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.