Production of superoxide radicals and hydrogen peroxide by NADH-ubiquinone reductase and ubiquinol-cytochrome c reductase from beef-heart mitochondria

Dedicated to Professor Luis F. Leloir on the occasion of his seventieth birthday.
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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) H2O2 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 nonenzymatically 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 · s−1 and H2O2 with k4 values of 0.009 to 4.3 m−1 · s−1.

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    Permanent address: Department of Physiology and Biochemistry, The University of Southampton, Southampton S09 3TU, England.

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