A Possible Site of Superoxide Generation in the Complex I Segment of Rat Heart Mitochondria

Ohnishi, S. Tsuyoshi; Ohnishi, Tomo̧ko; Muranaka, Shikibu; Fujita, Hirofumi; Kimura, Hiroko; Uemura, Kenichiro; Yoshida, Ken; Utsumi, Kozo

doi:10.1007/s10863-005-4117-y

Cited by 117 publications

(93 citation statements)

References 89 publications

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“…Proposals have included the flavin (11)(12)(13)35), bound reduced nucleotide (14), FeS clusters N2 (15) and N1a (16), and a semiquinone radical (17,18). Here, we demonstrate that fully reduced flavin, on the matrix side of the inner membrane, is a significant source of superoxide in isolated complex I.…”

Section: Discussionmentioning

confidence: 66%

“…Many previous studies have addressed the question of how superoxide is produced by complex I (11)(12)(13)(14)(15)(16)(17)(18). Most of these studies examined intact mitochondria or submitochondrial particles, in which it is difficult to correlate observations directly to complex I, or to define and control the conditions precisely (NADH, NAD ϩ , ubiquinone, and ubiquinol concentrations, redox status, proton motive force, and pH).…”

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confidence: 99%

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The mechanism of superoxide production by NADH:ubiquinone oxidoreductase (complex I) from bovine heart mitochondria

Kußmaul

Hirst

2006

Proc. Natl. Acad. Sci. U.S.A.

644

561

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NADH:ubiquinone oxidoreductase (complex I) is a major source of reactive oxygen species in mitochondria and a significant contributor to cellular oxidative stress. Here, we describe the kinetic and molecular mechanism of superoxide production by complex I isolated from bovine heart mitochondria and confirm that it produces predominantly superoxide, not hydrogen peroxide. Redox titrations and electron paramagnetic resonance spectroscopy exclude the iron-sulfur clusters and flavin radical as the source of superoxide, and, in the absence of a proton motive force, superoxide formation is not enhanced during turnover. Therefore, superoxide is formed by the transfer of one electron from fully reduced flavin to O2. The resulting flavin radical is unstable, so the remaining electron is probably redistributed to the iron-sulfur centers. The rate of superoxide production is determined by a bimolecular reaction between O2 and reduced flavin in an empty active site. The proportion of the flavin that is thus competent for reaction is set by a preequilibrium, determined by the dissociation constants of NADH and NAD ؉ , and the reduction potentials of the flavin and NAD ؉ . Consequently, the ratio and concentrations of NADH and NAD ؉ determine the rate of superoxide formation. This result clearly links our mechanism for the isolated enzyme to studies on intact mitochondria, in which superoxide production is enhanced when the NAD ؉ pool is reduced. Therefore, our mechanism forms a foundation for formulating causative connections between complex I defects and pathological effects.flavin ͉ iron-sulfur cluster ͉ semiquinone ͉ oxidative stress T he production of reactive oxygen species, such as superoxide, by mitochondria is a major cause of cellular oxidative stress. It contributes to many pathological conditions such as Parkinson's and other neurodegenerative diseases, ischemia reperfusion injury, atherosclerosis, and aging (1-3). In mammalian mitochondria, most of the superoxide originates from NADH:ubiquinone oxidoreductase (complex I) and ubiquinol: cytochrome c oxidoreductase (complex III) of the electron transport chain, but there is increasing evidence that superoxide production by complex I, into the mitochondrial matrix, is predominant (4, 5). Indeed, complex I deficiencies have been identified across a wide spectrum of pathologies and linked to enhanced superoxide production as well as to deficiencies in energy production (6-10). Therefore, it is imperative to define how, why, and when superoxide is produced by complex I to formulate causative connections with pathological effects and rational proposals for how defects may be addressed.Many previous studies have addressed the question of how superoxide is produced by complex I (11-18). Most of these studies examined intact mitochondria or submitochondrial particles, in which it is difficult to correlate observations directly to complex I, or to define and control the conditions precisely (NADH, NAD ϩ , ubiquinone, and ubiquinol concentrations, redox status, proton motive...

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Section: Discussionmentioning

confidence: 66%

mentioning

confidence: 99%

The mechanism of superoxide production by NADH:ubiquinone oxidoreductase (complex I) from bovine heart mitochondria

Kußmaul

Hirst

2006

Proc. Natl. Acad. Sci. U.S.A.

644

561

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“…1 and 2). However, this amount was clearly detectable, an observation that which differs from isolated muscle, heart, and brain mitochondria (41,44,49), which generate very little or no H 2 O 2 when complex I substrates are oxidized. Complex I substrates in muscle, heart, and brain mitochondria generate more H 2 O 2 when rotenone is added, possibly due to downstream inhibition of a Q-cycle type mechanism in complex I, similar to the known effect of antimycin at complex III (44).…”

Section: Discussionmentioning

confidence: 95%

Reactive Oxygen and Targeted Antioxidant Administration in Endothelial Cell Mitochondria

O’Malley

Fink

Ross

et al. 2006

Journal of Biological Chemistry

115

103

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We used fluorescent probes and EPR to study the mechanism(s) underlying reactive oxygen species (ROS) production by endothelial cell mitochondria and the action of mitoquinol, a mitochondria-targeted antioxidant. ROS measured by fluorescence resulted from complex I superoxide released to the matrix and converted to H 2 O 2 . In contrast, EPR largely detected superoxide generated at complex III and effluxed outward. ROS fluorescence by mitochondria fueled by the complex II substrate, succinate, was substantial but markedly inhibited by rotenone. Superoxide, detected by EPR, in succinate-fueled mitochondria was not inhibited by rotenone and likely derived from semiquinone formation at complex III. Mitoquinol decreased H 2 O 2 fluorescence by succinate-fueled mitochondria but had little effect on the EPR signal for superoxide. This was not associated with a detectable decrease in membrane potential. Mitoquinol markedly enhanced ROS fluorescence in mitochondria fueled by the complex I substrates, glutamate and malate. Inhibitor studies suggested that this occurred in complex I, at one or more Q binding pockets. The above effects of mitoquinol were determined in mitochondria isolated and subsequently exposed to the targeted antioxidant. However, similar effects were observed in mitochondria after antecedent exposure to mitoquinol/mitoquinone in culture, suggesting that the agent is retained after isolation of the organelles. In conclusion, ROS production in bovine aortic endothelial cell mitochondria results largely from reverse transport to complex I and through the Q cycle in complex III. Mitoquinol blocks ROS from reverse electron transport but increases superoxide production derived from forward transport. These effects likely occur at one or more Q binding sites in complex I. Control of reactive oxygen species (ROS)2 in endothelial cells is particularly important, because endothelial dysfunction, which may be triggered by ROS (1), is a major factor contributing to the development of atherosclerotic heart disease (2-4). Moreover, mitochondrial oxidative damage may underlie problems, including the vascular and neurologic complications of diabetes, cell damage in degenerative diseases, and aging (5-9). Reactive oxygen derived from endothelial cell mitochondria has also been implicated in the metabolic pathways leading to the microvascular complications of diabetes (6).Mitochondrial electron transport generates substantial amounts of superoxide derived from electron leaks as substrates are metabolized (10). The process may have adverse consequences as evidenced by observations in mice lacking the mitochondrial enzyme manganese superoxide dismutase. These mice develop dilated cardiomyopathy and live less than 2 weeks (11). The major sites of superoxide production have been somewhat controversial, but there is evidence that most derive from complexes I and III (12). There is also evidence that complex I superoxide is released exclusively to the matrix side of the inner membrane, whereas complex III likely generates su...

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“…Two regions of the enzyme complex are hypothesized to be responsible for generating the superoxide anion radical (O 2 ·-). One is located on the FMN cofactor and is modulated by its binding protein moiety (4,5,7), while the other is likely located on the ubiquinone-binding site and probably acts in the mediation of ubiquinone reduction (8,9).…”

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Site-Specific S-Glutathiolation of Mitochondrial NADH Ubiquinone Reductase

et al. 2007

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The generation of reactive oxygen species in mitochondria acts as a redox signal in triggering cellular events such as apoptosis, proliferation, and senescence. Overproduction of superoxide (O 2 ·-) and O 2 ·--derived oxidants change the redox status of the mitochondrial GSH pool. An electron transport protein, Mitochondrial Complex I, is the major host of reactive/regulatory protein thiols. An important response of protein thiols to oxidative stress is to reversibly form protein mixed disulfide via S-glutathiolation. Exposure of Complex I to oxidized GSH, GSSG, resulted in specific Sglutathiolation at the 51 kDa and 75 kDa subunits. Here, to investigate the molecular mechanism of S-glutathiolation of Complex I, we prepared isolated bovine Complex I under non-reducing conditions and employed the techniques of mass spectrometry and EPR spin trapping for analysis. LC/MS/MS analysis of tryptic digests of the 51 kDa and 75 kDa polypeptides from glutathiolated Complex I (GS-NQR) revealed that two specific cysteines (C 206 and C 187 ) of the 51 kDa subunit and one specific cysteine (C 367 ) of the 75 kDa subunit were involved in redox modifications with GS binding. The electron transfer activity (ETA) of GS-NQR in catalyzing NADH oxidation by Q 1 was significantly enhanced. However, O 2 ·-generation activity (SGA) mediated by GS-NQR suffered a mild loss as measured by EPR spin trapping, suggesting the protective role of Sglutathiolation in the intact Complex I. Exposure of NADH dehydrogenase (NDH), the flavin subcomplex of Complex I, to GSSG resulted in specific S-glutathiolation on the 51 kDa subunit. Both ETA and SGA of S-glutathiolated NDH (GS-NDH) decreased in parallel as the dosage of GSSG increased. LC/MS/MS analysis of a tryptic digest of the 51 kDa subunit from GS-NDH revealed that C 206 , C 187 , and C 425 were glutathiolated. C 425 of the 51 kDa subunit is a ligand residue of the 4Fe-4S N3 center, suggesting that destruction of 4Fe-4S is the major mechanism involved in the inhibiton of NDH. The result also implies that S-glutathiolation of the 75 kDa subunit may play a role in protecting the 4Fe-4S cluster of the 51 kDa subunit from redox modification when Complex I is exposed to redox change in the GSH pool.Mitochondrial Complex I (EC 1.6.5.3. NADH:ubiquinone oxidoreductase) is the first energyconserving segment of the electron transport chain (ETC) (1-3). The enzyme catalyzes electron transfer from NADH to ubiquinone coupled with the translocation of four protons across the

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A Possible Site of Superoxide Generation in the Complex I Segment of Rat Heart Mitochondria

Cited by 117 publications

References 89 publications

The mechanism of superoxide production by NADH:ubiquinone oxidoreductase (complex I) from bovine heart mitochondria

The mechanism of superoxide production by NADH:ubiquinone oxidoreductase (complex I) from bovine heart mitochondria

Reactive Oxygen and Targeted Antioxidant Administration in Endothelial Cell Mitochondria

Site-Specific S-Glutathiolation of Mitochondrial NADH Ubiquinone Reductase

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