Voltage-dependent Anion Channels Control the Release of the Superoxide Anion from Mitochondria to Cytosol

Several reactions in biological systems contribute to maintain the steady-state concentrations of superoxide anion (O 2⨪) and hydrogen peroxide (H2O2). The electron transfer chain of mitochondria is a well documented source of H2O2; however, the release of O 2⨪ from mitochondria into cytosol has not been unequivocally established. This study was aimed at validating mitochondria as sources of cytosolic O 2⨪, elucidating the mechanisms underlying the release of O 2⨪ from mitochondria into cytosol, and assessing the role of outer membrane voltage-dependent anion channels (VDACs) in this process. Isolated rat heart mitochondria supplemented with complex I or II substrates generate an EPR signal ascribed to O 2⨪. Inhibition of the signal in a concentration-dependent manner by both manganese-superoxide dismutase and cytochromec proteins that cannot cross the mitochondrial membrane supports the extramitochondrial location of the spin adduct. Basal rates of O 2⨪ release from mitochondria were estimated at ∼0.04 nmol/min/mg protein, a value increased ∼8-fold by the complex III inhibitor, antimycin A. These estimates, obtained by quantitative spin-trapping EPR, were confirmed by fluorescence techniques, mainly hydroethidine oxidation and horseradish peroxidase-basedp-hydroxyphylacetate dimerization. Inhibitors of VDAC, 4′-diisothiocyano-2,2′-disulfonic acid stilbene (DIDS), and dextran sulfate (in a voltage-dependent manner) inhibited O 2⨪ production from mitochondria by ∼55%, thus suggesting that a large portion of O 2⨪ exited mitochondria via these channels. These findings are discussed in terms of competitive decay pathways for O 2⨪ in the intermembrane space and cytosol as well as the implications of these processes for modulating cell signaling pathways in these compartments. Several reactions in biological systems contribute to maintain the steady-state concentrations of superoxide anion (O 2⨪) and hydrogen peroxide (H2O2). The electron transfer chain of mitochondria is a well documented source of H2O2; however, the release of O 2⨪ from mitochondria into cytosol has not been unequivocally established. This study was aimed at validating mitochondria as sources of cytosolic O 2⨪, elucidating the mechanisms underlying the release of O 2⨪ from mitochondria into cytosol, and assessing the role of outer membrane voltage-dependent anion channels (VDACs) in this process. Isolated rat heart mitochondria supplemented with complex I or II substrates generate an EPR signal ascribed to O 2⨪. Inhibition of the signal in a concentration-dependent manner by both manganese-superoxide dismutase and cytochromec proteins that cannot cross the mitochondrial membrane supports the extramitochondrial location of the spin adduct. Basal rates of O 2⨪ release from mitochondria were estimated at ∼0.04 nmol/min/mg protein, a value increased ∼8-fold by the complex III inhibitor, antimycin A. These estimates, obtained by quantitative spin-trapping EPR, were confirmed by fluorescence techniques, mainly hydroethidine oxidation and horseradish peroxidase-basedp-hydroxyphylacetate dimerization. Inhibitors of VDAC, 4′-diisothiocyano-2,2′-disulfonic acid stilbene (DIDS), and dextran sulfate (in a voltage-dependent manner) inhibited O 2⨪ production from mitochondria by ∼55%, thus suggesting that a large portion of O 2⨪ exited mitochondria via these channels. These findings are discussed in terms of competitive decay pathways for O 2⨪ in the intermembrane space and cytosol as well as the implications of these processes for modulating cell signaling pathways in these compartments. Mitochondria from various aerobic organisms have been recognized as effective sources of hydrogen peroxide (H2O2) (1Chance B. Sies H. Boveris A. Physiol. Rev. 1979; 59: 527-605Google Scholar, 2Boveris A. Oshino N. Chance B. Biochem. J. 1972; 128: 617-630Google Scholar). H2O2produced by mitochondria has been suggested to regulate several signal transduction pathways, including c-Jun N-terminal kinase (JNK1) activity (3Sauer H. Wartenberg M. Hescheler J. Cell. Physiol. Biochem. 2001; 11: 173-186Google Scholar, 4Nemoto S. Takeda K. Yu Z.X. Ferrans V.J. Finkel T. Mol. Cell. Biol. 2000; 20: 7311-7318Google Scholar). Alterations in mitochondrial H2O2 steady-state levels by genetic modulation of catalase expression in the mitochondrial matrix is associated with changes in cell proliferation (3Sauer H. Wartenberg M. Hescheler J. Cell. Physiol. Biochem. 2001; 11: 173-186Google Scholar, 5Rodriguez A.M. Carrico P.M. Mazurkiewicz J.E. Melendez J.A. Free Radic. Biol. Med. 2000; 29: 801-813Google Scholar), tumor necrosis factor (TNF) response (6Bai J. Cederbaum A.I. J. Biol. Chem. 2000; 275: 19241-19249Google Scholar), and apoptosis (7Bai J. Rodriguez A.M. Melendez J.A. Cederbaum A.I. J. Biol. Chem. 1999; 274: 26217-26224Google Scholar).A two-step model that accounts for mitochondrial H2O2 production has become widely accepted (8Boveris A. Adv. Exp. Med. Biol. 1977; 78: 67-82Google Scholar,9Cadenas E. Davies K.J.A. Free Radic. Biol. Med. 2000; 29: 222-230Google Scholar). The first step, shown below in Reaction 1, UQ⨪+O2→UQ+O2⨪REACTION 1 entails the autoxidation of ubisemiquinone in the respiratory chain to generate O 2⨪, which is released into the mitochondrial matrix (10Boveris A. Cadenas E. Stoppani A.O.M. Biochem. J. 1976; 156: 435-444Google Scholar, 11Cadenas E. Boveris A. Ragan C.I. Stoppani A.O.M. Arch. Biochem. Biophys. 1977; 180: 248-257Google Scholar). The second step entails the conversion of O 2⨪to H2O2 catalyzed by manganese superoxide dismutase (Mn-SOD), which resides in the mitochondrial matrix. H2O2 diffuses rapidly through membranes (12Antunes F. Cadenas E. FEBS Lett. 2000; 475: 121-126Google Scholar), and the release of H2O2 from mitochondria to cytosol reflects the balance between H2O2production and consumption reactions, with the latter mainly involving reduction of the hydroperoxide to H2O via matrix glutathione peroxidase. Mitochondria contribute ∼20–30% to the cytosolic steady-state concentration of H2O2 (∼10−8m) (13Boveris A. Cadenas E. Mazzaro D. Clerch L. Oxygen, Gene Expression, and Cellular Function. Marcel Dekker Inc., New York1997: 1-25Google Scholar).Two recent findings have modified and extended this two-step mechanism for mitochondrial production of H2O2. First, part of the O 2⨪ generated during mitochondrial electron transfer is vectorially released into the intermembrane space (14Han D. Williams E. Cadenas E. Biochem. J. 2000; 353: 411-416Google Scholar). Evidence for this, obtained with mitoplasts (mitochondria devoid of the outer membrane), consisted of abrogation of the EPR spin adduct signal by superoxide dismutase, competitive inhibition by cytochromec, and broadening of the signal by membrane-impermeable spin-broadening agents (14Han D. Williams E. Cadenas E. Biochem. J. 2000; 353: 411-416Google Scholar). The mechanism underlying the release of O 2⨪ into the intermembrane space considers the formation of ubisemiquinone (Reaction 1) at two sites in the ubiquinone pool, the QI site that lies near the matrix and the QOsite in the vicinity of the intermembrane space (15de Vries S. J. Bioenerg. Biomembr. 1986; 18: 196-224Google Scholar, 16Sharp R.E. Moser C.C. Gibney B.R. Dutton P.L. J. Bioenerg. Biomembr. 1999; 31: 225-233Google Scholar). Autoxidation of ubisemiquinone at the QO site (UQ O⨪) results in release of O 2⨪ into the cytosolic side of the mitochondrial inner membrane. Of note, O 2⨪ cannot cross membranes except in the protonated form (a small fraction of the O 2⨪ pool at physiological pH; pK a = 4.8) (17Gus'kova R.A. Ivanov I. Akhobadze V.V. Rubin A.R. Biochim. Biophys. Acta. 1984; 778: 579-583Google Scholar). Taken together, this finding that H2O2 both at the intermembrane space and matrix from O 2⨪ generated the the release of O 2⨪ the intermembrane space in to the of a superoxide dismutase activity in this the of a in the intermembrane space of mitochondria has been confirmed both in rat A. I. J. Biol. Chem. 2001; Scholar, and K. J. Biol. Chem. 2001; Scholar). These findings a the the of in the intermembrane space R.A. I. J. Biol. Chem. Scholar), which was the of a by J. Biol. Chem. release of O 2⨪ into the intermembrane space the of O 2⨪ generated in this manner into the cytosol the outer membrane and both contribute to the cytosolic steady-state levels of this and in the of cell signaling The intermembrane space several O pathways and the intermembrane space as well as for O 2⨪ the outer in the voltage-dependent anion VDAC, voltage-dependent anion superoxide acid EPR, electron acid VDAC, voltage-dependent anion superoxide acid EPR, electron acid as the of and proteins between the intermembrane space and Rev. Biochem. Mol. Biol. Scholar, J. Biol. role both in the release of the and in the mitochondrial has been M. Biochem. J. 1999; Scholar, M. S. A. 2000; Scholar). The by has a of and is for the of J. Biol. Scholar). membranes have been shown to an anion through which O 2⨪ J. Biol. as a that the of O 2⨪ from the intermembrane space to the of the outer membrane of This study was aimed at validating mitochondria as sources of cytosolic O 2⨪, elucidating the mechanisms underlying the release of O 2⨪ from mitochondria into cytosol, and assessing the role of outer membrane in this mitochondrial respiratory chain has been recognized as an effective source of H2O2 from the of O 2⨪. of O 2⨪ from mitochondria is the of two to generate O 2⨪ into the intermembrane and of O 2⨪ from the intermembrane space to the through of oxidation as the source of O 2⨪ in the intermembrane space is by the mechanism underlying the of the complex III antimycin and The electron transfer from to the inner ubiquinone pool as thus through Reaction in an of by the as in Reaction of the electron transfer (15de Vries S. J. Bioenerg. Biomembr. 1986; 18: 196-224Google Scholar, A. Arch. Biochem. Biophys. Scholar). The to the QO site of the ubiquinone pool, electron transfer from or to 1986; Scholar), as in Reaction in the inhibition of formation (15de Vries S. J. Bioenerg. Biomembr. 1986; 18: 196-224Google Scholar, A. Arch. Biochem. Biophys. M. FEBS Lett. Scholar). The of antimycin and are with this electron transfer and the QO site of the ubiquinone pool in O 2⨪ by role for in O 2⨪ from the intermembrane space to the is by the of is the in the outer membrane for the of and proteins between the intermembrane space and the The that as a O is not in membranes an anion that the of O 2⨪ has been R.E. I. J. Biol. Chem. Scholar). this two and dextran O 2⨪ production (in a voltage-dependent manner) in heart inhibition by is inhibition by dextran sulfate in a manner is a that has been for M. S. A. Scholar). the with that the and of the of generated O 2⨪ into the physiological of are not and the of O an for of this from the intermembrane space in the of an intermembrane space First, the of for has been to in the of the M. J. Bioenerg. Biomembr. Scholar). to the of in O 2⨪, in O 2⨪. the inner membrane O 2⨪ is to the the inner membrane and outer membrane are the of intermembrane space is estimated to S. S. J. Biol. Scholar). near the the inner and outer membrane the inner membrane lies to the that the of O 2⨪, generated from the respiratory is in the into the of F. A. R.E. Free Radic. Biol. Med. Scholar). the estimated O 2⨪ is to the intermembrane O 2⨪ generated the intermembrane space the outer near the dextran inhibited O by mitochondria This that the to VDAC, or that O through channels in the outer membrane The latter is by the of two channels in to in the outer of the outer membrane and the both of which are to in Rev. Biochem. Mol. Biol. Scholar). has been suggested that through the outer membrane is by is N. M. H. J. Bioenerg. Biomembr. 2001; Scholar). and sources below N. M. H. J. Bioenerg. Biomembr. 2001; Scholar). has been suggested that as a N. M. H. J. Bioenerg. Biomembr. 2001; Scholar, E. J. M. M. Mol. Cell. Biol. of O 2⨪ from mitochondria into cytosol is to to O 2⨪, as oxidation or and from with mitochondrial this obtained with spin-trapping EPR, which signal supports the that O vectorially released from mitochondria into cytosol at rates of this was by antimycin A. The of O 2⨪ release from mitochondria is an and a portion of the O 2⨪ that is generated the intermembrane The levels of O 2⨪ in this is by several to cytosol through and outer membrane with cytochromec and to H2O2 that is = or catalyzed by intermembrane space = the latter the for O 2⨪. the of intermembrane space has been in and A. I. J. Biol. Chem. 2001; Scholar, K. J. Biol. Chem. 2001; Scholar), concentration and in heart mitochondria at of a the between and O O 2⨪ in the intermembrane of is to to the inner J. Biol. and to O physiological and implications for O the intermembrane space and cytosol to O 2⨪ released into the from mitochondria an role in cell O 2⨪ has been in several signaling and to O 2⨪ of O 2⨪ released from mitochondria I. J. Biol. Chem. Scholar). decay of O 2⨪ released from mitochondria a with to the This O 2⨪ decay of in the intermembrane of and that in the intermembrane space have been to to apoptosis by mechanism N. M. K. B. J. Biol. 2001; Scholar). O 2⨪ generated the intermembrane space an role in of these through formation is of superoxide dismutase activity is in the Biochem. J. Scholar), is in to proteins from mitochondrial O 2⨪ as well as from O 2⨪ Mitochondria from various aerobic organisms have been recognized as effective sources of hydrogen peroxide (H2O2) (1Chance B. Sies H. Boveris A. Physiol. Rev. 1979; 59: 527-605Google Scholar, 2Boveris A. Oshino N. Chance B. Biochem. J. 1972; 128: 617-630Google Scholar). H2O2produced by mitochondria has been suggested to regulate several signal transduction pathways, including c-Jun N-terminal kinase (JNK1) activity (3Sauer H. Wartenberg M. Hescheler J. Cell. Physiol. Biochem. 2001; 11: 173-186Google Scholar, 4Nemoto S. Takeda K. Yu Z.X. Ferrans V.J. Finkel T. Mol. Cell. Biol. 2000; 20: 7311-7318Google Scholar). Alterations in mitochondrial H2O2 steady-state levels by genetic modulation of catalase expression in the mitochondrial matrix is associated with changes in cell proliferation (3Sauer H. Wartenberg M. Hescheler J. Cell. Physiol. Biochem. 2001; 11: 173-186Google Scholar, 5Rodriguez A.M. Carrico P.M. Mazurkiewicz J.E. Melendez J.A. Free Radic. Biol. Med. 2000; 29: 801-813Google Scholar), tumor necrosis factor (TNF) response (6Bai J. Cederbaum A.I. J. Biol. Chem. 2000; 275: 19241-19249Google Scholar), and apoptosis (7Bai J. Rodriguez A.M. Melendez J.A. Cederbaum A.I. J. Biol. Chem. 1999; 274: 26217-26224Google Scholar). two-step model that accounts for mitochondrial H2O2 production has become widely accepted (8Boveris A. Adv. Exp. Med. Biol. 1977; 78: 67-82Google Scholar,9Cadenas E. Davies K.J.A. Free Radic. Biol. Med. 2000; 29: 222-230Google Scholar). The first step, shown below in Reaction 1, UQ⨪+O2→UQ+O2⨪REACTION 1 entails the autoxidation of ubisemiquinone in the respiratory chain to generate O 2⨪, which is released into the mitochondrial matrix (10Boveris A. Cadenas E. Stoppani A.O.M. Biochem. J. 1976; 156: 435-444Google Scholar, 11Cadenas E. Boveris A. Ragan C.I. Stoppani A.O.M. Arch. Biochem. Biophys. 1977; 180: 248-257Google Scholar). The second step entails the conversion of O 2⨪to H2O2 catalyzed by manganese superoxide dismutase (Mn-SOD), which resides in the mitochondrial matrix. H2O2 diffuses rapidly through membranes (12Antunes F. Cadenas E. FEBS Lett. 2000; 475: 121-126Google Scholar), and the release of H2O2 from mitochondria to cytosol reflects the balance between H2O2production and consumption reactions, with the latter mainly involving reduction of the hydroperoxide to H2O via matrix glutathione peroxidase. Mitochondria contribute ∼20–30% to the cytosolic steady-state concentration of H2O2 (∼10−8m) (13Boveris A. Cadenas E. Mazzaro D. Clerch L. Oxygen, Gene Expression, and Cellular Function. Marcel Dekker Inc., New York1997: 1-25Google Scholar). recent findings have modified and extended this two-step mechanism for mitochondrial production of H2O2. First, part of the O 2⨪ generated during mitochondrial electron transfer is vectorially released into the intermembrane space (14Han D. Williams E. Cadenas E. Biochem. J. 2000; 353: 411-416Google Scholar). Evidence for this, obtained with mitoplasts (mitochondria devoid of the outer membrane), consisted of abrogation of the EPR spin adduct signal by superoxide dismutase, competitive inhibition by cytochromec, and broadening of the signal by membrane-impermeable spin-broadening agents (14Han D. Williams E. Cadenas E. Biochem. J. 2000; 353: 411-416Google Scholar). The mechanism underlying the release of O 2⨪ into the intermembrane space considers the formation of ubisemiquinone (Reaction 1) at two sites in the ubiquinone pool, the QI site that lies near the matrix and the QOsite in the vicinity of the intermembrane space (15de Vries S. J. Bioenerg. Biomembr. 1986; 18: 196-224Google Scholar, 16Sharp R.E. Moser C.C. Gibney B.R. Dutton P.L. J. Bioenerg. Biomembr. 1999; 31: 225-233Google Scholar). Autoxidation of ubisemiquinone at the QO site (UQ O⨪) results in release of O 2⨪ into the cytosolic side of the mitochondrial inner membrane. Of note, O 2⨪ cannot cross membranes except in the protonated form (a small fraction of the O 2⨪ pool at physiological pH; pK a = 4.8) (17Gus'kova R.A. Ivanov I. Akhobadze V.V. Rubin A.R. Biochim. Biophys. Acta. 1984; 778: 579-583Google Scholar). Taken together, this finding that H2O2 both at the intermembrane space and matrix from O 2⨪ generated the compartments. the release of O 2⨪ the intermembrane space in to the of a superoxide dismutase activity in this the of a in the intermembrane space of mitochondria has been confirmed both in rat A. I. J. Biol. Chem. 2001; Scholar, and K. J. Biol. Chem. 2001; Scholar). These findings a the the of in the intermembrane space R.A. I. J. Biol. Chem. Scholar), which was the of a by J. Biol. Chem. Scholar). The release of O 2⨪ into the intermembrane space the of O 2⨪ generated in this manner into the cytosol the outer membrane and both contribute to the cytosolic steady-state levels of this and in the of cell signaling The intermembrane space several O pathways and the intermembrane space as well as for O 2⨪ the outer in the voltage-dependent anion VDAC, voltage-dependent anion superoxide acid EPR, electron acid VDAC, voltage-dependent anion superoxide acid EPR, electron acid as the of and proteins between the intermembrane space and Rev. Biochem. Mol. Biol. Scholar, J. Biol. role both in the release of the and in the mitochondrial has been M. Biochem. J. 1999; Scholar, M. S. A. 2000; Scholar). The by has a of and is for the of J. Biol. Scholar). membranes have been shown to an anion through which O 2⨪ J. Biol. Scholar). as a that the of O 2⨪ from the intermembrane space to the of the outer membrane of This study was aimed at validating mitochondria as sources of cytosolic O 2⨪, elucidating the mechanisms underlying the release of O 2⨪ from mitochondria into cytosol, and assessing the role of outer membrane in this process. mitochondrial respiratory chain has been recognized as an effective source of H2O2 from the of O 2⨪. of O 2⨪ from mitochondria is the of two to generate O 2⨪ into the intermembrane and of O 2⨪ from the intermembrane space to the through of oxidation as the source of O 2⨪ in the intermembrane space is by the mechanism underlying the of the complex III antimycin and The electron transfer from to the inner ubiquinone pool as thus through Reaction in an of by the as in Reaction of the electron transfer (15de Vries S. J. Bioenerg. Biomembr. 1986; 18: 196-224Google Scholar, A. Arch. Biochem. Biophys. Scholar). The to the QO site of the ubiquinone pool, electron transfer from or to 1986; Scholar), as in Reaction in the inhibition of formation (15de Vries S. J. Bioenerg. Biomembr. 1986; 18: 196-224Google Scholar, A. Arch. Biochem. Biophys. M. FEBS Lett. Scholar). The of antimycin and are with this electron transfer and the QO site of the ubiquinone pool in O 2⨪ by role for in O 2⨪ from the intermembrane space to the is by the of is the in the outer membrane for the of and proteins between the intermembrane space and the The that as a O is not in membranes an anion that the of O 2⨪ has been R.E. I. J. Biol. Chem. Scholar). this two and dextran O 2⨪ production (in a voltage-dependent manner) in heart inhibition by is inhibition by dextran sulfate in a manner is a that has been for M. S. A. Scholar). the with that the and of the of generated O 2⨪ into the physiological of are not and the of O an for of this from the intermembrane space in the of an intermembrane space First, the of for has been to in the of the M. J. Bioenerg. Biomembr. Scholar). to the of in O 2⨪, in O 2⨪. the inner membrane O 2⨪ is to the the inner membrane and outer membrane are the of intermembrane space is estimated to S. S. J. Biol. Scholar). near the the inner and outer membrane the inner membrane lies to the that the of O 2⨪, generated from the respiratory is in the into the of F. A. R.E. Free Radic. Biol. Med. Scholar). the estimated O 2⨪ is to the intermembrane O 2⨪ generated the intermembrane space the outer near the dextran inhibited O by mitochondria This that the to VDAC, or that O through channels in the outer membrane The latter is by the of two channels in to in the outer of the outer membrane and the both of which are to in Rev. Biochem. Mol. Biol. Scholar). has been suggested that through the outer membrane is by is N. M. H. J. Bioenerg. Biomembr. 2001; Scholar). and sources below N. M. H. J. Bioenerg. Biomembr. 2001; Scholar). has been suggested that as a N. M. H. J. Bioenerg. Biomembr. 2001; Scholar, E. J. M. M. Mol. Cell. Biol. of O 2⨪ from mitochondria into cytosol is to to O 2⨪, as oxidation or and from with mitochondrial this obtained with spin-trapping EPR, which signal supports the that O vectorially released from mitochondria into cytosol at rates of this was by antimycin A. The of O 2⨪ release from mitochondria is an and a portion of the O 2⨪ that is generated the intermembrane The levels of O 2⨪ in this is by several to cytosol through and outer membrane with cytochromec and to H2O2 that is = or catalyzed by intermembrane space = the latter the for O 2⨪. the of intermembrane space has been in and A. I. J. Biol. Chem. 2001; Scholar, K. J. Biol. Chem. 2001; Scholar), concentration and in heart mitochondria at of a the between and O O 2⨪ in the intermembrane of is to to the inner J. Biol. and to O physiological and implications for O the intermembrane space and cytosol to O 2⨪ released into the from mitochondria an role in cell O 2⨪ has been in several signaling and to O 2⨪ of O 2⨪ released from mitochondria I. J. Biol. Chem. Scholar). decay of O 2⨪ released from mitochondria a with to the This O 2⨪ decay of in the intermembrane of and that in the intermembrane space have been to to apoptosis by mechanism N. M. K. B. J. Biol. 2001; Scholar). O 2⨪ generated the intermembrane space an role in of these through formation is of superoxide dismutase activity is in the Biochem. J. Scholar), is in to proteins from mitochondrial O 2⨪ as well as from O 2⨪ The mitochondrial respiratory chain has been recognized as an effective source of H2O2 from the of O 2⨪. of O 2⨪ from mitochondria is the of two to generate O 2⨪ into the intermembrane and of O 2⨪ from the intermembrane space to the through The of oxidation as the source of O 2⨪ in the intermembrane space is by the mechanism underlying the of the complex III antimycin and The electron transfer from to the inner ubiquinone pool as thus through Reaction in an of by the as in Reaction of the electron transfer (15de Vries S. J. Bioenerg. Biomembr. 1986; 18: 196-224Google Scholar, A. Arch. Biochem. Biophys. Scholar). The to the QO site of the ubiquinone pool, electron transfer from or to 1986; Scholar), as in Reaction in the inhibition of formation (15de Vries S. J. Bioenerg. Biomembr. 1986; 18: 196-224Google Scholar, A. Arch. Biochem. Biophys. M. FEBS Lett. Scholar). The of antimycin and are with this electron transfer and the QO site of the ubiquinone pool in O 2⨪ by role for in O 2⨪ from the intermembrane space to the is by the of is the in the outer membrane for the of and proteins between the intermembrane space and the The that as a O is not in membranes an anion that the of O 2⨪ has been R.E. I. J. Biol. Chem. Scholar). this two and dextran O 2⨪ production (in a voltage-dependent manner) in heart inhibition by is inhibition by dextran sulfate in a manner is a that has been for M. S. A. Scholar). the with that the and of the of generated O 2⨪ into the physiological of are not and the of O an for of this from the intermembrane space in the of an intermembrane space First, the of for has been to in the of the M. J. Bioenerg. Biomembr. Scholar). to the of in O 2⨪, in O 2⨪. the inner membrane O 2⨪ is to the the inner membrane and outer membrane are the of intermembrane space is estimated to S. S. J. Biol. Scholar). near the the inner and outer membrane the inner membrane lies to the that the of O 2⨪, generated from the respiratory is in the into the of F. A. R.E. Free Radic. Biol. Med. Scholar). the estimated O 2⨪ is to the intermembrane O 2⨪ generated the intermembrane space the outer near the dextran inhibited O by mitochondria This that the to VDAC, or that O through channels in the outer membrane The latter is by the of two channels in to in the outer of the outer membrane and the both of which are to in Rev. Biochem. Mol. Biol. Scholar). has been suggested that through the outer membrane is by is N. M. H. J. Bioenerg. Biomembr. 2001; Scholar). and sources below N. M. H. J. Bioenerg. Biomembr. 2001; Scholar). has been suggested that as a N. M. H. J. Bioenerg. Biomembr. 2001; Scholar, E. J. M. M. Mol. Cell. Biol. of O 2⨪ from mitochondria into cytosol is to to O 2⨪, as oxidation or and from with mitochondrial this obtained with spin-trapping EPR, which signal supports the that O vectorially released from mitochondria into cytosol at rates of this was by antimycin A. The of O 2⨪ release from mitochondria is an and a portion of the O 2⨪ that is generated the intermembrane The levels of O 2⨪ in this is by several to cytosol through and outer membrane with cytochromec and to H2O2 that is = or catalyzed by intermembrane space = the latter the for O 2⨪. the of intermembrane space has been in and A. I. J. Biol. Chem. 2001; Scholar, K. J. Biol. Chem. 2001; Scholar), concentration and in heart mitochondria at of a the between and O O 2⨪ in the intermembrane of is to to the inner J. Biol. and to O 2⨪. The physiological and implications for O the intermembrane space and cytosol to O 2⨪ released into the from mitochondria an role in cell O 2⨪ has been in several signaling and to O 2⨪ of O 2⨪ released from mitochondria I. J. Biol. Chem. Scholar). decay of O 2⨪ released from mitochondria a with to the This O 2⨪ decay of in the intermembrane of and that in the intermembrane space have been to to apoptosis by mechanism N. M. K. B. J. Biol. 2001; Scholar). O 2⨪ generated the intermembrane space an role in of these through formation is of superoxide dismutase activity is in the Biochem. J. Scholar), is in to proteins from mitochondrial O 2⨪ as well as from O 2⨪