COMPLEX I IS THE MAJOR SITE OF MITOCHONDRIAL SUPEROXIDE PRODUCTION BY PARAQUAT*

Helena M. Cochemé and Michael P. Murphy From the Medical Research Council Dunn Human Nutrition Unit, Wellcome Trust/MRC Building,

Hills Road, Cambridge CB2 0XY, UK Running Title: Interaction of paraquat with mitochondria

Address correspondence to: Michael P. Murphy, Medical Research Council Dunn Human Nutrition Unit, Wellcome Trust/MRC Building, Hills Road, Cambridge CB2 0XY, United Kingdom. Tel: +44 (0)1223 252 900. Fax: +44 (0)1223 252 905. E-mail: mpm@mrc-dunn.cam.ac.uk

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 which 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 which 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 (1,1′-dimethyl-4,4′-bipyridinium dichloride; PQ) is used to increase superoxide (O2

•-) flux when investigating oxidative stress (reviewed in 1,2). The paraquat dication (PQ2+)

accepts an electron from a reductant to form the paraquat monocation radical (PQ•+), which then rapidly reacts with O2 (k ~7.7 x 108 M-1.s-1) (3) to produce O2

•- and regenerate PQ2+ (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 O2•- 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); RNAi-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 experimental models of Parkinson disease to generate mitochondrial oxidative damage (30). Consequently, there is considerable interest in identifying the sites of PQ2+ reduction associated with mitochondria and in determining whether O2

•- production by PQ occurs within

1

http://www.jbc.org/cgi/doi/10.1074/jbc.M708597200The latest version is at JBC Papers in Press. Published on November 26, 2007 as Manuscript M708597200

Copyright 2007 by The American Society for Biochemistry and Molecular Biology, Inc.

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mitochondria, or if it takes place outside and then passes into the matrix. The very negative reduction potential of PQ (PQ2+/PQ•+, E0 = –446 mV) (1) severely restricts its pool of possible intracellular reductants. However, there are a number of proposed sites for PQ2+ reduction both inside and outside mitochondria including NAD(P)H-dependent flavoenzymes, such as microsomal NADPH-cytochrome P450 reductase (31,32), NADH-cytochrome b5 oxidoreductase and NADH-coenzyme Q oxidoreductase of the mitochondrial outer membrane (28,33), and complex I of the mitochondrial inner membrane (34). Here we show that PQ2+ is taken up across the mitochondrial inner membrane by a carrier-mediated and membrane potential (∆ψm)-dependent process, and that once in the matrix PQ2+ is reduced to PQ•+ by complex I in mammalian mitochondria and by NADPH dehydrogenases in yeast. The PQ•+ then reacts with oxygen to form O2

•- and cause mitochondrial oxidative stress. EXPERIMENTAL PROCEDURES

Chemicals - Coelenterazine (CLZ; 2-(p-hydroxybenzyl)-6-(p-hydroxyphenyl)-8-benzyl-imidazo[1,2-a]pyrazin-3-(7H)-one) was from Calbiochem. Amplex Red was from Molecular Probes. 1-Methyl-4-phenylpyridinium iodide (MPP+) was from Research Biochemicals, Inc. (USA). [3H]-TPMP+

(specific activity 60 Ci.mmol-1, 1 mCi.mL-1) and [14C]-PQ (specific activity 55 mCi.mmol-1, 0.1 mCi.mL-1) were from American Radiolabeled Chemicals, Inc. (USA). All other reagents were supplied by Sigma-Aldrich or BDH.

Yeast Strains and Growth - The Saccharomyces cerevisiae strains used were: wild-type EG103 (MATα his3 leu2 trp1 ura3) (18) and CEN.PK2-1C (MATα his3 leu2 trp1 ura3) (35), and the MnSOD knock-out (∆sod2) EG110 (EG103, sod2∆::TRP1) (36). The S. cerevisiae deletion library (Open Biosystems) was derived from the wild-type strain BY4741 (MATa his3 leu2 met15 ura3). Yeast were cultured in the following liquid media: lactate (2% (v/v) DL-lactic acid, 0.3% yeast extract, 0.05% glucose, 0.05% CaCl2⋅2H2O, 0.05% NaCl, 0.06% MgCl2⋅6H2O,

0.1% KPi, 0.1% NH4Cl (all w/v); pH 5.5 NaOH), YPD (1% (w/v) yeast extract, 2% (w/v) peptone, 2% (w/v) glucose) and YPG (1% (w/v) yeast extract, 2% (w/v) peptone, 3% (v/v) glycerol). For plates, liquid media were supplemented with 2% (w/v) agar. YPG+E plates contained 1 mg.mL-1 erythromycin. For growth assays, 10 mL yeast cultures at A600 ~0.1 were treated with PQ (0.01–10 mM) and incubated at 30ºC with shaking at 250 rpm. A600 was measured after 48 h and was expressed as a percentage of the appropriate control without PQ. To screen for PQ-resistant yeast mutants, YPG medium containing a concentration gradient of PQ was prepared in 10 cm square Petri dishes (Sarstedt). A slant of YPG + 1 mM PQ was first poured and once set was overlaid with standard YPG to form a flat surface. Yeast suspensions were then spotted (~30,000 cells) in rows along the PQ concentration gradient (0–1 mM), incubated for 1 week a 30ºC, and growth was compared against a wild-type control on each plate.

Mitochondrial Preparations - Yeast mitochondria were isolated from lactate-grown wild-type (strain EG103) and ∆sod2 cultures as described previously (37,38). The protein concentration was measured by the bicinchoninic acid method with BSA as a standard (39). Aliquots of the mitochondrial preparation mixed with 10 mg.mL-1 fatty acid-free BSA were snap-frozen and stored at –80°C and before use were thawed rapidly (~30 s at 30ºC), pelleted by centrifugation (2 min at 16,000 x g), and washed once in mannitol buffer (0.6 M mannitol, 10 mM Tris maleate, 5 mM KPi, 0.5 mM EDTA; pH 6.8 KOH). These mitochondria can maintain a ∆ψm indistinguishable from that of freshly isolated mitochondria (data not shown). Mitochondria from rat liver and rat heart were prepared fresh by homogenization and differential centrifugation (40). The protein concentration was determined by the biuret method with BSA as a standard (41). Rat liver and heart mitochondrial incubations were performed in KCl buffer (120 mM KCl, 10 mM HEPES, 1 mM EGTA; pH 7.2 KOH). When required, mitochondria were disrupted with a sonicator (Misonix 3000, setting 3) for 3 x 5 s periods at 30 s intervals on ice. Bovine heart mitochondrial membranes were prepared by mechanical disruption of isolated bovine heart

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mitochondria (42), and were resuspended in KPi buffer (50 mM KPi, 1 mM EGTA, 100 µM DTPA, 100 µM neocuproine; pH 7.2 KOH).

Measurement of Mitochondrial Membrane Potential - Mitochondrial membrane potential (∆ψm) was measured from the uptake of [3H]-methyl triphenylphosphonium (TPMP+) (43). Yeast mitochondria (0.1–0.4 mg protein.mL-1) were incubated for 3 min at 30°C in 1 mL mannitol buffer with substrate and 1 µM TPMP+ including 25 nCi.mL-1 [3H]-TPMP+, and then pelleted by centrifugation (2 min at 16,000 x g). Radioactivity in the pellet and supernatant was measured by liquid scintillation analysis (44), and the ∆ψm was derived from the Nernst equation (43). The mitochondrial matrix volume was taken as 1.8 µL.mg protein-1, and data were corrected for the 60% of TPMP+ assumed to be membrane-bound (published by 45; calculated according to the method in 43).

Superoxide and H2O2 Assays - Aconitase activity was measured spectrophotometrically by a coupled enzyme assay (46), and O2

•- production was inferred from the rate of aconitase inactivation (15). O2

•- was also assayed by chemiluminescence of coelenterazine (CLZ) in a luminometer (Berthold AutoLumatPlus LB 953) over 5 min with cumulative readings for 5 s every 30 s (47,48) and was expressed as relative light units (RLU).s-1. Samples were incubated in a 1 mL volume at 30ºC with 2 µM CLZ. H2O2 efflux from heart mitochondria was assayed using a fluorometer (Shimadzu RF-5301) by incubating heart mitochondria at 37ºC in a stirred 2.5 mL volume with 5 U.mL-1 horseradish peroxidase and 50 µM Amplex Red (15), and was calibrated against H2O2 standards.

Yeast Mitochondrial DNA Damage Assays - Cytoplasmic petite mutants were identified by the colorimetric tetrazolium overlay technique (49). Yeast were cultured overnight in 5 mL YPD medium, from A600 ~0.1 until A600 ~10. An aliquot was diluted into H2O, spread onto YPD plates and incubated at 30ºC for 2 days until colonies formed. Plates were then overlaid with 20 mL of 1.5% (w/v) agar dissolved in 67 mM NaPi (pH 7.0) and supplemented with 0.1% (w/v) 2,3,5-triphenyltetrazolium chloride, incubated for a further 1 h at 30ºC and then scored for red

(= respiration-competent) or white (= petite) colonies. Approximately 5,000 colonies were scored for each condition.

Mitochondrial DNA (mtDNA) point mutations were measured by the erythromycin resistance (EryR) assay, based on the principle that specific point mutations in the mtDNA-encoded 21S ribosomal gene can confer EryR (50,51). Wild-type yeast (strain CEN.PK2-1C) were streaked onto YPG agar and incubated at 30ºC for ~2–3 days until single colonies formed. Yeast cultures were then prepared by inoculating 5 mL YPG with a single colony and incubating ± 100 µM PQ at 30ºC for 48 h. A 20 µL sample was removed, diluted into H2O, spread in triplicate onto YPG agar plates and incubated at 30ºC for 2 days to establish the number of respiration-competent cells. The remainder of the culture was washed in H2O, resuspended in ~300 µL H2O and divided between two YPG+E agar plates, which were scored for EryR colonies after ~7 days at 30ºC. The proportion of EryR cells was expressed per 108 respiration-competent cells.

Detection of the PQ•+ Radical - The PQ•+ radical was detected either by EPR (52) or spectrophotometrically at 603 nm (3). For EPR experiments, an EMX 10/12 EPR spectrometer and an AquaX cell (Bruker, Germany) were used at room temperature with the following instrument settings: microwave frequency, 9.85 GHz; microwave power, 10 mW; modulation frequency, 100 kHz; modulation amplitude, 0.8 G; sweep time, 83.886 s; time constant, 163.84 ms; receiver gain, 6.32 x 104. The PQ•+ radical was generated in vitro from PQ2+ by reduction with freshly prepared sodium dithionite. Hypoxic conditions were established by purging samples with nitrogen. Quantitative determinations were based on the height of the maximum peak (Fig. 7A), which gave a linear standard curve in the range tested (0–250 µM; Fig. 7B). The EPR signal of the SO2

•- radical in the dithionite solution did not interfere with quantitation of the PQ•+ radical (data not shown). PQ•+ formation by hypoxic mitochondria was monitored by following an increase in A603 over time with a spectrophotometer (DW-2000 SLM-Aminco), equipped with stirring and thermostatted at 30ºC. The suspension was rendered anaerobic by purging with argon and sealing the 3 mL quartz cuvette. Following a 5–10

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min incubation, samples were subjected to absorbance readings (500–700 nm), firstly while still under anaerobic conditions and then following exposure to air. Difference spectra ± O2 were then determined to diagnose PQ•+ radical formation.

Mitochondrial Uptake of PQ - Ion-selective electrodes were constructed and used as described (53,54), except that the plasticizer dioctyl phthalate was replaced by 2-fluoro-2′-nitrodiphenyl ether (55), which greatly improved membrane selectivity for PQ. Electrodes were characterized for either PQ or 1-methyl-4-phenylpyridinium (MPP+) by filling and soaking overnight in a 10 mM aqueous solution. Incubations were performed in a stirred 3 mL chamber, thermostatted at 30ºC. The PQ-electrode

response was linear with the log10[PQ2+], with a slope of 29.4 ± 1.0 mV per decade (mean ± sd; n = 5, with at least three different PQ-electrodes) which is consistent with the 30.02 mV predicted by the Nernst equation for a dication species at 30ºC. Alternatively, uptake of PQ by mitochondria was measured by centrifuging the incubations (2 min at 16,000 x g) and quantifying the amount of PQ in the mitochondrial pellet, either by EPR (under anaerobic conditions following conversion to PQ•+ with excess sodium dithionite), or by using radiolabelled [14C]-PQ with liquid scintillation analysis. In some cases, dual isotope counting ([3H]-TPMP and [14C]-PQ) was performed to control for any effect of PQ uptake on ∆ψm. RESULTS AND DISCUSSION

PQ Increases Mitochondrial Matrix O2•-

within Intact Yeast - Growth of wild-type yeast on the non-fermentable substrate glycerol was more PQ-sensitive than on the fermentable substrate glucose (Fig. 2A), indicating that PQ disrupted

mitochondrial function. This disruption was due to O2

•- within mitochondria, as growth of yeast lacking mitochondrial SOD (∆sod2) on the non-fermentable substrate lactate was far more PQ-sensitive than that of the wild-type strain (Fig. 2B) (56). To see if PQ caused damage in the mitochondrial matrix, we studied its effect on the accumulation of specific point mutations in mitochondrial DNA (mtDNA) using the

erythromycin resistance (EryR) assay (50,57), and found that PQ elevated mtDNA damage (Fig. 2C).

The damage to mtDNA was due to O2•- within the

mitochondrial matrix, as PQ only increased the formation of petite colonies in the ∆sod2 yeast strain and not the wild type (Fig. 2D). Together these data confirm that PQ causes mitochondrial oxidative damage by increasing matrix O2

•-. Matrix O2

•- Production by PQ in Isolated Yeast Mitochondria Requires a Membrane Potential and a Respiratory Substrate - We next tested the effect of PQ on O2

•- production in isolated yeast mitochondria as measured by the O2

•--specific chemiluminescence of the membrane-permeant probe coelenterazine (CLZ). In the presence of the respiratory substrate ethanol, PQ caused a dramatic dose-dependent increase in O2

•- production by ∆sod2 mitochondria, but not wild-type mitochondria (Fig. 3A). Surprisingly, this O2

•- production was completely blocked by abolishing the mitochondrial membrane potential (∆ψm) with the uncoupler carbonyl cyanide p-trifluoro-methoxyphenylhydrazone (FCCP) (Fig. 3A). Addition of exogenous SOD did not affect PQ-induced O2

•- production (Fig. 3B), consistent with PQ generating O2

•- inside the mitochondrial matrix.

The CLZ assay did not detect matrix O2•-

production in wild-type yeast mitochondria (Fig. 3A), because CLZ cannot compete for O2

•- against MnSOD (k ~105 and 109 M-1.s-1 respectively) (58,59). Therefore to assess O2

•- formation in wild-type yeast mitochondria, we measured the inactivation of the matrix enzyme aconitase, which is significantly more reactive with O2

•- (k ~107 M-1.s-1) (60) than CLZ and will not react with O2

•- outside the mitochondrial matrix. Aconitase inactivation also showed a dose-dependent increase in O2

•- production with PQ

within isolated yeast mitochondria that required a respiratory substrate, was blocked by FCCP (Fig. 3C) and was unaffected by exogenous SOD (Fig. 3D).

A similar pattern of O2•- production was

obtained by both the CLZ and the aconitase assays when glycerol-3-phosphate (5 mM) was used as a respiratory substrate (data not shown). Together these data indicate that PQ produces O2

•- inside the mitochondrial matrix in isolated yeast mitochondria in the presence of a respiratory

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substrate and a ∆ψm. The requirement for a ∆ψm could be to enhance the uptake of PQ into the mitochondrial matrix and/or to assist in its reduction by the respiratory chain.

PQ Increases O2•- Production by

Mammalian Mitochondria - To see if the interaction of PQ with mammalian and yeast mitochondria was similar, we next investigated its effects on O2

•- production within isolated rat heart mitochondria. As with yeast mitochondria, there was a dose-dependent inactivation of aconitase by PQ that required a respiratory substrate, was blocked by FCCP (Fig. 3E), and was unaffected by exogenous SOD (Fig. 3F). Thus PQ-induced O2

•- production by mammalian mitochondria is similar to that of yeast mitochondria. Interestingly, the rate of PQ-induced aconitase inactivation was greater for heart mitochondria respiring on succinate than on the complex I-linked substrates glutamate/malate, and the complex I inhibitor rotenone prevented O2

•- formation by PQ in the presence of succinate (Fig. 3E).

To investigate the intriguing substrate- and ∆ψm-dependence of PQ-induced O2

•- production in real time, we measured H2O2 efflux, derived from matrix O2

•-, in heart mitochondria (61). In the absence of PQ, energizing heart mitochondria with glutamate/malate resulted in negligible H2O2 efflux (Fig. 4A) that increased substantially when succinate was the substrate (Fig. 4B). This increase with succinate was due to the high ∆ψm and fully reduced coenzyme Q pool driving reverse electron transport through complex I that led to O2

•- production (61). O2•- production

from succinate was blocked by abolishing the ∆ψm with FCCP (Fig. 4B), with the complex III inhibitor stigmatellin (data not shown), or by inhibiting electron entry from the coenzyme Q pool into complex I with rotenone (Fig. 4D).

Addition of PQ to mitochondria respiring on glutamate/malate increased H2O2 efflux (Fig. 4C) and this was decreased by abolishing the ∆ψm with rotenone (Fig. 4C) or stigmatellin (data not shown), however these inhibitors were only effective when present from the beginning of the incubation; when they were added after H2O2 production was well established they increased H2O2 efflux (Fig. 4C and data not shown). In contrast, FCCP blocked the PQ-induced H2O2

efflux when added early or late in the incubation (Fig. 4A). This difference may occur because both rotenone and stigmatellin are respiratory inhibitors that cause a build-up of electrons within complex I, as well as abolishing the ∆ψm. In contrast, the uncoupler FCCP abolishes the ∆ψm while oxidizing complex I. These findings are consistent with the uptake of PQ into mitochondria requiring a ∆ψm and the PQ in the matrix then interacting with a reduced complex I to generate O2

•-. Addition of PQ to mitochondria respiring

on succinate led to a greater efflux of H2O2 than when glutamate/malate was used as a respiratory substrate (Fig. 4E), consistent with the aconitase inactivation data (Fig. 3E). This H2O2 efflux caused by PQ in the presence of succinate was inhibited by FCCP (Fig. 4B), rotenone (Fig. 4D) and stigmatellin (data not shown). However, in contrast to when glutamate/malate was the respiratory substrate, these compounds were equally effective at preventing H2O2 efflux when added at the beginning or in the middle of the incubation (Figs. 4B & 4D, and data not shown). Stigmatellin and FCCP abolish the ∆ψm and would thus prevent any putative ∆ψm-dependent PQ uptake as well as reverse electron transport through complex I. However, rotenone would not abolish the ∆ψm and in these experiments would only prevent the movement of electrons into complex I driven by reverse electron transport. This shows that complexes II and III are not involved in the production of O2

•- by PQ. These findings indicate that in mammalian mitochondria respiring on succinate, O2

•- production by PQ occurs at complex I and requires a ∆ψm both to cause the accumulation of PQ into mitochondria and to drive electrons from the coenzyme Q pool into complex I by reverse electron transport. Thus the main site of PQ reduction in mammalian mitochondria is likely to be on complex I.

Sites of O2•- Production by PQ within

Mitochondria - To further investigate the sites of PQ•+ generation in mammalian mitochondria and to determine whether a ∆ψm was essential for this, we measured O2

•- production by bovine heart mitochondrial membranes using the CLZ chemiluminescence assay. In this system, the respiratory complexes are exposed to PQ without requiring uptake into the mitochondrial matrix and

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reverse electron transport into complex I is not possible.

PQ did not enhance O2•- production by

bovine heart mitochondrial membranes respiring on succinate (Fig. 5A), although it was possible to stimulate O2

•- production by complex III with the inhibitor antimycin A. This confirms that complex III does not interact with PQ2+ to produce O2

•-. Similar results were obtained when measuring H2O2 efflux from freeze-thawed or sonicated rat heart mitochondria oxidizing succinate (data not shown). These findings and the FCCP- and rotenone-sensitive O2

•- production in intact mitochondria respiring on succinate in the presence of PQ (Figs. 4B & 4D) indicate that succinate induces O2

•- production from PQ via complex I through ∆ψm-dependent reverse electron transport (61).

Incubation of bovine heart mitochondrial membranes with NADH led to a dramatic PQ dose-dependent stimulation of O2

•- production (Fig. 5B). In this situation rotenone stimulated O2

•- production by increasing the reduction state of complex I, consistent with the H2O2 assay results obtained for intact heart mitochondria (Fig. 4C). The flavoprotein inhibitor diphenyleneiodonium (DPI) prevented the PQ-induced O2

•- production (Fig. 5B). Furthermore NADPH resulted in negligible O2

•- production compared to that by NADH (Fig. 5B). These data indicate that complex I can reduce PQ2+ to PQ•+ thereby producing O2

•-, and are consistent with an earlier report with isolated complex I (34). We cannot entirely eliminate the possibility that there are other sites of PQ2+ reduction in mammalian mitochondria. However, the data in Fig. 4D show that rotenone almost completely abolished H2O2 efflux for mitochondria respiring on succinate in the presence of PQ. Under these conditions the mitochondrial NADH and NADPH pools are reduced, the ∆ψm is high and only access to complex I is prevented. Furthermore, when sonicated heart mitochondria were incubated with NADPH there was negligible O2

•- production compared to the rotenone-sensitive O2

•- production in the presence of NADH (Fig. 5C). These data are consistent with complex I being the major site of PQ2+ reduction within mammalian mitochondria.

We next investigated the potential sites of PQ-induced O2

•- production within yeast mitochondria, which do not contain complex I. For this ∆sod2 yeast mitochondria were broken by sonication and then incubated with the electron donors NADH, NADPH, succinate or glycerol-3-phosphate along with PQ, and O2

•- production was measured (Fig. 5D and data not shown). This indicated that the main substrate for PQ2+ reduction was NADPH and that NADH had a negligible role. This O2

•- production was completely abolished by the flavin inhibitor DPI indicating that reduction occurred through a flavin-containing NADPH dehydrogenase (Fig. 5D). Therefore within mitochondria, PQ in the presence of NAD(P)H will lead to the formation of O2

•- and the main sites for this are complex I in mammalian mitochondria and intramitochondrial NADPH dehydrogenases in yeast mitochondria. There is no requirement for a ∆ψm to reduce PQ2+ to PQ•+ once the PQ is within mitochondria and when NADH is used as the substrate; however, for mammalian mitochondria respiring on succinate, PQ reduction requires a ∆ψm to drive electrons from the coenzyme Q pool into complex I.

Investigating the ∆ψm-dependence of PQ2+ Uptake by Mitochondria - The above findings indicate that PQ2+ in the mitochondrial matrix can increase matrix O2

•- production in the absence of a ∆ψm. This suggests that the ∆ψm-dependence of PQ-induced O2

•- production occurs because the ∆ψm drives PQ uptake into the matrix. Many lipophilic cations and dications are taken up electrophoretically into mitochondria by direct passage through the phospholipid bilayer driven by the ∆ψm in response to the Nernst equation (44,62). Significantly, the structurally similar compound 1-methyl-4-phenylpyridinium (MPP+; Fig. 1B) is accumulated by mitochondria in a ∆ψm-dependent manner by direct passage through the inner membrane (54). We confirmed the ∆ψm-dependent uptake of MPP+ into energized yeast mitochondria using a MPP+-selective electrode and showed that this uptake could be further stimulated by the lipophilic anion tetraphenylborate (TPB–; Fig. 6A), which facilitates the direct passage of lipophilic cations across the membrane (63). However similar

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experiments using a PQ-selective electrode showed no measurable PQ uptake into energized yeast mitochondria (Fig. 6B) or mammalian mitochondria (data not shown). Furthermore, sub-stoichiometric amounts of TPB– did not stimulate accumulation into mitochondria (data not shown). Therefore there is no bulk uptake of PQ2+ into mitochondria, unlike the structurally related compound MPP+. The reason for this marked difference is presumably the double charge on PQ2+ relative to MPP+ which will cause a 4-fold increase in the Born energy for its movement across the membrane and thereby result in a far larger activation energy required for the movement of PQ2+ through biological membranes relative to MPP+ (64).

However while the PQ-selective electrode eliminates the possibility of bulk movement of PQ2+ across the membrane, it will only detect large-scale redistributions. To see if there was a low capacity uptake system that led to ∆ψm-dependent uptake of PQ2+ into mitochondria, we utilized more sensitive detection methods based on either EPR (Figs. 7A & 7B) or radiolabeled PQ and were able to show time-dependent uptake of PQ into yeast mitochondria (Fig. 7C). This uptake was ∆ψm-sensitive as the presence of the uncoupler FCCP or the complex III inhibitor myxothiazol at the start of the incubation completely prevented PQ uptake (Fig. 7D). Furthermore the K+/H+ antiporter nigericin (1 µM), which increases ∆ψm by converting the mitochondrial pH gradient into a ∆ψm, stimulated PQ uptake (data not shown). Addition of FCCP at the end of the incubation did not lead to immediate reversal of the accumulation (Fig. 7D), unlike the situation with MPP+ (Fig. 6A). PQ uptake into yeast mitochondria was also driven by the respiratory substrates succinate and glycerol-3-phosphate (data not shown). Qualitatively similar data were obtained for mammalian mitochondria, which also showed ∆ψm-dependent uptake of PQ that was prevented, but not reversed by uncoupling (Fig. 7E).

When the initial rate of uptake of PQ2+ into yeast mitochondria was plotted against the initial [PQ2+] (1.25–20 mM) an apparent saturation was found with half maximal uptake at about 3.5 mM (Fig. 7F). While these data are consistent

with a saturable, low affinity, low capacity mediated uptake system, a definitive interpretation of these kinetic data is not possible. This is because incubating mitochondria with increasing PQ2+ concentrations from above ~1 mM for yeast mitochondria gradually lowers the ∆ψm (data not shown). Consequently the decrease in uptake with increasing [PQ2+] may be due to a combination of saturation and of the progressive decrease in ∆ψm as the PQ2+ concentration increases. This interpretation is supported by the lack of fit of these data to any of the standard transformations of Michaelis-Menten kinetics.

Nature of the Mitochondrial PQ Uptake System - The data in Fig. 7 are consistent with, but not proof of, carrier-mediated uptake of PQ into mitochondria. Carrier-mediated PQ transport has been reported in lung alveolar epithelial cells by the polyamine transport system, which is inhibited by putrescine (65), and across the blood-brain barrier via the neutral amino acid transporter, which is inhibited by L-valine (66). To explore whether PQ uptake into mitochondria occurs through a carrier in the mitochondrial inner membrane we focussed on yeast as the activity was greater, deletion libraries were available and many mitochondrial carriers are similar in yeast and mammals.

If uptake is carrier-mediated then structurally similar compounds such as MPP+, diquat and putrescine (Fig. 1B) might inhibit PQ uptake. To ensure that any effects of these compounds on PQ uptake were not due to effects on ∆ψm, we measured the uptake of [14C]-PQ and [3H]-TPMP+ simultaneously by dual label scintillation counting. MPP+, diquat and putrescine all decreased PQ uptake in a concentration-dependent manner without severely affecting the ∆ψm (Fig. 8A). Neither vanadate (an inhibitor of ABC transporters) or oligomycin (an inhibitor of ATP synthase) affected PQ uptake into mitochondria (Fig. 8B), suggesting that uptake does not depend on intramitochondrial ATP. The thiol alkylating agent N-ethyl maleimide (NEM) inhibits many mitochondrial transporters (e.g. 67) and it had a considerably greater effect on PQ uptake than it did on ∆ψm (Fig. 8C). These data are consistent with a mitochondrial transporter mediating ∆ψm-

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dependent, but ATP-independent, uptake of PQ into mitochondria. Possible candidates are the mitochondrial carrier family which has 35 members in yeast mitochondria (68,69). We hypothesized that yeast lacking the putative mitochondrial PQ carrier would show resistance to PQ on non-fermentable medium. We screened the growth on fermentable medium supplemented with PQ of 29 yeast strains in which the genes for proven or putative mitochondrial carriers had been deleted. The 0–1 mM PQ gradient in the plates showed the PQ sensitivity of the wild-type strain and detected a number of strains such as ∆ctp1 that were clearly resistant to PQ (Fig. 8D). Of the 29 deletion strains tested, 8 were resistant to PQ, however PQ uptake by mitochondria isolated from these strains was indistinguishable from wild-type (Table 1). Thus deletion of these carriers protects against mitochondrial damage by PQ through other mechanisms. For example, deletion of the citrate transporter Ctp1p may protect by increasing the matrix citrate concentration thereby preventing O2

•- damage to the sensitive iron-sulfur center in the active site of aconitase. Therefore it is not possible to use a simple PQ screen alone to identify the putative PQ uptake system, making its identification significantly more difficult. Furthermore, as the apparent affinity of PQ for its putative carrier was around 3 mM, while the affinities of substrates for other carriers are often in the 10–100 µM range (70), it is likely that PQ is a poor substrate for its carrier or carriers, making it more challenging to identify. Even though the putative PQ carrier(s) remain to be identified, the data in Figs. 3, 4 & 7F are consistent with a low affinity carrier-mediated uptake process driven by the ∆ψm, and have eliminated a number of candidates.

Contribution of PQ•+ Formation Outside Mitochondria to PQ Toxicity - The experiments in Figs. 3–5 show that PQ2+ can be reduced to PQ•+ within mitochondria. To see if PQ2+ reduction to PQ•+ on the outer membrane or intermembrane space of mitochondria could contribute to its toxicity, we measured the formation of PQ•+ from PQ2+ by anaerobic yeast and heart mitochondria in the presence of various electron donors. The PQ•+ radical is only stable in the absence of O2 and has a distinctive visible absorption spectrum (Fig. 9A). When yeast mitochondria were incubated

anaerobically with NADPH there was some formation of PQ•+ as indicated by the increase in absorption at 603 nm (Fig. 9B, inset right) and by the difference spectrum in the presence and absence of O2 (Fig. 9B). However, there was no PQ2+ reduction by ethanol, NADH (Fig. 9B), or NADP (data not shown). In rat heart mitochondria there was some formation of PQ•+ in the presence of NADH, but not by succinate or NADPH (Fig. 9C). Therefore under conditions where there was extensive O2

•- production within mitochondria (Figs. 3 & 4), there was negligible reduction of PQ2+ to PQ•+ and consequent O2

•- production outside mitochondria. Exogenous NADH and NADPH cannot cross the intact mitochondrial inner membrane and may reduce PQ2+ via enzymes such as NADH-cytochrome b5 oxido-reductase and NADH-coenzyme Q oxidoreductase of the mitochondrial outer membrane (28,33). However in our intact heart mitochondrial preparations, ~90% of NADH consumption was inhibitable by rotenone (data not shown), indicating that most of the observed extramitochondrial PQ reduction by NADH was due to a small proportion of damaged, NADH-permeable mitochondria. This further limits the contribution of PQ reduction and O2

•- formation outside mammalian mitochondria. Thus while there may be some NADH/NADPH-dependent PQ•+ formation within cells contributing to O2

•- formation, this seems likely to be far less significant than O2

•- formation within mitochondria.

Extramitochondrial reduction of PQ2+ to PQ•+ could contribute to O2

•- production within mitochondria by enhancing the net uptake of PQ into mitochondria. The PQ2+ dication is not taken up into energized mitochondria directly through the mitochondrial inner membrane because its double charge greatly increases its activation energy for movement through the hydrophobic core of the membrane (44,64). However the Born energy component of this activation energy is proportional to the square of its charge and for the PQ•+ radical cation will be 4-fold lower than the PQ2+ dication. The structure and charge of the PQ•+ monocation is very similar to the MPP+ monocation (Fig. 1B), which is taken up directly through the membrane (54 and Fig. 6A). Thus the PQ•+ cation could probably accumulate into

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mitochondria by direct movement through the membrane driven by the ∆ψm. However, its competing rapid reaction with O2 (7.7 x 108 M-1. s-1) even at 30 µM O2, a plausible oxygen concentration in cells (e.g. 71), means that its half-life is only ~30 µs which may make this pathway negligible. To see if PQ•+ uptake by energized mitochondria could contribute to net PQ uptake, we incubated energized yeast mitochondria + NADPH, to enhance PQ•+ formation outside mitochondria (Fig. 9B). Under these conditions, no increase in net PQ uptake was observed (data not shown), even when PQ•+ was generated in the vicinity of mitochondria. To summarize, the increase in mitochondrial O2

•- caused by PQ is predominantly due to O2

•- generated within mitochondria.

Conclusions - Here we have demonstrated how PQ can act to increase intramitochondrial O2

•- production (Fig. 10). The major mode of O2

•- production requires the ∆ψm-dependent uptake of the PQ2+ dication by energized mitochondria. This uptake does not seem to involve movement of the dication through the phospholipid bilayer, but instead is carrier-mediated by a putative PQ carrier protein or proteins, which require a ∆ψm to drive PQ accumulation into the mitochondria. Once within mitochondria, the PQ is largely retained, even after the ∆ψm has been abolished. This ∆ψm-dependence of PQ uptake into mitochondria may help explain findings in the literature that flies with targeted expression of human uncoupling protein 2 in the mitochondria of neurons show increased resistance to PQ (25) and that treatment of yeast cultures with the complex III inhibitor antimycin A causes PQ resistance (72), presumably because the inhibition of ∆ψm prevents mitochondrial uptake.

The PQ within mitochondria then goes on to produce O2

•- through its reduction by intramitochondrial NAD(P)H dehydrogenases to the PQ•+ radical cation which reacts rapidly with O2 to form O2

•- and regenerates the PQ2+ dication. In yeast this reduction is NADPH-dependent and involves intramitochondrial NADPH dehydro-genases. In mammalian mitochondria the only major source of reduction of PQ2+ to PQ•+ is complex I. Once PQ2+ is accumulated into mitochondria driven by the ∆ψm, it can interact

directly with complex I respiring on NADH-linked substrates by reduction at complex I. This rate of PQ2+ reduction and consequent O2

•- formation is enhanced by rotenone, presumably due to greater reduction of the electron donating sites upstream of the rotenone inhibition site. In contrast, mitochondria also produce extensive amounts of O2

•- when respiring on succinate. However, this O2

•- production does not occur in mitochondrial membranes respiring on succinate, requires a ∆ψm even after significant amounts of PQ have been accumulated by mitochondria and is inhibited by rotenone. Therefore this PQ2+ reduction is due to the action of complex I.

The requirement for a ∆ψm and blocking by rotenone is similar to the production of O2

•- from complex I by reverse electron transport and suggests that under conditions of a reduced coenzyme Q pool and a high ∆ψm, electrons are forced into complex I where reduction of PQ2+ takes place. Whether this is the same site as that of O2

•- production by complex I during reverse electron transport or if this site is the same for PQ2+ reduction during NADH oxidation is currently unclear, but investigation of this point may help shed light on the mechanisms of O2

•- production by complex I. That complex I is the major site of O2

•- production within mitochondria exposed to PQ, and that complex I damage is a major factor in idiopathic Parkinson disease, strongly supports PQ toxicity as a useful model for Parkinson disease (73).

While this manuscript was in preparation another investigation of the interaction of PQ with mammalian mitochondria was published by Castello et al. (74). That paper also reported that mitochondria are a major site of ∆ψm-dependent reactive oxygen species formation by PQ. However, their suggestion that complex III in the respiratory chain is the major site of PQ reduction contrasts with our findings that complex I is the only significant site of PQ reduction within mammalian mitochondria. Furthermore, as the reduction potential for PQ2+/PQ•+ (–446 mV) is far lower than the potentials spanned by complex III (~0 mV to +250 mV) and the lowest Em'7 for a Q cycle intermediate (the ubiquinolate radical anion/ubiquinone couple) is only –160 mV (75), complex III is unlikely to reduce PQ. Another

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difference between Castello et al. and our findings is their report that succinate and PQ addition to isolated mitochondria leads to H2O2 production which is only partly inhibited by rotenone (74), whereas we find complete inhibition. It may be that brain mitochondrial preparations contain endogenous, NADH-linked substrates that increase rotenone-sensitive PQ reduction. The other difference is that we show uptake of PQ into mitochondria, which is ∆ψm-dependent in both yeast and mammalian mitochondria, while Castello et al. report that PQ uptake by mitochondria is not dependent on ∆ψm. These differences may arise from the greater sensitivity of the two methods we employed, namely the uptake of radiolabelled compound and analysis of

uptake by EPR, compared to the HPLC determination used by Castello et al. (74).

In conclusion, we have shown that PQ causes mitochondrial oxidative damage in mammalian systems following its ∆ψm-dependent accumulation into the mitochondrial matrix. Within the matrix, PQ2+ is reduced by complex I either via electrons from NADH-linked substrates or from the coenzyme Q pool driven into complex I by reverse electron transport. The PQ•+ radical thus formed rapidly reacts with O2 to give O2

•-, and due to its short half-life, PQ•+ will preferentially generate O2

•- close to complex I. Thus within mammalian systems mitochondria are a major site of O2

•- formation and complex I is likely to be both a site and target for damage of this O2

•- production.

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FOOTNOTES *This work was supported by the Medical Research Council (UK) and Research into Ageing (UK). We thank Meredith Ross, Thomas Hurd, Andrew James, Edmund Kunji and Martin Brand for helpful discussions on the manuscript. We are grateful to Tracy Prime, Jan Trnka and Angela Logan for technical assistance, and to Edith Gralla and Catherine Clarke (UCLA) for supplying yeast strains.

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The abbreviations used are: ∆ψm, mitochondrial membrane potential; CLZ, coelenterazine; DPI, diphenyleneiodonium; EryR, erythromycin resistant (or erythromycin resistance); FCCP, carbonyl cyanide p-trifluoromethoxyphenylhydrazone; MnSOD, manganese superoxide dismutase; MPP+, 1-methyl-4-phenylpyridinium cation; mtDNA, mitochondrial DNA; NEM, N-ethyl maleimide; O2

•-, superoxide; PQ, paraquat (1,1′-dimethyl-4,4′-bipyridinium dichloride); PQ2+, paraquat dication; PQ•+, paraquat monocation radical; RLU, relative light units; SOD, superoxide dismutase; TPB–, tetraphenylborate anion; TPMP+, methyl triphenylphosphonium cation.

FIGURE LEGENDS

Fig. 1. Paraquat and its Mechanism of Redox Cycling. A. The paraquat dication (PQ2+) undergoes

univalent reduction to generate the paraquat radical (PQ•+), which then reacts rapidly with O2 to produce superoxide (O2

•-). B. Structures of the neurotoxin 1-methyl-4-phenylpyridinium (MPP+), the pesticide 1,1′-ethylene-2,2′-bipyridinium (diquat) and the diamine putrescine.

Fig. 2. Effect of PQ on Yeast Cultures and Induction of mtDNA Damage. A. Wild-type (CEN.PK2-1C) yeast were cultured in either YPD (glucose) or YPG (glycerol) medium, with a range of PQ concentrations (0.01–10 mM). The initial cell density was adjusted to A600 ~0.1, and growth was measured spectrophotometrically after 48 h. Data are expressed as percentage of the appropriate controls without PQ and are the means ± range of two independent experiments. B. As for A, except wild-type (EG103) and ∆sod2 strains were cultured in lactate medium. C. Induction of mtDNA point mutations. Wild-type (CEN.PK2-1C) yeast were cultured in YPG medium ± PQ (100 µM). Point mutations in the mtDNA were determined by the erythromycin resistance (EryR) assay. Data are the means ± SEM of 10 independent cultures. The number of EryR colonies is expressed per 108 respiration-competent cells. D. Effect of MnSOD deletion and PQ exposure on petite formation. Wild-type (EG103) and ∆sod2 yeast were cultured in YPD medium ± PQ (100 µM). Petite mutants were identified by the tetrazolium overlay technique. Data are the means ± SD of three independent experiments. Statistical significance was calculated with a Student’s two-tailed t-test. n/s (not significant), P > 0.05; *, P < 0.05; **, P < 0.01.

Fig. 3. O2•- Production by PQ in Yeast and Mammalian Mitochondria - Effect of Respiratory

Substrate, Uncoupler and Exogenous SOD. A. O2•- production was measured by the coelenterazine (CLZ)

chemiluminescence assay in either wild-type or ∆sod2 mitochondria. Mitochondria (50 µg protein.mL-1) in mannitol buffer were energized with substrate (5 mM ethanol), treated with PQ (1 or 10 mM) and uncoupled with FCCP (1 µM) as indicated. Data are the means ± SD of four determinations. B. PQ-induced mitochondrial O2

•- production as determined by the CLZ chemiluminescence assay is insensitive to exogenous SOD. Experiments were performed as for A in ∆sod2 mitochondria with PQ (10 mM) and SOD (100 U.mL-1) as indicated. Data are the means ± SD of four determinations. C. As for A, except that O2

•- production was determined by the aconitase inactivation assay in wild-type mitochondria (0.3 mg protein.mL-1). Data are the means ± SD of four determinations. D. PQ-induced mitochondrial O2

•- production, as inferred from aconitase inactivation, is insensitive to exogenous SOD. Experiments were performed as for C with PQ (10 mM) and SOD (50 U.mL-1) as indicated. Data are the means ± range of two determinations. E. O2

•- production in rat heart mitochondria was determined by the aconitase inactivation assay. Heart mitochondria (2 mg protein.mL-1) were incubated for 10 min at 37ºC in KCl buffer supplemented with 0.1% (w/v) BSA. Substrate (5 mM glutamate/malate, or 5 mM succinate ± 4 µg.mL-1 rotenone), uncoupler (1 µM FCCP) and PQ (0.1 or 1 mM) were present as indicated. Data are the means ± SD of three determinations. F. PQ-induced O2

•- production in rat heart mitochondria, as determined by the aconitase inactivation assay, is insensitive to exogenous SOD. As for E, except that the

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incubations were supplemented with SOD (50 U.mL-1) where indicated. Data are the means ± SD of three–four determinations.

Fig. 4. H2O2 Production by PQ in Mammalian Mitochondria - Effect of Respiratory Substrate, Uncoupler and Respiratory Inhibitors. A–D. H2O2 production from rat heart mitochondria was determined fluorometrically by the Amplex Red assay. Heart mitochondria (0.2 mg protein.mL-1) were incubated at 37ºC in KCl buffer supplemented with 0.01% (w/v) BSA. Substrate (5 mM succinate or 5 mM glutamate/malate) and PQ (0.1 or 1 mM) were added as indicated. Data are representative of typical traces, repeated at least twice. A & B. Effect of the uncoupler FCCP (1 µM). C & D. Effect of the complex I inhibitor rotenone (4 µg.mL-1). E. Rates of H2O2 efflux from rat heart mitochondria, determined from the above traces. Data are the means ± SD of three–four determinations.

Fig. 5. Potential Sites of PQ2+ Reduction in Mitochondria. A–D. O2•- production was measured by

the coelenterazine (CLZ) chemiluminescence assay. A. Bovine heart mitochondrial membranes (0.2 mg protein.mL-1) were incubated in KPi buffer at 30ºC ± exogenous SOD (100 U.mL-1). Substrate (5 mM succinate), PQ (0.1 or 1 mM) and antimycin A (AA, 1 µM) were present as indicated. B. As for A, except the bovine heart mitochondrial membranes were incubated with NADH (1 mM) ± rotenone (4 µg.mL-1) or NADPH (1 mM), ± diphenyleneiodonium (DPI, 50 µM). Note the change of scale in the y axis. C. Sonicated rat heart mitochondria (0.2 mg protein.mL-1) were incubated with substrate (1 mM NADH or NADPH) ± rotenone (4 µg.mL-1), PQ (1 mM), DPI (50 µM) and exogenous SOD (100 U.mL-1) as indicated. D. Sonicated ∆sod2 yeast mitochondria (50 µg protein.mL-1) were incubated with substrate (1 mM NADH or NADPH), PQ (1 mM), DPI (50 µM) and exogenous SOD (100 U.mL-1) as indicated. All data are the means ± SD of three determinations.

Fig. 6. Investigating Mitochondrial Uptake of PQ with an Ion-Selective Electrode. A. ∆ψm-dependent uptake of the structurally similar 1-methyl-4-phenylpyridinium ion (MPP+; Fig. 1B) by yeast mitochondria is illustrated as a positive control. The MPP+-selective electrode was calibrated with three successive additions of 10 µM MPP+ (arrowheads). Isolated wild-type yeast mitochondria (Mitos) were incubated at 0.4 mg protein.mL-1 in mannitol buffer, energized with substrate (5 mM ethanol) and then uncoupled with FCCP (1 µM). The presence of TPB– (5 µM; dashed line) both accelerates and increases the extent of MPP+ uptake. Data are representative of typical traces. B. As for A, except that a PQ-selective electrode was calibrated with three successive additions of 10 µM PQ2+ (arrowheads). The presence of TPB– had no effect (data not shown).

Fig. 7. Uptake of PQ into Yeast and Mammalian Mitochondria. A. Typical EPR spectrum of the PQ•+ radical (100 µM) generated in vitro by reduction of PQ2+ with a 2-fold excess of sodium dithionite. B. Standard curve for the quantification of PQ•+ by EPR based on the height of the maximum peak (arrows in A), which gave a linear relationship for the range tested (0–250 µM). C. Time course of PQ uptake by yeast mitochondria. Wild-type yeast mitochondria (0.4 mg protein.mL-1) were energized with substrate (5 mM ethanol) and incubated in the presence of 10 mM PQ for 0–15 min at 30ºC. The amount of PQ in the mitochondrial pellet at each time point was measured by the EPR method. Data were corrected for the t=0 control and are the means ± range of duplicate determinations. D. PQ uptake into yeast mitochondria is dependent on ∆ψm. Wild-type yeast mitochondria (0.4 mg protein.mL-1) were incubated for 10 min at 30ºC in the presence of 10 mM PQ ± 5 mM ethanol ± 1 µM FCCP ± 1 µM myxothiazol (myxo). Data were obtained by EPR quantification and are the means ± SD of four determinations. Statistical significance was calculated with a Student’s two-tailed t-test. *, P < 0.05; ***, P < 0.001. E. Time course of PQ uptake by mammalian mitochondria. Rat liver mitochondria (1 mg protein.mL-1) in KCl buffer + 0.01% BSA were energized with 5 mM glutamate/malate and incubated in the presence of 0.1 mM PQ for 0–10 min at 37ºC. To test the ∆ψm-dependence of PQ uptake,

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controls were performed with 1 µM FCCP added either at the start or end of the 5 min incubations. Data were based on scintillation counting of [14C]-PQ, were corrected for the t=0 control, and are the means ± range of duplicate determinations. F. Kinetics of PQ uptake into yeast mitochondria. Wild-type yeast mitochondria (0.4 mg protein.mL-1) were energized with 5 mM ethanol and incubated for 1 min at 30ºC in the presence of 0–20 mM PQ. Data were obtained by the EPR method, were corrected for the t=0 control and are the means ± range of duplicate determinations.

Fig. 8. Characterization of PQ Uptake. A. Competitive inhibition of PQ uptake by structurally similar compounds. Wild-type yeast mitochondria (0.4 mg protein.mL-1) were energized with 5 mM ethanol and incubated for 5 min at 30ºC. PQ (10 or 100 µM, spiked with 0.1 µCi.mL-1 [14C]-PQ), diquat, putrescine (putr.), MPP+ and L-valine (either 100 or 500 µM) were present as indicated. Mitochondrial membrane potential (∆ψm) was measured simultaneously by uptake of [3H]-TPMP+. Data were corrected for FCCP-independent counts and are the means ± range/SD of two–three determinations. B. Testing the ATP-dependence of PQ uptake. Mitochondria were incubated as for A, in the presence of 100 µM PQ and treated with either 1 µM oligomycin (inhibitor of ATP synthase) or 1 µM vanadate (inhibitor of ABC transporters). C. Investigating the effect of N-ethyl maleimide (NEM). Mitochondria were incubated as for A, in the presence of 100 µM PQ and a range of NEM concentrations (0.01–1 mM). D. Screening for PQ resistance in mitochondrial carrier mutants from the yeast deletion library. Wild-type and mutant strains were spotted in rows onto YPG agar containing a 0–1 mM PQ gradient. Photograph of a typical plate after incubation at 30ºC for 7 days. The results of the screen are summarized in Table 1.

Fig. 9. PQ•+ Radical Formation by Anaerobic Mitochondria. A. Typical absorbance spectrum of the PQ•+ radical (20 µM) generated in vitro by reduction of PQ2+ with excess sodium dithionite. B. PQ•+ radical formation by anaerobic yeast mitochondria. Wild-type yeast mitochondria (0.4 mg protein.mL-1) were incubated anaerobically (argon-purged) for 10 min at 30ºC in the presence of 1 mM PQ ± ethanol (5 mM), NADH (1 mM) or NADPH (1 mM). Samples were then subjected to absorbance readings (500–700 nm), firstly while still under anaerobic conditions and then following exposure to air, and the difference spectra ± O2 were determined. The inset shows a typical set of original raw data for the incubation with NADPH. C. PQ•+ radical formation by anaerobic mammalian mitochondria. Rat heart mitochondria (1 mg protein.mL-1) were incubated anaerobically in KCl buffer + 0.01% BSA for 5 min at 37ºC in the presence of 1 mM PQ ± NADH (1 mM), NADPH (1 mM) or succinate (5 mM). Difference spectra ± O2 to diagnose PQ•+ radical formation were performed as described for B.

Fig. 10. Scheme of Proposed PQ Interactions with Mammalian Mitochondria. The paraquat dication PQ2+ enters mitochondria via a putative carrier-mediated pathway driven by the mitochondrial membrane potential (∆ψm). Once inside the matrix, PQ2+ is reduced to the monocation radical PQ•+ at complex I in the respiratory chain by electrons donated from NADH. Alternatively, in the presence of a ∆ψm, PQ•+ can be generated at complex I by reverse electron transport whereby electrons from the CoQ pool (e.g. from the substrate succinate) are driven into complex I by the ∆ψm. By analogy with the structurally similar MPP+, PQ•+ generated outside mitochondria may be taken up into the matrix by direct passage across the membrane. However the short lifetime of PQ•+ at physiological [O2] probably limits the contribution of this process to PQ2+ uptake into mitochondria.

Table 1. Summary of the screen for PQ resistance in mutants from the yeast deletion library. Experiments were performed as described in the methods section. A typical plate is shown in Fig. 8D. For each mutant, the deleted gene (a), corresponding ORF (b), and a description of the carrier/transporter function/substrate (c) are listed. This information was compiled from the Saccharomyces Genome Database (www.yeastgenome.org) and a review (68). Candidates have been grouped as belonging to the mitochondrial carrier family, and other transporters (mainly ABC transporters). To assess PQ resistance,

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growth of mutant strains was compared against the isogenic wild-type control on each plate (d). –, more sensitive to PQ than wild-type; = same as wild-type; +/++/+++, more resistant to PQ than wild-type (low/medium/high). Note that it was not possible to screen all mitochondrial carriers and transporters, because some deletion strains are lethal and others are non-viable on non-fermentable media. Mitochondria were isolated from selected strains, and PQ uptake ± FCCP was tested by the EPR technique. In all cases, mitochondria isolated from carrier mutants showed ∆ψm-dependent uptake of PQ which was indistinguishable from that of wild-type mitochondria. n.d., not determined.

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MitochondrialCarrierFamily

Gene (a) ORF (b) Description (c)PQ Resistance

againstWild-Type (d)

PQ Uptake by Isolated

Mitochondria (e)

AAC1 YMR056C ADP/ATP exchanger = n.d.

AAC3 YBR085W ADP/ATP exchanger = n.d.

AGC1 YPR021C glutamate importer, aspartate/glutamate exchanger = yes

CRC1 YOR100C carnitine transporter = yes

CTP1 YBR291C citrate transporter +++ yes

DIC1 YLR348C dicarboxylate/phosphate exchanger = n.d.

MRS3 YJL133W iron transporter = n.d.

MRS4 YKR052C iron transporter = n.d.

NDT1/YEA6 YEL006W NAD+ importer = n.d.

NDT2/YIA6 YIL006W NAD+ importer – n.d.

OAC1 YKL120W oxaloacetate, sulfate, and thiosulfate transporter + yes

ODC1 YPL134C 2-oxoadipate and 2-oxoglutarate exporter = n.d.

ODC2 YOR222W 2-oxoadipate and 2-oxoglutarate exporter = n.d.

ORT1 YOR130C ornithine exporter + yes

PIC2 YER053C phosphate importer = n.d.

SAL1 YNL083W MgATP transporter + yes

SFC1 YJR095W succinate/fumarate exchanger ++ yes

TPC1 YGR096W thiamine pyrophosphate importer + yes

YHM2 YMR241W function unknown + n.d.

YMC1 YPR058W function unknown = yes

YMC2 YBR104W function unknown = n.d.

YFR045W function unknown = n.d.

YPR011C function unknown = n.d.

YMR166C function unknown ++ yes

OtherTransporters

SMF2 YHR050W divalent metal ion transporter – n.d.

MDL1 YLR188W ABC transporter = n.d.

VMR1 YHL035C ABC transporter = n.d.

YDR061W ABC transporter = n.d.

YNR070W putative ABC transporter = n.d.

Table 1

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•H3C CH

3N N+

H3C CH

3+N N+

PQ2+

O2•-

O2

PQ•+

e-

k ~7.7 x 108 M-1.s-1 E0 = –446 mV

Figure 1

A)MPP+

CH3N+

B)

N++N

diquat

NH2

H2N

putrescine

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0

2

4

6

8

PQ

Ery

R C

olon

ies

(per

108

resp

iratio

n-co

mpe

tent

cel

ls)

– +

*

wild-type

C) D)

Yea

st G

row

th(%

of c

ontr

ol w

ithou

t PQ

)

glucose

glycerol0

20

40

60

80

100

0 0.01 0.1 1 10

[PQ] (mM)

0

20

40

60

80

100

[PQ] (mM)0 0.01 0.1 1 10

wild-type

∆sod2

Yea

st G

row

th(%

of c

ontr

ol w

ithou

t PQ

)

A) B)

Figure 2

0

1

2

3

4

– + – +wild-type ∆sod2

Pet

ite C

olon

ies

(%)

PQ

*

**

n/s

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C)

Aco

nita

se In

activ

atio

n(P

seud

o F

irst O

rder

Rat

e C

onst

ant,

min

-1)

A)

CLZ

Che

milu

min

esce

nce

(RLU

.s-1)

–10 100 0

0

500

1,000

1,500

2,000

––

+

wild-type ∆sod2

FCCP[PQ] (mM)

substrate––

++

+–

10 10 1010 0 0

CLZ

Che

milu

min

esce

nce

(RLU

.s-1)B)

Aco

nita

se In

activ

atio

n(P

seud

o F

irst O

rder

Rat

e C

onst

ant,

min

-1)

D)– SOD+ SOD

0

0.05

0.10

0.15

0.20

0.25

PQ

substrate––

++

+–

Figure 3

Aco

nita

se In

activ

atio

n(P

seud

o F

irst O

rder

Rat

e C

onst

ant,

min

-1)F)E)

0.0

0.5

1.0

1.5

2.0

Aco

nita

se In

activ

atio

n(P

seud

o F

irst O

rder

Rat

e C

onst

ant,

min

-1)

0 1

FCCP

[PQ] (mM)

– –0 1

+0 1 0

––0 0.1 1

+0 1

succinate

rotenone– – – +– –0.0

0.2

0.4

0.6

0.8

1.0

1.2

0 1 0 [PQ] (mM)glutamate/

malate succinate

0

500

1,000

1,500

2,000

PQsubstrate–

–++

+–

∆sod2

– SOD+ SOD

0.00

0.05

0.10

0.15

0.20

FCCP[PQ] (mM)

substrate––

++

+–

10 10 1010 0 0

wild-typewild-type

heart

heartglutamate/malate substrate

substrate0.1 0.1

0.1

– SOD+ SOD

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0.5

µM

H2O

2

A)

D)

no additions

0.1 mM PQ

1 mM PQ

1 mM PQ + FCCP

1 mM PQ (+ FCCP)glutamate/

malate

( )

FCCP

0.1 mM PQ

no additions

1 min

( ) 0.1 mM PQ (+ rotenone)

succinate 0.1 mM PQ + rotenonerotenone

glutamate/malate

1 mM PQ (+ rotenone)

1 mM PQ + rotenone

1 mM PQ

B) 1 mM PQ

0.1 mM PQ

no additions

1 mM PQ + FCCP

( ) 0.1 mM PQ (+ FCCP)

succinate

FCCP

C)

no additionsrotenone

( )

Figure 4

0

1

2

3

4

0 0.1 0.5 1 0 0.1 0.5 1

H2O

2 (n

mol

.min

-1.m

g pr

otei

n-1)

[PQ] (mM)glutamate/

malate succinate

E)

substrateheart

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0

5,000

10,000

15,000

20,000

25,000

0 0 0.1 1 0 0.1 1 0 0.1 1

nosubs. NADH

NADH + rotenone NADPH

no additions+ SOD+ DPI

B)A)

CLZ

Che

milu

min

esce

nce

(RLU

.s-1)

0

250

500

750

1,000

1,250

1,500

0 1 0 0.1 1 0

no substrate succinate

succ.+ AA

CLZ

Che

milu

min

esce

nce

(RLU

.s-1)

[PQ](mM)

– SOD+ SOD

Figure 5

D)

0

1,000

2,000

3,000

4,000

5,000

6,000

DPISOD

nosubstrate NADH NADPH

– PQ+ PQ

–– +

+–

– –– +

+–

– –– +

+–

–

CLZ

Che

milu

min

esce

nce

(RLU

.s-1)

0

5,000

10,000

15,000

20,000

25,000

30,000

nosubstrate NADH

NADH + rotenone NADPH

–– +

+–

– –– +

+–

– –– +

+–

– –– +

+–

– DPISOD

– PQ+ PQ

CLZ

Che

milu

min

esce

nce

(RLU

.s-1)

C)

[PQ](mM)

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10 m

V

1 min

substrate FCCP

Mitos

B)

30 m

V

1 min

FCCPsubstrate

Mitos

[MP

P+]

A)

+ TPB

– TPB

3 x 10 µM MPP+

3 x 10 µM PQ2+

Figure 6

[PQ

2+]

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B)

D)

Figure 7

0.00

0.05

0.10

0.15

0.20

0 2 4 6 8 10

Incubation Time (min)

PQ

Upt

ake

(nm

ol.m

g pr

otei

n-1)

– FCCP+ FCCP (start)+ FCCP (end)

C)

E) F)

0

20

40

60

80

100

0 5 10 15 20

[External PQ] (mM)

PQ

Upt

ake

(nm

ol.m

in-1.m

g pr

otei

n-1)

A)

3480 3490 3500 3510 3520 3530 3540 3550

(G)

100 µM PQ•+

R2 = 0.996

00 50 100 150 200 250

[PQ•+] (µM)

Max

imum

Pea

k H

eigh

t (A

rbitr

ary

Uni

ts)

50,000

100,000

150,000

200,000

substratePQ

myxo.

+ + +

–++–

––FCCP+––

+

–+

+

+

–+

+

+

–

++

start end(30 s)

end(5 min)

PQ

Upt

ake

(nm

ol.m

g pr

otei

n-1)

***

0

50

100

150

200

*

0

50

100

150

0 5 10 15

PQ

Upt

ake

(nm

ol.m

g pr

otei

n-1)

1 2.5

Incubation Time (min)

200

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A)

Figure 8

wild-type

∆aac1

∆aac3

∆agc1

∆crc1

∆ctp1

∆dic1

[PQ]

% o

f Con

trol

D)

0

20

40

60

80

100

putr. MPP+ L-valine

+ competitor (100 µM)

PQ(100 µM)

PQ(10 µM)

diquat putr. MPP+

+ competitor (100 µM)

diquat putr. MPP+

+ competitor (500 µM)

diquat

C)

0.01 0.1 1

% o

f Con

trol

PQ UptakeΔψ

m

PQ(100 µM)

+ NEM (mM)

0

20

40

60

80

100

PQ UptakeΔψ

m

B)

0

20

40

60

80

100

oligomycin vanadate

% o

f Con

trol

PQ UptakeΔψ

m

PQ(100 µM)

+ inhibitor (1 µM)

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500 550 600 650 700Wavelength (nm)

NADH

NADPHethanol

A)

0.02

∆A

bsA

bsor

banc

e

Wavelength (nm)

500 550 600 650 700

0.1

Abs

– O2

+ O2

Wavelength (nm)

0.00

0.05

0.10

0.15

0.20

500 550 600 650 700

Figure 9

B)

C)

500 550 600 650 700

NADH

0.02

∆A

bs

NADPHsuccinate

Wavelength (nm)

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+

-

I III IV

respiratory chain

O2•-O

2

cyt c

succinate(+ )

NADH

PQ•+ PQ2+

PQ•+

carrier ∆ψm?

PQ2+

O2•-O

2

e-

IICoQ

matrix

intermembrane space

cytoplasm

mitochondrial inner membrane

mitochondrial outer membrane

∆ψm

Figure 10

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Helena M. Cochemé and Michael P. MurphyComplex I is the major site of mitochondrial superoxide production by paraquat

published online November 26, 2007J. Biol. Chem. 

  10.1074/jbc.M708597200Access the most updated version of this article at doi:

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