Control of mitochondrial superoxide production by reverse electron transport at complex I

Control of mitochondrial superoxide production by reverse electron transport at complex I Received for publication, April 23, 2018, and in revised form, May 8, 2018 Published, Papers in Press, May 9, 2018, DOI 10.1074/jbc.RA118.003647 Ellen L. Robb ‡ , Andrew R. Hall ‡ , Tracy A. Prime ‡ , Simon Eaton §1 , Marten Szibor ¶储 , Carlo Viscomi ‡ , Andrew M. James ‡ , and XMichael P. Murphy ‡2 From the ‡ Medical Research Council Mitochondrial Biology Unit, Hills Road, University of Cambridge, Cambridge CB2 0XY, United Kingdom, the § UCL Great Ormond Street Institute of Child Health, London WC1N 1EH, United Kingdom, the ¶ Faculty of Medicine and Life Sciences, University of Tampere, Tampere FI-33014, Finland, and the 储 Max-Planck-Institute for Heart and Lung Research, Ludwigstrasse 43, 61231 Bad Nauheim, Germany Edited by Ruma Banerjee The generation of mitochondrial superoxide (O 2 .) by reverse electron transport (RET) at complex I causes oxidative damage in pathologies such as ischemia reperfusion injury, but also provides the precursor to H 2 O 2 production in physiological mitochondrial redox signaling. Here, we quantified the factors that determine mitochondrial O 2 .production by RET in isolated heart mitochondria. Measuring mitochondrial H 2 O 2 production at a range of proton-motive force (⌬p) values and for several coenzyme Q (CoQ) and NADH pool redox states obtained with the uncoupler p-trifluoromethoxyphenylhydrazone, we show that O 2 .production by RET responds to changes in O 2 concentration, the magnitude of ⌬p, and the redox states of the CoQ and NADH pools. Moreover, we determined how expressing the alternative oxidase from the tunicate Ciona intestinalis to oxidize the CoQ pool affected RET-mediated O 2 .production at complex I, underscoring the importance of the CoQ pool for mitochondrial O 2 .production by RET. An analysis of O 2 .production at complex I as a function of the thermodynamic forces driving RET at complex I revealed that many molecules that affect mitochondrial reactive oxygen species production do so by altering the overall thermodynamic driving forces of RET, rather than by directly acting on complex I. These findings clarify the factors controlling RET-mediated mitochondrial O 2 .production in both pathological and physiological conditions. We conclude that O 2 .production by RET is highly responsive to small changes in ⌬p and the CoQ redox state, indicating that complex I RET represents a major mode of mitochondrial redox signaling. Superoxide (O 2 .) 3 is the proximal reactive oxygen species (ROS) formed within mitochondria, with most O 2 .being very rapidly converted to H 2 O 2 by manganese superoxide dismutase (MnSOD) within the matrix (1, 2). As well as contributing to oxidative damage, H 2 O 2 acts as a redox signal, both within the mitochondria and in the cytosol (3–6). This mode of signal transduction arises via the reversible oxidation of protein thiols that pass on the modification to effector proteins as a redox relay (3–6). Whereas there are a number of potential mitochondrial sources of O 2 .(2, 7, 8), respiratory chain complex I is considered to be a major contributor (1). The production of O 2 .at complex I can be driven by reverse electron transport (RET) by a highly reduced coenzyme Q (CoQ) pool and a large protonmotive force (⌬p), which together drive electrons backward through complex I and lead to a dramatic increase in O 2 . production (Fig. 1)(9). This process has been known since the 1960s (10) but was tacitly assumed to be an in vitro curiosity of no physiological relevance (1, 11, 12). However, there is now considerable evidence that RET at complex I is a physiological process that underlies mitochondrial redox signaling in a range of situations (11, 13) while also leading to pathological oxidative damage during ischemia–reperfusion injury (1, 14, 15). A particularly intriguing aspect of RET is that it does not require damage to, or inhibition of, the respiratory chain (1, 9). As RET responds sensitively to physiological variables, O 2 .production by complex I can be modulated under physiological conditions (1, 9). Hence, there is considerable interest in understanding the mechanisms by which mitochondria regulate O 2 . production at complex I by RET as a physiological signaling process. Here, we quantified the factors that determine RET in isolated heart mitochondria. To do this, we measured H 2 O 2 generation as a function of the membrane potential (⌬ ␺ ) and of the reduction state of the CoQ and NADH pools. This was done by altering ⌬ ␺ with the uncoupler carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) and also oxidizing the CoQ pool by ectopic expression within heart mitochondria of the alternative oxidase (AOX) from Ciona intestinalis (16). Next, we assessed the dependence of RET O 2 .production on O 2 concentration, [O 2 ], and last, we deterThis work was supported by the Medical Research Council UK (Grants MC_U105663142 (to M. P. M.) and MC_UU_00015/5 (to C. V.)) and by a Wellcome Trust Investigator award (110159/Z/15/Z) (to M. P. M). The authors declare that they have no conflicts of interest with the contents of this article. Author’s Choice—Final version open access under the terms of the Creative Commons CC-BY license. 1 Supported in part by the Great Ormond Street Hospital Children’s Charity andtheNIHRBiomedical ResearchCentre atGreat OrmondStreet Hospital. 2 To whom correspondence should be addressed: MRC Mitochondrial Biology Unit, Hills Road, Cambridge CB2 0XY, United Kingdom. Tel.: 44-1223252-900; E-mail: [email protected]. 3 The abbreviations used are: O 2 ., superoxide; ROS, reactive oxygen species; SOD, superoxide dismutase; RET, reverse electron transport; CoQ, coenzyme Q; ⌬p, proton-motive force; ⌬ ␺ , membrane potential; FCCP, carbonyl cyanide p-trifluoromethoxyphenylhydrazone; AOX, alternative oxidase; TPMP, methyltriphenylphosphonium; HRP, horseradish peroxidase; TPP, triphenylphosphonium; ANOVA, analysis of variance; CN-POBS, N-cyclohexyl-4-(4-nitrophenoxy)benzenesulfonamide. cro ARTICLE Author’s Choice J. Biol. Chem. (2018) 293(25) 9869–9879 9869 © 2018 Robb et al. Published by The American Society for Biochemistry and Molecular Biology, Inc. at University of Tampere on July 5, 2018http://www.jbc.org/Downloaded from mined how compounds known to alter mitochondrial ROS production affect RET. Together, these data provide a complete description of mitochondrial O 2 .production by RET and indicate how this process contributes to oxidative damage and redox signaling. Results Dependence of complex I O 2 .production by RET on ⌬p and CoQ redox state To assess the factors that determine O 2 .production by RET at complex I, we used isolated heart mitochondria. H 2 O 2 efflux from mitochondria is proportional to O 2 .production, assuming that O 2 .is converted to H 2 O 2 by matrix MnSOD and that H 2 O 2 degradation by peroxidases is similar across all conditions assessed (1). Whereas H 2 O 2 movement across the plasma membrane is facilitated by aquaporins (17–19), these carriers are not present in mitochondria (20); hence, H 2 O 2 efflux is probably unmediated, albeit facilitated by the large surface area of the inner membrane. Thus, measuring H 2 O 2 efflux enables us to infer how O 2 .production by RET at complex I responds to mitochondrial state. Three factors are critical in determining O 2 .production by RET: the ⌬p and the redox states of the mitochondrial CoQ and NADH pools (Fig. 1)(1). The O 2 concentration is also likely to affect O 2 .production (1); however, these experiments were in air-saturated incubation medium ([O 2 ]⬃200 ␮ M); the dependenceofO 2 .productionon[O 2 ]wasdeterminedlater.Thedependence of mitochondrial H 2 O 2 production on these three variables was measured in parallel (Fig. 2). ⌬pwas measured as ⌬ ␺ from the distribution of the lipophilic methyltriphenylphosphonium (TPMP) cation (21)(Fig. 2B). Whereas this does not measure the pH gradient (⌬pH) across the inner membrane, ⌬pH is a smaller (⬃10–20%) component of ⌬p than ⌬ ␺ and here is assumed to be invariant. The redox state of the CoQ pool was assessed by measuring its percentage reduction by reverse-phase HPLC (Fig. 2C). The redox state Figure 1. Mitochondrial O 2 .production from complex I by RET. The top panel shows conventional forward electron transport by mitochondria. The bottom panel shows mitochondrial O 2 .production by RET. This occurs when the ⌬p (a combination of the ⌬ ␺ and the ⌬pH) is high and the CoQ pool is reduced. Q, ubiquinone; QH 2 , ubiquinol. Complex I reverse electron transport 9870 J. Biol. Chem. (2018) 293(25) 9869–9879 at University of Tampere on July 5, 2018http://www.jbc.org/Downloaded from of the NADH pool was inferred from NAD(P)H fluorescence. Although this cannot distinguish between NADH and NADPH, the mitochondrial NADPH pool is thought to contribute less to changes in this variable (22); hence, this method gives a reasonable assessment of the NADH pool redox state (Fig. 2D). The addition of succinate to heart mitochondria led to extensive H 2 O 2 production (Fig. 2A), a large ⌬ ␺ (Fig. 2B), and highly reduced CoQ (Fig. 2C) and NAD(P)H (Fig. 2D) pools. The addition of the complex I inhibitor rotenone decreased H 2 O 2 production without affecting ⌬ ␺ (Fig. 2B) or the CoQ pool (Fig. 2C), but there was oxidation of the NAD(P)H pool (Fig. 2D). These findings are consistent with H 2 O 2 production originating from complex I by RET (1). To explore the dependence of RET on ⌬ ␺ and the redox state of the CoQ and NAD(P)H pools, we measured these variables with increasing amounts of the uncoupler FCCP. The gradual decrease in H 2 O 2 production that resulted (Fig. 2A) was associated with a decrease in ⌬ ␺ (Fig. 2B) and oxidation of the CoQ (Fig. 2C) and NAD(P)H (Fig. 2D) pools. The matrix pH has been suggested to alter RET directly at complex I, independently of its role as the ⌬pH component of ⌬p( 23, 24). Hence, we next used the K ⫹ /H ⫹ exchanger nigericin to decrease the matrix pH from ⬃7.7 to that of the incubation medium (pH 7.4), thereby abolishing the ⌬pH component of ⌬p. Importantly, the magnitude of ⌬p will not change, due to a compensatory increase of ⬃20 mV in ⌬ ␺ (Fig. 2B). Nigericin resulted in an oxidation of the CoQ (Fig. 2C) and NAD(P)H pools (Fig. 2D) and a decrease in H 2 O 2 efflux (Fig. 2A). Together, these data show a strong dependence of RET at complex I on the magnitude of ⌬p and on the CoQ and NAD(P)H redox states. Effect of the AOX on O 2 .production by RET To analyze the effects of CoQ pool redox state on O 2 .production by RET, independently of effects on ⌬p, we utilized mice expressing the AOX from C. intestinalis (16). AOX transfers electrons from CoQH 2 directly to O 2 , bypassing complex IV, and thus acts as a safety valve to prevent the excessive reduction of the CoQ pool (25, 26). The AOX protein was present in heart mitochondria from AOX ⫹/⫺ knock-in mice (Fig. 3A) and was catalytically active, as respiration in mitochondria from WT mice was inhibited by cyanide, whereas mitochondria from AOX mice continued to respire, but this residual respiration was sensitive to the AOX inhibitor N-propyl gallate (Fig. 3B). Mitochondrial H 2 O 2 efflux during succinate oxidation was decreased by AOX expression as shown previously (16)( Fig. 3C). This was not due to a decrease in ⌬ ␺ (Fig. 3D); however, the CoQ pool was more oxidized in mitochondria containing AOX (27)(Fig. 3E). These findings show that the expression of AOX affects O 2 .production by RET at complex I by oxidizing the CoQ pool and further support the importance of CoQ pool redox state in determining mitochondrial O 2 .production by RET. Effect of [O 2 ]onO 2 .production by complex I RET As the generation of O 2 .by mitochondria requires that O 2 react with a protein-bound electron carrier, the rate of O 2 .production will probably depend on the [O 2 ](1). To assess this dependence, we measured mitochondrial H 2 O 2 efflux at different [O 2 ](Fig. 4). As the apparent K m of cytochrome oxidase for O 2 is very low (⬍1 ␮ M)(28), ⌬p and the redox state of the CoQ and NADH pools will not vary with [O 2 ]. The assessment of H 2 O 2 efflux at different [O 2 ] relies on the conversion of Amplex Red to its fluorescent product resorufin catalyzed by horseradish peroxidase (HRP); hence, we first demonstrated that the Nig Rot A B C D H2O2 (pmol/min/mg protein) CoQ (% reduced)NAD(P)H (% reduced) Δψ (mV) 160 120 80 40 0 160 140 120 100 80 180 60 80 40 0 20 60 80 40 0 20 [FCCP] (nM) 0.5 5 25 50 1000 + Nigericin Figure 2. Parallel measurement of mitochondrial H 2 O 2 efflux, ⌬ ␺ , and the redox status of the NAD(P)H and CoQ pools. Rat heart mitochondria were incubated in the presence of potassium succinate. Where indicated, FCCP, nigericin (1 ␮ M), or rotenone (5 ␮ M) was added, and the indicated variables were measured. A,H 2 O 2 efflux. B,⌬ ␺ .C, CoQ redox status. D, NAD(P)H redox status. Data are the mean ⫾S.E. (error bars) from 3–4 independent mitochondrial preparations. *, p⬍0.05 compared with succinate-only conditions by ANOVA. Complex I reverse electron transport J. Biol. Chem. (2018) 293(25) 9869–9879 9871 at University of Tampere on July 5, 2018http://www.jbc.org/Downloaded from fluorescence of resorufin (Fig. 4A,inset) and the conversion of Amplex Red to resorufin (Fig. 4A) were both independent of [O 2 ]. We then measured mitochondrial H 2 O 2 generation by RET and showed that it was proportional to [O 2 ] and was abolished by excess FCCP (Fig. 4B). In contrast, production of H 2 O 2 by NADH-linked substrates in the presence of rotenone (Fig. 4C) or from complex III upon inhibition of succinate respiration by antimycin (Fig. 4C) also showed increased H 2 O 2 production with [O 2 ], but in this case ROS production reached a plateau as [O 2 ] increased. Thus, production of O 2 .by RET at complex I is proportional to [O 2 ]. Effects of therapeutic compounds on complex I RET A number of potentially therapeutic compounds are thought to act, at least in part, by decreasing mitochondrial ROS production. Therefore, we set out to assess some of these compounds to determine whether they altered RET at complex I. The compounds tested were as follows: MitoQ, a mitochondria-targeted antioxidant based on ubiquinone (29); decylTPP, which contains the mitochondria-targeting triphenylphosphonium (TPP) cation and which is frequently used as a control compound to correct for nonspecific effects of MitoQ (21); SS31, a peptide composed of D-Arg-Dmt-Lys-Phe-NH 2 (30) (where Dmt represents 2,6-dimethyltyrosine), whose therapeutic effects are thought to be due to its interactions with mitochondria; CN-POBS, an inhibitor of mitochondrial O 2 .production by RET at complex I (12); and the antidiabetic biguanides AB αAOX αVDAC WT AOX 37 25 37 25 Mw (kDa) CD E 80 60 40 20 0 CoQ (% reduced) WT AOXWT AOX Δψ (mV) 200 150 100 50 0 200 100 0 300 WT AOX H2O2 (pmol/min/mg protein) Succinate KCN Propyl gallate 200 150 100 [O2] (µM) 0 5 10 15 Time (min) WT AOX Propyl gallate Figure 3. Effect of AOX expression on mitochondrial H 2 O 2 production. A, AOX expression in mouse heart mitochondria. Heart mitochondria from AOX ⫹/⫺ mice or WT littermate controls were analyzed by Western blotting for AOX using the mitochondrial outer membrane protein voltage-dependent anion channel as a loading control. Mitochondria from three separate AOX ⫹/⫺ and WT mice were assessed. B,O 2 consumption by mitochondria from AOX ⫹/⫺ and WT mice. Mitochondria (200 ␮ g of protein/ml) were incubated at 37 °C in an oxygen electrode and respiration was initiated by the addition of succinate (10 mM) followed by KCN (1 mM) and propyl gallate (50 ␮ M). Traces are typical of experiments repeated with at least three independent mitochondrial preparations for each condition. C,H 2 O 2 production by heart mitochondria from AOX ⫹/⫺ and WT mice. Mitochondria (200 ␮ g of protein/ml) were incubated with succinate (10 mM), and H 2 O 2 production was assessed. n⫽3 (WT) or 6 (AOX). D,⌬ ␺ of heart mitochondria from AOX and WT mice. Mitochondria (500 ␮ g of protein/ml) were incubated at 37 °C for 5 min with succinate (10 mM), and ⌬ ␺ was assessed. n⫽4. E, CoQ redox state of heart mitochondria from AOX and WT mice. Mitochondria (1 mg of protein/ml) were incubated at 37 °C for 2 min with succinate (10 mM), and the CoQ redox state was assessed. n⫽4. *, p⬍0.05; Error bars, S.E. [O2] (µM) Fluorescence (AU) [O2] (µM) 400 300 200 100 0 H2O2 (nmol/min/mg protein) FCCP [O2] (µM) 400 300 200 100 0 H2O2 (nmol/min/mg protein) 050 100 150 200 [O2] (µM) 0 50 100 150 200 C Succinate Succinate Antimycin Rotenone/glu/mal Fluorescence (AU) Figure 4. Dependence on mitochondrial H 2 O 2 generation on [O 2 ]. A, conversion of Amplex Red to resorufin by HRP at different [O 2 ]. The [O 2 ]ofKCl buffer containing Amplex Red, HRP, and SOD was decreased by bubbling with N 2 . Then known amounts H 2 O 2 were injected, and fluorescence was measured. The inset shows the fluorescence of resorufin (25 ␮ M), which was added to an incubation of mitochondria respiring on succinate in the presence of rotenone (5 ␮ M), and resorufin fluorescence was measured as the [O 2 ] decreased due to mitochondrial respiration. B, dependence on [O 2 ] of mitochondrial H 2 O 2 generation by RET. Rat heart mitochondria were incubated in an oxygen electrode, and H 2 O 2 generation was measured at various [O 2 ] set by bubbling with N 2 , and ROS production was measured over a dynamic range of O 2 tensions. Where indicated, 500 nMFCCP was present. Data are the mean ⫾S.E. (error bars). n⫽11. C, comparison of [O 2 ] dependence of different modes of mitochondrial H 2 O 2 generation. Mitochondria were assessed as in B, except that the respiratory substrate was glutamate/malate (5 mMeach) in the presence of rotenone (4 ␮ g/ml). n⫽6, or mitochondria were respiring on succinate with antimycin (1 ␮ M) present. Data are the mean ⫾S.E. (n⫽5) and compared with the trace from C. Complex I reverse electron transport 9872 J. Biol. Chem. (2018) 293(25) 9869–9879 at University of Tampere on July 5, 2018http://www.jbc.org/Downloaded from metformin and phenformin, which are known to interact with complex I (31, 32). We first assessed whether these compounds affected H 2 O 2 efflux by RET at complex I. All of the compounds, except SS31, decreased mitochondrial O 2 .production by RET to some extent (Fig. 5A). As RET at complex I is very sensitive to the magnitude of ⌬ ␺ and redox state of the CoQ pool, these compounds may affect RET indirectly by altering these bioenergetic variables rather than by directly interacting with complex I to block RETdependent O 2 .production. Consistent with this interpretation, all of the compounds decreased ⌬ ␺ (Fig. 5B), and some affected the redox states of the CoQ and NAD(P)H pools (Fig. 5, Cand D). Together, these findings suggest that the effects of these compounds on RET at complex I may be indirect due to effects on ⌬ ␺ and perhaps on the redox states of the NAD(P)H and CoQ pools. Dependence of RET on ⌬ ␺ and the redox state of the CoQ pool Measuring H 2 O 2 production by mitochondria in parallel with ⌬ ␺ and the redox state of the CoQ pool indicated that O 2 .production by RET at complex I was very sensitive to both variables (Figs. 2 and 3). To illustrate the dependence of RET on ⌬ ␺ and redox state of the CoQ pool, we replotted the data from Fig. 2 to show H 2 O 2 production as a function of both ⌬ ␺ and CoQ redox state (Fig. 6). This 3D plot makes evident the very steep dependence of H 2 O 2 production on ⌬ ␺ and on CoQ redox state, confirming the exquisite sensitivity of O 2 . production by RET at complex I to these two physiological variables. The thermodynamic driving force for RET at complex I Whereas Fig. 6 shows clearly that decreasing ⌬ ␺ and oxidizing the CoQ pool lowers RET, it does not provide a quantitative basis to allow us to infer whether the effects of nigericin shown in Fig. 2Aand those of the various compounds shown in Fig. 5A are due to direct interactions with complex I itself or are indirect effects due to altering the driving forces of RET. Therefore, we next determined how O 2 .production during RET depends on the overall thermodynamic driving force across complex I. The direction of electron flow at complex I is determined by the balance of thermodynamic driving forces across the complex (Fig. 1). During forward electron transfer at complex I, two electrons are passed from NADH to CoQ. NADH ⫹CoQ 7NAD⫹⫹CoQH2 Reaction 1 The driving force for the transfer of two electrons from NADH to CoQ is ⌬E h . H2O2 (pmol/min/mg protein) Δψ (mV) CoQ (% reduced)NAD(P)H (% reduced) Control MitoQ DecylTPP Phenformin Metformin CNPOBS SS31 200 150 100 50 0 150 100 50 0 100 80 60 40 20 0 80 60 40 20 0 A B C D Figure 5. Effects of compounds that interact with mitochondria on H 2 O 2 generation by RET. Rat heart mitochondria were incubated for 10 min in the presence of 10 mMsuccinate and 1 ␮ MMitoQ, decylTPP, CN-POBS, SS31, phenformin, and metformin and assessed as in Fig. 2. Data are the mean ⫾ S.E. (error bars), n⫽4. A,H 2 O 2 efflux. B,⌬ ␺ .C, CoQ redox status. D, NAD(P)H redox status. *, p⬍0.05. 20 140 60 100 120 160140100 Membrane potential (mV) 20 40 60 CoQ (% reduced) H2O2 (pmol.min-1.mg protein-1) Figure 6. Dependence of mitochondrial H 2 O 2 production by RET upon ⌬ ␺ and the redox state of the CoQ pool. The data from Fig. 2 (A–C) are plotted together to show the relationship between mitochondrial H 2 O 2 production, ⌬ ␺ , and CoQ redox state. Complex I reverse electron transport J. Biol. Chem. (2018) 293(25) 9869–9879 9873 at University of Tampere on July 5, 2018http://www.jbc.org/Downloaded from ⌬Eh⫽Eh 冉 NAD⫹ NADH 冊 ⫺Eh 冉 CoQ CoQH2 冊 (Eq. 1) This driving force, 2⌬E h , is used to pump four protons across the mitochondrial inner membrane against the ⌬p. Hence, for forward electron movement to occur, the thermodynamic requirement is as follows. 2⌬Eh⬎4⌬p(Eq. 2) However, when the ⌬p is high and/or the ⌬E h is decreased, RET can occur provided the following is true. 2⌬Eh⬍4⌬p(Eq. 3) We can thus calculate the thermodynamic driving force (⌬G) for RET, where Fis the Faraday constant (33). ⌬G⫽2F⌬Eh⫺4F⌬p(Eq. 4) Rearranging to express the thermodynamic driving force for RET as a positive number in V gives the following. ⫺⌬G/F⫽4⌬p⫺2⌬Eh(Eq. 5) From this, we can use the data from the FCCP titration in Fig. 2to calculate ⫺⌬G/F,the driving force for RET (see “Experimental procedures”). This analysis yields a plot of H 2 O 2 production by RET as a function of the thermodynamic driving force across complex I (Fig. 7A). This shows that when ⫺⌬G/F⬍0, there is a residual background level of ROS production, but as soon as the driving force for RET passes a threshold and ⫺⌬G/F⬎0, there is a dramatic and steep increase in O 2 .production by RET. This analysis confirms that the O 2 .production by RET requires a sufficient thermodynamic force to reverse electron transport at complex I and further shows the steep dependence of O 2 .production on this driving force. A benefit of describing H 2 O 2 production by RET at complex I as a function of its overall thermodynamic driving force is that it enables us to quantify whether compounds that affect O 2 . production by RET do so by altering the drivers of this process or by acting directly on complex I. A compound that only affects O 2 .production by RET indirectly through altering ⌬p and ⌬E h would lie on the curve shown in Fig. 7A. In contrast, a compound that directly affected complex I independently of the thermodynamic drivers of RET, would lie below this curve. We first applied this analysis to nigericin, which decreases H 2 O 2 production by RET (Fig. 2A) but which also led to a more oxidized CoQ pool (Fig. 2C) without affecting ⌬p. Carrying out this analysis, including accounting for changes in matrix pH on E h of the NADH and CoQ pools (see “Calculations”) indicated that the decrease in H 2 O 2 production by nigericin was due to its effects on the CoQ pool redox state (Fig. 7A). In contrast, the decrease of H 2 O 2 production by the complex I inhibitor rotenone (Fig. 2A) was not due to changes in the thermodynamic driving forces for RET, as these data lay below the trend line in Fig. 7A. If the compounds that affect mitochondrial ROS production assessed in Fig. 5 decrease mitochondrial ROS production independently of the drivers of RET, then they should lie below the trend line shown in Fig. 7A, as was the case for rotenone. When the data from Fig. 5 were analyzed to show the effect of these compounds on the thermodynamic driving forces for RET, the results lay above the trend line, showing the dependence of H 2 O 2 production on the thermodynamic driving forces across complex I (Fig. 7B). Hence, these data suggest that the effects of the compounds analyzed in Fig. 5 on ROS production by RET are more likely to be accounted for by their effects on ⌬p and/or ⌬E h rather than due to specific inhibitory effects on complex I. Β H2O2 (nmol/min/mg protein) +CNPOBS +SS31 +Metformin +Phenformin +DecylTPP + MitoQ +Nigericin -100 +Nigericin Α H2O2 (nmol/min/mg protein) -ΔG/F (mV) 150 100 50 0 -250 -200 -50 0-150 50 150 100 50 0 -100 -ΔG/F (mV) -250 -200 -50 0-150 50 2ΔEh > 4Δp2ΔEh < 4Δp Rotenone Rotenone 100 150 100 150 Figure 7. Thermodynamic driving forces across complex I during RET. A, dependence of mitochondrial H 2 O 2 efflux on the thermodynamic driving force for RET across complex I. Data from Fig. 2 were used to calculate ⫺⌬G/F, as this was varied by titration of the uncoupler FCCP (black circles)orinthe presence of FCCP (red circle) or rotenone (green circle). Data are the mean ⫾ S.E. (error bars)(n⫽3–4). B, the effects of compounds that interact with mitochondria on H 2 O 2 efflux and the thermodynamic driving force for RET. Data from Fig. 5 were used to calculate ⫺⌬G/F, and this was plotted against mitochondrial H 2 O 2 efflux (blue circles), along with the data from A. Complex I reverse electron transport 9874 J. Biol. Chem. (2018) 293(25) 9869–9879 at University of Tampere on July 5, 2018http://www.jbc.org/Downloaded from Discussion We investigated the dependence of O 2 .production by RET at complex I within isolated mitochondria on E h of the NAD(P)H and CoQ pools, ⌬p, matrix pH, and [O 2 ]. This approach confirmed that O 2 .production by RET at complex I is favored by a high ⌬p and a reduced CoQ pool, with exquisite sensitivity to small changes in these two drivers (Fig. 6). This analysis was extended to show that O 2 .production by RET at complex I was also highly responsive to small changes in the overall thermodynamic driving force for RET across the complex (Fig. 7A). Two sites have been proposed for O 2 .production by complex I during RET: the FMN of the complex I NADH-binding site (9, 34, 35) or the CoQ-binding site (36, 37). We favor the FMN site as the source of O 2 .production by complex I during RET. This is because the penetration of O 2 to the CoQ site is difficult to envisage from the structure of complex I (38, 39). In addition, the generation of the negatively charged O 2 .from the CoQ site would require its thermodynamically unfavorable formation within the hydrophobic core of the membrane bilayer (38, 39). Furthermore, the FMN site is well established as a source of O 2 .production by rotenone-inhibited complex I (38, 39). During this process, NADH reduces the FMN to FMNH ⫺ , which is then readily accessed by O 2 to form O 2 .(38, 39). In addition, any O 2 .formed at this site is released directly into the aqueous phase (9, 35, 40). The relationship between [O 2 ] and RET was linear over the physiological [O 2 ] range, consistent with O 2 .production being driven by the second-order reaction between O 2 and FMNH ⫺ on complex I when it is free from bound NAD ⫹ or NADH (9, 35, 40). During rotenone inhibition, the FMN/FMNH ⫺ ratio is set by a rapid pre-equilibration with the matrix NAD ⫹ /NADH pool and thereby determines the rate of O 2 .production (9, 35). We favor FMNH ⫺ as the donor of an electron to O 2 for O 2 .production by complex I during RET. However, if this is the case then the greater O 2 .production during RET compared with rotenone-inhibited complex I has to be explained, because under both conditions, O 2 .production is determined by the FMN/ FMNH ⫺ ratio (9, 23, 24, 37). The most likely reason why RETdriven O 2 .production is greater than that upon rotenone inhibition is because the large thermodynamic driving force for electron movement backward through complex I during RET holds the FMN/FMNH ⫺ ratio at a more negative E h than is possible by equilibrium with the NAD ⫹ /NADH pool (9). The more negative midpoint potential of the FMN/FMNH ⫺ couple (E m,7.5 ⫽⫺380 mV) (41) compared with the NAD ⫹ /NADH couple (E m,7.5 ⫽⫺335 mV) is consistent with this hypothesis. Other factors that could contribute to the elevated O 2 .production during RET compared with rotenone inhibition include differential access of O 2 to the FMNH ⫺ due to alterations in the NADH and NAD ⫹ binding or differences in the activity of the peroxidases that degrade H 2 O 2 within the mitochondrial matrix. One further consideration is that during O 2 .production, FMNH ⫺ donates one electron to O 2 to form a semiquinone radical, FMN䡠, which is then thought to rapidly redistribute its unpaired electron throughout the iron–sulfur centers on complex I (40). However, the FMN䡠radical also reacts very rapidly with O 2 to form O 2 ., so if electron redistribution were slowed during RET, the enhanced lifetime of FMN䡠would also enhance O 2 .production (40). A further point to note is that the term RET is often interpreted as requiring NAD ⫹ reduction at the FMN site of complex I. This is not the case, as O 2 .production by RET at complex I occurs when the NAD ⫹ /NADH pool is highly reduced and there is no net electron flow from complex I into this pool (e.g. Fig. 2D). Thus, we favor a model in which O 2 reacts with a FMNH ⫺ to generate O 2 ., whereas the FMN still exchanges electrons with the matrix NAD ⫹ /NADH pool, although there is no net electron transfer (Fig. 8). The generation of O 2 .by RET at complex I could be described as a function of the thermodynamic driving forces, ⌬p and ⌬E h , across the complex. As well as illustrating the factors that drive RET, this analysis allowed us to integrate the effects of the forces driving RET. Applying this to the decrease in RET when the matrix pH decreased suggested that the lower rate of RET could be accounted for by the change in the ⌬E h between the CoQ and NADH pools and may not be due to a direct effect of pH on complex I itself (24). Furthermore, this approach suggests that compounds such as MitoQ and metformin that affect mitochondrial ROS metabolism may do so indirectly, rather than by specific interactions with complex I. However, it is important to note that the calculation of ⫺⌬G/Frequires a number of assumptions and combines several technically challenging experimental measurements; hence, systematic errors will affect accuracy and precision. In addition, we have assumed that the CoQ pool interacts to the same extent with all complexes, and any effects of supercomplex formation on this were not considered (42). Nevertheless, our work does indicate novel approaches for determining how compounds impact mitochondrial ROS production and has implications for the interpretation of experiments using these compounds. For example, MitoQ is widely used in vitro and in vivo, where it acts as a chain-breaking antioxidant decreasing oxidative damage (29, 43). As the excessive accumulation of hydrophobic TPP compounds disrupts mitochondrial function (44, 45), controls with compounds with matched physicochemical properties are essential to correct for nonspecific effects. Thus, some “antioxQ QH O2 2 ATP demand FMN/FMNH4H+ 4H+ CoQ CoQH2 Electron supply O2 Oxygen concentration NADH/NAD Δp 2 eComplex I Redox signal /oxidative damage Metabolism Work Figure8. Model ofthefactors that determinethe production ofO 2 .byRET at complex I. Complex I reverse electron transport J. Biol. Chem. (2018) 293(25) 9869–9879 9875 at University of Tampere on July 5, 2018http://www.jbc.org/Downloaded from idant” effects of lipophilic cations may be due to mild disruption of ⌬p that lowers mitochondrial O 2 .production by RET. Similarly, some of the beneficial effects of less hydrophobic cations, such as metformin in vivo, may be associated with limiting O 2 .production by RET, in addition to stimulating AMP-activated protein kinase by inhibiting complex I (46, 47). Mitochondrial O 2 .production by RET was initially investigated in vitro, and at that time its relevance to in vivo physiology was not considered (10). Recently, mitochondrial ROS production by RET has been demonstrated in multiple situations in vivo. For example, during ischemia, high levels of succinate accumulate, and it is oxidized upon reperfusion to drive a burst of ROS via RET (14, 48). More generally, RET at complex I occurs as a redox-signaling pathway in inflammation (13), contributes to lifespan in flies (49), and is part of the oxygen-sensing mechanism of the carotid body (49). The linear dependence of ROS production by RET on O 2 (Fig. 4), which has been shown previously by others (50–53), further adds to its appeal as a potential component of the carotid body O 2 sensor (49). One possibility is that O 2 .production by RET at complex I accounts for most of the redox signaling from the mitochondrion to the rest of the cell (3–6). The appeal of RET as a mitochondrial redox signal is illustrated in Figs. 6 and 7, which show the tremendous sensitivity of RET to ⌬p and the redox status of the CoQ pool. The magnitude of ⌬p is directly linked to ATP demand, whereas the redox state of the CoQ pool reflects electron supply to the respiratory chain. Thus, RET provides a sensitive mechanism for the real-time feedback of the most critical aspects of mitochondrial status to the rest of the organelle and to the cell (Fig. 8). In summary, we have provided a thermodynamic underpinning to O 2 .production by RET at complex I. This analysis highlights the potential for this mechanism to play a key role in mitochondrial redox signaling and demonstrates how to investigate the effects of RET on various physiological and pathological processes. Experimental procedures Mitochondria isolation All procedures were performed in accordance with the UK Guide for the Care and Use of Laboratory Animals (PPL: 70/7538). Rat hearts were collected from 10–12-week-old female Wistar rats (Charles River). C57Bl/6 mice carrying a single copy of the C. intestinalis AOX gene in the Rosa26 locus were generated as described (16). AOX mice and their WT littermate controls of both sexes were used at 8–12 weeks of age to prepare heart mitochondria. Rats were killed by stunning followed by cervical dislocation. Mice were killed by cervical dislocation only. Hearts were removed into ice-cold STE buffer (250 mMsucrose, 5 mMTris-HCl (pH 7.4, KOH), and1m MK-EGTA, supplemented with 0.1% (w/v) fatty acid–free BSA. Heart mitochondria were isolated by homogenization and differential centrifugation (700 ⫻gfor 3 min; 3⫻5,500 ⫻gfor 10 min) at 4 °C. Mitochondrial protein content was determined using the bicinchoninic acid assay with BSA as a standard. Hydrogen peroxide efflux H 2 O 2 efflux from mitochondria was assayed using a plate reader fluorometer (SpectraMax GeminiXS, Molecular Devices; used at medium sensitivity). Resorufin (the product of Amplex Red oxidation) fluorescence was detected using ␭ ex ⫽ 570 nm and ␭ em ⫽585 nm. Mitochondria (2 mg protein/ml) were incubated with 2.5 ␮ MAmplex Red (Invitrogen), 5 units/ml HRP in STE with 10 mMpotassium succinate at 37 °C. H 2 O 2 production rates were linear over 10 min but were measured from 0 to 2 min to facilitate comparison with other measurements. The H 2 O 2 response was calibrated using freshly prepared H 2 O 2 standards ( ⑀ 240 ⫽43.5 M ⫺1 cm ⫺1 ) that were added sequentially to mitochondrial incubations lacking only succinate to generate a linear calibration curve. Mitochondriatargeted test compounds were added 30 s before measurement. Mitochondrial ⌬ ⑀ Mitochondrial ⌬ ⑀ was measured by the uptake of radiolabeled [ 3 H]TPMP as described (21). Mitochondria (2 mg protein/ml) were incubated at 37 °C with 10 mMsuccinate, 500 nM TPMP supplemented with [ 3 H]TPMP (50 nCi/ml) with the test compounds for 2 min in 250 ␮ l of medium in Eppendorf tubes. Mitochondria were pelleted by centrifugation (10,000 ⫻gfor 30 s). Supernatant (200 ␮ l) was removed, and the pellets were dried with a rolled-up tissue and then solubilized in 40 ␮ lof20% (v/v) Triton X-100. Both the supernatant and pellets were then added to scintillant (Ultima-Gold liquid scintillant, PerkinElmer Life Sciences) and incubated for1hatroom temperature and then vortexed, and [ 3 H]TPMP content was assessed using a TriCarb LCS counter (PerkinElmer Life Sciences) counter with appropriate quench controls. To calculate the ⌬ ⑀ , first the accumulation ratio was calculated assuming a mitochondrial matrix volume of 0.6 ␮ l/mg protein, and the mitochondrial ⌬ ⑀ was then calculated from the Nernst equation, assuming 40% binding of TPMP and that this was independent of mitochondrial ⌬ ⑀ and consistent across all conditions (21). CoQ extraction and detection Mitochondria (2 mg of protein/ml) were incubated in 500– 700 ␮ l of STE with 10 mMpotassium succinate at 37 °C on a shaking heat block for 2 min. At the end of the incubation, mitochondria were rapidly pelleted by centrifugation (10,000 ⫻ gfor 30 s), the supernatant was removed, and pellets were snapfrozen in a dry ice/ethanol bath and stored at ⫺80 °C until analysis. Immediately before HPLC analysis, the pellets were homogenized in 0.5 ml of ice-cold, nitrogen-purged 1-propanol in an ice-cold glass-on-glass homogenizer, 100 ␮ l of ice-cold H 2 O was added, and the samples were centrifuged (16,000 ⫻g at 4 °C for 5 min). Supernatants (200 ␮ l) were immediately analyzed by HPLC on a 150 ⫻4.6-mm, 3 ␮ Hypersil ODS column (Thermo). Solvent A was MeOH, 50 mMNaClO 4 ; solvent B was EtOH, 50 mMNaClO 4 . The gradient was 60% to 50% A over 15 min at a flow rate of 0.8 ml/min at 45 °C. Identity was established by retention time compared with authentic standards at the absorbance maxima for CoQ and CoQH 2 (260 and 290 nm, respectively). CoQ 9 redox state was determined from the peak areas of ubiquinone and ubiquinol at 292.5 nm, the isosbestic point for oxidized and reduced CoQ 9 . The percentage reducComplex I reverse electron transport 9876 J. Biol. Chem. (2018) 293(25) 9869–9879 at University of Tampere on July 5, 2018http://www.jbc.org/Downloaded from tion of the CoQ 9 pool was calculated as area of the reduced peak divided by the sum of both peak areas. Control incubations under conditions designed to maximally oxidize and reduce the CoQ pool demonstrated that this approach accurately reported the CoQ redox state, as the percentage reduction increased to ⬃80% in the presence of cyanide or anoxia, whereas inhibition of the respiratory chain with malonate decreased the percentage reduction to ⬃20%. Redox state of mitochondrial NAD(P)H/NAD(P) pools The redox state of the NAD(P)H/NAD(P) ⫹ pool was determined by monitoring NAD(P)H fluorescence using a plate reader fluorimeter (SpectraMax GeminiXS; Molecular Devices) using ␭ ex ⫽365 nm and ␭ em ⫽450 nm. Mitochondria were incubated as described for measuring H 2 O 2 efflux. The signal was calibrated by subtraction of background fluorescence (mitochondria with no additions), and maximal reduction of the pool was set by incubating mitochondria with 5 mMmalate and 5 ␮ Mrotenone for 5 min. Combined respirometry and H 2 O 2 production measurements Combined respiration and H 2 O 2 production by mitochondria was assessed using an Oxygraph2K (O2K) respirometer (Oroboros, Innsbruck, Austria) with a fluorescence LED module attachment. To assess the effect of O 2 concentration on H 2 O 2 production, mitochondria (200–250 ␮ g of protein/ml) were suspended in 2 ml of KCl buffer (120 mMKCl, 10 mM Hepes, 1 mMEGTA, pH 7.2) supplemented with 50 units/ml SOD, 4 units/ml HRP, 0.2 mg/ml fatty acid–free BSA, 25 ␮ M Amplex Red with stirring at 37 °C. Respiration was initiated by the addition of either 5 mMglutamate and 5 mMmalate; 10 mM succinate; or 10 mMsuccinate with 5 ␮ Mrotenone. Where indicated, incubations were supplemented at the start with 5 ␮ M rotenone, 500 nMFCCP, 1 ␮ Mantimycin A, or 1 ␮ Mnigericin. The concentration of O 2 was adjusted by bubbling the buffer with N 2 . Amplex Red fluorescence was measured via the O2K fluorometer, and the corresponding voltage changes were calibrated via titration of known amounts of H 2 O 2 (500 nMto 5 ␮ M) in the presence of mitochondria, SOD, fatty acid–free BSA, HRP, and Amplex Red. Western blotting Mitochondrial pellets (⬃250 ␮ g of protein) were solubilized on ice in 50 ␮ l of lysis buffer (100 mMTris, 300 mMNaCl, 0.05% Nonidet P-40, pH 7.4, supplemented with protease and phosphatase inhibitors (Roche Applied Science). Protein was then quantified by the BCA assay and diluted in 4⫻loading buffer (Invitrogen), and 10 ␮ g of protein was separated by SDS-PAGE on a 10% gel and transferred to polyvinylidene difluoride membrane. Membranes were incubated with a 1:20,000 dilution of rabbit serum raised against two AOX peptides (FKIETNDSTDEPNIEVENFPC and CVNHDLGSRKPDEQNPYPPGQ (49)) and a mouse monoclonal antibody against the voltage-dependent anion channel (1:1,000; Abcam ab14734)) and visualized using a LI-COR Odyssey flatbed scanner with anti-mouse and anti-rabbit secondary antibodies conjugated to IRDye 680RD and IRDye 800CW, respectively. Calculations This section describes calculations required to determine the thermodynamic driving force for RET from the data in Figs. 2 and 5to generate the graphs shown in Fig. 7. The thermodynamic driving force for RET is derived from Equation 5. In mV, it is as follows. ⌬p⫽⌬ ␺ ⫺61.5⌬pH (Eq. 6) In the presence of nigericin, ⌬p is unchanged, whereas ⌬pH ⫽0. Hence, ⌬pH ⫽⌬ ␺ (⫹nigericin) ⫺⌬ ␺ (⫺nigericin). From Fig. 2C, 61.5⌬pH ⫽⫺18.1 mV and the matrix pH ⫽7.7. ⌬p⫽⌬ ␺ ⫹18.1 (Eq. 7) As the matrix pH is 7.7, E h for the NAD ⫹ /NADH couple in mV is as follows. Eh共NAD⫹/NADH兲⫽⫺341 ⫹30.5log10共NAD⫹/NADH兲 (Eq. 8) In the presence of nigericin, the matrix pH will be 7.4, and under those conditions, the following will be true. Eh共NAD⫹/NADH兲⫽⫺332 ⫹30.5log10共NAD⫹/NADH兲 (Eq. 9) As the matrix pH is 7.7, E h for the CoQ/CoQH 2 couple in mV is as follows. Eh共CoQ/CoQH2兲⫽⫺38 ⫹30.5log10共CoQ/CoQH2兲 (Eq. 10) In the presence of nigericin, the matrix pH will be 7.4, and under those conditions, the following will be true. Eh共CoQ/CoQH2兲⫽⫺20 ⫹30.5log10共CoQ/CoQH2兲 (Eq. 11) Statistical analysis and experimental design Data were expressed as mean ⫾S.E., with pvalues calculated using a two-tailed Student’s ttest for pairwise comparisons whereas one-way analysis of variance (ANOVA) followed by Tukey’s post hoc test was used for multiple comparisons. Statistical analyses were performed using GraphPad Prism version 7 software. Author contributions—E. L. R. and A. R. H. designed and carried out the experiments, with assistance from T. A. P. S. E. carried out the CoQ assays. M. S. and C. V. provided the AOX mice. A. M. J. provided advice and helped with data interpretation. M. P. M. directed the project and wrote the manuscript, with assistance from all other authors. Acknowledgment—We are grateful to Judy Hirst for helpful discussions and suggestions. References 1. Murphy, M. P. (2009) How mitochondria produce reactive oxygen species. Biochem. J. 417, 1–13 CrossRef Medline Complex I reverse electron transport J. Biol. Chem. (2018) 293(25) 9869–9879 9877 at University of Tampere on July 5, 2018http://www.jbc.org/Downloaded from