This study examined the antioxidant mechanisms of glutathione, L-cysteine, and methionine in preventing oxidative DNA damage caused by the Fenton reaction of iron and copper. Cyclic voltammetry studies showed that while the antioxidants bound to the metals, they did not shift the redox potential enough to prevent the Fenton reaction. Additionally, the antioxidants were not electrochemically active, indicating they were not acting as reactive oxygen species scavengers. Therefore, the primary mechanism of antioxidant activity for these molecules is metal coordination, not ROS scavenging.

1. ElectrochemicalStudies of the
Coordination of Antioxidant
Molecules with Iron and
Copper
Kayla DiMarco, Matthew Zimmerman
Clemson University
May 1, 2013
ABSTRACT: Reactive oxidative species (ROS)
often created through the Fenton Reaction is a
well-known cause of oxidative damage to DNA.
Cell death and mutation contribute to many
diseases as well as aging. Antioxidants
containing sulfur and selenium can prevent this
through various mechanisms. Glutathione
(GSH), L-Cysteine, and Methionine were all
effective antioxidants toward DNA damage at
low concentrations.. Cyclic voltammetry (CV)
studies became the next step in determining the
mechanisms used by these antioxidants. The
results showed that the antioxidants were not
ROS scavengers and used metal coordination as
its mechanism of preventing DNA damage.
These studies further the understanding of
antioxidant mechanisms.
Introduction
Figure 1 The Fenton Reaction
[2-4]
The Hydroxyl radical produced by the Fenton
reaction is a powerful oxidant, which can
destroy DNA and lead to disease and aging. In
fact, the metal mediated *OH formation is the
leading cause of cell death. [5-6] The Fenton
reaction generates a radical incredibly close to
DNA through the oxidation of iron(II) or copper
(I) then the reduction of hydrogen peroxide, as
depicted in figure 1. The Fenton reaction as it
occurs in living organisms can only run when in
the range of -160 mV to 460 mV (at a pH of 7).
[1] One possible mechanism of sulfur and
selenium antioxidant activity may be caused by
an ability to shift the oxidation (or reduction) of
iron and copper out of this range.
Results and Discussion
Combined copper or iron in metal:ligand
samples. For example, copper (which has 4
binding sites) was combined with methionine
(Met) in 600 micromolar increasing
concentrations. 600 micromolar Copper was
paired with 600 micromolar Met, then 1200
micromolar met, then 1800 micromolar Met and
so on until reaching a 1:5 ratio.
Iron was combined with all three ligands in
this same way. To keep the samples in as close
to biological conditions as possible, the samples
were treated with buffers (MOPS for copper and
MES for iron) which kept the solution at pH 7.
Each sample was prepared as quickly as
possible to ensure that they did not oxidize or
reduce during the process of prepping the
samples (in other words, each sample was made
completely within 45 minutes).
Parameters for CV
Initial 1 V
High 1 V
Low -1 V
Polarity negative
Scan rate .1s
Sleep 4s
Interval .001 V
Quiet time 5s
Sensitivity 1e -005

2. Electrochemical Results
Figure 2 L-Cysteine and copper
Epa = 0.387 V CuI/II
Figure 2
Figure 3 L-Cysteine and iron
Epc = -0.257 V FeII/III
The Gel electrophoresis studies conducted previously by other members of the team determined
that Cysteine and methionine prevent DNA damage through copper coordination. They used
[Cu(bipy)2]+ to fully coordinate with copper and measured the effectiveness of the antioxidants if they
were unable to coordinate. The results showed that they could not even in high concentrations. When
[Fe(EDTA)]2- was studied, the results showed that despite the inability of the ligands to coordinate with
the iron, there was still damage prevention, although significantly less. They suspected that at higher
concentrations, the DNA inhibition was most likely be due to ROS scavenging.
-1.0 -0.5 0.0 0.5 1.0
-0.00004
-0.00002
0.00000
0.00002
0.00004
0.00006
0.00008
Current,I(A)
Potential, E(V)
-1.0 -0.5 0.0 0.5 1.0
-0.000015
-0.000010
-0.000005
0.000000
0.000005
0.000010
0.000015
Current,I(A)
Potential, E(V)

3. 3
For Glutathione, the group determined through DNA damage assays that its antioxidant activity
is not attributed to GPx activity. Based on the results, metal-antioxidant binding was the primary
mechanism and ROS scavenging was a secondary mechanism for iron-mediated damage. [7]
The results of the L-Cysteine on copper and iron are depicted in figures 2 and 3. These results
were similar for all 3 ligands. Even with the addition of the ligands and high concentrations, oxidation
of each of these metals is within the range for the Fenton reaction to still occur. There was a slight shift
of no more than .05 V from the oxidation of iron or copper without the ligands. This shows that there
was still binding of the ligands, but not a significant amount. The previous studies suspected that the
ligands may participate in ROS scavenging. When measuring the electrochemical activity of the
ligands, they showed that they were not electrochemically active. This prevents them from being likely
candidates for ROS scavenging.
Conclusions
The gel studies showed that the antioxidants studied were effective at preventing DNA damage. The
CV studies show that ROS scavenging was not the reason for this. Metal coordination would be the
likely antioxidant mechanism for methionine, glutathione, and L-cysteine.
References
[1] Pierre, J. L.; Fontecave, M. Biometals 1999, 12, 195-9.
[2] Rai, P.; Wemmer, D.; Linn, S. Nucleic Acid Res.
2005, 33, 497-510.
[3] Bar-Or, D.; Thomas, G. W.; Rael, L. T.;
Lau, E. P.; Winkler, J. V. Biochem. Biophys.
Res. Commun. 2001, 282, 356-60.
[4] Rae, T. D.; Schmidt, P. J.; Pufahl, R. A.; Culotta,
V. C.; O'Halloran, T. V. Science 1999, 284, 805-8.
[5] D. Bar-Or, G. W. Thomas, L. T. Rael, E. P. Lau and J. V. Winkler, Biochem. Biophys. Res.
Commun., 2001, 282, 356–360.
[6] S. Park and J. A. Imlay, J. Bacteriol., 2003, 185, 1942–1950.
[7] Metallomics, 2011, 3, 503–512