title: Differential effects of mitochondrial Complex I inhibitors on production of reactive oxygen species.
abstract: We have investigated the production of reactive oxygen species (ROS) by Complex I in isolated open bovine heart submitochondrial membrane fragments during forward electron transfer in presence of NADH, by means of the probe 2',7'-Dichlorodihydrofluorescein diacetate. ROS production by Complex I is strictly related to its inhibited state. Our results indicate that different Complex I inhibitors can be grouped into two classes: Class A inhibitors (Rotenone, Piericidin A and Rolliniastatin 1 and 2) increase ROS production; Class B inhibitors (Stigmatellin, Mucidin, Capsaicin and Coenzyme Q(2)) prevent ROS production also in the presence of Class A inhibitors. Addition of the hydrophilic Coenzyme Q(1) as an electron acceptor potentiates the effect of Rotenone-like inhibitors in increasing ROS production, but has no effect in the presence of Stigmatellin-like inhibitors; the effect is not shared by more hydrophobic quinones such as decyl-ubiquinone. This behaviour relates the prooxidant CoQ(1) activity to a hydrophilic electron escape site. Moreover the two classes of Complex I inhibitors have an opposite effect on the increase of NADH-DCIP reduction induced by short chain quinones: only Class B inhibitors allow this increase, indicating the presence of a Rotenone-sensitive but Stigmatellin-insensitive semiquinone species in the active site of the enzyme. The presence of this semiquinone was also suggested by preliminary EPR data. The results suggest that electron transfer from the iron-sulphur clusters (N2) to Coenzyme Q occurs in two steps gated by two different conformations, the former being sensitive to Rotenone and the latter to Stigmatellin.
authors.affiliation: Dipartimento di Biochimica G. Moruzzi, University of Bologna, Via Irnerio 48, 40126 Bologna, Italy.
authors.forename: Romana
authors.initials: R
authors.lastname: Fato
authors.forename: Christian
authors.initials: C
authors.lastname: Bergamini
authors.forename: Marco
authors.initials: M
authors.lastname: Bortolus
authors.forename: Anna Lisa
authors.initials: AL
authors.lastname: Maniero
authors.forename: Serena
authors.initials: S
authors.lastname: Leoni
authors.forename: Tomoko
authors.initials: T
authors.lastname: Ohnishi
authors.forename: Giorgio
authors.initials: G
authors.lastname: Lenaz
mesh_terms: D000818:Animals; D002417:Cattle; D004578:Electron Spin Resonance Spectroscopy; D042967:Electron Transport Complex I / Q000037:antagonists & inhibitors* / Q000187:drug effects / Q000378:metabolism; D005663:Furans / Q000494:pharmacology; D006861:Hydrogen Peroxide / Q000378:metabolism; D007306:Insecticides / Q000494:pharmacology; D007700:Kinetics; D008928:Mitochondria / Q000378:metabolism; D008929:Mitochondria, Heart / Q000378:metabolism; D021122:Protein Subunits / Q000378:metabolism; D011725:Pyridines / Q000494:pharmacology; D017382:Reactive Oxygen Species / Q000378:metabolism*; D012402:Rotenone / Q000494:pharmacology; D013367:Submitochondrial Particles / Q000378:metabolism*; D013482:Superoxide Dismutase / Q000378:metabolism; D014451:Ubiquinone / Q000378:metabolism
chemical_list: D005663:Furans; D007306:Insecticides; D021122:Protein Subunits; D011725:Pyridines; D017382:Reactive Oxygen Species; D012402:Rotenone; C061022:bullatacin; D014451:Ubiquinone; C100213:piericidin A; D006861:Hydrogen Peroxide; D013482:Superoxide Dismutase; D042967:Electron Transport Complex I