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Graham N. George
X-ray Absorption Spectroscopy of Molybdenum Enzymes
Graham N. George
Graham N. George
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.
Graham N. George
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.
Graham N. George
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 ΔR≈π/2k – generally about 0.15 Å.
• Little or no geometrical information.
• Analysis not always reliable (especially with black box software).
Graham N. George
X-ray Absorption near-edge spectra – sensitivity
Se-methionine
elemental Se
selenate
2-
selenite
2-
Graham N. George
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.
Graham N. George
DMSO reductase
S
O
CH3 CH3
2H+, 2e-
SCH3 CH3
H2O
Catalyses the two-electron reduction of dimethylsulfoxide to dimethylsulfide.
Prototypical member of the DMSO reductase family of Mo enzymes
Graham N. George
DMSO reductase
Oxidized enzyme – Active site
Graham N. George
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.
Graham N. George
DMSO reductase – interaction with substrates and products
Ser147
S
Mo
S
O
Bound DMSO
McAlpine, A. S.; McEwan, A. G.; Bailey, S. (1998) J. Mol. Biol. 275, 613-623.
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.
Graham N. George
Interaction of DMSO reductase with dimethyl sulfide
Open questions:
• Is it an oxidized or a reduced species? Suggestions include:1. A fully reduced MoIV site.1
2. A partly reduced site MoV-O-S(CH3)2.2
3. 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 S=O 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, 9810-98203. Bennett, B. et al. (unpublished)
Graham N. George
EXAFS of (CH3)2S bound DMSO reductase
data
fit
Mo-S + Mo-O
Mo-O
EXAFS indicates4 Mo-S at 2.37 Å1 Mo-O at 2.23 Å1 Mo-O at 1.98 Å(no short Mo=O)Cannot see DMSO
George et al. (1999) J. Am. Chem. Soc. 121, 1256-1266.
Graham N. George
Interaction with alternative products
Dimethylsulfide – ~5mM (CH3)2S
Dimethylselenide – ~60mM (CH3)2Se forms analogous species
Trimethylarsine – 1:1 (CH3)3As (stoichiometric) with enzyme. Trimethylphosphine – ~5mM (CH3)3P yellow species forms.
Graham N. George
Mo K near-edge spectra
oxidized(CH3)2S
(CH3)2Se
(CH3)2As
• 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)
Graham N. George
Mo K-edge EXAFS Fourier Transforms
oxidized
(CH3)2S
(CH3)2Se
(CH3)2As
mono-oxo tetrathiolate
des-oxo tetrathiolate species No EXAFS observed for (CH3)2S sulfur
Mo=O
Mo-S
Mo····As
Mixed with oxidized enzyme, Mo···Se observed
stoichiometric, Mo···As observed
(CH3)2S, (CH3)2Se and (CH3)3As appear to form structurally related species.
Graham N. George
As K near-edge spectra
(CH3)3As
(CH3)3AsO
DMSOR + (CH3)3As
• Arsenic is oxidized to AsV in (CH3)3As bound enzyme
Graham N. George
As K-edge EXAFS
data fit
As
Mo
Mo-O
Mo-S
Mo····As
data
fitAs=O
As-C
As····Mo
• EXAFS shows (CH3)3As located at Mo site.• Both As=O and As-C interactions are clearly resolved.
Graham N. George
EXAFS of (CH3)3As-bound DMSO reductase
• Arsenic is oxidized (AsV)
• Molybdenum is reduced (MoIV)
• As=O bond-length is within normal range – no particular distortion is present.
2.37 Å
3.44 Å
2.23 Å2.01 Å
1.70 Å
MoS
As
OSer147
Graham N. George
DFT of (CH3)3As-bound DMSO reductase
• (CH3)3As remains bound but with longer than observed Mo-O=As 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
Graham N. George
DFT Calculation – (CH3)2S=O leaves active site…
Active site pocket must be important in stabilizing bound form
Graham N. George
The Future – High Resolution EXAFS
Graham N. George
Effect of k-range on EXAFS resolution
Mo
N
Mo
N
Graham N. George
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.
Graham N. George
High resolution EXAFS of sulfite oxidase
High-resolution EXAFS Scan
Ordinary EXAFS Scan
2002 9-3, 55min.
1996 7-3, 35min.k=14 Å-1
k=25 Å-1
• 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.
Graham N. George
High resolution EXAFS of sulfite oxidase
Graham N. George
Graham N. George
Graham N. George
Graham N. George
Graham George / Ingrid Pickering Group
Graham N. George
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.
The National Institutes of Health GM57375
Acknowledgements
Graham N. George
• 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
Future Directions…
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