Some Fundamentals of Mossbauer Spectroscopy and...

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Some Fundamentals of Mossbauer Spectroscopy and Relations to EPR.Examples Illustrating the Power of Combining the Two Techniques

Eckard Munck, Carnegie Mellon University

Penn State Bioinorganic Workshop 2012 May 20 version

Fundamentals (30 min)

Applications

Coupled Chromophores of Sulfite Reductase

Discovery of 3Fe-4S clusters

Breaking News: A novel FeV =O complex

A Superoxo intermediate in a dioxygenase

14.4 KeV

136 KeV

57Co 270 daysEC

The Radiation Source

4velocity [mm/s]

420‐2‐4

absorptio

Quadrupole Splitting

Source Absorber

Isomer shiftδ

Zero field Mossbauer spectra

‐3/2

Magnetic Dipole Splitting

20151050‐5‐10‐15‐20velocity [mm/s]

absorptio

‐1/2

+1/2‐1/2

Doppler shift ΔE = v/c Eγ

1 mm/s 3 ×1011 mm/s

14.4 ×103 eV = 4.8 × 10‐8 eV

Counter

57CoSource

Sample

Counter

10‐7 eV

mg = ‐1/2

mg = +1/2

Ig = 1/2

14.4 keV

Where Is the Nuclear Excited State??The excited state is at the Sun, 150 million km away.

43210‐1‐2‐‐3‐‐4velocity [mm/s]

absorptio

+1/2-1/2

FeV FeIV FeIV FeIII FeIII FeII FeII

S=1/2 S=1 S=2 S=1/2 S=5/2 S=0 S=2

Common spin states of mononuclear Fe complexes

Isomer shift

‐e2/r

R>≈ 10‐4 Å

RBohr = 0.53 Å

δ ≈ 10‐8 eV

Ψ depends mainly on the s electron density at nucleus which in turn is influenced by the d electron density through shielding

δ = 2/3π Z e2 {|Ψ(0)|2Abs ‐|Ψ(0)|2Source]} {<r2 >exc ‐ <r2 >grd }

Electronic property Property of 57 Fe nucleus

Book: Gutlich, Link and Trautwein, Mossbauer spectroscopy and Transition Metal Chemistry

Radial Distribution Function = r2 [Rnl (r)]2

Plot from J. M. Standard handout

420-2-4

Velocity (mm/s)

S=2 FeII

S=0 FeII

S=2 FeIV =O

S=1 FeIV =O

S=1/2 FeV =O

TPA ligand

TAML ligand

TauD J

dioxygenase

Representative Quadrupole Doublets

1.51.00.50.0

δ (mm/s)

FeII S=2FeIII S=5/2FeIV S=2

FeII S=0FeIII S=1/2

FeIV S=1FeV S=1/2

‐0.5

Isomer shift ranges for mononuclear octahedral complexes(vs Fe metal) Diagram is very approximate and incomplete

Note: Overlapping ranges can generally be sorted out by magnetic properties (spin)

Experimental errors are typically ± 0.01 mm/s

Isomer shift depends on

Oxidation stateSpin State

Nature of LigandsCoordination Number

GeometryCovalency

Quadrupole Splitting

HQ = (eQVzz /12) { 3Iz2 - 15/4 + η (Ix

2 - Iy 2 ) }

Vxx , Vyy , Vzz : principal components of the Electric Field Gradient (EFG) tensor

η = (Vxx - Vyy )/Vzz = asymmetry parameter

Nuclear quadrupole interactions

V(x,y,z) is the potential generated by the electronic environment at the 57 Fe nucleusVzz = {∂2 V/∂z2 }0

Vzz and η (and often more) can be determined experimentally

ΔEQ = (eQVzz /2) √ (1 + η2 /3)

M = +3/2

M = + 1/2

Spin around the vertical Q > 0 for 57 Fecigar shapednot pancake

M = +3/2

M = + 1/2

± 3/2

± 1/2

I = 3/2

± 1/2

± 3/2

I = 3/2

ΔEQ < 0

ΔEQ > 0

Sign of ΔEQ

d(xy)Positive charge

420-2-4

Velocity (mm/s)

S=2 FeII

S=0 FeII

S=2 FeIV =O

S=1 FeIV =O

S=1/2 FeV =O

TPA ligand

TAML ligand

TauD J

dioxygenasealso s*&# iron

Typical quadrupole doublets

Comments on Electronic Properties

Electronic Zeeman TermS= ½ and isotropic g-tensor: gx =gy =gz =g0

H = g0 β B Sz

S=1/2g0 βB

+1/2 Spin up

-1/2 Spin down

Expectation value of Sz

<Ψspin-down |Sz |Ψspin-down > = <Sz > = -1/2

<Sx> = < Sy > = 0

Call direction of B the z axis

= displacementd = c F

displacement = c × force

Isotropic System

Stiff cy

Soft cx Soft cx

Principal axes of c tensor

Anisotropic System

d = c Fc is a 2 x 2 tensor

B’=<S>

B = <S>

H = g0 β S•B

H = β S•g•B = β S•B’

isotropic g: gx =gy =gz =g0

anisotropic g: gx ≠gy ≠gz

For spin S = ½ we always have <SB’ > = ± 1/2

B’ = g•B

Isotropic gz = gx

gz > gx

gz >> gx ≈ 0

<S> is parallel to B’

Consider only x‐y plane

Locked near molecular z axis

Intensity at gz∼ gx2 +gy2 Aasa, Vanguard 1976

H = gzβBzSz + (1/2)gxβ(S++S-)B1x (microwaves)

EPR intensity ∼ |<Ψspin-up|gxS-B1x|Ψspin-down>|2

2Sx = (S+ + S- )

H = gzβBzSz + (1/2) gxSxB1x (microwaves)

when gx =0 and gy = 0 there is no EPR and <S> is locked along z

Zero-field splitting in High-spin FeIII

H = D {Sz 2 - 35/4 + (E/D) (Sx

2 - Sy2 )} + g0 β S●B

E/D = 0 = axial symmetry; often the case for hemes

± 5/2

± 3/2

± 1/2

M gx gy gz

0 0 10

effective g‐values

S = 5/2

2D ≈ 15 cm-1

The ± 5/2 and ± 3/2 levelsare EPR‐silent. ΔM = 5 and ΔM = 3 transitions are forbidden

H = S•D•S more generally

A typical heme

Comments on Magnetic Hyperfine interactions

Spin-dipolar

Fermi Contact

orbital

Bint = Bcontact + Bspin-dipolar + B orbital

All lumped together in S • A • IBint = -<S> • A/gNβN

electron

Magnetic moment of 57 Fe nucleus

-B ·μ = - B · (gNβN I)nuclear

magnetic moment

Nuclear Zeeman term

+S·A·I

He = βS·g·B + S·D·S Electronic terms

magnitudes

HN = - gNβNI·(Bint + B)

HN = - gNβNI ·Beff

S·A·I = - <S>·A· (-gNβNI)

He = βS·g·B + S·D·S + S·A·I - gNβNB·I

Bint = - <S>·A /gNβN

The 57 Fe nucleus sees Beff

Nuclear Zeeman term

There is more to He **

He = ∑ {βSn·gn·B + Sn·D·Sn} + J S1·S2 + d·S1xS2n=1,2

All these terms influence <S> and thus Bint

Isotropic exchange

Antisymmetric exchange

** But we have enough of it, haven’t we?

Half‐Integral Spin

Integer or Zero Spin

4.2 K Mössbauer Spectra for B = 0

e. g. FeIII FeI FeV

FeIII FeII and FeIVFeIII

FeIIFeIV

FeIII FeIII

Kramers doublets

Generally singlets<S> = 0 in zero field

Bint = - <S> •A0 /gN βN

For isotropic system: Bint is parallel to <S>, which is parallel to B

Bint is parallel to B

Isotropic system

Isotropic

somewhat anisotropic

Very anisotropic favoring z

Bint locked along molecular z axis

Bint is parallel to B

Bint not parallel to B

γ‐rays

Δm = +1

Δm = 0 Δm= 0

Δm= ‐1

Δm= ±1

+1/2-1/2

Δm = ±1 ~ 1 + cos2θ

Δm = 0 ~ sin2θ

Intensities

Magnetic dipole transition (property of 57Fe)

detectorsample57 Co

Bint Bapplied

γ rays

Bint = -<S>•A0 /gn βn

For an isotropic system

Quadrupole doublets cancel for “parallel minus transverse”

500 gauss applied field

When you observe

You MUST observe

Difference spectrum

EPR spectrum

E. coli Sulfite Reductase

What can we do with that?

Siroheme 4Fe‐4S

L. M. Siegel, P. Janick, J. Christner, E. Munck

10 K X-band EPR6.82

1.985.24

0.95 spins/heme

Sulfite reductase

Integral

Double integral0.80 mM heme

0.76 mM spin S = 5/2

0.95 spins/heme

0.76 mM spins

δ = 0.45 ± 0.01 mm/s

absorptio

velocity [mm/s]

420‐2‐4

S = 1/2 S = 0 S = 1/2

3+ 2+ 1+0.40 0.500.30 0.60 δ[mm/s]

190‐K Mössbauer Spectrum of the [4Fe‐4S] Cluster

Absorptio

‐8 ‐6 ‐4 ‐2 0 2 4 6 8

Velocity (mm/s)

[4Fe-4S]2+ doublet

Spectrum of SiR should look like this

B = 6 T

B = 50 mTparallel

B = 50 mTTransverse Siroheme

absorptio

1050‐5

velocity [mm/s]

But doesn’t

51Christner,J.;P.;Münck, E.;Janick,P.A.;Siegel,L.M. J. Biol. Chem. 1982, 356, 2089

Conclusion[4Fe-4S] cluster must be associated with the EPR signal.

Δm = 0 lines

absorptio

50‐5velocity [mm/s]

‐0.3

[4Fe-4S]2+ Cluster of Sulfite ReductaseAfter removal of heme spectrum

Solid: parallelHashed: transverse

Mössbauer Study 1982

X‐ray structure: D. E. McRee et al. J. Biol. Chem. 1986, 261,10277

Siroheme 4Fe‐4S

Coupled Chromophores !!

Paramagnetism of [4Fe‐4S]2+ Cluster

Sheme=5/2

Admixture of S=1 excited state in the S=0 ground state.

by Mössbauer Analysis 1997Xia, J., Hu, X., Popescu, C., Lindahl, P.A., Münck, E. JACS, 1997,119, 8301

Acetyl Synthase A‐cluster

C. Darnault et al. Nat. Struct. Biol. 2003, 10, 271

by X‐ray Crystallography 2003

2,3 homoprotocatechuate dioxygenase

2,3 HPCD

M. M. Mbughuni,a M. Chakrabarti,b J. A.Hayden,b E. L. Bominaar,b M. P. Hendrich,b E. Münck,b and J. D. Lipscomba1 PNAS 2010

Work with the group of J. D. Lipscomb

Emerson, et. al., PNAS 2008

Fe or Mn

His200

Asn157

Glu267

Tyr269

Tyr257

His248

His214

His155

Arg293

Arg243

2,3 HPCD active site

H200N mutant allows to trap Int‐1

4NC is a slow substrate

2,3 HPCD + 4‐nitrocatechol

Zero field Mossbauer Spectra of H200N mutant

2,3 HPCD + 4‐nitrocatechol

Add O2 and freeze at 10 s

Int‐1

Zero field Mossbauer Spectra at 4.2 K H200N mutant

2,3 HPCD + product

Int ‐2 at 10 min

δ = 0.50 mm/s suggests S= 5/2 FeIIIbut behaves like integer spin species

δ = 1.12 mm/s

Mbughuni M M et al. PNAS 2010;107:16788-16793

In applied fields, Int‐1 sure looks like high‐spin (S=5/2) species

Full splitting for B = 0.6 Tsuggests two closely <0.3 cm‐1)electronic levels. Try EPR

The g = 8.17 feature is characteristic of an S = 2 system

Parallel mode EPR of Int‐1

T = 10 K

T = 10 K + 17O

Int‐2

0 1000 2000 3000 4000 5000

-0.117

S = 2D = ‐ 0.5 cm‐1 small !E/D = 0.2

S = SFe + SR

H = J SFe •SR

S = 3 multiplet

S = 2 multipletwith zero‐field splitting

3 J = 18 cm‐1

g=11.6

g=8.17

SFe = 5/2

SR = 1/2

SFe = 5/2

S = 1/2

SFe = 5/2 antiferromagnetically coupled to SR = ½ radical

The g = 8.17 feature is characteristic of an S = 2 system

Parallel mode EPR of Int‐1

T = 10 K

T = 10 K + 17O

Int‐2

Nearly 400 gauss17 O splitting

A(17O) = 180 MHz

Michael Hendrich did EPRSpinCount software

Blue: α spin densityGreen: β spin density

Superoxide radical centered on distal O

You are here

DFT geometry optimization of Int-1.

Mbughuni M M et al. PNAS 2010;107:16788-16793

2,3 HPCD states studied

Discovery of 3Fe-4S clusters

72X‐ray Structure: Nature 1979

Azotobacter vinelandii Ferredoxin

S = 0 S = ½ g =1.94

Azotobacter cluster

+ 1 electronFe3+ Fe3+ Fe3+ Fe2+

S = ½ g =2.01

+ 1 electronFe3+ Fe2+

Integer Spin

Typical [2Fe‐2S] cluster

Oxidized

Reduced

No EPR (yet)

g=2.01

Mossbauer spectra of oxidized state

Mossbauer spectra of oxidized state Obviously a 2Fe center

g = 2.01

Oxidized

Reduced

No EPR (yet)

absorptio

420‐2‐4velocity [mm/s]

B=0.5 kG

A ‐ B2:1

[4Fe‐4S]2+ plus X

B=0.5 kG

absorptio

420‐2‐4velocity [mm/s]

01 2 3

B [kG]

Magnetization

Simulation with S = 2 Spin Hamiltonian

Why did we miss 3rd iron in oxidized state ?

Fe2.5+

[3Fe‐4S]+ Cluster in reduced S=2 state

Exchangeable Fe

CysCys

citrate

Aconitase with Substrate

Radical SAM enzymes

Pyruvate Formate Lyase as seen on Joan Broderick’s home page

Radical SAM Enzymes

A novel FeV =O complex

O=FeIV (NCCH3)(TMC)

acetonitrile

Stable at room temperature

complex 1

TMC, 1,4,8,11‐tetramethyl‐1,4,8,11‐tetraazacyclotetradecane

2‐H+

Add HOOtBu and base to 1 at – 44 C

Yes, we can

X‐Band EPR of 2 and 2‐H+

2 and 2‐H+ are S=1/2 species.

T = 20K

gy=2.01

gx=2.045

gz=1.97

X-Band EPR of 2-H+:A closer look at the N-hyperfine structure

360350340330320

Signal

B (mT)

14N Hyperfine (A-tensors)1 : 1 : 1

Ax = 28.5 MHzAy = 11 MHzAz = 11 MHz

gx = 2.045

15N Hyperfine (A-tensors)1 : 1

Ax = 40 MHzAy = 15 MHzAz = 15 MHz

1:3 15NCCH3 : CH2Cl2

1:3 NCCH3 : butyronitrile

The largest splitting is along x.

X‐Band EPR of 2‐H+:17O=FeIV (TMC) hyperfine structure

360350340330320B (mT)

Signal

gy = 2.01

100% enrichment

30% enrichment

Note: The large splitting is along y.

17O Hyperfine (A‐tensors):Ax = ~35 MHzAy = 133 MHzAz = ~22 MHz

For an isotropic species the magnetic hyperfine field Bint isparallel to the applied field Bapplied for all molecules in the sample

detectorsample57 Co

Bint Bapplied

γ rays

Bint = ‐<S>•A /gn βn

Quadrupole doublets cancel for “parallel minus transverse”

500 gauss applied field

Parallel MB of 2

Parallel minus perpendicular of 2

Transverse MB of 2

6420‐2‐4‐6

Absorptio

Velocity (mm/s)

∆EQ (mm s‐1) = ‐0.50

57Fe: Ax = ‐47MHzAy = ‐17 MHz Az = 0 MHz

Green: O=FeIV ‐NCCH3

Blue: O=FeIV ‐OHRed: 2 (55% of Fe)

δ (mm s‐1) = 0.10(4)

Proposed Mechanism for generation of 2 and 2-H+

Purpose of base:‐O‐O‐tBu

H+ goes here FeV or FeIV (aminyl)●

In this project, the Mossbauer isomer shift and the resonance Raman spectrum mislead us for almost 2 years

At the Brown Bag Lunch on Thursday, Katie Meier will address this points and showhow everything fits at the end.

Mossbauer

Three knights have to come to the rescue

Schulz et al , Biochemistry 1984, Horseradish peroxidase; MossbauerB. M. Hoffman et al Horseradish peroxidase; ENDOR

A‐tensors

CompoundAx,y,z (57Fe) (MHz) Ax,y,z (14Naxial) (MHz) Ax,y,z (17O) (MHz)

x y z x y z x y zHRP Cpd I -26 -26 -8 - - - 35 35 nd

(TMC)FeIV =O,1 -30 -30 -4 - - - -27 -27 61

2 or 2-H+ -47 -17 0 29 11 11 35 133 22

T. Jackson et al FeIV =O complexes of TMC ligand

Compound I ChloroperoxidaseFeIV + porphyrin radical

17 O of 1 from DFT

FeIV =O 57Fe and 17 O A-tensors are axial

Compoundg A (57Fe) A (17O) A (14N)

x y z x y z x y z x y z

HRP Cpd I − − − -26 -26 -8 35 36 nd − − −

1 − − − -28 -28 -4 -27 -27 +61 − − −

2 2.05 2.01 1.97 -47 -17 0

2-H+ 2.05 2.01 1.97 -47 -17 0

[FeV(O)-(TAML)]1-

1.99 1.97 1.74 -49.3 -1.5 ≈0a − − − − − −

aafter orbital correctionThis is a bona fide FeV =O complex

Results for 2 and 2-H+

[FeV (O)(TAML)‐1

TAML ligand of Terry Collins

BP86 functional

unpaired spin density

Blue: positiveRed: negative

An [O=FeV=NR]+ Center Formed by One‐Electron Oxidation of an Oxoiron(IV) Complex

better view

z N(px)

yaminylradicalimido

FeVbetter view

An [O=FeV=NR]+ Center Formed by One‐Electron Oxidation of an Oxoiron(IV) Complex

y O (py)oxo

imidox

better view

FeV(O)(TAML)a 1ox (FeV) 2 1 (FeIV ) 2 (FeIV N●) cFe-dxz 0.07 0.15 0.23 0.58 0.58Fe-dyz 0.57 0.63 0.53 0.58 0.58

Naxial-px -0.02 -0.30 -0.85

Calculated unpaired spin populations: BP86

1ox = 1minus one electron

H+ goes here[FeV(O)(TMC)(NC(O)CH3 )]+

supported by mass‐spec

Emily and Otto

Preliminary assessment of this talk

Some Fundamentals of Mossbauer Spectroscopy and...

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