Spectroelectrochemical Determination of the Temperature

3 Spectroelectrochemical Determination of the Temperature Dependence of Reduction Potentials

Downloaded by CORNELL UNIV on May 18, 2017 | http://pubs.acs.org Publication Date: June 1, 1982 | doi: 10.1021/ba-1982-0201.ch003

Tris(1,10-phenanthroline) Complexes of Iron and Cobalt with c-Type Cytochromes VERNON T. TANIGUCHI, WALTHER R. ELLIS, JR., VINCE CAMMARATA, JOHN WEBB, FRED C. ANSON, and HARRY B. GRAY 1

California Institute of Technology, Arthur Amos Noyes Laboratory, Pasadena, CA 91125 Recent advances in applying thin-layer spectroelectrochemical methods to determine formal reduction potentials (E°) and electron stoichiometries (n-values) of metalloproteins have been extended to the study of the temperature dependences of the E°-values of c-type cytochromes. Isothermal and nonisothermal electrochemical cells are described briefly and the conventions used to assign electron transfer reaction entropies, ∆S°rc, to redox half-cell reactions are discussed. Calibration of the nonisothermal behavior of the spectroelectrochemical cells is performed using Fe(1,10-phenanthroline) 3 and Co(1,10-phenanthroline) 3, two redox couples for which both isothermal and nonisothermal results are available. A detailed study of the temperature dependence of the formal reduction potential of horse heart cytochrome c yields the following thermodynamic parameters: ΔΗ°, -14.5 kcal/mol; ΔS°, -28.5 eu; and ΔS° , -12.9 eu. Preliminary ΔS° values for several other c-type cytochromes are as follows: tuna cytochrome c, -10.3 eu; Rhodospirillum rub rum cytochrome c , -9.6 eu; Pseudomonas aeruginosa cytochrome c , -16.2 eu; Rhodospirillum rubrum cytochrome c', +0.5 eu; and Rhodopseudomonas palus tris cytochrome c', -6.0 eu. 3+/2+

3+/2+

rc

rc

2

551

1

Current address: Murdoch University, School of Mathematical and Physical Sci ences, Murdoch, Perth, W.A. 6150, Australia. 0065-2393/82/0201-0051$06.00/0 © 1982 American Chemical Society Kadish; Electrochemical and Spectrochemical Studies of Biological Redox Components Advances in Chemistry; American Chemical Society: Washington, DC, 1982.

52

BO ILOGC IAL REDOX COMPONENTS l u c i d a t i o n o f t h e t h e r m o d y n a m i c s o f m e t a l l o p r o t e i n e l e c t r o n trans-

-•"^fer r e a c t i o n s

(J-15)

is a matter

o f fundamental

importance i n

b i o c h e m i s t r y (16). T h e e n t h a l p i e s a n d e n t r o p i e s o f m e t a l l o p r o t e i n e l e c t r o n transfer reactions a r e i n f l u e n c e d b y changes i n p r o t e i n c o n f o r m a t i o n a n d s o l v a t i o n as w e l l as o t h e r s t r u c t u r a l a n d m e d i u m effects (2-11,13-15). S u c h effects a r e i n v o l v e d (16,17) i n t h e m e c h a n i s m s o f metalloprotein

b i n d i n g a n d e l e c t r o n transfer

to membrane-bound

c o m p l e x e s . T h i s chapter describes experiments that use spectroelec t r o c h e m i c a l m e t h o d s to d e t e r m i n e m e t a l l o p r o t e i n e l e c t r o n t r a n s f e r e n thalpies a n d entropies.


Variable Temperature Electrochemical Cells T h e r m o d y n a m i c parameters for m e t a l l o p r o t e i n e l e c t r o n transfer reactions have b e e n o b t a i n e d from measurements o f the temperature d e p e n d e n c e s o f t h e e q u i l i b r i u m c o n s t a n t s for r e a c t i o n s o f m e t a l l o p r o t e i n r e d o x c o u p l e s w i t h i n o r g a n i c c o m p l e x e s ( 1 - 3 , 6 - 8 , 9, 14), from c a l o r i m e t r i c m e a s u r e m e n t s (4, 5 , 9,14), a n d f r o m i n d i r e c t c a l c u l a t i o n s ( 9 , 1 2 , 14). R e c e n t e x p e r i m e n t s s h o w (18-22) t h a t s p e c t r o e l e c t r o c h e m i c a l m e t h o d s e m p l o y i n g t h i n - l a y e r e l e c t r o l y s i s c e l l s (23-25) w i t h o p t i c a l l y t r a n s p a r e n t e l e c t r o d e m a t e r i a l s (26) e n a b l e t h e r a p i d a n d accurate determination o f metalloprotein reduction potentials ( E ° ) . T h e r e l a t i v e e a s e w i t h w h i c h s u e h E ° v a l u e s c a n b e o b t a i n e d [as c o m p a r e d to t h e m o r e c o m m o n l y e m p l o y e d c h e m i c a l ( J -9,12,14) a n d p o t e n t i o m e t r i c (27-30) p r o c e d u r e s ] m a k e s t h e s t u d y o f t h e t e m p e r a t u r e d e p e n d e n c e s o f m e t a l l o p r o t e i n E° v a l u e s a m u c h m o r e t r a c t a b l e problem. H o w e v e r , u n l i k e the classical e q u i l i b r i u m , kinetic, a n d calorimet ric experiments, studies o f the temperature dependences o f properties of electrochemical cells involve certain added complications. I n addi tion to the r e d o x half-reaction o f interest, one m u s t d e a l e x p e r i m e n t a l l y w i t h at l e a s t o n e o t h e r h a l f - c e l l r e a c t i o n t o c o m p l e t e t h e e l e c t r o c h e m i c a l c e l l . A s u i t a b l e reference e l e c t r o d e half-cell is u s u a l l y e m p l o y e d a n d , as a r e s u l t , p r o p e r t i e s o f t h e r e f e r e n c e e l e c t r o d e h a l f - r e a c t i o n a l s o can contribute to the observed temperature dependent properties o f the c o m p l e t e c e l l r e a c t i o n . A n a r r a n g e m e n t c o m m o n l y e m p l o y e d is d e s c r i b e d b y the f o l l o w i n g c e l l m +

Hg|Hg Cl (sat.), KCl(sat.)l|KCl(sat.)HM , 2

2

(m

n)+

M ~ \M^

T(varied) i n w h i c h a s a t u r a t e d c a l o m e l e l e c t r o d e ( S C E ) i s u s e d as t h e r e f e r e n c e half-cell, M and M ~ represent the o x i d i z e d a n d r e d u c e d halves of the redox c o u p l e o f interest, a n d M ' represents t h e w o r k i n g elec trode material. S u c h so-called "isothermal" cell arrangements, i n m+

(m

n)+

Kadish; Electrochemical and Spectrochemical Studies of Biological Redox Components Advances in Chemistry; American Chemical Society: Washington, DC, 1982.

3. TANG IUCHI ET AL.Temperature and Reduction Potentials

53

w h i c h t h e t e m p e r a t u r e o f t h e e n t i r e e l e c t r o c h e m i c a l c e l l is v a r i e d , w e r e u s e d i n m o s t o f t h e e a r l y r e d o x t h e r m o d y n a m i c s t u d i e s (31-38) on i n o r g a n i c c o m p l e x e s i n a q u e o u s s o l u t i o n . The t e m p e r a t u r e d e p e n d e n c e o f the o v e r a l l c e l l p o t e n t i a l i n s u c h isothermal cell experiments provides ground-state enthalpy ( Δ Η ° ) a n d e n t r o p y ( A S ° ) c h a n g e s for t h e c o m p l e t e c e l l r e a c t i o n (33-35, 37) M

m

+

+ (n/2) · H

2

=M

( m

"

n ) +

+

+η ·H

(1)


w h i c h n o w is r e f e r e n c e d to t h e s t a n d a r d h y d r o g e n gas e l e c t r o d e ( N H E ) , u s i n g the f o l l o w i n g expression for the temperature d e p e n d e n c e o f t h e S C E (35, 39) EICE(T)

= 0.2444 - 0.00066(T - 25)

(2)

w h e r e Τ is t e m p e r a t u r e i n degrees C e n t i g r a d e (20° < Τ < 35°) a n d E S C E ( T ) is t h e p o t e n t i a l i n v o l t s o f t h e S C E (vs. N H E ) at t e m p e r a t u r e T. F o r t h e n e t e n t r o p y c h a n g e for t h e c o m p l e t e c e l l r e a c t i o n A S ° = (S°

reu

- S° ) + ( n · S ° ox

H+

- n / 2 · S° ) H2

(3)

w h e r e t h e S ° terms represent p a r t i a l m o l a l entropies, t h e e n t r o p y dif ference d u e to t h e r e d o x h a l f - c e l l o f interest c a n b e separated from that due to the reference electrode half-cell b y t h i r d - l a w considerations (40). T h e p a r t i a l m o l a l e n t r o p y o f d i h y d r o g e n , H , i s 3 1 . 2 e u (41). W h e r e a s some p r e c e d e n t exists for a n " a b s o l u t e " e n t r o p y scale w i t h SH+ a p p r o x i m a t e l y e q u a l t o - 5 . 5 e u ( 3 5 , 3 7 , 4 2 , 4 3 ) , m o s t o f t h e c u r r e n t l i t e r a t u r e o n a q u e o u s i n o r g a n i c c o m p l e x e s (31 -37) a n d m e t a l l o p r o t e i n r e d o x c o u p l e s (2-4, 6, 7, 9, 14, 15) e m p l o y s t h e s o - c a l l e d " p r a c t i c a l " s c a l e (37,41,44), w h i c h assigns a v a l u e o f z e r o t o SH+. A d h e r i n g t o t h e practical entropy scale convention, rearrangement o f E q u a t i o n 3 i n d i cates t h a t for a o n e - e l e c t r o n r e d o x p r o c e s s (n = 1) 2

S?ed - S °

x

= A S ° + 15.6 e u

(4)

T h u s , the difference i n partial m o l a l entropies b e t w e e n the r e d u c e d a n d o x i d i z e d h a l v e s o f a o n e - e l e c t r o n r e d o x c o u p l e [often c a l l e d t h e " r e a c t i o n e n t r o p y " , AS° (43,45)] is e q u a l t o t h e n e t e n t r o p y c h a n g e for the c o m p l e t e c e l l reaction, A S ° , p l u s 15.6 e u i n a n isothermal c e l l experiment. C

A l t h o u g h isothermal e l e c t r o c h e m i c a l c e l l s are straightforward on a c o n c e p t u a l a n d t h e r m o d y n a m i c basis, certain p r a c t i c a l considerations m a k e t h e m u n s u i t a b l e for r o u t i n e u s e i n e x t e n s i v e i n v e s t i g a t i o n s . T h e difficulties e n c o u n t e r e d relate to t h e a m o u n t o f t i m e r e q u i r e d for sev eral o f the c o m m o n reference electrodes to reach true temperature


54

BO ILOGC IAL REDOX COMPONENTS

e q u i l i b r i u m . T h e a t t a i n m e n t o f t h e r m a l p h a s e e q u i l i b r i a (e.g., s o l i d v s . l i q u i d ) o f t e n is n e c e s s a r y i n c e r t a i n r e f e r e n c e

electrodes

[ S C E and

A g ( A g C I ) ] . I n the e a r l y i s o t h e r m a l c e l l e x p e r i m e n t s ( 3 3 - 3 5 , 37) sepa rate S C E s w e r e p r e p a r e d a n d e q u i l i b r a t e d to different t e m p e r a t u r e s for at l e a s t 2 d a y s b e f o r e u s e . A c e l l a r r a n g e m e n t o f the t y p e m+

(m

H g | H g C l ( s a t . ) , K C l ( s a t . ) [ | K C l ( s a t . ) ' | f C l ( s a t . ) | \M , 2


Τ ι( fixe d)

nn

f

M ~ \M

2

Τ (varied) 2

c a n b e u t i l i z e d effectively to c i r c u m v e n t the p r o b l e m s associated w i t h i s o t h e r m a l e l e c t r o c h e m i c a l c e l l s (45). I n s u c h a n o n i s o t h e r m a l c e l l a r r a n g e m e n t , o n l y t h e t e m p e r a t u r e o f t h e r e d o x h a l f - c e l l o f i n t e r e s t is v a r i e d . T h e t e m p e r a t u r e o f t h e r e f e r e n c e e l e c t r o d e h a l f - c e l l is m a i n t a i n e d at s o m e fixed, c o n s t a n t t e m p e r a t u r e . A s d e s c r i b e d p r e v i o u s l y (45), i f t h e t e m p e r a t u r e c o e f f i c i e n t s o f c e r t a i n t h e r m a l j u n c t i o n p o t e n t i a l s c a n b e m a d e e i t h e r n e g l i g i b l e o r c o n s t a n t r e l a t i v e to t h e o v e r a l l t e m p e r a t u r e c o e f f i c i e n t o f t h e n o n i s o t h e r m a l c e l l (dE°/dT) reaction e n t r o p i e s for r e d o x c o u p l e s c a n b e d e t e r m i n e d d i r e c t l y f r o m nonisothermal cell measurements 9

AS?

C

= S?ed - S°

ox

= nF(dE°/dT)

(5)

w h e r e F is t h e F a r a d a y c o n s t a n t a n d η is t h e n u m b e r o f e l e c t r o n s trans ferred. S u c h

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

from isothermal e x p e r i m e n t s o n l y w h e n it can b e demonstrated that the v a r i o u s t h e r m a l j u n c t i o n potentials d o not c o n t r i b u t e s i g n i f i c a n t l y to the temperature coefficient o f the nonisothermal c e l l potential. S u c h of

the

n o n i s o t h e r m a l AS? , b e c a u s e the t h e r m a l j u n c t i o n potentials are

considerations

do

not,

however,

affect

the

relative values

the

C

same i n e a c h case. T o s t u d y the t h e r m o d y n a m i c s o f m e t a l l o p r o t e i n e l e c t r o n

transfer

reactions, w e e m p l o y e d s p e c t r o e l e c t r o c h e m i c a l m e t h o d s u s i n g t h i n l a y e r e l e c t r o l y s i s c e l l s a n d o p t i c a l l y t r a n s p a r e n t e l e c t r o d e s ( O T E s ) to which

the

nonisothermal

electrochemical

cell

configuration

was

adapted.

Experimental Materials. Horse heart cytochrome c (type VI), from Sigma Chem ical Co., was purified on a CM-cellulose column according to published procedures (46) prior to use. Ruthenium(III) mediator-titrant: [Ru(NH ) py] (C10 ) was prepared as described previously (47). The sodium salt (indicator grade) of 2,6-dichloroindophenol (Aldrich Chemical Co.) was used with3

4

3


5

3. TANG IUCHI ET AL.Temperature and Reduction Potentials

out further purification. I o n i c strength 0.1 M , p H 7.0 s o d i u m phosphate buffer (1.97 x 1 0 " M N a H P 0 - H 0 , 2.68 x 10~ M N a H P 0 ) was pre pared from analytical grade reagents. A l l protein and buffer solutions were prepared u s i n g d e i o n i z e d water purified b y a Barnstead N A N O p u r e water purifier. G o l d electroformed mesh ( m i n i g r i d , 6 0 % transmittance) was used as the w o r k i n g electrode material i n the thin-layer cells ( B u c k b e e - M e a r s C o . ) . Teflon tape spacer ( D i l e c t r i c Corp.) was 0.1 m m thick. E p o x i - P a t c h epoxy (Dexter Corp.) was used to seal the w o r k i n g thin-layer c e l l compartment. R o s i n Solder C r e a m soft solder, A r c h e r brand from R a d i o Shack, was used to make the external electrical contact to the m i n i g r i d electrode. O p t i c a l w i n dows were o f h i g h quality quartz (0.15-cm thickness, transparency to 170 nm). Apparatus. A n a e r o b i c optically transparent thin-layer electrolysis ( O T T L E ) cells were s i m i l a r i n design to cells already d e s c r i b e d (21). T h e w o r k i n g O T T L E c e l l compartment ( 2 x 2 cm) was supported i n a L u c i t e or K e l - F (Teflon) c e l l b o d y (1.2 x 2.5 x 7.0 cm) i n the manner d e s c r i b e d i n Reference 21. O p t i c a l path lengths v a r i e d from 0.1 to 0.4 m m (with Teflon tape spacers), w i t h two opposing m i n i g r i d s used i n the thicker cells. E l e c t r i c a l contact to the m i n i g r i d was through an 18-gauge copper w i r e soldered to a portion of the m i n i g r i d left exterior to the t h i n c e l l (21). W o r k i n g O T T L E c e l l compartments were sealed permanently a n d attached to the c e l l bodies u s i n g epoxy cement. C e l l bodies were d e s i g n e d to a l l o w the w o r k i n g c e l l c o m partments to be r e b u i l t s i m p l y b y cutting away the epoxy sealant w i t h a sharp scalpel. 2

2

2


55

4

2

2

4

T w o Τ 7/25 inner tapers were m a c h i n e d lengthwise into the c e l l bodies and connected to the thin-layer cavity through two 2 x 7 m m filling ports. O u r nonisothermal salt b r i d g e consisted of a 5 m m x 25 c m Pyrex tube filled w i t h deoxygenated saturated potassium c h l o r i d e solution. T h e bottom e n d was ter minated i n a Τ 7/25 outer g r o u n d glass j o i n t w i t h a p l a t i n u m j u n c t i o n and a S a r g e n t - W e l c h c a l o m e l reference electrode (miniature, p l a t i n u m j u n c t i o n , # S - 3 0 0 8 0 - 1 7 ) resided at the top. A p l a t i n u m w i r e auxiliary electrode was sealed into a tube of soft glass w i t h a Τ 5/20 outer ground glass joint. T h i s apparatus was supported i n a compartment consisting o f a Τ 5/20 inner g r o u n d glass joint on top of a Τ 7/25 outer g r o u n d glass joint terminated b y a (fine) porous glass frit. T h i s compartment contained deoxygenated supporting elec trolyte a n d served to isolate the auxiliary electrode from the protein solution. M e t h o d s . Protein solutions were deoxygenated prior to use b y gentle vacuum/argon c y c l i n g on a vacuum/purified argon d o u b l e m a n i f o l d . Protein solutions were loaded into the O T T L E cells under anaerobic conditions u s i n g either rubber septum caps a n d syringe techniques or w i t h i n a V a c u u m A t m o spheres C o . H E - 4 3 - 2 D r i - L a b plus H E - 4 9 3 D r i - T r a i n inert atmosphere box. T h e reference and auxiliary electrode apparatus was sealed into the appropri ate O T T L E c e l l b o d y compartments w i t h A p i e z o n Η grease. T h e fully l o a d e d O T T L E cells had a dead solution v o l u m e of about 0.7 m L . T h e actual v o l u m e s of the thin-layer cavities v a r i e d from about 30 to 90 μL·. Formal reduction potentials at different temperatures for the metalloproteins were d e t e r m i n e d u s i n g the O T T L E cells i n a nonisothermal elec trochemical c e l l configuration. Potentials were a p p l i e d across the thin-layer cells w i t h a Princeton A p p l i e d Research M o d e l 174A polarographic analyzer and were measured accurately w i t h a K e i t h l y 177 m i c r o v o l t D M M digital multimeter. C e l l temperatures were v a r i e d u s i n g a s p e c i a l l y constructed vari able temperature c e l l holder a n d constant temperature water bath a n d mea-



56


siired directly w i t h an O m e g a E n g i n e e r i n g , Inc. precision m i c r o thermocouple (plus F l u k e 2175A D i g i t a l Thermometer, ±0.2°) situated i n the protein solu tion i n close p r o x i m i t y to the thin-layer cavity. T h e S C E reference electrode was separated from the thermostated c e l l b y the nonisothermal salt b r i d g e a n d m a i n t a i n e d at ambient room temperature. A l l U V - v i s i b l e spectra were ob t a i n e d w i t h a C a r y 219 r e c o r d i n g spectrophotometer. Formal reduction potentials were d e t e r m i n e d b y sequentially a p p l y i n g a series o f potentials, Ε (applied), across the thin-layer c e l l . E a c h potential was m a i n t a i n e d u n t i l electrolysis ceased so that the e q u i l i b r i u m value of the ratio of concentrations o f o x i d i z e d to r e d u c e d forms o f a l l redox couples i n solution, [ 0 ] / [ R ] , was established as defined b y the Nernst equation. C o m p l e t e elec trolysis occurred r a p i d l y as a result o f the short diffusional path length created b y the t h i n solution layer. A " s m a l l m o l e c u l e , " e l e c t r o c h e m i c a l l y reversible redox c o u p l e (mediator-titrant) w i t h an E° close to that of the metalloprotein was i n c l u d e d to facilitate electron transfer between the metalloprotein redox c o u p l e and the w o r k i n g electrode (48, 49). Redox couples were converted i n increments from one oxidation state to the other b y the series of a p p l i e d poten tials, for w h i c h each value of [ 0 ] / [ R ] was d e t e r m i n e d from the corresponding overlay spectra. F o r m a l reduction potentials a n d η - v a l u e s were d e t e r m i n e d from plots of E ( a p p l i e d ) vs. l o g [ 0 ] / [ R ] . A t least seven data points were i n c l u d e d i n each Nernst plot.

Calibration of the Nonisothermal OTTLE Cells A n e a r l i e r p u b l i c a t i o n (15) d e s c r i b e d t h e e m p i r i c a l a p p r o a c h u s e d to c a l i b r a t e o u r n o n i s o t h e r m a l e l e c t r o c h e m i c a l c e l l s . T h e r e d o x t h e r m o d y n a m i c s o f the F e ( p h e n ) i and Co(phen)i couples (where phen represents 1,10-phenanthroline) were d e t e r m i n e d u s i n g o u r apparatus a n d the results w e r e c o m p a r e d w i t h p u b l i s h e d values from isothermal a n d nonisothermal experiments. + / 2 +

+ / 2 +

F i g u r e s 1 a n d 2 i l l u s t r a t e results that are t y p i c a l o f the data o b t a i n e d for t h e F e ( p h e n ) | and Co(phen)i couples, respectively, u s i n g thin-layer spectroelectrochemistry. T h e results o b t a i n e d w h e n s u c h e x p e r i m e n t s are p e r f o r m e d as a f u n c t i o n o f t e m p e r a t u r e a r e p r e s e n t e d i n T a b l e s I a n d I I a n d i l l u s t r a t e d i n F i g u r e s 3 a n d 4. T h e t e m p e r a t u r e coefficients o f the nonisothermal c e l l potentials, dE°/dT, w e r e d e t e r m i n e d f r o m t h e s l o p e o f a l i n e a r l e a s t s q u a r e s fit to t h e E ° v s . t e m p e r a t u r e d a t a . B e c a u s e w e w e r e t e s t i n g a n o n i s o t h e r m a l c e l l ar rangement, reaction entropies (AS? ) w e r e c a l c u l a t e d d i r e c t l y from E q u a t i o n 5. C o m p l e t e c e l l e n t r o p i e s a d j u s t e d to t h e N H E s c a l e ( A S ° ) w e r e d e t e r m i n e d f r o m E q u a t i o n 4. S t a n d a r d f r e e - e n e r g y c h a n g e s for t h e c o m p l e t e c e l l r e a c t i o n w e r e c a l c u l a t e d f r o m t h e E° v a l u e s ( V v s . N H E ) at 2 5 ° C a n d t h e s t a n d a r d e n t h a l p y c h a n g e s ( Δ Η ° ) w e r e d e t e r m i n e d from the c o r r e s p o n d i n g A G ° a n d A S ° values. + / 2 +

+ / 2 +

C

Table III summarizes our entropy results (15) for the Fe(phen)i and Co(phen)i couples along w i t h p u b l i s h e d values from isothermal and nonisothermal experiments. E x c e l l e n t agreement is o b t a i n e d for t h e e n t r o p i e s for F e ( p h e n ) | from the nonisothermal + / 2 +

+ / 2 +

+ / 2 +



3.

TANiGUCHi ET AL.

Temperature

400

and Reduction

Potentials

57

500 WAVELENGTH (nm)

600

3

12

Figure la. Thin-layer spectroelectrochemistry of Fe(phen) }* * (sulfate salt), 0.4 mM, pH 2.33 (HN0 ); ionic strength, 0.1 Μ (NaN0 ), 14.4°C. Overlay spectra at different values of the applied potential, E , in V vs. NHE. 3

3

appl

ι—ι

•

1

1—I

1

1.134I.II4UJ X i.094Ζ 1.074> 1.054-

> 1.0341.014c I

I -1.0

Figure

j -0.5

lb.

I 0.0 LOG [ 0 ] / [ R ]

ι +0.5

Nernst plot of the data in Figure

I I +1.0

la.

O T T L E e x p e r i m e n t s a n d the isothermal p o t e n t i o m e t r i c e x p e r i m e n t s that w e r e p e r f o r m e d u n d e r i d e n t i c a l conditions o f p H a n d i o n i c s t r e n g t h . F u r t h e r m o r e , t h e e n t r o p i e s for b o t h F e ( p h e n ) i and Co(phen)i from nonisothermal O T T L E a n d c y c l i c v o l t a m m e t r y ex p e r i m e n t s a n d i s o t h e r m a l p o t e n t i o m e t r i c e x p e r i m e n t s are a l l i n satis+ / 2 +

+ / 2 +



58


240

260

280

WAVELENGTH (nm)

300

12

Figure 2a. Thin-layer spectre-electrochemistry of Co(phenf * * (perchlorate salt), 0.2 mM; ionic strength, 0.1 M, pH 7 sodium phosphate buffer, 22.2°C. Overlay spectra at different values of E | , in mV vs. NHE. 3

W P

factory a g r e e m e n t , p a r t i c u l a r l y i f r e a s o n a b l e a l l o w a n c e s a r e m a d e for the differences i n e x p e r i m e n t a l conditions. T h e agreement b e t w e e n the AS° values determined from isothermal a n d nonisothermal experiments indicates that the tempera ture coefficients o f the various t h e r m a l j u n c t i o n potentials i n o u r n o n i s o t h e r m a l O T T L E c e l l s d o n o t c o n t r i b u t e s i g n i f i c a n t l y to t h e o b s e r v e d t e m p e r a t u r e coefficient o f the o v e r a l l c e l l p o t e n t i a l . T h e r e f o r e , E q u a t i o n 5 c a n b e u s e d to d e t e r m i n e A S ? d i r e c t l y for r e d o x c o u p l e s C

C

t h a t are s u i t a b l e for s t u d y w i t h o u r n o n i s o t h e r m a l O T T L E c e l l s . R e s u l t s for t h e c o r r e s p o n d i n g r e d u c t i o n p o t e n t i a l s , free e n e r g i e s , a n d e n t h a l p i e s are g i v e n i n T a b l e I V .

Redox Thermodynamics for Horse Heart Cytochrome c T h e t h i n - l a y e r s p e c t r o e l e c t r o c h e m i c a l r e s u l t s o b t a i n e d for h o r s e h e a r t c y t o c h r o m e c a r e d i s p l a y e d i n F i g u r e 5. A n y d e s i r e d r a t i o o f


TANiGUCHi


3.

ET AL.

59

Temperature and Reduction Potentials

-0.5

0.0

+0.5

LOG [0]/[R] Figure 2b.

Nernst plot of the data in Figure 2a.

T a b l e I. Temperature D e p e n d e n c e o f the F o r m a l R e d u c t i o n P o t e n t i a l , E ° , for F e ( p h e n ) | Using Nonisothermal T h i n - L a y e r Spectroelectrochemistry + / 2 +

o0

RT/F°

C O

( V vs. NHE)

(mV)

5.2 9.8 11.2 14.4 15.1 15.7 20.9

1.077 1.078 1.074 1.078 1.075 1.074 1.072

55 58 58 59 61 59 60

E

0.998 0.999 0.998 0.999 0.999 0.998 0.999

+/2+

Note: Fe(phen)§ (sulfate salt), 0.4 mM, pH 2.33 (HN0 ); ionic strength, 0.1 M (NaN0 ). ±0.2°. ±0.002 V. Experimental Nernst slope at temperature T. Linear correlation coefficient. 3

3

a

b

c

d

o x i d i z e d to r e d u c e d p r o t e i n , [ 0 ] / [ B ] , w a s e s t a b l i s h e d e a s i l y a n d m a i n tained merely b y adjusting the overall solution potential v i a the potentiostat. E l e c t r o l y s i s t o e q u i l i b r i u m w a s a c c e l e r a t e d b y t h e a d d i t i o n o f suitable redox mediators. Both R u ( N H ) p y (where p y represents p y r i d i n e ) a n d 2 , 6 - d i c h l o r o i n d o p h e n o l w e r e u s e d to ascertain the i n d e p e n d e n c e o f t h e p r o t e i n E° v a l u e s o n t h e p a r t i c u l a r m e d i a t o r - t i t r a n t e m p l o y e d . A region o f the cytochrome c absorption spectrum i n w h i c h the mediators s h o w e d n e g l i g i b l e absorption i n b o t h r e d u c e d a n d o x i d i z e d states w a s m o n i t o r e d . 3 + / 2 +

3

5


60

BO ILOGC IAL REDOX COMPONENTS T a b l e I I . T e m p e r a t u r e D e p e n d e n c e o f the F o r m a l R e d u c t i o n P o t e n t i a l , E ° , for C o ( p h e n ) § Using Nonisothermal Thin-Layer Spectroelectrochemistry + / 2 +


RT/F (°C)

(mV vs. NHE)

(mV)

8.8 9.6 13.3 13.5 16.9 18.6 18.9 22.2 25.3 28.0

361 365 368 368 372 374 368 374 377 378

56 55 57 58 56 54 56 57 57 64

C

1.000 0.999 0.999 0.998 0.999 0.999 1.000 1.000 0.999 1.000

+/2+

Note: Co(phen)i (perchlorate salt), 0.2 mM; ionic strength, 0.1 M, pH 7 sodium phosphate buffer. ±0.2°. ± 2 mV. Experimental Nernst slope at temperature T. Linear correlation coefficient. α b c

d

I.090h

ω,

.080

> > l.070h

l.060h

10

15

20

TEMPERATURE °C +I2+

Figure 3. Temperature dependence of E ° for Fe(phenf using nonisothermal thin-layer spectroelectrochemistry where E ° (25°) = 1.073 V (NHE) and the slope = -1.90 x 10~ V/°C. 3

4

H o r s e heart c y t o c h r o m e c e x h i b i t e d reversible, one-electron N e r n s t i a n b e h a v i o r i n t h e s e e x p e r i m e n t s . E l e c t r o l y s i s to e q u i l i b r i u m o c c u r r e d s i g n i f i c a n t l y faster w h e n c h r o m a t o g r a p h i c a l l y p u r e (46) S i g m a t y p e V I h o r s e h e a r t c y t o c h r o m e c w a s e m p l o y e d (cf. t h e u p p e r r i g h t i n s e t i n F i g u r e 5). I n a d d i t i o n , t h e e x p e r i m e n t a l l y d e t e r m i n e d


Temperature

and Reduction

3. TANG IUCHI ET AL.


τ

1

1

61

Potentials 1

1

Γ

TEMPERATURE °C +l2+

Figure 4. Temperature dependence of E ° for Co(phen)3 using nonisothermal thin-layer spectroelectrochemistry where E ° (25°) = 377 mV (NHE) and the slope = +7.92 x 10~ V/°C. 4

t h e r m o d y n a m i c p a r a m e t e r s ( d i s c u s s e d later) w e r e s l i g h t l y d i f f e r e n t f r o m t h o s e o b t a i n e d for t h e n o n p u r i f i e d S i g m a t y p e V I p r o t e i n (15). R e s u l t s for t h e t e m p e r a t u r e d e p e n d e n c e o f t h e c y t o c h r o m e c for m a l r e d u c t i o n p o t e n t i a l are p r e s e n t e d i n T a b l e V a n d i l l u s t r a t e d i n F i g u r e 6 [E° ( 2 5 ° C ) = 2 6 0 ± 2 m V ( N H E ) a n d dE°/dT = - 5 . 6 1 ± 0.5 x 1 0 " V / ° C for the g i v e n p H a n d s u p p o r t i n g e l e c t r o l y t e c o n d i t i o n s ] . T h e r m o d y n a m i c p a r a m e t e r s are as f o l l o w s : A S ° , - 2 8 . 5 ± 1.2 e u ; A S ? , - 1 2 . 9 ± 1.2 e u ; A G ° , - 6 . 0 0 ± 0 . 0 5 k c a l / m o l ; a n d ΔΗ°, - 1 4 . 5 ± 0.4 k c a l / m o l . T h e s e v a l u e s are i n e x c e l l e n t a g r e e m e n t w i t h p u b l i s h e d p a r a m e t e r s ( 6 , 7) for h o r s e h e a r t c y t o c h r o m e c ( p H 7, μ = 0.1 M ) : A S ° , - 2 8 ± 5 eu; AS? , - 1 3 ± 5 eu; AG°, - 6 . 0 kcal/mol; and Δ Η ° , - 1 4 . 5 ± 1.5 k c a l / m o l . 4

C

C

Reaction Entropies for Other Cytochromes c T h e t e m p e r a t u r e d e p e n d e n c e s o f the f o r m a l r e d u c t i o n p o t e n t i a l s o f s e v e r a l o t h e r c - t y p e c y t o c h r o m e s w e r e s t u d i e d (51). E v e n at t h i s e a r l y stage i n o u r w o r k , c e r t a i n i n t e r e s t i n g c o m p a r i s o n s c a n b e m a d e . A l l the c y t o c h r o m e s e x h i b i t either essentially zero or s l i g h t l y n e g a t i v e e l e c t r o n transfer r e a c t i o n e n t r o p i e s . T h e s e A S ? v a l u e s d o n o t c o r r e l a t e w i t h t h e o v e r a l l c h a r g e o n t h e p r o t e i n (cf. T a b l e V I ) , as f o u n d w i t h s i m p l e i n o r g a n i c c o m p l e x e s o f i r o n [e.g., c o n s i d e r t h e f o l l o w i n g A S ? values: F e ( C N ) - ~ , - 4 9 e u (35); F e ( H 0 ) ? , + 4 3 e u (45)]. I n d e e d , e v i d e n c e (7) i n d i c a t e s t h a t t h e r e a c t i o n e n t r o p i e s r e f l e c t r a t h e r s m a l l C

C

3

/ 4

6

+ / 2 +

2



Co(phen)i

c

6

12

22 ± 3

18.3 ± 3

6.4 ± 4

2.7 ± 3 C

-4.4 ± 2°

-20.0 ± 2

-3.6

3 ± 2

-12.6 ± 2

(eu)

Kd

( = S°

-5.2 ± 2

C

-20.8 ± 2

(eu)

Values of A S ° and A S ?

3

3

3

p h o s p h a t e buffer μ = 0.1 M p H 7.0

0.05 M K C 1 + 25 m M phen

Μ = 0.01 M

3

3

4.5 x Ι Ο " M H N O 3 μ = 0.1 M ( N a N 0 )

0.05 M K C 1 + 25 m M phen

4.5 x 1 0 " M H N 0 M = 0.1 M ( N a N 0 )

Electrolyte

m

- S° ) for the F e ( p h e n ) i

From E° vs.temperature data collected in the range 5-16°C. This work. From E° vs. temperature data collected in the range 7-30°C.

+ , 2 +

Fe(phen)i

a

+ / 2 +

Couple

Table III.

+/2+

+

2 +

b

45

50

b

45

34

Reference

Redox Couples

isothermal potentiometric Pt electrode nonisothermal cyclic voltammetry Pt electrode nonisothermal OTTLE A u electrode isothermal potentiometric Pt electrode nonisothermal cyclic voltammetry Pt electrode nonisothermal OTTLE A u electrode

Method

and C o ( p h e n ) § '



+

+ / 2 +

0.377 (±0.002)

0.387

0.399

1.073° (±0.002)

1.114

1.099

E ° (25°C) (V vs. NHE)

- 8 . 7 ± 0.1

-8.9

-9.2

- 2 4 . 7 ± 0.1

-25.7

- 2 5 . 3 ± 0.1

AG° (kcal/mol)

- 9 . 5 ± 0.3

-9.2

-10.3

- 3 0 . 8 ± 1.3

-29.3

- 3 1 . 5 ± 0.5

ΔΗ° (kcal/mol)

Additional Thermodynamic Parameters for the F e ( p h e n ) |

+/2+

b

a

Note: Supporting electrolytes are the same as those given in Table III. The value for £ ° at 25°C was extrapolated from the experimentally determined dE°ldT. This work.

Co(phen)i

Fe(phen)i

Couple

Table IV.

+

2+

45

50

45

34

Reference

Redox Couples

isothermal potentiometric Pt electrode nonisothermal cyclic voltammetry Pt electrode nonisothermal OTTLE Au e l e c t r o d e isothermal potentiometric Pt electrode nonisothermal cyclic voltammetry Pt electrode nonisothermal OTTLE Au e l e c t r o d e

Method

and C o ( p h e n ) i '


GO

O

fi» o"

2

3

Ω S w H > r

c

52 g Ο


Figure 5a. Thin-layer spectroelectrochemistry of horse heart cyto chrome c, 0.13 mM; ionic strength, 0.1 M, pH 7 sodium phosphate buffer, 25.0°C; [Ru(NH ) py(Cl0 ) ], 0.65 mM. Overlay spectra and absorbance changes at 550 nm as a function of time at different values of the applied potential, E , in mV vs. NHE. 3

5

4

3

appl

c h a n g e s i n p r o t e i n s t r u c t u r e i n t h e v i c i n i t y o f t h e h e m e c sites. O n e p o s s i b i l i t y is t h a t t h e s e s m a l l s t r u c t u r a l c h a n g e s a r e r e l a t e d t o t h e degree o f e x p o s u r e o f the h e m e c g r o u p to water m o l e c u l e s , b e c a u s e t h e c ' p r o t e i n s , w h o s e sites a r e e x p o s e d t o a d e g r e e , e x h i b i t m o r e p o s i t i v e Δ S ? - v a l u e s . W e a s s o c i a t e s o l v e n t e x p o s u r e w i t h less t i g h t e n i n g o f the p r o t e i n framework structure o n r e d u c t i o n ; i n other words, less p r o t e i n c o n f o r m a t i o n a l c h a n g e is n e e d e d t o a c c o m m o d a t e t h e i n sertion o f an e l e c t r o n i f the h e m e c g r o u p i n q u e s t i o n c a n interact w i t h water molecules. c

S u c h a s i m p l e interpretation cannot a c c o u n t for a l l redox m e t a l loprotein reaction entropies. Proteins w i t h relatively exposed heme c g r o u p s e x h i b i t m o r e n e g a t i v e AS° -values than can b e accommodated s a t i s f a c t o r i l y b y o u r c r u d e m o d e l (e.g., c y t o c h r o m e c i f r o m Fseudomonas aeruginosa). M o r e r e s e a r c h w i l l b e r e q u i r e d b e f o r e t h e t h e r m o d y n a m i c s o f s i n g l e r e d o x center m e t a l l o p r o t e i n e l e c t r o n trans fer r e a c t i o n s c a n b e u n d e r s t o o d i n a q u a n t i t a t i v e s e n s e . c

5 5


3.

TANiGucHi ET AL.


ι

Temperature and Reduction Potentials

1

1

1

1

r —

65 ι

LOG [0]/[R] Figure

5b.

Nernst plot of the data in Figure

5a.

Table V. Temperature Dependence of the Formal Reduction Potential, JE°, for Horse Heart Cytochrome c Using Nonisothermal Thin-Layer Spectroelectrochemistry E

(°C)

8.6 9.2 15.5 16.8 19.8 20.2 24.4 29.8 30.6 35.2 39.4

o0

RT/F

C

(mV vs. NHE)

(tnV)

269 270 267 265 262 263 262 257 258. 254 253

56 56 57 58 56 58 59 60 59 61 59

e

2

Spectroelectrochemical Determination of the Temperature

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