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2005 when x rays modify-protein_structure_radiationd_amage at work
1. When X-rays modify the protein
structure: radiation damage at work
Oliviero Carugo1,2
and Kristina Djinovic´ Carugo3
1
Department of General Chemistry, University of Pavia, Via Taramelli 12, 27100 Pavia, Italy
2
National Laboratory TASC, INFM, Area Science Park Basovizza, 34012 Basovizza, Trieste, Italy
3
Max F. Perutz Laboratories, University Departments at Vienna Biocenter, Institute for Biomolecular Structural Chemistry,
University of Vienna, Campus Vienna Biocenter 6/1, Rennweg 95b, A-1030 Vienna, Austria
The majority of 3D structures of macromolecules are
currently determined by macromolecular crystallogra-
phy, which employs the diffraction of X-rays on single
crystals. However, during diffraction experiments, the
X-rays can damage the protein crystals by ionization
processes, especially when powerful X-ray sources at
synchrotron facilities are used. This process of radiation
damage generates photo-electrons that can get trapped
in protein moieties. The 3D structure derived from such
experiments can differ remarkably from the structure of
the native molecule. Recently, the crystal structures of
different oxidation states of horseradish peroxidase and
nickel-containing superoxide dismutase were deter-
mined using crystallographic redox titration performed
during the exposure of the crystals to the incident X-ray
beam. Previous crystallographic analyses have not
shown the distinct structures of the active sites
associated with the redox state of the structural features
of these enzymes. These new studies show that, for
protein moieties that are susceptible to radiation
damage and prone to reduction by photo-electrons,
care is required in both the design of the diffraction
experiment and the analysis and interpretation.
Introduction
X-ray crystallography is rapidly evolving as a standard
technique in biochemistry and molecular biology. Impress-
ive progress has been made during the past two decades,
including recombinant-DNA technology and other tech-
niques for sample preparation, computational procedures
to solve the phase problem (see Glossary) and refine 3D
structures, and tunable synchrotron beam lines to collect
diffraction data [1]. Crystallography is becoming a source
of information not only to interpret but also to predict
biological features of proteins, such as function [2] and
interaction networks [3]. Sequence analyses and data-
bases are increasingly being replaced by 3D-structure
analyses and databases owing to their high information
content at atomic level [4,5]. However, large macromol-
ecular assemblies and membrane proteins [6] remain
problematic because they are difficult to handle and
crystallize and the necessary diffraction quality is often
difficult to obtain. Several structural genomics initiatives
have been launched with the dual aim of determining the
3D structures of entire proteomes [7,8] and of developing
new technologies to enable high-throughput analyses
[9,10]. Use of synchrotron X-ray sources has made a
collection of extremely good diffraction data available,
which might result in detailed stereochemical character-
izations of the proteins: in a few cases, when atomic
resolutions (1.2 A˚ or better) are reached [11], very detailed
3D structural information can be obtained.
More than ten years ago, Henderson estimated that a
crystal cryo-cooled at 77 K would survive approximately
one day in an X-ray beam at a second generation
synchrotron facility and five years on a rotating anode
source, corresponding to an absorbed dose of 2!107
Gy
[12]. Because the X-ray beams generated at modern
synchrotron facilities deliver such a dose in standard
data-collection experiments [13], radiation damage is a
prominent issue in modern structural biology, and X-ray
beams at synchrotron radiation facilities often need to be
attenuated or defocused [14].
Several papers have been published on the observation
of radiation damage during the diffraction experiments
[15–18] and on the possible use of the radiation damage
itself to determine the crystal structures of biological
macromolecules (radiation-damage-induced phasing) [19].
However,experimentalartefactsarestillpossibleandcanbe
caused by the interaction between the incident X-ray
photons and the molecules from which the crystals are built.
A special case of radiation damage is photo-reduction of
metals in metalloproteins. Photo-reduction of metals in
proteins and of free metal ions in an aqueous solution was
studied by extended X-ray absorption fine structure
(EXAFS) and by X-ray absorption near-edge structure
(XANES) [20–24], which showed that these events take
place when samples are irradiated with intense X-ray
beams. The stereochemistry of a metal centre, which
accepts electrons emitted by the protein and the solvent
atoms in the crystal as a consequence of their excitation by
the intense incident X-ray beam, cannot be determined by
routine crystallographic experiments [25]. A protein
moiety is progressively reduced during the data collection
and the resulting 3D model can be considerably different
from the native structure.
A modification of the standard X-ray diffraction data-
collection procedures has had recent success [25] – it
Corresponding authors: Carugo, O. (carugo@tasc.infm.it), Carugo, K.D.
(kristina.djinovic@univie.ac.at).
Available online 8 March 2005
Review TRENDS in Biochemical Sciences Vol.30 No.4 April 2005
www.sciencedirect.com 0968-0004/$ - see front matter Q 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.tibs.2005.02.009
2. enables structural characterization of an intact, non-
reduced protein structure. This technique, termed here
‘multi-crystal data-collection strategy’ (Figure 1), has been
applied to study the various redox states that are
accessible to the haem moiety in horseradish peroxidase
and to the nickel in a nickel-containing superoxide
dismutase [25–26].
Here, we review radiation damage and its impact on the
biological information extracted from the crystal struc-
tures of horseradish peroxidase and nickel-containing
superoxide dismutase.
X-rays on crystals: radiation damage
When a crystal is exposed to an X-ray beam, the latter can
be absorbed and diffracted (Rayleigh scattering) by the
ordered and periodical arrangement of the electrons of the
atoms present in the crystal [27]. It is this phenomenon
that enables determination the 3D structure of proteins
and other molecules once the intensities of the diffracted
Glossary
B factors: In addition to the spatial position of the atoms, crystallography
enables the determination of the atomic-displacement parameters, usually
referred to as B or thermal factors, which monitor the average positional
spreading of the atom around its equilibrium position.
Compton scattering: Inelastic scattering of an X-ray photon by an electron.
X-rays transfer part of their energy to the electrons and the scattered photons
have wavelengths longer than the photons of the incident X-rays.
Incident X-rays: TheX-rayswithwhichproteincrystalsareirradiatedindiffraction
experiments. X-rays interact with electrons in atoms and an X-ray photon is
deflected away from its trajectory because of its collision with an electron.
Phase problem: The interaction between the incident X-rays and the crystal
gives rise to characteristic diffraction patterns that are unique to the crystal.
Such patterns are characterized by the intensities of the diffracted beams, which
are measured experimentally, and their associated phases, which are not
measurable. The phase information that is lost during the diffraction
experiment must be recovered by computational and/or experimental
methods; this is termed the phase problem. It is the fundamental problem of
any crystallographic structure determination.
Photo-reduction: A reduction reaction induced by light. Sometimes the
expression ‘photo-reduction’ is also used to indicate reductions in which the
substrate is not electronically excited by light. In these cases the expression
photo-induced reduction is more appropriate.
Rayleigh scattering: Elastic (or coherent) scattering of an X-ray photon by an
electron. X-rays do not transfer part of their energy to the electrons and the
scattered photons have the same wavelength of the incident X-rays
Sealed tubes: In a sealed tube, a cathode emits electrons that get accelerated
because of the high potential with respect to the metal anode. They travel in
vacuum and reach the anode (copper or molybdenum speed. Most of the
electron energy is converted into heat, which is removed by cooling with water.
A small amount of the energy is emitted as X-rays, the sharp peaks in the
spectrum are due to electron transitions between orbitals of the anode material.
Rotating anode: The heating of the anode caused by the electron beam limits
the maximum power of the tube. Power loading can be improved if the anode is
a rotating cylinder instead of a fixed piece of metal.
Tunable X-ray sources at synchrotron radiation facilities: In a synchrotron ring,
the charged particles (electrons or positrons) circulate in high vacuum at
velocities close to the speed of light and emit a spectrum of electromagnetic
radiation when they pass through a magnetic field. X-rays are electromagnetic
radiation in the wavelength range 1000–0.1 A˚ . A tunable X-ray source at a
synchrotron radiation facility enables selection of desired wavelengths.
Radiation-damage-induced phasing: This method exploits specific structural
changes caused by powerful X-ray beams at synchrotron sources in biological
macromolecules. These changes are employed to derive phase information
needed to solve the phase problem.
Photo-ionization: This is the process in which an atom or a molecule is ionized
by photons.
Non-Bragg diffuse scattering: The non-Bragg reflections, or diffuse scattering,
are produced by the non-periodical components of the protein crystal and
contains information about the inter- and intra-molecular motion in the crystal.
Cryo-cooling data-collection methods: The single crystal is flash-cooled to a
temperature of w100 K before being exposed to the incident radiation and kept
under cryogenic conditions during the whole experiment using a cold nitrogen
stream. The chemical reactions, and diffusion of the products, which are
responsible for the radiation damage, are slowed down and, in some cases,
entirely stopped at low temperatures, resulting in an increased lifetime of a
single specimen in the X-ray beam.
Multi-crystal data-collection strategy: A series of diffraction experiments is
performed on a number of crystals. The resulting datasets are then combined to
obtain a set of complete datasets, each corresponding to a nearly constant degree
of absorbed X-ray irradiation. Thus, it is possible to refine a 3D model for many
steps of the reaction induced by the irradiation, similar to a redox titration taking
place in a test tube. By monitoring the oxidation state of the active site by
electronic absorption microspectroscopy [33] it is then possible to assign the
oxidation state to each of the 3D structures that have been refined.
EXAFS: Extended X-ray absorption fine structure is a technique that uses X-ray
absorption measurements in the region up to 1000 eV over the absorption edge
of an atom to yield information about the local coordination environment of the
atom under study.
XANES: X-ray absorption near-edge structure is a technique that uses X-ray
absorption measurements in the region near the absorption edge (within
several tens of eV) of an atom to determine the local structure around the atom
under study.
TiBS
Incident X-rays
Protein
crystal
Diffracted X-rays
Subset 1
time t1
dose d1
Subset 2
time t2
dose d2
Susbset 3
time t3
dose d3
3
2
1
Crystal
Time t3
Dose d3
subset 1
Dose d1
Dose d2
Time t2
Time t1
Time t2
Dose d2
subset 2
Dose d3
Dose d1
Time t1
Time t3
Time t1
Dose d1
subset 3
Dose d2
Dose d3
Time t3
Time t2
(a)
(b)
Figure 1. Multi-crystal data-collection strategy. (a) In a crystallographic data-
collection procedure, the X-rays diffracted by the crystal are measured in a
sequential manner. The crystal is rotated in such a way that its orientation, relative
to the detector (which is fixed), changes. Therefore, some of the diffraction data
(subset 1) are measured at time t1, others (subset 2) at time t2 and others (subset 3)
at time t3. We limit this example to three subsets of data for simplicity, although
much higher values can be used. This implies that the diffraction data of subset 1
experience radiation dose d1, those of subset 2 take up dose d2, and those of subset
3 dose d3. (b) By using three different crystals it is possible to make three complete
data collections in such a way that all subsets of diffracted X-rays (subsets 1, 2
and 3) can be measured at the same time (t1, t2 or t3). In other words, it is possible to
construct three complete datasets, the first of which experiences radiation dose d1,
the second radiation dose d2 and the third radiation dose d3. Therefore, it is possible
to have an entire dataset collected at time t1 that experienced radiation dose d1
(pink), an entire dataset collected at time t2 which experienced radiation dose d2
(blue), and another entire dataset collected at time t3 that took up radiation dose
d3 (orange). Each of these reconstructed datasets enables, through crystallographic
analyses, the structural effects of the radiation damage occurring during the
exposure of the crystals to the incident X-rays to be monitored.
Review TRENDS in Biochemical Sciences Vol.30 No.4 April 2005214
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3. beams have been experimentally measured. The
interaction between X-rays and solid-state matter is
nevertheless a more complex phenomenon.
Besides the elastic Rayleigh scattering that causes the
diffraction, the incident X-ray photons can lose some of
their energy upon interaction with matter (inelastic
Compton scattering) and they can also transfer their
energy and ionize atoms (photo-ionization). In this case,
the resulting photo-electrons, which become solvated,
initiate photochemical processes that chemically modify
the molecules within the crystal, which typically contains
w50% of water. Solvated electrons might cause breakage
of X–H (XZS, O, N or C) bonds of the molecules in the
crystal, with consequent formation of highly reactive
species such as hydroxyl and hydrogen radicals, which
can initiate a myriad of reactions. An insidious effect of
these solvated electrons travelling within the crystal
specimen occurs when they are trapped within molecular
moieties that can be reduced to stable products. For
example, if a metal ion in a high oxidation state is present
in the crystal, it might easily accept these solvated
electrons and, thus, reduce its oxidation state. The
resulting 3D structure could reflect an altered sample,
different from the original material used at the beginning
of the experiment.
Incident photons have sufficient energy to photo-ionize
atoms within the crystal and cause degradation. Degra-
dation of the crystal can be monitored by, for example,
periodically measuring reference diffraction intensities,
which are often observed to decrease with time. Such
diffraction decay is caused by radiation damage and
depends on the incident flux and energy of the radiation,
and is faster when highly intense X-ray sources at
synchrotron radiation facilities are used. It is, to a lesser
extent, also observed in experiments with low intensity
X-ray sources that are used in smaller laboratories (sealed
tubes or rotating anodes).
Apart from the Compton and Rayleigh scattering,
some of the ‘non-diffraction’ phenomena that occur in a
crystal exposed to X-rays can be exploited to extract
biological information: for example, the non-Bragg
diffuse scattering can provide information about intra-
molecular dynamics [28,29].
Several stratagems can be used to reduce radiation
damage during data collection, the most common being
cryo-cooling data-collection methods [30], which are
routinely used in macromolecular crystallography. Nota-
bly, cryo-cooling methods are key features, together with
tunable X-ray sources at synchrotron radiation facilities,
that enable the collection of diffraction data at several
wavelengths for use in phasing procedures. These exper-
iments require long exposures to X-rays and have had
considerable impact on state-of-the-art macromolecular
crystallography during the past decade [31,32].
Systematic analyses of the radiation damage
Although cryo-cooling significantly reduces the radiation
damage during X-ray diffraction experiments, protein
crystals still decay when exposed to ionizing radiation.
Several studies have been devoted to the analysis of
radiation damage in protein crystals, and have revealed
that this is not a stochastic phenomenon; rather, it
concentrates at particular sites of the protein molecule.
Thus far, several proteins have been systematically
analysed: Torpedo californica acetylcholinesterase, hen
egg white lysozyme, winged bean chymotripsin inhibitor
and myrosinase [15–17]. A series of X-ray diffraction
datasets were taken consecutively from the same crystal
and the structures given by each dataset were determined.
The comparison of the refined structures with increasing
exposures showed that there are two main types of
residues that are affected by the ionizing radiation.
Disulfide bonds progressively break, and glutamate and
aspartate side-chains have a tendency to decarboxylate.
The results agreed with radio- and photochemical studies
of both proteins and model compounds in solution [33],
showing that the ionizing radiation produces the same
kind of structural modifications in samples, both in
solution and crystalline aggregate state. The photo-
electrons directly generated by X-ray absorption either
in the bulk solvent or in the protein [16] seem to be
responsible for such degradation and have been shown to
be mobile at cryo-temperatures within proteins [34]. Upon
irradiation, the disulfide bond (RSSR) forms a radical
anion (RSSR%K
) that might subsequently be protonated
(RSSRH%
). This dissociates into a thiol (RSH) and thiyl
radical (RS%
) [35]. Other modifications, such as the loss of
hydroxyl groups from tyrosine and of the methylthio group
from methionine, were also observed [15,16]. The solvent
accessibility does not seem to be directly correlated to
specific radiation damage on protein residues per se,
although there are semi-quantitative indications that
flexible regions are more affected than those that adopt
ordered a-helical or b-stranded secondary structure [18].
It also seems that less-stable disulfide bonds dissociate
more easily [16]. It has been suggested that disulfide
bonds or metal bio-sites in structures deposited in the
Protein Data Bank [36] require re-investigation because
they might be severely affected by the exposure of protein
crystals to X-rays [16].
Thorough analyses have shown structural changes
and/or chemical modifications in bacteriorhodopsin crys-
tals exposed to the X-ray radiation during diffraction
experiments [37]. A diffraction experiment conceptually
similar to the multi-crystal data-collection strategy, but
using several zones on a single crystal, was employed to
minimize the extent of radiolysis during the exposure to
X-rays; this enabled the study of only selected light-
induced conformational changes of bacteriorhodopsin.
Careful experimentalists, aware of possible subtle
structural changes of photo-active yellow protein (PYP)
caused by radiation damage, have used experimental
phasing techniques [38], together with the computational
corrections for radiation damage, based on the use of a
control dataset. This elegant method has identified a new
photo-intermediate of PYP.
X-ray titrations of protein structures
The specific radiation damage described might hinder the
correct description of the molecular details. For example,
the decarboxylation of glutamate and aspartate side-
chains during data collection might influence their atomic
Review TRENDS in Biochemical Sciences Vol.30 No.4 April 2005 215
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4. displacement parameters (B factors) or result in a locally
inaccurate stereochemistry with a concomitant risk of
misinterpretation of the results. Careful X-ray diffraction
and spectroscopic experiments on green fluorescent protein
(GFP) have shown that illumination with visible or UV light
causes a photo-conversion of GFP in a one-photon process,
which is complemented by decarboxylation of the glutamic
residue in the active site [39]. To confirm that the
decarboxylation was not due to X-ray-induced structural
change, a diffraction dataset on a low intensity X-ray source
was collected to compare the structure with that obtained
form the X-ray high-intensity experiment.
Another relevant problem arises in redox enzymes, in
which the oxidation state of the metal ions at the active site
could be affected by photo-electrons or free radicals,
modifying the active site stereochemistry. Changes to
stereochemistry caused by altered oxidation state can be
followed by X-ray diffraction experiments that employ the
multi-crystal data-collection strategy [25] (Figure 1). In this
way, it is possible to obtain complete diffraction datasets by
collecting subsets ofdata acquired fromseveralcrystals that
have taken up the same radiation dose. The complete
dataset obtained at low dose enables the analysis of the
material that did not suffer radiation damage. Complete
datasets, which bear increasing radiation doses, can be
assembled in a similar manner and used to analyse the
progressive effects of the radiation damage on the 3D
features. This sort of X-ray titration can also be elegantly
complemented by microspectrophotometric experiments, in
which optical spectra are recorded in the course of the X-ray
data collection or ‘off line’: the polarized absorption studies
provide a rigorous experimental tool to correlate the
observed structures and fine structural changes with their
physico-chemical features [40].
Unstable horseradish peroxidase redox intermediates
Horseradish peroxidase is an archetypal haem-containing
enzyme that uses hydrogen peroxide (H2O2) or alkyl
peroxide to oxidize a variety of organic and inorganic
compounds. The stable oxidation states of this enzyme are
illustrated in Figure 2; there are five stable intermediates
in two possible catalytic cycles [25]. Although there are no
severe experimental problems in determining the crystal
structures of the chemically reduced ferrous form or the
ground-state ferric form, the structures of intermediate
states – compound I, II and III – have not been determined
until recently: by applying the multi-crystal data-collec-
tion strategy, the structure of the catalytic pathway of
horseradish peroxidase has been analysed for the first
time. In classical data-collection experiments, compound
III is rapidly reduced to the ferrous form once exposed to
X-rays; analogously, compound I is reduced to compound
II, and compound II to the ferric form.
The Fe–O distances determined crystallographically
(1.7, 1.8 and 1.8 A˚ for compound I, II and III, respectively)
agree well with those derived from EXAFS (1.64, 1.93 and
1.72 A˚ for compound I, II and III, respectively) [40].
Although the region above the haem plane does not host
solvent molecules in the ferric and ferrous forms, in
compound III, a superoxide radical anion binds to the
metal with one of its oxygen atoms in a bent conformation
and is stabilized by three hydrogen bonds that its other
oxygen atom forms with Arg38, His42 and a water
molecule. In compounds I and II, a ferryl oxygen atom is
coordinated by the iron in the haem group, and forms two
hydrogen bonds with Arg38 and a water molecule, which,
in turn, interacts with His42 via another hydrogen bond.
The structures of active sites of the five stable oxidation
states of are illustrated in Figure 3.
The analysis of compound III also revealed the
mechanism of the four-electron reduction of oxygen to
water, which causes the transformation of compound III to
the ferrous form of the horseradish peroxidase (Figure 4).
The O–O bond of the superoxide ligand breaks and the
resulting water molecule forms a hydrogen bond with the
ferryl-like oxygen that keeps its coordination position close
totheiron.AtahigherX-raydose,theFe–Obondbreaksand
a penta-coordinated iron centre forms, with the iron slightly
moving below the haem plane. Interestingly, these are the
onlymodificationsintheproteinthatareobserved.Disulfide
bonds, for example, are unaffected by any measurable radi-
ation damage, suggesting that the metal bio-site is much
more reactive than the rest of the molecule.
Nickel superoxide dismutase: war and peace between
spectroscopy and crystallography
Oxygen-metabolizing organisms have to face the toxicity
of superoxide radicals (O2
K
) [41] that are generated by a
single-electron transfer to dioxygen. Superoxide dismutases
(SOD) enzymes, which are active in the defence against
oxidative stress, keep the concentration of O2
K
at controlled
low levels, thus, protecting biological molecules from oxi-
dative damage. SODs are classified according to the metal
species that acts as the redox-active centre to catalyse the
dismutation reaction 2O2
K
C2HC
/O2CH2O2. Three metal
TiBS
Fe (III)
Ferric form
(ground state)
Fe(II)
Ferrous form
Fe(III)
Compound III
O
O•–
HOO•
Fe(IV)
Compound I
π radical cation
O
•+
Fe(IV)
Compound II
O
H+
H+
H+
e–, H+
e–, H+
e–, H+
H2O
H2O
H2O2
O2
Figure 2. Stable oxidation states of the active site of horseradish peroxidase. The
peroxidase catalytic cycle, via compounds I and III, is depicted on the right side
(purple arrows). The oxidase catalysis cycle, via the ferrous form and compound III,
is shown on the left side (green arrows).
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5. species are found at the active site of SOD [42]; copper-
and zinc-containing SODs (CuZnSODs) have copper as the
catalytically active metal, whereas manganese- and iron-
containing SODs (MnSODs and FeSODs, respectively)
host manganese and iron, respectively. Recently, a new
SOD with Ni3C
at its active site (NiSOD) was purified
from several Streptomyces species [43–45]. X-ray absorp-
tion spectroscopy [46] and electron paramagnetic reson-
ance (EPR) spectroscopy on the resting enzyme revealed
the involvement of sulfur ligands and at least one axial
nitrogen-ligand in the Ni coordination. The structure of
NiSOD (from S. seoulensis) in two independent crystal
forms [47], determined by classical X-ray diffraction
experiments, showed a square-planar Ni coordination for
the amino group of His1, the amide group of Cys2 and two
thiolate groups Cys2 and Cys6 (Figure 5). The imidazole of
His1 was not found to coordinate the Ni but was, instead,
involved in hydrogen-bonding interactions to neighbour-
ing residues. By contrast, EPR spectra confirmed the
presence of an axial N-ligand at pHz5. A subsequent
multi-crystal data-collection strategy revealed the active
site of NiSOD in three stages of its oxidation state:
oxidized (Ni3C
), intermediate and reduced (Ni2C
)
(Figure 6). Similarly, the crystal structure of a homologous
NiSOD from S. coelicolor, determined to high resolution
(1.3 A˚ ) in a standard X-ray diffraction experiment [48],
showed a partially reduced metal site, with the His1 side-
chain in a double conformation corresponding to liganding
and non-liganding orientations, implying oxidized and
reduced states of the nickel ion in the active site. This
finding suggests that the application of multi-crystal data-
collection strategy would enable the ‘isolation’ of the pure
oxidized and reduced form of the enzyme.
Concluding remarks: final perplexities and caveats
Detailed studies have shown that the initial effects
radiation damage do not affect the overall diffraction
quality of the crystals, which therefore do not show
symptoms of lethal illness. The photo-electrons and the
radicals formed by the absorption of photons by the crystal
can initiate chemical reactions that are not spread
stochastically within the crystal but tend to cluster in
protein moieties, causing specific damage observed as
ablation of carboxylate groups from aspartate and gluta-
mate residues, breakage of disulfide bonds and reduction
of metal centres. A thorough understanding of these
processes becomes particularly relevant when some of the
afflicted residues are involved in crucial functional roles of
the protein, such as, for example, in GFP [39].
The case of redox enzymes is also pertinent. There is
evidence that some of the oxidation states that are stable
in horseradish peroxidase and NiSOD cannot be
structurally characterized with routine crystallographic
techniques because the oxidized forms can be quickly
reduced, probably by photo-electrons. The resulting 3D
models could, in such cases, correspond to one ore more
states that have evolved during exposure to the X-ray
beam. An ingenious multi-crystal data-collection strategy
has been proposed, and successfully applied, to determine
distinct redox forms of horseradish peroxidase and,
subsequently of NiSOD, the metal centres of which are
too sensitive to radiation damage to be treated routinely.
Arg38
Arg38
Arg38 Arg38 Arg38His42 His42 His42His42His42
His170 His170 His170 His170 His170
Acetate
Water Water
Water
Compound III (1h57) Compound II (1h55)Compound I (1hch)Ferric form (1h5a) Ferrous form (1h58)
Figure 3. Crystal structures of the active site of horseradish peroxidase in the various stable oxidation states. The PDB codes are given in parentheses. The iron cation is
indicated by a magenta sphere and the water molecules are indicated by red spheres. Hydrogen bonds are indicated by broken lines.
Fe(III)
His170
H+
H+
Fe(III)
His170
•+
Fe(III)
His170
OH•
His170
Fe(III)
+ 2H2O
Fe(II)
His170
e– + H+
e– + H+
e– + H+
e– + H+
H2OH2O
H2O
H2O
O•
TiBS
O2
–
Figure 4. Mechanism of reduction of horseradish peroxidase from compound III to
the ferrous form.
Review TRENDS in Biochemical Sciences Vol.30 No.4 April 2005 217
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6. These observations indicate that caution is required in
using crystallographic results in biochemistry and mol-
ecular biology, in particular when molecules under study
are highly sensitive to radiation. Many inaccurate results
have been deposited in the Protein Data Bank; is it, thus,
justified to take the PDB files as a standard of truth? Some
results might host experimental artefacts and could
propagate errors in the Protein Data Bank.
What we study and even what we teach could be, at
least in part, wrong. For example, interpretation of the
structural features of the blue copper protein active sites
might need to be revisited. Their oxidized, copper(II) forms
are considered strong oxidizing agents because their metal
bio-sites are constrained to adopt a stereochemistry that
is more appropriate for the reduced, copper(I) state [49].
It can be hypothesized that the oxidized, copper(II)
forms are, at least partially, reduced during diffraction
experiments [23,24].
Obviously, this does not imply that the entatic state
theory that relates reactivity with stereochemical strain
[50] must be reconsidered; we would like to point out that
caution should be applied when using crystallographic
information in biological interpretations. Despite exciting
progress in theory and methods, experimental artefacts
are still possible, and synergy between crystallographic
and spectroscopic methods is certainly necessary [51].
Limiting and controlling radiation damage remains, of
course, essential to crystallography. Scavengers can
intercept photo-induced radical species and reduce radi-
ation damage when introduced appropriately in the
crystal sample [52], and nanotechnology-based templates
can give higher resistance to intense beams [53]. Radi-
ation damage can, however, be an intrinsically interesting
phenomenon that might give crucial functional infor-
mation when exploited suitably, but could be misleading
when ignored or overlooked.
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