title: Superoxide activates mitochondrial uncoupling protein 2 from the matrix side. Studies using targeted antioxidants.
abstract: Superoxide activates nucleotide-sensitive mitochondrial proton transport through the uncoupling proteins UCP1, UCP2, and UCP3 (Echtay, K. S., et al. (2002) Nature 415, 1482-1486). Two possible mechanisms were proposed: direct activation of the UCP proton transport mechanism by superoxide or its products and a cycle of hydroperoxyl radical entry coupled to UCP-catalyzed superoxide anion export. Here we provide evidence for the first mechanism and show that superoxide activates UCP2 in rat kidney mitochondria from the matrix side of the mitochondrial inner membrane: (i) Exogenous superoxide inhibited matrix aconitase, showing that external superoxide entered the matrix. (ii) Superoxide-induced uncoupling was abolished by low concentrations of the mitochondrially targeted antioxidants 10-(6'-ubiquinonyl)decyltriphenylphosphonium (mitoQ) or 2-[2-(triphenylphosphonio)ethyl]-3,4-dihydro-2,5,7,8-tetramethyl-2H-1-benzopyran-6-ol bromide (mitoVit E), which are ubiquinone (Q) or tocopherol derivatives targeted to the matrix by covalent attachment to triphenylphosphonium cation. However, superoxide-induced uncoupling was not affected by similar concentrations of the nontargeted antioxidants Q(o), Q(1), decylubiquinone, vitamin E, or 6-hydroxy-2,5,7,8-tetramethylchroman 2-carboxylic acid (TROLOX) or of the mitochondrially targeted but redox-inactive analogs decyltriphenylphosphonium or 4-chlorobutyltriphenylphosphonium. Thus matrix superoxide appears to be necessary for activation of UCP2 by exogenous superoxide. (iii) When the reduced to oxidized ratio of mitoQ accumulated by mitochondria was increased by inhibiting cytochrome oxidase, it induced nucleotide-sensitive uncoupling that was not inhibited by external superoxide dismutase. Under these conditions quinols are known to produce superoxide, and because mitoQ is localized within the mitochondrial matrix this suggests that production of superoxide in the matrix was sufficient to activate UCP2. Furthermore, the superoxide did not need to be exported or to cycle across the inner membrane to cause uncoupling. We conclude that superoxide (or its products) exerts its uncoupling effect by activating the proton transport mechanism of uncoupling proteins at the matrix side of the mitochondrial inner membrane.
authors.affiliation: Medical Research Council Dunn Human Nutrition Unit, Hills Road, Cambridge CB2 2XY, UK.
authors.forename: Karim S
authors.initials: KS
authors.lastname: Echtay
authors.forename: Michael P
authors.initials: MP
authors.lastname: Murphy
authors.forename: Robin A J
authors.initials: RA
authors.lastname: Smith
authors.forename: Darren A
authors.initials: DA
authors.lastname: Talbot
authors.forename: Martin D
authors.initials: MD
authors.lastname: Brand
mesh_terms: D000154:Aconitate Hydratase / Q000378:metabolism; D000818:Animals; D000975:Antioxidants / Q000494:pharmacology*; D002412:Cations; D002462:Cell Membrane / Q000378:metabolism; D002839:Chromans / Q000494:pharmacology; D003486:Cyanides / Q000378:metabolism; D005260:Female; D007473:Ion Channels; D007668:Kidney / Q000378:metabolism; D007700:Kinetics; D008564:Membrane Potentials; D026901:Membrane Transport Proteins; D051379:Mice; D018345:Mice, Knockout; D008928:Mitochondria / Q000378:metabolism; D024101:Mitochondrial Proteins; D008956:Models, Chemical; D011485:Protein Binding; D011506:Proteins / Q000378:metabolism*; D011522:Protons; D051381:Rats; D017208:Rats, Wistar; D017382:Reactive Oxygen Species; D013481:Superoxides / Q000378:metabolism*; D013997:Time Factors; D014451:Ubiquinone / Q000378:metabolism; D000071246:Uncoupling Protein 2; D014810:Vitamin E / Q000494:pharmacology
chemical_list: D000975:Antioxidants; D002412:Cations; D002839:Chromans; D003486:Cyanides; D007473:Ion Channels; D026901:Membrane Transport Proteins; D024101:Mitochondrial Proteins; D011506:Proteins; D011522:Protons; D017382:Reactive Oxygen Species; C000604389:UCP2 protein, human; C000604390:Ucp2 protein, mouse; C000604391:Ucp2 protein, rat; D000071246:Uncoupling Protein 2; D013481:Superoxides; D014451:Ubiquinone; D014810:Vitamin E; D000154:Aconitate Hydratase; C010643:6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid