Fast reduction of a copper center in laccase by nitric oxide and formation of a peroxide intermediate

Article abstract of DOI:10.1021/ja016107j

The rapid reduction of one of the copper atoms (type 2) of tree laccase by nitric oxide (NO) has been detected. Addition of NO to native laccase in the presence of oxygen leads to EPR changes consistent with fast reduction and slow reoxidation of this metal center. These events are paralelled by optical changes that are reminiscent of formation and decay of the peroxide intermediate in a fraction of the enzyme population. Formation of this species is only possible if the trinuclear copper cluster (type 2 plus type 3) is fully reduced. This condition can only be met if, as suggested previously, a fraction of the enzyme contains both type 3 coppers already reduced before addition of NO. Our data are consistent with this assumption. We have suggested recently that fast reduction of copper is the mechanism by which NO interacts with the oxidized dinuclear center in cytochrome c oxidase. The present experiments using laccase strongly support this view and suggest this reaction as a general mechanism by which copper proteins interact with NO. In addition, this provides an unexploited way to produce a stable peroxide intermediate in copper oxidases in which the full complement of copper atoms is present. This enables the O-O scission step in the catalytic cycle to be studied by electron addition to the peroxide derivative through the native electron entry site, type 1 copper.

Full text of DOI:10.1021/ja016107j

Published on Web 01/19/2002
Fast Reduction of a Copper Center in Laccase by Nitric Oxide
and Formation of a Peroxide Intermediate
Jaume Torres,†,‡ Dimitri Svistunenko,† Bo Karlsson,§ Chris E. Cooper,† and
Michael T. Wilson*,†
Contribution from the Department of Biological Sciences, UniVersity of Essex,
WiVenhoe Park CO4 3SQ, Colchester, Essex, U.K., and Department of Medical Laboratory
Science, Gothenburg UniVersity of Health Sciences, Medicinaregatan 8,
B 41390 Gothenburg, Sweden
Received April 30, 2001. Revised Manuscript Received November 2, 2001
Abstract: The rapid reduction of one of the copper atoms (type 2) of tree laccase by nitric oxide (NO) has
been detected. Addition of NO to native laccase in the presence of oxygen leads to EPR changes consistent
with fast reduction and slow reoxidation of this metal center. These events are paralelled by optical changes
that are reminiscent of formation and decay of the peroxide intermediate in a fraction of the enzyme
population. Formation of this species is only possible if the trinuclear copper cluster (type 2 plus type 3) is
fully reduced. This condition can only be met if, as suggested previously, a fraction of the enzyme contains
both type 3 coppers already reduced before addition of NO. Our data are consistent with this assumption.
We have suggested recently that fast reduction of copper is the mechanism by which NO interacts with the
oxidized dinuclear center in cytochrome c oxidase. The present experiments using laccase strongly support
this view and suggest this reaction as a general mechanism by which copper proteins interact with NO. In
addition, this provides an unexploited way to produce a stable peroxide intermediate in copper oxidases in
which the full complement of copper atoms is present. This enables the O-O scission step in the catalytic
cycle to be studied by electron addition to the peroxide derivative through the native electron entry site,
type 1 copper.
Introduction
We have tested this model using laccase (p-diphenol:dioxygen
oxidoreductase, EC 1.10.3.2), the simplest of the family of the
Nitric oxide is known to act as a signal transducer in
neurotransmission1 and vasodilation2 by interacting with iron
in the heme protein guanylyl cyclase. However, nitric oxide
may also exert potent physiological effects through interaction
with copper. We have recently reported very fast reactions
between nitric oxide and cytochrome c oxidase,3,4 and we have
explained these through a mechanism in which NO acts as a
one-electron reductant of the enzyme. We have proposed that
reduction occurs at CuB(II), the oxidized copper in the dinuclear
center, and that, once reduced to CuB(I), this rapidly equilibrates
with other redox centers in the enzyme. This rapid interaction
of NO with oxidized copper has been suggested to play a key
role in the control of the activity of cytochrome c oxidase4 and
is possibly relevant to the control of a number of other copper
containing enzymes. However, the evidence for the reaction of
NO with this metal is indirect and only inferred from the
observed electron transfer to other redox sites.
multicopper oxidases, which catalyzes the four-electron reduc-
tion of dioxygen to water. Laccase contains four copper atoms,
classified mainly according to their EPR features:5 type 1 (T1)
or blue, which has a small parallel hyperfine coupling (A| ∼
(40-70) × 10-4 cm-1 or 43-75 G), type 2 (T2) with normal
EPR features, and a dinuclear EPR-undetectable site termed type
3 (T3). Optically, T1 has a relatively strong band in the visible
region (ꢀ615 ∼ 5700 M-1 cm-1), T3 absorbs at 330 nm (ꢀ330
∼
3600 M-1 cm-1),6 whereas T2 is practically undetectable.
Electron entry from the physiological substrate into the enzyme
is at T1, which transfers electrons to a trinuclear copper center
formed by T2 and T3.7,8 It is at this center, analogous to the
trinuclear center found in ascorbate oxidase9 or ceruloplasmin,10
where oxygen binds and is reduced.
The understanding of the function of this enzyme has been
greatly improved by the use of two derivatives. One of these is
type 2 depleted (T2D) laccase, in which one of the copper atoms
* Corresponding author. Telephone: +44 1206 872538. Fax: +44 1206
872592. E-mail: wilsmt@essex.ac.uk.
(5) Reinhammar, B.; Malmstro¨m, B. G. In Copper Proteins; Spiro, T. G., Ed.;
Wiley-Interscience: New York, 1981; Chapter 3..
† University of Essex.
‡ Present address: Department of Biochemistry, 80 Tennis Court Rd.,
University of Cambridge, Cambridge CB2 1GA, U.K.
§ Gothenburg University of Health Sciences.
(6) Kau, L.-S.; Spira-Solomon, D. J.; Penner-Hann, J. E.; Hodgson, K. O.;
Solomon, E. I. J. Am. Chem. Soc. 1987, 109, 6433-6442.
(7) Allendorf, M. D.; Spira, D. J.; Solomon, E. I. Proc. Natl. Acad. Sci. U.S.A.
1985, 82, 3063-3067.
(1) Garthwaite, J.; Boulton, C. L. Annu. ReV. Physiol. 1995, 57, 683-706.
(2) Moncada, S.; Higgs, A. N. Engl. J. Med. 1993, 329, 2002-2012.
(3) Torres, J.; Cooper, C. E.; Wilson, M. T. J. Biol. Chem. 1998, 273, 8756-
8766.
(8) Reinhammar, B. Chem. Scr. 1985, 25, 172-176.
(9) Messerschmidt, A.; Luecke, H.; Huber, R. J. Mol. Biol. 1993, 230, 997-
1014.
(4) Cooper, C. E.; Torres, J.; Sharpe, M.; Wilson, M. T. FEBS Lett. 1997,
414, 281-284.
(10) Zaitseva, I.; Zaitsev, V.; Card, G.; Moshov, K.; Bax, B.; Ralph, A.; Lindley,
P. J. Biol. Inorg. Chem. 1996, 1, 15-23.
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A R T I C L E S
Torres et al.
(T2) is removed by a chelator.11 Studies using this derivative
showed that, in contrast to the oxygen carrier hemocyanin, the
dinuclear T3 reduced site in laccase does not bind oxygen12 in
contrast with previous reports.13 A second derivative14 is T1Hg,
in which the copper atom in the T1 center has been substituted
by the redox-inactive Hg(II). Using this method, it is possible
to obtain an enzyme reduced with only three electrons, all of
them in the trinuclear center. The combination of fully reduced
(three electrons) T1Hg with oxygen results in the formation of
a species which is described as a hydroperoxide, bridged
between an oxidized T3 copper and reduced T2.12,15 This
intermediate has been suggested by these authors to precede
the formation of the native intermediate8 in the normal catalytic
cycle.
The reaction of NO with tree laccase has been studied
previously.16 In these experiments, performed in the absence
of oxygen and under a saturating NO concentration, the enzyme
was fully reduced in a process that was reported to be very
slow (t1/2 ∼ 70 min). These experiments, however, were made
with a low temporal resolution (e.g., the first EPR measurement
was collected 5 min after the addition of NO). On the other
hand, the redox state of type 2 copper cannot be assessed
optically and EPR measurements are complicated by changes
in the redox potential of the metal centers upon freezing the
samples.16 Therefore, an accurate measurement of the level of
reduction of type 2 copper in the first seconds was not given.
In the present work, we present evidence that NO can reduce
T2 copper in laccase in a very fast reaction (t1/2 < 1 s).
Figure 1. Spectral changes after addition of NO to laccase as prepared in
the presence of oxygen. Difference spectra relative to the resting oxidized
enzyme (as prepared) after the addition of an aliquot of 2 mM NO (final
concentration ∼ 100 µM) to a solution containing ∼70 µM laccase in 100
mM HEPES, pH 6.0. The first spectrum (A) was collected immediately
after the addition of NO, and spectra were collected every 2 min thereafter.
Spectrum B corresponds to the sample after 80 min. A vertical dotted line
is plotted to indicate the blue shift of the band as the intermediate decays.
Addition of NO to an anaerobic sample in the same conditions generates
spectrum C. Insert: Time course followed at 335 nm (O) fitted to an
exponential decay (t1/2 ∼ 15 min) (s). The features of spectrum C at 400-
425 nm and the small bands in the visible region (530 and 565 nm)
correspond to the formation of a ferric-NO complex of lacquer peroxidase,19
a contaminant (less than 0.5% of the laccase concentration) that is common
in these preparations. These features decayed rapidly at pH 6 (t1/2 ∼ 30 s)
and were not present at the time points shown (spectrum A and below).
The trough at ∼420 nm at 80 min (spectrum B) is caused by a further blue
shift of the Soret band with respect to the oxidized enzyme.
to liquid nitrogen where they were stored until use. The oxidation of
the type 2 copper was monitored using the intensity of the low-field
component18 at g ) 2.47, measured using conditions in which the signal-
to-noise ratio was maximized (i.e., averaging many scans at low
temperature (8 K)). After the addition of NO, the band shape of this
component was slightly different from the control, although it was
unchanged thereafter. Due to this fact, the average intensity corre-
sponding to the samples after 1 h was taken as 100% type 2 copper
oxidation.
Nitric oxide was obtained from a Kipps apparatus maintained in a
certified hood (due to the potential toxicity of NO gas). Sulfuric acid
(1 M) was mixed with sodium nitrite. The gaseous product(s) of this
reaction were passed through a series of traps (NaOH, H2SO4, H2O,
KI, and dry ice) to remove nitrogen oxides other than NO. Finally NO
was collected in a gastight syringe and injected into an anaerobic
solution. The NO concentration was measured with an NO electrode
(Iso-NO Mark II, World Precision Instruments). The electrode was
precalibrated by the addition of a standard sodium nitrite solution to
excess acidified potassium iodide; this generates NO stoichiometric to
the added nitrite. Additions of NO were made using a gastight Hamilton
syringe.
Experimental Section
Laccase was obtained and purified according to the method of
Reinhammar17 from acetone powder of Rhus vernicifera (Saito & Co.
Ltd. Tokyo, Japan). The concentration of laccase was determined using
ꢀ614 ) 5700 M-1 cm-1.6 Static spectra were collected with a Cary 5E
UV-vis-near-IR spectrophotometer. The experiments were performed
at 20 °C in 0.1 M HEPES (N-[2-hydroxyethyl]piperazine-N′-[2-
ethanesulfonic acid]) at pH 6 or pH 7.4. The pH was altered by passage
through a Sephadex G-25 column equilibrated with buffer at the desired
pH. Anaerobic laccase was prepared by degassing a solution in a cuvette
presealed with a rubber cap. This was purged with N2 gas, and after a
few cycles the sample was immediately used.
Stopped-flow experiments were performed using a SX-18MV
stopped-flow apparatus (Applied Photophysics, Leatherhead, U.K.).
Laccase incubated anaerobically with NO was rapidly mixed with buffer
containing oxygen at known concentrations. This was obtained by
mixing anaerobically the degassed solution with the required volume
of oxygen equilibrated buffer.
EPR spectra were measured on a Bruker EMX spectrometer with
an ER 041XG microwave bridge (X-band). An ER 4122SP cavity was
used. The temperature was controlled using an Oxford Instrument
helium system. A Bruker WINEPR (v. 2.11) package was used for
spectral analysis. Aliquots of the enzyme or reaction mixture were
placed in 3 mm i.d. EPR “precision” tubes (Wilmad PQ) and frozen in
ethanol thermostated in dry ice. Once frozen, the tubes were transferred
It should be remembered that on exposure to air NO reacts
spontaneously and rapidly to form toxic NO2 gas. Due care should thus
be exercised when handling NO, and all transfers should be undertaken
in a fume hood.
Results
Addition of NO to resting oxidized tree laccase (as prepared)
in the presence of oxygen resulted in rapid spectral changes
depicted in Figure 1 (spectrum A). This difference spectrum
shows prominent positive features at 335 nm, 470 nm, and a
trough at 610 nm. These positive features are strongly remi-
(11) Graziani, M. T.; Morpurgo, L.; Rotilio, G.; Mondovi, B. FEBS Lett. 1976,
70, 87-90.
(12) Cole, J. L.; Tan, G. O.; Yang, E. K.; Hodgson, K. O.; Solomon, E. I. J.
Am. Chem. Soc. 1990, 112, 2243-2249.
(13) Reinhammar, B.; Oda, Y. J. Inorg. Biochem. 1979, 11, 115-117.
(14) Morie-Bebel, M. M.; Morris, M. C.; Menzie, J. L.; McMillin, D. R. J. Am.
Chem. Soc. 1984, 106, 3677-3678.
(15) Cole, J. L.; Ballou, D. P.; Solomon, E. I. J. Am. Chem. Soc. 1991, 113,
8544-8546.
(18) Va¨nngård, T. In Magnetic Resonance in Biological Systems; Ehrenberg,
A., Malmstro¨m, B. G., Va¨nngård, T., Eds.; Pergamon: Oxford, 1967; pp
(16) Martin, C. T.; Morse, R. H.; Kanne, R. M.; Gray, H. B.; Malmstro¨m, B.
G.; Chan, S. I. Biochemistry 1981, 20, 5147-5155.
(17) Reinhammar, B. Biochim. Biophys. Acta 1970, 205, 35-47.
213-219,
(19) Suzuki, S.; Yoshimura, T.; Sakurai, T. J. Inorg. Biochem. 1991, 44, 267-
276.
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Fast Reduction of a Copper Center in Laccase
A R T I C L E S
Figure 2. Time course of the changes generated following addition of
oxygen to laccase incubated anaerobically with NO. Time courses followed
at 330 nm in the stopped flow apparatus after mixing anaerobic laccase
(65 µM) incubated with ∼100 µM NO for 20 s with buffer at known oxygen
concentrations. Oxygen concentration after mixing (bottom to top): 25,
45, 70, and 120 µM. The traces were fitted to a double-exponential increase.
Insert: Plot of the observed pseudo-first-order rate constant of the fast
component versus the oxygen concentration.
Figure 3. Time course of the events described in Figure 1 followed by
EPR. EPR spectra of oxidized resting laccase at pH 6 (65 µM) measured
at 77 K. Microwave power, 3.2 mW; microwave frequency, 9.4859 GHz;
modulation frequency, 100 kHz; modulation amplitude, 1 G; time constant,
0.041 s; sweep rate, 9.15 G/s. The low-field component of the type 2 copper
signal (g ) 2.47) is indicated. Inset: Intensity of this component changing
during the experiment and normalized with respect to the intensity obtained
after 1 h. The first two points (before addition of 100 µM NO) were taken
as a control, and the following measurements were taken at different time
points (0.5, 1, 10, 11, 60, and 68 min) after the addition of NO. The line
represents an exponential fit to the data.
niscent of the difference spectrum of a laccase intermediate.20
This intermediate has been described as a peroxide, bridging
oxidized T3 and reduced T2, and usually obtained by mixing
oxygen with the three electron-reduced derivative T1Hg.20
Addition of NO to anaerobic laccase generated only minor
optical changes (Figure 1, spectrum C), but after addition of
oxygen, spectrum A, was generated (Figure 1). Addition of
oxygen to anaerobic laccase in the absence of NO did not
produce any optical change. Similarly, addition of sodium nitrite
(up to 5 mM) did not elicit the spectrum seen in Figure 1.
The amplitude of the change at 335 nm was the same at pH
7.4 or pH 6. At pH 6, the decay of this difference spectrum
(Figure 1 and insert) was slow (t1/2 ∼ 15 min). At pH 7.4 this
decay rate fell ∼5-fold (t1/2 ∼ 75 min). Both the absolute rate
of decay and its pH dependency are similar to those observed
for the decay of the peroxide intermediate obtained using the
T1Hg species.20 Using the reported extinction coefficient for
the intermediate, relative to the oxidized enzyme (∆ꢀ335 ∼ 5000
M-1 cm-1),20 the amplitude of the decay at 335 nm (Figure 1,
insert) is consistent with a concentration of ∼18 µM for this
species (i.e., 25% of the total laccase concentration used in the
experiment). The final spectrum (Figure 1, spectrum B and
insert), after the decay of the band at 335 nm, shows a remaining
positive band at 330 nm which is consistent with the oxidation
incubated in 0.2 mM potassium ferricyanide for 1 h, in 1 mM
fluoride for 30 min, or in 1 mM azide for 1 min.
The rate constant for the formation of the band at 335 nm in
Figure 1 (spectrum A) was obtained by mixing oxygen
containing buffer with an anaerobic solution containing the
native enzyme plus NO (Figure 2). The time courses were fitted
to a double-exponential curve.
The fastest of these processes was found to be oxygen
concentration dependent, yielding a value of 3.5 × 106 M-1
s-1 for the second-order rate constant (Figure 2, insert), similar
to that found for the formation of the peroxide intermediate in
T1Hg.15 The second process was slower (kobs ∼ 30 s-1) and
independent of the oxygen concentration. Use of sodium nitrite
(5 mM) instead of NO did not elicit any changes.
Since the redox state of type 2 copper itself is difficult to
detect optically, the changes occurring after addition of NO were
monitored by EPR spectroscopy (Figure 3). This figure shows
that the intensity of the low field component (g ) 2.47)
corresponding to the oxidized type 2 copper (see Materials and
Methods) drops ∼30-40% after the addition of NO (Figure 3,
insert). This is followed by slow reoxidation. Note, however,
that the first two measurements were obtained after 30 and 60
s after the addition of NO, and the percentage of T2 reduction
could be even higher (see figure legend). This suggests that the
reduction of the T2 coppers may not be restricted to the putative
population of laccase having T3 reduced (23%). In fact, when
the enzyme in the presence of oxygen was titrated with NO
and the absorbance at 335 nm corresponding to the putative
peroxide intermediate was followed optically, it was observed
(Figure 4) that the maximum amplitude of the absorbance
change was obtained when the molar ratio NO/laccase was 1:1.
This indicates that a complete formation of the putative peroxide
intermediate is only obtained at equimolar concentrations of NO
and laccase. Since the formation of the peroxide intermediate
requires the reduction of T2, we conclude therefore that the
affinity of T2 for NO may be similar, irrespective of the redox
state of T3.
of ∼25% reduced dinuclear T3 center (∆ꢀ335 ∼ 3600 M-1 cm-1
;
see above), present in the native enzyme. A similar difference
spectrum was observed (not shown) after addition of H2O2 to
laccase (H2O2/laccase molar ratio, 50:1), as reported previ-
ously.21 Addition of NO to the sample incubated with H2O2
did not generate any optical change. Thus, the appearance of
the features in spectrum A (Figure 1) are accompanied by
changes consistent with oxidation of a fraction of T3. This is
also expected for the formation of the peroxide intermediate.20
Appearance of spectrum A (Figure 1) upon NO addition was
also observed (not shown) when the enzyme had been previously
(20) Shin, W.; Sundaram, V. M.; Cole, J. L.; Zhang, H. H.; Hedman, B.;
Hodgson, K. O.; Solomon, E. I. J. Am. Chem. Soc. 1996, 118, 3202-
3215.
(21) Penner-Hann, J. E.; Hedman, B.; Hodgson, K. O.; Spira, D. J.; Solomon,
E. I. Biochem. Biophys. Res. Commun. 1984, 119, 567-574.
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A R T I C L E S
Torres et al.
observed in the appearance of the band at 335 nm (Figure 4).
The obvious alternative is that a fraction of T3 is already reduced
before addition of NO, as suggested previously.21 Indeed, our
data are consistent with 25% of the molecules of the native
enzyme containing reduced T3 that become oxidized upon NO
addition and oxygen binding. Similar spectral changes occurred
after addition of hydrogen peroxide, an oxidant for T3 copper,
consistent with oxidation of 25% of T3, also reinforcing this
hypothesis. Furthermore, addition of NO to this fully oxidized
sample did not give rise to optical changes. Also, upon mixing
oxygen with the enzyme anaerobically incubated with NO, the
band at 335 nm is formed with a second-order rate constant of
3.5 × 106 M-1 s-1. This value is identical, within experimental
error, to 2 × 106 M-1 s-1, reported for the formation of the
peroxide intermediate when mixing oxygen with fully reduced
T1Hg,15 and also similar to 5 × 106 M-1 s-1, obtained for the
formation of the native intermediate when mixing oxygen with
the fully reduced enzyme.22 In the last case, although the
formation of the native intermediate is associated with oxidation
of T1, the same kinetic process should be observed (e.g.,
oxidation of T3 coppers), as oxidation of T3 is the rate-limiting
step.20
Figure 4. Effect of substoichiometric additions of NO to laccase in the
presence of oxygen. Titration corresponding to the changes at 330 nm after
the addition of alliquots of NO to 8.5 µM laccase in the presence of oxygen.
The time interval between NO addition and measurement was less than 5
s. The vertical dotted line indicates the enzyme concentration.
Discussion
On addition of NO to an aerobic solution of laccase or
addition of oxygen to anaerobic solutions of laccase in the
presence of NO, rapid absorbance changes occur, as depicted
in Figures 1 and 2. The question arises as to whether these
changes are due to the direct reaction of NO and O2 with laccase
or indirectly to the reaction of the products of the reaction
between NO and O2 with the enzyme.
In aerobic aqueous solutions nitrite is essentially the only
product of the reaction between NO and O2.22 Not only does
addition of nitrite to oxidized laccase not generate the spectra
observed, but the formation of nitrite is in any case much too
slow to account for our observations.23 Although higher nitrogen
oxides are formed as intermediates in the reaction of NO and
O2, the calculated concentrations22 are far too small to be
responsible for the observed stoichiometric changes in the
laccase copper centers. The concentrations of N2O3 and NO2
may be calculated to be 1 and 0.4 nM, respectively, versus a
laccase concentration of 65 µM. We conclude therefore that
the spectra we have described are due directly to the action of
NO and not to the products of its reaction with O2.
In fact, the changes observed after addition of NO to laccase
in the presence of oxygen can be interpreted as due to the
formation of the peroxide intermediate in a fraction (25%) of
the molecules. However, to achieve this, a fully reduced
trinuclear center (T3 and T2) must be present,6 which upon
reaction with oxygen would produce the peroxide intermediate.
Whereas we observe reduction of T2 by NO (see Figure 2), it
seems unlikely that NO also reduces T3. First, because reduction
of T3 by NO has been reported to be very slow,16 and second,
because of the 1:1 molar stoichiometry between NO and laccase
The fact that the spectral changes attributed to the peroxide
intermediate are observed upon NO addition, even after pre-
incubation of laccase with ferricyanide or fluoride, is consistent
with the fact that ferricyanide cannot oxidize the dinuclear center
T3.21 Therefore, the population of molecules containing reduced
T3 is still able to form the peroxide intermediate. Fluoride, on
the other hand, is a strong ligand (K ∼ 104 M-1 24
) of oxidized
type 2 copper, which might preclude the formation of the
intermediate. Fluoride, however, does not bind T2 when the
dinuclear center T3 is reduced.24 The observation that the
intermediate is formed also after incubation of laccase with azide
is intriguing, as azide has been reported to bind preferentially
to T2 when T3 is reduced.7 The reaction may still be possible,
however, if NO can reduce T2 and the peroxide group bridges
between T2 and the T3 copper that is not bound to azide. This
reaction possibly displays different kinetics.
The fact that the change in amplitude at 335 nm is complete
at a stoichiometry of 1:1 NO/laccase suggests that all the T2
sites are reduced at the end of the titration, or at least bind NO.
Thus, although the affinity of NO for T2 copper may be different
depending on the redox state of T3, as for azide7 or fluoride,24
both fractions of the enzyme bind NO with an affinity > 106
M-1. Furthermore, the fact that other centers (T3 or T1) did
not become reduced suggests that only one NO is bound per
molecule, at T2. The absence of bleaching of the bands at 614
or 330 nm after addition of NO to the anaerobic enzyme
provides further evidence that binding of NO and reduction of
Cu must occur at the optically undetectable T2 copper, confirm-
ing previous results16 which describe this center as the electron
entry site in the reduction of laccase by NO. Furthermore,
although the EPR results (Figure 3) suggest that only a fraction
(30-40%) of the T2 copper becomes reduced, it is possible
that low-temperature effects leading to electron redistribution16,25
between T2 copper and NO may mask the true percentage of
reduced T2 copper at room temperature. Formation of the native
(22) Lewis, R. S.; Deen, W. M. Chem. Res. Toxicol. 1994, 7, 568-574.
(23) For example in Figure 2 an anaerobic solution of 65 µM laccase/100 µM
NO was mixed with 25 µM oxygen and the spectrum described in Figure
1 appeared at a rate of approximately 50 s-1. The rate equation for the
reaction of NO with O2 may be written as d[NO2-]/dt ) 4k1[NO]2[O2],
where k1 ) 2 × 106 M-2 s-1. Given the second-order nature of this process
in [NO], the chemical oxidation of NO is far too slow to produce species
relevant to the laccase reactions observed here. The calculated initial rate
for nitrite formation in the above case is 2 × 10-6 M s-1, which indicates
that under the concentration regime employed the half-time for the reaction
is >40 s, whereas the reaction with the laccase has a half-time of some 14
ms.
(24) Andre´asson, L.-E.; Bra¨nde´n, R.; Reinhammar, B. Biochim. Biophys. Acta
1976, 438, 370-379.
(25) Dooley, D. M.; McGuirl, M. A.; Brown, D. E.; Turowski, P. N.; McIntire,
W. S.; Knowles, P. F. Nature 1991, 349, 262-264.
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Fast Reduction of a Copper Center in Laccase
A R T I C L E S
intermediate, as a product of the decay of the peroxide
intermediate, was not detected, either by optical means or by
EPR. This intermediate, which displays a band at 360 nm
relative to the oxidized enzyme,22 is formed upon electron
transfer from T1 to the peroxide intermediate and has been
associated with an oxygen radical which is detected only at low
temperature.26 Although such a species may be formed in our
sample as the product of the decay of the peroxide intermediate,
the fact that in our system the peroxide intermediate must be
reduced by T2 (t1/2 of minutes), and not by T1 (>1000 s-1),
suggests that its formation is probably paralleled by its decay,
and a significant population never accumulates.
In summary, we have observed rapid reduction of a type 2
copper in laccase upon NO addition. This has been observed
directly by EPR and also by optical means through a subpopu-
lation of the enzyme (∼25%) which contains a dinuclear T3
center already fully reduced (2 electrons). The kinetics of the
formation of the peroxide intermediate are virtually identical
to those reported for the formation of the native intermediate
using fully reduced enzyme, or the formation of the peroxide
intermediate using fully reduced T1Hg. The rate of decay of
the species described is very slow and similar to the decay of
the peroxide intermediate, showing also a similar pH depen-
dency. We conclude that the peroxide intermediate has been
formed via reduction of T2 copper by NO in a fast reaction.
We suggest that this reaction may not only hold implications
for the regulation, by NO, of enzymes containing trinuclear
copper centers (e.g. the major copper protein in blood plasma,
ceruloplasmin), but also could be relevant to the regulation of
copper enzymes in general. In addition, the method we report
here for the formation of the peroxide intermediate in laccase
molecules which possess the full complement of copper atoms
should prove useful to the study of the catalytic cycle of this
enzyme. For example, it permits one to investigate the O-O
scission step by the addition of a single electron through the
native T1 copper. This reaction cannot be so easily isolated by
other methods.
Acknowledgment. This work was supported by the Spanish
government (J.T.). The authors gratefully acknowledge Dr.
Bengt Reinhammar for useful discussions and the generous gift
of tree laccase.
(26) Aasa, R.; Bra¨nde´n, R.; Deinum, J.; Malmstro¨m, B. G.; Reinhammar, B.;
Va¨nngård, T. FEBS Lett. 1976, 61, 115-119.
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