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

2. 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.

3. 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.

4. 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).

5. Graham N. George X-ray Absorption near-edge spectra – sensitivity Se-methionine elemental Se selenate 2- selenite 2-

6. 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 Dmol 3 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.

7. Graham N. George DMSO reductase 2H +, 2e - H2OH2O Catalyses the two-electron reduction of dimethylsulfoxide to dimethylsulfide. Prototypical member of the DMSO reductase family of Mo enzymes

8. Graham N. George DMSO reductase Oxidized enzyme – Active site

9. 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.

10. 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.

11. 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 Mo IV site. 1 2.A partly reduced site Mo V -O  -S(CH 3 ) 2. 2 3.An oxidized Mo VI 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, 9810-9820 3.Bennett, B. et al. (unpublished)

12. Graham N. George EXAFS of (CH 3 ) 2 S bound DMSO reductase data fit Mo-S + Mo-O Mo-O 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 Mo=O) Cannot see DMSO George et al. (1999) J. Am. Chem. Soc. 121, 1256-1266.

13. Graham N. George Interaction with alternative products Dimethylsulfide – ~5mM (CH 3 ) 2 S Dimethylselenide – ~60mM (CH 3 ) 2 Se forms analogous species Trimethylarsine – 1:1 (CH 3 ) 3 As (stoichiometric) with enzyme. Trimethylphosphine – ~5mM (CH 3 ) 3 P yellow species forms.

14. Graham N. George Mo K near-edge spectra oxidized (CH 3 ) 2 S (CH 3 ) 2 Se (CH 3 ) 2 As Near-edge spectra are shifted to lower energy with respect to oxidized enzyme. Consistent with a relative reduction of the metal site (e.g. Mo IV vs. Mo VI oxidized)

15. Graham N. George Mo K-edge EXAFS Fourier Transforms oxidized (CH 3 ) 2 S (CH 3 ) 2 Se (CH 3 ) 2 As mono-oxo tetrathiolate des-oxo tetrathiolate species No EXAFS observed for (CH 3 ) 2 S sulfur Mo=O Mo-S Mo····As Mixed with oxidized enzyme, Mo···Se observed stoichiometric, Mo···As observed (CH 3 ) 2 S, (CH 3 ) 2 Se and (CH 3 ) 3 As appear to form structurally related species.

16. Graham N. George As K near-edge spectra (CH 3 ) 3 As (CH 3 ) 3 AsO DMSOR + (CH 3 ) 3 As Arsenic is oxidized to As V in (CH 3 ) 3 As bound enzyme

17. Graham N. George As K-edge EXAFS data fit As Mo Mo-O Mo-S Mo····As data fit As=O As-C As····Mo EXAFS shows (CH 3 ) 3 As located at Mo site. Both As=O and As-C interactions are clearly resolved.

18. Graham N. George EXAFS of (CH 3 ) 3 As-bound DMSO reductase Arsenic is oxidized (As V ) Molybdenum is reduced (Mo IV ) As=O bond-length is within normal range – no particular distortion is present. 2.37 Å 3.44 Å 2.23 Å 2.01 Å 1.70 Å Mo S As O Ser147

19. Graham N. George DFT of (CH 3 ) 3 As-bound DMSO reductase (CH 3 ) 3 As remains bound but with longer than observed Mo-O=As distance. DFTMo-S 2.41, Mo-O(Ser) 1.95, Mo-O(AsMe 3 ) 2.45, Mo-As 3.56 EXAFSMo-S 2.37, Mo-O(Ser) 2.01, Mo-O(AsMe 3 ) 2.23, Mo-As 3.44

20. Graham N. George DFT Calculation – (CH 3 ) 2 S=O leaves active site… Active site pocket must be important in stabilizing bound form

21. Graham N. George The Future – High Resolution EXAFS

22. Graham N. George Effect of k-range on EXAFS resolution Mo N N

23. Graham N. George Sulfite Oxidase Sulfite Oxidase Crystal Structure Initially, the enzyme was in the fully-oxidized Mo VI form Photoreduction (probably) during data acquisition reduced enzyme to Mo IV via Mo V. Data likely arises from of a mixture of all three oxidation states.

24. 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.

25. Graham N. George High resolution EXAFS of sulfite oxidase

26. Graham N. George

29. Graham George / Ingrid Pickering Group

30. 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

31. 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…