ARCHIVES

OF BlOCHEMlSTRY

AND BIOPHYSICS

Vol. 288, No. 1, July, pp. 2933301, 1991

Oxidation and Reduction of Exogenous Cytochrome by the Activity of the Respiratory Chain N. E. Lofrumento,’ Department

D. Marzulli,

of Biochemistry

and Molecular

L. Cafagno, Biology,

Received December 5, 1990, and in revised form February

G. La Piana,

and CNR Research

and T. Cipriani Unit,

University

of Bari,

Bari,

Italy

26,1991

Oxidation of exogenous NADH by isolated rat liver mitochondria is generally accepted to be mediated by endogenous cytochrome c which shuttles electrons from the outer to the inner mitochondrial membrane. More recently it has been suggested that, in the presence of added cytochrome c, NADH oxidation is carried out exclusively by the cytochrome oxidase of broken or damaged mitochondria. Here we show that electrons can be transferred in and out of intact mitochondria. It is proposed that at the contact sites between the inner and the outer membrane, a “bi-trans-membrane” electron transport chain is present. The pathway, consisting of Complex III, NADH-b5 reductase, exogenous cytochrome c and cytochrome oxidase, can channel electrons from the external face of the outer membrane to the matrix face of the inner membrane and viceversa. The activity of the pathway is strictly dependent on both the activity of the respiratory o lssl Academic PESS, IUC. chain and mitochondrion integrity.

In 1951 Lehninger reported that isolated intact mammalian mitochondria do not oxidize NADH except when they are preincubated in a hypotonic medium or when cytochrome c is added (1). In the latter condition the rotenone-insensitive NADH dehydrogenase, located on the external mitochondrial membrane, catalyzes the transfer of reducing equivalents from NADH to cytochrome c (24). However, it is still a matter of debate as to whether the exogenous cytochrome c in Lehninger’s experimental protocol is oxidized solely by the freely accessible molecules of the cytochrome oxidase of broken and/or damaged mitochondria (5), or by intact ones (3,4,6-11). We have obtained data which indicate that electrons from the exogenous ferrocytochrome c enter the inner-membrane electron transport chain of intact mitochondria and pro’ To whom the correspondence should be addressed at Department of Biochemistry and Molecular Biology, University of Bari, Via Amendola, 165/A 70126 BARI, Italy. Fax 039-80-243317. 000%9861/91 $3.00 Copyright 0 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.

c

mote the reduction of molecular oxygen. Furthermore, reducing equivalents generated inside the mitochondria by the oxidation of respiratory substrates can be utilized to reduce exogenous ferricytochrome c. We propose that an electron “bi-trans-membrane” pathway, which channels electrons through both the inner and the outer membrane, exists in rat liver mitochondria. A preliminary account of this work has already been given (22). MATERIALS

AND

METHODS

Preparation of mitochondria and incubation conditions. Liver mitochondria from male Wistar rats weighing 200-250 g were isolated in 0.25 M sucrose by a standard centrifugation procedure and washed once more for 10 min at 4000g. Mitochondrial preparations contained 0.14 nmol of cytochrome aa per milligram protein as determined by the method reported in Ref. (5). Protein was determined by the biuret method. Incubation of the mitochondrial suspension (2 mg protein/ml) was carried out at 25°C in a standard medium at pH 7.8 consisting of 220 mM sucrose, 15 mM KCl, 1 mM EDTA and 20 mM Hepes. Under these conditions the mitochondria had a respiratory control index of 9.0 and an ADP/O ratio of 1.9 with succinate (t rotenone) as substrate. The activities of both rotenone-insensitive NADH dehydrogenase (see Figs. 2 and 6Al and monoamino oxidase (121, as marker enzymes of outer mitochondrial membrane, were determined; for monoamino oxidase a value of 20 nmol benzaldehyde formed/min/mg protein was found. Oxygen consumption was determined in a thermostated and closed chamber, equipped with a Clark-type electrode. Spectrophotometric oxidation of NADH and the redox state of cytochrome c were determined using a dual wavelength HitachiiPerkinElmer spectrophotometer Model 557 at 340-374 nm (EmM = 4.28 cm- ‘1 and 548-540 nm (I&., = 21 cm-‘), respectively. Lktermination of mitochortirial integrity. Since the inner membrane of intact mitochondria is impermeable to NADH (11, this compound, added to the incubation medium, cannot be oxidized either by the rotenone-sensitive NADH dehydrogenase, which is accessible to NADH only from the inner side of the inner membrane, or by the rotenoneinsensitive NADH dehydrogenase which is spatially separated from endogenous cytochrome c and cytochrome oxidase, both of which are required for the oxidation process. Therefore the oxidation of added NADH, in the absence of rotenone, must be the expression of the amount of damaged mitochondria present in a given preparation. The integrity of the mitochondria present in our preparation was determined by the water-treatment method as follows: mitochondria (6 mg protein) were added to 1.5 ml of Hz0 and after 1 min of continuous stirring 1.5 ml of double-concentrated standard medium was added (see also Ref. (8)); in

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parallel samples mitochondria were directly incubated for 1 min in 3 ml of standard medium; 2 min later 0.2 mM NADH was added and its oxidation determined either spectrophotometrically at 340-374 nm or by oxygen uptake (in this case 1 mM NADH was added); exogenous cytochrome c was not added. As a consequence of the damage produced by water treatment exogenous NADH can penetrate the matrix space and endogenous cytochrome c is released into the incubation medium; therefore added NADH can be oxidized by the rotenone-sensitive NADH dehydrogenase and/or by the reduction-oxidation cycles of endogenous cytochrome c which is then able to react with both the rotenone-insensitive NADH dehydrogenase present on the outer membrane fragments and the cytochrome oxidase present in the inner mitochondrial membrane. Determinations made in parallel samples have shown that in water-treated mitochondria, more than 50% of endogenous cytochrome c was released into the incubation medium. The percentage of intact mitochondria present in a given preparation can be rapidly determined from the relative rate of exogenous NADH oxidation before and after water treatment (see Fig. 1). The contribution of the bi-trans-membrane electron transport process of intact mitochondria is negligible compared to that of other methods (Refs. 7, 8) and Fig. 3B). Chemicals. NADH, horse heart cytochrome c, antimycin A, FCCP,’ myxothiazol, trypsin, soybean trypsin inhibitor, and aprotinin (serine protease inhibitor from bovine lung) were purchased from Boehringer Biochemia Robin Spa (Milano, Italy). All the other reagents were of commercially available analytical grade.

RESULTS

Knowledge of the integrity of the mitochondrial preparation is a crucial point for the purpose of this paper. Figure 1 shows that in water-treated rat liver mitochondria (trace c) the oxidation rate of added NADH was increased from 2 in the control to 200 nmol/min/mg. From these values it can be calculated that 99.0% of mitochondria have both the inner and the outer membrane intact. The test of integrity carried out before starting each experiment revealed that in our mitochondrial preparations the percentage of damaged mitochondria was never higher than 1.5%. It is well known that the external mitochondrial membrane can be damaged and removed by low concentrations of digitonin (13). Consistent with this, trace b shows that in the presence of 100 pg digitonin/mg protein, the rate of NADH oxidation is only slightly increased. Such an effect can be ascribed to the release into the incubation medium of endogenous cytochrome c but not to damage of the inner membrane. Consistent with this, we found that in digitonin-treated mitochondria up to 30% of endogenous cytochrome c was released into the incubation medium. Further support to this view is given by the finding that the addition of 1 yM cytochrome c resulted in a five- to sixfold increase in the rate of NADH oxidation in both the absence and the presence of digitonin, but the same addition only increased the rate obtained with watertreated mitochondria from 200 (trace C) to 240 nmol/min/ mg protein (trace d). These results are consistent with the finding that the NADH oxidation rate, determined ’ Abbreviations phenylhydrazone;

used: FCCP, carbonyl cyanide-p-trifluoromethoxyTMPD, N,iV,N’,N’-tetramethyl-p-phenylenediamine.

t NiDH

FIG. 1. Exogenous NADH oxidation by intact and water-treated mitochondria. In traces a and b rat liver mitochondria (6 mg protein) were incubated at 25°C in 3 ml of standard medium and in the absence of rotenone. After 1 min 100 ng digitonin/mg protein were added only in trace b and the reaction started after a further 2 min with the addition of 0.2 mM NADH. In traces c and d mitochondria were added to 1.5 ml of Hz0 and after 1 min 1.5 ml of double-concentrated standard medium was added; after a further 2 min NADH alone (trace c) or together with 1 pM cytochrome c (trace d) was added. The integrity of mitochondrial preparation was determined as described under Materials and Methods. In traces a and b at the time indicated, 1 PM cytochrome c was added. The values in the figure represent the rate of NADH oxidation (nmoles of NADH oxidized/min/mg protein).

before the addition of exogenous cytochrome c, was inversely correlated to the intactness of mitochondrial membranes. As reported in (1, 4, 5) and also shown in Fig. 1, the oxidation of exogenous NADH by isolated mitochondria can be greatly increased by the addition of catalytic amounts of cytochrome c which can be considered as an obligatory intermediate in the NADH oxidation process. Therefore we utilized the redox state of added cytochrome c (Fig. 2) to determine the oxidation rate of small amounts of NADH. However since cytochrome c remains in the reduced form until NADH is completely oxidized, this method gives a measure of the rate of reactions involved in the oxidation of exogenous ferrocytochrome c. As illustrated in Fig. 2A, on the addition of 100 /.LM NADH, 95% of all the added cytochrome c undergoes a very abrupt oxidized + reduced -+ oxidized transition. The time course of each redox cycle, which can be repeated several times, is dependent on both the oxidation capacity of the system (i.e., the number of mitochondria present in the suspension) and the NADH concentration. Identical values of the rate of NADH oxidation were obtained

BI-TRANS.MEMBRANE

ELECTRON

TRANSPORT

295

CHAIN

c

cyt. CZ’

c cyt.c2*

4W 1

,54 ,58 ,62

4vM I

lrnlrl

cba NADH

t NADH

FIG. 2. Exogenous cytochrome c redox cycles induced by pulses of NADH oxidation. Rat liver mitochondria (2 mg protein/ml) were incubated at 25°C in 3 ml of standard medium with 3 FM rotenone, 20 yM cytochrome c and in (B) 100 pg digitonin/mg protein was also present. After 2 min incubation 0.1 mM NADH was added. (A and B) Additions made 30s before NADH: trace a, none; trace b, 1 mM ADP and 1 mM Pi; trace c, protein) determined on the basis 2 pM FCCP. The values in the figure represent the rate of NADH oxidation (nmoles of NADH oxidized/min/mg of the time course of each cytochrome c redox cycle. Exactly the same results were obtained when 0.8 pM antimycin A and 6 pM myxothiazol were also present in the incubation medium.

by the following three methods: cytochrome c redox cycle; oxygen consumption; absorbance decrease at 340-374 nm. The oxidation of exogenous NADH was greatly stimulated by the addition of ADP and the uncoupler FCCP as revealed by the consistent decrease in the time-span of the cytochrome c redox cycle. As shown in Fig. 2A these two compounds increased the oxidation rate by 1.6 and 3.5 times, respectively. It was found that in both the presence and the absence of ADP and FCCP, NADH oxidation was insensitive not only to rotenone but also to antimycin A and myxothiazol (see Fig. 2 and Refs. (4,5)). The stimulation induced by ADP and FCCP indicates that the oxidation of NADH is controlled by the existence of a permeability barrier to protons (14). This is consistent with the view that, mediated by the externally added cytochrome c molecules, reducing equivalents-in the form of electrons-are transferred from exogenous NADH to molecular oxygen inside the mitochondria. While it has been proven that the reduction of added cytochrome c is mediated by the rotenone-insensitive NADH dehydrogenase present on the outer mitochondrial membrane (2-4), the pathway along which electrons are transferred from exogenous ferrocytochrome c to molecular oxygen is still to be defined. The first possibility to be considered is that the cytochrome oxidase molecules of damaged mitochondria may interact with free ferrocytochrome c molecules present in the incubation medium and contribute to the oxidation of NADH; this process, however, would be insensitive to both ADP and FCCP. Even if one assumes that, in the control sample of Fig. 2A, the oxidation of NADH is mediated exclusively by the freely accessible cytochrome aa molecules, it can be calculated, from the observed increase in the oxidation rate, that in the presence of ADP and FCCP, 38% [(21-

13) X 100/21] and 71% [(46-13) X 100/46], respectively, of the reducing equivalents are transferred from extramitochondrial cytochrome c to the molecular oxygen inside the mitochondria. The possibility that this transfer could be a “one-trans-membrane” rather than a bi-transmembrane process has to be considered. Exogenous cytochrome c, reduced at the external side of the outer membrane of intact mitochondria, can be reoxidized in part by the freely accessible cytochrome oxidase molecules of the 1% damaged mitochondria (see Fig. 1) and in part, as illustrated in the scheme of Fig. 7A, by the cytochrome oxidase of those mitochondria with the inner membrane intact but with the external one freely permeable to cytochrome c. In the latter case electrons are transferred from the outer to the inner side of the inner membrane and this process would be the one stimulated by ADP and FCCP. However, such a possibility is not consistent with the findings reported in Fig. 2B which show that by increasing the permeability of the external membrane by digitonin-treatment (Fig. 1 and Ref. (13)), the rate of NADH oxidation was increased but the stimulation brought about by ADP and FCCP was almost completely lost. The results of Fig. 2 are consistent with the finding that cytochrome c, both in mammalian and plant mitochondria, is not able to permeate the outer membrane (35, 7, 8, 11, 13). Even if at first glance and on the basis of the current point of view it is difficult to accept, the possibility that electrons can be transferred from the outside to the inside of intact mitochondria and utilized for the reduction of molecular oxygen must be considered. This transfer may take place along the chain consisting of: exogenous NADH; rotenone-insensitive NADH dehydrogenase; exogenous cytochrome c molecules reduced by NADH-b,,

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system and bound to the outer membrane; and the cytochrome oxidase functionally linked to the inner mitochondrial membrane. In order for this process to take place it is not necessary to invoke a shuttle function for the endogenous cytochrome c molecules (6); the bi-transmembrane electron channeling system can utilize the bridges set up by the contact points between the outer and the inner mitochondrial membrane (15,16). Very recently it has been shown that purified fractions of “contact sites” from mouse liver are characterized by the presence of both monoamine oxidase and cytochrome c oxidase activities (17). The idea that the bi-trans-membrane electron transport chain may transfer electrons not only from outside the mitochondria to inside, but also in the reverse direction, has been substantiated by the experiments reported in Fig. 3A. They show that redox cycles of exogenous cytochrome c can be induced by pulses of reducing equivalents generated inside the mitochondria by the oxidation of small amounts of succinate. Trace a shows that, in the course of the oxidation of 0.2 mM succinate, added cytochrome c is first 40% reduced and then reoxidized. In experiments not reported here the reduction of exogenous cytochrome c was also obtained in the presence of piruvate or malate + glutamate, but in this case a concentration higher than 0.5 mM of each respiratory substrate, was required. Traces b-d show that the incubation of mitochondria with increasing concentrations of digitonin from 33 to 100 pg/mg protein, promoted a progressive decrease

ET AL.

in the reduction level of exogenous cytochrome c. The reduction of added cytochrome c was very low in mitochondria treated with 100 pg digitoninlmg protein (trace d) and almost completely absent in water-treated ones (trace e). Trace f shows that simply increasing the preincubation time from 2 to 10 min before the addition of succinate decreases the reduction level of exogenous cytochrome c. Oxygen consumption determined in parallel incubations showed that in all the conditions reported in Fig. 3A the rate of succinate oxidation remained constant except in trace e where a higher rate was obtained. This indicates that the flow of reducing equivalents from succinate to oxygen is not influenced by digitonin, whereas the reduction of exogenous cytochrome c becomes quantitatively less pronounced or completely disappears as the mitochondrial membranes become increasingly damaged. These results are not in agreement with the assumption, also illustrated in the scheme of Fig. 7A, that with succinate as the donor of reducing equivalents, the reduction of added cytochrome c would occur exclusively at the external side of the inner membrane of those mitochondria with a damaged external membrane. According to this assumption it would be expected that by promoting the damage of the external membrane of intact mitochondria, still present in our preparation, and/or increasing the permeability of the mitochondria already damaged, the extent of the reduction of exogenous cytochrome c would be further increased; the results reported in Fig. 3A show that the opposite is the case. This suggests that the oxi-

d A

t cyt c2+ 1.5 VM

conditions were those reported in Fig. FIG. 3. Exogenous cytochrome c redox cycles induced by pulses of succinate oxidation. Experimental 2A. Before the addition of 0.2 mM succinate (Succ), the mitochondria ((A) 2 mg protein/ml; (B) 0.2 mg protein/ml) were preincubated for 2 min. (A) of 33 (trace b), 66 (trace c), and 100 pg/mg protein (trace d), was present in the incubation medium; trace a, \.., Dieitonin. ~o...~~~~~,at a concentration control sample; in trace e, mitochondria were water treated with the same procedure used in trace c of Fig. 1; in trace f the preincubation time was 10 min. (B) Cyanide (1 mM) was also present in the incubation medium; trace a, control sample; trace b, 10.min preincubation; trace c, digitonin 100 pg/mg protein; trace d, water-treated mitochondria. The values in the figure represent the reduction rate of cytochrome c (nmoles/ min/mg protein).

BI-TRANS-MEMBRANE

ELECTRON

dation but not the reduction rate of exogenous cytochrome c is greatly increased in damaged mitochondria. Figure 3B shows the results obtained when the experiment in Fig. 3A was carried out in the presence of 1 mM cyanide. In this case the activity of succinate exogenous cytochrome c oxidoreductase was determined. It has been proposed that such an activity be used as a parameter to evaluate the integrity of mitochondrial preparations (7). The added cytochrome c was then 99.5% reduced and the reduction rate obtained increased from 8 in the control (trace a) to 14 nmol/min/mg in the presence of 100 pg digitonin/mg protein (trace c); with osmotically shocked mitochondria (trace d) the rate obtained was 90 nmol/ min/mg. From these values it can be calculated that the permeability of mitochondria is increased from 9% in the control (trace a) to 15% in the presence of digitonin (trace c), assuming a 100% value for water-treated mitochondria (trace d). The apparent discrepancy between the 9% permeability obtained for the control sample in Fig. 3B and the 1% in Fig. 1 is explained by the fact that the 9% value, in addition to the activity of damaged mitochondria, contains also the rate of electron transfer from succinate to exogenous cytochrome c occurring in intact mitochondria and mediated by the bi-trans-membrane system. The 1% value, on the other hand, is the expression of damaged mitochondria only, since intact ones are expected to be unable to oxidize exogenous NADH. The results reported in Fig. 3 show that there is a direct correlation between the increase in the permeability of mitochondrial membranes and the decrease in the level of exogenous cytochrome c reduction state. This last process appears to be so strictly dependent on mitochondrion integrity that a slight increase in the permeability from 9 to 11% , obtained by prolonging the preincubation time up to 10 min (Fig. 3B: trace b), gives a 45% decrease in the reduction level of cytochrome c (compare traces f and a in Fig. 3A). This last observation is consistent with the results reported in Fig. 4 which show that the cytochrome c redox cycles, promoted by the succinate oxidation, are prevented or abolished by ADP, FCCP, antimycin A, or myxothiazol depending on whether they are added before or after succinate. This suggests that the respiratory chain of intact mitochondria is involved in the reduction process of exogenous cytochrome c. It is well known that ADP and FCCP greatly increase the turnover of each respiratory component in intact mitochondria; thus electrons are forced to flow to the oxygen and therefore cannot be transferred outside for the reduction of exogenous cytochrome c molecules. Antimycin and myxothiazol, interacting with the b-c1 Complex, inhibit one of the first steps on the substrate side of the inner-membrane electron transport pathway, so that both the oxygen uptake and the reduction of extramitochondrial cytochrome c are blocked. This indicates that Complex III may function as the branching point for electron flow to oxygen or to the external mitochondrial compartment. This is further

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CHAIN

CAodptrO’ f

FCC P

I

FIG. 4. Exogenous cytochrome c redox cycles induced by pulses of succinate oxidation: effect of respiratory chain inhibitors and activators. Experimental conditions were those reported in Fig. 2A. Before the first addition of 0.2 mM succinate (Succ), mitochondria were preincubated for 2 min. At the time indicated by the arrows the following additions were made: 1 mM ADP (+ 1 mM Pi) or 2 FM FCCP or 0.8 pM antimycin A (AA) or 6 FM myxothiazol (Myxo); subsequently, when the first cytochrome c redox cycle was completed, 1.5 mM KCN was added in all cases, followed by a second “pulse” of 0.2 mM succinate (Succ).

supported by the results, also reported in Fig. 4, which show that once the first redox cycle has been completedthat is, when the small amount of added succinate (0.2 mM) has been completely oxidized-and the activity of cytochrome aa has been blocked by cyanide, all the cytochrome c present in the incubation medium becomes completely reduced on the subsequent addition of 0.2 mM succinate. Under these conditions, the ox-red-ox cycle is not obtained and as expected, ADP and FCCP have no effect and furthermore both antimycin and myxothiazol completely prevent the reduction of exogenous cytochrome c. The sensitivity of the cytochrome c redox cycle to both the activators and the inhibitors of the respiratory chain, reported in Fig. 4, also indicates that only the activity of intact mitochondria succinate dehydrogenase can contribute to the reduction of exogenous cytochrome c. Additional evidence that electrons can be transferred from the inside to the outside of mitochondrial compartments is given by the results shown in Fig. 5. To increase the capacity of electron flow along the bi-trans-membrane chain, the activity of the cytochrome c oxidase was partially inhibited with 0.5 mM azide. Under these conditions all the exogenous cytochrome c molecules were involved in the redox cycles which were induced by pulses of reducing equivalents from inside the mitochondria. Butylmalonate, a well-known inhibitor of the carrier of dicarboxylic acids (18), prevented or completely reversed the reduction of cytochrome c depending on whether it was added before or after succinate. These results demonstrate

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.yL+ .yL+ ~PM ~PM 1 1 3min

FIG. 5. Inhibitory effect of butylmalonate on the exogenous cytochrome c redox cycles induced by pulses of succinate oxidation. Experimental conditions were those reported in Fig. 2A except that 0.5 mM azide was also present in the incubation medium and the concentration of cytochrome c was 5 pM. Mitochondria were preincubated for 2 min before the addition of 0.2 mM succinate (Succ). As indicated by the (BM) was added after (trace b) or before arrows, 5 mM butylmalonate (trace c) the succinate.

that in order to reduce exogenous cytochrome c, succinate must first penetrate intact mitochondria. The finding that in the presence of butylmalonate no redox cycle of cytochrome c is observed gives further support to the data reported in Figs. 3A and 4; i.e., succinate dehydrogenase present in mitochondrial fragments or damaged mito-

ET AL.

chondria does not contribute to the redox cycle of exogenous cytochrome c. Further support to the existence of the bi-trans-membrane electron transport system is given by the results of experiments reported in Fig. 6. Trace (b) of Fig. 6A shows that the rate of NADH oxidation was 73% inhibited in trypsin-treated mitochondria, as can be calculated from the increase in the time-course of the cytochrome c redox cycle. Until NADH was completely oxidized, only 24% of added cytochrome c remained in the reduced state compared to the 95% in the control sample (trace a). These results are consistent with the well documented findings (19) that trypsin readily deactivates the rotenone-insensitive NADH-cytochrome c oxidoreductase system. They also suggest that trypsin inhibits the reduction of cytochrome c but may have no effect on its oxidation process. Among the many well-known protein inhibitors (for review see Ref. (20)), we have used the soybean trypsin inhibitor and the bovine lung trypsin inhibitor (aprotinin) to prevent or abolish the proteinase activity of trypsin. The data obtained indicate that the inhibitory effect of trypsin was linked to its proteinase activity as already observed in Ref. (19) and directly shown by the finding that trypsin, pretreated with an excess of soybean inhibitor, had no effect on the cytochrome c redox cycle (trace a). As shown in trace c NADH oxidation was inhibited by 84% when mitochondria were incubated in the presence of trypsin-aprotinin complex. However in this case the exogenous cytochrome c was rapidly and almost completely reduced in a manner similar to that in the control sample, suggesting that trypsin-aprotinin complex inhibited the oxidation of cytochrome c but not its reduction catalyzed by NADH-cytochrome c oxidoreductase. No ef-

FIG. 6. Effect of trypsin and trypsin-aprotinin complex on the exogenous cytochrome c redox cycles induced by pulses of both NADH and succinate oxidation. Rat liver mitochondria (2 mg protein/ml) were incubated at 25°C in 3 ml of standard medium with 3 PM rotenone. Before the addition of 10 pM (A) and 20 PM (B) cytochrome c, mitochondria were preincubated for 3 min in the presence of the following additions: traces a, alternatively none, 40 pg aprotinin/mg protein, 500 pg soybean inhibitor/mg protein, and 100 gg trypsin + 500 pg soybean inhibitor/mg protein; traces b 100 pg trypsin/mg protein; traces c, 100 pg trypsin + 40 jrg aprotinin/mg protein; trypsin + soybean inhibitor and trypsin + aprotinin were added to the incubation medium 5 min before the mitochondria to promote the formation of the protease-inhibitor complex. (A) NADH (45 FM) and (B) 0.2 mM succinate (Succ) were added, 1 min after the addition of cytochrome c.

BI-TRANS.MEMBRANE

ELECTRON

feet at all was observed in the presence of aprotinin (again see trace a). In experiments not reported here, it has been found that the oxidation of exogenous ferrocytochrome c, in the absence of any reducing system, was inhibited by the trypsin-aprotinin complex but not by aprotinin or by trypsin alone. Consistent with the results reported in Fig. 6A we found that oxygen uptake, supported by exogenous NADH oxidation, was inhibited by both trypsin and trypsin-aprotinin complex but not by aprotinin. We also confirmed the finding reported in Ref. (19) that oxygen uptake by succinate oxidation was not affected by trypsin and found that this is also true when mitochondria were treated with trypsin-aprotinin complex, trypsin-soybean inhibitor complex, aprotinin, and soybean inhibitor. This suggests that inner membrane activities involved in the succinate oxidation are resistant to all these compounds. Figure 6B shows that aprotinin, soybean, and trypsinsoybean complex had no effect (trace a) but trypsin alone (trace b) decreased the redox cycle of exogenous cytochrome c supported by succinate oxidation. In the presence of trypsin-aprotinin complex the reduction level as well as the time course of the redox cycle were both greatly increased (trace c). The effect of trypsin can be ascribed to a decrease in the rate of succinate-exogenous cytochrome c electron transfer, whereas the effect of trypsinaprotinin complex can be explained by an inhibition of exogenous cytochrome c oxidation. Altogether the results reported in Fig. 6 strongly suggest that trypsin-aprotinin complex is a rather specific inhibitor of the exogenous cytochrome c oxidation and that trypsin inhibits the reduction of exogenous cytochrome c supported by both succinate and exogenous NADH oxidation. Evidence has been reported suggesting that intact mitochondrial outer membrane is impermeable to trypsin (19). Therefore we may assume that the same is true for trypsin-aprotinin complex. This observation together with the finding that in succinate oxidation, trypsin and trypsin-aprotinin had no effect on oxygen uptake (not shown), but affected the exogenous cytochrome c redox cycle, further substantiates the view that the bi-transmembrane electron transport chain can be considered a branch pathway, which is not involved in the succinate oxidation supported by the one-trans-membrane respiratory chain activity. DISCUSSION

The data we obtained indicate that, with isolated rat liver mitochondria and in the presence of exogenous ferricytochrome c, reducing equivalents, in the form of electrons, can be transferred from exogenous NADH to the inside of the mitochondria and utilized by the cytochrome c oxidase for the reduction of molecular oxygen. The pathway does not appear to be unidirectional since electrons can also be transferred from inside to outside mi-

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tochondria. The existence of a bi-trans-membrane electron transport pathway which channels electrons in and out of intact mitochondria through both the inner and the outer membrane is supported by the following observations: (i) exogenous NADH is only poorly oxidized by intact mitochondria unless a catalytic amount of cytochrome c is added (Refs. (1,4,5), Fig. 1); (ii) the oxidation of exogenous NADH mediated by exogenous cytochrome c is greatly stimulated when the electrochemical membrane potential is dissipated by the addition of ADP (+Pi) or the uncoupler FCCP (Fig. 2A); (iii) exogenous ferricytochrome c is reduced by electrons originating from succinate oxidation which occurs at the inner side of inner membrane (Figs. 3A, 4, 5, and 6B); (iv) the inhibition by butylmalonate of succinate transport from outside to inside the mitochondria prevents or abolishes the redox cycle of exogenous cytochrome c (Fig. 5); (v) antimycin and myxothiazol have no effect on the redox cycles of exogenous cytochrome c induced by the addition of NADH (Fig. 2A) but inhibit completely those cycles promoted by succinate oxidation (Fig. 4); (vi) the removal of the external membrane permeability barrier by preincubation with digitonin (Ref. (13), Fig. 1) increases the rate of NADH oxidation and abolishes the stimulatory effect of ADP and FCCP (Fig. 2B); (vii) the digitonin treatment also abolishes the redox cycles of exogenous cytochrome c induced by succinate oxidation (Fig. 3A); (viii) in cyanide-inhibited respiration the low rate of exogenous cytochrome c reduction by succinate is only slightly increased by digitonin treatment compared to the high value obtained after water treatment (Fig. 3B); (ix) the redox cycle of exogenous cytochrome c induced by succinate oxidation is almost completely absent in mitochondria damaged by water-treatment (Fig. 3A); (x) as expected, from the activity of the succinate exogenous cytochrome c oxidoreductase (Fig. 3B) a higher percentage of damaged mitochondria has been calculated since with this method the rate of electron transfer from inside to outside intact mitochondria is also determined; (xi) trypsin as a nonpenetrant compound (19) inhibited the reduction of exogenous cytochrome c supported by both NADH (Ref. (19), Fig. 6A) and succinate oxidation (Fig. 6B); (xii) trypsin-aprotinin complex inhibited the oxidation of exogenous cytochrome c maintained in its reduced state either by NADH or by succinate oxidation (Fig. 6). Observations (ii), (iv), (vi), (vii), (ix), (x), (xi), and (xii) are not consistent with the possibility, illustrated in Fig. 7A, that the redox cycles of exogenous cytochrome c promoted either by NADH or by succinate oxidation are due solely to the small fraction of completely damaged mitochondria (not higher than 1.5%, according to the integrity test of Fig. 1) and/or to those with an outer membrane damaged to an extent which is just sufficient to make it permeable to cytochrome c. It should also be noted that cytochrome oxidase molecules either derived from damaged mitochondria or still present in the intact inner

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ET AL.

02

NADH

NADH

FIG. 7. Proposed mechanisms for the oxidation of exogenous cytochrome e in isolated rat liver mitochondria. (A) Endogenous cytochrome c (C.), present in the intermembrane space (IMS), can shuttle electrons between Complex III (b-c,) and cytochrome oxidase (a-a,) or between exogenous cytochrome c (Co) [bound to NADH-b5 reductase (NADH-b5) and in equilibrium with the molecules present outside the mitochondria] and cytochrome oxidase. In mitochondria with the external membrane damaged or broken and the inner one intact, exogenous ferrocytochrome c is oxidized directly by the cytochrome oxidase. (B) One contact site between the outer and the inner membrane is shown. Complex III, in contact with NADH-bS reductase, mediates, through Complex I or II (I/II), the transfer of electrons from reduced respiratory substrates (SH,) present inside the mitochondria, to the exogenous cytochrome c bound to the NADH-b5 system. Furthermore the cytochrome oxidase of the contact site (17) can reduce molecular oxygen by accepting electrons directly from exogenous ferrocytochrome c, one of its two binding sites being freely accessible from the outside. At the contact sites, electrons can flow in and out of the mitochondria through a “bi-trans-membrane” pathway, the components of which are in part located in the inner membrane and in part in the outer membrane. Endogenous cytochrome c may not be involved in the oxidation of exogenous cytochrome c by cytochrome oxidase. IM, inner mitochondrial membrane; OM, outer mitochondrial membrane; e, electron flow.

membrane, when interacting with cytochrome c would so rapidly catalyze its oxidation that no redox cycles could be observed. Indeed, in digitonin-treated and watertreated mitochondria, the cytochrome c redox cycles, induced by “pulses” of succinate oxidation, are greatly decreased (Fig. 3A). As a working hypothesis it is suggested that the bitrans-membrane pathway may be situated at the contact sites which have been shown to be present between the inner and outer membranes (15-17). As illustrated in Fig. 7B, it is proposed that at the contact sites, the b-c1 complex of the inner membrane may be functionally linked to the NADH-cytochrome b5 reductase so that electrons can be transferred from succinate to the cytochrome c present outside the mitochondria. Cytochrome oxidase molecules present in the contact sites (17) could also be directly linked to the exogenous cytochrome c by means of a binding site which, in agreement with the existence on the oxidase of two distinct binding sites (21), need not be the same as that utilized by the endogenous cytochrome c molecules. Therefore cytochrome oxidase may catalyze the oxidation of exogenous cytochrome c without the involvement of endogenous cytochrome c. In the scheme of Fig. 7B it is tentatively proposed that the pathway of electron flow from inside to outside may be not the same as when the flow occurs in the opposite direction. The involvement of different components of the outer membrane in inward and outward electron flow, respectively, is suggested by the differential inhibitory effects of trypsin and trypsin-aprotinin complex on cytochrome c redox cycles. In the presence of electron carriers other than

added cytochrome c, the oxidation of exogenous NADH may or may not occur at the contact sites and in both cases endogenous cytochrome c may be involved. Such a possibility would be consistent with the findings reported by Wikstrom and Casey (5) which showed that on anaerobiosis all endogenous cytochrome aa was rapidly (within 10s) reduced when TMPD was used as redox mediator for the oxidation of NADH; with exogenous cytochrome c as electron carrier only 10% was rapidly reduced and the remaining 90% of cytochrome aa was extremely slowly reduced. From the data reported in Figs. 1 and 2 it emerges that the rate of electron transport from outside to inside mitochondria is very low and is strictly dependent on the amount of exogenous cytochrome c. This indicates that the bi-trans-membrane electron transport process does not interfere with the activity of the respiratory chain, thus agreeing with the findings (not reported here) that the rate of succinate oxidation by intact mitochondria is not influenced by the addition of 20 PM cytochrome c. In epatocytes the bi-trans-membrane electron transport chain may have the function of providing a link between the redox processes occurring in the mitochondrial compartment and those of the cytosol without affecting the energy conservation capacity of the cell. The data obtained show that very few nmoles of cytochrome c, present at the external side of the outer mitochondrial membrane, are required to catalyze such an activity. ACKNOWLEDGMENTS The authors are grateful to Mr. L. Gargano for his technical assistance.

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