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TRANSCRIPT
The strength of EPR and ENDOR techniques in revealing
structure–function relationships in metalloproteins
Sabine Van Doorslaer* and Evi Vinck
Received 1st February 2007, Accepted 11th May 2007
First published as an Advance Article on the web 6th June 2007
DOI: 10.1039/b701568b
Recent technological and methodological advances have strongly increased the potential of
electron paramagnetic resonance (EPR) and electron nuclear double resonance (ENDOR)
techniques to characterize the structure and dynamics of metalloproteins. These developments
include the introduction of powerful pulsed EPR/ENDOR methodologies and the development of
spectrometers operating at very high microwave frequencies and high magnetic fields. This
overview focuses on how valuable information about metalloprotein structure–function relations
can be obtained using a combination of EPR and ENDOR techniques. After an overview of the
historical development and a limited theoretical description of some of the key EPR and ENDOR
techniques, their potential will be highlighted using selected examples of applications to iron-,
nickel-, cobalt-, and copper-containing proteins. We will end with an outlook of future
developments.
1. Introduction
Since the introduction of electron paramagnetic resonance
(EPR) spectroscopy in 1944 by Zavoisky1 and of the related
electron nuclear double resonance (ENDOR) spectroscopy in
1956 by Feher,2 both techniques have been applied extensively
in the fields of biology, medicine, chemistry, physics and
material sciences. The techniques can be used to determine
the structure and dynamics of paramagnetic systems (i.e.
systems containing one or more unpaired electrons), and can
also be applied to diamagnetic systems using spin probes3 or
spin labels.4 Until the end of the 1980s, the vast majority of the
EPR/ENDOR studies were performed using continuous wave
(CW) techniques at X-band microwave (mw) frequencies
(B9.5 GHz). Although the use of these CW-EPR and
ENDOR techniques have proven to be invaluable for many
analyses, the amount of information that can be obtained is
limited by factors inherent to the CW approach. In the 1960s,
it was shown that the complementary use of pulsed EPR and
ENDOR may overcome these problems,5–7 but it was only
with the introduction of fast electronics and the commerciali-
zation of pulsed-EPR/ENDOR spectrometers in the late 1980s
that these techniques started to be applied on a larger scale.
Since then, the field of EPR has been revolutionized by new
developments both in pulsed-EPR methodology and in types
of applications, similar to the evolution observed in the field of
nuclear magnetic resonance (NMR), EPR’s (more famous)
sister technique. Furthermore, the progresses in EPR have
been paralleled by rapid instrumental developments, amongst
which is the construction of so-called ‘high-field EPR spectro-
meters’. These spectrometers operate at mw frequencies of
95 GHz and higher, and their development was initiated by the
seminal work of Lebedev.8 Details on the new methodological
and instrumental developments in EPR and on various appli-
cations are reported in the reference book on pulsed EPR by
Schweiger and Jeschke9 and in recent review papers,10–19
including two recent Invited Articles in this journal on the
applications of high-field EPR18 and high-field ENDOR.19 It
should be noted that EPR and ENDOR are by no means
routinely applicable techniques and the interpretation of EPR
and ENDOR spectra is often very complex.
For decades, EPR has been one of the methods of choice to
study paramagnetic metalloproteins. Indeed, many of the
metal ions binding to proteins are paramagnetic: Fe(III),
Cu(II), Ni(I), Ni(III), Co(II), Mn(II), VO(II), etc., and thus form
ideal intrinsic probes for EPR. The recent instrumental and
methodological developments in EPR and ENDOR have
made these techniques unparalleled tools for unravelling the
electronic and local geometric structure of the metal centre.
In this Invited Article, we will highlight some of the
possibilities of advanced EPR and ENDOR techniques for
the study of metalloproteins. The manuscript is structured as
follows. After an introduction to EPR and ENDOR spectro-
scopy, we show how the recent developments in EPR and
ENDOR techniques have given a new impetus to the elucida-
tion of metalloprotein structure and function. We hereby focus
on examples from our own research work, but extend the
discussion with work of others for those applications we feel
exemplify the field, even though they are not part of our
current research. We will conclude with indicating some of
the challenges for the future. Note that this overview is not
meant to be exhaustive and we apologize to our colleagues
whose work we may have inadvertently failed to quote.
SIBAC Laboratory, University of Antwerp, Universiteitsplein 1,B-2160 Wilrijk-Antwerp, Belgium. E-mail:[email protected]; Fax: +32 3 820 24 70; Tel: +32 3820 24 61
4620 | Phys. Chem. Chem. Phys., 2007, 9, 4620–4638 This journal is �c the Owner Societies 2007
INVITED ARTICLE www.rsc.org/pccp | Physical Chemistry Chemical Physics
2. Theoretical background of EPR spectroscopy
2.1. What information can be obtained using EPR
spectroscopy?
The spin Hamiltonian, H, describing the unpaired electron(s)
coupled to nuclear spins in an external magnetic field, forms
the basis for understanding the outcome of EPR experiments.
For an unpaired electron (S = 1/2), coupled to different
nuclear spins (Ik), the spin Hamiltonian is given by9,20–23
H ¼ be ~B0gS=hþXk
ð ~SAkIk � bngn;k ~B0Ik=hÞ
þX
k;Ik41=2
~IkPkIk ð1Þ
where be is the Bohr magneton, h is Planck’s constant, bn is thenuclear magneton and B0 is the external magnetic field. We
make use of the symbol B to indicate the transpose of a
vector.
The g tensor in the field-dependent electron Zeeman term
(first term of eqn (1)) contains information about the local
symmetry of the paramagnetic site and its electronic state. The
hyperfine interactions between the unpaired electron and the
surrounding nuclear spins are described by the second Hamil-
tonian term in eqn (1). The hyperfine matrix A can be written
as the sum of the isotropic, Fermi contact interaction, aiso,
originating in the finite probability for the unpaired electron to
be found at the nucleus, and of the dipole–dipole coupling, T.
For some cases, in which the electron–nucleus distance, r, is
larger than 0.26 nm and the spin delocalization over the ligand
is negligible, the latter term can be approximated by a
point–dipolar interaction that allows for a direct determina-
tion of r,24
Tij ¼m04p
1
r3gnbnbegið3rirj � dijÞ ði; j ¼ x; y; zÞ ð2Þ
with rx, ry and rz the direction cosines defining the orientation
of the magnetic field in the g tensor frame.
For transition-metal centers, knowledge of the metal hyper-
fine tensor (for metal isotopes with nuclear spin) in combina-
tion with the g tensor often provides a fingerprint to identify
the metal ion, its oxidation state and the local symmetry of the
metal site.
The third term in eqn (1) represents the nuclear Zeeman
interactions, whereby the nuclear g factors, gn,k, are identifying
the type of nucleus interacting with the unpaired electron. The
nuclear Zeeman interaction can be considered isotropic for
most EPR experiments, although in some particular cases a
pseudo-nuclear Zeeman interaction needs to be considered
(see also section 3.1.1).
The fourth term in eqn (1) represents the nuclear quadru-
pole interaction found for nuclei with spins I 4 1/2. It reflects
the interaction between the non-spherical charge distribution
of the nucleus with the electric field gradient, eq, caused by the
electrons and nuclei in the environment of the nucleus. The
principal components of the traceless nuclear quadrupole
tensor, P, are usually described as
Pz ¼e2qQ
2Ið2I � 1Þh ; Z ¼Px � Py
Pzð3Þ
where Q is the nuclear electrical quadrupole moment, Z is the
asymmetry parameter, and |Px| o |Py| o |Pz|.
For high-spin systems with S 4 1/2, the spin Hamiltonian
in eqn (1) will be extended with a field-independent fine-
structure term, the zero-field splitting,
HZFS = SDS (4)
The D tensor is the traceless, symmetric zero-field interaction
describing the dipole–dipole coupling between the electron
spins and a contribution from the spin–orbit coupling. It gives
direct information on the ground state and spin state of the
system at hand.
Finally, if the unpaired electrons of two neighbouring
paramagnetic sites are weakly interacting, as can be found in
biradicals or in cases of high concentration of paramagnetic
molecules, the spin Hamiltonian will be the sum of the two
individual spins added with an exchange-coupling term and a
dipole–dipole coupling term, revealing information on the
spin–spin distances. More details on this matter can be found
elsewhere.9
In short, a set of EPR experiments can in principle provide
insight into the following structural characteristics of a
metalloprotein. The g tensor and the zero-field interaction
can reveal important information on the ground state, oxida-
tion state and local symmetry of the metal center. Knowledge
of the hyperfine interaction between the unpaired electron(s)
and the transition-metal nucleus (I Z 1/2) can corroborate
this derivation. Furthermore, analysis of the hyperfine inter-
action of the surrounding nuclei allows one to unravel the spin
distribution in the system (via the Fermi contact term) and to
determine electron–nucleus distances and spatial information
(dipole–dipole contribution). The nuclear quadrupole interac-
tion reflects the electric-field gradient at the different quad-
rupolar nuclei. This gradient depends on the full electronic
structure. Put together, these EPR parameters allow for the
determination of both the local geometric and electronic
structure of the metal site. Finally, since most of the EPR
parameters are tensors (see eqn (1)), a detection of the time
averaging of these tensors will give direct information about
the dynamics of the system under study.
2.2. How to obtain this information
2.2.1. General. In order to determine the set of parameters
described in the previous section, the EPR spectroscopist can
use a multitude of CW and pulsed methods. The applicable
techniques can differ in many ways. They can make use of a
single mw frequency (e.g. X-band CW-EPR), multiple micro-
wave frequencies (e.g. electron double resonance (ELDOR)
spectroscopy), a combination of mw and radio frequencies (rf)
(ENDOR) or a combination of mw frequencies and optical
frequencies (e.g. optically detected magnetic resonance
(ODMR)). In order to enhance the obtainable information,
the spectroscopist may choose to combine results of experi-
ments performed at different mw frequencies. While the
majority of experiments is still performed at X-band mw
frequencies (B9.5 GHz), the impact of so-called high-field
EPR and ENDOR experiments, performed at frequencies
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from 95 GHz (W-band) upwards, has been growing exten-
sively in recent years.10,16,18,19
In practice, at all times the EPR spectroscopist will have to
combine a number of techniques in order to obtain an as-
complete-as-possible set of parameters. Although the selection
of experiments is in general not trivial, we outline a rough
guideline for the study of transition metal complexes by means
of the example of a histidine-bound Cu(II). This scheme will
help the reader to understand the examples given in section 3.
Note that this guideline only includes the major techniques
highlighted in this paper, but does not pretend to represent the
full arsenal of techniques that can be used. In general, field-
swept EPR techniques, such as CW-EPR, can reveal informa-
tion about the g tensor, the zero-field tensor, the strongest
hyperfine interactions and possible electron spin–spin interac-
tions. The type of information that can be retrieved is spin-
system dependent (see section 2.1). In the case of the
Cu(II)–His example, the CW-EPR experiment will provide
information about the g and copper hyperfine tensor. Further-
more, ENDOR and ELDOR-detected NMR techniques can
reveal the hyperfine, nuclear Zeeman and nuclear quadrupole
couplings of nearby nuclei (Cu(II)–His: information about the
directly coordinating 14N of His and the nearby 1H). Finally,
electron spin echo envelope modulation (ESEEM) techniques
allow for an identification of these parameters for the nuclei
that are even further away from the unpaired electron(s)
(Cu(II)–His: remote nitrogen of the His and protein backbone
nitrogens). In short, as we move further away from the
unpaired electron(s), we will shift from field-swept EPR
techniques to ENDOR and ELDOR-detected NMR tools,
and finally to ESEEM techniques. In practice, the application
area of the different techniques will overlap partially and the
choice of the appropriate techniques is also nucleus dependent
(e.g. protons at a distance of 0.4 nm away from the unpaired
electron are perfectly detectable with X-band ENDOR, where-
by nitrogens at the same distance need to be probed with
X-band ESEEM techniques).
In the following sections we outline in more detail the most
important classes of techniques that are mentioned above and
that will recur in section 3.
2.2.2. Field-swept EPR techniques. Any EPR analysis will
start with the measurement of the CW-EPR spectrum of the
sample under study. In CW-EPR the absorption of continu-
ously irradiated microwaves with a constant frequency is
monitored as a function of the magnetic field, B0. For techni-
cal reasons, the derivative of the absorption signal is mea-
sured. For a simple S = 1/2 system, the magnetic field will lift
the degeneracy of the mS manifolds (see electron Zeeman term
in eqn (1)), and the state of the electron spin can be changed by
mw irradiation with an energy equal to the energy difference
between the two spin states. In general, the allowed EPR
transitions follow the selection rules |DmS| = 1, |DmI| = 0.
The field at which the resonance occurs links to the g value as
described by eqn (1). The resonance depends on the orienta-
tion of the magnetic field versus the molecular frame, since the
g tensor in eqn (1) has, in its most general case, non-equivalent
principal values. Since, in a powder or a frozen solution, the
paramagnetic molecule will take on all possible orientations
versus the static magnetic field, its CW-EPR spectrum is the
sum of the resonances of all these orientations. Because of the
magnetic-field dependence of the electron Zeeman interation
(eqn (1)), small differences in the principal values can be
resolved at high mw frequencies, where they may remain
unnoticed at the standard X-band frequency. The increased
g resolution is therefore one of the important advantages of
going to higher mw frequencies.
The CW-EPR spectra become more complex if hyperfine
interactions with the transition-metal nucleus or surrounding
strongly coupled nuclei are (partially) resolved. Furthermore,
the zero-field splitting will additionally complicate the spec-
trum for high-spin systems. If the zero-field interaction is
considerably larger than the electron Zeeman term, it may
become impossible to derive all parameters from the CW-EPR
spectrum. Again, experiments at higher mw frequencies can
help, since the field dependence of the electron Zeeman term
allows for a fine-tuning of the ratio between the two terms.
CW-EPR can be used not only to determine the static
structure of the paramagnetic molecule, but it is also a
valuable tool for measuring dynamical aspects. Molecular
motions that are very fast compared to the applied mw
frequency will cause a total averaging of the orientation-
dependent contributions and result in a so-called ‘isotropic’
spectrum (fast motion limit), whereas motions that are con-
siderably slower than the mw frequency are observed as static
(‘powder-like’ spectrum). All intermediate motions will give
rise to complex spectral features that allow the determination
of the nature and correlation time of the motion. Change of
the mw frequency thus shifts the reference to which the motion
is compared and a multi-frequency approach facilitates largely
the interpretation of the complex spectra. Further details on
CW-EPR can be found in the standard text books on this
topic.20–23
In principle, similar information can be obtained from
electron spin echo (ESE)-detected EPR. In these experiments,
a spin echo, usually a Hahn echo or a primary echo, is
generated using a specific mw pulse sequence and the echo
intensity is monitored as a function of the external magnetic
field. For an S = 1/2 system, this approach generates a
spectrum matching the integrated form of the corresponding
CW-EPR spectrum. In cases where the unpaired electron(s)
are interacting with surrounding nuclear spins, the ESE-de-
tected EPR spectrum may be distorted due to echo modula-
tions (see later) and special precautions should be taken when
interpreting these data.9 ESE-detected EPR may be preferred
over CW-EPR at high mw frequencies at which echo modula-
tions are shallow and the poor phase stability of the CW-EPR
experiments can cause severe spectral deformations. Further
details about the advantages of ESE-detected EPR and alter-
native mw pulse sequences can be found in ref. 9.
2.2.3. ENDOR spectroscopy. Although in some cases the
hyperfine interactions of the surrounding nuclei can partially
be resolved by field-swept EPR experiments, other techniques
need to be applied to map out the hyperfine and nuclear
quadrupole couplings in detail. In particular, the spectro-
scopist will look for methods to directly probe the nuclear
transitions (|DmS| = 0, |DmI| Z 1). Fig. 1a shows the energy
4622 | Phys. Chem. Chem. Phys., 2007, 9, 4620–4638 This journal is �c the Owner Societies 2007
levels for a S = 1/2, I = 1/2 system (e.g. an unpaired electron
coupled to a proton). The nuclear frequencies are in this case
given by9
nmS¼1=2 ¼ na ¼ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiðA=2þ nI Þ2 þ B2=4
q
nmS¼�1=2 ¼ nb ¼ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffið�A=2þ nI Þ2 þ B2=4
q ð5Þ
where A and B are related to the secular (Azz) and pseudo-
secular (Azx and Azy) hyperfine couplings: A = Azz and
B = (Azx2 + Azy
2)1/2. nI = �bngnB0/h is the nuclear Lamor
frequency. For isotropic hyperfine values and cases where |nI|c |A|, B, the nuclear frequencies reduce to
na,b = |nI � A/2| (6)
In the case where |nI| 4 |A/2|, one refers to the nuclei as being
weakly coupled, whereas |nI| o |A/2| is typical for strongly
coupled nuclei. The |nI| E |A/2| case is called the cancellation
condition.
For nuclei with I4 1/2, the nuclear frequencies additionally
depend on the nuclear quadrupole interactions.9,20,21,23
In order to detect the nuclear frequencies, ENDOR experi-
ments can be performed. In CW ENDOR, introduced by
Feher2 in 1956, the CW-EPR signal at a given field is saturated
by an increase in the mw power. The recovery of this signal is
then monitored as a function of the frequency of continuously
irradiated radiofrequency waves. Indeed, as the radio fre-
quency matches a nuclear frequency, the populations of all
energy levels will be changed and a net mw absorption can be
detected. The typical ENDOR spectra for the cases described
by eqn (6) are shown in Fig. 1b. Inspection of Fig. 1b reveals
immediately the advantage of ENDOR at high mw frequen-
cies. Indeed, higher mw frequencies imply the use of higher
magnetic fields and lead, through the magnetic-field depen-
dence of nI, to a better peak separation in the ENDOR
spectra. In many cases, a multi-frequency approach, combin-
ing ENDOR results obtained at different mw frequencies,
proves to be favourable. Note that the ENDOR spectrum will
only reflect the nuclear frequencies corresponding to the
orientations selected by the magnetic-field setting at which
the ENDOR experiment is performed. In order to reconstruct
the full hyperfine and nuclear quadrupole tensors, experiments
at different magnetic-field settings (‘observer positions’)
should be performed.
One of the most often used pulsed-ENDOR techniques is
Davies ENDOR spectroscopy25 (Fig. 2a). The first selective
mw p pulse inverts the population of a particular EPR
transition. Each time the frequency of the subsequent fre-
quency-swept rf p pulse becomes equal to a nuclear frequency,
the population of the EPR transitions will be affected. This
change is monitored via a two-pulse-echo detection scheme
(last two mw pulses). Mims ENDOR6 (Fig. 2b) offers an
alternative scheme for recording the nuclear frequencies
whereby the rf-induced changes in the grated polarization
created by the first two mw p/2 pulses are detected.
Pulsed ENDOR schemes offer the advantage over CW
ENDOR that certain ENDOR signals can be selectively
suppressed. An example of this is commonly used in Davies
ENDOR in order to disentangle overlapping ENDOR signals
of weakly coupled protons and strongly coupled nitrogens.
The former signals can be largely suppressed by the use of
short mw pulses (so-called hyperfine contrast selective
ENDOR9). Furthermore, more complex pulsed-ENDOR
schemes allow for a correlation of the nuclear frequencies
with other parameters in a second dimension. The HYEND
technique, in which ENDOR frequencies are directly corre-
lated to the hyperfine splitting, is a good example of this.26
Since a recent Invited Article in this journal focused on
pulsed ENDOR techniques,19 we refer to this work and other
reviews and standard text books for further details on CW and
pulsed ENDOR.9,11,19,27–29
2.2.4. HYSCORE spectroscopy. The nuclear frequencies
can also be determined using ESEEM techniques. In many
cases these techniques give complementary information to the
ENDOR experiments. Low-frequency signals (o5 MHz) are
best detected using ESEEM. In this way, ESEEM will be the
method of choice for the study of weakly coupled nitrogens,
as are often found for protein-embedded transition-metal
complexes.
Fig. 1 (a) Energy level for an S=1/2, I=1/2 system, whereby nI o 0, A, B4 0 and |nI|4 |A/2|. (b) The ENDOR spectrum described by eqn (6)
for the weak-coupling case |nI| 4 |A/2| (top spectrum) and the strong-coupling case |nI| o |A/2| (bottom spectrum).
This journal is �c the Owner Societies 2007 Phys. Chem. Chem. Phys., 2007, 9, 4620–4638 | 4623
In ESEEM experiments a sequence of mw pulses is applied
that generates an electron spin echo. The modulation of the
intensity of this echo is then monitored as a function of one of
the pulse intervals.9 Fourier transformation of this time signal
results in frequency spectra reflecting the nuclear frequencies
similar to an ENDOR spectrum. The origin of this echo
modulation is explained in detail in the standard text books9,30
and will not be treated here.
Here, we will only focus on the basic interpretation of
hyperfine sublevel correlation (HYSCORE) experiments.31
HYSCORE is a four-pulse ESEEM technique (Fig. 2c) where-
by the two interpulse distances t1 and t2 are varied indepen-
dently. Two-dimensional Fourier transformation leads to
frequency-domain spectra where the nuclear frequencies be-
longing to different mS manifolds are correlated. The
HYSCORE sequence is based on the three-pulse ESEEM
experiment, that consists of three p/2 mw pulses whereby the
primary spin echo is detected as a function of the time between
the last two pulses.
For the S = 1/2, I = 1/2 system described earlier, the
HYSCORE spectrum is characterized by a correlation pattern
as shown in Fig. 3a. For the weak-coupling case, the cross
peaks are found in the (++) quadrant, whereas they appear
in the (�+) quadrant for the strong-coupling case. As can be
seen from Fig. 3a, the hyperfine value can easily be estimated
from the peak positions. Furthermore, the correlation pattern
considerably facilitates spectral assignment for complex sys-
tems where the electron is interacting with many nuclei. For
disordered systems, e.g. frozen solutions of proteins, each
magnetic-field setting corresponds to a selection of molecular
orientations (so-called orientation selection) and the
HYSCORE signals will no longer be sharp cross peaks but
extended ridges representing the sum of the cross peaks
corresponding to each of these orientations. This is shown in
Fig. 3b, where a proton HYSCORE spectrum of a frozen
solution of a Cu(II)-bound prion protein (PrP(23-231)) is
depicted. The intersection of the diagonal and the anti-diag-
onal (dashed line) represents the proton Larmor frequency for
this field. This is a typical example of the weak-coupling case
(signals in (++) quadrant). Analysis of the HYSCORE
spectra of disordered systems requires spectral simulation
and is non-trivial.
For an S = 1/2, I = 1 system (e.g. an unpaired electron
interacting with a 14N nucleus), the correlation pattern be-
comes more complicated. In cases where only one molecular
orientation is selected by the magnetic field setting (single-
crystal case), six nuclear frequencies can be detected: four
single-quantum frequencies (|DmI| = 1) and two double-
quantum (DQ) frequencies (|DmI| = 2) (Fig. 3c). The corre-
sponding HYSCORE spectrum will then reveal information
about the nine correlations linking the nuclear frequencies of
the two mS manifolds. For cases with a considerable nuclear
quadrupole contribution (which is generally the case for 14N
nuclei), the HYSCORE spectra are dominated by the double-
quantum (DQ) cross peaks, linking the frequencies32
nDQa ;b ¼ 2
ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffinI �
a
2
� �2þ e2qQ
4h
� �2
ð3þ Z2Þ
sð7Þ
where a is the hyperfine coupling at the particular observer
position. These DQ cross peaks will appear in the (++)
quadrant for a weak-coupling case and in the (�+) quadrant
for a strong-coupling case. The other cross peaks can be found
spread over both quadrants. This is exemplified in Fig. 3d,
showing the 14N HYSCORE spectrum of a VO(II) salen-type
molecule taken at an observer position known to excite all
molecular orientations in the ligand plane. This example was
chosen because it nicely represents some of the essential
characteristics of nitrogen HYSCORE spectra of disordered
systems. Similar spectra have been reported for VO(II) model
systems of the enzyme bromoperoxidase.33 Similar to the case
depicted in Fig. 3b, the HYSCORE features in Fig. 3d are
broad, because several molecular orientations are excited. The
SQ ridges are clearly broader than the DQ cross peaks. This
results from the fact that the SQ nuclear frequencies depend in
first order on the nuclear quadrupole interaction, whereby the
DQ nuclear frequencies only depend in second order on this
coupling (eqn (7)), resulting in less extended and hence more
intense DQ ridges. Using eqn (7) and the position and width of
the DQ cross peaks in Fig. 3d, we can immediately estimate
that the in-plane 14N principal hyperfine values will lie
Fig. 2 Pulsed EPR and ENDOR sequences: (a) Davies ENDOR, (b)
Mims ENDOR, (c) HYSCORE. In matched HYSCORE, the second
and third p/2 pulse are replaced by matched pulses, (d) SMART
HYSCORE, and (e) ELDOR-detected NMR.
4624 | Phys. Chem. Chem. Phys., 2007, 9, 4620–4638 This journal is �c the Owner Societies 2007
between 5.2 and 6.1 MHz and that 1.77 MHz r |e2qQ/h| r2.05 MHz for 1 Z Z Z 0, which will largely facilitate the
further spectral analysis and simulations.
In the exact cancellation case, the effective field experienced
by the nucleus in one of the two mS manifolds is approxi-
mately zero. The ESEEM frequencies within this manifold are
then close to the true nuclear quadrupole frequencies34
n0 ¼e2qQ
2hZ; n� ¼
e2qQ
4hð3� ZÞ; nþ ¼
e2qQ
4hð3þ ZÞ ð8Þ
In the HYSCORE spectra, sharp cross-peaks will be observed
linking the above frequencies with the DQ nuclear frequency
from the other mS manifold.
The nuclear quadrupole couplings of 2H nuclei are in
general very small, so that in this case, the HYSCORE spectra
are dominated by cross peaks between the basic frequencies,
rather than by the DQ frequencies. The typical forms of these
HYSCORE spectra are described in ref. 35.
Significant sensitivity enhancement can be obtained in
ESEEM experiments by the use of matched mw pulses.36
Matched pulses are high turning angle (HTA) pulses that
enhance the efficiency of forbidden transfers and thus drasti-
cally increase the intensity of basic- and combination-fre-
quency transitions. In the case of three-pulse ESEEM and
HYSCORE spectroscopy, the second and third p/2 pulse will
be matched to increase the efficiency of the forbidden transfers
between allowed electron coherence and nuclear coherence. If
one wants to enhance the ESEEM signals of a weakly coupled
nucleus, the field strength of the matching mw pulses, n1match,
should be taken to be equal to the nuclear Zeeman frequency
of this nucleus. For a strongly coupled nucleus, the field is
matched on the hyperfine coupling, which, in practice, usually
means that maximal mw power needs to be applied. The
optimal length of the HTA pulse then has to be determined
experimentally using a 2D three-pulse ESEEM experiment,
where the pulse length of the second and third p/2 pulse is
varied simultaneously in one dimension, and the time between
these two pulses is varied in the second dimension (normal
three-pulse ESEEM dimension).36 Fig. 4 shows a nice example
of the effect of mw pulse matching on the three-pulse ESEEM
spectra of an organic radical in polymethylmetacrylate. The
matching is done on weakly coupled protons (n1match = 15.125
MHz). Fig. 4a shows the echo modulation for a standard
three-pulse ESEEM (all mw pulse lengths equal to 16 ns)
showing shallow proton modulations. Fig. 4b depicts the huge
change in the modulation pattern when optimal matching
pulses are used (tp/2, match = 48 ns). This difference is also
reflected in the corresponding frequency-domain spectrum
(figure inset), showing now intense proton signals. Not only
is a clear signal centered around nH observed, but also the low
frequency component of a proton hyperfine coupling of
B28 MHz is now clearly visible (indicated by an asterisk in
the inset). Similar effects can be observed for the correspond-
ing HYSCORE spectra (not shown).
It is evident that the use of matched mw pulses has largely
increased the potential of the different ESEEM techniques.
Fig. 3 (a) Schematic representation of a HYSCORE spectrum of an S = 1/2, I = 1/2 system, with nI o 0, A, B 4 0 and B oo A, nI. For theweak-coupling case, the cross-peaks between the basic frequencies,na and nb, will appear in the (++) quadrant (squares), in the strong-coupling
case, the cross peaks are found in the (�+) quadrant (circles). (b) Experimental proton HYSCORE spectrum of a frozen solution of Cu(II)-bound
PrP(23–231) taken at an observer position corresponding to g= 2.37. t was 96 ns, and the pulse lengths, tp/2 = 16 ns and, tp = 16 ns, were taken.
(c) Schematic representation of the energy levels for an S = 1/2, I = 1 system indicating the double-quantum (DQ) and single-quantum (SQ)
nuclear frequencies. The example represents a strong coupling case. (d) Experimental 14N HYSCORE spectrum of a frozen toluene solution of
(R,R)-N,N0-bis(3,5-di-tert-butylsalicylidene)-1,2-cyclohexane-diamino vanadyl (II) at g = 2.056 (strong-coupling case). The DQ and SQ cross
peaks are indicated. t was 176 ns, and the pulse lengths, tp/2 = 16 ns and, tp = 16 ns, were taken.
This journal is �c the Owner Societies 2007 Phys. Chem. Chem. Phys., 2007, 9, 4620–4638 | 4625
Where ESEEM analyses would have been abandoned before
because of lack of significant echo modulations, the relevant
nuclear information can now be obtained.
Matched pulses can also be used to overcome one of the
most annoying problems of HYSCORE experiments, namely
the fact that the intensity of the HYSCORE signals depends
strongly on the time t (so-called t-dependent blind spots).
Because of this, HYSCORE spectra with different interpulse
distances, t, should be recorded and added for each observer
position. This considerably increases the measurement time.
The blind spots can be avoided by the use of an alternative
SMART HYSCORE pulse sequence37 (Fig. 2d). In this
scheme, the original p/2–t–p/2 nuclear-coherence generator
is replaced by a one-pulse nuclear-coherence generator
(matched pulse), avoiding the t-dependent blind spots. The
second p pulse in the sequence is needed to refocus the free
induction decay (FID) signal and allow echo detection.
Finally, since the modulation depth is proportional to
I(I + 1), the echo modulation tends to be dominated by the
modulations of the nuclei with the highest nuclear spin. In
complex situations where the unpaired electron is interacting
with several nuclei, this may lead to cross-suppression of the
weaker modulations.38 This can be circumvented by the use of
matching pulses36 or more extended ESEEM schemes.38
Further information on HYSCORE, ESEEM and other
pulsed EPR sequences can be found in ref. 9.
2.2.5. ELDOR-detected NMR. Although ELDOR-de-
tected NMR (Fig. 2e) is by far not as commonly applied as
the different pulsed-ENDOR or ESEEM sequences described
above, its use can sometimes be very advantageous.39,40 The
basics of the method are quite simple. The selective p pulse
with mw frequency 1 inverts the population of an EPR
transition. Whenever the variable microwave frequency of
the mw2 pulse matches one of the allowed or forbidden
EPR transitions, the population in the observed transition
will change. Observation of the FID signal as a function of the
difference between the two mw frequencies again results in a
spectrum reflecting the nuclear frequencies Alternatively, the
mw1 p pulse can be replaced by a p/2–t–p scheme whereby the
spin echo is then monitored as a function of the varying mw
frequency 2. A drawback of the ELDOR-detected NMR
method is the fact that the central hole (around nmw1 =
nmw2) will cover the signals at low frequencies. Fig. 5a shows
an extreme example of a broad central hole, but also in more
favourable cases, the central hole can still be significant
(Fig. 5b). The ELDOR-detected NMR approach is therefore
Fig. 5 (a) Q-Band ELDOR-detected NMR spectrum of a frozen
solution of Cu(II) tetraphenylporphyrin taken at an observer position
corresponding to g = 2.054. The spectrum is detected using a 60 ms(HTA, mw2) - 2.5 ms (delay) – 32 ns (mw1) - t - 64 ns (mw1) - t - echosequence, with t= 820 ns. The experiment was performed at 11 K. (b)
W-Band ELDOR-detected NMR spectrum of a frozen solution of
Cu(II)-bound peptide taken at an observer position corresponding to
g = 2.0465. The spectrum is detected using a 3 ms (HTA, mw2) - 5
ms(delay) – 120 ns (mw1) – FID sequence. The experiment was
performed at 10 K.
Fig. 4 Three-pulse ESEEM modulation pattern for the propagating radical of polymethylmetacrylate polymers. (a) Standard three-pulse
ESEEM, 16–t–16–t–16–t–echo, t = 200 ns. (b) Matched three-pulse ESEEM, 16–t–48(HTA)–t–48(HTA)–t—echo, n1match = 15.125 MHz, t =200 ns. The inset shows the corresponding frequency-domain spectra obtained after Fourier transformation. The signals marked with an asterisk
are discussed in the text.
4626 | Phys. Chem. Chem. Phys., 2007, 9, 4620–4638 This journal is �c the Owner Societies 2007
best suited to the detection of strongly coupled nuclei. Alter-
natively, the experiments can be performed at higher mw
frequencies (making use of the increase in the nuclear Zeeman
frequencies). The experiment depicted in Fig. 5a was per-
formed at Q-band mw frequencies. The proton Zeeman fre-
quency lies here at B50 MHz and the proton frequencies are
clearly separated from the central hole. At X-band mw
frequencies, nH lies around 15 MHz and a similar experiment
would then not allow an analysis of the proton interactions.
Note that the width of the central hole can be reduced by pulse
shaping and/or going to lower temperatures. It depends on the
local concentration of the paramagnetic centres (links to the
phase-memory time). The example in Fig. 5a can thus be
further optimized, but was chosen here to illustrate the
worst-case scenario.
At W-band mw frequencies, the ELDOR-detected NMR
approach can lead to better signal-to-noise ratios than pulsed
ENDOR, especially in the case of strongly coupled nuclei.40
Detection of these interactions implies the use of Davies
ENDOR, and the detection of the ENDOR spectrum of
strongly coupled nitrogen nuclei is notoriously difficult (and
sometimes impossible) at W-band, requiring thousands of scans.
The corresponding W-band ELDOR-detected NMR spectrum
can usually be detected in a few scans. The explanation for this is
probably manifold and factors such as sample heating due to the
rf pulse in the Davies scheme, the different relaxation paths
involved in the two experiments and the smaller amount of
pulses in the ELDOR-detected scheme should be considered.
Fig. 5b shows an example of a single-scan W-band ELDOR-
detected NMR spectrum of a Cu(II)-bound small His-containing
peptide. The spectral contributions of the directly binding
nitrogen of the histidine ligand can be readily identified after
only one scan. The full hyperfine and nuclear quadrupole tensor
can in this way be determined by recording the W-band
ELDOR-detected NMR spectra at different magnetic-field set-
tings (Maria Fittipaldi, work in progress).
2.3. From spectrum to model
In order to extract the EPR parameters from the different
experimental results, adequate simulation and fitting programs
are imperative. Due to the complexity and large diversity of
the EPR experiments and the spin systems that can be
addressed by them, an accurate simulation of the spectra is
not trivial. Although simulation programs targeted at the
simulation of specific EPR problems have been developed
over the years,41–47 it is only recently that attempts to develop
software packages that can tackle CW and pulsed EPR/
ENDOR simulations on a general level have been started.48
The matlab-based EasySpin program48 is at present the most
extended and flexible simulation package for both CW and
pulsed EPR spectra, but it still does not cover everything. It is
evident that the further development and generalization of the
simulation programs will remain one of the most important
aims in the field.
Once the EPR parameters have been determined experimen-
tally, the key question is how to interpret the different spin
Hamiltonian parameters in terms of useful electronic and geo-
metric structural information. In section 2.1. we already outlined
how the individual EPR parameters can in principle be linked to
different structural and electronic parameters. In practice, this
step proves to be very difficult. Let us take the example of the
hyperfine coupling. The Amatrix can be split in an isotropic and
an anisotropic part. While the isotropic part (Fermi contact
part) can still be easily linked to the spin density on the nucleus,
the interpretation of the anisotropic part is often less straight-
forward. This part contains orbital contributions and an elec-
tron–nuclear-distance dependence that are not easily separated.
Although several useful approaches have been explored and
used for many decades,9,20–24 such as the point–dipolar approx-
imation (see eqn (2)), there are many cases reported in which the
full information hidden in the A matrix could not be satisfacto-
rily decoded. This has, in combination with the fast increase in
EPR methodology and its linked increase in experimental data,
triggered the development of different quantum chemical ap-
proaches to compute the different spin Hamiltonian para-
meters.49,50 In this, a symbiotic relation has now been formed
between the theoretical and experimental approaches, as will
also become clear from some of the examples in section 3.
Experimental EPR data provide the ultimate control for the
quality of the quantum chemical computation. Although for
some cases density functional theory (DFT) and other ab initio
methods have proven to generate theoretical spin Hamiltionian
parameters that agree quantitatively with the experiment, a
qualitative agreement is, for the majority of cases, still the best
one can currently achieve, especially for transition metal com-
plexes.49,50 Even with these limitations, the quantum chemical
computations are starting to play an essential role in EPR
analysis. As mentioned in the previous section, the spectral
simulation of EPR/ENDOR spectra can become very complex
and at some points only a fraction of the hyperfine and nuclear
quadrupole information can be extracted in a first analysis. If
these parameters are then used to evaluate different theoretical
models, valuable hints about the principal values and directions
of the A and P tensors of the remaining magnetic nuclei can be
derived from the quantum chemical computations. This infor-
mation can then be used again as starting values to optimize the
spectral simulations in order to derive the full set of spin
Hamiltonian parameters for the system under study.51,52
Furthermore, once a satisfying agreement is found between
the experimental data and the theoretical model, not only
important structural information can be obtained from the
best-matching model, but also the computed ground-state
wavefunction can be linked to the inner working of the
transition-metal site. Indeed, important characteristics, such
as redox functions or ligand binding, are not only governed by
the geometric structure of the site, the electronic state plays an
equally important factor. Knowledge of the electronic struc-
ture is therefore essential if one wants to understand, for
instance, why apparently small changes can induce function
loss in one protein, and why seemingly more drastic changes
still lead to (partial) preservation of the metalloprotein’s
function in another case. Once a good theoretical model is
found, the effect of changes in the structure (e.g. mutagenesis)
can be probed theoretically. Thus, the amount of further
experiments can be reduced to a selective and well-targeted
set. In this way, the final outcome of the study exceeds what
could possibly be reached by a separate approach. One of the
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essential goals for the future is therefore a flawless matching of
the two approaches, whereby the earlier-mentioned experi-
mental and theoretical problems have to be overcome.
2.4. Advantages and drawbacks of using EPR to study
metalloproteins
One of the important advantages of EPR is the large variety of
experiments that can be performed. For many paramagnetic
transition metal complexes, CW-EPR spectra can be obtained
in a large temperature range up to room temperature. For
these complexes, EPR can reveal important information about
the molecular dynamics on timescales between 10 ps and a few
ms.53,54 Information on the static structure of the transition-
metal site can be obtained from the different advanced pulsed-
EPR/ENDOR methods described in the previous sections.
However, in order to be able to use the latter methods, the
samples need to be cooled to very low temperatures (o25 K).
The predominant reason for the latter is the need to slow down
different relaxation pathways.9 Cooling is generally done using
liquid helium and together with the high cost of the EPR
equipment, this makes running an EPR facility expensive.
However, other advanced techniques, such as XRD, neutron
diffraction or NMR, are also very costly. A second drawback
is the fact that the spectral complexity requires specialist’s
input and a full structural analysis using EPR cannot be done
on a high through-put basis.
The amount of (protein) material needed for the analysis
depends on many factors, including the specific spin Hamilto-
nian parameters, the sensitivity of the specific spectrometer at
hand, and even the time (and money) the particular spectro-
scopist is willing to invest (measurement time and liquid-helium
consumption). In the most favourable cases, protein concentra-
tions of 50–100 mM are sufficient. This is the case when the
paramagnetic centre is characterised by a g tensor exhibiting
little anisotropy and when the (metal) hyperfine coupling is
small. An example of this is the NO-ligated ferrous form of a
heme protein. In extreme cases, protein concentrations in the
mM range are needed. This is the case for systems exhibiting
large g anisotropy and/or large hyperfine couplings. The EPR
study of low-spin ferric heme proteins requires concentrations of
1–3 mM (see section 3.1.1.). Note that the spin–spin relaxation
will become faster as the concentration of paramagnetic mole-
cules increases. At a certain concentration, the relaxation will be
so fast that no spin-echo signal will be detectable. Hence, an
increase in the concentration of a protein does not necessarily
lead to a higher echo intensity and the optimum concentration
needs to be established for each system separately. The volume
needed for the experiment depends on the mw frequency. While
X-band EPR measurements require about 100 ml of the sample,
only 1 ml is needed at W-band mw frequencies. In practice, the
available material will thus partially determine the type of
experiments that can be performed.
In many cases, EPR provides valuable (and sometimes
essential) information complementary to other structure char-
acterization methods. In NMR, the presence of the unpaired
electron(s) will usually cause large shifts and line broadenings,
making it difficult if not impossible to gain structural informa-
tion in the neighbourhood of the unpaired electron. EPR can
provide this essential information and thus complete the
experimental data. Single-crystal X-ray diffraction structure
analyses and neutron-diffraction analyses have proven their
extreme value in the determination of (metallo)protein struc-
tures. However, EPR studies can also provide unique com-
plementary information that may put XRD results in another
perspective, as will be exemplified in sections 3.1.1 and 3.2.1.
Other spectroscopic methods, such as extended X-ray absorp-
tion fine structure (EXAFS), optical and laser spectroscopies
are also often used complementarily to EPR spectroscopy.
3. Applications
In this section, we will describe a few examples of EPR and
ENDOR studies of iron-, nickel-, cobalt-, and copper-contain-
ing proteins. The examples are chosen so as to illustrate the
potential of state-of-the-art EPR and are not meant to be
exhaustive.
3.1. Iron-containing proteins
A glance in one of the many standard books on bioinorganic
chemistry reveals that various proteins contain iron-binding
sites or have an iron co-factor, many of them being para-
magnetic. We will focus in this section on examples of ferric
forms of heme proteins from our own research and on
iron–sulfur proteins. The latter proteins are not studied by
us, but their importance in biological processes merits some
attention in this overview.
3.1.1. Ferric form of heme proteins. The most well-known
iron-containing co-factor is probably the heme group (Fig.
6a), which is for instance found in globins. The iron ion of the
heme group is reported to occur in the ferrous, ferric and ferryl
form. The (paramagnetic) ferric (Fe(III)) forms of cyto-
chromes, peroxidases and catalases are biologically active.
The ferric or so-called ‘met-form’ of the globins was long
thought to be biological inactive, because oxygen can only
reversibly bind at the ferrous heme iron to realize the globin’s
most documented functions of oxygen storage and transport.
However, recently discovered new functions of vertebrate
myoglobin55 and the discovery of new globin-type proteins
in all kingdoms of nature with unknown functions56 have
renewed the interest in the ferric form of globins.
Ferric heme proteins can have different spin states: a low-
spin (LS) state (S= 1/2), or a high-spin (HS) state (S= 3/2 or
S = 5/2). The spin states are governed by the strength of the
axial ligands. All globins have a conserved histidine at position
F8 that binds to the heme iron (proximal side), whereby the
distal ligand can vary. A weak axial ligand, such as a distal
water, will lead to a HS state, which is typically observed for
the met form of vertebrate myo- and hemoglobin. Coordina-
tion of a strong axial ligand, such as an endogenous histidine,
then leads to a LS iron(III) state, as found for the recently
discovered vertebrate neuroglobin and cytoglobin.57 The
X-band CW-EPR spectra of frozen solutions of LS and HS
ferric heme proteins differ considerably. The LS ferric heme
proteins are characterized by a highly rhombic g tensor, as
exemplified by the principal g values of ferric mouse neuro-
globin: gx = 1.29, gy = 2.15, gz = 3.12.58 The X-band
4628 | Phys. Chem. Chem. Phys., 2007, 9, 4620–4638 This journal is �c the Owner Societies 2007
CW-EPR of HS globins can be described by the effective g
values (gx,eff E gy,eff E 6 and gz,eff E 2) (see later). X-Band
CW-EPR thus offers a fast and easy method to determine the
spin state of the iron.
Peisach and Blumberg systemized the analysis of the
CW-EPR spectra of several biological molecules and related
model systems. They constructed the so-called ‘‘truth tables’’,
which provide a means to relate the EPR parameters of a
paramagnetic site to structural information. This approach
was applied successfully to LS ferric heme proteins59 whereby
the g values give indications on the type and orientation of the
axial ligands binding to the heme iron. Although these tables
are based on crude assumptions, they can nevertheless give
important initial information on the system. Indeed, using this
simple approach, we could show for the case of ferric neuro-
globin that the iron center is bis-histidine coordinated, and the
histidine planes are not eclipsing the Fe–pyrrole nitrogen
bonds. Furthermore, the EPR parameters indicate that a
conformation in between parallel and near to perpendicular
alignment of the histidine planes is present.60 This was later
confirmed by the X-ray data.61,62
In 1986, Scholes and co-workers used CW-ENDOR to
study frozen solutions of bis(imidazole)-ligated LS ferric heme
complexes.63 Although they could derive interesting informa-
tion on the electronic and geometric structure of the heme
environment from this study, there were several method-
inherent limitations to the work, mainly due to the high degree
of spectral overlap of the contributions of the individual
nuclei. The same problems were observed for X-band Davies
ENDOR64 and three-pulse ESEEM65,66 studies of ferric heme
proteins, since in all cases the spectra are one-dimensional and
the spectral contributions of different nuclei will show a
similar degree of overlap.
Hoffman and co-workers showed that some of the above-
mentioned problems can be solved by using a combination of
CW, Davies- and Mims-ENDOR techniques at Q-band
(35 GHz) mw frequencies.67,68 Due to the magnetic-field
dependence of the Larmor frequency, nI, the spectral contri-
butions of the different nuclei can then be better separated at
higher fields. In this way, the authors established that the
allylbenzene-bound heme of inactivated chloroperoxidase is in
fact an N-alkylhemin metallocycle with a carbon of allylben-
zene bonded to the pyrrole nitrogen.68
Raitsimring, Walker and Astashkin showed in a series of
papers that it is advantageous to perform two- or four-pulse
ESEEM experiments at different settings of the magnetic field
and different mw frequencies.69–71 From the magnetic field
dependence of the proton combination peaks, detailed informa-
tion about the orientation of the proton hyperfine tensor in the g
tensor frame can then be obtained. For the nearby protons of the
axial imidazole ligands, this information can be directly linked to
the orientation of imidazole ligands in the heme pocket.
The first two-dimensional ESEEM experiments on ferric LS
heme proteins were performed by Garcıa-Rubio et al.72 They
compared the HYSCORE spectra of a bis(imidazole) ligated
heme compound, with selective isotopic substitution of the
nitrogens, to those of cytochrome b559. In this way the
HYSCORE peaks of cytochrome b559 could be assigned. The
large spectral similarity between cytochrome b559 and the
model system led to a detailed identification of the heme-
ligand orientations.
In our recent work on a ferric porphyrin model system, we
combined X-band CW-EPR, HYSCORE, and four-pulse
ESEEM experiments to maximize the information that can
be obtained from an EPR study of ferric heme complexes.73
The strategy consists of three steps. In a first step, the
Fig. 6 Schematic representation of (a) Fe(II) protoporphyrin IX (heme), (b) Rieske-type [2Fe–2S] centre, (c) coenzyme F430, (d) vitamin B12
(R = CN) or coenzyme B12 (R = 50-deoxy-50-adenosyl), (e) a copper site in azurin.
This journal is �c the Owner Societies 2007 Phys. Chem. Chem. Phys., 2007, 9, 4620–4638 | 4629
orientation of the hyperfine and nuclear quadrupole tensors of
the pyrrole nitrogens is determined in the g tensor frame by the
simulation of the nitrogen HYSCORE spectra. For all metal-
loporphyrin complexes the highest nuclear quadrupole value
of the pyrrole nitrogen is found to lie in the porphyrin plane
perpendicular to the metal–nitrogen bond. This provides a
means to determine the orientation of the g tensor frame in the
molecular frame. The analysis of the proton HYSCORE and
combination-peak experiments then provides the hyperfine
tensors of the imidazole protons. These parameters can be
decoded into the proton–iron distance and orientation of the
Fe–H vector in the g tensor frame. Using the results of the first
step, this information is then linked to the orientation of the
imidazole planes versus the porphyrin. In a final control step,
the imidazole nitrogen hyperfine and nuclear quadrupole
tensors are determined from the nitrogen HYSCORE spectra.
The in-plane rotation of the principal axes of these tensors is
linked to the orientation of the imidazole plane and should
match the earlier proton data. In a later work, this methodo-
logy was extended with pulsed ENDOR experiments and
applied to ferric mouse neuroglobin.74 Fig. 7a shows a typical
nitrogen HYSCORE spectrum of ferric mouse neuroglobin.
The DQ cross peaks of the heme and histidine nitrogens are
well separated. As outlined in section 2.2.4., initial spin
Hamiltonian parameters can be derived from the positions
of these cross-peaks using eqn (7), and this can then be refined
by spectral simulation. The structural parameters obtained by
this approach for the F8His and E7His of ferric mouse
neuroglobin matched the earlier X-ray data. A similar proce-
dure was also successfully applied to unravel the direct heme
environment of tomato hemoglobin, a globin for which no
prior structural information was available.75 In our recent
study of ferric cytoglobin, the strategy could also be used to
solve an ambiguity between two conflicting X-ray studies
(I. Ioanitescu, to be published). Although the error margins
on the structural parameters are larger than for those obtained
from X-ray diffraction studies, the method does not require
single crystals and can thus be used to study every ferric LS
heme protein without prior crystallization. The methodology
now also opens the possibility of identifying disulfide-bridge-
induced changes in the heme pocket of human neuroglobin. It
was earlier found that most neuroglobins, with the exception
of Rodentia and zebrafish neuroglobins, can form a disulfide
bridge between the cysteines on positions CD7 and D5.76
Formation of this disulfide bridge is linked to a change in
the oxygen affinity of the ferrous heme center. We showed that
the CW-EPR spectrum of ferric human neuroglobin is char-
acterised by two LS components with slightly different princi-
pal g values and that one of these components is clearly linked
to the disulfide bridge formation.58 Up until now, all efforts to
isolate and crystalize the latter form of the protein have failed.
The combined pulsed EPR/ENDOR approach may well pro-
vide the only way to identify the structural change occurring in
the heme pocket upon disulfide bridge formation. A large part
of the ferric forms of heme proteins are in a HS state. The mw
quantum energy at X-band (B9.5 GHz) is much smaller than
the zero field splitting (5–10 cm�1), so that the observed EPR
spectrum only arises from the transitions of the lower Kramers
doublet and can be described as an Seff = 1/2 system whereby
in second order the effective spin Hamiltonian is given by
Heff ¼ be~B0geffSeff=h
þXk
ð~SeffAk;effIk � bngn;k;eff ~B0Ik=hÞ
þX
k;Ik41=2
~IkPk;effIk ð9Þ
whereby the effective spin Hamiltonian parameters link to the
general spin Hamiltonian values of eqn (1) and (4) by
gk;x;eff ¼ gk;x 3� 12E
D
� �;
gk;y;eff ¼ gk;y 3þ 12E
D
� �;
gk;z;eff ¼ gk;z;
ð10Þ
Ak;x;eff ¼ Ak;xð3�12E
DÞ;
Ak;y;eff ¼ Ak;yð3þ12E
DÞ;
Ak;z;eff ¼ Ak;z �2Ak;zAk;y
D;
ð11Þ
Fig. 7 (a) Standard X-band HYSCORE spectrum of a frozen solu-
tion of ferric mouse neuroglobin taken at an observer position
corresponding to g = 1.86. t was taken to be 96 ns. The prominent
DQ cross-peaks stemming from the directly coordinating heme and
histidine nitrogens are indicated. (b) Matched X-band HYSCORE of a
frozen solution of the ferric form of the E7Q mutant of human
neuroglobin taken at an observer position corresponding to g =
3.48. The matching pulse sequence 8–t–24(HTA)–t1–16–t2–24(HTA)–
t echo, with n1match = 31.25 MHz and t = 96 ns, was used.
4630 | Phys. Chem. Chem. Phys., 2007, 9, 4620–4638 This journal is �c the Owner Societies 2007
gn;k;x;eff ¼ gn;k þ2gxbeAk;x
bnD;
gn;k;y;eff ¼ gn;k þ2gybeAk;y
bnD;
gn;k;z;eff ¼ gn;k:
ð12Þ
where D and E are the tetragonal and rhombic zero-field
splitting. The effective nuclear quadrupole tensor, Peff, usually
shows only minor deviations from the P tensor in eqn (1).77
As mentioned before, the X-band CW-EPR spectrum of HS
ferric heme compounds can be simulated with an effective
electron spin of 1/2, and effective g values. Unfortunately, the
X-band CW-EPR spectra of different HS ferric heme proteins
tend to be alike and reveal little information on the system
other than the identification of the spin state. A direct
determination of the zero-field splitting using CW-EPR is only
possible at high mw frequencies.78
In 1982, Scholes et al. published a seminal paper on the
CW-ENDOR analysis of a single crystal of aquometmyoglo-
bin, unravelling the hyperfine and nuclear quadrupole tensors
of the heme and histidine nitrogens.79 Since then, only a few
ESEEM or pulsed ENDOR studies of HS heme compounds
have been reported,80 contrasting with the recent evolution in
the field of EPR. There are two reasons for this. First of all, the
effective hyperfine values of the heme and imidazole nitrogens
reach values of 30–45 MHz for orientations in the heme
plane.79 These interactions cannot be observed using standard
ESEEM techniques. Furthermore, the pseudo-nuclear contri-
butions to the nuclear Zeeman term given in eqn (12) con-
siderably complicate the analysis of ENDOR and ESEEM
spectra, especially when no single crystals are available. In a
recent study, we showed how some of these problems can be
circumvented by using matched HYSCORE spectroscopy.77
Fig. 7b shows a matched X-band nitrogen HYSCORE spec-
trum of the ferric form of the E7Q mutant of neuroglobin
taken at an observer position g = 3.48. Because of the
sensitivity enhancement by the matched mw pulses mentioned
earlier, the nitrogen DQ cross peaks can readily be detected in
this spectrum. These signals are not revealed in the standard
HYSCORE experiment. Furthermore, clear ridges stemming
from nearby protons can be recognized in the (++) quadrant.
The ridges in the (�+) quadrant at frequencies lower than
15 MHz are complex to interpret and agree with the SQ
nitrogen frequencies and different combination frequencies
(nitrogen–nitrogen and nitrogen–proton).
The HYSCORE study of the ferric form of the E7Q-mutant
of human neuroglobin also revealed that this protein lacks the
characteristic distal water at neutral pH and that the lysine at
position E10 coordinates to the iron at high pH.77 Our current
work now focuses on testing the performance of different
advanced pulsed-EPR and ENDOR techniques, e.g.
HYSCORE, ELDOR-detected NMR, and HYEND, at dif-
ferent mw frequencies for the analysis of HS ferric heme
proteins (work in progress). The ELDOR-detected NMR
technique at W-band (95 GHz) mw frequencies in particular
promises to become a valuable tool in the analysis of ferric HS
systems.
3.1.2. Iron–sulfur proteins. Another important class of iron
proteins comprises the so-called iron–sulfur proteins. These
proteins have different functions, but are most frequently
involved in electron transport. Different iron–sulfur clusters
([2Fe–2S], [3Fe–4S], and [4Fe–4S]) are observed. Character-
istic of iron–sulfur proteins is the coordination of the iron ion
by cysteines, or occasionally by other amino acids of the
protein side chains, and, for polynuclear Fe–S centers, the
remarkable ligation with bridging ‘inorganic’ sulfide ions. It is
safe to say that EPR has played a crucial role in iron–sulfur
protein research. Indeed, based on their CW-EPR analysis,
Bertrand et al., could propose the existence of a new class of
[2Fe–2S] proteins, the so-called ‘‘Rieske-type’’ proteins (Fig.
6b), which are characterized by a larger g tensor anisotropy
compared to the classical ferredoxin type (gav B 1.91 instead
of gav B 1.96).81 They pointed out that the unusual g values of
this new type of [2Fe–2S] protein are consistent with the
presence of ligands at the ferrous site that are more electro-
negative than sulfide, which is in contrast to the classical
ferredoxin-type clusters where all ligation is provided by
sulfide. In recent years, more advanced pulsed EPR techniques
have been used successfully to identify the direct environment
of the iron–sulfur clusters as exemplified in ref. 82–84. A
detailed overview of the EPR and ENDOR work done on
iron–sulfur proteins is presented in ref. 84 and 85.
3.2. Nickel-containing proteins
Ni(I) (d9) and Ni(III) (d7) are paramagnetic ions. In many
cases, identification of the oxidation state of the nickel ion is
not straightforward and the nickel centre of many nickel-
containing enzymes is poorly characterized, as will become
apparent from the following examples. We focus here on the
EPR analyses of different forms of methyl-coenzyme M
reductase (MCR) to which one of us (S.V.D) also contributed.
In the second part, we also summarize some of the EPR work
done in field of [FeNi] hydrogenases. This is not our own
research field, but we feel it illustrates the potential and also
future challenges of EPR in the analysis of metal-containing
proteins nicely and it can therefore not been left out of this
overview.
3.2.1. Methyl-coenzyme M reductase (MCR). MCR plays
a crucial role in the methane-forming step of the energy
metabolism of archaea, since it catalyzes the reduction of
methyl-coenzyme M (2-(methylthio)ethane-sulfonate,
CH3SCoM) with coenzyme B (7-thioheptanoyl-threonine-
phospate, HSCoB) to methane and CoMS-SCoB.86 The en-
zyme contains the nickel-centred porphinoid co-factor, F430
(Fig. 6c). Different forms of the enzyme and their interconver-
sion relationships have been reported.87 Since many of these
forms are paramagnetic (including the enzymatically active
(‘ready’) forms), X-band CW-EPR played an important role in
differentiating the MCR forms.
The CW-EPR and ENDOR spectra of the isolated F430 co-
factor and of the active form of the enzyme, MCRred1, are very
alike and typical for a d9 Ni(I) complex with the unpaired
electron residing in the dx2�y2 orbital.88,89 The principal g
values are given in Table 1. Although the hyperfine couplings
of the four pyrrole nitrogens show small differences, they are
This journal is �c the Owner Societies 2007 Phys. Chem. Chem. Phys., 2007, 9, 4620–4638 | 4631
of the same order and typical for strongly coupled nitrogens
(Aiso E 28.5 MHz for MCRred1 and Aiso E 27.8 MHz89 for
Ni(I) F43088). Fig. 8a shows the X-band Davies ENDOR
spectra of the Ni(I) form of F430 pentamethyl ester. The top
spectrum was recorded using selective, soft mw pulses. It is
quasi-symmetrical around the proton Larmor frequency, nH,and is dominated by the spectral contributions of the nearby
protons of the F430 macrocycle. When using hard mw pulses
(bottom spectrum in Fig. 8a), the spectrum changes comple-
tely. The proton signals are now suppressed and the under-
lying signals of the strongly coupled nitrogens become
apparent. Since at this observer position all in-plane orienta-
tions are excited, the nitrogen ENDOR spectrum is complex,
stemming from many orientations. The two groups of four
lines in Fig. 8a indicate the basic nuclear frequencies corre-
sponding to the two in-plane principal directions of the
hyperfine and nuclear quadrupole tensor (Px E �1.85 MHz,
Py E 1.1 MHz88). The extra features stem from the other in-
plane orientations. Fig. 8a illustrates the hyperfine contrast
selective ENDOR effect mentioned in section 2.2.3.
In the presence of coenzyme M (2-mercaptoehanesulfonate,
HSCoM) and HSCoB, MCRred1 is converted into MCRred2
(maximum conversion is 50%). The CW-EPR spectrum of this
form is characterized by an unusual, highly rhombic g tensor,
whereby the broadening of the EPR line widths observed for61Ni-labeled MCRred2 reveals that the paramagnetic centre is
nickel based.87 Combination of X-band and Q-band ENDOR
and HYSCORE spectroscopy allowed us to determine the
hyperfine and nuclear quadrupole parameters of the pyrrole
nitrogens. Contrasting the case of MCRred1, two sets of
nitrogens with hyperfine values differing by about a factor of
two could be identified for MCRred290 (Aiso E 23.9 MHz and
Aiso E 13.8 MHz). The smaller hyperfine coupling could be
detected using Q-band HYSCORE (Fig. 8b). It was assigned
to the nitrogen of pyrrole A. The pyrrole ring A is not
p-conjugated and therefore more flexible than the other pyr-
role rings (Fig. 6c). Reduction of the nitrogen hyperfine values
therefore indicates that pyrrole A is slightly bent out of the
macrocyle plane. The Q-band HYSCORE spectrum of33S-labeled coenzyme M revealed additional features (signals
marked by the arrows in Fig. 8b) that stem from the 33S
interactions. The two cross-peaks in the (�+) quadrant at
(�10.8, 31.8) MHz and (�31.8, 10.8) MHz are assigned to the
triple-quantum transitions. The appearance of the 33S-related
cross-peaks proved unambiguously the coordination of CoM
to the nickel center in MCRred2.91
MCRox1 is an enzymatically inactive but ‘ready’ form of
MCR, which can be readily reduced to the active MCRred1.
The oxidation state of MCRox1 is highly debated, Ni(I) (d9),89,92
Ni(III) (d7),93 a Ni(III)-thiolate and a high-spin Ni(II) ion (S= 1)
antiferromagnetically coupled to a thiyl radical94 have all been
proposed. The fact that MCRox1 is converted to MCRred1 after
addition of Ti(III) citrate at pH 9 and 60 1C indicates that
MCRox1 is a Ni(III) complex.95 On the other hand, MCRox1 can
also be generated by cryoreduction of MCRox1-silent, an enzy-
matically inactive EPR-silent Ni(II) form, which makes a Ni(I)
oxidation state more probable. The g values (Table 1) and
pyrrole nitrogen hyperfine values (Aiso E 25 MHz) are typical
of an S = 1/2 species with an unpaired electron predominantly
residing in the nickel dx2�y2 orbital. Since both Ni(I) and Ni(III)
complexes with the unpaired electron in the dx2�y2 orbital have
been reported, this does not shed light on the oxidation state of
nickel in MCRox1. Furthermore, the X-ray absorption (XAS)
spectroscopy,96 UV-Vis97 and magnetic circular dichroism98
data indicate a close resemblance of MCRox1 to the EPR-silent
Ni(II) states.
Fig. 8 (a) X-Band Davies ENDOR spectra of the Ni(I) form of F430
pentamethyl ester taken at an observer position corresponding to g Egx (B0 = 337.4 mT). The top spectrum was taken using selective mw
pulses (tp/2 = 200 ns, tp = 400 ns) and a long rf p pulse (10 ms), thebottom spectrum was taken using hard mw pulses (tp/2 = 26 ns, tp =
52 ns) and a shorter rf pulse (5.4 ms). The meaning of the two groups of
four lines is explained in the text. (b) Q-Band HYSCORE spectrum
recorded at 25 K of MCR in the MCRred2 state with H33S-CoM,
observer position g = 2.28. The arrows identify the peaks that
originate from 33S interactions. The remaining cross-peaks stem from
an interaction with the nitrogen of pyrrole A. The spectrum was
reproduced with permission from C. Finazzo, J. Harmer, C. Bauer,
B. Jaun, E. C. Duin, F. Mahlert, M. Goenrich, R. K. Thauer, S. Van
Doorslaer and A. Schweiger, J. Am. Chem. Soc., 2003, 125, 4988–4989.
Copyright 2003 American Chemical Society.
Table 1 Principal g values of coenzyme F430 and different MCRforms
Complex gx gy gz Ref.
Ni(I)–F430 2.063 2.063 2.244 88MCRred1 2.060 2.070 2.2485 89MCRred2 2.1753 2.2313 2.2869 90MCRox1 2.1527 2.1678 2.2312 94
4632 | Phys. Chem. Chem. Phys., 2007, 9, 4620–4638 This journal is �c the Owner Societies 2007
Only recently, our multi-frequency EPR, ENDOR and
HYSCORE approach in combination with 61Ni, 33S and 2H
isotope labeling led to an identification of MCRox1 as a Ni(III)
thiolate in resonance with a thiyl radical/high-spin Ni(II)
complex.94 Analysis of the 61Ni and 33S hyperfine values
proved that Ni–S coordination was present in which 7 � 3%
of the spin density is on the sulfur. The spin-density distribu-
tion and Ni–S bonding show the non-innocent electron donat-
ing character of the sulfur ligand on the oxidation state and
can explain the apparent Ni(II)-like spectra observed with
XAS, UV-Vis and MCD. Deuteration of the CoM ligand
revealed that the distance between the nickel centre and b-protons of CoM is very similar to those of Ni(II)-MCRox1-silent
for which an X-ray study exists.99 Exchange experiments in
D2O also suggest that the binding of the SO3� group of CoM
may be stabilized by hydrogen bonding with two nearby
tyrosines (Tyra333 and Tyrb367).94
In the context of the previous descriptions it is worthwhile
to mention that all known X-ray structures of MCR describe
inactive, oxidized (Ni(II)) forms. At present, no crystals of the
important paramagnetic forms have been grown, so the EPR
technique provides the main characterization method for the
active centre of MCR.
3.2.2. [NiFe] hydrogenases. Hydrogenases catalyze the het-
erolytic splitting of molecular hydrogen. The metal-containing
hydrogenases are usually classified by the type of metal in their
active site: [NiFe] and [Fe]-only hydrogenases, of which the
former class is the largest.100 The active site of the [NiFe]
hydrogenases consists of a heterobimetallic cluster, in which
the nickel and iron atoms are bridged by the sulfur atoms of
two cysteines. Furthermore, two additional cysteines bind
terminally to the Ni atom. The iron atom is ligated by three
non-protein di-atomic molecules (two CN� and one CO). At
least seven oxidation states have been reported for [NiFe]
hydrogenases. Advanced EPR techniques have played and are
still performing a crucial role in the elucidation of the structure
and activation mechanisms of hydrogenases.101
In the oxidized form, two EPR-active forms co-exist: the
‘unready’ Ni–A and the ‘ready’ Ni–B form, which can be
activated by reduction under a H2 atmosphere in a few
minutes (Ni–B) or after incubation for hours (Ni–A).102 In
the aerobically ‘as-isolated’ enzyme (Ni–A) an additional
bridging of the Ni and Fe atom is achieved by (presumably)
an oxygen species as could be inferred from 17O ENDOR at
Q-band mw frequencies.103 Combination of the EPR and
ENDOR data with density functional theory (DFT) calcula-
tions lead to two possible models: (i) OH� as bridging ligand
and (ii) the bridging ligand is a diatomic-type ligand.104 A
similar approach for Ni–B allowed for the identification of an
OH� bridging ligand.105
Reduction of the enzyme by two electrons leads to the
catalytically active Ni–C state. Illumination at low tempera-
ture causes the conversion of Ni–C into another paramagnetic
state Ni–L. Using 2H2O exchange experiments in combination
with ENDOR and HYSCORE spectroscopy the presence of a
hydride as a bridging ligand could be proven for a regulatory
hydrogenase106 and for a catalytic hydrogenase.107 After
illumination at 10 K, the strong hyperfine coupling assigned
to the bridging hydride is lost, indicating a cleavage of the
metal–hydride bond.106
The EPR story of [NiFe] hydrogenases can, in our view, be
considered a textbook example of the potential of modern-day
EPR. Not only are the experimental data gathered using a
variety of multi-frequency EPR and ENDOR techniques101
revealing information unattainable by other spectroscopic
methods, the interpretation of the EPR data is also corrobo-
rated by DFT computations.104,108 As stated earlier, one of the
great challenges is the theoretical modelling of the magnetic
parameters extracted from the experimental data in order to
obtain direct structural information about the system at hand.
The example of the study of [NiFe] hydrogenases shows the
potential of an integrated EPR-DFT approach. Note that we
focused in this section on the Ni-related centres in [NiFe]
hydrogenases. However, these proteins contain 11 more iron
atoms besides the one in the Ni–Fe subunit. These additional
Fe atoms are part of three iron–sulfur clusters and can also be
studied by EPR (see section 3.1.2).
3.3. Cobalt-containing proteins. Cobalt(II) is found in nat-
ural proteins, such as some active forms of B12 enzymes, but is
also a good functional mimic of other metal ions. Further-
more, Co(II) is an excellent spectroscopic probe since it is both
optically and EPR active. For these reasons, the unique
oxygen binding properties of Co(II) porphyrins have obtained
considerable attention in biochemical studies, because these
complexes can be used as model systems for the ligand-binding
characteristics of globins.109 Because of the fast autoxidation
rates, the analysis of the oxygenated native heme proteins is
often very difficult. Cobaltous-substituted porphyrins can then
offer a way to study both the oxy and deoxy species using CW
and pulsed EPR and ENDOR.110,111 Similarly, the EPR
analysis of Co(II)-substituted zinc-dependent aminopeptidases
revealed essential structural information that was not obtained
by other techniques.112 We here focus on the study of the
EPR-active forms of B12 proteins.
3.3.1. Cobaltous forms of B12 proteins. B12 derivatives are
involved in unique organometallic biological reactions.113 The
B12 co-factors are essentially cobalt-containing corrin com-
plexes (Fig. 6d). Cobalt(II) corrins such as cob(II)alamine (B12r)
are often relevant intermediates in the reaction cycles of B12
enzymes, but they are only transiently formed and therefore
difficult to observe. Cob(II)alamin complexes in which the
dimethylbenzimidazole (DBI) group is coordinated to the
cobalt atom are named base-on forms, whereas, if such a
coordination is absent or replaced by an exogeneous ligand,
these complexes are referred to as base-off complexes.
For isolated B12 co-factors in solution at neutral pH and in
the solid state, the base-on form is the most stable constitu-
tion. In contrast, at low pH, a base-off form is found. This
transition between the two forms is clearly reflected in the
X-band CW-EPR spectra. In the EPR spectrum of the base-on
form, a clear three-fold splitting of the high-field lines can be
observed, due to the hyperfine interaction with the 14N nucleus
(I = 1) of the coordinating DBI ligand. This disappears in the
EPR spectrum of the base-off form, since DBI is no longer
coordinating. In a recent work, we studied the solvent effects
This journal is �c the Owner Societies 2007 Phys. Chem. Chem. Phys., 2007, 9, 4620–4638 | 4633
on base-off Co(II) corrinates.114 Previous literature addressed
Co(II) corrinates in solution often as four-coordinated Co(II)
corrins.115 Using pulsed EPR and ENDOR in combination
with 2H2O exchange experiments we could clearly prove axial
solvent ligation for Cob(II)ester (heptamethyl cobyrinate per-
chlorate) and the base-off form of cob(II)alamin (B12r) in polar
solvents.114 Fig. 9 shows the difference HYSCORE spectrum
of Cob(II)ester in CD3OD and CH3OH. The 2H cross-peaks
reveal direct information on the hyperfine coupling to the
methanol deuterons proving axial coordination of the solvent.
The additional cross-peaks stem from combination between
the 2H basic frequency and one of the DQ frequencies of the
ligand nitrogens as indicated in Fig. 9. Since only combina-
tions between nuclear frequencies within the same mS mani-
fold can occur, the appearance of these cross-peaks give direct
information on the relative sign of the 2H and 14N hyperfine
couplings.114
In addition, the axial ligation of Cob(II)ester in methanol
was shown to be influenced by the phase transition of the
solvent. Axial ligation of one methanol molecule is observed in
the a-crystalline phase, whereas six-coordination becomes
possible in the glassy state. In a methanol : H2O mixture, only
five-coordination of Cob(II)ester is observed. Addition of
water to methanol changes its phase-transition properties
and leads to frozen solutions with a large degree of crystalline
structure. Differences in the solvent ligation could be related to
changes in the network formed by the solvent molecules and
the ring substituents. Furthermore, we could show that in
apolar solvents Cob(II)ester forms a contact-ion pair with its
counter ion, perchlorate. No evidence of pure four-coordi-
nated CoII corrins in solution is found. These observations
were corroborated by DFT computations, whereby again the
power of a combined EPR-DFT approach could be proven.
In addition, the hyperfine and nuclear quadrupole para-
meters of the magnetic nuclei in the vicinity of the unpaired
electron could be determined. Combined with our earlier study
on the base-on form of cob(II)alamin,116 these results can be
used as reference data to study in detail the electronic structure
of the Co(II) forms of B12 proteins.
The initial EPR studies on B12 proteins focused on the
analysis of the cobalamin form and of possible intermediately
generated radicals using X-band CW-EPR. The base-on bind-
ing mode has been found in diol dehydratase (DD)117 and in
B12-dependent ribonucleotide reductase (RNR).118 For other
B12 proteins, the intramolecular dimethylbenzimidazole base is
replaced by the imidazole side chain of a protein histidine
residue. One such example is glutamate mutase from Closti-
dium cochlearium. The EPR spectrum of this protein shows a
typical base-on pattern with the three-fold splitting of the
high-field line.119 Upon binding of cobalamin to the 15N-
labeled protein, the three-fold splitting changed into a two-
fold splitting (15N, I = 1/2), indicating that the cobalt is
coordinating to a nitrogen of the enzyme. Furthermore, the
EPR spectrum of a sample of the 15N-labelled protein in which
only the histidines were 14N labeled, showed again the char-
acteristic three-fold splitting. This EPR study was the first
unambiguous proof that in the carbon-skeleton rearranging
mutase a histidine residue of the enzyme rather than DBI
coordinates to the cobalt center. Finally, the base-off form of
cob(II)alamin is found to occur as an intermediate state in a
number of proteins. As an example, methanol:5-hydroxy-
benzimidazolylcobamide methlytransferase (MT1) is the first
of two enzymes playing a role in the transmethylation of
methanol to 2-mercaptoethanesulfonic acid inMethanosarcina
barkeri. Binding of MT1 to the methyl group of CH3OH only
occurs when the cobalt corrin is reduced to the Co(I) state.
This reduction requires H2, hydrogenase, methyltransferase
activation protein and ATP. Using CW-EPR, it could be
shown that the as-isolated oxidized Co(III) state is reduced to
a base-on cob(II)alamin (DBI internal coordination) by addi-
tion of H2 and hydrogenase.120 Subsequent addition of
methyltransferase activation protein and ATP leads to base-
off cob(II)alamin.
At present, advanced pulsed EPR studies on B12 proteins
are still very scarce. The Co(II)-product radical pair state of
ethanolamine deaminase is the B12 protein that is by far the
most studied by pulsed-EPR methods,121 but even for this
system only X-band three-pulse ESEEM techniques and no
advanced multi-dimensional pulsed EPR methods were ap-
plied. In our opinion, a large potential to understand the
inner workings of these proteins is in this way left aside and
we share the surprise of Bennet112 that relatively few EPR
studies of Co(II)-containing or Co(II)-substituted enzymes are
reported.
3.4. Copper-containing proteins
Undoubtedly, the majority of the EPR work on metallopro-
teins is performed on copper-containing proteins. Cu(II) (d9,
S = 1/2) is an ideal EPR probe. The ion is detectable by
CW-EPR in a broad range of temperatures, making it a
perfect tool to determine the dynamics and static structure
of the protein via the study of the temperature dependence of
the g and copper hyperfine values (63Cu, I = 3/2, abundance
69.15%, 65Cu, I= 3/2, natural abundance 30.85%). Although
Fig. 9 Difference HYSCORE spectrum of cob(II)ester in CD3OD and
in CH3OH obtained by subtraction of the spectra after normalization
to the DQ cross peak of the corrin nitrogen interaction. The observer
position corresponds to g||, mI = �7/2. From the analysis of the 2H
cross peaks in addition to W-band deuterium Mims ENDOR experi-
ments the axial ligation of the solvent could be proven.114 The other
cross-peaks in the (++) quadrant originate from combination fre-
quencies between the nitrogen DQ and deuterium basic frequencies.
4634 | Phys. Chem. Chem. Phys., 2007, 9, 4620–4638 This journal is �c the Owner Societies 2007
the examples of EPR studies of copper-containing proteins are
legion, including our own work on the Cu(II)-binding of prions
and prion analogues,122–125 we will here focus only on the
study of azurin using multi-frequency EPR techniques,126–136
because we think it offers the reader a clear insight into the
possibilities of what can be obtained by these advanced EPR
techniques.
3.4.1. Unravelling the structure of azurin. Although a quick
scan through the EPR/ENDOR literature shows that the
majority of the studies on copper proteins are performed at
X-band mw frequencies, the use of high-field EPR techniques
has great potential. This is exemplified by the study of wild-
type azurin from Pseudomonas aeruginosa and some of its
mutants. The Cu(II)-binding site in azurin is a typical type-I
copper centre characterized by the strong ligation of two
histidines and a cysteine and a weak ligation of commonly a
methionine residue (Fig. 6e). In order to understand the
electron-accepting and electron-donating processes occurring
in this blue-copper protein, the electronic structure of the
copper sites should be mapped out. EPR is one of the
techniques of choice. However, at X-band frequencies, it is
impossible to accurately determine the principal g values. This
high resolution can be obtained when using 95 GHz EPR
spectroscopy. The EPR lines in the W-band ESE-detected
EPR spectra of single crystals of wild-type azurin could be
related to the 16 protein molecules in the unit cell.126 Similar
analyses for the M121Q and M121H mutants of azurin
revealed clear spectral changes that could be assigned to
admixture of dyz (M121H) or dz2(M121Q) character into the
wave function.127,128 W-Band nitrogen ENDOR and X-band
ESEEM studies allowed for a complete determination of the
hyperfine and nuclear quadrupole tensors of the remote
nitrogens Ne of the histidine ligands.129–133 In a seminal work,
Groenen and co-workers detected for the first time single-
crystal W-band ESEEM spectra arising from the directly
coordinated nitrogens of the histidines that ligate copper in
azurin.134 The set of experimental data was completed by a
recent W-band deuterium ENDOR study of single crystals of
azurin selectively deuterated at the Cb position of the copper-
binding cysteine-112.135 The magnitude of the isotropic hy-
perfine coupling of the b deuterons can directly be related to
the spin density on the cysteine sulfur. All these EPR studies
have not only provided a direct insight into aspects of the
electronic structure of the copper site in azurin, they also form
a critical and necessary test for the different quantum-chemical
studies performed on azurin.136–138 Only a good qualitative
agreement between theory and experiment gives confidence in
the quantum-chemical approach and in the deduced conclu-
sions on the inner working of the protein.
3.5. Other metal-containing proteins
The above paragraphs highlighted the type of metal-contain-
ing proteins we are familiar with from our own research.
However, the list of paramagnetic ions reported to bind in
vivo to proteins is much larger (Mn(II), Mo(V), ...) and so are
the type of EPR studies performed on them. For a detailed
overview of these systems, we refer to a number of recent
reviews.15,84,89,139,140
4. Conclusions and challenges for the future
Recent methodological advances in EPR, amongst which are
the development of different microwave pulse sequences and
the construction of high-performance CW and pulsed EPR
and ENDOR spectrometers operating at high mw frequencies,
have largely increased the potential of applying these techni-
ques to different problems. However, EPR remains a specia-
list’s tool and because of this, the technique is still relatively
unknown to other scientific communities despite the recent
booming of the field. In this overview we have tried to give the
non-specialist reader an insight into the advantages and dis-
advantages of using EPR to study metalloproteins. It should
be noted, however, that the application domains of EPR reach
much further than the field of metalloproteins, comprising
applications in biology, chemistry, material sciences, physics,
medicine and pharmacology.
In our opinion, the most important challenges for the future
include instrumental aspects, such as improvement of sensi-
tivity and spectral resolution through new spectrometer design
at high mw frequencies, the further development and automa-
tion of adequate simulation programs, and the improvement
of the quantum chemical strategies for computation of EPR
parameters (see section 2.3). The ultimate goal is to create an
integrated toolbox that includes the spectral derivation of the
different spin Hamiltonian parameters up to the interpretation
of these magnetic interaction parameters in term of (geometric
and electronic) structure and dynamics of the complex under
study.
Acknowledgements
The majority of the own work cited in this manuscript stems
from the corresponding author’s research work from the last
10 years done at the ETH Zurich (1996–2001) and at the
University of Antwerp (2002 till now). Many students, post-
doctoral researchers and co-workers have contributed or are
contributing to the research on metalloproteins. Many thanks
go to all of them. The high-level technical support of Jorg
Forrer (ETH Zurich) and Paul Casteels (Antwerp) is highly
appreciated and was essential for many experiments. Special
thanks and thoughts go to the recently deceased Arthur
Schweiger, who was the corresponding author’s mentor in
Zurich and initiated and trained her in pulsed EPR. In general,
Arthur Schweiger has played a key role in the development of
advanced pulsed EPR techniques. He is missed as a great
scientist and a good friend.
In addition, S.V.D. has been inspired for the present article
through the many contacts and collaborations with fellow
EPR spectroscopists throughout the world, not in the least
within the COST P15 action ‘Advanced Paramagnetic Reso-
nance Methods in Molecular Biophysics’, chaired by the
author. Furthermore, S.V.D. wants to thank the many (bio)-
chemists she worked with in the course of her work on
metalloproteins. S.V.D. gratefully acknowledges the
This journal is �c the Owner Societies 2007 Phys. Chem. Chem. Phys., 2007, 9, 4620–4638 | 4635
University of Antwerp and the Fund for Scientific Research-
Flanders (FWO) for funding. E.V. is a research assistant of the
FWO.
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