Mitochondrial glutathione depletion reveals a novel role for the pyruvate dehydrogenase complex as a key H2O2-emitting source under conditions of nutrient overload

Abstract

Once regarded as “byproducts” of aerobic metabolism, the production of superoxide/H2O2 is now understood to be a highly specialized and extensively regulated process responsible for exerting control over a vast number of thiol-containing proteins, collectively referred to as the redox-sensitive proteome. Although disruptions within this process, secondary to elevated peroxide exposure, have been linked to disease, delineation of the sources and mechanisms regulating increased peroxide burden remain poorly defined and as such difficult to target using pharmacotherapy. Here we identify the pyruvate dehydrogenase complex (PDC) as a key source of H2O2 within skeletal muscle mitochondria under conditions of depressed glutathione redox buffering integrity. Treatment of permeabilized myofibers with varying concentrations of the glutathione depleting agent 1-chloro-2,4-dinitrobenzene (CDNB) led to a dose-dependent increase in pyruvate-supported JH2O2 emission, with emission rates eventually rising to exceed those of all substrate combinations tested. This striking sensitivity to glutathione depletion was observed in permeabilized fibers prepared from multiple species and was specific to PDC. Physiological oxidation of the cellular glutathione pool following high fat feeding in rodents was found to elevate PDC JH2O2 emission, as well as increase the sensitivity of the complex to GSH depletion. These findings reveal PDC as a potential major site of H2O2 production that is extremely sensitive to mitochondrial glutathione redox status.

Progress in understanding the molecular oxygen paradox – function of mitochondrial reactive oxygen species in cell signaling

“…However, recent work by our group and others found that it serves as a GSH and GSSG sensor much like OGDH (O'Brien et al, 2017). Using purified PDH and permeabilized muscle fibers, Fisher-Wellman and coworkers found that depletion of glutathione pools or inhibition of NADPH production augmented pyruvate-driven O 2˙− / H 2 O 2 production (Fisher-Wellman et al, 2013. It was then postulated by the same group that PDH was able to sense changes in mitochondrial redox buffering capacity, potentially through S-glutathionylation, which alters ROS production (Fisher-Wellman et al, 2013).…”

“…Using purified PDH and permeabilized muscle fibers, Fisher-Wellman and coworkers found that depletion of glutathione pools or inhibition of NADPH production augmented pyruvate-driven O 2˙− / H 2 O 2 production (Fisher-Wellman et al, 2013. It was then postulated by the same group that PDH was able to sense changes in mitochondrial redox buffering capacity, potentially through S-glutathionylation, which alters ROS production (Fisher-Wellman et al, 2013). However, based on the experimental design it was unclear whether or not PDH was actually sensing any changes in mitochondrial redox buffering capacity or if the increase was simply due to the CDNB-mediated depletion of glutathione pools.…”

Lipoic acid metabolism and mitochondrial redox regulation

“…This glutathionylation can increase ROS generation from the E3 subunit since FAD cannot be oxidized by the lipoic acid moiety and reduces O2 by one electron to produce superoxide. Importantly, glutathionylation decreases ROS production when pyruvate is being oxidized, and depletion of glutathione leads to increased ROS production from PDH (71). PDH has also been shown to interact with Grx2 suggesting that the reversible glutathionylation regulates PDH through similar mechanisms to OGDH (70).…”

The 2-Oxoacid Dehydrogenase Complexes in Mitochondria Can Produce Superoxide/Hydrogen Peroxide at Much Higher Rates Than Complex I

“…, branched-chain 2-oxoacid dehydrogenase (BCKDH) complex (22), and pyruvate dehydrogenase (PDH) complex (20,23). Proline dehydrogenase has also been implicated (24).…”

“…However, other sites within the matrix can also be important producers of superoxide/H 2 O 2 under these conditions (19,20,23). Together with site I F , these matrix sites (including the OGDH, BCKDH, and PDH complexes) comprise the NADH/NAD ϩ isopotential group, i.e.…”