Mössbauer Spectroscopy. Carsten Krebs Department of Chemistry Department of Biochemistry and Molecular Biology The Pennsylvania State University

Transcription

1 Mössbauer Spectroscopy Carsten Krebs Department of Chemistry Department of Biochemistry and Molecular Biology The Pennsylvania State University

2 Recommended Literature P. Gütlich, E. Bill, A. X. Trautwein Mössbauer Spectroscopy and Transition Metal Chemistry Springer, 2011 E. Münck Aspects of 57 Fe Mössbauer Spectroscopy Chapter 6 in Physical Methods in Bioinorganic Chemistry L. Que, Jr. (editor) University Science Books, 2000

3 General remarks Outline 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

4 The Electromagnetic Spectrum

5 Recoil Effect in Free Atoms nucleus recoil energy: E R = E 2 0 / 2Mc 2 M γ-photon E γ = E nuc - E R E R 5-6 orders of magnitude greater than natural linewidth no resonance possible => Nuclear γ-resonance cannot be observed with gases and liquids!

6 Recoil Effect in Free Atoms.. too short! 7

7 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 (ffactor) 1961: Receives the Nobel Prize in Physics

8 Mössbauer periodic table

9 Periodic table of life

10 Mössbauer spectroscopy The light source : Decay scheme of 57 Co Nuclear Spin I = 5/2 Electron capture 57 Co kev 3,300 times the energy of a 285-nm UV photon recoil imparts significant change of energy of the photon 9% 91% emitting and absorbing nuclei must be embedded in solid lattice I = 3/ kev there is recoil-less emission and absorption of -photons (f-factor) I = 1/2 57 Fe at low temperatures, all Fe species have same f-factor fraction of Fe species in sample is proportional to area of Mössbauer subspectrum

11 Mössbauer spectroscopy The light source : Decay scheme of 57 Co Nuclear Spin Electron capture 57 Co I = 5/ kev I = 3/2 9% 91% 14.4 kev v DE = c E v = source velocity c = speed of light I = 1/2 57 Fe Doppler effect allows the energy of the photon to be varied slightly

12 Mössbauer spectroscopy The light source : Decay scheme of 57 Co absorption Nuclear Spin Electron capture 57 Co I = 5/ kev 9% 91% 0 Doppler velocity I = 3/ kev I = 3/2 I = 1/2 57 Fe I = 1/2 57 Fe (sample) Photon can be absorbed by a 57 Fe nucleus in the sample

13 Experimental setup (transmission geometry) v = 1 mm/s => DE = ev = 11.6 MHz = cm -1 = 5.6 K

14 Low-Field Mössbauer spectrometer Velocity Transducer 57 Co source Sample Detector

15 High-Field Mössbauer spectrometer

16 High-Field Mössbauer spectrometer Magnetic field -beam

17 General remarks Outline 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

18 absorption [%] Types of Mössbauer spectra: 1) Quadrupole Doublet Source Absorber M I = 3/2 I = 3/2 DE Q M I = 1/2 E S E A I = 1/2 Isomer shift (δ) M I = 1/2 Quadrupole Splitting (ΔE Q ) 0 6 δ DE Q velocity [mm/s] 2 4

19 absorption [%] Types of Mössbauer spectra: 1) Quadrupole Doublet M I = 3/2 How long are the black arrows if the red double arrow is 1 m? DE Q M I = 1/2 M I = 1/2 Quadrupole Splitting (ΔE Q ) 0 6 δ DE Q velocity [mm/s] 2 4

20 absorption [%] Types of Mössbauer spectra: 1) Quadrupole Doublet M I = 3/2 How long are the black arrows if the red double arrow is 1 m? DE Q M I = 1/2 ΔE = 2 mm/s = ev E = 14.4 kev ΔE/E = M I = 1/2 Quadrupole Splitting (ΔE Q ) 150,000,000,000 m (distance earth to sun) 0 6 δ DE Q velocity [mm/s] 2 4

21 Isomer shift electron density at nucleus properties of 57 Fe nucleus δ = ( sample (0) 2 - source (0) 2 ) 4/5 π Ze 2 R 2 (ΔR/R) ΔR/R is the change of radius in ground and excited state (negative for 57 Fe). (0) 2 is the probability to find an electron at the 57 Fe nucleus only s-electrons have non-zero probability to be at nucleus d-electrons affect s-electron density by shielding nucleus atom 3d - electrons shield the nuclear potential for s - orbitals r(bohr)

22 Typical isomer shift values for various spin- and oxidation states of iron Fe(I) S=3/2 Fe(I) S=1/2 Fe(II) S=2 Fe(II) S=1 Fe(II) S=0 Fe(III) S=5/2 Fe(III) S=3/2 Fe(III) S=1/2 Fe(IV) S=2 Fe(IV) S=1 Fe(IV) S=0 Fe(V) S=3/2 (adapted from Gütlich, Bill, Trautwein Mössbauer Spectroscopy and Transition Metal Chemistry, Springer 2011) Fe(V) S=1/2 Fe(VI) S=1 (relative to -iron at 300 K ) Fe(VI) S=0

23 Isomer shift correlations Oxidation state: (Fe IV ) < (Fe III ) < (Fe II ) number of d-electrons increases shielding of s-electrons increases (0) 2 decreases increases (because ΔR/R is negative) Spin state: Ligands: (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) (S-ligands) < (N,O-ligands) (4-coordinate) < (6-coordinate)

24 Quadrupole splitting Nuclei with I > 1/2 have an electric quadrupole moment Q, which has different energies in an electric field gradient E (efg). E E electric field lines Q

25 Quadrupole splitting - theoretical '2D' model: the four model charges ±q generate an inhomogeneous field with an electric field gradient (efg) unfavorable orientation favorable orientation

26 Electric charge distribution and EFG tensor between 0 and 1 only two independent comp. Ĥ q = I Q I = eqv zz /12 [ 3 I z 2 I (I + 1) + (I x 2 I y2 )] DE Q = eqv zz /2 [ /3] 1/2 = (V xx V yy ) / V zz (asymmetry parameter)

27 Expectation values of the efg tensor elements (V ii ) val / e<r -3 > for d-electrons orbital V xx V yy V zz d x2-y2-2/7-2/7 4/7 0 d z2 +2/7 +2/7-4/7 0 d xy -2/7-2/7 +4/7 0 d xz -2/7 +4/7-2/7 +3 d yz +4/7-2/7-2/7-3 to convert V ii in ΔE Q multiply by 4.2 mms -1 / 4/7 e <r-3> (for <r -3 >=5a 0-3, Q=0.15b) (Gütlich, Bill, Trautwein, Mössbauer Spectroscopy and Transition Metal Chemistry, Springer 2011 ) for a general 3d n valence electron configuration: add up the individual contributions for all d-electrons

28 Typical values of and DE Q for biological samples Oxidation state Spin state Ligands (mm/s) DE Q (mm/s) Fe(II) S = 2 heme Fe-(O/N) Fe/S S = 0 heme < 1.5 Fe(III) S = 5/2 heme Fe-(O/N) Fe/S < 1.0 S = 3/2 heme S = 1/2 heme Fe-(O/N) Fe(IV) S = 2 Fe-(O/N) S = 1 heme Fe-(O/N) Adapted from E. Münck, Physical Methods in Bioinorganic Chemistry, L. Que, Jr. (ed) 2000

29 Calculation of and DE Q 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.

30 General remarks Outline 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

31 Types of Mössbauer spectra: 2) Magnetic Spectra I=3/2 Dm /2 +1/2-1/2-3/2 I=1/2-1/2 +1/2 Splitting of the six lines increases as the magnetic field experienced by the 57 Fe nucleus (the effective magnetic field) increases

32 Types of Mössbauer spectra: 2) Magnetic Spectra I=3/2 Dm /2 +1/2-1/2-3/2 I=1/2-1/2 +1/2 Splitting of the six lines increases as the magnetic field experienced by the 57 Fe nucleus (the effective magnetic field) increases

33 Types of Mössbauer spectra: 2) Magnetic Spectra Intensity ratio of the six lines depends on the orientation of the effective magnetic field to the propagation direction of the beam. I=3/2 Selection rule Δm = 0, 1 Intensity of Δm = 1 lines (1 +cos 2 ) Intensity of Δm = 0 lines sin 2 +3/2 +1/2-1/2-3/2 B effective -beam 3:4:1:1:4:3 B effective -beam 3:0:1:1:0:3 Powder spectrum 3:2:1:1:2:3 I=1/2-1/2 +1/2 Dm

34 Types of Mössbauer spectra: 2) Magnetic Spectra I=3/2 +3/2 +1/2-1/2-3/2 I=1/2-1/2 +1/2 inner four lines are shifted relative to the outer two lines

35 What causes the large field sensed by the 57 Fe nucleus? The paramagnetism of the Fe ions! High-spin Fe 2+ High-spin Fe 3+ S = 2 S = 5/2 High-spin Fe 4+ S = 2 Low-spin Fe 2+ Low-spin Fe 3+ S = 0 S = 1/2 Low-spin Fe 4+ S = 1

36 General remarks Outline 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

37 Spin Hamiltonian for EPR Spectroscopy Ĥ = μ B S g B + S D S + S A I electron Zeeman zero field splitting (ZFS) hyperfine coupling = μ B S g B + D (S z 2 S(S+1)/3) + E (S x 2 S y2 ) + 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

38 EPR and Mössbauer spectroscopy are complementary Electron Spin Method EPR Half-Integer Spin S = 1/2, 3/2, 5/2, EPR-active Integer Spin S = 0, 1, 2, 3, EPR-silent (in most cases)

39 Energy (cm -1 ) Energy (cm -1 ) EPR and Mössbauer spectroscopy are complementary Electron Spin Method EPR Half-Integer Spin S = 1/2, 3/2, 5/2, EPR-active Integer Spin S = 0, 1, 2, 3, EPR-silent (in most cases) B B

40 Energy (cm -1 ) Effective g-values for an S = 5/2 spin system with ZFS z B x B y B z Calculated with D = 2 cm -1 and E/D = 0 g eff g eff g eff y z x E/D x y z x y

41 Energy (cm -1 ) Effective g-values for an S = 5/2 spin system with ZFS B x B y B z Calculated with D = 2 cm -1 and E/D = 1/ g eff g eff g eff y z x E/D z x y z x y

42 Powder EPR spectra of species with anisotropic g-values g = [GHz] / B [G] Taken from G. Palmer, Physical Methods in Bioinorganic Chemistry, L. Que (ed) 2000

43 Effective g-values for an S = 5/2 spin system with ZFS rhombic 4.3-signal 4.3 protocatechuate-3,4-dioxygenase 200 B (mt) Adapted from G. Palmer, Physical Methods in Bioinorganic Chemistry, L. Que (ed) 2000 g eff g eff g eff y z x E/D z x y z x y

44 Spin Hamiltonian for Mössbauer Spectroscopy electron spin hyperfine coupling nuclear spin Ĥ = μ B S g B + S D S + S A I - g N μ N B I + I Q I electron Zeeman zero field splitting hyperfine 57 Fe nuclear Zeeman quadrupole splitting In small external magnetic fields (e.g. 10 mt) the first two term are much larger than hyperfine coupling

45 Spin Hamiltonian for Mössbauer Spectroscopy electron spin hyperfine coupling nuclear spin Ĥ = μ B S g B + S D S + S A I - g N μ N B I + I Q I electron Zeeman zero field splitting hyperfine 57 Fe nuclear Zeeman quadrupole splitting = S A I - g N μ N B I + I Q I S is spin expectation value; it contains information of electronic structure

46 Spin Hamiltonian for Mössbauer Spectroscopy electron spin hyperfine coupling nuclear spin Ĥ = μ B S g B + S D S + S A I - g N μ N B I + I Q I electron Zeeman zero field splitting hyperfine 57 Fe nuclear Zeeman quadrupole splitting = S A I - g N μ N B I + I Q I S is spin expectation value; it contains information of electronic structure = - g N μ N [ - S A/ g N μ N + B ] I + I Q I B int B ext B eff

47 Spin Hamiltonian for Mössbauer Spectroscopy electron spin hyperfine coupling nuclear spin Ĥ = μ B S g B + S D S + S A I - g N μ N B I + I Q I electron Zeeman zero field splitting hyperfine 57 Fe nuclear Zeeman quadrupole splitting = S A I - g N μ N B I + I Q I S is spin expectation value; it contains information of electronic structure = - g N μ N [ - S A/ g N μ N + B ] I + I Q I B int B ext B eff The internal magnetic field, B int, depends on the spin expectation value, S, and the hyperfine coupling tensor, A.

48 A( 57 Fe) Hyperfine Coupling Tensor A = A Fermi-contact + A dipole + A orbit 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)

49 A( 57 Fe) Hyperfine Coupling Tensor A = A Fermi-contact + A dipole + A orbit 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).

50 Spin expectation values for half-integer spin systems S ~ de/db Have the full expectation even in small external fields S g eff /4 Correlation between EPR and Mössbauer!

51 Spin expectation values for half-integer spin systems S ~ de/db Have the full expectation even in small external fields S g eff /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

52 Energy (cm -1 ) S Spin expectation values for S = 1/2 3 S = 1/ B B S is (nearly) isotropic [i.e. the same in the x, y, and z-direction

53 S S Energy (cm -1 ) Spin expectation values for S = 5/2 Calculated with D = 2 cm -1 and E/D = 1/ B (T) ground doublet B (T) B (T) z x 0-1 middle doublet x, y, z B (T) y B (T)

54 S Spin expectation values for integer spin systems Calculated for S = 2 with D = 10 cm -1 and E/D = 1/3 x y z B (T) B (T) B (T) Have in most cases S 0 for B ext = 0 0 z Small B ext may result in small S [depends on ZFS parameters] -1 x Large B ext results in sizeable S B (T) y

55 EPR and Mössbauer spectroscopy are complementary Electron Spin Method EPR Half-Integer Spin S = 1/2, 3/2, 5/2, EPR-active Integer Spin S = 0, 1, 2, 3, EPR-silent (in most cases) Mössbauer Sizeable B int in small B ext Magnetically split spectra Small B int in small B ext Quadrupole doublets (analysis complex, but facilitated using results from EPR) (in most cases, but not always*) * (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)

56 General remarks Outline 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

57 Orientation of B int relative to B ext 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

58 Orientation of B int relative to B ext ray B external ray B external The internal field is aligned antiparallel to the external field

59 Orientation of B int relative to B ext ray or B external 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)

60 General remarks Outline 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

61 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).

62 Energy (cm -1 ) Calculate S for each electronic state Slow relaxation limit 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 B

63 Energy (cm -1 ) Fast relaxation limit Calculate S for each electronic state Calculate the average spin expectation value, S av, from the individual S values according to their Boltzmann factors Calculate Mössbauer spectrum using S av. There is only one subspectrum associated with all electronic states In small magnetic fields S av 0, therefore no hyperfine interactions, i.e. spectrum is a quadrupole doublet B

64 Cases when S is zero S = 0 B int =0 quadrupole doublet for small B ext 1. Diamagnetic compounds 2. Paramagnetic compounds with integer spin ground state for B ext = 0 (or B ext small) 3. Compound in fast relaxation limit in small magnetic field (then S av 0, therefore no hyperfine interactions, i.e. spectrum is a quadrupole doublet

65 EPR and Mössbauer spectroscopy are complementary Electron Spin Method EPR Half-Integer Spin S = 1/2, 3/2, 5/2, EPR-active Integer Spin S = 0, 1, 2, 3, EPR-silent (in most cases) Mössbauer Magnetically Split Spectra (at low T) (analysis complex, but facilitated using results from EPR) Quadrupole doublets at high temperatures Quadrupole doublets in small B ext (in most cases) (analysis straightforward) Magnetically Split Spectra for large B ext

66 General remarks Outline 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

67 Example 1 The high-spin Fe(III) site in transferrin (S = 5/2)

68 The high-spin Fe(III) site in transferrin (S = 5/2) g eff doublet 1 doublet 2 doublet E/D g eff E/D g eff E/D

69 The high-spin Fe(III) site in transferrin (S = 5/2) D = 0.25 cm mt 50 mt perp 0.5 T 2 T E/D = 0.3 g = 2.0 δ = 0.54 mm/s ΔE Q = 0.30 mm/s η = 1.0 A/g n n = (-22.3, -21.9, -22.3) T 6 T Kretchmar, et al. Biol. Metals 1988 (1) 26

70 The high-spin Fe(III) site in transferrin (S = 5/2) 50 mt 50 mt perp 0.5 T 2 T 6 T Kretchmar, et al. Biol. Metals 1988 (1) 26

71 General remarks Outline 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

72 Example 2 The exchange-coupled high-spin Fe 2 (II/III) cofactor of myo-inositol oxygenase

73 Energy The spin-coupled Fe 2 II/III cluster in myo-inositol oxygenase The active form of myo-inositol oxygenase harbors an antiferromagnetically coupled dinuclear site with a high-spin Fe 3+ ion (S 1 = 5/2) and a high-spin Fe 2+ ion (S 2 = 2). It has an EPR-active S = 1/2 ground state. S = 9/2 S = S 1 + S 2 g = (1.95, 1.81, 1.81) 4.5 J Ĥ HDvV = J S 1 S 2 S = 7/2 E(S) = J/2 S (S + 1) 3.5 J 2.5 J 1.5 J S = 5/2 S = 3/2 S = 1/2 EPR-spectroscopy probes the total ground spin state of a coupled cluster.

74 The spin-coupled Fe 2 II/III cluster in myo-inositol oxygenase = 1.09 mm/s DE Q = 2.86 mm/s high-spin Fe(II) = 0.48 mm/s DE Q = 1.10 mm/s high-spin Fe(III) Mössbauer-spectroscopy probes the local spin/oxidation state of each 57 Fe-labeled site of a coupled cluster. At 120 K in zero field: fast-relaxation limit quadrupole doublets intrinsic Fe oxidation state

75 The spin-coupled Fe 2 II/III cluster in myo-inositol oxygenase Mössbauer-spectroscopy probes the local spin/oxidation state of each 57 Fe-labeled site of a coupled cluster. At 4.2 K: slow-relaxation limit magnetically split spectra S = 1/2 S isotropic field-orientation-dependence

76 Spin projection factors Ĥ hf = S 1 A 1 I 1 + S 2 A 2 I 2 hyperfine 1 hyperfine 2 = S tot (c 1 A 1 ) I 1 + S tot (c 2 A 2 ) I 2 Spin projection factors c i = [S(S+1) + S i (S i +1) S j (S j +1)] / [2S(S+1)] For S = 1/2 ground state, c 1 = +7/3 and c 2 = -4/3 for S 1 = 5/2 and S 2 = 2 See A. Bencini and D. Gatteschi, EPR of Exchange Coupled Systems, Springer, 1989 for derivation of spin coupling coeff.

77 Spin Hamiltonian of an exchange-coupled cluster Ĥ hf = S 1 A 1 I 1 + S 2 A 2 I 2 hyperfine 1 hyperfine 2 = S tot (c 1 A 1 ) I 1 + S tot (c 2 A 2 ) I 2 A 1 and A 2 (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 c 1 A 1 and c 2 A 2 to be determined. by determining A 1 and A 2, one can estimate c 1 and c 2 and therefore determine the nature of the spin coupling of the cluster. if hyperfine coupling is resolved in EPR, then c 1 A 1 and c 2 A 2 can be determined, but not the sign of c 1 and c 2.

78 The spin-coupled Fe 2 II/III cluster in myo-inositol oxygenase B internal > B external Fe(III) site has typical field dependence (B int antiparallel to B ext for ground state, i.e. B eff decreases with increasing B ext ) Fe(II) site has atypical field dependence (B int parallel to B ext for ground state) this behavior is due to opposite sign of spin coupling coefficients

79 General remarks Outline 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

80 Example 3 The exchange-coupled high-spin diiron cofactors of the class Ia ribonucleotide reductase from E. coli

81 Class I Ribonucleotide Reductase from E. coli Stubbe, et al. Chem. Rev. 2003, Proposed PCET (Proton Coupled Electron Transfer) Pathway

82 Cofactor generation of E. coli ribonucleotide reductase

83 Spectroscopic signatures of the active Fe 2 III/III -Y122 form S = 1/2 for Tyr

84 Spectroscopic signatures of the active Fe 2 III/III -Y122 form Two quadrupole doublets in Mössbauer Suggests integer spin ground state 4.2K 53 mt

85 Spectroscopic signatures of the active Fe 2 III/III -Y122 form Spectrum reveals that B ext = 6 T B eff = B ext = 6 T B int = 0, S = 0

86 Spectroscopic signatures of the active Fe 2 III/III -Y122 form BUT how do we pair the lines? = 0.45 mm/s DE Q = 2.43 mm/s = 0.54 mm/s DE Q = 1.63 mm/s

87 Spectroscopic signatures of the active Fe 2 III/III -Y122 form BUT how do we pair the lines? = 0.69 mm/s DE Q = 1.94 mm/s = 0.29 mm/s DE Q = 2.11 mm/s

88 Spectroscopic signatures of the active Fe 2 III/III -Y122 form BUT how do we pair the lines? = mm/s DE Q = 0.48 mm/s = 1.51 mm/s DE Q = 0.31 mm/s

89 Site-specific Labeling with 57 Fe

90 Site-specific Labeling with 57 Fe Bollinger, et al. JACS 1997, 5976

91 General remarks Outline 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

92 Example 4 The Fe(IV)-oxo intermediate in taurine:2- oxoglutarate dioxygenase (TauD) αkg His His Asp/Glu

93 Generalized Reaction Catalyzed by the Fe(II)- and -Ketoglutarate-Dependent Dioxygenases R H O O O - + O O 2 + -O O - R OH + O - + CO 2 O O αkg His His Asp/Glu

94 Mechanism of Taurine:αKG Dioxygenase (TauD) > 3 x 10 4 M -1 s -1 fast 2.5 s x 10 5 M -1 s -1 fast fast k H = 13 s -1 k D =0.25 s -1 fast

95 Evidence for an Fe(IV) Intermediate by Mössbauer Spectroscopy = 1.16 mm/s DE Q = 2.76 mm/s high-spin Fe(II)

96 Evidence for an Fe(IV) Intermediate by Mössbauer Spectroscopy = 1.16 mm/s DE Q = 2.76 mm/s = 0.30 mm/s DE Q = 0.90 mm/s Fe(IV)

97 Evidence for an Fe(IV) Intermediate (J) by Mössbauer Spectroscopy J (mm) = 1.16 mm/s DE Q = 2.76 mm/s = 0.30 mm/s DE Q = 0.90 mm/s 0.2 TauD Fe(II) αkg Taurine Time (s) 2.5 s -1 2 nd Intermediate Fe(II) O 2 13 s x 10 5 M -1 s -1 J Fe(IV)

98 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

99 Mössbauer Evidence that the Intermediate has an Integer Spin Ground State with S = 2 +3/2 8 T S = 2 I = 3/2 +1/2-1/2-3/2 S = 1 I = 1/2-1/2 +1/2

100 Further Characterization of J from DFT Calculations resonance Raman Fe=O = 821 cm -1 (Proshlyakov, et al. JACS 2004, 126, 1022) EXAFS d Fe=O = 1.62 Å exp calc (mm/s) DE Q (mm/s) A/g N N (T) Comparison of experimentally determined, spectroscopic parameters to those calculated by DFT methods provides detailed structural information.

101 General remarks Outline 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

102 Example 5 A mononuclear {Fe(NO) 2 } 9 complex with S = 1/2 A. L. Speelman, et al., Inorg. Chem. 2016

103 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

104 Mössbauer Spectroscopy of the {Fe(NO) 2 } 9 complex g = 2.0 δ = 0.37 mm/s ΔE Q = mm/s η = 0.3 A/g n n = (-26.2, -23.4, -4.6) T

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

106 A little bit of fine-print for low-field spectra g = 2.0 δ = 0.37 mm/s ΔE Q = mm/s η = 0.3 A/g n n = (-26.2, -23.4, -4.6) T S is isotropic All directions are probed A is very anisotropic B int anisotropic

107 A mononuclear {Fe(NO) 2 } 9 complex with S = 1/2 High-field spectra reveal that the slow-relaxation limit applies

108 General remarks Outline 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

109 Mössbauer spectroscopy A few final remarks Standard conditions: ~0.4 ml frozen solution with 1 mm 57 Fe Natural abundance of 57 Fe is 2.2% (you need to enrich with 57 Fe, 4-5 $ per mg of 57 Fe) If you can make samples with 3 mm 57 Fe 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, CH 2 Cl 2 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

110 Acknowledgements