The Thiol System: Life and Death in the Numbers

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The Thiol System Life and Death in the Numbers
Garry R. Buettner and Freya Q. Schafer Free
Radical and Radiation Biology Program and ESR
Facility The University of Iowa Iowa City, IA
52242-1101 Tel [phone] Email
garry-buettner_at_uiowa.edu
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Sulfur in Biology
Found in two amino acids Cysteine and
Methionine
pKa (S-H) 8.3
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Some oxidation states of sulfur
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Oxidation of Cysteine
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Cystine, i.e. cysteine-disulfide, and its
oxidation
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Disulfides
Disulfide formation is an important redox
reaction of cysteine 2 RSH ? RSSR 2H
2e- Used to form structure of proteins as
redox buffer in signaling to change
activity of enzymes
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Protein Disulfides
When cysteine residues are oxidized to
disulfides, they can form
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Glutathione, an unusual tripeptide
Glutathione (GSH)
Standard Peptide Bond
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Some general reactions of thiols
RSH GSSG RSSG GSH
thiol/disulfide exchange 2RSH
RSSR 2H 2e- two-electron transfer RSH
RS? H e- single-electron
transfer RSH RS? H?
hydrogen-atom transfer RSOH GSH RSSG
H2O nucleophilic substitution RSH
RCHO RSCH(OH)R nucleophilic
addition RSCH(OH)R ? RSC(O)R 2H 2e-
RSH R-X R-SR HX
S-conjugation (R-X is
electrophilic)
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The thiol-disulfide exchange reaction
Example RSH GSSG RSSG GSH
These reactions are probably the most common
reactions of cysteine residues in vivo.
However, the actual reaction may be better
written as RS- GSSG H
RSSG GSH It is the ionized form of a thiol
that is often the dominant player in reactions
because of its greater electron density, i.e.
nucleophilicity.
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Some two-electron reduction potentials
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Vicinal thiols, Vicinal
Vicinal from Latin vicinalis, neighboring. In
chemistry, vicinal means neighboring groups, e.g.
vicinal alcohol groups.
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Vicinal thiols
In a protein, vicinal implies two intervening
amino acids, e.g., -CXXC- would be two
cysteines separated by two intervening amino
acids. The -CXXC- motif of thioldisulfide
oxidoreductases is now widely recognized as being
essential for the catalysis of thiol redox
reactions Thioredoxin -Cys-Gly-Pro-Cys- G
lutaredoxin -Cys-Pro-Try-Cys- PDI
-Cys-Gly-His-Cys- DsbA
-Cys- Pro-His-Cys-
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The pKa and in turn the reduction potential of
the active thiol in proteins with vicinal thiols
is key in the function of di-thiol proteins
(circles) DsbA (triangles) Trx
pK1 of the active thiol
Chivers PT, Prehoda KE, Raines RT. (1997) The
CXXC motif a rheostat in the active site.
Biochemistry. 364061.
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An important, perhaps urgent, function of the
thiol system is to remove hydroperoxides
ROOH 2(-Cys-SH) ? ROH H2O
-(Cys-S)2
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GSH system and peroxide removal
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A Major Player,
GSH and Redox State
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Definition of Redox State
The redox state of a redox couple is defined by
the half-cell reduction potential and the
reducing capacity of that couple.
A convenient notation for the status of a redox
pair, such as GSSG/2GSH, would be Ehc(GSH)
GSH, e.g. ?265 mV (GSH) 3.5 mM.
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Redox State vs Redox Environment
Redox State should be reserved for referring to
the status of a specific redox couple. Redox
Environment should be used to refer to the
overall redox status of cells, tissues, fluids,
etc. Redox is an adjective, not a noun thus, a
statement such as Cellular redox is important.
is a grammatical faux pas.
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The Nernst Equation
The voltage of an electrochemical cell is
calculated with the Nernst equation. For a
reaction such as Red1 Ox2 ? Ox1 Red2, the
Nernst eqn will have the form
This uses the standard convention for each half
cell Ehc Eo (RT/nF)
? ln(Red/Ox)
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2e--Processes
The NADP/NADPH system NADP 2e- H?
NADPH E?' ?315 mV at 25?C, pH 7 Ehc
?315 ? (59.1/2) log (NADPH/NADP) mV
at 25?C, pH 7  
Jensen, M.A. Elving, P.J. Nicotinamide adenine
dinucleotide (NAD) formal potential of the
NAD/NAD? couple and NAD? dimerization rate.
Biochim. Biophys. Acta. 764 310-315 1984.
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The NADP/NADPH couple
The half-cell potential for the NADP/NADPH
couple is independent of the absolute
concentration of either species, only the ratio
is needed to estimate the reduction potential.
In cells and tissues the NADPH/NADP ratio tends
to be approximately 1001. Reich, J.G. Selkov,
E.E. Energy metabolism of the cell A
theoretical treatise. Chapter IV p. 135 Academic
Press, NY, New York 1981.
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The Glutathione System
GSSG 2H 2e- ? 2GSH Ehc
?240 ? 30 log (GSH2/GSSG) mV at
25?C, pH 7.0.
!!!
Schafer FQ, Buettner GR. (2001) Redox state of
the cell as viewed though the glutathione
disulfide/glutathione couple. Free Radic Biol
Med. 301191-1212. Schafer FQ, Buettner GR.
(2003) Redox state and redox environment in
Biology. In Signal Transduction by Reactive
Oxygen and Nitrogen Species Pathways and
Chemical Principles. Eds Forman HJ, Torres M,
Fukuto J. Kluwer academic Publishers, Dordrecht,
Netherlands, Chapter 1, pp. 1-14.
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The GSSG/GSH couple
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Cellular Redox Systems
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Reduction Potential and Biological Status of Cells
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Table Continued
Do you need this part?
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Model of how Ehc for the GSSG/2GSH couple may be
associated with the different physiological states
I range of proliferation II range of
differentiation III range of apoptosis IV range
of necrosis
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Thiol Systems
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Thiols as switches
Thiols are major contributors to the redox
environment of cells. They are involved in the
function and control of many proteins.
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Thiols and disulfides as nano-switches
A nano-switch is a very small, operating on a
nanometer scale. For example, the distance
between two sulfhydryls in a protein with two
intervening amino acids, such as in
thioredoxin. The redox environment of the cell,
or regions within a cell, could be viewed as a
means to activate a cellular switchboard, thereby
changing the message of cellular signals. By
changing the reduction potential, a series of
nano-switches are activated/de-activated that
move the cell from proliferation through various
stages of proliferation, differentiation or into
apoptosis. Necrosis is the complete loss of
the ability to activate/de-activate and respond
to changes in these nano-switches.
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Type I switch
For the GSSG, 2H/2GSH couple Ehc ?240 ?
(59.1/2) log (GSH2/GSSG) mV at 25?C, pH 7.0
If GSH 5 mM and GSSG 25 ?M, then Ehc
?240 mV. If GSH decreases to 2.5 mM and
GSSG increases to 100 ?M, a 35 mV change will
occur resulting in Ehc ?205 mV. The
PSSG/PSH ratio will change by a factor of 8,
resulting in an 8-fold change in the status of
the switch.
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Type II switch
Using the example for the Type I switch, this
same 35 mV change in the GSSG/2GSH couple will
result in a change of the PSS/P(SH)2 ratio by
a factor of 16. Thus, a Type II switch
translates changes in reduction potential into a
2-fold greater change in the status of the
nano-switch, compared to a Type I switch.
Schafer FQ, Buettner GR. (2001) Redox state of
the cell as viewed though the glutathione
disulfide/glutathione couple. Free Radic Biol
Med. 301191-1212. Schafer FQ, Buettner GR.
(2003) Redox state and redox environment in
Biology. In Signal Transduction by Reactive
Oxygen and Nitrogen Species Pathways and
Chemical Principles. Eds Forman HJ, Torres M,
Fukuto J. Kluwer academic Publishers, Dordrecht,
Netherlands, Chapter 1, pp. 1-14.
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Summary
In a cell there is are many copies of proteins,
and several components to a signal. Thus, the
state of a switch is due to the ensemble. It
is the ensemble that results in an action like a
rheostat.
Overviews Nathan, Carl (2003) Specificity of a
third kind reactive oxygen and nitrogen
intermediates in cell signaling. J. Clin. Invest.
111769778. Cooper CE, Patel RP, Brookes PS,
Darley-Usmar, VM, (2002) Nanotransducers in
cellular redox signaling modification of thiols
by reactive oxygen and nitrogen species, Trends
in Biochemical Sciences. 27 489-492.
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Reduction potential driven nano-switches move
cells through different biological stages.