Evidence for Two Sites of Superoxide Production by Mitochondrial NADH-Ubiquinone Oxidoreductase (Complex I)

Jason R Treberg,Casey L Quinlan,Martin D Brand

doi:10.1074/jbc.m111.252502

Jason R Treberg, Casey L Quinlan + Show 1 more

Open Access

https://doi.org/10.1074/jbc.m111.252502

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Abstract

Complex I (NADH-ubiquinone oxidoreductase) can form superoxide during forward electron flow (NADH-oxidizing) or, at sufficiently high protonmotive force, during reverse electron transport from the ubiquinone (Q) pool (NAD(+)-reducing). We designed an assay system to allow titration of the redox state of the superoxide-generating site during reverse electron transport in rat skeletal muscle mitochondria: a protonmotive force generated by ATP hydrolysis, succinate:malonate to alter electron supply and modulate the redox state of the Q pool, and inhibition of complex III to prevent QH(2) oxidation via the Q cycle. Stepwise oxidation of the QH(2)/Q pool by increasing malonate concentration slowed the rates of both reverse electron transport and rotenone-sensitive superoxide production by complex I. However, the superoxide production rate was not uniquely related to the resultant potential of the NADH/NAD(+) redox couple. Thus, there is a superoxide producer during reverse electron transport at complex I that responds to Q pool redox state and is not in equilibrium with the NAD reduction state. In contrast, superoxide production during forward electron transport in the presence of rotenone was uniquely related to NAD redox state. These results support a two-site model of complex I superoxide production; one site in equilibrium with the NAD pool, presumably the flavin of the FMN moiety (site I(F)) and the other dependent not only on NAD redox state, but also on protonmotive force and the reduction state of the Q pool, presumably a semiquinone in the Q-binding site (site I(Q)).

Highlights

Complex I can produce superoxide at very high rates under conditions that drive the reduction of NADϩ by reverse electron transport
An important distinction is that the rate of superoxide production by isolated mitochondria during reverse electron transport can be severalfold higher than the maximum rate when the flavin is fully reduced by the addition of NADHgenerating substrates plus rotenone or other complex I Q site inhibitors [10, 20]
Support for the two-site model comes from three key observations: (i) there is a higher superoxide production rate during reverse electron transport compared with forward electron transport; (ii) this higher rate is not accompanied by a greater NADH/NADϩ ratio in isolated mitochondria as would be predicted by a onesite (IF) model, but instead by slightly lower NAD reduction [22]; and (iii) this higher rate is highly sensitive to dissipation of ⌬pH [10]

Summary

Introduction

Complex I can produce superoxide at very high rates under conditions that drive the reduction of NADϩ by reverse electron transport. We test the one-site model by measuring the relationship between superoxide production and the potential of the NADH/NADϩ redox couple during reverse electron transport in isolated mitochondria.

Results

Conclusion