mössbauer spectroscopy · mössbauer spectroscopy the “light source”: decay scheme of 57co...

Mössbauer Spectroscopy

Carsten Krebs

Department of Chemistry

Department of Biochemistry and Molecular Biology

The Pennsylvania State University

Recommended Literature

E. Münck “Aspects of 57Fe Mössbauer Spectroscopy”

Chapter 6 in Physical Methods in Bioinorganic Chemistry

L. Que, Jr. (editor)

University Science Books, 2000

P. Gütlich, E. Bill, A. X. Trautwein

“Mössbauer Spectroscopy and Transition Metal Chemistry”

Springer, 2011

Outline • General remarks

• Quadrupole doublet spectra (isomer shift, quadrupole splitting)

• Magnetically split spectra (spin expectation value, hyperfine tensor)

• The correlation between EPR and Mössbauer spectroscopies

(effective g-values and spin expectation values)

• How is the internal field oriented relative to the external field?

• How does the fluctuation rate of the electronic states affect the Mössbauer spectrum?

• Example 1: EPR and Mössbauer of the high-spin Fe(III) center in transferrin

• Example 2: EPR and Mössbauer of the Fe(II)/Fe(III) cluster in myo-inositol oxygenase

(incl. magnetic Mössbauer of dinuclear clusters)

• Example 3: Mössbauer studies of the Fe(III)/Fe(III) cluster E. coli RNR

• Example 4: The high-spin Fe(IV)-oxo intermediate in TauD

• Example 5: A mononuclear Fe-dinitrosyl complex with S = 1/2

• Considerations for sample preparation

The Electromagnetic Spectrum

nucleus

recoil energy:

ER = E02 / 2Mc2

Eγ = Enuc - ER

γ-photon

M

example: 57Fe

Eγ = 14.4 keV

ER = 2*10-3 eV

ΔE = 4.6*10-9 eV

Recoil Effect in “Free” Atoms

ER 5-6 orders of magnitude greater than natural linewidth

no resonance possible

=> Nuclear γ-Resonance cannot be observed with gases and liquids !

7

.. too short !

Recoil Effect in “Free” Atoms

Rudolf L. Mössbauer

• 1958: Discovers the “recoilless

nuclear resonance absorption of γ-

radiation”

• emitting and absorbing nuclei

must be embedded in solid

lattice

• there is recoil-less emission and

absorption of -photons (f-

factor)

• 1961: Receives the Nobel Prize in

Physics

Mössbauer periodic table

Periodic table of life

Mössbauer spectroscopy

The “light source”: Decay scheme of 57Co

57Co

57Fe

I = 5/2

I = 3/2

I = 1/2

Nuclear

Spin

136.4 keV

14.4 keV

• 3,300 times the energy

of a 285-nm UV photon

• recoil imparts significant

change of energy of the photon

• emitting and absorbing nuclei

must be embedded in solid lattice

• there is recoil-less emission and

absorption of -photons (f-factor)

• at low temperatures, all Fe species

have same f-factor

• fraction of Fe species in sample is

proportional to area of

Mössbauer subspectrum

Electron capture

91% 9%



57Co

57Fe

I = 5/2

I = 3/2

I = 1/2

Nuclear

Spin

136.4 keV

14.4 keV

Electron capture

91% 9%

• Doppler effect allows the energy of the photon to be varied slightly

DE = E

v

c

v = source velocity

c = speed of light



57Co

57Fe

I = 5/2

I = 3/2

I = 1/2

Nuclear

Spin

136.4 keV

14.4 keV

Electron capture

91% 9%

57Fe (sample)

0 Doppler velocity

abso

rpti

on

I = 3/2

I = 1/2

• Photon can be absorbed by a 57Fe nucleus in the sample

Experimental setup (transmission geometry)

v = 1 mm/s => DE = 4.8 10-8 eV

= 11.6 MHz

= 3.9 10-4 cm-1

= 5.6 K

Low-Field Mössbauer spectrometer

Sample Detector Velocity Transducer 57Co source

High-Field Mössbauer spectrometer

High-Field Mössbauer spectrometer

Magnetic field

-beam

Types of Mössbauer spectra: 1) Quadrupole Doublet

velocity [mm/s]

4 2 0 -2 -4

abso

rpti

on [

%]

6

0

DEQ

δ

I = 3/2

I = 1/2

DEQ

Quadrupole Splitting (ΔEQ)

ES EA

Source Absorber

Isomer shift (δ)

MI = 3/2

MI = 1/2

MI = 1/2


velocity [mm/s]

4 2 0 -2 -4

abso

rpti

on [

%]

6

0

DEQ

δ

DEQ


MI = 3/2

MI = 1/2

MI = 1/2

How long are the black arrows if

the red double arrow is 1 m?


velocity [mm/s]

4 2 0 -2 -4

abso

rpti

on [

%]

6

0

DEQ

δ

DEQ


MI = 3/2

MI = 1/2

MI = 1/2

How long are the black arrows if

the red double arrow is 1 m?

ΔE = “2 mm/s” = 9.6 10-8 eV

E = 14.4 keV

ΔE/E = 6.7 10-12

150,000,000,000 m

(distance earth to sun)

Isomer shift

δ = ( |sample(0)|2 - | source(0)|2 ) 4/5 π Ze2 R2 (ΔR/R)

• ΔR/R is the change of radius in ground and excited state (negative for 57Fe).

• |(0)|2 is the probability to find an electron at the 57Fe nucleus

• only s-electrons have non-zero probability to be at nucleus

• d-electrons affect s-electron density by shielding

properties of 57Fe nucleus electron density at nucleus

r(bohr)

nucleus 3d - electrons shield the nuclear potential for s - orbitals

atom

Typical isomer shift values for

various spin- and oxidation

states of iron

Fe(III) S=1/2

Fe(III) S=3/2

Fe(III) S=5/2

Fe(II) S=0

Fe(II) S=1

Fe(II) S=2

Fe(I) S=1/2

Fe(I) S=3/2

Fe(V) S=1/2

Fe(V) S=3/2

Fe(IV) S=0

Fe(IV) S=1

Fe(IV) S=2

Fe(VI) S=0

Fe(VI) S=1 (relative to -iron at 300 K ) (adapted from Gütlich, Bill,

Trautwein Mössbauer Spectroscopy

and Transition Metal Chemistry,

Springer 2011)

Isomer shift correlations

• Oxidation State - number of 3d valence electrons at the iron

(δ increases with 3d population)

• Spin State - bond lengths influence the covalency

(lower δ for low-spin than for high-spin …)

• Nature of Ligands - covalency of chemcial bonds

(δ decreases with higher covalency)

δ (4-coordination) < δ (6-coordination)

δ (sulfur ligands) < δ (nitrogen ligands)

Oxidation state: (FeIV) < (FeIII) < (FeII)

number of d-electrons increases

shielding of s-electrons increases

|(0)|2 decreases

increases (because ΔR/R is negative)

Spin state: (low-spin) < (high-spin)

low-spin complexes have shorter, more covalent M-L bonds

less d-electron density

shielding of s-electrons decreases

|(0)|2 increases

decreases (because ΔR/R is negative)

Ligands: (S-ligands) < (N,O-ligands)

(4-coordinate) < (6-coordinate)

Quadrupole splitting

Q

∇E

E

electric field

lines

Nuclei with I > 1/2 have an

electric quadrupole moment Q,

which has different energies in an

electric field gradient ∇E (efg).

Quadrupole splitting

- theoretical '2D' model: the four model charges ±q generate an inhomogeneous

field with an electric field gradient (efg)

favorable orientation unfavorable orientation

Electric charge distribution and EFG tensor

between 0 and 1

only two independent comp.

Ĥq = I • Q • I

= eQVzz/12 [ 3 Iz2 – I (I + 1) + (Ix

2 – Iy2)]

DEQ = eQVzz/2 [ 1 + 2/3]1/2

= (Vxx – Vyy) / Vzz (asymmetry parameter)

Expectation values of the efg tensor elements

(Vii)val / e<r-3> for d-electrons

orbital Vxx Vyy Vzz

dx2-y2 -2/7 -2/7 4/7 0

dz2 +2/7 +2/7 -4/7 0

dxy -2/7 -2/7 +4/7 0

dxz -2/7 +4/7 -2/7 +3

dyz +4/7 -2/7 -2/7 -3

to convert Vii in ΔEQ multiply by 4.2 mms-1/ 4/7 e <r-3>

(for <r-3>=5a0-3, Q=0.15b)

(Gütlich, Bill, Trautwein, Mössbauer Spectroscopy and Transition Metal Chemistry, Springer 2011 )

for a general 3dn valence electron configuration:

add up the individual contributions for all d-electrons

Typical values of and DEQ for biological samples

Oxidation state Spin state Ligands (mm/s) DEQ (mm/s)

Fe(II) S = 2 heme 0.85 - 1.0 1.5 - 3.0

Fe-(O/N) 1.1 - 1.3 2.0 - 3.2

Fe/S 0.60 - 0.70 2.0 - 3.0

S = 0 heme 0.30 - 0.45 < 1.5

Fe(III) S = 5/2 heme 0.35 – 0.45 0.5 – 1.5

Fe-(O/N) 0.40 – 0.60 0.5 – 1.5

Fe/S 0.20 – 0.35 < 1.0

S = 3/2 heme 0.30 – 0.40 3.0 – 3.6

S = 1/2 heme 0.15 – 0.25 1.5 – 2.5

Fe-(O/N) 0.10 – 0.25 2.0 – 3.0

Fe(IV) S = 2 Fe-(O/N) 0.0 – 0.35 0.5 – 1.5

S = 1 heme 0.0 – 0.10 1.0 – 2.0

Fe-(O/N) -0.20 – 0.10 0.5 – 4.3

Adapted from E. Münck, Physical Methods in Bioinorganic Chemistry, L. Que, Jr. (ed) 2000

Calculation of and DEQ using DFT

• Recent important advances by Neese and co-workers showed that Mössbauer

parameters can be predicted well computationally using DFT methods

• Isomer shifts and quadrupole splittings are predicted to within 0.1 mm/s and

0.5 mm/s, respectively

• One can evaluate hypothetical structures and compare them to the

experimentally determined Mössbauer parameters

e.g. F. Neese, (2002) Inorg. Chim. Acta 337C, 181.

Types of Mössbauer spectra: 2) Magnetic Spectra

-1/2

+1/2

+1/2

-1/2

-3/2

+3/2

I=3/2

I=1/2

Dm -1 0 +1 -1 0 +1

• Splitting of the six lines increases as the magnetic field experienced by the 57Fe

nucleus (the effective magnetic field) increases


-1/2

+1/2

+1/2

-1/2

-3/2

+3/2

I=3/2

I=1/2

• Intensity ratio of the six lines depends on the orientation of the effective

magnetic field to the propagation direction of the beam.

Dm -1 0 +1 -1 0 +1

Powder spectrum 3:2:1:1:2:3

Beffective || -beam 3:0:1:1:0:3

Beffective -beam 3:4:1:1:4:3

Intensity of Δm = 1 lines (1 +cos2)

Intensity of Δm = 0 lines sin2

Selection rule Δm = 0, 1


-1/2

+1/2

+1/2

-1/2

-3/2

+3/2

I=3/2

I=1/2

• inner four lines are shifted relative to the outer two lines

What causes the large field sensed by the 57Fe nucleus?

The paramagnetism of the Fe ions!

High-spin Fe3+

Low-spin Fe2+

High-spin Fe2+

Low-spin Fe3+

S = 2 S = 5/2

S = 0 S = 1/2

High-spin Fe4+

Low-spin Fe4+

S = 2

S = 1

Spin Hamiltonian for EPR Spectroscopy

Ĥ = μB S • g • B + S • D • S + S • A • I

electron Zeeman zero field splitting (ZFS) hyperfine coupling

= μB S • g • B + D (Sz2 – S(S+1)/3) + E (Sx

2 – Sy2) + S • A • I

• ZFS removes the (2S + 1)-fold degeneracy of the spin

• Only observed for systems with S 1

• D and E are axial and rhombic ZFS parameters

• E/D also known as “rhombicity”

• E/D can take values between 0 and 1/3

EPR and Mössbauer spectroscopy are complementary

EPR

Integer Spin Half-Integer Spin

S = 1/2, 3/2, 5/2, … S = 0, 1, 2, 3, …

• EPR-silent • EPR-active

(in most cases)

Electron

Spin

Method


EPR


S = 1/2, 3/2, 5/2, … S = 0, 1, 2, 3, …


(in most cases)

Electron

Spin

Method

-3

0

3

0 0.25 0.5 0.75 1-3

0

3

0 0.25 0.5 0.75 1

B

Ener

gy

(cm

-1)

B

Ener

gy

(cm

-1)

Effective g-values for an S = 5/2 spin system with ZFS

Bx By Bz

0

2

4

6

8

10

12

0 0.1 0.2 0.3

0

1

2

3

4

5

6

7

0 0.1 0.2 0.3

0

2

4

6

8

10

12

0 0.1 0.2 0.3

E/D g

eff

gef

f g

eff

0

6

12

0

3

6

0 0.1 0.2 0.3

0 0.1 0.2 0.3

12

6

0

0 0.1 0.2 0.3

6

0

0

6

0

0

2

10

6

Calculated with D = 2 cm-1 and E/D = 0

Ener

gy

(cm

-1)

z

x y

z

x y

z x

y

0 0.5 1 0 0.5 1 0 0.5 1


Bx By Bz

0.9

0.9

4.3

9.6

0.6

4.3

0.6

9.6

4.3

0

2

4

6

8

10

12

0 0.1 0.2 0.3

0

1

2

3

4

5

6

7

0 0.1 0.2 0.3

0

2

4

6

8

10

12

0 0.1 0.2 0.3

E/D g

eff

gef

f g

eff

0

6

12

0

3

6

0 0.1 0.2 0.3

0 0.1 0.2 0.3

12

6

0

0 0.1 0.2 0.3

Calculated with D = 2 cm-1 and E/D = 1/3

Ener

gy

(cm

-1)

z

x y

z

x y

z x

y

0 0.5 1 0 0.5 1 0 0.5 1

Powder EPR spectra of species with anisotropic g-values

Taken from G. Palmer, Physical Methods in Bioinorganic Chemistry, L. Que (ed) 2000

g = 714.484 [GHz] / B [G]


0

2

4

6

8

10

12

0 0.1 0.2 0.3

0

1

2

3

4

5

6

7

0 0.1 0.2 0.3

0

2

4

6

8

10

12

0 0.1 0.2 0.3

E/D g

eff

gef

f g

eff

0

6

12

0

3

6

0 0.1 0.2 0.3

0 0.1 0.2 0.3

12

6

0

0 0.1 0.2 0.3

50

9.7 4.3

400 300 200 100

B (mT)

protocatechuate-3,4-dioxygenase

Adapted from G. Palmer, Physical Methods in Bioinorganic Chemistry,

L. Que (ed) 2000

rhombic 4.3-signal z

x y

z

x y

z x

y

Spin Hamiltonian for Mössbauer Spectroscopy

Ĥ = μB S • g • B + S • D • S + S • A • I - gNμN B • I + I • Q • I

electron Zeeman zero field splitting hyperfine 57Fe nuclear Zeeman quadrupole splitting

nuclear spin electron spin hyperfine coupling

In small external magnetic fields (e.g. 10 mT) the first two term are much larger

than hyperfine coupling




= S • A • I - gNμN B • I + I • Q • I

S is spin expectation value; it contains information of electronic structure





= S • A • I - gNμN B • I + I • Q • I


= - gNμN [ - S • A/ gNμN + B ] • I + I • Q • I

Bint Bext

Beff





= S • A • I - gNμN B • I + I • Q • I


= - gNμN [ - S • A/ gNμN + B ] • I + I • Q • I

Bint Bext

Beff

The internal magnetic field, Bint, depends on


• the spin expectation value, S, and the

• hyperfine coupling tensor, A.

A(57Fe) Hyperfine Coupling Tensor

a.) Fermi - Contact Contribution

Exchange interaction affords polarization of the filled inner s-shells.

(different radial distribution of spin-up and spin-down electrons)

- in general the largest contribution to A

- isotropic, negative sign (-20 to -22 T)

A = AFermi-contact + Adipole + Aorbit

A(57Fe) Hyperfine Coupling Tensor

A = AFermi-contact + Adipole + Aorbit

Dipole - Contribution, → Adipole

Arises from non-spherical distribution of the electronic spin density.

Orbital - Contribution, → Aorbit

Arises from non-quenched orbital momentum of the electronic state due to

spin-orbit coupling (SOC).

Spin expectation values for half-integer spin systems

• S ~ dE/dB

• Have the “full” expectation even in small external fields

• S ≈ geff/4

• Correlation between EPR and Mössbauer!

Spin expectation values for half-integer spin systems

• S ~ dE/dB

• Have the “full” expectation even in small external fields

• S ≈ geff/4

• Correlation between EPR and Mössbauer!

• Each electronic state has a S associated with it

• First we look at properties of S first (magnitude, anisotropy,

orientation relative to external field);

• Next, we take into consideration that more than one state is

populated

Spin expectation values for S = 1/2

S is (nearly) isotropic [i.e. the same in the

x, y, and z-direction

-3

0

3

0 0.25 0.5 0.75 1

Ener

gy

(cm

-1)

S

B B

0 0.5 1.0

0.5

0

-0.5

S = 1/2

0 0.5 1

Calculated with D = 2 cm-1 and E/D = 1/3

Spin expectation values for S = 5/2 S

x

z

y

0.9

0.9

4.3

9.6

0.6

4.3

0.6

9.6

4.3

Ener

gy

(cm

-1)

S x, y, z

ground doublet middle doublet

0 0.5 1 B (T)

0 0.5 1 0 0.5 1 B (T) B (T)

-1

-2

0

0 0.5 1 B (T)

0 0.5 1 B (T)

Spin expectation values for integer spin systems

Have in most cases S 0 for Bext = 0

Small Bext may result in small S

[depends on ZFS parameters]

Large Bext results in sizeable S

Calculated for S = 2 with D = 10 cm-1 and E/D = 1/3

x

x y

y

z

z B (T)

0 4 8 0 4 8

B (T)

0 4 8

B (T)

0 4 8

S

0

-1

-2

B (T)


• Small Bint

in small Bext

• Sizeable Bint in

small Bext Mössbauer

EPR


S = 1/2, 3/2, 5/2, … S = 0, 1, 2, 3, …


(in most cases)

* (there are exceptions, such as high-spin Fe(III)-superoxo complexes or the [3Fe-4S]0 cluster,

see Eckard Münck’s PSU workshop talk in 2014and Mike Hendrich’s section in Palmer chapter in Que book)

(analysis complex, but facili-

tated using results from EPR)

Electron

Spin

Method

(in most cases, but not always*)

Magnetically split spectra Quadrupole doublets

Orientation of Bint relative to Bext

Fe Representation of a Fe-containing protein

Representation of an isotropic S of an electronic state

of the Fe-containing protein (e.g. middle Kramers

doublet of a mononuclear rhombic ferric site

Representation of an anisotropic S of an electronic

state of the Fe-containing protein (e.g. ground Kramers

doublet of a mononuclear rhombic ferric site


ray

ray

Bexternal

Bexternal

• The internal field is aligned antiparallel to the external field


ray

Bexternal

or

• The internal field is oriented along the axis with the greatest component of S

• The orientation of S depends on molecular frame; thus, because molecules are frozen

randomly, the internal fields are oriented randomly (powder averaged spectrum)

Relaxation of the electronic states and

their effect on the Mössbauer spectrum

Paramagnetic Fe-sites have more than one electronic state; fluctuation rate between

electronic states needs to be considered for such systems.

Three cases are possible:

• The relaxation between electronic states is slow compared to the time scale of

Mössbauer spectroscopy (10-7 s).

(typically encountered for metalloproteins at 4.2 K)

• The relaxation between electronic states is fast compared to the time scale of

Mössbauer spectroscopy.

(encountered at “high” temperatures; depends on system under

consideration)

• The relaxation between electronic states is comparable to the time scale of

Mössbauer spectroscopy. This case is more difficult to treat and one tries to

avoid it by choosing different experimental conditions (temperature, external

field).

Slow relaxation limit

• Calculate S for each electronic state

• Calculate Mössbauer spectrum for each electronic state

• Add the subspectra of all electronic states according to their Boltzmann

population factors [~exp(-E/kT)]

• The resulting spectrum contains multiple subspectra (one for every electronic

state)

• The subspectra are magnetically split

-3

0

3

0 0.25 0.5 0.75 1

Ener

gy

(cm

-1)

B

Fast relaxation limit

• Calculate S for each electronic state

• Calculate the average spin expectation value, Sav, from the

individual S values according to their Boltzmann factors

• Calculate Mössbauer spectrum using Sav. There is only one

subspectrum associated with all electronic states

• In small magnetic fields Sav ≈ 0, therefore no hyperfine

interactions, i.e. spectrum is a quadrupole doublet

-3

0

3

0 0.25 0.5 0.75 1

Ener

gy

(cm

-1)

B

Cases when S is zero

• S = 0 Bint =0 quadrupole doublet for small Bext

1. Diamagnetic compounds

2. Paramagnetic compounds with integer spin ground state for

Bext = 0 (or Bext small)

3. Compound in fast relaxation limit in small magnetic field (then

Sav ≈ 0, therefore no hyperfine interactions, i.e. spectrum is a

quadrupole doublet


• Quadrupole doublets

in small Bext

• Magnetically Split

Spectra (at low T)

Mössbauer

EPR


S = 1/2, 3/2, 5/2, … S = 0, 1, 2, 3, …


(in most cases)

(in most cases) (analysis complex, but facili-

tated using results from EPR) (analysis straightforward)

Electron

Spin

Method

• Magnetically Split

Spectra for large Bext

• Quadrupole doublets

at high temperatures

Example 1

The high-spin Fe(III) site in transferrin (S = 5/2)


0

2

4

6

8

10

12

0 0.1 0.2 0.3

0

1

2

3

4

5

6

7

0 0.1 0.2 0.3

0

2

4

6

8

10

12

0 0.1 0.2 0.3

doublet 1 doublet 2 doublet3

E/D 0 0.1 0.2 0.3

E/D 0 0.1 0.2 0.3

E/D 0 0.1 0.2 0.3

gef

f

gef

f

gef

f

0

6

12

0

6

12

0

3

6


Kretchmar, et al. Biol. Metals 1988 (1) 26

D = 0.25 cm-1

E/D = 0.3

g = 2.0

δ = 0.54 mm/s

ΔEQ = 0.30 mm/s

η = 1.0

A/gnn = (-22.3, -21.9, -22.3) T

50 mT

50 mT

perp

0.5 T

2 T

6 T


Kretchmar, et al. Biol. Metals 1988 (1) 26

50 mT

50 mT

perp

0.5 T

2 T

6 T

Example 2

The exchange-coupled high-spin Fe2(II/III) cofactor of myo-inositol oxygenase

The spin-coupled Fe2II/III cluster in myo-inositol oxygenase

• The active form of myo-inositol oxygenase harbors an antiferromagnetically coupled

dinuclear site with a high-spin Fe3+ ion (S1 = 5/2) and a high-spin Fe2+ ion (S2 = 2).

• It has an EPR-active S = 1/2 ground state.

Ener

gy

S = 9/2

S = 7/2

S = 5/2

S = 3/2

S = 1/2 1.5 J

2.5 J

3.5 J

4.5 J

• EPR-spectroscopy probes the total ground spin state of a coupled cluster.

g = (1.95, 1.81, 1.81)

ĤHDvV = J S1•S2

S = S1 + S2

E(S) = J/2 S (S + 1)

• Mössbauer-spectroscopy probes the local spin/oxidation state of each

57Fe-labeled site of a coupled cluster.

• At 120 K in zero field: fast-relaxation limit quadrupole doublets

intrinsic Fe oxidation state


= 1.09 mm/s

DEQ = 2.86 mm/s

high-spin Fe(II)

= 0.48 mm/s

DEQ = 1.10 mm/s

high-spin Fe(III)

• Mössbauer-spectroscopy probes the local spin/oxidation state of each 57Fe-labeled site of a coupled cluster.

• At 4.2 K: slow-relaxation limit magnetically split spectra

• S = 1/2 S isotropic field-orientation-dependence


Spin projection factors

Ĥhf = S1•A1•I1 + S2•A2•I2

hyperfine 1 hyperfine 2

= Stot• (c1 · A1) •I1 + Stot• (c2 · A2) •I2

Spin projection factors

ci = [S(S+1) + Si(Si+1) – Sj(Sj+1)] / [2S(S+1)]

For S = 1/2 ground state, c1= +7/3 and c2= -4/3 for S1 = 5/2 and S2 = 2

See A. Bencini and D. Gatteschi, EPR of Exchange Coupled Systems, Springer, 1989 for derivation of spin coupling coeff.

Spin Hamiltonian of an exchange-coupled cluster

Ĥhf = S1•A1•I1 + S2•A2•I2

hyperfine 1 hyperfine 2

= Stot• (c1 · A1) •I1 + Stot• (c2 · A2) •I2

• A1 and A2 (the intrinsic A-tensors given with respect to the local spin)

are dominated by the Fermi contact term, which is ~ -20 to -22 T.

• Analysis of field-dependent Mössbauer spectra allows c1 · A1 and c2 · A2 to

be determined.

• by determining A1 and A2, one can estimate c1and c2 and therefore determine

the nature of the spin coupling of the cluster.

• if hyperfine coupling is resolved in EPR, then |c1 · A1| and |c2 · A2| can be

determined, but not the sign of c1 and c2.

• Binternal > Bexternal

• Fe(III) site has “typical” field dependence (Bint antiparallel to Bext

for ground state, i.e. Beff decreases with increasing Bext)

• Fe(II) site has “atypical” field dependence (Bint parallel to Bext for

ground state)

• this behavior is due to opposite sign of spin coupling coefficients


Example 3

The exchange-coupled high-spin diiron cofactors of the class Ia ribonucleotide reductase from E. coli

Class I Ribonucleotide

Reductase from E. coli

Stubbe, et al. Chem. Rev. 2003,

2167-2202.

Proposed PCET (Proton Coupled

Electron Transfer) Pathway

Cofactor generation of E. coli

ribonucleotide reductase

Spectroscopic signatures of the active Fe2III/III-Y122• form

S = 1/2 for Tyr


Two quadrupole doublets in Mössbauer

Suggests integer spin ground state 4.2K

53 mT


Spectrum reveals that

Beff = Bext = 6 T

Bint = 0,

S = 0

Bext = 6 T


BUT … how do we pair the lines?

= 0.45 mm/s

DEQ = 2.43 mm/s

= 0.54 mm/s

DEQ = 1.63 mm/s



= 0.69 mm/s

DEQ = 1.94 mm/s

= 0.29 mm/s

DEQ = 2.11 mm/s



= -0.52 mm/s

DEQ = 0.48 mm/s

= 1.51 mm/s

DEQ = 0.31 mm/s

Site-specific Labeling with 57Fe

Site-specific Labeling with 57Fe

Bollinger, et al. JACS 1997, 5976

Example 4

The Fe(IV)-oxo intermediate in taurine:2-oxoglutarate dioxygenase (TauD)

αKG

His

His

Asp/Glu

Generalized Reaction Catalyzed by the Fe(II)- and

-Ketoglutarate-Dependent Dioxygenases

O

O

OO

O

O

OO

O

CO2O2 - --

-+ + + +HR OHR

αKG

His

His

Asp/Glu

> 3 x 104 M-1s-1 fast

2.5 s-1

fast

fast

fast

1.5 x 105 M-1s-1

kH = 13 s-1

kD=0.25 s-1

Mechanism of Taurine:αKG Dioxygenase (TauD)

Evidence for an Fe(IV) Intermediate by Mössbauer Spectroscopy

= 1.16 mm/s

DEQ = 2.76 mm/s high-spin Fe(II)

Evidence for an Fe(IV) Intermediate by Mössbauer Spectroscopy

= 0.30 mm/s

DEQ = 0.90 mm/s

= 1.16 mm/s

DEQ = 2.76 mm/s

Fe(IV)

Evidence for an Fe(IV) Intermediate (J) by Mössbauer Spectroscopy

0.1 0.2 0.3

Time (s)

J (

mM

)

0.2

0.4

0.6

= 0.30 mm/s

DEQ = 0.90 mm/s

= 1.16 mm/s

DEQ = 2.76 mm/s

TauDFe(II)αKGTaurine

O2

J 2nd Intermediate

1.5 x 105 M-1 s-1

13 s-1

2.5 s-1

Fe(IV) Fe(II)

High-Field Mössbauer of the Fe(IV) Intermediate

0 ms

20 ms

• Magnetic spectra of the Fe(II) reactant complex not well understood

• Experimental spectrum collected under the same experimental conditions is

used without any simulation for deconvolution of data

Mössbauer Evidence that the Intermediate has an Integer Spin Ground State with S = 2

8 T

S = 1

S = 2 I = 3/2

I = 1/2 -1/2

+1/2

+1/2

-1/2

-3/2

+3/2

Further Characterization of J from DFT Calculations

• Comparison of experimentally determined, spectroscopic parameters to

those calculated by DFT methods provides detailed structural information.

(mm/s)

DEQ (mm/s)

0.30 0.27

-0.90 -0.65

exp calc

-18.4

-17.6

-31.0

A/gNN (T)

-22.2

-21.4

-34.4

• resonance Raman Fe=O = 821 cm-1 (Proshlyakov, et al. JACS 2004, 126, 1022)

• EXAFS dFe=O = 1.62 Å

Example 5

A mononuclear {Fe(NO)2}9 complex with S = 1/2

A. L. Speelman, et al., Inorg. Chem. 2016

EPR-Spectroscopy of the {Fe(NO)2}9 complex

• Intense S = 1/2 signal

• S virtually isotropic

• Magnetically split Mössbauer spectra expected

with strong field-orientation dependence

Mössbauer Spectroscopy of the {Fe(NO)2}9 complex

g = 2.0

δ = 0.37 mm/s

ΔEQ = +1.77 mm/s

η = 0.3

A/gnn = (-26.2, -23.4, -4.6) T

A little bit of fine-print for low-field spectra …

A little bit of fine-print for low-field spectra …

• S is isotropic

All directions are probed

• A is very anisotropic

Bint anisotropic

g = 2.0

δ = 0.37 mm/s

ΔEQ = +1.77 mm/s

η = 0.3

A/gnn = (-26.2, -23.4, -4.6) T

A mononuclear {Fe(NO)2}9

complex with S = 1/2

• High-field spectra reveal

that the slow-relaxation

limit applies

Mössbauer spectroscopy – A few final remarks

• “Standard” conditions: ~0.4 mL frozen solution with 1 mM 57Fe

• Natural abundance of 57Fe is 2.2% (you need to enrich with 57Fe, 4-5 $ per mg of 57Fe)

• If you can make samples with 3 mM 57Fe you should do so

• Sample composition matters (purity, number of different species)

• If you have more than one Fe site, think about selective enrichment

• Prepare a parallel EPR sample (in particular if you anticipate species with half-integer S)

• Avoid high concentrations of relatively heavy atoms (Cl, S, P) due to scattering (100 mM

phosphate buffer not a problem, CH2Cl2 solvent is problematic)

• Data collection takes a long time (on average 1 to 1.5 days per spectrum);

longest spectrum (in our lab) was 6 days collection time

longest sample queue (in our lab) was about 5-6 weeks

• $100 per day operation costs for cryogens and source

Acknowledgements

mössbauer spectroscopy · mössbauer spectroscopy the “light source”: decay scheme of 57co...

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