REVIEWS ROS as signalling molecules: mechanisms that generate specificity in ROS homeostasis Benoît D’Autréaux and Michel B. Toledano Abstract | Reactive oxygen species (ROS) have been shown to be toxic but also function as signalling molecules. This biological paradox underlies mechanisms that are important for the integrity and fitness of living organisms and their ageing. The pathways that regulate ROS homeostasis are crucial for mitigating the toxicity of ROS and provide strong evidence about specificity in ROS signalling. By taking advantage of the chemistry of ROS, highly specific mechanisms have evolved that form the basis of oxidant scavenging and ROS signalling systems. Iron–sulphur clusters Reactive oxygen species (ROS) is a collective term use of ROS sensors that ‘measure’ the intracellular Iron–sulphur clusters are metal that describes the chemical species that are formed concentration of ROS by a redox-based mechanism centres that consist of sulphide upon incomplete reduction of oxygen and includes the and proportionally set the expression of ROS-specific (S2–) and iron. The most – superoxide anion (O2 ), hydrogen peroxide (H2O2) and scavengers, thereby maintaining the concentration of common structures are the • diamond [2Fe–2S] and the hydroxyl radical (HO ). ROS are thought to mediate ROS below a toxic threshold. These pathways regulate the cubane [4Fe–4S] clusters. the toxicity of oxygen because of their greater chemical a physiological response that is fitted to a ROS signal, Iron–sulphur clusters are reactivity with regard to oxygen. They also operate as in which the ROS signal is the agonist and the sensor is usually coordinated by Cys or intracellular signalling molecules, a function that has a ROS-specific receptor. His residues at the iron atoms. been widely documented but is still controversial. This In this Review, we discuss the redox mechanisms of scepticism stems from the apparent paradox between such pathways in microbial models and their potential the specificity that is required for signalling and the equivalents in mammals. We focus on pathway speci- reactive nature of ROS that renders them indiscrimi- ficity and on the mechanistic features that generate nate and potentially lethal oxidants. Specificity in specificity. Finally, we compare these mechanisms with signalling is achieved through the non-covalent other types of ROS signalling pathways. binding of a ligand to its cognate receptor by virtue of the complementarity of macromolecular shapes. By ROS chemistry sets target specificity contrast, ROS operate in signalling through chemical Chemical reactivity distinguishes ROS from other reactions with specific atoms of target proteins that signalling molecules (BOX 1; BOX 2). ROS have distinct lead to covalent protein modifications1. Therefore, ROS biological properties, which include chemical reactivity, molecular recognition occurs at the atomic and not half-life and lipid solubility2. HO• has indiscriminate at the macromolecular level, which necessarily expands reactivity towards biological molecules, whereas – the potential number of ROS-specific receptors because O2 and H2O2 each have preferred biological targets. CEA, IBITECS, SBIGEM, the atomic targets of ROS are the amino-acid building This preference is best exemplified by the striking Laboratoire Stress Oxydants blocks of numerous proteins. So, how is specificity early observation of the use of the redox-sensitive et Cancer, Batiment 142, Commissariat à l’Energie achieved in ROS signalling? transcription factors SoxR and OxyR, which enable Atomique-Saclay, To provide answers to this question, we consider Escherichia coli to discriminate and regulate distinct 91191 Gif-sur-Yvette, France. particular ROS signalling pathways that control ROS responses towards ROS (see below). Such chemical dis- Correspondence to M.B.T. intracellular homeostasis. Studies of these pathways crimination is a hallmark of the high atomic reactivity e-mail have provided (and are still providing) the fundamental of O – with iron–sulphur clusters ([Fe–S] clusters), which [email protected] 2 doi:10.1038/nrm2256 molecular principles of ROS-based redox regulation constitute the SoxR redox centre, and of the reactivity Published online that constitute solid demonstrations of high specificity of H2O2 with Cys residues, which constitute the OxyR 12 September 2007 in ROS signalling. These pathways generally make redox centre. NATURE REVIEws | MOLECULAR CELL BIOLOGY VOLUME 8 | octobER 2007 | 813 © 2007 Nature Publishing Group REVIEWS Box 1 | ROS sources and biochemical properties The Cys residue is ideally suited for reacting with per- H2O2; it constitutes the catalytic centre of the thiol oxidase [Fe–S] cluster (oxidation) class of H2O2 scavengers and the main regulatory target of H2O2. With the exception of the transcription factor peroxide operon regulator (PerR; a member of the [Fe–S] cluster (Fe release) – 0.16 V O2 Fur family), which uses an iron centre, all characterized Radical (NO•, semiquinones) e– 4e– H2O2-specific regulatory mechanisms involve the oxida- Flavin, quinone Respiration tion of unique Cys residues by H2O2. Two unique chemical – O2 NADPH oxidase H2O features endow the Cys residue with its redox-regulatory properties: the exquisite H2O2 chemical reactivity it can Flavin, quinone reach (depending on its protein context), and its ability e– SOD (BOX 2) + 0.94 V [Fe–S] cluster + 2.33 V to cycle between different stable redox forms . Cys e– residues are not equal in their ability to undergo redox ROS targets ROS origins modifications4–6, which provides the basis for selectiv- ity and specificity7–9. For example, it is often overlooked • H2O2 Fenton reaction HO (Fe2+, Cu+) that the low molecular weight thiol glutathione (GSH) 5 e– cannot react with ROS in vivo ; it only participates in [Fe–S] cluster (Fe release) Lipid (peroxidation) peroxide scavenging by assisting glutathione peroxi- Metallo-enzyme (oxidation) DNA (oxidation) Cys, Met (oxidation) Myeloperoxidase + 0.46 V Amino acid (oxidation) dases (GPXs) or by forming S-glutathionylated adducts CI– with protein-sulphenic acids (SOHs). Selective reactivity explains why the Cys residue is not a main target of H2O2 HOCI toxicity (BOX 1). Indeed, proteome-wide identification of oxidized protein thiols shows that H O does not cause Although molecular oxygen is a di-radical and rather unreactive, its univalent reduction 2 2 Nature Reviews | Molecular Cell Biology random protein thiol oxidation10. leads to the formation of chemically more reactive species (known as reactive oxygen – Cys can also serve in redox regulation by coordinating species; ROS). These are the superoxide anion (O2 ), hydrogen peroxide (H2O2) and the hydroxyl radical (HO•). Because of their intrinsic chemical properties, each ROS reacts zinc, which provides a redox control of metal binding and 11–13 with preferred biological targets (coloured boxes; see figure). As a rule, ROS reactivity metal control of Cys redox reactivity . As a Lewis dictates toxicity while decreasing signalling ability. acid, zinc lowers the pKa of its coordinating thiol and – O2 is a by-product of respiration and is produced by NADPH oxidases. Due to high potentially modifies its reactivity. Reciprocally, Cys oxi- – attraction, O2 oxidizes iron–sulphur ([Fe–S]) clusters at a rate that is almost diffusion- dation leads to zinc release, which results in a change of – limited, and releases iron. O2 can react with thiols in vitro, but the slow reaction rates conformation that alters protein function. 5 mean that this cannot occur in vivo . In Escherichia coli, the steady-state Met residues are another H O target and can be oxidi- concentration of O – is very low (~10–11 M)2, which reflects its instability; this is not only 2 2 2 zed at the sulphur atom to form methionine sulphoxide; due to its reaction with the [Fe–S] cluster, but also to spontaneous and superoxide- however, the slow reaction rates are not compatible dismutase-mediated O – dismutation to H O . The instability of O – and its inability to 2 2 2 2 (BOX 1) diffuse through membranes because of its negative charge make this ROS a poor with an in vivo regulatory function . By contrast, signalling molecule. selenocysteine, which is more reactive with H2O2 than • Cys, may operate in H O -based redox regulation; for H2O2 toxicity is essentially the consequence of its reduction to HO by metal- 2 2 3 catalysed Fenton chemistry . H2O2 is actually a poor oxidant and reacts mildly with example, the thiol oxidase function of the selenothiol- [Fe–S] (rate constant of 102–103 M–1.s–1) and loosely bound metals (103–104 M–1.s–1), based hydroperoxide glutathione peroxidase GPX4 and very slowly with glutathione and free Cys (2–20 M–1.s–1) and with Met residues (REFS 14,15). (10–2 M–1.s–1)3,5. By contrast, its reactivity towards Cys residues can significantly increase to 10–106 M–1.s–1 depending on the protein environment (BOX 2). As a Prokaryotic ROS homeostatic pathways corollary, H O is relatively stable (cellular half-life ~1 ms, steady-state levels ~10–7 M). 2 2 Prokaryotic pathways of ROS signalling constitute pre- Diffusion of H O might be modulated by changes in membrane permeability or by 2 2 cise, fast and highly dynamic adjustment mechanisms transport through aquaporins114. Its selective reactivity and diffusibility makes H O 2 2 (FIG. 1) fit for signalling. of intracellular ROS homeostasis . A key feature The highly toxic HO• has high indiscriminate reactivity, which limits its diffusion to is feedback regulation: this originates from the nature of –9 2 • the pathway outputs, namely, ROS scavengers that extin- sites of production (half-life 10 s) . Despite this, HO seems to operate in H2O2 sensing. guish ROS input signals. Tracking pathway outputs and physiological function is key to establishing the exact nature of the chemical(s) that are sensed, the knowledge – The avidity of O2 for [Fe–S] clusters is a consequence of which is essential to understand the specificity of a of its high electrostatic attraction, which is not seen with redox mechanism.