Glutamine Metabolism Links ATP-Dependent Complex IV Regulation to Oxidative Stress in the Heart

All articles of this category (opens in new window) Background: Cytochrome c oxidase (complex IV of the mitochondrial respiratory chain, CIV) is the gatekeeper of oxidative phosphorylation and a key regulator of cellular energy supply. By allosteric ATP-inhibition, CIV precisely adjusts oxygen consumption to match the cellular energy demand, thereby limiting the formation of excessive reactive oxygen species (ROS). Disruption of this regulatory mechanism leads to mitochondrial dysfunction and oxidative stress, both implicated in cardiovascular and metabolic diseases. Given that amino acid metabolism influences the ATP/ADP ratio and redox state, we examined its impact on the ATP-dependent regulation of CIV. Methods: CIV activity was measured in intact rat heart mitochondria using Seahorse extracellular flux analysis. Sequential injections of reduced cytochrome c stimulated electron transfer between complexes III and IV, resulting in changes of the oxygen consumption rate (OCR) that served as a readout for CIV activity. In parallel, mitochondrial ROS release (MitoSOX) and membrane potential (TMRE) were quantified by flow cytometry. To explore metabolic regulation, selected TCA cycle metabolites and inhibitors of complexes I and II were applied to assess their impact on ATP-dependent CIV inhibition. Results: ATP dose-dependently suppressed CIV activity, reduced OCR, and lowered ROS release while increasing membrane potential. ADP showed opposite effects, restoring OCR and elevating ROS. Complex I inhibition with rotenone enhanced the ATP-mediated suppression, but strikingly increased ROS formation, whereas complex II inhibition with malonate attenuated ATP-dependent CIV activity without major changes in ROS release. Among TCA intermediates, α-ketoglutarate and glutamate weakened the ATP-mediated effects on OCR ( p < 0.001), causing enhanced ROS production ( p < 0.05). In contrast, succinate uniquely abolished ATP-dependent CIV regulation ( p < 0.001), maintaining high OCR under ATP-rich conditions without affecting ROS. Conclusion: Our findings demonstrate that individual TCA cycle substrates differentially modulate CIV activity, thereby shaping mitochondrial respiration, ROS generation, and membrane potential. Such substrate-specific regulations provide a mechanistic link between metabolic state and mitochondrial oxidative stress in the heart, and highlight CIV as a potential target for metabolic interventions in cardiovascular disease. Publication History Article published online: 17 February 2026 © 2026. Thieme. All rights reserved. Georg Thieme Verlag KG Oswald-Hesse-Straße 50, 70469 Stuttgart, Germany