Mitochondrial Glutathione Depletion Reveals A Novel Role For Pyruvate Dehydrogenase As A Key H2o2 Emitting Source

Pyruvate dehydrogenase (PDH) is one of the seven potential sites for O 2 −• generation within mammalian mitochondria; however its role in contributing to H 2 O 2 emission in vivo has historically been viewed as negligible. To determine if pyruvate‐stimulated H 2 O 2 emission is sensitive to an oxidative shift in redox environment, saponin permeabilized fibers were prepared from red gastrocnemius muscle of rats in the presence of varying concentrations of the glutathione (GSH) depleting agent 1‐chloro‐2,4‐dinitrobenzene (CDNB). Compared to GSH‐replete fibers, an exponential increase in pyruvate‐stimulated H 2 O 2 emission was observed following a 25% depletion in GSH (Ctrl 1.08 ± 0.11, CDNB 61.36 ± 12.34; pmoles/min/mg, mean ± SE), with peak rates of emission reaching 265.65 ± 19.68 in maximally (98%) depleted fibers. This striking sensitivity to GSH depletion was specific to PDH as GSH depletion had minimal effect on H 2 O 2 emission in fibers energized with glutamate, α‐ketoglutarate, or succinate alone. Pyruvate‐stimulated H 2 O 2 emission in GSH‐depleted fibers was significantly lower in the presence of the PDH kinase inhibitor dichloroacetic acid (− DCA 273.90 ± 22.14, + DCA 51.76 ± 4.55; P<0.001), implying that pyruvate‐stimulated O2 −• is sensitive to PDH phosphorylation status. These data suggest PDH may be a significant source of H 2 O 2 production when the mitochondrial redox environment is in a more oxidized state.