Electron Transfer in Biology and the Solid State - American Chemical

9 Electron-Transfer Kinetics of Singly Labeled Ruthenium(II) Polypyridine Downloaded by UNIV OF OKLAHOMA on October 28, 2014 | http://pubs.acs.org Publication Date: May 5, 1989 | doi: 10.1021/ba-1990-0226.ch009

Cytochrome c Derivatives Bill Durham, Lian Ping Pan, Seung Hahm, Joan Long, and Francis Millett Department of Chemistry and Biochemistry, University of Arkansas, Fayetteville, AR 72701

Cytochrome c was labeled at specific lysine groups with Ru(bipyridine) (dicarboxybipyridine) [Ru(bpy) (dcbpy)]. Singly labeled derivatives at lysines 7, 8, 13, 25, 72, 86, and 87 were isolated. Laser flash photolysis experiments showed that the excited state of the ruthenium complex undergoes an electron-transfer reaction with the heme in some of the derivatives. The first-order rate constants are 16 X 10 and 26 X 10 s for the forward and backward electron -transfer reactions, respectively, in the derivative labeled at lysine 13. Derivatives also were prepared by allowing Ru(bpy) CO to react with cytochrome c at pH 7, followed by reaction with imidazole. Purified derivatives containing Ru(bpy) (imidazole) linked to histidines26 and 33 were obtained. 2

6

2

6

-1

2

2

3

2+

SMALL METAL COMPLEXES COVALENTLY LINKED TO METALLOPROTEINS have p r o v e n to b e i n v a l u a b l e i n t h e study o f electron-transfer reactions. M u c h o f t h e p i o n e e r i n g w o r k i n this area has b e e n d o n e b y G r a y , I s i e d , a n d co-workers, w h o used R u ( N H ) 3

5

2 +

b o u n d to t h e naturally o c c u r r i n g

h i s t i d i n e residues o f c y t o c h r o m e c (cyt c) (1-5), m y o g l o b i n (6), a n d o t h e r proteins (7-9). I n these examples, t h e distances b e t w e e n t h e r e d o x centers are w e l l d e f i n e d , as are t h e d r i v i n g forces for these reactions. A r m e d w i t h this i n f o r m a t i o n , these researchers have b e e n able to c o m p a r e t h e o b s e r v e d rate constants for electron transfer to those p r e d i c t e d b y t h e t h e o r e t i c a l treatments p r e s e n t e d b y M a r c u s (10-13). 0065-2393/90/0226-0181$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.

182

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

Downloaded by UNIV OF OKLAHOMA on October 28, 2014 | http://pubs.acs.org Publication Date: May 5, 1989 | doi: 10.1021/ba-1990-0226.ch009

F r o m these comparisons, it has b e c o m e e v i d e n t that the distance d e p e n d e n c e for electron transfer t h r o u g h p r o t e i n can b e d e s c r i b e d as an ex p o n e n t i a l decrease w i t h distance scaled b y a t e r m ($ (typically about 0 . 9 - 1 . 2 A"1). T h e free energy d e p e n d e n c e of these reactions appears to b e w e l l d e s c r i b e d b y the relations d e v e l o p e d b y M a r c u s . F e w examples o f reactions have d r i v i n g forces o f m a g n i t u d e comparable to o r larger t h a n the r e o r g a n i zational b a r r i e r s , b u t such reactions are o f considerable interest. Q u e s t i o n s about the nature o f the m o l e c u l a r m a t e r i a l b e t w e e n redox centers a n d h o w it m a y affect the rate o f e l e c t r o n transfer are c u r r e n t l y b e i n g actively p u r s u e d . M a n y c l e v e r strategies have b e e n u s e d to systematically v a r y the d r i v i n g forces i n these systems, i n c l u d i n g c h a n g i n g the m e t a l i n the h e m e p o r t i o n (14) of the p r o t e i n a n d p e r m u t a t i o n o f the ligands o n the r u t h e n i u m (15). T h e z i n c - s u b s t i t u t e d proteins (6) p r o v i d e l o n g - l i v e d e x c i t e d states w i t h w h i c h reactions w i t h h i g h d r i v i n g forces can b e e x p l o r e d . W e have d e v e l o p e d a synthetic m e t h o d (16) that allows the attach m e n t o f Ru(bpy) (dcbpy) to the a m i n e e n d o f l y s i n e residues (bpy is b i p y r i d i n e ; d c b p y is the deprotonated f o r m o f 4 , 4 ' - d i c a r b o x y - 2 , 2 ' - b i p y r i d i n e ) . R u ( b p y ) ( d c b p y ) is e s s e n t i a l l y e q u i v a l e n t to t h e w e l l - k n o w n c o m p l e x Ru(bpy) (17). R u ( b p y ) has a strongly o x i d i z i n g o r r e d u c i n g e x c i t e d state w i t h a l i f e t i m e o f about 600 ns. It has b e e n u s e d i n the study o f e l e c t r o n transfer reactions w i t h m a n y different reagents, i n c l u d i n g c y t c a n d several other proteins (13, 18, 19). T h e distance d e p e n d e n c e q u e s t i o n has b e e n e x p l o r e d w i t h esterifled derivatives o f R u ( b p y ) i n s y n t h e t i c p o l y m e r s (20). 2

2

3

2 +

3

2 +

3

2 +

T h e reactions o f R u ( b p y ) (i.e., the excited-state a n d ground-state 1 + and 3 + forms) are c h a r a c t e r i z e d b y h i g h d r i v i n g forces a n d l o w r e o r g a n i zational b a r r i e r s . T h u s , the ( b p y ) R u ( d c b p y - c y t c) derivatives m a y p r o v i d e further o p p o r t u n i t i e s to o b t a i n i n f o r m a t i o n about e l e c t r o n transfer i n the barrier-free r e g i m e . A n a d d e d advantage is that the electron-transfer p r o p erties o f the native h e m e i r o n can be e x p l o r e d i n these studies. P r o t e i n s d e r i v a t i z e d w i t h Ru(bpy) (dcbpy) w i l l have reasonably w e l l - d e f i n e d g e o m etries from w h i c h the distances b e t w e e n electron-transfer centers can b e obtained. 3

2 +

2

2

I n the process o f d e v e l o p i n g a practical synthetic route to the ( b p y ) R u ( d c b p y - c y t c) derivatives, a v a l u a b l e side reaction was d i s c o v e r e d . T h i s side reaction can u l t i m a t e l y b e m a d e to p r o d u c e (bpy) (imid)Ru((His)cyt c) d e rivatives ( i m i d is imidazole) i n w h i c h the two c o o r d i n a t i o n sites not taken u p b y b i p y r i d i n e are o c c u p i e d b y a s i m p l e i m i d a z o l e a n d the i m i d a z o l e o f a h i s t i d i n e r e s i d u e . T h e s e derivatives show l o n g - l i v e d r o o m - t e m p e r a t u r e emission that p r e s u m a b l y w i l l u n d e r g o photoredox reactions. 2

2

Synthesis of (bpy) Ru(dcbpy-cyt c) Derivatives 2

T h e general synthetic scheme u s e d to p r e p a r e the ( b p y ) R u ( d c b p y - c y t 2

c)

derivatives is shown i n S c h e m e I. T h e N - h y d r o x y s u c c i n i m i d e ester of d c b p y


DUHHAM ET AL.

Ru(II) Polypyridine Cytochrome c Derivatives


9.


183

184


was p r e p a r e d b y reaction w i t h the partially d e p r o t o n a t e d d c b p y i n d i m e t h y l f o r m a m i d e ( D M F ) i n the presence of d i c y c l o h e x y l c a r b o d i i m i d e . T h e ester was f o u n d to react w i t h the lysine groups of cyt c i n a few h o u r s at r o o m t e m p e r a t u r e . T h e major separation step p e r f o r m e d at this stage is i l l u s t r a t e d i n F i g u r e 1. T h e p u r i f i e d fractions w e r e a l l o w e d to react w i t h about a 10-fold excess of R u ( b p y ) C 0 (presumably R u ( b p y ) ( H 0 ) i n so 2

3

2

2

2 +


lution) a n d t h e n c h r o m a t o g r a p h e d to r e m o v e u n r e a c t e d starting m a t e r i a l . A few of the o r i g i n a l l y p u r i f i e d fractions c o n t a i n e d m i x t u r e s of closely related derivatives that c o u l d b e separated b y f u r t h e r chromatography. H i g h performance l i q u i d chromatographs ( H P L C s ) of t r y p t i c digests o f t h e r u t h e n i u m - c o n t a i n i n g derivatives a n d subsequent a m i n o a c i d analysis of the appropriate fractions w e r e u s e d to identify the location of each l a b e l . A representative example is i l l u s t r a t e d i n F i g u r e 2. T h e p u r i f i e d derivatives h a d h e m e redox potentials i n the range of native cyt c, 2 5 0 - 2 6 0 m V . T h e derivatives also r e t a i n e d the 6 9 5 - n m absorption b a n d . T h i s r e t e n t i o n i n d i cated that m e t h i o n i n e 80 was not p e r t u r b e d .

Synthesis of (bpy) (imid)Ru((His)cyt c) Derivatives 2

It was d i s c o v e r e d d u r i n g the preparation of the ( b p y ) R u ( d c b p y - c y t c) d e rivatives that R u ( b p y ) C 0 was able to specifically m o d i f y the h i s t i d i n e r e s idues o n cyt c u p o n p r o l o n g e d i n c u b a t i o n at p H 7. T h e reaction is reasonably efficient a n d does not r e q u i r e a large excess o f reagent. It was possible to separate two singly l a b e l e d ( b p y ) ( H 0 ) R u ( ( H i s ) c y t c) derivatives b y c h r o matography ( C M - 3 2 ) (data not shown). T h e s e d e r i v a t i v e s , as w e l l as the p a r e n t c o m p o u n d R u ( b p y ) ( i m i d ) ( H 0 ) , w e r e not l u m i n e s c e n t . H o w e v e r , one of us ( D u r h a m ) h a d p r e v i o u s l y o b s e r v e d that s o l u t i o n - p h a s e Ru(bpy) (imid) h a d a n e m i s s i o n c e n t e r e d at 660 n m w h e n excited at 450 n m . T h e r e f o r e , the c r u d e reaction m i x t u r e of ( b p y ) ( H 0 ) R u ( ( H i s ) c y t c) derivatives was i n c u b a t e d w i t h 1 M i m i d a z o l e to p r e p a r e ( b p y ) (imid)Ru((His)cyt c) derivatives. T h e reaction, f o l l o w e d b y m o n i t o r i n g t h e e m i s s i o n at 660 n m , was f o u n d to b e c o m p l e t e after 18 h . C h r o m a t o g r a p h y ( C M - 3 2 ) r e s u l t e d i n the separation s h o w n i n F i g u r e 3. T h e U V - v i s i b l e spectra o f fractions 1 a n d 2 w e r e e q u a l to the s u m of the spectra o f one equivalent of R u ( b p y ) ( i m i d ) a n d one e q u i v a l e n t o f native cyt c ( F i g u r e 4); fraction 3 c o n t a i n e d 2 equivalents of R u ( b p y ) ( i m i d ) . 2

2

3

2

2

2

2

2

2

2 +

2 +

2

2

2

2

2

2 +

2

2

2 +

F r a c t i o n s 1 a n d 2 w e r e r e c h r o m a t o g r a p h e d ( C M - 3 2 ) , a n d an aliquot o f each was digested w i t h t r y p s i n a n d c h r o m a t o g r a p h e d o n a reverse-phase H P L C c o l u m n , as d e s c r i b e d b y P a n et a l . (16). I n the c h r o m a t o g r a m o f fraction 2, the native p e p t i d e 2 8 - 3 8 was c o m p l e t e l y m i s s i n g , a n d a R u ( b p y ) ( i m i d ) - c o n t a i n i n g p e p t i d e that e l u t e d a little later i n the c h r o matogram h a d t h e same a m i n o a c i d c o m p o s i t i o n as 2 8 - 3 8 . T h e r e w e r e n o other changes i n the H P L C chromatogram relative to that of native cyt c. T h e r e f o r e , fraction 2 is singly l a b e l e d at H i s 33. I n a s i m i l a r fashion, fraction 1 was f o u n d to b e singly l a b e l e d at H i s 26. 2

2


9.

D U R H A M E T AL.

185

RU(I1) Polypyridine Cytochrome c Derivatives


0.40

100

150

200

250

300

Fraction

30

Fraction

Fraction

Figure 1. Purification of the dcbpy-cyt c and (bpy) Ru(dcbpy-cyt c) derivatives. A, The crude reaction mixture of dcbpy-cyt c (500 mg) was chromatographed on a 2.5- x 70-cm column (Bio-Rex 70) with an exponential gradient from 50 mM ammonium phosphate, pH 7.2, to 160 mM ammonium phosphate, pH 7.2. The flow rate was 25 mL/h and the fraction size was 3.8 mh. Absorbance was measured at 542 nm. B, Fraction 1 from A was rechromatographed on a 1.5- X 25-cm column (Whatman sulfopropyl SE-53) with an exponential gradient from 20 to 250 mM sodium phosphate, pH 6.0. The fraction size was 1 mh and the absorbance was measured at 542 nm. C, Repurified fraction 4 was treated with Ru(bpy) C0 and chromatographed on a 0.6- X 45-cm column (Whatman CM-32) with a gradient from 20 to 400 mM sodium phosphate, pH 6.0. The fraction size was 1 mL and absorbance was measured at 542 nm. The fraction marked 4U contained unmodified dcbpy-cyt c. (Reproduced from ref. 16. Copyright 1988 American Chemical Society.) 2

2

3


186


14 22 7


0.01

A

210

290

—

10

i

—

—

20

—

J

— —

30 %

50

ί

40

METHANOL

Figure 2. HPLC of the tryptic digest of (bpy) Ru(dcbpy-cyt c) fraction 4A. The tryptic digest (50 \Lg) was eluted on a column (Dynamax 300-A) with a linear gradient from 0.019c trifluoroacetic acid to 100% methanol. The native and ruthenium-containing peptides were identified by amino acid analysis and indicated on theftgure. Small peptides such as 23-25 and 26-27 eluted in the void volume of the column. (Reproduced from ref. 16. Copyright 1988 Amer ican Chemical Society.) 2

T h e e m i s s i o n spectra o f Ru(bpy) (imid) 2

2

2 +

,

fractions

1 a n d 2 w e r e v e r y s i m i l a r to that o f

w i t h a m a x i m u m at 670 n m at 298 Κ ( F i g u r e 4) a n d 619

n m at 77 K .

Electron Transfer in (bpy) Ru(debpy-cyt c) 2

A l l e x p e r i m e n t a l e v i d e n c e supports the c o n t e n t i o n that t h e excited-state a n d ground-state parameters o f R u ( b p y ) ( d c b p y ) l i n k e d to cyt c are n e a r l y i d e n 2

tical to those o f the free c o m p l e x . Specifically, b o t h have

luminescence

m a x i m a at 662 n m at 298 Κ that shift to 606 n m at 77 K . T h e l i f e t i m e o f the free c o m p l e x a n d the l i f e t i m e o f a d e r i v a t i v e m a d e from l y s o z y m e , a p r o t e i n w i t h n o h e m e i r o n , are also i d e n t i c a l . T h e absorption spectra o f the d e r i v atives of cyt c are the s u m of the cyt c a n d r u t h e n i u m c o m p l e x spectra. Cherry and Henderson

(17) e x a m i n e d t h e e x c i t e d - s t a t e

R u ( b p y ) ( d c b p y ) a n d s h o w e d i t to b e s i m i l a r to R u ( b p y ) 2

3

2 +

decay

of

. T h e addition of


9.

D U R H A M ET AL.

Ru(ll) Polypyridine Cytochrome c Derivatives

187


Native

0

40

80

120

Fraction Figure 3. Purification of (bpy) (imid)Ru((His)cyt c) derivatives. The mixture was chromatographed on 1.5- x 30-cm column (Whatman CM-32) with an exponential gradient from 50 to 200 mM sodium phosphate, pH 6. The fraction size was 1 mh, and the absorbance was measured at 542 nm. The sample was prepurified by chromatography onal- x 10-cm column (Riogel P-2) with 20 mM sodium phosphate, pH 7. 2

an electron-transfer-quenching pathway complicates t h e decay of the e x c i t e d state as i n d i c a t e d b y S c h e m e I I . I n this s c h e m e , a l l decay paths that d o n o t i n v o l v e e l e c t r o n transfer are r e p r e s e n t e d b y rate constant k , e l e c t r o n trans fer from the excited state b y k a n d back e l e c t r o n transfer o f the r e s u l t i n g ground-state molecules b y k . T h e rate constant k contains radiative a n d nonradiative t e r m s , i n a d d i t i o n to possible c o n t r i b u t i o n s from e n e r g y - t r a n s fer q u e n c h i n g . Solution-phase R u ( b p y ) * has b e e n s h o w n to b e q u e n c h e d b y e l e c t r o n transfer b y cyt c (2). E x p e r i m e n t a l l y , h o w e v e r , i t is difficult to r u l e o u t t h e possibility that there is some c o n t r i b u t i o n from e n e r g y transfer. It cannot b e r u l e d out o n spectroscopic grounds. d

i9

2

d

3

2 +

O u r i n i t i a l studies s h o w e d that ( b p y ) R u ( d e b p y - e y t c) derivatives ex 2

hibited quenching of both the luminescence intensity and lifetime. T h e m a g n i t u d e of the q u e n c h i n g appeared to decrease w i t h i n c r e a s i n g r u t h e n i u m c o m p l e x to h e m e distance. I n a subsequent investigation, transient absorption measurements m a d e w i t h laser flash photolysis e q u i p m e n t w e r e c a r r i e d o u t w i t h t h e R u ( b p y ) ( d e b p y - e y t c) derivatives (22). T h e derivatives l a b e l e d at lysines 86, 87, a n d 8 s h o w e d n o l u m i n e s c e n c e q u e n c h i n g , a n d no transients i n d i c a t i v e 2



188


450

350

*

550

650

Wavelength

Figure 4. Absorbance and luminescence spectra of (bpy) (imid)Ru((His)cyt c). Absorbance spectra shown for fraction 2 (—), native cyt c (—and Ru(bpy) (imid) (- - -)• The uncorrected luminescence spectrum of Ru(bpy) (imid) is shown on the right of the figure. 2

2

2+

2

2

2+

2

of e l e c t r o n transfer w e r e found w i t h these derivatives. T h e d e r i v a t i v e w i t h a m o d i f i e d l y s i n e 13, h o w e v e r , s h o w e d a transient at 550 n m that c l e a r l y indicates the formation of an Fe(II) i n t e r m e d i a t e ( F i g u r e 5). F u r t h e r m o r e , the transient difference s p e c t r u m for the region a r o u n d 550 n m shows m i n i m a at 542 a n d 556 n m that arc isosbestic points i n the c o n v e r s i o n of o x i d i z e d to r e d u c e d cyt c. A s e x p e c t e d , the transient is s h o r t - l i v e d because of the back-reaction w i t h ground-state Ru(III). T h e l u m i n e s c e n c e decay a n d t r a n sient absorbance measurements at 550 a n d 440 n m w e r e simultaneously fit


9.

DURHAM ET AL.

RU(H) Polypyridine

189

Cytochrome c Derivatives


0.205

0.155

ι—ι—ι—ι—r

-100

0

100

200

300

400

T i m e (usee) Figure 5. Transient absorbance of (bpy) Ru(dcbpy-(lysine 13)cyt c) recorded at 550 nm following a 25-ns pulse at 460 nm (Nd:YAG pumped coumarin 460 dye laser). Sample was purged with nitrogen. 2

to the f o l l o w i n g equations:

J (Ru(II)*, emission) = I e^

(1)

h+ki)t

0

I (Fe(II), 550 nm) = A[e~ * * (

1+

d)f

ί (Ru(II),440nm) = àz oC [l U

0

-

e~ ]

-

Be~

(2)

ht

(fcl+

*

d)i

+ Ce~ ]

(3)

ht

w h e r e C is the concentration o f Ru(II) excited state p r o d u c e d b y laser p u l s e , I is the signal i n t e n s i t y at any t i m e , I is the signal i n t e n s i t y at t = 0, A = k^e^Co/ikz-k^kt), Β = ( V * d ) / ( V f c i - f c d ) > C = kJikf-kr-kà, a n d the rate constants are as i n d i c a t e d i n S c h e m e I I . C o r r e c t i o n s w e r e also m a d e for the finite rise a n d fall t i m e o f the laser p u l s e as d e s c r i b e d b y D e m a s (23). T h e values for t h e first-order rate constants (k = 8 ± 3 Χ 1 0 s" , ^ = 16 ± 3 Χ 1 0 s"\ k = 2 6 ± 5 Χ 1 0 s" ) w e r e o b t a i n e d for the l y s i n e 13 derivative (22). D e r i v a t i v e s m o d i f i e d at lysines 25, 2 7 , 7 2 , a n d 7 w e r e also f o u n d to have transients associated w i t h e l e c t r o n transfer. T h e m e a s u r e d first-order rate constants k i n these cases w e r e 1.5 ± 0.3 Χ 1 0 , 30 ± 5 Χ 1 0 , 24 ± 5 Χ 1 0 , a n d 0 . 6 ± 0.2 Χ 1 0 s , r e s p e c t i v e l y (22). 0

0

6

d

6

6

2

6

2

6

1

1

6

6

1

Because b o t h c y t c a n d R u ( b p y ) have b e e n e x a m i n e d extensively, i t is informative to compare the o b s e r v e d rate constants w i t h those p r e d i c t e d b y theory. I n the context o f the semiclassical treatment suggested b y M a r c u s 3

2 +


190

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

a n d S u t i n (13), the f o l l o w i n g relation b e t w e e n free energy, distance, a n d rate is expected. ι

10

*

13

exp [-β(ά

~

el

-

3)] exp [ - ( X

+

AG) ] 2

4KRT

W

I n this expression, A G is the free energy of reaction, k \ is the rate constant for e l e c t r o n transfer, d is separation distance, λ is the reorganizational b a r rier, a n d β is the scaler that describes h o w fast the rate decreases w i t h separation distance. T h e t e r m (d - 3), w h e r e d is the i n t e r a u c l e a r separation, is i n c l u d e d to a l l o w for a v a n d e r Waals contact distance o f 3 Â , at w h i c h distance the first exponential t e r m is assumed to b e u n i t y . Studies of the Ru(bpy) self-exchange rate suggest that contact is best m e a s u r e d w i t h respect to the l i g a n d atoms because of the d e r e a l i z a t i o n of m e t a l - e l e c t r o n density onto the b i p y r i d i n e s . A x u p et al. (6) m a d e use of a s i m i l a r a s s u m p t i o n i n t h e i r analysis o f e l e c t r o n transfer i n ( N H ) R u ( H i s ) M b . I n t h e i r case, t h e distance b e t w e e n the nearest l i g a n d atoms was taken as a measure of d (i.e., h e m e o r h i s t i d i n e ring atoms a r o u n d Z n a n d h i s t i d i n e ring atoms o r a m i n e nitrogens o n the r u t h e n i u m complex).


e

3

2 +

3

5

E s t i m a t e s of the reorganizational b a r r i e r for the reaction of R u ( b p y ) w i t h h e m e Fe(II) can b e m a d e from self-exchange data for c y t c a n d R u ( b p y ) . T y p i c a l values g i v e n i n the l i t e r a t u r e are 100 a n d 55 k j , r e s p e c t i v e l y (13, 21). T h e reorganizational b a r r i e r for e l e c t r o n transfer w i t h i n a d e r i v a t i v e based o n these values is 78 k j . T h e d r i v i n g force for f o r w a r d a n d b a c k w a r d e l e c t r o n transfer reactions u s i n g r e d u c t i o n potentials E°(2 + * / 3 + ) = - 0 . 7 2 V a n d £ ° ( 2 + / 3 + ) = 1.31 V for R u ( b p y ) ( d c b p y ) are 0.98 a n d 1.05 V , respectively. Values of β are somewhat v a r i e d , b u t 0.9 A " appears to b e representative. I n the case of the l y s i n e 13 d e r i v a t i v e , w e estimate d to b e i n the range of 6 - 1 0 Â o n the basis of constraints of the l y s i n e t a i l , 9 - 1 6 Â for lysines 25 a n d 7, 6 - 1 2 Â for l y s i n e 27, a n d 8 - 1 6 Â for l y s i n e 7. F i g u r e 6 shows the locations of the l a b e l e d lysines. 3

3

3 +

2 +

2

1

B y u s i n g the parameters d e s c r i b e d , e q 4 can be u s e d to p r e d i c t a distance d e p e n d e n c e for the reaction of R u ( b p y ) ( d e b p y ) w i t h the Fe(II) of the h e m e . T h e r e l a t i o n is i l l u s t r a t e d i n F i g u r e 7. T h e best-fit l i n e was o b t a i n e d w i t h β = 0.9 Â " a n d λ = 43 k j . 2

3+

1

Electron Transfer in (bpy) (imid)Ru((His)cyt c) 2

A q u e n c h i n g scheme i d e n t i c a l to that d e s c r i b i n g the reactions of the Ru(bpy) (dcbpy-cyt c) derivatives can b e u s e d w i t h the h i s t i d i n e derivatives. 2

T h e l u m i n e s c e n c e decay rates for fractions 1 a n d 2 w e r e f o u n d to b e 10 Χ 1 0 a n d 12 Χ 1 0 s , respectively. T h e s e c o m p a r e d w e l l w i t h a rate constant of 14 Χ 1 0 s" m e a s u r e d w i t h solution-phase R u ( b p y ) ( i m i d ) . N o absorption transients i n d i c a t i v e of electron transfer w e r e o b s e r v e d . I n 6

6

6

_1

1

2


2

2 +


9.

DURHAM ET AL.

Ru(ll) Polypyridine Cytochrome c Derivatives

191

Figure 6. Schematic diagram of horse-heart cyt c viewed from the front side of the heme crevice. The approximate positions of the β carbon atoms at the lysine residues are indicated by closed circles and dashed circles for the residues located on the front and bach of cyt c, respectively. (Reproduced from ref 22. Copyright 1989 American Chemical Society.) these derivatives, it appears that the rate of e l e c t r o n transfer from the e x c i t e d state o f r u t h e n i u m to the h e m e m u s t be too small to c o m p e t e w i t h the natural l u m i n e s c e n c e decay rate.

Experimental Methods Preparation of (bpy) (imid)Ru((His)cyt c) Derivatives. Horse-heart cyt c (2 mM Sigma type VI in 2 mL of 20 mM sodium phosphate, pH 7) was incubated with 4 mM Ru(bpy) C0 (22) for 16 h at 25 °C in the dark under anaerobic conditions. Imidazole, 1 M , was then added to the solution, and the incubation continued for an additional 18 h. The solution was oxidized with potassium ferricyanide and passed through a 1- X 10-cm column (Biogel P-2) to remove excess reagent and equilibrate the modified cyt c with 20 m M sodium phosphate, pH 7. The sample was eluted from a 1.5- X 30-cm column (Whatman CM-32) with an exponential gradient from 50 to 200 mM sodium phosphate, pH 6. Visible spectra of eachfractionwere recorded on a diode array spectrophotometer (Hewlett-Packard HP8452A). Fluorescence spec tra were recorded on a spectrometer (Perkin-Elmer 650-40) using excitation at 450 nm. Luminescence lifetimes were measured as described by Pan et al. (16). 2

2

3

Identification of the Residue Modified. Each derivative was dialyzed into 0.1 M bicine, pH 8, at a concentration of 1 μg/μL and digested with 50 n g ^ L TPCKtreated trypsin for 15 h. (TPCK is tosylamide-2-phenylethyl chloromethyl ketone.)


192



10

d (angstroms) Figure 7. Plot of natural logarithm of the rate constant vs. separation distance d. Best-fit line calculated with β = 0.9 A and λ = 43 kj. Data points for denvatives modified at lysines 7 (A), 13( + ), 25 (M), 27 (%), and 72(x)at the minimum and maximum estimated distance. (Reproduced from ref 22. Copyright 1989 American Chemical Society.) 1

The tryptic digests were separated on a reverse-phase H P L C column (Dynamax 300 A) with a linear gradient from 0.01% trifluoroacetic acid to 100% methanol. The eluent was monitored at 210 and 290 nm by using two H P L C detectors in series. The amino acid composition of each peptide was determined as described by Pan et al. (16).

References 1. Yocom, K. M . ; Shelton, J. B.; Shelton, J. R.; Schroeder, W. Α.; Worosila, G . ; Isied, S. S.; Bordignon, E.; Gray, Η. B. Proc. Natl. Acad. Sci. U.S.A. 1982, 79, 7052-7055. 2. Winkler, J. R.; Nocera, D . G . ; Yocom, Κ. M . ; Bordignon, E.; Gray, Η. B. J. Am. Chem. Soc. 1982, 104, 5798-5800. 3. Nocera, D. G . ; Winkler, J. R.; Yocom, Κ. M . ; Bordignon, E.; Gray, Η. B. J. Am. Chem. Soc. 1984, 106, 5145-5150. 4. Isied, S. S.; Worosila, G . ; Atherton, S. J. J. Am. Chem. Soc. 1982, 104, 7659-7661. 5. Isied, S. S.; Kuehn, C.; Worosila, G. J. Am. Chem. Soc. 1984, 106, 1722-1726. 6. Axup, A. W.; Albin, M . ; Mayo, S. L . ; Crutchley, R. J . ; Gray, Η. Β.J.Am. Chem. Soc. 1988, 110, 435-439. 7. Kostic, N. M . ; Margalit, R.; Che, C . - M . ; Gray, Η. B. J. Am. Chem. Soc. 1983, 105, 7765-7767.



9.

DURHAM ET AL.

RU(II) Polypyridine Cytochrome c Derivatives

193

8. Jackman, M . P.; McGinnis, J . ; Powls, R . ; Salmon, G. Α.; Sykes, A. G.J.Am. Chem. Soc. 1988, 110, 5880-5887. 9. Che, C . - M . ; Margalit, R.; Huey-Jenn, C.; Gray, H . B. Inorg. Chim. Acta 1987, 135, 33-35. 10. Marcus, R. A. Disc. Faraday Soc. 1960, 29, 21-31. 11. Marcus, R. A. J. Phys. Chem. 1963, 67, 853-857. 12. Marcus, R. A. J. Chem. Phys. 1965, 43, 679-701. 13. Marcus, R. Α.; Sutin, N. Biochim. Biophys. Acta. 1985, 811, 265-322. 14. Karas, J. L.; Liebe, C. M . ; Gray, H . B. J. Am. Chem. Soc. 1988, 110, 599-600. 15. Bechtold, R . ; Kuehn, C.; Lepre, C.; Isied, S. S. Nature 1986, 322, 286-288. 16. Pan, L . P.; Durham, B.; Wolinska, J . ; Millet, F. Biochemistry 1988, 27, 7180-7184. 17. Cherry, W. R . ; Henderson, L. J., Jr. Inorg. Chem. 1984, 23, 983-986. 18. English, A. M . ; Lum, V. R.; Delaive, P. J.; Gray, H . B. J. Am. Chem. Soc. 1982, 104, 870-871. 19. Brunschwig, B. S.; Delaive, P. J . ; English, A. M . ; Goldberg, M . ; Gray, H . B.; Mayo, S. L . ; Sutin, N. Inorg. Chem. 1985, 24, 3743-3749. 20. Guarr, T.; McGuire, M . E.; McLendon, G. J. Am. Chem. Soc. 1985, 107, 5104-5111. 21. Brown, G. M . ; Sutin, N. J. Am. Chem. Soc. 1979, 101, 883-892. 22. Durham, B.; Pan, L. P.; Long, J . ; Millett, F. Biochemistry 1989, 28, 86598665. 23. Demas, J. N. Excited State Lifetime Measurements; Academic: New York, 1983. RECEIVED for review May 1, 1989. A C C E P T E D revised manuscript August 10, 1989.


Electron Transfer in Biology and the Solid State - American Chemical

Recommend Documents