X-ray Absorption Spectroscopy of Molybdenum Enzymes

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X-ray Absorption Spectroscopy of Molybdenum
Enzymes
Graham N. George
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Overview

• Strengths and Limitations of X-ray Absorption
  Spectroscopy.
• XAS studies of enzymes of DMSO reductase.
• High resolution EXAFS spectroscopy.
• Combined approach use EXAFS spectroscopy and
  Density Functional Theory.

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X-ray Absorption Spectroscopy

• EXAFS (Extended X-ray Absorption Spectroscopy)
  oscillations in X-ray absorption Gives a Radial
  Structure
• Examine Fourier transform peaks occur at
  inter-atomic distances (usually not interpreted
  directly).
• Fit theoretical model to EXAFS spectra.
• Modern ab initio codes (e.g. FEFF) are very
  accurate little requirement for standards.

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X-ray Absorption Spectroscopy Strengths and
Limitations

• Examines all of a particular element in a sample.
• Can examine any phase (solids, solutions etc.).
• Accurate bond-lengths (better than ?0.02 Å).
• Approximate coordination numbers atomic number
  (?15).
• Oxidation state (often only relative).
• Poor resolution ?Rp/2k generally about 0.15 Å.
• Little or no geometrical information.
• Analysis not always reliable (especially with
  black box software).

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X-ray Absorption near-edge spectra sensitivity
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Density Functional Calculations

• Modern codes are simple to use and run.
• Inexpensive computer systems (e.g. we use an 8 x
  2.8GHz Xenon processor Linux cluster).
• EXAFS analysis run on same computers.
• Absolute accuracy of bond-lengths is poor our
  bond-lengths are up to about 0.1Å too long for
  functionals used.

Density Functional theory calculations used the
Dmol3 Materials Studio V2.2. The Becke exchange
and Perdew correlation functionals were used to
calculate both the potential during the SCF, and
the energy. Double numerical basis sets included
polarization functions for all atoms.
Calculations were spin-unrestricted and all
electron core potentials were used.
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DMSO reductase
Prototypical member of the DMSO reductase family
of Mo enzymes
Catalyses the two-electron reduction of
dimethylsulfoxide to dimethylsulfide.
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DMSO reductase
Oxidized enzyme Active site
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Active Site Structure - Perspective

• Previously there has been much debate about
  structure of active site.
• Many crystal structures have been published with
  chemically impossible arrangements of atoms at
  the active site (e.g. active site too crowded).
• All DMSO reductase crystal structures published
  to date have some sort of problem of this nature.
  
• This has been attributed to multiple species
  co-crystallizing.

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DMSO reductase interaction with substrates and
products
DMSO reductase binds dimethylsulfide to form a
pink-purple species. The exact nature of this
novel species is very interesting as it is likely
to be important in developing an understanding of
catalytic mechanism.
McAlpine, A. S. McEwan, A. G. Bailey, S. (1998)
J. Mol. Biol. 275, 613-623.
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Interaction of DMSO reductase with dimethyl
sulfide

• Open questions
• Is it an oxidized or a reduced species?
  Suggestions include
• A fully reduced MoIV site.1
• A partly reduced site MoV-O-S(CH3)2.2
• An oxidized MoVI site.3
• Is the S-O bond longer than normal?
• Crystallography indicates 1.7 Å, which compares
  with the value of 1.53 Å for DMSO bound to Mo in
  models, and 1.50 Å for free DMSO. Suggested that
  binding to enzyme weakens the SO double bond.

1. McAlpine, A. S. McEwan, A. G. Bailey, S. (1998)
   J. Mol. Biol. 275, 613-623.
2. Bray et al. (2001) Biochemistry 40, 9810-9820
3. Bennett, B. et al. (unpublished)

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EXAFS of (CH3)2S bound DMSO reductase
EXAFS indicates 4 Mo-S at 2.37 Å 1 Mo-O at 2.23
Å 1 Mo-O at 1.98 Å (no short MoO) Cannot see DMSO
George et al. (1999) J. Am. Chem. Soc. 121,
1256-1266.
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Interaction with alternative products
Dimethylsulfide 5mM (CH3)2S Dimethylselenide
60mM (CH3)2Se forms analogous
species Trimethylarsine 11 (CH3)3As
(stoichiometric) with enzyme. Trimethylphosphine
5mM (CH3)3P yellow species forms.
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Mo K near-edge spectra

• Near-edge spectra are shifted to lower energy
  with respect to oxidized enzyme. Consistent with
  a relative reduction of the metal site (e.g. MoIV
  vs. MoVI oxidized)

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Mo K-edge EXAFS Fourier Transforms
Mo-S
MoO
mono-oxo tetrathiolate
des-oxo tetrathiolate species No EXAFS observed
for (CH3)2S sulfur
Mixed with oxidized enzyme, MoSe observed
MoAs
stoichiometric, MoAs observed
(CH3)2S, (CH3)2Se and (CH3)3As appear to form
structurally related species.
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As K near-edge spectra

• Arsenic is oxidized to AsV in (CH3)3As bound
  enzyme

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As K-edge EXAFS

• EXAFS shows (CH3)3As located at Mo site.
• Both AsO and As-C interactions are clearly
  resolved.

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EXAFS of (CH3)3As-bound DMSO reductase
Ser147

• Arsenic is oxidized (AsV)
• Molybdenum is reduced (MoIV)
• AsO bond-length is within normal range no
  particular distortion is present.

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DFT of (CH3)3As-bound DMSO reductase

• (CH3)3As remains bound but with longer than
  observed Mo-OAs distance.
• DFT Mo-S 2.41, Mo-O(Ser) 1.95, Mo-O(AsMe3) 2.45,
  Mo-As 3.56
• EXAFS Mo-S 2.37, Mo-O(Ser) 2.01, Mo-O(AsMe3)
  2.23, Mo-As 3.44

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DFT Calculation (CH3)2SO leaves active site
Active site pocket must be important in
stabilizing bound form
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The Future High Resolution EXAFS
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Effect of k-range on EXAFS resolution
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Sulfite Oxidase

• Sulfite Oxidase Crystal Structure
• Initially, the enzyme was in the fully-oxidized
  MoVI form
• Photoreduction (probably) during data acquisition
  reduced enzyme to MoIV via MoV.
• Data likely arises from of a mixture of all three
  oxidation states.

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High resolution EXAFS of sulfite oxidase

• Modern high-intensity beamlines and detector
  systems allow us to significantly extend the
  range of the data.
• This allows data to be collected at higher
  resolution.
• Technical issues Problems with data
  acquisition (beamline stability). Problems
  using ab initio theory at very high k.

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High resolution EXAFS of sulfite oxidase
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Graham George / Ingrid Pickering Group
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Acknowledgements
The National Institutes of Health 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.
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Future Directions

• Spectroscopy at low temperatures !
• As of Summer 2003, Graham George Ingrid
  Pickering Canada Research Chairs in X-ray
  Absorption Spectroscopy and Molecular
  Environmental Science at University of
  Saskatchewan, home of the Canadian Light Source