The biological chemistry of thiols:

1. The biological chemistry of thiols: reactions with biologically-relevant oxidants reactions of radicals formed on oxidation Peter Wardman University of Oxford, Gray Cancer Institute Supported by

2. Overview • Thiol oxidation products & reactivity of oxidants • thiol ionization a key property: pH-sensitive chemistry • non-enzyme-based oxidants are mainly radicals • enzyme-based oxidants utilize H2O2 cofactor • Thiyl radical is a key precursor of products • a moderately strong oxidant in its own right • Addition/transformation routes of thiyl radicals • conjugation with thiolate constitutes a ‘redox switch’ • isomerization of cis fatty acids to trans • intramolecular transformation of GSH thiyl radicals

3. Biological thiols and their oxidation products

4. Biologically-important thiols Adult human has ~ 30 g glutathione (typical cytosolic concentration is 5–10 mM) • Cysteine is the most abundant thiol moiety • Glutathione is a cysteinyl peptide and the most abundant non-protein thiol • Lipoate is an example of a dithiol, can reduce GSH

5. Oxidation of thiols and radical intermediates

6. A key property in thiol chemistry: dissociation to thiolate

7. Thiol ionization (dissociation of S–H):the most important single property? • The S–H bond of thiols dissociates with pKa (pH where 50% dissociated) in the range ~ 7–10:RSH Á RS– + H+thiolpKaH2S ~7·1 (to form HS–)cysteine ~8·5, 8·9 (-NH3+, -NH2) glutathione ~8·9, 9·1 (-NH3+, -NH2) • About 3% of glutathione (GSH) is dissociated to the thiolate form (GS–) at pH 7·4 • Many reactions of thiols with oxidants will be pH-dependent around physiological pH because the thiolate form is usually oxidized much faster than the undissociated thiol

8. Effects of thiol dissociation on rates of reactions in the physiological pH range

9. Example of thiolate(pH)-dependent reactivity Reactivity of GSH increased exactly 100–fold between pH 6–8 • Acetaminophen (paracetamol, Tylenol) oxidized in the liver to a quinoneimine • GSH adds rapidly to double bond, protects against adduct forming with protein thiols • Only thiolate anion reactive, reaction pH-dependent at pH < thiol pKa • Reaction accelerated by glutathione-S-transferases Coles et al. 1988

10. pH-Dependent reaction of NO2• with thiols NO2• + RS–® NO2– + RS• • Half-life of NO2• in presence of 5 mM GSH is only ~ 7 µs at pH 7·4 cysteine glutathione Estimates of rate constants from simplified analysis that under-estimated reactivity at higher pH, hence slope < 1 Ford et al. 2002

11. Oxidation of thiols (thiolate) to form thiyl radicals

12. There are several potential oxidants of thiols free-radical oxidants non-radical oxidants N2O3 orCu(I) /Cu(II) NO• •OH NO2• •OH ~30% H+ orAscH– Fe(III) O2 O2•– ONOOH ~70% NO2–MPO NO• Fe(II) H+ NO3– Cl–, MPO NO2• ONOO– HOCl H2O2 ~65% × 2 O2•– ONOOCO2– ± SOD O2•– ~35% •OH ~65% CO2 CO3•–

13. Oxidant Rate constant / M–1 s–1 (glutathione)at pH 7.4, room temperature •OH 1·3 ´1010 (Quinitiliani et al. 1977) NO2• 1·9 ´107 (Ford et al. 2002) CO3•– 5·3 ´106 (Chen & Hoffman 1973) O2•– 2·2 ´102(Jones et al. 2002) Reactivity of oxidants forming thiyl radicals Oxidant + RSH/RS–® product + RS• (+ H+) Half-life of radical is about0.7 / (k [GSH]) seconds

14. Nitric oxide (without O2) oxidizes GSH with concentration-dependent kinetics • GSSG and nitrous oxide are products • Higher reactivity is reported at high [NO•] (Aravindakumar et al. 2002) compared to low [NO•] (Hogg et al. 1996) • This can be explained by an equilibrium step(Folkes & Wardman 2004):GSH Á GS– + H+GS– + NO• (+ H+) Á GSN•OH 2 GSN•OH ® GSSG + HONNOH HONNOH ® N2O + H2O • The rate is then proportional to [NO•]2[GSH]

15. Reaction of ~ 9 µM NO• with GSH, ~ 27°C Anaerobic solutions! Folkes & Wardman 2004

16. Thiyl radicals as a route to nitrosothiols • Formation of S-nitrosothiols can be envisaged to occur by a two-step process: RSH + oxidant ® RS• + product RS• + NO•® RSNO • Radical-coupling reaction is poorly characterized • rate constant 2·8 ´107 M–1 s–1 reported(Hofstetter et al. 2006) • if correct, reaction would be too slow to compete with reaction of RS• with ascorbate in tissue and/or urate in plasma (at least in cytoplasmic/aqueous compartments)

17. Reactions of non-radical oxidants Oxidant Rate constant / M–1 s–1 (glutathione)at pH 7.4, 25 °C (*37°C) HOCl >1·0 ´107 (Folkes et al. 1995) N2O3 ~6·6 ´107 (Keshive et al. 1996) ONOO– ~6·0 ´102 (Koppenol et al. 1992) H2O2 * ~0·9 ´100 (Winterbourn & Metodiewa 1999) • Oxygen can be incorporated into products: GS– + H2O2® GSOH + OH– GSH + ONOO–® GSOH + NO2– GSOH + GSH ® GSSG + H2O • HOCl can give a sulfonamide and ‘dehydro’ GSH (Harwood et al. 2006) and GSCl and GS• (Davies & Hawkins 2000)

18. Enzyme-based oxidants use H2O2 cofactor and often generate thiyl radicals • e.g. Horseradish peroxidase, prostaglandin H synthase • Cpd I and II intermediates are one-electron oxidants • Thiyl radical spin-trapped (Harman et al. 1986) • No thiyl radicals from GSH peroxidase

19. Thiyl radicals a product of radical ‘repair’ (including drug radicals) Carbon-centred Oxygen-centred Sulfur-centred

20. Thiyl radicals are oxidizing agents and react with ascorbate and urate

21. Reaction of GS• with ascorbate • Absorption of ascorbate radical at 360 nm after generating GS• by pulse radiolysis:GS• + AscH–® GSH + Asc•– • Rate constant 6·0 ´108M–1 s–1 at pH 7 (Forni at al. 1983) implies half-life of GS• is ~ 3 µs with 0·4 mM ascorbate • Thiyl radicals products of general radical ‘repair’, so radical from the ascorbate ‘sink’ is an indicator of radical stress ESR signal from Asc•– in human skin illuminated with UVA light (Haywood et al. 2003)

22. Reaction of GS• with urate and stepwise radical transformation • Thiyl radicals from GSH oxidize urate (UH2–): GS• + UH2–® GSH + UH•–k ~ 3 ´107 M–1 s–1 at pH 7·4(Ford et al. 2002) • In turn the urate radical oxidizes ascorbate: UH•– + AscH–® UH2– + Asc•–k ~ 1·4 ´107 M–1 s–1(Willson et al. 1985) Urate and ascorbate are the dominant radical scavengers in blood plasma because the GSH concentration is only ~ 1 µM

23. Thiyl radicals react with thiolate and oxygen to ‘switch’ or modulate redox properties

24. Conjugation (addition) reactions of thiyl radicals with thiolate and oxygen • Addition reactions can act as a redox ‘switch’, or to weaken the oxidizing power of thiyl radicals • thiolate addition to form disulfide radical-anion (a reductant and source of superoxide) GS• + GS–Á (GSSG)•– (GSSG)•– + O2® GSSG + O2•– • oxygen addition to form less reactive peroxyl radical GS• + O2Á GSOO• • These reactions can be important in cells in vitro but might be less important in vivo because GS• reacts with ascorbate preferentially • but protein thiol groups in proximity may enhance S–S bond formation (cf. oxyR + H2O2, Demple 1999)

25. GSOO• is a weaker oxidant than GS• GS• + CPZ ® GS– + CPZ•+ (CPZ = chlorpromazine)GS• + O2Á GSOO•K ~ 3200 M–1 (Tamba et al. 1986)GSOO• + CPZ ® GSOO– + CPZ•+ • Oxygen slows down rate of oxidation of chlorpromazine by GS• (Wardman 1990) • GSOO• is at least 10-fold less reactive than GS• kobs = kGS/(1+K[O2])

26. Thiyl radicals add to double bonds – can catalyse isomerization

27. Cis/trans isomerization of fatty acids • Thiyl radical is a catalyst

28. Some thiyl radicals can also switch from oxidizing to reducing by intramolecular rearrangement

29. Base-catalysed intramolecular transformation of GS• • Deprotonation of –NH3+ moiety renders the adjacent C–H group susceptible to H abstraction by –S• • Half-life ~ 0.5 ms at pH 7.4 (Grierson et al. 1992) • Resulting carbon-centred radical reducing, will add O2 to form superoxide via a peroxyl radical-adduct

30. Conclusions • Thiols are important antioxidants by: • scavenging oxidizing radicals directly in some circumstances • ‘repairing’ free radical damage • However, the thiyl radical products of radical scavenging or ‘repair’ by thiols are not inert: • thiyl radicals are oxidizing agents • they act as damage transfer agents to O2, urate and (especially) ascorbate radical ‘sinks’ • they catalyse cis/trans isomerization of fatty acid double bonds

31. Some books, reviews & illustrative references While some of these are now dated, they still provide a good overview of the key chemistry of thiyl radical generation and fate Chatgilialoglu, C; Ferreri. Trans lipids: the free radical path., C., Acc. Chem. Res., 2005, 38, 441-8. Folkes, L. K.; Wardman, P. Kinetics of the reaction between nitric oxide and glutathione: implications for thiol depletion in cells. Free Radic. Biol. Med. 37: 549-556; 2004. Ford, E. et al. Kinetics of the reactions of nitrogen dioxide with glutathione, cysteine, and uric acid at physiological pH. Free Radic. Biol. Med. 32: 1314-1323; 2002. S-Centered Radicals (Alfassi, Z. B., ed.), Wiley: Chichester, 1999. ISBN: 0-471-98687-9 Biothiols in Health and Disease (Packer, L.; Cadenas, E., eds.), Marcel Dekker: New York, 1995. ISBN: 0-8247-9654-3 Wardman, P.; von Sonntag, C. Kinetic factors that control the fate of thiyl radicals in cells. Methods Enzymol., 251: 31-45; 1995. Schöneich, C. et al. Oxidation of polyunsaturated fatty acids and lipids through thiyl and sulfonyl radicals: reaction kinetics, and influence of oxygen and structure of thiyl radicals. Arch. Biochem. Biophys. 292: 456-467; 1992. Sulfur-Centred Reactive Intermediates in Chemistry and Biology (Chatgilialoglu, C.; Asmus, K.-D., eds.), Plenum Press: New York, 1990. ISBN: 0-306-43723-6