Thiol-based antioxidants elicit mitochondrial oxidation via respiratory complex III

Abstract

Excessive oxidation is widely accepted as a precursor to deleterious cellular function. On the other hand, an awareness of the role of reductive stress as a similar pathological insult is emerging. Here we report early dynamic changes in compartmentalized glutathione (GSH) redox potentials in living cells in response to exogenously supplied thiol-based antioxidants. Noninvasive monitoring of intracellular thiol-disulfide exchange via a genetically encoded biosensor targeted to cytosol and mitochondria revealed unexpectedly rapid oxidation of the mitochondrial matrix in response to GSH ethyl ester or N-acetyl-l-cysteine. Oxidation of the probe occurred within seconds in a concentration-dependent manner and was attenuated with the membrane-permeable ROS scavenger tiron. In contrast, the cytosolic sensor did not respond to similar treatments. Surprisingly, the immediate mitochondrial oxidation was not abrogated by depolarization of mitochondrial membrane potential or inhibition of mitochondrial GSH uptake. After detection of elevated levels of mitochondrial ROS, we systematically inhibited multisubunit protein complexes of the mitochondrial respiratory chain and determined that respiratory complex III is a downstream target of thiol-based compounds. Disabling complex III with myxothiazol completely blocked matrix oxidation induced with GSH ethyl ester or N-acetyl-l-cysteine. Our findings provide new evidence of a functional link between exogenous thiol-containing antioxidants and mitochondrial respiration.

Extending roGFP Emission via Förster-Type Resonance Energy Transfer Relay Enables Simultaneous Dual Compartment Ratiometric Redox Imaging in Live Cells

“…At the level of the population average, we observed that both the cytosol and mitochondrial matrix experienced an initial reduction in redox potential upon addition of NAC; however, a small oxidative rebound was observed in the mitochondria after several minutes, which was absent in the cytosol (Figure 4). Our population measurement is in agreement with the previous population measurements, and the smaller magnitude of the oxidative rebound likely reflects cell-type differences 40,42 . Furthermore, our measurements also reveal a reductant-specific difference in the stress response because, while NAC-induced stress causes an oxidative rebound in mitochondria only, DTT-induced stress causes an oxidative rebound in both the cytosol and the mitochondria.…”

“…In our initial redox studies (Figure 3), we observed that the Neuro2A cells exhibited an oxidative rebound following DTT addition during the final calibration phase, which indicates that Neuro2A cells respond to reductive stress. It has previously been observed that reductive stress causes a paradoxical oxidative response in HEK293, H9c2, and other cell types 40–42 . NAC is a cell-permeant reductant that increases the levels of cytosolic reduced glutathione, but NAC is mitochondrial-lyimpermeant 40,42 .…”

“…It has previously been observed that reductive stress causes a paradoxical oxidative response in HEK293, H9c2, and other cell types 40–42 . NAC is a cell-permeant reductant that increases the levels of cytosolic reduced glutathione, but NAC is mitochondrial-lyimpermeant 40,42 . Interestingly, NAC-induced reductive stress causes an oxidative response in mitochondria but not the cytosol 40,42 .…”

“…In order to quantify the cell-to-cell heterogeneity, we scored the difference in compartment-specific responses by measuring the ratio of the mito-roGFP1-mApple percent sensor oxidation to the cytosolic roGFP1 percent sensor oxidation, which reflects the change in mitochondrial redox relative to cytosolic redox upon NAC treatment (Figure 4). The heterogeneity reflects cell-to-cell differences in compartmentalized redox buffering capacity as well as cross-compartment redox coupling 9,40–42 . Thus, our roGFP-RFP FRET relay redox sensors enable differential compartment-specific dynamics to be quantified, showing that cytosolic stress can affect mitochondrial redox dynamics.…”

Distinct responses of compartmentalized glutathione redox potentials to pharmacologic quinones targeting NQO1

“…MitoSOX Red dye was used to analyze the extent of ROS generation by mitochondria via confocal microscopy as described previously [22]. …”