Fatty acid oxidation drives mitochondrial hydrogen peroxide production by α-ketoglutarate dehydrogenase

In the present study, we examined the mitochondrial hydrogen peroxide (mH₂O₂) generating capacity of α-ketoglutarate dehydrogenase (KGDH) and compared it to components of the electron transport chain using liver mitochondria isolated from male and female C57BL6N mice. We show for the first time there are some sex dimorphisms in the production of mH₂O₂ by electron transport chain complexes I and III when mitochondria are fueled with different substrates. However, in our investigations into these sex effects, we made the unexpected and compelling discovery that 1) KGDH serves as a major mH₂O₂ supplier in male and female liver mitochondria and 2) KGDH can form mH₂O₂ when liver mitochondria are energized with fatty acids but only when malate is used to prime the Krebs cycle. Surprisingly, 2-keto-3-methylvaleric acid (KMV), a site-specific inhibitor for KGDH, nearly abolished mH₂O₂ generation in both male and female liver mitochondria oxidizing palmitoyl-carnitine. KMV inhibited mH₂O₂ production in liver mitochondria from male and female mice oxidizing myristoyl-, octanoyl-, or butyryl-carnitine as well. S1QEL 1.1 (S1) and S3QEL 2 (S3), compounds that inhibit reactive oxygen species generation by complexes I and III, respectively, without interfering with OxPhos and respiration, had a negligible effect on the rate of mH₂O₂ production when pyruvate or acyl-carnitines were used as fuels. However, inclusion of KMV in reaction mixtures containing S1 and/or S3 almost abolished mH₂O₂ generation. Together, our findings suggest KGDH is the main mH₂O₂ generator in liver mitochondria, even when fatty acids are used as fuel.