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      Superoxide Is Produced by the Reduced Flavin in Mitochondrial Complex I : A SINGLE, UNIFIED MECHANISM THAT APPLIES DURING BOTH FORWARD AND REVERSE ELECTRON TRANSFER *

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          Abstract

          NADH:ubiquinone oxidoreductase (complex I) is a major source of reactive oxygen species in mitochondria and a contributor to cellular oxidative stress. In isolated complex I the reduced flavin is known to react with molecular oxygen to form predominantly superoxide, but studies using intact mitochondria contend that superoxide may result from a semiquinone species that responds to the proton-motive force (Δp) also. Here, we use bovine heart submitochondrial particles to show that a single mechanism describes superoxide production by complex I under all conditions (during both NADH oxidation and reverse electron transfer). NADH-induced superoxide production is inhibited by complex I flavin-site inhibitors but not by inhibitors of ubiquinone reduction, and it is independent of Δp. Reverse electron transfer (RET) through complex I in submitochondrial particles, driven by succinate oxidation and the Δp created by ATP hydrolysis, reduces the flavin, leading to NAD + and O 2 reduction. RET-induced superoxide production is inhibited by both flavin-site and ubiquinone-reduction inhibitors. The potential dependence of NADH-induced superoxide production (set by the NAD + potential) matches that of RET-induced superoxide production (set by the succinate potential and Δp), and they both match the potential dependence of the flavin. Therefore, both NADH- and RET-induced superoxide are produced by the flavin, according to the same molecular mechanism. The unified mechanism describes how reactive oxygen species production by complex I responds to changes in cellular conditions. It establishes a route to understanding causative connections between the enzyme and its pathological effects and to developing rational strategies for addressing them.

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          The sites and topology of mitochondrial superoxide production.

          Mitochondrial superoxide production is an important source of reactive oxygen species in cells, and may cause or contribute to ageing and the diseases of ageing. Seven major sites of superoxide production in mammalian mitochondria are known and widely accepted. In descending order of maximum capacity they are the ubiquinone-binding sites in complex I (site IQ) and complex III (site IIIQo), glycerol 3-phosphate dehydrogenase, the flavin in complex I (site IF), the electron transferring flavoprotein:Q oxidoreductase (ETFQOR) of fatty acid beta-oxidation, and pyruvate and 2-oxoglutarate dehydrogenases. None of these sites is fully characterized and for some we only have sketchy information. The topology of the sites is important because it determines whether or not a site will produce superoxide in the mitochondrial matrix and be able to damage mitochondrial DNA. All sites produce superoxide in the matrix; site IIIQo and glycerol 3-phosphate dehydrogenase also produce superoxide to the intermembrane space. The relative contribution of each site to mitochondrial reactive oxygen species generation in the absence of electron transport inhibitors is unknown in isolated mitochondria, in cells or in vivo, and may vary considerably with species, tissue, substrate, energy demand and oxygen tension. Copyright (c) 2010 Elsevier Inc. All rights reserved.
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            The mechanism of superoxide production by NADH:ubiquinone oxidoreductase (complex I) from bovine heart mitochondria.

            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.
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              Mitochondrial alpha-ketoglutarate dehydrogenase complex generates reactive oxygen species.

              Mitochondria-produced reactive oxygen species (ROS) are thought to contribute to cell death caused by a multitude of pathological conditions. The molecular sites of mitochondrial ROS production are not well established but are generally thought to be located in complex I and complex III of the electron transport chain. We measured H(2)O(2) production, respiration, and NADPH reduction level in rat brain mitochondria oxidizing a variety of respiratory substrates. Under conditions of maximum respiration induced with either ADP or carbonyl cyanide p-trifluoromethoxyphenylhydrazone,alpha-ketoglutarate supported the highest rate of H(2)O(2) production. In the absence of ADP or in the presence of rotenone, H(2)O(2) production rates correlated with the reduction level of mitochondrial NADPH with various substrates, with the exception of alpha-ketoglutarate. Isolated mitochondrial alpha-ketoglutarate dehydrogenase (KGDHC) and pyruvate dehydrogenase (PDHC) complexes produced superoxide and H(2)O(2). NAD(+) inhibited ROS production by the isolated enzymes and by permeabilized mitochondria. We also measured H(2)O(2) production by brain mitochondria isolated from heterozygous knock-out mice deficient in dihydrolipoyl dehydrogenase (Dld). Although this enzyme is a part of both KGDHC and PDHC, there was greater impairment of KGDHC activity in Dld-deficient mitochondria. These mitochondria also produced significantly less H(2)O(2) than mitochondria isolated from their littermate wild-type mice. The data strongly indicate that KGDHC is a primary site of ROS production in normally functioning mitochondria.
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                Author and article information

                Journal
                J Biol Chem
                jbc
                jbc
                JBC
                The Journal of Biological Chemistry
                American Society for Biochemistry and Molecular Biology (9650 Rockville Pike, Bethesda, MD 20814, U.S.A. )
                0021-9258
                1083-351X
                20 May 2011
                10 March 2011
                10 March 2011
                : 286
                : 20
                : 18056-18065
                Affiliations
                [1]From the Medical Research Council Mitochondrial Biology Unit, Wellcome Trust/MRC Building, Hills Road, Cambridge, CB2 0XY, United Kingdom
                Author notes
                [1 ] To whom correspondence should be addressed. Tel.: 44-1223-252810; E-mail: jh@ 123456mrc-mbu.cam.ac.uk .
                Article
                M110.186841
                10.1074/jbc.M110.186841
                3093879
                21393237
                7ee2466d-d61d-4df4-b183-1a47f1b4a6c3
                © 2011 by The American Society for Biochemistry and Molecular Biology, Inc.

                Author's Choice—Final version full access.

                Creative Commons Attribution Non-Commercial License applies to Author Choice Articles

                History
                : 20 September 2010
                : 23 February 2011
                Categories
                Bioenergetics

                Biochemistry
                mitochondria,respiratory chain,membrane energetics,enzyme mechanisms,bioenergetics,oxidative stress,reactive oxygen species (ros),flavoproteins,proton pumps,nadh:ubiquinone oxidoreductase

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