Wilfred R. Hagen Introduction to Biomolecular Electron ...duinedu/summercourse/eprslides.pdf · Introduction to Biomolecular Electron Paramagnetic Resonance Theory E.C. Duin 1

1

Introduction to

Biomolecular

Electron

Paramagnetic

Resonance

Theory

E.C. Duin

1

EPR, the Technique….

• Molecular EPR spectroscopy is a method to look at the

structure and reactivity of molecules.

• EPR is limited to paramagnetic substances (unpaired

electrons). When used in the study of metalloproteins not the

whole molecule is observed but only that small part where the

paramagnetism is located.

• This is usually the central place of action – the active site of

enzyme catalysis.

• Sensitivity: 10 μM and up.

• Naming: Electron paramagnetic resonance (EPR), electron

spin resonance (ESR), electron magnetic resonance (EMR)

2

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.

Wilfred R. Hagen

Paper:

EPR spectroscopy as a probe of metal centres in

biological systems (2006) Dalton Trans. 4415-4434

Book:

Biomolecular EPR Spectroscopy (2009)

3

Spectral Simulations

• The book ‘Biomolecular EPR spectroscopy’ comes with a

suite of programs for basic manipulation and analysis of EPR

data which will be used in this class.

Simple Spectrum

Hyperfine Spectrum

Isotropic Radicals

EPR File Converter

EPR Editor

Visual Rhombo

Single Integer Signal

GeeStrain-5

4

2

Discovery

E.K. Zavoisky’s first EPR system

First EMR on CuCl2.2H2O

4.76mT @ 133MHz

In 1944, E.K. Zavoisky discovered magnetic

resonance. Actually it was EPR on CuCl2.

5

EPR Theory

A Free Electron in Vacuo

Free, unpaired electron in space:

electron spin - magnetic moment6

A Free Electron in a Magnetic Field

• An electron with spin S = ½ can have two orientations in a

magnetic field B0 labeled by mS = +½ or mS = −½.

• The unpaired electron will have a state of lowest energy when

the moment of the electron is aligned with the magnetic field

and a state of highest energy when aligned against the

magnetic field.7


• The energy of each orientation is the product of µ and B0. For

an electron µ = msgeβ, where β is a conversion constant

called the Bohr magneton and ge is the spectroscopic g-factor

of the free electron and equals 2.0023192778 (≈ 2.00).

Therefore, the energies for an electron with ms = +½ and ms =

-½ are, respectively: ½ geβB0 and -½ geβB08

3


The electronic-Zeeman energies are E = mSgβB

+½: E = +½geβB0

-½: E = -½geβB0

∆𝐸 = ℎ𝜈 = 𝑔𝑒𝛽𝐵0

The electron can change orientation

by absorbing electromagnetic

radiation which energy should exactly

equal the state energy difference ΔE,

and this defines the resonance

condition:

9


Two fundamental constants:

- Planck’s constant: h

- Bohr magneton: β

Two experimental parameters:

- Microwave frequency: ν

- Magnetic field: B0

ℎ𝜈 = 𝑔𝑒𝛽𝐵0

A constant of proportionality: g-value

Property of matter, for the free electron: g = ge = 2.00232

10

Spin-orbit Coupling

Resonance condition: 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 g = ge + δg contains the chemical information on the

nature of the bond between the electron and the molecule, the

electronic structure of the molecule.

The value of g can be taken as a fingerprint of the molecule.

11

Anisotropy

Example: compound with axial paramagnetic anisotropy. This will

have a different dg-value for different orientations dependent on

the alignment of B0 along the z-axis or the y- or x-axes.

Anisotropy: the fact that molecular

properties, such as δg are angular

dependent and reflect the 3D

electronic structure of the paramagnet.

12

4

Powder 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.)

13

Angular Dependency of g-Value

Defining the orientation of the magnetic field (a vector) with

respect to the coordinates of the molecule (and vice versa).

Two polar angles, θ and ϕ, where θ is the angle between the

vector B and the molecular z-axis, and ϕ is the angle between

the projection of B onto the xy-plane and the x-axis.14


Inclusion of the angular terms into the resonance equation

gives:

ℎ𝜈 = 𝑔 𝜃, 𝜙 𝛽𝐵0

A spin Hamiltonian that considers any arbitrary orientation

of B relative to g is given by:

𝐻 = 𝛽𝐵𝑇 ∙ 𝑔 ∙ 𝑆

With 𝑆 being the Pauli spin operator vector with individual

components 𝑆𝑥, 𝑆𝑦, and 𝑆𝑧.

15


B can be aligned along any of the principle g tensor axes

and the individual components of B are defined in terms of

the polar angles (θ, φ).

Bx = B sinθ cosϕ, By = B sinθ sinϕ, and Bz = B cosθ

Filling in these values provides a more detailed matrix

representation of the spin Hamiltonian equation:

𝐻 = 𝛽 ∙ 𝐵𝑥 𝐵𝑦 𝐵𝑧 ∙

𝑔𝑥𝑥 0 00 𝑔𝑦𝑦 0

0 0 𝑔𝑧𝑧

∙

𝑆𝑥

𝑆𝑦

𝑆𝑧

16

5


Combining with the polar angles equations for the

individual components of B:

𝐻 = 𝛽(𝐵𝑠𝑖𝑛𝜃𝑐𝑜𝑠𝜙 ∙ 𝑔𝑥𝑥 ∙ 𝑆𝑥 + 𝐵𝑠𝑖𝑛𝜃𝑠𝑖𝑛𝜙 ∙ 𝑔𝑦𝑦 ∙ 𝑆𝑦 + 𝐵𝑐𝑜𝑠𝜃 ∙ 𝑔𝑧𝑧 ∙ 𝑆𝑧)

The effect of the 𝑆𝑥, 𝑆𝑦, 𝑆𝑧 operators can be represented by

the 2 × 2 Pauli spin matrices quantized in units of ℏ as:

𝑆𝑥 =1

20 11 0

, 𝑆𝑦 =1

20 −𝑖−𝑖 0

, 𝑆𝑧 =1

21 00 −1

17


The matrix of the Hamiltonian has the form:

𝐻 = 𝛽1

2𝐵

𝑔𝑧𝑧𝑐𝑜𝑠𝜃 𝑔𝑥𝑥𝑠𝑖𝑛𝜃𝑐𝑜𝑠𝜙 + 𝑖𝑔𝑦𝑦𝑠𝑖𝑛𝜃𝑠𝑖𝑛𝜙

𝑔𝑥𝑥𝑠𝑖𝑛𝜃𝑐𝑜𝑠𝜙 − 𝑖𝑔𝑦𝑦𝑠𝑖𝑛𝜃𝑠𝑖𝑛𝜙 −𝑔𝑧𝑧𝑐𝑜𝑠𝜃

The two resulting eigenvalues are:

𝐸1 = −1

2𝛽𝐵 𝑔𝑥𝑥

2 𝑠𝑖𝑛2𝜃𝑐𝑜𝑠2𝜙 + 𝑔𝑦𝑦2 𝑠𝑖𝑛2𝜃𝑠𝑖𝑛2𝜙 + 𝑔𝑧𝑧

2 𝑐𝑜𝑠2𝜃

𝐸2 =1

2𝛽𝐵 𝑔𝑥𝑥

2 𝑠𝑖𝑛2𝜃𝑐𝑜𝑠2𝜙 + 𝑔𝑦𝑦2 𝑠𝑖𝑛2𝜃𝑠𝑖𝑛2𝜙 + 𝑔𝑧𝑧

2 𝑐𝑜𝑠2𝜃

18


For resonance absorption to occur:

𝐸1 − 𝐸2 = ℎ𝜈 = 𝑔 𝜃, 𝜙 𝛽𝐵0

As a result the value of g(θ, φ) now becomes:

𝑔 𝜃, 𝜙 = 𝑔𝑥𝑥2 𝑠𝑖𝑛2𝜃𝑐𝑜𝑠2𝜙 + 𝑔𝑦𝑦

2 𝑠𝑖𝑛2𝜃𝑠𝑖𝑛2𝜙 + 𝑔𝑧𝑧2 𝑐𝑜𝑠2𝜃

This equation gives the effective anisotropic g value for any

orientation (θ, φ) with respect to the applied magnetic field

B.

19


For axial spectra: 𝑔𝑎𝑥 𝜃 = 𝑔𝑥𝑦2 𝑠𝑖𝑛2𝜃 + 𝑔𝑧

2𝑐𝑜𝑠2𝜃

Plot of the angle θ and the axial g-value versus the

resonance field (gz = 2.050 and gxy = 2.002, ν = 9500 MHz)20

6

Line Shape of EPR Spectra

Note: g// = gz and g = gxy21

g-Value

Two fundamental constants:

- Planck’s constant: h

- Bohr magneton: β

For X-band:

ℎ𝜈 = 𝑔𝛽𝐵0

𝑔 = 0.7145𝜈 (𝑀𝐻𝑧)

𝐵 (𝐺𝑎𝑢𝑠𝑠)

22

Correlation Between Line Shape and Structure

EPR symmetry g and A tensors Molecular point

symmetry

Isotropic gxx = gyy = gzz

Axx = Ayy = Azz

Oh, Td, O, Th, T

Axial gxx = gyy ≠ gzz

Axx = Ayy ≠ Azz

D4h, C4v, D4, D2d, D6h,

C6v, D6, D3h, D3d, C3v,

D3

Rhombic gxx ≠ gyy ≠ gzz

Axx ≠ Ayy ≠ Azz

D2h, C2v, D2

23

Correlation Between Line Shape and Structure

• Does the shape of the EPR signal, ‘isotropic’, ‘axial’, or

‘rhombic’ reflect the symmetry of a coordination site in a

metalloprotein?

• Most of the time the answer is: No!

• Example: the Cu(II) spectrum of plastocyanin is virtually axial

(gx ≈ gy) even when recorded at higher frequency for

increased g-value resolution.

• Crystallographic analysis, however, reveals a highly distorted

tetrahedral site essentially with no symmetry at all!

24

7

S=1/2 Systems

25

+½: E = +½geβB0

-½: E = -½geβB0

Resonance condition:


For X-band:

𝒈 = 𝟎. 𝟕𝟏𝟒𝟓𝝂 (𝑴𝑯𝒛)

𝑩 (𝑮𝒂𝒖𝒔𝒔)

What is this !?!

26

Hyperfine Interactions

Additional splitting can be observed in EPR

signals:

Hyperfine interaction

Interaction of the electron spin with the nuclear

spin of the metal ion nucleus

Super hyperfine interaction

Interaction of the electron spin with first

coordinate sphere ligands nuclei

Spin-spin interaction

Interaction of the electron spin with other

electron spins within 10 Å distance.

27


• Interaction of the electron spin (S = ½) with the nuclear spin

of the metal ion nucleus (I = ½)

• Four different situations: Four new energy levels28

8


• With the four new energy

levels there are two field

positions where the

resonance conditions are

met: |Δms| = 1, |ΔmI| = 0

• The signal is in principle

split in two.

• The original, unsplit, peak

would have been exactly

in the middle of the two

hyperfine lines.

29

Quantum Mechanical Description

• A full quantum mechanical description of the

spectroscopic EPR event is not possible due to the

complexity of the systems under study.

• In Biomolecular EPR spectroscopy we use the concept

of the spin Hamiltonian. This describes a system with

an extremely simplified form of the Schrödinger wave

equation that is a valid description only of the lowest

electronic state of the molecule plus magnetic

interactions.

𝐻𝑠𝜓𝑠 = 𝐸𝜓𝑠

With: Hs, spin Hamiltonian; ys, the spin functions; E,

energy values of the ground state spin manifold.30


• For an isolated system with a single unpaired electron

and no hyperfine interaction the only relevant interaction

is the electronic Zeeman term, so the spin Hamiltonian is

𝐻𝑠 = 𝛽𝐵𝛽(𝑠𝑖𝑛𝜃𝑐𝑜𝑠𝜙 ∙ 𝑔𝑥𝑥 ∙ 𝑆𝑥 + 𝑠𝑖𝑛𝜃𝑠𝑖𝑛𝜙 ∙ 𝑔𝑦𝑦 ∙ 𝑆𝑦 + 𝑐𝑜𝑠𝜃 ∙ 𝑔𝑧𝑧 ∙ 𝑆𝑧)

A shorter way of writing this is

𝐻𝑠 = 𝛽𝐵 • 𝑔 • 𝑆

Solving this we get the equation we saw earlier for the

angular dependency of the g-value

𝑔 𝜃, 𝜙 = 𝑔𝑥𝑥2 𝑠𝑖𝑛2𝜃𝑐𝑜𝑠2𝜙 + 𝑔𝑦𝑦

2 𝑠𝑖𝑛2𝜃𝑠𝑖𝑛2𝜙 + 𝑔𝑧𝑧2 𝑐𝑜𝑠2𝜃

31


• More terms can be added to the Hamiltonian when

needed.

• For hyperfine interactions Hs becomes

𝐻𝑠 = 𝛽𝐵 • 𝑔 • 𝑆 + 𝑆 • 𝐴 • 𝐼

where A is the anisotropic hyperfine tensor.

• For multi-electron (high-spin) systems Hs becomes

𝐻𝑠 = 𝛽𝐵 • 𝑔 • 𝑆 + 𝑆 • 𝐷 • 𝑆

where D is the zero-field interaction.

• When both are present, both terms will have to be

added!

32

9


• These simplified wave equations will sometimes, under

strict conditions, give analytical solutions.

• It is important to realize that a lot of the tools and

simulations software used in biomolecular EPR

spectroscopy can only be used when certain conditions

are met.

• In most systems we will encounter, we can use these

tools without any problem. There are specific cases,

however, where we cannot use these tools.

33


• Hyperfine interaction: 𝐻𝑠 = 𝛽𝐵 • 𝑔 • 𝑆 + 𝑆 • 𝐴 • 𝐼

• Assumption 1: the Zeeman interaction is much larger

(two orders of magnitude or more) than the hyperfine

interaction which can therefore be treated as a

perturbation of the larger Zeeman interaction.

• Assumption 2: Just like the Zeeman interaction, the

hyperfine interaction will be anisotropic. It is assumed

that g and A are collinear.34


• Using these assumptions the resonance condition

becomes

ℎ𝜐 = 𝑔𝛽𝐵0 + ℎ𝐴𝑚𝐼

where A is called the Hyperfine Coupling Constant

and mI is the magnetic quantum number for the nucleus.

• This describes most hyperfine patterns we will

encounter.

• Exceptions can be found for example for Cu-ion spectra

(A-values of 30-200 Gauss) measured at lower

frequencies (L-band). In some Cu spectra the g and A

tensors are not linear.

• Other examples are Mn2+ spectra where D is small.

35


• How to determine the hyperfine coupling constant A:

A

A A36

Interaction

with I = 1/2

Interaction

with I = 1

10


• With the four new energy

levels there are two field

positions where the

resonance conditions are

met.

• The signal is in principle

split in two.

• The original, unsplit, peak

would have been exactly

in the middle of the two

hyperfine lines.

37


• Bio transition metal nuclear spin and their hyperfine structure

38


Bio ligand atom nuclear spins and their

EPR superhyperfine patterns

39

Organic Radicals

• Effect of hyperfine splitting on anisotropic signals can be

very complex

• Organic radicals

• Measured in solution.

• Fast tumbling motions of the molecules averages the

axial anisotropy of the g-factors resulting in the

detection of only a narrow isotropic EPR signal with a

single g-value, ‘giso’.

• Line shape is pure isotropic.

40

11


• Since there are 2I + 1 possible values of mI (mI = I, I-1,

…, 0, …,-I+1, -I), the hyperfine interaction terms splits

the Zeeman transition into 2I + 1 lines of equal intensity.

No interaction

I = ½ → 2 lines

I = 1 → 3 lines

I = 3/2 → 4 lines

I = 2 → 5 lines

41


• Different patterns with lines with different intensities are

obtained when the unpaired electron interacts with more

than one nucleus with the same nuclear spin.

No interaction

1x I = ½

2x I = ½

3x I = ½

4x I = ½

42


• Use a stick diagram or Pascal triangle to predict the

relative intensities of each line.

43


• Different patterns with lines with different intensities are

obtained when the unpaired electron interacts with more

than one nucleus with the same nuclear spin.

No interaction

1x I = 1

2x I = 1

3x I = 1

4x I = 1

44

12


• Interaction with 4 N atoms with I = 1 results in a

spectrum with 9 lines.45

• Use a stick diagram or

Pascal triangle to

predict the relative

intensities of each line.

Organic Radicals: Localized

• The unpaired electron density or spin density is close to

unity on one particular atom in the molecule.

• The unpaired electron will not interact to a significant

amount for nuclei further than two bonds away from the

atom with the unpaired electron.

• Example: ethanol radical HOCH2CH2•

• For this ethanol radical you would only expect to see

splittings from the protons of the two CH2 groups.

• These protons are termed α- and β-protons, counting

from the atom with the unpaired electron.

• Protons that are further away (e.g., the OH proton) will

be expected to give a negligible hyperfine coupling

constant.46


• Note that due to the fact that the OH proton is

exchangeable there is no interaction observed. However,

since only the α- and β-protons are contributing virtually

the same spectrum is generated by making a n-propyl

radical (CH3CH2CH2•) or a n-butanyl radical

(CH3CH2CH2CH2•).

47


• The EPR spectrum of the ethanol radical shows a triplet

of triplets

48

HO-C(Hβ)2-C(Hα)2•

13

Organic Radicals: Delocalized

• The unpaired electron in an orbital that can be

delocalized over the whole molecule or a large section.

• In delocalized radicals, hyperfine interaction affects spin-

active nuclei attached to atoms in the α- and β-position

with respect to the unpaired electron.

• Nuclei in identical chemical environments are equivalent.

• Example: benzene radical anion.

• Due to the delocalization, the spin density is spread

around the whole molecule and all the protons interact

with the unpaired electron.

• In the benzene anion radical all six hydrogen atoms are

equivalent and they will cause a split of the EPR signal

into a septet. 49

Organic Radicals: Delocalized

• In the benzene anion radical all six hydrogen atoms are

equivalent and they will cause a split of the EPR signal

into a septet.

50

Benzyl Radical

• The electron is still delocalized but the HOMO has

different densities on the different C atoms

• The two ortho-protons are attached to carbons with the

same spin density and their chemical environment is the

same – hence they are equivalent and will split EPR

signal into a 1:2:1 triplet

• Meta-protons are also equivalent and yield triplet

splitting.51

Benzyl Radical

• The fast rotation across C-C bond linking the aromatic

ring to the methylene group makes the two protons Hα

equivalent – hence triplet splitting from this group.

• The only para-proton will split the signal into a doublet.

• The EPR spectrum of benzyl radical should thus give a

triplet (Hα) of triplets (Hortho) of triplets (Hmeta) of doublets

(Hpara) resulting in 3×3×3×2 = 54 lines.

52

14

How to Analyze Splitting Patterns

• Four of the triplets around 338 mT and 339.7 mT overlap

hence the total number of lines in the spectrum is 48

rather than 54.

53


i. Make sure the overall spectrum is symmetrical. If this is

not the case you are dealing with more than one

paramagnetic species. You have to look for measuring

conditions that allow the spectra to be measured with

different ratios. If that is possible you could try to isolate

each species by a series of subtractions. If this is not

possible you will have to try to insulate and analyze

both spectra separately.

54


ii. If the structure of the molecule is available, identify all

nuclei with a nuclear spin in the α- and β-position

relative to the unpaired electron. Use splitting diagrams

to reproduce the splitting pattern and use a Pascal

triangle and to determine the relative intensity of the

hyperfine lines for each environment.

55


iii. To get to the first multiplet (multiplet A), take the

distance between the first two lines at the low field (e.g.,

left side) in the spectrum. Measure out this same

distance to detect if there are more lines that belong to

this multiplet. Keep in mind that that the multiplet might

not be symmetrical due to overlap with lines from other

multiplets.

56

15


iv. To find the second multiplet, measure the distance from

the outermost line to the first line which does not form

part of multiplet A. This distance corresponds to the

hyperfine coupling constant of the next multiplet

(labelled B). Just as with multiplet A keep on measuring

the same distance towards higher field until all the lines

of multiplet B have been identified.

57


v. Since multiplet B has a larger hyperfine coupling

constant A than that of multiplet the hyperfine pattern of

multiplet A will be superimposed on each component of

multiplet B.

vi. Repeat steps 6-9 until all multiplets have been

identified. If you somehow get stuck on the left side also

start from the right side and try to get to the middle of

the spectrum.

58


59

Solution vs. powder spectra

• With small molecules in dilute solutions fast tumbling

motions averages the anisotropy of the g-factor resulting in

the detection of a narrow isotropic EPR signal with one

apparent g-value, giso. In this case the splitting patterns will

all have their origin in this central isotropic signal.

• With large molecules like proteins that show slow tumbling,

or molecules in frozen or powder samples, all g-values will

be displayed. Now each peak will have its own splitting

pattern since the hyperfine interaction A is anisotropic.

• An average g value can be calculated gav = (gx + gy + gz)/3

for the ‘powder’ spectra. Note that the giso obtained at room

temperature and the gav obtained on frozen or powder

samples can differ slightly due to the influence of solvent at

ambient temperatures.60

16

Hyperfine Splitting on Anisotropic Spectra

• The hyperfine splitting pattern will be the same for each

of the principle g-factors

• A is anisotropic and therefore the magnitude of the

splitting can differ significantly for the different g-factors

• In some cases the patterns on the different g-factors can

even overlap partly or completely making it much more

difficult to analyze these patterns.

61

Hyperfine Splitting on Anisotropic Spectra

62

Resolved

hyperfine on

the gz peak for

the Cu nucleus

(I = 3/2)

Unresolved

hyperfine on

the gxy peak

Type Identification - Metals

• Which redox state is EPR active?

Prepare different samples: 1) as such

2) reduced (dithionite)

3) oxidized (ferricyanide)

• How many unpaired electrons? Different spin states!

Metal Ion Electron Configuration Spin State

Fe2+ d6 S = 0 (ls) or S = 2 (hs)

Fe3+ d5 S = 5/2 (hs)

Ni1+ d9 S = ½

Ni2+ d8 S = 0 or S = 1

Ni3+ d7 S = ½

Cu1+ d10 S = 0

Cu2+ d9 S = ½

63


• Has the metal a nuclear spin?

Is the signal going to be split into 2 I + 1 lines?

• In general: The spin–orbit coupling parameter is positive for

systems with less than half filled outer shells and negative for

those with more than half filled shells, which means that the

former have g<ge and the latter have g>ge.

Atom Isotope Spin (abundance)

V 50, 51 50V, 6 (0.25); 51V, 𝟕𝟐 (99.75)

Mn 55 𝟓𝟐

Fe 54, 56, 57, 58 𝟏𝟐 (2.119)

Co 59 𝟕𝟐

Ni 58, 60, 61, 62 𝟑𝟐 (1.14)

Cu 63, 65 63Cu, 𝟑𝟐 (69.17); 65Cu, 𝟑

𝟐 (30.83)

Mo 92, 94, 95, 96, 97, 98, 100 95Mo, 𝟓𝟐 (15.92); 97Mo, 𝟓

𝟐 (9.55)

W 180, 182, 183, 184, 186 𝟏𝟐 (14.3)

64

17


65

Nickel

Hydrogenase.

The iron is low-spin Fe2+

The signal is due to the nickel

g>ge: gxyz = 2.32, 2.24, 2.01

Ni3+, d7

66

Nickel

Methyl-coenzyme-M reductase

g>ge: gxyz = 2.252, 2.073, 2.064

Hyperfine structure due to N-

ligands detectable

Ni1+, d9

67

Ni1+, d9

Type Identification - Nickel

What if you are not certain about

the origin of the spectrum:

labeling studies with nuclear

isotopes

Spectra of hydrogenase

(A) growth was performed with

natural Ni (natural

abundance of 61Ni is

1.19%);

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

A

B

C

D

68

18

Type Identification - Nickel

Methyl-coenzyme-M reductase

from Methanothermobacter

marburgensis

(C) natural Ni

(D) growth in the presence of 61Ni (86 %)

A

B

C

D

69

Cobalt

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

g>ge

70

The Microwave Frequency

• An increase in the strength of the

magnetic field B0 will result in a larger

separation of the two energy levels.

• As a result there will be an increased

population difference between the

ground and excited state resulting in

higher signal amplitude.

• To be able to meet the resonance

conditions the frequency will also

have to be increased according to

𝑔 = 0.7145𝜈 (𝑀𝐻𝑧)

𝐵 (𝐺𝑎𝑢𝑠𝑠)

71


• Starting at 133 MHz just like NMR spectroscopy

researchers have been pushing to get better resolution

and better sensitivity.

• For both technical and fundamental reasons it turned out

that the optimum sensitivity in EPR is reached in the

8-12 GHz range and X-band is right there in the middle

of that range.

• There are cases, however, that the information obtained

at X-band frequencies is limited and a higher frequency

is needed.

72

19


• Note that there is a theoretical limit of maximal resolution

enhancement by frequency increase. In practical cases

the enhancement is usually less or in some cases there

is no enhancement at all.

• EPR absorption lines can

have a width that is

independent of the used

frequency and the

corresponding resonance

field. As a consequence,

the resolution of two

partially overlapping lines

will increase with increasing

frequency.

73


• The Zeeman interaction is field dependent

• The linewidth is generally not field dependent (with the

exception of g-strain).

• The (super) hyperfine interactions are also independent

of the magnetic field.

• Therefore, changing the microwave frequency means

changing the relative weight of the B-dependent and B-

independent interactions and so the shape (and

information content) of the spectrum changes with

frequency.

• Note that by doing this, for example, the description of

the high-spin systems is no longer valid.

74

250 275 300 325 350 375 400 425

Field (mT)

2.3

1.8

9


• Comparison of a Vitamin B12

spectrum at X-band (9.5

GHz) and Q-band (35 GHz).

• In this example, the

superhyperfine lines are still

resolved at Q-band, but are

not overlapping with the gx-

and gy-peaks anymore. Now

the 8 hyperfine lines can be

detected without any

problems

X-band

Q-band

75


• Comparison of the MCRred1 spectrum at X-band (9.5

GHz) and W-band (90 GHz).

• Note that the linewidth does not change on a linear Gauss-

scale (both scales cover 3000 Gauss).

X-band

W-band

76

20


• When the spectra are both plotted on the same g-scale is

seems like the linewidth is much smaller, and small

differences in g-values can be detected.

• In this example, the superhyperfine lines are not resolved

at W-band.

X-band

W-band

77

CobaltMethyltransferase from M.

marburgensis.

(A) Protein as isolated.

(B) Computer simulation

gxyz = 2.2591, 2.2530, 2.00659

The position of the 8 hyperfine

lines are indicated in the

figure. Each line, in turn, is

split due to interaction with a

N-ligand

A

B

Co+, d7, I = 7/2

g>ge

78

Identification of Ligands

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

79

Vanadium

Vanadium-containing

chloroperoxidase from the

fungus Curvularia inaqualis

V4+

d1

I = 7/2

80

21

Vanadium

Spectrum is due to V4+

d1, I = 7/2

The spectrum is axial:

g// = 1.95 and g = 1.98 (g<ge)

Both lines are split into 8

hyperfine lines.

The hyperfine splitting A is very large and the hyperfine lines of

one peak will be on the other side of the other peak. This

causes an effect called overshoot: The orientation and shape

of these lines will change.

81

Molybdenum

A

B

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).

Note that we have g-values

above and below ge

Mo5+, d1

82

Tungsten

A

B

Formyl-methanofuran

dehydrogenase (FDH II) from

cells grown on tungstate.

(A) gxyz = 2.0488, 2.0122,

1.9635.

(B) 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%.

W5+, d1

83

Copper

Cu2+ in Cu(ClO4)2

d9, I = 3/2

Axial signal

(g>ge)

84

22

Copper

85

Comparison of EPR spectra and structures for copper centres in

azurin. a, Type 1 (wildtype). b, Type 0 (C112D/M121L). c, Type 2

(C112D).(Rosenzweig (2009) Nature Chemistry, 1, 684 – 685)

Iron-sulfur Clusters

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

86

S=1/2 Systems

87



ΔE

Absorption

B0

1 Derivativest

High-Spin Systems

• A system with n unpaired electrons has a spin equal to S

= n/2. Such a system has a spin multiplicity:

ms = 2S + 1

• This value is equal to the number of spin energy levels.

• All the spin levels together are called the spin multiplet.

• An essential difference between S = ½ systems and

high-spin or S ≥ 1 systems is that the latter are subject to

an extra magnetic interaction namely between the

individual unpaired electrons.

• Unlike the electronic Zeeman interaction this interaction

is always present and is independent of any external

field. Another name for this interaction therefore is zero-

field interaction.88

23

(non-)Kramers’ Systems

Systems with more than one unpaired electron

Half-integer / Kramers’ systems

• S = 3/2, 5/2, 7/2, 9/2

• All systems detectable in perpendicular-mode EPR

Integer / non-Kramers’ systems

• S = 1, 2, 3, 4

• Detection in parallel-mode EPR

• In biochemistry only relevant for S = 2 systems of

[3Fe-4S]0 and Heme-Fe2+

• The Ni2+ in MCR is S =1 but does not display a

spectrum89

Examples for Fe3+/S = 5/2

Rubredoxin

(Photosystem II)

KatG

Example 1: Fe3+ in

rubredoxins. The ion is

coordinated by the

thiolate groups of four

Cys residues.

Example 2: Fe3+ in the

heme group of KatG.

The ion is coordinated

by the four nitrogen

ligands from the heme

ring.

90

Half-Integer / Kramers’ systems

• The spin Hamiltonian becomes

𝐻𝑠 = 𝛽𝐵 • 𝑔 • 𝑆 + 𝑆 • 𝐷 • 𝑆

• Solving the wave equations it can be shown that in zero

field, the sub levels of a half-integer spin multiplet group

in pairs (Kramer pairs) and these pairs are separated by

energy spacings significantly greater than the X-band

microwave energy hν.

• These spacing are also called zero-field splittings, or

ZFS.

91

Energy Levels for S = 5/2 System

• The S = 5/2 multiplet forms three Kramers’ doublets that are

separated from the others by energies significantly larger than

the ≈ 0.3-cm-1 microwave quantum (X-band).

|mS = ±1/2>

|mS = ±3/2>

|mS = ±5/2>

92

24


• The degeneracy between the pairs is lifted in an external field.

• Since the zero field splitting is very large, the external field-

induced splitting only allows for the occurrence of EPR

transitions within each (split) pair of levels.

Only intra-doublet transitions observed in EPR.

|mS = ±1/2>

|mS = ±3/2>

|mS = ±5/2>

93


• There is no crossing over and mixing of the energy levels.

• For Kramers’ systems each Kramer pair can give rise to its own

resonance.

• Each of these can be described in terms of an effective S = ½

spectrum with three effective g-values.

|mS = ±1/2>

|mS = ±3/2>

|mS = ±5/2>

94

Half-Integer / Kramers’ Systems

ℎ𝜈 = 𝑔𝑒𝑓𝑓𝛽𝐵

• geff encompasses the real g-values plus the effect of the

zero-field interaction.

• Just like the g-value and A-values also the zero-field

interaction parameter can be anisotropic and have three

values Dx, Dy, and Dz.

95


ℎ𝜈 = 𝑔𝑒𝑓𝑓𝛽𝐵

• In contrast to g and A, however, the three Di’s are not

independent because Dx2 + Dy

2 + Dz2 = 0, and so they

can be reduced to two independent parameters by

redefinition:

𝐷 =3𝐷𝑧

2and 𝐸 =

𝐷𝑥−𝐷𝑦

2

• We can also define a rhombicity

𝜂 = 𝐸𝐷 with 0 ≤ 𝜂 ≤ 1 3

96

25


• From the complete energy matrix it can be derived that

under the so-called weak-field limit (Zeeman

interaction << zero-field interaction) the three

elements of the real g-tensor, gx, gy, and gz, can be fixed

at 2.00 and that the shape of the EPR spectra, the

effective g-values, is a function of the rhombicity E/D.

• The relationship of the effective g-values versus the

rhombicity can be plotted in two-dimensional graphs, so-

called rhombograms.

97

Rhombogram

E/D = 0.315

S = 1/2S = 3/2

|mS = ±1/2>

|mS = ±3/2>

|mS = ±5/2>4.5 K

98

Rhombogram & Simulations

|mS = ±1/2>

|mS = ±3/2>

|mS = ±5/2>

Axial component(s)

E/D = 0.00

Rhombic component

E/D = 0.03

–– spectrum

–– simulation

10 K

99

Mn2+

• The somewhat simplified description of the EPR spectra

of the different Fe3+ systems and iron-sulfur-cluster

containing proteins was possible due to the fact that they

all fall within the weak-field limit (Zeeman interaction <<

zero-field interaction).

• It is also possible that the zero-field interaction is much

weaker than the Zeeman interaction, and this “strong-

field limit” hold for six-coordinate Mn2+, which is not only

biologically relevant as a site in some manganese

proteins, but also because this is a very common

contaminant of biological preparations.

100

26

Mn2+

• The electronic spin state of Mn2+ is S = 5/2.

• Six energy states with the electron spin magnetic

quantum number, ms = 5/2, 3/2, 1/2. -1/2, -3/2, and -5/2

arise due to the Zeeman effect.

101

Mn2+

• Pure cubic (e.g., octahedral) situation: D and E are zero,

g is isotropic, because of the high spin a new zero-field

energy term exists that produces EPR anisotropy.

• The zero-field splitting for Bǁz are shown.

102

Mn2+

• Due to the nuclear spin magnetic quantum number, all

lines will be further split into six hyperfine lines, m = 5/2,

3/2, 1/2. -1/2, -3/2, and -5/2.

• Note that due to second-order effects the energy level

splitting by the Zeeman effect is not linear.103

Mn2+

• The energy level diagram

predicts that the

spectrum is dominated by

the ms = +1/2 ↔ -1/2

transition and shows the

presence of six hyperfine

lines each split by a small

anisotropy induced by the

zero-field splitting.

104

• In between the six hyperfine lines there are five pairs of weak

lines from forbidden ΔmI = ± 1 transitions with an order of

magnitude lower intensity than the main lines.

• This whole ms = ±1/2 spectrum is on top of a very broad,

rather structureless feature that is the sum of all the other five

Δms =1 transitions (e.g., ms = -3/2 ← -5/2).

Mn2+

d7

I = 5/2

(S = 5/2)

27

Integer / non-Kramers’ Systems

• Non-Kramers’ systems or integer systems are systems

with S = 1, 2, 3, 4.

• These systems are very seldom observed in biological

systems.

• One of the reasons is that just as in the Kramers’

systems the energy levels are organized in doublets

(and one singlet, |0).

• These doublets, however, are split even at zero field and

this splitting is generally greater than the energy of the

X-band radiation.

• This means that in most cases the signals cannot be

detected.

105

S = 2 System

• For E = 0, the effective g-values are gxyz = 0, 0, 4 (for

|±1 doublet) and gxyz = 0, 0, 8 (for |±2 doublet), purely

axial.

• This means that the signals are not detectable

106

S = 2 System

• For E ≠ 0, the signals become more rhombic and mixing

of the wave function takes place

• Signal can be detected in perpendicular-mode EPR:

|Δms| = 1 or in parallel-mode EPR |Δms| = 0

• Since the levels are already split at B0 = 0 the peaks will

be shifted to higher g-values. 107

S = 2 System

• Due to several mechanisms the EPR signals are very

broad and deformed and not much information can be

obtained from the signals itself.

• For most systems however this zero-filed splitting is

larger than the microwave energy at X-band and no

signal will be detected. 108

28

S = 2 System

• Example of the spectra detected for the S = 2 [3Fe-4S]0

cluster from hydrogenase from Allochromatium vinosum.109


Hyperfine interaction

Interaction of the electron spin with the

nuclear spin of the metal ion nucleus

Super hyperfine interaction

Interaction of the electron spin with first

coordinate sphere ligands nuclei

Spin-spin interaction

Interaction of the electron spin with other

electron spins within 10 Å distance.

110

Spin-Spin Interaction

• In principle one could expect to see two signals for each

paramagnetic species present and both signal would be

split due to the spin-spin interaction.

• The distance of the split peaks would be dependent on

the distance between the two species in the molecule

• This would only happen when the g-tensors of both

species are linear.

• When the g-tensors are

not parallel, however,

the spectra will change

significantly.

111


A) [4Fe-4S]+ signal detected in

a ferredoxin from Bacillus

stearothermophilus.

B) Signal detected in a so-

called 8Fe ferredoxin from

Clostridium pasteurianum.

In this sample two [4Fe-4S]+

clusters are present.

Spectrum B does not look like two overlapping signals.

A more complex signal is now detected. The broad

wings in the EPR spectrum (indicated by the arrows)

are typically found for two interacting clusters.

112

29


• Adenosylcobalamin (coenzyme B12)-dependent

isomerases.

• Catalyze skeletal rearrangements via a radical

mechanism.

• The reaction starts with the generation of the 5’-

deoxyadenosyl radical and cob(II)alamin from enzyme-

bound adenosylcobalamin by homolysis of the

coenzyme’s cobalt-carbon σ-bond in the presence of a

substrate molecule SH.113


• Stereospecific hydrogen abstraction from the substrate

molecule by the 5’-deoxyadenosyl radical gives 5’-

deoxyadenosine and a substrate radical S•.

• At this point two paramagnetic species are present in the

enzyme, the Co2+ species and the S• radical species.

114


A) EPR spectrum of the Co2+

species by itself (gav ≈2.18)

B) EPR of the radical species

by itself (g = 2.0023)

C-E) The actual observed EPR

spectra for different types of

isomerases.

115


Different types of interactions dependent on the distance

between the two interacting paramagnets.

• Through-space dipole-dipole interaction: anisotropic

interaction that follows a 1/r3 dependence on the spacing

between the interacting centers

• Exchange interaction that depends on orbital overlap

and spin polarization effects: isotropic interaction, which

falls off approximately exponentially with the distance

between the partners.116

30

Weakly Coupled Spin Systems

• At distances greater than approximately 9 Å, the

exchange interaction creates a doublet splitting in

the EPR spectrum of each partner

• At closer distances, the exchange interaction mixes the

two spin systems, such that their g-values become

averaged and eventually converge to a triplet state at

interspin separations of <7 Å.

117

Weakly Coupled Spin Systems

• EPR spectrum of

hydroxyethylhydrazine-

inactivated ethanolamine

ammonia-lyase showing the

presence of features

corresponding to B12r and a

companion radical species

with absorption near g = 2.0.

118

• The signals from the low-spin Co2+ and the partner

radical were split by a combination of exchange and

dipole-dipole coupling. This can be detected as the

additional hyperfine splitting of the Co2+ signal.

• The amplitude of the signal of the radical centered at g

= 2.0 is off scale.

Strongly Coupled Spin Systems

• At distances greater than approximately 9 Å, the

exchange interaction creates a doublet splitting in the

EPR spectrum of each partner

• At closer distances, the exchange interaction mixes

the two spin systems, such that their g-values

become averaged and eventually converge to a

triplet state at interspin separations of <7 Å.

119


Simulation of signals:

• For distances larger than 4-5 Å both

paramagnets can be considered point

dipoles.

• The zero-field splitting is described as a

traceless tensor with an axial, D, and a

rhombic, E, term. In the commonly used

point-dipole approximation, E ≡ 0.

• The principal axis of the zero-field

splitting normally contains the interspin

vector. In simulations, Euler rotations (θ)

are required to relate the axis system of

the zero-field splitting tensor to a

reference system, such as the g-axis of

Co2+.120

31


• When there is a strong coupling between the cobalt and

the radical species, the EPR spectra becomes a hybrid

of both the cobalt and the radical EPR signals and

exhibit an average g-value of ≈ 2.1 that arises from

coupling between a carbon centered radical (g = 2.0023)

with cob(II)alamin (gav ≈ 2.18).

• The signals are due to a ‘hybrid’ triplet spin system

comprising both paramagnets.

121


(C) Signal of the coupled

Co2+/radical species in

ethanolamine ammonia

lyase after reacting with

ethanolamine. The

interspin distance is 8.7 Å

and θ = 25°

(D) Coupled Co2+/radical

species in lysine-5,6-

aminomutase after reacting

with 4-thialysine. The

interspin distance is 7.0 Å

and θ = 43°.

122

Very Strongly Coupled Spin Systems

• When there is a very strong coupling the EPR spectrum

does not resemble that of Co2+ or a radical species.

• However, it is still consistent with a rhombic triplet-state

species. A prominent half-field transition at around g = 4

can be detected (not shown).

• Note that the EPR spectrum covers a wide area.

• The close spacing of the unpaired electrons, together

with the spin delocalization within the allylic radical,

requires a higher level of treatment than the point-dipole

approximation.123

Very Strongly Coupled Spin Systems

(E) Coupled Co2+/radical species

in diol dehydratase after

reacting with 5’-deoxy-3’,4’-

anhydroadenosylcobalamin.

The interspin distance is 3.5 Å

and θ = 75°.

124

32

g-Strain

• We know from folding studies and from structural NMR

and X-ray studies that samples of proteins come with a

distribution of conformations.

• For EPR this means that the paramagnet in each

molecule has a slightly different structural surrounding

and thus a slightly different g-value.

• This structural inhomogeneity or g-strain is reflected in

the spectroscopy in the form of an inhomogeneous line

shape.

• This normally results in a change from a Lorentzian to

Gaussian line shape. An important consequence of this

g-value anisotropy is that the line width, W, is in general,

also isotropic.

125

g-Strain

• Most of the time we do not have to worry about this, but

particularly in the EPR spectra of the iron-sulfur clusters

g-strain can have a big effect on the shape of the EPR

spectrum and therefore on the simulation and

interpretation of the EPR data.

126

g-Strain

• This effect is shown in the figure for the [4Fe-4S] cluster

detected in spinach-leaf ferredoxin. The line width is very

similar in the range of 35 to 3.3 GHz.

• At 1.1 GHz a broadening is detected due to unresolved

hyperfine coupling.

• The most noticeable

difference is now that

the linewidth, plotted

on a g-scale does not

change when the

spectra are measured

at higher frequency.

127

ENDOR

• Nuclear hyperfine splitting might not always be resolved

but might be hidden in the EPR peaks.

• Techniques have been developed to detect these

interactions: Electron-Nuclear Double Resonance

(ENDOR), Electron Spin Echo Envelope Modulation

(ESEEM), and HYperfine Sublevel CORrElation

(HYSCORE) spectroscopies.

• In transition metal complexes and metalloproteins,

magnetic nuclei such as 1H, 2H, 13C, 14N, 15N, 17O, 31P

and 33S, in the vicinity (2-12 Å) of the paramagnetic

metal ion can be detected by these techniques.

• Identification of the presence of a particular ligand

nucleus, and under favorable circumstances metal-

ligand nuclei distances and angles can be obtained.128

33

ENDOR

• The red lines show the allowed EPR transitions and the

stick EPR spectrum.

• The blue lines show the NMR (ENDOR) transitions and

the stick ENDOR spectrum.

• Energy level

diagram for an

S = ½, I = ½

spin system.

• The g-value

and hyperfine

coupling are

assumed to be

isotropic.

129

ENDOR

• The energy levels for an S = ½, I = ½ spin system

• Using low MW powers, resonance absorption occurs as,

for example, the EPR II transition frequency E1 ↔ E3 is

induced but rapid relaxation ensures that the excited

spins return quickly to the ground state leading to an

unsaturated EPR signal (a).

130

ENDOR

• Using higher MW power, the E1 ↔ E3 transition becomes

saturated such that the spin population in both levels

equalizes (b), a situation that should be avoided in regular

CW EPR spectroscopy since the signal intensity will

decrease to almost zero.

• To restore the EPR signal intensity, a population difference

between the levels E1 and E3 needs to be created.131

ENDOR

• Two mechanisms: One way is by ‘pumping’ the RF

transition E3 ↔ E4 (NMR I) using a saturating RF field

(c). An enhancement of the EPR absorption is detected if

the irradiated RF frequency is resonant with this

transition. This enhancement represents the first

ENDOR signal.

132

34

ENDOR

• In an alternative way, the RF transition E1 ↔ E2 (NMR II)

can be pumped (Fig. 39d). This also creates an

enhancement and represents the second ENDOR

resonance line.

133

A) EPR spectrum obtained for IspG upon incubation with

the substrate MEcPP and the reductant dithionite

B) Proposed structure for the reaction intermediate

ENDOR

134

ENDOR

C) 31P-ENDOR spectra. Spectra were collected at the fields

and g-values indicated, and are shown alongside the

respective pulse-echo detected EPR spectra.

A cluster-31P distance of 6.6 Å was calculated135

Pulsed ENDOR

• The Davies ENDOR sequence (πMW-πRF-π/2MW-τ-πMW-τ-

echo) is the pulsed equivalent of a CW ENDOR

experiment.

• The effect of this sequence on the occupation of the

energy levels for the two-spin system (S = ½, I = ½) is

shown. 136

35

Pulsed ENDOR

• The first selective πMW pulse inverts the electron spin

populations in the E1 and E3 manifolds (a) inducing the

transition EPR II.

• This is followed by a πRF pulse that inverts the nuclear

spin populations upon resonance with an NMR transition

(NMR I, b). 137

Pulsed ENDOR

• The remaining sequence consists of either the selective

(c) or non-selective (d) electron-spin echo detection

pulses.

138

ESEEM

• Electron Spin Echo Envelope Modulation (ESEEM) is an

important technique for measuring the hyperfine

interaction parameters between electron spins and

nearby nuclear spins.

• From the analysis of the ESEEM signals detailed

information about electron spin density distribution,

distances and bonding angles is gained.

139

ESEEM

• With ESEEM the NMR frequencies are observed

indirectly through observation of the mixing of allowed

and (formally) forbidden EPR transitions as modulations

superimposed on a time-decaying spin-echo.

• Echo envelope modulation occurs when state mixing of

hyperfine levels occurs.

• Fourier transformation of the modulated time trace

results in spectra in the frequency domain containing the

nuclear frequencies.140

36

2D-HYSCORE

• An extension of ESEEM is HYperfine Sub-level

CORrElation (2D-HYSCORE).

• This technique is essentially a two dimensional ESEEM

experiment in which correlation is transferred from one

electron spin manifold to another.

• HYSCORE allows one to take a complicated ESEEM

spectrum and extend the data into a second dimension.

141

2D-HYSCORE

• The HYSCORE pulse sequence is a four-pulse MW

sequence in which an additional mixing π pulse is

inserted between the second and third π/2 pulse of the

three-pulse ESEEM Experiment.

• The two inter-pulse delays, t1 and t2, are varied

independently to produce a two-dimensional (2D) time

delay array.

142

2D-HYSCORE

• The nuclear coherence generated by the first two π/2

pulses undergoes free evolution during time t1 with

frequency ω12 (ω34). The mixing π pulse then transfers

populations in one ms manifold to the other and similarly

transfers the nuclear coherence between manifolds so

that it evolves with frequency ω34 (ω12) during time t2.

• The modulated time decay data is subsequently Fourier

transformed in both dimensions (i.e. t1 and t2) to produce

a 2D frequency-domain spectrum (with axes ν1 and ν2).

143

2D-HYSCORE

• The nuclear frequencies from the different ms manifolds

are correlated and appear as cross-peaks at the

frequencies (ν1, ν2), (ν2, ν1), and (ν1, -ν2), (ν2, -ν1) in the

(+,+) and (+,-) quadrants of the 2D spectrum,

respectively.

• As strong cross peaks can only be observed between

NMR frequencies of the same nucleus, HYSCORE

spectra can be significantly simplified compared to three-

pulse ESEEM.

144

37

2D-HYSCORE

• Another advantage of HYSCORE spectroscopy is that

the frequencies from weakly-coupled nuclei (|aiso| < 2|νL|)

appear as cross-peaks in the (+, +) quadrant, whereas

strongly-coupled nuclei (|aiso| > 2|νL|) are observed in the

(+,–) quadrant (a). This facilitates spectral interpretation

for systems containing many interacting nuclei such as

metalloenzymes or proteins.145

2D-HYSCORE

• For disordered systems with broad ESEEM features, the

correlation peaks broaden into ridges, as illustrated for

the two-spin system (S = ½, I = ½) with an axial

hyperfine tensor (b). The anisotropy of the dipolar

hyperfine interaction, T, can be determined from the

maximum curvature of the ridges away from the anti-

diagonal, labelled ωmax, and the magnitude of aiso can be

found from the ridge end points or from simulation. 146

2D-HYSCORE

13C and 17O HYSCORE Spectra

of the FeSA species in IspG

147

Summary

+½: E = +½geβB0

-½: E = -½geβB0



For X-band:

𝒈 = 𝟎. 𝟕𝟏𝟒𝟓𝝂 (𝑴𝑯𝒛)

𝑩 (𝑮𝒂𝒖𝒔𝒔)

148

38


• Simplified Schrödinger wave equation

𝐻𝑠𝜓𝑠 = 𝐸𝜓𝑠

𝐻𝑠 = 𝛽𝐵 • 𝑔 • 𝑆 + 𝑆 • 𝐴 • 𝐼 + 𝑆 • 𝐷 • 𝑆 + … … …

Zeeman

interaction Hyperfine

interaction

Zero field

splitting

or

spin-spin

interaction

• Can sometimes be solved under a set of assumptions

• Solutions sometimes analytical, sometimes need for

numerical approach

• Cannot allows be solved149

Line Shape of EPR Spectra for S = ½ Systems

• Four basic shapes.

• When more than three

peaks are detected the

signal could be split due

to spin interaction or

there could be more than

one signal present.

150

Line Shape of EPR Spectra for High-Spin Systems

• Half-integer/non-Kramers:

S = 3/2, 5/2, 7/2, 9/2

• Rhombograms will help with the identification of the spin

state, determination of which spin doublets are

detectable and determination of the E/D value.

151

Examples for Fe3+/S = 5/2

Rubredoxin

(Photosystem II)

KatG

|mS = ±1/2>

|mS = ±3/2>

|mS = ±5/2>

152

39

Practical Aspects of EPR Spectrometry

1) Metal-Ion Type Identification

2) Optimal Measuring Conditions (T,P)

3) The X-band EPR Spectrometer

4) Spectrometer Parameters

5) Spin Intensity

6) Redox Titrations

7) Freeze-Quench Experiments

8) Simulation of EPR Spectra

9) EPR on Whole Cells/Cell Extract

10) Site-Directed Spin Labeling (SDSL) EPR

153

1) Metal-Ion Type Identification

• Which redox state is EPR active?

Prepare different samples: 1) as such

2) reduced (dithionite)

3) oxidized (ferricyanide)

• How many unpaired electrons? Different spin states!

Metal Ion Electron Configuration Spin State

Fe2+ d6 S = 0 (ls) or S = 2 (hs)

Fe3+ d5 S = 5/2 (hs)

Ni1+ d9 S = ½

Ni2+ d8 S = 0 or S = 1

Ni3+ d7 S = ½

Cu1+ d10 S = 0

Cu2+ d9 S = ½

154

Metal-Ion Type Identification

• Has the metal a nuclear spin?

Is the signal going to be split into 2 I + 1 lines?

• In general: The spin–orbit coupling parameter is positive for

systems with less than half filled outer shells and negative for

those with more than half filled shells, which means that the

former have g<ge and the latter have g>ge.

Atom Isotope Spin (abundance)

V 50, 51 50V, 6 (0.25); 51V, 𝟕𝟐 (99.75)

Mn 55 𝟓𝟐

Fe 54, 56, 57, 58 𝟏𝟐 (2.119)

Co 59 𝟕𝟐

Ni 58, 60, 61, 62 𝟑𝟐 (1.14)

Cu 63, 65 63Cu, 𝟑𝟐 (69.17); 65Cu, 𝟑

𝟐 (30.83)

Mo 92, 94, 95, 96, 97, 98, 100 95Mo, 𝟓𝟐 (15.92); 97Mo, 𝟓

𝟐 (9.55)

W 180, 182, 183, 184, 186 𝟏𝟐 (14.3)

155

2) Optimal measuring Conditions (T, P)

• There is a need to measure at lower temperatures!

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

range!

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

This effect is absent in frozen samples.

Do-it-yourself

microwave source

156

40

The Need for Lower Temperatures

The energy difference between the

two energy level due to the Zeeman

splitting is very small, ~0.3 cm-1 for

X-band EPR.

Based on the Boltzmann

distribution

it can be shown that only at low

temperatures there will be enough difference in the

population of the S = -½ level (n0) and the S = ½ level

(n1) to create a signal.

𝑛1 = 𝑛0𝑒−

∆𝐸𝑘𝑇

157

Spin-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!158

Heisenberg Uncertainty Principle

• 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

sample is, 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 themselves as

a constant broadening of the EPR line independent of

the temperature.

159

What to Do?

• 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 Plot

2) Make Power Plots

TEMPERATURE

BROADENINGPOWER

SATURATION

OPTIMAL

CONDITIONS

160

41

Power Plots

• 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

161

Power Plots

Relationship between the amplitude, gain and the power in

dB:

𝑎𝑚𝑝𝑙𝑖𝑡𝑢𝑑𝑒

𝑔𝑎𝑖𝑛∙ 10 −𝑑𝐵

20 = constant

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.162

Power Plot (Copper Perchlorate)

163

Power Plot (Copper Perchlorate)

• The relaxation rate increases

with increasing temperature.

• Therefore if a signal does not

saturate at a certain power at

a certain temperature it will

also not saturate at the same

power at a higher

temperature.

• The temperature behavior

or Curie behavior will be

different for different species.

164

42

Curie Plot (Copper Perchlorate)

In, normalized value for

the intensity;

I0, observed intensity;

T, absolute temperature

in K;

dB, reading of the

attenuator;

gain, gain

𝐼𝑛 =𝐼0 ∙ 𝑇 ∙ 10 −𝑑𝐵

20

𝑔𝑎𝑖𝑛

165

Curie Plot

166

Line Shape

• The basic form of an EPR

peak is described by the

Lorentz distribution. The

Lorentzian line shape is

also frequently called the homogeneous line shape.

• In biological samples the paramagnet in each molecule

has a slightly different structural surrounding and thus a

slightly different g-value.

• This structural inhomogeneity is reflected in the form of

an inhomogeneous line shape in addition to the

Lorentzian shape.

• At low temperature the contribution from homogeneous

broadening is small and the line shape can be described

by the Gaussian distribution. 167

Line Shape

• At relatively high

temperature a Lorentzian

line shape will be

observed, while a

Gaussian line will be

observed at relatively low

temperatures

• The Gaussian shape will

be broader.

• Preference to measure at

the higher temperature

end of Curie plot

• Practice better signal-to-

noise at the lower end.168

43

Power Plots

• Note that there are different

ways to compose power plots.

• Plot A shows the signal intensity

vs. power (-dB) with no

correction.

• Plot B show the signal intensity

versus 𝑃 (in mW). In this case

there is a linear relationship as

long as the sample does not

saturate (indicated by the

straight line).

• Plot C uses the earlier described

method on slide 165.

169

3) The X-band EPR Spectrometer

• In 1944, E.K. Zavoisky discovered magnetic resonance.

Actually it was EPR on CuCl2.

E.K. Zavoisky’s first EPR system

170

EPR Spectrometer

Varian E3

Varian E4

Bruker ElexSys X-band

Bruker ElexSys W-band

171

X-Band EPR Spectrometer

172

44


Microwave

bridge

173

• On the left is a monochromatic source of microwaves of

constant output (200 mW) and slightly (10%) tunable

frequency.

174

• The produced radiation is transferred by means of a

rectangular, hollow wave guide to an attenuator where the

200 mW can be reduced by a factor between 1 and 106.

175

• The output of the attenuator is transferred with a waveguide

to a circulator that forces the wave into the resonator/cavity.

• The entrance of the resonator is marked by the iris, a device

to tune the amount of radiation reflected back out of the

resonator. 176

45

• The reflected radiation returns to the circulator and is

directed to the diode for the detection of microwave

intensity.

177

• Any remaining radiation that reflects back from the

detector is forced by the circulator into the upward

waveguide that ends in a wedge to convert the radiation

into heat.178

• A small amount of the 200 mW source output is directed

through the reference arm directly to the detector to produce

a constant working current.

• The reference arm contains a port that can be closed and a

device to shift the phase of the wave. 179


• Most EPR spectrometers are

reflection spectrometers.

• They measure the changes

(due to spectroscopic

transitions) in the amount of

radiation reflected back from

the microwave cavity

containing the sample.

• The detector should only detect

the microwave radiation

coming back from the cavity.

180

46

Cavity/EPR Resonator

• A microwave cavity is simply a

metal box with a rectangular or

cylindrical shape which resonates

with microwaves much as an

organ pipe resonates with sound

waves.

• The resonator is designed to set

up a pattern of standing

microwaves in its interior.

• Standing electromagnetic waves

have their electric and magnetic

field components exactly out of

phase - where the magnetic field

is maximum, the electric field is

minimum.181


• Resonance means that the

cavity stores the microwave

energy; therefore, at the

resonance frequency of the

cavity, no microwaves will be

reflected back, but will remain

inside the cavity.

• Energy can be lost to the side

walls of the cavity because

the microwaves generate

electrical currents in the side

walls of the cavity which in

turn generates heat. 182


• We can measure Q factors easily:

Q = (νres)/(Δν)

where νres is the resonant frequency of the cavity and Δν

is the width at half height of the resonance.

• Cavities are

characterized by their

Q or quality factor,

which indicates how

efficiently the cavity

stores microwave

energy.

183


• In order for the microwaves to

enter the cavity one of its end walls

must have an opening: iris.

• The size of the iris controls the

amount of microwaves which will

be reflected back from the cavity

and how much will enter the cavity.

• Just before the iris is a small metal

plate (attached to the iris screw).

Moving this plate up or down

changes the amount of coupling.

• Only for one unique position is the

cavity critically coupled: all waves

enter the cavity, and no radiation is

reflected out.184

47


• How do all of these properties of a cavity give rise to an

EPR signal? When the sample absorbs the microwave

energy, the Q is lowered because of the increased

losses and the coupling changes.

• The cavity is therefore no longer critically coupled and

microwaves will be reflected back to the bridge, resulting

in an EPR signal.

185

186

Tuning the Microwave Cavity and Bridge

• Locate and center the “dip”

on the display.

• The pattern is a display of the

microwave power reflected from

the cavity and the reference

arm power as a function of the

microwave frequency.

• The dip corresponds to the microwave power absorbed

by the cavity and thus is not reflected back to the detector

diode.

• By centering the dip on the display monitor, the

microwave source is set to oscillate at the same

frequency as the cavity resonant frequency187

Tuning the Microwave Cavity and Bridge

• Tune the signal (reference) phase. Adjust the Signal

Phase until the depth of the dip is maximized and looks

somewhat symmetric.

• Adjust the bias level. Adjust the Bias until the Diode

meter needle is centered.

• Critical coupling of the cavity. Power is increased and

the iris screw is adjusted to keep the diode current in the

center.188

48

• Center Field and Sweep Width

• For initial broad scans, a Sweep Width around 5000

Gauss is recommended. Set the Center Field value to

2600 Gauss. This means that the scan will start at 100

Gauss and stops at 5100 Gauss (2500 Gauss below and

2500 Gauss above the Center Field value).

• This scan will cover the complete area available with our

magnet where signals might be detectable.

4) Spectrometer Parameters

189

Spectrum Settings

• Points

• Standard 1024 points. Can be increased to 4096 for

wide scans to keep the resolution.

• It is advisable, however, to rescan the interesting parts of

a wide scan.

• Subtractions are not possible if the amounts of points

between the two spectra are different.

190

Spectrum Settings

• Gain

• Use the full range of the

digitizer (a), coincides with

the screen display.

• If the receiver gain is too low

the effect of digitization will

be evident in the spectrum

(b)

• At too high gain the signals

will be clipped due to an

overload in the signal

channel (c).191

Microwave Bridge Parameters

• Microwave power level. The EPR signal intensity grows

as the square root of the microwave power in the

absence of saturation effects. When saturation sets in,

the signals broaden and become weaker. Several

microwave power levels should be tried to find the

optimal microwave power.

192

49

Phase Sensitive Detection

• Enhancement of the sensitivity of the spectrometer: less

noise from the detection diode and the elimination of

baseline instabilities due to the drift in DC electronics.

• The magnetic field at the site of the sample is modulated

(varied) sinusoidally at the modulation frequency. If there

is an EPR signal, the field modulation quickly sweeps

through part of the signal and the microwaves reflected

from the cavity are amplitude modulated at the same

frequency.

• Only the amplitude modulated signals are detected. Any

signals which do not fulfill these requirements (i.e, noise

and electrical interference) are suppressed.

193

Phase Sensitive Detection

amplitude proportional to the slope of the signal.

• As a result the first derivative of the signal is measured.

• Two new parameters: modulation amplitude, and

frequency.

• For an EPR signal

which is approximately

linear over an interval

as wide as the

modulation amplitude,

the EPR signal is

transformed into a sine

wave with an

194

Field Modulation

• A good compromise between signal intensity and signal

distortion occurs when the amplitude of the magnetic field

modulation is equal to the width of the EPR signal. Also, if we

use a modulation amplitude greater than the splitting between

two EPR signals, we can no longer resolve the two signals.

• With more magnetic field

modulation, the intensity of

the detected EPR signals

increases; however, if the

modulation amplitude is too

large (larger than the

linewidths of the EPR

signal), the detected EPR

signal broadens and

becomes distorted.

195

Signal Channel Parameters

• Modulation frequency: normally set to 100 kHz

• Modulation amplitude: You can start with 6 Gauss. The

larger this value the lower the value needed for the

Receiver Gain, which means less noise. Excessive field

modulation, however, broadens the EPR lines and does

not contribute to a bigger signal. As a rule-of-thumb this

value has to be smaller than the line width of your signal.

196

50

Time Constant

• To further improve the sensitivity, a time constant is

used to filter out more of the noise.

• Time constants filter out noise by slowing down the

response time of the spectrometer. As the time constant

is increased, the noise levels will drop. If we choose a

time constant which is too long for the rate at which we

scan the magnetic field, we can distort or even filter out

the very signal which we are trying to extract from the

noise. Also, the apparent field for resonance will shift.197

Signal Channel Parameters

• Time Constant and Conversion Time: If the Time

Constant is too large in comparison with the Conversion

Time (the rate at which the field is scanned) the signals we

want to detect will get distorted or will even be filtered out.

• A longer Conversion Time, however, also improves the

signal to noise ratio in a different way: The signal channel

incorporates an integrating ADC (Analog to Digital

Converter) to transfer the analog EPR spectra to the digital

data acquisition system. An important side effect of using

the integration method for the conversion is that it

integrates the noise out of the signal.

• With a sweep width off about 1000 Gauss a Conversion

Time of 163.84 msec and a Time Constant of 163.84 msec

can be used.198

Signal Averaging

• Very weak signals might get lost in the noise. You can

increase your signal to noise ratio by signal averaging. The

resultant signal to noise is proportional to N, where N is

the number of scans.

• With a perfectly stable laboratory environment and

spectrometer, signal averaging and acquiring a spectrum

with a long scan time and a long time constant are

equivalent. Unfortunately perfect stability is impossible to

attain. Slow variations result in baseline drifts. For a slow

scan (>15 min) the variations can cause broad features in

the spectrum dependent on the sample concentration and

the gain used. If you were to signal average the EPR signal

with a scan time short compared to the variation time, these

baseline features could be averaged out. 199

Spectrometer Parameters

• Center Field, Sweep Width, Gain, Microwave power level:

sample dependent

• Modulation frequency: normally set to 100 kHz

• Modulation amplitude: normally set to 6 Gauss.

• Time Constant and Conversion Time: same value!

sweep width off 1000 Gauss; both 163.84 msec

• Number of X-Scans: normally set to 1

200

51

5) Spin Intensity

• Also known as spin counting

• 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:

201

where

In normalized double integral

I0 observed intensity

d distance between the starting and ending points (in

Gauss)

T absolute temperature in K

dB reading of the attenuator

f tube calibration factor

a gain

and

Normalized Signal Intensity

𝐼𝑛 =𝐼0 ∙ 𝑑2 ∙ 𝑇 ∙ 10 −𝑑𝐵

20

𝑔𝑝𝑎𝑣 ∙ 𝑓 ∙ 𝑎

𝑔𝑝𝑎𝑣 =

2

3

𝑔𝑥2 +𝑔𝑦

2 +𝑔𝑧2

3+

𝑔𝑥 +𝑔𝑦 +𝑔𝑧

9

202

Signal Integration

Step 1: select spectrum

203

Signal Integration

Step 2: select area to integrate

(shown is the first integral)

204

52

Signal Integration

Step 3: get the value for the double integral

205

Comparison with ‘Spin’ Standard

• Keep measuring conditions the same: temperature,

modulation amplitude, sweep time, amount of points,

amount of scans (These are not averaged!)

• Measure samples on the same day!

• Correct for spin: S(S+1)

𝐶𝑢 =𝐼𝑛(𝑢) ∙ 𝐶𝑠𝑡

𝐼𝑛(𝑠𝑡)

𝐼𝑛 =𝐼0 ∙ 𝑑2 ∙ 𝑇 ∙ 10 −𝑑𝐵

20

𝑔𝑝𝑎𝑣 ∙ 𝑓 ∙ 𝑎

206

Signal Intensity???

Clostridium

pasteurianum

[2Fe-2S]2+

S = 9/2

(D < 0)

207

Signal Intensity???

• 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.

208

53

Signal Intensity ???

Simulations will be needed to get the

signal intensity of a signal when

more than one signal is present, the

signal intensity is too low (too much

noise), or the baseline is not linear.

Spectrum

Simulation

Difference

209

6) Redox Titrations

• With species that are only paramagnetic at a certain

redox potential it is possible to do a redox titration and

obtain the midpoint potential (Em) of the redox couple.

• This is particular useful if you are studying proteins that

are involved in electron transfer pathways.

• In these experiments the protein is titrated in both the

oxidative direction with ferricyanide and in the reductive

direction with dithionite. The potential can be measured

with a combination Ag/AgCl electrode,

• A mixture of redox dyes is added to stabilize the redox

potential outside the Em region

210

Redox Titrations

• When there is only a single paramagnetic species present

the intensity of this signal can be determined directly.

• When more than one species are present you have to look

for unique features, or the different components have to be

simulated and their intensities determined.

• Plots of the intensity vs. the potential are generated.

• The points in the plot can be fitted with the Nernst

equation:

𝐸 = 𝐸0 +𝑅𝑇

𝑛𝐹𝑙𝑛

[𝑜𝑥]

[𝑟𝑒𝑑]

R (gas constant) = 8.314 J K−1 mol−1; F (Faraday constant)

= 9.649×104 C mol−1; n is the number of moles of electrons

211

Redox Titrations

Heterodisulfide Reductase/Hydrogenase Complex:

• Found in the cytosol of some Methanogens

• Electrons from hydrogen are used to break down the

heterodisulfide CoB-S-S-CoM which allows the reuse of

both cofactors.

• A proton gradient is generated in this process

• Except for one, all clusters are involved in electron

transport.

212

54

Redox Titrations

One unique cluster can either bind CoM or CoB

Labeling studies

with H33S-CoM

(33S: I = 3/2)

213

Redox Titrations

Cluster with bound

HS-CoM behaves like a

[4Fe-4S]2+/3+ cluster

(paramagnetic when

oxidized)

Em = -60 mV, pH 7.6, n = 1

214

7) Freeze-Quench Experiments

• To follow a reaction involving

paramagnetic species freeze-

quench experiments can be

performed.

• In this experiment enzyme is

mixed with substrate and

other compounds and EPR

samples are made by rapid

mixing and freezing.

• Multiple samples have to be

made to get insight into the

formation/disappearance of an

EPR signal.

215

Role of IspG in Isoprene Synthesis

216

55


• IspG contains a single

[4Fe-4S] cluster.

• The cluster is very

unstable.

• Cluster falls apart when

exposed to molecular

oxygen.

• Instability probably

caused by incomplete

coordination.

217

Cys

Cys

Cys

Cys

• The reaction is a reductive elimination of a hydroxyl

group involving 2 electrons.

• A [4Fe-4S] cluster can only donate 1 electron at-a-time.

• Formation of radical species expected.


218

Freeze-Quench Experiments with IspG

• A transient isotropic signal is detected with maximal intensity

at 90 ms.

• A transient rhombic signal, FeSA, reaches maximal intensity

at 30 s.

• A second rhombic signal, FeSB, accumulates over time and

reaches maximal intensity at 4 min. 219

Freeze-Quench Experiments

radical

speciesFeSA

FeSB

220

56

A) EPR spectrum obtained for IspG upon incubation with

the substrate MEcPP and the reductant dithionite

B) Proposed structure for the reaction intermediate

ENDOR

221

2D-HYSCORE

13C and 17O HYSCORE Spectra

of the FeSA species in IspG

222

• There are several reason why you might

want to simulate an EPR signal.

• For example to obtain the intensity of a

particular signal in a mixture of signals.

Spectrum

Simulation

Difference

8) Simulation of EPR spectra

223

224

57

• Estimates of the effective g-values

Visual Rhombo

225

Simulation

Clostridium

pasteurianum

[2Fe-2S]2+

S = 9/2

(D < 0)

226

9) EPR on Whole Cells/Cell Extract

• CO2-reducing pathway

of methanogenesis,

which uses H2 and

CO2 as substrates.

• The reduction of CO2

to CH4 proceeds via

coenzyme-bound C1-

intermediates,

methanofuran (MFR),

tetrahydromethanopter

in (H4MPT), and

coenzyme M (HS-

CoM).

CO2

CHO MFR

CHO H4MPT

H4MPTCH

CH3 H4MPT

CH2 H4MPT

CH3 S CoM

CH4

MFR

H4MPT

H4MPT

CoMHS CoBHS+

S S CoBCoM

H2

H2

H2

H2

MFR

FormylmethanofuranDehydrogenaseHydrogenase

Methyl-H4MPT:coenzyme M

Methyltransferase

Methyl-coenzyme MReductase

HeterodisulfideReductase

227

EPR on Whole Cells

• Overview of all

paramagnetic species

• Behavior under

different growth

conditions

• Estimates of the

amount of species

present (simulations

and integration)

2800 3000 3200 3400 3600 3800

Field (Gauss)

5 K

20 K

70 K

228

58

EPR on Whole Cells

A: 80% H2/20% CO2

B: 80% N2/20% CO2

1) MCR (red2 form)

2) MCR (red1 form)

3) Hydrogenase (Ni-C form)

4) Heterodisulfide reductase

5) Hydrogenase (Ni-A form)

6) MCR (ox1 form)

280 290 300 310 320 330 340 350 360

2.4 2.3 2.2 2.1 2.0 1.9

Field (mT)

77 K

B

A

65

43

2

1

229

10) Site-Directed Spin Labeling (SDSL) EPR

Provides specific information on the location and

environment of an individual residue within large and

complex protein structures.

A. Motion: Determine rotational mobility of label at

different protein sites.

B. Accessibility: An amino acid can be on the surface of a

protein and accessible to water, or it can be placed

inside the structure and is less accessible or not

accessible at all. An amino acid can also be deep in the

membrane space.

C. Distance: Measure the distance between 2 or more

amino acids in one system or between systems. 230

The Technique

• Specific Cys residues are labeled with a spin label:

2,2,5,5-tetramethyl-1-oxyl-3-methyl

methanethiosulfonate (MTSL).

• Drawback: Cys residues have to be introduced in the

structure at the positions of interest. All other accessible

Cys residues need to be deleted.

231

Nitroxide Spin Labels

232

Hyperfine Interactions: TEMPO

59

Nitroxyl Lineshapes

• Conventional X-band

EPR spectra are

sensitive to rotational

motion in the range 0.1

to ~100 nsec.

• In the fast motional limit

(~0.1 nsec) three lines

of approximately equal

height are observed.

233Thomas Stockner (2105) Biochm. Soc. Trans. 43:1023-1032

Freely

tumbling

Weakly

immobilized

Strongly

immobilized

Nitroxyl Lineshapes

• In the motional

narrowing region the

three lines become

broader and since the

total intensity does not

change the line

amplitude decreases.

• These changes,

however, vary for each

of the three lines.

234

Freely

tumbling

Weakly

immobilized

Strongly

immobilized

Nitroxyl Lineshapes

gx=2.0091, gy=2.0061, gz=2.0023

The field shift between the X- and Z- orientations is

H=h/gx- h/gz hg/4~11G

I=1: Ax= 6.2, Ay = 6.3, Az=33.6

9.4 GHz

235

Nitroxyl Lineshapes

In the motional narrowing region, the dependence of the

width of an individual hyperfine line on the nuclear spin

state (mI) can be expressed as

ΔB(mI) = A + B mI + C mI2

The dependence of the linewidth on the nuclear spin state

(mI) indicates that each line will have a different width. The

‘A’ term broadens all lines equally; the ‘C’ term broadens

the low-and high-field lines but does not affect the center

line (for which mI = 0); the ‘B’ term is negative and causes

the high-field (mI = -1) line to broaden and the low-field (mI

= 1) line to narrow.236

60

Nitroxyl Lineshapes

237

As the molecule

tumbles, the smaller

splitting for mI = 0 is

averaged more

effectively than the

larger splittings, which

causes differences in

the linewidths of the

three hyperfine lines.

Nitroxyl Lineshapes

• Strongly immobilized

corresponds to the slow

motion limit (>100

nsec). The spectrum is

very similar to the

‘powder’ or ‘rigid limit’

spectrum that is

obtained for any

nitroxide in the absence

of rotational motion and

for a dilute powder or

frozen solution 238

Freely

tumbling

Weakly

immobilized

Strongly

immobilized

Nitroxyl Lineshapes

• High field EPR

spectroscopy is the g-

resolved spectroscopy,

the regions corresponding

to different orientations of

the magnetic axis relative

to the external magnetic

field do not overlap.

• Note that using a different

frequency will change the

time scale of the

experiment.

239

170 GHz

gx=2.0091, gy=2.0061, gz=2.0023

I=1, Ax= 6.2, Ay = 6.3, Az=33.6

A. Motion

Attachment of the spin label to even a small unstructured

peptide can result in some degree of motional restriction,

and this restriction increases significantly in the presence of

local secondary structure.

240

61

MTSL-15 AA peptide

The motion of the spin label side

chain is sensitive to tertiary contacts

and protein structure in the local

environment of the spin label.

A. dilute solution of MTSL fast

motional limit (~0.1 nsec)

B. attached to 15 AA peptide with

random coil structure

C. attached to same peptide with an

α-helical structure

D. No motion (slow motion limit,

>100 nsec) or in frozen solution241

Spin Label Mobility Based on EPR Spectra Line Shape

(A) motionally restricted spin label of a buried side chain of

a helix and (B) increased mobility of an exposed side chain

of the same helix forming β spectrin lipid-binding domain

The separation between the outer hyperfine extrema (2Apar)

and the peak-to-peak separation of the central line

width (ΔH0) provide a measure of label mobility.

242Czogalla, A. (200&) Acta Biochim Polonica 54: 235-244

Spin Label Mobility Based on EPR Spectra Line Shape

(C) Mobility map constructed as a plot of the inverse

second moment of the EPR spectrum (<H2>–1) versus

inverse central line width (ΔH0–1), which indicates the

correlation between the measured parameters and regions

of protein topology.

243

Mobility Map

244

62

B. Accessibility

An amino acid can be located on a solvent-exposed

surface, buried within a protein, or within a membrane

bilayer.

245

Power Saturation Studies

• Under nonsaturating conditions, the amplitude of the

spectral lines are proportional to the incident microwave

power, increasing linearly with the square root of the

incident power, 𝑃 or P1/2.

• Under saturation conditions the increase becomes less

than linear.

• When paramagnetic relaxation reagents interact with the

spin label, they enhance the relaxation rate and allow the

sample to absorb more power before becoming

saturated.

246


Three common reagents:

Oxygen

• small and hydrophobic

• generally found in the center of lipid bilayers of

membranes and in hydrophobic pockets of proteins

• Only present to a small extent in solution

Ni(II) ethylendiaminediacetate (NiEDDA)

• Neutral/Water soluble

Chromium Oxalate (CROX)

• Negative/Water soluble

247


248

The spin label is in a water-

soluble environment. CROX

and NiEDDA prevent

saturation (in comparison

with the N2 curve), while O2

does have a much smaller

effect.

63

T4 lysozyme

• Oxygen accessibility

and probe mobility

were measured as a

function of sequence

number for spin

labels attached to T4

lysozyme (T4L) and

cellular retinol binding

protein (CRBP).

249

• The correlation between the two parameters indicates

that the most mobile sites are also the most oxygen

accessible.

• The repeat period of about 3.6 for T4L is consistent with

the α-helical structure of this segment of the protein.

Depth Parameter

250

• [O2] is highest in the center of the membrane and lowest

at the surface. The opposite is true for the [NiEDDA].

• The natural log of this ratio yields Φ, a parameter with a

linear dependence on depth into the bilayer.

C. Distances

• Measure the distance between 2 spin labels.

• This can be intramolecular distances between two labels

in the same monomer or intermolecular distances

between sites on different proteins.

251

• Binding processes,

conformational changes

• In the range ~8-20 Å,

interactions between

the two paramagnets

give rise to distance-

dependent line

broadening in CW EPR.

Example: spin labeled Gramicidin A

3/2 re

dipolar, MHz,

2/][]Å[

102.53

4

MHzr

dipolar

Å.930Interspin distance=

Distances: Pulse EPR

DEER (Double Electron-Electron Resonance) is a pulse EPR technique

that measures the dipolar frequency between two spins. Deer can

cancel out all interactions resulting in an EPR spectrum except the

dipole interaction in spin pairs. The dipolar Pake pattern shown is an

FT of the DEER echo.

252

64

Solutions

253

Radicals

1) Frequency = 9500 MHz

B-min = 3300 Gauss

B-max = 3500 Gauss.

g = 2, W = 3, A = 0

2) I-spin = 2/2

A-value = 25 Gauss

A-value = 15 Gauss

A-value = 5 Gauss

A-value = 2 Gauss

.

254

As the value of A come close to the line width of the signal, the

hyperfine lines start to cancel each other (A = 5, green line) or only a

small broadening of the signal is detectable which is called unresolved

hyperfine splitting (A = 2, purple line). The A = 0 spectrum is overlain

(dash/dot) for comparison

Effect of Deuterium

3) Three protons (I = ½) with A = 40 G

4) Three deuterium (I = 1) with A = 12.4 G.

255

Unresolved Hyperfine Effects

5) Proton 1: A = 20 G

Proton 2: A = 10 G

Proton 3: A = 5 G

Just as in exercise 2, the splitting between the lines is very close to

the line width. (You can try a much smaller line width (like 1 or 0.5

Gauss) to show the complete hyperfine pattern

256

65

Hyperfine Patterns

6) Frequency, 9500 MHz; B-min, 3300 Gauss; B-max, 3500 Gauss.

Rh-diazo radical: Two N interactions with identical A.

For nitrogen 1: A-value = 10 G


MNP-C•: Interaction with 1 N (large A) and 1 H (smaller A)

For nitrogen: A-value = 15 G

For hydrogen: A-value = 4 G

257

Hyperfine Patterns

DMPO-C•: Interaction with 1 H (large A) and 1 N (smaller A)

For nitrogen: A-value = 30 G


DMPO-N•: Similar interaction with 1 H (large A),

1 N (smaller A) and 1 N (even smaller A)




258

Ethanol Radical

7) Signal is split into four due to the three Hβ. There is an

additional split into two due to the single Hα.259

CH3CH•OH

Hyperfine Patterns

• The first multiplet (A) induces a small two-fold split due to one I = ½

nucleus

• The second (B) is a triplet due to two equal I = ½ nucleus

• The third (C) is again a two-fold split due to one I = ½ nucleus

• The fourth (D) is a triplet due to a I = 1 nucleus260

66

Hyperfine Patterns

9) B-min: 3100 gauss, B-max: 3700 gauss.

For Co: A-value = 50 G, for nitrogen: A-value = 20 G, for

hydrogen: A-value = 10 G261

Dimethyl Nitroxyl Radical.

• Frequency = 9766.13 MHz, B-min = 3380 gauss, B-max

= 3580 gauss.

• g = 2.005, W = 0.7, AN = 17.0 (1x), AH = 14.8 (6x)

• To get the pattern right AN needs to be just a bit larger

than AH

262

Simulations – Not Enough Orientations

263

Simulations – Not Enough Orientations

264

67

2Fe Ferredoxin

3200 3300 3400 3500 3600 3700 3800

9630.00/3005/4005

Gxyz

: 2.0115, 1.9605, 1.9270

Wxyz

: 9, 13.5, 12.5

Field (Gauss)

Cp2Fe_spec

simulation

Cp 2Fe Fd

265

Overshoot

Overshoot

266

Cobalamin

250 275 300 325 350 375 400 425

Field (mT)

2.3

1.8

9

X-band

Q-band

gxyz = 2.275, 2.220, and 2.006Wxyz = 25, 25, and 7.0ACo

xyz = 11, 11, and 111AN

xyz = 18, 18, and 18

267

Simulations

268

68


4 x S=1

In this structure the

interaction with four

nitrogen ligands (I = 1)

would result in 9

superhyperfine lines.

269

Simulations

2900 3000 3100 3200 3300 3400

Field (Gauss)

MCRox1

MCRred1

1

2

34

5

6

78

9

10

270

Simulations

MCRox1 gxyz = 2.2305, 2.1665, and 2.1530.Wxyz = 3.5, 4.0, and 4.0AN

xyz = 8.0, 9.7, and 9.7

MCRred1 gxyz = 2.2495, 2.0720, and 2.0625.Wxyz = 4.5, 3.5, and 5.0AN

xyz = 8.8, 9.9, and 9.9 271

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Wilfred R. Hagen Introduction to Biomolecular Electron ...duinedu/summercourse/eprslides.pdf · Introduction to Biomolecular Electron Paramagnetic Resonance Theory E.C. Duin 1