Cookies help us deliver our services. By using our services, you agree to our use of cookies. More information

Mailloux 2021 Redox Biol

From Bioblast
Publications in the MiPMap
Mailloux RJ (2021) An update on methods and approaches for interrogating mitochondrial reactive oxygen species production. Redox Biol 45:102044. https://doi.org/10.3390/antiox9060472

» PMID: 34157640 Open Access

Mailloux RJ (2021) Redox Biol

Abstract: The chief ROS formed by mitochondria are superoxide (O2·-) and hydrogen peroxide (H2O2). Superoxide is converted rapidly to H2O2 and therefore the latter is the chief ROS emitted by mitochondria into the cell. Once considered an unavoidable by-product of aerobic respiration, H2O2 is now regarded as a central mitokine used in mitochondrial redox signaling. However, it has been postulated that O2·- can also serve as a signal in mammalian cells. Progress in understanding the role of mitochondrial H2O2 in signaling is due to significant advances in the development of methods and technologies for its detection. Unfortunately, the development of techniques to selectively measure basal O2·- changes has been met with more significant hurdles due to its short half-life and the lack of specific probes. The development of sensitive techniques for the selective and real time measure of O2·- and H2O2 has come on two fronts: development of genetically encoded fluorescent proteins and small molecule reporters. In 2015, I published a detailed comprehensive review on the state of knowledge for mitochondrial ROS production and how it is controlled, which included an in-depth discussion of the up-to-date methods utilized for the detection of both superoxide (O2·-) and H2O2. In the article, I presented the challenges associated with utilizing these probes and their significance in advancing our collective understanding of ROS signaling. Since then, many other authors in the field of Redox Biology have published articles on the challenges and developments detecting O2·- and H2O2 in various organisms [1-3]. There has been significant advances in this state of knowledge, including the development of novel genetically encoded fluorescent H2O2 probes, several O2·- sensors, and the establishment of a toolkit of inhibitors and substrates for the interrogation of mitochondrial H2O2 production and the antioxidant defenses utilized to maintain the cellular H2O2 steady-state. Here, I provide an update on these methods and their implementation in furthering our understanding of how mitochondria serve as cell ROS stabilizing devices for H2O2 signaling. Keywords: Methods for measuring ROS, Mitochondria, Peroxide detectors, Reactive oxygen species, Superoxide probes Bioblast editor: Reiswig R

Selected quotes and comments

Communicated by Gnaiger E (2022-12-19)
  • Section 2.1: Fuel oxidation, chemiosmotic coupling, and oxidative phosphorylation (OXPHOS) rely on electron transferring redox active centers embedded in mitochondrial dehydrogenases and multi-subunit complexes inserted in the mitochondrial inner membrane.
Comment: Chemiosmotic coupling and OXPHOS rely not only on electron transferring redox active centers (in the electron transfer system ETS of the mitochondrial inner membrane mtIM) but on the posphorylation system including the F1FO-ATPase.
  • The concentration of O2 in air-saturated solution is ~200 μM whereas it has been estimated to occur at ~3 μM inside mitochondria [19: Murphy MP (2009) How mitochondria produce reactive oxygen species. Biochem J 417:1-13. https://doi.org/10.1042/BJ20081386]. This discrepancy has caused some to question if mitochondria truly are important ROS sources given that [O2]cytoplasm > [O2]matrix.
Comment: The major 'driver' for the lower [O2] relevant for mitochondria in their intracellular environment in the living organism compared to the concentration of O2 in air-saturated solution is the respiratory cascade from nose/lung to mitochondria. [O2]matrix may not be relevant, since the oxygen concentration in the lipid phase of the mtIM is higher at identical partial pressure.
  • the proximal ROS formed and emitted from mitochondria to the cell is H2O2.
  • Section 3: most of the studies have focused exclusively on using isolated mitochondria. This approach limits the physiological relevance of the results collected since estimation of O2.-/H2O2 production is being conducted in the presence of atmospheric O2 levels.
  • Studies using permeabilized muscle fibers and the Oroboros O2k system have been conducted, providing some important physiologically relevant information on ROS production by the “unconventional” sources. .. Additionally, new approaches need to be developed to measure the rate of ROS production by the 12 sites in a cellular context. This could either be with using permeabilized fibers or tissues in the O2k system or developing genetically modified cell lines that allow for the interrogation of ROS production by these sites.
Comment: It is important to note that the use of atmospheric O2 levels imposes hyperoxic conditions not only on isolated mitochondria (imt) but on isolated living cells (including genetically modified cell lines) and permeabilized cells. It is thus not a feature of imt, but application of tissue-specific physiological intracellular oxygen regimes in studies with imt is simple and is imperative when aiming at investigating physiological H2O2 production. Implementing physiologically relevant oxygen regimes in permeabilized fibers, however, is nearly impossible due to uncontrollable oxygen diffusion gradients in fiber bundles after disruption of microcirculation. See: Gnaiger E (2003) Oxygen conformance of cellular respiration. A perspective of mitochondrial physiology. https://doi.org/10.1007/978-1-4419-8997-0_4


Labels: MiParea: Respiration 

Stress:Oxidative stress;RONS 




HRR: Oxygraph-2k