title: Protein S-glutathionylation alters superoxide/hydrogen peroxide emission from pyruvate dehydrogenase complex.
abstract: Pyruvate dehydrogenase (Pdh) is a vital source of reactive oxygen species (ROS) in several different tissues. Pdh has also been suggested to serve as a mitochondrial redox sensor. Here, we report that O2•-/ H2O2emission from pyruvate dehydrogenase (Pdh) is altered by S-glutathionylation. Glutathione disulfide (GSSG) amplified O2•-/ H2O2production by purified Pdh during reverse electron transfer (RET) from NADH. Thiol oxidoreductase glutaredoxin-2 (Grx2) reversed these effects confirming that Pdh is a target for S-glutathionylation. S-glutathionylation had the opposite effect during forward electron transfer (FET) from pyruvate to NAD+lowering O2•-/ H2O2production. Immunoblotting for protein glutathione mixed disulfides (PSSG) following diamide treatment confirmed that purified Pdh can be S-glutathionylated. Similar observations were made with mouse liver mitochondria. S-glutathionylation catalysts diamide and disulfiram significantly reduced pyruvate or 2-oxoglutarate driven O2•-/ H2O2production in liver mitochondria, results that were confirmed using various Pdh, 2-oxoglutarate dehydrogenase (Ogdh), and respiratory chain inhibitors. Immunoprecipitation of Pdh and Ogdh confirmed that either protein can be S-glutathionylated by diamide and disulfiram. Collectively, our results demonstrate that the S -glutathionylation of Pdh alters the amount of ROS formed by the enzyme complex. We also confirmed that Ogdh is controlled in a similar manner. Taken together, our results indicate that the redox sensing and ROS forming properties of Pdh and Ogdh are linked to S-glutathionylation.
authors.affiliation: Department of Biochemistry, Memorial University of Newfoundland, 230 Elizabeth Ave, St. John's, Newfoundland, Canada A1B 3X9.
authors.forename: Marisa
authors.initials: M
authors.lastname: O'Brien
authors.affiliation: Department of Biochemistry, Memorial University of Newfoundland, 230 Elizabeth Ave, St. John's, Newfoundland, Canada A1B 3X9.
authors.forename: Julia
authors.initials: J
authors.lastname: Chalker
authors.affiliation: Department of Biochemistry, Memorial University of Newfoundland, 230 Elizabeth Ave, St. John's, Newfoundland, Canada A1B 3X9.
authors.forename: Liam
authors.initials: L
authors.lastname: Slade
authors.affiliation: Department of Biochemistry, Memorial University of Newfoundland, 230 Elizabeth Ave, St. John's, Newfoundland, Canada A1B 3X9.
authors.forename: Danielle
authors.initials: D
authors.lastname: Gardiner
authors.affiliation: Department of Biochemistry, Memorial University of Newfoundland, 230 Elizabeth Ave, St. John's, Newfoundland, Canada A1B 3X9. Electronic address: rjmailloux@mun.ca.
authors.forename: Ryan J
authors.initials: RJ
authors.lastname: Mailloux
mesh_terms: D000818:Animals; D042967:Electron Transport Complex I / Q000378:metabolism; D054477:Glutaredoxins / Q000378:metabolism; D005978:Glutathione / Q000378:metabolism*; D006861:Hydrogen Peroxide / Q000378:metabolism; D007655:Ketoglutarate Dehydrogenase Complex / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D008931:Mitochondria, Muscle / Q000378:metabolism; D010084:Oxidation-Reduction; D018384:Oxidative Stress; D011768:Pyruvate Dehydrogenase Complex / Q000235:genetics / Q000378:metabolism*; D017382:Reactive Oxygen Species / Q000378:metabolism*; D013481:Superoxides / Q000378:metabolism
chemical_list: C516009:Glrx2 protein, mouse; D054477:Glutaredoxins; D011768:Pyruvate Dehydrogenase Complex; D017382:Reactive Oxygen Species; D013481:Superoxides; D006861:Hydrogen Peroxide; D007655:Ketoglutarate Dehydrogenase Complex; D042967:Electron Transport Complex I; D005978:Glutathione
keywords: Mitochondria; Pyruvate dehydrogenase; Reactive oxygen species; Redox signaling; S-glutathionylation