Electron attachment to uracil: effective destruction at subexcitation
energies
G. Hanel
a
, B. Gstir
a
, S. Denifl
a
, P. Scheier
a
, M. Probst
a
, B. Farizon
b
, M. Farizon
b
,
E. Illenberger
c
and T. D. Märk
a,d
a
Institut für Ionenphysik der Universität Innsbruck, Technikerstrasse 25,
A-6020 Innsbruck, Austria
b
Institut de Physique Nucléaire de Lyon, IN2P3-CNRS et Université Claude Bernard,
F-69622 Villeurbanne
c
Institut für Chemie – Physikalische und Theoretische Chemie, Freie Universität Berlin,
D-14195 Berlin
d
also adjunct Professor at Department of Plasmaphysics, Comenius University,
SK-84248 Bratislava, Slovak Republic
Abstract
We demonstrate that electrons at energies below the threshold for electronic
excitation (< 3eV) effectively decompose gas phase uracil generating a mobile
hydrogen radical and the corresponding closed shell uracil fragment anion (U-H)
-
.
The reaction is energetically driven by the large electron affinity of the (U-H)
radical. This observation has significant consequences for the molecular picture of
radiation damage, i. e. genotoxic effects or damage of living cells due to the
secondary component of high energy radiation.
To be published in Phys.Rev.Letter
2
The interaction of high energy radiation (α-, β-, γ- rays or heavy ions) with living
cells does not in general directly lead to DNA strand breaks. The primary interaction
essentially removes electrons from the components of the complex molecular network,
i. e., electrons from valence states of the chemical bonds but also electrons from
localized inner shells of the individual atoms. As a result of the subsequent charge
transfer and energy dissipating processes, chemical bonds can be ruptured generating
neutral or ionic radicals as additional secondary species. Electrons as the most abundant
secondary species, are created with an estimated quantity of ≈ 4 x 10
4
electrons per
MeV primary quantum deposited [1]. The larger majority possesses initial kinetic
energies up to about 20 eV [2]. In the course of successive inelastic collisions within the
medium they are thermalized within 10
-12
s before they reach some stage of solvation,
then as chemical rather inactive species. Moreover, damage of the genom in a living cell
by ionizing radiation is about one third direct and two third indirect [3]. Direct damage
concerns reactions directly in the DNA and its closely bound water molecules and
indirect damage results from energy deposition in water molecules and other
biomolecules in the surrounding of the DNA. It is believed that almost all the indirect
damage is due to the attack of the highly reactive hydroxyl radical [4,5].
The importance of reactions of presolvated electrons with amino acids and
nucleotides has already been pointed out more than 2 decades ago by time resolved
pulse radiolysis experiments [6]. More recently, the ability of free ballistic electrons (3-
20 eV) to efficiently induce single and double strand breaks in supercoiled DNA has
clearly been shown by Sanche and co-workers [7]. In these studies it was demonstrated
that the DNA strand breaks were initiated by the formation and decay of transient
negative ion (TNI) states, localized on the various DNA components (base, phosphate,
deoxyribose or hydration water). Resonances in DNA strand break curves were
observed in the energy range around 10 eV, similar to those TNI states formed by these
components in the gas phase or in homogeneous films as exemplified in ref. [7] in the
form of measured electron energy-dependent desorption yields of energetic H
-
from
thymine showing a strong peak at around 10 eV.
In order to distinguish between intrinsic molecular effects and environmental
effects, recently a number of studies about the interaction of primary and secondary
radiation (species) with isolated nucleic acid bases has been carried out utilizing recent
advances in crossed molecular beam techniques. In addition, several theoretical
investigations on the properties of these various DNA components (electron affinities,
ionisation energies etc. [8-13]) have been performed. When considering electron
attachment studies two series of experiments are noteworthy. Using Rydberg electron
attachment Schermann and co-workers [14-16] produced gas phase uracil, thymine and
3
adenine molecular anions ascribed to the existence of dipole-bound parent anions. In
addition, using as target a mixed uracil-argon cluster beam they were also able to
observe weakly bound valence monomer uracil anions U
-
. From calculations and
experimental evidence they derived the valence adiabatic electron affinity of uracil to be
small but positive (≈ 70 meV in contrast to an earlier value of 400 meV by Sevilla et al.
[10]). This value is close to the calculated value of 86 meV [9] and measured value of
93±7 meV [17] and 54±35 meV [15] of the dipole bound electron affinity. Moreover,
minute amounts of uracil anions outside of the peaked Rydberg n-dependencies were
interpreted to be due to background anions (U-H)
-
produced by spurious uncontrolled
free electron interactions. In contrast, Illenberger, Sanche and co-workers [18] using a
trochoidal electron monochromator in conjunction with a quadrupole mass spectrometer
reported recently for thymine and cytosine strong zero energy parent anion signals for
free electron attachment to these nucleic bases. Similar results, i.e., the observation of
the parent anion, were reported by the same group for various 5-halouracils (5-X-U;
with X = Cl, Br and I) [19,20].
Here we demonstrate that under isolated conditions the RNA base uracil is
effectively damaged (dissociated) by very low energy free electrons, i. e., below the
threshold for electronic excitation (< 3eV). This is an energy region which was not
covered by the previous experiments of Sanche and co-workers where single and double
strand breaks in supercoiled DNA induced by free electrons was studied between 3 and
20 eV [7]. Dissociative electron attachment (yielding (U-H)
-
) observed here proceeds
via a C-H bond rupture generating a mobile and reactive hydrogen radical. Energetically
this is accomplished by the surprisingly high electron affinity of the (U-H) radical
leading to .
The present experiment is performed in a crossed electron/molecular beam
arrangement recently constructed in our laboratory and described in more detail in refs.
[21,22]. A highly monochromatized electron beam (best values achieved lie at around
30 meV; in the present case set to energy resolutions between 80 and 120 meV in order
to allow working at low target gas pressures), generated by an electrostatic
hemispherical electron monochromator, interacts perpendicularly with an effusive beam
of uracil molecules. The uracil beam is generated by heating the uracil powder sample
in a Knudsen type oven to 185°C and effusing the sublimated molecules through a 1
mm capillary directly into the collision region. The anions resulting from the electron –
molecule collisions are extracted from the collision region by a weak extraction
potential (at maximum 200 meV/cm) and focused to a high resolution quadrupole mass
filter (mass range 2000 amu) where they are mass analyzed and detected by single pulse
counting electronics. The electron energy scale and energy resolution has been
4
determined by measuring electron attachment to uracil and to CCl
4
or SF
6
and using the
resonant “zero energy peaks “ thus obtained for calibration and reference [23,24] (see as
an example the curve given in Fig.1).
Figure 2 (top) shows the cross section for the formation of the closed shell anion
(U-H)
-
which is produced mainly at energies below about 5 eV. By a comparing anion
currents measured under defined pressure conditions in the target region and using the
accurately known DEA cross section in CCl
4
at 0.8 eV [25,26] we can estimate the
DEA cross section in uracil leading to hydrogen radical abstraction to have a value of
≈3x10
-20
m
2
at the peak maximum. At higher electron energies, in the range between ≈ 3
- 12 eV we observe further products (CN
-
, OCN
-
and C
3
OH
2
N
-
), however, at significant
lower cross sections. These smaller product anions arise from complex decomposition
processes involving cleavage of the aromatic ring.
If U assigns the undissociated target molecule uracil, then (U-H)
-
is the most
abundant fragment anion produced via
e
-
+ U → U
#-
→ (U-H)
-
+ H
•
(1)
Dissociation of uracil to yield (U-H)
-
at these very low electron energies is a remarkable
observation as in Rydberg electron transfer from highly excited atoms only
undissociated uracil radical anions were detected [14-16]. Owing to the high dipole
moment of uracil (4.3 D) they were ascribed as weakly-bound dipole bound states. It
should be noted that in the present experiment we in fact observe a small ion signal at
112 amu (in addition to the signal at 111 which is attributed to the closed shell anion
(U-H)
-
). The signal at 112 amu, however, can fully be accounted for by the contribution
of the
13
C isotope in (U-H)
-
in its natural abundance. We can hence conclude that the
undissociated uracil anion is not formed at any measurable amount in the present
crossed beam single collision experiment. Moreover, we also conclude that this
behavior is in contrast to recent results obtained for free electron attachment to thymine
and cytosine [18], where the major reaction channel was the production of the
undissociated nucleic base parent anion, with CN
-
and O
-
being the major fragment ions
observed though with a factor of 100 lower probability.
The resonances in the shape of the (U-H)
-
cross section curve indicate that it is
formed by resonant dissociative electron attachment (DEA) where U
#-
is the transient
negative ion generated by the initial Franck-Condon transition. DEA is in fact the only
mechanism to induce a bond cleavage at such low electron energies. The ion yield curve
indicates that presumably different negative ion states of the precursor ion U
#-
are
involved. It is interesting to note that Burrow and co-workers [11] using electron
5
transmission (ET) spectroscopy reported the occurrence of three transient anion states at
0.2 eV, 1.6 eV and 5.0 eV assigning each of these states to the accommodation of the
extra electron into the antibonding π* system. Some of the structure presently obeserved
may also arise from anion states whose lifetime is sufficiently long to allow nuclear
motion [27].
Moreover, reaction (1) can generate 4 different isomeric anions (U-H)
-
. We have
carried out high level ab initio calculations using the G”MP2 [28] method to get
information on the energy threshold to generate these different isomeric anions. The
computational method is considered to be accurate to better than ± 0.1 eV, see, for
example refs. [28,29]. The calculated energy thresholds are E(1) = 0.8 eV, E(3) = 1.4
eV, E(5) = 2.7 eV and E(6) = 2.2 eV where the number in parentheses assigns the N or
C atom from which H abstraction takes place (see Fig. 1). These numbers are based on
the relation E(n) = D(n) – EA(n) where D assigns the binding energy of the hydrogen
atom at the particular site n = 1, 3, 5, 6 and EA the electron affinity of the
corresponding uracil radical. The explicit numbers are D(1) = 4.4 eV, EA (1) = 3.6 eV,
D(3) = 5.4 eV, EA (3) = 4.0 eV, D(5) = 5.2 eV, EA (5) = 2.5 eV, and D(6) = 5.0 eV, EA
(6) = 2.8 eV. It is interesting to note that a very narrow peak is observed close to zero
eV which is below the threshold for the energetically lowest channel. The origin of this
peak is not completely clear as its intensity also depends on the presence of the
calibration gas (CCl
4
). There is, however some (U-H)
-
signal present close to zero eV
also in the case when the calibration gas CCl
4
is completely absent in the target region.
It is well known that endothermic DEA reactions can exhibit distinct zero energy
peaks due to transitions from vibrationally excited states of the neutral molecule (hot
band transitions). Due to the particular conditions in DEA (reciprocal energy
dependence of the cross section, etc.) these threshold peaks can appreciably contribute
to the process even at very moderate population of vibrationally excited levels [30]. It is
important to note that if the zero eV contribution is due to a hot band, it would probably
not have any significance in a real biological environment due to the much lower
temperature.
We note that in DEA experiments to 5-bromouracil [19] and further 5-halouracils
[19,20] in fact very effective pathways of dissociative attachment at virtually 0 eV were
identified. In the case of bromouracil (U
Br
) the following complementary reactions:
e
-
(0 eV) + U
Br
→ U
Br
#-
→ (U
Br
-Br)
•
+ Br
-
(2a)
→ (U
Br
-Br)
-
+ Br
•
(2b)