Interaction of α-Phenyl-N-tert-Butyl Nitrone and Alternative Electron Acceptors with Complex I Indicates a Substrate Reduction Site Upstream from the Rotenone Binding Site

Abstract: Mitochondrial complexes I, II, and III were studied in isolated brain mitochondrial preparations with the goal of determining their relative abilities to reduce O₂ to hydrogen peroxide (H₂O₂) or to reduce the alternative electron acceptors nitroblue tetrazolium (NBT) and diphenyliodonium (DPI). Complex I and II stimulation caused H₂O₂ formation and reduced NBT and DPI as indicated by dichlorodihydrofluorescein oxidation, nitroformazan precipitation, and DPI-mediated enzyme inactivation. The O₂ consumption rate was more rapid under complex II (succinate) stimulation than under complex I (NADH) stimulation. In contrast, H₂O₂ generation and NBT and DPI reduction kinetics were favored by NADH addition but were virtually unobservable during succinate-linked respiration. NADH oxidation was strongly suppressed by rotenone, but NADH-coupled H₂O₂ flux was accelerated by rotenone. α-Phenyl- N-tert -butyl nitrone (PBN), a compound documented to inhibit oxidative stress in models of stroke, sepsis, and parkinsonism, partially inhibited complex I-stimulated H₂O₂ flux and NBT reduction and also protected complex I from DPI-mediated inactivation while trapping the phenyl radical product of DPI reduction. The results suggest that complex I may be the principal source of brain mitochondrial H₂O₂ synthesis, possessing an "electron leak" site upstream from the rotenone binding site (i.e., on the NADH side of the enzyme). The inhibition of H₂O₂ production by PBN suggests a novel explanation for the broad-spectrum antioxidant and antiinflammatory activity of this nitrone spin trap.