INTERACTION OF ALPHA-PHENYL-N-TERT-BUTYL NITRONE AND ALTERNATIVE ELECTRON-ACCEPTORS WITH COMPLEX-I INDICATES A SUBSTRATE REDUCTION SITE UPSTREAM FROM THE ROTENONE BINDING-SITE

Mitochondrial complexes I, II, and III were studied in isolated brain mitochondrial preparations with the goal of determining their relative abilities to reduce Ca to hydrogen peroxide (H2O1) or to reduce the a lternative electron accepters nitroblue tetrazolium (NBT) and diphenyl iodonium (DPI). Complex I and II stimulation caused H2O2 formation and reduced NET and DPI as indicated by dichlorodihydrofluorescein oxidat ion, nitro-formazan precipitation, and DPI-mediated enzyme inactivatio n. The O-2 consumption rate was more rapid under complex II (succinate ) stimulation than under complex I (NADH) stimulation. In contrast, H2 O2 generation and NET and DPI reduction kinetics were favored by NADH addition but were virtually unobservable during succinate-linked respi ration. NADH oxidation was strongly suppressed by rotenone, but NADH-c oupled H2O2 flux was accelerated by rotenone. alpha-Phenyl-N-tert-buty l nitrone (PBN), a compound documented to inhibit oxidative stress in models of stroke, sepsis, and parkinsonism, partially inhibited comple x I-stimulated H2O2 flux and NET reduction and also protected complex I from DPI-mediated inactivation while trapping the phenyl radical pro duct of DPI reduction. The results suggest that complex I may be the p rincipal source of brain mitochondrial H2O2 synthesis, possessing an ' 'electron leak'' site upstream from the rotenone binding site (i,e., o n the NADH side of the enzyme). The inhibition of H2O2 production by P EN suggests a novel explanation for the broad-spectrum antioxidant and antiinflammatory activity of this nitrone spin trap.