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J. Biol. Chem., Vol. 280, Issue 22, 21295-21312, June 3, 2005

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Interactions of Mitochondria-targeted and Untargeted Ubiquinones with the Mitochondrial Respiratory Chain and Reactive Oxygen Species

IMPLICATIONS FOR THE USE OF EXOGENOUS UBIQUINONES AS THERAPIES AND EXPERIMENTAL TOOLS^*

Andrew M. James, Helena M. Cochemé, Robin A. J. Smith¶, and Michael P. Murphy||

From the Medical Research Council Dunn Human Nutrition Unit, Wellcome Trust/MRC Building, Hills Road, Cambridge CB2 2XY, United Kingdom and the ^¶Department of Chemistry, University of Otago, P. O. Box 56, Dunedin, New Zealand

Received for publication, February 9, 2005

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Antioxidants, such as ubiquinones, are widely used in mitochondrial studies as both potential therapies and useful research tools. However, the effects of exogenous ubiquinones can be difficult to interpret because they can also be pro-oxidants or electron carriers that facilitate respiration. Recently we developed a mitochondria-targeted ubiquinone (MitoQ₁₀) that accumulates within mitochondria. MitoQ₁₀ has been used to prevent mitochondrial oxidative damage and to infer the involvement of mitochondrial reactive oxygen species in signaling pathways. However, uncertainties remain about the mitochondrial reduction of MitoQ₁₀, its oxidation by the respiratory chain, and its pro-oxidant potential. Therefore, we compared MitoQ analogs of varying alkyl chain lengths (MitoQ_n, n = 3–15) with untargeted exogenous ubiquinones. We found that MitoQ₁₀ could not restore respiration in ubiquinone-deficient mitochondria because oxidation of MitoQ analogs by complex III was minimal. Complex II and glycerol 3-phosphate dehydrogenase reduced MitoQ analogs, and the rate depended on chain length. Because of its rapid reduction and negligible oxidation, MitoQ₁₀ is a more effective antioxidant against lipid peroxidation, peroxynitrite and superoxide. Paradoxically, exogenous ubiquinols also autoxidize to generate superoxide, but this requires their deprotonation in the aqueous phase. Consequently, in the presence of phospholipid bilayers, the rate of autoxidation is proportional to ubiquinol hydrophilicity. Superoxide production by MitoQ₁₀ was insufficient to damage aconitase but did lead to hydrogen peroxide production and nitric oxide consumption, both of which may affect cell signaling pathways. Our results comprehensively describe the interaction of exogenous ubiquinones with mitochondria and have implications for their rational design and use as therapies and as research tools to probe mitochondrial function.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Mitochondria are the major site of reactive oxygen species generation (ROS)¹ within cells (1, 2). When ROS production exceeds the capacity of detoxification and repair pathways, oxidative damage to protein, DNA, and phospholipid occurs, disrupting mitochondrial oxidative phosphorylation and leading to cell damage and death. This contributes to a number of human pathologies including Parkinson disease, Alzheimer disease, Friedreich ataxia, ischemia-reperfusion injury, diabetes, and aging (2, 3). In addition to this pathological role, ROS can also act as redox signaling molecules (4, 5). Hence, mitochondrial ROS production and oxidative damage are attractive targets for pharmacological intervention for both therapeutic and investigative purposes (6–10).

Antioxidants have the potential to block oxidative damage and redox signaling, and exogenous ubiquinones have been widely used for this purpose in mitochondrial studies. These molecules are based on the predominant human form of endogenous ubiquinone, coenzyme Q₁₀ (CoQ₁₀; Fig. 1A), which is synthesized in the mitochondrial inner membrane and comprises a ubiquinone head group attached to a tail of 10 five-carbon isoprenoid units (11). The ubiquinone moiety is redoxactive, accepting two electrons and two protons in its reduction to a ubiquinol, while the extremely hydrophobic tail ensures that within the cell it is almost exclusively associated with phospholipid bilayers (Fig. 1B). The redox activity of the ubiquinone moiety enables it to act as a mobile electron carrier in the mitochondrial inner membrane where it is reduced to a ubiquinol by several membrane bound dehydrogenases and oxidized back to a ubiquinone by complex III. Furthermore, the reduced ubiquinol form of CoQ₁₀ has an important protective function as a chain breaking antioxidant, terminating lipid peroxidation in phospholipid bilayers (12, 13). Therefore, ubiquinone supplementation is an attractive therapeutic strategy in human pathology, as it could both stimulate oxidative phosphorylation by complementing any defects in respiration (14) and protect against oxidative damage (15). However, this duality complicates experimental interpretation, as any effects of CoQ₁₀ can result from its interaction with oxidative phosphorylation, oxidative damage, or redox signaling pathways.

View larger version (38K): [in this window] [in a new window]   FIG. 1. Ubiquinones used in this study. A, structures. B, octan-1-ol/PBS partition coefficients: a, calculated using Advanced Chemistry Development (ACD) Software Solaris v4.67 as described in Smith et al. (31); b, determined experimentally in this study; c, determined experimentally in Kelso et al. (29); d, determined experimentally in Asin-Cayuela et al. (30). C, a schematic of MitoQ10 in a phospholipid bilayer composed of C18 saturated fatty acids. The TPP cation and the alkyl chain favor the polar surface and hydrophobic core, respectively. The preferred position of the uncharged but moderately hydrophilic ubiquinone moiety is less clear.

Although decreasing hydrophobicity improves overall bioavailability, it would also be of benefit to target ubiquinones specifically to mitochondria, as they are the main site of ubiquinone utilization but represent only a small fraction of the cell volume. Lipophilic cations, such as methyltriphenylphosphonium (TPMP; Fig. 1A), are accumulated several hundred-fold by the large membrane potential (negative inside) generated by mitochondria during oxidative phosphorylation (6, 9). We have exploited this property by covalently attaching a ubiquinone moiety to the lipophilic triphenylphosphonium (TPP) cation generating a mitochondria-targeted ubiquinone (MitoQ₁₀; Fig. 1A), which is selectively accumulated within isolated mitochondria, and within mitochondria in cells and in vivo (9, 29–31). The interaction of amphipathic alkyltriphenylphosphonium cations with phospholipid bilayers occurs as follows: the TPP lipophilic cation is bound as a monolayer in a potential well at about the level of the phospholipid fatty acid carbonyls, while the hydrophobic alkyl chain is inserted into the hydrophobic core of the membrane (31–34). This is illustrated for MitoQ₁₀ in Fig. 1C. Although the large ionic radius and hydrophobicity of the TPP cation allows molecules such as MitoQ₁₀ to permeate phospholipid bilayers readily, their steady-state concentration within the hydrophobic core of the membrane is low. Furthermore, within energized mitochondria the membrane potential causes most MitoQ₁₀ to be adsorbed to the matrix surface of the inner membrane. MitoQ₁₀ is a particularly effective antioxidant against lipid peroxidation (29, 30) and has been used in a range of studies of mitochondrial dysfunction and oxidative stress where its mitochondrial localization has enabled the site of intracellular redox signaling to be inferred (35–42). However, the details of the mitochondrial processes affected by MitoQ₁₀ remain unclear because it could act as an antioxidant (thereby blocking oxidative damage and redox signaling) or as an electron carrier in the respiratory chain (thereby stimulating oxidative phosphorylation). A further consideration is that, while ubiquinols are potent antioxidants, there are also partially reduced and/or partially protonated intermediate forms that can act as pro-oxidants through interacting with oxygen to form superoxide () (13, 43, 44). The implications of autoxidation are unclear as stress response pathways that boost antioxidant defenses are often switched on by mild oxidative stress (a process termed hormesis). As a result autoxidation could paradoxically contribute to conditioning and protection against subsequent oxidative stress (4, 45, 46).

In summary, ubiquinone augmentation is an attractive therapy for degenerative diseases as it has the potential to both stimulate oxidative phosphorylation and protect against lipid peroxidation. However this combination of effects makes interpretation difficult, limiting the design of rational therapies. Furthermore, while MitoQ₁₀ has been widely used to infer the existence of mitochondrial ROS-dependent signaling pathways, aspects of its mechanism of action remain uncertain. To clarify these issues, we set out to determine how MitoQ₁₀ and a number of related short-chain exogenous ubiquinones interact with both the mitochondrial respiratory chain and ROS. For this we used a range of MitoQ analogs that differ in the number of carbons linking the ubiquinone to the TPP moiety (MitoQ_n; n = 3, 5, 10, or 15 CH₂ groups) (29, 30) and compared their interactions with those of the untargeted short-chain ubiquinone analogs, CoQ₂, decylQ, and idebenone (Fig. 1A). This work has led to a better understanding of how MitoQ and other exogenous ubiquinones interact with the respiratory chain and ROS and has considerable implications for their rational design and use as therapies and as tools to probe mitochondrial oxidative damage and redox signaling.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Yeast Incubations—The Saccharomyces cerevisiae strains used were WT (CEN.PK2–1C MATa his3 leu2 trp1 ura3) and COQ2 (CEN.PK2–1C coq2::HIS3), kindly supplied by Prof. Catherine Clarke, UCLA (47). Coq2 codes for the enzyme para-hydroxybenzoate:hexaprenyl transferase, which catalyzes the transfer of the polyisoprenoid chain to 4-hydroxybenzoic acid in CoQ biosynthesis. COQ2 is auxotrophic for CoQ and fails to grow on non-fermentable carbon sources, such as glycerol. Yeast were cultured in 10 ml of YPG (1% (w/v) yeast extract, 2% (w/v) peptone, 3% (v/v) glycerol). The initial cell density was adjusted to A₆₀₀ 0.1. To achieve a reproducible transition to a respiratory phenotype, the medium was supplemented with 0.05% (w/v) glucose. This allowed fermentative growth of COQ2 up to A₆₀₀ 0.4 after which growth rapidly ceased unless ubiquinone supplementation restored oxidative phosphorylation. Yeast were incubated in the dark at 30 °C with mechanical shaking at 250 rpm. Growth was monitored spectrophotometrically by measuring A₆₀₀ over 120 h. For all yeast experiments, ubiquinones and other hydrophobic compounds were added in Me₂SO to 1% (v/v) of the total culture volume, which did not affect the growth of the WT and COQ2 strains on glucose (data not shown).

COQ2 yeast cultures for mitochondrial isolation were grown aerobically at 30 °C to mid-logarithmic phase (A₆₀₀ 1) in lactate medium (2% (v/v) DL-lactic acid, 0.3% yeast extract, 0.2% glucose, 0.05% CaCl₂·2H₂O, 0.05% NaCl, 0.06% MgCl₂·6H₂O, 0.1% KH₂PO₄, 0.1% NH₄Cl (all w/v) (pH 5.5, NaOH). COQ2 yeast can use D-lactate as a respiratory substrate because it donates electrons to oxidative phosphorylation at complex IV via the reduction of cytochrome c (cyt c). For WT yeast, the level of glucose was kept at 0.05% (w/v). Mitochondria were isolated according to published protocols (48, 49). The protein concentration was measured by the bicinchoninic acid assay using BSA as a standard (50). Aliquots of the mitochondrial preparation were mixed with 10 mg·ml^–1 fatty acid-free BSA as a cryoprotectant, snap-frozen on dry ice, and stored at –80 °C. Upon thawing the mitochondria retained a membrane potential that was indistinguishable from that of freshly isolated yeast mitochondria as confirmed by the uncoupler-sensitive uptake of [³H]TPMP (data not shown) (29).

Oxygen consumption was measured with a Clark-type oxygen electrode (Rank Brothers, Bottisham, Cambridge, UK) in a 1-ml stirred chamber at 30 °C. Aliquots of frozen COQ2 yeast mitochondria were thawed rapidly, washed, and resuspended in mannitol buffer (0.6 M mannitol, 10 mM Tris maleate, 5 mM KP_i, 0.5 mM EDTA (pH 6.8, KOH)) at 0.2 mg protein·ml^–1. The mitochondria were energized with 5 mM glycerol 3-phosphate (G3P) and uncoupled with 1 µM carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP). Ubiquinones (1–20 µM in Me₂SO) were titrated in successively, followed by 1 µM myxothiazol to determine non-mitochondrial oxygen consumption. For some experiments mitochondria were sonicated (3 x 5 s, setting 4; Misonix XL-2020 with microtip) in an ice bath prior to the measurement of respiration and addition of ubiquinone. Uptake of MitoQ analogs by yeast mitochondria was measured using an electrode selective for TPP cations (30). For these experiments WT yeast mitochondria (0.4 mg protein·ml^–1) were incubated in 2.5 ml of mannitol buffer in the presence of 2 µM MitoQ analog at 30 °C, and the uptake of MitoQ in response to energization with 5 mM ethanol was measured.

Mammalian Mitochondrial Preparations—Bovine heart mitochondrial membranes (BHM) were prepared from isolated bovine heart mitochondria as described previously (51, 52). Rat liver mitochondria were prepared by homogenization followed by differential centrifugation as described previously (53). Rat heart mitochondria were prepared by tissue disruption using an Ultra-Turrax (5 s), followed by differential centrifugation as described previously (53). Protein concentration was determined using the biuret assay with BSA as a standard (54).

Ubiquinone Reduction and Oxidation by Respiratory Complexes— Assays were based on previously described methods for measuring respiratory complex activity (55). However, for all assays except complex III the spectrophotometric decrease in A₂₇₅ (at this wavelength ubiquinone absorbs more strongly than ubiquinol) was monitored instead. Assays were performed in KP_i buffer (50 mM KP_i-KOH, 100 µM EDTA, 100 µM diethylenetriaminepentaacetic acid (pH 7.8) unless stated otherwise) at 37 °C. For complex I, the buffer was supplemented with 100 µg protein·ml^–1 BHM, 100 µM NADH, and 2 mM KCN, and the reaction was started by addition of 50 µM ubiquinone. For complex II, the buffer was supplemented with 100 µg protein·ml^–1 BHM, 5 mM succinate, 8 µg·ml^–1 rotenone, and 2 mM KCN, and the reaction was started by addition of 50 µM ubiquinone. For glycerol-3-phosphate dehydrogenase (G3PDH), EDTA and diethylenetriaminepentaacetic acid (DTPA) were omitted as Ca²⁺ may be required for activity (56), and the buffer was supplemented with 200 µg protein·ml^–1 BHM, 2 mM KCN, and 50 µM ubiquinone. The reaction was started with the addition of 10 mM G3P. Rotenone (8 µg·ml^–1) and malonate (20 mM) did not affect the rate of ubiquinone reduction in the presence of G3P. For complex III, the buffer was supplemented with 50 µg protein·ml^–1 BHM, 50 µM bovine cyt c, 8 µg·ml^–1 rotenone, and 2 mM KCN, plus or minus 400 nM myxothiazol. The reaction was started with the addition of 50 µM ubiquinol, and cyt c reduction was measured by an increase at A₅₅₀ (55). The myxothiazol-insensitive rate was measured in parallel and subtracted as there is a significant non-enzymatic rate of cyt c reduction by ubiquinols (43).

Measurement of the Ubiquinone Redox State—Spectrophotometric measurements were made at 275 nm. The ubiquinone redox state was measured at 37 °C in KP_i buffer supplemented with 100 µg protein·ml^–1 BHM, 8 µg·ml^–1 rotenone, and 10 µM ubiquinone. BHM were also supplemented with 5 µM bovine cyt c, as this can be lost during membrane isolation. For succinate (5 mM) oxidation, fumarase (5 units·ml^–1) was also added as fumarate absorbs at A₂₇₅ (local maximum at 240 nm). Furthermore, a background trace in the absence of ubiquinone was subtracted as fumarase does not completely abolish fumarate accumulation. For NADH oxidation, NADH absorption interfered with ubiquinone redox changes at A₂₇₅, therefore an NADH regeneration system (5 mM lactate and 2 units·ml^–1 lactate dehydrogenase) was used. Nucleotides were added as NAD⁺ (50 µM) because unlike NADH, it rapidly reached a steady-state NADH/NAD⁺ ratio. Further additions of lactate dehydrogenase had no effect on this ratio. A background trace acquired in the absence of ubiquinone was subtracted. For HPLC measurements, the incubation was as above, but fumarase was omitted. Two min after the addition of succinate, 1 volume of dichloromethane:diethylether (1:2) was added, and the mixture was then vortexed under argon. The phases were separated by centrifugation (1 min at 1,000 x g), and the upper organic phase was removed to a test tube overgassed with N₂. The lower aqueous phase was re-extracted as before with dichloromethane:diethylether (1:2), and the second organic phase was combined with the first. This was evaporated to dryness under a flow of N₂, then resuspended in 1 ml of 45% (v/v) acetonitrile, 0.1% (v/v) trifluoroacetic acid. Samples were analyzed by reverse-phase HPLC (Gilson 321 pump, 1 ml·min^–1) on a C18 column (Jupiter 300 Å, Phenomenex), using a staged gradient (4.5% acetonitrile for 5 min, 4.5–54% acetonitrile over 5 min, 54–72% acetonitrile over 20 min, 72–90% acetonitrile over 5 min) containing 0.1% (v/v) trifluoroacetic acid and detected at 220 nm using a Gilson UV/VIS 151 spectrophotometer. Peaks were assigned by comparison with ubiquinone or ubiquinol standards. Spectrophotometric measurements of ubiquinone in rat liver mitochondria were made at 275 nm. Mitochondria (200 µg protein·ml^–1) were incubated at 25 °C in a stirred cuvette containing 250 mM sucrose, 5 mM Tris, 1 mM EGTA (pH 7.4, HCl) supplemented with 8 µg·ml^–1 rotenone and 5 µM ubiquinone. 5 mM succinate, 400 nM FCCP, and 400 nM myxothiazol were added as indicated.

Ubiquinol Oxidation by Peroxynitrite—Ubiquinol oxidation by ONOO^– was measured at 37 °C in KP_i buffer supplemented with 100 µg protein·ml^–1 BHM, 8 µg·ml^–1 rotenone, 5 µM cyt c, and 10 µM ubiquinone. 5 mM succinate was added to energize the BHM, and two additions of 50 µM ONOO^– were made. This was followed by 20 mM malonate and a further addition of 50 µM ONOO^–. Ubiquinone redox changes were measured at 275 nm. ONOO^– has a strong absorbance maximum at 302 nm (₃₀₂ = 1.67 mM^–1·cm^–1 (57)) leading to a transient spike in A₂₇₅ until it has decayed. The decay back to base-line absorbance took 8 s in KP_i buffer. For cis-parinaric acid (PA; Molecular Probes, Eugene, OR) oxidation, the incubation was performed using a Shimadzu RF-5301PC fluorimeter in a stirred 3-ml cuvette thermostatted at 37 °C. PA was excited at 324 nm, and its fluorescence was monitored at 413 nm. The assay was in KP_i buffer supplemented with 100 µg protein·ml^–1 BHM, 8 µg·ml^–1 rotenone, 5 µM cyt c, and 10 µM ubiquinone. BHM were supplied with 5 mM succinate, then after 1 min 2 µM PA was added, followed by 20 µM ONOO^– and each minute thereafter. ONOO^– was prepared as described previously (58).

Measurement of Reactive Oxygen Species and Autoxidation—Reduced MitoQ₁₀.was prepared as described previously (29). Ubiquinol oxidation by was measured spectrophotometrically at 275 nm in a stirred 3-ml cuvette. Oxidation of reduced MitoQ₁₀ (50 µM) by generated from 0.015 unit·ml^–1 xanthine oxidase and 5 mM acetaldehyde was measured at 37 °C in KP_i buffer. After 18 min 100 units·ml^–1 SOD was added. Oxidation of reduced MitoQ₁₀ (50 µM) by generated from KO₂ was measured at 37 °C in PBS-saturated octan-1-ol or KP_i buffer (pH 7.3). A saturated solution of KO₂ (10 mM) was prepared by dissolving 1.4 mg solid KO₂ in 2 ml of 10 mM 18-crown-6 ether in Me₂SO. A solution where KO₂ had degraded to H₂O₂ was prepared by mixing 10 mM KO₂ in 10 mM 18-crown-6 ether with 1 volume of H₂O followed by incubation for 2 min. Autoxidation of reduced MitoQ₁₀ (50 µM) was measured spectrophotometrically in KP_i buffer (pH 6.8, 7.8, and 8.3) at 275 nm in a strirred 3-ml cuvette at 37 °C. generation from reduced MitoQ₁₀ (50 µM) was measured using acetylated cytochrome c (cyt c_acet) reduction, which was measured spectrophotometrically at 550 nm and 37 °C. KP_i buffer was supplemented with 100 µg protein·ml^–1 BHM, 8 µg·ml^–1 rotenone, 50 µM cyt c_acet, 400 nM myxothiazol, 2 mM KCN, 10 µM ubiquinone, and 5 mM succinate. The reaction was repeated in the presence of 100 units·ml^–1 SOD to determine the SOD-sensitive rate. H₂O₂ production was measured using an Apollo-4000 H₂O₂ electrode (World Precision Instruments) in an open stirred chamber at 37 °C. KP_i buffer was supplemented with 200 µg protein·ml^–1 BHM, 8 µg·ml^–1 rotenone, 5 µM cyt c, and 100 units·ml^–1 SOD. To this, 400 nM myxothiazol, 50 µM CoQ₂, and 5 mM succinate were added as indicated. The electrode was calibrated with known amounts of H₂O₂. NO· was measured using a ISO-NO NO· electrode (World Precision Instruments) in an open stirred chamber at 37 °C. KP_i buffer was supplemented with 20 µg protein·ml^–1 BHM, 8 µg·ml^–1 rotenone, 5 µM cyt c, and 10 µM CoQ₂. 250 µM 3,3-bis(aminoethyl)-1-hydroxy-2-oxo-1-triazene (DETA-NONOate; 100 mM in 10 mM KOH), 5 mM succinate, 400 nM myxothiazol, and 100 units·ml^–1 SOD were added as indicated. The electrode was calibrated with known amounts of S-nitroso-N-acetyl penicillamine (SNAP) to saturated CuCl.

Aconitase inactivation within mitochondria was measured spectrophotometrically by a coupled enzyme assay linking isocitrate production by aconitase to NADP⁺ reduction by isocitrate dehydrogenase (₃₄₀ NADPH = 6.22 mM^–1·cm^–1) (59, 60). Aliquots of frozen WT (EG103 MAT his3 leu2 trp1 ura3) yeast mitochondria (61) were thawed rapidly, then washed in 0.6 M mannitol buffer and resuspended in this buffer at 0.3 mg of protein/ml. The mitochondria were incubated in the presence of 5 mM G3P for 30 min in a shaking water bath at 30 °C. Samples were removed every 5 min, snap-frozen on dry ice, and thawed prior to assaying. Aconitase activity was assayed in a 96-well plate with a 10-µl sample added to 190 µl of assay buffer (50 mM Tris-HCl, 0.6 mM MnCl₂, 5 mM sodium citrate, 0.2 mM NADP⁺, 0.1% v/v Triton X-100, and 0.4 unit·ml^–1 isocitrate dehydrogenase (pH 7.4)) at 30 °C. A₃₄₀ readings were carried out at 15 s intervals over 7 min in an EL_x808 Ultra Microplate Reader (Bio-Tek Instruments). Each time point was measured in quintuplicate, and plotting the natural logarithm of activity versus time linearized the time course of aconitase inactivation. The slope of the line corresponded to the pseudo-first order rate constant of aconitase inactivation. The background rate of NADPH formation was determined in the presence of fluorocitrate (100 µM), a competitive inhibitor of aconitase, and was always less than 10% of the initial rate.

Efflux of H₂O₂ from isolated rat heart mitochondria was measured using a Shimadzu RF-5301PC fluorimeter in a stirred 3-ml cuvette thermostatted at 25 or 37 °C. Mitochondria (200 µg protein·ml^–1) were incubated in 120 mM KCl, 3 mM HEPES-KOH, 1 mM EGTA, and 0.01% (w/v) fatty acid-free BSA (pH 7.2), containing 4 units·ml^–1 horseradish peroxidase, 50 µM Amplex Red (Molecular Probes) and 100 units·ml^–1 SOD. 5 mM succinate and 8 µg·ml^–1 rotenone were added as indicated. Amplex Red was excited at 560 nm, and its fluorescence was monitored at 590 nm.

Partition Coefficients—Octan-1-ol/PBS partition coefficients (the concentration of ubiquinone in octan-1-ol relative to the concentration in PBS) were determined as described previously (30). Membrane/PBS partition coefficients (the concentration of ubiquinone in membranes relative to the concentration in PBS) were estimated by incubating 2 ml of PBS containing 250 µg protein·ml^–1 BHM and 50 µM ubiquinone for 5 min at 37 °C. BHM were pelleted by centrifugation (30 min at 16,000 x g), after which the supernatant was removed to a test tube and back-extracted with 1 volume of octan-1-ol. One extraction was sufficient for all ubiquinones except MitoQ₅ and MitoQ₃, which were extracted with 2 ml of octan-1-ol two and three times, respectively. The pellet was fully aspirated to remove as much water as possible, and then all ubiquinones were extracted four times with 100 µl of octan-1-ol. To estimate the partition coefficient, the inner membrane surface area per unit volume of rat heart mitochondria (61 µm²·µm³) was used along with values for membrane thickness (6 nm) and mitochondrial volume (0.6 µl·mg protein^–1) (62). This gave an estimated membrane volume of 0.22 µl·mg protein^–1.

Thiol Oxidation—Thiol oxidation by H₂O₂ was measured at 37 °C in KP_i buffer containing 10 mM glucose, 500 µM GSH, 200 µM NADPH, 0.4 unit·ml^–1 glutathione reductase, and 0.02 unit·ml^–1 glutathione peroxidase. GSH oxidation was detected by glutathione reductase-dependent NADPH oxidation. After A₃₄₀ stabilized, 0.006 unit·ml^–1 glucose oxidase was added to generate a stable flux of H₂O₂. Subsequently ubiquinone or NaBH₄-reduced ubiquinol (50 µM) was added. The response was linear with H₂O₂ over the range 1–100 µM and with glucose oxidase from 0.003 to 0.03 unit·ml^–1. The rate of NADPH oxidation was low in the absence of glutathione peroxidase and glutathione reductase. Glutathione peroxidase activity (0.02 unit·ml^–1) was in excess of glucose oxidase activity, yet kept to a minimum to allow for reaction of H₂O₂ with MitoQ₁₀ and CoQ₂.

For opening of the mitochondrial permeability transition pore (PTP) by t-butylhydroperoxide (tBHP) in isolated rat liver mitochondria, the final centrifugation during mitochondrial isolation was in 250 mM sucrose, 5 mM Tris (pH 7.4, HCl). Measurements of extra-mitochondrial Ca²⁺ were made in a stirred 3-ml cuvette thermostatted at 25 °C using a Shimadzu RF-5301PC fluorimeter. Calcium green-5N (Molecular Probes), a membrane-impermeant Ca²⁺ probe, was excited at 506 nm, and emission was monitored at 532 nm. Fluorescence was measured in 250 mM sucrose, 5 mM Tris, 10 µM EGTA (pH 7.4, HCl) supplemented with 0.5 mg protein·ml^–1 rat liver mitochondria, 8 µg·ml^–1 rotenone, and 1 µM calcium-green-5N. Mitochondria were supplied with 5 mM succinate, and after 1 min 5 µM CaCl₂ ± 5 µM tBHP were added. 1–5 µM MitoQ₁₀ and/or 500 nM cyclosporin A (CsA) were also added to some incubations.

NMR—Reduced MitoQ₁₀ was prepared by reaction with NaBH₄ in methanol under argon. Excess NaBH₄ was quenched with 10% (v/v) methane sulfonic acid, and the resulting mixture was extracted with dichloromethane, washed with water, and the solvent evaporated in vacuo. The ¹H NMR spectra of the product indicated it was 90% reduced. Solutions of the ubiquinol (10 mM) in D₂O ± 10 mM H₂O₂ and/or 100 µM EDTA were prepared in 5-mm NMR tubes. Oxidation of reduced MitoQ₁₀ was monitored using ¹H NMR of the ring methoxy peaks at 3.9 and 4 ppm for the ubiquinol and ubiquinone, respectively. This showed that 1 day exposed to air oxidized 3–6% of the ubiquinol, and this was identical in the presence or absence of H₂O₂.

Visualization of Respiratory Complexes—Structural files of complex II from Escherichia coli (Protein Data Bank code 1NEK [PDB] ) (63) and complex III from Bos taurus (Protein Data Bank code 1NTZ [PDB] ) (64) were visualized with PyMOL (DeLano Scientific, San Carlos, CA). Structural files for MitoQ analogs were generated with CORINA (Computer Chemistry Center, University of Erlangen-Nuremberg, Erlangen, Germany). To position MitoQ derivatives relative to the enzymes, three carbon atoms from the ring of the ubiquinone head group were pair-fitted with three corresponding atoms from the ubiquinone or inhibitor bound in their active sites.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Mitochondrial Respiration in CoQ-deficient Yeast Is Restored by Untargeted Exogenous Ubiquinones but Not by MitoQ₁₀— The first question we addressed was whether MitoQ₁₀ could act as an effective electron carrier in the mitochondrial respiratory chain. For this we used yeast that cannot synthesize CoQ (COQ2); consequently oxidative phosphorylation is inactive, and this yeast strain cannot grow on non-fermentable media (47). This lack of growth could be rescued by supplementation with exogenous CoQ₆ (the endogenous form of CoQ in yeast) (Fig. 2A). Although supplemented growth was slower than for the WT strain, the maximum cell density achieved was similar. Therefore exogenous ubiquinones can reach mitochondria within cells and restore oxidative phosphorylation. The effectiveness of various CoQ analogs at restoring growth exhibited a dependence on concentration and on the length of the isoprenoid tail, with CoQ₆ being the most effective (Fig. 2B). Interestingly, ubiquinone analogs with a saturated alkyl tail did not restore cell growth (Fig. 2C). MitoQ₁₀ at concentrations of 100 nM to 10 µM also failed to stimulate cell growth. WT yeast growth was inhibited at 10 µM MitoQ₁₀, but as similar growth inhibition was observed with 10 µM decyl-TPP, but not with 10 µM TPMP, toxicity is probably due to the higher local concentration of more hydrophobic TPP cations within phospholipid bilayers (data not shown). CoQ₁₀, the predominant CoQ in humans, did not support cell growth in non-fermentable media at 5 and 50 µM (data not shown). However, this was caused by poor uptake of CoQ₁₀ probably due to its insolubility, as CoQ-deficient yeast engineered to synthesize CoQ₁₀ grow well on non-fermentable substrates (65).

It is possible that MitoQ₁₀, decylQ, and idebenone are actually effective respiratory substrates but that their mitochondrial accumulation by yeast is poor. Therefore we determined whether MitoQ₁₀, decylQ, idebenone, and the CoQ analogs could stimulate uncoupled respiration by mitochondria isolated from COQ2 yeast. CoQ₂ and decylQ were the most effective at restoring respiration, followed by idebenone and CoQ₁ (Fig. 2D). Therefore the failure of decylQ and idebenone to complement the respiratory defect in intact COQ2 yeast (Fig. 2C) was a result of insufficient mitochondrial accumulation. In contrast, MitoQ₁₀ was still ineffective at stimulating respiration in isolated COQ2 mitochondria, even though it was rapidly accumulated by energized yeast mitochondria (data not shown). This confirms that MitoQ₁₀ is an ineffective electron carrier for respiration and explains its failure to restore yeast growth in non-fermentable media. Surprisingly, CoQ₆ was also ineffective at restoring respiration, despite being able to restore growth. However, this was due to its slow uptake by isolated mitochondria as respiration more than doubled (+110% versus intact mitochondria with CoQ₆) if mitochondria were sonicated before CoQ₆ additions were made (data not shown). Sonication did not increase respiration with CoQ₀ or MitoQ₁₀. Therefore, CoQ₆ is presumably too hydrophobic to diffuse rapidly through the mitochondrial outer membrane and stimulate respiration in these short term experiments. In summary we observe three classes of exogenous ubiquinone interaction: CoQ₁, CoQ₂, and CoQ₆, which all migrate to mitochondria in intact yeast and restore respiration; decylQ and idebenone, which can restore respiration but do not accumulate sufficiently in mitochondria in yeast cells; and MitoQ₁₀, which fails to restore respiration even after it accumulates in mitochondria. We next focussed on determining why MitoQ₁₀ was unable to complement the respiratory defect in the CoQ-deficient mitochondria.

MitoQ Analogs Are Not Oxidized by Complex III but Are Reduced by Complex II and Glycerol-3-Phosphate Dehydrogenase—The failure of MitoQ₁₀ to stimulate respiration in CoQ-deficient mitochondria was general to both yeast and mammals, as it did not complement respiration in CoQ-deficient human fibroblasts, which could be rescued by decylQ.² Therefore MitoQ₁₀ is either poorly oxidized by complex III or poorly reduced by mitochondrial ubiquinone reductases (Fig. 3A). To find out which we determined whether the reduced form of MitoQ₁₀ and other short-chain ubiquinols were oxidized by complex III in bovine heart mitochondrial membranes (BHM). The ubiquinol form of MitoQ₁₀ was an ineffective substrate for complex III, while those of CoQ₂, decylQ, and idebenone were all rapidly oxidized (Fig. 3B). We then measured how effectively the oxidized forms of MitoQ₁₀ CoQ₂, decylQ, and idebenone were reduced by complex I, complex II, and G3PDH. MitoQ₁₀ was an ineffective substrate for complex I (Fig. 3C) but was well reduced by complex II (Fig. 3D) and G3PDH (Fig. 3E). In contrast, CoQ₂, decylQ, and idebenone were effective substrates for all three ubiquinone reductases (Fig. 3, C–E).

View larger version (27K): [in this window] [in a new window]   FIG. 2. Utilization of ubiquinone analogs by CoQ-deficient (COQ2) yeast. Yeast growth was monitored by following A600 over time. Cultures were prepared in YPG + 0.05% glucose, and the initial cell density was adjusted to A600 0.1. Supplementation of the glycerol medium with 0.05% glucose allows fermentative growth of COQ2 yeast to A600 0.4 (indicated by the horizontal line in B and C). A, growth of COQ2 yeast can be restored by supplementation with CoQ6. a–c, growth curves for WT yeast (a), COQ2 yeast (b), and COQ2 yeast supplemented with 50 µM CoQ6 (c). Traces are means ± ranges of duplicate determinations for a typical experiment, which was repeated three times. B, effect of CoQ isoprenoid chain length on growth of COQ2 yeast. A600 was measured after 120 h, by which time maximum cell density had been reached for all growth conditions. The values are the means ± ranges of two independent experiments. C, MitoQ and non-isoprenoid untargeted ubiquinones are ineffective at restoring growth in COQ2 yeast. Conditions are the same as described for B. D, MitoQ10 cannot restore respiration in mitochondria isolated from COQ2 yeast. Mitochondria from COQ2 yeast were incubated with 5 mM G3P and 1 µM FCCP. a–g, the myxothiazol-insensitive respiration rate determined in the presence of the indicated concentrations of CoQ2 (a), decylQ (b), idebenone (c), CoQ1 (d), MitoQ10 (e), CoQ6 (f), and CoQ0 (g). Data are the means ± S.D. of three independent experiments.

Reduction of MitoQ₁₀ by succinate is via complex II as it is completely inhibited by the competitive inhibitor malonate and other electron carriers, such as , do not mediate it as reduction occurred at a similar rate under anaerobic conditions and in the presence of SOD (data not shown). Direct electron transfer from the reduced endogenous CoQ₁₀ pool is also unlikely to contribute to the reduction of MitoQ₁₀ as electron transfer between a ubiquinol and a ubiquinone occurs by sequential deprotonation/electron transfer reactions that cannot occur within the phospholipid bilayer (66). Therefore we conclude that the predominant sources of electrons for MitoQ₁₀ in mitochondria are the active sites of ubiquinone reductases. In summary, MitoQ analogs cannot complement defects in respiration because they are poorly oxidized by complex III. The longer chain MitoQ analogs are extensively reduced by complex II and G3PDH but not by complex I.

MitoQ₁₀ Remains Reduced under Conditions Where CoQ₂, DecylQ, and Idebenone Are Oxidized—In addition to transferring electrons in oxidative phosphorylation, the ubiquinol form of CoQ₁₀ also acts as a chain breaking antioxidant in lipid peroxidation through donation of a hydrogen atom to a carbon or oxygen-centered radical (12). Therefore if an exogenously added ubiquinone is to be an effective antioxidant, its redox state is critical. The poor reactivity of MitoQ₁₀ with complex III implies that MitoQ₁₀ may be persistently reduced and consequently a better antioxidant than CoQ₂, decylQ, or idebenone. To investigate this, we measured the steady-state ubiquinone/ubiquinol ratio for exogenous ubiquinones in the presence of BHM respiring on succinate (Fig. 4A). Under these conditions CoQ₂ remained largely oxidized (Fig. 4A, trace a), as did decylQ and idebenone (data not shown). In contrast, MitoQ₁₀ and MitoQ₁₅ (Fig. 4A, traces d and e) were rapidly reduced. MitoQ₃ and MitoQ₅ (Fig. 4A, traces b and c) were slowly reduced, consistent with their lower rate of reduction by complex II (Fig. 3D). For CoQ₂ and MitoQ₁₀, the relative amounts of ubiquinol and ubiquinone were determined by HPLC (Fig. 4, B and C), and the ratios of their peak areas are shown in Fig. 4D (open bars, CoQ₂; filled bars, MitoQ₁₀). CoQ₂ and MitoQ₁₀ were both largely in the oxidized form on incubation with BHM alone, but on addition of substrate, CoQ₂ remained oxidized, while MitoQ₁₀ was reduced to its ubiquinol form. The complex III-inhibitor myxothiazol caused CoQ₂ to become reduced but had no effect on MitoQ₁₀ as it was already in the ubiquinol form. The ratio of reduced to oxidized idebenone was qualitatively similar to CoQ₂ (data not shown), but co-eluting peaks present in BHM alone prevented precise quantification at 220 nm by HPLC.

View larger version (24K): [in this window] [in a new window]   FIG. 3. Reactivity of MitoQ and other ubiquinone analogs with respiratory chain complexes. A, interaction of respiratory chain enzymes with the mitochondrial ubiquinone pool. DHAP, dihydroxyacetone phosphate; UQ, ubiquinone; UQH2, ubiquinol; Cyt cox and Cyt cred, oxidized and reduced cyt c, respectively. B, oxidation of ubiquinol analogs (50 µM) by complex III. C–E, reduction of ubiquinone analogs (50 µM) by complex I (C), complex II (D), and G3PDH (E). Activity was measured in BHM as described under "Materials and Methods." The activities of the various ubiquinone analogs are given relative to that of CoQ2. The values are the means ± S.E. of three independent experiments. DQ, decylQ; Ide, idebenone; MQ3–15, MitoQ3–15.

View larger version (28K): [in this window] [in a new window]   FIG. 4. Ubiquinone redox state on reduction by mitochondrial membranes. A, MitoQ analogs, unlike other short-chain ubiquinones, are predominantly in a reduced form in succinate-energized uninhibited BHM. BHM were supplemented with rotenone, cyt c, fumarase, and 10 µM CoQ2 (a), MitoQ3 (b), MitoQ5 (c), MitoQ10 (d), or MitoQ15 (e). Succinate was added where indicated. The background rate in the absence of ubiquinone was subtracted. B, BHM were supplemented with rotenone, cyt c, and either ethanol (a), 10 µM CoQ2 (b), succinate, myxothiazol, and 10 µM CoQ2 (c), or succinate and 10 µM CoQ2 (d), then incubated for 2 min, extracted, and run on an HPLC that measured A220. C, same as described for B but supplemented with 10 µM MitoQ10 instead of CoQ2. D, reduction state of CoQ2 or MitoQ10 on incubation with BHM. The ratios of the ubiquinol to ubiquinone peak areas for MitoQ10 (filled bars) and CoQ2 (open bars) were determined by HPLC as shown in B and C. Data are means ± S.E. of three independent experiments. E, MitoQ analogs and untargeted ubiquinones remain predominantly oxidized in the presence of an NADH regeneration system. BHM were supplemented with cyt c, NAD+, lactate, and either 10 µM CoQ2 (a), MitoQ3 (b), MitoQ5 (c), MitoQ10 (d), or MitoQ15 (e). Lactate dehydrogenase (LDH) and cyanide were added where indicated. A background rate in the absence of ubiquinone was subtracted. F, the redox state of exogenous ubiquinones in intact liver mitochondria. Mitochondria (200 µg protein·ml–1) were supplemented with rotenone and a 5 µM concentration of either idebenone (a), CoQ2 (b), or MitoQ10 (c). Succinate, FCCP, and myxothiazol were added as indicated. A decrease in A275 indicates ubiquinone reduction, whereas an increase indicates ubiquinol oxidation.

We next investigated whether the membrane potential in intact rat liver mitochondria largely prevented the oxidation of exogenous ubiquinones by complex III. In contrast to BHM, CoQ₂, idebenone, and MitoQ₁₀ were all largely reduced in mitochondria energized with succinate (Fig. 4F). When the membrane potential was collapsed with the uncoupler FCCP, CoQ₂ and idebenone rapidly became oxidized but were re-reduced on addition of myxothiazol. DecylQ behaved in a manner similar to CoQ₂ and idebenone (data not shown). Therefore the reduced state of exogenous ubiquinones in coupled mitochondria was due to the membrane potential slowing their oxidation by complex III. This behavior was in marked contrast to MitoQ₁₀ as its negligible oxidation by complex III meant that it remained reduced even when the mitochondria were uncoupled. Interestingly, a partial decrease in the membrane potential upon addition of ADP (State 3) did not lead to large scale oxidation of CoQ₂ (data not shown), thus ATP synthesis does not cause extensive oxidation of exogenous untargeted ubiquinones, but complete collapse of the membrane potential does.

In summary, the equilibrium redox state of an exogenous ubiquinone is determined by its relative rates of reduction and oxidation (Fig. 3A). The oxidation of MitoQ₁₀ by complex III is negligible, while it is rapidly reduced by complex II, hence MitoQ₁₀ is fully reduced under most conditions. This is not the case for CoQ₂, decylQ, and idebenone: although they are fully reduced in coupled mitochondria, they are rapidly oxidized by complex III when the membrane potential is low. This may be critical during pathological conditions where depolarization occurs such as during ischemic injury or following induction of the PTP.

The Ineffective Oxidation of MitoQ₁₀ by Complex III Enhances Antioxidant Protection against Peroxynitrite—The antioxidant efficacy of MitoQ₁₀ is due to its conversion to a ubiquinol, as the ubiquinone is inactive (29, 30). To see if the greater tendency of MitoQ₁₀ to remain in the reduced form enhanced its antioxidant ability, we examined its interaction with the biologically significant oxidant peroxynitrite (ONOO^–), which is produced in vivo by the reaction of with nitric oxide (NO·) (69). Among the oxidizing reactions of ONOO^– is the one electron oxidation of ubiquinol to a ubisemiquinone radical which then dismutates (70) (Reactions 1 and 2).

(REACTIONS 1 and 2)

An advantage of using ONOO^– as the ubiquinol oxidant is that at pH 7 it decays within a few seconds to unreactive end products (69), leading to a pulse of ubiquinol oxidation after which regeneration of its antioxidant function by re-reduction can be assessed. CoQ₂ incubated with BHM respiring on succinate remained in its ubiquinone form (Fig. 5A, trace a), while MitoQ₁₀ was reduced by the respiratory chain (Fig. 5A, trace b). MitoQ₅ was also reduced but to a lesser extent due to its slower reaction with complex II (Fig. 5A, trace c). Addition of ONOO^– led to a sharp upward spike in A₂₇₅ for all three ubiquinones. For CoQ₂ (Fig. 5A, trace a) the transient increase in A₂₇₅ was solely due to the absorbance of ONOO^– itself (₃₀₂ = 1.67 mM^–1·cm^–1 (57)), which decayed away over 8 s. For MitoQ₅ (Fig. 5A, trace c) there was a transient spike in A₂₇₅ due to both ONOO^– itself and the formation of oxidized MitoQ₅. After the ONOO^– had decayed away, A₂₇₅ decreased as the ubiquinone was slowly reduced back to the ubiquinol. For MitoQ₁₀, there was also a dramatic spike in A₂₇₅, due to both ONOO^– and ubiquinol oxidation, but in this case the ubiquinone was reduced back to the ubiquinol rapidly by complex II (Fig. 5A, trace b). This oxidation and re-reduction of MitoQ₁₀ by ONOO^– could be repeated several times and two such reaction cycles are shown in Fig. 5A. The re-reduction of the ubiquinone was by complex II, as malonate prevented reduction of MitoQ₅ and MitoQ₁₀ after addition of ONOO^– (Fig. 5A).

The slower reduction of MitoQ₅ by complex II enabled ONOO^– decay and ubiquinone reduction to be easily distinguished in Fig. 5A as a biphasic change in A₂₇₅ after addition of ONOO^–. The biphasic nature of MitoQ₁₀ re-reduction after ONOO^– addition was not obvious so the traces from Fig. 5A were expanded to clearly show that the decay in ONOO^– differs from the re-reduction of MitoQ₁₀ (Fig. 5B). The base lines of the traces have been aligned to emphasize the relative changes in A₂₇₅. For CoQ₂ ± malonate (traces c and d) and for MitoQ₁₀ + malonate (trace b), the addition of ONOO^– leads to an increase in A₂₇₅ that decays back to base line over 8 s due to the breakdown of ONOO^–. In contrast, addition of ONOO^– to MitoQ₁₀ in the presence of uninhibited BHM (trace a) is biphasic with an initial decay in A₂₇₅ due to ONOO^– that is followed by a slower decrease in A₂₇₅ due to reduction of the ubiquinone formed by ONOO^– oxidation. Idebenone behaved in the same way as CoQ₂ (data not shown).

To confirm that the ubiquinol forms of idebenone and CoQ₂ could also react with ONOO^–, we repeated these experiments in myxothiazol-inhibited BHM. Addition of succinate led to the complete reduction of idebenone (Fig. 5C, trace a) and MitoQ₁₀ (Fig. 5C, trace b). ONOO^– rapidly oxidized these ubiquinols, and this was reversed by the respiratory chain within 20sin a malonate-sensitive fashion. CoQ₂ behaved similarly to idebenone (data not shown). Thus, the ubiquinol forms of all exogenous ubiquinones can be oxidized by ONOO^– and then recycled by the respiratory chain.

View larger version (30K): [in this window] [in a new window]   FIG. 5. Ubiquinone oxidation by ONOO–. A, MitoQ analogs are oxidized by ONOO– and re-reduced in uninhibited BHM. BHM were supplemented with rotenone, cyt c, and either 10 µM CoQ2 (a), MitoQ10 (b), or MitoQ5 (c). Succinate, ONOO– (50 µM) and malonate were added where indicated. The background change in absorbance due to fumarate production was subtracted. B, expansion of the period after ONOO– addition in A. a–d, MitoQ10 before (a) and after malonate (b); CoQ2 before (c) and after malonate (d). C, short-chain ubiquinones are oxidized by ONOO– in myxothiazol-inhibited BHM. BHM were supplemented with rotenone, myxothiazol, and either 10 µM idebenone (a), MitoQ10 (b), or MitoQ5 (c). Succinate, ONOO– (50 µM) and malonate were added where indicated. D, MitoQ10 prevents PA oxidation. BHM were supplemented with rotenone, myxothiazol, cyt c, and either 10 µM MitoQ10 (a and d) or idebenone (b and e), or EtOH carrier (c and f). PA (2 µM) was added to all traces as indicated. ONOO– (50 µM) was added to traces a–c as indicated. Succinate was added 1 min prior to PA.

In summary, the reduced form of MitoQ₁₀ is an effective antioxidant against ONOO^–, and its slow oxidation by complex III makes it a more effective antioxidant than untargeted ubiquinone analogs. Importantly, the respiratory chain can reduce MitoQ₁₀ repeatedly recycling it back to its active antioxidant form after it has detoxified ONOO^–.

View larger version (30K): [in this window] [in a new window]   FIG. 6. Consumption of by ubiquinols. A, proposed reactions through which ubiquinols both generate and consume . Ubiquinol (UQH2) directly consumes in the membrane or aqueous phase to generate H2O2 generation is dependent on deprotonation of ubiquinol to the ubiquinolate anion (UQH–). UQ, ubiquinone; , ubisemiquinone anion; UQH·, ubisemiquinone radical. The deprotonation reaction occurs in the aqueous phase and is thus inhibited by ubiquinol hydrophobicity. B, ubiquinols are slowly oxidized by in aqueous solution. Reduced MitoQ (50 µM) was incubated in KPi buffer with 0.015 unit·ml–1 xanthine oxidase at: pH 7.8 (a) or pH 6.8 (b). Acetaldehyde (5 mM) and SOD (100 units·ml–1) were added as indicated. C, ubiquinols are rapidly oxidized by in organic solvent. 25 µl of 10 mM KO in 10 mM 18-crown-6 ether (a) or 25 µl of 10 mM 18-crown-6 ether (b) or 50 µl of 1:1 10 mM KO2 in 10 mM 18-crown-6 ether:H2O incubated for 2 min (c) was added to 50 µM reduced MitoQ10 in 2.5 ml of PBS-saturated octan-1-ol. 25 µl of 10 mM KO2 in 10 mM 18-crown-6 ether was added to a 50 µM concentration of the reduced form of MitoQ10 in 2.5 ml of KPi (pH 7.4) (d). Inset, before and after the addition of KO2 to MitoQ10 in made trace a. KO2 or equivalent additions were as indicated.

There is the possibility that ubiquinols can consume , probably by reaction with its protonated form (HO^·₂,pK_a 4.8), in a mechanism analogous to their chain terminating reaction in lipid peroxidation.

(REACTION 3)

To see if Reaction 3 could lead to a direct antioxidant effect of exogenous ubiquinols on we first measured oxidation of the ubiquinol form of MitoQ₁₀ at 275 nm by generated from acetaldehyde and xanthine oxidase in aqueous buffer. Generation of caused slow oxidation of reduced MitoQ₁₀ that could be fully blocked by SOD (Fig. 6B). Although can react with ubiquinol in aqueous buffer, spontaneous dismutation to hydrogen peroxide (H₂O₂) appears to dominate as the rate of ubiquinol oxidation was low relative to the rate of production. Reaction 3 is also likely to occur within phospholipid bilayers and could thereby provide a mechanism for detoxifying HO^·₂ that is inaccessible to SOD. This would be expected to be important in tissues such as the heart, where cristae phospholipids occupy a volume similar to the aqueous mitochondrial matrix (62). To investigate this we added 100 µM potassium superoxide (KO₂) to a 50 µM concentration of the reduced form of MitoQ₁₀ in PBS-saturated octan-1-ol. KO₂ rapidly oxidized reduced MitoQ₁₀ (Fig. 6C, trace a). Oxidation of reduced MitoQ₁₀ was specific to as it was not oxidized by carrier or by KO₂ that was previously decomposed to H₂O₂ (Fig. 6C, traces b and c). Furthermore, reduced MitoQ₁₀ in aqueous buffer was not oxidized by KO₂ due to its rapid dismutation to H₂O₂ (Fig. 6C, trace d). Therefore these results show that ubiquinols are likely to be effective scavengers of when it diffuses into phospholipid bilayers as HO^·₂. While several variations of Reaction 3 with different protonation states of the reactants could contribute to this, their net effect would be similar: ubiquinol oxidation, H₂O₂ generation, and a lower steady-state concentration of .

Ubiquinol Autoxidation Requires Deprotonation and Is Decreased by Ubiquinol Hydrophobicity—While the ubiquinol form of MitoQ₁₀ is not oxidized by complex III, like all other exogenous ubiquinols it can be oxidized directly by oxygen to form in vitro. The transfer of electrons from ubiquinol to cyt c has been studied extensively (43, 66, 72). From this it can be concluded that the direct donation of an electron by ubiquinol to oxygen is unlikely (UQH ⁺₂/UQH₂, E_m,7 > 850 mV (66)). Instead, electron transfer from ubiquinol requires an initial deprotonation to a ubiquinolate anion (pK_a 11.3 (66)), and this is the likely electron donor to oxygen (UQH·/UQH^–, E_m,7 = 190 mV (66); see Reactions 4 and 5).

(REACTIONS 4 and 5)

The ubisemiquinone radical formed can also react with oxygen to form , or it can dismutate, but it is the initial reaction between the ubiquinolate and oxygen that is likely to be rate-limiting for autoxidation (Fig. 6A). To see if this model would account for ROS production by exogenous ubiquinols, we assessed the pH sensitivity of ubiquinol autoxidation by measuring the increase in ubiquinone absorption at 275 nm. There was negligible oxidation of the reduced form of MitoQ₁₀ in aqueous solution at pH 6.8, but autoxidation increased a little at pH 7.8 and dramatically at pH 8.3 (Fig. 7A). Consistent with Fig. 6A, ubiquinol autoxidation was SOD-insensitive. To demonstrate that generation occurred during ubiquinol autoxidation we used acetylated cyt c (cyt c_acet), which is readily reduced by one electron transfer from (73) and ubisemiquinones (43, 72) but whose reduction and oxidation by the respiratory chain are limited (73). In aqueous buffer the ubiquinol, but not the ubiquinone, forms of CoQ₂ and MitoQ₁₀ reduced cyt c_acet, and this reduction occurred primarily via as the rate was 80–90% SOD-sensitive (data not shown). To demonstrate generation during autoxidation of complex II-reduced MitoQ₁₀, we measured cyt c_acet reduction in myxothiazol-inhibited BHM (Fig. 7B). This showed that reduction of MitoQ₁₀ by complex II also caused cyt c_acet reduction and that this rate increased with pH from pH 6.8 to 8.3. This pH dependence was not due to changes in MitoQ₁₀ reduction by complex II as this rate was identical at pH 6.8 and 8.3 (data not shown). Therefore production from exogenous ubiquinols produced chemically or by mitochondrial respiration is pH-dependent, consistent with ubiquinol deprotonation being critical for autoxidation.

As deprotonation creates two charged species, autoxidation will predominantly occur in the aqueous phase rather than within phospholipid bilayers and its rate should be inversely proportional to ubiquinol hydrophobicity. To investigate this we measured ubiquinol autoxidation in myxothiazol-inhibited BHM using a range of MitoQ and non-targeted ubiquinone analogs with a spectrum of hydrophobicities (Fig. 7C). All the exogenous ubiquinones reduced cyt c_acet (Fig. 7C), and in all cases this rate of reduction was about 50% inhibitable by SOD (data not shown). The rate of production from autoxidizing untargeted ubiquinones was inversely proportional to their octan-1-ol/PBS partition coefficients (Fig. 1B). The lowest levels of were generated by the most hydrophobic ubiquinol, decylQ, with the most water-soluble, idebenol, producing the most and CoQ₂ being intermediate. There was a similar inverse correlation with hydrophobicity for the MitoQ analogs from MitoQ₅ to MitoQ₁₅. MitoQ₃ was an exception to this trend, possibly due to its slow reduction by complex II leading to a lower ubiquinol concentration (Fig. 3C). While this inverse relationship between autoxidation and hydrophobicity held within the two groups, the MitoQ analogs were less prone to autoxidation than more hydrophobic non-targeted ubiquinones (e.g. MitoQ₁₀ versus idebenone). MitoQ analogs are charged cations, while untargeted ubiquinones are neutral, so partitioning into octan-1-ol may not accurately reflect binding to phospholipid bilayers. Therefore we measured the relative binding of MitoQ analogs and untargeted ubiquinones to mitochondrial membranes (Fig. 7D) and found that increased binding of MitoQ analogs to phospholipid bilayers could explain their lower rate of autoxidation when compared with equivalent untargeted ubiquinols.

In summary, all ubiquinols are autoxidized, but this requires an initial deprotonation to a ubiquinolate anion, which is unfavorable within the hydrophobic environment of a phospholipid bilayer. Therefore there are two major determinants of the degree to which ubiquinol will autoxidize: its extent of reduction and its hydrophobicity. Furthermore, the charged TPP moiety of MitoQ analogs may be a better way of lowering overall hydrophobicity and improving pharmacokinetics without enhancing the tendency to autoxidation, as it leads to adsorption onto phospholipid bilayers and insertion of the ubiquinol moiety into the hydrophobic core of the membrane.

Ubiquinol Autoxidation Produces Superoxide, Which Can Dismutate to Hydrogen Peroxide or Consume Nitric Oxide—In the above analysis, generation during ubiquinol autoxidation was assessed using cyt c_acet reduction, but cyt c_acet can be reduced by and by the ubisemiquinone radical (43, 66, 72, 73), both of which may form during ubiquinol autoxidation. Furthermore, even though the reduction of cyt c_acet was 50% SOD-sensitive, it is still difficult to conclude a dominant role for , as the steady-state concentration of the ubisemiquinone radical decreases as a consequence of consuming by dismutation (74). In addition, cyt c_acet itself may act as a sink and thus distort the relative rates and routes of ubiquinol autoxidation. Therefore to confirm that autoxidation of exogenous ubiquinol produced , we used a H₂O₂ electrode to measure the production of H₂O₂ from the dismutation of (4). Measurement of H₂O₂ is of further significance as MitoQ₁₀ has been shown to block a number of redox signaling and apoptotic pathways, but the mechanism remains unclear (35–37, 39–42). BHM alone did not produce H₂O₂ when inhibited by myxothiazol (Fig. 7E, trace a) nor did BHM supplemented with CoQ₂ and succinate (Fig. 7E, trace b). However, inhibition of ubiquinol oxidation with myxothiazol (Fig. 7E, trace c) or cyanide (data not shown) led to a build up of ubiquinol and consequent H₂O₂ production. MitoQ₁₀ also produced H₂O₂ but did so in the absence of myxothiazol as it was already fully reduced (data not shown). Therefore autoxidation of ubiquinols does produce that will dismutate to H₂O₂, and this could be important in redox signaling.

View larger version (22K): [in this window] [in a new window]   FIG. 7. Generation of by ubiquinols. A, ubiquinol autoxidation increases with pH. Oxidation of the ubiquinol form of MitoQ10 in KPi buffer at: pH 8.3 (a), pH 7.8 (b), or pH 6.8 (c). NaBH44-reduced MitoQ10 (50 µM) was added as indicated. SOD (100 units·ml–1) was added as indicated to trace a. B, ubiquinol-dependent reduction of cyt cacet increases with pH. a–d, reduction of cyt cacet by BHM supplemented with myxothiazol, KCN, rotenone, cyt cacet, and 10 µM MitoQ10 at: pH 8.3 (a), pH 7.8 (b), pH 7.3 (c), or pH 6.8 (d). C, increasing ubiquinol hydrophobicity decreases reduction of cyt cacet. a–g, reduction of cyt cacet by inhibited BHM supplemented with myxothiazol, KCN, rotenone, and cyt cacet and 10 µM idebenone (a), CoQ2 (b), decylQ (c), MitoQ3 (d), MitoQ5 (e), MitoQ10 (f), or MitoQ15 (g). Succinate was added as indicated. C, ubiquinol-dependent reduction of cyt cacet is partially SOD-sensitive. D, MitoQ analogs partition more strongly into BHM than into octan-1-ol. Ubiquinones (50 µM) were incubated in PBS (2 ml) containing BHM (500 µg of protein) at 37°C for 5 min followed by centrifugation. The relative concentration in each phase was determined after extraction into octan-1-ol. BHM phospholipid volume was taken as 0.22 µl·mg protein–1. BHM/PBS partition coefficients are the means ± range (n = 2). E, ubiquinone autoxidation generates H2O2. a–c, H2O2 production from BHM supplemented with rotenone, cyt c, SOD (100 units·ml–1) and either myxothiazol (a), 50 µM CoQ2 (b), or myxothiazol and 50 µM CoQ2 (c). Succinate was added where indicated. F, ubiquinone autoxidation consumes NO·. a and b, NO· consumption by BHM (20 µg protein·ml–1) supplemented with rotenone, cyt c, and either ethanol (a) or 10 µM CoQ2 (b). DETA-NONOate, succinate, myxothiazol, and SOD were added as indicated.

View larger version (13K): [in this window] [in a new window]   FIG. 8. MitoQ autoxidation causes H2O2 efflux from mitochondria but only MitoQ3 damages aconitase. A, MitoQ analogs generate H2O2 within intact mitochondria. Isolated rat heart mitochondria (0.2 mg protein·ml–1) were incubated at 25 °C with rotenone, Amplex Red, horseradish peroxidase, SOD, and either ethanol (a), 1 µM MitoQ3 (b), 1 µM MitoQ5 (c), 1 µM MitoQ10 (d), or 1 µM MitoQ15 (e). Succinate was added where indicated. B,H2O2 generation from MitoQ3 compared with other mechanisms of H2O2 production. Isolated rat heart mitochondria (0.2 mg protein·ml–1) were incubated at 37 °C with Amplex Red, horseradish peroxidase, SOD, and either rotenone (a), ethanol (b), 1 µM MitoQ3 (c), 1 mM Paraquat (d), or 100 µM Paraquat (e). Succinate was added where indicated. C, aconitase inactivation was plotted as the natural logarithm of activity versus time and given a linear fit. Isolated wild-type yeast mitochondria (0.3 mg protein·ml–1) were incubated at 30 °C with aliquots taken every 5 min for 30 min. White bar, Background rate of aconitase inactivation in unenergized WT yeast mitochondria. Black bars, energized with 5 mM G3P and incubated with either 0–10 mM Paraquat, 1 µM FCCP, 1 µM MitoQ3, 1 µM MitoQ5, 1 µM MitoQ10, or 1 µM decyl-TPP. Aconitase inactivation was plotted as time versus the natural logarithm of activity and given a linear fit. The slope of this line corresponds to the pseudo-first order rate constant of aconitase inactivation. Data are the mean ± S.D. of three independent experiments. Statistical significance was calculated relative to G3P alone using a Student's one-tailed t test. *, p < 0.05; ***, p < 0.001.

MitoQ Analogs Cause Efflux of Hydrogen Peroxide from Mitochondria, but Only MitoQ₃ Damages Aconitase—The above findings show that all exogenous ubiquinols have the potential to autoxidize and form in aqueous solution. To determine whether ubiquinol autoxidation led to significant generation within intact mitochondria, we measured the efflux of H₂O₂ from isolated rat heart mitochondria in the presence of 1 µM exogenous ubiquinol. H₂O₂ efflux is due to the dismutation of to H₂O₂, which can then diffuse through the mitochondrial inner membrane (81) and be detected using Amplex Red and horseradish peroxidase (Fig. 8A). A significant rate of H₂O₂ efflux was observed in the presence of rotenone and a 1 µM concentration of either MitoQ₃, MitoQ₅, or MitoQ₁₀ (Fig. 8A, traces b–d). As expected, the presence of exogenous SOD had no effect on H₂O₂ efflux from MitoQ₃ (data not shown). In BHM exogenous ubiquinols inhibit the horseradish peroxidase detection system (data not shown), consequently the accumulation of ubiquinol may cause the apparent decrease in H₂O₂ efflux with time. Therefore, the initial rate of H₂O₂ efflux is the best indication of autoxidation, and this is significantly lower for the more hydrophobic MitoQ analogs. To gauge the relative magnitude of MitoQ₃ autoxidation, we compared it with other known mechanisms for generating H₂O₂ efflux at 37 °C (Fig. 8B). This showed that H₂O₂ generation by 1 µM MitoQ₃ was equivalent to high micromolar concentrations of the redox cycler Paraquat and greater than the proton motive force-dependent and rotenone-sensitive efflux of H₂O₂ from succinate-energized mitochondria (82) (Fig. 8B).

To determine whether this level of intramitochondrial production was damaging, we incubated intact yeast mitochondria with MitoQ analogs and studied their effects on aconitase activity, the iron-sulfur center of which is particularly sensitive to (60). The pseudo-first order rate constant for aconitase inactivation within mitochondria was taken as an indication of the steady-state matrix concentration (Fig. 8C). Aconitase inactivation responded appropriately to factors that decrease (no substrate, uncoupler) or increase (Paraquat), therefore this system can detect variations in endogenous level (Fig. 8C). Addition of 1 µM MitoQ₃ caused a small but statistically significant increase in aconitase inactivation above that of substrate alone. In contrast, 1 and 5 µM MitoQ₁₀ decreased the rate of aconitase inactivation, but this was due to mild uncoupling as decyl-TPP gave a similar result (Fig. 8C and data not shown). Therefore, although MitoQ analogs can generate within mitochondria, this rate appears too low to cause significant damage to mitochondrial enzymes.

Exogenous Ubiquinones Do Not React with Peroxides— MitoQ₁₀ blocks H₂O₂-induced apoptosis and cell death, but the details of how this is achieved remain unclear (29, 41, 42). H₂O₂ can cause oxidative damage through Fenton chemistry with Fe²⁺ or Cu⁺ as well as being a diffusible signaling molecule that reacts with protein thiols (4, 5). As direct reaction of H₂O₂ or alkyl peroxides with ubiquinones and ubiquinols has not been reported under physiological conditions, it was important to clarify whether they react with MitoQ₁₀ and other exogenous ubiquinones. A lack of direct reaction of the ubiquinol form of MitoQ₁₀ with H₂O₂ was confirmed by ¹H NMR measurement of the oxidation of 10 mM reduced MitoQ₁₀ in acidified D₂O. Even incubation with 10 mM H₂O₂ under air over several days did not result in H₂O₂-sensitive oxidation of reduced MitoQ₁₀ (data not shown).

As many of the signaling effects of H₂O₂ within mitochondria occur through its interactions with thiols, we next determined whether MitoQ₁₀ could affect the oxidation of reactive biological thiols by H₂O₂. To do this we incubated the ubiquinol form of MitoQ₁₀ in the presence of reduced GSH and glutathione peroxidase and exposed it to a flux of H₂O₂ generated by glucose oxidase. The H₂O₂ from glucose oxidase oxidized GSH to GSSG catalyzed by glutathione peroxidase, and GSSG was detected through NADPH consumption by glutathione reductase (Fig. 9A). Neither the reduced nor oxidized forms of MitoQ₁₀ (Fig. 9A and data not shown) or CoQ₂ (data not shown) affected GSH oxidation (Fig. 9A) confirming that exogenous ubiquinols neither directly scavenge H₂O₂ nor interfere with the reaction of H₂O₂ with biological thiols. We next investigated the induction of the PTP by Ca²⁺ and tBHP as this involves oxidation of critical protein thiols (83). PTP opening in Ca²⁺-loaded rat liver mitochondria was induced by 5 µM tBHP and led to the release of accumulated Ca²⁺ (Fig. 9B, trace b). This process could be blocked by CsA (Fig. 9B, trace c). When the experiment was repeated in the presence of either 1 µM (Fig. 9C) or 5 µM MitoQ₁₀ (data not shown), PTP opening was still triggered by 5 µM tBHP (Fig. 9C, trace b) and blocked with CsA (Fig. 9C, trace c). The extent of Ca²⁺ uptake was decreased compared to the system without MitoQ₁₀, but the kinetics of PTP induction were similar. Thus MitoQ₁₀ cannot scavenge alkyl peroxides and prevent PTP opening caused by thiol oxidation.

View larger version (10K): [in this window] [in a new window]   FIG. 9. MitoQ10 does not prevent peroxide-induced thiol oxidation. A, MitoQ10 does not prevent glutathione oxidation by H2O2. H2O2 was generated by glucose oxidase (0.006 units·ml–1) and 10 mM glucose in the presence of glutathione peroxidase (0.02 units·ml–1), 500 µM GSH, NADPH, and glutathione reductase. Reduced MitoQ10 was added as indicated. B, the mitochondrial PTP is triggered by tBHP. Liver mitochondria were incubated in the presence of succinate, rotenone, calcium green-5N and either no additions (a), 5 µM tBHP (b), or 5 µM tBHP and 500 nM CsA (c). 5 µM Ca2+ was added where indicated. C, induction of the PTP by tBHP is not blocked by MitoQ10. Liver mitochondria were incubated in the presence of succinate, rotenone, calcium green-5N, 1 µM MitoQ10, and either no addition (a), 5 µM tBHP (b), or 5 µM tBHP and 500 nM CsA (c). 5 µM Ca2+ was added where indicated.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
This study has clarified the redox, antioxidant, and prooxidant properties of a series of mitochondria-targeted and untargeted exogenous ubiquinones. While CoQ₁, CoQ₂, decylQ, CoQ₆, and idebenone could all restore respiration in mitochondria lacking CoQ, MitoQ₁₀ could not. The ability of the CoQ analogs to restore respiration was consistent with their fast reduction by ubiquinone reductases and their rapid oxidation by complex III. In contrast, none of the MitoQ analogs could act as electron carriers in respiration because they were not oxidized by complex III. While all the MitoQ analogs rapidly migrate through the mitochondrial inner membrane (30), the hydrophobic core is still a significant activation energy barrier for their transport, albeit a far lower one than that for equivalent hydrophilic cations (32, 33). Furthermore, although MitoQ₁₀ has a similar octan-1-ol/PBS partition coefficient to idebenone, MitoQ analogs are amphipathic and consequently not evenly distributed within phospholipid bilayers (Fig. 1C) (32, 33). Thus for MitoQ analogs the steady-state concentration of the ubiquinone moiety at a particular distance from the membrane surface will be related to the length of the alkyl chain. This is in marked contrast to the untargeted ubiquinones, which will be freely soluble within the hydrophobic core. This affinity of MitoQ analogs for the surface of the phospholipid bilayer is simply demonstrated by its limited solubility in cyclohexane, an organic solvent that mimics the membrane core: MitoQ₁₀ formed a separate, orange-red, oily phase, with only some slight discoloration of the cyclohexane. In contrast idebenone was freely soluble up to at least 4 mM in cyclohexane, as were MitoQ₁₀ and idebenone in octan-1-ol, a more polar solvent that mimics the membrane surface. Therefore, the significantly decreased reactivity of all MitoQ analogs with complex III may be a consequence of the low concentration of their ubiquinone moieties in the active sites of complex III (Fig. 10A) (64, 84). These are near the center of the phospholipid bilayer, and for MitoQ analogs to dock into them, the TPP cation must be located in the hydrophobic core (Fig. 10A). In addition, enzyme shape and dimerization may further increase the effective distance between these binding sites and the surface of the phospholipid bilayer (64, 84). Therefore the steady-state concentration of MitoQ analogs within the membrane core is decreased relative to other ubiquinones, and this is likely to explain their low reactivity with complex III.

View larger version (104K): [in this window] [in a new window]   FIG. 10. Modeling of the binding of MitoQ analogs to the active sites of complexes II and III. A, MitoQ10 (red) bound to the ubiquinone reduction (left) and ubiquinol oxidation (right) binding sites in complex III from B. taurus. B, MitoQ10 (red) bound to the ubiquinone reduction site of complex II from E. coli. C and D, binding of MitoQ3 (C) and MitoQ5 (D) to the ubiquinone reduction site of complex II. Val, Phe, Met, Ile, Leu, Tyr, and Trp amino acid residues were considered hydrophobic and colored yellow to indicate the approximate position of the phospholipid bilayer; all other residues are blue.

The effects of MitoQ analogs are not due to their ability to complement respiration. In contrast, untargeted ubiquinones should be able to supplement respiratory defects in vivo provided they can be delivered to mitochondria within cells. In our experiments only the ubiquinones with isoprenoid tails, CoQ₆, CoQ₂, and CoQ₁, could restore non-fermentative growth in CoQ-deficient yeast, while the exogenous ubiquinones with simple saturated carbon chains were ineffective. As this difference is not due to poor electron transfer in the respiratory chain, there may be lower mitochondrial accumulation of the saturated relative to the isoprenoid exogenous ubiquinones. This is not due to differences in passive diffusion as CoQ₆ is several orders of magnitude more hydrophobic than decylQ, which in turn has an octan-1-ol/PBS partition coefficient 1,000-fold higher than CoQ₁ (Fig. 1B) (31). Supporting this, the diffusion of CoQ₆ through the mitochondrial outer membrane was far slower than for decylQ or idebenone (Fig. 2D). As uptake does not appear to be passive, we speculate that in yeast there is a process for trafficking exogenous ubiquinones from the plasma membrane to mitochondria and that it is selective for ubiquinones with isoprenoid tails. Such a process is suggested by some earlier work on yeast where accumulation of exogenous CoQ₆ in mitochondria was deficient in some strains (85). It is unclear whether a similar pathway would exist in mammalian cells; however, in a clinical case where CoQ₁₀ supplementation ameliorated the symptoms of CoQ deficiency, subsequent switching to idebenone caused clinical and metabolic worsening, which disappeared on return to CoQ₁₀ supplementation (86). As the final steps of CoQ synthesis are in mitochondria, it is not clear why a putative CoQ uptake system should exist. However, it is possible there is a pathway that allows the export mitochondrial CoQ to the plasma membrane, and in the presence of exogenous CoQ it can operate in reverse to insert CoQ into mitochondria. This may explain why dietary supplementation with CoQ₁₀ leads to only small increases in mitochondrial CoQ₁₀ concentration except when there is an underlying CoQ₁₀ deficiency (20–24, 87). Our results suggest that short chain ubiquinone analogs containing isoprenoid side chains may accumulate more effectively within mitochondria than saturated exogenous ubiquinones and are worth exploring as potential therapies for human diseases with CoQ deficiencies.

The autoxidation of ubiquinols is well established, but its significance for exogenous short-chain ubiquinones is uncertain. Here we confirm that autoxidation of exogenous ubiquinones occurs by enzymatic reduction to the ubiquinol followed by deprotonation to the ubiquinolate anion, which reacts with oxygen to produce . Critically, ubiquinol autoxidation will not occur when the ubiquinol head group is within the phospholipid bilayer and is therefore proportional to ubiquinol hydrophilicity. However, while it was possible to detect autoxidation of ubiquinols in BHM and isolated mitochondria, the autoxidation of MitoQ₁₀ or idebenone did not lead to a significant increase in damage to matrix aconitase. This lack of autoxidation-dependent damage is consistent with clinical trials using idebenone that have shown it is well tolerated for at least 2 years (28, 88–90), even though it has a high tendency to autoxidize in vitro (71). While the focus of studies on ROS is often their toxicity, it is possible that autoxidation of exogenous ubiquinones generates subtoxic levels of ROS. This may even be beneficial as ROS can increase the expression of antioxidant pathways via hormesis (4, 91). The complex nature of ROS interactions is illustrated by the paradoxical fact that 20 µM H₂O₂, autoxidation-resistant ascorbate derivatives, and MitoQ₁₀ all extend the lifespan of cultured cells and decrease telomere shortening (39, 91). Therefore, it remains unclear whether exogenous ubiquinone autoxidation is detrimental in vivo as it is associated with a large increase in membrane associated antioxidant capacity, against both lipid peroxidation and , and could lead to the induction of antioxidant enzymes via redox-sensitive transcription factors (92–94). Whether a large increase in protection against lipid peroxidation is on balance beneficial when coupled to a small increase in is beyond the scope of this work and would require long term animal studies looking at pathological markers. Even so, it is apparent that increasing hydrophobicity limits the "bad" aqueous autoxidation of ubiquinols without diminishing its "good" organic phase antioxidant functions (Fig. 6). This could provide a teleological explanation for the extreme hydrophobicity of endogenous CoQ₁₀ (Fig. 1B).

MitoQ₁₀ has been shown to block a number of redox signaling pathways as well as cell death induced by exogenous H₂O₂ (29, 41, 42); however, the details of the processes affected are uncertain. MitoQ₁₀ does not react significantly with H₂O₂ or other alkyl peroxides (Fig. 9) but is very effective against lipid peroxidation (29, 30). This suggests that the effects of MitoQ₁₀ against H₂O₂ are primarily due to blocking the consequences of OH· presumably formed from iron-catalyzed Fenton chemistry. The presence OH· within mitochondria results in lipid peroxidation, which has a number of deleterious effects on mitochondrial function and leads to breakdown products, such as the reactive aldehyde 4-hydroxy-2-nonenal, which may have a role in cell damage and signaling (95). This work shows that while MitoQ₁₀ could interact with ROS in several ways in vivo, the most likely way is that it blocks the effects of exogenous H₂O₂ by acting downstream of H₂O₂ to inhibit peroxidative chain reactions.

Here we have investigated how a series of exogenous ubiquinone analogs interact with oxidative phosphorylation and with ROS metabolism. We found that while the type of reactions that MitoQ and untargeted ubiquinone analogs underwent with ROS were similar, ubiquinone hydrophobicity dramatically affected the extent of these reactions. This work clearly shows that the antioxidant reactions of exogenous ubiquinones will predominantly occur within phospholipid bilayers, while the pro-oxidant reactions require an aqueous environment. Therefore, the relative rates of these reactions can be fine-tuned by hydrophobicity, allowing a rational approach to the design of therapeutic ubiquinone-based antioxidants (e.g. MitoQ₁₀) or mitochondria-targeted redox cyclers (e.g. MitoQ₃). Furthermore, there was a sharp distinction between how MitoQ analogs and other short-chain ubiquinones interacted with the mitochondrial respiratory chain. This occurred because the TPP cation decreased the oxidation of MitoQ analogs by complex III. Thus MitoQ analogs function only as antioxidants, while other short-chain ubiquinones can also be oxidative phosphorylation substrates. When used in combination they may provide a way for separating cause and effect in mitochondrial DNA diseases, Parkinson disease, and aging itself where both a decline in oxidative phosphorylation and an increase in ROS may overlap to cause pathology.

	FOOTNOTES

 
^* This work was supported by the Medical Research Council, Foundation for Research, Science and Technology New Zealand and by Antipodean Biotechnology. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

This article was selected as a Paper of the Week.

Recipient of a Ph.D. Studentship from Research into Ageing, United Kingdom.

^|| To whom correspondence should be addressed. Tel.: 44-1223-252900; Fax: 44-1223-252905; E-mail: mpm{at}mrc-dunn.cam.ac.uk.

¹ The abbreviations used are: ROS, reactive oxygen species; COQ2, coenzyme Q-deficient yeast strain; cyt c, cytochrome c; cyt c_acet, acetylated cytochrome c; BHM, bovine heart mitochondrial membranes; CoQ₀, 2,3-dimethoxy-5-methyl-1,4-benzoquinone; CoQ_1–10, coenzyme Q with a tail of 1–10 isoprenoid units (2,3-dimethoxy-5-methyl-6-polyprenyl-1,4-benzoquinone); CsA, cyclosporin A; DETA-NONOate, 3,3-bis(aminoethyl)-1-hydroxy-2-oxo-1-triazene; FCCP, carbonyl cyanide p-trifluoromethoxyphenylhydrazone; G3P, glycerol 3-phosphate; G3PDH, glycerol-3-phosphate dehydrogenase; MitoQ, ubiquinone linked to a triphenylphosphonium cation by an alkyl chain of unspecified length; MitoQ_3–15, ubiquinone linked to a triphenylphosphonium cation by an alkyl chain of 3–15 carbons; PA, cis-parinaric acid; PBS, phosphate-buffered saline; PTP, mitochondrial permeability transition pore; SOD, superoxide dismutase; tBHP, t-butyl-hydroperoxide; TPP, triphenylphosphonium cation; WT, wild-type yeast strain; TPMP, methyltriphenylphosphonium; BSA, bovine serum albumin; HPLC, high performance liquid chromatography.

² P. Rustin, personal communication.

	ACKNOWLEDGMENTS

 
We thank D. R. Gauntlett for technical assistance and Dr. Abdul-Rahman B. Manas for the supply of synthetic chemicals.

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