Differential effects of mitochondrial Complex I inhibitors on production of reactive oxygen species |
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Authors: | Romana Fato Marco Bortolus Serena Leoni Giorgio Lenaz |
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Institution: | a Dipartimento di Biochimica “G. Moruzzi”, University of Bologna, Via Irnerio 48, 40126 Bologna, Italy b Johnson Research Foundation, Department of Biochemistry and Biophysics, School of Medicine, University of Pennsylvania, Philadelphia, PA, USA c Dipartimento di Scienze Chimiche, University of Padova, via Marzolo 1, 35131 Padova, Italy |
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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 Q2) prevent ROS production also in the presence of Class A inhibitors. Addition of the hydrophilic Coenzyme Q1 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 CoQ1 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. |
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Keywords: | DCFDA 2&prime 7&prime -Dichlorodihydrofluorescein diacetate NADH β-Nicotinamide adenine dinucleotide ROS Reactive oxygen species MTT 3-[4 5-dimethyl-thiazol-2-yl]-2 5-diphenyl-tetrazolium bromide FMN Flavin mononucleotide BHM Bovine heart mitochondria BSA Bovine serum albumine DOC Deoxycholate SMP Submitochondrial particles from bovine heart DPI Diphenylene iodonium DB Decyl-ubiquinone DCIP 2 6-dichloroindo-phenol CoQ1 2 3-Dimethoxy-5-methyl-6-(3-methyl-2-butenyl)-1 4-benzoquinone FCCP Carbonylcyanide-p-trifluoromethoxyphenylhydrazone AAPH α α&prime -Azodiisobutyramidine dihydrochloride |
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