The Contribution of Nicotinamide Nucleotide Transhydrogenase to Peroxide Detoxification Is Dependent on the Respiratory State and Counterbalanced by Other Sources of NADPH in Liver Mitochondria

J Biol Chem. 2016 Sep 16;291(38):20173-87. doi: 10.1074/jbc.M116.730473. Epub 2016 Jul 29.


The forward reaction of nicotinamide nucleotide transhydrogenase (NNT) reduces NADP(+) at the expense of NADH oxidation and H(+) movement down the electrochemical potential across the inner mitochondrial membrane, establishing an NADPH/NADP(+) ratio severalfold higher than the NADH/NAD(+) ratio in the matrix. In turn, NADPH drives processes, such as peroxide detoxification and reductive biosynthesis. In this study, we generated a congenic mouse model carrying a mutated Nnt(C57BL/6J) allele from the C57BL/6J substrain. Suspensions of isolated mitochondria from Nnt(+/+), Nnt(+/-), and Nnt(-/-) mouse liver were biochemically evaluated and challenged with exogenous peroxide under different respiratory states. The respiratory substrates were also varied, and the participation of concurrent NADPH sources (i.e. isocitrate dehydrogenase-2, malic enzymes, and glutamate dehydrogenase) was assessed. The principal findings include the following: Nnt(+/-) and Nnt(-/-) exhibit ∼50% and absent NNT activity, respectively, but the activities of concurrent NADPH sources are unchanged. The lack of NNT activity in Nnt(-/-) mice impairs peroxide metabolism in intact mitochondria. The contribution of NNT to peroxide metabolism is decreased during ADP phosphorylation compared with the non-phosphorylating state; however, it is accompanied by increased contributions of concurrent NADPH sources, especially glutamate dehydrogenase. NNT makes a major contribution to peroxide metabolism during the blockage of mitochondrial electron transport. Interestingly, peroxide metabolism in the Nnt(+/-) mitochondria matched that in the Nnt(+/+) mitochondria. Overall, this study demonstrates that the respiratory state and/or substrates that sustain energy metabolism markedly influence the relative contribution of NNT (i.e. varies between nearly 0 and 100%) to NADPH-dependent mitochondrial peroxide metabolism.

Keywords: NADPH; c57bl/6j; hydrogen peroxide; mitochondria; oxidation-reduction (redox); peroxidase; respiratory chain; transhydrogenase; tricarboxylic acid cycle (TCA cycle) (Krebs cycle).

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Energy Metabolism / physiology*
  • Mice
  • Mice, Mutant Strains
  • Mitochondria, Liver / enzymology*
  • Mitochondria, Liver / genetics
  • Mitochondrial Proteins / genetics
  • Mitochondrial Proteins / metabolism
  • NADP / genetics
  • NADP / metabolism*
  • NADP Transhydrogenase, AB-Specific / genetics
  • NADP Transhydrogenase, AB-Specific / metabolism*
  • Oxygen Consumption / physiology*
  • Peroxides / metabolism*


  • Mitochondrial Proteins
  • Peroxides
  • NADP
  • NADP Transhydrogenase, AB-Specific
  • Nnt protein, mouse