Sulfur K-edge X-ray Absorption Spectroscopy From Molecules to

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					                Sulfur K-edge X-ray Absorption Spectroscopy:
                        From Molecules to Whole Cells

                                                        Ingrid J. Pickering

Department of Geological Sciences, University of Saskatchewan, 114 Science Place, Saskatoon
SK, Canada S7N 5E2

I acknowledge the invaluable assistance of my coworkers Graham George, Eileen Yu Sneeden
and Hugh Harris of SSRL and Roger Prince of ExxonMobil. Much of the work presented was
supported by NIH GM57375. The Stanford Synchrotron Radiation Laboratory is a national user
facility operated by Stanford University on behalf of the U.S. Department of Energy, Office of
Basic Energy Sciences. The SSRL Structural Molecular Biology Program is supported by the
Department of Energy, Office of Biological and Environmental Research, and by the National
Institutes of Health, National Center for Research Resources, Biomedical Technology Program.
These notes are adapted from a presentation at the CAMD Workshop on Biological Applications
of Synchrotron Radiation, June 2-6, 2003.

A. Introduction.....................................................................................................................2
B. Examples .........................................................................................................................3
    • Identification of solution species in model of biological active site........................3
    • Determination of sulfur forms in whole blood ........................................................4
    • Biodesulfurization....................................................................................................5
    • Transformations of sulfur species in wasabi and horseradish .................................6
D. Further Reading...............................................................................................................8

Ingrid J. Pickering                                                    1                     Sulfur K-edge X-ray Absorption Spectroscopy
A. Introduction
Sulfur is an essential biological element, yet its biochemistry is only partially understood because
there are so few tools for studying the element in biological systems. Indeed, it has even been
called a "spectroscopically silent" element. Sulfur K-edge X-ray absorption spectroscopy (XAS)
provides a unique approach for determining the chemical forms of sulfur in intact biological
samples, and for quantifying their contributions.

Note that while this talk is specific to the sulfur K-edge, much of what is discussed is applicable
to other absorption edges.

No pretreatment required. Unlike many other analytical techniques which require extensive
pretreatments, XAS can be applied essentially without pretreatment so the sample can be
examined in its native form. Moreover, the beam interrogates all forms of the element in the
sample however they are incorporated, including in solution, in crystalline or amorphous solids
and in gases. (This of course is generally applicable to other elements, not just to the S K-edge.)
Hence, it is a unique probe of such complex samples as intact biological tissues or cell cultures,
in addition to solutions, purified materials, etc.

Rich structure in near-edge spectra. The near-
edge region of any spectrum is dominated by
dipole-allowed (∆l = ±1) bound-state
transitions of the 1s electron (for a K-edge) to
vacant molecular orbitals of substantial p-
orbital character. Hence, the near-edge                                                          SO42-
spectrum provides a sensitive probe of
electronic structure and thus of chemical form.                                                  RSO3-
Sulfur shows particularly rich near-edge
spectra, due to valence p-orbitals, the relatively
sharp line-widths at these energies and the
large chemical shift range (of some 14 eV)
over its range of oxidation states (-2 to +6).                                                   RSO2-
The figure (right) shows examples of
biologically relevant S K-edge spectra.
Note that, rather than identifying specific
compounds, we are identifying classes of                    x2                                   RS-Me
compounds, for example aliphatic thiols rather                                                   RS-H
than cysteine itself. While small variations in
structure may allow us to be more specific, in              x2                                   RS-SR
many complex systems (see below) there are                  x2                                   S8
too many things to identify individual species.

Ingrid J. Pickering                                  2           Sulfur K-edge X-ray Absorption Spectroscopy
B. Examples
Identification of solution species in model of biological active site. The species
[LMoOS(OPh)] [L = hydrotris(3-isopropylpyrazol-1-yl)borate] was synthesized as a possible
model of the [MoVIOS]2+ active site of the hydroxylase molybdenum enzymes [P. D. Smith, D.
A. Slizys, G. N. George, C. G. Young, J. Am. Chem. Soc. 122(12), 2946-2947 (2000)]. In the
solid it exists as the S-S bonded MoV dimer, as shown by the crystal structure and confirmed by
S K-edge XAS. However, in solution XAS reveals the presence of the monomer with the sulfur
present as the MoVI=S thio group. This metal coordination gives rise to the unusually low energy
peaks shown in the solution spectrum (below). A small contribution of the monomer can also be
seen in the solid spectrum. The synthesis of the monomeric oxo-thio molybdenum site had
eluded synthetic chemists for more than 20 years, and its identification by XAS was a
breakthrough for the understanding of this group of molybdenum enzymes.



Ingrid J. Pickering                            3              Sulfur K-edge X-ray Absorption Spectroscopy
Determination of sulfur forms in whole blood. Sulfur K-edge XAS was used to determine the
sulfur forms in packed erythrocytes and plasma [I. J. Pickering, R. C. Prince, T. Divers, G. N.
George, FEBS Lett. 441, 11-14 (1998)]. The percentages of different forms are shown in the
table below. The plasma shows considerable protein disulfide component which is expected as
crystalline horse serum albumin contains 34 disulfide sulfurs, and only one cysteine and no
methionines. In contrast, intracellular cysteine is expected to be mainly present as the thiol, as is

Analysis of the chemical forms of sulfur in
packed erythrocytes (A) and plasma (B) from
horse blood. Spectra were fit to the sum of the
spectra of five model compounds, all in
aqueous solution at pH 7.0, representing the
different chemical forms that are likely to be
present: (a) oxidized glutathione (R-S-S-R), (b)
cysteine (R-S-H), (c) methionine (R-S-R), (d)
methionine sulfoxide (RSOR) and (e) sulfate
(SO42-) The points show the experimental data
and the overlaid solid line the results of the fit,
with the residual beneath. The individual
components shown as broken lines have been
scaled according to their percentages in the fit

                      R-S-S-R          R-S-H              R-S-R            RSOR                 SO42-
Erythrocytes            21              55                 21                2                   1
Plasma                  76              21                  0                0                   3

Ingrid J. Pickering                                   4           Sulfur K-edge X-ray Absorption Spectroscopy
Biodesulfurization. Oil refineries remove most of the sulfur in fuels by hydrodesulfurization,
where sulfur-containing species react with high-pressure hydrogen, in the presence of a catalyst,
to produce H2S and desulfurized products. Sterically hindered dibenzothiophenes are amongst
the most resistant species, but biodesulfurization, where sulfur is removed by an enzymatic
process that yields sulfate, may provide an alternative or perhaps complementary technology for
attaining low sulfur fuels.



The sulfur in most crude oils is thiophenic and aliphatic. The latter is readily removed during
processing, whereas hindered thiophenes are particularly recalcitrant to hydrodesulfurization.
Organisms have been isolated which are able to remove the sulfur in hindered dibenzothiophenes
(DBT) to leave hydroxy-biphenyls.

Sulfur K-edge X-ray absorption spectroscopy was used to examine the effect of one strain of
Rhodococcus [M. J. Grossman, M. K. Lee, R. C. Prince, K. K. Garrett, G. N. George and I. J.
Pickering, Appl. Environ. Microbiol. 65, 181-188 (1999)]. This Rhodococcus oxidizes much of
the thiophenic sulfur, but some oxidized forms accumulate. This experiment used an unprocessed
diesel cut of a crude oil, in which the sulfur was initially approximately 50% thiophenic. In this
experiment, 30% of the sulfur was removed as sulfate, and 50% of the remainder was oxidized to
the sulfone and sultine.

Ingrid J. Pickering                             5                Sulfur K-edge X-ray Absorption Spectroscopy
Transformations of sulfur species in wasabi and horseradish. Sulfur K-edge XAS has been
used to determine the chemical identity of the sulfur-containing species in the intensely flavored
plant roots horseradish (Armoracia laphthifolia) and wasabi (Wasabia japonica) in situ, before
and after cell disruption [E. Y. Yu, I. J. Pickering, G. N. George, R. C. Prince, Biochim. Biophys.
Acta, 1527(3), 156-160, (2001)]. The major sulfur-containing species in the intact root is
sinigrin (1-thio-β-D-glucopyranose 1-N-(sulfoxy)-3-buteneimidate) and related congeners.
Disrupting the cells by applying local pressure allowed the conversion of the sulfur moieties in
sinigrin to the intensely lachrymatory allyl isothiocyanate and sulfate in approximately
equimolar amounts. In contrast to previous suggestions, no detectable thiocyanates were formed,
but an unusual thio intermediate, the precursor to the Lossen rearrangement, may have been
observed for the first time.

                                                                                    allyl isothiocyanate

                                                                                       allyl thiocyanate


Above: Schematic of the degradation of β-D-S-                                               methionine
glucosides (I) by myrosinase (β-thioglucoside
glucohydrolase) and related biochemistry. The
initial hydrolysis of (I) gives rise to glucose (II) and
(III), which will be in equilibrium with (IV). (IV) is
then thought to undergo a Lossen rearrangement to                                                sulfate
produce the isothiocyanate (V) and sulfate. Right: S
K-edge spectra of standard species relevant to
horseradish and wasabi. Allyl isothiocyanate and
ethyl thiocyanate were in toluene solution, and all
other compounds were in aqueous solutions at
neutral pH.

Ingrid J. Pickering                                6            Sulfur K-edge X-ray Absorption Spectroscopy
                                                      Results of curve-fitting analyses of sulfur
         After                                        K-edge X-ray absorption spectra of a
         bruising                                     section of horseradish root. Spectra are
                                                      shown before (lower) and after (upper)
                                                      disruption of the cells by bruising. The data
                                                      are shown as filled circles, the best fit as a
                                                      solid line, and standards, scaled according
                                                      to their fitted contributions to the
                                                      horseradish spectra, as broken lines (sini. –
         Before                                       sinigrin; RNCS – allyl isothiocyanate; RSR
         bruising                                     – methionine; SO42- – sulfate). The residual
                                                      (observed minus calculated) spectrum is
                                                      shown beneath. The asterisk indicates a
                                                      minor component present only in the
                                                      bruised data set that was not modeled by the
                                                      curve-fitting analysis. Numerical results are
                                                      shown below in terms of percentage of total
                                                      sulfur. Values in parentheses are 95%
                                                      confidence limits, estimated from the
                                                      diagonal elements of the covariance matrix.
Sulfur                         Horseradish                                 Wasabi
Species               Intact    Bruised    ∆% per S     Intact      Bruised             ∆% per S
sinigrin              53(4)      15(5)       -38        45(2)         7(2)                -38
sulfate               31(1)      47(1)        16        29(1)        49(1)                 20
isothiocyanate        13(8)      35(6)       22         13(3)        31(3)                 18
thioether             3(12)       2(7)        -1        14(3)        12(5)                 -2

                                              Spectroscopic identification of a species with novel
                                              sulfur coordination in bruised horseradish. A close-
                                              up of the spectrum of the bruised horseradish
                                              sample is shown in (a), clearly indicating a small
                                              peak of unknown origin at about 2468 eV. Pseudo
                                              Voigt peak deconvolution of this spectrum yielded
                                              a peak energy of 2468.2 eV (a'). The spectrum of
                                              the thioketone, 2,2,4,4-tetramethyl-1,3-
                                              cyclobutanedithione, is compared in (b) (peak
                                              energy is 2467.5 eV). Ab initio calculations indicate
                                              that the peak energy of the thioketone model should
                                              be 0.5 eV lower than that of the proposed
                                              intermediate, in agreement with the assignment of
                                              this feature.

Ingrid J. Pickering                              7               Sulfur K-edge X-ray Absorption Spectroscopy
C. Further Reading

The following is a (personally-biased!) list of possible further reading:

First reports of sulfur spectroscopy of complex mixtures:
C. L. Spiro, J. Wong, F. W. Lytle, R. B. Greegor, D. H. Maylotte, S. H. Lamson, "X-ray
Absorption Spectroscopic Investigation of Sulfur Sites in Coal: Organic Sulfur Identification",
Science, 226(4670) 48-50 (1984).

Sulfur in biological systems:
I. J. Pickering, R. C. Prince, T. Divers and G. N. George, "Sulfur K-edge X-ray Absorption
Spectroscopy for Determining the Chemical Speciation of Sulfur in Biological Systems",
Federation of European Biochemical Societies (FEBS) Letters, 441, 11-14 (1998).

Sulfur in bacteria and self-absorption (thickness effects):
I. J. Pickering, G. N. George, E. Y. Yu, D. C. Brune, C. Tuschak, J. Overmann, J. T. Beatty and
R. C. Prince, "Analysis of sulfur biochemistry of sulfur bacteria using X-ray absorption
spectroscopy", Biochemistry, 40(27), 8138 -8145, (2001).

Sulfur in plant tissues:
E. Y. Yu, I. J. Pickering, G. N. George and R. C. Prince, "In situ observation of the generation of
isothiocyanates from sinigrin in horseradish and wasabi", Biochim. Biophys. Acta, 1527(3), 156-
160, (2001).

Sulfur in tunicate blood:
P. Frank, B. Hedman, K. O. Hodgson, "Sulfur Allocation and Vanadium-Sulfate Interactions in
Whole Blood Cells from the Tunicate Ascidia ceratodes, Investigated Using X-ray Absorption
Spectroscopy" Inorg. Chem. 38(2) 260-270 (1999).

Ligand covalency and electronic structure:
E. Anxolabehere-Mallart, T. Glaser, P. Frank, A. Aliverti, G. Zanetti, B. Hedman, K. O.
Hodgson, E. I. Solomon, "Sulfur K-Edge X-ray Absorption Spectroscopy of 2Fe-2S Ferredoxin:
Covalency of the Oxidized and Reduced 2Fe Forms and Comparison to Model Complexes", J.
Am. Chem. Soc. 123(23); 5444-5452 (2001).

Ingrid J. Pickering                              8               Sulfur K-edge X-ray Absorption Spectroscopy