Inhibitors of the Quinone-binding Site Allow Rapid Superoxide Production from Mitochondrial NADH:Ubiquinone Oxidoreductase (Complex I)

Adrian J Lambert,Martin D Brand

doi:10.1074/jbc.m406576200

Adrian J Lambert, Martin D Brand

Open Access

https://doi.org/10.1074/jbc.m406576200

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Abstract

Neither the route of electron transport nor the sites or mechanism of superoxide production in mitochondrial complex I has been established. We examined the rates of superoxide generation (measured as hydrogen peroxide production) by rat skeletal muscle mitochondria under a variety of conditions. The rate of superoxide production by complex I during NADH-linked forward electron transport was less than 10% of that during succinate-linked reverse electron transport even when complex I was fully reduced by pyruvate plus malate in the presence of the complex III inhibitor, stigmatellin. This asymmetry was not explained by differences in protonmotive force or its components. However, when inhibitors of the quinone-binding site of complex I were added in the presence of ATP to generate a pH gradient, there was a rapid rate of superoxide production by forward electron transport that was as great as the rate seen with reverse electron transport at the same pH gradient. These observations suggest that quinone-binding site inhibitors can make complex I adopt the highly radical-producing state that occurs during reverse electron transport. Despite complete inhibition of NADH: ubiquinone oxidoreductase activity in each case, different classes of quinone-binding site inhibitor (rotenone, piericidin, and high concentrations of myxothiazol) gave different rates of superoxide production during forward electron transport (the rate with myxothiazol was twice that with rotenone) suggesting that the site of rapid superoxide generation by complex I is in the region of the ubisemiquinone-binding sites and not upstream at the flavin or low potential FeS centers.

Highlights

Removed from the cytosol, as in the case of copper/zinc superoxide dismutase (SOD) nullizygous mice, the effects are not lethal, an increase in sensitivity to oxidative stress is apparent [3]
The rate of superoxide production by complex I during NADH-linked forward electron transport was less than 10% of that during succinate-linked reverse electron transport even when complex I was fully reduced by pyruvate plus malate in the presence of the complex III inhibitor, stigmatellin
In agreement with previous studies (10 –15), we show that superoxide production by complex I during reverse electron transport is huge compared with forward electron transport under similar conditions

Summary

EXPERIMENTAL PROCEDURES

Materials—Piericidin A was a kind gift from Dr Mauro Degli Esposti (University of Manchester, UK). Superoxide production rate was assessed by measurement of hydrogen peroxide generation rate, determined fluorometrically by measurement of oxidation of p-hydroxyphenylacetic acid (PHPA) coupled to the enzymatic reduction of H2O2 by horseradish peroxidase. Certain compounds employed in the experiments (such as myxothiazol and ATP) caused significant quenching of the fluorescent signals; careful calibration with standard curves generated for all conditions was essential to obtain the correct rates of H2O2 production. Measurement of Mitochondrial Protonmotive Force (⌬p)—Mitochondrial membrane potential, ⌬␺, was determined using an electrode sensitive to TPMPϩ as described [24]. Skeletal muscle mitochondria were incubated under the same conditions as for superoxide production at 37 °C in standard buffer with PHPA, horseradish peroxidase, and SOD. The significance of differences between means was assessed by unpaired Student’s t test; p values Ͻ 0.05 were taken to be significant

RESULTS

40 Ϯ 5 34 Ϯ 4

DISCUSSION