Transcript Physical Methods - Bryn Mawr College

Physical Methods In Bioinorganic Chemistry
1. X-ray spectroscopy: EXAFS, XANES
2. Resonance spectroscopy
Electron paramagnetic resonance - EPR
Pulsed EPR: ESEEM, ENDOR
Resonance Raman - RR
3. Magnetic Spectroscopy
Magnetic Circular Dichroism
4. Mossbauer Spectroscopy
Physical Methods In Bioinorganic Chemistry
1.
•
•
•
•
2.
X-ray spectroscopy: EXAFS, XANES
Gives M-L distances to high precision
Gives identities and numbers of M and L
Gives some information on geometry
No info on angles, conformations
Resonance spectroscopy
• Electron paramagnetic resonance - EPR
• Pulsed EPR: ESEEM, ENDOR
• Resonance Raman - RR
3. Magnetic Spectroscopy
Magnetic Circular Dichroism
4. Mossbauer Spectroscopy
Physical Methods In Bioinorganic Chemistry
1. X-ray spectroscopy: EXAFS, XANES
2. Resonance spectroscopy
Electron paramagnetic resonance - EPR
Gives info on metal identity, donor atoms,
and 2nd sphere atoms
Some info on bonding character
Pulsed EPR: ESEEM, ENDOR
Resonance Raman - RR
3. Magnetic Spectroscopy
Magnetic Circular Dichroism
4. Mossbauer Spectroscopy
Physical Methods In Bioinorganic Chemistry
1. X-ray spectroscopy: EXAFS, XANES
2. Resonance spectroscopy
Electron paramagnetic resonance - EPR
Pulsed EPR: ESEEM, ENDOR
Resonance Raman - RR
Gives info on vibrations and bond order
Reveals coupled electronic and vibrational states
3. Magnetic Spectroscopy
Magnetic Circular Dichroism
4. Mossbauer Spectroscopy
Physical Methods In Bioinorganic Chemistry
1. X-ray spectroscopy: EXAFS, XANES
2. Resonance spectroscopy
Electron paramagnetic resonance - EPR
Pulsed EPR: ESEEM, ENDOR
Resonance Raman - RR
3. Magnetic Spectroscopy
Magnetic Circular Dichroism
Correlates e- transitions and MO’s by symmetry
4. Mossbauer Spectroscopy
Physical Methods In Bioinorganic Chemistry
1. X-ray spectroscopy: EXAFS, XANES
2. Resonance spectroscopy
Electron paramagnetic resonance - EPR
Pulsed EPR: ESEEM, ENDOR
Resonance Raman - RR
3. Magnetic Spectroscopy
Magnetic Circular Dichroism
4. Mossbauer Spectroscopy
Gives oxidation state of Fe ions
Usefulness?
Inorganic Chemistry Vol. 44, No. 4: February 21, 2005"Functional Insight from Physical
Methods on Metalloenzymes"
Edward I. Solomon
pp 723 - 726;
XAS techniques:
Get your bearings
in energy:
Physical Methods In Bioinorganic Chemistry
1. X-ray spectroscopy: EXAFS, XANES
2. Resonance spectroscopy
• Electron paramagnetic resonance - EPR
• Pulsed EPR: ESEEM, ENDOR
• Resonance Raman - RR
3. Magnetic Spectroscopy
• Magnetic Circular Dichroism
4. Mossbauer Spectroscopy
Gives oxidation state of Fe ions
Usefulness?
Inorganic Chemistry Vol. 44, (2005) pp 723 - 726
"Functional Insight from Physical Methods on Metalloenzymes"
Edward I. Solomon - Stanford University
X-RAY ABSORPTION SPECTROSCOPY: XAS, EXAFS, XANES
When an atom is bombarded by X-rays:
- an electron from a core level is excited to the unoccupied states of the system
- changing the X-ray excitation energy changes the unoccupied state the electron can reach
- EXAFS: extended X-ray absorption Fine Structures
- XANES: X-ray Absorption Near Edge Structure
When a photoelectron is ejected:
EXAFS “Ripples” from
interference of neighbors
Energy needed to
eject core electron
RAWDATA
http://www.haverford.edu/chem/Scarrow/EXAFS123/Plotting%20Graphs.htm
FITTING http://www.haverford.edu/chem/Scarrow/EXAFS123/FITTING.htm
REFINING
http://www.haverford.edu/chem/Scarrow/EXAFS123/REFINING.htm
Considering the wave nature of the ejected
photoelectron and regarding the atoms as point
scatterers a simple picture can be seen in which
the backscattered waves interfere with the
forward wave to produce either peaks or troughs.
Can’t do this at home; requires an intense X-ray source
-> Synchrotron Radiation
1. SSRL: Stanford Synchrotron Radiation Lab
The Stanford Synchrotron Radiation Laboratory, a division of Stanford Linear Accelerator Center, is operated
by Stanford University for the Department of Energy. SSRL is a National User Facility which provides
synchrotron radiation, a name given to x-rays or light produced by electrons circulating in a storage ring at
nearly the speed of light. These extremely bright x-rays can be used to investigate various forms of matter
ranging from objects of atomic and molecular size to man-made materials with unusual properties. The
obtained information and knowledge is of great value to society, with impact in areas such as the
environment, future technologies, health, and education.
2. Advanced Photon Source - The
Advanced Photon Source at
Argonne National Laboratory is a
national synchrotron-radiation light
source research facility funded by
the U.S. Department of Energy,
Office of Science, Office of Basic
Energy Sciences. Using highbrilliance x-ray beams, well over
3000 individual users conducted
research at the APS. When all 70
beamlines are operational, that
number is expected to grow to more
than 4000 annually.
Mn-O ~ 1.4 Å
Mn-O ~ 2.2 Å
Mn-Mn ~ 3.0 Å
Mn-Ca ~ 3.0 Å
Various
Intramoecular
Distances in the
Tetra-Mn cluster
of Photosystem II,
the O2 evolving
center in
Photosynthesis,
as seen by EXAFS.
The K-edge XANES spectrum measured at 10K
and low X-ray dose for intact PSII samples (A)
is similar to corresponding edges for dimeric
Mn(IV,IV) or Mn(III,III) model complexes (B).
After exposure to various doses of x-rays under
‘crystallographic’ conditions the edge energy is
shifting to lower energies and the edge shape
transforms into that observed for Mn2+ in
solution (compare A and B).
XANE
S
The EXAFS measurements in panel C show
that this reduction process severely affects the
integrity of the Mn4OxCa cluster. The blue top
trace shows the FT spectrum of the intact
cluster. The second FT peak, which reflects the
bis oxo bridged Mn-Mn interactions at 2.7-2.8 Å
is already reduced significantly after reduction
of 25% Mn to Mn2+ (green trace).
Concomitantly the first peak moves to longer
distances reflecting the conversion of μ-oxo
bridges into terminal water ligands. The red
trace reflects the structure of the Mn4OxCa
complex at the average reduction level of ~70%
that is reached during crystallographic
experiments
Dr. Johannes Messinger, MPI für Bioanorganische Chemie, Mülheim an der Ruhr
http://ewww.mpi-muelheim.mpg.de/bac/mitarbeiter/messinger/messinger_en.php
EXAF
S
XANES simulations of the [CuSMo] active site in CO dehydrogenase:
Most of the structural information derived by XAS is obtained from the oscillatory high-energy part of a XAS
spectrum (EXAFS). However, the structural details obtained are in most cases limited to radial models
because the EXAFS signal is dominated by single scattering processes of the photoelectron after the X-ray
absorption. In contrast, for the absorption edge region of the spectrum (XANES) multiple scattering events
are very important and they depend on the 3D arrangement of the atoms around the excited atom. Using the
program FEFF8, I performed an extensive Mo- and Cu-K-edge XANES analysis for various forms of the
metalloenzyme CODH (unpublished data).
Research of Manuel Gnida
Department of Pediatrics
Stanford University School of Medicine
EXAFS data for Tyrosinase
Biochimica et Biophysica Acta (BBA) - Protein Structure and Molecular Enzymology
Volume 788, Issue 2, 31 July 1984, Pages 155-161
Electron Paramagnetic Resonance (EPR)
or
Electron Spin Resonance (ESR)
a
Ms = +1/2
a
b
No magnetic field
B=0
b
DE = g mb B
where the g-value
gives characteristic info.
DE is microwave region
Ms = -1/2,
antiparallel to B more stable
Magnetic field
B≠0
Resonance
Measurement
EPR spectrometer is:
•constant frequency
o X-band: 9-10 Giga Hertz
(GHz)
o Q-band (high field): 35 GHz
• vary B field (~3500 Gauss)
to bring into resonance;
• DE is absorbed by the sample
when the frequency of the
radiation is appropriate to the
energy difference between two
states of the electrons in the
sample
(10,000 Gauss = 1 Tesla)
Interpreting EPR.1
The Derivative Signal
Interpreting EPR.2
(Nuclear) Hyperfine Coupling
. CH
3
AH
radical
e- localized on C
Hyperfine coupled to 3H (quartet)
AH
. CH (OCH )
2
3
radical
e- localized on C
AH
Larger hyperfine (AH) coupled to 2H
(triplet) and smaller hyperfine (AH) to
3H (quartet)
Interpreting EPR.3
Isotropic vs Anisotropic Spectra
giso
Depends on sample type:
- liquid solution room temp
- frozen solution
- powder
- single crystal (oriented)
Depends on symmetry around metal ion
g||
g
gyy
gxx
cubic,
octahedral,
Oh
tetragonal,
D4h
rhombic,
lower symmetry
The EPR spin Hamiltonian operator with x,y,z tensors:
gzz
Mo EPR Spectroscopy: The First Spectroscopic Technique
Characterizing the Mo site in Enzymes
Mix of Isotopes:
92Mo 15%
94Mo 9%
95Mo 16%
95Mo 17%
97Mo 9%
98Mo 24%
100Mo 10%
92Mo, 94Mo, 96Mo, 98Mo
95Mo
and 100Mo have I = 0, give one hyperfine signal
and 97Mo (total 25%) have I = 5/2, give six hyperfine signals with A(95,97Mo)
Isotropic Mo EPR spectrum
EPR Spectra of Model Complexes. The
EPR spectrum of LMoVO(bdt) (1) exhibits
a rhombic g tensor and an unusual
A(95,97Mo) matrix that consists of two large
components (A1 A3) at the extremes of the
spectrum and one small component in the
center, as shown in Figure 4 and Table 4.
The point group symmetry of a metal
complex determines which metal d orbitals
are allowed to intermix. Such intermixing
will determine whether or not the principal
axes of the g and A(95,97Mo) tensors
coincide. Complexes with no symmetry
elements (C1) or with an inversion center
(Ci) are not required to have any of the
principal g and A(95,97Mo) axes coincident,
whereas complexes with C2, Cs, or C2h
point group symmetry are required to have
one of the principal g and A(95,97Mo) axes
coincident.38-40 In the case of oxomolybdenum(V) complexes of the type
LMoOX2, which closely approximate Cs
symmetry, an Euler angle (30-40) for the
rotation of the g- and A(95,97Mo) )-tensor
elements has typically been observed.38 An
unusual feature of 1 is that the g and A
tensors are nearly coincident in this lowsymmetry (Cs) complex, where such
coincidence between principal g and
A(95,97Mo) ) tensors is not required.
(a) EPR spectra of
LMoO(bdt):
experimental frozensolution X-band
spectrum (top) and
simulated spectrum (I
= 0 component only,
bottom).
(b) EPR spectra of
LMoO(bdt):
experimental
frozen-solution
(top) and simulated
spectrum (I = 5/2
component only,
bottom).
(c) EPR spectra of
LMoO(bdt) (1):
experimental
frozen-solution (top)
and composite
simulated spectrum
(bottom).
Model Spectroscopy
EPR parameters indicate similar Mo
environments in Tp*MoO(S2DIFPEPP)
and Tp*MoO(bdt)
simulation
S
O
N
HN
piv-HN
S
experimental
N
N
F
S
S2DIFPEPP
S
F
bdt
Sample
g1
g2
g3
<g>b
A1
A2
A3
<A>b
c
c
c
hpH SO
1.990
1.966
1.954
1.970
54.4
21.0
11.3
28.9
0
14
22
lpH SO
2.007
1.974
1.968
1.983
56.7
25.0
16.7
32.8
0
18
0
Tp*MoO(S2PEPP)
2.006
1.976
1.936
1.973
46.7
3.3
50.4
33.5
0
0
0
Tp*MoO(S2DIFPEPP)
2.006
1.976
1.936
1.973
47.3
3.3
51.0
33.9
0
0
0
Tp*MoO(bdt)
2.004
1.972
1.934
1.971
50.0
11.4
49.7
37.0
0
0
0
Cu-substituted Alcohol Dehydrogenase
Replacement of the catalytic Zn(II) in horse liver alcohol
dehydrogenase (HLADH) with copper produces a
mononuclear Cu(II) chromophore with a ligand set consisting
of two cysteine sulphurs, one histidine nitrogen plus one
further atom. The fourth ligand to the metal ion and the
conformation of the protein may be altered by addition of
exogenous ligands and/or the cofactor NADH.
The spectra obtained clearly fall into two categories: Figure (A), (B),
(C) and (E), where there is some rhombic distortion with g1 > g2 > g3
and the copper hyperfine splitting of g1 is relatively small (which we
take as evidence for a high copper-thiolate covalence and extensive
ground-state copper–sulphur orbital mixing), and Figure (D), the binary
complex with pyrazole, which is the only truly axial species with g1 >
g2 = g3 and where the copper hyperfine splitting of the g1 line is clearly
much greater. For the binary complex with pyrazole the g (g2, g3) line
is split into eight equally spaced hyperfine lines, which is most easily
explained by equivalent interaction of the electron with both the copper
nucleus and the two nitrogen ligands in the XY plane.
Copper(II) is a 3d9 ion, i.e. it has four filled and one singly occupied 3d
orbitals. Before any analysis of the optical spectrum can be undertaken
it is necessary to establish the nature of the ground-state hole-orbital.
The EPR spectrum shows that the ground state approximates to one of
axial symmetry with g1 > g2 . The g-values and anisotropies of the
Cu(II)-HLADH complexes are not very different from those of typical
blue copper proteins
g3
g2
g1
Pulse EPR and 55Mn-ENDOR Experiments
The chemistry of photosynthetic water oxidation can not be understood without knowing the electronic structure of all
intermediate states. The S2 and S0 states are paramagnetic (S = 1⁄2) and display perpendicular mode EPR signals
(Figure 7).
A direct analysis of the EPR signals involves too many variables and therefore does not lead to satisfying insights
into the electronic structure of the S2 and S0 states. Application of pulse 55Mn-ENDOR spectroscopy allows a
precise determination of the effective isotropic hyperfine interaction parameters (Ai,iso). The experimental spectra
and simulations are shown in Figure 8.
Figure 7: EPR multiline signals of the S0 (top; Messinger et al., Biochemistry 1997, 36, 11055-11060) and the S2 state
(bottom; Dismukes and Siderer, PNAS 1981, 78, 274-278).
S = 1⁄2 on a Mn(3+),Mn(4+) unit, a d4-d3
antiferromagnetically couple dimer.
Each Mn has I- 5/2 (each alone produces 6 lines),
2 Mn produce 16 lines); see p. 309 text)
Proposed S = 5⁄2 state of Mn cluster
Raman Spectroscopy
A scattering technique
 Reveals vibrational levels
 Complementary selection rules to Infrared Spectroscopy
 IR: Ddipole moment, ∫ Yg.s. me Ye.s. dt,

where me has symmetry of x,y,z
 Raman: Dpolarizability moment, ∫ Yg.s.P Ye.s. dt,

where P has symmetry of Rx, Ry, Rz
 Good for aqueous biological samples; no strong O-H absorption

Laser source
Stokes & Anti-Stokes
Resonance Raman (RR) = Raman + electronic spectroscopy
If the wavelength of the exciting laser coincides with an electronic absorption of a
molecule, the intensity of Raman-active vibrations associated with the absorbing
chromophore are enhanced by a factor of 100 to 10,000. This resonance enhancement or
resonance Raman effect can be extremely useful, not just in significantly lowering the
detection limits, but also in introducing electronic selectivity.
RR of [U=O]2+ ion showing
symmetric mode at 835 cm-1 is
dependent on excitation energy
RR gives detailed orbital
and energy information
about two Mo=O model
systems
#1.
#2.
Figure 4. Gaussian resolved electron absorption
spectrum of 1 in acetonitrile, and solid state rR
excitation profiles. These vibrational modes have been
assigned as intraligand vibrations that possess
dominant quinoxoline character (1345 cm-1, red circles)
and C=C + quinoxaline character (1551 cm-1, blue
circles). (Inset) Electron density difference map that
details the nature of the intraligand transition in 1 (red:
electron density loss in transition, green: electron
density gain in transition; H-atoms omitted for clarity).
Magnetic Circular Dichroism (MCD)
Examples of questions that can be answered
* What is the metal center oxidation state and spin state?
* What are the effects of inhibitors/substrate/mutations on the electronic and magnetic
properties of the metal center(s)?
* What are the axial ligands on low-spin ferric heme centers?
Major advantages
* All matter exhibits MCD
* Improved resolution of electronic transitions compared to absorption measurements
* Selective determination of the electronic properties of paramagnetic metal centers via
temperature-dependent studies
* Selective investigation of magnetic properties of individual metal centers via
temperature and magnetic field dependence studies of discrete transitions
Magnetic Circular Dichroism (MCD)
MCD of 2p-3d excitation: In the presence of the applied magnetic field H, there are some
empty down spin 3d states. Only the 2p electrons with down spin can be excited into the 3d
states because of the conservation of spins. When the orbital motion of the 2p states is in the
same direction as the circular motion of the incident light the transition probability is larger,
while when the two motions are in opposite directions the probability is small. As a result the
spectrum shown in the figure (b) is obtained as the difference in the absorption of right- and
left- circularly polarized light (LCP and RCP).
Comparison of MCD Spectrum
and
Absorption Spectrum.
Note additional features of
MCD compared to
absorption spectrum
Note how two MCD have
distinct differences
Whereas Absorption spectra
are nearly identical.
MCD
absorption
Comparison of deconvoluted MCD
and
Resolved Absorption spectra.
Pulse EPR and 55Mn-ENDOR Experiments
The chemistry of photosynthetic water oxidation can not be understood without knowing the electronic structure of all
intermediate states. The S2 and S0 states are paramagnetic (S = 1⁄2) and display perpendicular mode EPR signals
(Figure 7).
A direct analysis of the EPR signals involves too many variables and therefore does not lead to satisfying insights
into the electronic structure of the S2 and S0 states. Application of pulse 55Mn-ENDOR spectroscopy allows a
precise determination of the effective isotropic hyperfine interaction parameters (Ai,iso). The experimental spectra
and simulations are shown in Figure 8.
Figure 7: EPR multiline signals of the S0 (top; Messinger et al., Biochemistry 1997, 36, 11055-11060) and the S2 state
(bottom; Dismukes and Siderer, PNAS 1981, 78, 274-278).
S = 1⁄2 on a Mn(3+),Mn(4+) unit, a d4-d3
antiferromagnetically couple dimer.
Each Mn has I- 5/2 (each alone produces 6 lines),
2 Mn produce 16 lines); see p. 309 text)
Proposed S = 5⁄2 state of Mn cluster
Biochem. J. (1996) 314 (421–426)
Mode of action and active site of an extracellular
peroxidase from Pleurotus ostreatus
Young-Hoon HAN*, Kwang-Soo SHIN†, Hong-Duk YOUN*,
Yung Chil HAH* and Sa-Ouk KANG*‡
Seoul National University, Seoul Korea and †Department of Microbiology, College of
Sciences, Taejon University, Taejon 300-716, Republic of Korea
The properties of the haem environment of a peroxidase
from Pleurotus ostreatus were studied by electronic
absorption spectroscopy. A high-spin ferric form was
predominant in the native enzyme and a high-spin ferrous
form in the reduced enzyme. Cyanide was readily bound to
the haem iron in the native form, thereby changing the
enzyme to a low-spin cyano adduct. Compound III of the
enzyme was formed after the addition of an excess of H2O2
to the native enzyme, and thereafter spontaneously reverted
to the native form. The enzyme oxidized a spin trap (shown
in A) in the presence of H2O2 to produce its radical product.
Free radicals were detected as intermediates of the enzymemediated oxidation of 1-(3,5-dimethoxy-4-hydroxyphenyl)-2(2-methoxyphenoxy)-1,3-dihydroxypropane and
acetosyringone. These results can be explained by the
mechanisms involving an initial one-electron oxidation of the
lignin substructure. This radical may undergo Ca-Cb
cleavage, Ca-oxidation and alkyl-phenyl cleavage.
Figure 4. EPR spectra of the free radicals produced
upon oxidation of (A) the model compound (i) and (C)
acetosyringone by PoP
Model Spectroscopy
EPR parameters indicate similar Mo
environments in Tp*MoO(S2DIFPEPP)
and Tp*MoO(bdt)
simulation
S
O
N
HN
piv-HN
S
experimental
N
N
F
S
S2DIFPEPP
S
F
bdt
Sample
g1
g2
g3
<g>b
A1
A2
A3
<A>b
c
c
c
hpH SO
1.990
1.966
1.954
1.970
54.4
21.0
11.3
28.9
0
14
22
lpH SO
2.007
1.974
1.968
1.983
56.7
25.0
16.7
32.8
0
18
0
Tp*MoO(S2PEPP)
2.006
1.976
1.936
1.973
46.7
3.3
50.4
33.5
0
0
0
Tp*MoO(S2DIFPEPP)
2.006
1.976
1.936
1.973
47.3
3.3
51.0
33.9
0
0
0
Tp*MoO(bdt)
2.004
1.972
1.934
1.971
50.0
11.4
49.7
37.0
0
0
0
Model Spectroscopy
Magnetic Circular Dichroism (MCD) indicates subtle differences between
Tp*MoO(pterin-dithiolene) and Tp*MoO(benzene-dithiolene)
-2000
-1000
same -1700 same -1400
Low temperature (5K)
MCD spectrum of
TpMoO(DIFPEPP) (red).
Gaussian resolved bands
are presented as dashed
lines and the resultant
spectral simulation is
given in blue. Numbers
(cm-1) under peaks
indicate change between
Tp*MoO(S2DIFPEPP) as
compared to Tp*MoO(bdt)
Model Spectroscopy
MCD Band Assignmnets
xy
Gordon Research Conference on Mo & W Enzymes Lucca, Italy 2009
Quinoxalyl Dithiolene model system
From the ML Kirk Lab: Isodensity Density Plots of HOMO & LUMO
LUMO
localized on quinoxaline
Note: asymmetric electron density
on dithiolene
HOMO
localized on Mo d(xy)
Mössbauer Spectroscopy
From: Introduction to Mössbauer Spectroscopy:
http://www.rsc.org/Membership/Networking/InterestGroups/MossbauerSpect/Intropart1.asp
Fig5: Elements of the periodic table which have known
Mössbauer isotopes (shown in red font).
Those which are used the most are shaded with black
Process: gamma radiation from source element identical to that under study is reabsorbed by sample
nuclei.
Measured as isomer shift, , mm/sec and quadrupole splitting, DEq
Process: gamma radiation from an excited source element is reabsorbed by sample nuclei (of same
element) by resonance since the energies of source and sample nuclei match.
However, energy lost to recoil of nuclei prevents resonance and must be corrected. This is
accomplished by putting sample in solid matrix which dampens any movement.
Recoiling nucleus
emitted g ray
Matrix-embedded nucleus, emits g ray
without recoil
Entire process at right:
emitter nucleus emits g ray, absorbed by same type of
nucleus in sample. Detected as decrease in g ray
intensity, shown as descending peak in plot.
Now, want to observe the hyperfine interactions of nucleus environment, a tiny energy perturbation on
the g ray absorption. Likened to: For the most common Mössbauer isotope, 57Fe, this linewidth is 5x10-9ev.
Compared to the Mössbauer gamma-ray energy of 14.4keV this gives a resolution of 1 in 1012, or the
equivalent of a small speck of dust on the back of an elephant or one sheet of paper in the distance between
the Sun and the Earth. (!)
Such miniscule variations of the original gamma-ray are quite easy to achieve by the use of the doppler effect. In
the same way that when an ambulance's siren is raised in pitch when it's moving towards you and lowered when
moving away from you, the gamma-ray source can be moved towards and away from the absorber. This is most
often achieved by oscillating a radioactive source with a velocity of a few mm/s and recording the spectrum in
discrete velocity steps. Fractions of mm/s compared to the speed of light (3x1011mm/s) gives the minute energy
shifts necessary to observe the hyperfine interactions. For convenience the energy scale of a Mössbauer spectrum
is thus quoted in terms of the source velocity, as shown in Fig1.
Mossbauer epctroscpy is threfore
measured as isomer shift, , mm/sec.
57Fe
Mossbauer most useful in bioinorganic for
oxidation state and spin state identification.
Note that this requires 57Fe site labeling.
Fe(2+) high spin — ~1.3 mm/sec
Fe(2+) low spin — ~0.1 mm/sec
Resonance peak at 0 m/sec
when source identical to sample
Fe(3+) high spin — ~0.5-0.7
mm/sec
Fe(3+) low spin — ~0 mm/sec
Nuclei in states with an angular momentum quantum number I>1/2 have a non-spherical charge
distribution. This produces a nuclear quadrupole moment. In the presence of an asymmetrical electric
field (produced by an asymmetric electronic charge distribution or ligand arrangement) this splits the
nuclear energy levels.
Quadrupole splitting, measured as DEq in mm/sec, indicates 57Fe site symmetry
We utilized this apparent enhanced lability of one iron of the [4Fe-4S]
cluster to achieve site-specific labeling of the unique site with 57Fe (above).
After the [4Fe-4S]-PFL-AE had been exposed to oxidant, the released iron
was removed by gel filtration chromatography and the [3Fe-4S]+ formed
was quantified by EPR spectroscopy. An equimolar equivalent of 57Fe(II)
and a small excess of dithiothreitol (DTT) was then added, and the resulting
protein, which was EPR-silent, was examined by Mössbauer spectroscopy
in the absence and presence of S-adenosylmethionine SAM (Figure 8).
The results show that the added 57Fe(II) is incorporated into the cluster, as
spectrum A is a typical quadrupole doublet for iron in a [4Fe-4S]2+ cluster (
= 0.42 mm/s, EQ = 1.12 mm/s). The Mössbauer spectrum is dramatically
perturbed, however, upon addition of SAM, as shown by spectrum B and
the difference spectrum C in Figure 8. A new quadrupole doublet appears
with parameters ( = 0.72 mm/s, EQ = 1.15 mm/s) that are inconsistent with
the typical iron environment in a [4Fe-4S]2+ cluster and suggest an
increase in coordination number and/or binding of more ionic ligands to the
unique site iron.80 Significantly, when a [357Fe-4S]+ cluster is generated in
57Fe -enriched PFL-AE and natural-abundance Fe(II) and DTT are added,
no perturbation of the Mössbauer spectrum is observed upon addition of
SAM, consistent with the selective binding of the added iron to the unique
site. These results clearly demonstrated for the first time the presence of a
unique iron site in the [4Fe-4S] cluster of PFL-AE and provided evidence for
interaction of SAM with the unique iron site.
Figure 8 Mössbauer spectra of PFL-AE
site-specifically labeled at the unique iron
site with 57Fe. (A) 356Fe157Fe4S]2+ in
the absence of SAM. (B)
[356Fe157Fe4S]2+ in the presence of
SAM. (C) Difference spectrum B - A. (D)
Difference spectrum of spectra recorded
at high field.