epr

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ElectronElectronParamagneticParamagneticResonanceResonance

E. DuinE. Duin

2Paper:

‘‘EPR spectroscopy as a probe of metal centres in biological EPR spectroscopy as a probe of metal centres in biological systems’systems’

Dalton Trans. (2006) 4415-4434Dalton Trans. (2006) 4415-4434

Wilfred R. HagenWilfred R. Hagen

Fred Hagen completed his PhD on EPR of metalloproteins at the University of Amsterdam in 1982 with S.P.J. Albracht and E.C. Slater.

3A Free Electron A Free Electron in Vacuoin Vacuo

Free, unpaired electron in space:electron spin - magnetic moment

4A Free Electron in a Magnetic FieldA Free Electron in a Magnetic Field

5A Free Electron in a Magnetic FieldA Free Electron in a Magnetic Field

+½: E = +½geβB0

-½: E = -½geβB0

ΔE = geβB0

β: Bohr magentonB0: magnetic fieldge: g value

For a free electron:g = ge = 2.00232

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Resonance condition: ΔE = hν = geβB0

When the electron is bound to one, or more nuclei, then a virtual observer on the electron would experience the nucleus (nuclei) as an orbiting positive charge producing a second magnetic field, δB, at the electron.

hν = geβ(Be + δB)

Since only the spectrometer value of B is known:

hν = (ge + δg)βB = gβB

The quantity ge + δg contains the chemical information on the nature of the bond between the electron and the molecule, the electronic structure of the molecule.

Spin-orbit CouplingSpin-orbit Coupling

7Spin-orbit CouplingSpin-orbit Coupling

Example: compound with axial paramagnetic anisotropy.This will have a different g value for different orientations dependent on the alignment of B along the z axis or the y or x axes.

8Powder SpectrumPowder Spectrum

A sample of realistic size consists of randomly oriented molecules, resulting in a so-called ‘powder spectrum’.

In the example of the compound with axial paramagnetic anisotropy, the spectrum has axial EPR absorption.

(Higher chance of having the B vector anywhere in the xy plane than parallel to the z axis.)

9Line Shape of EPR SpectraLine Shape of EPR Spectra

10Hyperfine InteractionsHyperfine Interactions

Interactions of the electron spin with the nuclear spin of the metal ion nucleus or first coordinate sphere ligands nuclei or other electron spins within 10 Ǻ distance cause additional splitting.

11Hyperfine InteractionsHyperfine Interactions

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• Bio transition metal nuclear spins (I): 2 I + 1 EPR lines(Called hyperfine structure)

I = 0 → 1 line

I = ½ → 2 lines

I = 1 → 3 lines

I = 3/2 → 4 lines

Hyperfine InteractionsHyperfine Interactions

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• Bio transition metal nuclear spins

• The spin-orbit coupling parameter is positive (g<ge) for systems with less than half filled outer shells and negative (g>ge) for those with more than half filled shells (Generally!)

• With redox state is EPR active?• How many unpaired electrons present?

Type Identification - MetalsType Identification - Metals

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Hydrogenase – gxyz = 2.32, 2.24, 2.01

Methyl-coenzyme-M reductasegxyz = 2.252, 2.073, 2.064


Ni3+, d7

Ni1+, d9

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Hydrogenase (A, B) and methyl-coenzyme-M reductase (C, D) from Methanothermobacter marburgensis grown on different isotope mixtures:

(A) growth was performed with natural Ni (natural abundance of 61Ni is 1.19%);

(B) growth in the presence of 61Ni (I = 3/2)

(C) natural Ni(D) growth in the presence of

61Ni.

A

B

C

D


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Methyltransferase from M. marburgensis. (A) Protein as isolated. (B) Computer simulation. gxyz = 2.2591, 2.2530, 2.00659

A

B

Co+, d7, I = 7/2


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Vanadium-containing chloroperoxidase from the fungus Curvularia inaqualis

V4+, d1, I = 7/2(g// = 1.95 and g = 1.98)


18Type Identification - MetalsType Identification - Metals

A

B

C

D

Methanobacterium wolfei formyl-methanofuran dehydrogenase (FDH I) isolated from cells grown on molybdate(A) Two signals with gxyz = 2.003, 1.989,

1.955 and gxyz = 2.00, 1.984, 1.941(B) Cells grown in the presence of 97Mo-

molybdate (I = 5/2).FDH II from cells grown on tungstate.(C) gxyz = 2.0488, 2.0122, 1.9635.(D) Simulation of C based on the natural

abundance of the tungsten isotopes:I = 0: 180W, 0.14%; 182W, 26.4%; 184W, 28.4% and I = 1/2: 183W, 14.4%.

Mo5+, d1

W5+, d1

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Bio ligand atom nuclear spins and their EPR superhyperfine patterns

Identification of LigandsIdentification of Ligands

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No interaction

1 x S=1/2

2 x S=1/2

3 x S=1/2

4 x S=1

Identification of LigandsIdentification of Ligands

21Identification of LigandsIdentification of Ligands

Free electron in dx2-y2 orbital Free electron in dz2 orbital

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20-70 K

4-10 K

4-20 K

4-10 K

[2Fe-2S]1+ S = ½

[2Fe-2S]2+ S = 0

[3Fe-4S]0 S = 2

[3Fe-4S]1+ S = ½

[4Fe-4S]1+ S = ½

[4Fe-4S]2+ S = 0

(HiPIP)[4Fe-4S]2+ S = 0

[4Fe-4S]3+ S = ½

Type Identification – Iron-sulfur ClustersType Identification – Iron-sulfur Clusters

23Type Identification - MetalsType Identification - Metals

Cu2+, d9, = 3/2

Mn2+, d7, I = 5/2(S = 5/2)

24EPR SpectrometerEPR Spectrometer

25The Need for Lower TemperaturesThe Need for Lower Temperatures

EPR frequencies (1-100 GHz) are in the microwave range!

Aqueous solutions will warm up in the EPR cavity at RT!

Do-it-yourself microwave source

26The Need for Lower TemperaturesThe Need for Lower Temperatures

The energy difference between the two energy level due to the Zeeman splitting is very small, ~0.3 cm-1 forX-band EPR.

Based on the Boltzmann distribution

kTEΔ

01 ennit can be shown that only at low temperatures there will be enough difference in the population of the S = -1/2 level (n0) and the S = ½ level (n1) to create a signal.

27Spin-lattice RelaxationSpin-lattice Relaxation

EPR on metalloproteins:• the relaxation rate decreases with decreasing

temperature; and• the relaxation rate is anisotropic (i.e. is different for

different parts of the spectrum).

When too much power is applied the signal will saturate: Power saturation!

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Due to the uncertainty principle the EPR spectra will broaden beyond detection at higher temperatures. At lower temperatures the spectra will sharpen up.

This sharpening up of the spectrum by cooling the sampleis, however, limited by a temperature-independent process:inhomogeneous broadening.

The protein or model molecules in dilute frozen solutions are subject to a statistical distribution in conformations, each with slightly different 3D structures and, therefore, slightly different g values, which manifest themselvesas a constant broadening of the EPR line independent of the temperature.

Heisenberg Uncertainty PrincipleHeisenberg Uncertainty Principle

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Optimal measuring conditions (T,P) are determined by the interplay of the Boltzmann distribution, the Heisenberg uncertainty relation, the spin–lattice relaxation rate, and the conformational distribution of molecular structure.

How do I find the correct measuring condition?1) Make a Curie Plot2) Make Power Plots

What to do?What to do?TEMPERATUREBROADENING

POWERSATURATION

OPTIMALCONDITIONS

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• The power in EPR is expressed in decibels (dB) attenuation

• X-band microwave sources have a constant output that is usually leveled off at 200 mW (= 0 dB):

P(dB) = −10 × log(0.2/P(W))

• logarithmic scale: every 10 dB attenuation means an order-of-magnitude reduction in power.

• A good X-band bridge operates at power levels between 0 and 60 dB

Power PlotsPower Plots

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Both power and gain scales are logarithmic!

Need for low temperatures and high power, but this could lead to power saturation!

Practical rule: the amplitude of a non-saturated EPR signal does not change if a reduction in power by 1 dB is compensated by an increase in gain by one step.

Power PlotsPower Plots

Relationship between the amplitude, gain and the power in dB:

constant10gainamplitude

20dB

32Power Plot (Copper Perchlorate)Power Plot (Copper Perchlorate)

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• The relaxation rate decreases with decreasing temperature.

• If the signal is not broadened it should be easier to find a power that does not saturate the signal at higher temperatures.

• This ‘temperature behavior’ or ‘Curie behavior’ will be different for different species.

Power Plot (Copper Perchlorate)Power Plot (Copper Perchlorate)

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In, normalized value for the intensity; I0, observed intensity; T, absolute temperature in K; dB, reading of the attenuator; gain, gain

Curie Plot (Copper Perchlorate)Curie Plot (Copper Perchlorate)

35Curie PlotCurie Plot

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To calculate the amount of signal in a protein sample, the spin intensity can be compared with that of a standard with a known concentration (Copper perchlorate: 10 mM)

Since an EPR spectrum is a first derivative, we have to integrate twice to obtain the intensity (I0 = area under the absorption spectrum).

In addition, corrections are needed for a number of parameters, to ‘normalize’ the spectra. Only then a direct comparison of double integral values of standard and unknown is possible:

Signal Intensity/Spin countingSignal Intensity/Spin counting

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a)fg10TdI ( I av

p

20dB20

n

()

933

2 zyx2z

2y

2xav

p

ggggggg

whereIn normalized double integralI0 observed intensityd distance between the starting and ending points (in

Gauss)T absolute temperature in KdB reading of the attenuatorf tube calibration factora gain

and

Normalized Signal IntensityNormalized Signal Intensity

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n(st)

stn(x)x

avp

20dB20

n

ICI

C

a)fg10TdI ( I

()

Using a ‘Spin’ StandardUsing a ‘Spin’ Standard

• Keep measuring conditions the same: temperature, modulation amplitude, sweep time, amount of points, amount of scans (These are not averaged!)

• Measures samples on the same day.• Correct for spin: S(S+1)

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Half-integer/non-Kramers• S = 3/2, 5/2, 7/2, 9/2• All systems detectable in perpendicular-mode EPR

Integer/Kramers• S = 1, 2, 3, 4• Detection in parallel-mode EPR• In biochemistry only relevant for S = 2 systems of

[3Fe-4S]1+ and Heme-Fe2+

High-spin SystemsHigh-spin Systems

40Examples for FeExamples for Fe3+3+//SS = 5/2 = 5/2

Rubredoxin(Photosystem II)

KatG

41Energy Levels for S = 5/2 SystemEnergy Levels for S = 5/2 System

• Zero-field splitting effects in S = 5/2 systems with a zero field splitting parameter (D) that is large compared to the microwave frequency.

• Only intra-doublet transitions observed in EPR.

|mS = ±1/2>

|mS = ±3/2>

|mS = ±5/2>

42RhombogramRhombogram

E/D = 0.315

S = 1/2S = 3/2

|mS = ±1/2>

|mS = ±3/2>

|mS = ±5/2>4.5 K

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|mS = ±1/2>

|mS = ±3/2>

|mS = ±5/2>

Axial component(s)E/D = 0.00

Rhombic componentE/D = 0.03

Rhombogram & SimulationsRhombogram & Simulations

–– spectrum–– simulation

10 K

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x

y

z

45SimulationsSimulations

Important when more than one signal is present, the

signal intensity is too low, or the baseline is not linear.

Spectrum

Simulation

Difference

46Signal Intensity???Signal Intensity???

Clostridiumpasteurianum

[2Fe-2S]2+

S = 9/2

(D < 0)

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The effective spin-Hamiltonian suggests an easy way for quantification of high-spin spectra: one simply applies the double-integration procedure to the effective Seff = 1/2 spectrum as if it were a real S = 1/2 spectrum, however, with a correction for the fractional population of the relevant doublet. (Most of the time not possible!)

Exception: For high spin ferric hemoproteins (D ≈ +10 cm−1) in X-band at T = 4.2 K the fractional population of the |mS = ±1/2> doublet is very close to unity (0.999) therefore, quantification of the spectrum does not require a depopulation correction.

(Correction for spin needed S(S+1))

Signal Intensity???Signal Intensity???

epr

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b cells

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fe4s0 s

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b methyltransferase