Physical Methods in

Bioinorganic Chemistry

1
The electronic
properties and high
electron densities of
metal ions as a class of
functional groups
render them especially
amenable to study by
physical techniques
such as EXAFS,
Mössbauer, EPR, and
magnetic susceptibility.
 Magnetic Resonance Methods
When a particle with a magnetic moment is placed in a magnetic field, the states that
have this moment oriented in different directions with respect to the applied field have
different energies. Transitions between these states may be induced by the absorption of
light of the appropriate frequency. This phenomenon is called magnetic resonance.

Electron Paramagnetic Resonance Nuclear Magnetic Resonance

(EPR) (NMR)
If the particles are electrons, the If the particles are nuclei, the
appropriate light frequencies are in appropriate frequencies are in the
the microwave range. radiofrequency region.
 Nuclear Magnetic Resonance
Nuclear magnetic resonance (NMR) is a pre-eminent technique for molecular-level
understanding because it exquisitely displays differences in and connections between
chemical environments of atoms in a molecule. Many biologically relevant nuclei – 1H, 13C,
31P, 15N, 14N, 2H, 23Na, 17O, 19F and more – can be usefully observed by NMR and only a few

micrograms are sufficient for proton NMR work. In the past 25 years with vastly improved
sensitivity and sophistication of imaging capability, numerous areas of biological sciences
have taken advantage of the power of NMR to observe selected components of organisms,
both in vivo and in vitro.
Intensity: number of
1H NMR of ethylbromide: chemically 0 ppm reference
equivalent nuclei compound
Line splitting:
Through bond
Chemical shift coupling between
(ppm): chemically different
Every chemically nuclei
different nucleus will
have a different
chemical shift
depending on its
chemical nature
 Nuclear Magnetic Resonance
NMR studies of metal nuclei can provide useful biochemical information.
The most useful are metal nuclei having a spin I = ½ for which no quadrupolar line
broadening occurs. However, the very low resonance frequencies of most naturally
occurring metal isotopes lead to poor sensitivity and inability to observe signals at
achievable protein concentrations. Moreover, apart from 57Fe, which suffers from a low
sensitivity, all metal nuclei are quadrupolar (I > ½) and are therefore susceptible to line
broadening:

Natural Natural
Element Spin Element Spin
Abundance (%) Abundance (%)
23Na 3/2 100 57Fe 1/2 2.19
25Mg 5/2 10.13 59Co 7/2 100
39K 3/2 93.10 61Ni 3/2 1.19
43Ca 7/2 0.145 63Cu 3/2 69.09
53Cr 3/2 9.55 67Zn 5/2 4.11
55Mn 5/2 100 99Tc 9/2 Radioactive
 Nuclear Magnetic Resonance
39K NMR surface/free K+ cations

• 39K (93.12%) en 41K (6.82%)

• quadrupolar.
• low sensitivity compared to 7Li and 23Na NMR

channel K+ cations

Variable temperature 39K NMR spectra of 0.53 M

Na2(5’-GMP) in presence of 0.10 M K+
 Nuclear Magnetic Resonance
43Ca NMR calcium bound to the
single high-affinity
calcium site

• 43Ca (0.145%)
• spin I = 7/2
• quadrupolar

43Ca NMR spectra of the titration of 0.33 mM equine apolysozyme with 43Ca2+ at pH 6.0:
(a) 0.23 equiv. of 43Ca2+; (b) 0.46 equiv. of 43Ca2+; (c) 0.69 equiv. of 43Ca2+; (d) 0.92 equiv. of
43Ca2+; (e) 1.15 equiv. of 43Ca2+.
 Nuclear Magnetic Resonance
59Co NMR
• 59Co (100%)
• spin I = 7/2
• quadrupolar

59Co NMR spectra of (A) vitamin B12, (B) B12 coenzyme, (C) methylcobalamin and (D)
dicyanocobyrinic acid. FWHH = full with at half height.
 Nuclear Magnetic Resonance
Other biochemically useful nuclei, except for Fe, which have spin I = ½ are 111Cd, 113Cd,
203Tl, 205Tl, 195Pt, and 207Pb. Examples include the 195Pt NMR (33.83% natural abundance)

structural studies of platinum anticancer drugs and their adducts with DNA and 113Cd NMR
spectral investigations of metallothionein:
Cisplatin

Time dependent 195Pt NMR spectra of the reaction between cisplatin and chicken-
erythrocyte DNA at 37 °C. As cisplatin is consumed, new resonances appear at 150–220
ppm and 300–350 ppm upfield of the cisplatin resonance. The chemical shifts of these
resonances were consistent with the presence of mono- and bifunctional adducts.
 Nuclear Magnetic Resonance
Metallothioneins constitute a class of ubiquitously occurring low molecular mass proteins
(6–7 kDa) possessing two cysteine thiolate-based metal clusters (usually formed by the
preferential binding of d10 metal ions such as Zn(II) and Cd(II). The three-dimensional
solution structure of mammalian proteins has been determined by two-dimensional NMR
spectroscopy of 113Cd7-metallothionein. The structure shows two protein domains
encompassing the M3(CysS)9- and M4(CysS)11-cluster with each metal ion being
tetrahedrally coordinated by thiolate ligands. The application of 113Cd NMR (22.19% natural
abundance) proved to be indispensable in the structural studies of metallothioneins:

M4(CysS)11-cluster M3(CysS)9-cluster

Biodegradation, 1998, 9, 501.

 Nuclear Magnetic Resonance

Each of the seven Cd ions (I to VIII) is represented by an individual 113Cd NMR peak:

V, VI

II VII
III IV
I

One-dimensional 113Cd NMR (1H-decoupled) spectrum at 27 °C of isolated recombinant sea

urchin Cd7-MT at natural abundance. The 113Cd signals in are numbered according to
decreasing chemical shift.
 Nuclear Magnetic Resonance
Subsequent 113Cd-113Cd COSY provided the information about the cluster organization in
this protein. This information has been derived from the cross-peak pattern. Thus, the cross
peaks associated with 113Cd resonances IV, V, VI, and VII and those with resonances I, II and
III suggested the presence of a two cluster structure.

Two-dimensional homonuclear 113Cd COSY spectrum at 27 °C of sea urchin 113Cd7-MT.

 Nuclear Magnetic Resonance
Until a decade agon, metalloproteins containing paramagnetic metal ions were not thought
to be suitable for the application of NMR techniques because the presence of
paramagnetic centers destroys the resolution of the spectrum.
However, this broadening of the signals is less severe when the paramagnetic center
exhibits a fast electronic relaxation. As a result: nearby proton resonances can still be
recognized. The longer the distance between the proton and the paramagnetic center, the
smaller the shift:
+Eu3+

-Eu3+
 Nuclear Magnetic Resonance
The replacement of diamagnetic and NMR-silent (I = 0) metal ions by suitable paramagnetic
metal ions can provide additional structural and mechanistic information, such as, the
identification of amino acid side chain ligands. For example, the incorporation of
lanthanides in Ca2+ binding sites of proteins:

Overlap of 15N–1H HSQC spectra of CaM-CD2-III-5G-52 at a selective region at different La3+

concentrations in 20 mM PIPES-20 mM KCl, pH 6.8. Upon the addition of La3+, E67 moves
upfield and the intensity of D58 decreases, while I114 (corresponding to I97 in CD2) and
T37 have only minor changes.
J. Inorg. Biochem. 2005, 99, 1376.
 Mössbauer Spectroscopy
The Mössbauer effect as a spectroscopic method probes transitions within an atom’s
nucleus and therefore requires a nucleus with low-lying excited states. The effect has
been observed for 43 elements. For applications in biochemistry, the 57Fe nucleus has the
greatest relevance. Unfortunately, the low natural abundance of 57Fe (2.19%) necessitates
the enrichment either by reconstitution of the metalloprotein from the apo form or, if this
route is not feasible, by isolation from a growth medium containing 57Fe enriched iron
salts. Other possible nuclei are 40K, 61Ni, and 67Zn.

Mössbauer spectroscopy requiers (a) the emission of γ-rays from the source element in a
excited nuclear state and (b) the absorption of these γ-rays by the same element in the
sample under study.
 Mössbauer Spectroscopy
Mössbauer spectroscopy probes high-energy transitions in the atomic nucleus and is based
on the phenomenon of recoil-free γ-ray resonance absorption.
The energy levels that are being probed in Mössbauer spectroscopy are influenced by their
surrounding environment, both electronic and magnetic, which can change or split these
energy levels. These changes in the energy levels can provide information about the atom's
local environment within a system and ought to be observed using resonance-fluorescence.

Environment dependent electronic and magnetic interactions result is a splitting of the

energy levels: Mössbauer spectroscopy is an excellent tool to probe the molecular
environment of the metal ion under study.
 Mössbauer Spectroscopy
There are, however, two major obstacles in obtaining this information: the 'hyperfine'
interactions between the nucleus and its environment are extremely small, and the recoil
of the nucleus as the gamma-ray is emitted or absorbed prevents resonance.
Under normal conditions, atomic nuclei recoil (transfer of momentum) when they emit or
absorb gamma rays, and the wavelength varies with the amount of recoil.
In a free nucleus during emission or absorption of a gamma ray it recoils due to
conservation of momentum, just like a gun recoils when firing a bullet, with a recoil energy
ER. The emitted gamma ray has ER less energy than the nuclear transition but to be
resonantly absorbed it must be ER greater than the transition energy due to the recoil of
the absorbing nucleus. To achieve resonance the loss of the recoil energy must be
overcome in some way.
 Mössbauer Spectroscopy

As the atoms will be moving due to random thermal motion the gamma-ray energy has a
spread of values ED caused by the Doppler effect. To produce a resonant signal the two
energies need to overlap and this is shown in the red-shaded area. This area is shown
exaggerated as in reality it is extremely small, a millionth or less of the gamma-rays are in
this region, and impractical as a technique.

Recoil Effect

Doppler Effect

Transition Energy
 Mössbauer Spectroscopy
Rudolf Mössbauer found that at a sufficiently low temperature, a significant fraction of
the nuclei embedded in a crystal lattice may emit or absorb gamma rays without any
recoil.

The strictly monochromatic gamma radiation emitted from the excited nucleus of a
suitable isotope during a radioactive decay pathway can therefore be absorbed by the
same isotope in the sample.
 Mössbauer Spectroscopy
Mössbauer spectroscopy is limited by the need for a suitable gamma-ray source. Usually,
this consists of a radioactive parent that decays to the desired isotope. For example, the
source for 57Fe consists of 57Co, which decays by electron capture to an excited state of
57Fe, then subsequently decays to a ground state emitting the desired gamma-ray (14.41

keV).
The radioactive cobalt is prepared on a foil, often of rhodium. Ideally the parent isotope
will have a sufficiently long half-life to remain useful, but will also have a sufficient decay
rate to supply the required intensity of radiation.
 Mössbauer Spectroscopy
So far we have seen one Mössbauer spectrum: a single line corresponding to the
emitting and absorbing nuclei being in identical environments. As the environment of the
nuclei in a system we want to study will almost certainly be different to our source the
hyperfine interactions between the nucleus and the its environment will change the
energy of the nuclear transition. To detect this we need to change the energy of our
probing gamma-rays
The energy changes caused by the hyperfine interactions we will want to look at are very
small, of the order of billionths of an electron volt. Such miniscule variations of the
original gamma-ray are quite easy to achieve by the use of the Doppler effect. In the same
way that when an ambulance's siren is raised in pitch when it's moving towards you and
lowered when moving away from you, our gamma-ray source can be moved towards and
away from our absorber. This is most often achieved by oscillating a radioactive source
with a velocity of a few mm/s and recording the spectrum in discrete velocity steps.
With an oscillating source we can now modulate the energy of the gamma-ray in very
small increments. Where the modulated gamma-ray energy matches precisely the energy
of a nuclear transition in the absorber the gamma-rays are resonantly absorbed and we
see a peak.
 Mössbauer Spectroscopy

The absorption peak occurs at 0 mm/s, where

source and absorber are identical.

For convenience the energy scale of a Mössbauer spectrum is thus quoted in terms of the
source velocity
 Mössbauer Spectroscopy
The energy levels in the absorbing nuclei can be modified by their environment in three
main ways: by the Isomer Shift (IS), Quadrupole Splitting (QS), and Magnetic Splitting.
 Mössbauer Spectroscopy
The isomer shift (δ) is a measure of the s-electron density at the iron nucleus. Changes in
the s population of the valence shell or shielding effects caused by increasing or decreasing
p or d electron density influence this parameter. Any difference in the s-electron
environment between the source and absorber thus produces a shift in the resonance
energy of the transition. This shifts the whole spectrum positively or negatively depending
upon the s-electron density, and sets the center of the spectrum.
As the shift cannot be measured directly it is quoted relative to a known absorber. For
example 57Fe Mössbauer spectra will often be quoted relative to alpha-iron at room
temperature.
 Mössbauer Spectroscopy

The isomer shift is useful for determining oxidation states, spin states, ligand bonding
states, electron shielding and the electron-drawing power of electronegative groups.
For example, the electron configurations for Fe2+ and Fe3+ are (3d)6 and (3d)5 respectively.
The ferrous ions, Fe(II) have less s-electron density at the nucleus due to the greater
screening of the d-electrons. Thus ferrous ions Fe(II) have larger positive isomer shifts than
ferric ions Fe(III):
 Mössbauer Spectroscopy

Quadrupolar splitting: Nuclei in states with an angular momentum quantum number

I>1/2 have a non-spherical charge distribution. This produces a nuclear quadrupole
moment. In the presence of an asymmetrical electric field (produced by an asymmetric
electronic charge distribution or ligand arrangement) this splits the nuclear energy levels.
In the case of an isotope with a I=3/2 excited state, such as 57Fe , the excited state is split
into two substates mI=±1/2 and mI=±3/2.
 Mössbauer Spectroscopy

The quadrupole splitting of the signal, ΔEQ , which reveals the assymmetry of the electric
field surrounding the metal center.
In general, the higher the symmetry of the electric field around the nucleus, the smaller
the value of ΔEQ. For instance, ΔEQ (Fe(II) > ΔEQ (Fe(III) for the high spin case because d5
high-spin Fe(III) (S=5/2) has spherical symmetry and the d6 high-spin Fe(II) (S = 2)does not.
 Mössbauer Spectroscopy
Comparison of ΔEQ values for heme ligands confirm this trend. For high spin heme Fe(III)
we have ΔEQ = 0.5-1.5, while for high –spin Fe(II) heme we have ΔEQ = 1.5-3.0.
 Mössbauer Spectroscopy
Mössbauer spectra for a model system of the iron core in iron-oxo proteins which contain
short μ-oxo-iron(III) bonds. This leads to an axial distortion of the electric field, producing
large quadrupole splittings (≈ 1.5-2.0 mm sec-1). Protonation of this μ-oxo bond to from μ-
hydroxo species lengthens the Fe-O distance to ≈ 1.96 Å with a concomitant diminuation in
ΔEQ to ≈ 0.5 mm sec-1.

δ δ

HYDROXO-bridged
OXO-bridged
 Mössbauer Spectroscopy
Magnetic hyperfine interactions: In the presence of a magnetic field the nuclear spin
moment experiences a dipolar interaction with the magnetic field ie Zeeman splitting.
This magnetic field splits nuclear levels with a spin of I into (2I+1) substates. Transitions
between the excited state and ground state can only occur where mI changes by 0 or 1. This
gives six possible transitions for a 3/2 to 1/2 transition.
 Magnetic Circular Dichroism

Both Circular Dichroism (CD) and MCD spectroscopies measure the difference in
intensity between left and right circular polarized light. However, in contrast to
CD, MCD spectroscopy is performed in a strong magnetic field parallel to the
direction of propagation of the circular polarized light. Typical experimental
conditions for LT MCD measurements are temperatures between 1.5 - 30 K (liquid
helium) and magnetic fields of 0 - 7 T.

Magnetic circular dichroism (MCD) is the differential absorption of left and right
circularly polarized (LCP and RCP) light, induced in a sample by a strong magnetic
field oriented parallel to the direction of light propagation.
MCD measurements:
§ can detect transitions which are too weak to be seen in conventional optical
absorption spectra.
§ can probe paramagnetic properties and the symmetry of the electronic levels of
the studied systems, such as metal ion sites.
 Magnetic Circular Dichroism
The major difference between MCD spectroscopy, on the one hand, and the most
widely used spectroscopic techniques that are based on an applied field, such as
NMR and EPR spectroscopy, on the other, is that MCD spectroscopy is not based
on resonance between spin states, but is instead based on the wavelength
dependent absorption of circularly polarized light to form electronic excited
states. The UV-visible absorption and MCD spectra of a molecular complex,
therefore, contain the same set of spectral bands, but the band morphologies are
different:
 Magnetic Circular Dichroism
In the 1840s, Michael Faraday observed that plane polarized beams of light are rotated
during transmission through certain substances by the application of an applied magnetic
field, due to the differential absorbance of lcp and rcp light which is induced by the applied
field.
In contrast with natural CD, which is relatively rare, the Faraday effect is a property of all
matter. MCD spectroscopy, which is the spectroscopic method normally used to study
magnetic optical activity, therefore differs significantly from CD spectroscopy, since a signal is
observed even for compounds that lack a chiral centre.
 Magnetic Circular Dichroism

The MCD signal ΔA is derived via the absorption of the LCP and RCP light as:

with A- and A+ the absorption coefficients for right and left circularly polarized
light, respectively. The spectra are a representation of ΔA vs wavelength. Often,
ΔA is recorded as a function of the applied field (up to ) and the temperature.
 Example
“De Novo Design, Synthesis and Characterisation of MP3, A New Catalytic Four-
Helix Bundle Hemeprotein”
Chem. Eur. J. 2012, 18, 15960 – 15971

A new artificial metalloenzyme, MP3 (MiniPeroxidase 3), designed by combining

the excellent structural properties of four-helix bundle protein scaffolds with the
activity of natural peroxidases, was synthesised and characterised.
CD measurements revealed the high helix-forming propensity of the peptide,
confirming the appropriateness of the model procedure.
UV/Vis, MCD and EPR experiments gave insights into the coordination geometry
and the spin state of the metal.
Kinetic experiments showed that FeIII–MP3 posseses peroxidase-like activity
comparable to R38A–hHRP, highlighting the possibility of mimicking the functional
features of natural enzymes.
 Magnetic Circular Dichroism
Miniaturisation design process. a) A cartoon representation of a dimer subunit of
bacterioferritin [PDB code 1BRF]; the heme group and metal ions are represented with
space filling, and the coordinating residues are highlighted as sticks. b) Heme-facing helices
of BFR used as a scaffold to design MP3: residues 14–28 and 44–58 were connected by a
loop (in red) to obtain a minimal a2/heme/a2 structure.

Chem. Eur. J. 2012, 18, 15960 – 15971

 Magnetic Circular Dichroism
Structure of bacterioferritin (BFR), a natural protein that forms a roughly spherical, hollow
shell from 12 dimeric identical subunits. Dimeric subunits are composed of two four-helix
bundle each containing a di-iron site, and incorporate a heme group with a methionine
from each monomer providing the axial ligand (Figure 1 a).
Focused on the heme-facing helices of BFR. Each helix in BFR is about 30 residues long and
the connecting loop in the helix–loop–helix motif (α2-motif) is three residues long (red on
Figure 1b)). To downsize this structure, the minimum number of residues necessary to
completely cover heme and to allow a juxtaposition of the connecting three-residue loop
was selected. A “rose-like” αL-β loop was engineered to obtain a single chain from two
antiparallel helices(Figure 1 b).

Chem. Eur. J. 2012, 18, 15960 – 15971

 Magnetic Circular Dichroism
The sequence of BFR was
modified, by defining key residues
firstly to covalently bind the
porphyrin to the peptide chain,
and secondly for the construction
of the distal and proximal sites:
§ protoporphyrin IX was
substituted with deuteroporphyrin
IX
§ one of the two coordinating Met
residues was replaced by a
histidine, mimicking the HRP
proximal site, and a position along
the helices was selected to insert
an aspartate residue, forming an
ion pair with the coordinating
histidine.

Chem. Eur. J. 2012, 18, 15960 – 15971

 Magnetic Circular Dichroism
§ The second Met residue was
replaced by the smaller serine
residue, which is unable to
coordinate the iron ion, to create a
distal site. A position along the
helices was selected to insert an Arg
residue as found in the distal site of
HRP (corresponding to Arg38 of
HRP). The distal site was then
completed with smaller residues to
create a cavity large enough to
accommodate an organic substrate,
and to leave sufficient
conformational mobility for the
catalytic Arg to bind and transport
the peroxide ion to the active site,
as observed in natural peroxidases.

Chem. Eur. J. 2012, 18, 15960 – 15971

 Magnetic Circular Dichroism
To gain more structural insights into the species involved in the heme coordination, and to
compare them with natural proteins, MCD and EPR spectroscopy was used.

a) Soret and b) visible regions of the MCD spectra of FeIII–MP3 in water at different pH
values, at 25 °C.

Chem. Eur. J. 2012, 18, 15960 – 15971

 Magnetic Circular Dichroism

To investigate the axial ligation in FeIII–MP3 and the high-spin ↔ low-spin

alkaline transition, MCD experiments were performed at different pH values.
The MCD spectroscopy has been frequently used to characterize
metalloporphyrins because of its sensitivity to changes in axial ligation, spin state,
and the oxidation state of heme complexes.
Ligation assignments are made through comparisons of the spectra of the
structurally uncharacterized hemeprotein with those of structurally defined heme
centres.
MCD spectra of FeIII–MP3 were compared with metMb (oxidized form of
Myoglobine, having FeIII; and ferric (FeIII) -horse radish peroxidase, both of
which also have a proximal histidine ligand.

Chem. Eur. J. 2012, 18, 15960 – 15971

 Magnetic Circular Dichroism
The clearest correlation between the heme electronic state and the MCD spectrum is seen
in the Soret spectra in which an intense derivative-shaped MCD curve is associated with
low-spin forms, whereas high-spin forms show a much weaker MCD spectrum.
By increasing the pH from 2.0 to 4.8, the binding of the proximal histidine alters the high-
spin ↔ low-spin equilibrium towards the latter. Nevertheless, the high-spin form remains
predominant at high pH.

The MCD spectrum of FeIII–

MP3 at pH 2.0 is
characteristic of ferric high-
spin hemeproteins

Chem. Eur. J. 2012, 18, 15960 – 15971

Evidence for the pentacoordinate high-spin complex is further confirmed from the visible
region of the MCD spectra.

At pH 4.8 and 6.7, the spectra show a dominant trough at 568 nm, which is also the
dominant feature of high-spin ferric-HRP. At intermediate pH values the progressive
redshift of this trough is consistent with a change in the proximal axial ligation but a very
small trough at 585 nm is observed at pH 9.3. This is typical of low-spin alkaline HRP (a
histidine/hydroxyl ligated heme). This main difference suggests that, at room temperature,
FeIII–MP3 does not undergo the alkaline transition from the high-spin form at neutral pH
to the low-spin form at alkaline pH.

Chem. Eur. J. 2012, 18, 15960 – 15971

To further prove the absence of the alkaline pH-dependent high-spin ↔ low-spin
transition, low-temperature EPR spectroscopy was used. The results show the presence of
the high-spin form at all of the investigated pH values.

The spectra show a pattern of signals

with g values around 5.9 and 2.0, which
are typical of a (near)-axial, high-spin
species, at all pH values. This species is
also predominant at pH 11.8, and no low-
spin signal in the region around g≈3 is
observed.

Chem. Eur. J. 2012, 18, 15960 – 15971