). S u c h h i g h stereoselectivity indicates an i n t i m a t e interaction i n w h i c h the coordination spheres of the reactants i n t e r p e n e t r a t e . Stereoselectivities of this m a g n i t u d e w o u l d facilitate the d e t e c t i o n of c h i r a l i n d u c t i o n i n reactions w i t h metalloproteins. It is of interest t h e n to i d e n t i f y w h e t h e r the interactions w i t h a m e t a l l o p r o t e i n w i l l p r o v i d e a large or m o d e s t stereoselectivity, a n d thus to d e t e r m i n e the specificity of the i n t e r a c t i o n . -

3

2 +

3

2 +


13.


MARUSAK E T AL.

245

T h e reaction

Oxidation o f H o r s e C y t o c h r o m e c(II) b y [Co(ox) ] 3

b e t w e e n horse c y t o c h r o m e c(II) a n d [Co(ox) ]*~ is a w e l l - c h a r a c t e r i z e d single3

electron transfer that has b e e n t h e subject o f t w o previous k i n e t i c i n v e s tigations (15, 16). U n d e r pseudo-first-order conditions w i t h a n excess o f oxidant a n d at p H 7.0, oxidation o f c y t o c h r o m e c(II) b y [ C o ^ x ^ ] " is a first3

o r d e r process for greater than three half-lives. Pseudo-first-order rate c o n Downloaded by UNIV OF CALIFORNIA SANTA BARBARA on September 3, 2015 | http://pubs.acs.org Publication Date: May 5, 1989 | doi: 10.1021/ba-1990-0226.ch013

stants are p r e s e n t e d i n Table I. A plot o f the first-order rate constant,

fc , obs

against [Co(III)] is linear, w i t h n o e v i d e n c e for rate saturation to indicate Table I. Rate Constants for the Reaction of [CoCox^] with Horse Cytochrome c(II) pH

JO [Co(III)], M

10 k , s3

3

ofes

1

k , M-V so

4.10

E

1.50

13.3

4.44

E

1.44

11.3

7.9

4.98

E

1.43

10.4

7.3

5.28

E

1.37

10.7

7.8

5.99

E

1.39

11.3

5.97*

1.42

6.46

8.1

8.47

6.0 6.0

1.40

8.33

6

1.61

9.40

5.9

6.88

6

1.41

8.47

6.0

8.07

5.8

7.90*

1.40

5.55

E

1.43

10.1

7.1

6.11

E

1.44

10.8

7.5

6.58

E

1.51

11.3

7.5

6.70

D

1.61

11.1

6.9

10.2

6.72

RF

1.45

7.17*"

1.45

7.68

8.92

7.0 6.1

1.60

10.4

6.5

7.99

E

1.77

10.5

5.9

8.57

E

1.78

10.4

5.8

9.02

E

1.78

9.33

7.0#

0.36

2.74

7.7

7.0(K

0.64

3.76

5.9

7.00^

1.13

6.66

5.9

7.0(K

1.68

9.33

5.6

7.0(K

2.39

14.3

6.0

6.78*

1.61

10.1

6.3

6.80

Λ

1.61

9.70

6.0

6.80*

1.61

9.40

5.8

RF

1

8.9

6.81

FE

3-

5.2

NOTE: Reaction conditions were 2 5 . 0 ° C , 0 . 1 0 M ionic strength (KC1), and [cyt (II)] = 5 Χ 1 0 " M . 5 X 1 0 " M acetate. L7 x ΙΟ" M phosphate. 5 x ÎO" M M E S . 5 x ÎO" M H E P E S . •1.7 X ÎO" M borate. '3.3 X 10~ M phosphate. 5.5 x ΙΟ" M phosphate and 5 X 1 0 " M H E P E S . 8.3 x ΙΟ" M phosphate and 5 Χ 1 0 " M H E P E S . 1 . 1 1 Χ ΙΟ" M phosphate and 5 x 1 0 " M H E P E S . C

Α

6

3

b

3

C

3

D

3

3

3

G

4

3

Λ

4

3

3

3


246


the presence of k i n e t i c a l l y i m p o r t a n t i o n - p a i r formation. Rate saturation has not b e e n detected i n previous w o r k o n this reaction or i n reactions w i t h o t h e r negatively charged reagents such as [ F e i C N ) ^ " (18, 24). 3

I n w e a k l y c o o r d i n a t i n g buffers, the second-order rate constant for the e l e c t r o n transfer is almost i n d e p e n d e n t of p H o v e r the range 4 . 5 - 7 . 5 ( F i g u r e 6), w i t h a value of 7.1 ± 0.4 M s" , c o m p a r e d to the p r e v i o u s l y r e p o r t e d Downloaded by UNIV OF CALIFORNIA SANTA BARBARA on September 3, 2015 | http://pubs.acs.org Publication Date: May 5, 1989 | doi: 10.1021/ba-1990-0226.ch013

_

1

1

15 I

«

1

10-

0j

1

1

4

1

S

1—ι

1

6

7

1

1 8

pH Figure 6. Dependence of k on pH for the reaction of horse cytochrome c(II) with /CofoxjaJ " at 25.0 °C and 0.10 M ionic strength (KCl). Key: O , acetate buffer; · , MES (2-[N-morpholino]ethanesulfonic acid); • , HEPES (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid). so

3

value o f 5.5 M " s" i n 0.5 M phosphate at p H 7.0 (15). B e l o w p H 4.5, the reaction shows a slight t r e n d , increasing w i t h decreasing p H , perhaps r e s p o n d i n g to changes i n the p r o t e i n charge. A b o v e p H 8, a m o r e m a r k e d rate r e d u c t i o n is expected o n t h e r m o d y n a m i c grounds. I n the presence of c h l o r i d e i o n , the reaction shows i n h i b i t i o n b y phosphate to the extent o f about 2 0 % of the reaction rate ( F i g u r e 7), a feature n o t e d p r e v i o u s l y (16). Phosphate i o n is k n o w n to b i n d at sites o n the p r o t e i n surface that are u s e d b y a l l anions (25). A p p a r e n t l y [Co(ox) ] ~ is able to o x i d i z e b o t h c h l o r i d e a n d p h o s p h a t e - b o u n d forms of the p r o t e i n at a p p r o x i m a t e l y the same rate. 1

1

3

3



13.

MARUSAK ET AL.

S

S


J

0.000

1

1

0.001

247

1

0.002

0.003

0.004

[phosphate] (M) Figure 7. Dependence ofk on the total phosphate concentration at pH 6.8 for the reaction of horse cytochrome c(II) with [Co(ox) ] ~ at 25.0 °C and 0.10 M ionic strength (KCl). so

3

3

Equilibrium. W h e n horse c y t o c h r o m e c(III) is d i a l y z e d against a so l u t i o n c o n t a i n i n g [ C o i o x ^ ] , e q u i l i b r a t i o n o f the b i n d i n g o f the c o m p l e x i o n w i t h t h e p r o t e i n takes place. O n e i s o m e r i c f o r m of the i o n b i n d s p r e f e r e n t i a l l y to t h e p r o t e i n , a n d its concentration is d e p l e t e d i n t h e b u l k solution. D e tection o f stereoselectivity is a c h i e v e d b y e x a m i n i n g t h e c i r c u l a r d i c h r o i s m s p e c t r u m of the b u l k solution. It is also possible to examine t h e e n a n t i o m e r i c e n h a n c e m e n t o f t h e p r o t e i n b i n d i n g b y dialysis o f the e q u i l i b r a t e d p r o t e i n solution w i t h buffer a n d examination of the c o m p l e x released. T h e t w o m e t h ods are c o m p l e m e n t a r y i n that t h e enantiomer e n h a n c e d i n t h e first exper i m e n t is t h e opposite o f that e n h a n c e d i n t h e second e x p e r i m e n t . 3 -

T r u e e q u i l i b r i u m conditions cannot b e established because [ C o f a x ^ ] " racemizes w i t h i n a f e w hours i n solution, e v e n at 0 ° C . F i g u r e 8 shows t h e circular d i c h r o i s m s p e c t r u m of the b u l k [ C o ^ x ^ ] after dialysis. T h e c i r c u l a r d i c h r o i s m o b t a i n e d b y d i a l y z i n g t h e e q u i l i b r a t e d dialysis sack w i t h buffer has b e e n subtracted. Q u i t e clearly, this s p e c t r u m shows that t h e p r e s e n c e of t h e Δ i s o m e r is e n h a n c e d i n the b u l k solution a n d indicates p r e f e r e n t i a l b i n d i n g to t h e p r o t e i n b y t h e Λ isomer. W i t h a n estimate o f the e q u i l i b r i u m constant for association o f racemic [Co(ox) ] ~ w i t h horse c y t o c h r o m e c(III) of 200 M (18), t h e ratio K /$ is calculated to b e i n excess o f 1.04. 3

3 -

3

1

A

3

à

American Chemical Society Library 1155 15th St., State; N.W. Johnson, M., et al.; In Electron Transfer in Biology and the Solid Advances in Chemistry; American Chemical Society: Washington, DC, 1989. Washington» D.C. 20036


248


400

600

500 wavelength (nm)

Figure 8. Circular dichroism spectrum (φ, millidegrees) of 1.5 x ΙΟ" M [Coiox)^ - after dialysis with 1 x ΙΟ" M cytochrome c(UI) in 10~ M HEPES buffer at pH 7.0 and 0.10 M ionic strength (KCl). 3

3

2

3

Stereoselectivity. T h e stereoselectivity of the electron-transfer reaction b e t w e e n horse c y t o c h r o m e c(II) a n d [ C o ( o x ) ] " can b e e x a m i n e d b y d e t e r m i n i n g the optical activity p r o d u c e d i n solutions of [ C o f a x ^ ] " w h e n it is reacted w i t h a s t o i c h i o m e t r i c deficiency of the p r o t e i n . A t y p i c a l s p e c t r u m o b t a i n e d i n this m a n n e r is s h o w n i n F i g u r e 9. T h e s p e c t r u m , that of the Δ isomer, indicates that the [ A - C o i o x ^ ] " reacts p r e f e r e n t i a l l y w i t h the p r o t e i n . T h e stereoselectivity, l i t t l e affected b y p H o v e r the range 4 . 2 - 7 . 6 , is r e l a 3

3

3

3

400

500

600

wavelength (nm) Figure 9. Circular dichroism spectrum (φ, millidegrees) of 3.85 X 10^ M [Co(ox) ] ~ obtained in the reaction of cytochrome c(II) (2.3 X 10~* M) with excess [Co(ox) ]*~ (1.53 X 10 M) in HEPES buffer, pH 6.97, at 23 °C and 0.10 M ionic strength (KCl). 3

3

3

3


13.


MARUSAK ET AL.

249

t i v e l y insensitive to the presence of phosphate i o n a n d the s u p p o r t i n g e l e c trolyte, w i t h kjk±

averaging 1.19 (Table II). T h i s i n s e n s i t i v i t y i m p l i e s that

the b i n d i n g of phosphate is not i m p o r t a n t i n s t a b i l i z i n g the i o n p a i r w i t h [ C o f a x ^ ] , a feature that is i n general agreement w i t h the s m a l l effect of 3-


phosphate o n the reaction rate. Table II. Stereoselectivity in the Oxidation of Horse Cytochrome c(II) by [ C o M J 3

10 [cytc(iï)], M

10

4

pH 4.43 6.97 7.01 6.73 6.87"" 4.70 FE

C

E

RF

E

4

2.2 2.4 3.0

UCo(oxhl^l, M 14.3 15.3 11.3 15.3 15.0 14.2

1.6 1.2 2.4

ee\ % 7 6 11 10

± 2 ± 2 ± 1 ± 1

10 ± 1 4 ± 2

kA/kA 1.15 1.13 1.25 1.22 1.22 1.08

NOTE: Reaction conditions were 23 ° C and 0.10 M ionic strength (KC1). ee is enantiomeric excess; Λ form in all cases. 5 x Ι Ο M acetate. 5 Χ ΙΟ" M H E P E S . Ί . 7 x ΙΟ" M phosphate. 'No supporting electrolyte. a

fe

3

c

3

3

Stereoselectivity i n the electron-transfer reaction b e t w e e n c y t o c h r o m e c(II) a n d [ C o i o x J J is s m a l l , a fact suggesting that the interactions i n v o l v e d are not p a r t i c u l a r l y specific. H o w e v e r , a qualitative i n t e r p r e t a t i o n o f the data is not uninformative. A l t h o u g h there is no k i n e t i c e v i d e n c e for a n i n t e r m e d i a t e i n the reaction, the o v e r a l l process can b e separated i n t o two k i n e t i c a l l y distinct steps: formation of an electron-transfer p r e c u r s o r c o m p l e x b e t w e e n the p r o t e i n a n d the oxidant, followed b y e l e c t r o n transfer w i t h i n this assembly. 3 -

B o t h of these steps can p r o v i d e a source of stereoselectivity, b u t ex p e r i e n c e w i t h reactions b e t w e e n m e t a l - i o n complexes suggests that p r e cursor c o m p l e x formation assumes a d o m i n a n t role (5). A l t h o u g h i t is not possible i n this instance to examine stereoselectivity d i r e c t l y i n the p r e c u r s o r c o m p l e x , the process can be m o d e l e d b y the e q u i l i b r i u m dialysis e x p e r i ments i n w h i c h the o n l y difference is the oxidation state of the p r o t e i n . S o m e caution is r e c o m m e n d e d i n this i n t e r p r e t a t i o n because the p r o t e i n does u n d e r g o a s m a l l conformational change d u r i n g the redox process. T h i s change is k n o w n to affect the conformation at the h e m e edge, the p r e s u m e d site of electron transfer (18). Binding Sites. N M R spectroscopic investigations of the b i n d i n g of the paramagnetic analogue [Cr(ox) ] ~ to c y t o c h r o m e c(III) r e v e a l the location of at least three a n i o n - b i n d i n g sites o n the p r o t e i n surface ( F i g u r e 10). T w o 3

3



250


Figure 10. Representation of cytochrome c, showing the heme edge and anionbinding sites as indicated in the text. Site 1 is on the top side of the protein, away from the heme edge; sites 2 and 3 are close to the heme edge.

of these sites are close to the h e m e edge. H o w e v e r , a t h i r d (weak) b i n d i n g site o n the opposite side o f the p r o t e i n , distant f r o m the h e m e edge, can b e i g n o r e d i n the present argument. O f the two sites close to the h e m e edge, site 3 has the highest affinity for [ C ^ o x ^ ] a n d has b e e n s h o w n to be k i n e t i c a l l y i m p o r t a n t i n the oxidation b y [ F e ( C N ) ] " (25). It seems l i k e l y that it is the p r i m a r y b i n d i n g site for [ C o i o x ^ ] " i n the e q u i l i b r a t i o n a n d electron-transfer studies. Phosphate is k n o w n to b i n d m o s ^ s t r o n g l y at sites 1 a n d 2 (26), so that the i n t e r a c t i o n of [ C o i o x ^ ] " at site 3 m i g h t b e e x p e c t e d to be r e l a t i v e l y insensitive to the presence of phosphate, as has b e e n f o u n d . 3 -

6

3

3

3

T h e d e t a i l e d information available about the i n t i m a t e electron-transfer m e c h a n i s m makes this p a r t i c u l a r reaction i d e a l for e x p l o r i n g the i n t e r p r e tation of stereoselectivity data. I n general, w h e n m e t a l - i o n c o m p l e x e s are capable of b i n d i n g to different sites o n the surface of m e t a l l o p r o t e i n s , a n i m p o r t a n t question i n d e t e r m i n i n g the d e t a i l e d m e c h a n i s m is the r o l e of the precursor i n the o v e r a l l reaction. T h e r e are two k i n e t i c a l l y i n d i s t i n g u i s h a b l e possibilities: m e c h a n i s m A (eqs 7 a n d 8), w h e r e the p r e c u r s o r is active a n d forms an i n t e r m e d i a t e o n the reaction profile, a n d m e c h a n i s m Β (eqs 9 a n d 10), w h e r e the p r e c u r s o r is inactive, the so-called " d e a d - e n d " m e c h a n i s m . A

cyt c(II) + [ C o ( o x ) ] - cyt c(III) + "[Co(ox) ]*-~ 3

3

Β

3

cyt c(II) + [ C o ( o x ) ] "

Chiral Recognition by Metal-Ion Complexes in Electron-Transfer

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