Induction of mitochondrial reactive oxygen species production by GSH mediated S-glutathionylation of 2-oxoglutarate dehydrogenase

2-Oxoglutarate dehydrogenase (Ogdh) is an important mitochondria redox sensor that can undergo S-glutathionylation following an increase in H(2)O(2) levels. Although S-glutathionylation is required to protect Ogdh from irreversible oxidation while simultaneously modulating its activity it remains unknown if glutathione can also modulate reactive oxygen species (ROS) production by the complex. We report that reduced (GSH) and oxidized (GSSG) glutathione control [Formula: see text] /H(2)O(2) formation by Ogdh through protein S-glutathionylation reactions. GSSG (1 mM) induced a modest decrease in Ogdh activity which was associated with a significant decrease in [Formula: see text] /H(2)O(2) formation. GSH had the opposite effect, amplifying [Formula: see text] /H(2)O(2) formation by Ogdh. Incubation of purified Ogdh in 2.5 mM GSH led to significant increase in [Formula: see text] /H(2)O(2) formation which also lowered NADH production. Inclusion of enzymatically active glutaredoxin-2 (Grx2) in reaction mixtures reversed the GSH-mediated amplification of [Formula: see text] /H(2)O(2) formation. Similarly pre-incubation of permeabilized liver mitochondria from mouse depleted of GSH showed an approximately ~3.5-fold increase in Ogdh-mediated [Formula: see text] /H(2)O(2) production that was matched by a significant decrease in NADH formation which could be reversed by Grx2. Taken together, our results demonstrate GSH and GSSG modulate ROS production by Ogdh through S-glutathionylation of different subunits. This is also the first demonstration that GSH can work in the opposite direction in mitochondria-amplifying ROS formation instead of quenching it. We propose that this regulatory mechanism is required to modulate ROS emission from Ogdh in response to variations in glutathione redox buffering capacity.