title: ROS generation and multiple forms of mammalian mitochondrial glycerol-3-phosphate dehydrogenase.
abstract: Overproduction of reactive oxygen species (ROS) has been implicated in a range of pathologies. Mitochondrial flavin dehydrogenases glycerol-3-phosphate dehydrogenase (mGPDH) and succinate dehydrogenase (SDH) represent important ROS source, but the mechanism of electron leak is still poorly understood. To investigate the ROS production by the isolated dehydrogenases, we used brown adipose tissue mitochondria solubilized by digitonin as a model. Enzyme activity measurements and hydrogen peroxide production studies by Amplex Red fluorescence, and luminol luminescence in combination with oxygraphy revealed flavin as the most likely source of electron leak in SDH under in vivo conditions, while we propose coenzyme Q as the site of ROS production in the case of mGPDH. Distinct mechanism of ROS production by the two dehydrogenases is also apparent from induction of ROS generation by ferricyanide which is unique for mGPDH. Furthermore, using native electrophoretic systems, we demonstrated that mGPDH associates into homooligomers as well as high molecular weight supercomplexes, which represent native forms of mGPDH in the membrane. By this approach, we also directly demonstrated that isolated mGPDH itself as well as its supramolecular assemblies are all capable of ROS production.
authors.affiliation: Institute of Physiology Academy of Sciences of the Czech Republic v.v.i., Vídeňská 1083, 14220 Prague 4, Czech Republic.
authors.forename: Tomáš
authors.initials: T
authors.lastname: Mráček
authors.forename: Eliška
authors.initials: E
authors.lastname: Holzerová
authors.forename: Zdeněk
authors.initials: Z
authors.lastname: Drahota
authors.forename: Nikola
authors.initials: N
authors.lastname: Kovářová
authors.forename: Marek
authors.initials: M
authors.lastname: Vrbacký
authors.forename: Pavel
authors.initials: P
authors.lastname: Ješina
authors.forename: Josef
authors.initials: J
authors.lastname: Houštěk
mesh_terms: D000818:Animals; D004579:Electron Transport; D005292:Ferricyanides / Q000378:metabolism; D005993:Glycerolphosphate Dehydrogenase / Q000737:chemistry* / Q000378:metabolism; D005994:Glycerophosphates / Q000378:metabolism; D006861:Hydrogen Peroxide / Q000378:metabolism; D008322:Mammals; D008928:Mitochondria / Q000201:enzymology / Q000378:metabolism*; D051381:Rats; D017382:Reactive Oxygen Species / Q000378:metabolism*; D013385:Succinate Dehydrogenase / Q000737:chemistry / Q000378:metabolism; D014451:Ubiquinone / Q000378:metabolism
chemical_list: D005292:Ferricyanides; D005994:Glycerophosphates; D017382:Reactive Oxygen Species; D014451:Ubiquinone; D006861:Hydrogen Peroxide; D005993:Glycerolphosphate Dehydrogenase; D013385:Succinate Dehydrogenase
keywords: 2,6-dichlorophenolindophenol; 2-n-heptyl-4-hydroxyquinoline N-oxide; AA; BAT; CoQ; DCPIP; E.C. 1.1.5.3; FeCN; GP; HAR; HQNO; HRP; In-gel ROS detection; MXT; Mitochondrial glycerol-3-phosphate dehydrogenase; OXPHOS; ROS; ROS production; SDH; Supercomplex; antimycin A; brown adipose tissue; coenzyme Q; ferricyanide, potassium hexacyanoferrate(III); hexaammineruthenium(III) chloride; horseradish peroxidase; mGPDH; mitochondrial FAD-dependent glycerol-3-phosphate dehydrogenase; myxothiazol; oxidative phosphorylation; reactive oxygen species; sn-glycerol-3-phosphate; succinate dehydrogenase