Biologically Relevant Thiol Modifications Effects on Protein Function Christine Winterbourn Christchurch School of Medicine, University of Otago, New Zealand Society for Free Radical Biology and Medicine Workshop on Thiol Groups in Oxidative Stress and Redox Signaling Denver, November 2006

Thiols are among the most oxidant-sensitive biological molecules. All thiols are not equally reactive. All oxidants do not give the same products. Identification of biological targets. Consequences of thiol protein oxidation.

Most oxidants react with the anionic form of thiols Most oxidants react with the anionic form of thiols. Compounds with low pKa are more reactive. Hydrogen Peroxide Taurine Chloramine Hypochlorous Acid kGSH H2O2 1 M-1s-1 TauCl 115 HOCl >107 Winterbourn & Metodiewa (1999) FRBM 27:322 Peskin et al (2001) FRBM 30:572

Relative sensitivities of creatine kinase (pK 5 Relative sensitivities of creatine kinase (pK 5.5) and glyceraldyde-3-phosphate dehydrogenase (pK 7.5) SDS-PAGE after labelling reduced thiols with iodoacetamide-fluorescein HOCl, M 0 10 20 50 100 CK GAPDH Taurine chloramine, M 0 5 10 20 100 CK > GAPDH CK = GAPDH Peskin & Winterbourn (2006) FRBM 40:45

Identifying Physiological Targets Determined by reaction rates and concentrations. Reactions occur in competition. Reactions that occur in isolation will not all be sufficiently favorable to occur in cells. For two substrates, the ratio of the amounts of oxidant reacting with each is given by k1 [substrate 1] k2 [substrate 2]

A physiological example for H2O2 kH2O2 M-1s-1 Conc. % of H2O2 reacting with target GSH 1 2 mM 0.04 Protein tyrosine phosphatases PTP1B 20 1 µM 0.0004 Cdc25 150 1 µM 0.003 GAPDH ~500 100 µM 1 Peroxiredoxins >100,000 50 µM 99 Some proteins that are easily oxidized by H2O2 in isolation are unlikely to be directly oxidized in a cell -until more favorable targets are oxidized

Diffusion distance of H2O2 in 50 µM peroxiredoxin: 20 µm Diffusion Distance and Site Localisation Diffusion distance = For H2O2 D ~2x10-5 cm2/s Diffusion distance of H2O2 in 50 µM peroxiredoxin: 20 µm Requirements for localized oxidant action: Local area of high substrate concentration or Association between oxidant generator and target Barrier to diffusion eg membrane

Some cellular thiol proteins become oxidized even though they are not particularly reactive. Could this oxidation be indirect?

Thiol Oxidation 1 electron RSH RS thiyl radical 2 electron RSH RSOH RSO2H RSO3H sulfenic sulfinic sulfonic RSH RSSR disulfide

Products of GSH oxidation: LC/MS total Ion Chromatograms Hypochlorite 1:1 GSH GSSG GSSG GSSG is the major product but not necessarily the only one. A: Sulfinic and sulfonic acids B: dehydroglutathione C: Sulfonamide D: Various C D B Taurine chloramine 0.5:1 Peroxynitrite 1:1 D B D A C Harwood et al Biochem J 399,161 (2006)

Products of Protein Thiol Oxidation Usual candidates Mixed disulfide R-S-S-G Sulfenic acid R-S-OH Interchain disulfide R-S-S-R Internal disulfide (vicinal thiol) R S S More recently recognized Nitrosothiols -S-NO Sulfinic (or sulfonic) acid eg “overoxidized” peroxiredoxins -SO2H Sulfenamide eg PTP1B S-N(amide) Sulfinamide eg HOCl modification S-N(amine)

Distinguishing Different Oxidation Products Reversible Anti-GSH antibodies Use selective reductants Arsenite -SOH Ascorbate -SNO Glutaredoxin -SG Irreversible

Sulfenic Acids : Low MW forms unstable but can be stabilised within Protein Albumin treated with H2O2 Detection 1. Spectral change on reaction with NBD* Thiol lmax ~420 nm Sulfenate lmax ~350 nm H2O2-treated native 2. Reaction with dimedone and MS of tryptic digest *7Cl-4NO2benzo-2-oxa-1,3-diazone Carballal et al (2003) Biochemistry 42:9906 Ellis & Poole (1997) Biochemistry 36:15013

Consequences of Protein Thiol Oxidation Antioxidant protection Cell signaling Metabolic regulation Toxicity

Mechanisms Removal of oxidant Enzyme inactivation Mixed disulfide formation Conformational change Crosslinking/aggregation

Protection Removal of oxidant Enzyme inactivation Mixed disulfide formation Conformational change Crosslinking/aggregation Metabolic regulation Signaling Toxicity

GSH and Thioredoxin Ultimate sinks for cellular oxidations Antioxidant activity Disulfide reduction Often not direct targets for oxidants Usually not in redox equilibrium and reactivity dictated by kinetics Recycled by NADPH

Labeled Oxidized Thiol Proteins in Jurkat Cells control 200 M H2O2 GAPDH Peroxiredoxin 2 Proteins extracted, reduced, labelled with fluorescein-iodoacetamide and separated by 2D SDS-PAGE (Baty et al (2005) Biochem J 389:785)

OxyR: A redox-activated genetic switch Prokaryotic transcription factor Thiol oxidation to disulfide induces conformational change to activate DNA binding kH2O2 ~2x105 M-1s-1 Pomposiello & Demple (2001) Trends Biotech 19:109

HSP33 – A redox-regulated molecular chaperone Prokaryote chaperone Activated by oxidative stress Graf & Jakob (2002) CMLS 59:1624

Peroxiredoxins Ubiquitous class of antioxidant or signaling proteins Present in high copy numbers Highly reactive with H2O2 (k>105 M-1s-1) 2-cys and 1-cys forms

Prx2 : A 2-Cys Peroxiredoxin Conventional peroxiredoxin / thioredoxin cycle H2O2 H2O2 SpH SrH SOH SrH SO2H SrH SrH SpH SrH SOH SrH SO2H Overoxidation Thioredoxin, Thioredoxin reductase, NADPH S S S S Kang et al (2005) Trends Mol Med 11:571

Model for Mammalian 2-Cys Peroxiredoxin Oxidation Wood, Poole & Karplus, Science 2003

High reactivity of Prx2 with H2O2 6 µM Prx2 H2O2 M 0 0.8 1.5 3 Reagents mixed for 20 s and separated by non-reducing PAGE dimer monomer A. Peskin et al (2006) submitted

Prx2 and catalase react with H2O2 at similar rates Prx2 0.15 mg/ml H2O2 (5 µM) - + + + + + + Catalase (mg/ml) 0 2 catalase Prx2 dimer Prx2 monomer Catalase rate constant 5 x 106 M-1 s-1 A. Peskin et al (2006) submitted

Prx2 is abundant (~250 µM) in the erythrocyte It is oxidized by very low H2O2 concentrations despite active catalase and GPx It forms reversible dimers but does not undergo irreversible oxidation [H2O2] µM 0 1 2 5 10 20 50 100 200 Prx2 dimer Prx2 monomer F Low et al (2006) Blood in press

Summary Thiol oxidation is important in antioxidant defense and redox signaling. Thiols differ in reactivity depending on pKa and molecular environment. Absolute reactivity and selectivity vary for different oxidants. For H2O2, only a few proteins have shown sufficient reactivity to be direct targets. Conformation change as well as enzyme inactivation are important regulatory mechanisms.