Physical Methods In Bioinorganic Chemistry 1. X-ray spectroscopy: EXAFS, XANES 2. Resonance...

Physical Methods In Bioinorganic Chemistry

1. X-ray spectroscopy: EXAFS, XANES2. Resonance spectroscopy

Electron paramagnetic resonance - EPRPulsed EPR: ESEEM, ENDORResonance Raman - RR

3. Magnetic SpectroscopyMagnetic Circular Dichroism

4. Mossbauer Spectroscopy


1. 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, conformations2. Resonance spectroscopy

• Electron paramagnetic resonance - EPR• Pulsed EPR: ESEEM, ENDOR• Resonance Raman - RR





Electron paramagnetic resonance - EPRGives info on metal identity, donor atoms,

and 2nd sphere atomsSome info on bonding characterPulsed EPR: ESEEM, ENDORResonance Raman - RR





Electron paramagnetic resonance - EPRPulsed EPR: ESEEM, ENDORResonance Raman - RRGives info on vibrations and bond orderReveals coupled electronic and vibrational states






3. Magnetic SpectroscopyMagnetic Circular DichroismCorrelates e- transitions and MO’s by symmetry






4. Mossbauer SpectroscopyGives oxidation state of Fe ions

Usefulness?

Inorganic Chemistry Vol. 44, No. 4: February 21, 2005"Functional Insight from Physical Methods on Metalloenzymes"Edward I. Solomonpp 723 - 726;

XAS techniques:Get your bearingsin energy:



• Electron paramagnetic resonance - EPR• Pulsed EPR: ESEEM, ENDOR• Resonance Raman - RR

3. Magnetic Spectroscopy• Magnetic Circular Dichroism

4. Mossbauer SpectroscopyGives 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:

Energy needed to eject core electron

EXAFS “Ripples” from interference of neighbors

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.

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

http://www.haverford.edu/chem/Scarrow/EXAFS123/Plotting%20Graphs.htm

http://www.haverford.edu/chem/Scarrow/EXAFS123/FITTING.htm

Can’t do this at home; requires an intense X-ray source-> Synchrotron Radiation

1. SSRL: Stanford Synchrotron Radiation LabThe 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 high-brilliance 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.

http://en.wikipedia.org/wiki/Synchrotron

http://www-ssrl.slac.stanford.edu/

Mn-O ~ 1.4 Å Mn-O ~ 2.2 Å

Mn-Mn ~ 3.0 Å

Mn-Ca ~ 3.0 Å

Various Intramoecular Distances in theTetra-Mn clusterof 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).

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

XANES

EXAFS

Dr. Johannes Messinger, MPI für Bioanorganische Chemie, Mülheim an der Ruhr

http://ewww.mpi-muelheim.mpg.de/bac/mitarbeiter/messinger/messinger_en.php

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 GnidaDepartment of PediatricsStanford University School of Medicine

EXAFS data for TyrosinaseBiochimica et Biophysica Acta (BBA) - Protein Structure and Molecular EnzymologyVolume 788, Issue 2, 31 July 1984, Pages 155-161

Electron Paramagnetic Resonance (EPR)or

Electron Spin Resonance (ESR)

Ms = -1/2,antiparallel to B more stable

Ms = +1/2

E = g b B

where the g-valuegives characteristic info.E is microwave regionNo magnetic field

B = 0

Magnetic fieldB ≠ 0

Resonance Measurement

EPR spectrometer is:

•constant frequencyo X-band: 9-10 Giga Hertz (GHz)o Q-band (high field): 35 GHz

• vary B field (~3500 Gauss) to bring into resonance;• E 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.1The Derivative Signal

Interpreting EPR.2(Nuclear) Hyperfine Coupling

. CH3 radical

e- localized on C

Hyperfine coupled to 3H (quartet)

. CH2(OCH3) radical

e- localized on C

Larger hyperfine (AH) coupled to 2H (triplet) and smaller hyperfine (AH) to 3H (quartet)

AH

AH

AH

Interpreting EPR.3Isotropic vs Anisotropic Spectra

Depends on sample type:

- liquid solution room temp- frozen solution- powder - single crystal (oriented)

Depends on symmetry around metal ion

cubic,octahedral,Oh tetragonal,

D4h

rhombic,lower symmetry

The EPR spin Hamiltonian operator with x,y,z tensors:

giso

g||

g

gxx

gyy

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 and 100Mo have I = 0, give one hyperfine signal

95Mo 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 oxo-molybdenum(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 low-symmetry (Cs) complex, where such coincidence between principal g and A(95,97Mo) ) tensors is not required.

(a) EPR spectra of LMoO(bdt):

experimental frozen-solution 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).

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

EPR parameters indicate similar Mo environments in Tp*MoO(S2DIFPEPP) and Tp*MoO(bdt)

S S

bdt

S

SN

N

HN

N

O

piv-HN F

F

S2DIFPEPP

simulation

experimental

Cu-substituted Alcohol DehydrogenaseReplacement 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

g1g2g3

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: dipole moment, ∫ g.s. e e.s. dt, where me has symmetry of x,y,z Raman: polarizability moment, ∫ g.s.P e.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

isdependent on excitation

energy

RR gives detailed orbital and energy information about two Mo=O model systems #1.

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).

#2.

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 andAbsorption Spectrum.

Note additional features of MCD compared to absorption spectrum

MCD

absorption

Note how two MCD have distinct differences

Whereas Absorption spectra are nearly identical.

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

Figure 4. EPR spectra of the free radicals produced Figure 4. EPR spectra of the free radicals produced upon oxidation of (A) the model compound (i) and (C) upon oxidation of (A) the model compound (i) and (C)

acetosyringone by PoP acetosyringone by PoP

Biochem. J. (1996) 314 (421–426) Mode of action and active site of an extracellular peroxidase from Pleurotus ostreatusYoung-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 enzyme-mediated 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.

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

EPR parameters indicate similar Mo environments in Tp*MoO(S2DIFPEPP) and Tp*MoO(bdt)

S S

bdt

S

SN

N

HN

N

O

piv-HN F

F

S2DIFPEPP

simulation

experimental

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

-2000 -1700 same-1000 same -1400

Magnetic Circular Dichroism (MCD) indicates subtle differences between Tp*MoO(pterin-dithiolene) and Tp*MoO(benzene-dithiolene)

Model Spectroscopy

xy

MCD Band Assignmnets

Quinoxalyl Dithiolene model system

HOMOlocalized on Mo d(xy)

LUMOlocalized on quinoxaline

Note: asymmetric electron density on dithiolene

From the ML Kirk Lab: Isodensity Density Plots of HOMO & LUMO

Gord

on

Rese

arc

h C

onfe

ren

ce o

n M

o &

W E

nzy

mes

Lucc

a,

Italy

200

9

Mössbauer SpectroscopyFrom: 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, Eq

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 ray Matrix-embedded nucleus, emits raywithout recoil

Entire process at right:emitter nucleus emits ray, absorbed by same type of nucleus in sample. Detected as decrease in rayintensity, shown as descending peak in plot.

Now, want to observe the hyperfine interactions of nucleus environment, a tiny energy perturbation on the 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.

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/secFe(2+) low spin — ~0.1 mm/sec Fe(3+) high spin — ~0.5-0.7 mm/sec Fe(3+) low spin — ~0 mm/sec

Resonance peak at 0 m/sec when source identical to sample

Mossbauer epctroscpy is threfore measured as isomer shift, , mm/sec.

Quadrupole splitting, measured as Eq in mm/sec, indicates 57Fe site symmetry

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.

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.

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.

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Physical Methods In Bioinorganic Chemistry 1. X-ray spectroscopy: EXAFS, XANES 2. Resonance...

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