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20
RADIATION CHEMISTRY AND PHYSICS, RADIATION TECHNOLOGIES
performed DFT (density functional theory) computations
for optimizations and energy calculations
of the parent molecules c-(L-Met-L-Met) and its
simplified model c-(L-Met-Gly). In c-(L-Met-Gly),
one of the Met side chains is replaced by a hydrogen
atom, and the other Met side chain contains
either a Met(S∴O) + radical cation or a Met(S∴N)
radical derived from the Met side chain.
The • OH-induced reaction pathways in
c-(L-Met-L-Met) have been characterized by the
pulse radiolysis measurements coupled to time-resolved
UV-VIS spectroscopy. The optical spectrum
recorded 2 µs after pulse irradiation can be
resolved into contributions from the following components
(Chart 1): the hydroxysulfuranyl radical
(1), the intramolecular sulfur-nitrogen three-electron-bonded
radical (2), the intramolecular sulfur-sulfur
three-electron-bonded radical cation (3),
and two α-(alkylthio)alkyl radicals (5a/5b) (Fig.1A).
The sum over all component spectra with their respective
yields resulted in an excellent fit to the
experimental spectrum. The G-value of radical cations
(3), G 3
=2.6, matched perfectly the G-value of
radical ions, G(ions)=2.6, measured by time-resolved
conductivity experiments (Fig.1B). It is important
to note that an excellent spectral resolution
was achieved using the intermediates 1 and 3,
the intramolecular sulfur-oxygen three-electron-
-bonded radical cation (4) and 5a/5b with the respective
yields for 3 and 4: 2.4 and 2.4. However,
in contrast to the previous spectral resolution the
yield of the sulfur radical cations G 3+4
=4.8, does
not match G(ions)=2.6 determined by conductivity
measurements.
In this report, we have provided an experimental
proof that one-electron oxidation by • OH radicals
of simple cyclic dipeptide containing two-methionine
residues leads to efficient formation of
the Met(S∴N) radicals. This is a strongly competitive
process to the formation of the Met(S∴S) +
and Met(S∴O) + radical cations in spite of the close
proximity of sulfur atoms located in the side chains
of Met residues and the close proximity of sulfur
atoms and oxygen atoms located in the peptide
bonds.
References
[1]. Schöneich C.: Biochim. Biophys. Acta, 1703, 111-119
(2005).
[2]. Glass R.S.: Top. Curr. Chem., 205, 1-87 (1999).
[3]. Davies M.: Biochim. Biophys. Acta, 1703, 93-109
(2005).
[4]. Schöneich C., Pogocki D., Hug G.L., Bobrowski K.: J.
Am. Chem. Soc., 125, 13700-13713 (2003).
RADIATION-INDUCED OXIDATION OF DIPEPTIDES
CONTAINING TYROSINE AND METHIONINE – INFLUENCE
OF AMINO ACID SEQUENCE AND pH
Gabriel Kciuk, Jacek Mirkowski, Gordon L. Hug 1/ , Krzysztof Bobrowski
1/
Radiation Laboratory, University of Notre Dame, USA
One-electron induced oxidation by hydroxyl radicals
( • OH) of two dipeptides, tyrosyl-methionine
(Tyr-Met) and methionyl-tyrosine (Met-Tyr), were
carried out by means of pulse radiolysis. The formation
of tyrosyl radicals (TyrO • ) in these compounds
occurs via dehydration of dihydroxycyclohexadienyl
radicals (Tyr • (OH)OH) – reaction (2).
This latter radical is the product of an addition of
•
OH radicals to the aromatic ring of a tyrosine residue
– reaction (1):
Tyr + • OH → Tyr • (OH)OH (1)
Tyr • (OH)OH + H + → TyrO • + H 2 O (2)
The reaction of – OH elimination is catalyzed by
protons (reaction 2) and is particularly efficient at
pH~1. The other possible reaction pathway that
leads to TyrO • radicals is an electron transfer between
an intact tyrosine residue and sulfur-centered
radicals located in the methionine residue.
This reaction pathway was previously identified
during Br 2
•–
oxidation of oligopeptides containing
both methionine and tyrosine residues [1,2]. In our
experiments, the • OH radical was used since this
radical is responsible for oxidative stress in living
cells. Therefore, our results can be extrapolated to
biological systems. Since the rate constants of the
reactions between the • OH radical and both amino
acids are very similar – k=1.2×10 10 dm 3 mol –1 s –1 ,
the primary • OH radical attack should be divided
almost equally between the two amino acid residues
of each dipeptide. Our studies were aimed at
the identification of the sulfur-centered radicals
that are involved in electron transfer, leading to
the formation of TyrO • radicals.
Oxidation of Tyr-Met by • OH radicals
Transient spectra recorded during the oxidation
of Tyr-Met at pH 6.6 (Fig.1) were assigned to
dihydroxycyclohexadienyl (Tyr • (OH)OH-Met) and
tyrosyl (TyrO • -Met) radicals. Concentration profiles
for both radicals were created after resolution of
absorption spectra at any desired time delay following
the electron pulse. Similar yields of both detected
radicals Tyr • (OH)OH-Met and TyrO • -Met
were observed in the first 5 µs. The radiation chemical
yield of each radical amounted to half of the
total yield of • OH radicals. After several hundred
microseconds, the concentration of TyrO • -Met
radicals was stable, but the Tyr • (OH)OH-Met radicals
had decayed by half (Fig.1, inset). A complementary
experiment, performed on solutions containing
tyrosine-glycine (Tyr-Gly) under similar
conditions (pH, concentration), revealed that the
decay kinetics of dihydroxycyclohexadienyl radicals
in both compounds (Tyr-Met and Tyr-Gly) were
identical. However, in solutions containing Tyr-Gly