Chemistry and biology of reactive oxygen species in signaling or stress responses

Abstract

Reactive oxygen species (ROS) are a family of molecules that are continuously generated, transformed and consumed in all living organisms as a consequence of aerobic life. The traditional view of these reactive oxygen metabolites is one of oxidative stress and damage that leads to decline of tissue and organ systems in aging and disease. However, emerging data show that ROS produced in certain situations can also contribute to physiology and increased fitness. This Perspective provides a focused discussion on what factors lead ROS molecules to become signal and/or stress agents, highlighting how increasing knowledge of the underlying chemistry of ROS can lead to advances in understanding their disparate contributions to biology. An important facet of this emerging area at the chemistry-biology interface is the development of new tools to study these small molecules and their reactivity in complex biological systems.

Figure 1. Reactions of primary ROS with functional groups on proteins

A one-electron reduction of molecular oxygen, either from the electron transport chain (ETC) or through the action of NADPH oxidases (Nox), yields superoxide ([O₂]^•−). Superoxide is converted to hydrogen peroxide (H₂O₂) by superoxide dismutase (SOD) or by dismutation in aqueous solution. H₂O₂ can react with various functional groups; for example, this ROS can oxidize cysteine residues to form sulfenic acids or histidine residues to form 2-oxo-histidines. Sulfenic acids can then go on to form disulfide bonds or be further oxidized to sulfinic and then sulfonic acids by a second and third equivalent of H₂O₂, respectively. H₂O₂ can also be converted to hydroxy radical ([OH]^•) by catalysis with redox-cycling metals such as Fe²⁺ and Cu²⁺, which can then oxidize functional groups such as methionine residues to form methionine sulfoxides or other amino acids such as lysine, arginine, proline and histidine to form protein carbonyls. The enzyme myeloperoxidase (MPO) can convert H₂O₂ to the highly reactive hypochlorous acid (HOCl), which can oxidize cysteine residues to form sulfenic acids or tyrosine residues to form chlorotyrosine. Oxidized products in blue are those with known pathways to reverse the redox modification, whereas those products highlighted in red are thought to be irreversibly oxidized.

Figure 2. Potential layers of regulation for membrane-localized H₂O₂ signaling

Receptor activation, often by growth factors (GF) or other ligands, leads to superoxide ([O₂]^•−) generation at the cellular membrane by Nox proteins, with subsequent production of H₂O₂ by dismutation or action of SOD. H₂O₂ can then pass through specific aquaporins (AQP) to reach the intracellular cytosol. Concomitantly, receptor activation also leads to localized Prx1 phosphorylation and deactivation, decreasing the redox-buffering capacity near the cell membrane. Localized rises in intracellular H₂O₂ levels can cause further deactivation of Prx2 by overoxidation. These various points of regulation can work together to lead to transient rises in H₂O₂ concentrations and the subsequent oxidation of local redox targets.

Figure 3. ROS signaling in physiology

(a) ROS have recently been discovered as second-messenger signaling agents used to control growth and maintenance of neural stem cells located in both the subgranular zone of the hippocampus as well as the subventricular zone of the lateral ventricles. (b) ROS have also been discovered as signaling agents at both the biochemical and whole-organism level to trigger chemotaxis and recruitment of leukocytes to damaged tissue. (c) Finally, the oxidation state of peroxiredoxins (Prx) have been shown to be modulated between reduced (Prx-SH) and oxidized (Prx-SO₂H) forms to regulate circadian rhythms in the absence of transcription or translation.

Figure 4. Chemical tools to study redox biology

(a) The conversion of boronates to phenols by H₂O₂ has been used to create a suite of novel fluorescent probes with various properties, such as red-shifted emission (Peroxy Orange 1, PO1), mitochondrial localization (Mitochondria Peroxy Yellow 1, MitoPY1) and enhanced sensitivity through cytosolic trapping groups (Peroxyfluor-6 acetoxymethyl ester, PF6-AM). (b) A mitochondrial-targeted MS probe, which similarly uses the conversion of a boronic acid to a phenol, allows ratiometric detection and quantification of H₂O₂ in vivo by analysis of the ion count ratios between the protected and deprotected form of the probe, which can be distinguished by differences in mass to charge (m/z) ratios. (c) Dimedone-based reactivity probes can trap oxidized cysteine residues from a sulfenic acid and when coupled to purification or labeling groups, allow the identification of the redox-modified target.