title: Thiol-based antioxidants elicit mitochondrial oxidation via respiratory complex III.
abstract: Excessive oxidation is widely accepted as a precursor to deleterious cellular function. On the other hand, an awareness of the role of reductive stress as a similar pathological insult is emerging. Here we report early dynamic changes in compartmentalized glutathione (GSH) redox potentials in living cells in response to exogenously supplied thiol-based antioxidants. Noninvasive monitoring of intracellular thiol-disulfide exchange via a genetically encoded biosensor targeted to cytosol and mitochondria revealed unexpectedly rapid oxidation of the mitochondrial matrix in response to GSH ethyl ester or N-acetyl-l-cysteine. Oxidation of the probe occurred within seconds in a concentration-dependent manner and was attenuated with the membrane-permeable ROS scavenger tiron. In contrast, the cytosolic sensor did not respond to similar treatments. Surprisingly, the immediate mitochondrial oxidation was not abrogated by depolarization of mitochondrial membrane potential or inhibition of mitochondrial GSH uptake. After detection of elevated levels of mitochondrial ROS, we systematically inhibited multisubunit protein complexes of the mitochondrial respiratory chain and determined that respiratory complex III is a downstream target of thiol-based compounds. Disabling complex III with myxothiazol completely blocked matrix oxidation induced with GSH ethyl ester or N-acetyl-l-cysteine. Our findings provide new evidence of a functional link between exogenous thiol-containing antioxidants and mitochondrial respiration.
authors.forename: Vladimir L
authors.initials: VL
authors.lastname: Kolossov
authors.forename: Jessica N
authors.initials: JN
authors.lastname: Beaudoin
authors.forename: Nagendraprabhu
authors.initials: N
authors.lastname: Ponnuraj
authors.forename: Stephen J
authors.initials: SJ
authors.lastname: DiLiberto
authors.forename: William P
authors.initials: WP
authors.lastname: Hanafin
authors.forename: Paul J A
authors.initials: PJ
authors.lastname: Kenis
authors.forename: H Rex
authors.initials: HR
authors.lastname: Gaskins
mesh_terms: D000818:Animals; D000975:Antioxidants / Q000494:pharmacology*; D015374:Biosensing Techniques; D016466:CHO Cells; D003412:Cricetulus; D014450:Electron Transport Complex III / Q000037:antagonists & inhibitors / Q000378:metabolism*; D054477:Glutaredoxins / Q000235:genetics / Q000378:metabolism; D005978:Glutathione / Q000378:metabolism; D045325:HCT116 Cells; D057809:HEK293 Cells; D006801:Humans; D053078:Membrane Potential, Mitochondrial / Q000187:drug effects; D008928:Mitochondria / Q000187:drug effects* / Q000378:metabolism; D010084:Oxidation-Reduction; D018384:Oxidative Stress / Q000187:drug effects*; D017382:Reactive Oxygen Species / Q000378:metabolism; D013438:Sulfhydryl Compounds / Q000494:pharmacology*; D013552:Swine; D013997:Time Factors; D014162:Transfection
chemical_list: D000975:Antioxidants; D054477:Glutaredoxins; D017382:Reactive Oxygen Species; D013438:Sulfhydryl Compounds; D014450:Electron Transport Complex III; D005978:Glutathione