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Electrochemical Studies of Biological Systems Downloaded from pubs.acs.org by HONG KONG UNIV SCIENCE TECHLGY on 09/16/18. For personal use only.
Bioelectrochemical Modelling of Cytochrome c CHARLES C. Y. TING and JOSEPH JORDAN 152 Davey Laboratory, Department of Chemistry, Pennsylvania State University, University Park, Penn. 16802 MAURICE GROSS Laboratoire d'Electrochimie et Chimie-Physique du Corps Solide, Université Louis Pasteur, BP 296 R/8, 67008 Strasbroug, France Numerous papers have in recent years been devoted to electrochemical studies of porphyrins and metalloporphyrins. Paradoxically, an investigation of heme c (the prosthetic group of cytochrome c) is conspicuous by its absence. Heme c was first prepared from the naturally occurring protein in a classical piece of work by Theorell (1). Subsequently both heme c and its equatorial ligand (porphyrin c) became accessible by the synthetic route (2,3) outlined in Figure 1. The salient feature of porphyrin c is the bis-cysteinated substitution on the ring, which is unique in cytochrome c among hemoproteins (4). The corresponding substituents in hemoglobin and myoglobin are vinyl groups (5,6). In the present paper, we report some preliminary findings on the electrochemical behavior of porphyrin c and heme c. Experimental Materials. Porphyrin c and heme c were synthesized ad hoc using procedures referred to earlier in this write-up. Yields and elemental analyses are summarized in Table I. Table I Compound
Mol. % 1 Wt. Yield
Porphyrin c 805.0 40 Heme c 917.8 90
% Theory 0 6.08
Elemental Analysis Fe %S % Ν Actual Theory Actual Theory Actual 8.05 10.44 10.10 0.22 7.97 8.94 9.16 6.05 6.99 6.81
Authenticity was verified with the aid of the spectra illustrated in Figure 2 recorded with the aid of a Bausch and Lomb 505 Spectro photometer and quartz cells whose optical pathlength was 1 and 0.1 cm. P.C. Polarography. Current-voltage curves were recorded at a conventional (dropping mercury electrode (dme) which had 26
2.
TING E T A L .
Bioelectrochemical
the f o l l o w i n g c h a r a c t e r i s t i c s : c i r c u i t ) = 4.55 s e c o n d s .
Modelling
m
=
1.66
of Cytochrome
c
27
mg p e r s e c o n d ; t ( o p e n
C y c l i c V o l t a m m e t r y . K e m u l a ' s h a n g i n g chrop m e r c u r y e l e c t r o d e (hdme) s e r v e d a s i n d i c a t o r e l e c t r o d e . P o t e n t i a l s c a n r a t e s i n a r a n g e b e t w e e n 0.01 and 5 0 v o l t * s e c " w e r e u s e d . 1
Coulometry. Current-time i n t e g r a l s were determined a t appro p r i a t e c o n t r o l l e d p o t e n t i a l s , corresponding to well-defined polar o g r a p h i c d i f f u s i o n c u r r e n t d o m a i n s . The c a t h o d e was a m e r c u r y p o o l o f 2.60 s q . cm. Instrumentation, Solvents, Supporting E l e c t r o l y t e s , etc. A l l e x p e r i m e n t s w e r e c a r r i e d o u t a t 25°C. On s o l u b i l i t y c o n s i d e r a t i o n s DMF and w a t e r w e r e u s e d a s s o l v e n t s f o r heme c and p o r p h y r i n c r e s p e c t i v e l y . 0.1 M p e r c h l o r i c a c i d was t h e s u p p o r t i n g e l e c t r o l y t e i n a l l experiments. Three-electrode systems were employed t h r o u g h o u t , u s i n g a s a t u r a t e d a q u e o u s £alomel r e f e r e n c e e l e c t r o d e (SCE) a n d a p l a t i n u m f o i l a u x i l i a r y c o u n t e r e l e c t r o d e . A l l e l e c t r o c h e m i c a l measurements were performed w i t h the a i d o f a m u l t i purpose instrument equipped w i t h advanced s o l i d s t a t e o p e r a t i o n a l a m p l i f i e r a n d f e e d b a c k c i r c u i t s , v i z , t h e M o d e l 170 E l e c t r o c h e m i c a l S y s t e m s u p p l i e d by £ r i n c e t o n A p p l i e d R e s e a r c h (PAR) C o r p o r a t i o n , P r i n c e t o n , N.J. O u t p u t s i g n a l s were a u t o m a t i c a l l y c o r r e c t e d f o r i R d r o p s a n d r e c o r d e d o n a b u i l t - i n X-Y pen r e c o r d e r a n d / o r w i t h t h e a i d o f a d i g i t a l o s c i l l o s c o p e ( M o d e l 1090 w i t h M o d e l 9 0 p l u g - i n u n i t , N i c o l e t Instrument Corporation, Madison, Wisconsin). The s c o p e was e q u i p p e d w i t h a mi n i - c o m p u t e r w h i c h had c a p a b i l i t i e s o f s t o r i n g d a t a i n a 4 0 9 6 χ 4 0 9 6 a r r a y memory a s f a s t as 5 y s e c p e r d a t a p o i n t . The c o u p l i n g o f t h e PAR i n s t r u m e n t w i t h t h e o s c i l l o s c o p e a l l o w e d measurement o f f a s t l i n e a r sweep voltammograms w i t h an a c c u r a c y o f 1 p e r c e n t . Whenever a p p r o p r i a t e , t o t a l c u r r e n t s were c o r r e c t e d f o r r e s i d ual c u r r e n t s to y i e l d the corresponding f a r a d a i c c u r r e n t s . Poten t i a l s are r e p o r t e d i n accordance w i t h the Stockholm Sign Conven t i o n o f t h e I n t e r n a t i o n a l U n i o n o f P u r e and A p p l i e d C h e m i s t r y ( 7 ) , i . e . , t h e more c a t h o d i c ( r e d u c i n g ) a p o t e n t i a l t h e more n e g a t i v e i t s assignment. R e s u l t s and
Discussion
Experimental 1.
f i n d i n g s are summaried below.
E l e c t r o a n a l y t i c a l Chemistry of Porphyrin c. D.C. p o l a r o g r a m s o f p o r p h y r i n c y i e l d e d two c a t h o d i c w a v e s w i t h w e l l - d e f i n e d l i m i t i n g c u r r e n t s whose c h a r a c t e r i s t i c s a r e l i s t e d i n Table I I .
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ELECTROCHEMICAL STUDIES O F BIOLOGICAL SYSTEMS
Table I I Half-Wave Limiting Current Potential P o t e n t i a l Domain J idt (faradays-mole ) ( v o l t vs. SCE) (volt) - 1
-0.525 -0.730
-0.56 -0.88
2.02 ± 0.04 3.94 ± 0.02
t o -0.62 t o -1.0
Wave A n a l y s i s Slope (volt)
7
0.030 0.054
* S l o p e o f p l o t o f l o g [ ( i - i ) / i ] v e r s u s E. H
2. 3.
4.
Coulometry a t -0.615 v o l t v e r s u s SCE ( o n t h e f i r s t l i m i t i n g current plateau) substantiated a two-electron transfer, i . e . , two f a r a d a y s p e r m o l e o f p o r p h y r i n c . C o u l o m e t r y a t -0.945 v o l t v e r s u s SCE ( o n t h e second l i m i t i n g current plateau) i n d i c a t e d t h e occurrence o f an o v e r a l l f o u r e l e c t r o n t r a n s f e r , i . e . , a t o t a l o f f o u r f a r a d a y s p e r mole o f porphyrin c. C y c l i c V o l t a m m e t r y a t t h e hdme y i e l d e d t h e f o l l o w i n g r e s u l t s . ( a ) A t r e l a t i v e l y f a s t p o t e n t i a l s c a n r a t e s (10
