An update on methods and approaches for interrogating mitochondrial reactive oxygen species production

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.