Redox Modulation by Reversal of the Mitochondrial Nicotinamide Nucleotide Transhydrogenase
Authors: Michael P. Murphy
Journal: Cell Metabolism (2015)

The mitochondrial nicotinamide nucleotide transhydrogenase (NNT) uses the protonmotive force to transfer electrons from NADH to NADP, thereby maintaining the NADP/NADPH pool reduced to protect mitochondria from oxidative damage. In this issue, Nickel et al., 2015Nickel A.G. von Hardenberg A. Hohl M. Löffler J.R. Kohlhaas M. Becker J. Reil J.-C. Kazakov A. Bonnekoh J. Stadelmaier M. et al.Cell Metab. 2015; 22 (this issue): 472-484Abstract Full Text Full Text PDF Scopus (221) Google Scholar show that NNT also operates in reverse, oxidizing the NADP/NADPH pool, thereby disrupting antioxidant defense. The mitochondrial nicotinamide nucleotide transhydrogenase (NNT) uses the protonmotive force to transfer electrons from NADH to NADP, thereby maintaining the NADP/NADPH pool reduced to protect mitochondria from oxidative damage. In this issue, Nickel et al., 2015Nickel A.G. von Hardenberg A. Hohl M. Löffler J.R. Kohlhaas M. Becker J. Reil J.-C. Kazakov A. Bonnekoh J. Stadelmaier M. et al.Cell Metab. 2015; 22 (this issue): 472-484Abstract Full Text Full Text PDF Scopus (221) Google Scholar show that NNT also operates in reverse, oxidizing the NADP/NADPH pool, thereby disrupting antioxidant defense. The way we have been taught inevitably influences how we think of metabolic processes—I always remember the Krebs cycle as a complete circle that only goes clockwise! Of course, the textbook presentation is just a way of compiling information concisely, and in vivo metabolic networks can be quite different. Similar simplifications affect our view of mitochondrial proton translocating complexes. A good example is the FoF1-ATP synthase that utilizes the protonmotive force across the inner membrane to drive ATP synthesis during oxidative phosphorylation. However, if the protonmotive force is too low then the protein can turn into an ATPase, hydrolysing ATP and pumping protons out of the mitochondria. This occurs in vivo, for example during ischemia when respiration is prevented, thereby draining away the ATP synthesized by glycolysis (St-Pierre et al., 2000St-Pierre J. Brand M.D. Boutilier R.G. Proc. Natl. Acad. Sci. USA. 2000; 97: 8670-8674Crossref PubMed Scopus (129) Google Scholar). Mitochondrial respiratory complexes can also be driven "backwards," compared to their conventional direction, depending on the balance of thermodynamic forces. For example, a recent study to which I contributed suggested that complexes I and II may operate in reverse and contribute to ischemia-reperfusion injury (Chouchani et al., 2014Chouchani E.T. Pell V.R. Gaude E. Aksentijević D. Sundier S.Y. Robb E.L. Logan A. Nadtochiy S.M. Ord E.N. Smith A.C. et al.Nature. 2014; 515: 431-435Crossref PubMed Scopus (1529) Google Scholar). During ischemia, complex II utilizes the reduced Coenzyme Q pool to convert fumarate to succinate, then upon reperfusion the accumulated succinate drives reversal of complex I, thereby producing damaging reactive oxygen species (ROS) (Chouchani et al., 2014Chouchani E.T. Pell V.R. Gaude E. Aksentijević D. Sundier S.Y. Robb E.L. Logan A. Nadtochiy S.M. Ord E.N. Smith A.C. et al.Nature. 2014; 515: 431-435Crossref PubMed Scopus (1529) Google Scholar). More interesting than the reversal of respiratory complexes in pathology is the possibility that such unconventional operation may occur in normal physiology. For example, the reversal of complex II during anoxia may generate succinate as a hypoxic signal, complex I going backward could be a physiological ROS signal, and ATP hydrolysis by the FoF1-ATP synthase might enable maintenance of a protonmotive force independently of the respiratory chain (Brown et al., 2006Brown S.V. Hosking P. Li J. Williams N. Eukaryot. Cell. 2006; 5: 45-53Crossref PubMed Scopus (85) Google Scholar, Chouchani et al., 2014Chouchani E.T. Pell V.R. Gaude E. Aksentijević D. Sundier S.Y. Robb E.L. Logan A. Nadtochiy S.M. Ord E.N. Smith A.C. et al.Nature. 2014; 515: 431-435Crossref PubMed Scopus (1529) Google Scholar). In this issue, Nickel et al. show that the latest proton pumping mitochondrial complex that may operate in two directions is the nicotinamide nucleotide transhydrogenase (NNT) (Nickel et al., 2015Nickel A.G. von Hardenberg A. Hohl M. Löffler J.R. Kohlhaas M. Becker J. Reil J.-C. Kazakov A. Bonnekoh J. Stadelmaier M. et al.Cell Metab. 2015; 22 (this issue): 472-484Abstract Full Text Full Text PDF Scopus (221) Google Scholar). The NNT is a mitochondrial inner membrane protein that takes electrons from NADH to reduce NADP to NADPH, utilizing the protonmotive force across the inner membrane to drive the process (Figure 1A). This coupling of electron transfer to the protonmotive force is essential to maintain the difference in redox state between the mitochondrial NADP/NADPH and NAD/NADH pools, despite their identical midpoint potentials (Rydström, 2006Rydström J. Biochim. Biophys. Acta. 2006; 1757: 721-726Crossref PubMed Scopus (179) Google Scholar). This redox difference is essential for the quite different biological roles of the two nicotinamide nucleotides. The NAD/NADH pool mainly acts a conduit for electrons from substrates to the respiratory chain and is thus only partially reduced (Eh ∼ −300 mV). In contrast, the mitochondrial NADP/NADPH pool supplies electrons to glutathione reductase, to maintain the mitochondrial glutathione/glutathione disulfide ratio high, and to thioredoxin reductase 2, which keeps the matrix thioredoxin 2 pool reduced (Murphy, 2012Murphy M.P. Antioxid. Redox Signal. 2012; 16: 476-495Crossref PubMed Scopus (251) Google Scholar). Consequently, the mitochondrial NADP/NADPH pool is far more reduced (Eh ∼ −415 mV) than the NAD/NADH pool. The only known role for the NNT is to use the protonmotive force to help maintain the redox difference between mitochondrial NADP/NADPH and NAD/NADH (Figure 1A). While the NADP-dependent isocitrate dehydrogenase and malic enzyme also contribute, this function of NNT is important, as is illustrated by a C57BL/6 mouse strain (6J) that has an inactive NNT (Freeman et al., 2006Freeman H. Shimomura K. Horner E. Cox R.D. Ashcroft F.M. Cell Metab. 2006; 3: 35-45Abstract Full Text PDF PubMed Scopus (127) Google Scholar, Toye et al., 2005Toye A.A. Lippiat J.D. Proks P. Shimomura K. Bentley L. Hugill A. Mijat V. Goldsworthy M. Moir L. Haynes A. et al.Diabetologia. 2005; 48: 675-686Crossref PubMed Scopus (307) Google Scholar). The 6J strain has increased oxidative damage and disrupted insulin signaling when compared to a control strain (6N) with a functional NNT (Freeman et al., 2006Freeman H. Shimomura K. Horner E. Cox R.D. Ashcroft F.M. Cell Metab. 2006; 3: 35-45Abstract Full Text PDF PubMed Scopus (127) Google Scholar, Toye et al., 2005Toye A.A. Lippiat J.D. Proks P. Shimomura K. Bentley L. Hugill A. Mijat V. Goldsworthy M. Moir L. Haynes A. et al.Diabetologia. 2005; 48: 675-686Crossref PubMed Scopus (307) Google Scholar). This view of the NNT has now been extended by the fascinating work of Nickel et al., 2015Nickel A.G. von Hardenberg A. Hohl M. Löffler J.R. Kohlhaas M. Becker J. Reil J.-C. Kazakov A. Bonnekoh J. Stadelmaier M. et al.Cell Metab. 2015; 22 (this issue): 472-484Abstract Full Text Full Text PDF Scopus (221) Google Scholar, who suggest that under some conditions the NNT can act in reverse, passing electrons from NADPH to NAD while pumping protons out of the mitochondrial matrix (Figure 1B). This hypothesis arose from studying a model of heart failure, which unexpectedly showed that mice lacking NNT had less oxidative damage to the heart than controls with an active NNT. This was surprising, given the role of NNT in protecting against oxidative damage: how could inactivating the NNT decrease oxidative damage? The authors hypothesized that the stress to the heart in this model was causing the NNT to run backward, sucking electrons from NADPH and feeding them into the NAD/NADH pool so as to maintain oxidative phosphorylation. This oxidation of the NADP/NADPH pool in turn undermined antioxidant defense by the glutathione and thioredoxin systems, leading to oxidative damage (Figure 1B). In the mouse strains lacking NNT, this reversal could not occur, the NADP/NADPH pool was maintained, and they had less oxidative damage than the controls with an active NNT. This elegant model was shown to be plausible in vitro, but of course just because a process can occur in vitro does not mean that it will take place in vivo. My view is that the evidence presented for NNT reversal contributing to oxidative damage in vivo is strong, but not yet definitive. Further work will be necessary to explore this intriguing hypothesis. For example, one limitation is that the in vivo data are based on comparison of two mouse strains; future studies on mice where NNT is conditionally deleted are required to further test this hypothesis, as is acknowledged by the authors. This work does introduce the fascinating possibility that the NNT can be reversed from its "normal" direction in vivo under pathological conditions. Could there also be a physiological role for NNT reversal? One possibility is that by altering the NADP/NADPH ratio, and thus the activity of the glutathione and thioredoxin systems, the activity of thiol-dependent enzymes in the matrix could be modulated, adding a further nuance to redox regulation. Another idea worth exploring is that the NNT provides a link between the NADP/NADPH and NAD/NADH pools, and the protonmotive force, thereby potentially coordinating changes in these key determinants of mitochondrial activity (Gameiro et al., 2013Gameiro P.A. Laviolette L.A. Kelleher J.K. Iliopoulos O. Stephanopoulos G. J. Biol. Chem. 2013; 288: 12967-12977Crossref PubMed Scopus (85) Google Scholar). Finally, this stimulating study reminds us that the direction of biochemical reactions in vivo is determined by local thermodynamics, not textbooks, potentially providing fresh insights into pathology and metabolic regulation. Reversal of Mitochondrial Transhydrogenase Causes Oxidative Stress in Heart FailureNickel et al.Cell MetabolismAugust 6, 2015In BriefThe mitochondrial transhydrogenase (Nnt) normally regenerates NADPH from NADH to bolster antioxidative capacity. Nickel et al. show that pathological workload reverses the Nnt reaction and depletes NADPH in the mouse heart, which increases ROS production and leads to maladaptive cardiac remodeling in heart failure. Full-Text PDF Open Archive