Andrew M James

Publications (31)145.22 Total impact

• Dataset: Prime PNAS 2009

  Tracy A Prime, Frances H Blaikie, Cameron Evans, Sergiy M Nadtochiy, Andrew M James, Christina C Dahm, Dario A Vitturi, Rakesh P Patel, C Robin Hiley, Irina Abakumova, Raquel Requejo, Edward T Chouchani, Thomas R Hurd, John F Garvey, Cormac T Taylor, Paul S Brookes, Robin A J Smith, Michael P Murphy
• Dataset: Prime PNAS 2009

  Tracy A Prime, Frances H Blaikie, Cameron Evans, Sergiy M Nadtochiy, Andrew M James, Christina C Dahm, Dario A Vitturi, Rakesh P Patel, C Robin Hiley, Irina Abakumova, Raquel Requejo, Edward T Chouchani, Thomas R Hurd, John F Garvey, Cormac T Taylor, Paul S Brookes, Robin A J Smith, Michael P Murphy
• Article: Mitochondrial accumulation of a lipophilic cation conjugated to an ionisable group depends on membrane potential, pH gradient and pK (a): implications for the design of mitochondrial probes and therapies.

  Peter G Finichiu, Andrew M James, Lesley Larsen, Robin A J Smith, Michael P Murphy
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  ABSTRACT: Mitochondria play key roles in a broad range of biomedical situations, consequently there is a need to direct bioactive compounds to mitochondria as both therapies and probes. A successful approach has been to target compounds to mitochondria by conjugation to lipophilic cations, such as triphenylphosphonium (TPP), which utilize the large mitochondrial membrane potential (Δψ(m), negative inside) to drive accumulation. This has proven effective both in vitro and in vivo for a range of bioactive compounds and probes. However so far only neutral appendages have been targeted to mitochondria in this way. Many bioactive functional moieties that we would like to send to mitochondria contain ionisable groups with pK (a) in the range that creates an assortment of charged species under physiological conditions. To see if such ionisable compounds can also be taken up by mitochondria, we determined the general requirements for the accumulation within mitochondria of a TPP cation conjugated to a carboxylic acid or an amine. Both were taken up by energised mitochondria in response to the protonmotive force. A lipophilic TPP cation attached to a carboxylic acid was accumulated to a greater extent than a simple TPP cation due to the interaction of the weakly acidic group with the pH gradient (ΔpH). In contrast, a lipophilic TPP cation attached to an amine was accumulated less than the simple cation due to exclusion of the weakly basic group by the ΔpH. From these data we derived a simple equation that describes the uptake of lipophilic cations containing ionisable groups as a function of Δψ(m), ΔpH and pK (a). These findings may facilitate the rational design of additional mitochondrial targeted probes and therapies.
  Journal of Bioenergetics 11/2012; · 2.81 Impact Factor
• Source

  Available from: Alexander V Peskin

  Article: A Mitochondria-Targeted Macrocyclic Mn(II) Superoxide Dismutase Mimetic.

  Geoffrey F Kelso, Andrej Maroz, Helena M Cochemé, Angela Logan, Tracy A Prime, Alexander V Peskin, Christine C Winterbourn, Andrew M James, Meredith F Ross, Sally Brooker, Carolyn M Porteous, Robert F Anderson, Michael P Murphy, Robin A J Smith
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  ABSTRACT: Superoxide (O(2)(⋅-)) is the proximal mitochondrial reactive oxygen species underlying pathology and redox signaling. This central role prioritizes development of a mitochondria-targeted reagent selective for controlling O(2)(⋅-). We have conjugated a mitochondria-targeting triphenylphosphonium (TPP) cation to a O(2)(⋅-)-selective pentaaza macrocyclic Mn(II) superoxide dismutase (SOD) mimetic to make MitoSOD, a mitochondria-targeted SOD mimetic. MitoSOD showed rapid and extensive membrane potential-dependent uptake into mitochondria without loss of Mn and retained SOD activity. Pulse radiolysis measurements confirmed that MitoSOD was a very effective catalytic SOD mimetic. MitoSOD also catalyzes the ascorbate-dependent reduction of O(2)(⋅-). The combination of mitochondrial uptake and O(2)(⋅-) scavenging by MitoSOD decreased inactivation of the matrix enzyme aconitase caused by O(2)(⋅-). MitoSOD is an effective mitochondria-targeted macrocyclic SOD mimetic that selectively protects mitochondria from O(2)(⋅-) damage.
  Chemistry & biology 10/2012; 19(10):1237-46. · 6.52 Impact Factor
• Article: Inactivation of pyruvate dehydrogenase kinase 2 by mitochondrial reactive oxygen species.

  Thomas R Hurd, Yvonne Collins, Irina Abakumova, Edward T Chouchani, Bartlomiej Baranowski, Ian M Fearnley, Tracy A Prime, Michael P Murphy, Andrew M James
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  ABSTRACT: Reactive oxygen species are byproducts of mitochondrial respiration and thus potential regulators of mitochondrial function. Pyruvate dehydrogenase kinase 2 (PDHK2) inhibits the pyruvate dehydrogenase complex, thereby regulating entry of carbohydrates into the tricarboxylic acid (TCA) cycle. Here we show that PDHK2 activity is inhibited by low levels of hydrogen peroxide (H(2)O(2)) generated by the respiratory chain. This occurs via reversible oxidation of cysteine residues 45 and 392 on PDHK2 and results in increased pyruvate dehydrogenase complex activity. H(2)O(2) derives from superoxide (O(2)()), and we show that conditions that inhibit PDHK2 also inactivate the TCA cycle enzyme, aconitase. These findings suggest that under conditions of high mitochondrial O(2)() production, such as may occur under nutrient excess and low ATP demand, the increase in O(2)() and H(2)O(2) may provide feedback signals to modulate mitochondrial metabolism.
  Journal of Biological Chemistry 08/2012; 287(42):35153-60. · 4.77 Impact Factor
• Article: Mitochondrial oxidative stress and the metabolic syndrome.

  Andrew M James, Yvonne Collins, Angela Logan, Michael P Murphy
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  ABSTRACT: The current epidemic of the metabolic syndrome in the developed world is largely due to overnutrition and lack of physical activity. However, the underlying causes by which chronic overnutrition interacts with genotype and physical inactivity to generate the metabolic syndrome phenotype are complex, and include multiple metabolic and physiological alterations. Mitochondrial oxidative stress has been suggested to contribute to the metabolic syndrome, but the mechanisms and significance are unclear. Here we review how disruption of mitochondrial metabolism and increased oxidative stress may occur during overnutrition coupled with limited physical activity. From this we suggest a unifying hypothesis to integrate what is known about mitochondrial involvement in the metabolic syndrome that points to testable hypotheses and novel therapeutic approaches.
  Trends in Endocrinology and Metabolism 07/2012; 23(9):429-34. · 8.11 Impact Factor
• Article: Using the mitochondria-targeted ratiometric mass spectrometry probe MitoB to measure H2O2 in living Drosophila.

  Helena M Cochemé, Angela Logan, Tracy A Prime, Irina Abakumova, Caroline Quin, Stephen J McQuaker, Jigna V Patel, Ian M Fearnley, Andrew M James, Carolyn M Porteous, Robin A J Smith, Richard C Hartley, Linda Partridge, Michael P Murphy
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  ABSTRACT: The role of hydrogen peroxide (H(2)O(2)) in mitochondrial oxidative damage and redox signaling is poorly understood, because it is difficult to measure H(2)O(2) in vivo. Here we describe a method for assessing changes in H(2)O(2) within the mitochondrial matrix of living Drosophila. We use a ratiometric mass spectrometry probe, MitoB ((3-hydroxybenzyl)triphenylphosphonium bromide), which contains a triphenylphosphonium cation component that drives its accumulation within mitochondria. The arylboronic moiety of MitoB reacts with H(2)O(2) to form a phenol product, MitoP. On injection into the fly, MitoB is rapidly taken up by mitochondria and the extent of its conversion to MitoP enables the quantification of H(2)O(2). To assess MitoB conversion to MitoP, the compounds are extracted and the MitoP/MitoB ratio is quantified by liquid chromatography-tandem mass spectrometry relative to deuterated internal standards. This method facilitates the investigation of mitochondrial H(2)O(2) in fly models of pathology and metabolic alteration, and it can also be extended to assess mitochondrial H(2)O(2) production in mouse and cell culture studies.
  Nature Protocol 05/2012; 7(5):946-58. · 8.36 Impact Factor
• Article: Mitochondrial redox signalling at a glance.

  Yvonne Collins, Edward T Chouchani, Andrew M James, Katja E Menger, Helena M Cochemé, Michael P Murphy
  Journal of Cell Science 02/2012; 125(Pt 4):801-6. · 6.11 Impact Factor
• Article: Selective uncoupling of individual mitochondria within a cell using a mitochondria-targeted photoactivated protonophore.

  Susan Chalmers, Stuart T Caldwell, Caroline Quin, Tracy A Prime, Andrew M James, Andrew G Cairns, Michael P Murphy, John G McCarron, Richard C Hartley
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  ABSTRACT: Depolarization of an individual mitochondrion or small clusters of mitochondria within cells has been achieved using a photoactivatable probe. The probe is targeted to the matrix of the mitochondrion by an alkyltriphenylphosphonium lipophilic cation and releases the protonophore 2,4-dinitrophenol locally in predetermined regions in response to directed irradiation with UV light via a local photolysis system. This also provides a proof of principle for the general temporally and spatially controlled release of bioactive molecules, pharmacophores, or toxins to mitochondria with tissue, cell, or mitochondrion specificity.
  Journal of the American Chemical Society 01/2012; 134(2):758-61. · 9.91 Impact Factor
• Article: Measurement of H2O2 within living Drosophila during aging using a ratiometric mass spectrometry probe targeted to the mitochondrial matrix.

  Helena M Cochemé, Caroline Quin, Stephen J McQuaker, Filipe Cabreiro, Angela Logan, Tracy A Prime, Irina Abakumova, Jigna V Patel, Ian M Fearnley, Andrew M James, Carolyn M Porteous, Robin A J Smith, Saima Saeed, Jane E Carré, Mervyn Singer, David Gems, Richard C Hartley, Linda Partridge, Michael P Murphy
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  ABSTRACT: Hydrogen peroxide (H(2)O(2)) is central to mitochondrial oxidative damage and redox signaling, but its roles are poorly understood due to the difficulty of measuring mitochondrial H(2)O(2) in vivo. Here we report a ratiometric mass spectrometry probe approach to assess mitochondrial matrix H(2)O(2) levels in vivo. The probe, MitoB, comprises a triphenylphosphonium (TPP) cation driving its accumulation within mitochondria, conjugated to an arylboronic acid that reacts with H(2)O(2) to form a phenol, MitoP. Quantifying the MitoP/MitoB ratio by liquid chromatography-tandem mass spectrometry enabled measurement of a weighted average of mitochondrial H(2)O(2) that predominantly reports on thoracic muscle mitochondria within living flies. There was an increase in mitochondrial H(2)O(2) with age in flies, which was not coordinately altered by interventions that modulated life span. Our findings provide approaches to investigate mitochondrial ROS in vivo and suggest that while an increase in overall mitochondrial H(2)O(2) correlates with aging, it may not be causative.
  Cell metabolism 03/2011; 13(3):340-50. · 17.35 Impact Factor
• Source

  Available from: PubMed Central

  Article: Proteomic approaches to the characterization of protein thiol modification.

  Edward T Chouchani, Andrew M James, Ian M Fearnley, Kathryn S Lilley, Michael P Murphy
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  ABSTRACT: Protein cysteine residues are central to redox signaling and to protection against oxidative damage through their interactions with reactive oxygen and nitrogen species, and electrophiles. Although there is considerable evidence for a functional role for cysteine modifications, the identity and physiological significance of most protein thiol alterations are unknown. One way to identify candidate proteins involved in these processes is to utilize the proteomic methodologies that have been developed in recent years for the identification of proteins that undergo cysteine modification in response to redox signals or oxidative damage. These tools have proven effective in uncovering novel protein targets of redox modification and are important first steps that allow for a better understanding of how reactive molecules may contribute to signaling and damage. Here, we discuss a number of these approaches and their application to the identification of a variety of cysteine-centered redox modifications.
  Current opinion in chemical biology 02/2011; 15(1):120-8. · 8.30 Impact Factor
• Article: Quantification and identification of mitochondrial proteins containing vicinal dithiols.

  Raquel Requejo, Edward T Chouchani, Andrew M James, Tracy A Prime, Kathryn S Lilley, Ian M Fearnley, Michael P Murphy
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  ABSTRACT: Vicinal dithiols may play a role in mitochondrial antioxidant defences and in redox signalling. We quantified protein vicinal dithiols within mammalian mitochondria using the vicinal dithiol-specific reagent phenylarsine oxide (PAO). We found 5-15% of thiols exposed on mitochondrial proteins were vicinal dithiols and that these thiols were particularly sensitive to oxidation by hydrogen peroxide. To visualise these proteins we used PAO to block vicinal dithiols, followed by alkylation of other thiols with N-ethylmaleimide (NEM). The PAO was then removed with 2,3-dimercapto-1-propanesulfonic acid (DMPS) and the exposed vicinal dithiols were labelled with iodoacetamide-biotin. To identify these proteins, we developed a selective proteomic methodology, based on Redox difference in gel electrophoresis (Redox-DIGE). Vicinal dithiol proteins were selectively labelled with a red fluorescent thiol-reactive Cy5 maleimide and mixed with Cy3 maleimide labelled protein in which vicinal dithiols remained untagged. Individual proteins were resolved by 2D gel electrophoresis and fluorescent scanning revealed vicinal dithiol proteins by the increase in Cy5 red fluorescence. These proteins were identified by peptide mass fingerprinting and mass spectrometry. These findings are consistent with roles for mitochondrial vicinal dithiol proteins in antioxidant defence and redox signalling and these methodologies will enable these roles to be explored.
  Archives of Biochemistry and Biophysics 12/2010; 504(2):228-35. · 2.93 Impact Factor
• Article: Complementation of coenzyme Q-deficient yeast by coenzyme Q analogues requires the isoprenoid side chain.

  Andrew M James, Helena M Cochemé, Masatoshi Murai, Hideto Miyoshi, Michael P Murphy
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  ABSTRACT: The ubiquinone coenzyme Q (CoQ) is synthesized in mitochondria with a large, hydrophobic isoprenoid side chain. It functions in mitochondrial respiration as well as protecting membranes from oxidative damage. Yeast that cannot synthesize CoQ (DeltaCoQ) are viable, but cannot grow on nonfermentable carbon sources, unless supplied with ubiquinone. Previously we demonstrated that the isoprenoid side chain of the exogenous ubiquinone was important for growth of a DeltaCoQ strain on the nonfermentable substrate glycerol [James AM et al. (2005) J Biol Chem280, 21295-21312]. In the present study we investigated the structural requirements of exogenously supplied CoQ(2) for growth on glycerol and found that the first double bond of the initial isoprenoid unit is essential for utilization of respiratory substrates. As CoQ(2) analogues that did not complement growth on glycerol supported respiration in isolated mitochondria, discrimination does not occur via the respiratory chain complexes. The endogenous form of CoQ in yeast (CoQ(6)) is extremely hydrophobic and transported to mitochondria via the endocytic pathway when supplied exogenously. We found that CoQ(2) does not require this pathway when supplied exogenously and the pathway is unlikely to be responsible for the structural discrimination observed. Interestingly, decylQ, an analogue unable to support growth on glycerol, is not toxic, but antagonizes growth of DeltaCoQ yeast in the presence of exogenous CoQ(2). Using a DeltaCoQ double-knockout library we identified a number of genes that decrease the ability of yeast to grow on exogenous CoQ. Here we suggest that CoQ or its redox state may be a signal for growth during the shift to respiration.
  FEBS Journal 03/2010; 277(9):2067-82. · 3.79 Impact Factor
• Article: Consequences of long-term oral administration of the mitochondria-targeted antioxidant MitoQ to wild-type mice.

  Sergio Rodriguez-Cuenca, Helena M Cochemé, Angela Logan, Irina Abakumova, Tracy A Prime, Claudia Rose, Antonio Vidal-Puig, Anthony C Smith, David C Rubinsztein, Ian M Fearnley, Bruce A Jones, Simon Pope, Simon J R Heales, Brian Y H Lam, Sudeshna Guha Neogi, Ian McFarlane, Andrew M James, Robin A J Smith, Michael P Murphy
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  ABSTRACT: The mitochondria-targeted quinone MitoQ protects mitochondria in animal studies of pathologies in vivo and is being developed as a therapy for humans. However, it is unclear whether the protective action of MitoQ is entirely due to its antioxidant properties, because long-term MitoQ administration may alter whole-body metabolism and gene expression. To address this point, we administered high levels of MitoQ orally to wild-type C57BL/6 mice for up to 28 weeks and investigated the effects on whole-body physiology, metabolism, and gene expression, finding no measurable deleterious effects. In addition, because antioxidants can act as pro-oxidants under certain conditions in vitro, we examined the effects of MitoQ administration on markers of oxidative damage. There were no changes in the expression of mitochondrial or antioxidant genes as assessed by DNA microarray analysis. There were also no increases in oxidative damage to mitochondrial protein, DNA, or cardiolipin, and the activities of mitochondrial enzymes were unchanged. Therefore, MitoQ does not act as a pro-oxidant in vivo. These findings indicate that mitochondria-targeted antioxidants can be safely administered long-term to wild-type mice.
  Free radical biology & medicine 10/2009; 48(1):161-72. · 5.42 Impact Factor
• Source

  Available from: C Robin Hiley

  Article: A mitochondria-targeted S-nitrosothiol modulates respiration, nitrosates thiols, and protects against ischemia-reperfusion injury.

  Tracy A Prime, Frances H Blaikie, Cameron Evans, Sergiy M Nadtochiy, Andrew M James, Christina C Dahm, Dario A Vitturi, Rakesh P Patel, C Robin Hiley, Irina Abakumova, Raquel Requejo, Edward T Chouchani, Thomas R Hurd, John F Garvey, Cormac T Taylor, Paul S Brookes, Robin A J Smith, Michael P Murphy
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  ABSTRACT: Nitric oxide (NO(*)) competitively inhibits oxygen consumption by mitochondria at cytochrome c oxidase and S-nitrosates thiol proteins. We developed mitochondria-targeted S-nitrosothiols (MitoSNOs) that selectively modulate and protect mitochondrial function. The exemplar MitoSNO1, produced by covalently linking an S-nitrosothiol to the lipophilic triphenylphosphonium cation, was rapidly and extensively accumulated within mitochondria, driven by the membrane potential, where it generated NO(*) and S-nitrosated thiol proteins. MitoSNO1-induced NO(*) production reversibly inhibited respiration at cytochrome c oxidase and increased extracellular oxygen concentration under hypoxic conditions. MitoSNO1 also caused vasorelaxation due to its NO(*) generation. Infusion of MitoSNO1 during reperfusion was protective against heart ischemia-reperfusion injury, consistent with a functional modification of mitochondrial proteins, such as complex I, following S-nitrosation. These results support the idea that selectively targeting NO(*) donors to mitochondria is an effective strategy to reversibly modulate respiration and to protect mitochondria against ischemia-reperfusion injury.
  Proceedings of the National Academy of Sciences 07/2009; 106(26):10764-9. · 9.68 Impact Factor
• Article: Chapter 19 Measuring redox changes to mitochondrial protein thiols with redox difference gel electrophoresis (redox-DIGE).

  Thomas R Hurd, Andrew M James, Kathryn S Lilley, Michael P Murphy
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  ABSTRACT: Low levels of reactive oxygen and nitrogen species (ROS and RNS) produced by the mitochondrial respiratory chain and ROS and RNS from other sources act as redox signals by oxidizing thiols on specific proteins. Because these thiol modifications occur on a relatively small number of proteins in the absence of bulk thiol changes, it is necessary to use sensitive methods to discover them. Recently, a number of methods have been developed to help facilitate the identification and characterization of redox-sensitive thiol proteins. In this chapter we describe one such method, redox difference gel electrophoresis (redox-DIGE), in which oxidized thiol proteins in redox-challenged samples are labeled with a thiol-reactive fluorescent tag and compared with those in control samples labeled with a different tag on the same 2-D gel. This enables the sensitive detection of redox-sensitive thiol proteins by measuring changes in the relative fluorescence of the two tags within a single protein spot, followed by protein identification by mass spectrometry. With this method we have been able to identify several mitochondrial proteins whose thiol state and activity are altered by low levels of ROS from the respiratory chain, which may be an important and unexplored mode of mitochondrial redox signaling. Importantly, this method is not only applicable to studies in isolated mitochondria but can also be applied to more complicated systems such as intact cells and perhaps even whole organisms.
  Methods in enzymology 02/2009; 456:343-61. · 1.90 Impact Factor
• Article: Mitochondria-targeted antioxidants in the treatment of disease.

  Robin A J Smith, Victoria J Adlam, Frances H Blaikie, Abdul-Rahman B Manas, Carolyn M Porteous, Andrew M James, Meredith F Ross, Angela Logan, Helena M Cochemé, Jan Trnka, Tracy A Prime, Irina Abakumova, Bruce A Jones, Aleksandra Filipovska, Michael P Murphy
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  ABSTRACT: Mitochondrial oxidative damage is thought to contribute to a wide range of human diseases; therefore, the development of approaches to decrease this damage may have therapeutic potential. Mitochondria-targeted antioxidants that selectively block mitochondrial oxidative damage and prevent some types of cell death have been developed. These compounds contain antioxidant moieties, such as ubiquinone, tocopherol, or nitroxide, that are targeted to mitochondria by covalent attachment to a lipophilic triphenylphosphonium cation. Because of the large mitochondrial membrane potential, the cations are accumulated within the mitochondria inside cells. There, the conjugated antioxidant moiety protects mitochondria from oxidative damage. Here, we outline some of the work done to date on these compounds and how they may be developed as therapies.
  Annals of the New York Academy of Sciences 01/2009; 1147:105-11. · 3.15 Impact Factor
• Article: Rapid and extensive uptake and activation of hydrophobic triphenylphosphonium cations within cells.

  Meredith F Ross, Tracy A Prime, Irina Abakumova, Andrew M James, Carolyn M Porteous, Robin A J Smith, Michael P Murphy
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  ABSTRACT: Mitochondria-targeted molecules comprising the lipophilic TPP (triphenylphosphonium) cation covalently linked to a hydrophobic bioactive moiety are used to modify and probe mitochondria in cells and in vivo. However, it is unclear how hydrophobicity affects the rate and extent of their uptake into mitochondria within cells, making it difficult to interpret experiments because their intracellular concentration in different compartments is uncertain. To address this issue, we compared the uptake into both isolated mitochondria and mitochondria within cells of two hydrophobic TPP derivatives, [3H]MitoQ (mitoquinone) and [3H]DecylTPP, with the more hydrophilic TPP cation [3H]TPMP (methyltriphenylphosphonium). Uptake of MitoQ by mitochondria and cells was described by the Nernst equation and was approximately 5-fold greater than that for TPMP, as a result of its greater binding within the mitochondrial matrix. DecylTPP was also taken up extensively by cells, indicating that increased hydrophobicity enhanced uptake. Both MitoQ and DecylTPP were taken up very rapidly into cells, reaching a steady state within 15 min, compared with approximately 8 h for TPMP. This far faster uptake was the result of the increased rate of passage of hydrophobic TPP molecules through the plasma membrane. Within cells MitoQ was predominantly located within mitochondria, where it was rapidly reduced to the ubiquinol form, consistent with its protective effects in cells and in vivo being due to the ubiquinol antioxidant. The strong influence of hydrophobicity on TPP cation uptake into mitochondria within cells facilitates the rational design of mitochondria-targeted compounds to report on and modify mitochondrial function in vivo.
  Biochemical Journal 06/2008; 411(3):633-45. · 4.90 Impact Factor
• Article: Mitochondrial targeting of quinones: therapeutic implications.

  Helena M Cochemé, Geoffrey F Kelso, Andrew M James, Meredith F Ross, Jan Trnka, Thabo Mahendiran, Jordi Asin-Cayuela, Frances H Blaikie, Abdul-Rahman B Manas, Carolyn M Porteous, Victoria J Adlam, Robin A J Smith, Michael P Murphy
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  ABSTRACT: Mitochondrial oxidative damage contributes to a range of degenerative diseases. Ubiquinones have been shown to protect mitochondria from oxidative damage, but only a small proportion of externally administered ubiquinone is taken up by mitochondria. Conjugation of the lipophilic triphenylphosphonium cation to a ubiquinone moiety has produced a compound, MitoQ, which accumulates selectively into mitochondria. MitoQ passes easily through all biological membranes and, because of its positive charge, is accumulated several hundred-fold within mitochondria driven by the mitochondrial membrane potential. MitoQ protects mitochondria against oxidative damage in vitro and following oral delivery, and may therefore form the basis for mitochondria-protective therapies.
  Mitochondrion 07/2007; 7 Suppl:S94-102. · 3.62 Impact Factor
• Article: Interaction of the mitochondria-targeted antioxidant MitoQ with phospholipid bilayers and ubiquinone oxidoreductases.

  Andrew M James, Mark S Sharpley, Abdul-Rahman B Manas, Frank E Frerman, Judy Hirst, Robin A J Smith, Michael P Murphy
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  ABSTRACT: MitoQ(10) is a ubiquinone that accumulates within mitochondria driven by a conjugated lipophilic triphenylphosphonium cation (TPP(+)). Once there, MitoQ(10) is reduced to its active ubiquinol form, which has been used to prevent mitochondrial oxidative damage and to infer the involvement of reactive oxygen species in signaling pathways. Here we show MitoQ(10) is effectively reduced by complex II, but is a poor substrate for complex I, complex III, and electron-transferring flavoprotein (ETF):quinone oxidoreductase (ETF-QOR). This differential reactivity could be explained if the bulky TPP(+) moiety sterically hindered access of the ubiquinone group to enzyme active sites with a long, narrow access channel. Using a combination of molecular modeling and an uncharged analog of MitoQ(10) with similar sterics (tritylQ(10)), we infer that the interaction of MitoQ(10) with complex I and ETF-QOR, but not complex III, is inhibited by its bulky TPP(+) moiety. To explain its lack of reactivity with complex III we show that the TPP(+) moiety of MitoQ(10) is ineffective at quenching pyrene fluorophors deeply buried within phospholipid bilayers and thus is positioned near the membrane surface. This superficial position of the TPP(+) moiety, as well as the low solubility of MitoQ(10) in non-polar organic solvents, suggests that the concentration of the entire MitoQ(10) molecule in the membrane core is very limited. As overlaying MitoQ(10) onto the structure of complex III indicates that MitoQ(10) cannot react with complex III without its TPP(+) moiety entering the low dielectric of the membrane core, we conclude that the TPP(+) moiety does anchor the tethered ubiquinol group out of reach of the active site(s) of complex III, thus explaining its slow oxidation. In contrast the ubiquinone moiety of MitoQ(10) is able to quench fluorophors deep within the membrane core, indicating a high concentration of the ubiquinone moiety within the membrane and explaining its good anti-oxidant efficacy. These findings will facilitate the rational design of future mitochondria-targeted molecules.
  Journal of Biological Chemistry 06/2007; 282(20):14708-18. · 4.77 Impact Factor