Vol. 65, No. 1, 1975
BIOCHEMICALAND BIOPHYSICAL RESEARCH COMMUNICATIONS
CONTROLLED REDUCTION OF CYTOCHROME b IN SUCCINATE-CYTOCHROME c REDUCTASE COMPLEX BY SUCClNATE IN THE PRESENCE OF ASCORBATE AND ANTIMYCIN
Bernard L. Trumpower and Aspandiar
Katki
Department of B i o c h e m i s t r y Dartmouth Medical School Hanover, N.H. 03755
Received May 6,1975 Summary More than 75 percent of the cytochrome b of r e s o l v e d s u c c i n a t e - c y t o c h r o m e c reductase complex is r a p i d T y reduced upon a d d i t i o n of s u c c i n a t e ~ In the presence of ascorbate and a n t i m y c i n , the r e d u c t i o n of b by s u c c i n a t e is markedly i n h i b i t e d such t h a t electron transfer-into b proceeds as a p s e u d o - f i r s t order r e a c t i o n w i t h a r a t e c o n s t a n t o f - 3 x lO-2/min at 25°C. This i n h i b i t i o n is c o n s i s t e n t w i t h a model in which the t o t a l s u c c i n a t e - r e d u c i b l e b is r e g u l a t e d by the redox s t a t u s of cytochrome ~I or a n o t h e r , c u r r e n t l y u n i d e n t i f i e d component of the cytochrome b - c l complex whose redox s t a t u s c l o s e l y p a r a l l e l s t h a t of cytochrome ! l . Introduction Cytochrome b of the m i t o c h o n d r i a l has been i n t e n s i v e l y its
discovery
studied
during
electron
questions
regarding
(I).
(I)
transfer,
proton
the b i o l o g i c a l
what is the sequence of e v e n t s ,
associated with
transport, electron
(2) does cytochrome b p a r t i c i p a t e conservation
by which o x i d a t i o n
Of p a r t i c u l a r
interest
causes an increased
Copyright © 1975 by Academic Press, Inc. All rights o f reproduction in any form reserved..
function fashion. electron
structure,
to and from cytochrome b, in the primary
is coupled
and
step of energy
to p h o s p h o r y l a t i o n ?
is the phenomenon in which a d d i t i o n
02 to anaerobic m i t o c h o n d r i a substrate
including
and changes in p r o t e i n
transfer
since
However, two of
of cytochrome b have y e t to be answered in unequivocal These are
chain
the 50 year i n t e r v a l
in heart muscle fragments
the most fundamental
transport
in the presence of a n t i m y c i n reduction 16
and
of cytochrome b ( 2 - 5 ) .
of
Vol. 65, No. 1, 1975
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
This "oxidant-induced reduction of b" is c u r r e n t l y not understood, but i t
appears that antimycin induces a r e g u l a t o r y e f f e c t s i m i l a r
to one which e x i s t s i n t r i n s i c a l l y reductase complex (6).
in the succinate-cytochrome c
To date the only proposed mechanisms f o r
the oxidant-induced reduction of b have r e l i e d on postulated changes in the midpoint p o t e n t i a l of cytochrome b (cf 7). We r e p o r t here experiments with resolved succinate-cytochrome c reductase complex which demonstrate the inverse of the oxidantinduced reduction of b and which suggest that r e g u l a t i o n of electron t r a n s f e r into b is a k i n e t i c event spanning the second coupling s i t e . Materials and Methods Succinate-cytochrome c reductase complex was prepared from bovine heart mitochondria (8).
Cytochrome c reductase a c t i v i t y
was measured at 30°C, using 30 ~M cytochrome c (8). For measurement of cytochrome spectra and rates of b reduction, reductase complex was suspended at 1.42 mg per ml in O.l M sodium phosphate-O.5 mM EDTA, pH 7.4 and d i f f e r e n c e spectra recorded on a Cary ll8C Spectrophotometer.
Cytochrome b was measured against
a reference in which cytochrome Cl was reduced with ascorbate (9). Additions to the sample and reference cuvettes are recorded in the f i g u r e legends.
Reduction
of b was followed by r e p e t e t i v e
scanning of the wavelength region 585 ÷ 555 nm at a scan rate of 0.5 nm per sec and amounts of reduced b were calculated from the absorbance d i f f e r e n c e at 563 nm minus 577 nm (9). Results and Discussions Resolved succinate-cytochrome ~ reductase complex catalyzes reduction of cytochrome c by succinate with a zero order rate of 6-8 ~mole cytochrome C reduced per min per mg and, as shown below, t h i s a c t i v i t y is f u l l y
s e n s i t i v e to antimycin.
17
The reduc-
Vol. 65, No. 1, 1975
tase
BIOCHEMICAL A N D BIOPHYSICAL RESEARCH C O M M U N I C A T I O N S
complex c o n t a i n s
cytochrome ~I
per mg p r o t e i n .
dithionite-reducible shown).
Thus t h i s
and v e r y
little
b is
Figure
cytochrome b i s
I,
rapidly
before
reactive
b (cf
half-time
by s u c c i n a t e
in r e s o l v e d
1
I
of
I
I 600
(a)
(data
not
activity
9). is
added to the r e d u c t a s e , which
can be o b t a i n e d .
This
than
reductase
I
of the
by high
in a r e a c t i o n
less
I 500
I0 p e r c e n t
characterized
when s u c c i n a t e
a spectrum
b and 1.3 nmole of
to carbon monoxide
is
reduced
the r e p o r t e d
1 500
Less than
preparation
denatured
As shown in
completion
2.3 nmole of cytochrome
I
I00 msec f o r complex
I
I 600
agrees w i t h
reduction
I0).
M i 500
comes to
of
The e x t e n t
of
i---I 564 t
600
(b)
(c) / .i
565.2
T OI A
532
? I
564
T
.01A
Figure 1 Absorption difference s p e c t r a of cytochrome _b in r e s o l v e d s u c c i n a t e - c y t o c h r o m e c r e d u c t a s e complex. Spectrum (a) is o f the cytochrome b reduced by s u c c i n a t e . Spectrum (b) i s of the cytochrome b reduced by s u c c i n a t e in the presence of 1 mM a s c o r b a t e and 2 ~g antimycin. Spectrum (c) i s of the cytochrome b reduced by d i t h i o n i t e , which was i d e n t i c a l in the presence or absence o f a s c o r b a t e and antimycin.
18
Vol. 65, No. 1, 1975
b reduction
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
by s u c c i n a t e
dithionite-reducible cient
for
b,
the t o t a l
reduction
addition
there
is
succinate 2 min.
ascorbate
the r a p i d l y
inhibition
Figure (II),
in
reduced b.
antimycin
and f o r
In agreement w i t h
of one a n t i m y c i n
It
that
ascorbate-dependent Figure is
2 also
It
inhibition
was of p a r t i c u l a r
dependent
inhibition
of a b s p e c i e s Figure
inhibits
the r a p i d
and coworkers
by a n t i m y c i n
site
site
by adding
represents
or a change in
shown in
of S l a t e r
3, a l t h o u g h
is
involved
reduction
of
plus
of ~ I . the
by s u c c i n a t e .
This
which effect
excess f e r r i c y a n i d e . if
the a s c o r b a t e -
the m i d p o i n t
potential
of b r e d u c t i o n .
As
antimycin
b by s u c c i n a t e ,
19
in
of b reduction
a change in
ascorbate
reflects
per m o l e c u l e
to e s t a b l i s h
the k i n e t i c s
transfer
as shown in
in the absence of a s c o r b a t e .
interest
caused a
of e l e c t r o n
to be i d e n t i c a l
binding
caused
the a s c o r b a t e - d e p e n d e n t
inhibition
was not e l i m i n a t e d
reoxida-
as shown below.
of b r e d u c t i o n
shows the s l i g h t
caused by a n t i m y c i n
of antimycin
for
the same b i n d i n g inhibition
added a f t e r
minus a n t i m y c i n
c reductase
a stoichiometry
If
minus a s c o r b a t e
the r e s u l t s
of cytochrome
seems l i k e l y
be added in e i t h e r
by s u c c i n a t e
required
and
When added p r i o r
caused no d e t e c t a b l e
Ascorbate
of b reduction
inhibits
of a n t i m y c i n
was m a n i f e s t e d .
the b-c I complex were found
inhibition
could
no
completely
the presence
and a n t i m y c i n
of b r e d u c t i o n
2.
coeffi-
and a s c o r b a t e ,
in v i r t u a l l y
ascorbate
and a n t i m y c i n
The amounts of a n t i m y c i n
through
resulted
Thus,
of b r e d u c t i o n ;
inhibition
antimycin
between ~562 and ~566.
ascorbate
no i n h i b i t i o n slight
with
and the same i n h i b i t i o n
succinate, of
of
no d i s t i n c t i o n
sequence
of the
a homogenous e x t i n c t i o n
treated
of b by s u c c i n a t e
to s u c c i n a t e ,
tion
assuming
is
of b w i t h i n
the r e d u c t i o n
from 72 to 80 p e r c e n t
b pool.
When the r e d u c t a s e subsequent
varied
there
completely is
a slow
Vol. 65, No. 1, 1975
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
J
80
8
~
c
40 EL 20
~/
(cL..............
o/
//"
0.2
/(b)
.o-. . . . . . . . . . . . . . . . . .
0.4
0.6
0.8
Antimycin/Cytochrome cI (mole/mole)
Figure 2 Effect of antimycin on cytochrome c reductase a c t i v i t y and reduction of cytochrome b by succinate in the presence of ascorbate. Curve (a) depicts the extent of i n h i b i t i o n of succinatecytochrome c reductase a c t i v i t y . In the absence of antimycin the reductase complex had a c t i v i t y of 6.9 umole cytochrome C reduced/ min-mg. Curve (b) depicts the extent to which antimycin i n h i b i t s reduction of cytochrome b by succinate in the presence of 250 uM ascorbate. In the absence of antimycin, 2.8 nmole of b was reduced by succinate. Curve (c) shows the effect of antimycin-on reduction of cytochrome b by succinate in the absence of ascorbate. Prior to addition of succinate, 500 ~M ferricyanide was added to the sample cuvette.
reduction
of b such that all
of the succinate-reducible b is
eventually reduced. The inhibited b reduction
proceeds as a
pseudo-first order reaction, as shown in Figure 4, with a rate constant of 2.96 x lO-2/min, 23 m i n .
which corresponds to a half-time of
The small portion of succinate-reducible b whose i n h i b i -
tion is caused by antimycin minus ascorbate also goes slowly reduced with the same rate constant as the f u l l y inhibited b (data not shown). Figure 3 also shows the rapid reduction
of b which is induced
when the ascorbate-dependent i n h i b i t i o n is reversed by addition of ferricyanide.
This response is analogous to the oxidant-
induced bLreduction
(2-5).
The small amount of b whose rapid
20
VoI. 65, No. 1, 1975
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
t=GO
+ Fe (CN)6 0
E c
0
E
0
£ ~
1.0
0 u "0 Q)
,5
2'o
4'O
5'o
Reaction Time (min)
Figure 3 Time-course of the r e d u c t i o n of cytochrome b by s u c c i n a t e in the presence of ascorbate and a n t i m y c i n . Succinate~cytochrome c reductase complex, c o n t a i n i n g 3 nmole of d i t h i o n i t e r e d u c i b l e b, was p r e i n c u b a t e d 5 min w i t h 1.5 nmole of a n t i m y c i n and 1 ~mole of ascorbate in a volume of 1 ml. The r e a c t i o n was i n i t i a t e d by a d d i t i o n of 20 ~mole of s u c c i n a t e . E a c h p o i n t i n d i c a t e s the amount of reduced cytochrome b at the i n d i c a t e d time i n t e r v a l s a f t e r a d d i t i o n of s u c c i n a t e . -The r a p i d r e d u c t i o n of b r e s u l t i n g from a d d i t i o n of 1.5 ~mole of f e r r i c y a n i d e is i n d i c a t e d by the dashed l i n e .
reduction
i s not induced by f e r r i c y a n i d e
whose i n h i b i t i o n
occurs w i t h
be seen by comparing We conclude
that
caused by a n t i m y c i n of b ( 2 - 5 ) . activity
Figures
minus a s c o r b a t e ,
the a s c o r b a t e - d e p e n d e n t is r e l a t e d
Our f i n d i n g s
or another
cytochrome b-c I complex.
inhibitory
to the o x i d a n t - i n d u c e d
support the h y p o t h e s i s
as can
21
reduction
(4) t h a t
the of
component of the
Mechanisms p r e v i o u s l y reduction
effect
by the redox s t a t u s
ascorbate-reducible
the o x i d a n t - i n d u c e d
to the b
2 and 3.
of cytochrome b is r e g u l a t e d
cytochrome ~I
explain
antimycin
corresponds
proposed to
of b have invoked a change
Vol. 65, No. 1 , 1 9 7 5
BIOCHEMICAL A N D BIOPHYSICAL RESEARCH C O M M U N I C A T I O N S
x
2.0 "0 rY
q,)
E
I.O
¢o
I
~ 0.5 n "0
"0
E
8
!
I
I
/
20 4,0 Reaction Time (min)
I
Figure 4 Semilogarithmic p l o t of the reduction of cytochrome b by succinate in the presence of antimycin and ascorbate. Each point represents the amount of s u c c i n a t e - r e d u c i b l e b remaining unreduced at the indicated time. A t o t a l of 2.60 ~mole-of b was reduced by succinate at " i n f i n i t e time." The f i r s t order rate constant f o r the reaction shown is 2.96 x lO-L/min.
in the midpoint p o t e n t i a l of cytochrome b and have r e s t r i c t e d t h i s r e g u l a t i o n to the postulated energy transducing species, cytochrome AT.
The i n h i b i t i o n which we observe involves a l l
of
the succinate-reducible b, including ~562 and ~566, and our r e s u l t s demonstrate that the regulation of b reduction can be explained as a change in the k i n e t i c s of electron t r a n s f e r into cytochrome b.
This does not exclude the p o s s i b i l i t y of a
t r a n s i e n t change in the reduction p o t e n t i a l of cytochrome b, but such a change would have to occur in the continuous presence of ascorbate.
Since t h i s
i n h i b i t i o n r e s u l t s in a 14,000 f o l d
22
Vol. 65, No. 1, 1975
increase practical
BIOCHEMICALAND BIOPHYSICAL RESEARCH COMMUNICATIONS
in the h a l f - t i m e difference
dynamic e v e n t ,
for
b reduction,
between k i n e t i c
except that
there
regulation
o n l y the l a t t e r
is
little
and a thermo-
fulfills
the c r i t e r i a
whereby energy m i g h t be conserved in cytochrome b.
Acknowledgements This investigation was s u p p o r t e d by a N a t i o n a l Institutes of H e a l t h Research G r a n t , I-ROI-GM20379, and by a Cottrell Research Grant from Research C o r p o r a t i o n .
References I. 2. 3. 4. 5. 6. 7. 8. 9. I0. II.
K e i l i n , D. (1925) Proc. Roy. Soc. ( L o n d o n ) , Ser. B, 9__8_8,312-324. Baum, H., R i e s k e , J . S . , S i l m a n , H . I . , and L i p t o n , S.H. (1967) Proc. Nat. Acad. S c i . U . S . , 57, 798-S05. W i l s o n , D . F . , Koppelman, M., E r e c i n s k a , M., and D u t t o n , P.L. (1971) Biochem. B i o p h y s . Res. Comm., 44, 759-766. R i e s k e , J . S . (1971) Arch. Biochem. B i o p h y s . , 145, 179-193. W i l s o n , D . F . , E r e c i n s k a , M., L e i g h , J r . , J . S . , and Koppelman, M. (1972) Arch. Biochem. B i o p h y s . , 151, 112-121. E r e c i n s k a , M. (1972) Proc. Fed. Am. Soc. Exp. B i o l . A b s t r . , 1106, 415. Wikstrom, M.K.F. (1973) B i o c h i m . B i o p h y s . A c t a , 301, 155-193. Y a m a s h i t a , S . , and Racker, E. (1969) J. B i o l . chem., 244, 1220-1227. Berden, J . A . , and S l a t e r , E.C. (1970) B i o c h i m . B i o p h y s . A c t a , 216, 237-249. E r e c i n s k a , M., and W i l s o n , D.F. (1972) FEBS L e t t s . , 24, 269-272. S l a t e r , E.C. (1973) B i o c h i m . B i o p h y s . A c t a , 301, 129--T54.
23
BIOCHEMICALAND BIOPHYSICAL RESEARCH COMMUNICATIONS
CONTROLLED REDUCTION OF CYTOCHROME b IN SUCCINATE-CYTOCHROME c REDUCTASE COMPLEX BY SUCClNATE IN THE PRESENCE OF ASCORBATE AND ANTIMYCIN
Bernard L. Trumpower and Aspandiar
Katki
Department of B i o c h e m i s t r y Dartmouth Medical School Hanover, N.H. 03755
Received May 6,1975 Summary More than 75 percent of the cytochrome b of r e s o l v e d s u c c i n a t e - c y t o c h r o m e c reductase complex is r a p i d T y reduced upon a d d i t i o n of s u c c i n a t e ~ In the presence of ascorbate and a n t i m y c i n , the r e d u c t i o n of b by s u c c i n a t e is markedly i n h i b i t e d such t h a t electron transfer-into b proceeds as a p s e u d o - f i r s t order r e a c t i o n w i t h a r a t e c o n s t a n t o f - 3 x lO-2/min at 25°C. This i n h i b i t i o n is c o n s i s t e n t w i t h a model in which the t o t a l s u c c i n a t e - r e d u c i b l e b is r e g u l a t e d by the redox s t a t u s of cytochrome ~I or a n o t h e r , c u r r e n t l y u n i d e n t i f i e d component of the cytochrome b - c l complex whose redox s t a t u s c l o s e l y p a r a l l e l s t h a t of cytochrome ! l . Introduction Cytochrome b of the m i t o c h o n d r i a l has been i n t e n s i v e l y its
discovery
studied
during
electron
questions
regarding
(I).
(I)
transfer,
proton
the b i o l o g i c a l
what is the sequence of e v e n t s ,
associated with
transport, electron
(2) does cytochrome b p a r t i c i p a t e conservation
by which o x i d a t i o n
Of p a r t i c u l a r
interest
causes an increased
Copyright © 1975 by Academic Press, Inc. All rights o f reproduction in any form reserved..
function fashion. electron
structure,
to and from cytochrome b, in the primary
is coupled
and
step of energy
to p h o s p h o r y l a t i o n ?
is the phenomenon in which a d d i t i o n
02 to anaerobic m i t o c h o n d r i a substrate
including
and changes in p r o t e i n
transfer
since
However, two of
of cytochrome b have y e t to be answered in unequivocal These are
chain
the 50 year i n t e r v a l
in heart muscle fragments
the most fundamental
transport
in the presence of a n t i m y c i n reduction 16
and
of cytochrome b ( 2 - 5 ) .
of
Vol. 65, No. 1, 1975
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
This "oxidant-induced reduction of b" is c u r r e n t l y not understood, but i t
appears that antimycin induces a r e g u l a t o r y e f f e c t s i m i l a r
to one which e x i s t s i n t r i n s i c a l l y reductase complex (6).
in the succinate-cytochrome c
To date the only proposed mechanisms f o r
the oxidant-induced reduction of b have r e l i e d on postulated changes in the midpoint p o t e n t i a l of cytochrome b (cf 7). We r e p o r t here experiments with resolved succinate-cytochrome c reductase complex which demonstrate the inverse of the oxidantinduced reduction of b and which suggest that r e g u l a t i o n of electron t r a n s f e r into b is a k i n e t i c event spanning the second coupling s i t e . Materials and Methods Succinate-cytochrome c reductase complex was prepared from bovine heart mitochondria (8).
Cytochrome c reductase a c t i v i t y
was measured at 30°C, using 30 ~M cytochrome c (8). For measurement of cytochrome spectra and rates of b reduction, reductase complex was suspended at 1.42 mg per ml in O.l M sodium phosphate-O.5 mM EDTA, pH 7.4 and d i f f e r e n c e spectra recorded on a Cary ll8C Spectrophotometer.
Cytochrome b was measured against
a reference in which cytochrome Cl was reduced with ascorbate (9). Additions to the sample and reference cuvettes are recorded in the f i g u r e legends.
Reduction
of b was followed by r e p e t e t i v e
scanning of the wavelength region 585 ÷ 555 nm at a scan rate of 0.5 nm per sec and amounts of reduced b were calculated from the absorbance d i f f e r e n c e at 563 nm minus 577 nm (9). Results and Discussions Resolved succinate-cytochrome ~ reductase complex catalyzes reduction of cytochrome c by succinate with a zero order rate of 6-8 ~mole cytochrome C reduced per min per mg and, as shown below, t h i s a c t i v i t y is f u l l y
s e n s i t i v e to antimycin.
17
The reduc-
Vol. 65, No. 1, 1975
tase
BIOCHEMICAL A N D BIOPHYSICAL RESEARCH C O M M U N I C A T I O N S
complex c o n t a i n s
cytochrome ~I
per mg p r o t e i n .
dithionite-reducible shown).
Thus t h i s
and v e r y
little
b is
Figure
cytochrome b i s
I,
rapidly
before
reactive
b (cf
half-time
by s u c c i n a t e
in r e s o l v e d
1
I
of
I
I 600
(a)
(data
not
activity
9). is
added to the r e d u c t a s e , which
can be o b t a i n e d .
This
than
reductase
I
of the
by high
in a r e a c t i o n
less
I 500
I0 p e r c e n t
characterized
when s u c c i n a t e
a spectrum
b and 1.3 nmole of
to carbon monoxide
is
reduced
the r e p o r t e d
1 500
Less than
preparation
denatured
As shown in
completion
2.3 nmole of cytochrome
I
I00 msec f o r complex
I
I 600
agrees w i t h
reduction
I0).
M i 500
comes to
of
The e x t e n t
of
i---I 564 t
600
(b)
(c) / .i
565.2
T OI A
532
? I
564
T
.01A
Figure 1 Absorption difference s p e c t r a of cytochrome _b in r e s o l v e d s u c c i n a t e - c y t o c h r o m e c r e d u c t a s e complex. Spectrum (a) is o f the cytochrome b reduced by s u c c i n a t e . Spectrum (b) i s of the cytochrome b reduced by s u c c i n a t e in the presence of 1 mM a s c o r b a t e and 2 ~g antimycin. Spectrum (c) i s of the cytochrome b reduced by d i t h i o n i t e , which was i d e n t i c a l in the presence or absence o f a s c o r b a t e and antimycin.
18
Vol. 65, No. 1, 1975
b reduction
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
by s u c c i n a t e
dithionite-reducible cient
for
b,
the t o t a l
reduction
addition
there
is
succinate 2 min.
ascorbate
the r a p i d l y
inhibition
Figure (II),
in
reduced b.
antimycin
and f o r
In agreement w i t h
of one a n t i m y c i n
It
that
ascorbate-dependent Figure is
2 also
It
inhibition
was of p a r t i c u l a r
dependent
inhibition
of a b s p e c i e s Figure
inhibits
the r a p i d
and coworkers
by a n t i m y c i n
site
site
by adding
represents
or a change in
shown in
of S l a t e r
3, a l t h o u g h
is
involved
reduction
of
plus
of ~ I . the
by s u c c i n a t e .
This
which effect
excess f e r r i c y a n i d e . if
the a s c o r b a t e -
the m i d p o i n t
potential
of b r e d u c t i o n .
As
antimycin
b by s u c c i n a t e ,
19
in
of b reduction
a change in
ascorbate
reflects
per m o l e c u l e
to e s t a b l i s h
the k i n e t i c s
transfer
as shown in
in the absence of a s c o r b a t e .
interest
caused a
of e l e c t r o n
to be i d e n t i c a l
binding
caused
the a s c o r b a t e - d e p e n d e n t
inhibition
was not e l i m i n a t e d
reoxida-
as shown below.
of b r e d u c t i o n
shows the s l i g h t
caused by a n t i m y c i n
of antimycin
for
the same b i n d i n g inhibition
added a f t e r
minus a n t i m y c i n
c reductase
a stoichiometry
If
minus a s c o r b a t e
the r e s u l t s
of cytochrome
seems l i k e l y
be added in e i t h e r
by s u c c i n a t e
required
and
When added p r i o r
caused no d e t e c t a b l e
Ascorbate
of b reduction
inhibits
of a n t i m y c i n
was m a n i f e s t e d .
the b-c I complex were found
inhibition
could
no
completely
the presence
and a n t i m y c i n
of b r e d u c t i o n
2.
coeffi-
and a s c o r b a t e ,
in v i r t u a l l y
ascorbate
and a n t i m y c i n
The amounts of a n t i m y c i n
through
resulted
Thus,
of b r e d u c t i o n ;
inhibition
antimycin
between ~562 and ~566.
ascorbate
no i n h i b i t i o n slight
with
and the same i n h i b i t i o n
succinate, of
of
no d i s t i n c t i o n
sequence
of the
a homogenous e x t i n c t i o n
treated
of b by s u c c i n a t e
to s u c c i n a t e ,
tion
assuming
is
of b w i t h i n
the r e d u c t i o n
from 72 to 80 p e r c e n t
b pool.
When the r e d u c t a s e subsequent
varied
there
completely is
a slow
Vol. 65, No. 1, 1975
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
J
80
8
~
c
40 EL 20
~/
(cL..............
o/
//"
0.2
/(b)
.o-. . . . . . . . . . . . . . . . . .
0.4
0.6
0.8
Antimycin/Cytochrome cI (mole/mole)
Figure 2 Effect of antimycin on cytochrome c reductase a c t i v i t y and reduction of cytochrome b by succinate in the presence of ascorbate. Curve (a) depicts the extent of i n h i b i t i o n of succinatecytochrome c reductase a c t i v i t y . In the absence of antimycin the reductase complex had a c t i v i t y of 6.9 umole cytochrome C reduced/ min-mg. Curve (b) depicts the extent to which antimycin i n h i b i t s reduction of cytochrome b by succinate in the presence of 250 uM ascorbate. In the absence of antimycin, 2.8 nmole of b was reduced by succinate. Curve (c) shows the effect of antimycin-on reduction of cytochrome b by succinate in the absence of ascorbate. Prior to addition of succinate, 500 ~M ferricyanide was added to the sample cuvette.
reduction
of b such that all
of the succinate-reducible b is
eventually reduced. The inhibited b reduction
proceeds as a
pseudo-first order reaction, as shown in Figure 4, with a rate constant of 2.96 x lO-2/min, 23 m i n .
which corresponds to a half-time of
The small portion of succinate-reducible b whose i n h i b i -
tion is caused by antimycin minus ascorbate also goes slowly reduced with the same rate constant as the f u l l y inhibited b (data not shown). Figure 3 also shows the rapid reduction
of b which is induced
when the ascorbate-dependent i n h i b i t i o n is reversed by addition of ferricyanide.
This response is analogous to the oxidant-
induced bLreduction
(2-5).
The small amount of b whose rapid
20
VoI. 65, No. 1, 1975
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
t=GO
+ Fe (CN)6 0
E c
0
E
0
£ ~
1.0
0 u "0 Q)
,5
2'o
4'O
5'o
Reaction Time (min)
Figure 3 Time-course of the r e d u c t i o n of cytochrome b by s u c c i n a t e in the presence of ascorbate and a n t i m y c i n . Succinate~cytochrome c reductase complex, c o n t a i n i n g 3 nmole of d i t h i o n i t e r e d u c i b l e b, was p r e i n c u b a t e d 5 min w i t h 1.5 nmole of a n t i m y c i n and 1 ~mole of ascorbate in a volume of 1 ml. The r e a c t i o n was i n i t i a t e d by a d d i t i o n of 20 ~mole of s u c c i n a t e . E a c h p o i n t i n d i c a t e s the amount of reduced cytochrome b at the i n d i c a t e d time i n t e r v a l s a f t e r a d d i t i o n of s u c c i n a t e . -The r a p i d r e d u c t i o n of b r e s u l t i n g from a d d i t i o n of 1.5 ~mole of f e r r i c y a n i d e is i n d i c a t e d by the dashed l i n e .
reduction
i s not induced by f e r r i c y a n i d e
whose i n h i b i t i o n
occurs w i t h
be seen by comparing We conclude
that
caused by a n t i m y c i n of b ( 2 - 5 ) . activity
Figures
minus a s c o r b a t e ,
the a s c o r b a t e - d e p e n d e n t is r e l a t e d
Our f i n d i n g s
or another
cytochrome b-c I complex.
inhibitory
to the o x i d a n t - i n d u c e d
support the h y p o t h e s i s
as can
21
reduction
(4) t h a t
the of
component of the
Mechanisms p r e v i o u s l y reduction
effect
by the redox s t a t u s
ascorbate-reducible
the o x i d a n t - i n d u c e d
to the b
2 and 3.
of cytochrome b is r e g u l a t e d
cytochrome ~I
explain
antimycin
corresponds
proposed to
of b have invoked a change
Vol. 65, No. 1 , 1 9 7 5
BIOCHEMICAL A N D BIOPHYSICAL RESEARCH C O M M U N I C A T I O N S
x
2.0 "0 rY
q,)
E
I.O
¢o
I
~ 0.5 n "0
"0
E
8
!
I
I
/
20 4,0 Reaction Time (min)
I
Figure 4 Semilogarithmic p l o t of the reduction of cytochrome b by succinate in the presence of antimycin and ascorbate. Each point represents the amount of s u c c i n a t e - r e d u c i b l e b remaining unreduced at the indicated time. A t o t a l of 2.60 ~mole-of b was reduced by succinate at " i n f i n i t e time." The f i r s t order rate constant f o r the reaction shown is 2.96 x lO-L/min.
in the midpoint p o t e n t i a l of cytochrome b and have r e s t r i c t e d t h i s r e g u l a t i o n to the postulated energy transducing species, cytochrome AT.
The i n h i b i t i o n which we observe involves a l l
of
the succinate-reducible b, including ~562 and ~566, and our r e s u l t s demonstrate that the regulation of b reduction can be explained as a change in the k i n e t i c s of electron t r a n s f e r into cytochrome b.
This does not exclude the p o s s i b i l i t y of a
t r a n s i e n t change in the reduction p o t e n t i a l of cytochrome b, but such a change would have to occur in the continuous presence of ascorbate.
Since t h i s
i n h i b i t i o n r e s u l t s in a 14,000 f o l d
22
Vol. 65, No. 1, 1975
increase practical
BIOCHEMICALAND BIOPHYSICAL RESEARCH COMMUNICATIONS
in the h a l f - t i m e difference
dynamic e v e n t ,
for
b reduction,
between k i n e t i c
except that
there
regulation
o n l y the l a t t e r
is
little
and a thermo-
fulfills
the c r i t e r i a
whereby energy m i g h t be conserved in cytochrome b.
Acknowledgements This investigation was s u p p o r t e d by a N a t i o n a l Institutes of H e a l t h Research G r a n t , I-ROI-GM20379, and by a Cottrell Research Grant from Research C o r p o r a t i o n .
References I. 2. 3. 4. 5. 6. 7. 8. 9. I0. II.
K e i l i n , D. (1925) Proc. Roy. Soc. ( L o n d o n ) , Ser. B, 9__8_8,312-324. Baum, H., R i e s k e , J . S . , S i l m a n , H . I . , and L i p t o n , S.H. (1967) Proc. Nat. Acad. S c i . U . S . , 57, 798-S05. W i l s o n , D . F . , Koppelman, M., E r e c i n s k a , M., and D u t t o n , P.L. (1971) Biochem. B i o p h y s . Res. Comm., 44, 759-766. R i e s k e , J . S . (1971) Arch. Biochem. B i o p h y s . , 145, 179-193. W i l s o n , D . F . , E r e c i n s k a , M., L e i g h , J r . , J . S . , and Koppelman, M. (1972) Arch. Biochem. B i o p h y s . , 151, 112-121. E r e c i n s k a , M. (1972) Proc. Fed. Am. Soc. Exp. B i o l . A b s t r . , 1106, 415. Wikstrom, M.K.F. (1973) B i o c h i m . B i o p h y s . A c t a , 301, 155-193. Y a m a s h i t a , S . , and Racker, E. (1969) J. B i o l . chem., 244, 1220-1227. Berden, J . A . , and S l a t e r , E.C. (1970) B i o c h i m . B i o p h y s . A c t a , 216, 237-249. E r e c i n s k a , M., and W i l s o n , D.F. (1972) FEBS L e t t s . , 24, 269-272. S l a t e r , E.C. (1973) B i o c h i m . B i o p h y s . A c t a , 301, 129--T54.
23
