Mitochondrial hydrogen peroxide production by pyruvate dehydrogenase and α-ketoglutarate dehydrogenase in oxidative eustress and oxidative distress

Abstract

Pyruvate dehydrogenase (PDH) and α-ketoglutarate dehydrogenase (KGDH) are vital entry points for monosaccharides and amino acids into the Krebs cycle and thus integral for mitochondrial bioenergetics. Both complexes produce mitochondrial hydrogen peroxide (mH₂O₂) and are deactivated by electrophiles. Here, we provide an update on the role of PDH and KGDH in mitochondrial redox balance and their function in facilitating metabolic reprogramming for the propagation of oxidative eustress signals in hepatocytes and how defects in these pathways can cause liver diseases. PDH and KGDH are known to account for ∼45% of the total mH₂O₂ formed by mitochondria and display rates of production several-fold higher than the canonical source complex I. This mH₂O₂ can also be formed by reverse electron transfer (RET) in vivo, which has been linked to metabolic dysfunctions that occur in pathogenesis. However, the controlled emission of mH₂O₂ from PDH and KGDH has been proposed to be fundamental for oxidative eustress signal propagation in several cellular contexts. Modification of PDH and KGDH with protein S-glutathionylation (PSSG) and S-nitrosylation (PSNO) adducts serves as a feedback inhibitor for mH₂O₂ production in response to glutathione (GSH) pool oxidation. PSSG and PSNO adduct formation also reprogram the Krebs cycle to generate metabolites vital for interorganelle and intercellular signaling. Defects in the redox modification of PDH and KGDH cause the over generation of mH₂O₂, resulting in oxidative distress and metabolic dysfunction-associated fatty liver disease (MAFLD). In aggregate, PDH and KGDH are essential platforms for emitting and receiving oxidative eustress signals.

Keywords: fatty liver disease; hydrogen peroxide; mitochondria; oxidative distress; oxidative eustress; pyruvate dehydrogenase; redox signaling; α-ketoglutarate dehydrogenase.

Figure 1

The entry of carbon into the Krebs cycle through PDH and KGDH, the catalytic cycle of the α-ketoacid dehydrogenases, and the sites for redox regulation and mH₂O₂generation.A, the entry of monosaccharides (glucose) and amino acids into mitochondria and the Krebs cycle through pyruvate dehydrogenase (PDH) and α-ketoglutarate dehydrogenase (KGDH). Acetyl-CoA and succinyl-CoA formed by PDH and KGDH are oxidized further by the rotation of the Krebs cycle, producing the electron carrier NADH which is metabolized by the electron transport chain (ETC) to generate ATP by oxidative phosphorylation (OxPhos). PDH and KGDH catalyze high energy reactions in the Krebs cycle and are thus important sites for allosteric activation and deactivation (denoted by red × or green triangle). B, the catalytic pathway for the keto acid dehydrogenases using KGDH as an example. α-ketoglutarate is decarboxylated on the C1 position (in blue) by α-ketoglutarate decarboxylase (E1), transferring the succinyl group to thiamine pyrophosphate (TPP). The dihydrolipoamide succinyltransferase (or acyltransferase: E2, DLAT) catalyzes the formation of a high-energy thioester bond via the transfer of the succinyl moiety from TPP to coenzyme A (CoASH). The dihydrolipoamide is then oxidized by the dehydrogenase activity of the E3 subunit (dihydrolipoamide dehydrogenase or DLD), reducing FAD to FADH₂ and then producing NADH. Electrons leak from the FAD through side reactions that generate semiflavin radicals, flavin hydroperoxides, and oxy-flavin radicals, which form mH₂O₂. Note that the FAD can generate both mitochondrial superoxide (mO₂^•−) and mitochondrial (mH₂O₂), reactions that depend on the redox state of the flavin and its interactions with molecular oxygen (O₂). The mO₂^•− is dismutated to mH₂O₂ by superoxide dismutase (SOD; intermembrane space = SOD1, matrix = SOD2). The E2 dihydrolipoamide is a target for redox modifications by mH₂O₂ and various electrophiles and soft acid metals (e.g., arsenic) which block the activity of the enzyme but also inhibit mH₂O₂ generation. The figure was generated with Biorender Software (Agreement Number: NR25TFIEU4).

Figure 2

Oxidative eustress and oxidative distress and the role of pyruvate dehydrogenase (PDH) and α-ketoglutarate dehydrogenase (KGDH) in supplying mitochondrial (mH₂O₂) for optimal liver health. Spatio-temporal control over mH₂O₂ generation triggers oxidative eustress pathways (left) which activate proliferation, adaptation, growth, and repair cascades required for the restoration of normal hepatic function and maintaining optimal liver health. The mH₂O₂ levels oscillate in the 1 to 100 nM range, which is achieved, in part, through the regulation of its production through activation and inhibition of PDH and KGDH. Defects in the regulation of mH₂O₂ generation by PDH and KGDH result in its sustained overproduction. This sustained overgeneration of mH₂O₂ is due to factors that promote mitochondrial dysfunction (e.g., toxins or poor nutrition). This promotes mH₂O₂ accumulation, triggering oxidative distress. This oxidative distress (right) is characterized by defective oxidative eustress signals, over-oxidation of antioxidant defenses, and cell damage and death. These effects result in inflammation, hepatic ballooning, and metabolic dysfunction causing irreversible liver damage (cirrhosis) and the development of hepatocellular carcinoma. Note that the early stages of MAFLD is reversible. However, sustained oxidative distress induced by the continued accumulation of mH₂O₂ causing progressive cell damage and perturbed eustress leads to irreversible liver disease. The figure was generated with Biorender Software (Agreement number: KN25TFIEZ2).

Figure 3

Pyruvate dehydrogenase (PDH) and α-ketoglutarate dehydrogenase (KGDH) as major mitochondrial (mH₂O₂) sources during reverse electron transfer (RET) from the ubiquinone pool. Under oxidative eustress conditions (left), electron flow from succinate, glycerol-3-phosphate, proline, and other sources generates NADH through proton return at complex I. Under these conditions, mitochondria are operating normally and the back fluxes in electrons are controlled. The NADH is then oxidized by PDH or KGDH (represented by the E1:E2:E3 subunits) and the electrons drive mH₂O₂ generation, which is transmitted into the cell to trigger proliferation and growth, cell adaptation, and tissue regeneration (e.g., in the case of liver recovery from injury). Oxidative distress (right) is triggered by mitochondrial dysfunction and metabolic gridlock. This can result in the accumulation of ubiquinone metabolites (e.g., succinate, glycerol-3-phosphate, proline), which drive the overgeneration of NADH by complex I through RET. This overloads PDH and KGDH with electrons resulting in the sustained overproduction of mH₂O₂. The resulting oxidative distress is related to the nonspecific and over-oxidation of protein cysteine thiols and the disruption of redox signaling circuits like protein S-glutathionylation (PSSG). The overgeneration of mH₂O₂ also triggers macromolecular damage and the induction of cell death. The figure was generated with Biorender Software (Agreement number: IC25TFIF36).

Figure 4

The protein S-glutathionylation cycle and the control of mH₂O₂production by PDH and KGDH through the reversible addition and removalof GSH to and from the E2 subunit.A, reversible protein S-glutathionylation (PSSG) occurs in response to changes in mH₂O₂ availability and the generation of NADPH, which drive reduced glutathione (GSH) pool oxidation to glutathione disulfide (GSSG) and its reduction through peroxidases and reductases. The decrease in the GSH/GSSG ratio activates the glutathionyltransferase activity of glutaredoxin-2 (Glrx2), resulting in the formation of PSSG. Reduction of the GSH pool and the increase in GSH/GSSG induces Glrx2 deglutathionylase activity restoring protein function. In this way, glutathionylation regulates proteins in response to changes in redox tone. Figure was generated using Biorender Software (Agreement number: YM25TFIF86). B, the dihydrolipoamide of the E2 subunit of PDH and KGDH is targeted for reversible glutathionylation. This increases and decreases mH₂O₂ generation in response to changes in redox tone, protecting cells from oxidative distress but also simultaneously modulating cell oxidative eustress signals. Figure was generated using Biorender Software (Agreement number: XE25TFIFB9).