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
3
I=5/2
3/2
1/2
14.4 KeV
136 KeV
57Co 270 daysEC
The Radiation Source
57Fe
4velocity [mm/s]
420‐2‐4
absorptio
n [%
]
6
0
ΔEQ
δ
I=3/2
I=1/2
±3/2
±1/2
±1/2
ΔEQ
Quadrupole Splitting
ESEA
Source Absorber
Isomer shiftδ
Zero field Mossbauer spectra
5
me
I=3/2
I=1/2
+3/2
‐3/2
+1/2
EA
Magnetic Dipole Splitting
20151050‐5‐10‐15‐20velocity [mm/s]
5
0
absorptio
n [%
]
‐1/2
+1/2‐1/2
mg
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
7
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.
1m
+3/2
-3/2
+1/2
43210‐1‐2‐‐3‐‐4velocity [mm/s]
5
0
absorptio
n [%
]
-1/2
+1/2-1/2
1 m
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
V(r)
∼
r=0
‐e2/r
r
V(r)
R>
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
2
1
0
420-2-4
2
1
0
2
1
0
2
1
0
2
1
0
Abs
orpt
ion
(%)
Velocity (mm/s)
S=2 FeII
S=0 FeII
S=2 FeIV =O
S=1 FeIV =O
S=1/2 FeV =O
Cyt c
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
z
x
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
z
x
ΔEQ < 0
ΔEQ > 0
Sign of ΔEQ
d(z2)
d(xy)Positive charge
2
1
0
420-2-4
2
1
0
2
1
0
2
1
0
2
1
0
Abs
orpt
ion
(%)
Velocity (mm/s)
S=2 FeII
S=0 FeII
S=2 FeIV =O
S=1 FeIV =O
S=1/2 FeV =O
Cyt c
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
MS
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
<Sz>
ACME
force
= displacementd = c F
displacement = c × force
Isotropic System
c
c
c
c
y
ACME
Stiff cy
Stiff cy
Soft cx Soft cx
force
Principal axes of c tensor
Anisotropic System
d = c Fc is a 2 x 2 tensor
∼
∼
y
x
S
B’=<S>
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
z
z
z
x
x
x
Isotropic gz = gx
gz > gx
gz >> gx ≈ 0
<S>
B
<S> is parallel to B’
Consider only x‐y plane
Locked near molecular z axis
B
28
Intensity at gz∼ gx2 +gy2 Aasa, Vanguard 1976
H = gzβBzSz + (1/2)gxβ(S++S-)B1x (microwaves)
gz
gxgy
S
zBz
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
z x
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
0 0 6
6 6 2
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
Bint
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
32
-B ·μ = - B · (gNβN I)nuclear
magnetic moment
Nuclear Zeeman term
33
+S·A·I
He = βS·g·B + S·D·S Electronic terms
magnitudes
34
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
gNβN
35
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?
36
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
S=3/2
S = 2
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
z
z
z
x
x
x
Isotropic
somewhat anisotropic
Very anisotropic favoring z
B
Bint locked along molecular z axis
Bint is parallel to B
Bint not parallel to B
39
θ
Bint
γ‐rays
Δm = +1
Δm = 0 Δm= 0
Δm= ‐1
Δm= ±1
+3/2
+1/2
-3/2
-1/2
+1/2-1/2
I=3/2
I=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
this
You MUST observe
that
Difference spectrum
EPR spectrum
43
E. coli Sulfite Reductase
What can we do with that?
44
Siroheme 4Fe‐4S
L. M. Siegel, P. Janick, J. Christner, E. Munck
10 K X-band EPR6.82
1.985.24
B
0.95 spins/heme
Sulfite reductase
6.82
5.24
1.98
Integral
Double integral0.80 mM heme
0.76 mM spin S = 5/2
0.95 spins/heme
0.76 mM spins
48
δ = 0.45 ± 0.01 mm/s
absorptio
n[%]
velocity [mm/s]
4
0
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
49
50
40
30
20
10
0
Absorptio
n (%
)
‐8 ‐6 ‐4 ‐2 0 2 4 6 8
Velocity (mm/s)
heme
[4Fe-4S]2+ doublet
Spectrum of SiR should look like this
50
B = 6 T
B = 50 mTparallel
B = 50 mTTransverse Siroheme
1.5
0.0
absorptio
n [%
]
1050‐5
velocity [mm/s]
1.0
0.0
1.5
0.0
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
n [%
]
50‐5velocity [mm/s]
‐0.3
0.0
0.3
1.5
0.0
[4Fe-4S]2+ Cluster of Sulfite ReductaseAfter removal of heme spectrum
Solid: parallelHashed: transverse
52
Mössbauer Study 1982
X‐ray structure: D. E. McRee et al. J. Biol. Chem. 1986, 261,10277
Siroheme 4Fe‐4S
Coupled Chromophores !!
54
Paramagnetism of [4Fe‐4S]2+ Cluster
S=2
S=2
S=5/2
S=5/2
S=0
Sheme=5/2
Admixture of S=1 excited state in the S=0 ground state.
55
Ni
by Mössbauer Analysis 1997Xia, J., Hu, X., Popescu, C., Lindahl, P.A., Münck, E. JACS, 1997,119, 8301
Acetyl Synthase A‐cluster
Nip
Nid
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
HPCA
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
©2010 by National Academy of Sciences
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= 2K
T = 10 K
T = 10 K + 17O
Int‐2
0 1000 2000 3000 4000 5000
B (G)
2.458
-0.117
0.398
0.913
1.428
1.943
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
S=3
SFe = 5/2
S = 1/2
S = 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= 2K
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
©2010 by National Academy of Sciences
2,3 HPCD states studied
Discovery of 3Fe-4S clusters
72X‐ray Structure: Nature 1979
S=0
Azotobacter vinelandii Ferredoxin
73
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
S=0
S=1/2
Reduced
S=0
No EPR (yet)
g=2.01
Mossbauer spectra of oxidized state
Mossbauer spectra of oxidized state Obviously a 2Fe center
g = 2.01
Oxidized
S=0
S=1/2
Reduced
S=0
No EPR (yet)
78
4
0
absorptio
n [%
]
420‐2‐4velocity [mm/s]
3
0
1
0
B=0
B=0.5 kG
A
B
A ‐ B2:1
[4Fe‐4S]2+ plus X
79
B=0
B=0.5 kG
3
0
absorptio
n[%]
420‐2‐4velocity [mm/s]
2
0
3
01 2 3
B [kG]
Magnetization
40
Bint
Simulation with S = 2 Spin Hamiltonian
S=1/2
Why did we miss 3rd iron in oxidized state ?
81
Fe3+
Fe2.5+
[3Fe‐4S]+ Cluster in reduced S=2 state
82
Exchangeable Fe
CysCys
Cys
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
2
2-H+
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
4
2
0
6420‐2‐4‐6
4
2
0
0
Absorptio
n (%
)
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
EPR
DFT
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
xy
yz
xz
x
z
FeIV
z
y
y
x
y
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
xy
yz
xz
y
zx
unpaired spin density
Blue: positiveRed: negative
An [O=FeV=NR]+ Center Formed by One‐Electron Oxidation of an Oxoiron(IV) Complex
xy
yz
xz
x
z
FeIV
x
z
z
yz
y
y
better view
xy
yz
xz
x
z
FeIV
x
z N(px)
z
yz
y
yaminylradicalimido
FeVbetter view
An [O=FeV=NR]+ Center Formed by One‐Electron Oxidation of an Oxoiron(IV) Complex
xy
yz
xz
z
z
y O (py)oxo
FeV
N(px)
imidox
z x
z
better view
y
y
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 =
FeV
1ox = 1minus one electron
B3LYP
H+ goes here[FeV(O)(TMC)(NC(O)CH3 )]+
supported by mass‐spec
Emily and Otto
Preliminary assessment of this talk
