A spectroscopic investigation of the interaction-between nitrogen monoxide and copper sites of the fungal laccase from Rigidoporus lignosus

Article abstract of DOI:10.1039/b313630b

The interaction of NO, with the copper centres of the laccase secreted by Rigidoporus lignosus was studied under both aerobic or anaerobic conditions. The reduction of the T1 site was always observed, as detected by the disappearance of the characteristic optical band at 604 nm (T3 presents probably the same behaviour because of the decreasing of the band at 330 nm) and the absence of its characteristics EPR signal, while T2 undergoes an initial partial and transitory reduction, its EPR signal intensity totally restoring after 24 h interaction. Different magnetic parameters of the T2 site have been detected, evidencing an increase of the hyperfine coupling constant. Furthermore, the number of superhyperfine lines on the fourth line of T2 copper was also found to increase from seven in the native to nine in the NO-treated laccase, this fact implying the coordination of a nitrogenous species to the T2 site. It was also shown that nitrite can be a source of NO, thus, paralleling the behaviour of NO-donor molecules or NO gas, but after longer interaction times. The nitrogenous species coordinated to T2 site is probably NO₂^-, which arises indirectly by NO oxidation. In order to understand the mechanistic pathway of this interaction, some experiments were also carried out in the presence of azide to study the interaction of NO with this laccase having its trinuclear cluster blocked by the presence of an exogenous ligand as N₃^-. After the addition of NO-donor molecules to the azide-treated laccase, a new EPR signal appeared at low temperatures, which is ascribable to the partially reduced T3 site, while the T1 and T2 sites were found to be totally reduced. The mechanistic pathway of the NO interaction seems to proceed through the reduction of T1 and T3 copper sites, followed by the coordination of nitrogenous species to T2.

Full text of DOI:10.1039/b313630b

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A spectroscopic investigation of the interaction between nitrogen
monoxide and copper sites of the fungal laccase from
Rigidoporus lignosus
R. P. Bonomo,*a B. M. G. Castronovoa and A. M. Santorob
a Dipartimento di Scienze Chimiche, Università di Catania, Viale A. Doria 6, 95125 Catania,
Italy. E-mail: rbobomo@unict.it
b Istituto di Biostrutture e Bioimmagini del CNR (sezione di Catania), Viale A. Doria 6,
95125 Catania, Italy
Received 28th October 2003, Accepted 4th November 2003
First published as an Advance Article on the web 25th November 2003
The interaction of NO, with the copper centres of the laccase secreted by Rigidoporus lignosus was studied
under both aerobic or anaerobic conditions. The reduction of the T1 site was always observed, as detected by
the disappearance of the characteristic optical band at 604 nm (T3 presents probably the same behaviour because
of the decreasing of the band at 330 nm) and the absence of its characteristic EPR signal, while T2 undergoes an
initial partial and transitory reduction, its EPR signal intensity totally restoring after 24 h interaction. Different
magnetic parameters of the T2 site have been detected, evidencing an increase of the hyperfine coupling constant.
Furthermore, the number of superhyperfine lines on the fourth line of T2 copper was also found to increase from
seven in the native to nine in the NO-treated laccase, this fact implying the coordination of a nitrogenous species
to the T2 site. It was also shown that nitrite can be a source of NO, thus, paralleling the behaviour of NO-donor
molecules or NO gas, but after longer interaction times. The nitrogenous species coordinated to T2 site is probably
NO2Ϫ, which arises indirectly by NO oxidation. In order to understand the mechanistic pathway of this interaction,
some experiments were also carried out in the presence of azide to study the interaction of NO with this laccase
having its trinuclear cluster blocked by the presence of an exogenous ligand as N3Ϫ. After the addition of NO-donor
molecules to the azide-treated laccase, a new EPR signal appeared at low temperatures, which is ascribable to the
partially reduced T3 site, while the T1 and T2 sites were found to be totally reduced. The mechanistic pathway of the
NO interaction seems to proceed through the reduction of T1 and T3 copper sites, followed by the coordination of
nitrogenous species to T2.
action with metal sites of other multicopper proteins, as ceru-
Introduction
plasmin, was also investigated,8 the results of the study showing
that the T1 copper() site is reduced, through the formation of
a reversible diamagnetic charge-transfer complex, which led to
the disappearance of its UV-Vis and EPR spectroscopic
features.
Nitric oxide is a labile, diffusible and short-lived messenger
molecule involved in many biological phenomena of the
cardiovascular, nervous, and immune systems.1,2 Although NO
is relatively stable as a free radical, it rapidly reacts with free
radicals and metals in metalloproteins, such as Fe heme
proteins (hemoglobin, guanylyl cyclase, cytochrome P450).3–6
In fact, it has often been utilised as a spin probe in the study
of metal centres of metalloproteins.
In this paper we report about the interaction of NO with
copper sites of the laccase isoform (PoxB)15 secreted by the
fungus, Rigidoporus lignosus, either under anaerobic or aerobic
conditions. This interaction was followed by means of spectro-
scopic techniques (UV-Vis and EPR spectroscopy), and using
either NO-donor molecules, as an easily handily source of
NO,16 or highly purified gaseous NO. NO-donor molecules
Recently,7 it was demonstrated that NO may also exert
potent physiological effects through the interaction with
copper: the interaction of NO with divalent copper has been
suggested to play a key role in the control activity of some
copper enzymes (e.g. ceruloplasmin,8 cytochrome c oxidase,9
dopamine β-hydroxylase10).
Laccase is the simplest multicopper oxidase containing four
copper atoms, arranged in three different sites: the T1 (or blue
copper) is the electron acceptor site characterised by peculiar
spectroscopic features, such as an intense optical absorption
at 600 nm and a small parallel hyperfine coupling constant
(A|| ∼40–95 × 10Ϫ4 cmϪ1); the T2 site contains a copper ion,
devoid of any detectable optical absorption feature, but with
normal EPR parameters (g|| ∼2.230–2.245, A|| ∼185–200 × 10Ϫ4
cmϪ1); the T3 site contains two copper ions which are anti-
ferromagnetically coupled with an optical absorption band at
330 nm.11 These two latter sites (T2 and T3) are arranged in a
unique trinuclear cluster capable of binding oxygen.12
are interesting compounds because they could release NO
ϩ
in different redox states (NO , NO or NOϪ) with different
ؒ
kinetics depending upon their structural features and the
experimental conditions in which the dissociation reaction
is forced to occur.17 In particular, S-nitrosocysteamine
(CSNO) and 3-(2-hydroxy-2-nitroso-1-propylhydrazino)-1-pro-
panamine (PAPANONOate), which release NO, have been
used. Furthermore, sodium nitrite has been also tested in
the interaction with PoxB to see whether the observed
changes are to be ascribed to nitrite itself, which is known to be
one of the by-products, when NO is reacting in aqueous
solutions.
In other words, exploiting the NO (used as a spin and redox
probe) reactivity towards the laccase copper sites we tried to
gain information on their structural features, reactivity and
electron transfer processes. Moreover, since in the recent liter-
ature reports, the possibility of NO playing a role as biological
messenger through the modification of the activity of the
copper metal protein7 was invoked, the action of NO with
respect to laccase biological activity was also monitored.
The reaction of NO with laccases was previously investigated
by Martin et al.13 and recently by Torres et al.14 In their recent
note, these last authors have suggested that NO interaction with
tree laccase induces the T2 reduction and peroxide intermediate
formation in the trinuclear cluster. Previously the NO inter-
D a l t o n T r a n s . , 2 0 0 4 , 1 0 4 – 1 1 2
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known to have a relevant physiological role as in vivo NO car-
Results and discussion
riers, often augments the complexity of the overall system, since
they always contain small amounts of free thiols which can
have reducing properties towards the laccase metal centres.
NONOates are easier to handle and, in particular, in order to
explore the various protein/NO-donor molar ratios (from 1 : 2
to 1 : 20), PAPANONOate has been preferably used. When
under anaerobic conditions, PoxB interacts with NO obtained
by the decomposition reaction of PAPANONOate, the absorb-
ance of the band at 604 nm decreases with time, as a function of
the NO amount produced in solution, which, on the other
hand, is also a function of the initial PAPANONOate concen-
tration. For the complete reduction of the T1 site a large excess
of PAPANONOate is required, in particular, the band at
604 nm completely disappears when using a 1 : 20 protein/NO
ratio (PoxB/PAPANONOate ratio 1 : 10). At longest interaction
time (after 5 h and up to 24 h, the pattern remains almost
constant), the absorbance of the band at 330 nm is also found
to decrease, therefore, suggesting that NO interacts with the T3
copper site also (see Fig. 2(a) and (b)).
The laccase isoform PoxB from Rigidoporus lignosus has the
typical spectroscopic characteristics of laccases extracted from
fungi. In particular, its optical absorption spectrum shows an
intense band at 280 nm which can be attributed to electronic
transitions (n π* and π π*) occurring within the protein
aromatic groups, another band at 330 nm (it generally appears
as a shoulder) which originates from CT transitions within the
T3 copper site (LMCT transitions from π-type imidazole and
hydroxide to copper d orbitals) and the typical band at 604 nm
due to the CT transition from the cysteine sulfur to copper d
orbitals of the T1 site.18 In addition, its frozen-solution EPR
spectra show the usual copper signals due to T1 and T2 sites,
having the following magnetic parameters: T1; g|| = 2.204(5), A||
= 93(4) × 10Ϫ4 cmϪ1, g⊥ = 2.041(5); T2; g|| = 2.242(3), A|| = 193(4)
× 10Ϫ4 cmϪ1, g⊥ = 2.038(5).15,19 Furthermore, frozen-solution
spectra registered at temperatures below 125 K, show a well
resolved superhyperfine structure on the fourth line of T2
copper site superimposed on the envelope of the overall per-
pendicular part. Seven lines with a coupling constant AN
=
⊥
14.3 (0.6) × 10Ϫ4 cmϪ1 can be attributed to a coordination
sphere which contains three nitrogen donor atoms in the copper
equatorial plane (see Fig. 1). At room temperature the EPR
spectrum only reveals the T1 features with the following mag-
netic parameters: g|| = 2.191(5), A|| = 84(5) × 10Ϫ4 cmϪ1, g⊥ =
2.045(5). The slightly lower g|| and A|| values and the absence of
the T2 signal, ascribable to the protein auto-reduction phen-
omena, are well known facts already reported in the literature.20
Fig. 2 UV-Vis absorption spectra of 50 µM PoxB in 100 mM sodium
phosphate buffer at pH 7 and (a) 0.5 mM PAPANONOate, (b) 1 mM
CSNO at different incubation times: 0 h (——), 1 h (- -), 5 h ( ؒ ؒ ؒ ).
ؒ
Low-temperature EPR spectra scanned at different times
(1, 2, 3, 5, 24 h) show a progressive T1 reduction up to a com-
plete disappearance of its features, therefore confirming what
has been found by optical spectral measurements. The T2 site
undergoes an initial partial and transitory reduction (from 10
up to 40%), but after a period which is dependent on the NO
molar excess, its signal intensity is restored to the initial value
(see Fig. 3). The g values remain practically constant, g||
=
2.239(3) and g⊥ = 2.042(5), while A|| increases from 193 to
202(4) × 10Ϫ4 cmϪ1. Even the superhyperfine pattern substanti-
Fig. 1 (a) Frozen-solution EPR spectra at 77 K of 0.6 mM PoxB
solution in 100 mM sodium phosphate buffer at pH 7; (b) the
perpendicular part in an enlarged scale showing the superhyperfine
structure of the T2 copper highest field feature; (c) its second derivative
mode: the first seven lines have an average shf constant of 1.50(6) mT,
the eighth, not belonging to this group, being at 1.90 mT.
Reaction of oxidised laccase with NO donors (PAPANONOATE
or CSNO)
A parallel behaviour can be observed by using NO-donor
molecules belonging to different classes (NONOates and
S-nitrosothiols), and experimental results with PAPANONOate
parallel those with CSNO. The use of S-nitrosothiols, which are
Fig. 3 Reduction percentages calculated from the integrated signals of
Ϫ3/2 (T2) (᭞) and Ϫ1/2 (T1) (᭢) transitions from the frozen-solution
EPR spectrum of 0.3 mM PoxB in 100 mM sodium phosphate buffer at
pH 7 and 1 mM PAPANONOate.
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ally changes, and at the highest interaction time, 24 h, can be
observed more clearly, but it was resolved enough after 5 h. In
fact, from the seven-shf-line pattern observed in the frozen-
solution EPR spectrum of native PoxB, a clear shf structure
sample after 24 h, presents features similar to those observed
under anaerobic conditions, the signal of T1 copper site being
partially or fully reduced (and this fact parallels with the
experimental observations of other workers, who found an ease
of reduction of multi-copper oxidases at low temperatures21),
while that of T2 is characteristic of a copper species with
four-nitrogen coordination, as suggested by the nine-line super-
hyperfine structure (see Fig. 5).
In this connection, a quantitative assay of the nitrite content
by means of the Griess assay in comparison with a blank, con-
taining the same amount of NO-donor dissolved in the buffer
solution, shows that the amount of nitrite determined in the
consisting of nine lines, showing AN = 14.6(0.7) × 10Ϫ4 cmϪ1
⊥
can be easily detected (see Fig. 4(a), (b) and 5). In other words,
the T2 copper ion now experiences the presence of more than
three nitrogen atoms in its in-plane coordination, nine shf lines
being ascribable to the presence of four nitrogen atoms bound
to the T2 copper site. Under aerobic conditions, optical absorp-
tion spectra of PoxB in the presence of PAPANONOate in the
same ratio reveal a T1 site partial reduction, but after 45 min
the blue band is restored to the same native laccase absorbance
values. However, the frozen-solution EPR spectrum of the same
protein solution is about 14
7% less (we can not exclude
that part of NO is converted to other NOx species, therefore,
escaping from the nitrite determination).
Reaction of oxidised laccase with gaseous NO
PoxB-gaseous NO interaction reveals UV-Vis and EPR
spectroscopic changes very similar to that obtained with NO
donors. In fact, UV-Vis spectra show the intensity reduction of
the bands at 330 and 604 nm, giving the clear indication of
the T3 and T1 copper site reduction, respectively. Moreover,
frozen-solution EPR spectra show the initial intensity decreas-
ing of T1 and T2 copper features, too, but, a complete re-
oxidation of the T2 site and the complete reduction of T1 site
can be observed after 24 h interaction. Also in this case the
nine-shf-line pattern is quite evident, therefore suggesting no
difference between experiments with the use of NO-donor
molecules and gaseous NO.
Interaction of PAPANONOate with fully reduced/oxidised
laccase
In order to test whether NO released by PAPANONOate could
have oxidising properties towards the fully reduced form of the
PoxB laccase, NO-donors were added in the same proportion
to this laccase previously reduced by ascorbate. As usual, the
reduced laccase is lacking its spectroscopic UV-Vis and EPR
features and after the NO-donor addition, no spectroscopic
change (UV-Vis/EPR) is observed. Furthermore, to exclude
that spectroscopic changes can be attributed to a laccase con-
taining partially reduced metal sites, experiments were carried
out on fully oxidised PoxB laccase, by insufflating molecular
oxygen, and removing its excess with cycles of vacuum and
purging with N2, before the addition of NO-donor molecules,
in order to avoid large concentrations of NOx. In these condi-
tions, the addition of PAPANONOate or CSNO gives rise
exactly to the same experimental results.
Fig. 4 Frozen-solution EPR spectra at 125 K of 0.3 mM PoxB
solutions in 100 mM sodium phosphate buffer at pH 7 and (a) 3 mM
PAPANONOate, (b) 6 mM CSNO and (c) 6 mM sodium nitrite, after
an interaction time of 24 h.
Reaction of oxidised laccase with sodium nitrite
In a first step of this work, the addition of sodium nitrite to the
PoxB solution was considered as a control experiment, since it
is well known that NO in water, under aerobic or anaerobic
conditions, could transform to nitrite. The ability of nitrite to
act as an NO source in vivo was recently reported in the liter-
ature,10 the disproportion reaction of nitrites to NO being
ؒ
strongly dependent on the acidity of the medium:
3NO2Ϫ ϩ 2H3Oϩ
2NO ϩ NO3Ϫ ϩ 3H2O
Actually, the addition of nitrites to the PoxB solutions gives
rise to a behaviour similar to that described when discussing the
laccase interaction with NO-donor molecules. UV-Vis spectra
of PoxB solutions after the addition of an excess (twenty-fold)
of sodium nitrite, under anaerobic conditions, give rise to no
changes within the first 3 h. However, after this time, a gradual
reduction of the absorbance of the band at 604 nm can be
detected. After 5 h, this reduction is about 60% of that of the
native laccase and becomes complete after 24 h (see Fig. 6). At
Fig. 5 (a) Perpendicular part of a frozen-solution EPR spectrum at
77 K run on 0.3 mM PoxB solution in 100 mM sodium phosphate
buffer at pH 7 after the addition of 6 mM CSNO, showing the nine-line
superhyperfine structure of the T2 copper highest field feature; (b) its
second derivative mode: the first nine lines have an average shf constant
of 1.53(8) mT, the tenth, not belonging to this group, being at 1.0 mT
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Reaction of oxidised laccase with azide and PAPANONOate or
ascorbate
In order to reach a significant insight into the possible mechan-
ism of this interaction, experiments were carried out on PoxB
previously treated with sodium azide. It is well known that
the azide anion, N3Ϫ, is an inhibitor of the multicopper oxid-
ases,20,23 since it binds to the trinuclear cluster altering the redox
processes with molecular oxygen and, therefore, blocking the
overall catalytic cycle. Although several papers on the use of
azide in the reaction with multicopper oxidases have been pub-
lished, it is not easy to gain a simple picture of the azide inter-
action with the trinuclear cluster.24,25 Our goal is to verify the
NO reactivity with PoxB, having its trinuclear cluster blocked
by the presence of azide and, therefore, we assume, as a simpli-
Ϫ
fied picture, that one N3 molecule can contemporarily bind
both T2 and T3 copper sites, owing to the bridging nature of
Fig. 6 UV-Vis absorption spectra of 50 µM PoxB in 100 mM sodium
phosphate buffer at pH 7 and (a) 1 mM sodium nitrite solution under
anaerobic conditions and (b) 1 mM sodium nitrite solution under
aerobic conditions at different incubation times: 0 (—), 3 h ( ؒ ؒ ؒ ),
N3 .
Ϫ 18 When PoxB is incubated with azide under aerobic condi-
tions, in several PoxB/azide ratios and at different incubation
times, it can be easily seen through enzymatic activity assays
towards ABTS (2.2Ј-azinobis(3-ethylbenzothiazolin-6-sulfonic
acid diammonium salt) that the inhibition reaction depends
both on incubation time and azide concentration. A real inhib-
ition (about 98%) is obtained at the incubation time of 24 h, by
using a 1 : 50 PoxB/azide ratio.
5 h (---), 24 h (- -).
ؒ
pH 4 (this acidity value does not disturb the stability of
PoxB 22), the disproportion reaction proceeds more quickly and
the absorbance reduction of the bands of T1 at 604 nm and at
330 nm of T3 is complete within 30 min.
In these conditions, PoxB UV-Vis spectra show deep modifi-
cations in the 300–500 nm region, in which two new bands at
340 and 420 nm, grow in intensity, as shown by other authors in
the literature.20 The frozen-solution EPR spectrum shows the
usual T1 features, while the T2 signal broadens and its intensity
decreases with respect to that observed in the native protein.
When the addition of nitrite is done under aerobic condi-
tions, the T1 site absorbance reduction is slower, but the results
at the end are very similar to those observed under anaerobic
conditions, especially for the EPR spectroscopic behaviour at
low temperatures. However, oxidation phenomena of the pro-
tein can be observed, as denoted by the yellowish colour which
its solution assumes, after 3 h of incubation a new band appear-
ing at about 470 nm and increasing with time (see Fig. 6). Still
the frozen EPR solution spectra (see Fig. 4(c)) run in these
conditions show the same spectroscopic characteristics, thus
indicating this “new” UV-Vis band is not involved in the pro-
tein metal centres. This behaviour was observed when using the
NO-donor molecules under aerobic conditions, too, but only
after several days of incubation.
Ϫ
This could be a good indication of the N3 bridge-binding
between T2 and T3 copper sites. When PAPANONOate is
added (PoxB : NO 1 : 10) to a PoxB sample pre-treated with
azide in 50 fold molar excess after an incubation time of 24 h,
the UV-Vis optical spectrum run immediately after the addition
of NO-donor, shows the usual reduction of T1 copper site.
Other spectroscopic changes can be observed, such as the dis-
appearance of the band at 420 nm and the appearance of the
band at 485 nm (see Fig. 7(a)) which increases its intensity with
time. Frozen-solution EPR spectra show the disappearance of
the T1 and T2 signals and a new clear signal appears (g|| =
2.270(3), A|| = 99(4) × 10Ϫ4 cmϪ1, g⊥ = 2.070(5) see Fig. 8, with a
rhombic perturbation being clearly visible). This pattern is simi-
lar to that observed following a control reduction experiment of
the azide-treated PoxB laccase with ascorbate in which the same
EPR spectrum appears (g|| = 2.268(3), A|| = 94(4) × 10Ϫ4 cmϪ1, g⊥
NO Influence on enzymatic activity of PoxB laccase
In order to test eventual modifications on the laccase activity
after the interaction with NO-donor molecules or nitrites in
aerobic conditions, enzymatic activity assays against DMP
(2,6-dimethoxyphenol) were carried out on samples of PoxB
incubated with PAPANONOate or sodium nitrite always in
the same ratios and on a control sample made by an equal
volume of buffer. In both the treated samples, the protein
loses its characteristic blue colour, the enzymatic activity
decreases 40% after 24 h (residual activity 60%). However,
after washing the protein with pH 7 buffer, its blue colour is
restored and it is possible to see about 80% of the initial
catalytic activity. In the case of partial oxidation of the protein
by nitrite or NO, after washing, its residual catalytic activty is
below 20%.
NO Influence on polypeptide chain integrity of PoxB laccase
In order to verify whether phenomena of protein fragmentation
have been occurred on PoxB as a consequence of the treatment
with NO, MALDI mass spectra (not shown) have been per-
formed on both wild PoxB laccase and the same sample after 24
h incubation with NO. The spectra always show two peaks only,
the first one at m/z 27874 60 Da corresponding to the double
charged ion (M2ϩ), and the second at 55530 82 Da corre-
sponding to the molecular ion (Mϩ). No other relevant signal
due to a possible fragmentation pattern has been observed in
the mass range from 10 to 70 kDa.
Fig. 7 UV-Vis absorption spectra of 50 µM PoxB in 100 mM sodium
phosphate buffer at pH 7: (a) native (—), 2.5 mM sodium azide (24 h)
(- -), and 0.5 mM PAPANONOate under anaerobic conditions ( ؒ ؒ ؒ );
ؒ
(b) native (—), 2.5 mM sodium azide (24 h) (- -) and 0.5 mM sodium
ؒ
ascorbate under anaerobic conditions ( ؒ ؒ ؒ ).
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copper atoms belonging to the T3 site are bound to three
histidines with another water molecule bridging them. We were
puzzled about these results, but we could not reject experi-
mental facts. In the recent literature there has been a certain
debate about the identity of the paramagnetic copper (T2)
within the trinuclear cluster and we share the ideas of some
authors, who assert that the T2 copper site could be any of the
three copper atoms arranged in the triangular cluster.35,36 On
the other hand, a multi-frequency EPR study37 carried out on
a frozen-solution of a T1 mercury-containing laccase estab-
lished for the T2 copper centre a CuN3O chromophore, these
experimental results perfectly matching with ours.
These discrepancies can not be justified by suggesting con-
formational changes of the protein on freezing or, equivalently,
asserting that structural changes could come from the crystal
packing forces, which could make the protein assume a particu-
lar conformation in the solid state. Interestingly, the picture of
the T2 copper site coming from almost all these diffractometric
studies, a CuN2O chromophore in a trigonal planar configur-
ation, is far away from EPR results which predict for it an
approximate square planar environment in most of the fungal
and plant laccases. Should we conclude that frozen-solution
spectroscopic EPR and X-ray crystal diffractometric studies are
seeing different “objects”? Should we consider that conform-
ational changes of the protein on passing from the crystalline to
aqueous solution state (additional histidines being not present
in the protein sequence involved in the coordination to copper
atoms) could favour the involvement of other nitrogen donor
atoms (peptidic?) in the coordination to the T2 copper site? At
the moment there are no definite answers to these questions,
although it is hard to believe conformational changes would
supply the energy required to the formation of a new copper–
nitrogen bond. It is possible to hypothesise that the water mole-
cule connecting the two crystallographically equivalent copper
atoms of the T3 site can easily change its position, bridging in
solution the “crystallographic” T3 copper and T2 copper
atoms, the breakage of a copper–oxygen bond being com-
pensed by the formation of a new one. The T3 site would be
now formed by copper atoms which are always anti-
ferromagnetically coupled, so as to make this site EPR silent,
however, it hard to believe that they are equivalent, as, on the
other hand, was already suggested in a previous study by other
scientists.38 The copper atom of the “new” T2 site in aqueous
solution would be now involved in the coordination with three
histidine residues and a water molecule, in a more regular
geometry, which is exactly what we see in our spectroscopic
studies. Therefore, we conclude asserting that at least in our
laccase, the spectroscopically characterised T2 copper site in
solution is not the T2 site which is revealed by diffractometric
studies at the solid state. We would suggest that great care is
necessary in using diffractometric data to explore the catalytic
nature of a protein metal site. Perhaps, many of the conclusions
about the cataytic mechanism of plant and fungal laccase which
were drawn out from a comparison between data obtained from
protein aqueous solutions and the molecular picture depicted
by diffractometric studies need to be rethought.
Fig. 8 Frozen-solution EPR spectra at 125 K of 0.3 mM PoxB
solutions in 100 mM sodium phosphate buffer at pH 7 in of 15 mM
sodium azide and 1.5 mM PAPANONOate under anaerobic conditions,
after 24 h incubation times.
= 2.063(5)). In other words, while T1 and T2 sites are strongly
reduced, only a partial reduction of the T3 dinuclear site can be
achieved by the interaction of the azide-treated laccase with
NO. Therefore, the new band at 485 nm, following the sugges-
tion of Rotilio and co-workers26 can be probably assigned to a
Ϫ
CT transition due to N3 adduct formation with the dinuclear
T3 centre, in which the two copper atoms have a half-met redox
state. This result is confirmed by the low-temperature EPR
spectrum, in which the signals pertaining to T1 and T2 sites
disappear and the new EPR signal could probably be ascribed
to one of the two copper atoms of the T3 centre, its magnetic
parameters matching quite well with a supposed tetrahedral
geometry of the copper atoms in this site. This could be a fur-
ther indication that only one molecule of N3Ϫ anion is involved
in the bridge-binding between T2 and T3 copper sites.
Conclusions
By using two different classes of NO-donors, NONOates and
S-nitrosothiols, or gaseous NO similar effects can be detected.
Moreover, the behaviour observed in the case of the interaction
of PoxB with nitrite under anaerobic conditions is revealed to
be an interesting surprise, because an experimental proof that
nitrite can also behave as an NO donor when interacting with
a metallo-protein, was obtained.27 Tables 1 and 2 summarise
the experimental shifts of magnetic parameters, optical and
enzymatic activity changes which are determined in this study
after the interaction of Pox B laccase with NO, azide or ascor-
bate has occurred.
It is important to stress that our starting point is a T2 site
which presents three nitrogen donor atoms involved in the cop-
per coordination as predicted by its magnetic parameters28 and
by the well resolved shf structure on the ϩ3/2 T2 copper line.
We are aware that this result is in contrast with what has been
previously accepted on the basis of the crystal structures of
both ascorbate oxidase29 from Cucurbita pepo and the T2
depleted laccase from Coprinus cinereus,30 which attributed only
two histidine nitrogen atoms to the T2 copper site. Also some
recent results on the crystal structures of other laccases (Tram-
etes versicolor and Melanocarpus albomyces) have confirmed the
conclusions of the preceding diffractometric studies, that is, the
coordination around the T2 copper site involves the presence of
only two histidine residues.31–33 Moreover, a recent report on
crystals of Rigidoporus lignosus laccase at a resolution factor of
1.7 Å confirmed these diffractometric results,34 namely, the T2
copper atom in fungal laccases is always found to be in the
environment of two histidines with a water molecule com-
pleting its tri-coodination, and, furthermore, each of the two
The important events in the NO-PoxB laccase interaction
are (i) the partial or total reduction of T1 (bleaching of the
protein), an initial reduction of T2 (which is capable of reoxid-
ation) as well as the reduction of T3 copper sites (gradual dis-
appearance of the 330 nm band), and (ii) the coordination of a
nitrogenous species to the T2 site. It is important to point out
that these events are not contemporary: the former is a fast
phenomenon (under anaerobic conditions often occurring in
a few minutes), the latter can be seen after a few hours (with
nitrite under aerobic or anaerobic conditions, within the first
hour). As we stated above, our starting point is a T2 copper site
which is bound to three nitrogen donor atoms arranged around
the equatorial plane, the fourth position probably being occu-
pied by a water molecule or a hydroxyl group. Since the evident
D a l t o n T r a n s . , 2 0 0 4 , 1 0 4 – 1 1 2
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Table 1 Shifts of the parallel and perpendicular magnetic parameters obtained from the frozen-solution EPR spectra of the Pox B laccase from
Rigidoporus lignosus at a temperature below 125 K or directly in liquid nitrogen, as a consequence of the interaction with NO or with azide and NO
or azide and ascorbate
g||
A|| × 10Ϫ4/cmϪ1
g⊥
104AN⊥/cmϪ1
No. shf lines
Wild laccase (EPR silent T3 site)a
T1
T2
T1 (RT)
2.204(5)
2.242(3)
2.191(5)
93(4)
193(4)
84(5)
2.041(5)
2.038(5)
2.045(5)
–
–
7
–
14.3 (0.6)
–
Laccase/NO (EPR silent T1 and T3 sites)b
T2 2.239(3) 202(4)
Laccase/azide/NO (EPR silent T1 and T2 sites)c
T3 2.270(3) 99(4)
Laccase/azide/ascorbate (EPR silent T1 and T2 sites)c
T3 2.268(3) 94(4) 2.063(5)
2.042(5)
2.070(5)
14.6 (0.7)
9
–
–
–
–
Values in parentheses represent the presumed errors on the last figure of each parameter. a The T2 site is not visible in the room-temperature spectra
probably because of auto-reduction phenomena. b The T1 copper site was reduced by NO and, therefore is not visible any more in the EPR spectrum.
These values are average values from all the experiments carried out with NO-donors, NO(g) or nitrite. c Both T1 and T2 copper sites were reduced by
NO or ascorbate when the native laccase is pre-treated with an 50-fold excess of azide.
Table 2 UV-Vis changes of PoxB from Rigidoporus lignosus in the presence of NO, sodium nitrite, azide and ascorbate in anaerobic conditions.
The residual activity (R.A.) was determined with assays against DMP after 24 h incubation time (i.t.) in the presence of NO or sodium nitrite before
and after having washed laccase with phosphate 0.1 M buffer (pH 7)
R.A at
24 h i.t.
Interaction of NO, NO2Ϫ, azide,
ascorbate with Pox B laccase
% Absorbance reduction of
the band at λmax330 nm
% Absorbance reduction of
the band at λmax604 nm
New bands,
λmax/nm
R.A. at
after
24 h i.t.a
washinga
Wild PoxB
0
0
12
15
18
43
50
86
88
63
72
83
7
100
100
PoxB/NO (1 : 1)a
PoxB/NO (1 : 2)a
PoxB/NO (1 : 5)a
PoxB/NO (1 : 10)a
PoxB/NO (1 : 15)a
PoxB/NO (1 : 20)a
PoxB/NO (1 : 20)b
PoxB/NO2Ϫ (1 : 20)
PoxB/NO2Ϫ (1 : 20)c
PoxB/NO2Ϫ (1 : 20) pH 4d
PoxB/N3Ϫ (1 : 50)
nde
nde
nde
nde
nde
40
60
nde
20
78
nde
nde
nde
nde
nde
38
nde
470
340
420
485
485
2
nde
PoxB/N3Ϫ (1 : 50) ϩ NO (1 : 20)
60
98
95
100
nde
nde
nde
nde
PoxB/N3Ϫ (1 : 50) ϩ Ascorbate(1 : 10)
All these reduction percentages have a presumed errors about 15 %. It is quite difficult to determine the absorbance reduction of the shoulder at
330 nm. a Interaction of laccase with CSNO measuring the absorbance reduction of the optical band at 604 nm after 1h. b From all the sources of
NO, as CSNO, PAPANONOate or NO(g) after 24h i.t. c In aerobic conditions. d After 30 min. i.t. e n.d. not determined.
nine-line shf pattern cannot come from a contribution of nitro-
gen donor atoms apically linked to copper39 (in this eventuality,
the shf pattern would have been remained unchanged, i.e. a
seven-line shf structure, and the absolute value of the parallel
hyperfine coupling constant would have largely decreased), the
T2 copper site should undergo a substitution reaction, H2O
or OHϪ in the fourth coordination position of the copper
equatorial plane being gradually replaced by a nitrogen
containing species.
All these results lead to a set of questions about the inter-
action mechanism between NO and the copper sites of PoxB
laccase. A possible mechanism accounts for a first direct inter-
action of NO with the T1 and T2 copper sites, whose spectro-
scopic features tend to disappear (decrease of the optical
absorbance at 604 nm and of both EPR signals, the T1 signal
becoming completely silent at the longest interaction times).
This experimental behaviour, which is partly analogous to what
occurs when NO is made to interact with other fungal lac-
cases13 or other blue copper proteins, such as ceruloplasmin,40
can be ascribed to a direct T1 (and initially T2, too) copper site
reduction via a long range outer-sphere mechanism. This event
could be accompanied by complex formation between T2 and
probably NOϩ or other species containing nitrogen donor
atoms (the appearance of the nine-line shf structure on the
EPR spectral features is the evident proof ). This mechanism
could be justified taking into account the different times which
are necessary to observe the complete reduction of the T1 site
and the appearance of the shf pattern of T2. We are aware that
this hypothesis is in contrast with the thermodynamics of the
electron transfer process. In other words, the redox potential of
the NOϩ/NO couple (EЊ = 1.21 V)41 is more positive than that
of the T1 Cu2ϩ/Cuϩ couple (EЊ = 0.70 V),19 and the overall
D a l t o n T r a n s . , 2 0 0 4 , 1 0 4 – 1 1 2
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process would have been occurred by oxidation of Cuϩ and
reduction of NOϩ. This could mean that the overall process
is not under thermodynamic control, but a kinetic control is
operating, something similar to what occurs in the case of
laccase mediators.42 A different version of this mechanism, as
suggested by Martin et al.,13 takes into account the oxidation of
NO directly to nitrite (it has the same redox state of NOϩ)
through a one-electron reduction process. The redox potential
value for the couple NO2Ϫ/NO would have a smaller value than
that of the T1 site (a value of 0.37 V is reported by Ford and
Lorkovic43). In this perspective, the nitrogenous ligand which
would bind to the T2 copper site could be reasonably enough
NO2Ϫ, involved in a nitro coordination (also the results of the
Griess assay seem to suggest an involvement of NO2Ϫ). The
following scheme summarises the discussion of the principal
mechanism of this interaction:
be achieved. A nice EPR spectrum (with different magnetic
parameters with respect to T1 and T2 sites) from one of the
copper atoms belonging to the T3 site has been obtained (we
are not able to say which of the two is reduced). Moreover, no
clear shf structure is detected at 24 h after the addition of
NO-donor to the azide-pre-treated PoxB, probably because T2
is found to be linked to a molecule of azide, this fact preventing
it bonding to the nitrogenous species formed by the oxidation
of NO.
If we compare our results with those of other reports on the
same subject, all the evidence makes it clear that a difference
between tree and fungal laccase reactivity genuinely exists. This
different reactivity could probably be ascribable both to the
higher T1 redox potential (in tree laccases, T1, T2 and T3 have
almost similar redox potentials45) and to different pocket char-
acteristics that affect molecular recognition processes through
the presence of negative or positive charged residues. The latter
difference should be a negligible factor when using neutral or
slightly polar large molecules, but it could become important
when small charged molecules or radicals are interacting with
the catalytic site.19
[CuII(T1)]2ϩ ϩ NO ϩ H2O
[CuI(T1)]ϩ ϩ NO2Ϫ ϩ 2Hϩ (1)
It would be possible to write analogous reactions for the
reduction of T2 and T3 copper sites, but we cannot exclude that
their reduction occurs through the mediation of T1 site, namely
that they are reduced because T1 delivers excess electrons to
them. Then we have to consider the coordination of nitrite to
T2 site.
Experimental
Materials
Cysteamine hydrochloride, (CSH), sodium nitrite, sodium
ascorbate and sodium azide were purchased from Sigma,
sodium phosphate from BDH, tert-butyl nitrite (tBuONO)
from Fluka, 2.2Ј-azinobis(3-ethylbenzothiazolin-6-sulfonic
acid) diammonium salt (ABTS) and DMP (2,6-dimethoxy-
phenol) from Aldrich and Griess reagent from Merck. 3-(2-
hydroxy-2-nitroso-1-propylhydrazino)-1-propanamine (PAPA-
NONOate, (whose half-life at 25 ЊC is 77 min) was purchased
from Alexis. Milli-Q ultrapure water has been used in all
experiments.
Ϫ
[CuII(T2)(H2O)]2ϩ ϩ NO2
[CuII(T2)(NO2Ϫ)]ϩ ϩ H2O (2.1)
We cannot exclude either the possibility that T2 “complex-
ation” is an event which occurs on T2 reduced copper. Since
this hypothesised NO2Ϫ adduct does not form immediately (but
at least within the first hour) when nitrite is used, it could imply
that its formation could be the result of a series of reactions
which involve the reduction of T2 copper site and the delivery
of electrons to T1 and T3 sites. The fact that the nine-line shf
structure appears only at the longest interaction times rather
Protein purification and laccase activity assays
Ϫ
than being an indication of a NO2 concentration dependence
The main laccase isoform from Rigidoporus lignosus, PoxB, was
purified from a liquid growth culture medium as previously
described.15 The purity of these samples was checked by 12.5%
SDS-PAGE, (sodium dodecyl sulfate–polyacrylamide gel elec-
trophoresis) according to the procedure reported by Laemmli,46
and by measuring the absorbance ratio A280/A600. The concen-
tration of laccase was determined by measuring ε604 (pH 7) =
5000 MϪ1 cmϪ1 and performing the Bradford47 assay quanti-
fication for proteins. Spectrophotometric assays of the laccase
activity at 25 ЊC were carried out in 2 ml final volume contain-
ing McIlvaine buffer (pH 6) and 1 mM of DMP or citrate
buffer (pH 3) and 2 mM ABTS. Oxidation of DMP was fol-
on the formation of a possible NO2Ϫ–T2 copper adduct, it
could also suggest the following complicated mechanistic
pattern:
Ϫ
[CuI(T2)(H2O)]ϩ ϩ NO2
[CuI(T2)(NO2)] ϩ H2O (2.2)
[CuII(T2)(NO2)]ϩ ϩ eϪ
[CuI(T2)(NO)]ϩ ϩ H2O (2.4)
[CuI(T2)(NO2)]
(2.3)
[CuI(T2)(H2O)]ϩ ϩ NO
[CuI(T2)(NO)]ϩ ϩH2O
[CuII(T2)(NO2Ϫ)]ϩ ϩ 2Hϩ ϩ 2eϪ, (2.5)
lowed by monitoring the absorbance increase at 476 nm (ε476
=
14800 MϪ1 cmϪ1) and of ABTS at 420 nm (ε420 = 36000 MϪ1
Ϫ
in other words, a labile NO or NO2 adduct could form with
cmϪ1).
reduced T2 copper and subsequently the oxidation of both
copper and/or NO leads to the formation of the T2 copper
NO2Ϫ-adduct, with electrons delivered to T1 and T3 sites.
The experiment in the presence of azide was devised with
the specific aim to see what would have been occurred in this
CSNO Preparation
S-Nitrosocysteamine (CSNO) was synthesised in a degassed
water solution by reacting 1 M cysteamine hydrochloride with
1.1 equivalent of tBuONO for 15 min at room temperature
according to the method described by Roy et al.48 Its concen-
tration can be evaluated by measuring the intensity of the vari-
laccase–NO interaction, when the trinuclear cluster has been
blocked by the presence of an exogenous ligand such as N3
Ϫ
,
and therefore, to have an indirect confirmation about the par-
ticular pathway followed by the NO interaction with the copper
sites. Again there are discrepancies between EPR spectroscopic
and diffractometric data44 on the nature of the azide binding
site: is the azide anion interconnecting T2 and T3 sites, as sug-
gested by the spectroscopic data18 or is only binding to one of
the T3 copper atoms, as evidenced by diffractometric studies?
This experiment seems to lead to the definite conclusion that
both T1 and T2 centres are susceptible of reduction by NO or
ascorbate, but only a partial reduction of T3 dinuclear site can
ous absorption bands. We used either the band at 333 nm (ε333
=
793 MϪ1 cmϪ1) or that at 545 nm (ε545 = 15 MϪ1 cmϪ1). All the
solutions containing CSNO were freshly prepared, kept on ice
in the dark and always used in experiments of the same day.
NO Gaseous apparatus
Gaseous nitric oxide was purchased by Air Liquide and was
used by washing it through a series of traps (NaOH, H2O). The
D a l t o n T r a n s . , 2 0 0 4 , 1 0 4 – 1 1 2
110

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NO concentration was determined measuring the aqueous solu-
tion nitrite content. Additions of NO to the solution containing
PoxB laccase were made using a gastight Hamilton syringe.
follows: microwave frequency: 9.344–9.376 GHz; modulation
frequency: 100 kHz; modulation amplitude: 0.2–0.6 mT; time
constant: 164–327 ms; sweep time: 2.8 min; microwave power:
20–40 mW; receveir gain: 1 × 104–2 × 105. To increase the
signal-to-noise ratio of the EPR spectra, several scans of the
same sample were often accumulated. Magnetic parameters of
these mononuclear copper species were obtained directly from
the experimental frozen-solution spectra, calculated from the
second and third lines, in order to remove second order effects.50
MALDI mass spectra were acquired by using a Voyager-DE
MALDI-TOF instrument (Perseptive Biosystem, Framingham,
MA, USA) and α-cyano-4-hydroxycinnamic acid as matrix,
according to the thin layer procedure.51
Nitrite determination
Nitrite content at different times was determined in the buffer
solution in which laccase is left to interact with NO-donor
molecules, following the procedure suggested by Green et al.49
100 µl of Griess reagent was added to 900 µl solution obtained
by ultrafiltration (Centricon Millipore cutoff 10 kDa) of a
laccase solution previously incubated with NO-donor mole-
cules (parallel experiments with the same amounts were carried
out on buffer solutions containing NO-donor molecules only)
and the absorbance at 546 nm was measured after 30 min. A
calibration straight line was obtained by using sodium nitrite as
standard.
Acknowledgements
We gratefully acknowledge Dr G. Scicolone, Dr G. Tabbì and
Dr P. Malvagna for their technical help in finding the best
conditions to run UV-Vis, EPR spectra and MALDI spectra.
This work was supported by PRIN 2000 MM05188234-007,
CNR PF Biotecnologie 0100246PF49
Interaction involving laccase, NO-donors, azide and ascorbate
The interaction of NO-donor molecules or nitrite with PoxB
was followed both under aerobic and anaerobic conditions, by
using PoxB samples in 100 mM sodium phosphate at pH 7,
considering various ratios of the protein and the NO-donor
molecules or nitrite and monitoring the spectroscopic (UV-Vis
and EPR) changes. In all the UV-Vis spectrophotometric
measurements the addition of the NO-donor was done directly
to 50 µM protein samples. Frozen-solution EPR spectra were
run in quartz tubes on 300 µM protein samples to which the
NO-donor was previously added to the eppendorf.
Separate experiments were carried out adding both cyste-
amine, 1,3-propandiamine and propylamine (which can be con-
sidered as by-products of the NO-molecule decomposition)
under aerobic and anaerobic conditions to the PoxB containing
solutions. No changes are observed with respect to the spectro-
scopic features of native PoxB either in the UV-Vis absorption
experiments at room temperature or in the frozen-solution EPR
spectra. In all experiments dealing with the azide addition,
sodium azide was added to the laccase solution under aerobic
conditions 24 h before the addition of the NO-donor molecule
by using a 1 : 50 PoxB/azide ratio. In the experiment in which
the laccase was reduced with ascorbate, the addition of sodium
ascorbate to the laccase solution (1 : 10 PoxB/ascorbate ratio)
was made under anaerobic conditions.
References
1 L. J. Ignarro, Annu. Rev. Pharmacol. Toxicol., 1990, 30, 535.
2 C. J. Lowennstein and S. H. Snyder, Cell, 1992, 70, 705.
3 Q. H. Gibson and F. J. W. Roughton, J. Physiol. (London), 1957,
136, 507.
4 R. Cassoly and Q. H. Gibson, J. Mol. Biol, 1975, 91, 301.
5 V. S. Sharma, T. G. Traylor, R. Gardiner and H. Mizukami,
Biochemistry, 1987, 26, 3837.
6 D. P. Ballau, Y. Zhao, P. E. Brandish and M. A. Marletta, Proc. Natl.
Acad. Sci. USA., 2002, 99(19), 12097.
7 C. E. Cooper, J. Torres, M. A. Sharpe and M. T. Wilson, FEBS Lett.,
1997, 414, 218.
8 G. Musci, S. Di Marco, M. C. Bonaccorsi di Patti and L. Calabrese,
Biochemistry, 1991, 30, 9866.
9 J. Torres, C. E. Cooper and M. Wilson, J. Biol. Chem., 1998, 273(15),
8756.
10 X. Zhou, M. G. Espey, J. X. Chen, L. J. Hofseth, K. M. Miranda,
S. P. Hussain, D. A. Winks and C. C. Harris, J. Biol. Chem., 2000,
275(28), 21241.
11 E. I. Solomon, U. M. Sundaram and T. E. Machonkin, Chem. Rev.,
1996, 96, 2563.
12 J. L. Cole, P. A. Clark and E. I. Solomon, J. Am. Chem. Soc., 1990,
112(26), 9534.
Anaerobic laccase and NO-donor solutions were prepared by
degassing them with a few cycles of vacuum and purging them
with ultrapure N2 gas in an eppendorf presealed with a rubber
cap. Aliquots of NO-donor solutions were added by using
Hamilton gas-tight mycrosyringes in a home made glove box
under nitrogen flux.
13 C. T. Martin, R. H. Morse, R. M. Kanne, H. B. Gray, B. G.
Malmstrom and S. I. Chan, Biochemistry, 1981, 20, 5147.
14 J. Torres, D. Svinstunenko, B. Karlsson, C. Cooper and M. T.
Wilson, J. Am. Chem. Soc., 2002, 124(6), 963.
15 R. P. Bonomo, A. M. Boudet, R. Cozzolino, E. Rizzarelli, A. M.
Santoro, R. Sterjiades and R. Zappalà, J. Inorg. Biochem., 1998, 71,
205 (magnetic parameters obtained on a 63Cu PoxB laccase at
150 K).
16 N. S. Kishnani and H. L. Fung, Methods Enzymol, 1996, 268A,
Spectroscopic measurements
259–265.
17 M. Feelisch and J. S. Stamler, in Methods in Nitric Oxide Research,
ed. M. Feelisch and J. J. Stamler, Wiley & Sons, Chichester, UK,
1996, pp. 71–115.
18 E. I. Solomon, T. E. Machonkin and U. M. Sundaram, in
Multi-Copper Oxidases, ed. A. Messerschimidt, World Scientific
Publishing, Singapore, 1997, pp. 103–127.
19 A. Garzillo, M. C. Colao, V. Buonocore, R. Oliva, L. Falcigno,
M. Saviano, A. M. Santoro, R. Zappalà, R. Bonomo, G. Palmieri,
P. Giardina, C. Bianco and G. Sannia, J. Protein Chem., 2001, 20,
191.
20 A. C. F. Gorren, E. De Boer and R. Wever, Biochim. Biophys. Acta,
1987, 916, 38.
21 R. P. Bonomo, G. Cennamo, R. Purrello, A. M. Santoro and
R. Zappalà, J. Inorg. Biochem., 2000, 83, 67.
22 D. J. Spira-Solomon, M. D. Allendorf and E. I. Solomon, J. Am.
Chem. Soc., 1986, 108, 5318.
23 T. Sakurai and J. Takahashi, Biochem. Biophys. Res. Commun., 1995,
215(1), 235.
24 J. C. Severns and D. R. McMillin, Biochemistry, 1990, 29, 8592.
25 I. Gromov, A. Marchesini, O. Farver, I. Pecht and D. Goldfarb,
Eur. J. Biochem., 1999, 266, 820.
Optical absorption spectra in the UV-Vis region were recorded
at 25 ЊC on a Varian UV-Vis near IR Cary 500 spectrophoto-
meter in 1 cm path length quartz cells.
All EPR spectra of frozen laccase solution, at protein con-
centration of 0.3–0.6 mM, dissolved in 0.1 M phosphate buffer,
pH 7.0, were recorded at liquid-nitrogen temperature (77 K)
through the use of a quartz cold finger or at 125 K with a
variable temperature apparatus. A conventional Bruker ER
200 D spectrometer operating at X-band (9.3–9.7 GHz), driven
by the ESP 3220 and a Bruker Elexsys E500CW-ESR spectro-
meter driven by PC running XEpr program under Linux and
equipped with a Super X-band microwave bridge operating at
9.3–9.5 GHz and a SHQE cavity were both used throughout
this work. Aliquots of the reaction mixture at different times
were placed in 3 mm inner diameter EPR quartz tubes and
frozen at liquid-nitrogen temperature, then the tubes were
transferred to the apparatus and the EPR spectra registered.
Instrumental setting of EPR spectrum recordings were as
D a l t o n T r a n s . , 2 0 0 4 , 1 0 4 – 1 1 2
111

View Article Online
26 L. Morpurgo, A. Desideri and G. Rotilio, in The Coordination
Chemistry of Metalloenzymes, ed. I. Bertini, R. S. Drago and
C. Luchinat, D. Reidel Publishing Company, Dordrecht, Holland,
1983, pp. 207–213.
27 D. L. H. Williams, Methods Enzymol., 1996, 268A, 299.
28 I. Peisach and W. E. Blumberg, Arch. Biochem. Biophys., 1974, 165,
691.
29 A. Messerschmidt, R. Ladenstein, R. Huber, L. Bolognesi,
R. Petruzzelli, A .Rossi and A. Finazzi-Agrò, J. Mol. Biol., 1992,
224, 179.
30 V. Ducros, A. M. Brzozwski, K. S. Wilson, S. H. Brown,
P. Østergaard, P. Schneider, D. S. Yaver, A. H. Pedersen and
G. J .Davies, Nat. Struct. Biol., 1998, 5(4), 310.
31 K. Piontek, M. Antorini and T. Choinowski, J. Biol. Chem., 2002,
277(40), 37663.
36 T. L. Fraterrigo, C. Miller, B. Reinhammar and D. R. McMillin,
J. Biol. Inorg. Chem., 1999, 4, 183.
37 M. M. Morie-Bebel, D. R. McMillin and W. E. Antholine, Biochem.
J., 1987, 235, 415.
38 J. L. Cole, L. Avigliano, L. Morpurgo and E. Solomon, J. Am.
Chem. Soc., 1991, 113, 9080.
39 R. P. Bonomo, F. Riggi and A. J. Di Bilio, Inorg. Chem., 1988, 27,
2519.
40 L. Calabrese and G. Musci, in Multi-copper Oxidases,
ed. A. Messerschmidt, World Scientific Publishing, Singapore, 1997,
pp. 307–354.
41 W. H. Koppenol, Methods Enzymol., 1996, 268A, 7.
42 R. Bourbonnais and M. G. Paice, FEBS Lett., 1990, 267, 99.
43 P. C. Ford and I. M. Lorkovic, Chem Rev., 2002, 102, 993–1017.
44 H. Beinert, J. Inorg. Biochem, 1996, 64, 79.
32 N. Hakulinen, L. L. Kiiskinen, K. Kruus, M. Saloheimo,
A. Paananen, A. Koivula and J. Rouvinen, Nat. Struct. Biol., 2002,
9(8), 601.
33 T. Bertrand, C. Jolivat, P. Briozzo, E. Caminade, N. Joly, C. Mazdak
and C. Mougin, Biochemistry, 2002, 41, 7325.
34 M. Miglio, S. Garavaglia, M. T. Cambria, S. Ragusa, A. Cambria
and M. Rizzi,, Scuola Nazionale di Biofisica, Bressanone, 8–10 Sept.,
2003.
35 J. Li, D. R. McMillin and W. E. Antholine, J. Am. Chem. Soc., 1992,
114, 725.
45 B. Reinhammar, in Copper Proteins and Copper Enzymes, ed. R.
Lontie, CRC Press, Boca Raton, FL, USA, 1984, vol III, pp. 1–35.
46 U. K. Leammli, Nature, 1970, 227, 680.
47 M. M. Bradford, Anal. Biochem., 1976, 72, 248–254.
48 B. Roy, A. M. D’Hardemare and M. Fontecave, J. Org. Chem., 1994,
59, 7019.
49 L. C. Green, D. A. Wagner, J. Glogowski, P. L. Skipper, J. S.
Wishnok and S. R. Tannenbaum, Anal. Biochem., 1982, 126, 131.
50 A. Lund and T. Vanngard, J. Chem. Phys., 1969, 50, 2979.
51 O. Vorm, P. Roepstrorff and M. Mann, Anal. Chem., 1994, 66, 3281.
D a l t o n T r a n s . , 2 0 0 4 , 1 0 4 – 1 1 2
112