ROS as Signalling Molecules?

Scientists have long had evidence that ROS and other free radicals are toxic substances that are the cause of diseases and aging (Stadtman, et al., 2008). This led, perhaps inescapably, to the idea that ROS are solely harmful to the cell, the bad guys if you will, and antioxidants which serve to limit their destruction as the good guys. However, recent discoveries have shown evidence that ROS could also act as signalling molecules (Hancock, Desikan, & Neill, 2001) and their production within the cell is necessary in its normal physiological function. In order to act as a signalling molecule, target specificity is needed. However, ROS is well known for its damaging effects; specificity is the last thing in mind when ROS is considered. To resolve this apparent contradiction, the biochemistry of ROS must be reconsidered.

ROS Biochemistry The ROS family consist of three different types of molecules, namely superoxide (O 2-), hydrogen peroxide (H2O2), and the hydroxyl radical (OH). These are generally produced by electron transfer reactions, which can be accidentally or deliberately mediated by enzymatic or nonenzymatic reactions. Due to electron spin restrictions, oxygen is commonly reduced by adding electrons one at a time. Thus, the reduction of O2 to 2H2O requires four electrons, and this univalent pathway creates intermediates which are more reactive than O 2 itself, and these intermediates of oxygen reduction are commonly termed as reactive oxygen species (ROS). O2 + e- O2O2- + e-+ 2H+ H2O2 H2O2 + e- +H+ H2O + OH OH + e- + H+ H2O Superoxide and hydroxyl radical possesses an unpaired electron in their valence shell, and hence are termed free radicals. Hydrogen peroxide lacks an unpaired electron and is not a free radical.

Superoxide The superoxide anion created from molecular oxygen by the addition of an electron is, in spite of being a free radical, not highly reactive (Faraggi & Houee-Levin, 1999). Most of its chemical reactions are as a one-electron oxidant or one-electron reductant. This allows it to

react univalently with transition metal ions, changing the metal ions oxidation state, which could lead to inhibitory or damaging effects (Kono & Fridovic, 1982). The radical is produced in significant amounts intracellularly, both in the mitochondria, mainly due to the electron leakage from the respiratory chain and in cytosol via flavoenzymes, such as xanthine oxidase. Other superoxide-producing enzymes are lipoxygenase, cyclooxygenase and NADPH oxidase. In cells, superoxide molecules are rapidly disposed of by the superoxide dismutates (SOD) metalloenzymes family. The enzyme brings two superoxide molecules to react together to form hydrogen peroxide and oxygen. This is a dismutation reaction and is catalysed by the Superoxide Dismutates protein family at an extremely fast rate constant of 210 9 M-1 s-1 (Faraggi & Houee-Levin, 1999). SOD---M(n+1)+ + O2- SOD---Mn+ + O2 SOD---Mn+ + O2- + 2H+ SOD---M(n+1)+ + H2O2 where M = Cu (n=1) ; Mn (n=2) ; Fe (n=2) ; Ni (n=2).

Hydrogen peroxide Hydrogen peroxide can be generated from the two electron reduction of oxygen, and is not a free radical but an oxidizing agent that is not particularly reactive.

In cells, hydrogen peroxide is rapidly disposed of by a variety of enzymes such a catalase (Rate constant = 2.3107 M-1 s-1, (Strothera & Ackerman, 1961)), glutathione (GSH) peroxidase (Rate constant = 1108 M-1 s-1, (Floh, Loschen, Gnzler, & Eichele, 1972)) and peroxiredoxin (Prx(SH)2)(Rate constant = 1.3107 M-1 s-1, (Peskin, Low, Paton, Maghzal, & Hampton, 2007)). 2 H2O2
Catalase

2 H2O + O2

GSH 2 H2O + GSSG peroxidase 2 H2O2 + Prx(SH)2 Peroxiredoxin 2 H2O + PrxS2

2 H2O2 + 2 GSH

Hydroxyl Radical

The hydroxyl radical is an extremely reactive oxidizing radical that will react with most biomolecules including proteins, lipids and DNA in its vicinity at diffusion controlled rates. Therefore, it is probably more detrimental to biological systems than any other ROS. Hydroxyl radicals are usually produced in vivo from hydrogen peroxide in a reaction catalysed by metal ions (e.g., Fe2+ or Cu+), often bound in complex with different proteins (e.g., ferritin) or other molecules (Kehrer, 2000). This is known as the Fenton reaction: H2O2 + Fe2+ OH + OH- + Cu2+/Fe3+ Superoxide plays an important role in connection with the Fenton reaction by recycling the metal ions which are crucial in the formation of hydroxyl radicals, by reducing it. Fe3+ + O2- Cu+/ Fe2+ + O2 Together, these reactions are known as the Haber-Weiss reaction.

ROS Signalling The first free radical to be identified as a signalling molecule was Nitric Oxide (NO). Interest in this simple diatomic molecule first started when the endothelium-derived relaxing factor (EDRF), first proposed by Furchgott and Zawadski in 1980, (Furchgott & Zawadzki, 1980) was identified in 1987 by Palmer et al (Palmer, Ferrige, & Moncada, 1987) as NO. In 1992, interest was such that NO was voted "molecule of the year" by Science and earned Robert F. Furchgott, Louis J. Ignarro, and Ferid Murad the Nobel Prize in Physiology or Medicine in 1998 for their discoveries concerning "nitric oxide as a signalling molecule in the cardiovascular system." The possibility that a free radical could act as a signalling molecule raised a possibility that ROS could also act as a signalling molecule. Later evidence has shown that ROS can indeed function as a signalling molecule.

Physiologic/Homeostatic
Agonist or stimulus-coupled

Stress/Adaptive
May or may not be agonist or stimulus-coupled Often compartmentalized, but not a requirement May be targeted precisely, often to one protein that acts as the sensor and/or transducer of a response

Maladaptive/Injurious
Driven by exogenous agents or substances produced at pathophysiological levels Generally not compartmentalized Spatiotemporally promiscuous

Compartmentalized (spatiotemporally restricted) Coordinately regulated at multiple steps of the transduction pathways (multiple targets) Targeted precisely (highly substrate specific)

 Dynamic and reversible Operates on physiological time scales, usually milliseconds to minutes Examples: Regulation of trafficking of AMPA receptors, epidermal growth factor receptors and adrenergic receptors by S-nitrosylation (7, 8) Regulation of endothelial cell (9) and platelet granule exocytosis (10), and insulin secretion (11) by S-nitrosylation Regulation of ventilatory drive (12) by S-nitrosylation-based signaling

Often reversible, though not a necessary requirement Operates on lengthy time scales, usually hours to days Examples: Bacterial response to nitrosative and oxidative stresses by Snitrosylation and S-oxidation of OxyR, a prokaryotic transcription factor (13, 14) Anchorage and growth of transformed cells by ROSdependent Src oxidation (15) S-oxidation and S-nitrosylation of Tyr phosphatases by growth factor (16) and stress signals (17)

Often irreversible Timescale may range from acute to chronic depending on level of insult Examples: Neurodegeneration promoted by S-nitrosylation of protein disulfide isomerase (18) and Parkin, a ubiquitin-ligase (19), or by Tyr nitration of synuclein (20) Dysregulation of skeletal muscle 2+ Ca signaling by Tyr nitration and thiol oxidation of the sarcoplasmic 2+ reticulum Ca ATPase (21) Increases in Tyr nitration in ARDS (22) and other inflammatory disorders (23)

Table 1. The many effects of ROS on the cell (Forrester & Stamler, 2007).

REFERENCES
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Forrester, M. T., & Stamler, J. S. (2007). A Classification Scheme for Redox-Based Modifications of Proteins. American Journal of Respiratory Cell and Molecular Biology, 36: 135-137. Furchgott, R. F., & Zawadzki, J. V. (1980). The obligatory role of endothelial cells in the relaxation of arterial smooth muscle by acetylcholine. Nature, 228: 373376. Hancock, J., Desikan, R., & Neill, S. (2001). Role of reactive oxygen species in cell signalling pathways. Biochemical Society Transactions, 29: 345-350. Kehrer, J. P. (2000). The HaberWeiss reaction and mechanisms of toxicity. Toxicology, 149: 43-50. Kono, Y., & Fridovic, I. (1982). Superoxide Radical Inhibits Catalase. Journal of Biological Chemistry, 257: 5751-5754. Palmer, R., Ferrige, A., & Moncada, S. (1987). Nitric oxide release accounts for the biological activity of endothelium-derived relaxing factor. Nature, 327: 524 - 526. Peskin, A. V., Low, F. M., Paton, L. N., Maghzal, G. J., & Hampton, M. B. (2007). The high reactivity of peroxiredoxin 2 with H(2)O(2) is not reflected in its reaction with other oxidants and thiol reagents. Journal of Biological Chemistry, 282:11885-11892. Stadtman, E. R., Lou, M. F., Crabb, J. W., Rozanski, G. J., Veeramani, S., Lin, M.-F., et al. (2008). Pathological Processes Related to Redox. In R. Banerjee, D. Becker, M. Dickman, V. Gladyshev, & S. Ragsdale, Redox Biochemistry (pp. 183-225). New Jersey: Wiley. Strothera, G., & Ackerman, E. (1961). Physical factors influencing catalase rate constants. Biochimica et Biophysica Acta, 47: 317-326.