BIOCHIMIE, 1978, 60, 321-325.
EPR determination of the oxidation-reduction potentials of the hemes in cytochrome c3 from Desulfovibrio vulgaris. D. V. D E R V A R T A N I A N ,), A. V. X A V I E R b) a n d J. L E G A L L ¢).
~) Dept. o[ Biochemistry, University o[ Georgia, Athens, Georgia 30602 (U.S.A.) ; b) Centro da Quimica Estrutural da Universidade de Lisboa, I.S.T., Lisboa 1 (Portugal) ; c) Laboratoire de Chimie Bact~rienne, C.N.R.S., 1327~ Marseille Cedex 2 (France).
Rdsum6.
Summary.
La spectroscopie P,PE, en conjonction a v e c la potentiom6trie r6dox, a 6t6 utilis6e pour d6terminer les potentiels de demi-r6duction de chacun des quatre h ~ m e s du cytochrome Ca : c o m m e des travaux ant6rieurs le laissaient pr6voir, ces potentiels sont diff6rents. Les valeurs de EM sont les suivantes : h 6 m e I, - - 2 8 4 m V " h ~ m e II : - - 3 1 0 m V ~ h~me III : - - 3 2 4 m V et h~me IV : - - 3 1 9 mV. Dans c h a q u e cas, la valeur de n est proche de 1.
EPR s p e c t r o s c o p y in conjunction with oxidation-reduction potentiometry h a s b e e n u s e d to determine the half-reduction potentials of the four heroes of cytochrome c3. As predicted, the four heroes of cytochrome ca h a v e different mid-point potentials. The EM v a l u e s are : Heine L - - 2 8 4 raV : Heine IL - - 3 1 0 m V " Heine HI, - - 3 2 4 m V and Heine IV, - - 3 1 9 raV. The nv a l u e s in e a c h c a s e w a s near one.
Introduction.
resonances potentials.
C y t o c h r o m e c a f r o m D. vulgaris is a s m a l l m o l e c u l a r w e i g h t h e m o p r o t e i n (M r = 13 000) w h i c h c o n t a i n s f o u r h e m e s p e r m o l e c u l e , is a u t o x i d i z a b l e a n d h a s b e e n r e p o r t e d ¢o h a v e a n e g a t i v e o x i d a tion-reduction potential based on light-absorption s p e c t r o s c o p y [c[. ref. 1]. E P R a n d NMR s t u d i e s [1-5] o n t h e f u n c t i o n i n g of t h e f o u r h e r o e s d u r i n g reduction and reoxidation suggested that the four hemes might have four different oxidation-reduct i o n p o t e n t i a l s . E P R a n d N M R s t u d i e s [2-5] h a v e i m p l i c a t e d h i s t i d i n e as t h e p r o b a b l e 5 t h a n d 6 t h b i o l o g i c a l l i g a n d s of t h e h e m e i r o n . E P R s p e c t r o s c o p y h a s d e t e r m i n e d [1] f o u r d i s t i n c t sets of g - v a l u e s c o r r e s p o n d i n g to t h e f o u r h e m e m o i e t i e s located in different environments within the protein. It is t h e p u r p o s e of t h i s s t u d y to d e t e r m i n e t h e m i d - p o i n t p o t e n t i a l s of t h e f o u r l l e m e s b y E P R s p e c t r o s c o p y . It is r e p o r t e d i n t h i s s t u d y t h a t t h e f o u r h e r o e s h a v e n o t o n l y f o u r d i s t i n c t sets of E P R
but
also
four
unique
half-reduction
Methods. G r o w t h of D. vulyaris and purification of cytochrome c~ was carried out as previously reported [1]. EP'R spectroscopy was performed as previously described w i t h a V a r i a n model V-4501A spectrometer [1]. Liquidh e l i u m m e a s u r e m e n t s were made w i t h a n Air Products system LTD-3~110. Oxidation- r e d u c t i o n m e a s u r e m e n t s of cytochrome ca (3.3 m g / m l in 50 mM Tris buffer, pH 8.0) were p e r f o r m e d i n a system s i m i l a r to t h a t described b y D u t t o n [6], w i t h s i m i l a r oxidationreduction m e d i a t o r s a n d at s i m i l a r c o n c e n t r a t i o n s of mediators. The o x i d a t i o n - r e d u c t i o n potential was measured w i t h a F i s h e r Accumet Model 520 digital m e t e r and adjusted w i t h small a d d i t i o n s of sodium dithionite or p o t a s s i u m f e r r i c y a n i d e solutions. After a sample was e q u i l i b r a t e d for a p p r o x i m a t e l y 4 m i n at a set potential, it was rapidly frozen at 77°K for the s u b s e q u e n t EPR m e a s u r e m e n t s . E,PR spectra were directly i n s e r t e d via the Data General Corporation Nova 2 m i n i c o m p u t e r onto a magnetic disc system
D. V. DerVartanian, A. V. Xavier and J. Le Gall.
322
(Ball C o m p u t e r ) f o r a p e r m a n e n t r e c o r d a n d s u b s e q u e n t u s e o f e x p e r i m e n t a l d a t a . C o m p u t e r fits o f t h e experimental data were performed with a computer p r o g r a m [7] of t h e N e r n s t e q u a t i o n f o r d i r e c t d e t e r m i -
n a t i o n of t h e m i d - p o i n t p o t e n t i a l point values are ± 5 mV.
a n d n - v a l u e . Mid-
Results. g = 3,22296
2.48 2~34
2.04 I
F i g u r e 1 illustrates the E P R s p e c t r a of f e r r i c y t o c h r o m e c 3 f r o m D. vulgaris at four r e p r e s e n l a t i v e o x i d a t i o n - r e d u c t i o n potentials. As p r e v i o u s l y des40"
ItU[ i
J
20-
-3Pro
0° 140 °
BE
II
\
A 70-
[ia.-3|OllY " ~
-31711
\
0. 70"
I[lll[ Ill
\
3S-
\ -331 my
O°
70"
HEM[ IV
~x
3So.
H-- (GAUSS)
Fie. 1. - - EPR spectra at IO°K of c!ltochrome c~ f r o m D. v u l g a r i s at f o u r r e p r e s e n t a t i v e o x i d a t i o n - r e d u c t i o n potentials. D e t a i l s on r e c o r d i n g t h e E P R s p e c t r a o n t h e m a g n e t i c disc s y s t e m m a y b e f o u n d in t h e M e t h o d s section. The EPR poteutiometric titration procedures a r e a l s o in t h e M e t h o d s section. E P R c o n d i t i o n s : m i c r o w a v e f r e q u e n c y , 9.187 GHz ; m i c r o w a v e p o w e r , 10 m W ; t i m e c o n s t a n t , 0.1 sec ; s c a n n i n g r a t e , 1000 G per min. Samples were stored under anaerobic condit i o n s i n f r e q u e n c y - m a t c h e d E P R t u b e s of 4 m m o u t e r diameter. BIOCHIMIE, 1978, 60, n ° 3.
,, • -.,~.
O- I
o
-z~o
-z~o
-3;o
-~o
[h(mV)
Fro. 2. - - C o m p u t e r f i t s of n o r m a l i z e d signal ampliludes at y+ f o r h e m e s I-IV as a f u n c t i o n of m e a s u r e d o x i d a t i o n - r e d a c t i o n potentials (Eh). Eu a n d n - v a l u e s a r e l i s t e d i n t a b l e II.
Redox potentials of cytochrome cs.
323
c h r o m e c 3 w h i c h is f u l l y o x i d i z e d at - - 3 7 mV, 27 p e r c e n t r e d u c e d at - - 3 0 7 mV, 47 p e r c e n t red u c e d at - - 3 3 ' 1 m V a n d 73 p e r c e n t r e d u c e d at
c r i b e d [1] f e r r i c y t o c h r o m e c 3 exists in a l o w spin ferric heme configuration with four different sets of g - v a l u e s c o r r e s p o n d i n g to h e m e s I, II, I I I
TABLE I.
Degree o[ reduction of heroes of cytochromes c3 at some representative oxidation-reduction potentials. Heroes (g, "} Eh (blillivolts)
1
II
III
IV
- 37 ( ' " )
30 (0) (") 11 (63) 11 (63)
124 (0) 79 (36) 73 (41)
70 (0) 44 (37) 42 (40)
40 (47) 34 (55)
199 (0) 172 (14) 145 (27)
8 (73) 4 (83)
44 (65) 3 2 (74)
40 (43) 3 2 (54)
33 ( 5 6 ) 27 ( 6 4 )
106 (53) 80 (60)
2 (93) 0 (100)
20 (84) 12 (90)
21 (70) 16 (77)
22 (71) 14 (81)
54 (73) 31 (84)
- 292 - 307 (***) - 331 (***) - 343 - 362 (~**) - 386
75 (0)
{gy ")
(*) Intensities are normalized signal amplitudes. (**) Values given (in parentheses) are in percentage X 1{)0 Reduced, relative to the initial low-spin ferric normalized intensities. (***) EPR spectra at these oxidation-reduction potentials are shown in figure 1. TABLE II.
Summary o[ G-values, mid-poin! potentials and N-values [or the four heroes o[ cytochrome %. Heroes
gz value E~t n-value
I
II
III
IV
3.127 - 284 mV 1.1
2.971 - 310 mV 1.0
2. 821 - 324 mV 1.1
2.479 - 319 mV 1.2
a n d IV, i n d i c a t i n g t h e n o n - e q u i v a l e n c e of the f o u r l o w - s p i n h e i n e m o i e t i e s . D e t e r m i n a t i o n of t h e m i d p o i n t p o t e n t i a l s ~v.as c a r r i e d out b y m e a s u r i n g at each discrete oxidation-reduction potential the gz (*) s i g n a l a m p l i t u d e for h e r o e s I t h r o u g h IV a n d s u b s t r a c t i o n of the a p p r o p r i a t e b a s e - l i n e corr e c t i o n . T h i s is a v a l i d p r o c e d u r e s i n c e no ch, anges in s i g n a l s h a p e o r w i d t h w e r e o b s e r v e d in t h e gz s i g n a l i n t e n s i t i e s d u r i n g r e d u c t i o n - r e o x i d a t i o n c y c l i n g e i t h e r in t h e a b s e n c e o r p r e s e n c e of added redox mediators. B a s e d on m e a s u r e d a m p l i t u d e s at gy ( ' ) , t h e s p e c t r a p r e s e n t e d in F i g u r e 1 c o r r e s p o n d to c y t o (*) gz and g~ are defined as the end m a x i m u m and mid-point crossing, respectively, of the first derivative EPR absorption.
BIOCHIMIE, 1978, 60, n ° 3.
- - 362 mV. T h e p e r c e n t r e d u c t i o n of e a c h h e m e at t h e s e o x i d a t i o n - r e d u c t i o n p o t e n t i a l s is s h o w n in t a b l e I. T h e d e g r e e of r e d u c t i o n of t h e i n d i v i d u a l h e m e s of c y t o c h r o m e c 3 as s h o w n in t a b l e I i n d i c a t e s t h a t h e i n e I is p r e f e r e n t i a l l y r e d u c e d a n d as p r e v i o u s l y r e p o r t e d [1] is also t h e h e i n e w h i c h is t h e last h e m e to be r e o x i d i z e d . H e m e II is t h e n e x t c o m p o n e n t to be r e d u c e d a f t e r r e d u c t i o n of h e m e I. H e m e I I I is t h e last h e m e to be r e d u c e d w h i l e h e i n e IV is i n t e r m e d i a t e in reducti.on a n d reoxidation properties (.approximately between h e m e s I I a n d III). It is c l e a r t h a t t h e p e r c e n t a g e r e d u c t i o n as m e a s u r e d f r o m n o r m a l i z e d s i g n a l a m p l i t u d e s at gy d o e s n o t b e a r a c l e a r r e l a t i o n s h i p to the r e d u c t i o n states of H e m e s I-IV e v e n t h o u g h gy is a s u p e r i m p o s i t i o n of c o n t r i b u t i o n s f r o m all f o u r h e m e s . It is o n l y at - - 331 m V a n d m o r e nega-
324
D. V. DerVartanian,
A. V. Xavier
tive potentials that t h e r e is some c o r r e l a t i o n of the r e d u c t i o n state of Oy - w i l h those of heroes III and IV (see table I) but a p o o r c o r r e l a t i o n w i t h hemes I and II. In the range of - - 3 7 mV to - - 3 0 7 mV there is little c o r r e l a t i o n of the degree of r e d u c t i o n at ~y ,~ to any heme. F i g u r e 2 shows the c o m p u t e r fits for the Nernst equation a p p l i e d to the e x p e r i m e n t a l l y d e t e r m i ned a m p l i t u d e s of the g~ values for the four individual heroes as a f u n c t i o n of m e a s u r e d o x i d a t i o n r e d u c t i o n p o t e n t i a l in the range of 0 mV to - - 400 inV. The c o m p u t e r d e t e r m i n e d m i d - p o i n t potentials and n-values are listed in table H. The n-value for each h e m e system w e r e f o u n d to be on'e.
Discussion
of
Results.
The h a l f - r e d u c t i o n potentials d e t e r m i n e d for h e m e s I, H, III and IV i n d i c a t e that the four h e m e s w h i c h are detected by E P R s p e c t r o s c o p y definitely have four u n i q u e m i d - p o i n t o x i d a t i o n - r e d u c tion potentials. These E M values are consistent w i t h the o b s e r v e d r e d u c t i o n and r e o x i d a t i o n t~PR b e h a v i o r found for the four hemes. The m a x i m u m difference in m i d - p o i n t p o t e n t i a l is found b e t w e e n heroes I and III w i t h a value of 40 inV. The m i d - p o i n t potential values f o u n d in this study for the four heroes of c y l o c h r o m e c~ are not as large in potential d i f f e r e n c e as has been r e p o r t e d for the i r o n - s u l f u r centers of c o m p l e x r e s p i r a t o r y p r o t e i n s such as NADH d e h y d r o g e n a s e [8, 9] hut a p p e a r to be m o r e r e l a t e d in values to the m i d - p o i n t potentials for c y t o c h r o m e s b-561 and b-566 d e t e r m i n e d by E P R s p e c t r o s c o p y by Leigh and E r e c i n s k a [101. These latter w o r k e r s r e p o r t e d a d i f f e r e n c e in m i d - p o i n t p o t e n t i a l of 100 mV b e t w e e n c y t o c h r o m e b-561 (E M = 100 raM) and b-566 (E~ = 0 mV). The results found h e r e should be c o n t r a s t e d to s i m i l a r E P R p o t e n l i o m e t r i c titr,ations for cytoc h r o m e c~ f r o m D e s u l f o v i b r i o gigas [11]. In the case of that c y t o c h r o m e c~ only t w o low-spin f e r r i c heine r e s o n a n c e s are detected but the potent i o m e t r i c titrations reveal that for e a c h g~ h e m e r e s o n a n c e as a f u n c t i o n of m e a s u r e d o x i d a t i o n r e d u c t i o n potential, t w o u n i q u e m i d - p o i n t potentials m a y be r e s o l v e d by s i m i l a r c o m p u t e r fitting w i t h the Nernst e q u a t i o n of the ,experimental data. The m i d - p o i n t potentials d e t e r m i n e d for each heine c o n t r i b u t i o n vary b e t w e e n - - 2 3 5 mV and - - 3 2 9 inV. The d i f f e r e n c e in m i d - p o i n t potentials in the case of c y t o c h r o m e c a from D. gigas is BIOCHIMIE, 1978, 60, n ° 3.
a n d J . L e Gall.
a p p r o x i m a t e l y 80 mV w h i c h is closer to the potential gap found for c y t o c h r o m e s b-561 and b-566. F o l l o w i n g the r e c o v e r y rate of the heine m e t h y l NMR resonances, McDonald et al. [2] h a v e observed the d e v e l o p m e n t of t h r e e d i f f e r e n t o x i d a t i o n states w h i c h ,agree w i t h our o x i d a t i o n - r e d u c t i o n p o t e n t i a l m e a s u r e m e n t s . Recent p o t e n t i o m e t r i c t i t r a t i o n s [12] by c y c l i c v o l t m e t r y give a formal oxidation-reduction potential o f 270 ___ 40 mV for the four heroes of c y t o c h r o m e s c a from D. oul.qaris (Miyazaki strain) w h i c h are c o m p a r a b l e to the values given in table II. The question as to w h y t h e r e is a d i f f e r e n c e in m i d - p o i n t potentials for the heroes of c y t o c h r o m e c:~ m a y be found in the biological i m p o r t a n c e of o x i d a t i o n - r e d u c t i o n i n t e r m e d i a t e s i n v o l v i n g one t h r o u g h four el.ectrons. A h a l f - r e d u c e d i n t e r m e diate possibly i n v o l v i n g a t w o e l e c t r o n r e d u c t i o n state has been f o u n d by NMR [2-4] and also by E P R s p e c t r o s c o p y [1] in the case of c y i o c h r o m e c 3 f r o m D. v u l g a r i s . In a d d i t i o n w h e n cytoc h r o m e c 3 f r o m D. v u l g a r i s w a s r e d u c e d w i t h a large excess of s o d i u m d i t h i o n i t e i n the p r e s e n c e of h y d r o x y l a n f i n e (resulting in a m m o n i a format i o n on subsequent r e o x i d a t i o n of c y t o c h r o m e Ca), all four h e m e s of the c y t o c h r o m e could not be c o m p l e t e l y r e d u c e d . In fact heroes I, II, III, and IV w e r e 75 p e r cent, 64 p e r cent, 43 p e r c e n t and 59 p e r cent r e d u c e d r e s p e c t i v e l y ; the r e d u c t i o n state of these h e m e s is related to the d e t e r m i n e d F ~ values f o u n d in table II. This o b s e r v a t i o n in turn suggests that the r e d u c t i o n level of the heroes is c r i t i c a l to the biological reaction(s) in w h i c h c y t o c h r o m e c a is involved.
Acknowledgements. W e grate[ully acknowledge the excellent technical assistance of Mrs. Frances Strickland. W e also t h a n k Dr. John W a m p l e r [or his generous and n u m e r o u s contributions to the computer acquisition of data and subsequent calculations. D. V. D. gralefull!l acknowledges research support f r o m the Nalional I n s t i t u t e for General Medical Sciences (Research grant no. GM-18895) and the National Science F o u n d a t i o n (Research grant no. PCM 7622~10). A. V. X. acknowledges support f r o m S F group grant PCM 76-82502 to the Fresh W a t e r Biological Institute, Unioersil!l o[ Minnesota.
REFERENCES. 1. DerVartanian, D. V. ,~ LeGall, d. (1974) Biochim. Biophys. Acta, ~6, 79-99. 2. McDonald, C. C., Phillips, W. D. ~ LeGall, J. (1974) Biochemistry, 13, 1952-1959. 3. Dobson, C. M., Hoyle, iN. J., Geraldes, C. F., Wright, P. E., Williams, R. J. P. ~ LeCrall, J. (1974) Nature, 24,9, 425-429. 4. Monra, J. J. G., Xavier, A. V., Cookson, D. J., Moore, G. R., Williams, R. J. P., Bruschi, M. ~ LeGall, J. (1977) FEBS Letters, 81, 275-280.
R e d o x p o t e n t i a l s o f c y t o c h r o m e c3. 5. DerVartanian, D. V. ~ LeGall, J., Biochim. Biophys. Acta (Bioenergetics) in press. 6. Dutton, P. L. (1971) Biochim. Biophys. Acta, 226, 63-80. 7. Wampler, J., u n p u b l i s h e d results. 8. Orme-Johnson, N. R., Orme-Johnson, W. H., Hansen, R. E., Beinert, H. ~ Hatefi, Y. (1971) Biochem. Biophys. Res. Commun., 44, 446452.
BIOCHIMIE, 1978, 60, n ° 3.
325
9. Ohnishi, T., Wilson, D. F., Asakura, T. ~ Chance, B. (1972) Biochem. Biophys. Res. Commun., 46, 1631-1637. 10. Leigh, J. S. ~ Erecinska, M. (1975) Biochim. Biophys. Acta, 387, 95-106. 11. Xavier, A. V., Moura, J. J. G., LeGall, J. & DerVartanian, D. V., m a n u s c r i p t in preparation. 12. Niki, K., personal c o m m u n i c a t i o n to A. V. X.
EPR determination of the oxidation-reduction potentials of the hemes in cytochrome c3 from Desulfovibrio vulgaris. D. V. D E R V A R T A N I A N ,), A. V. X A V I E R b) a n d J. L E G A L L ¢).
~) Dept. o[ Biochemistry, University o[ Georgia, Athens, Georgia 30602 (U.S.A.) ; b) Centro da Quimica Estrutural da Universidade de Lisboa, I.S.T., Lisboa 1 (Portugal) ; c) Laboratoire de Chimie Bact~rienne, C.N.R.S., 1327~ Marseille Cedex 2 (France).
Rdsum6.
Summary.
La spectroscopie P,PE, en conjonction a v e c la potentiom6trie r6dox, a 6t6 utilis6e pour d6terminer les potentiels de demi-r6duction de chacun des quatre h ~ m e s du cytochrome Ca : c o m m e des travaux ant6rieurs le laissaient pr6voir, ces potentiels sont diff6rents. Les valeurs de EM sont les suivantes : h 6 m e I, - - 2 8 4 m V " h ~ m e II : - - 3 1 0 m V ~ h~me III : - - 3 2 4 m V et h~me IV : - - 3 1 9 mV. Dans c h a q u e cas, la valeur de n est proche de 1.
EPR s p e c t r o s c o p y in conjunction with oxidation-reduction potentiometry h a s b e e n u s e d to determine the half-reduction potentials of the four heroes of cytochrome c3. As predicted, the four heroes of cytochrome ca h a v e different mid-point potentials. The EM v a l u e s are : Heine L - - 2 8 4 raV : Heine IL - - 3 1 0 m V " Heine HI, - - 3 2 4 m V and Heine IV, - - 3 1 9 raV. The nv a l u e s in e a c h c a s e w a s near one.
Introduction.
resonances potentials.
C y t o c h r o m e c a f r o m D. vulgaris is a s m a l l m o l e c u l a r w e i g h t h e m o p r o t e i n (M r = 13 000) w h i c h c o n t a i n s f o u r h e m e s p e r m o l e c u l e , is a u t o x i d i z a b l e a n d h a s b e e n r e p o r t e d ¢o h a v e a n e g a t i v e o x i d a tion-reduction potential based on light-absorption s p e c t r o s c o p y [c[. ref. 1]. E P R a n d NMR s t u d i e s [1-5] o n t h e f u n c t i o n i n g of t h e f o u r h e r o e s d u r i n g reduction and reoxidation suggested that the four hemes might have four different oxidation-reduct i o n p o t e n t i a l s . E P R a n d N M R s t u d i e s [2-5] h a v e i m p l i c a t e d h i s t i d i n e as t h e p r o b a b l e 5 t h a n d 6 t h b i o l o g i c a l l i g a n d s of t h e h e m e i r o n . E P R s p e c t r o s c o p y h a s d e t e r m i n e d [1] f o u r d i s t i n c t sets of g - v a l u e s c o r r e s p o n d i n g to t h e f o u r h e m e m o i e t i e s located in different environments within the protein. It is t h e p u r p o s e of t h i s s t u d y to d e t e r m i n e t h e m i d - p o i n t p o t e n t i a l s of t h e f o u r l l e m e s b y E P R s p e c t r o s c o p y . It is r e p o r t e d i n t h i s s t u d y t h a t t h e f o u r h e r o e s h a v e n o t o n l y f o u r d i s t i n c t sets of E P R
but
also
four
unique
half-reduction
Methods. G r o w t h of D. vulyaris and purification of cytochrome c~ was carried out as previously reported [1]. EP'R spectroscopy was performed as previously described w i t h a V a r i a n model V-4501A spectrometer [1]. Liquidh e l i u m m e a s u r e m e n t s were made w i t h a n Air Products system LTD-3~110. Oxidation- r e d u c t i o n m e a s u r e m e n t s of cytochrome ca (3.3 m g / m l in 50 mM Tris buffer, pH 8.0) were p e r f o r m e d i n a system s i m i l a r to t h a t described b y D u t t o n [6], w i t h s i m i l a r oxidationreduction m e d i a t o r s a n d at s i m i l a r c o n c e n t r a t i o n s of mediators. The o x i d a t i o n - r e d u c t i o n potential was measured w i t h a F i s h e r Accumet Model 520 digital m e t e r and adjusted w i t h small a d d i t i o n s of sodium dithionite or p o t a s s i u m f e r r i c y a n i d e solutions. After a sample was e q u i l i b r a t e d for a p p r o x i m a t e l y 4 m i n at a set potential, it was rapidly frozen at 77°K for the s u b s e q u e n t EPR m e a s u r e m e n t s . E,PR spectra were directly i n s e r t e d via the Data General Corporation Nova 2 m i n i c o m p u t e r onto a magnetic disc system
D. V. DerVartanian, A. V. Xavier and J. Le Gall.
322
(Ball C o m p u t e r ) f o r a p e r m a n e n t r e c o r d a n d s u b s e q u e n t u s e o f e x p e r i m e n t a l d a t a . C o m p u t e r fits o f t h e experimental data were performed with a computer p r o g r a m [7] of t h e N e r n s t e q u a t i o n f o r d i r e c t d e t e r m i -
n a t i o n of t h e m i d - p o i n t p o t e n t i a l point values are ± 5 mV.
a n d n - v a l u e . Mid-
Results. g = 3,22296
2.48 2~34
2.04 I
F i g u r e 1 illustrates the E P R s p e c t r a of f e r r i c y t o c h r o m e c 3 f r o m D. vulgaris at four r e p r e s e n l a t i v e o x i d a t i o n - r e d u c t i o n potentials. As p r e v i o u s l y des40"
ItU[ i
J
20-
-3Pro
0° 140 °
BE
II
\
A 70-
[ia.-3|OllY " ~
-31711
\
0. 70"
I[lll[ Ill
\
3S-
\ -331 my
O°
70"
HEM[ IV
~x
3So.
H-- (GAUSS)
Fie. 1. - - EPR spectra at IO°K of c!ltochrome c~ f r o m D. v u l g a r i s at f o u r r e p r e s e n t a t i v e o x i d a t i o n - r e d u c t i o n potentials. D e t a i l s on r e c o r d i n g t h e E P R s p e c t r a o n t h e m a g n e t i c disc s y s t e m m a y b e f o u n d in t h e M e t h o d s section. The EPR poteutiometric titration procedures a r e a l s o in t h e M e t h o d s section. E P R c o n d i t i o n s : m i c r o w a v e f r e q u e n c y , 9.187 GHz ; m i c r o w a v e p o w e r , 10 m W ; t i m e c o n s t a n t , 0.1 sec ; s c a n n i n g r a t e , 1000 G per min. Samples were stored under anaerobic condit i o n s i n f r e q u e n c y - m a t c h e d E P R t u b e s of 4 m m o u t e r diameter. BIOCHIMIE, 1978, 60, n ° 3.
,, • -.,~.
O- I
o
-z~o
-z~o
-3;o
-~o
[h(mV)
Fro. 2. - - C o m p u t e r f i t s of n o r m a l i z e d signal ampliludes at y+ f o r h e m e s I-IV as a f u n c t i o n of m e a s u r e d o x i d a t i o n - r e d a c t i o n potentials (Eh). Eu a n d n - v a l u e s a r e l i s t e d i n t a b l e II.
Redox potentials of cytochrome cs.
323
c h r o m e c 3 w h i c h is f u l l y o x i d i z e d at - - 3 7 mV, 27 p e r c e n t r e d u c e d at - - 3 0 7 mV, 47 p e r c e n t red u c e d at - - 3 3 ' 1 m V a n d 73 p e r c e n t r e d u c e d at
c r i b e d [1] f e r r i c y t o c h r o m e c 3 exists in a l o w spin ferric heme configuration with four different sets of g - v a l u e s c o r r e s p o n d i n g to h e m e s I, II, I I I
TABLE I.
Degree o[ reduction of heroes of cytochromes c3 at some representative oxidation-reduction potentials. Heroes (g, "} Eh (blillivolts)
1
II
III
IV
- 37 ( ' " )
30 (0) (") 11 (63) 11 (63)
124 (0) 79 (36) 73 (41)
70 (0) 44 (37) 42 (40)
40 (47) 34 (55)
199 (0) 172 (14) 145 (27)
8 (73) 4 (83)
44 (65) 3 2 (74)
40 (43) 3 2 (54)
33 ( 5 6 ) 27 ( 6 4 )
106 (53) 80 (60)
2 (93) 0 (100)
20 (84) 12 (90)
21 (70) 16 (77)
22 (71) 14 (81)
54 (73) 31 (84)
- 292 - 307 (***) - 331 (***) - 343 - 362 (~**) - 386
75 (0)
{gy ")
(*) Intensities are normalized signal amplitudes. (**) Values given (in parentheses) are in percentage X 1{)0 Reduced, relative to the initial low-spin ferric normalized intensities. (***) EPR spectra at these oxidation-reduction potentials are shown in figure 1. TABLE II.
Summary o[ G-values, mid-poin! potentials and N-values [or the four heroes o[ cytochrome %. Heroes
gz value E~t n-value
I
II
III
IV
3.127 - 284 mV 1.1
2.971 - 310 mV 1.0
2. 821 - 324 mV 1.1
2.479 - 319 mV 1.2
a n d IV, i n d i c a t i n g t h e n o n - e q u i v a l e n c e of the f o u r l o w - s p i n h e i n e m o i e t i e s . D e t e r m i n a t i o n of t h e m i d p o i n t p o t e n t i a l s ~v.as c a r r i e d out b y m e a s u r i n g at each discrete oxidation-reduction potential the gz (*) s i g n a l a m p l i t u d e for h e r o e s I t h r o u g h IV a n d s u b s t r a c t i o n of the a p p r o p r i a t e b a s e - l i n e corr e c t i o n . T h i s is a v a l i d p r o c e d u r e s i n c e no ch, anges in s i g n a l s h a p e o r w i d t h w e r e o b s e r v e d in t h e gz s i g n a l i n t e n s i t i e s d u r i n g r e d u c t i o n - r e o x i d a t i o n c y c l i n g e i t h e r in t h e a b s e n c e o r p r e s e n c e of added redox mediators. B a s e d on m e a s u r e d a m p l i t u d e s at gy ( ' ) , t h e s p e c t r a p r e s e n t e d in F i g u r e 1 c o r r e s p o n d to c y t o (*) gz and g~ are defined as the end m a x i m u m and mid-point crossing, respectively, of the first derivative EPR absorption.
BIOCHIMIE, 1978, 60, n ° 3.
- - 362 mV. T h e p e r c e n t r e d u c t i o n of e a c h h e m e at t h e s e o x i d a t i o n - r e d u c t i o n p o t e n t i a l s is s h o w n in t a b l e I. T h e d e g r e e of r e d u c t i o n of t h e i n d i v i d u a l h e m e s of c y t o c h r o m e c 3 as s h o w n in t a b l e I i n d i c a t e s t h a t h e i n e I is p r e f e r e n t i a l l y r e d u c e d a n d as p r e v i o u s l y r e p o r t e d [1] is also t h e h e i n e w h i c h is t h e last h e m e to be r e o x i d i z e d . H e m e II is t h e n e x t c o m p o n e n t to be r e d u c e d a f t e r r e d u c t i o n of h e m e I. H e m e I I I is t h e last h e m e to be r e d u c e d w h i l e h e i n e IV is i n t e r m e d i a t e in reducti.on a n d reoxidation properties (.approximately between h e m e s I I a n d III). It is c l e a r t h a t t h e p e r c e n t a g e r e d u c t i o n as m e a s u r e d f r o m n o r m a l i z e d s i g n a l a m p l i t u d e s at gy d o e s n o t b e a r a c l e a r r e l a t i o n s h i p to the r e d u c t i o n states of H e m e s I-IV e v e n t h o u g h gy is a s u p e r i m p o s i t i o n of c o n t r i b u t i o n s f r o m all f o u r h e m e s . It is o n l y at - - 331 m V a n d m o r e nega-
324
D. V. DerVartanian,
A. V. Xavier
tive potentials that t h e r e is some c o r r e l a t i o n of the r e d u c t i o n state of Oy - w i l h those of heroes III and IV (see table I) but a p o o r c o r r e l a t i o n w i t h hemes I and II. In the range of - - 3 7 mV to - - 3 0 7 mV there is little c o r r e l a t i o n of the degree of r e d u c t i o n at ~y ,~ to any heme. F i g u r e 2 shows the c o m p u t e r fits for the Nernst equation a p p l i e d to the e x p e r i m e n t a l l y d e t e r m i ned a m p l i t u d e s of the g~ values for the four individual heroes as a f u n c t i o n of m e a s u r e d o x i d a t i o n r e d u c t i o n p o t e n t i a l in the range of 0 mV to - - 400 inV. The c o m p u t e r d e t e r m i n e d m i d - p o i n t potentials and n-values are listed in table H. The n-value for each h e m e system w e r e f o u n d to be on'e.
Discussion
of
Results.
The h a l f - r e d u c t i o n potentials d e t e r m i n e d for h e m e s I, H, III and IV i n d i c a t e that the four h e m e s w h i c h are detected by E P R s p e c t r o s c o p y definitely have four u n i q u e m i d - p o i n t o x i d a t i o n - r e d u c tion potentials. These E M values are consistent w i t h the o b s e r v e d r e d u c t i o n and r e o x i d a t i o n t~PR b e h a v i o r found for the four hemes. The m a x i m u m difference in m i d - p o i n t p o t e n t i a l is found b e t w e e n heroes I and III w i t h a value of 40 inV. The m i d - p o i n t potential values f o u n d in this study for the four heroes of c y l o c h r o m e c~ are not as large in potential d i f f e r e n c e as has been r e p o r t e d for the i r o n - s u l f u r centers of c o m p l e x r e s p i r a t o r y p r o t e i n s such as NADH d e h y d r o g e n a s e [8, 9] hut a p p e a r to be m o r e r e l a t e d in values to the m i d - p o i n t potentials for c y t o c h r o m e s b-561 and b-566 d e t e r m i n e d by E P R s p e c t r o s c o p y by Leigh and E r e c i n s k a [101. These latter w o r k e r s r e p o r t e d a d i f f e r e n c e in m i d - p o i n t p o t e n t i a l of 100 mV b e t w e e n c y t o c h r o m e b-561 (E M = 100 raM) and b-566 (E~ = 0 mV). The results found h e r e should be c o n t r a s t e d to s i m i l a r E P R p o t e n l i o m e t r i c titr,ations for cytoc h r o m e c~ f r o m D e s u l f o v i b r i o gigas [11]. In the case of that c y t o c h r o m e c~ only t w o low-spin f e r r i c heine r e s o n a n c e s are detected but the potent i o m e t r i c titrations reveal that for e a c h g~ h e m e r e s o n a n c e as a f u n c t i o n of m e a s u r e d o x i d a t i o n r e d u c t i o n potential, t w o u n i q u e m i d - p o i n t potentials m a y be r e s o l v e d by s i m i l a r c o m p u t e r fitting w i t h the Nernst e q u a t i o n of the ,experimental data. The m i d - p o i n t potentials d e t e r m i n e d for each heine c o n t r i b u t i o n vary b e t w e e n - - 2 3 5 mV and - - 3 2 9 inV. The d i f f e r e n c e in m i d - p o i n t potentials in the case of c y t o c h r o m e c a from D. gigas is BIOCHIMIE, 1978, 60, n ° 3.
a n d J . L e Gall.
a p p r o x i m a t e l y 80 mV w h i c h is closer to the potential gap found for c y t o c h r o m e s b-561 and b-566. F o l l o w i n g the r e c o v e r y rate of the heine m e t h y l NMR resonances, McDonald et al. [2] h a v e observed the d e v e l o p m e n t of t h r e e d i f f e r e n t o x i d a t i o n states w h i c h ,agree w i t h our o x i d a t i o n - r e d u c t i o n p o t e n t i a l m e a s u r e m e n t s . Recent p o t e n t i o m e t r i c t i t r a t i o n s [12] by c y c l i c v o l t m e t r y give a formal oxidation-reduction potential o f 270 ___ 40 mV for the four heroes of c y t o c h r o m e s c a from D. oul.qaris (Miyazaki strain) w h i c h are c o m p a r a b l e to the values given in table II. The question as to w h y t h e r e is a d i f f e r e n c e in m i d - p o i n t potentials for the heroes of c y t o c h r o m e c:~ m a y be found in the biological i m p o r t a n c e of o x i d a t i o n - r e d u c t i o n i n t e r m e d i a t e s i n v o l v i n g one t h r o u g h four el.ectrons. A h a l f - r e d u c e d i n t e r m e diate possibly i n v o l v i n g a t w o e l e c t r o n r e d u c t i o n state has been f o u n d by NMR [2-4] and also by E P R s p e c t r o s c o p y [1] in the case of c y i o c h r o m e c 3 f r o m D. v u l g a r i s . In a d d i t i o n w h e n cytoc h r o m e c 3 f r o m D. v u l g a r i s w a s r e d u c e d w i t h a large excess of s o d i u m d i t h i o n i t e i n the p r e s e n c e of h y d r o x y l a n f i n e (resulting in a m m o n i a format i o n on subsequent r e o x i d a t i o n of c y t o c h r o m e Ca), all four h e m e s of the c y t o c h r o m e could not be c o m p l e t e l y r e d u c e d . In fact heroes I, II, III, and IV w e r e 75 p e r cent, 64 p e r cent, 43 p e r c e n t and 59 p e r cent r e d u c e d r e s p e c t i v e l y ; the r e d u c t i o n state of these h e m e s is related to the d e t e r m i n e d F ~ values f o u n d in table II. This o b s e r v a t i o n in turn suggests that the r e d u c t i o n level of the heroes is c r i t i c a l to the biological reaction(s) in w h i c h c y t o c h r o m e c a is involved.
Acknowledgements. W e grate[ully acknowledge the excellent technical assistance of Mrs. Frances Strickland. W e also t h a n k Dr. John W a m p l e r [or his generous and n u m e r o u s contributions to the computer acquisition of data and subsequent calculations. D. V. D. gralefull!l acknowledges research support f r o m the Nalional I n s t i t u t e for General Medical Sciences (Research grant no. GM-18895) and the National Science F o u n d a t i o n (Research grant no. PCM 7622~10). A. V. X. acknowledges support f r o m S F group grant PCM 76-82502 to the Fresh W a t e r Biological Institute, Unioersil!l o[ Minnesota.
REFERENCES. 1. DerVartanian, D. V. ,~ LeGall, d. (1974) Biochim. Biophys. Acta, ~6, 79-99. 2. McDonald, C. C., Phillips, W. D. ~ LeGall, J. (1974) Biochemistry, 13, 1952-1959. 3. Dobson, C. M., Hoyle, iN. J., Geraldes, C. F., Wright, P. E., Williams, R. J. P. ~ LeCrall, J. (1974) Nature, 24,9, 425-429. 4. Monra, J. J. G., Xavier, A. V., Cookson, D. J., Moore, G. R., Williams, R. J. P., Bruschi, M. ~ LeGall, J. (1977) FEBS Letters, 81, 275-280.
R e d o x p o t e n t i a l s o f c y t o c h r o m e c3. 5. DerVartanian, D. V. ~ LeGall, J., Biochim. Biophys. Acta (Bioenergetics) in press. 6. Dutton, P. L. (1971) Biochim. Biophys. Acta, 226, 63-80. 7. Wampler, J., u n p u b l i s h e d results. 8. Orme-Johnson, N. R., Orme-Johnson, W. H., Hansen, R. E., Beinert, H. ~ Hatefi, Y. (1971) Biochem. Biophys. Res. Commun., 44, 446452.
BIOCHIMIE, 1978, 60, n ° 3.
325
9. Ohnishi, T., Wilson, D. F., Asakura, T. ~ Chance, B. (1972) Biochem. Biophys. Res. Commun., 46, 1631-1637. 10. Leigh, J. S. ~ Erecinska, M. (1975) Biochim. Biophys. Acta, 387, 95-106. 11. Xavier, A. V., Moura, J. J. G., LeGall, J. & DerVartanian, D. V., m a n u s c r i p t in preparation. 12. Niki, K., personal c o m m u n i c a t i o n to A. V. X.
