A Photochemically Generated Selenyl Free Radical Observed by High Energy Resolution Fluorescence Detected X-ray Absorption Spectroscopy

Article abstract of DOI:10.1021/acs.inorgchem.8b01522

Selenium-based selenyl free radicals are chemical entities that may be involved in a range of biochemical processes. We report the first X-ray spectroscopic observation of a selenyl radical species generated photochemically by X-ray irradiation of low-temperature solutions of l-selenocysteine. We have employed high energy resolution fluorescence detected X-ray absorption spectroscopy (HERFD-XAS) and electron paramagnetic resonance (EPR) spectroscopy, coupled with density functional theory calculations, to characterize and understand the species. The HERFD-XAS spectrum of the selenyl radical is distinguished by a uniquely low-energy transition with a peak energy at 12659.0 eV, which corresponds to a 1s → 4p transition to the singly occupied molecular orbital of the free radical. The EPR spectrum shows the broad features and highly anisotropic g-values that are expected for a selenium free radical species. The availability of spectroscopic probes for selenyl radicals may assist in understanding why life chooses selenium over sulfur in selected biochemical processes.

Full text of DOI:10.1021/acs.inorgchem.8b01522

Article
pubs.acs.org/IC
Cite This: Inorg. Chem. 2018, 57, 10867−10872
A Photochemically Generated Selenyl Free Radical Observed by
High Energy Resolution Fluorescence Detected X‑ray Absorption
Spectroscopy
Susan Nehzati,† Natalia V. Dolgova,† Dimosthenis Sokaras,‡ Thomas Kroll,‡ Julien J. H. Cotelesage,†
Ingrid J. Pickering,†,§ and Graham N. George*,†,§
†Molecular and Environmental Sciences Group, Department of Geological Sciences, University of Saskatchewan, Saskatoon,
Saskatchewan S7N 5E2, Canada
‡Stanford Synchrotron Radiation Lightsource, SLAC National Accelerator Laboratory, Stanford University, Menlo Park, California
94025, United States
§Department of Chemistry, University of Saskatchewan, Saskatoon, Saskatchewan S7N 5C9, Canada
ABSTRACT: Selenium-based selenyl free radicals are chemical entities that may be
involved in a range of biochemical processes. We report the first X-ray spectroscopic
observation of a selenyl radical species generated photochemically by X-ray irradiation of
low-temperature solutions of L-selenocysteine. We have employed high energy resolution
fluorescence detected X-ray absorption spectroscopy (HERFD-XAS) and electron
paramagnetic resonance (EPR) spectroscopy, coupled with density functional theory
calculations, to characterize and understand the species. The HERFD-XAS spectrum of
the selenyl radical is distinguished by a uniquely low-energy transition with a peak energy
at 12 659.0 eV, which corresponds to a 1s → 4p transition to the singly occupied
molecular orbital of the free radical. The EPR spectrum shows the broad features and
highly anisotropic g-values that are expected for a selenium free radical species. The
availability of spectroscopic probes for selenyl radicals may assist in understanding why
life chooses selenium over sulfur in selected biochemical processes.
radicals are notoriously difficult to observe directly,5,13−15 and
1. INTRODUCTION
most frequently indirect methods such as spin-trapping have
The chemistries of sulfur and selenium have many similarities.1
been used.8 To our knowledge, there are currently no reports
Both elements are important in biology, with sulfur being one
of direct observations of selenyl radicals.
of the six elements that are central to all life, and selenium
Photochemical modification of elements of interest in X-ray
playing important roles in redox homeostasis. Selenium is
absorption spectroscopy (XAS) experiments is a well-
distinguished among the elements of life as having the lowest
established problem16 against which various experimental
crustal abundance,2 and is incorporated into biological
strategies have been developed;17 we recently exploited this
molecules at tremendous metabolic cost.3 It is further
photochemistry to enable a study of thiyl radicals using sulfur
distinguished as the element with the smallest differential
K-edge XAS.13 We report herein the first direct X-ray
between a dietary level that is appropriate for good human
spectroscopic observation of photochemically generated
health, and a toxic excess; a phenomenon that has caused Jukes
selenyl radicals in cryogenic aqueous solutions of the
to dub selenium as an “essential poison”.4 The physiologically
biologically relevant selenol, selenocysteine.
important selenolate (R−Se−), found as the L-selenocystei-
neate form of the amino acid, is possibly the strongest
nucleophile to be found in living systems, but many open
2. MATERIALS AND METHODS
2.1. Sample Preparation. Reagents were purchased from Sigma-
questions remain as to why it is so biologically important.
Aldrich (Oakville, ON) and were of the highest quality available.
Thiyl radicals (R−S·) are well-known in both chemistry and
Aqueous solutions of L-selenocysteine (1 mM final concentration)
biology; they have been exploited in synthetic organic
were prepared by reduction of the diselenide L-selenocystine with
chemistry,5 while in biology, they have been implicated in a
range of biological processes,6−8 such as the catalytic
mechanism of ribonucleotide reductases.9 In contrast to thiyl
radicals, the analogous selenium-based selenyl radicals (R−
Se·),10 sometimes called selanyl radicals, have been implicated
only rarely.11 Differences in reactivity between thiyl and selenyl
radicals have been used to explain why nature uses selenium in
preference to sulfur in some biological molecules.12 Thiyl
sodium borohydride as described by Pickering et al.,18 loaded into 2
mm thick polyacetal cuvettes closed with a mylar tape window, and
then frozen by immersion into a partly frozen isopentane slurry with a
temperature of approximately 120 K. Samples were transported and
stored at liquid nitrogen temperatures until data acquisition.
Received: June 1, 2018
Published: August 22, 2018
© 2018 American Chemical Society
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2.2. High Energy Resolution Fluorescence Detected X-ray
Absorption Spectroscopy. High energy resolution fluorescence
detected (HERFD) XAS measurements were carried out at the
Stanford Synchrotron Radiation Lightsource (SSRL) using beamline
6-2 with the SPEAR3 storage ring containing 500 mA at 3.0 GeV. A
Si(311) double crystal monochromator having an energy resolution at
the Se K-edge of ∼0.4 eV was used for the incident beam, together
with a 6-element array of Si(844) crystal analyzers to record the Se
Kα1 emission line,19 with an overall energy resolution of 0.95 eV
measured using scans of the elastic scattering. The incident and
transmitted X-rays were monitored using gas ionization chambers
filled with helium and nitrogen, respectively. An in-hutch photon
shutter was employed to prevent exposure of the sample when data
were not being actively recorded to ensure accurate exposure times,
and aluminum filters upstream of the incident ion chamber were used
to adjust X-ray exposures. Samples were maintained at a temperature
of 10 K using a helium flow cryostat (Oxford instruments, Abingdon,
U.K.) and were inclined at an angle of 45° to the incident X-ray beam
to facilitate measurement of X-ray fluorescence, giving an incident X-
ray path length of 2.8 mm. Energy calibration of the monochromator
was relative to the lowest-energy inflection of a gray hexagonal
selenium foil, which was assumed to be 12 658.0 eV. Data reduction
and analysis was carried out as previously described20 using the
EXAFSPAK suite of computer programs (http://ssrl.slac.stanford.
edu/exafspak.html).
lifetime broadening from the 1s core-hole, enormously
enhancing the spectral resolution. The residual observed
broadening of the HERFD-XAS spectra is dominated by
core-hole lifetime broadening of the 2p3/2 hole that is created
with the formation of the Kα1 fluorescent photon,32
convoluted with broadening functions arising from the energy
resolution of the emission spectrometer and to a small extent
from the beamline. The HERFD-XAS method requires a near
90° analyzer Bragg angle θB [Si(844) has θB ≈ 85.2° at the Se
Kα1 energy of 11 224 eV] and the use of a tightly focused
incident X-ray beam (SSRL beamline 6-2 has a beam size of
0.3 × 0.1 mm2 at 12.6 keV). During experiments designed to
characterize the capabilities of HERFD-XAS for biological
studies, we observed striking photochemical changes in
solutions of L-selenocysteine at a pH of 2.0, chosen to ensure
protonation of the selenol which has a pKa of 5.47;33 at this
pH, both the carboxylic acid and the amino group of the amino
acid also will be in their protonated states. For convenience in
discussion of the chemistry around selenium, we abbreviate L-
selenocysteine as Cys−SeH, where, in this case, Cys− denotes
the fragment HO2C(H3N+)CHCH2−.
Figure 1 shows the progressive changes in the HERFD-XAS
spectra of a solution of Cys−SeH with increasing X-ray
2.3. Electron Paramagnetic Resonance (EPR) Spectroscopy.
EPR spectroscopy was carried out using a JEOL RE1X X-band
instrument (JEOL, Tokyo, Japan) located adjacent to the beamline at
SSRL, and employing a sample temperature of 80 K. Irradiated
samples were transported under liquid nitrogen directly from the
beamline to the EPR spectrometer, and their EPR spectra were
recorded immediately.
2.4. Density Functional Theory (DFT) Calculations. DFT
geometry optimizations were carried out using DMol3 and Biovia
Materials Studio Version 2017 R221,22 using the Perdew−Burke−
Ernzerhof functional both for the potential during the self-consistent
field procedure, and for the energy.23,24 DMol3 double numerical basis
sets included polarization functions for all atoms with all-electron
relativistic core treatments. Environmental effects were modeled using
the Conductor-like Screening Model (COSMO)25 in DMol3, with a
dielectric value representing water (ε = 78.54).
DFT simulations of near-edge spectra were calculated using the
StoBe-deMon code26 employing the half-core-hole approximation for
the core-hole, incorporating relaxation of selected excited states at the
absorption edge, and employing the coordinates from Dmol3
geometry optimizations. StoBe-deMon calculations employed the
nonlocal exchange function of Perdew and Wang27 and the Perdew
correlation functional approximation.28,29 The (6311/311/1) basis set
was used for carbon and (311/1) for hydrogen. For selenium, the
(63321/5321/41) basis set was employed. Interpolation of the
exchange-correlation potential employed the auxiliary basis sets
(5,5;5,5) for selenium, (5,2;5,2) for carbon, nitrogen and oxygen,
and (3,1;3,1) for hydrogen. Computed spectra for open-shell free
radical species were averages of α and β symmetry; convolution with
pseudo-Voigt line shape functions was conducted as previously
described.30
Figure 1. Changes with increasing X-ray dose in the Se K-edge
HERFD-XAS spectra of a 1 mM solution of L-selenocysteine at pH
2.0, corresponding to calculated absorbed radiation doses of 0.5 MGy
(green line), 1.0 MGy (yellow line), and 2.9 MGy (red line). The
arrows indicate the direction of the change in the spectra with
increasing dose.
exposure, with three individual 2 min sweeps superimposed. A
low-energy peak at approximately 12 656.0 eV is observed to
appear and to accumulate in intensity with increased X-ray
exposure. The difference spectrum, which should correspond
to approximately 100% of the photochemical product, is shown
in Figure 2 together with the spectrum of pure starting Cys−
SeH, also obtained by difference. The 12 656.0 eV peak energy
of this product species is unusually low for a selenium K-edge
spectrum, with the lowest-energy features for inorganic
selenides (Se2−) typically falling around 12 659 eV. The
spectrum bears a striking resemblance to the sulfur K-edge
conventional XAS spectra recently reported by us for thiyl
radicals,13 allowing for the approximately 10 189 eV difference
between the sulfur and selenium K-edge threshold energies.
The species with the 12 659.0 eV peak does not form upon
irradiation of the deprotonated L-selenocysteinate Cys−Se−
(not illustrated) prepared similarly but at a higher pH, and
instead a different species with a peak energy ∼ 1 eV higher is
produced at much lower rates. Because of these considerations,
3. RESULTS AND DISCUSSION
3.1. X-ray Absorption Spectroscopy. In the present
work, we employ HERFD-XAS,31 which uses a high-resolution
X-ray spectrometer to measure the X-ray fluorescence with
considerably better resolution than the natural line width. In
conventional K-edge XAS, the short lifetime of the 1s core-hole
causes a substantial broadening of the spectra, with a
consequent loss of detail and chemical sensitivity. Our Se K-
edge HERFD-XAS employs the Se Kα1 fluorescence line, and
effectively observes just a subset of the 2p3/2 → 1s transitions
that give rise to the Se Kα1. This eliminates most of the
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Figure 2. Difference selenium K-edge HERFD-XAS spectra
corresponding to 100% reactant Cys−SeH (R−SeH), and 100%
photochemical product Cys−Se·. The difference spectrum for Cys−
SeH was generated by subtracting a fraction of the high-dose
spectrum from Figure 1 from that of the lowest dose to eliminate the
low-energy feature. Similarly, the spectrum of Cys−Se· was obtained
by subtracting small increments of the low dose from the high-dose
spectrum, until just before the appearance of negative features
corresponding to the peak signal intensity of Cys−SeH.
Figure 3. DFT computed ground state spin density of Cys−Se·
showing the schematic structure (top) together with the energy
minimized geometry optimized structure and the spin density
isosurface corresponding to 0.05 spins per a.u.3 (bottom). Computed
atomic Mulliken spin populations are shown on the schematic
structure; populations with absolute values of less than 0.001 are
omitted.
we assign the observed 12 659.0 eV low-energy feature to the
selenyl radical Cys−Se·. We hypothesize that the photo-
chemical reaction proceeds either by a direct photochemical
process (eq 1) or through chemistry involving the photolysis
products of water (eqs 2−4):
alternative yielded enthalpies that were 82 kJ mol−1 higher
than the selenyl radical species, which is well outside the range
of any significant thermal population, indicating that such
tautomers are probably unimportant. In Cys−Se·, the spin
densities on the two protons of the β-carbons differ, and are
lower by an order of magnitude than for the analogous sulfur-
based radical previously investigated. Like the thiyl species, the
proton oriented closer to the node of the spin density on
selenium has a smaller density than that further from the node,
suggesting that distinct proton hyperfine couplings for these
protons would be observed in electron nuclear double
resonance experiments of Cys−Se· or related species.
3.3. Computed Spectra of the Selenyl Radical. Figure
4 compares the DFT StoBe-deMon computed spectra for
Cys−Se· and Cys−SeH together with the experimental spectra
generated by difference. StoBe-deMon calculations predict an
isolated low-energy peak in the XAS of Cys−Se·, in strong
support of our conclusion that we have observed X-ray
photochemically generated selenyl radical species, and the
overall match of computed and experimental spectra is
excellent. In addition, the computed spectrum of the selenol
Cys−SeH also agrees very well with experiment (Figure 4). As
expected, the low-lying transition for Cys−Se· involves the
singly occupied molecular orbital. Additionally, the next
highest transition at ∼12 261 eV can be assigned to a Se(1s)
→ (Se−C)σ* transition, while the 12 261 eV peak in the
selenol corresponds to close-lying Se(1s) → (Se−C)σ* and
Se(1s) → (Se−H)σ* transitions.
Cys−SeH + hν → Cys−Se· + H·
(1)
H2O + hν → H· + HO·
(2)
Cys−SeH + H· → Cys−Se· + H2
(3)
Cys−SeH + HO· → Cys−Se· + H2O
(4)
SSRL beamline 6-2 has a very high flux density of 1.3 × 1014
photons s−1 mm−2; at similar high flux densities, evolution of
bubbles of H2 from X-ray irradiation of room-temperature
aqueous solutions has previously been reported, probably
arising through combination of two H· radicals.34 The
capability of direct generation of alkylselenyl radicals by
breakage of Se−H bonds can be estimated from the Se−H
bond dissociation energies which, at about 330 kJ mol−1 (or
3.4 eV),35 are substantially less than the X-ray energy
employed in our experiments. The hydrogen radicals generated
in eqs 1 and 2 are likely to engage in further chemistry; in our
system, the large excess of water will likely form hydrated
electrons, which can reduce redox active species, and can be
clearly seen as a beam mark on the irradiated sample from
color centers arising from hydrated electrons (eq 5) trapped in
the ice matrix.17
−
H2O + H· → H3O+ + eaq
(5)
3.2. Ground State Density Functional Theory Calcu-
lations. Figure 3 shows the results of energy minimized
geometry optimized DFT of Cys−Se·, superimposed with the
spin density isosurface, together with a schematic structure of
the species, labeled with the computed atomic Mulliken spin
populations. The spin densities of the free radical species are
seen to be highly localized on the selenium, with the singly
occupied molecular orbital being an almost pure Se 4p orbital.
Abstraction of a proton from the amino group could
potentially yield tautomeric structures with −SeH and a
nitrogen-centered free radical. DFT computation of this
3.4. Electron Paramagnetic Resonance (EPR) Spec-
troscopy. EPR spectroscopy is often considered the method
of choice for the study of free radical species. Previous work
has discussed the difficulties with observing features of the
analogous sulfur-based thiyl radical,13,14,36 due to substantial
spin−orbit coupling to filled sulfur-based orbitals, with a very
broad gzz at the high value of 2.3. With the selenium-based
selenyl radical, this broadening and shift to high g-values will
be even more pronounced, so that reports of EPR spectra of
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outside the g ∼ 2.0 region. Examination of the EPR of
selenocysteine-containing samples prior to irradiation (Figure
5), and of control samples containing no selenocysteine,
showed essentially baseline spectra with no EPR signals apart
from the expected g ∼ 2.0 signal in the irradiated
selenocysteine-free sample. The un-irradiated sample did
contain a small g ∼ 2.0 signal (Figure 5); since this signal
was also present in the polyacetal cuvette (not illustrated), we
conclude that the un-irradiated sample itself is EPR silent. The
EPR spectrum of selenyl is expected to be highly anisotropic
from spin−orbit coupling with filled selenium-based p-orbitals,
and we assign the feature at g ∼ 2.9 as gzz from this species,
with gxx and gyy being expected closer to g ∼ 2.0. Naturally
abundant selenium has a single magnetic isotope 77Se at 7.6%
abundance with a nuclear spin I = 1/2 and a nuclear g-value of
1.074. A sizable 77Se hyperfine coupling is expected from the
near-unity spin population on selenium, and we therefore
expect a sizable and anisotropic 77Se hyperfine coupling. While
quantitative measurement of this must await experiments using
selenocysteine enriched with 77Se, it seems probable that some
of the structure in Figure 5 may be due to 77Se hyperfine. The
EPR signal reported here was stable for a period of hours at
100 K and below, indicating that selenyl is a stable commodity
in solution at these temperatures.
Figure 4. Comparison of the computed HERFD-XAS spectra (sim.,
black line) with the experimental difference spectra (expt.; gold line)
for both Cys−Se· and Cys−SeH. The stick spectra of StoBe-DeMon
computed transitions are also shown (vertical red lines).
putative selenyl radicals with substantially lower gzz than thiyl37
probably arise from other species. Thus, while there are reports
of selenium-based free radical systems,38 to date, there have
been no authenticated reports of selenyl radical EPR spectra.
Figure 5 shows the EPR spectrum of an X-ray irradiated
The present study comprises the first example of the direct
detection and characterization of isolated selenium-based free
radicals using X-ray absorption spectroscopy, and the first X-
ray spectroscopic observation of a selenyl radical. While selenyl
radicals may well have been generated with previous XAS
experiments,18 the core-hole broadening of conventional XAS
spectra means that such species might be difficult to resolve.
Figure 6 shows a comparison of conventional XAS with
Figure 5. Electron paramagnetic resonance spectra of a sample of
selenocysteine prior to (lower trace) and following (upper trace) X-
ray irradiation. The gray lines show zero for the different spectra.
Spectra were recorded at 80 K using 0.5 mT modulation amplitude
with a microwave frequency of 9.194 GHz. The vertical scale of the g
∼ 2.0 region of the irradiated spectrum has been scaled down by a
factor of 200 relative to both the outer spectrum and the un-irradiated
spectrum.
Figure 6. Comparison of XAS with HERFD-XAS for selenocysteine.
The XAS spectrum18 is shown as blue line and the HERFD-XAS by
the red line (spectrum of Figure 2). The dramatic improvement in
resolution arising from the HERFD-XAS method is clearly apparent.
HERFD-XAS, clearly showing the substantially enhanced
resolution of HERFD-XAS measurements. Thus, these
observations of the selenyl radical are enabled by high photon
flux intensity coupled with the exquisitely high spectral
resolution afforded by HERFD-XAS.
The characteristic spectrum of selenyl, with a pronounced
low-energy peak arising from the paramagnetic singly occupied
molecular orbital, shows some parallels with the well-studied
ligand K-edge spectroscopy of transition metal species.39,40
These were first understood more than three decades ago, by
Sigiura et al.41 and subsequently by others42−46 as due to
aqueous selenocysteine sample. As expected, the spectrum is
dominated by an intense g ∼ 2.0 signal arising from abundant
free radical species that are either trapped in the ice matrix or
contained in the polyacetal sample cuvette.16 The EPR signals
of selenyl are predicted to occur at either side of the g ∼ 2.0
signal; since a broad gzz at low field (high g-value) is expected,
Figure 5 shows a vertically expanded spectrum. The low signal
amplitude is not correlated with an overall low integrated
intensity and arises because of the broad nature of the signal
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covalency between ligand and metal d-orbitals giving partial
holes in ligand-based orbitals, resulting in intense dipole-
allowed transitions at lower energy than other spectral features.
This observation has been exploited extensively for a variety of
different chemical and biochemical systems as a direct probe of
ligand covalency.39,40 To date, such ligand K-edge features
have not been reported upon in detail for selenium
coordination, but the analogous transitions for sulfur ligands
have been very well studied.39,40 In general, coordination
geometries with more overlap between metal and ligand
orbitals tend to have more intense transitions at higher
transition energies, whereas those with less overlap tend to
have lower intensities, and to be more free-radical-like, arising
at lower transition energies. Like the thiyl radical for sulfur,13
the selenyl system reported here is expected to represent a low-
energy limit below which K-edge associated transitions are not
expected to arise.
The reasons behind life’s use of selenium in preference to
sulfur in some systems are still somewhat unclear.3 First, and as
briefly mentioned above, a tremendous metabolic effort is
expended by cells in order to incorporate selenium into
biological molecules,3 and the process of insertion of
selenocysteine is enormously more complex than it is for
cysteine or for any of the 19 other canonical amino acids.
Second, since selenium itself is also toxic,3,4 its use poses an
inherent biochemical risk. Selenium enzymes may be favored
as they are often much more effective in terms of catalytic rate
than their sulfur counterparts. However, another hypothesis as
to why selenium is used in preference to sulfur in antioxidant
enzymes such as the thioredoxin reductases relates to the
inherent differences between the chemistry of thiyl and selenyl
radicals. Thus, in these systems, accidental one-electron
transfer can generate either thiyl or selenyl radicals with sulfur
and selenium enzymes, respectively.12 The thiyl radical can
abstract hydrogen from the protein backbone,47 forming
carbon centered radicals that could reform thiyl or react with
O2 to form peroxy (O2·) radicals, resulting in further damage
and consequent enzyme inactivation.12 In contrast, selenyl will
not abstract hydrogen, minimizing the risks of inactivation
from side-reactions in these systems.12 Our studies reported
here provide both the first direct detection of and a
spectroscopic signature for selenyl free radicals, and may
provide important tools for directly addressing this chemistry.
grant in Health Research Using Synchrotron Techniques
(THRUST). N.V.D. and J.J.H.C. were THRUST Associates.
Use of the Stanford Synchrotron Radiation Lightsource, SLAC
National Accelerator Laboratory, is supported by the U.S.
Department of Energy, Office of Science, Office of Basic
Energy Sciences, under Contract No. DE-AC02-76SF00515.
The SSRL Structural Molecular Biology Program is supported
by the DOE Office of Biological and Environmental Research,
and by the National Institutes of Health, National Institute of
General Medical Sciences (including P41GM103393). The
contents of this publication are solely the responsibility of the
authors and do not necessarily represent the official views of
NIGMS or NIH.
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AUTHOR INFORMATION
Corresponding Author
*E-mail: g.george@usask.ca.
■
ORCID
Ingrid J. Pickering: 0000-0002-0936-2994
Graham N. George: 0000-0002-0420-7493
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work was supported by Discovery Grants from the
Natural Sciences and Engineering Research Council of Canada
(NSERC, I.J.P. and G.N.G.), an Innovation and Science Fund
of Saskatchewan grant (I.J.P.), a Canada Foundation for
Innovation John Evans Leader’s Fund award (I.J.P.) and by
Canada Research Chairs (I.J.P. and G.N.G.). S.N. acknowl-
edges funding from the Dr. Rui Feng Scholarship and was a
Fellow in the Canadian Institutes of Health Research-Training
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DOI: 10.1021/acs.inorgchem.8b01522
Inorg. Chem. 2018, 57, 10867−10872

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