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Are mitochondria a spontaneous and permanent source of reactive oxygen species?
Hans Nohl1, Lars Gille, Andrey Kozlov
1Fundamental Pharmacology and Toxicology, University of Veterinary Medicine Vienna, Vienna, Austria. hans.nohl@vu-wien.ac.at
Abstract:
The bioenergetic properties of mitochondria in combination with the high turnover rate of dioxygen qualify these organelles for the formation of reactive oxygen species (ROS). The assumption that mitochondria are the major intracellular source of ROS was essentially based on in vitro experiments with isolated mitochondria. The transfer of these data to the living cell may, however, be incorrect. Artefacts due to the preparation procedure or inadequate detection methods of ROS may lead to false positive results. Inhomogeneous results were found to be due to an interaction of the detection system with components of the respiratory chain which could be avoided by a recently developed non-invasive method. One of the most critical electron transfer steps in the respiratory chain is the electron bifurcation from ubiquinol to the cytochrome bc(1) complex. This electron bifurcation requires the free mobility of the head domain of the Rieske iron-sulfur protein. Inhibition of electron bifurcation by antimycin A causes leakage of single electrons to oxygen which results in the release of ROS. Hindrance of electron bifurcation was also observed following alterations of the physical state of membrane phospholipids in which the cytochrome bc(1) complex is inserted. Irrespective of whether the fluidity of the membrane was elevated or decreased, electron flow rates to the Rieske iron-sulfur protein were drastically reduced. Concomitantly superoxide radicals were released from these mitochondria, strongly suggesting the involvement of the ubiquinol/cytochrome bc(1) redox couple in this process.
Insights
Mitochondria generate reactive oxygen species (ROS), but in vitro studies may overstate their role. Non-invasive methods reveal the ubiquinol/cytochrome bc1 complex
Area of Science:
- Mitochondrial bioenergetics
- Cellular redox signaling
- Reactive oxygen species (ROS) production
Background:
- Mitochondria are implicated as major intracellular sources of reactive oxygen species (ROS).
- Previous studies relied on in vitro experiments with isolated mitochondria, potentially leading to artefacts.
- In vivo ROS detection in living cells is challenging due to methodological limitations.
Purpose of the Study:
- To investigate the role of mitochondria in ROS formation using a non-invasive method.
- To identify specific sites and mechanisms of ROS production within the mitochondrial respiratory chain.
- To assess the impact of membrane properties on mitochondrial ROS generation.
Main Methods:
- Utilized a recently developed non-invasive method to detect ROS in mitochondria.
- Investigated the electron bifurcation step from ubiquinol to the cytochrome bc1 complex.
- Examined the effect of antimycin A on electron bifurcation and ROS release.
- Altered the physical state (fluidity) of membrane phospholipids surrounding the cytochrome bc1 complex.
Main Results:
- A non-invasive method minimized artefacts associated with ROS detection.
- Inhibition of electron bifurcation at the ubiquinol/cytochrome bc1 complex by antimycin A led to ROS release.
- Altering membrane phospholipid fluidity, either increasing or decreasing it, reduced electron flow to the Rieske iron-sulfur protein.
- Reduced electron flow correlated with increased superoxide radical release from mitochondria.
Conclusions:
- The ubiquinol/cytochrome bc1 redox couple is critically involved in mitochondrial ROS production.
- Mitochondrial membrane properties significantly influence electron transfer and ROS generation.
- Non-invasive techniques are crucial for accurate assessment of mitochondrial ROS in vivo.