Complex I Is the Major Site of Mitochondrial Superoxide Production by Paraquat

Abstract

Paraquat (1,1-dimethyl-4,4-bipyridinium dichloride) is widely used as a redox cycler to stimulate superoxide production in organisms, cells, and mitochondria. This superoxide production causes extensive mitochondrial oxidative damage, however, there is considerable uncertainty over the mitochondrial sites of paraquat reduction and superoxide formation. Here we show that in yeast and mammalian mitochondria, superoxide production by paraquat occurs in the mitochondrial matrix, as inferred from manganese superoxide dismutase-sensitive mitochondrial DNA damage, as well as from superoxide assays in isolated mitochondria, which were unaffected by exogenous superoxide dismutase. This paraquat-induced superoxide production in the mitochondrial matrix required a membrane potential that was essential for paraquat uptake into mitochondria. This uptake was of the paraquat dication, not the radical monocation, and was carrier-mediated. Experiments with disrupted mitochondria showed that once in the matrix paraquat was principally reduced by complex I (mammals) or by NADPH dehydrogenases (yeast) to form the paraquat radical cation that then reacted with oxygen to form superoxide. Together this membrane potential-dependent uptake across the mitochondrial inner membrane and the subsequent rapid reduction to the paraquat radical cation explain the toxicity of paraquat to mitochondria.Paraquat (1,1Ј-dimethyl-4,4Ј-bipyridinium dichloride; PQ) 2 is used to increase superoxide (O 2 . ) flux when investigating oxidative stress (reviewed in Refs. 1 and 2). The paraquat dication (PQ 2ϩ ) accepts an electron from a reductant to form the paraquat monocation radical (PQ . . and regenerate PQ 2ϩ (Fig. 1A). This redox cycling is a proximal cause of PQ toxicity, as indicated by the protection against PQ by superoxide dismutase (SOD) overexpression or administration of SOD mimetics (4 -8), and by the PQ hypersensitivity caused by SOD deficiency (9 -11).PQ has been used to generate O 2 . in systems ranging from isolated mitochondria (12-15) and cultured mammalian cells (4,14,16,17), to whole organisms including Saccharomyces cerevisiae (18 -21), Caenorhabditis elegans (22, 23), Drosophila melanogaster (9, 24 -26), and rodents (6,11,27). In many of these studies, PQ increases mitochondrial oxidative damage; for example, mitochondrial expression of human peroxiredoxin 5 protects yeast more effectively against PQ toxicity than expression in the cytosol (19); flies overexpressing catalase in mitochondria are resistant to PQ, whereas enhancement of cytosolic catalase was not protective (24); RNA interference silencing of MnSOD (the isoform of superoxide dismutase located in the mitochondrial matrix) in flies causes hypersensitivity to PQ (9), mice heterozygous for MnSOD show greater sensitivity to PQ than wild-type (11), and mitochondrial swelling is one of the earliest ultrastructural changes upon PQ exposure in vivo (28,29). Therefore the interaction of PQ with mitochondria is an important component of its toxicity, and PQ is used in expe...

Response to the Letter to the Editor: “Mitochondria isolated from the striatum of the brain exhibit a higher degree of oxidative phosphorylation coupling, which shows that they are not subject to energetic dysfunction upon acute paraquat administration”

“…The results indicated that the herbicide treatment inhibited by 32 % the rotenonesensitive complex I-III activity, which was similar to the data reported in our published work, indicating that the external NADH cytochrome c reductase system does not significantly contribute to NADH oxidation in our assays. It is important to note that the inhibition of complex I and the increase in free radicals production by paraquat has been extensively described in different models of toxicity (Cocheme and Murphy 2008;Drechsel and Patel 2009;Fukushima et al 1993;Tawara et al 1996) and our results were in accordance with those studies.…”

Two Thioredoxin Reductases, trxr-1 and trxr-2, Have Differential Physiological Roles in Caenorhabditis elegans

“…One possible mechanism is that nearby amino acid residues may increase the nucleophilicity of the Cys 2 by lowering its pKa as previously proposed in Drosophila melanogaster TrxR (Williams et al, 2000). At a physiological pH, most Cys with pKa~8.3 will be found in the protonated and thereby rather inert forms, while Sec would mainly be deprotonated and thus more more prone to engage in chemical reaction (Cocheme and Murphy, 2008). However, the actual pKa values of Cys present in protein can be significantly lowered by the combined effects of a number of other residues in the protein, depending on the microenvironment of the polypeptide structure.…”

“…The Trx-reducing activity of mammalian TrxR is totally selenium-dependent, which implies that all of the Trx-dependent systems, in fact, are selenium-dependent (Cocheme and Murphy, 2008). The lower pKa of a selenol and the higher nucleophilicity of a selenolate confers a type of "catalytic advantage" over Cys, when attacked position of thiol-disulfide interchange reaction.…”

Nrf2 stabilization prevents critical oxidative damage in Down syndrome cells

“…The ability of mCAT and cCAT to protect against chronic mitochondrial oxidative stress was assayed by treating mCAT‐ or cCAT‐expressing HEK cells with 5 mM PQ for 48 hr (Supporting Information Figure S2B). PQ diverts electrons out from Complex I of the electron transport chain generating ROS in the mitochondrial compartment (Cochemé & Murphy, 2008). Both CAT constructs prevented PQ‐induced cell death (Figure 1h) and diminished oxidative damage (Supporting Information Figure S2).…”