An EPR, thermostability and pH-dependence study of wild-type and mutant forms of catechol 1,2-dioxygenase from Acinetobacter radioresistens S13

Article abstract of DOI:10.1007/s10534-012-9595-x

Intradiol dioxygenase are iron-containing enzymes involved in the bacterial degradation of natural and xenobiotic aromatic compounds. The wild-type and mutants forms of catechol 1,2-dioxygenase Iso B from Acinetobacter radioresistens LMG S13 have been investigated in order to get an insight on the structure-function relationships within this system. 4K CW-EPR spectroscopy highlighted different oxygen binding properties of some mutants with respect to the wild-type enzyme, suggesting that a fine tuning of the substrate-binding determinants in the active site pocket may indirectly result in variations of the iron reactivity. A thermostability investigation by optical spectroscopy, that reports on the state of the metal center, showed that the structural stability is more influenced by the type rather than by the position of the mutation. Finally, the influence of pH and temperature on the catalytic activity was monitored and discussed in terms of perturbations induced on the tertiary contact network of the enzyme. Springer Science+Business Media New York 2013.

Full text of DOI:10.1007/s10534-012-9595-x

Biometals
DOI 10.1007/s10534-012-9595-x
An EPR, thermostability and pH-dependence study
of wild-type and mutant forms of catechol 1,2-dioxygenase
from Acinetobacter radioresistens S13
Raffaella Caglio
Francesca Valetti
Elena Ghibaudi
•
Enrica Pessione
Carlo Giunta
•
•
•
Received: 24 July 2012 / Accepted: 29 October 2012
Ó Springer Science+Business Media New York 2012
Abstract Intradiol dioxygenase are iron-containing
enzymes involved in the bacterial degradation of
natural and xenobiotic aromatic compounds. The
wild-type and mutants forms of catechol 1,2-dioxy-
genase Iso B from Acinetobacter radioresistens LMG
S13 have been investigated in order to get an insight on
the structure–function relationships within this sys-
tem. 4K CW-EPR spectroscopy highlighted different
oxygen binding properties of some mutants with
respect to the wild-type enzyme, suggesting that a fine
tuning of the substrate-binding determinants in the
active site pocket may indirectly result in variations of
the iron reactivity. A thermostability investigation by
optical spectroscopy, that reports on the state of the
metal center, showed that the structural stability is
more influenced by the type rather than by the position
of the mutation. Finally, the influence of pH and
temperature on the catalytic activity was monitored
and discussed in terms of perturbations induced on the
tertiary contact network of the enzyme.
Keywords EPR spectroscopy Á Structure–function
relationships Á Intradiol dioxygenase Á Aromatic
compound degradation Á Protein stability Á
Iron-containing enzyme
Abbreviations
1,2-CTD
Catechol 1,2-dioxygenase
Ar-1,2-CTD Catechol 1,2-dioxygenase Iso B from
A. radioresistens S13
1,2-CCD
4-CCD
Chlorocatechol 1,2-dioxygenase
Chlorocatechol 1,2-dioxygenase from
R. opacus 1CP
Introduction
Non-heme Fe dioxygenase, that are classified into
extradiol and intradiol cleaving enzymes based on
their catalytic mechanism, are of paramount impor-
tance for the bacterial degradation of natural and
xenobiotic aromatic compounds (Furukawa 2000;
Milligan and Haggblom 1998; Nishizuka et al. 1962;
Ogawa et al. 2003; Pieper and Reineke 2000; Reineke
1
998).
R. Caglio Á E. Pessione Á F. Valetti Á C. Giunta
Both classes catalyze the dearomatization of the
Dipartimento di Scienze della Vita e Biologia dei Sistemi,
Universit a` di Torino, Via Accademia Albertina 13,
substrate by inserting molecular oxygen in the
aromatic ring and are characterized by the presence
II
of Fe and Fe as the catalytic center, respectively.
1
0123 Turin, Italy
III
E. Ghibaudi (&)
˚
The X-ray structures at 1.80 A resolution of wild-
Dipartimento di Chimica, Universit a` di Torino,
Via Giuria 7, 10125 Turin, Italy
e-mail: elena.ghibaudi@unito.it
type catechol 1,2-dioxygenase from Acinetobacter
radioresistens LMG S13 and of its A72G mutant have
123

Biometals
been recently deposited in the Protein Data Bank
activity have been checked in all variants and the
results have been compared and discussed in the light
of the structural data available on the enzyme.
(
entries 2XSV and 2XSU) (Micalella et al. 2011).
The catalytic center is a trigonal bipyramidal Fe
III
ion coordinated by two histidine and two tyrosine
residues and a water molecule in the equatorial plane
as the fifth ligand. The axial tyrosine and the water
ligand are displaced by catechol, allowing a direct
Experimental
III
coordination of the diol to the Fe , based on spectral
Chemicals
(
Horsman et al. 2005; Citadini et al. 2005) and
crystallographic evidence (Citadini et al. 2005; Ear-
hart et al. 2005; Ferraroni et al. 2004, 2006; Matera
et al. 2010; Vetting and Ohlendorf 2000). According
to the accounted models for the catalytic cycle, the
oxygen binds directly to the iron centre (Bugg 2003) in
an ordered sequential mechanism.
All reagents used in this study were analytical grade and
were purchased from Sigma-Aldrich (Milan, Italy).
Liquid helium was supplied by SIAD (Turin, Italy).
Protein expression and purification
A basic issue concerning this class of enzymes is
their substrate selectivity: although it is known to be
influenced by electronic factors, a number of investi-
gations prove that it is finely tuned by the active site
residues (Caglio et al. 2009; Ferraroni et al. 2004;
Ferraroni et al. 2006; Matera et al. 2010).
Ar-1,2-CTD was expressed in E. coli BL21(DE3) as
described elsewhere (Caglio et al. 2009). The concen-
tration of wt and recombinant protein was determined
through the absorbance peaks at 280 and 430 nm, by
using the extinctioncoefficients reported by Briganti et al.
1
mg/ml
1 mg/ml
1 mg/ml
(
2000) e
= 0.798 e430
.049. All UV–Vis measurements were performed on a
= 0.033 e440
=
280
Someresidueinthe catalyticpocket havebeenshown
crucial for catalysis (Ferraroni et al. 2004; Vetting and
Ohlendorf 2000): these are Asp52/Pro76, Ala53/Gly77,
Phe78/Leu109 and Cys224/Ala254 (4-CCD from R.
opacus/1,2-CTD Acinetobacter ADP1). Site-directed
mutagenesis applied to positions 69 and 72 of the
primary sequence of catechol 1,2-dioxygenase IsoB
from A. radioresistens LMG S13 (Caglio et al. 2009;
Caposio et al. 2002) brought to the expression of the
following mutants: Leu69Ala, Ala72Gly, Ala72Pro,
Ala72Ser, Ala72Asn, Ala72Asp and the double mutant
Leu69Gly Ala72Gly. It has been demonstrated (Caglio
et al. 2009) that mutants at pos.69 show a higher
substratespecificitytowards4-chlorinatedcatecholsand
are active towards 4,5-dichlorocatechol and tert-butyl-
catechol, which are never or rarely recognized by
0
diode-array spectrophotometer Agilent 8453 UV–Vis
equipped with a Peltier system for temperature control.
Electron spin resonance measurements
CW-EPR measurements were performed on a Bruker
300ESP instrument, equipped with a cylindrical cavity
and a cryostat for measurements at liquid Helium
(4 K) temperature. The protein samples dissolved in
50 mM HEPES buffer pH 8.0 were concentrated up to
1 mM on Amicon VIVASPIN ultrafilters and put into
quartz tubes (Ø = 3 mm), that were subsequently
frozen in liquid nitrogen and inserted into the cryostat
for measurements. The instrumental settings were as
follows: microwave power 2 mW; modulation ampli-
tude 2 G; modulation freq. 100 kHz; T = 4 K; freq.
9.42 GHz. Saturation curves were determined with the
same instrumental settings, except that microwave
power was varied in the 80 lW–10 mW range.
1
,2-chlorocatechol dioxygenase (1,2-CCD) (Potrawfke
et al. 2001) or 1,2-catechol dioxygenase (1,2-CTD).
Conversely mutations at pos. 72 results in higher catalytic
efficiency towards mono-chlorinated substrates.
In the present study, a CW-EPR investigation of the
wild-type and mutants forms of Ar-1,2-CTD from A.
radioresistens LMG S13 has been carried out with a
special focus on the oxygen binding mode of the
different variants. The thermostability of these vari-
ants has also been monitored by UV–Vis spectros-
copy, taking the iron centre as a marker. Finally the
optimal Temperature and pH values for the catalytic
Thermal unfolding studies
Thermal transitions were monitored in the 20–95 °C
range by using the absorption peak at 430 nm as a
marker of the integrity of the iron catalytic centre. The
wt and mutant samples in 50 mM HEPES buffer pH
1
23

Biometals
8
.0 were concentrated up to 0.13 mM by ultrafiltration
resulting data overlapped in order to confirm that there
were no different effects of buffering compounds on
the enzyme activity due to the chemical structure and
not on the pH.
on Amicon VIVASPIN ultrafilters. Each sample was
incubated for 3 min at the selected temperature and the
absorbance at 430 nm was subsequently recorded. The
same procedure was applied at each T.
Determination of the temperature of optimal
activity
Results
EPR measurements
The catalytic activity of wt and variants of Ar-1,2-
CTD was determined in the 10–60 °C range after
incubating each sample for 3 min at the chosen
temperature.
The wild-type (wt) and mutant forms of Ar-1,2-CTD
underwent EPR measurements at 4 K, in order to find
out whether mutations had a direct influence on the
iron coordination sphere. In fact, EPR spectroscopy is
extremely sensitive to the chemical and magnetic
environment of paramagnetic ions and may report on
distortions and/or changes occurring within the iron
coordination sphere.
The activity assay was performed spectrophoto-
metrically by following the absorbance increase at
-
1
-1
2
60 nm (e = 17,600 M cm ) due to the enzy-
matic conversion of catechol into cis–cis muconic
acid. 1 ml solution contained: 0.1 lM (final concen-
tration) Ar-1,2-CTD ? catechol (concentration ran-
ge = 1 lM–1 mM) in 50 mM HEPES buffer pH 8.0.
The following samples were examined: wt, L69A,
A72G, A72P, A72S, A72N, A72D and the double
mutant L69G A72G. The EPR spectra are reported in
Fig. 1a, b: this is the first EPR report of a whole series
of Ar-1,2-CTD mutants compared to the wt form.
All samples, with the only exception of A72D, were
characterized by a signal at g *4.29 that is consistent
The kinetic parameter (k ) was determined by fitting
cat
the experimental data to the Michaelis–Menten model
by a non-linear least-squares fitting program. In order
to ensure that the activity at increasing temperature
was not affected by changes in K the substrates were
M
III
always used in large excess (although comparison of
with the presenceofa Fe centrecoordinated to Tyr and
K at 20 and 30 °C on wt and mutants confirmed that
His in a ligand field rhombic (highly asymmetric)
environment, in agreement with the data previously
reported by Briganti et al. (2000). In the case of the wt
enzyme, the sample was concentrated enough to exhibit
the g *10 spectral minor component (data not shown)
arising from the lowest energy Kramers doublet.
No iron signal was highlighted in the A72D mutant:
this is indicative of a low level of iron incorporation
that results in an Fe concentration lower than the
sensitivity limit of EPR.
M
no changes were observed on this parameter upon
temperature increase).
Determination of the pH of optimal activity
The pH of optimal activity as well as the pH value
corresponding to 50 % residual activity of wt and
variants of Ar-1,2-CTD were found by determining
the kcat values at 30 °C over the 5.5–10.0 pH range,
according to the above-described assay. The experi-
mental conditions were the same as described above,
apart from the buffer which was changed in order to
cover the whole pH range. Twelve different buffers
made by three compounds, MES (pH 5.5–7.5), HEPES
Unlike the spectra reported by Briganti et al.
(1997), the EPR traces denounce the massive presence
of O either dissolved or in close interaction with the
2
iron center. O dissolved in the buffer is responsible
2
for the linebase distortion and does not imply any
specific interaction with the protein. Conversely, the
two additional signals that appear as shoulders of the
g *4.29 spectral line are typical of a magnetic
interaction between paramagnetic centers (Cappillino
et al. 2012). As the only paramagnetic species found in
(
pH 7.5–8.5), CHES (pH 8.5–10.0) were prepared
according to the result shown by an informatic tool
http://www.bioinformatics.org/JaMBW/5/4/index.
(
html), setting the preparation temperature at 20 °C and
the working temperature at 30 °C.
Buffer concentration was varied in order to keep a
constant ionic strength of 0.033 M. At pH 7.5 and 8.5
both available buffer systems were used and the
the samples are O and the Fe(III) ion, these signals are
2
likely to arise from the interaction between these two
species.
123

Biometals
Fig. 1 a, b 4K EPR spectra of Ar-1,2-CTD wt and variants in
0 mM HEPES solution pH 8.0 before degassing. The signal at
5
g = 4.29 raises from the iron coordination centre. The spectra
show signals assignable to oxygen either dissolved in the buffer
or in magnetic interaction with the iron. Experimental settings:
microwave power 2 mW; modulation amplitude 2 G; freq.
9
.42 GHz
Fig. 2 a, b 4K EPR spectra of Ar-1,2-CTD wt and variants in
5
0 mM HEPES solution pH 8.0 after degassing with Argon. The
In order to establish the nature and the degree of
signal at g = 4.29 raises from the iron coordination centre. The
oxygen dissolved in the buffer has disappeared and only L69A
and the double mutant forms still show the presence of oxygen
interacting with the iron. Experimental settings: microwave
power 2 mW; modulation amplitude 2 G; freq. 9.42 GHz
lability of such interaction, the samples were thawed
in a ice/water bath and degassed for several minutes
under an Argon flux.
The spectral traces recorded on the degassed
samples are reported in Fig. 2a, b.
is completely distorted as compared to wt: this finding
is consistent with a strong perturbation of the
environment of the iron centre that results in an
heterogeneous environment of the iron in this mutant.
In the attempt to further investigate the structural
features of the iron environment and to determine the
iron-ligand distances by pulsed EPR, the relaxation
behaviour of all samples was investigated and the
microwave saturation curves were determined at 4 K
(Fig. 4). Different magnetic environments usually
results in distinct relaxation behaviours: unfortu-
nately, the iron centre of Ar-1,2-CTD turned out to
be a very strong relaxing agent in all samples, thus
making undistinguishable the relaxation times at 4 K
and preventing the pulsed-EPR approach.
Dissolved oxygen was removed in all cases and, in
most samples, the O2 interacting with iron was
removed as well, giving rise to EPR spectra similar
to that reported by Briganti et al. (2000). Although, a
few exceptions were found. The shoulders of the
g *4.29 EPR signal are still visible in both the L69A
sample and the double mutant: this suggests a higher
stability of their Fe–O adduct as compared to the
2
other samples. Their magnified EPR spectra are shown
in Fig. 3. Further attempts to degas the sample did not
bring about any further change, thus showing that the
interaction between O and iron is relatively stable in
2
these two cases.
Interestingly, samples that have lost the shoulders
of the g *4.29 signal also underwent a colour change
from bright red to pale red. Conversely, those that
Thermal denaturation studies
exhibited a stronger interaction with O kept their
2
bright colour unchanged. This provides an indirect
A difference in the stability of the iron coordination
center of the different protein samples was detected as
samples were thawed and frozen again: the intensity of
the EPR signal arising from the iron centre was
negatively affected, suggesting an iron loss. In order to
evidence that O enters the active site and is spatially
2
very close to the iron.
The EPR spectra of the A72N mutant is also
reported in Fig. 3. The spectral pattern of this variant
1
23

Biometals
Fig. 4 Saturation plot of Ar-1,2-CTD wt and variants in
0 mM HEPES solution pH 8.0 after degassing with Argon,
based on the intensity of the g = 4.29 EPR signal recorded at
5
4
K
pH and temperature influence on the catalytic
activity
Fig. 3 4K EPR spectra of three variants of Ar-1,2-CTD in
5
0 mM HEPES solution pH 8.0 after degassing with Argon. The
spectra of the L69A and the L69G A72G forms highlight the
presence of oxygen in magnetic interaction with the iron; the
spectral pattern associated with the A72N form indicates a
strong perturbation of the iron coordination sphere
In order to make a comparison between the catalytic
performances of the wt and mutant forms of Ar-1,2-
CTD, their optimal temperature, i.e. the temperature
value corresponding to the highest enzyme activity
(here referred to as optimal T values) were deter-
mined. These results are reported in Table 1, together
with the corresponding kcat values (determined in the
presence of catechol).
quantify the thermal stability of the iron center, we
determined the melting temperatures of wt Ar-1,2-
CTD and its mutant by using the absorption band at
4
30 nm as a marker of the state of the metal center, as
this band reports specifically on the iron (Table 1).
Interestingly, we noticed meaningful stability dif-
ferences between the wt and the mutants. Ar-1,2-CTD
wt is the most stable form as it shows a melting
temperature of about 48 °C. All mutants were less
stable; no direct correlation between the mutated
position and the stability was found: for example, the
L69A and the A72S forms display the same thermal
stability, whereas A72G is slightly less stable. Inter-
estingly, a similar marked decrease in thermal stability
was observed in both the double mutant and the A72P
variant: these two forms were the less stable of the
whole family of mutants. No data were collected on
A72N due to the extreme instability of the sample, that
did not allow the application of the experimental
protocol.
As far as the optimal T values are concerned, no
significant differences were noticed between the wt
and the A72G or the A72P mutant. Conversely,
mutation at position 69 seems to lower the T of
optimal activity, as highlighted by the L69A and the
double mutant form, although this latter is far less
active. An opposite trend is followed by the A72S
mutant, whose optimal T is several degrees higher than
the wt form. Interestingly, based on the kcat values, this
is also the most active mutant, although still half-active
with respect to the wt. A possible stabilizing effect of
hydrogen bonds to the serine residue inserted by the
mutation could rationalise this finding.
In general, mutations at position 72 do not seem to
be disrupting towards the catalytic activity of the
enzyme, although in all cases their kcat values are
123

Biometals
Table 1 Thermal stability values, pH and T dependence of the wild-type and mutant forms of Ar-1,2-CTD
Protein variant
1
Melting
temperature (°C)
2
T of optimal activity
3
4
pH of optimal
activity (catechol)
pH50 %
(catechol)
-1
(°C) and kcat (s
)
a
a
a
WT
48.2 ± 0.2
45.4 ± 0.8
43.6 ± 0.2
36.8 ± 0.5
45.6 ± 0.2
37.9 ± 0.7
30 °C
8.0–8.5
8.0–9.0
9.0
6.89 ± 0.05
7.07 ± 0.07
7.44 ± 0.05
6.57 ± 0.02
7.53 ± 0.06
7.49 ± 0.25
3
0.03 ± 1.77
27 °C
.81 ± 0.10
30 °C
3.56 ± 2.61
30 °C
4.27 ± 0.16
37 °C
5.48 ± 0.51
23–27 °C
.04 ± 0.27
L69A
4
A72G
A72P
1
8.5–9.0
8.0–9.0
8.0
1
a
a
A72S
1
L69G A72G
2
Column: (1) Melting T of Ar-1,2-CTD wt and variants determined by monitoring the optical absorption at 430 nm; (2) T of optimal
activity and corresponding kcat value; (3) pH of optimal activity at 30 °C; (4) pH values corresponding to 50 % residual activity at
3
a
0 °C
Data adapted from Caglio et al. (2009)
significantly lower as compared to wt. Mutations at
position 69 affects the catalytic proficiency much
more deeply, but it is also to be considered that
mutation at that site enhances the recognition and
catalysis towards 4-chlorocatechol by inverting the
substrate preference (Caglio et al, 2009).
was not detected in blank samples (containing the
buffer, without the protein), it is clear that the
increased oxygen concentration in the protein sample
has to be related with the presence of the protein.
Moreover, two clearly distinct behaviours were
observed.
Table 1 also reports the pH-dependence ofkcat for the
wtandmutantsformsofAr-1,2-CTDenzyme,expressed
eitheraspHofoptimalactivityoraspHcorrespondingto
For most Ar-1,2-CTD variants, dioxygen could be
removed by simple degassing: this went along with a
marked colour change and the disappearance of the
shoulders of the g *4.29 signal, clearly indicating
that the interaction with dioxygen was extremely
weak. A few samples still exhibited the shoulders of
the g *4.29 signal even after repeated attempts to
degas the sample under an Argon pressure; in addition
these samples kept their color unchanged, providing
an indirect evidence that the interaction between O2
and iron is still in place and that it likely arises from the
close proximity of the two paramagnetic species. This
finding suggests an increased ability of the L69A and
the double mutant to interact with oxygen and to form
a moderately stable adduct.
5
0 % residual activity (measured at 30 °C).
The presence of mutations shifts or widens the pH
range of optimal activity towards higher values
(
8.5–9.0).
As far as pH50 % is concerned, its value is shifted
towards pH [ 7 in all cases, with the only exception of
the A72P mutant, whose behaviour resembles that of
wt. Once again, no correlation between the mutation
site and the pH-dependence of the activity is found,
whereas the type of mutation seems more crucial.
Discussion
These results may be interpreted in the light of the
reaction mechanism of intradiol catechol dioxygenase
and, in particular, of the detailed theoretical study by
Nakatani et al. (2009). It is well known that the
catalytic mechanism of intradiol catechol dioxygenase
requires the activation of oxygen through an electron-
Oxygen binding properties
The EPR results show the presence of consistent
oxygen amounts in the proteins samples. As oxygen
1
23

Biometals
3
10.6 A (L69A), respectively. Such finding is sup-
transfer from the substrate, coordinated to the iron.
Nakatani clearly shows that such charge-trasfer is
ported by the unique ability of the L69A mutant to
oxidise bulky substrates such as tert-butyl-catechol
(Caglio et al. 2009), which cannot be recognized by the
wt or by the mutants at position 72. All in all, two
factors may explain the increased ability of the L69A
possible only once the Fe(III)–O bond is established,
2
Ã
because it influences the energy level of the p orbital
p
of O . In other words, according to the ‘‘oxygen
2
activation mechanism’’, once the catechol is in the
active pocket, the dioxygen molecule directly attacks
the iron center to form a g1-end-on type Fe(III)–O2
adduct which is immediately converted into an
iron(III)-g1-end-on superoxo species thanks to the
charge-transfer from the catecholate moiety. The
superoxo species quickly attacks the catechol moiety
to give rise to the subsequent steps of the reaction
mechanism (Bugg and Ramaswamy 2008). Interest-
and the double mutant to interact with O , even in the
2
absence of catechol: (i) the higher accessibility of the
metal site, due to the increased pocket volume, that
may favour dioxygen diffusion towards the iron centre;
(ii) the perturbation of the tertiary contact networks,
that may displace some residues belonging to the
catalytic pocket and allow them to assist the formation
of the adduct, resulting in an unexpected stabilization
of the O –Fe adduct, even in the absence of catechol.
2
ingly the charge-transfer towards O seems to occur
2
A72N is the only mutant whose EPR spectral
pattern denounces a deep perturbation of the first
coordination sphere of the metal, although pos. 72 is
not directly coordinated to the metal. The structural
perturbation goes along with the peculiar catalytic
behaviour of the A72N mutant (Caglio et al. 2009),
that is almost inactive with respect to other variants at
the same sequence site. The replacement of Ala with
Asn is likely to deeply affect the network of tertiary
contacts surrounding position 72; the perturbation may
propagate through the structure and reach the metal
coordination site, thus explaining both the unusual
spectral pattern and the loss of catalytic proficiency of
this mutant.
without formation of iron(II) and may take place only
˚
when the Fe–O distance becomes 2.4 A or less. This
2
is the key process of the dioxygen activation and is
clearly influenced by the chemical environment of the
iron within the catalytic pocket.
Thus, it seems clear that, once the substrate bound,
the reaction of catechol dioxygenase implies a direct
interaction of dioxygen with iron(III), in spite of the
fact that—from a chemical point of view—this is
usually unfavoured with respect to Fe(II), due to the
ability of the latter to trigger the formation of an
Fe(III)–superoxide stable complex.
All these findings suggest that the Fe(III)–oxygen
interaction is possible and somehow assisted by the
chemical environment.
Thermal stability
Although in the absence of substrate, our experi-
mental evidence of O interaction with the iron center,
2
As far as the thermal stability issue is concerned, the
experimental data show that it seems more influenced
by the type than by the position of mutation. In fact,
mutants L69A, A72S and A72G exhibit almost the
same melting temperature in spite of the fact of
bearing mutations at different sequence sites. Replac-
ing Leu with Ala or Ala with Gly or Ser does not imply
deep sterical perturbations, as the mutated residues
occupy almost the same volume (or even a lower one)
than the native ones. Polarity is not significantly
affected, as well: the mutated residues are apolar or, in
the case of Ser, slightly polar. This may provide a
reason for their stability, which is only slightly lower
as compared to wt.
in two Ar-1,2-CTD variants may be explained in the
light of the structural perturbation induced by muta-
tions. According to the Ar-1,2-CTD structure reported
by Micalella et al. (2011), a weak water ligand is
coordinated to the iron center and it is H-bonded to
Arg216 in the wt form of the enzyme. Mutations at
positions 69 and 72 disrupt the H-bridge, further
weakening the bond between water and the metal
centre and promoting a perturbation of the tertiary
contacts within the catalytic pocket that may result in
the displacement of aminoacid residues. In addition, it
is worth noting that the mutants that bind oxygen more
strongly are those that, according to Micalella et al.
(
2011), are characterized by wider catalytic pockets.
Conversely, the A72P and the double mutant
forms have more chances to bring about significant
In fact, mutations at position 72 and 69 increases the
3
pocket volume from 1.5 A (wt) to 6.5 A (A72G) and
3
123

Biometals
perturbations of the local environment of the mutation
site. In fact, Pro is known to introduce abrupt torsions
in the peptide backbone; on the other hand, a double
mutation on two sites that are close in space is likely to
disrupt the local network of tertiary contacts. This
inevitably results in lower structural stability, as
witnessed by the lower melting temperature found in
these two systems.
optimal activity (37 °C rather than the 30 °C typical of
the wt and the other A72 variants). Although a
mechanistic explanation for this finding is not avail-
able, it seems possible that the presence of a Ser
residue instead of an Ala may contribute to the
stabilisation of the catalytic intermediates through the
establishment of bonding interactions between the
alcoholic function and the catechol or by a more
general stabilisation of the active site through a
rearranged hydrogen-bond network leading to stabi-
lised intermediates that supports catalysis.
The fact that a certain level of catalytic activity is
maintained in both mutants and that their EPR spectra
are quite similar to wt suggests that the perturbation
affects only mildly the topology of the first coordina-
tion sphere of the metal centre. Since the mutation
sites are located in a region of the catalytic pocket that
stabilizes the substrate by interacting with position 4
and 5 of the aromatic ring but are not very close to the
residues that coordinate the iron atom the observed
behaviour is consistent with a low impairment of the
melting temperature which is estimated on the basis of
the stability of the iron–protein interaction. The more
pronounced effect highlighted on the double mutant
can be affected by a significant decrease—in this
mutant only—of the hydrophobicity of the pocket
which can influence the iron retention, as discussed in
Di Nardo et al. (2004).
In general, most Ar-1,2-CTD samples are charac-
terized by melting T significantly higher than their T
of optimal activity. The only exception is represented
by A72P enzyme, that exhibits a melting T surpris-
ingly close to the T of optimal activity: apparently, in
this specific case, the bulk of the protein may be
destabilized to a good extent, without inducing the
breakdown of the metal active site. This suggests that
the metal center of Ar-1,2-CTD is one of the
determinants of the protein stability. This is often the
case in metalloproteins, where a catalytic core
relatively resistant to thermal denaturation is sur-
rounded by a more labile structural region (Boscolo
et al. 2009).
As for the effect of pH, experimental data once
again do not highlight direct correlations between the
position of mutations and the pH of optimal activity.
This might be related with the fact that Ala72 or Leu69
do not undergo acid–base equilibria, i.e. they are
insensitive to pH changes. The same is true for the
residues found at these positions in the mutants, within
the pH range scanned in our experiments.
Temperature and pH influence on the catalytic
activity
A comparison between the melting temperatures and
the T of optimal activity reported in Table 1 does not
highlight cross-correlations between the two sets of
values. The structural stability appears to be influ-
enced by the kind of residue introduced by mutation,
whereas both the T of optimal activity and the kcat
determined at that T seem more influenced by the
localization of the mutation site. Position 72 is less
crucial than pos. 69 in influencing the catalytic
proficiency. This agrees with the experimental find-
ings reported by Caglio et al. (2009), who found that
the L69A mutant was less reactive towards catechol as
compared to mutants on pos.72. This may also be
related with the fact that Leu69 is strictly conserved in
all intradiolic catechol dioxygenase.
If we look at the pH50 % values, the most original
behaviour belongs once again to A72P, which is
surprisingly similar to wt considering the highly
perturbing effect of Pro on the backbone structure.
All mutations are characterized by pH50 % values
0.5–1.0 pH unit higher than wt. As the mutated
residues are not involved in acid–base equilibria, this
change has to be related with modifications of the
tertiary interactions network which are likely to reflect
on the protonation equilibria of the aminoacid closer to
the iron site. A moderate and indirect effect, which is
nonetheless very difficult to rationalize, can involve
the action of pH on the substrate, since the Fe(III)
catecholate complex is also expected to be modulated
by the acid–base equilibria in the microenvironment
surrounding the catalytic site.
Interestingly, the peculiar behaviour of the A72S
enzyme highlighted by Caglio et al. (2009) is
confirmed by our findings. In fact, this is the most
active A72 mutant between those examined in this
paper and it is also characterized by the highest T of
1
23

Biometals
Conclusions
Bugg TDH (2003) Dioxygenase enzymes: catalytic mechanisms
and chemical models. Tetrahedron 59:7075–7101
Bugg TDH, Ramaswamy S (2008) Non-heme iron-dependent
dioxygenases: unravelling catalytic mechanisms for
complex enzymatic oxidations. Curr Opin Chem Biol
12:134–140
The comparative EPR investigation of a series of Ar-
1
,2-CTD mutants has allowed us to highlight the effect
of two mutations on oxygen binding properties of the
enzyme. Our data prove that a perturbation of the
environment of the catalytic pocket may result in an
increased ability of the enzyme to interact with
dioxygen.
Caglio R, Valetti F, Caposio P, Gribaudo G, Pessione E, Giunta
C (2009) Fine-tuning of catalytic properties of catechol
1,2-dioxygenase by active site tailoring. Chembiochem
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Caposio P, Pessione E, Giuffrida G, Conti A, Landolfo S, Giunta
C, Gribaudo G (2002) Cloning and characterization of
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Cappillino PJ, Miecznikowski JR, Tyler LA, Tarves PC,
McNally JS, Bala WL, Kasibhatla ST, Krzyaniak MD,
McCracken J, Wang F, Armstrong WH, Caradonna JP
A single mutant (A72N) denounces a structural
perturbation that affects directly the first coordination
sphere of the iron centre, whereas the A72D mutant
turn out to be unable to bind iron and provides no EPR
signal.
(
N
2012) Studies of iron(II) and iron(III) complexes with fac-
O, cis-N and N donor ligands: models for the
Finally, thermostability and pH studies shows that
the structural stability of mutants appears to be
influenced by the kind of residue introduced by
mutation, whereas the catalytic parameters seem more
influenced by the localization of the mutation site.
The experimental findings of this paper support
those published by Caglio et al. (2009) and Micalella
et al. (2011) and deepen our knowledge of the catechol
dioxygenase structure–function relationships. They
confirm the high degree of complexity of this enzyme,
where small structural changes may result in impres-
sive variations of the catalytic properties: this is
witnessed by the specificity inversion found by Caglio
et al. (2009) on the L69A mutant, which triggers the
conversion of a catechol-dioxygenase into a chloro-
catechol dioxygenase as for substrate selectivity.
Unraveling these aspects is of crucial importance
for an enzyme that raises a high biotechnological
interest due to its potential applications in the detec-
tion and removal of catechol derivatives from the
environment.
2
2
O
2
2 3
O
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