Partitioning of Superoxide and Hydrogen Peroxide Production by Mitochondrial Respiratory Complex I

Membrane-bound respiratory complex I in inside-out submitochondrial particles (SMP) catalyzes both superoxide and hydrogen peroxide formation in NADH- and/or succinate-supported reactions. At optimal NADH concentration (50μM), the complex I-mediated process results in a formation of two superoxide anions and H(2)O(2) as the reaction products in approximately 0.7 ratio. Almost the same ratio is found for purified complex I (0.6) and for the aerobic succinate-supported reverse electron transfer reaction. Superoxide production is depressed at high, more physiologically relevant NADH concentrations, whereas hydrogen peroxide formation is insensitive to the elevated level of NADH. The rates of H(2)O(2) formation at variable NAD(+)/NADH ratios satisfactorily fit the Nernst equation for a single reactive two-electron donor component equilibrated with ambient midpoint redox potential of -347mV (0.13 NAD(+)/NADH ratio, pH 8.0). Half-maximal superoxide production rate proceeds at significantly higher NAD(+)/NADH ratio (0.33). Guanidine strongly stimulates NADH-supported hydrogen peroxide and superoxide production at any NADH concentration and activates NADH:ferricyanide and inhibits NADH:hexaammineruthenium (III) reductase activities while showing no effects on NADH oxidase of SMP. In the low range of NADH concentration, superoxide production rate shows a simple hyperbolic dependence on NADH with apparent K(m)(NADH) of 0.5μM, whereas sigmoidal dependence of hydrogen peroxide production is seen with half-maximal rate at 25μM NADH. We interpret the data as to suggest that at least two sites participate in complex I-mediated ROS generation: FMNH(-) that produces hydrogen peroxide, and an iron-sulfur center (likely N-2) that produces superoxide anion.