spectroscopically challenged metals...xanes spectra are sensitive to ligation 0 100 200 300 400 9650...

XAS Applications 1

XAS ApplicationsJames E. Penner-Hahn

Department of Chemistry &Biophysics Program

The University of Michigan

XAS Applications 2

• Characterization of unknown protein

• Comparison with crystal structure• Determination of solution structure• When crystal structures are not

enough• Spatial localization

XAS Applications 3

Biological Zn sitesStructural

Zn

S(Cys)(Cys)S

(Cys)SS(Cys)

Zn(Cys)S S(Cys)

Storage

S(Cys)(Cys)S

Zn

Zne.g., Auld, Biometals, 2001

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, 8401-8409.

A case study in data under-determination

Zn EXAFS is remarkably insensitive to changes in ligation.

-10

-5

0

5

10

15

20

25

30

2 4 6 8 10 12

EXA

FS•k

3

k (Å-1)

ZnS2N

2

ZnS3N

ZnS4

0

10

20

30

40

50

0 1.5 3 4.5 6 7.5

FT M

agni

tude

R + (Å)

ZnS2N

2

ZnS3N

ZnS4

ZnSR

SR

L1

L2

XANES spectra are sensitive to ligation

0

100

200

300

400

9650 9660 9670 9680 9690 9700

Nor

mal

ized

Abs

orpt

ion

(cm

2 /g)

Energy (eV)

Peptide

ZnS2N

2

ZnS3N

ZnS4

0

100

200

300

400

9650 9660 9670 9680 9690 9700

Nor

mal

ized

Abs

orpt

ion

(cm

2 /g)

Energy (eV)

Inorganic

ZnS2N

2

ZnS3N

ZnS4

but show greater variation between different compounds than with changes in ligation.

Zn-S and Zn-N EXAFS signals are approximately out of phase

-3

-2

-1

0

1

2

3

2 4 6 8 10 12 14

k3 E

XA

FS

k (A-1)

Zn-S Zn-N

The observed EXAFS for mixed S/N sites is dominated by Zn-S scattering

-15

-10

-5

0

5

2 4 6 8 10 12 14

k3 E

XA

FS

k (A-1)

Zn-S Zn-N ZnS2N

2

One solution is to measure data over wide k range(ZnS2N2 inorganic)

0

10

20

30

40

50

60

70

0 1.5 3 4.5 6 7.5

FT M

agni

tude

R + (Å)

k=2-11 Å-1

k=2-12 Å-1

k=2-13 Å-1

k=2-14 Å-1

k=2-15 Å-1

k=2-16 Å-1

Note – R ~ 0.25 /2 k = 0.25 Å kmin ~ 6.3

High resolution EXAFS is required to reliably distinguish Zn-S from Zn-N …

-15

-10

-5

0

5

2 4 6 8 10 12 14

k3 E

XA

FS

k (A-1)

Zn-S Zn-N ZnS2N

2

…and even with high resolution data, extremely high signal/noise ratios are required

to detect Zn-N in the presence of Zn-S

-15

-10

-5

0

5

2 4 6 8 10 12 14

k3 E

XA

FS

k (A-1)

Zn-S Zn-N ZnS2N

2

XAS Applications 12

HomocysteineHomocysteine + Me-X = Methionine + HXE. coli has two methionine synthases

MetH – cobalamin dependent methionine synthase

3 C

H

NH2

COOHCH2CH2SCH

C

H

NH2

COOHCH2CH2HS

N

N N

N

CoMeR

N

N N

N

CoMe

MetE – cobalamin independent methionine synthase

XAS Applications 13

MetE (cobalamin independent MetSyn) contains Zn

Zn is tightly boundZn is required for activityIs Zn involved in reaction, or does it play a

structural role?

XAS Applications 14

The Zn site in MetE (cobalamin independent MetSyn) has ZnS2(O/N)2ligation.

-4

0

4

8

2 4 6 8 10 12

EXA

FS•k

3

k (Å-1)

0

5

10

15

20

0 1 2 3 4 5 6 7Four

ier T

rans

form

Mag

nitu

de

R + (Å)

Native+Hcy

Addition of homocysteine changes ligation to ZnS3(O/N).

XAS Applications 15

010203040506070

0 1 2 3 4 5 6 7

Four

ier T

rans

form

Mag

nitu

de

R + (Å)

MetE +/- L-homocysteine

MetH +/- L-homocysteine

MetH +/- D-homocysteine

EnzymeEnzyme + substrate

MetE Zn site changes on substrate binding

ZnS2(O/N)2

ZnS3(O/N)

ZnS3(O/N)

ZnS4

MetH (cobalamin dependent) also contains Zn

XAS Applications 16

Changes in ligation are due to homocysteine binding to Zn

0

10

20

30

40

50

60

0 1 2 3 4 5 6 7

Four

ier T

rans

form

Mag

nitu

de

R + (Å)

Native

+SeHcy+Hcy

MetH (ZnS3O)

MetE (ZnS2NO)

XAS Applications 17

Se EXAFS confirms structural picture of MetE and MetH sites

05

10152025303540

0 1 2 3 4 5 6 7Four

ier T

rans

form

Mag

nitu

de

R + (Å)

MetH

MetE

Se-CSe-Zn

Se•••SSe····S

XAS Applications 18

Combination of Zn + Se EXAFS consistent with only a small distortion from tetrahedral

geometry in substrate-bound enzyme

XAS Applications 19

Protein Farnesyl transferase

CaaX, X=S,M,Q,C,APeptide

FPP

C 15C 13 C 12

C 11 C 10C 8 C 7

C 6C 5

C 3 C 2C 1 O

P 1 OP 2 O

C 14 C 9 C 4 O O O O

-C-a-a-X

C15C13

C12C11

C10C8

C7C6

C5C3

C2C1

C14

C9

C4

XAS Applications 20

Protein Farnesyl transferase

XAS Applications 21

Zn-S Pep

Zn-S C299

Zn-O H2O

Zn-O D297

Zn-O D297

Zn-N H362

FTase -- 2.22 2.74 2.00 2.56 2.48 +FPP -- 2.27 3.22 1.99 2.03 2.10 -- 2.42 NA 2.38 3.05 2.64 Ternary 2.48 2.21 -- 1.90 2.45 2.24 2.40 2.26 -- 1.99 2.61 2.18 2.35 2.21 -- 2.08 2.55 2.17 2.41 2.33 -- 1.97 2.53 2.21 2.75 2.29 -- 2.22 2.67 2.34 Product 2.66 2.27 -- 2.06 2.42 2.18 Product+FPP

-- 2.30 -- 2.13 2.42 2.25

Ten FTase crystal structures are known

XAS Applications 22

-10

0

10

20

30

40

2 4 6 8 10 12 14 16 18k ( A-1 )

o

FTase

FTase + I2

Ternary complex

Product complex

XAS Applications 23

Zn is 4-coordinate;peptide sulfur binds only in ternary complex

0

2

4

6

8

10

0 1 2 3 4 5 6

FTaseFTase+I2TernaryProduct

R ( A)o

Zn-(N/O) =2.04 ÅZn-S =2.31 ÅZn-Sternary =2.33 Å

XAS Applications 24

Carboxylate shift may play an important role in activating peptide thiolate

OZn

OSCys

NHisAsp

Pep-SH OZn

O SCys

NHisAsp

PepS

+ H+

FPP

OZn

O SCys

NHisAsp

PepS FPPPepS-F + PP

FPP

XAS Applications 25

Biological Zn sites

Alkyl-transfer

Zn

S(Cys)L1

S(substrate)L2

Structural

Zn

S(Cys)(Cys)S

(Cys)SS(Cys)

XAS Applications 26

J. Am. Chem. Soc., 112 (10) 1990p. 4031-4032

p. 4032-4034

XAS Applications 27

EXAFS shows that CN–

does not remain bound

0 1.5 3 4.5 6 7.5

Four

ierT

rans

form

Mag

nitu

de

R + (Å)

CuCN•2LiCl

Cu-Nearest NeighborCu-C N

Cu-C N-Cu

CuCN•2LiCl + MeLi

CuCN•2LiCl + 2 MeLi

XAS Applications 28

Structures of cyanocuprates in THF

Cu C NN

CCu

Cu

Cl

MeLi

MeLi

Cu C NMe

Cu MeMe

C NLi Li

XAS Applications 29

Solution speciation of CuI+PhLiPhLi + CuI “phenylcopper”2PhLi+ CuI “diphenylcuprate”

1:1 Cu4Ph4(Me2S)2Cu5Mes5

1.2:1 [Cu5Ph6]–

1.5:1 [Cu4LiPh6]–

[Cu4MgPh6]

2:1 [CuPh2]–

[CuPh2Li]2[Cu3Li2Ph6]–

Crystalline phenyl:copper species

XAS Applications 30

0

50

100

150

200

250

300

8970 8980 8990 9000 9010 9020

Nor

mal

ized

Abs

orba

nce

Energy ( eV )

n=0.0n=0.2

n=0.4n=0.6

n=0.8n=1.0

n=1.1

n=1.2

Titration of CuI+ n PhLi shows isosbestic behavior up to 1.2 equivalents

XAS Applications 31

Titration of CuI+ n PhLi shows isosbestic behavior from 1.2-2.0 equivalents

0

50

100

150

200

250

300

8970 8980 8990 9000 9010 9020

Nor

mal

ized

Abs

orba

nce

Energy ( eV )

n=2.0n=1.8

n=1.7n=1.5n=1.4n=1.3n=1.2

XAS Applications 32

EXAFS data support XANES speciation

[Cu5Ph6]–

[CuI2]–

[CuPh2]–

0 0.5 1 1.5 20

0.2

0.4

0.6

0.8

1

PhLi / CuI Ratio

Cu

Com

posi

tion

1.2

CuI2-

Cu5Ph6-

CuPh2-

XAS Applications 33

Cu

Cl

Cl

Cl

Cl Cl

Cl

CuCl

Cl

Cl

ClCl

Cl

Cu

Cl

Cl

Cl

Cl Cl

Cl

+

Biological X-ray Absorption 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


• 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.


Common errors in EXAFS analysis

• Least-squares minimization• Fourier Filtering• Resolution


R

Carbonscattering

Iterative refinements are especially susceptible to multiple minima

Pd

P P

CCH2C

Me

ca b Me

N

Phosphorusscattering

Clot, et al., J. Am. Chem. Soc 2004, 126, 9079-9084


Minima give very different structures but nearly identical EXAFS

M-P

M-C

sum

Nfree vs. NobsNobs ~ 300 ( k = 0.05 Å; kmax= 15 Å)

Nfree ~ (2 k R ) /


M-O at 2.0 and 2.2 Å give two apparently well resolved peaks in FT

0

5

10

15

20

25

30

35

40

0 1 2 3 4Four

ier t

rans

form

mag

nitu

de

R + (Å)


Fitting each filtered peak gives the appearance of M-O and M-S EXAFS

-4

-2

0

2

4

6

8

10

4 6 8 10 12

EXA

FS•k

3

k (Å-1)

"M-O" peak "M-S" peak


EXAFS resolution is ~ /2 k =

0.13 Å

-2

0

2

4

6

8

10

12

0 2 4 6 8 10 12

EXA

FS•k

3

k (Å-1)

R1=1.75 Å, R

2=2.00 Å

R1=1.85 Å, R

2=2.00 Å

R1=1.85 Å, R

2=2.00 Å

R=1.875 Å

R=1.925 Å

R=1.925 Å,damped


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


0

2

4

6

8

10

12

14

0 1 2 3 4 5 6 7

Four

ier T

rans

form

Mag

nitu

de

Radius + (Å)

N

NH

Zn

Outer shell scattering can provide ligand identification and geometric information


Multiple scattering makes EXAFS sensitive to angular arrangement of ligands

N

NH

Zn


M CC

C

C

C

N

NN

N

N

CN

M N

O

O

N

O

OC

O R1

R2

R3

R4

1

2

Param

eters

R

Info

rmat

ion

However, ability to reliably determine geometry is limited


Dependence of XANES on Oxidation State

0

400

800

6535 6555 6575 6595Energy (eV)

Nor

mal

ized

Abs

orpt

ion

II/II

II/IIIIII/III

III/IV

XAS Applications 47

Mn XANES of PS II versus x-ray dose and XANES of inorganic model compounds.

Yano J et al. PNAS 2005;102:12047-12052

©2005 by National Academy of Sciences

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;102:12047-12052


X-ray fluorescence imaging

Korbas M et al. PNAS 2008;105:12108-12112


Elemental distributions in MeHg exposed and unexposed zebrafish.

Korbas M et al. PNAS 2008;105:12108-12112


Se K x-ray absorption near-edge spectrum of A.

Pickering I J et al. PNAS 2000;97:10717-10722


Data reduction scheme for the method of chemically specific imaging.



Chemically specific concentration images of different parts of A. bisulcatus.



spectroscopically challenged metals...xanes spectra are sensitive to ligation 0 100 200 300 400 9650...

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