Biochimica et Biophysica Acta, 1140 (1992) 169-174
169
© 1992 Elsevier Science Publishers B.V. All rights reserved 0005-2728/92/$05.00
BBABIO 43725
The effect of A H+ on the interaction of rotenone with Complex I of submitochondrial particles Alexander B. Kotlyar and Menachem Gutman Laser Laboratory for Fast Reactions in Biology, Department of Biochemistry, Tel Aviv University, Tel Aviv (Israel)
(Received 13 April 1992)
Key words: NADH-ubiquinone reductase; Respiratory chain; Electron transfer; Rotenone inhibition; (Bovine heart mitochondrion) The inhibition by rotenone of the forward (NADH-oxidase) and reverse (AtzH+-dependent succinate-NAD ÷ reductasc activities of submitochondrial vesicles was measured. The inhibition of NADH-oxidase, measured in the presence of uncoupler, followed a monophasic inhibition curve with K i ~_" I£, 60"
_I'--
40
20 ? 0
10
20
30
ROTENONE, I0 - 8 M F i g • 5 • ] ~h lbi~ ion by r o t e n o n e o f the N ~ H I
o x i d ase activity m e as I
ured with coupled and uncoupled submitochondrial vesicles. Curve 1: respiring particles were preincubated with rotenone for 4-8 min (until steady respiration was established) and the enzymatic activity was measured immediately after addition of gramicidin plus BSA to the reaction mixture (see Fig. 4). Curve 2: the steady rate of NADHoxidase was measured with uncoupled (by gramicidin) vesicles in presence of rotenone. 100% activity corresponds to 0.9 ~mol of NADH oxidized per min per mg protein.
the reaction mixture. Thus the uncoupler, which abolished zl~Ia., increased the sensitivity of the enzyme to rotenone. If the experiment is conducted in the presence of BSA, which sequesters all free rotenone, the rate measured in the presence of uncoupler is linear with time. Albumin added alone to the coupled rotenone-inhibited particles has no effect on the initial rate of respiration. This type of experiment was repeated with various rotenone concentrations and the results are summarized in Fig. 5. As seen in curve 1, in the coupled conditions about 15% of NADH-oxidase activity is insensitive to low rotenone concentrations and inhibition requires much higher concentrations. Addition of uncoupler to reaction medium leads to the transformation of the biphasic pattern of inhibition to a monophasic, highly sensitive one (see Fig. 5, curve 2), similar to that shown in Fig. 2, curve 2.
The effect of AIZH + on the affinity for rotenone The effect of A~.+ on affinity for rotenone may be due to a slower rate of rotenone binding or to the increased rate of dissociation. To evaluate the mechanism of this process we monitored the rate of rotenone dissociation from the enzyme. A concentrated suspension of particles (2 mg/ml) was preincubated in the presence of NADH with stoichiometric concentrations of rotenone. After equilibration, the samples were diluted in a reaction mixtures containing BSA and substrates. The catalysis was monitored over time, looking for the incremental rate due to the dissociation of
rotenone and its trapping by BSA. The results are summarized in Fig. 6. The release of NADH-oxidase (in presence of uncoupler) is a simple first-order reaction. On the other hand, the activation of the reverse reaction is biphasic. Approx. 70% of enzymatic activity is being liberated during a short time period (within 1 min) while the residual activity is being recovered much more slowly, the rate of both reactions reaching the level of a control sample after 15-20 min. Rapid activation of NAD +-reductase reaction is attributed to the rapid dissociation of rotenone from the enzyme in the presence of high electrochemical potential. The diminished affinity for rotenone detected in the coupled system may be due to the reduction of the quinone pool under a state of respiratory control. This possibility seems quite probable because the target of rotenone binding is located at or near the Q-binding site of the enzyme [17,19,28]. To check whether the redox state of ubiquinone has an effect on the rotenone binding we measured the sensitivity of uncoupled vesicles to rotenone when the ubiquinone pool is completely reduced. The particles were inhibited by KCN (1 mM) and mixothiazol (0.1 /xg/ml) to impose a total block of quinol oxidation, and the quinone pool was reduced by NADH (100 /xM)+ succinate (20 mM). The reduced vesicles were incubated for 4 min with rotenone; the reaction was initiated by addition of Qo and initial rate of NADH oxidation was measured
80 1 60
:2 ~
40
.
_-
100¢
Discussion
80
The recognition that the affinity of Complex I to rotenone varies with the magnitude of A/.~H+ is instrumental to understanding of the biphasic titration curves as shown in Figs. 3 and 5. To explain this phenomenon we assume that in uncoupled vesicles the sensitivity of Complex I is much higher (K i ~
60 o
1-40
2O
0
2
4
ROTENONE,
6
8
I0
I 0 -8 M
Fig. 7. Inhibition by rotenone of the NADH:Q-reductase measured with reduced and oxidized submitochondrial particles. (o) SMP (25 /~g/ml) were preincubated with rotenone in a reaction mixture containing: 0.25 M sucrose, 1 mg/ml BSA, 0.2 mM EDTA, 100 ,~M NADH, 20 mM succinate, 2 /~g/ml gramicidin, 1 mM CN-, 0.1 ~ g / m l mixopthiazol, 20 mM Hepes (potassium salts; pH 8.0) for 4 min at 30°C. NADH-Q reductase reaction was started by the addition of 400 ~M Qo and the initial rate of NADH oxidation was monitored. (e) the vesicles were treated with rotenone as above except that only Qo (400 /~M) was present during the incubation period.
(open symbols). In a parallel experiment, the incubation of the enzyme with rotenone was in presence of Qo (400 /xM) which lowered the reduction level of ubiquinone pool. The sensitivity to rotenone of the two systems is given in Fig. 7. In both cases the inhibition is established in the nanomolar range, independent on the redox state of the quinone, following a curve typical for uncoupled preparation (see Fig. 3, curve 2). Thus we conclude that reduction of the endogenous quinone in the membrane does not induce the state of low affinity, which is established only under conditions of high A/xr~+. TABLE I
Catalytic properties of the submitochondrial particle populations Sample
Particles a as prepared Population 1 Population 2
Sensitivity to rotenone
NADH-oxidase b activity (%)
Succinate_NAD + c reductase activity (%)
Respiratory d control
Low High
100 12 88
100 60 40
5.5 20 < 5.5
a Particles were pretreated with oligomycin (0.4/.~gfml), b NADH-oxidase reaction was assayed in the presence of gramicidin as described in Materials and Methods; 100% corresponds to the specific activity of 1.2/.tmole of NADH oxidized per min per mg of protein, c The energy-linked reduction of NAD + was measured as described in Materials and Methods; 100% corresponds to the specific activity of 0.14 /zmol of NAD + reduced per min per mg of protein, d Respiratory control is calculated as a ratio between the rates of NADH oxidation after and before the addition of gramicidin (see Fig. 4 curve 2).
174 ried out in absence of added cytochrome c, the contribution of these populations to the overall rate is negligible and cannot be identified with the less coupled population. The fact that rotenone binding is controlled by Atzn+ is an indication for a major change in the enzyme structure which follows the build-up or collapse of A/.~H+.The energy-rich state, having low affinity to rotenone, is effective in binding of the quinol molecule and its subsequent oxidation by Fe-S clusters. In contrast, the highly rotenone-sensitive non-energized state of enzyme is functioning during oxidation of NADH. In our opinion, stabilization of Complex I by rotenone in one of its conformation states may be the mechanism of inhibition.
Acknowledgements This research is supported by the US Navy N0001489-J 1622, the Israeli Ministry for Science and Technology and Koret Foundation. A.B.K. is a post-doctoral fellow of the Koret Foundation.
References 1 Hatefi, Y. and Rieske, J.S. (1967) Methods Enzymol. 10, 235-239. 2 Orme-Jonson, N.R., Hansen, R.E. and Beinert, H. (1974) J. Biol. Chem. 249, 1922-11927. 3 Albracht, S.P.J. and Subramaniam, J. (1977) Biochim. Biophys. Acta 462, 36-48. 4 Ohnishi, T. (1979) in Membrane Proteins in Energy Transduction (Capaldi, R.A., ed.), pp. 1-87, Marcel Dekker, New York. 5 Ingledew, W.J. and Ohnishi, T. (1980) Biochem. J. 186, 111-117. 6 Schatz, G. and Racker, E. (1966) J. Biol. Chem. 241, 1429-1438.
7 Lawford, H.G. and Garland, P.B. (1971) Biochem. J. 130, 10291044. 8 Ragan, C.I. and Racker, E. (1973) J. Biol. Chem. 248, 2563-2569. 9 Ragan, C.I. and Hinkle, P.C. (1975)J. Biol. Chem. 250, 8472-8480. 10 Ruzicka, F.J. and Crane, F.I. (1971) Biochim. Biophys. Acta 226, 221-223. 11 Klingenberg, M. and Slenczka, W. (1959) Biochem. Z. 331, 486517. 12 Chance, B. and Hollunger, G. (1960) Nature 185, 666-672. 13 Low, H. and Vallin, I. (1963) Biochim. Biophys. Acta 69, 361-374. 14 Hommes, F.A. (1963) Biochim. Biophys. Acta 77, 183-190. 15 Chance, B. and Hollunger, G. (1961) J. Biol. Chem. 236, 15341543. 16 Kotlyar, A.B. and Vinogradov, A.D. (1990) Biochim. Biophys. Acta 1019, 151-158. 17 Horgan, D.J., Ohno, H., Singer, T.P. and Casida, J.E. (1968) J. Biol. Chem. 243, 5967-5976. 18 Horgan, D.J. and Singer, T.P. (1967) Bicohem. J. 104, 50c-52c. 19 Horgan, D.J., Singer, T.P. and Casida, ,I.E. (1968) J. Biol. Chem. 243, 834-843. 20 Singer, T.P. and Gutman, M. (1971) Adv. Enzymol. 34, 79-153. 21 Bois, R. and Estabrook, R.W. (1969) Arch. Biochem. Biophys. 129, 362-369. 22 Van Belzen, R., Van Gaalen, M.C.M., Cuypers, P.A. and AIbracht, S.P.J. (1990) Biochim. Biophys. Acta 1017, 152-159. 23 Singer, T.P., Horgan, D.,I. and Casida, J.E. (1968) in Flavins and Flavoproteins (Yagi, K., ed.), pp. 192-215, University of Tokyo Press, Tokyo. 24 Gutman, M., Singer, T.P., Beinert, H. and Casida, J.E. (1970) Proc. Natl. Acad. Sci. USA 65, 763-770. 25 Ernster, L., Dallner, G. and Azzone, G.F. (1963) J. Biol. Chem. 238, 1124-1131. 26 Ohnishi, T. (1973) Biochim. Biophys. Acta 301, 105-128. 27 Gornall, A.G., Bardawill, C.S. and David, M.M. (1949) J. Biol. Chem. 177, 751-766. 28 Gutman, M., Coles, C.J., Singer, T.P. and Casida, J.E. (1971) Biochemistry 10, 2036-2043. 29 Huang, C.H., Keyhani, E. and Lee, C.P. (1973) Biochim. Biophys. Acta 305, 455-473. 30 Huang, C.H. and Lee, C.P. (1975) Biochim. Biophys. Acta 376, 398-414.
169
© 1992 Elsevier Science Publishers B.V. All rights reserved 0005-2728/92/$05.00
BBABIO 43725
The effect of A H+ on the interaction of rotenone with Complex I of submitochondrial particles Alexander B. Kotlyar and Menachem Gutman Laser Laboratory for Fast Reactions in Biology, Department of Biochemistry, Tel Aviv University, Tel Aviv (Israel)
(Received 13 April 1992)
Key words: NADH-ubiquinone reductase; Respiratory chain; Electron transfer; Rotenone inhibition; (Bovine heart mitochondrion) The inhibition by rotenone of the forward (NADH-oxidase) and reverse (AtzH+-dependent succinate-NAD ÷ reductasc activities of submitochondrial vesicles was measured. The inhibition of NADH-oxidase, measured in the presence of uncoupler, followed a monophasic inhibition curve with K i ~_" I£, 60"
_I'--
40
20 ? 0
10
20
30
ROTENONE, I0 - 8 M F i g • 5 • ] ~h lbi~ ion by r o t e n o n e o f the N ~ H I
o x i d ase activity m e as I
ured with coupled and uncoupled submitochondrial vesicles. Curve 1: respiring particles were preincubated with rotenone for 4-8 min (until steady respiration was established) and the enzymatic activity was measured immediately after addition of gramicidin plus BSA to the reaction mixture (see Fig. 4). Curve 2: the steady rate of NADHoxidase was measured with uncoupled (by gramicidin) vesicles in presence of rotenone. 100% activity corresponds to 0.9 ~mol of NADH oxidized per min per mg protein.
the reaction mixture. Thus the uncoupler, which abolished zl~Ia., increased the sensitivity of the enzyme to rotenone. If the experiment is conducted in the presence of BSA, which sequesters all free rotenone, the rate measured in the presence of uncoupler is linear with time. Albumin added alone to the coupled rotenone-inhibited particles has no effect on the initial rate of respiration. This type of experiment was repeated with various rotenone concentrations and the results are summarized in Fig. 5. As seen in curve 1, in the coupled conditions about 15% of NADH-oxidase activity is insensitive to low rotenone concentrations and inhibition requires much higher concentrations. Addition of uncoupler to reaction medium leads to the transformation of the biphasic pattern of inhibition to a monophasic, highly sensitive one (see Fig. 5, curve 2), similar to that shown in Fig. 2, curve 2.
The effect of AIZH + on the affinity for rotenone The effect of A~.+ on affinity for rotenone may be due to a slower rate of rotenone binding or to the increased rate of dissociation. To evaluate the mechanism of this process we monitored the rate of rotenone dissociation from the enzyme. A concentrated suspension of particles (2 mg/ml) was preincubated in the presence of NADH with stoichiometric concentrations of rotenone. After equilibration, the samples were diluted in a reaction mixtures containing BSA and substrates. The catalysis was monitored over time, looking for the incremental rate due to the dissociation of
rotenone and its trapping by BSA. The results are summarized in Fig. 6. The release of NADH-oxidase (in presence of uncoupler) is a simple first-order reaction. On the other hand, the activation of the reverse reaction is biphasic. Approx. 70% of enzymatic activity is being liberated during a short time period (within 1 min) while the residual activity is being recovered much more slowly, the rate of both reactions reaching the level of a control sample after 15-20 min. Rapid activation of NAD +-reductase reaction is attributed to the rapid dissociation of rotenone from the enzyme in the presence of high electrochemical potential. The diminished affinity for rotenone detected in the coupled system may be due to the reduction of the quinone pool under a state of respiratory control. This possibility seems quite probable because the target of rotenone binding is located at or near the Q-binding site of the enzyme [17,19,28]. To check whether the redox state of ubiquinone has an effect on the rotenone binding we measured the sensitivity of uncoupled vesicles to rotenone when the ubiquinone pool is completely reduced. The particles were inhibited by KCN (1 mM) and mixothiazol (0.1 /xg/ml) to impose a total block of quinol oxidation, and the quinone pool was reduced by NADH (100 /xM)+ succinate (20 mM). The reduced vesicles were incubated for 4 min with rotenone; the reaction was initiated by addition of Qo and initial rate of NADH oxidation was measured
80 1 60
:2 ~
40
.
_-
100¢
Discussion
80
The recognition that the affinity of Complex I to rotenone varies with the magnitude of A/.~H+ is instrumental to understanding of the biphasic titration curves as shown in Figs. 3 and 5. To explain this phenomenon we assume that in uncoupled vesicles the sensitivity of Complex I is much higher (K i ~
60 o
1-40
2O
0
2
4
ROTENONE,
6
8
I0
I 0 -8 M
Fig. 7. Inhibition by rotenone of the NADH:Q-reductase measured with reduced and oxidized submitochondrial particles. (o) SMP (25 /~g/ml) were preincubated with rotenone in a reaction mixture containing: 0.25 M sucrose, 1 mg/ml BSA, 0.2 mM EDTA, 100 ,~M NADH, 20 mM succinate, 2 /~g/ml gramicidin, 1 mM CN-, 0.1 ~ g / m l mixopthiazol, 20 mM Hepes (potassium salts; pH 8.0) for 4 min at 30°C. NADH-Q reductase reaction was started by the addition of 400 ~M Qo and the initial rate of NADH oxidation was monitored. (e) the vesicles were treated with rotenone as above except that only Qo (400 /~M) was present during the incubation period.
(open symbols). In a parallel experiment, the incubation of the enzyme with rotenone was in presence of Qo (400 /xM) which lowered the reduction level of ubiquinone pool. The sensitivity to rotenone of the two systems is given in Fig. 7. In both cases the inhibition is established in the nanomolar range, independent on the redox state of the quinone, following a curve typical for uncoupled preparation (see Fig. 3, curve 2). Thus we conclude that reduction of the endogenous quinone in the membrane does not induce the state of low affinity, which is established only under conditions of high A/xr~+. TABLE I
Catalytic properties of the submitochondrial particle populations Sample
Particles a as prepared Population 1 Population 2
Sensitivity to rotenone
NADH-oxidase b activity (%)
Succinate_NAD + c reductase activity (%)
Respiratory d control
Low High
100 12 88
100 60 40
5.5 20 < 5.5
a Particles were pretreated with oligomycin (0.4/.~gfml), b NADH-oxidase reaction was assayed in the presence of gramicidin as described in Materials and Methods; 100% corresponds to the specific activity of 1.2/.tmole of NADH oxidized per min per mg of protein, c The energy-linked reduction of NAD + was measured as described in Materials and Methods; 100% corresponds to the specific activity of 0.14 /zmol of NAD + reduced per min per mg of protein, d Respiratory control is calculated as a ratio between the rates of NADH oxidation after and before the addition of gramicidin (see Fig. 4 curve 2).
174 ried out in absence of added cytochrome c, the contribution of these populations to the overall rate is negligible and cannot be identified with the less coupled population. The fact that rotenone binding is controlled by Atzn+ is an indication for a major change in the enzyme structure which follows the build-up or collapse of A/.~H+.The energy-rich state, having low affinity to rotenone, is effective in binding of the quinol molecule and its subsequent oxidation by Fe-S clusters. In contrast, the highly rotenone-sensitive non-energized state of enzyme is functioning during oxidation of NADH. In our opinion, stabilization of Complex I by rotenone in one of its conformation states may be the mechanism of inhibition.
Acknowledgements This research is supported by the US Navy N0001489-J 1622, the Israeli Ministry for Science and Technology and Koret Foundation. A.B.K. is a post-doctoral fellow of the Koret Foundation.
References 1 Hatefi, Y. and Rieske, J.S. (1967) Methods Enzymol. 10, 235-239. 2 Orme-Jonson, N.R., Hansen, R.E. and Beinert, H. (1974) J. Biol. Chem. 249, 1922-11927. 3 Albracht, S.P.J. and Subramaniam, J. (1977) Biochim. Biophys. Acta 462, 36-48. 4 Ohnishi, T. (1979) in Membrane Proteins in Energy Transduction (Capaldi, R.A., ed.), pp. 1-87, Marcel Dekker, New York. 5 Ingledew, W.J. and Ohnishi, T. (1980) Biochem. J. 186, 111-117. 6 Schatz, G. and Racker, E. (1966) J. Biol. Chem. 241, 1429-1438.
7 Lawford, H.G. and Garland, P.B. (1971) Biochem. J. 130, 10291044. 8 Ragan, C.I. and Racker, E. (1973) J. Biol. Chem. 248, 2563-2569. 9 Ragan, C.I. and Hinkle, P.C. (1975)J. Biol. Chem. 250, 8472-8480. 10 Ruzicka, F.J. and Crane, F.I. (1971) Biochim. Biophys. Acta 226, 221-223. 11 Klingenberg, M. and Slenczka, W. (1959) Biochem. Z. 331, 486517. 12 Chance, B. and Hollunger, G. (1960) Nature 185, 666-672. 13 Low, H. and Vallin, I. (1963) Biochim. Biophys. Acta 69, 361-374. 14 Hommes, F.A. (1963) Biochim. Biophys. Acta 77, 183-190. 15 Chance, B. and Hollunger, G. (1961) J. Biol. Chem. 236, 15341543. 16 Kotlyar, A.B. and Vinogradov, A.D. (1990) Biochim. Biophys. Acta 1019, 151-158. 17 Horgan, D.J., Ohno, H., Singer, T.P. and Casida, J.E. (1968) J. Biol. Chem. 243, 5967-5976. 18 Horgan, D.J. and Singer, T.P. (1967) Bicohem. J. 104, 50c-52c. 19 Horgan, D.J., Singer, T.P. and Casida, ,I.E. (1968) J. Biol. Chem. 243, 834-843. 20 Singer, T.P. and Gutman, M. (1971) Adv. Enzymol. 34, 79-153. 21 Bois, R. and Estabrook, R.W. (1969) Arch. Biochem. Biophys. 129, 362-369. 22 Van Belzen, R., Van Gaalen, M.C.M., Cuypers, P.A. and AIbracht, S.P.J. (1990) Biochim. Biophys. Acta 1017, 152-159. 23 Singer, T.P., Horgan, D.,I. and Casida, J.E. (1968) in Flavins and Flavoproteins (Yagi, K., ed.), pp. 192-215, University of Tokyo Press, Tokyo. 24 Gutman, M., Singer, T.P., Beinert, H. and Casida, J.E. (1970) Proc. Natl. Acad. Sci. USA 65, 763-770. 25 Ernster, L., Dallner, G. and Azzone, G.F. (1963) J. Biol. Chem. 238, 1124-1131. 26 Ohnishi, T. (1973) Biochim. Biophys. Acta 301, 105-128. 27 Gornall, A.G., Bardawill, C.S. and David, M.M. (1949) J. Biol. Chem. 177, 751-766. 28 Gutman, M., Coles, C.J., Singer, T.P. and Casida, J.E. (1971) Biochemistry 10, 2036-2043. 29 Huang, C.H., Keyhani, E. and Lee, C.P. (1973) Biochim. Biophys. Acta 305, 455-473. 30 Huang, C.H. and Lee, C.P. (1975) Biochim. Biophys. Acta 376, 398-414.
