- Email: [email protected]
Electron transfer between outer and inner membranes in plant mitochondria
Plant Science Letters, 6 (1976) 215--221
215
© Elsevier Scientific Publishing Company, Amsterdam -- Printed in The Netherlands
ELECTRON TRANSFER BETWEEN OUTER AND INNER MEMBRANES IN PLANT MITOCHONDRIA
F. MOREAU Laboratoire de Biologie V}g@tale IV, Universit} Pierre et Marie Curie, 12, rue Cuvier, 75005 Paris (France)
(Received December 10th, 1975) (Accepted January 9th, 1976)
SUMMARY Experiments on dissociation and reassociation of outer and inner membranes of cauliflower (Brassica oleracea L.) mitochondria have been carried out in order to investigate the possible occurrence of an intermembrane electron transfer. With NADH as electron donor, it has been shown that electron transfer can take place between an antimycin-insensitive NADHcytochrome c reductase, on the outer membrane, and the cyanide-sensitive cytochrome oxidase, on the inner membrane, provided a mobile carrier such as cytochrome c is present in the intermembrane space. In intact plant mitochondria, part of the oxidation of exogenous NADH could be carried out by this pathway.
INTRODUCTION Intact plant mitochondria are normally able to carry out the oxidation of extramitochondrial NADH [1--3]. An NADH dehydrogenase, located on the outer face of the inner mitochondrial membrane, as well as the cytochromes of the respiratory chain are the normal components of this oxidation pathway [4--6]. Another NADH dehydrogenase, however, the antimycin-insensitive NADH-cytochrome c oxidoreductase, is also present in mitochondria, closely associated with the outer membrane [5--6]. In plant mitochondria also, the oxidation of extramitochondrial NADH bypasses the first energy conservation site [7--9]. It is only partially inhibited by antimycin A, a specific inhibitor of complex III [10]. Moreover, this inhibition can be partially overcome by the addition of exogenous cytochrome c [8-9]. In mammalian mitochondria, it has recently been shown that the electron transport between the two mitochondrial membranes can take place in the presence of added soluble cytochrome c or artificial electron carriers. The
216 participation of the NADH-cytochrome bs reductase system is implicated in this pathway [11--13]. In this work, the presence in cauliflower mitochondria of an oxidation mechanism for extramitochondrial NADH is demonstrated. It implies a transfer of reducing equivalents between the two mitochondrial membranes. This pathway requires the cooperation of a dehydrogenase associated with an electron transfer system on the outer membrane, of the cytochrome oxidase on the inner membrane and of a soluble intermediary carrier such as cytochrome c. MATERIALS A N D M E T H O D S
The techniques for the isolation of purified mitochondria from cauliflower buds (Brassica oleracea L., cv. Botrytis) as well as those for the isolation of outer and inner membranes by osmotic swelling have been described in full detail elsewhere [ 5]. Oxygen uptake was measured using a Clark oxygen electrode and a medium containing 300 mM mannitol, 10 mM phosphate buffer (pH 7,20), 10 mM KC1 and 5 mM MgC12. In order to eventually achieve a maximal electron flux, all membrane preparations were studied in the presence of 2 pM carbonyl cyanide m-chlorophenylhydrazone (m-C1-CCP) acting as an uncoupler. The NADH cytochrome c oxidoreductase was measured spectrophotometrically by following the reduction of oxidized cytochrome c at 550 nm [11], in a medium containing 300 mM mannitol, 1 mM NADH, 10 mM phosphate buffer (pH 7,20), 50 ~M cytochrome c, 1 mM KCN and 50 to 100 p g o f membrane proteins in a final volume of 3 ml. The reaction was started by the addition of NADH. When necessary antimycin A was added at a concentration of 1 pM. The succinate-cytochrome c oxidoreductase activity was measured using the same medium containing 10 mM succinate instead of NADH. Protein determinations were made according to Lowry et ai. [14] using bovine serum albumin as a standard. RESULTS
Oxidation of N A D H Table I shows that a high NADH oxidase activity is present in inner membranes of cauliflower mitochondria. This activity is highly sensitive to antimycin inhibition. The low activity found in outer membranes presumably results from the contamination (less than 10%) of this fraction by inner membrane vesicles [ 5]. By contrast, outer membranes, which have no or very little NADH oxidase activity, display a high level of NADH cytochrome c oxidoreductase activity which is highly resistant to antimycin inhibition. Action of cytochrome c on the oxidation of N A D H Fig. I A shows that N A D H is indeed actively oxidized by intact mitochondria. The addition of cytochrome c in trace amounts (5 ~M) induces an
217 TABLE I NADH OXIDASE AND NADH-CYTOCHROME c OXIDOREDUCTASE ACTIVITIES IN THE OUTER AND INNER MEMBRANES OF CAULIFLOWER MITOCHONDRIA
Inner membranes Outer membranes
NADH o x i d u e a
NADH-cytochrome c r e d u e t u e b
Antimycinsensitive
Antimycininsensitive
Antimycinsensitive
Antimycininsensitive
210 15
10 2
1300 60
25 350
anmoles 02 • rain-' • mg protein -I. bnmoles cytochrome c reduced • rain -~ - m g protein -z.
T
0
12
NADH (,~ Mit. M•I'. I"~/m-CI- CCP~/ -~~CmC i_nct:cCp(~ 292\Antimycin 5
216~Antimycin \/
10
Fig. I . Effect o f e y t o c h r o m e ¢ on the oxidation o f NADH and sueeinate b y eauUflower mitochondria. Compoeition of reaction medium: 300 mM mannitol, 10 mM phasphate b u f f e r (pH 7.2), 10 mM KCI, 5 ~ M MgCI 2. Final concentrations o f added reagents: 1 mM NADH, 10 mM sueeinate, 5 ~,M eytochrome ¢, 2 , M antimyein A, 1 mM KCN, 2 ~,M m-CI-CCP. Intact mitochondria (Mit): 0.'/5 mg protein/ml. The figures along the traces indicate O~ uptake in nmoles, rain-' •mg protein -I.
218
increase in the rate of oxidation of 20 to 50% on the average. This oxidation is strongly inhibited by antimycin A at a 2 #M concentration and then can be fully inhibited by 1 mM KCN. On the opposite, under the same conditions (Fig. 1B), the oxidation of succinate is only slightly increased in the presence of c y t o c h r o m e c, indicating that the mitochondria have not lost their cytochrome c and that the c y t o c h r o m e c-impermeable outer membrane [6] has not been damaged during the isolation procedure. In addition, Fig. 1C demonstrates that the stimulation of the oxidation of exogenous NADH by cytochrome c is insensitive to antimycin but is fully sensitive to cyanide. On the other hand, c y t o c h r o m e c is quite ineffective in stimulating succinate oxidation when antimycin is present (Fig. 1D).
Reassociation of mitochondrial membranes Fig. 2 describes an experiment in which isolated outer membranes are added to purified inner membranes in order to reconstruct a system roughly equivalent to intact mitochondria. When the three participating elements: inner membranes, outer membranes and c y t o c h r o m e c are added together (Fig. 2), a picture very similar to that given by intact mitochondria (Fig. 1C) can be reconstructed.
"-~. /
rn-Cl-CCP ~
5 ~ ~
210\ Antimycin
T
m-Cl-CCP
232\ Anl'imycin
8
8
Fig. 2. Cytochrome c-induced electron transfer between inner and outer mitochondrial membranes. Same medium as in Fig. 1. Inner membranes (IM): 0.6 mg protein/ml; outer membranes (OM): 0.3 mg protein/ml.
When outer membranes are added to inner membranes in the presence of antimycin, an increase in NADH oxidation can be observed (Fig. 2A). Taking into account the low antimycin-insensitive NADH oxidase activity of the outer membranes (Table I) and the amounts of these membranes used in the experiment, one can reasonably think that the increase in activity is not due to the outer membranes added to the medium, but more correctly reflects an interaction between outer and inner membranes (see DISCUSSION).The subsequent addition of c y t o c h r o m e c in trace amounts induces a further increase of the NADH oxidation which reaches a level comparable to that observed in intact mitochondria (cf. Fig. 1C).
219
When c y t o c h r o m e c is added first to inner membranes in the presence of antimycin (Fig. 2B), it has only a very slight action on the stimulation o f N A D H oxidation b y comparison with what is observed with intact mitochondria (cf. Fig. 1C). The subsequent addition o f outer membranes, induces a threefold increase in the rate of N A D H oxidation. This oxidation is insensitive t o antimycin b u t is strongly inhibited b y cyanide. When similar experiments are carried o u t with succinate as substrate, the combined additions of outer membranes and c y t o c h r o m e c do n o t increase the rate o f oxidation of this substrate. Therefore, from Figs. 2A and B, one can draw the conclusion that the presence of both c y t o c h r o m e c and outer membranes are responsible for the increase in the rate o f NADH oxidation b y pure inner membranes. This experiment demonstrates that electron transfer can take place in vitro b e t w e e n the t w o mitochondrial membranes through soluble c y t o c h r o m e c. It also suggests that the same process may occur in intact mitochondria as evidenced b y the comparison of Figs. 1C and 2B.
Permeability o f outer membranes to cytochrome c It is generally considered that exogenous soluble c y t o c h r o m e c cannot move across the outer mitochondrial membrane [6,15] although this point is still controversial with plant mitochondria [16]. In order to determine if the intermemhrane electron transfer observed with intact mitochondria (Figs. 1A and C) really occurs in organelles whose outer membranes have n o t been damaged, parallel experiments were performed with broken mitochondria. Table II shows that broken mitochondria have indeed a much higher succinate-cytochrome c reductase activity than intact mitochondria, indicating that the outer membrane prevents free access of c y t o c h r o m e c to t h e inner membrane. On the opposite, with N A D H as substrate, the intermembrane electron transfer induced b y addition of soluble c y t o c h r o m e c is n o t significantly different in intact and broken mitochondria. The results with the sucTABLE II EFFECT OF INTEGRITY OF THE OUTER MEMBRANE ON THE PERMEABILITY TO CYTOCHROME c AND ON THE INTERMEMBRANE ELECTRON TRANSFER Mitochondda
Intact
Brokenc
Succinate cytochrome c reductase a
NADH oxidaseb Antimycinsensitive
Antimyein-insensitive
Control
+ Cyt. c 28 20
25
220
6
350
170
5
anmoles cytoehrome c reduced, rain -1 *mg protein -1. bnmolu 02 • rain -z *mg protein -z. c M i t o c h o n d r i a were b r o k e n b y a light s o n i c a t i o n .
220
cinate-cytochrome c reductase activity indicate that c y t o c h r o m e c cannot move across the outer membrane of intact mitochondria in sufficient amounts to act as an efficient electron acceptor at the level of the inner mitochondrial membrane, in accordance with other observations [6,15]. On the other hand, as suggested b y Figs. 1A and C, c y t o c h r o m e c must nevertheless penetrate in catalytic amounts in the intermembrane space of intact mitochondria since, b y being alternatively reduced and oxidized, it is able to act as a mobile cartier b e t w e e n the N A D H - c y t o c h r o m e c reductase system of the outer membrane and the c y t o c h r o m e oxidase system of the inner membrane. DISCUSSION
A mechanism for the oxidation of extramitochondrial N A D H via a reductase located on the outer mitochondrial m e m b r a n e has been demonstrated. The intermembrane electron transfer which takes place requires the presence of a mobile carrier in the intermembrane space, for which soluble c y t o c h r o m e c seems to be the best candidate (Fig. 3). However, it cannot be excluded that direct contacts b e t w e e n the t w o mitochondrial membranes may also occur so that the electron transfer takes place in the absence of a mobile carrier, as it has been recently suggested in the case of interactions between mitochondria and microsomes [17]. This possibility is also indicated in the experiments on the reassociation o f the t w o mitochondrial membranes b y the increase in the rate o f electron transfer observed u p o n addition o f outer membranes, before c y t o c h r o m e c is added (Fig. 2A). As shown in Fig. 3, this intermemhrane electron p a t h w a y is specific for NADH. In contrast with the main p a t h w a y for NADH oxidation, which takes place through a flavin dehydrogenase (FPIM) l o c a t e d on the outer face of the
)
NADH
Cyt. C
OM IS
IM HA
_j
-c,,..c#
FP,.'-..~ b . . A ~ c " ~ Antimycin
o _1+.o~ KCN
Fig. 3. Electron transfer pathways during oxidation of extramitochondrial NADH by cauliflower mitochondria. OM: outer membrane; IM: inner membrane; IS: intermembrane space; MA: matrix. FpnM-bOM dehydrogenase complex o f the outer membrane containing both a flavoprotein and a b-type cytochrome. FPi M: dehydrogenase located on the outer face of the inner membrane; b, c, a, as: cytochrom-es of the respiratory chain.
221 inner membrane [6], this electron p a t h w a y probably includes the participation of another flavin dehydrogenase (FPoM) [6] and of a b-type c y t o c h r o m e (boM) [5], both of t h e m located on the outer membrane. Thus, this p a t h w a y appears be b o t h antimycin-insensitive and cyanidesensitive. Moreover, although it cannot account for more than 10% of the NADH oxidation in intact mitochondria, it could also act as a by-pass of the antimycin and rotenone-sensitive site, leaving but one phosphorylation site (site III) active. This c o u l d p r o v i d e an explanation for the strong resistance to inhibitors and the low P/O ratios generally observed in the oxidation of NADH by plant mitochondria [8,18]. The question also arises of whether or n o t the intermembrane electron transfer observed on isolated mitochondria can also take place in mitochondria within the cell. ACKNOWLEDGEMENTS This work was supported by a grant (RCP 223) from the Centre National de la Recherche Scientifique. REFERENCES 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18
W.D. Bonner and D.O. Voss, Nature, 191 (1961) 682. D.P. Hackett, Plant Physiol., 36 (1961) 445. T. Ohnishi, L. Kawaguehi and B. I-Iagihara,J. Biol. Chem., 241 (1966) 1797. G. Von Jagow and M. Kligenberg, Eur. J. Biochem., 12 (1970) 583. F. Moreau and C. Lance, Biochimie, 54 (1972) 1335. R. Douee, C.A. Mannella and W.D. Bonnet, Biochim. Biophys. Acta, 292 (1973) 105. I-L Ikuma and W.D. Bonnet, Plant Physiol., 42 (1967) 67. R.H. Wilson and J.B. Hanson, Plant Physiol., 44 (1969) 1335. D.D. Day and J.T. Wiskieh, Plant Physiol., 53 (1974) 104. J.A. Berden and E.C. Slater, Bioehim. Biophys. Acta, 216 (1970) 237. G.L. Sottocasa, B. Kuylenstierna, L. Er~ter and A. Bergstrand, J. Cell Biol., 32 (1967) 415. P. Nicholls, E. Mochan and H.K. Kimelberg, FEBS Letters, 3 (1969) 242. P.J. O'Brien, Mitochondrial intermembrane shuttles, in E. Quagliariello, S. Papa and C.S. Rossi (Eds.), Energy Transduction in Respiration and Photosynthesis, Adriatiea, London, 1971, p. 361. O.H. Lowry, N.J. Rosebrough, A.L. Farr and R.J. Randall, J. Biol. Chem., 193 (1951) 265. L. Wojtczak and H. Zaluska, Biochim. Biophys. Acta, 193 (1969) 64. J.M. Palmer and B.I. Kirk, Biochem. J., 140 (1974) 79. A.I. Archakov, A.V. Karlakin and V.P. Skulachev, Dokl. Aead. Nauk SSSR, 217 (1974) 1423. C. Lance, Physiol. V6g., 9 (1971) 259.
215
© Elsevier Scientific Publishing Company, Amsterdam -- Printed in The Netherlands
ELECTRON TRANSFER BETWEEN OUTER AND INNER MEMBRANES IN PLANT MITOCHONDRIA
F. MOREAU Laboratoire de Biologie V}g@tale IV, Universit} Pierre et Marie Curie, 12, rue Cuvier, 75005 Paris (France)
(Received December 10th, 1975) (Accepted January 9th, 1976)
SUMMARY Experiments on dissociation and reassociation of outer and inner membranes of cauliflower (Brassica oleracea L.) mitochondria have been carried out in order to investigate the possible occurrence of an intermembrane electron transfer. With NADH as electron donor, it has been shown that electron transfer can take place between an antimycin-insensitive NADHcytochrome c reductase, on the outer membrane, and the cyanide-sensitive cytochrome oxidase, on the inner membrane, provided a mobile carrier such as cytochrome c is present in the intermembrane space. In intact plant mitochondria, part of the oxidation of exogenous NADH could be carried out by this pathway.
INTRODUCTION Intact plant mitochondria are normally able to carry out the oxidation of extramitochondrial NADH [1--3]. An NADH dehydrogenase, located on the outer face of the inner mitochondrial membrane, as well as the cytochromes of the respiratory chain are the normal components of this oxidation pathway [4--6]. Another NADH dehydrogenase, however, the antimycin-insensitive NADH-cytochrome c oxidoreductase, is also present in mitochondria, closely associated with the outer membrane [5--6]. In plant mitochondria also, the oxidation of extramitochondrial NADH bypasses the first energy conservation site [7--9]. It is only partially inhibited by antimycin A, a specific inhibitor of complex III [10]. Moreover, this inhibition can be partially overcome by the addition of exogenous cytochrome c [8-9]. In mammalian mitochondria, it has recently been shown that the electron transport between the two mitochondrial membranes can take place in the presence of added soluble cytochrome c or artificial electron carriers. The
216 participation of the NADH-cytochrome bs reductase system is implicated in this pathway [11--13]. In this work, the presence in cauliflower mitochondria of an oxidation mechanism for extramitochondrial NADH is demonstrated. It implies a transfer of reducing equivalents between the two mitochondrial membranes. This pathway requires the cooperation of a dehydrogenase associated with an electron transfer system on the outer membrane, of the cytochrome oxidase on the inner membrane and of a soluble intermediary carrier such as cytochrome c. MATERIALS A N D M E T H O D S
The techniques for the isolation of purified mitochondria from cauliflower buds (Brassica oleracea L., cv. Botrytis) as well as those for the isolation of outer and inner membranes by osmotic swelling have been described in full detail elsewhere [ 5]. Oxygen uptake was measured using a Clark oxygen electrode and a medium containing 300 mM mannitol, 10 mM phosphate buffer (pH 7,20), 10 mM KC1 and 5 mM MgC12. In order to eventually achieve a maximal electron flux, all membrane preparations were studied in the presence of 2 pM carbonyl cyanide m-chlorophenylhydrazone (m-C1-CCP) acting as an uncoupler. The NADH cytochrome c oxidoreductase was measured spectrophotometrically by following the reduction of oxidized cytochrome c at 550 nm [11], in a medium containing 300 mM mannitol, 1 mM NADH, 10 mM phosphate buffer (pH 7,20), 50 ~M cytochrome c, 1 mM KCN and 50 to 100 p g o f membrane proteins in a final volume of 3 ml. The reaction was started by the addition of NADH. When necessary antimycin A was added at a concentration of 1 pM. The succinate-cytochrome c oxidoreductase activity was measured using the same medium containing 10 mM succinate instead of NADH. Protein determinations were made according to Lowry et ai. [14] using bovine serum albumin as a standard. RESULTS
Oxidation of N A D H Table I shows that a high NADH oxidase activity is present in inner membranes of cauliflower mitochondria. This activity is highly sensitive to antimycin inhibition. The low activity found in outer membranes presumably results from the contamination (less than 10%) of this fraction by inner membrane vesicles [ 5]. By contrast, outer membranes, which have no or very little NADH oxidase activity, display a high level of NADH cytochrome c oxidoreductase activity which is highly resistant to antimycin inhibition. Action of cytochrome c on the oxidation of N A D H Fig. I A shows that N A D H is indeed actively oxidized by intact mitochondria. The addition of cytochrome c in trace amounts (5 ~M) induces an
217 TABLE I NADH OXIDASE AND NADH-CYTOCHROME c OXIDOREDUCTASE ACTIVITIES IN THE OUTER AND INNER MEMBRANES OF CAULIFLOWER MITOCHONDRIA
Inner membranes Outer membranes
NADH o x i d u e a
NADH-cytochrome c r e d u e t u e b
Antimycinsensitive
Antimycininsensitive
Antimycinsensitive
Antimycininsensitive
210 15
10 2
1300 60
25 350
anmoles 02 • rain-' • mg protein -I. bnmoles cytochrome c reduced • rain -~ - m g protein -z.
T
0
12
NADH (,~ Mit. M•I'. I"~/m-CI- CCP~/ -~~CmC i_nct:cCp(~ 292\Antimycin 5
216~Antimycin \/
10
Fig. I . Effect o f e y t o c h r o m e ¢ on the oxidation o f NADH and sueeinate b y eauUflower mitochondria. Compoeition of reaction medium: 300 mM mannitol, 10 mM phasphate b u f f e r (pH 7.2), 10 mM KCI, 5 ~ M MgCI 2. Final concentrations o f added reagents: 1 mM NADH, 10 mM sueeinate, 5 ~,M eytochrome ¢, 2 , M antimyein A, 1 mM KCN, 2 ~,M m-CI-CCP. Intact mitochondria (Mit): 0.'/5 mg protein/ml. The figures along the traces indicate O~ uptake in nmoles, rain-' •mg protein -I.
218
increase in the rate of oxidation of 20 to 50% on the average. This oxidation is strongly inhibited by antimycin A at a 2 #M concentration and then can be fully inhibited by 1 mM KCN. On the opposite, under the same conditions (Fig. 1B), the oxidation of succinate is only slightly increased in the presence of c y t o c h r o m e c, indicating that the mitochondria have not lost their cytochrome c and that the c y t o c h r o m e c-impermeable outer membrane [6] has not been damaged during the isolation procedure. In addition, Fig. 1C demonstrates that the stimulation of the oxidation of exogenous NADH by cytochrome c is insensitive to antimycin but is fully sensitive to cyanide. On the other hand, c y t o c h r o m e c is quite ineffective in stimulating succinate oxidation when antimycin is present (Fig. 1D).
Reassociation of mitochondrial membranes Fig. 2 describes an experiment in which isolated outer membranes are added to purified inner membranes in order to reconstruct a system roughly equivalent to intact mitochondria. When the three participating elements: inner membranes, outer membranes and c y t o c h r o m e c are added together (Fig. 2), a picture very similar to that given by intact mitochondria (Fig. 1C) can be reconstructed.
"-~. /
rn-Cl-CCP ~
5 ~ ~
210\ Antimycin
T
m-Cl-CCP
232\ Anl'imycin
8
8
Fig. 2. Cytochrome c-induced electron transfer between inner and outer mitochondrial membranes. Same medium as in Fig. 1. Inner membranes (IM): 0.6 mg protein/ml; outer membranes (OM): 0.3 mg protein/ml.
When outer membranes are added to inner membranes in the presence of antimycin, an increase in NADH oxidation can be observed (Fig. 2A). Taking into account the low antimycin-insensitive NADH oxidase activity of the outer membranes (Table I) and the amounts of these membranes used in the experiment, one can reasonably think that the increase in activity is not due to the outer membranes added to the medium, but more correctly reflects an interaction between outer and inner membranes (see DISCUSSION).The subsequent addition of c y t o c h r o m e c in trace amounts induces a further increase of the NADH oxidation which reaches a level comparable to that observed in intact mitochondria (cf. Fig. 1C).
219
When c y t o c h r o m e c is added first to inner membranes in the presence of antimycin (Fig. 2B), it has only a very slight action on the stimulation o f N A D H oxidation b y comparison with what is observed with intact mitochondria (cf. Fig. 1C). The subsequent addition o f outer membranes, induces a threefold increase in the rate of N A D H oxidation. This oxidation is insensitive t o antimycin b u t is strongly inhibited b y cyanide. When similar experiments are carried o u t with succinate as substrate, the combined additions of outer membranes and c y t o c h r o m e c do n o t increase the rate o f oxidation of this substrate. Therefore, from Figs. 2A and B, one can draw the conclusion that the presence of both c y t o c h r o m e c and outer membranes are responsible for the increase in the rate o f NADH oxidation b y pure inner membranes. This experiment demonstrates that electron transfer can take place in vitro b e t w e e n the t w o mitochondrial membranes through soluble c y t o c h r o m e c. It also suggests that the same process may occur in intact mitochondria as evidenced b y the comparison of Figs. 1C and 2B.
Permeability o f outer membranes to cytochrome c It is generally considered that exogenous soluble c y t o c h r o m e c cannot move across the outer mitochondrial membrane [6,15] although this point is still controversial with plant mitochondria [16]. In order to determine if the intermemhrane electron transfer observed with intact mitochondria (Figs. 1A and C) really occurs in organelles whose outer membranes have n o t been damaged, parallel experiments were performed with broken mitochondria. Table II shows that broken mitochondria have indeed a much higher succinate-cytochrome c reductase activity than intact mitochondria, indicating that the outer membrane prevents free access of c y t o c h r o m e c to t h e inner membrane. On the opposite, with N A D H as substrate, the intermembrane electron transfer induced b y addition of soluble c y t o c h r o m e c is n o t significantly different in intact and broken mitochondria. The results with the sucTABLE II EFFECT OF INTEGRITY OF THE OUTER MEMBRANE ON THE PERMEABILITY TO CYTOCHROME c AND ON THE INTERMEMBRANE ELECTRON TRANSFER Mitochondda
Intact
Brokenc
Succinate cytochrome c reductase a
NADH oxidaseb Antimycinsensitive
Antimyein-insensitive
Control
+ Cyt. c 28 20
25
220
6
350
170
5
anmoles cytoehrome c reduced, rain -1 *mg protein -1. bnmolu 02 • rain -z *mg protein -z. c M i t o c h o n d r i a were b r o k e n b y a light s o n i c a t i o n .
220
cinate-cytochrome c reductase activity indicate that c y t o c h r o m e c cannot move across the outer membrane of intact mitochondria in sufficient amounts to act as an efficient electron acceptor at the level of the inner mitochondrial membrane, in accordance with other observations [6,15]. On the other hand, as suggested b y Figs. 1A and C, c y t o c h r o m e c must nevertheless penetrate in catalytic amounts in the intermembrane space of intact mitochondria since, b y being alternatively reduced and oxidized, it is able to act as a mobile cartier b e t w e e n the N A D H - c y t o c h r o m e c reductase system of the outer membrane and the c y t o c h r o m e oxidase system of the inner membrane. DISCUSSION
A mechanism for the oxidation of extramitochondrial N A D H via a reductase located on the outer mitochondrial m e m b r a n e has been demonstrated. The intermembrane electron transfer which takes place requires the presence of a mobile carrier in the intermembrane space, for which soluble c y t o c h r o m e c seems to be the best candidate (Fig. 3). However, it cannot be excluded that direct contacts b e t w e e n the t w o mitochondrial membranes may also occur so that the electron transfer takes place in the absence of a mobile carrier, as it has been recently suggested in the case of interactions between mitochondria and microsomes [17]. This possibility is also indicated in the experiments on the reassociation o f the t w o mitochondrial membranes b y the increase in the rate o f electron transfer observed u p o n addition o f outer membranes, before c y t o c h r o m e c is added (Fig. 2A). As shown in Fig. 3, this intermemhrane electron p a t h w a y is specific for NADH. In contrast with the main p a t h w a y for NADH oxidation, which takes place through a flavin dehydrogenase (FPIM) l o c a t e d on the outer face of the
)
NADH
Cyt. C
OM IS
IM HA
_j
-c,,..c#
FP,.'-..~ b . . A ~ c " ~ Antimycin
o _1+.o~ KCN
Fig. 3. Electron transfer pathways during oxidation of extramitochondrial NADH by cauliflower mitochondria. OM: outer membrane; IM: inner membrane; IS: intermembrane space; MA: matrix. FpnM-bOM dehydrogenase complex o f the outer membrane containing both a flavoprotein and a b-type cytochrome. FPi M: dehydrogenase located on the outer face of the inner membrane; b, c, a, as: cytochrom-es of the respiratory chain.
221 inner membrane [6], this electron p a t h w a y probably includes the participation of another flavin dehydrogenase (FPoM) [6] and of a b-type c y t o c h r o m e (boM) [5], both of t h e m located on the outer membrane. Thus, this p a t h w a y appears be b o t h antimycin-insensitive and cyanidesensitive. Moreover, although it cannot account for more than 10% of the NADH oxidation in intact mitochondria, it could also act as a by-pass of the antimycin and rotenone-sensitive site, leaving but one phosphorylation site (site III) active. This c o u l d p r o v i d e an explanation for the strong resistance to inhibitors and the low P/O ratios generally observed in the oxidation of NADH by plant mitochondria [8,18]. The question also arises of whether or n o t the intermembrane electron transfer observed on isolated mitochondria can also take place in mitochondria within the cell. ACKNOWLEDGEMENTS This work was supported by a grant (RCP 223) from the Centre National de la Recherche Scientifique. REFERENCES 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18
W.D. Bonner and D.O. Voss, Nature, 191 (1961) 682. D.P. Hackett, Plant Physiol., 36 (1961) 445. T. Ohnishi, L. Kawaguehi and B. I-Iagihara,J. Biol. Chem., 241 (1966) 1797. G. Von Jagow and M. Kligenberg, Eur. J. Biochem., 12 (1970) 583. F. Moreau and C. Lance, Biochimie, 54 (1972) 1335. R. Douee, C.A. Mannella and W.D. Bonnet, Biochim. Biophys. Acta, 292 (1973) 105. I-L Ikuma and W.D. Bonnet, Plant Physiol., 42 (1967) 67. R.H. Wilson and J.B. Hanson, Plant Physiol., 44 (1969) 1335. D.D. Day and J.T. Wiskieh, Plant Physiol., 53 (1974) 104. J.A. Berden and E.C. Slater, Bioehim. Biophys. Acta, 216 (1970) 237. G.L. Sottocasa, B. Kuylenstierna, L. Er~ter and A. Bergstrand, J. Cell Biol., 32 (1967) 415. P. Nicholls, E. Mochan and H.K. Kimelberg, FEBS Letters, 3 (1969) 242. P.J. O'Brien, Mitochondrial intermembrane shuttles, in E. Quagliariello, S. Papa and C.S. Rossi (Eds.), Energy Transduction in Respiration and Photosynthesis, Adriatiea, London, 1971, p. 361. O.H. Lowry, N.J. Rosebrough, A.L. Farr and R.J. Randall, J. Biol. Chem., 193 (1951) 265. L. Wojtczak and H. Zaluska, Biochim. Biophys. Acta, 193 (1969) 64. J.M. Palmer and B.I. Kirk, Biochem. J., 140 (1974) 79. A.I. Archakov, A.V. Karlakin and V.P. Skulachev, Dokl. Aead. Nauk SSSR, 217 (1974) 1423. C. Lance, Physiol. V6g., 9 (1971) 259.