Thiol-based antioxidants elicit mitochondrial oxidation via respiratory complex III

Excessive oxidation is widely accepted as a precursor to deleterious cellular function. However, the role of reductive stress as a similar pathological insult is only beginning to be understood. Prior research has shown that glutathione (GSH) redox potentials are critical for cellular homeostasis. Yet, the dynamic response of compartmentalized GSH to exogenous antioxidants remains unclear. No prior work had resolved how mitochondrial GSH responds to thiol-based compounds. This gap motivated the current investigation into the effects of thiol-based antioxidants on mitochondrial redox states. The cytosolic and mitochondrial compartments were known to differ in their redox regulation. However, the specific mechanisms linking exogenous thiols to mitochondrial oxidation were not established. This uncertainty drove the need to explore the early dynamic changes in GSH redox potentials in real time. The study aimed to clarify whether mitochondrial oxidation could be triggered by exogenously supplied thiol-based antioxidants.

The purpose of the study was to investigate the early dynamic changes in compartmentalized glutathione (GSH) redox potentials in response to exogenously supplied thiol-based antioxidants. The specific problem addressed was the lack of understanding regarding how mitochondrial redox states are affected by these compounds. The motivation stemmed from the emerging awareness of reductive stress as a pathological insult. The study sought to determine whether mitochondrial oxidation could be induced by GSH ethyl ester or N-acetyl-l-cysteine. It also aimed to identify the mechanisms through which these antioxidants influence mitochondrial redox dynamics. The researchers proposed that mitochondrial oxidation could be a consequence of respiratory complex III activity. By monitoring intracellular thiol-disulfide exchange, the study aimed to provide new insights into the functional link between exogenous thiols and mitochondrial respiration. The findings could help clarify the role of thiol-based antioxidants in cellular redox regulation.

The study employed a genetically encoded biosensor to monitor intracellular thiol-disulfide exchange in real time. The biosensor was specifically targeted to both the cytosol and mitochondria. This allowed for noninvasive tracking of glutathione (GSH) redox potentials in living cells. The researchers used GSH ethyl ester and N-acetyl-l-cysteine as exogenous thiol-based antioxidants. They observed the effects of these compounds on mitochondrial matrix oxidation. The experiments were conducted in a concentration-dependent manner to assess dose-response relationships. A membrane-permeable ROS scavenger, tiron, was used to evaluate the role of reactive oxygen species in the observed effects. The study also involved systematic inhibition of mitochondrial respiratory chain complexes to identify downstream targets of thiol-based compounds.

The administration of GSH ethyl ester or N-acetyl-l-cysteine led to rapid oxidation of the mitochondrial matrix. This oxidation occurred within seconds and was concentration-dependent. The cytosolic sensor did not show a similar response to these treatments. The mitochondrial oxidation was attenuated when tiron was used as a ROS scavenger. Surprisingly, depolarization of mitochondrial membrane potential did not prevent the oxidation. Inhibition of mitochondrial GSH uptake also failed to abrogate the effect. The study found elevated levels of mitochondrial ROS following treatment with thiol-based antioxidants. Systematic inhibition of respiratory chain complexes revealed that complex III was a downstream target of these compounds. Disabling complex III with myxothiazol completely blocked the oxidation induced by GSH ethyl ester or N-acetyl-l-cysteine. These findings suggest a direct link between exogenous thiols and mitochondrial respiration.

The study provides new evidence of a functional link between exogenous thiol-based antioxidants and mitochondrial respiration. The authors propose that these compounds can trigger rapid oxidation of the mitochondrial matrix. This oxidation occurs within seconds and is concentration-dependent. The cytosolic compartment did not respond similarly to the same treatments. The researchers suggest that the observed oxidation is mediated by respiratory complex III. Disabling this complex with myxothiazol completely blocked the oxidation effect. The findings indicate that mitochondrial ROS levels are elevated in response to thiol-based antioxidants. The study highlights the importance of monitoring intracellular redox states in real time. The authors emphasize the need for further research into the mechanisms underlying this functional link. These results may have implications for understanding the effects of antioxidant therapies on mitochondrial function.