The sites and topology of mitochondrial superoxide production

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Abstract

Mitochondrial superoxide production is an important source of reactive oxygen species in cells, and may cause or contribute to ageing and the diseases of ageing. Seven major sites of superoxide production in mammalian mitochondria are known and widely accepted. In descending order of maximum capacity they are the ubiquinone-binding sites in complex I (site IQ) and complex III (site IIIQo), glycerol 3-phosphate dehydrogenase, the flavin in complex I (site IF), the electron transferring flavoprotein:Q oxidoreductase (ETFQOR) of fatty acid beta-oxidation, and pyruvate and 2-oxoglutarate dehydrogenases. None of these sites is fully characterized and for some we only have sketchy information. The topology of the sites is important because it determines whether or not a site will produce superoxide in the mitochondrial matrix and be able to damage mitochondrial DNA. All sites produce superoxide in the matrix; site IIIQo and glycerol 3-phosphate dehydrogenase also produce superoxide to the intermembrane space. The relative contribution of each site to mitochondrial reactive oxygen species generation in the absence of electron transport inhibitors is unknown in isolated mitochondria, in cells or in vivo, and may vary considerably with species, tissue, substrate, energy demand and oxygen tension.

Introduction

In the mitochondrial free radical theory of ageing (Harman, 1972), mitochondrial reactive oxygen species (ROS) generation is an inevitable consequence of oxidative ATP production, and the primary cause of macromolecular damage. Some damage is not repaired, causing progressive failure of cellular machinery, ageing-related diseases, and ageing. There is considerable evidence both in favour and against the theory (Beckman and Ames, 1998, Finkel and Holbrook, 2000, Golden et al., 2002, Muller et al., 2007, Sanz et al., 2006). Most authors think that mitochondria-generated oxidative stress is important, particularly in ageing-related diseases, but is not the sole cause of ageing. According to a recent review (Muller et al., 2007), mitochondrial ROS convincingly determines lifespan in fungi (hyphal senescence in Podospora and chronological ageing in Saccharomyces) and reasonably convincingly in Caenorhabditis elegans. The case is tentative in Drosophila but inconclusive in mouse and human, and better tests are required.

Seven specific sites that are involved in ROS generation have been defined in isolated mammalian mitochondria using electron transport chain inhibitors (Andreyev et al., 2005, Brand et al., 2004, Jezek and Hlavata, 2005, Murphy, 2009, Raha and Robinson, 2000, Turrens, 2003), but we lack reliable measurements of their rates in the absence of such inhibitors, and there is no consensus on their relative importance. In cells our knowledge of which sites are important is even worse. It is based mostly on measurements using inhibitors of electron transport. Typically, if rotenone (a complex I inhibitor) raises ROS production in cells, endogenous ROS generation is inferred to be from complex I, but this inference is unjustifiable, and new approaches are needed.

Section snippets

The mitochondrial free radical theory of ageing

An association between mitochondrial ROS generation and age-related disease is generally accepted, although it is also agreed that the mitochondrial free radical theory’s explanation of ageing is incomplete (Beckman and Ames, 1998, Finkel and Holbrook, 2000, Golden et al., 2002, Muller et al., 2007, Sanz et al., 2006). Since age is the primary risk factor for many diseases, understanding the mechanisms of ageing may allow us to significantly reduce the burden of disease and increase human

Mitochondria as a source of ROS

The respiratory chain produces superoxide when single electrons leak to O2 as electron pairs flow down the chain (Chance et al., 1979). Interest in mitochondrial ROS began more than 40 years ago (Boveris and Chance, 1973, Boveris et al., 1972, Hinkle et al., 1967, Jensen, 1966) and has been well-reviewed (Andreyev et al., 2005, Brand et al., 2004, Jezek and Hlavata, 2005, Murphy, 2009, Raha and Robinson, 2000, Turrens, 2003). There are currently seven separate sites of mammalian mitochondrial

Measurement of ROS production in isolated mitochondria

Superoxide does not cross the mitochondrial inner membrane, so bulk phase assays using isolated mitochondria do not register matrix superoxide. However, matrix Mn–SOD converts most of the matrix superoxide to H2O2, which diffuses to the medium for assay. The standard assay of extramitochondrial H2O2 is to add horseradish peroxidase (to reduce H2O2 to H2O) and a substrate (e.g. amplex red) that is oxidized to a fluorescent product (resorufin). The method underestimates matrix superoxide

Sites of mitochondrial ROS production

The relative importance of each site to total superoxide production in isolated mitochondria is contentious, partly because of different assays, different substrates and different sources of mitochondria. Most assays of superoxide production from defined sites measure maximal capacities for superoxide production, and the actual rate from each site in the absence of inhibitors is not known. During reverse electron transport from succinate to NAD+, complex I can produce superoxide at high rates (

Topology of mitochondrial superoxide production

The topology of ROS production is crucial for which sites might most damage mtDNA. In intact mitochondria superoxide that is produced to the matrix is converted by matrix Mn–SOD to H2O2, which diffuses out and is assayed in the extramitochondrial medium. However, superoxide that is produced to the intermembrane space requires added SOD to be fully converted to H2O2 for assay, so H2O2 production that is dependent on added SOD is produced to the intermembrane space not to the matrix. This assay

ROS production by mitochondria in cells

Mitochondria are frequently asserted to be the main producers of ROS in cells (Skulachev, 1996), but this conclusion is not thoroughly established. In some cells, other sources, such as cytochrome P450 (Caro and Cederbaum, 2004) or plasma membrane NADPH oxidases (Babior et al., 2002), produce large amounts of ROS (Jezek and Hlavata, 2005), and it remains to be established what proportion of cellular ROS production is mitochondrial in different cells. Since mitochondrial ROS production may

Conclusion

Mitochondrial ROS production is clearly involved in ageing and the diseases of ageing, but its exact roles and importance remain ambiguous. We now know the identity of seven sites within mitochondria that generate superoxide, and have a good idea of their topology and their relative maximum capacities (Fig. 1). What we do not know is the rate at which these sites run under different physiological conditions of substrate supply and energy demand in isolated mitochondria, in intact cells, and in

Acknowledgements

Supported by grants from the National Institutes of Health (P01 AG025901, PL1 AG032118, P30 AG025708 and R01 AG033542) and the Keck Foundation.

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