Mitochondrial diaphorases as NAD⁺ donors to segments of the citric acid cycle that support substrate-level phosphorylation yielding ATP during respiratory inhibition

Abstract

Substrate-level phosphorylation mediated by succinyl-CoA ligase in the mitochondrial matrix produces high-energy phosphates in the absence of oxidative phosphorylation. Furthermore, when the electron transport chain is dysfunctional, provision of succinyl-CoA by the α-ketoglutarate dehydrogenase complex (KGDHC) is crucial for maintaining the function of succinyl-CoA ligase yielding ATP, preventing the adenine nucleotide translocase from reversing. We addressed the source of the NAD(+) supply for KGDHC under anoxic conditions and inhibition of complex I. Using pharmacologic tools and specific substrates and by examining tissues from pigeon liver exhibiting no diaphorase activity, we showed that mitochondrial diaphorases in the mouse liver contribute up to 81% to the NAD(+) pool during respiratory inhibition. Under these conditions, KGDHC's function, essential for the provision of succinyl-CoA to succinyl-CoA ligase, is supported by NAD(+) derived from diaphorases. Through this process, diaphorases contribute to the maintenance of substrate-level phosphorylation during respiratory inhibition, which is manifested in the forward operation of adenine nucleotide translocase. Finally, we show that reoxidation of the reducible substrates for the diaphorases is mediated by complex III of the respiratory chain.

Keywords: DT-diaphorase; adenine nucleotide translocase; reducing equivalent; succinyl-CoA ligase.

Figure 1.

Reconstructed time courses of safranin O signal calibrated to ΔΨ_m (solid traces) and parallel measurements of oxygen concentration in the medium (dotted traces) in isolated mouse liver mitochondria. Effect of cATR (2 μM) or oligomycin (oligo, 5 μM) on ΔΨ_m during anoxia (A–C, E) or during compromised respiratory chain by poisons (D), in the presence of different substrate combinations. ADP (2 mM) was added where indicated. Substrate concentrations were glutamate (glut; 5 mM), malate (mal; 5 mM), succinate (succ; 5 mM), and β-hydroxybutyrate, (βOH; 1 or 2 mM, as indicated). Substrate concentrations were the same for all subsequent experiments. At the end of each experiment, 1 μM SF 6847 was added to achieve complete depolarization, except for the orange trace in panel A, where 250 nM SF 6847 was added.

Figure 2.

A) Reconstructed time courses of safranin O signal calibrated to ΔΨ_m (solid traces) and parallel measurements of oxygen concentration in the medium (dotted traces) in isolated mouse liver mitochondria. Effect of cATR (2 μM) on ΔΨ_m of mitochondria during anoxia in the presence or absence of 2 mM NaAsO₂ is shown. ADP (2 mM) was added where indicated. Respiration rates in nanomoles per minute per milligram protein are indicated on the dotted lines. B) Reconstructed time courses of safranin O signal calibrated to ΔΨ_m in isolated mouse liver mitochondria in an open chamber. Effect of cATR (2 μM) on ΔΨ_m of mitochondria treated with rotenone (rot; 1 μM, where indicated) in the presence or absence of 2 mM NaASO₂ (2 mM, red trace) is shown. ADP (2 mM) was added where indicated. At the end of each experiment, 1 μM SF 6847 was added to achieve complete depolarization. C) Rates of acidification in the suspending medium of mitochondria respiring on the various substrate combinations indicated, on addition of different bioenergetic poisons. a-Kg, α-ketoglutarate; glut, glutamate; mal, maleate; pyr, pyruvate; succ, succinate.

Figure 3.

Reconstructed time courses of safranin O signal calibrated to ΔΨ_m (solid traces) and parallel measurements of oxygen concentration in the medium (dotted traces) in isolated mouse liver mitochondria supported by glutamate and malate. Effect of diaphorase inhibitors (doses are color-coded in panel A) on cATR-induced changes in ΔΨ_m during anoxia (A–D) or under complex I inhibition by rotenone (E–H). Gray traces in panels E–H show the effect of vehicles (either DMSO or ethanol). At the end of each experiment, 1 μM SF 6847 was added to achieve complete depolarization.

Figure 4.

A–H) Reconstructed time courses of safranin O signal calibrated to ΔΨ_m (solid traces) and parallel measurements of oxygen concentration in the medium (dotted traces) in isolated mouse liver mitochondria, demonstrating the effect of diaphorase substrates on cATR-induced changes in ΔΨ_m during anoxia (A–C) or complex I inhibition by rotenone (rot; D–H). Substrate combinations are indicated in the panels. I) Reconstructed time course of TPP signal (in volts) in isolated mouse liver mitochondria (mitos) supported by glutamate (glut) and malate (mal). Additions were as indicated by arrows. α-Kg, α-ketoglutarate; βOH, β-hydroxybutyrate. At the end of each experiment, 1 μM SF 6847 was added to achieve complete depolarization.

Figure 5.

Effect of the diaphorase substrates menadione (10 μM; A–C), DQ (50 μM; D) and mitoQ (0.5 μM; D), on cATR-induced changes of ΔΨ_m after complex III inhibition by stigmatellin (stigma; E) or complex IV by KCN (F). Reconstructed time courses of safranin O signal calibrated to ΔΨ_m in isolated mouse liver mitochondria and oxygen consumption (E, dotted lines) are shown. Mitochondria respired on different substrates, as shown. α-Kg, α-ketoglutarate; βOH, β-hydroxybutyrate; FerrCyan, ferricyanide; glut, glutamate; mal, maleate; Additions were as indicated by the arrows. At the end of each experiment, 1 μM SF 6847 was added to achieve complete depolarization.

Figure 6.

A–C) Reconstructed time courses of safranin O signal calibrated to percentage (solid traces; y axes of all panels are as shown in E) reflecting ΔΨ_m in isolated pigeon liver mitochondria, demonstrating the effect of various mitochondrial and diaphorase substrates on cATR-induced changes in ΔΨ_m after complex I inhibition by rotenone, with different respiratory substrates used, as indicated. D) Diaphorase inhibitors were present as indicated (dicoumarol, 5 μM; chrysin, 5 μM; diOH-flavone, 20 μM; and phenindione, 10 μM), and mitochondria were supported by glutamate and malate. E–G) Oxygen concentrations in the medium (dotted traces) were measured. Effects of DQ (50 μM; E), various substrates (F), and inhibitors atpenin A5 and KCN (G) are shown. α-Kg, α-ketoglutarate; βOH, β-hydroxybutyrate; AcAc, acetoacetate; glut, glutamate; mal, maleate; rot, rotenone; succ, succinate. At the end of each experiment. 1 μM SF 6847 was added to achieve complete depolarization.

Figure 7.

Illustration of the pathway linking ATP production by the succinyl-CoA ligase reaction to KGDHC activity, diaphorase activity, reoxidation of diaphorase substrates by complex III, reoxidation of cytochrome c, and rereduction of a cytosolic oxidant.