Reprint of: Production of Superoxide Radicals and Hydrogen Peroxide by NADH- Ubiquinone Reductase and Ubiquinol-Cytochrome c Reductase from Beef-Heart Mitochondria

Abstract

Complex I (NADH-ubiquinone reductase) and Complex III (ubiquinol-cytochrome c reductase) supplemented with NADH generated O2−at maximum rates of 9.8 and 6.5 nmol/min/mg of protein, respectively, while, in the presence of superoxide dismutase, the same systems generated H2O2 at maximum rates of 5.1 and 4.2 nmol/min/mg of protein, respectively. H2O2 was essentially produced by disproportionation of O2−, which constitutes the precursor of H2O2. The effectiveness of the generation of oxygen intermediates by Complex I in the absence of other specific electron acceptors was 0.95 mol of O2− and 0.63 mol of H2O2/mol of NADH. A reduced form of ubiquinone appeared to be responsible for the reduction of O2 to O2−, since (a) ubiquinone constituted the sole common major component of Complexes I and III, (b) H202 generation by Complex I was inhibited by rotenone, and (c) supplementation of Complex I with exogenous ubiquinones increased the rate of H2O2 generation. The efficiency of added quinones as peroxide generators decreased in the order Q1 > Q0 > Q2 > Q6 = Q10, in agreement with the quinone capacity of acting as electron acceptor for Complex I. In the supplemented systems, the exogenous quinone was reduced by Complex I and oxidized nonenzymati- cally by molecular oxygen. Additional evidence for the role of ubiquinone as peroxide generator is provided by the generation of O2− and H2O2 during autoxidation of quinols. In oxygenated buffers, ubiquinol (Q0H2), benzoquinol, duroquinol and menadiol generated O2−with k3 values of 0.1 to 1.4 M-1 s-1 and H2O2 with k4 values of 0.009 to 4.3 m-1·s-1