Topology of Superoxide Production from Different Sites in the Mitochondrial Electron Transport Chain

Abstract

We measured production of reactive oxygen species by intact mitochondria from rat skeletal muscle, heart, and liver under various experimental conditions. By using different substrates and inhibitors, we determined the sites of production (which complexes in the electron transport chain produced superoxide). By measuring hydrogen peroxide production in the absence and presence of exogenous superoxide dismutase, we established the topology of superoxide production (on which side of the mitochondrial inner membrane superoxide was produced). Mitochondria did not release measurable amounts of superoxide or hydrogen peroxide when respiring on complex I or complex II substrates. Mitochondria from skeletal muscle or heart generated significant amounts of superoxide from complex I when respiring on palmitoyl carnitine. They produced superoxide at considerable rates in the presence of various inhibitors of the electron transport chain. Complex I (and perhaps the fatty acid oxidation electron transfer flavoprotein and its oxidoreductase) released superoxide on the matrix side of the inner membrane, whereas center o of complex III released superoxide on the cytoplasmic side. These results do not support the idea that mitochondria produce considerable amounts of reactive oxygen species under physiological conditions. Our upper estimate of the proportion of electron flow giving rise to hydrogen peroxide with palmitoyl carnitine as substrate (0.15%) is more than an order of magnitude lower than commonly cited values. We observed no difference in the rate of hydrogen peroxide production between rat and pigeon heart mitochondria respiring on complex I substrates. However, when complex I was fully reduced using rotenone, rat mitochondria released significantly more hydrogen peroxide than pigeon mitochondria. This difference was solely due to an elevated concentration of complex I in rat compared with pigeon heart mitochondria. We measured production of reactive oxygen species by intact mitochondria from rat skeletal muscle, heart, and liver under various experimental conditions. By using different substrates and inhibitors, we determined the sites of production (which complexes in the electron transport chain produced superoxide). By measuring hydrogen peroxide production in the absence and presence of exogenous superoxide dismutase, we established the topology of superoxide production (on which side of the mitochondrial inner membrane superoxide was produced). Mitochondria did not release measurable amounts of superoxide or hydrogen peroxide when respiring on complex I or complex II substrates. Mitochondria from skeletal muscle or heart generated significant amounts of superoxide from complex I when respiring on palmitoyl carnitine. They produced superoxide at considerable rates in the presence of various inhibitors of the electron transport chain. Complex I (and perhaps the fatty acid oxidation electron transfer flavoprotein and its oxidoreductase) released superoxide on the matrix side of the inner membrane, whereas center o of complex III released superoxide on the cytoplasmic side. These results do not support the idea that mitochondria produce considerable amounts of reactive oxygen species under physiological conditions. Our upper estimate of the proportion of electron flow giving rise to hydrogen peroxide with palmitoyl carnitine as substrate (0.15%) is more than an order of magnitude lower than commonly cited values. We observed no difference in the rate of hydrogen peroxide production between rat and pigeon heart mitochondria respiring on complex I substrates. However, when complex I was fully reduced using rotenone, rat mitochondria released significantly more hydrogen peroxide than pigeon mitochondria. This difference was solely due to an elevated concentration of complex I in rat compared with pigeon heart mitochondria. The free radical theory of aging states that it is the mitochondrial production of reactive oxygen species (ROS), 1The abbreviations used are: ROS, reactive oxygen species; MLSP, maximum lifespan; ETF, electron transfer flavoprotein; QOR, quinone oxidoreductase; UCPs, uncoupling proteins; SOD, superoxide dismutase; BSA, bovine serum albumin. such as superoxide and hydrogen peroxide, and the resulting accumulation of damage to macromolecules that causes aging and determines maximum lifespan (MLSP) (1Harman D. J. Gerontol. 1956; 2: 298-300Google Scholar, 2Harman D. J. Am. Geriatr. Soc. 1972; 20: 145-147Google Scholar). Comparative approaches have shed considerable light on the relationship between ROS and MLSP. Notably, the rate of superoxide production by submitochondrial particles (3Sohal R.S. Svensson I. Sohal B.H. Brunk U.T. Mech. Ageing Dev. 1989; 49: 129-135Google Scholar) and the rate of H2O2 production by mitochondria (4Sohal R.S. Svensson I. Brunk U.T. Mech. Ageing Dev. 1990; 53: 209-215Google Scholar) are inversely related to MLSP in different species. A complicating factor is the association of longer MLSP with lower metabolic rates within mammals or other groups, but this complication has been resolved by the observation that birds tend to have longer MLSP than mammals with the same metabolic rate. Thus pigeons (long MLSP) have a lower rate of mitochondrial H2O2 production than rats (shorter MLSP), even though these two species have similar standard metabolic rates (5Ku H.H. Sohal R.S. Mech. Ageing Dev. 1993; 72: 67-76Google Scholar, 6Barja G. Cadenas S. Rojas C. Perez-Campo R. Lopez-Torres M. Free Radic. Res. 1994; 21: 317-327Google Scholar, 7Herrero A. Barja G. Mech. Ageing Dev. 1997; 98: 95-111Google Scholar, 8Herrero A. Barja G. J. Bioenerg. Biomembr. 1997; 29: 241-249Google Scholar). Similarly, canaries and parakeets (budgerigars) (long MLSP) have lower rates of mitochondrial H2O2 production than mice (shorter MLSP), although all three species have similar standard metabolic rates (9Herrero A. Barja G. Mech. Ageing Dev. 1998; 103: 133-146Google Scholar). Despite numerous studies reporting that mitochondria release H2O2, there is some controversy as to whether mitochondria are an important source of ROS under physiological and pathological conditions (10Forman H.J. Azzi A. FASEB J. 1997; 11: 374-375Google Scholar). In agreement with these concerns, Staniek and Nohl (12Staniek K. Nohl H. Biochim. Biophys. Acta. 2000; 1460: 268-275Google Scholar) reported that mitochondria respiring on complex I and complex II substrates do not generate H2O2 except in the presence of the complex III inhibitor antimycin A. They proposed that unspecific interactions between the commonly used methods of H2O2detection and mitochondria cause artificial rates of H2O2 production (11Staniek K. Nohl H. Biochim. Biophys. Acta. 1999; 1413: 70-80Google Scholar, 12Staniek K. Nohl H. Biochim. Biophys. Acta. 2000; 1460: 268-275Google Scholar). Two principal sites of superoxide generation have been identified in mitochondria: complex I and complex III. The relative importance of these two sites seems to vary with experimental conditions and between tissues and species (13Barja G. J. Bioenerg. Biomembr. 1999; 31: 347-366Google Scholar). There is no clear consensus in the literature about which side of the mitochondrial inner membrane superoxide is generated by complex I and complex III. In the traditional view, complex III generates superoxide on the matrix side of the mitochondrial inner membrane (14Turrens J.F. Biosci. Rep. 1997; 17: 3-8Google Scholar). The semiquinone at centero of complex III of heart mitochondria was shown to be the main producer of superoxide based on inhibitor studies (15Turrens J.F. Alexandre A. Lehninger A.L. Arch. Biochem. Biophys. 1985; 237: 408-414Google Scholar). However, the x-ray structure of complex III reveals that center o is oriented toward the intermembrane space (16Iwata S. Lee J.W. Okada K. Lee J.K. Iwata M. Rasmussen B. Link T.A. Ramaswamy S. Jap B.K. Science. 1998; 281: 64-71Google Scholar, 17Zhang Z. Huang L. Shulmeister V.M. Chi Y.I. Kim K.K. Hung L.W. Crofts A.R. Berry E.A. Kim S.H. Nature. 1998; 392: 677-684Google Scholar), suggesting that superoxide production by complex III is directed toward the cytoplasm and not toward the matrix. In support of this view, a recent study has reported that antimycin A-supplemented mitoplasts (mitochondria devoid of portions of outer membrane and cytochrome c) can release superoxide (18Han D. Williams E. Cadenas E. Biochem. J. 2001; 353: 411-416Google Scholar). In complex I, either the iron-sulfur centers (7Herrero A. Barja G. Mech. Ageing Dev. 1997; 98: 95-111Google Scholar, 19Genova M.L. Ventura B. Giuliano G. Bovina C. Formiggini G. Parenti Castelli G. Lenaz G. FEBS Lett. 2001; 505: 364-368Google Scholar) or the active site flavin (20Liu Y. Fiskum G. Schubert D. J. Neurochem. 2002; 80: 780-787Google Scholar) are thought to be mainly responsible for superoxide production. There is no x-ray crystal structure of complex I, but all of these centers are likely to face the matrix side of the membrane. 30 years ago, it was shown that the oxidation of palmitoyl carnitine by mitochondria leads to the generation of H2O2(21Boveris A. Oshino N. Chance B. Biochem. J. 1972; : 617-630Google Scholar, 22Boveris A. Chance B. Biochem. J. 1973; 134: 707-716Google Scholar). These results received little attention and, to our knowledge, no study has examined how lipid metabolism could cause ROS generation in mammalian mitochondria. The oxidation of fatty acids involves the electron transfer flavoprotein (ETF) and the electron transfer flavoprotein quinone oxidoreductase (ETF-QOR) that could act as potential sources of ROS production. The role of lipid metabolism in the generation of ROS by mitochondria gained our attention recently when it was shown that the expression of mitochondrial uncoupling proteins (UCPs) correlates with the use of lipid as fuel substrates (23Samec S. Seydoux J. Dulloo A.G. Faseb J. 1998; 12: 715-724Google Scholar, 24Cadenas S. Buckingham J.A. S. Seydoux J. N. Dulloo A.G. FEBS Lett. 1999; Scholar) and that are by superoxide D. J. Cadenas S. J.A. J.A. A. S. Nature. 2002; Scholar). is that the elevated expression of on lipid metabolism is a of a to of ROS production the oxidation of fatty In light of the controversy the production of H2O2 by mitochondria and its to studies to the generation of ROS by mitochondria are The of the study to the production of ROS by intact mitochondria from different to the electron transport sites in ROS to the topology of ROS on which side of the inner membrane ROS are and to in the production of ROS by heart mitochondria from rat and species with similar standard metabolic rate but different MLSP. The superoxide and hydrogen peroxide from The substrates and palmitoyl inhibitors antimycin and and bovine serum from rats between and by muscle, heart, and liver mitochondria as Biochim. Biophys. Acta. 1994; Scholar), in standard and at and on Mitochondria of in rate in the presence of an was determined using the A.G. J. Scholar) with as The rate of mitochondrial production of H2O2 was determined by its with acid in the presence of G. in Scholar) using a and pigeon mitochondria at at and in standard The to the standard at the in of from at and acid and a was for mitochondrial an inhibitor of the was to of H2O2 was by and for complex in the presence of for complex II or palmitoyl carnitine in the presence of as a of palmitoyl carnitine generates amounts of which the electron transport chain at complex I, and which the and by amounts of H2O2 to in the presence of the acid and They in the absence and presence of mitochondria to whether mitochondrial with the to H2O2 Mitochondria the the of the standard in the presence of skeletal muscle, and heart mitochondria and of the of the mitochondria the of various that we to the rates of H2O2 production using skeletal muscle as an and in in the the concentration of these did not the results not of mitochondrial H2O2 production using the standard with mitochondria than using the standard the and There significant rates of in the absence of mitochondria using the standard SOD, at the rate of of cytochrome by in a with and at at in a significant to the the under all conditions and The rates of H2O2 production in by the rates measured in the absence of mitochondria mitochondria: from the rates measured in the presence of mitochondria with the results fully rates of H2O2 of H2O2 production in the presence of and and The in the absence and presence of mitochondria the of with the inhibitors of electron transport used to more the sites of ROS production G. in Scholar). complex I, at and antimycin A at centero and center of complex III which side of the mitochondrial inner membrane superoxide was we measured the rate of H2O2 production in the presence and absence of exogenous mitochondria produce on the cytoplasmic face of the inner membrane, of exogenous the rate of (and that by or and with other side to an elevated rate of H2O2 production. mitochondria generate on the matrix side of the inner membrane, of it be to the matrix H2O2 and there be no difference in the rates of H2O2 in the presence and absence of exogenous Complex I concentration was measured as by H. Scholar). using The H2O2 production rate of mitochondria from a with a substrate was compared between different experimental conditions using of and the a between rat and pigeon heart mitochondria using a The of The rate of H2O2 production by skeletal muscle or heart mitochondria with and was and was not by of A and However, the of to mitochondria respiring on and a significant rate of A This H2O2 production from the matrix side of the inner membrane the was to of exogenous A and These results that complex I in skeletal muscle and heart mitochondria can generate superoxide on the matrix side of the inner membrane when it is fully reduced and by but that the rate measured when the complex is not by is of by rat heart mitochondria. are as as palmitoyl carnitine as in is by different There was no rate of H2O2 production when skeletal muscle or heart mitochondria the presence of in the absence or presence of and There was little or no rate of H2O2 production in the presence of in the absence or presence of that complex II the can produce significant amounts of on either side of the membrane when are of antimycin A to a but measurable rate of H2O2 production that was significantly by of exogenous and These results that center o of complex III can generate on the cytoplasmic face of the inner membrane of skeletal muscle or heart mitochondria when it is reduced of the complex at center by antimycin A but that the rate measured when the complex is not by antimycin A is There was some H2O2 production in the presence of antimycin A even exogenous SOD, which that center o can produce on the matrix side of the inner membrane. However, even mitochondria produce on the cytoplasmic face of the membrane, there be a rate of H2O2 production exogenous of or and the of In other a rate of H2O2 production has a and a we can that the from the cytoplasmic face of the membrane but we be whether the the rate of from the cytoplasmic face of the membrane or H2O2 from the matrix side of the membrane. muscle or heart mitochondria respiring on palmitoyl carnitine a significant rate of H2O2 production that was not significantly by of This that oxidation of palmitoyl carnitine oxidation of and or leads to significant ROS production and that this ROS is produced on the matrix side of the inner membrane. The of to a in the rate of H2O2 production that did not and was The rates of H2O2 production in the presence of palmitoyl carnitine and similar to in the presence of and A and suggesting that complex I was the source of this ROS with either substrate when complex I was fully reduced in the presence of In the absence of rotenone, perhaps complex I is more reduced with palmitoyl carnitine as substrate to electron transport and with for than it is with and to ROS production from complex I with palmitoyl carnitine. In the presence of palmitoyl the of to an in the rate of H2O2 production that did not in skeletal muscle mitochondria and was these conditions complex I, complex and the all be H2O2 production with palmitoyl carnitine was not than with palmitoyl carnitine this was it be that and can produce some on the matrix side of the membrane. Complex II and the not be the source of such there was no in the presence of and and of antimycin A to skeletal muscle or heart mitochondria with palmitoyl carnitine the rate of H2O2 production This rate was by of skeletal muscle that of it was due to production of on the cytoplasmic face of the inner membrane. these conditions complex I, complex the and center o of complex III all be In the presence of SOD, H2O2 production with palmitoyl carnitine antimycin A to be than the of the from complex I, complex the and center o of complex III. The for this is In the rates of of liver mitochondria lower than of skeletal muscle and heart with or with palmitoyl carnitine as substrate mitochondria respiring on and produced than mitochondrial in the absence or presence of The of did not H2O2 but to the rate of H2O2 production in the presence of These results to that complex I from liver mitochondria generates ROS on the cytoplasmic face of the inner membrane as as on the matrix but of the we to be mitochondria respiring on did not generate H2O2 except perhaps for a in the presence of and antimycin A or mitochondria respiring on palmitoyl carnitine did not produce H2O2 except perhaps for a in the presence of rotenone, antimycin or However, the rates of H2O2 production it to In a of rat pigeon heart mitochondria generated measurable amounts of H2O2 when with and The of the rate of H2O2 production in species In the presence of rotenone, the H2O2 production rate of mitochondrial was with rat mitochondria than pigeon mitochondria The of complex I in the two of mitochondria was measured to whether the of rat heart mitochondria to produce ROS in the presence of was by a concentration of complex I. Complex I was significantly in rat mitochondria than in pigeon mitochondria. that the different for ROS production between rat and pigeon heart mitochondria when H2O2 production rate was of complex I. Mitochondria from rat skeletal muscle, heart, and liver respiring on substrates to complex I or complex II in the absence of other inhibitors generated little or no measurable H2O2 and The absence of significant generation of H2O2 from mitochondria respiring on complex I and complex II substrates results from Staniek and Nohl (12Staniek K. Nohl H. Biochim. Biophys. Acta. 2000; 1460: 268-275Google Scholar) a of H2O2 production from rat heart mitochondria respiring on and J. Bioenerg. Biomembr. 1997; 29: Scholar) and (20Liu Y. Fiskum G. Schubert D. J. Neurochem. 2002; 80: 780-787Google Scholar) reported rates of H2O2 production from rat heart, or liver mitochondria with or as substrates. In the absence of rotenone, considerable ROS production from complex I by electron transport Y. Fiskum G. Schubert D. J. Neurochem. 2002; 80: 780-787Google this was not in the However, other studies have reported significant rates of H2O2 production in mitochondria in G. J. Bioenerg. Biomembr. 1999; 31: 347-366Google Scholar). of these studies used in the G. Cadenas S. Rojas C. Perez-Campo R. Lopez-Torres M. Free Radic. Res. 1994; 21: 317-327Google Scholar, 7Herrero A. Barja G. Mech. Ageing Dev. 1997; 98: 95-111Google Scholar, 8Herrero A. Barja G. J. Bioenerg. Biomembr. 1997; 29: 241-249Google Scholar, G. A. J. Bioenerg. Biomembr. 1998; Scholar). We considerable rates of H2O2 generation in the presence of we did not for some of the in production of H2O2 by mitochondria respiring on complex I and II substrates between various studies be due to the presence or absence of inhibitors of electron transport or to as by and Azzi (10Forman H.J. Azzi A. FASEB J. 1997; 11: 374-375Google Scholar) and Staniek and Nohl (12Staniek K. Nohl H. Biochim. Biophys. Acta. 2000; 1460: 268-275Google Scholar). In rat skeletal muscle and heart mitochondria produced H2O2 at measurable rates when respiring on palmitoyl carnitine with no inhibitors and This H2O2 was produced on the matrix side of the membrane, it was not significantly by of exogenous The H2O2 production with palmitoyl carnitine than with complex I substrates could be complex I is more reduced with palmitoyl due to electron transport and with for of complex I to matrix ROS production with palmitoyl carnitine as it could be that and can produce on the matrix side of the membrane when palmitoyl carnitine is from in the mitochondrial matrix of the is reduced to the semiquinone and more to the fully reduced suggesting that is the electron to M. Biochem. J. Scholar). can be fully reduced by three but it two when is the electron 1985; Scholar). was proposed that the of between the and semiquinone M. Biochem. J. Scholar). These that and could act as of superoxide to presence in reduced states lipid on the production of H2O2 by mitochondria that rat liver and pigeon heart mitochondria release H2O2 when respiring on palmitoyl carnitine A. Oshino N. Chance B. Biochem. J. 1972; : 617-630Google Scholar, 22Boveris A. Chance B. Biochem. J. 1973; 134: 707-716Google Scholar). These results gained little attention rates of H2O2 production. However, the lower rates of H2O2 production with palmitoyl carnitine in these the absence of carnitine and the use of of palmitoyl carnitine. is to that production of ROS by mitochondria lipid metabolism to an in the expression and of to the of ROS on mitochondria. In and expression is when fatty acids are (23Samec S. Seydoux J. Dulloo A.G. Faseb J. 1998; 12: 715-724Google Scholar, 24Cadenas S. Buckingham J.A. S. Seydoux J. N. Dulloo A.G. FEBS Lett. 1999; Scholar). acids and superoxide uncoupling by UCPs, and it was recently that the role of and be for ROS D. J. Cadenas S. J.A. J.A. A. S. Nature. 2002; Scholar). is commonly that of electron flow mitochondrial rise to H2O2 B. H. A. Scholar). J. Bioenerg. Biomembr. 1997; 29: Scholar) reported of free radical in the of for heart mitochondria respiring on physiological of than we our results with palmitoyl carnitine of of mitochondrial for skeletal muscle mitochondria of electron flow rise to conditions with a rate of of of mitochondrial This estimate of free radical be lower at physiological of the rate of H2O2 production by mitochondria with oxygen A. Chance B. Biochem. J. 1973; 134: 707-716Google Scholar). be even lower in more conditions of palmitoyl carnitine and lower mitochondrial membrane potential due to our upper estimate of free radical is to two of magnitude lower than the cited values. The results in this that rat heart and skeletal muscle mitochondria rates of H2O2 production than liver mitochondria either in the absence or presence of inhibitors, with the idea that tissues generate more ROS accumulation of damage S. Biochem. 1997; Scholar). A for these results is that liver mitochondria have a reduced of mitochondrial electron transport chain compared with heart and skeletal muscle Sohal R.S. Arch. Biochem. Biophys. 2000; Scholar). rat liver mitochondria have complex I and complex III than heart or skeletal muscle mitochondria Sohal R.S. Arch. Biochem. Biophys. 2000; Scholar). the potential sites of mitochondrial ROS production and the topology of ROS production from we used inhibitors of complex I and III of the electron transport chain. The inhibitors different complexes and cause to generate ROS to the of mitochondria. This to which complex has the to generate The presence and absence of the of the topology of ROS production. We that center o of complex III antimycin can generate more ROS than complex I and o of complex III generates superoxide in and on the cytoplasmic face of the mitochondrial inner membrane, whereas complex I ROS solely on the matrix side. using mitoplasts that complex III can release superoxide on the cytoplasmic face of the inner membrane (18Han D. Williams E. Cadenas E. Biochem. J. 2001; 353: 411-416Google Scholar). studies using intact mitochondria have reported in in the presence of antimycin A and (12Staniek K. Nohl H. Biochim. Biophys. Acta. 2000; 1460: 268-275Google Scholar, G. J. Bioenerg. Biomembr. 1999; 31: 347-366Google Scholar, 22Boveris A. Chance B. Biochem. J. 1973; 134: 707-716Google Scholar, J. Bioenerg. Biomembr. 1997; 29: Sohal R.S. Arch. Biochem. Biophys. 1998; Scholar). However, of these studies examined the topology of ROS production. Mitochondria respiring on complex I substrates H2O2 production in the presence of (13Barja G. J. Bioenerg. Biomembr. 1999; 31: 347-366Google Scholar, J. Bioenerg. Biomembr. 1997; 29: Scholar). the rates in the presence of in these studies are similar or than in the presence of it is that the ROS generated in the presence of from complex I. the in the rate of by mitochondria with palmitoyl carnitine and leads to that and produce ROS on the matrix side of the inner membrane and a physiological our results H2O2 production by mitochondria respiring on complex I and II substrates in the absence of inhibitors to support for the that there is an relationship between MLSP and H2O2 production by mitochondria from various species (4Sohal R.S. Svensson I. Brunk U.T. Mech. Ageing Dev. 1990; 53: 209-215Google Scholar) or that pigeon mitochondria respiring on complex I or II substrates generate H2O2 than rat mitochondria (5Ku H.H. Sohal R.S. Mech. Ageing Dev. 1993; 72: 67-76Google Scholar, 6Barja G. Cadenas S. Rojas C. Perez-Campo R. Lopez-Torres M. Free Radic. Res. 1994; 21: 317-327Google Scholar, 7Herrero A. Barja G. Mech. Ageing Dev. 1997; 98: 95-111Google Scholar, 8Herrero A. Barja G. J. Bioenerg. Biomembr. 1997; 29: 241-249Google Scholar, G. A. J. Bioenerg. Biomembr. 1998; Scholar). we that heart mitochondria from pigeons and rats respiring on did not generate measurable amounts of In the presence of rotenone, rat mitochondria produced more H2O2 of mitochondrial than did pigeon mitochondria that the of rat mitochondria to generate ROS is This was by a of complex I in rat heart mitochondria The rates of ROS production of complex I not in rat suggesting that complex I from pigeon heart mitochondria not have a for ROS production. These that the maximum of pigeon heart mitochondria to generate ROS from complex I is than in but to support for the theory that the elevated MLSP of pigeons compared with rats is due to lower mitochondrial production of be to mitochondrial H2O2 production between of different MLSP using palmitoyl carnitine. our results not be as that mitochondria produce no ROS under physiological the substrates be from or chain fatty from lipid Our results that complexes I and III of mitochondria do produce ROS oxidation of the these ROS are by and little or no H2O2 the the other fatty acid to release of ROS, from complex I on the matrix side of the inner membrane, which can to H2O2 the However, it is important to that the complex I, complex and perhaps and do have the to generate A of to oxygen that the metabolism be to cause accumulation of resulting in We and for of complex I and for