1. Wednesday, March 17, 2004Graham N. George When Good Crystallography Goes Bad: The Active Site Structure of Mo Enzymes Graham N. George Department of Geological Sciences, University of Saskatchewan.

2. Wednesday, March 17, 2004Graham N. George Overview Introduction to X-ray Absorption SpectroscopyIntroduction to X-ray Absorption Spectroscopy Introduction to Molybdenum EnzymesIntroduction to Molybdenum Enzymes The Active Site Structure of DMSO ReductaseThe Active Site Structure of DMSO Reductase Recent workRecent work

3. Wednesday, March 17, 2004Graham N. George E  Absorbance  = log e (I 0 /I 1 ) Ion chamber detector sample I0I0 I1I1 Experiment: Scan the X-ray energy while monitoring the X-ray absorption. This can only be done using a synchrotron X-ray source. What is X-ray Absorption Spectroscopy?

4. Wednesday, March 17, 2004Graham N. George h continuum Emitted photo-electron 1s Auger electron h fluorescent photon X-ray Absorption Spectroscopy – Basic Physics 2p 2s

5. Wednesday, March 17, 2004Graham N. George K-edge XAS of some first transition elements. X-ray absorption spectroscopy is element-specific

6. Wednesday, March 17, 2004Graham N. George Setup includes a liquid He cryostat and 30 element Ge detector array. Protein sample showing image of beam (X-ray induced color centers). XAS – Experimental Setup.

7. Wednesday, March 17, 2004Graham N. George E  Insufficient energy to eject core-electron Just enough energy to eject core-electron (low energy photo-electron results). core-electron easily ejected (high energy photo-electron) transitions to bound-states near-edge EXAFS oscillations What is X-ray Absorption Spectroscopy? h continuum Emitted photo-electron 1s

8. Wednesday, March 17, 2004Graham N. George Near-edge spectrum EXAFS oscillations (k 3 -weighted) X-ray Absorption Spectroscopy

9. Wednesday, March 17, 2004Graham N. George h continuum Emitted photo-electron 1s Auger electron h fluorescent photon X-ray Absorption Spectroscopy – Basic Physics 2p 2s

10. Wednesday, March 17, 2004Graham N. George E  E  Bromine atomBr 2 molecule Schematics diagrams of final state wave functions. Photo-electron DeBroglie wave. EXAFS – Basic Physics

11. Wednesday, March 17, 2004Graham N. George EXAFS oscillations (k 3 -weighted) Fourier transform The EXAFS Fourier Transform X-ray Absorption Spectroscopy

12. Wednesday, March 17, 2004Graham N. George Phase-corrected Fourier transform EXAFS oscillations (k 3 -weighted) The EXAFS Fourier Transform X-ray Absorption Spectroscopy

13. Wednesday, March 17, 2004Graham N. George The EXAFS Fourier Transform X-ray Absorption Spectroscopy Mo S Mo-S [MoS 4 ] 2-

14. Wednesday, March 17, 2004Graham N. George The EXAFS Fourier Transform X-ray Absorption Spectroscopy Mo S Mo-S Fe Mo····Fe Cl [MoS 4 FeCl 2 ] 2- [MoS 4 ] 2-

15. Wednesday, March 17, 2004Graham N. George Near-edge Spectra – Excitation to bound states Se-methionine elemental Se selenate 2- selenite 2-

16. Wednesday, March 17, 2004Graham N. George X-ray Absorption Spectroscopy Examines all of a particular element in a sample No sample pre-treatment required (don’t need crystals etc.) Near edge spectrum – gives information on electronic structure (oxidation state etc.) EXAFS (Extended X-ray Absorption Spectroscopy) oscillations in X-ray absorption Gives a Radial Structure.

17. Wednesday, March 17, 2004Graham N. George Strengths and Limitations of EXAFS Structural parameters that are available from EXAFS analysis: Average bond-lengths, R Coordination Numbers, N Debye-Waller factors, σ 2 σ 2 is the mean-square displacement of the bond-length from the average value R. It has components from atomic vibration and disorder, and can be thought of as being similar to a crystallographic temperature factor. It differs from the temperature factor in that it is due to relative displacement of atoms. Geometric information is generally unavailable, although multiple scattering sometimes allows bond-angle determination. Data analysis is not always a routine matter.

18. Wednesday, March 17, 2004Graham N. George Strengths and Limitations of EXAFS The Debye-Waller factor is not a total unknown The Debye-Waller is a sum of static disorder and vibrational components. σ 2 = σ 2 stat. + σ 2 vib. σ 2 vib. – can be computed accurately for a given bond-length (using force constants derived from vibrational spectroscopy or density functional theory). σ 2 stat. – upper and lower limits for this can be computed from the k-range of the data and the coordination number. 2Mo-S at ~ 2.4 Å0.0063 Å 2 > σ 2 > 0.0020 Å 2 2Mo-O at ~ 2.0 Å0.0068 Å 2 > σ 2 > 0.0025 Å 2

19. Wednesday, March 17, 2004Graham N. George EXAFS vs. Crystallography Comparison with small molecule X-ray crystal structures – Use crystallographic bond-lengths and coordination numbers Refine Debye-Waller factors σ 2 within reasonable bounds Expt. Calc. Mo=O Mo-S There is excellent agreement between the two techniques σ 2 values match well with ab-initio calculated values (e.g. for Mo=O 0.0018(2) vs. 0.0017 Å 2 ).

20. Wednesday, March 17, 2004Graham N. George Molybdenum Enzymes All molybdenum enzymes contain an organic cofactor. This is called “molybdopterin”. Almost all catalyze two-electron redox reactions involving oxygen transfer between Mo and substrate. Either one or two molybdopterin cofactors can be coordinated to the metal via the dithiolene linkage.

21. Wednesday, March 17, 2004Graham N. George 2H + H2OH2O Catalyses the two-electron reduction of dimethylsulfoxide (DMSO) to dimethylsulfide (DMS). Mo is oxidized from Mo 4+ to Mo 6+ formal oxidation state in the process. The Prototypical member of the DMSO reductase family of Mo enzymes. The best studied DMSO reductases are those of Rhodobacter capsulatus and Rhodobacter sphaeroides. These have nearly identical sequences and properties. DMSO reductase DMSODMS Mo 4+ Mo 6+ ++

22. Wednesday, March 17, 2004Graham N. George Structural studies of DMSO reductase active site 7 February 1996- first EXAFS 14 June 1996- first crystallography 18 October 1996- more crystallography 15 September 1997- some more crystallography 1 December 1997- still more crystallography 30 January 1998- even more crystallography 30 January 1998- more EXAFS (a different group) 27 November 1998- crystallography of a closely related enzyme 17 February 1999 - more EXAFS 16 August 2000- yet more crystallography Prior to the 1999 EXAFS study there was a lot of confusion and debate about the active site structure. To some extent the debate still continues.

23. Wednesday, March 17, 2004Graham N. George First EXAFS of DMSO reductase “X-ray Absorption Spectroscopy of Dimethyl Sulfoxide Reductase from Rhodobacter sphaeroides” G. N. George, J. Hilton and K. V. Rajagopalan J. Am. Chem. Soc. 1996, 118(5), 1113-1117 7 February 1996

24. Wednesday, March 17, 2004Graham N. George First EXAFS of DMSO reductase “X-ray Absorption Spectroscopy of Dimethyl Sulfoxide Reductase from Rhodobacter sphaeroides” G. N. George, J. Hilton and K. V. Rajagopalan J. Am. Chem. Soc. 1996, 118(5), 1113-1117 7 February 1996 Mono-oxo Mo 6+ and des-oxo Mo 4+ Four Mo-S indicated two cofactors Postulated oxo-transfer mechanism Suggested that one cofactor might dissociate. “It seems likely that the active site can adopt at least two different structures”

25. Wednesday, March 17, 2004Graham N. George First Crystallography of DMSO reductase “Crystal Structure of DMSO Reductase: Redox-Linked Changes in Molybdopterin Coordination” H. Schindelin, C. Kisker, J. Hilton, K. V. Rajagopalan and D. C. Rees Science, 1996, 272 1615-1621 14 June 1996 Gave us our first look at the 3-dimensional structure of the protein.

26. Wednesday, March 17, 2004Graham N. George First Crystallography of DMSO reductase “Crystal Structure of DMSO Reductase: Redox-Linked Changes in Molybdopterin Coordination” H. Schindelin, C. Kisker, J. Hilton, K. V. Rajagopalan and D. C. Rees Science, 1996, 272 1615-1621 14 June 1996 Big changes in Mo coordination on changing metal oxidation state! Mo 6+ Mo 4+ mono-oxo Mo 6+ des-oxo Mo 4+ Ser147

27. Wednesday, March 17, 2004Graham N. George Proposed Catalytic Mechanism

28. Wednesday, March 17, 2004Graham N. George More crystallographic results followed… “Crystal Structure of Dimethyl Sulfoxide Reductase from Rhodobacter capsulatus at 1.88 Å Resolution” F. Schneider, J. Löwe, R. Huber, H. Schindelin, C. Kisker and J. Knäblein J. Mol. Biol. 1996 263, 53-69 18 October 1996 The structure was different! – a dioxo Mo 6+ site with only one of two cofactors bound.

29. Wednesday, March 17, 2004Graham N. George And more crystallography followed those … “Molybdenum active centre of DMSO reductase from Rhodobacter capsulatus: crystal structure of the oxidised enzyme at 1.82-Å resolution and the dithionite-reduced enzyme at 2.8-Å resolution” A. S. McAlpine, A. G. McEwan, A. L. Shaw and S. Bailey JBIC, 1997, 2, 690-701 1 December 1997 The structures were different again! di-oxo Mo 6+ mono-oxo Mo 4+

30. Wednesday, March 17, 2004Graham N. George Even more crystallography followed … “The High Resolution Crystal Structure of DMSO Reductase in Complex with DMSO” A. S. McAlpine, A. G. McEwan and S. Bailey J. Mol. Biol. 1998, 275, 613-623 30 January 1998 This work found that if the product (DMS) is added to oxidized enzyme then a pink-purple species was formed which had DMSO bound to the active site. Oxidized Mo 6+ + DMS (gray-green)DMSO-bound form (pink purple) This DMSO-bound form was studied by X-ray crystallography.

31. Wednesday, March 17, 2004Graham N. George Even more crystallography followed … “The High Resolution Crystal Structure of DMSO Reductase in Complex with DMSO” A. S. McAlpine, A. G. McEwan and S. Bailey J. Mol. Biol. 1998, 275, 613-623 30 January 1998 A seven-coordinate mono-oxo molybdenum site, with DMSO covalently bound with an unusually long S=O bond length (1.7 Å).

32. Wednesday, March 17, 2004Graham N. George Lots of different structures for the same active site... Mo 6+ Mo 4+ Mo 6+ Mo 4+ DMSO All protein folds were essentially identical

33. Wednesday, March 17, 2004Graham N. George And then more EXAFS followed … The conclusions of this study totally supported the crystal structures of Bailey and co-workers… “X-ray absorption spectroscopy of dimethylsulfoxide reductase from Rhodobacter capsulatus” P. E. Baugh, C. D. Garner, J. M. Charnock, D. Collison, E. S. Davies, A. S. McAlpine, S. Bailey, I. Lane, G. R. Hanson and A. G. McEwan JBIC, 1998, 2, 634-643. 30 January 1998 And another mechanism was postulated…

34. Wednesday, March 17, 2004Graham N. George Mo 4+ DMSO Exptl. Calc. EXAFS vs. protein crystallography The crystal structure does not agree with our EXAFS data. Comparison with protein X-ray crystal structures – Use crystallographic bond-lengths and coordination numbers Refine Debye-Waller factors σ 2 within reasonable bounds

35. Wednesday, March 17, 2004Graham N. George EXAFS vs. small molecule crystallography Comparison with small molecule X-ray crystal structures – Use crystallographic bond-lengths and coordination numbers Refine Debye-Waller factors σ 2 within reasonable bounds Expt. Calc. Mo=O Mo-S There is excellent agreement between the two techniques σ 2 values match well with ab-initio calculated values (e.g. for Mo=O 0.0018(2) vs. 0.0017 Å 2 ).

36. Wednesday, March 17, 2004Graham N. George EXAFS vs. protein crystallography Mo 6+ Mo 4+ Mo 6+ Mo 4+ DMSO None of the crystal structures fitted our EXAFS data…

37. Wednesday, March 17, 2004Graham N. George Resonance Raman does agree with our EXAFS data “Active Site Structures and Catalytic Mechanism of Rhodobacter sphaeroides Dimethyl Sulfoxide Reductase as Revealed by Resonance Raman Spectroscopy” S. D. Garton, J. Hilton, H. Oku, B. R. Crouse, K. V. Rajagopalan and M. K. Johnson J. Am. Chem. Soc. 1997, 119, 12906-12916 31 December 1997 Resonance Raman spectroscopy indicated a mono-oxo Mo 6+ species for the oxidized enzyme Both Mo=O and Mo-S frequencies agreed quantitatively with our EXAFS-derived bond-lengths

38. Wednesday, March 17, 2004Graham N. George The first structure was unusual… More than one half of the metal coordination sphere is empty! Caltech Rees group, oxidized form

39. Wednesday, March 17, 2004Graham N. George A 3-coordinate Mo 4+ site – a totally unknown coordination Mo-O-C bond angle close to 180º More than ½ the metal coordination sphere totally empty The other crystal structures were odd too… Caltech Rees group, reduced form

40. Wednesday, March 17, 2004Graham N. George The other crystal structures were odd too… The O=Mo=O bond angle is impossibly small. Martinsried Huber group, oxidized form

41. Wednesday, March 17, 2004Graham N. George The other crystal structures were odd too… Many supposedly non-bonded atoms with overlapping Van der Waals radii O=Mo=O bond-angle impossibly small (70º) Daresbury Bailey group, oxidized form

42. Wednesday, March 17, 2004Graham N. George The other crystal structures were odd too… Daresbury Bailey group, oxidized form Overlapping Van der Waals radii with non-bonded atoms – the Mo 6+ structure showed six of these. All the other Bailey structures had the same problem. So did crystal structures of related enzymes (TMAO reductase). Too many atoms, not enough room.

43. Wednesday, March 17, 2004Graham N. George Were all of the structures wrong? On close inspection ALL of the structures had some chemically implausible or impossible features. Impossibly crowded atoms Impossibly acute or unlikely bond-angles Unusual bond-lengths (this is expected) We concluded that ALL crystal structures were either wrong or had major problems. We re-examined all the EXAFS (several times), including the DMSO bound form. We examined the EXAFS of other closely related Mo enzymes (biotinsulfoxide reductase, trimethylamineoxide reductase). They looked just like DMSO reductase.

44. Wednesday, March 17, 2004Graham N. George But what about the other EXAFS? The experimental data of Baugh et al. looks the same as ours – so why did they form such very different conclusions? – Physically impossible Debye-Waller factors. Some of the σ 2 values used by Baugh et. al. were so big that they effectively removed the EXAFS. Baugh et al. started from the crystal structure coordinates, then (probably) refined σ 2 values with no boundaries imposed, and finally refined bond- lengths etc. to obtain their final result. This gave conclusions that were heavily biased towards the crystal structure. “X-ray absorption spectroscopy of dimethylsulfoxide reductase from Rhodobacter capsulatus” P. E. Baugh, C. D. Garner, J. M. Charnock, D. Collison, E. S. Davies, A. S. McAlpine, S. Bailey, I. Lane, G. R. Hanson and A. G. McEwan JBIC, 1998, 2, 634-643.

45. Wednesday, March 17, 2004Graham N. George BondNR (Å)σ 2 (Å 2 ) Mo=O11.690.014 Mo-O11.910.001 Mo-O12.110.009 Mo-S22.280.016 Mo-S22.370.001 Mo-S12.810.013 Mo-C23.260.013 Mo-C23.410.001 Mo-C13.170.001 “X-ray absorption spectroscopy of dimethylsulfoxide reductase from Rhodobacter capsulatus” P. E. Baugh, C. D. Garner, J. M. Charnock, D. Collison, E. S. Davies, A. S. McAlpine, S. Bailey, I. Lane, G. R. Hanson and A. G. McEwan JBIC, 1998, 2, 634-643. But what about the other EXAFS? DMSO-bound form: A total of 9 components with 18 variables were refined.

46. Wednesday, March 17, 2004Graham N. George BondNR (Å)σ 2 (Å 2 ) Mo=O11.690.014 Mo-O11.910.001 Mo-O12.110.009 Mo-S22.280.016 Mo-S22.370.001 Mo-S12.810.013 Mo-C23.260.013 Mo-C23.410.001 Mo-C13.170.001 Too big “X-ray absorption spectroscopy of dimethylsulfoxide reductase from Rhodobacter capsulatus” P. E. Baugh, C. D. Garner, J. M. Charnock, D. Collison, E. S. Davies, A. S. McAlpine, S. Bailey, I. Lane, G. R. Hanson and A. G. McEwan JBIC, 1998, 2, 634-643. But what about the other EXAFS? Some of the σ 2 values were impossibly big. This reduces the EXAFS intensity of these components

47. Wednesday, March 17, 2004Graham N. George BondNR (Å)σ 2 (Å 2 ) Mo=O11.690.014 Mo-O11.910.001 Mo-O12.110.009 Mo-S22.280.016 Mo-S22.370.001 Mo-S12.810.013 Mo-C23.260.013 Mo-C23.410.001 Mo-C13.170.001 Too big Too small “X-ray absorption spectroscopy of dimethylsulfoxide reductase from Rhodobacter capsulatus” P. E. Baugh, C. D. Garner, J. M. Charnock, D. Collison, E. S. Davies, A. S. McAlpine, S. Bailey, I. Lane, G. R. Hanson and A. G. McEwan JBIC, 1998, 2, 634-643. But what about the other EXAFS? Other σ 2 values were impossibly small. This increases the EXAFS intensity of these components

48. Wednesday, March 17, 2004Graham N. George BondNR (Å)σ 2 (Å 2 ) Mo=O11.690.014 Mo-O11.910.001 Mo-O12.110.009 Mo-S22.280.016 Mo-S22.370.001 Mo-S12.810.013 Mo-C23.260.013 Mo-C23.410.001 Mo-C13.170.001 Too big Too small “X-ray absorption spectroscopy of dimethylsulfoxide reductase from Rhodobacter capsulatus” P. E. Baugh, C. D. Garner, J. M. Charnock, D. Collison, E. S. Davies, A. S. McAlpine, S. Bailey, I. Lane, G. R. Hanson and A. G. McEwan JBIC, 1998, 2, 634-643. But what about the other EXAFS? Ignore the very low intensity components in the fit, and those which cancel

49. Wednesday, March 17, 2004Graham N. George BondNR (Å)σ 2 (Å 2 ) Mo=O11.690.014 Mo-O11.910.001 Mo-O12.110.009 Mo-S22.280.016 Mo-S22.370.001 Mo-S12.810.013 Mo-C23.260.013 Mo-C23.410.001 Mo-C13.170.001 Too big Too small “X-ray absorption spectroscopy of dimethylsulfoxide reductase from Rhodobacter capsulatus” P. E. Baugh, C. D. Garner, J. M. Charnock, D. Collison, E. S. Davies, A. S. McAlpine, S. Bailey, I. Lane, G. R. Hanson and A. G. McEwan JBIC, 1998, 2, 634-643. But what about the other EXAFS?

50. Wednesday, March 17, 2004Graham N. George “Structure of the Molybdenum Site of Dimethyl Sulfoxide Reductase” G. N. George, J. Hilton, C. Temple, R. C. Prince and K. V. Rajagopalan J. Am. Chem. Soc. 1997, 121, 1256-1266. 17 February 1999 EXAFS and EPR spectroscopy This paper suggested that the conclusions of all of the crystallographic studies and the Daresbury EXAFS study were wrong. The structural conclusions presented in this work were essentially the same as in our original 1996 paper. We suggested that multiple species might be present in the crystals and that this caused erroneous conclusions.

51. Wednesday, March 17, 2004Graham N. George More crystallography… “The Crystal Structure Of Rhodobacter Sphaeroides Dimethylsulfoxide Reductase Reveals Two Distinct Molybdenum Coordination Environments” H-K Li, C. Temple, K. V. Rajagopalan, and H. Schindelin. J. Am. Chem. Soc. 2000, 122, 7673-7680. 16 August 2000 1.3 Å structure resolved two different active site structures within the crystals One of them looked quite like our solution structure!

52. Wednesday, March 17, 2004Graham N. George More crystallography… “The Crystal Structure Of Rhodobacter Sphaeroides Dimethylsulfoxide Reductase Reveals Two Distinct Molybdenum Coordination Environments” H-K Li, C. Temple, K. V. Rajagopalan, and H. Schindelin. J. Am. Chem. Soc. 2000, 122, 7673-7680. 16 August 2000 Most of this structure was in quantitative agreement with the EXAFS But it still had non-bonded atoms that were too close! Probably there are more than just two forms present.

53. Wednesday, March 17, 2004Graham N. George A combined approach – Use the information from EXAFS, crystallography and density functional calculations (DFT) to investigate nature of DMSO-bound form. DMSO-bound DMSO reductase has a highly characteristic electronic spectrum with absorption bands at 478 and 546 nm. Trimethylarsine [(CH 3 ) 3 As] forms a similar Mo 4+ complex with an essentially identical electronic spectrum in which trimethylarsineoxide [( CH 3 ) 3 As=O] is bound covalently to Mo. We will attempt to combine the information from the Mo and As EXAFS of this complex to help understand the coordination of DMSO.

54. Wednesday, March 17, 2004Graham N. George 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. Mo and As EXAFS

55. Wednesday, March 17, 2004Graham N. George Arsenic is oxidized (As 5+ ) and molybdenum is reduced (Mo 4+ ) As=O bond-length is within normal range – no particular distortion is present. Cofactor and Ser147 coordinates from crystal structure 2.37 Å 3.44 Å 2.23 Å 2.01 Å 1.70 Å Mo S As O Ser147 1.91 Å EXAFS of (CH 3 ) 3 As-bound DMSO reductase

56. Wednesday, March 17, 2004Graham N. George (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 DFT of (CH 3 ) 3 As=O-bound DMSO reductase

57. Wednesday, March 17, 2004Graham N. George 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 Estimate geometry of DMSO-bound enzyme from crystal structure and EXAFS with adjustments from (CH 3 ) 3 As=O experiment. EXAFS of (CH 3 ) 2 S=O bound DMSO reductase

58. Wednesday, March 17, 2004Graham N. George DMSO dissociates – rather than DMS – tendency to go in reverse. Active site pocket must be important in stabilization and direction. Future calculations will use hybrid approach to include protein. DFT Calculation – (CH 3 ) 2 S=O leaves active site…

59. Wednesday, March 17, 2004Graham N. George Future work and directions Protein crystal structure solution is traditionally aided by structural restraints of the amino acids derived from small molecule crystallography. For metalloproteins the information from crystallography should be supplemented by careful and independent spectroscopic studies. Modern computational techniques could aid in providing restraints. Crystallographers – don’t ignore the spectroscopy! Some sort of check for overlapping Van der Waals radii ought to be routinely applied to non-bonded “hetero-atoms”

60. Wednesday, March 17, 2004Graham N. George K.V. RajagopalanDuke University Medical Center Jim HiltonDuke University Medical Center Carrie TempleDuke University Medical Center Kimberly JohnsonDuke University Medical Center Eileen Yu Sneeden Stanford Synchrotron Radiation Lab. Hugh H. Harris SSRL (now University of Sydney) Roger C. PrinceExxonMobil Research & Eng. Co. Christian J. DoonanUniversity of Saskatchewan Ingrid J. Pickering University of Saskatchewan Acknowledgements

61. Wednesday, March 17, 2004Graham N. George Acknowledgements Canada Research Chairs Program University of Saskatchewan Provincial Government Canada Foundation for Innovation Canadian Light Source and its many sponsors www.lightsource.ca Stanford Synchrotron Radiation Laboratory, U.S. DOE and NIH