voltammetric studies of redox-active centers in metalloproteins adsorbed on electrodes

8/12/2019 Voltammetric Studies of Redox-Active Centers in Metalloproteins Adsorbed on Electrodes

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[ 18 ] VOLTAMMETRY OF REDOX-ACTIVE CENTERS 479

tions between excited spin levels. These signals are not easily correlatedwith the corresponding M6ssbauer spectrum because the electronic spinhas generally intermediate relaxation rates at temperatures 10-20 K)

where the excited spin levels are appreciably populated; for intermediaterelaxation rates the M6ssbauer spectra are broad and ill-resolved. M6ss-bauer spectra are observed in the slow fluctuation limit if the electronicspin relaxes with a rate slower than 106/sec Thus, if one observes aM6ssbauer spectrum in the slow fluctuation limit, one can be assured thatEPR spectra will be observed in the slow fluctuation limit as well.

A sur vey of the literature on M6ssbauer spectroscopy suggests that avariety of other iron-containing proteins are likely to exhibit integer spinEPR at either X- or Q-band frequencies. Based on the progress that has

been made in the last few years we anticipate that this technique will bea valuable tool for the b iochemist and biophysicist. By applying M6ssbauerand EP R spectroscopy to proteins with multiple iron-containing sites andusing the methodology discussed above, one should be able to untanglequite complex situations.

Acknowledgments

The work described here was supported by grants from the National Institutes of HealthGM-22701) and the National Science Foundation MCB-9096231).

[ 18] Vo l t a m m e t r i c S t u d i e s o f R e d o x - A c t i v e C e n t e r si n M e t a l l o p r o t e i n s A d s o r b e d o n E l e c t r o d e s

y FRASER A. ARMSTRONG, JULEA N. BUTT, and ARTUR SUCHETA

IntroductionIn recent years it has been shown that redox proteins can be induced

to interact directly with an electrode surface and display reversible electro-chemis try in the same way as many smaller molecules. ~,2 This has sug-gested the possibility of using dynamic electrochemical methods such ascyclic voltammetry to examine the many intricate functional propertiesof redox-active centers in proteins. In this chapter we describe a particularstrategy, thus far demonstrated to be applicable for investigating labile

I F. A. A rmstron g, Struct, Bonding Berlin) 72, 137 (1990).2 A. M. Bond and H. A. O. Hill, in Met al Ion s in Biological Sy st em s (H. Sigel and

A. Sigel, eds.), Dekker, New York, 1991.

Copyright 1993 by Academic Press, Inc.METHODS IN ENZYMOLOGY, VOL. 227 All rights of reproduction in any form reserved.


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480 PROBES OF METAL ION ENVIRONMENTS [18]

F e - S c l u s te r s , w h i c h c o u l d b e m o r e w i d e l y e x p l o it e d fo r de c i p h er in gc o m p l i c a t e d r e a c t i v i t y t h a t i s l i n k e d t o e l e c t r o n t r a n s f e r. O u r d i s c u s s i o nf o c u s e s o n a p p l ic a t io n ; t h e f u n d a m e n t a l p r in c i p le s a n d m e t h o d s o f d y -

n a m i c e l e c t r o c h e m i s t r y a r e d e s c r i b e d i n s e v e r a l e x c e l l e n t t e x t b o o k s . 3'4W e f ir st o u tl i n e s o m e g e n e r a l f e a t u r e s o f v o l t a m m e t r ic m e t h o d s t h a t a r ep o t e n t i a l l y u s e f u l f o r s t u d i e s o f r e d o x s i te s i n p r o t e i n s .

G e n e r a l I n fo r m a t i o n f r o m Vo l t a m m e t r i c E x p e r i m e n t s

C y c l i c v o l t a m m e t r y is a w i d e l y u s e d t e c h n i q u e t h at p r o v i d e s a b r o a ds n a p s h o t o f t h e r e d o x c h e m i s t r y o f m o l e c u l e s in s o lu t io n . B y a n a l o g y

w i t h a s p e c t r u m , in w h i c h t h e p r e s e n c e o f c e r ta i n s p e c i e s is e v id e n t f r o m

e n e rg y a b s o r p t i o n o r e m i s s i o n a t a c h a r a c t e r i s t i c e n e rg y, a v o l t a m m e t r i cr e s p o n s e a r i s e s f r o m e x c h a n g e o f e l e c t r o n s t h at i s m o s t r ea d i ly o b s e r v e da t s o m e c h a r a c t e r i s t i c p o t e n t i a l . T h e c u r r e n t - v o l t a g e p r o f i l e t h u s c o n s t i -t u t e s a s ig n a l w h i c h c a n b e a s s i g n e d t o a p a r t i c u l a r s p e c i e s ( r e d o x c o u p l e )t h a t is i d e n t if i a b le b y c o m p l e m e n t a r y s t u d i e s. V o l t a m m e t r y i s d y n a m i c a l l yi n t e r a c t i v e , t h a t i s , r e a c t i o n s c a n b e b o t h i n d u c e d b y a p p l ic a t i o n o f ap o t e n ti a l a n d a n a l y z e d t h ro u g h t h e c u r r e n t r e s p o n s e . C o m p l i c a te d a c t i v it ym a y b e v i s u a l i z e d i n b o t h t h e t i m e a n d p o t e n t i a l d o m a i n s . I n f o r m a t i o ni s o b t a i n e d o n r e d u c t i o n p o t e n t i a l s , e l e c t r o d e k i n e t i c s , a n d t h e r e l e v a n t

p a r a m e t e r s o f c o u p l e d r e a c t i o n s i n cl u di n g c a t a l y s i s ) '4 A v a r i e ty o f o t h e rd y n a m i c e l e c t ro c h e m i c a l m e t h o d s c a n b e e m p l o y e d t o r e v e a l m o r e d e t a il e dm e c h a n i s t i c in f o r m a t i o n . F o r e x a m p l e , i n p u l s e m e t h o d s , t h e c u r r e n t i sm o n i t o r e d ( o r m e a s u r e d a t s o m e t i m e i n t e r v a l ) a f t e r s t e p p i n g t h e a p p l i e dp o t e n t ia l t o a n e w v a l u e ; in h y d r o d y n a m i c m e t h o d s t h e c u r re n t i s m e a s u r e du n d e r v a r y i n g c o n d i t i o n s o f e n f o r c e d c o n v e c t i o n . I t i s i m p o r t a n t t o n o t et h e c o n t r a s t w i t h p o t e n t i o m e t r y, w h i c h i s a s t a t i c t e c h n i q u e c o n c e r n e dw i t h d e t e r m i n i n g t h e p o p u l a t i o n s o f s p e c i e s a t r e d o x e q u i l i b ri u m ( i .e . ,m e a s u r i n g t h e p o t e n t i a l a t w h i c h n o n e t c u r r e n t f l o w s ) .

T h e u s e f u l p o t e n ti a l r a n g e i s w i d e a n d c o n t i n u o u s , b e i n g r e s tr i c t e do n l y b y t h e p r a c t i c a l l im i t s f o r e l e c tr i c a l b r e a k d o w n o f e l e c t r o d e a n ds o l v e n t , n o t b y p r o p e r t i e s o f m e d i a t o r s a n d t i tr a n ts . To d a t e , t h e c h e m i s t r yo f b i o lo g i c al s p e c i e s g e n e r a t e d a t p o t e n ti a ls b e l o w a p p r o x i m a t e l y - 5 5 0m V [ v e r s u s s t a n d a r d h y d r o g e n e l e c t r o d e ( S H E ) ] , w h i c h i s t h e p r a c t i c a ll i m i t f o r d i t h i o n i t e a t p H 7 , h a s r e m a i n e d a l m o s t u n e x p l o r e d . T h i s i sd e s p i t e t h e l i k e l ih o o d t h a t s u c h s t r o n g r e d u c t a n t s m a y b e im p o r t a n t i n te r -

3 A. J. Bard and L. R. Faulkner, Elec troch emic al Methods , Fundament als and Applica-tio ns. Wiley, New York, 1980.

4 Southampton Electrochemistry Group, Instrumental Methods in Electroc hemistry.Ellis Horwood, Chichester, 1985.


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m e d i a t e s i n b i o l o g ic a l s y s t e m s ( w h i c h a r e r a r e l y a t e q u i li b ri u m ) . B y c o n -t r a s t , v o l t a m m e t r y w i t h n e u t r a l a q u e o u s s o l u t i o n s ( a t c a r b o n e l e c t r o d e s )i s f e a s i b l e a t p o t e n t i a l s b e l o w - 1 V a n d u p t o a t l e a s t + 8 0 0 m V.

A l t h o u g h v o l t a m m e t r y c a n y i e ld a n i n s tr u c ti v e p i c t u r e o f c o m p l e xr e a c t i v i t y, i t p r o v i d e s n o i n f o r m a t i o n o n s t r u c t u r e . E v e n s o , t h e t a s k o fc h a r a c t e r i z i n g a l a b il e a c t i v e s i te b y s p e c t r o s c o p i c m e t h o d s i s m a d e m u c he a s i e r s i n c e it is p o s s i b l e t o d e f in e t h e p r e c i s e c o n d i t i o n s ( r e a g e n t c o n c e n -t r a t ion , po ten t i a l ) under which a pa r t i cu la r s t a t e can ex i s t .

A d v a n t a g e s o f I m m o b i l iz i n g P r o t e i n M o l e c u l e s o n E l e c t r o d e s

I m p o r t a n t e x p e r i m e n t a l a n d in t e r p r e t a ti v e r e f in e m e n t s b e c o m e p o s s i -

b l e if w e c o n f i n e t h e m o l e c u l e s u n d e r s t u d y t o th e e l e c t r o d e s u r f a c e , a sdep ic t ed in F ig . 1 . The idea l i zed concep t i s a s fo l lows . (1 ) Redox-ac t ivep r o t e i n m o l e c u l e s a r e a d s o r b e d s t r o n g ly a t t h e s u r f a c e o f a n e l e c t r o d e .E l e c t r o n t r a n s f e r b e t w e e n t h e e l e c t r o d e a n d t h e p r o t e i n a c t i v e s i t e ( s ) i sr e v e r s i b l e i n t h e e l e c t r o c h e m i c a l s e n s e , t h a t i s , N e r n s t i a n e q u i l i b r i u m i sm a i n t a i n e d a t e a c h v a l u e o f t h e a p p l i e d p o t e n ti a l. (2 ) A d s o r p t i o n o c c u r sw i t h m i n i m a l d i s r u p t i o n o f t h e n a t i v e c o n f o r m a t i o n s o t h a t n a t i v e f u n c -t i o n a l p r o p e r t i e s a r e c o n s e r v e d . ( 3 ) T h e c o v e r a g e i s m o n o l a y e r o r l o w e r,a n d t h e a c t i v e s i t e in e a c h p r o t e i n m o l e c u l e a c t s i n d e p e n d e n t l y, b u t i d e nt i-

c a l ly, t o it s c o u n t e r p a r t s i n a d j a c e n t m o l e c u l e s . (4 ) F o r m a t i o n o f s u c h a na r r a y m a y d e p e n d c r i t i c a l l y o nco dsorptiono f o th e r c o m p l e x m o l e c u le s .T h e s e a r e r e p r e s e n t e d b y t h e s y m b o l A .

B e c a u s e t h e p r o t e i n m o l e c u l e s a r e a b s e n t f r o m t h e e l e c t r o l y t e , a n dn o n e e f f e c t i v e l y l e a v e t h e e l e c t r o d e s u r f a c e , a ll t h e c h a rg e t h a t c r o s s e s

e l e c t r o n sm e t a l i o n s

r ~ ) ~ l i g a n d s~ subs t r a t e s

ELECTRODE ELECTROLYTE

FIG. 1. Conc ept of the molecular arrangement formed by electron transfe r proteins andcoads orbate s at the surface of an electrode. Electrons can be freely exchanged betwee n theelectrode and redox ce nters within the protein. Binding sites in the protein which can beinfluenced or triggered by the status of the redox centers are available to reagents thatdiffuse freely in the solution.


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482 P R O B E S O F M E T A L I O N E N V I R O N M E N T S [ 8]

t h e i n t e r f a c e r e v e r s i b l y is a c c o u n t e d f o r in w h a t c a n b e r e g a r d e d a s a c l o s e ds y s t e m . A n a l y s i s o f th e v o l t a m m e t r y y i e ld s q u a n t it a ti v e i n fo r m a t io n o nr e a c t i v i t ie s o f s p e c if ic r e d o x s i t e s, i n f o r m a t i o n w h i c h f o r l a b il e s y s t e m s

m a y b e d i ff ic u lt t o o b t a i n b y c o n v e n t i o n a l m e t h o d s .We f i r s t c o n s i d e r a s i m p l e e l e c t r o n t r a n s f e r s y s t e m t h a t i s n o t c o m p l i -c a t e d b y c o u p l i n g t o f u r t h e r r e a c t i o n s s u c h a s r e v e r s i b l e b i n d in g o r c a t a ly -s is . I n t h is c a s e , t h e s p e c i f i c a d v a n t a g e s o f h a v i n g a ll th e p r o t e i n m o l e c u l e sc o n f i n e d t o t h e e l e c t r o d e s u r f a c e a r e a s f o l l o w s .

High Sample Economy. T h e a m o u n t o f m a t e r ia l r e q u i r e d t o f o rm am o n o l a y e r o n a t y p i c a l s m a l l e l e c t r o d e s u r f a c e c o r r e s p o n d s t o a p p r o x i -m a t e l y I p m o l . T h u s , a l arg e n u m b e r o f e x p e r i m e n t s c a n b e c o n d u c t e do n v a l u a b l e s a m p l e s i n l i m i t e d s u p p l y.

Precise Control of Redox Status of Centers. T h e s t a t u s o f a ll a c t i v es i te s c a n b e a d d r e s s e d a n d f i n e - t u n e d o n a r a p i d ti m e s c a l e . T h i s s t e m sf r o m t h e f a c t t h a t w a v e s o b s e r v e d f o r w e l l - b e h a v e dsurface confined sys -t e m s a r e c o m p a c t ( e v e n a t h i g h s c a n r a t e s ) , r e f l e c t i n g t h e e x h a u s t i v ec o n v e r s i o n o f r e d o x s p e c i e s. T h i s i s t o b e c o n t r a s t e d w i th t h e v o l t a m m e t r i cr e s p o n s e t y p i c a ll y o b s e r v e d f o r a p la n a r e l e c t r o d e c o n t a c t in g a th i c k l a y e ro f f r e e l y d i f f u si n g r e d o x - a c t i v e s p e c i e s , f o r w h i c h t h e c u r r e n t is fi ni te a n dd i ffus ion- l imi t ed a t a su ff i c i en t ly l a rge ove rpo ten t i a l . F igure 2 de sc r ib es thet h e o r e t i c a l w a v e f o r m ( d i s c u s s e d b y L a v i r o n 5) f o r th e i d e a l c a s e , n a m e l y, ar e v e r s i b l e d i f f u s i o n l e s s e l e c t r o n - t r a n s f e r r e a c t i o n t h a t i s u n c o m p l i c a t e db y s p e c i e s h e t e r o g e n e i t y, i n t e r si t e i n t e r a c t io n , o r c o u p l e d c h e m i s t r y. 5 T h es e p a r a t i o n A E p b e t w e e n o x i d a t i o n a n d r e d u c t i o n p e a k s (E pa - E p ) i s 0 m V ,and th e theore t i ca l ha l f -he igh t wid th 8 i s91/n m V a t 25 (wh ere n i s t hen u m b e r o f e l e c t r o n s t ra n s f e r r e d i n t h e e l e m e n t a r y p r o c e s s ). H o w e v e r ,e v e n i n th e l i m i t o f i rr e v e r s i b l e ( s lu g g is h ) e l e c t r o d e k i n e t ic s , t h e w a v ee n v e l o p e r e m a i n s c o m p a c t ; 8 f o r r e d u c t i o n b e c o m e s 6 2.5 /O m a m V [ o r 6 2 . 5 /(1 - a ) n a m V f o r o x i d a t i o n ] , w h e r e a i s th e t r a n s f e r c o e f f i c i e n t a n d n a i st h e n u m b e r o f e l e c t r o n s t r a n s f e r r e d i n t h e r a te - d e t e r m i n i n g s t e p .

Waveform Analysis. F o r a p r o t e i n m o l e c u l e i n s o l u t i o n , t h e v o l t a m -m e t r i c w a v e f o r m c a n b e c o m p l i c a t e d b y v a r i a t i o n i n t h e g e o m e t r y o fd i ff u s io n t o t h e e l e c t r o d e s u r fa c e . 2 S u c h e f f e c ts c a u s e w a v e s t o b r o a d e na n d f la t te n a s t h e n u m b e r a n d s i z e o f su i t a b le i n t e r a c ti o n s i te s o n t h ee l e c t r o d e s u r f a c e d e c r e a s e . T h i s i s n o t a p r o b l e m f o r a n i m m o b i l i z e dp r o t e i n . I n t h e c a s e o f s in g le , i s o l a t e d r e d o x c o u p l e s , t h e f o ll o w i n g e a s i lym e a s u r e d p a r a m e t e r s a r e a n a ly t ic a l ly i m p o r t an t : a v e r a g e p e a k p o t e n t ia l s[(Epa + Ep)/2 ] co r re sp on d to the r e du c t ion po ten t i a l E , a r eas o f wa ve sg i v e t h e f i n i t e c h a rg e t h a t i s e x c h a n g e d b e t w e e n e l e c t r o d e a n d p r o t e i n

5 E . L a v i r o n , J Electroanal Chem 1 0 1 , 1 9 1 9 7 9 ) .


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[1 8 ] VOLTAMM ETRY OF REDOX-ACTIVE CENTERS 483

E

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n E - E p e a k ) / m V

F I G . 2 . I d e a l cy c l i c vo l t ammo gram fo r t he ca se o f a r eve r s i b l e , su r f ace -bound r edoxco up l e w i th r ap id e l e c t r on t r a n s fe r k i n e t i c s , i n t he absen ce o f coup l ed chem ica l r e ac t ions andin t e r ac t i on s be tween r ed ox cen t e r s . The r e su l t i ng wave fo rm i s de sc r i bed by t he exp re s s ionI = - [ n E F 2 o A F / ( RT ) ] O ( 1 +0 ) - 2 , w h e r e ni s t he number o f e l e c t rons exchanged , F i s t heFa ra day c on s t an t 9 6 , 4 8 4 . 5cou lom bs pe r gr am-equ iva l en t) , ~ the s can r a te t aken a s ve loc i t y,i . e . , p o s i t i v e o rnega t i ve , depend ing on t he s can d i r ec t i on ) , F i s t he su r f ace concen t r a t i ono f r edox c en t e r, R i s t he ga s co ns t an t , T i s t h e abso lu t e t empe ra tu r e , and 0 = e x p [ n F ( E -E ) / ( RT ) ] . T h e wave f o rms r e su l t i ng f rom ac tua l expe r imen t s shou ld app roach t h i s i dea ls hape a t s c an r a te s s l o w e n o u g h t h a t t he e le c t ron exch ange r a te be tw een t he e l e c t rode andthe redox couple i s no t ra te - l imi t ing . The wid th a t ha l f -he ight 8 i s2 l n [ 3 + 2 ( 2 ) I / 2 ] RT / ( n F ) ,o r 8 2 . 8 / n m V a t 0 .

ac t ive s i tes , pea k sep ara t ions AEp ( i f ab ov e 0) can be used to der ive ra tecons t an t s fo r e l ec t ron excha nge , and the pa rame te r 3 / n i s an ind ica tor o fthe deg ree o f spec i e s he t e rogene i ty and in t e rs it e i n t e rac t ions . 5 Co m plexwa ve fo r m s a r i s e if two o r more r edox cou p le s have s imi l ar r educ t ionpo ten t ia l s . H ow ev e r, t he se ma y be decon vo lu t ed by com pu te r ca l cu la t i onssuch a s non l inea r r eg re s s ion o r by s imu la t ions ba sed o n the fundamen ta lparameters . Thus , fo r mul t icen te red pro te ins , one may read i ly de te rminethe s to ich io m et r ies o f spec i f ic cen te rs , the ir e lec t ron capac i ty, E va lues ,and e lec t ron t ransfer fac i l i t i es .

N ex t w e ex t end ou r d i s cus s ion to cou p led r eac t ions . I f e l ec tron t r ans fe rindu ces a rear rangem ent of the s t ruc ture of an ac t ive s i te , the vo l tam m o-g ram m ay be pe r tu rbed . Th e imm obi l i zed p ro t ein me thod be com es pa r ti cu -la rly usefu l for s tudying oxid a t ion s ta te -dependent in te rac t ions wi th ex t ra -neous mo lecu le s , s i nce t he p ro t e in -coa t ed e l ec t rode can be t r ans fe r r edbe tween so lu t ions conta in ing the reagent o f in te res t . Such reac t ions in -

c lude m eta l ion up take an d re lease , l igand b inding , and subs t ra te t ransfor-m at ion . Th is br ings us to ou t l ine further fea tures o f the sur face-con f inedvo l t ammet r i c me thod a s fo l l ows .


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4 8 4 P R OB E S O F M E TA L I ON E N V I R O N M E N T S [ 1 8 ]

Sens i ti v it y fo r S tudy o f In t e rac t ions be tween Ac t ive S i te s a nd Exogenous Reagen t s .T h e e x t r e m e l y s m a l l n u m b e r o f p r o t e i n m o l e c u l e s u n d e ro b s e r v a t i o n m a k e s i t p o s s i b l e t o o b s e r v e a n d q u a n t i f y r e a c t i o n s w h i c h

o c c u r b e t w e e n t h e p r o t e i n a c t i v e s i te a n d r e a g e n t s c o n t a i n e d i n t h e e l e c tr o -l y t e a t v e r y l o w c o n c e n t r a t i o n .Kine t ic Ana lys i s o f Coup led P roces se s .B e c a u s e t h e e l e c t r o d e a n d

t h e r e d o x c e n t e r s a r e i n d i r e c t e l e c t r o n i c c o m m u n i c a t i o n , a ll t h e re l a t e dc h e m i c a l r e a c t i v i ty c a n b e i n d u c e d a n d m o n i t o r e d in t h e ti m e - d o m a i n m o d et h ro u g h c o n t r o l o f t h e e l e c t r o d e p o t e n t ia l . B y a n a ly z i ng t h e w a v e f o r m o v e ra r a n g e o f sc a n r a t e s , i t i s p o s s i b l e t o p l o t t h e c o u r s e o f c o u p l e d r e a c t i o n s .D i g it a l s i m u l a t io n c a n b e e m p l o y e d t o o b t a i n r a t e c o n s t a n t s f o r t h e s ep r o c e s s e s . F o r a s u r f a c e - c o n f i n e d r e d o x e n z y m e , t h e c a t a l y t i c t u r n o v e r

o f su b s t r a t e g r e a t ly a m p l if ie s t he c u r r e n t - p o t e n t i a l r e s p o n s e . B e c a u s e t h ee n z y m e i s c o u p l e d d i r e c t l y t o a c o n t i n u o u s l y v a r i a b l e a n d t u n a b l e a p p l i e dp o t e n t i a l , a n a l y s i s o f t h e f o r m o f t h is r e s p o n s e u n d e r s t a t i o n a r y a n d r o t a t -i ng e l e c t r o d e c o n d i t i o n s l e a d s t o d e t e c t i o n a n d d e f i ni ti o n o f s u b t l e m e c h a -n i st ic p h e n o m e n a . T h e s e i n c l u d e t h e e f f e c t s o f e l e c t r o n d i s t r ib u t i o n o ro r d e r e d b i n d i n g t h a t m a y b e i m p o r t a n t i n r e g u l a t i o n .

B a s i c C o n s i d e r a ti o n s

T h e c e n t r a l p r o b l e m i s t o id e n t i f y t h e c o n d i t i o n s o f e l e c t r o d e s u r f a c e ,e l e c t r o l y te , a n d c o a d s o r b a t e ( if r e q u ir e d ) t h a t a r e n e c e s s a r y t o a c h i e v ea n d o p t im i z e a d s o r p t i o n o f t h e p r o te i n a n d o b s e r v a t i o n o f v o l t a m m e t r i cs igna ls . Th i s i s a com pl ic a ted , la rge ly un cha r t ed a rea , and a t r i a l ande r r o r s e a r c h , g u i d e d b y a v a il a b le i nf o r m a t io n o n th e p h y s ic a l p r o p e r t i e so f t h e p r o t e in , is e x e c u t e d . M a n y d e m a n d s m u s t b e m e t to a c h i e v e as t a b l e f ilm t h a t i s u s e f u l f o r s tu d i e s : t h e p r o t e i n m u s t r e m a i n c o n f o r m a t i o n -a l ly in t ac t w h i l e r e ta in ing in t e rna l m ob i l i t ie s tha t a re e s sen t i a l fo r func t ion ;e l e c t r o n e x c h a n g e b e t w e e n t h e a c t i v e s it es a n d t h e e l e c t r o d e m u s t b e fa s t;

t h e m i c r o e n v i r o n m e n t s o f a c t iv e s it e s s h o u ld b e u n i f o rm t h r o u g h o u t t h ea d s o r b e d p r o t e i n p o p u l a t io n ; a n d a c c e s s f o r e x t r a n e o u s r e a g e n t s m u s t b eu n i m p e d e d . I n f a v o r a b l e c a s e s i t i s p o s s i b l e t o o b t a i n a n i n d e p e n d e n ta s s e s s m e n t o f t h e d e g r e e o f c o n f o r m a t i o n a l c h a n g e i n d u c e d b y a d s o r p ti o n .B o w d e n a n d c o - w o r k e r s h a v e s h o w n t h a t a n e l e c t r o a c t i v e m o n o l a y e r o fc y t o c h r o m e c a d s o r b e d o n t in o x i d e (in th e p r e s e n c e o f p h o s p h a t e a t p H 7 )d i s p l a y s o p t i c a l a b s o r p t i o n b a n d s t h a t a r e c h a r a c t e r i s ti c o f t h e n a t i v eF e ( I I I ) a n d F e ( I I ) f o r m s . 6 I t s h o u l d a l s o b e e m p h a s i z e d t h a t t h e c a p a b i l i t yf o r fa c i le e l e c t r o n e x c h a n g e w i t h a n e l e c t r o d e is n o t s o s t ro n g l y c o r r e l a t e d

w i t h p r o t e i n s i z e a s is c o m m o n l y a s s u m e d . T w o e x a m p l e s i ll u st ra t in g t h i s

6 M . C o l l i n so n a n d E . F . B o w d e n ,Anal Chem 6 4 , 1 4 7 0 1 9 9 2 ) .


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[1 8 ] VOLTAMMETRY OF REDOX-ACTIVE CENTERS 4 8 5

point are the structurally related Fe-S flavoproteins fumarate reductaseand succinate dehydrogenase, the membrane-extrinsic forms of whichhave molecular weights of around 100,000. Each of the enzymes exhibits

a remarkably high level of electrocatalytic activity when adsorbed at car-bon electrodes. 7 8

Visualizing and Quantifying Complicated Redox Activities

To illustrate how information is obtained from these experiments, werefer to our own endeavors to study the complicated redox-linked activitiesof iron-sulf ur (Fe-S ) clusters in proteins.9 It is now recognized that thesecenters may perform vital roles aside from electron transfer and storage,roles that include acid-base catalysis and regulation of iron metabolismat the level o f mRNA. 1 11 We need to learn more about the factor s thatdetermine reactivities such as metal ion and ligand exchange as may occurat a labile cluster, that is, a cluster difficult to study by conventionalmeans. As an example, we consider some of the reactions that are possiblefor a protein-bound [3Fe-4S] cluster. These are indicated in Scheme I.

The [3Fe-4S] cluster is to be considered as a tridentate ligand thatmay exist in up to four oxidation levels) 2-~4 Binding of a single metal ionM may occur to the [3Fe-4S] core in one or more of the oxidation levels

to produce cubane-type products exemplified by the [4Fe-4S] cluster[M -- Fe(II)].10 The incoming metal ion will require fur ther coord inationby a ligand (X) that is possibly added to or exchanged readily for others(L). It is obvious that electron transfer, metal ion binding, and ligandbinding activities must be interlinked, and as such could provide the basisfor an intricate catalytic/regulatory system. The experimental challengeis to identify pathways and quantify the chemistry within this maze ofpossibilities. Voltammetry with an adsorbed protein is well suited to de-

7 A . S u c h e t a , B . A . C . A c k r e l l , B . C o c h r a n , a n d F. A . A r m s t r o n g ,Nature London) 356 ,361 (1992).

8 A . S u c h e t a , R . C a m m a c k , J . H . W e i n e r, a n d F. A . A r m s t r o n g ,Biochemistry 32, 5455(1993).

9 F. A . A r m s t r o n g ,in A d v a n c e s in I n o r g a n i c C h e m i s t r y ( A . G . S y k e s a n d R . C a m m a c k ,eds . ) , Vo l . 38 . A cad em ic P res s , S an Diego , 1992.

10 H . B e i n e r t a n d M . C . K e n n e d y ,Eur. J. Biochem. 186, 5 (1989).N R . C a m m a c k , in A d v a n c e s i n I n o rg a n ic C h e m i s t r y ( A . G . S y k e s a n d R. C a m m a c k ,

e d s . ) , Vo l . 3 8 . A c a d e m i c P r e s s , S a n D i e g o , 1 9 9 2 .12 S . C iu r l i an d R . H . H o lm ,lnorg. Chem. 30, 743 (1991).13 j . N . B u t t , F. A . A r m s t r o n g , J . B r e t o n , S . J . G e o rg e , A . J . T h o m s o n , a n d E . C . H a t c h i k i a n ,

J. Am. Chem. Soc. 113, 6663 (1991).14 j . N B u t t , A . S u c h e t a , F. A . A r m s t r o n g , J. B r e t o n , A . J , T h o m s o n , a n d E . C . H a t c h i k i a n ,

J. Am. Chem. Soc. 113, 8948 (1991).


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486 P R OB E S O F M E T A L I O N E N V I R O N M E N T S [ 8]

S S S

e

2 3S S S S S S

e e

M 5 M -~ 6 M 7i i

X X X Xv 8 v 9 v 1 0 v

s : . . q ' ~ . s ~ : ' ; ' , , . . s s , . , ; ', , . s Fe Pe ~Fe - ~ % 7 ~ * -

\ s f / - \ / - s - \ ~ / -e e e

l L -~ L . ~ LL~ ~ 11 12 13 1 4

i iX? L X? L X? L X? L

~ r 1 5 ~ v 1 6 ~ r 1 7 ~ v

e- e- e

o x i d a t io n le v e l o f [ 3 F e - 4 S ] c o r e

1 0 1 - 2 -SCHEME. Pathway s 1-17) o f redox , metal b inding, and ligand binding react ions basedon the [3Fe-4S] c lus ter. Sol id arrows indicate es tabl ished react ions or ones detected byv o l t a m m e t r y.

t e c t i n g a n d d e c i p h e r i n g c o u p l e d c h e m i s t r y t h a t a p p e a r s c h a o t i c w h e nv i e w e d b y m o r e c o n v e n t i o n a l m e t h o d s

Outline of Experimental Methods

We have found that a number of proteins adsorb spo ntaneously atedg e -or ien ted pyrolytic graphite (PGE) electrodes in the presence of

suitable coadsorbates such as neomycin or polymyxin. The PGE electrode

is a small block of pyro lyti c graphite mount ed on a brass rod and embed dedwith epoxy in a Teflon sheath. The block is oriented in such a way thatthe working surface is an ed g e plane perpend icular to the aromatic


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[ 1 8 ] V O LTA M M E T RY F REDOX-ACTIVE CENTERS 487

b a s a l p l a n e . A b r a s i o n o f t h e e d g e p l a n e b y c u tt i n g o r p o l is h i ng i n a irg e n e r a t e s h y d r o p h i l ic o x i d e s t h a t a r e w e a k l y a c i d ic . 15 N e o m y c i n a n do t h e r s i n t h e a m i n o c y c l i t o l f a m i l y o f a n t ib i o t ic s p o s s e s s a s p e c i fi c a r r a n g e -

m e n t o f v a r i o u s g r o u p s , p a r t i c u l a r lyN H 3

g r o u p s , l o c a t e d o n a c o m p l e x ,o f t en r igid , s t ruc tu re in such a w ay pe rh aps a s to fu lfill r equ i re m en t s o fp r e o rg a n iz a t io n in b i n di n g to c o m p l e x , c o m p l e m e n t a r y s u r f a c e s ) I n t h ep r e s e n c e o f t h e s e r e a g e n t s , f e r r e d o x in s a n d m a n y o t h e r p r o te i n m o l e c u l e sc o a d s o r b o n t h e P G E e l e c t r o d e s u r f a c e t o g iv e a n e l e c t r o a c t i v e film w i t ha c o v e r a g e o f o n e m o n o l a y e r o r l ow er.1 6

T h e e l e c t r o c h e m i c a l c e l l t h a t w e h a v e u s e d f r e q u e n t l y i s s h o w n i nF i g . 3 . T h e c e n t r a l w a t e r - j a c k e t e d c o m p a r t m e n t h o u s i n g t h e r e f e r e n c ee l e c t r o d e ( t y p i c a l l y s a t u r a t e d c a l o m e l ) i s l i n k e d v i a L u g g i n c a p i l l a r y

t i p s t o f o u r p o t s e a c h c o n t a i n i n g a p l a t i n u m ( P t ) w i r e o r g a u z e c o u n t e r -e l e c t r o d e . S u c h a n a r r a n g e m e n t a l l o w s t h e r e s p o n s e o f t h e p r o t e i n f ilmt o a v a r ie t y o f re a g e n t s t o b e o b s e r v e d ; t h e c o a t e d P G E e l e c t r o d e i ss i m p l y t r a n s f e r r e d f r o m o n e p o t t o a n o t h e r. T h e p o t s a r e t h e r m o s t a t t e db y i m m e r s in g t h e l o w e r h a l f o f t h e c e ll i n a c i rc u l a ti n g b a t h . F o r c y c l i cv o l t a m m e t r y, t h e e l e c t r o d e s a r e c o n n e c t e d t o a s t a n d a r d p o t e n t i o s t a t ,a n d d a t a a r e t y p ic a l ly r e c o r d e d o n a n X - Y r e c o r d e r. F o r d i g it al ly b a s e dt e c h n i q u e s s u c h a s s q u a r e - w a v e v o l t a m m e t r y, a c o m m e r c i a l i n s t r u m e n ts u c h a s t h e B A S 1 0 0 B e l e c t r o c h e m i c a l a n a l y z e r ( B i o a n a l y t i c a l S y s t e m s ,

We s t L a f a y e t t e , I N ) i s u s e f u l . We h a v e f a v o r e d a n a n a l o g - b a s e d s y s t e mw h e r e v e r t h e e x p l o r a t o r y n a t u r e o f a n e x p e r i m e n t h a s r e q u i r e d f le x i bi li ty( w h i c h i s t h e s i tu a t i o n i n m o s t c a s e s ) . T h e c a p a b i l i ty o f p e r f o r m i n gr o t a t i n g - d i s k v o l t a m m e t r y ( r e q u i r i n g a n e l e c t r o d e r o t a t o r ) i s a l s o t o b er e c o m m e n d e d s in c e r e a c ti o n s o f a d s o r b e d m o l e c u le s w it h re a g e n t s i ns o l u t i o n m a y d e p e n d c r i t i c a l l y o n m a s s t r a n s p o r t t o t h e e l e c t r o d e .

T h e f o l lo w i n g m e t h o d f o r p re p a r i n g a p r o te i n f il m h a s b e e n c o n s i s t e n t l ys u c c e s s f u l f o r a n u m b e r o f f e r r e d o x i n s , a n d w e o u t l in e i t h e r e w i t h t h eu n d e r s t a n d i n g t h a t w e h a v e n o t e n d e a v o r e d t o o p t i m i z e t h e t e c h n i q u e .

T h e P G E e l e c t r o d e i s f ir s t p o l i s h e d w i t h a n a q u e o u s s l u r r y o f 1 / x m A 1 203o n c o t t o n w o o l a n d t h e n s o n i c a t e d in c o l d w a t e r. I m m e d i a t e ly f o ll o w in gt h is , 1 /~ 1 o f ic e - c o l d p r o t e i n s o l u t i o n ( c o n ta i n in g c o a d s o r b a t e a s n e c e s s a r y )i s s p r e a d a c r o s s t h e e l e c t r o d e s u r f a c e u s i n g a g l a s s r o d d r a w n t o a f i n et ip . A t y p i c a l c o a t i n g s o l u t io n c o n t a i n s 1 0 0 / x M p r o t e i n a n d t h e r e q u i r e dc o n c e n t r a t i o n o f c o a d s o r b a t e i n 0 .1 M N a C I b u f f e r e d at p H 7 . T h e c o a t e de l e c t r o d e i s t h e n i n t r o d u c e d t o a p o t c o n t a in i n g b u f f e r - e l e c t r o l y t e a t t h e

15 F. A. Arm stro ng, P. A. Cox, H. A. O. Hill, V. J. Low e, and B. N. Oliver, J ElectroanalChem 217, 331 1987).

16 F. A. Arms trong, J. N. Butt, S. J. George, E. C. Hatchikian, and A. J. Thomson, FEBSLett 259, 15 1989).


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88 P R O B E S O F M E T A L I O N E N V I R O N M E N T S [ 8 ]

i

t ~

a:zz

g _ fFIG. 3. Diagram o f the electrochem ical cel l typical ly used in m etal loprotein experim ents .

The cent ra l ly loca ted reservoi r houses a re ference e lec t rode . I t i s water- jacke ted to m ain ta ina s tandard refere nce tempe rature. Liquid junct io ns, term inat ing in Luggin capi l lary t ips seeinse t) , connec t the re ference reserv oi r wi th each com ponent ce l l . P lat inum gauze a t tachedto the wal l o f each sample pot se rves as the countere lec t rode . Each sample pot can beindependent ly purged of a tmospher ic oxygen b y bubbl ing an iner t gas usua lly a rgon) througha f lexible tubing capi l lary gas l ine. This enters the pot through a s ide arm, which also servesas the passagew ay for the coun ter e lec t rode lead . A s ingle working e lec t rode can be rap id lyt ransfer red be tween pots , fac i li t a ting mul t i sample s tudies . By immers ing the lower par t o fthe cel l in a circulat ing bath, tem peratur e contro l can be acco mp lished.

d e s i r e d a p p l i e d p o te n t i a l , a n d t h e e x p e r i m e n t i s c o m m e n c e d . A n a l t e r n a -t i ve m e t h o d o f f i l m f o r m a t i o n t h a t h a s a l s o p r o v e d s u c c e s s f u l i s t o in t r o -d u c e a f r e s h l y p o l i s h e d e l e c t r o d e i n to a p o t c o n t a i n i n g a d i l u te - 1 / x M )s o l u t i o n o f t h e p r o t e i n a n d c o a d s o r b a t e i n t h e b u f f e r - e l e c t r o l y t e , t o c y c l e

t h e p o t e n t i a l o r p o i s e i t f o r a s h o r t t i m e i n a r e g i o n t h a t i s f a v o r a b l e f o ra d s o r p t i o n , a n d t h e n to t r a n s f e r t h e c o a t e d e l e c t r o d e to a s e c o n d p o t n o tc o n t a i n i n g t h e p r o t e i n ) f o r s t u d y .


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[ 8 ] V O L T A M M E T R Y O F R E D O X A C T I V E C E N T E R S 489

W e h a v e m a d e t h e f o ll o w i ng o b s e r v a t i o n s w i t h f e r re d o x i n s f r o mDesul-fovibrio africanus Azotobacter vinelandiia n d o t h e r s w h i c h t y p i c a l l yr e q u i re n e o m y c i n a s c o a d s o r b a t e : n o s ig n als a r e o b s e r v e d i f n e o m y c i n i s

a b s e n t f r o m t h e c o a t i n g s o l u t i o n ; i f n e o m y c i n i s p r e s e n t d u r in g c o a t i n gbu t absen t f rom the po t so lu t ion , the s igna l s d imin i sh r ap id ly ; and i fn e o m y c i n i s p r e s e n t i n b o t h t h e c o a t i n g a n d t h e p o t s o l u t i o n , t h e s i g n a l sp e r s i st . T h e r e s u l t s s h o w t h a t c o a d s o r p t i o n o f n e o m y c i n i s n e c e s s a r y f o ra d s o r p t i o n o f t h e p r o te i n . A s n e o m y c i n u n d e rg o e s n e t d e s o r p t i o n , a so c c u r s i f i t i s a b s e n t f r o m t h e s o l u t i o n , t h e p r o t e i n m o l e c u l e s a l s o d e s o r b .O n t h e o t h e r h a n d , l o s s o f si g n al s is s u p p r e s s e d i f n e o m y c i n i s p r e s e n t int h e s o l u ti o n . F r o m a n u m b e r o f s t u d i e s w i t h v a r i o u s p r o t e i n s , w e h a v en o t e d t h a t f ilm f o r m a t i o n d e p e n d s o n a n u m b e r o f f a c t o r s . S u c c e s s is m o r e

l ike ly i f t he ion ic s t r eng th i s 0 .2 M or lowe r, i f t he t e m per a tu r e i s l owtyp ica l ly 0), and i f t he sam ple o f in t e res t i s o f ve ry h igh pur i ty bec ause

t h e r e w o u l d b e c o m p e t i ti o n f o r o c c u p a n c y o f s it e s o n t h e e l e c tr o d e ) . W eh a v e a l s o f o u n d t h a t t h e e x t e n t o f a d s o r p t i o n a n d t h e r e d o x p r o p e r t i e s o ft h e c e n t e r s i n t h e a d s o r b e d p r o t e i n m o l e c u l e s m a y b e s e n s i t i v e t o t h ea p p l i e d p o t e n t i a l . A n y s u c h d e p e n d e n c e m u s t a l w a y s b e i d e n t i f i e d p r i o rt o m o r e d e t a i l e d i n v e s t i g a t io n s .

Ana lys i s and In te rp re ta t ion

Imaging Redox Centers in Proteins .F i g u r e 4 s h o w s v o l t a m m o g r a m so b t a i n e d f o r fo u r d i ff e re n t f e r re d o x i n s a b s o r b e d o n P G E e l e c t ro d e s . T h e s ea r e t y p i c a l l y m e a s u r e d o n t h e f o u r t h o r f i f t h c y c l e . A l t h o u g h d o m i n a t e db y t h e c o n t r i b u t io n f r o m c a p a c i t a n c e , t h e F a r a d a i c c o m p o n e n t s a r e r e a d il yr e v e a l e d a n d c a n b e s t u d i e d in g r e a t e r d e t a i l b y e x p a n d i n g t h e s c a l e . T h r e eo f t h e f e r r e d o x i n s d i s p l a y s e v e r a l d i s ti n c t iv e c o u p l e s , t h u s s h o w i n g t h a tt h e r e i s m u l t i p le e l e c t r o n - t r a n s f e r a c t i v i ty. T h e l o w p o t e n t i a l a t w h i c h a

n u m b e r o f t h e s e c o u p l e s o c c u r b e lo w - 5 5 0 m V ) s h o w s th a t t h e y a r ei n a c c e s s i b l e t o d i th i o n i te a n d h e n c e m a y n o t b e a d d r e s s e d b y s t ra i g h tf o r-w a r d c h e m i c a l t it r a ti o n s . F u r t h e r m o r e , i n c o n t r a s t t o m o r e c o n v e n t i o n a li n v e s t i g a t io n s o f s u c h s t ro n g r e d u c t a n t s , a s u ff i ci e n tl y a n a e r o b i c e n v i r o n -m e n t i s e a s y t o m a i n t a i n .

The f i r s t s t ep in ob ta in ing quan t i t a t ive in fo rmat ion f rom such re su l t sis t o s u p e r i m p o s e a b a s e l i n e o n th e v o l t a m m o g r a m . F o r a s im p l e e l e c tr o n -t r a n s f e r r e a c t i o n t h a t i s n o t c o m p l i c a t e d b y c o u p l e d c h e m i s t r y, t h e b a s e l in ec o n s i s ts o n l y o f th e c a p a c i ti v e c o m p o n e n t s o f t h e s w e e p . T o a r e a s o n a b l e

d e g r e e , t h e f o r m o f t h e b a s e l i n e c a n b e o b t a i n e d b y c a r r y i n g o u t a n i d e n ti c a lv o l t a m m e t r ic e x p e r i m e n t o n a f r e sh l y p o l i sh e d e l e c t r o d e p l a c e d d i r ec t lyi n t o e l e c t r o l y t e s o l u t i o n . A f t e r t a k i n g t h e b a s e l i n e i n t o c o n s i d e r a t i o n ,


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4 9 0 PROBES OF META L ION ENVIRONMENTS [1 8 ]

T b

T 4 A C

i [ [

-10 00 0 /mY vs. S.H.E.

F I G . 4 . A d s o r b e d f il m v o l t a m m e t r y o f f o u r fe r r e d o x i n s : a )Desulfovibrio africanus F dI I I, 2 00 m V / s e c e x p a n d e d v i e w o f o x i d a t iv e s w e e p is a l so s h o w n ) ; b )zotobacter vinelandiiF d I , 2 0 m V / s e c ; c )Thermodesulfobacterium commune F d , 2 0 0 m V / s e c ; d )Clostridiumpasteurianurn F d , 2 0 0 m V / s e c . F i l m s p r e p a r e d f r o m a c o a t in g s o l u t i o n c o n t a i n i n g 1 0 0 v. Mf e r r e d o x i n , 2 m M n e o m y c i n , a n d 1 00 p .M E G T A 0 .1 M N a C I , b u f f e re d a t p H 7 .4 ) w e r ep l a c e d i n t o a n e l e c tr o l y t e s o l u t io n a t 0 a l so c o n t a i n in g 2 m M n e o m y c i n a n d 1 0 0 / z M E G TA

0 .1 M N a C 1 , b u f f e r e d a t p H 7 .4 ) . R e d o x c o u p l e s a r e la b e l e d a c c o r d i n g t o t h e d i s c u s s i o nin the t ex t .

v a l u e s o f E , 8 , A E p a n d t h e r a t i oi p c i p ac n e a c h b e d e t e r m i n e d , a n d t h ea r e a s o f t h e w a v e s e s t i m a t e d .

A n i l l u s tr a t i o n o f th e p r o c e d u r e i s g i v e n i n F i g . 4 a , f o r t h e c a s e o ff e r r e d o x i n I I I ( F d I I I ) f r o mD esu l f o v i b r i o a f r i c a n u s A s i s o l a t e d , t h i sp r o t e in c o n t a i n s o n e [ 3 F e - 4 S ] a n d o n e [ 4 F e - 4 S ] c l u s te r , a n d w e r e fe r t oit a s 7 F e - F d III.1 7 A s r e c o r d e d i n b u f f e r - e l e c t r o l y t e c o n t a i n in g E G TA ,

17 F, A . A r m s t r o n g , S . J. G e o rg e , R . C a m m a c k , E . C . H a t c h i k i a n , a n d A . J . T h o m s o n ,B i o c h e m . J . 2 6 4 265 198 9 ) .


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[1 8 ] VOLTAMMETRY OF REDOX-ACTIVE CENTERS 491

t h e r a t io o f a r e a s u n d e r o x i d a t i v e w a v e s A , B , a n d C i s 1 : 1 : 2 [ t hep r i m e n o t a t i o n r e f e r s t o t h e f a c t t h a t t h e s e a r e a d s o r b e d r e d o x c o u p l e s ;t h e y m a y d i f f e r ( u s u a l l y m a rg i n a l l y ) i n p o t e n t i a l f r o m t h e s a m e c o u p l e

m ea sur ed in f r ee so lu t ion ] . 13 16 Co up le A wi thip c i p a1 and 8 = 100 m Vc o n f o r m s w e l l t o th e p r e d i c t i o n f o r a r e v e r s i b l e o n e - e l e c t r o n p r o c e s s w i t h ina h o m o g e n e o u s n o n i n te r a ct in g p o p u l a t io n o f r e d o x c e n t e rs . T h e f a c t t h atA Ep i s l e s s th a n 3 0 m V e v e n a t a s c a n r a t e o f 50 0 m V / s e c d e m o n s t r a t e st h a t e l e c t r o n t r a n s f e r i s f a s t . T h e a r e a s u n d e r t h e w a v e s a r e i n a c c o r dw i t h v a l u e s e x p e c t e d f o r a b o u t o n e e l e c t ro a c t i v e m o n o l a y e r o f p r o t e inm o l e c u l e s. T h e c o m p o n e n t w a v e s o f c o u p l e B a r e b r o a d e r , i n d ic a ti v e o fi n te r a c ti o n b e t w e e n r e d o x c e n t e r s o r h e t e r o g e n e i ty t h a t m i gh t b e d u e t ov a r y i n g m i c r o e n v i r o n m e n t s . C o u p l e C i s c l e ar ly m o r e c o m p l e x s in c e

ipc/ipai s l e s s t h a n l , a n d 8 f o r t h e o x i d a t i v e w a v e i s a p p r o x i m a t e l y 5 0 m V,c l o s e to t h e p r e d i c t e d v a l u e fo r a c o n c e r t e d t w o - e l e c t r o n p r o c e s s . F o r F dI I I , a n d f o r t h e 7 F e - f e r r e d o x i n f r o mA z o t o b a c t e r v i n e l a n d i i(Fig. 4b) ,c o u p l e s A a n d B h a v e b e e n e s t a b li s h e d to c o i n c i d e w i th c o u p l e s A a n dB o b s e r v e d f o r t h e p r o t e i n i n b u l k s o l u t i o n . T h e s e c o u p l e s h a v e b e e nass igned as [3F e-4 S] l+ / and [4F e-4 S] 2+/1+ re sp ec t iv e ly, on the b as i s o fc o u l o m m e t r i c t i t r a t i o n a n d e l e c t r o n p a r a m a g n e t i c r e s o n a n c e ( E P R ) a n dm agn e t i c c i r cu la r d i ch ro i sm (MC D) spe c t ro sco p ie s . 17-19

T h e f e r r e d o x i n f r o mT h e r m o d e s u l f o b a c t e r i u m c o m m u n ei s no t we l l

c h a r a c t e r i z e d a t t h e t im e o f w r i ti n g , b u t i ts v o l t a m m e t r y i s s h o w n i n F i g .4 c b e c a u s e i t i l l u s t r a t e s h o w t h e t e c h n i q u e p e r m i t s a c o m p l e x s i t u a t i o nt o b e v i s u a l i z e d . T h e p r o t e i n h a s b e e n r e p o r t e d t o c o n t a i n a [ 3 F e - 4 S ]c l u s t e r t h a t t a k e s u p F e ( l I ) t o f o r m [ 4 F e - 4 S ] . 2 F o l l o w i n g t h e i n f o r m a t i o ng i v e n b y t he v o l t a m m e t r y, E P R s p e c t r o s c o p i c e x a m i n a ti o n s s h o w e d th a tc o u p l e s A~ a n d A 2 a r e e a c h a s s o c i a t e d w i th t h e n o r m a l 1 + / 0 r e a c t i o no f [ 3 F e - 4 S ] c l u s t e r s ( r e a c t i o n I o f S c h e m e I ) , A 2 b e i n g o f u n u s u a l l yh i gh p o t e n t i a l (1 80 m V ) , w h e r e a s o n l y A I is t r a n s f o r m a b l e b y M ( I I) ( se eb e l o w ) . I t s e e m s , t h e r e fo r e , t h a t t h e p r o te i n c a n a c c o m m o d a t e t w o c h e m i -

c a l l y d i s t i n c t [ 3 F e - 4 S ] c l u s t e r s .T h e s im p l e s t v o l t a m m o g r a m is d i s p l a y e d b y th e f e r re d o x i n f r o mC l o s

t ri d iu m p a s t e u r i a n u m(F ig . 4d ) . The s ing le pa i r o f s ligh t ly d i s to r t ed w av es

18 F. A . A r m s t r o n g , S . J . G e o rg e , A . J . T h o m s o n , a n d M . G . Y a t e s ,F E B S L e t t . 2 3 4197 1988).

19 S . E . I i sm aa , A . E . V~izquez , G . M . Jense n , P. J . S t eph ens , J . N . B u t t , F. A . Ar m s t ron g ,a n d B . K . B u rg e s s ,J . B io l . Chem. 26621563 1991).

10 B . Gu ig l i a re l li , P. Be r t r an d , C . M ore , P. P apav ass i l i ou , E . C . H a tch ik i an , and J . P. G ayd a ,B i o c h i m . B i o p h y s . A c t a810 , 319 1985).


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49 2 PROBES OF ME TAL ION ENVIRONMENTS [1 8]

a r i s e s b e c a u s e t h e t w o [ 4 F e - 4 S ] 2+/1 + c l u s t e r s i n t h is p r o t e i n h a v e r e d u c t i o np o t e n t i a l s t h a t a r e v e r y s i m i l a r i n v a l u e . 2~

C o u p l e C , a s o b s e r v e d c l e a r l y f o r F d I I I a n d f o rAzotobac te r f e r r e -

d o x i n ( F i g . 4 a , b ), i s p a r t i c u l a r l y i n t r ig u i n g s i n c e th e E v a l u e i s p H d e p e n -d e n t a n d v e r y lo w ( in t h e re g io n o f - 7 0 0 t o - 8 0 0 m V a t p H 7 ). B yc o m p a r i n g th e a r e a o f C w a v e s w i th th e a r e a g e n e r a t e d b y t h e o n e - e l e c t ro nc o u p l e A , i t c a n b e d e t e r m i n e d t h a t t w o e l e c t r o n s a r e t ra n s fe rr e d. 1 6 F o rF d I II , th i s is m o s t e v i d e n t f r o m t h e o x i d a t i o n w a v e ; t h e b r o a d n e s s o ft h e r e d u c t i o n w a v e i n d i c a te s s o m e d e g r e e o f a d d i ti o n a l c o m p l e x i t y in th isp r o c e s s . ( O n a c a u t i o n a r y n o t e , c u r r e n t a r is i ng f r o m a n e l e c t ro n - t r a n s f e rr e a c t i o n t h a t is d e p e n d e n t o n a p r e c e d i n g o r i n t e r m e d i a r y c h e m i c a l p r o c e s sm a y n o t a p p e a r a s a d i s t i n c t i v e w a v e . T h i s s i t u a t i o n c a n u s u a l l y b e i d e n ti -

f ie d b y v a r y i n g t h e s c a n r a t e , a s a s l o w s c a n w il l e n a b l e a c h e m i c a l p r o e s st o r e m a i n k i n e t i c a ll y e f f e c t iv e a n d l e a d t o o b s e r v a t i o n o f a m o r e r e g u l a rw a v e f o r m . ) E v e n i n t h e c a seo f A z o t o b a c t e r f e r r e d o x i n , f o r w h i c h c o u p l eC l ie s v e r y c l o s e i n p o t e n t i a l t o c o u p l e B ( t h e [ 4 F e - 4 S ] 2+/1+ c l u s t e r i nt h is p r o t e i n h a s a n u n u s u a l l y l o w E v a l u e ) , th e n a r r o w s h a p e s o f t h e Cw a v e s m a k e i ts q u a n t if i c at i o n q u it e e a s y. F u r t h e r m o r e , in t h e c a s e o f F dI II , c o u p l e C d i s a p p e a r s i n c o n c e r t w i t h c o u p l e A w h e n th e [3 F e - 4 S ]c l u s t e r i s t r a n s f o r m e d i n to [ M 3 F e - 4 S ] b y u p t a k e o f M ( I I) . 13 W e h a v et h e r e f o r e s u g g e s t e d t h a t i t c o r r e s p o n d s t o t h e c o u p l e [ 3 F e - 4 S ] /2- w i t h

u p t a k e o f H + , t h a t i s, g e n e r a t i o n o f w h a t is fo r m a l l y a n a l l - F e (I I ) c l u s te r.C o u p l e s A a n d C t h u s c o r r e s p o n d t o r e a c t i o n s 1 a n d (2 + 3 ) o f S c h e m eI , w h e r e a s c o u p l e B c o r r e s p o n d s t o r e a c t i o n 9 w i t h M = F e a n d X =c y s t e i n e R S - . I t is l i k e ly th a t t h e c o u p l e o b s e r v e d a t v e r y l o w p o t e n t i a lf o r th e f e r r e d o x i n f r o mT. commune i s a l s o a s s o c i a t e d w i t h a [ 3 F e - 4 S ]c l u s t e r s i n c e r e a c t i o n w i t h v a r i o u s M ( I1 ) i o n s c a u s e s i t t o d i s a p p e a r i nc o n c e r t w i t h c o u p l eA I .

Investigating C oupled Chemical Reac tions That Are Slow on Voltam-metric Time Scale. A g o o d e x a m p l e o f a c o u p l e d r e a c t i o n t h a t is s lo w o n

t h e v o l t a m m e t r i c t i m e s c a l e i s g i v e n in F i g . 5, w h i c h s h o w s s u c c e s s i v ev o l t a m m o g r a m s ( p o s i t i v e s c a n d i r e c t i o n o n l y ) o f a f ilm o f F d I I I fo l lo w i n gt r a n s f e r t o a p o t c o n t a i n i n g F e ( I I ) , Z n ( I I ) , o r C d ( I I ) / 3 O v e r s e v e r a l s c a n sw e o b s e r v e a d e c r e a s e in t h e a m p l i tu d e s o f c o u p l e s A a n d C a n d a p p e a r -a n c e o f a n e w c o u p l e t h a t w e t e r m D . F o c u s i n g f i rs t o n t h e r e a c t i o n w i t hF e ( I I ), it w a s s h o w n b y c o u l o m e t r i c s tu d i e s a n d p r o d u c t a n a l y si s b y E P Ra n d M C D s t u d ie s t h a t t h e [ 3 F e - 4 S ] c l u s t e r t ak e s u p F e ( I I) t o f o r m as e c o n d [ 4 F e - 4 S ] 2+ c l u s t e r. 22 T h i s i s r e a c t i o n 5 o f S c h e m e I . T h i s n e w

21 E. T. Sm ith, D. W. B ennett, and B . A. Feinberg,Anal. Chim. Acta 251, 27 (1991).22 S. J. G eorge, F. A . Arm strong, E. C . Hatchikian, and A. J. Th om son,Biochem. J. 264

265 (1989).


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[ 1 8 ] V O LTA M M E T RY F REDOX-ACTIVE CENTERS 493

cluster has unusual magnetic properties, being a S = a system in thereduced state, and necessarily having noncysteinyl ligation, presumablyin the position of the new Fe subsite. The reduction potential of the new

cluster (couple D', reaction 9) is very similar to that of the indigenous,stable [4Fe-4S] z+/l+ cluster; hence , it appears as an increase in the ampli-tude of couple B'. Similar products (with different reduction potentials,however) were proposed for the reactions with Zn(II) and Cd(II). 13

This experiment provides a simple test for detecting redox-linked trans-format ions of clusters. Similar experiments with A z o t o b a c t e r ferredoxinhave shown that no such facile transformations occur. On the other hand,experiments with T c o m m u n e ferredoxin reveal easily that part of the[3Fe-4S] cluster population (AI' along with the broad couple at very low

potentia l, but not A2') transforms readily. 23In such a case the voltammetr ic data are analyzed as follows. 13 Inspec-

tion of the final voltammogram shows that the area of D' is equal to thearea of the initial wave A' before the reaction. This demonstrates 1 : 1interchange o f one-electron couples. By vertically displacing successivevoltammetric sweeps, the course o f the transformation can be monitored;in this case a plot of log[A/] (the difference in current as measured fortwo potential values) versus time is linear and has an identical slope forthe attenuation of couples A' and C' and the increase in amplitude of couple

D'. This establishes that the reaction conforms to first-order kinetics andthat the three processes are interrelated. However , one cannot determinereal rate constants in this way because the rate o f interconversion amongcenters may depend critically on oxidation level. In this case, for example,formation of [4Fe-4S] clusters occurs only when the [3Fe-4S] cluster isin the 0 oxidation level. This can be demonstrated readily by introducinga film of Fd III into M(II) at 0 mV, holding at this potential for around5 min, then rapidly cycling once. The voltammogram then observed isunchanged from that of the 7Fe form. In contrast, if the coated electrode

is introduced at a potential of -250 mV (i.e., switching the [3Fe-4S]cluster to the 0 oxidation level) and held for several seconds, a singlecycle reveals that reaction has occurred.

Given the importance of maintaining potential control over the variousredox centers, we can observe the ease with which this requirement canbe achieved with adsorbed protein voltammetry. For example, if we wishto study reaction 5 and determine the equilibrium constants for variousM(II) ions, it is essential to apply a potential that maintains [3Fe-4S] atthe 0 oxidation level while maintaining [M3Fe-4S] as 2 + . This

effectively isolates the chemical equilibrium that is of interest. The proce-23 j. N. Butt, F. A. Arms trong, J. Breton, and A. J. Thomson, unpublished observations

1992).


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494 PROBES OF METAL ION ENVIRONMENTS [1 8]

Fe

I

1

I

i

t

Z 1 p A

Cycle number

zn ~

2

,~ 1 I

I -4CI

Cd

1'

I

. , .; ..

I ] I 1 I i l I I

-500 0E/mV vs. SHE

1

o-2

II

I

I I

-1000

i i . i i j

1 2 3 4 5 6 7Cycle number

Cycle number


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[ 1 8 ] V O LTA M M E T RY O F R E D O X - A C T I V E C E N T E R S 495

d u r e i s st r a i g h t f o rw a r d f o r r e a c t i o n s t h a t a r e s l o w b y c o m p a r i s o n w i t hv o l t a m m e t r ic s c a n r a te s . U s i n g o u r d e s c r ib e d r e a c t io n s a s e x a m p l e s , t h e7 F e f e r r e d o x i n - c o a t e d e l e c t r o d e i s i n t r o d u c e d t o a s o l u t i o n c o n t a i n i n g a

k n o w n c o n c e n t r a t io n o f M ( I I) a t a b a s e p o t e n ti a l t h a t is m i d w a y b e -t w e e n t h e E ' v a l u e s o f r e a c t i o n s 1 a n d 9 . P o is in g t h e e l e c t r o d e a t t h isp o t e n t i a l p r e c i s e l y d e f i n e s t h e c o r r e c t o x i d a t i o n s t a t e s o f t h e c l u s t e r sinvo lved . A f te r s t i rr ing the so lu t ion fo r su ff i c ien t t ime to a l low equ i l ib r iumt o b e e s t a b l i s h e d , t h e s t a t u s o f t h e c l u s t e r s i s d e t e r m i n e d f r o m a r a p i dv o l t a m m e t r i c c y c l e . T h e r a t i o o f s u r f a c e p o p u l a t i o n s o f r e a c t a n t v e r s u sp r o d u c t a t e a c h c o n c e n t r a t io n o f M ( I I) c a n b e d e t e r m i n e d b y m e a s u ri n gt h e r e l a t iv e a m p l i t u d e s o f t h e r e s p e c t i v e v o l t a m m e t r i c s i g n al s. ( H o w t h isi s d o n e w i l l d e p e n d o n t h e w a v e s h a p e s o b s e r v e d . ) T h e d i s s o c i a t i o n c o n -

s t an t K d i s ca l cu la t ed by f it ti ng da ta to Eq s . (1 ) o r (2 ). A l thou gh{[M 3Fe -4S] 2+} _ { i ( I I ) }

1){[3F e-4S ] } K d

({[M3Fe-4S]2*}-~ = log{M(II)} - log K d (2)l o g \ { [ 3 F e _ 4 S ] 0} ]

t h e s c a t t e r o f d a t a m a y b e la rg e, m e a s u r e m e n t s a r e ra p i d , a n d t h e s a m p l ee c o n o m y m e a n s t h a t e x p e r i m e n t s c a n b e r e p e a t e d a l a rg e n u m b e r o f

t im e s w h e r e n e c e s s a r y. T h e s y s t e m c a n b e t e s t e d b y v a r y in g t h e b a s ep o t e n t i a l ; f o r e x a m p l e , i t i s p o s s i b l e t o c h e c k t h a t t h e e q u i l i b r i a b e i n gm e a s u r e d a r e v a l id p r o p e r t i e s o f t h e p r o t e i n a n d a r e n o t i n fl u e n c e d b yc h a n g e s i n t h e e l e c t r ic a l p r o p e r t i e s o f t h e e l e c t r o d e - s o l u t i o n i n t e r f a c e .

W i th t h e a b o v e p r o c e d u r e w e w e r e a b l e t o m e a s u r e t h e a f fi ni ty o f t h e[ 3 F e - 4 S ] c l u s t e r f o r v a r i o u s M ( I I) i o n s w i t h o u t i n t e r f e r e n c e f r o m c o u p l e dr e d o x r e a c t i o n s . R e s u l t s s h o w e d t h a t f o r F d I I I , t h e b in d i n g o f M ( I I ) to[ 3 F e - 4 S ] r u n s a c c o r d i n g t o t h e I r v i n g - W i l l ia m s s e r ie s , t h a t i s , C d ( II ) >Zn( I I ) >> Fe ( I I ) . A l thou gh the ide n t i ty o f the n onc lus te r l igand(s ) to M i s

FIG. 5 . Left) O x i d a t i v e s c a n s ( s u c c e s s i v e c y c l e s ) o f a fi lm o f 7 F e - f e rr e d o x i n I II o nt r a n s f e r t o s o l u t i o n s c o n t a i n i n g M ( I I) i o n s .Right) C o r r e s p o n d i n g s e m i l o g p l ot s s h o w i n gt i m e c o r r e s p o n d e n c e o f a p p e a r a n c e o f w a v e s D ' a n d d i s a p p e a r an c e o f w a v e s A ' . T h ep o t e n t ia l w a s h e l d b r ie f ly a t a p p r o x i m a t e l y + 5 0 m V p r i o r t o c o m m e n c e m e n t o f s c a n n i n g a t4 7 0 m V / s e c ( t e m p e r a t u r e 0 ). E a c h d e t e r m i n a t i o n i n v o l v e d m e a s u r e m e n t o f t h e d if fe r -e n c e i n c u r r e n t a t t w o p o t e n t i a l s a s i n d i c a t e d . T h e s e a r e a s f o l l o w s . F e 2+ ( 3 0 0 / x M ) : + ,i -129 rnV -- i -229 mY, co u p le A ' ; , i_393 mV - i -250 my, co u p le D ' ; O , i_651 m v - i -554 mv, co u pl eC ' . Z n 2+ (10 /.LM): + , L_129m - i -229mV, co up le A ' ; 0 , i_48 ,1m - i_570mv, co up le D ' ; O ,i_655 m - i_579mV, co up le C ' . C d 2+ (10 p .M): + , i_129m - i_229mv, co up le A ' ; I I , i_64]rnv -i-56s m v, c o m b i n a t i o n o f c o u p l e s C ' a n d D ' . [ R e p r i n te d w i t h p e r m i s s i o n f r o mJ . A m . C h e m .Soc . 11 3 , 6 6 6 3 (1 9 9 1) . C o p y r i g h t 19 91 A m e r i c a n C h e m i c a l S o c i e t y. ]


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4 9 6 P RO B ES O F M E TA L I O N E N V I R O N M E N T S [ 8 ]

yet to be es tablished, this study has indicated the biological feasibility ofFe-S clusters containing Zn.

I n v e s ti g a t in g C o u p l e d C h e m i c a l R e a c t i o n s T h a t A r e R a p i d o n Vo l t a m -

m e t r i c Ti m e S c a l e .If a redox-linked chemical reaction occurs on a timescale that is rapid com pared to the voltammet ric scan rate, it is not possibleto separate the coupled process from the electron transfer. A simple caseto consider is the rapid and reversible binding of a reagent to a center ineither o f two oxidation states. If the affinity for the reagent differs betweenoxidation states, then a single redox couple is observed with an E ' valuethat is dependent on the concentration of reagent. Because equilibriumis always established within the time scale of measurement, the result issimilar to that which would be obtained by potentiometry.

An example of such a system is given by the reaction o f the 3Fe cluste rof Fd III with a single thallium(l) ion.14 This reaction can be describedby a thermo dynamic cycle, in this case a bo x composed of reactions1, 5, 8, and 4 of Scheme I. With TI(I) concentr ations over the range 10 -5to 10 -1 M, and employing scan rates up to 500 mV/sec, the waves arisingfrom couple A' remain unchanged in shape or size as compared to theinitially form ed film, but the observed reduction potential shifts to a morepositive value E'obs. Equilibrium constants for reactions 4 and 5 areobtained by fitting data to Eq. (3):

E o b s = E ' + (2.3 R T / F ) log{1 + [Q]/Kdred)/(1 + [Q]/KdX)} (3)

in which Q represents the reagent that is binding to the cluster. A graphof E'obs against log[Q] is sigmoidal with asymptotic limits at the E valuesfor the two isolated couples, in this case reactions 1 and 8. The reduc tionpotential for reac tion 8 is now obtained from Eq. (4):

E'~s) = E'~1) + (2.3 R T / F ) lo g K d X /K d e d) (4)

For TI(I) binding to the [3Fe-4S] cluster of Fd III, at an ionic strengthof 0.5 M, such an analysis showed Kd ed = 1.5 /xM, Kd x = 34 mM,E = -1 77 mV, and E'xl = +81 mV. The results demons trate the point(to be illustrated furthe r below) that even weak binding of reagents to anactive site is detectable by the vol tammetri c method. On the basis of thesedata , a solution sample of oxidized Fd III (i. e., with [3 Fe- 4S] 1+) containingan appropriately high concentration of TI(I) was prepared for EPR spec-troscopy. The resulting spectrum showed that the g = 2.01 signal charac-teristic of the species [3Fe-4S] 1+ had been replaced by a rhombic siknal,

thus supporting the proposal that TI(I) interact s directly with the [3Fe-4S]core. The observation of near-ideal waveform even at quite high scanrates shows that entry and release of TI(I) are very rapid.


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[ 1 8 ] V O LTA M M E T R YF RED OX-ACTIVECENTERS 497

-l-o.1.A_ ~ ~ ~ - a

lo . A b

-800 -60 0 -40 0 -20o E /mV vs. S.H.E.

FIG. 6. Ad sorbed f i lm voltamm etry of a)D. africanus8Fe -FdlII , 10 mV /sec, 100/~MFe II). Th e film was transferred into a solution containing Fe II) and 347 m M mercaptoethanoland scan ned at b) 10, c) 200, and d) 500 m V/se c. The coating solution was as describedin Fig. 4. The electrolyte solution contained 2 mM neom ycin, 0 .2 M NaC 1, buffered at p H8, 0. Redox couples are labeled according to the discussion in the text.

E x t r a c t i n g K i n e t ic I n f o r m a t i o n .A f u r t h e r e x a m p l e i l l u s tr a t e s h o w k i -n e t i c d a t a c a n b e o b t a i n e d i f r a te s o f t h e c o u p l e d p r o c e s s e s a r e c o m p a r a b l et o t h e v o l t a m m e t r i c s c a n r a t e . T h e c a s e w e h a v e c h o s e n a g a in i n v o l v esF d I I I , a n d w e c o n s i d e r t h e r e v e r s i b l e b i n d i n g o f a l ig a n d , e t h a n o l 2 -t h io l a te , t o th e t r a n s f o r m e d [ 4 F e - 4 S ] c l u s t e r i n b o t h t h e 2 + a n d 1 + o x i d a -t i o n l e v e ls . 24 A s a t h e r m o d y n a m i c c y c l e , t h e s i t u a t io n is th a t o f a b o xc o m p r i s i n g r e a c t io n s9 13 16 a n d 12 o f S c h e m e I . To s t u d y t h is s y s t e m ,a fi l m o f th e 7 F e - f e r r e d o x i n is fi rs t t r a n s f o r m e d i n to t h e 8 F e f o r m a sd e s c r i b e d a b o v e , a n d t h e e l e c t r o d e i s t h e n t r a n s f e r r e d t o s o lu t i o n s c o n t a i n -in g v a r i o u s c o n c e n t r a t i o n s o f m e r c a p t o e t h a n o l o v e r t h e p H r a n g e 8 - 8 . 5 .R e s u l t s a r e s h o w n i n F i g . 6 . U n d e r s l o w s c a n c o n d i t io n s F i g. 6 b ), t h e

24 j. N. Butt, A. Su cheta, F . A . Arm strong , J. Breton, A . J. Tho m son, and E. C. Hatchikian,J. Am. Chem . Soc.11 5, 1413 1993).


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9 8 PROBES OF METAL ION ENVIRONMENTS [ 8]

single pair o f waves composed of overlaying couples B' and D' splits intotwo, with one remaining at around -390 mV and the other shifting to alower potential E'obs, the value of which is dependent on the calculated

concentrat ion of thiolate anion. No changes are observed if a film of theoriginal 7Fe-Fd III is scanned in the mercaptoethanol solution. It maytherefore be deduced that it is the transformed [4Fe-4S] cluster (not theindigenous [4Fe-4S] cluster) which reacts with thiolate. The variation inobserved E ' value with thiolate concen tration is of the same form asEq. (3).

If the vol tamm etry is performed at a fast scan rate (in this case typically500 mV/sec or higher), the position of the new couple becomes insensitiveto thiolate concentration, whereas the oxidation wave is observed to be

smaller than the reduction component. This occurs because the scan rateis now sufficiently fast to isolate the reversible redox couple (F ') arisingfrom the thiolate-ligated cluster, in this case reaction 6 of Scheme I. Thedistortion now observed on the low-potential side of the oxidation waveof couple B' arises because reoxidation of the remaining labile clusterpopulation is made more favorable by rapid recombination of the product[4Fe-4S] 2+ with thiolate .

Combining Eqs. (3) and (4), the equilibrium constants for reactions 2and 3 can be determined. From these studies we were able to determine

that binding of thiolate to the oxidized cluster [ K d L ) x = 28/zM] is muchstronger than binding to the r educed cluster [KarL) ed = 97 mM]. Thisfeature manifests itself clearly in the form o f the significant negative shiftin reduction potential from -396 mV (D') to -585 mV (F'). Interestingly,we found that we could not prepare a sample of the thiolate-ligated reducedcluster in solution for spectroscopic studies. This reflects the need foran intolerably high concentration of mercaptoethanol coupled with therequirement for a very low potential. As a voltammetric transient, how-ever, the vi rt ua l existence of the thiolate-ligated reduced cluster is

clearly demonstrated.With an intermediate scan rate, the kinetics of reactions 2 and 3 are

revealed more closely. A third oxidation wave ( * , Fig 6c) is now clearlyobserved, the position and size of which vary with scan rate and thiolateconcen tration. As the thiolate concentration increases, this wave shifts tomore negative potential but becomes smaller by comparison with oxidationwave F'. As the scan rate is increased, the wave becomes smaller andshifts to higher potential, merging with oxidative wave B'. Fur the r analysisof the voltammetry is now approached by computer simulation. In this

case an iterative program has been used, based on an effectively constantconcentrat ion of thiolate maintained at the electrode surface by the largebuffering capacity of the rapidly established thiol/thiolate equilibrium.


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118] VOLTAMMETRY F REDOX-ACTIVE CENTERS 499

( -

k

L )

O0(/)

C-OCOC

O

0 . 3 5

0 . 2 5

0 . 1 5

0 . 0 5

- 0 . 0 5

- 0 . 1 5

- 0 . 2 5

- 0 . 3 5- 4 5 0 - 3 5 0 2 5 0

- ~ .~ - ~ . . . ' ./ ~ N

- 2 5 0 - 1 5 0 - 5 0 5 0 1 5 0

/ X , E / m VFIG. 7. Simulated voltammogram showing behavior typical for a box c ompose d of reac-

tions 9, 13, 16,and 12 in Scheme I in which L react s reversibly with [4Fe-4S ] 2 and[4Fe-4S] 1 at an i ntermediate scan rate. Simulation is for experiments shown in Fig. 6 butwith a different ligand concent ration and scan rate v = 100 mV/se c. On rate constantsare 3.2 x 104 M -I sec -l for [4Fe-4S] 2 and 3.09 M -1 sec -1 for [ 4Fe-4S] l. Off ratecons tant s are 0.9 sec -1 for [4Fe-4S] 2 and 0.3 sec -l for [4Fe-4S] 1. The large on rate forthe oxidized cluster gives rise to the wave marked with an * in Fig. 6c. Electroc hemicalrate constants for couples B', D', and F' are also varied to optimize the fit. The potential(AE) axis is refere nced against the reduction potential of the transfo rmed [4Fe-4S] couple(D') as measured in the abs ence of ligand. Values of the current are present ed in dimensionlessform {I/[n2F2oAF/ RT)]}(see Fig. 2 for meaning of terms). C omponent signals arising fromcouples B' (heavy dashed line), F' (dotted line), and the highly distorted D' (light dashedline) reveal the nature of broadening observed for the total current (solid line) near thepotential characteristic for the reduction peak F'.

T h e c u r r e n t a t e a c h v a l u e o f a p p l i e d p o t e n t ia l i s d e t e r m i n e d b y a p p l y i n gt h e B u t l e r - Vo l m e r e q u a ti o n to e a c h re d o x c o u p le B , D , a n d F a n d b yc o n s i d e r i n g t h e c h a n g e i n p o p u l a t i o n s o f c l u s t e r s c a u s e d b y l ig a n d e x -c h a n g e . B y g e n e ra t in g s i m u l at e d v o l t a m m o g r a m s b a s e d o n v a r ie d r a t ec o n s t a n t s f o r t h e p r o c e s s e s i n v o l v e d , a b e s t f it o f k i n e ti c c o n s t a n t s c a nb e f o u n d . S u c h r a te c o n s t a n t s ( e le c t ro c h e m i c a l a s w e l l a s h o m o g e n e o u s )l e a d to t h e o p t i m u m r e p r o d u c t i o n o f e x p e r i m e n t a l t r a i ts ( e . g ., p o s i t i o na n d s iz e o f th e w a v e s ) o v e r th e r a n g e o f e x p e r i m e n t a ll y v a r i e d p a r a m e t e r s

s u c h a s r e a g e n t c o n c e n t r a t i o n s a n d s c a n r a t e . T h e r e s u l t o f o n e s u c hs im u l a t io n , d i sp l a yi n g e a c h c o m p o n e n t o f t h e v o l t a m m o g r a m , is s h o w ni n F i g . 7 . I t c a n b e n o t e d t h a t c o u p l e d e l e c t r o n t r a n s f e r r e a c t i o n s m a y


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5 PROBES OF METAL ION ENVIRONMENTS [18]

produce current contributions of sign contrary to simple expectation. Forexample, close inspection shows that one reaction yields a weak butnoticeable reduction peak in the direction of increasing potential.

From this particular set of experiments it was determined that thekinetic origin of the difference in affinities of [4Fe-4S] 2+ and [4Fe-4S] l+clusters for thiolate (and hence also of the decrease in reduction potentialin the presence of the ligand) lies in an approximately 104-fold increasein the rate of binding to the oxidized cluster.

Concluding Remarks

Although considerable effort may be required to establish conditions

for obtaining a stable, electroactive film, the voltammetric approach canlead to the detection and clarification of chemist ry that is not revealed byother methods. A wide spectrum of information on dynamic systems canbe derived, ranging from a rapid im ag e of the redox chemistry of centersin a protein to the determination of equilibrium and kinetic constants forcoupled reactions. It permits an extensive exploration o f reactivities withsmall amounts of material and is useful in the characterization of labilesystems for which critical conditions must be met for preparation o f spec-troscopic samples.

Acknowledgments

We thank Dr. Edmond Bowden for communicating results prior to publication. Thiswork has been supported by grants from the Exxon Education Foundation, The PetroleumResearch Fund administered by the American Chemical Society, and the National ScienceFoundation (MCB-9118772).

voltammetric studies of redox-active centers in metalloproteins adsorbed on electrodes

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