XAS Applications. James E. Penner-Hahn Department of Chemistry & Biophysics Program The University of Michigan. XAS Applications 1

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1 XAS Applications James E. Penner-Hahn Department of Chemistry & Biophysics Program The University of Michigan XAS Applications 1

2 Characterization of unknown protein Comparison with crystal structure Determination of solution structure When crystal structures are not enough Spatial localization XAS Applications 2

3 Biological Zn sites Structural (Cys)S (Cys)S Zn S(Cys) S(Cys) Storage S(Cys) (Cys)S Zn (Cys)S S(Cys) Zn XAS Applications 3 Zn e.g., Auld, Biometals, 2001

4 A case study in data under-determination Clark-Baldwin, et al. "The limitations of X-ray absorption spectroscopy for determining the structure of zinc sites in proteins. When is a tetrathiolate not a tetrathiolate?" J. Am. Chem. Soc. 1998, 120,

5 L 1 L 2 SR Zn SR Zn EXAFS is remarkably insensitive to changes in ligation. EXAFS k 3 FT Magnitude ZnS ZnS N ZnS N k (Å -1 ) ZnS 4 20 ZnS N R + (Å) ZnS 2 N 2

6 XANES spectra are sensitive to ligation but show greater variation between different compounds than with changes in ligation. Normalized Absorption (cm 2 /g) Normalized Absorption (cm 2 /g) Peptide ZnS 4 ZnS N 3 ZnS N Energy (ev) Inorganic ZnS 4 ZnS N 3 ZnS N Energy (ev)

7 Zn-S and Zn-N EXAFS signals are approximately out of phase k 3 EXAFS Zn-S Zn-N k (A -1 )

8 The observed EXAFS for mixed S/N sites is dominated by Zn-S scattering 5 Zn-S Zn-N ZnS 2 N 2 k 3 EXAFS k (A -1 )

9 One solution is to measure data over wide k range (ZnS 2 N 2 inorganic) FT Magnitude k=2-11 Å k=2-12 Å k=2-13 Å k=2-14 Å k=2-15 Å k=2-16 Å R + (Å) Note R ~ 0.25 /2 k = 0.25 Å k min ~ 6.3

10 High resolution EXAFS is required to reliably distinguish Zn-S from Zn-N 5 Zn-S Zn-N ZnS 2 N 2 k 3 EXAFS k (A -1 )

11 and even with high resolution data, extremely high signal/noise ratios are required to detect Zn-N in the presence of Zn-S 5 Zn-S Zn-N ZnS 2 N 2 k 3 EXAFS k (A -1 )

12 Homocysteine Homocysteine + Me-X = Methionine + HX E. coli has two methionine synthases MetH cobalamin dependent methionine synthase N N N N R Me Co Co N N N Me N HS CH 2 CH 2 NH 2 COOH C H H CH 2 CH 2 3 C NH 2 COOH XAS Applications 12 CH MetE cobalamin independent methionine synthase S

13 MetE (cobalamin independent MetSyn) contains Zn Zn is tightly bound Zn is required for activity Is Zn involved in reaction, or does it play a structural role? XAS Applications 13

14 The Zn site in MetE (cobalamin independent MetSyn) has ZnS 2 (O/N) 2 ligation. Addition of homocysteine changes ligation to ZnS 3 (O/N). Fourier Transform Magnitude EXAFS k Native +Hcy R + (Å) k (Å -1 ) XAS Applications 14

15 Fourier Transform Magnitude MetE Zn site changes on substrate binding MetH (cobalamin dependent) also contains Zn ZnS 2 (O/N) MetE +/- L-homocysteine MetH +/- L-homocysteine Enzyme Enzyme + substrate MetH +/- D-homocysteine R + (Å) ZnS 3 (O/N) ZnS 3 (O/N) ZnS XAS Applications 15 4

16 Fourier Transform Magnitude Changes in ligation are due to homocysteine binding to Zn 60 MetH (ZnS 3 O) MetE (ZnS NO) 2 Native 10 +Hcy +SeHcy R + (Å) XAS Applications 16

17 Se EXAFS confirms structural picture of Fourier Transform Magnitude MetE and MetH sites Se-C Se-Zn Se S Se S MetH MetE R + (Å) XAS Applications 17

18 Combination of Zn + Se EXAFS consistent with only a small distortion from tetrahedral geometry in substrate-bound enzyme XAS Applications 18

19 Protein Farnesyl transferase C 15 C 13 C 14 C 9 C 4 O O O O C 15 C 13 C12 C 11 C10 C 8 C7 C 6 C5 C 3 C2 C 1 O P 1 O P 2 O C 12 C 11 C 14 FPP C 10 C 8 C 7 C 6 C 9 CaaX, X=S,M,Q,C,A Peptide C 5 C 3 C 2 C 1 C 4 -C-a-a-X XAS Applications 19

20 Protein Farnesyl transferase XAS Applications 20

21 Ten FTase crystal structures are known Zn-S Pep Zn-S C 299 Zn-O H 2 O Zn-O D 297 Zn-O D 297 Zn-N H 362 FTase FPP NA Ternary Product Product+ FPP XAS Applications 21

22 40 Product complex 30 Ternary complex 20 FTase + I FTase k ( A -1 ) XAS Applications 22 o

23 Zn is 4-coordinate; peptide sulfur binds only in ternary complex FTase FTase+I2 Ternary Product Zn-(N/O) =2.04 Å Zn-S =2.31 Å Zn-S ternary =2.33 Å o XAS Applications R ( A) 23

24 Carboxylate shift may play an important role in activating peptide thiolate Asp O Zn O PepS-F + PP N His S Cys Pep-SH FPP Asp O O PepS Asp O O PepS Zn FPP N His S Cys Zn FPP N His S Cys + H + XAS Applications 24

25 Biological Zn sites Structural (Cys)S (Cys)S Zn S(Cys) S(Cys) Alkyl-transfer L 1 L 2 S(Cys) Zn S(substrate) XAS Applications 25

26 J. Am. Chem. Soc., 112 (10) 1990 p p XAS Applications 26

27 EXAFS shows that CN does not remain bound Fourier Transform Magnitude CuCN 2LiCl + 2 MeLi CuCN 2LiCl + MeLi CuCN 2LiCl Cu-C N Cu-C N-Cu Cu-Nearest Neighbor R+ (Å) XAS Applications 27

28 Structures of cyanocuprates in THF Cl Cu C N Cu C N MeLi Cu Me Cu C N MeLi Li C N Li Me Cu Me XAS Applications 28

29 Solution speciation of CuI+PhLi PhLi + CuI phenylcopper 2PhLi+ CuI diphenylcuprate Crystalline phenyl:copper species 1:1 Cu 4 Ph 4 (Me 2 S) 2 Cu 5 Mes 5 1.2:1 [Cu 5 Ph 6 ] 1.5:1 [Cu 4 LiPh 6 ] [Cu 4 MgPh 6 ] 2:1 [CuPh 2 ] [CuPh 2 Li] 2 [Cu 3 Li 2 Ph 6 ] XAS Applications 29

30 Titration of CuI+ n PhLi shows isosbestic behavior up to 1.2 equivalents 300 Normalized Absorbance n=0.0 n=0.2 n=0.4 n=0.6 n=0.8 n=1.0 n=1.1 n= Energy ( ev ) XAS Applications 30

31 Titration of CuI+ n PhLi shows isosbestic behavior from equivalents 300 Normalized Absorbance n=2.0 n=1.8 n=1.7 n=1.5 n=1.4 n=1.3 n= Energy ( ev ) XAS Applications 31

32 EXAFS data support XANES speciation Cu Composition CuI - CuPh Cu 5 Ph PhLi / CuI Ratio [CuI 2 ] [Cu 5 Ph 6 ] [CuPh 2 ] XAS Applications 32

33 Cl Cl Cl Cu Cl Cl Cl Cl Cl Cl Cl Cu + Cl Cl Cl Cl Cl Cl Cu Cl Cl XAS Applications 33

34 How to screw up you re the analysis of XAS data or some common errors, how to identify them, and how to (maybe) avoid them Biological X-ray Absorption 34

35 The job of a least-squares fitting program is to give you the best (smallest deviation) solution, not to give you the right solution. If you see something, it tells you something; if you see nothing, it tells you nothing. Biological X-ray Absorption 35

36 Common errors in EXAFS analysis Least-squares minimization Fourier Filtering Resolution Biological X-ray Absorption 36

37 Iterative refinements are especially susceptible to multiple minima Phosphorus scattering Carbon scattering R P P c Pd a b Me H 2 C C C Me Clot, et al., J. Am. Chem. Soc 2004, 126, Biological X-ray Absorption 37 N

38 Minima give very different structures but M-P nearly identical EXAFS N free vs. N obs M-C N obs ~ 300 ( k = 0.05 Å; k max = 15 Å) N free ~ (2 k R ) / sum Biological X-ray Absorption 38

39 M-O at 2.0 and 2.2 Å give two apparently well resolved peaks in FT Fourier transform magnitude R + (Å) Biological X-ray Absorption 39

40 Fitting each filtered peak gives the appearance of M-O and M-S EXAFS 10 8 "M-O" peak "M-S" peak EXAFS k k (Å -1 ) Biological X-ray Absorption 40

41 12 R 1 =1.75 Å, R 2 =2.00 Å R=1.875 Å 10 EXAFS resolution is ~ /2 k = 0.13 Å EXAFS k R 1 =1.85 Å, R 2 =2.00 Å R 1 =1.85 Å, R 2 =2.00 Å R=1.925 Å R=1.925 Å, damped k (Å -1 ) Biological X-ray Absorption 41

42 Summary The more variables you control, the more likely you are to obtain a unique solution Multiple data sets (elements, temperature, concentration, time, etc.) almost always help Conclusions are only as good as your model Biological X-ray Absorption 42

43 Outer shell scattering can provide ligand identification and geometric information Fourier Transform Magnitude Zn Radius + (Å) Biological X-ray Absorption 43 N NH

44 Multiple scattering makes EXAFS sensitive to angular arrangement of ligands Zn N NH Biological X-ray Absorption 44

45 However, ability to reliably determine geometry is limited Informa tion Pa rameters N N C C N C M C N O N C C N O M N Biological X-ray Absorption 45 R R3 R4 2 C O N R2 1 R1 O O

46 Dependence of XANES on Oxidation State Normalized Absorption II/II II/III III/III III/IV Energy (ev) Biological X-ray Absorption 46

47 XAS Applications 47

48 Mn XANES of PS II versus x-ray dose and XANES of inorganic model compounds. Yano J et al. PNAS 2005;102: by National Academy of Sciences

49 Spectral changes of PS II Mn EXAFS due to radiation damage with FTs (Left) and the k3-space EXAFS (Right). Yano J et al. PNAS 2005;102: by National Academy of Sciences

50 X-ray fluorescence imaging Korbas M et al. PNAS 2008;105: by National Academy of Sciences

51 Elemental distributions in MeHg exposed and unexposed zebrafish. Korbas M et al. PNAS 2008;105: by National Academy of Sciences

52 Se K x-ray absorption near-edge spectrum of A. Pickering I J et al. PNAS 2000;97: by National Academy of Sciences

53 Data reduction scheme for the method of chemically specific imaging. Pickering I J et al. PNAS 2000;97: by National Academy of Sciences

54 Chemically specific concentration images of different parts of A. bisulcatus. Pickering I J et al. PNAS 2000;97: by National Academy of Sciences