Transcript 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.

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
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
Biological thiols and their
oxidation products
Biologically-important thiols
+
H3N
CO2
-
CO2
-
H
+
SH
cysteine
O
N
H3N
O
Adult human
has ~ 30 g
glutathione
(typical
cytosolic
concentration
is 5–10 mM)
CO2
N
SH
-
H
glutathione
 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
SH
SH
CO2–
lipoate:
a dithiol
reduced
S
S
CO2–
oxidized
Oxidation of thiols and radical intermediates
thiol
RSH
thiyl radical
RS •
thiolate
RS–
thiyl peroxyl
radical
RS O O •
disulfide
RSSR
disulfide
radical-anion
(RSSR)•–
or (RSRS)–
sulfenic
acid
R
sulfinic
acid
S OH
O
R
S OH
sulfinyl
radical
O
R
O
O
sulfonic
acid
R
S OH
S•
sulfonyl
radical
R
S•
O
O
sulfonyl
peroxyl
radical
O
R
S O O•
O
A key property in thiol chemistry:
dissociation to thiolate
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  RS– + H+
thiol
pKa
H2S
~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
observed reactivity (100 = maximum)
Effects of thiol dissociation on rates of
reactions in the physiological pH range
100
10
dependence if undissociated
thiol reacts a factor of 10
slower than thiolate
1
0.1
dependence if undissociated
thiol is not measurably reactive
and only thiolate reacts
0.01
pKa of thiol
0.001
5
6
7
8
pH
9
10
11
Example of thiolate(pH)-dependent reactivity
H
N
COCH3
N
oxidation
COCH3
H
N
COCH3
GSH
SG
OH
O
OH
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 glutathioneS-transferases
Coles et al. 1988
pH-Dependent reaction of NO2• with thiols
NO2• + RS– NO2– + RS•
8.0
Estimates of rate
constants from
simplified analysis
that under-estimated
reactivity at higher
pH, hence slope < 1
7.5
cysteine
–1
–1
log (k / M s )
 Half-life of
NO2• in
presence of
5 mM GSH
is only ~ 7 µs
at pH 7·4
7.0
6.5
glutathione
6.0
5.5
Ford et al. 2002
6.0
6.5
7.0
pH
7.5
8.0
Oxidation of thiols (thiolate)
to form thiyl radicals
There are several potential oxidants of thiols
non-radical oxidants
or
Cu(I) /
Cu(II)
or
AscH–
O2
NO•
•OH
NO2•
~30%
•OH
Fe(III)
O2
•–
Fe(II)
HOCl
free-radical
oxidants
N2O3
Cl–, MPO
NO2–
MPO
NO•
ONOOH
H+
H+
~70%
NO3–
ONOO–
H2O2
~65%
NO2•
×2
O2•–
± SOD
O2
ONOOCO2– ~35%
•–
•OH
~65%
CO2
CO3•–
Reactivity of oxidants forming thiyl radicals
Oxidant + RSH/RS–  product + RS• (+ H+)
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)
Half-life of
radical is about
0.7 / (k [GSH])
seconds
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  GS– + H+
GS– + NO• (+ H+)  GSN•OH
2 GSN•OH  GSSG + HONNOH
HONNOH  N2O + H2O
 The rate is then proportional to [NO•]2[GSH]
Relative chemilumescence signal
Reaction of ~ 9 µM NO• with GSH, ~ 27°C
3
10 mM GSH
[GSH] / mM
0
2
pH 6.5
5
10
pH 7.4
1
pH 8.3
20
0
0
0.5 1.0 1.5 2.0 0
0.5
3
Time / 10 s
Folkes & Wardman 2004
1.0
1.5
Anaerobic
solutions!
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)
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)
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 spintrapped (Harman et
al. 1986)
 No thiyl radicals from
GSH peroxidase
•+
O
Fe
OO
IV
Fe
H2 O
cpd I
cpd III
H2O2
ROH
II
O2
•–
S
S
ROOH
•
•
S+
S+
III
IV
cpd II
O2
•
S+
•–
O2
HRP
reductant
S
O
Fe
Fe
H2O2
II
Fe
ferrous
Thiyl radicals a product of radical ‘repair’
(including drug radicals)
H
•
C
Carboncentred
+ RSH
+ RS •
C
O•
Oxygencentred
+ RS •
+ RSH
N
H
Sulfurcentred
OH
N
O
H
O
NMe2
N
•+
S
NMe2
Cl
N
Cl
+ RS •
+ RSH
S
Thiyl radicals are oxidizing
agents and react with
ascorbate and urate
Reaction of GS• with ascorbate
signal
 Absorption of ascorbate radical
at 360 nm after generating GS•
by pulse radiolysis:
GS• + AscH–  GSH + Asc•–
(a)
measured
ESR signal from Asc•– in human
H
skin illuminated with UVA light
(Haywood et al. 2003)
signal 0.5 mT
(a)
measured
 Thiyl radicals products of
general radical ‘repair’, so
radical from the ascorbate ‘sink’
is an indicator of radical stress
0.5 mT
e radical
ascorbate radical
ensity
signal intensity
 Rate constant 6·0  108 M–1 s–1
at pH 7 (Forni at al. 1983)
implies half-life of GS• is ~ 3 µs
with 0·4 mM ascorbate
(b)
light on
(b)
(c)
light off
0 light
2 4on 6 8 10 12 14
2
(c)
time (10 s)
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
Thiyl radicals react with
thiolate and oxygen to ‘switch’
or modulate redox properties
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–  (GSSG)•–
(GSSG)•– + O2  GSSG + O2•–
 oxygen addition to form less reactive peroxyl radical
GS• + O2  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)
GSOO• is a weaker oxidant than GS•
GS• + CPZ  GS– + CPZ•+
(CPZ = chlorpromazine)
GS• + O2  GSOO•
K ~ 3200 M–1 (Tamba et al. 1986)
GSOO• + CPZ  GSOO– + CPZ•+
7

GSOO•
is at least 10fold less reactive
than GS•
–1
5
8
–1
kobs / 10 M s
 Oxygen slows down
rate of oxidation of
chlorpromazine by
GS• (Wardman 1990)
6
4
3
2
1
0
kobs = kGS/(1+K[O2])
0
0.2
0.4
[O2] / mM
0.6
Thiyl radicals add to double bonds
– can catalyse isomerization
Cis/trans isomerization
of fatty acids
 Thiyl radical is a catalyst
Some thiyl radicals can also
switch from oxidizing to reducing
by intramolecular rearrangement
Base-catalysed intramolecular
transformation of GS•
CO2
 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
H
+
H 3N
O
N
H
O
N
S•
CO2
-
H
OH–
CO2
H 2N
-
O
N
H
CO2
H2 N
H
O
-
N
S•
H
O
-
H
O
N
•
CO2
N
SH
H
CO2
-
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
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: 549556; 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: 0-47198687-9
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