579th MEETING, LONDON
219
In experiments of this type on passive swelling, it is usual to add a Caz+-complexing agent, such as EDTA or EGTA, since CaZ+ions are known to cause swelling, possibly by activation of a phospholipase. Furthermore, Cleland, quoted in Robertson et a / . (1955), reported that EDTA greatly decreased the permeability of heart mitochondria to KCl. Fig. 2 [curves ( a ) and (c)] shows that EGTA markedly inhibits the entry of CIions, but curve (b) in Fig. 2 shows that this inhibition is time-dependent. Furthermore, the rate of entry of CI- is decreased after preincubation of the mitochondria (in the presence of rotenone and antimycin) in the absence of EGTA [Fig. 2(d)],and EGTA appears to serve merely to potentiate this effect [Fig. 2(e)]. This decrease in CI- permeability on preincubation and the time-dependence of the effect of EGTA are contrary to the view that, in this system, Ca2+ions produce damage and progressive increase in permeability. Estimations using a calcium-selective electrode show that during preincubation with respiratory inhibitors, Caz+ ions are leaking out of the mitochondria; this leakage will be accelerated by EGTA. It appears that operation of the anion-conducting pore requires free intramitochondrial Ca2+ions. Azzi, A. & Azzone, G. F. (1967) Biochim. Biophys. Acta 131, 468-478 Azzone, G. F. & Massari, S. (1973) Biochim. Biophys. Acfa 301, 195-226 Brierley, G. P. (1970) Biochemistry 9, 697-707 Brierley, G. P. & Stoner, C. D. (1970) Biochemistry 9, 708-713 Chappell, J. B. (1968) Br. Med. Bull. 24, 150-157 Mitchell, P. & Moyle, J. (1969) Eur. J . Biochem. 9, 149-155 Myers, V. B. & Haydon, D. A. (1972) Biochim. Biophys. Acta 274, 313-322 Robertson, R. N., Wilkins, M . J., Hope, A. B. & Nestel, L. (1955) Ausfr.J.Biol. Sci. 8, 164-185 Selwyn, M. J., Dawson, A. P., Stockdale, M. & Gains, N. (1970) Eur. J . Biochem. 14,120-126
Evidence that in Submitochondrial Particles Cytochrome Oxidase Translocates Protons M. CATIA SORGATO and STUART J. FERGUSON Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3 QU, and Department of Biochemistry, University of Birmingham, P.O. Box 363, Birmingham B15 2TT, U.K.
For some years it has been accepted that cytochrome oxidase is a transmembrane protein that probably translocates electrons from the outer (cytosolic) surface of the inner mitochondria1 membrane to the site of oxygen reduction at the matrix surface of the same membrane. This view has now been challenged by Wikstrom and colleagues (Wikstrom & Saari, 1977), who provided evidence that cytochrome oxidase has a proton-translocating capacity, although Moyle & Mitchell (1978) have recently reieterated their belief that cytochrome oxidase is not a proton pump [see also the response of Wikstrom & Krab (1978)l. The flow of electrons from cytochrome c through cytochrome oxidase to oxygen in submitochondrial particles, using external ascorbate plus NNN”’-tetramethyl-pphenylenediamine (AWN”’-tetramethyL1,Cbenzenediamine) as electron donor to cytochrome c, will only result in the development of a protonmotive force, positive charging and/or acidification of the particle lumen, if cytochrome oxidase translocates protons (Wikstrom & Saari, 1977; Sorgato et a/., 1978). We have found, by using a flow dialysis procedure for the assay of [14C]methylamineor S14CN- uptake. that oxidation of ascorbate plus NNN’N‘-tetramethyl-p-phenylenediamine(in the presence of sufficient antimycin to block electron transport between cytochromes b and cl) does result in positive charging and/or acidification of the lumen of the particles (Sorgato e t a / . , 1978; Sorgato & Ferguson, 1978). The demonstration of positive charging (membrane potential) alone might be taken to indicate the accumulation of the Wurster’s VOl. 7
220
BIOCHEMICAL SOCIETY TRANSACTIONS
Blue radical cation inside the particles, but the observation of a pH gradient implies that protons are translocated, giving rise to both the membrane potential and pH gradient. The interpretation of our experiments in terms of a proton-pumping capacity associated with cytochrome oxidase would be undermined if NNN’N’-tetramethyl-p-phenylenediamine did not, as commonly supposed, donate electrons solely to cytochrome c, but also to a proton-translocating component in the cytochrome blcoenzyme Q region of the respiratory chain. Inspection of the literature indicates that NNN’N’-tetramethylp-phenylenediamine (Em= +260mV) cannot directly reduce b cytochromes. Norling et al. (1972) showed that the reduction of b cytochromes by ascorbate plus NNNN’tetramethyl-p-phenylenediaminein submitochondrial particles in the presence of KCN was antimycin-sensitive, and the extent of b reduction was dependent on the concentration of NNN‘N’-tetramethyl-p-phenylenediamine. These data indicate that NNN”’tetramethyl-p-phenylenediamineequilibrates with cytochrome b via cytochromes c and c , . Afurther reduction of the b cytochromes can be observed when an energy supply is also provided (Tyler etal., 1966; Norling et al., 1972; Wikstrom, 1971). In the aerobic steady state the reduction of cytochrome b in mitochondria has been found to be entirely dependent on the supply of energy (Wikstrom, 1971). This last observation is worth considering in the context of a ‘Q-cycle’ (Mitchell, 1976). If NNN‘N‘-tetramethyl-pphenylenediamine could directly reduce ubiquinone to ubiquinol, then if a Q-cycle is operating, it might be expected that some reduction of cytochrome b in the aerobic steady state would be observed, as fully reduced ubiquinol is oxidized by both a b cytochrome and cytochrome cl according to the Q-cycle. Further evidence that N N N N tetramethyl-p-phenylenediamineinteracts with cytochrome c rather than cytochrome b was given by Caswell & Pressman (1968). Amongst bacteria, only those with a c-type cytochrome can oxidize reduced NN’-dimethyl-p-phenylenediamine(Nadi reaction) (Jurtshuk et al., 1978). This suggests that redox mediators of the phenylenediamine type do not readily interact with quinones and b-type cytochromes. This point is further emphasized by experiments of Marrs & Gest (1973), in which it was found that membranes from mutants of Rhodopseudomonas capsulata that were unable to oxidize ascorbate plus NNN’N’-tetramethyl-p-phenylenediamine were also blocked in cytochrome c oxidase activity, but not in cytochrome c reductase or in an alternative pathway for electrons from cytochrome blcoenzyme Q to oxygen. Furthermore, J. Willison & P. John (personal communication) have found that mutants of Paracossus denitriJicansthat lack cytochrome c cannot oxidize ascorbate plus NNN’N’-tetramethyl-p-phenylenediamine.In these mutants the b cytochromes and probably ubiquinone are not affected, and can donate electrons to an alternative oxidase Recalling that the cytochrome b and ubiquinone components of the respiratory chain in P . denitrificans closely resemble their mitochondrial counterparts, these findings with P . denitrij7cans again point to the interaction of NNN‘N-tetramethyl-p-phenylenediamine with c-type cytochromes. Various lines of evidence summarized here indicate that NNN‘N’-tetramethyl-pphenylenediamine donates electrons to a c-type cytochrome. Tyler et al. (1966) suggested that this cytochrome can be c , as well as c, but this would not affect our argument that ascorbate plus NNN’N’-tetramethyl-p-phenylenediamine can be used to test for the proton-translocating capacity of cytochrome oxidase (Sorgato & Ferguson, 1978), unless cytochrome c1 were itself able to translocate protons. We are not aware of any evidence that cytochrome c1 is proton-translocating, although there are indications that it spans the mitochondria1 membrane (Leung, 1977). We thank Dr. Philip John and Dr. Douglas Kell for valuable discussions. M. C. S. is a Research Fellow of the Consiglio Nazionale delle Ricerche, and was supported in Oxford by a Long-Term Fellowship from EMBO. Caswell, A. H. & Pressman, B. C . (1968) Arch. Biochem. Biophys. 125, 318-325 Jurtshuk, P., Mueller, T. J., McQuitty, D. N. & Riley, W. H. (1978) FEBS Meet. Proc. 11th 49, 99-121
1979
221
579th MEETING, LONDON
Leung, K. H. (1977) Ph.D. Thesis, Cornell University Marrs, B. & Gest, H. (1973) J . Bacteriol. 114, 1045-1051 Mitchell, P. (1976) J . Theor. Biol. 62, 327-367 Moyle, J. & Mitchell, P. (1978) FEBS Lett. 88, 268-272 Norling, B., Nelson, B. D., Nordenbrand, K. & Emster, L. (1972) Biochim. Biophys. Acta 275, 18-32
Sorgato, M. C. & Ferguson, S. J. (1978) FEES Lett. 90, 178-182 Sorgato, M. C., Ferguson, S . J., Kell, D. B. & John, P. (1978) Biochem. J. 174, 237-256 Tyler, D. D., Estabrook, R. W. & Sanadi, D. R. (1966) Arch. Biochem. Biophys. 114,239-251 Wikstrom, M. K. F. (1971) Biochim. Biophys. Acra 253, 332-345 Wikstrom, M. K. F. & Saari, H. T. (1977) Biochim. Biophys. Acta 462, 347-361 Wikstrom, M. K. F. & Krab, K. (1978) FEES Letr. 91, 8-14
A New Steady-State Method for Investigating Mitochondria1 Proton Transport MARTIN D. BRAND Department of Biochemistry, University of Cambridge, Cambridge C B 2 1Q W, U.K.
During energy conservation by mitochondria, protons are pumped across the inner membrane to form A p H + , an electrochemical gradient of H+. Return of protons to the matrix down this gradient may then be used to drive energy-requiring reactions, such as ATP synthesis or ion transport (Mitchell, 1966). Only a few methods have been employed to determine H+/O ratios, the number of H+ ejected during transport of 2e- from substrate to oxygen, or H+/ATP ratios, the number of H+ transported into the matrix per ATP synthesized. For a review see Brand (1977). These methods are (i) the 02-pulse technique of Mitchell & Moyle (1967) and its variant, the reductant pulse; (ii) measurement of movements of charge-compensating ions such as K+ or Ca2+;(iii) measurements of initial rates of H+ transport and their comparison with initial rates of e- flow; and (iv) comparison of A p H + with the phosphorylation potential of the ATP pool when the two are at equilibrium. Because of the lack of agreement between these various methods the H+/O ratio has yet to be unambiguously determined, For the span succinate + oxygen it has been given as 4 (Mitchell & Moyle, 1967), 6 (Brand et al., 1976; Nicholls, 1977) or 8 (Brand et al., 1976; Lehninger et al.. 1977). Thus an independent method of investigating H+/O ratios may prove valuable in resolving some of the problems that have been encountered in this field; such a method is described in this paper. In a steady-state the rates of H+ ejection and re-entry will be equal; the A p H + attained will depend on the conductance of the membrane to H+ (see Nicholls, 1974; Nicholls & Bernson, 1977). At constant conductance (set by addition of small amounts of uncoupler) A p H + will be proportional to the rate of H+ ejection, J H + O U T i.e.
k . A p H + =J H + O U T
Since JH+OUT = rate of oxygen consumption, V,x H+/O, then
A graph of A p H + against V therefore has slope (H+/O)/k.Since k is independent of the substrate involved, if a number of such graphs are plotted using different substrates, the ratio of the slopes will be the ratio of the H+/O quotients. This is the essence of the new method as used in this paper. V was measured with an oxygen electrode and was varied by addition of inhibitors (e.g. rnalonate for succinate oxidation) or by titration with substrate. A/,tH+ was more VOl. 7
219
In experiments of this type on passive swelling, it is usual to add a Caz+-complexing agent, such as EDTA or EGTA, since CaZ+ions are known to cause swelling, possibly by activation of a phospholipase. Furthermore, Cleland, quoted in Robertson et a / . (1955), reported that EDTA greatly decreased the permeability of heart mitochondria to KCl. Fig. 2 [curves ( a ) and (c)] shows that EGTA markedly inhibits the entry of CIions, but curve (b) in Fig. 2 shows that this inhibition is time-dependent. Furthermore, the rate of entry of CI- is decreased after preincubation of the mitochondria (in the presence of rotenone and antimycin) in the absence of EGTA [Fig. 2(d)],and EGTA appears to serve merely to potentiate this effect [Fig. 2(e)]. This decrease in CI- permeability on preincubation and the time-dependence of the effect of EGTA are contrary to the view that, in this system, Ca2+ions produce damage and progressive increase in permeability. Estimations using a calcium-selective electrode show that during preincubation with respiratory inhibitors, Caz+ ions are leaking out of the mitochondria; this leakage will be accelerated by EGTA. It appears that operation of the anion-conducting pore requires free intramitochondrial Ca2+ions. Azzi, A. & Azzone, G. F. (1967) Biochim. Biophys. Acta 131, 468-478 Azzone, G. F. & Massari, S. (1973) Biochim. Biophys. Acfa 301, 195-226 Brierley, G. P. (1970) Biochemistry 9, 697-707 Brierley, G. P. & Stoner, C. D. (1970) Biochemistry 9, 708-713 Chappell, J. B. (1968) Br. Med. Bull. 24, 150-157 Mitchell, P. & Moyle, J. (1969) Eur. J . Biochem. 9, 149-155 Myers, V. B. & Haydon, D. A. (1972) Biochim. Biophys. Acta 274, 313-322 Robertson, R. N., Wilkins, M . J., Hope, A. B. & Nestel, L. (1955) Ausfr.J.Biol. Sci. 8, 164-185 Selwyn, M. J., Dawson, A. P., Stockdale, M. & Gains, N. (1970) Eur. J . Biochem. 14,120-126
Evidence that in Submitochondrial Particles Cytochrome Oxidase Translocates Protons M. CATIA SORGATO and STUART J. FERGUSON Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3 QU, and Department of Biochemistry, University of Birmingham, P.O. Box 363, Birmingham B15 2TT, U.K.
For some years it has been accepted that cytochrome oxidase is a transmembrane protein that probably translocates electrons from the outer (cytosolic) surface of the inner mitochondria1 membrane to the site of oxygen reduction at the matrix surface of the same membrane. This view has now been challenged by Wikstrom and colleagues (Wikstrom & Saari, 1977), who provided evidence that cytochrome oxidase has a proton-translocating capacity, although Moyle & Mitchell (1978) have recently reieterated their belief that cytochrome oxidase is not a proton pump [see also the response of Wikstrom & Krab (1978)l. The flow of electrons from cytochrome c through cytochrome oxidase to oxygen in submitochondrial particles, using external ascorbate plus NNN”’-tetramethyl-pphenylenediamine (AWN”’-tetramethyL1,Cbenzenediamine) as electron donor to cytochrome c, will only result in the development of a protonmotive force, positive charging and/or acidification of the particle lumen, if cytochrome oxidase translocates protons (Wikstrom & Saari, 1977; Sorgato et a/., 1978). We have found, by using a flow dialysis procedure for the assay of [14C]methylamineor S14CN- uptake. that oxidation of ascorbate plus NNN’N‘-tetramethyl-p-phenylenediamine(in the presence of sufficient antimycin to block electron transport between cytochromes b and cl) does result in positive charging and/or acidification of the lumen of the particles (Sorgato e t a / . , 1978; Sorgato & Ferguson, 1978). The demonstration of positive charging (membrane potential) alone might be taken to indicate the accumulation of the Wurster’s VOl. 7
220
BIOCHEMICAL SOCIETY TRANSACTIONS
Blue radical cation inside the particles, but the observation of a pH gradient implies that protons are translocated, giving rise to both the membrane potential and pH gradient. The interpretation of our experiments in terms of a proton-pumping capacity associated with cytochrome oxidase would be undermined if NNN’N’-tetramethyl-p-phenylenediamine did not, as commonly supposed, donate electrons solely to cytochrome c, but also to a proton-translocating component in the cytochrome blcoenzyme Q region of the respiratory chain. Inspection of the literature indicates that NNN’N’-tetramethylp-phenylenediamine (Em= +260mV) cannot directly reduce b cytochromes. Norling et al. (1972) showed that the reduction of b cytochromes by ascorbate plus NNNN’tetramethyl-p-phenylenediaminein submitochondrial particles in the presence of KCN was antimycin-sensitive, and the extent of b reduction was dependent on the concentration of NNN‘N’-tetramethyl-p-phenylenediamine. These data indicate that NNN”’tetramethyl-p-phenylenediamineequilibrates with cytochrome b via cytochromes c and c , . Afurther reduction of the b cytochromes can be observed when an energy supply is also provided (Tyler etal., 1966; Norling et al., 1972; Wikstrom, 1971). In the aerobic steady state the reduction of cytochrome b in mitochondria has been found to be entirely dependent on the supply of energy (Wikstrom, 1971). This last observation is worth considering in the context of a ‘Q-cycle’ (Mitchell, 1976). If NNN‘N‘-tetramethyl-pphenylenediamine could directly reduce ubiquinone to ubiquinol, then if a Q-cycle is operating, it might be expected that some reduction of cytochrome b in the aerobic steady state would be observed, as fully reduced ubiquinol is oxidized by both a b cytochrome and cytochrome cl according to the Q-cycle. Further evidence that N N N N tetramethyl-p-phenylenediamineinteracts with cytochrome c rather than cytochrome b was given by Caswell & Pressman (1968). Amongst bacteria, only those with a c-type cytochrome can oxidize reduced NN’-dimethyl-p-phenylenediamine(Nadi reaction) (Jurtshuk et al., 1978). This suggests that redox mediators of the phenylenediamine type do not readily interact with quinones and b-type cytochromes. This point is further emphasized by experiments of Marrs & Gest (1973), in which it was found that membranes from mutants of Rhodopseudomonas capsulata that were unable to oxidize ascorbate plus NNN’N’-tetramethyl-p-phenylenediamine were also blocked in cytochrome c oxidase activity, but not in cytochrome c reductase or in an alternative pathway for electrons from cytochrome blcoenzyme Q to oxygen. Furthermore, J. Willison & P. John (personal communication) have found that mutants of Paracossus denitriJicansthat lack cytochrome c cannot oxidize ascorbate plus NNN’N’-tetramethyl-p-phenylenediamine.In these mutants the b cytochromes and probably ubiquinone are not affected, and can donate electrons to an alternative oxidase Recalling that the cytochrome b and ubiquinone components of the respiratory chain in P . denitrificans closely resemble their mitochondrial counterparts, these findings with P . denitrij7cans again point to the interaction of NNN‘N-tetramethyl-p-phenylenediamine with c-type cytochromes. Various lines of evidence summarized here indicate that NNN‘N’-tetramethyl-pphenylenediamine donates electrons to a c-type cytochrome. Tyler et al. (1966) suggested that this cytochrome can be c , as well as c, but this would not affect our argument that ascorbate plus NNN’N’-tetramethyl-p-phenylenediamine can be used to test for the proton-translocating capacity of cytochrome oxidase (Sorgato & Ferguson, 1978), unless cytochrome c1 were itself able to translocate protons. We are not aware of any evidence that cytochrome c1 is proton-translocating, although there are indications that it spans the mitochondria1 membrane (Leung, 1977). We thank Dr. Philip John and Dr. Douglas Kell for valuable discussions. M. C. S. is a Research Fellow of the Consiglio Nazionale delle Ricerche, and was supported in Oxford by a Long-Term Fellowship from EMBO. Caswell, A. H. & Pressman, B. C . (1968) Arch. Biochem. Biophys. 125, 318-325 Jurtshuk, P., Mueller, T. J., McQuitty, D. N. & Riley, W. H. (1978) FEBS Meet. Proc. 11th 49, 99-121
1979
221
579th MEETING, LONDON
Leung, K. H. (1977) Ph.D. Thesis, Cornell University Marrs, B. & Gest, H. (1973) J . Bacteriol. 114, 1045-1051 Mitchell, P. (1976) J . Theor. Biol. 62, 327-367 Moyle, J. & Mitchell, P. (1978) FEBS Lett. 88, 268-272 Norling, B., Nelson, B. D., Nordenbrand, K. & Emster, L. (1972) Biochim. Biophys. Acta 275, 18-32
Sorgato, M. C. & Ferguson, S. J. (1978) FEES Lett. 90, 178-182 Sorgato, M. C., Ferguson, S . J., Kell, D. B. & John, P. (1978) Biochem. J. 174, 237-256 Tyler, D. D., Estabrook, R. W. & Sanadi, D. R. (1966) Arch. Biochem. Biophys. 114,239-251 Wikstrom, M. K. F. (1971) Biochim. Biophys. Acra 253, 332-345 Wikstrom, M. K. F. & Saari, H. T. (1977) Biochim. Biophys. Acta 462, 347-361 Wikstrom, M. K. F. & Krab, K. (1978) FEES Letr. 91, 8-14
A New Steady-State Method for Investigating Mitochondria1 Proton Transport MARTIN D. BRAND Department of Biochemistry, University of Cambridge, Cambridge C B 2 1Q W, U.K.
During energy conservation by mitochondria, protons are pumped across the inner membrane to form A p H + , an electrochemical gradient of H+. Return of protons to the matrix down this gradient may then be used to drive energy-requiring reactions, such as ATP synthesis or ion transport (Mitchell, 1966). Only a few methods have been employed to determine H+/O ratios, the number of H+ ejected during transport of 2e- from substrate to oxygen, or H+/ATP ratios, the number of H+ transported into the matrix per ATP synthesized. For a review see Brand (1977). These methods are (i) the 02-pulse technique of Mitchell & Moyle (1967) and its variant, the reductant pulse; (ii) measurement of movements of charge-compensating ions such as K+ or Ca2+;(iii) measurements of initial rates of H+ transport and their comparison with initial rates of e- flow; and (iv) comparison of A p H + with the phosphorylation potential of the ATP pool when the two are at equilibrium. Because of the lack of agreement between these various methods the H+/O ratio has yet to be unambiguously determined, For the span succinate + oxygen it has been given as 4 (Mitchell & Moyle, 1967), 6 (Brand et al., 1976; Nicholls, 1977) or 8 (Brand et al., 1976; Lehninger et al.. 1977). Thus an independent method of investigating H+/O ratios may prove valuable in resolving some of the problems that have been encountered in this field; such a method is described in this paper. In a steady-state the rates of H+ ejection and re-entry will be equal; the A p H + attained will depend on the conductance of the membrane to H+ (see Nicholls, 1974; Nicholls & Bernson, 1977). At constant conductance (set by addition of small amounts of uncoupler) A p H + will be proportional to the rate of H+ ejection, J H + O U T i.e.
k . A p H + =J H + O U T
Since JH+OUT = rate of oxygen consumption, V,x H+/O, then
A graph of A p H + against V therefore has slope (H+/O)/k.Since k is independent of the substrate involved, if a number of such graphs are plotted using different substrates, the ratio of the slopes will be the ratio of the H+/O quotients. This is the essence of the new method as used in this paper. V was measured with an oxygen electrode and was varied by addition of inhibitors (e.g. rnalonate for succinate oxidation) or by titration with substrate. A/,tH+ was more VOl. 7
