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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
Author(s) -
Hensley Kenneth,
Pye Quentin N.,
Maidt Michael L.,
Stewart Charles A.,
Robinson Kent A.,
Jaffrey Fatima,
Floyd Robert A.
Publication year - 1998
Publication title -
journal of neurochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.75
H-Index - 229
eISSN - 1471-4159
pISSN - 0022-3042
DOI - 10.1046/j.1471-4159.1998.71062549.x
Subject(s) - rotenone , chemistry , submitochondrial particle , quenching (fluorescence) , electron transport chain , nitrone , photochemistry , mitochondrion , hydrogen peroxide , electron acceptor , stimulation , biochemistry , stereochemistry , biology , fluorescence , physics , quantum mechanics , cycloaddition , catalysis , neuroscience
Mitochondrial complexes I, II, and III were studied in isolated brain mitochondrial preparations with the goal of determining their relative abilities to reduce O 2 to hydrogen peroxide (H 2 O 2 ) or to reduce the alternative electron acceptors nitroblue tetrazolium (NBT) and diphenyliodonium (DPI). Complex I and II stimulation caused H 2 O 2 formation and reduced NBT and DPI as indicated by dichlorodihydrofluorescein oxidation, nitroformazan precipitation, and DPI‐mediated enzyme inactivation. The O 2 consumption rate was more rapid under complex II (succinate) stimulation than under complex I (NADH) stimulation. In contrast, H 2 O 2 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 H 2 O 2 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 H 2 O 2 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 H 2 O 2 synthesis, possessing an “electron leak” site upstream from the rotenone binding site (i.e., on the NADH side of the enzyme). The inhibition of H 2 O 2 production by PBN suggests a novel explanation for the broad‐spectrum antioxidant and antiinflammatory activity of this nitrone spin trap.