Phenazine Methosulfate Rewires Mitochondrial Redox Circuits to Restore Membrane Potential and ATP Synthesis Under ETC...

Mitochondrial electron transport chain (ETC) dysfunction is a major driver of bioenergetic failure, redox imbalance, and drug toxicity, yet strategies to restore oxidative phosphorylation under ETC blockade remain limited. Redox-active small molecules could, in principle, shuttle electrons from NADH to distal ETC components and oxygen, thereby modulating both respiration and reactive oxygen species (ROS) formation. Here, we show that the enzyme-independent redox cycler phenazine methosulfate (PMS) rewires mitochondrial redox circuits and restores respiration in human glioblastoma cells and cell-free systems under ETC inhibition. At subtoxic concentrations, PMS acutely increased oxygen consumption and mitochondrial superoxide generation via NADH-PMS-O₂ redox cycling, while restoring mitochondrial membrane potential and ATP synthesis under ETC blockade, and shifting metabolism away from glycolytic lactate production. This profile is consistent with a protective redox-bypass role, distinct from the pro-apoptotic effects reported following high-dose, prolonged PMS exposure. The PMS-driven restoration of electron flow, mitochondrial membrane potential, and respiratory ATP synthesis under inhibition of Complex I (rotenone), III (antimycin A and myxothiazol), and/or IV (cyanide) is consistent with direct cytochrome c reduction, as demonstrated herein, and engagement of multiple ETC redox centers, including coenzyme Q₁₀. In metformin-treated cells, PMS reversed suppression of respiration and lactate accumulation, outperforming existing redox-bypass strategies. These findings identify PMS-driven redox cycling as a previously unrecognized chemical redox-bypass mechanism that both regenerates mitochondrial bioenergetics and reshapes ROS production, suggesting a potential approach to counteract drug- and toxin-induced mitochondrial dysfunction and to exploit redox vulnerabilities in cancer.