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

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…