1. 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

2. 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.

3. 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 TauCl
HOCl >107
Winterbourn & Metodiewa (1999) FRBM 27:322
Peskin et al (2001) FRBM 30:572

4. 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 CK
GAPDH
Taurine chloramine, M
CK > GAPDH
CK = GAPDH
Peskin & Winterbourn (2006) FRBM 40:45

5. 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]

6. A physiological example for H2O2
kH2O2 M-1s-1 Conc. % of H2O2 reacting with target
GSH mM 0.04
Protein tyrosine phosphatases
PTP1B µM
Cdc µM
GAPDH ~ µM 1
Peroxiredoxins >100, 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

7. 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

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

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

10. 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)

11. 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)

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

13. 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

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

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

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

17. 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

18. 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)

19. 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

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

21. 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

22. 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

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

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

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

26. 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
Prx2 dimer
Prx2 monomer
F Low et al (2006) Blood in press

27. 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.