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 O 2 − 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 H 2O 2 at maximum rates of 5.1 and 4.2 nmol/min/mg of protein, respectively. H 2O 2 was essentially produced by disproportionation of O 2 −, which constitutes the precursor of H 2O 2. The effectiveness of the generation of oxygen intermediates by Complex I in the absence of other specific electron acceptors was 0.95 mol of O 2 − and 0.63 mol of H 2O 2/mol of NADH. A reduced form of ubiquinone appeared to be responsible for the reduction of O 2 to O 2 −, since (a) ubiquinone constituted the sole common major component of Complexes I and III, (b) H 2O 2 generation by Complex I was inhibited by rotenone, and (c) supplementation of Complex I with exogenous ubiquinones increased the rate of H 2O 2 generation. The efficiency of added quinones as peroxide generators decreased in the order Q 1 > Q 0 > Q 2 > Q 6 = Q 10, 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 nonenzymatically by molecular oxygen. Additional evidence for the role of ubiquinone as peroxide generator is provided by the generation of O 2 − and H 2O 2 during autoxidation of quinols. In oxygenated buffers, ubiquinol (Q 0H 2), benzoquinol, duroquinol and menadiol generated O 2 − with k 3 values of 0.1 to 1.4 m − · s −1 and H 2O 2 with k 4 values of 0.009 to 4.3 m −1 · s −1.