Lathyrus cicera copper amine oxidase reactions with tryptamine

Article abstract of DOI:10.1016/j.jinorgbio.2012.01.002

Lathyrus cicera copper amine oxidase (LCAO) rapidly formed the typical Cu(I)-TPQ semiquinone UV-visible spectrum, identical to that formed by other substrates, upon O₂ exhaustion by turnover with excess tryptamine. A new band at 630 nm formed more slowly, with intensity dependent on aldehyde and H₂O₂ concentrations. On prolonged incubation, all bands decayed in parallel, together with loss of enzymatic activity. The blue color disappeared on addition of KCN, a Cu(I) stabilizing agent, while the intensity of the radical visible bands increased. This shows that the 630 nm absorbing species is a Cu(II) derivative, as confirmed by the unchanged intensity of the EPR spectrum of the frozen blue solution from that of the native protein. Rapid kinetics experiments showed that this species derives from a reduced form of the protein, plus aldehyde and H₂O₂ and that it is not in dynamic equilibrium with the radical. Given the similar population of the semiquinone radical with all substrates, it is possible that the reaction with aldehyde and H₂O₂ occurs in all cases although substrates lacking the indole group only produce the Cu(I)-semiquinone band. The radical participation to the catalytic activity is demonstrated by the observation that its relative population (controlled by the pH) parallels changes in the reoxidation rate constant, while the 630 nm absorbing species is implied in the inactivation process, which depends on H₂O₂ and aldehyde concentration. The results of the paper are consistent with half-of-the-site reactivity, i.e. the two subunits of LCAO are kinetically and spectroscopically distinct from each other.

Full text of DOI:10.1016/j.jinorgbio.2012.01.002

Journal of Inorganic Biochemistry 109 (2012) 33–39
Contents lists available at SciVerse ScienceDirect
Journal of Inorganic Biochemistry
journal homepage: www.elsevier.com/locate/jinorgbio
Lathyrus cicera copper amine oxidase reactions with tryptamine
a,
a
b
a
a
Paola Pietrangeli ⁎, Andrea Bellelli , Paola Fattibene , Bruno Mondovì , Laura Morpurgo
a
Department of Biochemical Sciences, “A. Rossi Fanelli”, University of Rome “Sapienza”, P.le A. Moro 5, 00185 Rome, Italy
Dipartimento di Tecnologie e Salute, Istituto Superiore di Sanità,Viale Regina Elena 299, 00161 Rome, Italy
b
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 12 July 2011
Received in revised form 4 January 2012
Accepted 6 January 2012
Available online 12 January 2012
Lathyrus cicera copper amine oxidase (LCAO) rapidly formed the typical Cu(I)-TPQ semiquinone UV–visible
spectrum, identical to that formed by other substrates, upon O exhaustion by turnover with excess trypt-
amine. A new band at 630 nm formed more slowly, with intensity dependent on aldehyde and H concen-
trations. On prolonged incubation, all bands decayed in parallel, together with loss of enzymatic activity. The
blue color disappeared on addition of KCN, a Cu(I) stabilizing agent, while the intensity of the radical visible
bands increased. This shows that the 630 nm absorbing species is a Cu(II) derivative, as confirmed by the
unchanged intensity of the EPR spectrum of the frozen blue solution from that of the native protein. Rapid
kinetics experiments showed that this species derives from a reduced form of the protein, plus aldehyde
2
2 2
O
Keywords:
Trihydroxyphenylalanine quinone
Tryptamine
Visible bands
2 2
and H O and that it is not in dynamic equilibrium with the radical. Given the similar population of the semi-
Rapid kinetics
quinone radical with all substrates, it is possible that the reaction with aldehyde and H O2 occurs in all cases
2
although substrates lacking the indole group only produce the Cu(I)-semiquinone band. The radical partici-
pation to the catalytic activity is demonstrated by the observation that its relative population (controlled by
the pH) parallels changes in the reoxidation rate constant, while the 630 nm absorbing species is implied in
the inactivation process, which depends on H O and aldehyde concentration. The results of the paper are
2 2
consistent with half-of-the-site reactivity, i.e. the two subunits of LCAO are kinetically and spectroscopically
distinct from each other.
©
2012 Elsevier Inc. All rights reserved.
1
. Introduction
although the overall primary sequence identity is usually not high,
b25%. Notable exceptions are CuAOs purified from plants, such as lentil
and pea seedling, which share over 90% sequence identity [11], and the
BSAO/VAP1 couple which share 85% sequence identity [9]. The structur-
al homology and thirty-three residues fully conserved in the catalytic
site region [12], probably account for the similar, but not identical, reac-
tivity. They catalyze the oxidative deamination of primary amines by
Copper amine oxidases (CuAOs) share with other enzymes the
property of having a Cu-bound organic cofactor derived from the
post-translational modification of a conserved polypeptidic chain res-
idue [1]. The modified residue of CuAOs is a tyrosine which is oxidized
to 2,4,5-trihydroxyphenylalanine quinone (TPQ). In the copper pro-
tein lysyl oxidase, an enzyme required for cross-linking of collagen
and elastin, the cofactor lysine tyrosylquinone (LTQ) is derived from
an oxidized tyrosine linked to a conserved lysine. In alkylamine dehy-
drogenases from gram-negative soil bacteria the tryptophan trypto-
phylquinone (TTQ) is derived from two oxidized and condensed
tryptophan residues. In fungal galactose oxidase the cofactor is a
tyrosyl-cysteine radical. Amine oxidases are found in nearly all
forms of life, where they play important biological roles, namely nu-
trient catabolism in microorganisms [2], for which amines are the
sole source of carbon and nitrogen, wound healing in plants [3], bio-
genic amines catabolism and signaling in mammals [2].
2 2 2
the two electron reduction of O . Aldehyde, ammonia, and H O are
produced in the reaction which occurs with ping-pong mechanism
[2,13] (Scheme 1). The reductive phase of the ping-pong reaction con-
sists [14,15] of the initial formation of the substrate Schiff-base, a imino-
quinone, the subsequent conversion to the product Schiff-base, a
quinolaldimine, and the formation, upon release of the aldehyde, of
the reduced species Cu(II)-aminoresorcinol in equilibrium with Cu(I)-
semiquinone. The conversion is assisted by a conserved aspartate,
which helps proton abstraction from the α-carbon of the substrate
with formation of the carbanion. The mechanism of the reoxidation re-
action is not as well established as the reduction phase [15]. X-ray struc-
The X-ray structures, available for CuAOs of different sources [4–10],
show that the enzymes are dimeric, with extensively interacting sub-
units of 70–90 kDa. They display considerable structural homology,
tural data support the binding of O
between Cu(II) and the iminoquinone [16]. From this intermediate
is released and ammonia is hydrolyzed to restore TPQ. A debated
point remains, concerning the first electron transfer to O . Two different
mechanisms were proposed to overcome the energetically unfavorable
change of O spin multiplicity. According to mechanism 1, O binds
Cu(I)-semiquinone with transfer of one electron and formation of the
2 2 2
, probably in the form of H O ,
2 2
H O
2
⁎
Corresponding author. Tel.: +39 06 49910837; fax: +39 06 4440062.
2
2
E-mail address: paola.pietrangeli@uniroma1.it (P. Pietrangeli).
0162-0134/$ – see front matter © 2012 Elsevier Inc. All rights reserved.
doi:10.1016/j.jinorgbio.2012.01.002

34
P. Pietrangeli et al. / Journal of Inorganic Biochemistry 109 (2012) 33–39
E
E
E
O
O
O
+
RCH NH3
2
NH+
H2C
NH+
O
Cu(II)
Cu(II)
Cu(II)
O-
O-
O-
-
CH
R
R
NH4+
H O
H O
2
2
2
H O
2
2
O2
O2
E
E
E
HO
HO
HO
.
NH2
NH+
HC
NH2
Cu(II)
Cu(I)
Cu(II)
OH
O
OH
OH
R
C
H
R
Scheme 1. Scheme of the reaction of LCAO with amine substrate.
Cu(II)-superoxide adduct, while according to mechanism 2, the binding
of O occurs at a hydrophobic pocket adjacent to Cu(II). Then one elec-
tron is directly transferred to O
independent reactions, respectively implicated in the catalytic and
in the inactivation process.
2
2
from reduced TPQ (aminoresorcinol in-
−
termediate) to form the same Cu(II)-O
2
-semiquinone adduct as in the
previous case. Mechanism 1 was proposed for enzymes, such as plant
amine oxidases, which are able to form up to 40% of Cu(I)-semiquinone
on reduction by substrate under anaerobic conditions, and is supported
2. Experimental
2.1. Protein preparation
2
by the fast, single second order reaction with O . Mechanism 2 was pro-
posed for enzymes such as BSAO, which do not form appreciable
amounts of the semiquinone on reduction by substrate under anaerobic
conditions [17], and is supported by the catalytic activity of the Co(II)-
All chemicals were reagent grade and were used without further
purification. Substrates, catalase, aldehyde dehydrogenase (ADH), su-
peroxide dismutase (SOD), and horseradish peroxidase (HRP), were
purchased from Sigma–Aldrich S.r.l. (Milan, Italy). LCAO was purified
from Lathyrus cicera seedling by the method previously reported [24].
The purified proteins moved as single bands on SDS-PAGE. The con-
centration was measured using the molar extinction coefficient for
substituted derivative [18]. Furthermore, reduced BSAO reacts with O
2
with Michaelis–Menten mechanism, at a slower rate than the semiqui-
none forming enzymes. In conclusion, these properties were taken to
suggest that the mechanism might be different in the two types of en-
zymes [19].
CuAOs from animal and plant sources also differ because of the
specificity of the amine substrate. The natural mutation of a BSAO tyr-
osyl residue near the catalytic site, the so called “gate” residue, into a
phenylalanine residue in PSAO might be at least in part responsible
for the differences by increasing the hydrophobic nature of the site
−
1
− 1
phenylhydrazine, ε445nm =64 mM
cm , or benzylhydrazine,
−
1
− 1
ε380 nm =58 mM
cm , assuming a content of two TPQ per mole
enzyme. As previously discussed [20], the values obtained in this
way were in good agreement with those obtained by employing for
the protein the molar extinction coefficients reported for Pisum sati-
−
1
−1
vum AO [24,25], namely ε280 nm =300 mM
4.9 mM
cm
and ε500 nm =
−
1
− 1
[20]. However, it was shown that in Hansenula polymorpha amine ox-
cm . The copper content was assayed by atomic
idase a single-point mutation at a second sphere ligand to copper in-
creases the cofactor semiquinone level, on reduction by substrate
under anaerobic conditions, without altering the mechanism of O re-
2
duction [21]. The two types of enzymes also differ in the reactivity
with inhibitors which bind the TPQ carbonyl group. Plant AOs bind
two molecules of phenylhydrazine per dimer, while BSAO only
binds one molecule. Half-of-the-sites reactivity was suggested to
occur in the latter enzyme [22].
absorption spectrometry with a Perkin Elmer 3030 apparatus
equipped with a graphite furnace and by the biquinoline spectropho-
tometric method [26]. The AO activity was assayed at 25 °C with
1.0 mM putrescine in 0.1 M potassium phosphate buffer, pH 7.2, by
2 2
measuring the formation of H O from the absorbance of the pink ad-
−
1
−1
duct (ε515 nm=26 mM cm ) produced by the horseradish perox-
idase (HRP) catalyzed oxidation of aminoantipyrine, followed by
condensation with 3,5-dichloro-2-hydroxybenzensulfonic acid [27].
The present paper addresses these debated points by the study of
the reactions of LCAO with tryptamine and serotonin. Previous work
using various amine substrates had shown that this enzyme, like
2 2
The HRP system was also used for the quantitation of H O produced
at the end of inactivation experiments (see below). Samples with spe-
cific activity >70 IU/mg (micromoles of substrate oxidized/min)
were employed.
Potassium phosphate buffer (0.05 M) was used in the pH range
6.0–8.0 and 0.05 M sodium borate buffer in the pH range 8.4–9.2.
other AOs [23,24], is slowly inactivated by H
over, when left to incubate in the reduced state after O
The study of tryptamine-treated LCAO allows to follow two
2
O
2
produced on turn-
2
exhaustion.

P. Pietrangeli et al. / Journal of Inorganic Biochemistry 109 (2012) 33–39
35
The ionic strength of the solutions was kept constant at 120 mM by
proper addition of NaCl.
amines [24]. Complete inactivation of the enzyme and loss of reactiv-
ity with phenylhydrazine were eventually produced. The presence in
solution of either catalase or ADH protected in part the enzyme from
inactivation, since some residual activity and reactivity with phenyl-
hydrazine was found after a similar incubation time. After 15 min in-
cubation with tryptamine, the loss of catalytic activity was about 95%,
but did not exceed 40% in presence of either catalase or ADH. The in-
activation was completely prevented by the simultaneous presence of
2
.2. Inactivation experiments
Inactivation was achieved in two different ways i) by incubating
4
.0 μM LCAO with 2.0 mM substrate, in 0.1 M potassium phosphate
buffer pH 7.2 in a 1.0 mL test tube, open to air, using a 37 C°
thermostatted water bath. At given time intervals, aliquots of the so-
lutions were tested for activity with 1.0 m M putrescine at 25 °C, after
dilution to approximately 2.0 nM LCAO; ii) by incubating LCAO
both enzymes. This shows the involvement of both H
cetaldehyde in the inactivation process.
2 2
O and indolea-
In another series of experiments, the incubation with an excess of
tryptamine (1.0 mM) over O was carried out in a spectroscopic cu-
(
4.0–6.0 μM) with 1.0 mM substrate, at 37 °C, in an optical cuvette
provided with a Teflon stopper, thus limiting the amount of available
and allowing to monitor the UV–vis spectrum of reacting species.
The amount of H produced in the inactivation experiments was
2
vette, closed by a teflon stopper affording protection from oxygen
leaks. This allowed the observation of a spectrum with sharp absorp-
tion peaks at 464, 434, and 350 nm, typical of the Cu(I)-semiquinone
radical. Since this radical only forms under strictly anaerobic condi-
O
2
2 2
O
measured as described in the previous section by the HRP method,
after proper sample dilution.
tions [28], all O
atmospheric pressure) was consumed by turnover at this stage. An
equivalent amount of H was detected in solution with the HRP
2
initially available in solution (0.27 mM at 25 °C and
2
.3. Steady state kinetics
2 2
O
method described in Section 2.1. At difference from amine substrates
previously examined [24], an additional intense band around 630 nm
(Fig. 1) was formed by tryptamine. The maximum absorbance was
reached after 6 min (Fig. 2A, curve a), when the Cu(I)-semiquinone,
formed within mixing time, was already in the decreasing phase
(Fig. 2B, curve a). The presence of SOD had no effect on the formation
and decay of either the 630 nm band or the radical spectrum (Fig. 2A,
curve b). When LCAO was treated with tryptamine in presence of
ADH a much less intense 630 nm band was initially formed, which
then rapidly disappeared (Fig. 2A, curve c). A similar effect was pro-
duced by catalase (Fig. 2A, curve d). The seemingly slower decay of
the 630 nm band and the slower formation rate of 465 nm band in
presence of catalase (Fig. 2A, curve d and B, curve d, respectively) is
Kinetic parameters were obtained by measuring the velocity of
H
2
O
2
formation as described above for the enzymatic activity deter-
mination. K and kcat values were obtained by fitting the kinetic
data to the Michaelis–Menten equation v=Vmax [S]/(K +[S]) by
m
m
non-linear regression analysis using Microcal Origin 3.5 software.
The data were the average of three experiments, carried out at
2
5 °C, using at least eight amine concentrations. The standard devia-
values in the range pH 5.5–pH 9.1,
tion was ≤12%. kcat and kcat/K
m
and the dissociation constants of protonated groups that control the
dependence of kinetic parameters on the pH, Ka1 and Ka2 were mea-
sured as previously described [20].
2
.4. Rapid kinetics
2 2 2
imputable to the catalase reaction with H O , which releases O and
H
2
O. Catalase addition also produced the rapid bleaching of a fully de-
Rapid kinetic experiments were carried out using an Applied
veloped 630 nm band. A presumably similar band (not shown),
whose absorption is shifted to 650 nm and with slightly lower absor-
bance, was produced by 5-hydroxytryptamine (serotonin).
Photophysics MV17 stopped flow apparatus (Applied Photophysics,
Leatherhead, UK) equipped with either a monochromator and a
photomultiplier tube (for single wavelength time courses) or with a
photodiode array detector (for recording time resolved absorbance
spectra). When the photodiode array detector was used the intensity
of the 150 W lamp of the instrument was reduced to approximately
/4 by neutral filters to prevent possible photochemical damage of
the sample; this required increasing the exposure of the sensor to
ms/spectrum. The data collected by the instrument were trans-
The turnover reaction with 0.2 mM tryptamine, less than O
centration (0.27 mM), left LCAO in the oxidized state. Neither inacti-
vation nor optical effects occurred, although 0.2 mM H was
2
con-
2 2
O
detected in solution by the method described in Sections 2.1 and
2.2. A first important implication of this result is that the oxidized
protein does not form the Cu(I)-semiquinone spectrum neither the
1
5
ferred to a pentium IV based personal computer working under the
Linux operating system and analyzed using routines written for the
software package Octave.
0
0
.10
.08
2
.5. EPR measurements
EPR spectra were recorded with a Brucker ElexSys spectrometer,
0.06
operating in X band with a SHQ-type cavity. The sample was inserted
in a 3 mm I.D. Suprasil tube (Wilmad), 300 μl volume, for the mea-
surements at 150 K. EPR spectra were recorded at 1 mW microwave
0
0
0
.04
.02
.00
b
c
power, at 2 mT modulation amplitude, 0.4 s/mT sweep time to
sweep field ratio, 0.3 ms time constant. A Mn2+ standard sample,
a
d
e
fixed at the bottom of the cavity, was used for field reference. A HP
5
3
3
3150A frequency counter was used for frequency measurements.
. Results
300
400
500
600
700
800
Wavelength (nm)
.1. LCAO inactivation by tryptamine
Fig. 1. Optical spectra of tryptamine reacted LCAO in a closed spectrophotometer cu-
vette. 4.9 μM enzyme in 0.1 M K-phosphate buffer pH 7.2 plus excess tryptamine,
4
.0–6.0 μM LCAO was incubated with a large excess of tryptamine
2.0 mM) over O (0.27 mM) in a test tube open to air, as described in
a previous work carried out with several other aliphatic and aromatic
1
(
.0 mM, over O
c), 40 (d), and 90 (e) min. The arrow indicates the decrease of the 465 nm band in
the order a>b>c>d>e.
2
, recorded at 25 °C immediately after mixing (a), and after 12 (b), 20
(
2

36
P. Pietrangeli et al. / Journal of Inorganic Biochemistry 109 (2012) 33–39
0
0
0
0
0
.08
.06
.04
.02
.00
25
A
2
1
1
0
5
0
5
0
b
a
d
c
0.2
0.4
0.6
0.8
1.0
1.2
0
1
2
3
4
5
6
7
8
9
10
[
H O /indoleacetaldehyde] (mM)
2 2
Time (min)
Fig. 3. Dependence of the 630 nm band intensity on products (indoleacetaldehyde and
) concentration. 4.8 μM LCAO in 0.1 M K-phosphate buffer pH 7.2, at 25 °C, was
2 2
H O
0
0
0
0
0
0
0
0
0
.180
.175
.170
.165
.160
.155
.150
.145
.140
treated with 1.0 mM tryptamine in a closed cuvette after the addition to solutions stir-
red in air of 0, 1, 2, 3, 4 aliquots of 0.2 mM tryptamine, respectively. The points corre-
spond to 0.27, 0.47, 0.67, 0.87, 1.07 mM products.
B
a
630 nm band intensity (Fig. 4) showing that the large increase of Fig. 3
is mainly related to indoleacetaldehyde concentration. Fig. 4 also
shows that the initial intensity of the 465 nm radical band is not affected
d
2 2
by the presence of exogenous H O and that the loss of intensity with
time is faster, parallel to the 630 nm band decay. A different behavior
of the two sets of bands was also observed as a function of pH on a single
addition of 1.0 mM tryptamine. The intensity of the Cu(I)-semiquinone
spectrum changed with pH in the order pH 8.5bpH 7.2≈pH 6.5, while
the intensity of the 630 nm band was almost independent of pH
0
1
2
3
4
5
6
7
8
9
10
(Fig. 5). The latter band was instead substantially temperature depen-
Time (min)
dent. The rate of formation at 15 °C was lower than at 25 °C, but the
band reached a higher intensity and the decay time was longer. If the
Fig. 2. Time course of LCAO–tryptamine reaction in a closed spectrophotometer cu-
vette. 5.6 μM enzyme in 0.1 M K-phosphate buffer pH 7.2, at 25 °C, plus 1.0 mM trypt-
amine. Panel A: recorded at 630 nm (a), in presence of 2.3 μM SOD (b), or 60 units ADH
c), or 100 units catalase (d). Panel B: recorded at 465 nm in the absence (a) or in the
presence of catalase (d).
−1
−1
limiting absorbance of 22 mM cm is taken as the extinction coeffi-
cient of the derivative, the absorbance recorded in these experiments
accounted for about 30% and 50% of reacted molecules at 25 °C and
15 °C, respectively. An even higher ratio of 75% was measured immedi-
ately after thawing a frozen solution. The Cu(I)-TPQ semiquinone spec-
trum was unchanged in the 25–15 °C temperature range, but the
intensity was greatly reduced in the spectrum measured after thawing.
(
6
30 nm band with catalytic products. Re-oxygenation by stirring the
solution opened to air and subsequent addition of excess tryptamine
1.0 mM) produced a Cu(I)-semiquinone spectrum of the usual inten-
(
sity and a band at 630 nm, of approximately double intensity that
produced with a single 1.0 mM tryptamine treatment. The increased
intensity of the 630 nm band is apparently related to the accumula-
0.08
tion of catalytic products, H
by the value close to 0.47 mM H
amine addition. The 0.47 mM H
duced by the first tryptamine addition and 0.27 mM produced with
excess substrate, corresponding to the amount of O reintroduced
by stirring the solution in air. H concentrations measured at any
2 2
O and indoleacetaldehyde as confirmed
2
O
2
measured after the second trypt-
0.07
2 2
O value is the sum of 0.2 mM, pro-
4
65 nm
0
0
0
.06
.05
.04
2
H2O
2
2 2
O
H2O
2
stage of the tryptamine reaction were in good agreement with
expected values. The acetaldehyde concentration is assumed to be
6
30 nm
identical to that of H
products concentrations on the intensity of the 630 nm band, several
.2 mM tryptamine aliquots were added stepwise, stirring the solu-
2
2
O . In order to evaluate the effect of increasing
0.03
0
0
0
.02
.01
.00
0
tion in air. As in the case of a single 0.2 mM tryptamine addition, no
optical effects occurred. The final addition of excess tryptamine, in a
closed cuvette, required to obtain the Cu(I)-semiquinone cofactor in
the reduced state, showed a progressive increase of the intensity of the
0
1
2
3
4
5
6
7
8
9
10
−
1
−1
6
30 nm band, up to a limit value of approximately 22 mM cm
Fig. 3). The optical absorption of the Cu(I)-semiquinone was unaffected
throughout. In order to test the separate effect of each catalytic product,
the 1.0 mM tryptamine excess was used in presence of exogenous H
A high H concentration (2.0 mM) afforded a 20% increase of the
Time (min)
(
Fig. 4. Effect of exogenous H
a closed spectrophotometer cuvette. 4.8 μM enzyme in 0.1 M K-phosphate buffer pH
.2, at 25 °C, plus 1.0 mM tryptamine recorded at 465 nm (upper curves) or 630 nm
(lower curves) without and with 2.0 mM H O .
2 2
O on the time course of the LCAO–tryptamine reaction in
2 2
O .
7
2 2
O
2
2

P. Pietrangeli et al. / Journal of Inorganic Biochemistry 109 (2012) 33–39
37
0
0
0
0
0
0
.10
.08
.06
.04
.02
.00
0.10
0.08
0
0
0
0
.06
.04
.02
.00
d
c
b
a
b
a
c
4
00
500
600
700
800
d
Wavelength (nm)
300
400
500
600
700
800
Wavelength (nm)
Fig. 5. Dependence on pH of the optical spectra of tryptamine reacted LCAO in a closed
spectrophotometer cuvette.5.3 μM LCAO in 0.1 M K-phosphate buffer, at 25 °C, plus
Fig. 7. Effect of KCN on the reaction of LCAO with tryptamine in a closed spectropho-
tometer cuvette. 6.3 μM enzyme plus 1.0 mM tryptamine, at 25 °C, in Na-borate buffer
pH 8.5 (a), plus 1.0 mM KCN recorded immediately (b), after 9 min (c) and 18 min (d).
1.0 mM tryptamine, at pH 6.5 (dashed line), at pH 7.2 (dotted line), and in 0.1 M borate
buffer pH 8.5 (solid line). The spectra were recorded 2 min after tryptamine addition.
3
.2. EPR measurements
3.4. Steady state kinetics
EPR spectra were recorded at 100 K. The spectrum of 110 μM LCAO
Steady state kinetics were measured for tryptamine in the range
pH 6.5–pH 9.3. A bell shaped curve was obtained by plotting kcat
against hydrogen ion concentration. The curve showed a maximum
at pH 7.5 and apparent pKa1 =7.12± 0.03 and pKa2 =8.14± 0.02
values were calculated as reported in a previous paper [20]. Serotonin
kinetics were only measured at a single pH because of the very low
in 0.1 M phosphate buffer pH 7.2 was recorded before and after the
addition of 2.0 mM tryptamine. The intensity of the solution blue
color increased on freezing, while the EPR copper signal was almost
unaffected by the presence of tryptamine (Fig. 6). This may be taken
as an indication that the blue species is a Cu(II) derivative. It is well
known that the radical disappears at low temperature and that it
does not exceed 40% of total Cu at room temperature [28]. The EPR
spectra of Fig. 6 are very similar to those reported by Medda et al. at
low temperature [29]. This is not surprising as they were obtained be-
fore and after addition of excess tryptamine to lentil seedling amine
oxidase (LSAO) under comparable conditions.
−
1
k
cat value of 0.09 s
same conditions, was instead of 10.7 s , two orders of magnitude
higher. Serotonin and tryptamine K values, measured in 0.1 M K-
at pH 7.2. The kcat value for tryptamine, in the
−
1
m
phosphate buffer pH 7.2, were 0.095 mM and 1.12 mM, respectively.
3.5. Rapid kinetics
3
.3. Reactions in the presence of Cu binding inhibitors
Two types of rapid kinetic experiments were carried out, i.e. (i)
the characterization of the full catalytic cycle and (ii) the rapid mixing
In phosphate buffer pH 8.5, addition of 1.0 mM KCN, a reagent
2 2
of reduced LCAO with indoleacetaldehyde and H O . The steady state
known to stabilize the Cu(I) species [28], made the spectrum of the
radical substantially more intense while the 630 nm band of trypt-
process followed by stopped flow reveals three clearly distinguish-
able intermediates, namely the oxidized and reduced derivatives of
the enzyme and the putative enzyme-product complex which ab-
sorbs at 630 nm. The absorbance spectrum of the steady state mixture
cannot be described as a linear combination of the spectra of the three
species described above, indicating that at least a fourth spectroscopic
component is present, tentatively identified as the quinol-aldimine
enzyme substrate complex [30]. The fully reduced LCAO, which is de-
scribed as a mixture of the aminoresorcinol and Cu(I)-semiquinone
radical derivatives of the TPQ cofactor, is never obtained in these ex-
periments since the enzyme-product complex usually starts accumu-
lating before complete reduction of the enzyme (Fig. 8A, B). The time
courses recorded in this type of experiments can be satisfactorily de-
scribed using the reaction scheme already described for other plant
CuAOs (Scheme 1). In order to test whether or not the latter reaction
requires cycling, the following experiment was carried out: a 20 to
amine treated LCAO disappeared (Fig. 7). When NaN
the place of KCN both bands disappeared, while the addition of trypt-
amine to NaN treated LCAO only produced the reduction of the
00 nm band of the native protein.
3
was used in
3
5
a
b
2
5 μM solution of LCAO reduced with 1.0 mM putrescine was rapidly
mixed in the stopped flow apparatus with a solution of the indoleace-
taldehyde, produced using a minute (2.0 to 2.5 μM) concentration of
the same enzyme (Fig. 8C, D). Under these experimental conditions,
both solutions are strictly anaerobic and the enzyme is fully reduced;
no cycling is expected to occur. The enzyme product complex in these
2
60
280
300
320
340
360
Magnetic field (mT)
experiments formed in an exponential process, governed by a rate
Fig. 6. EPR spectra of native and tryptamine reacted LCAO at 150 K.130 μM LCAO in
.1 M potassium phosphate buffer, pH 7.2 before (a); after 2.0 mM tryptamine addition
b). Acquisition parameters: X-band, 1 mW microwave power, 1 mT modulation am-
plitude, 150 K temperature. A ElexSys spectrometer operating in X-band at 150 K
with a SHQ-type cavity was used.
−1
constant of 0.015 s
2 2
irrespective of the aldehyde or the H O con-
0
(
centrations. This result indicates unequivocally that the enzyme de-
rivative capable of reacting with the aldehyde of tryptamine is
reduced and that cycling is not required. We were puzzled by the

38
P. Pietrangeli et al. / Journal of Inorganic Biochemistry 109 (2012) 33–39
Fig. 8. Time courses of the appearance and decay of the inhibited intermediates.Experimental conditions: 0.1 M K-phosphate buffer pH 7.2, 25 °C.Panels A and B: oxidized LCAO
33 μM) was mixed with 1 mM tryptamine, 270 μM O (all concentrations after mixing). Only 50 of the original 500 spectra are shown in Panel A.Panels C and D: 21 μM LCAO re-
duced with 1.0 mM putrescine was mixed with 120 μM indoleacetaldehyde (produced by 2.1 μM LCAO in the presence of 120 μM tryptamine, 150 μM putrescine, 270 μM O and
.0 mM H ). The concentrations of the relevant reagents after mixing were: 11.6 μM LCAO, 60 μM indoleacetaldehyde, 770 μM H
(
2
2
1
2
O
2
2 2
O .
reaction rate constant being insensitive to the reagents' concentra-
tions. This probably indicates that a semi-stable reaction intermediate
is formed and that its monomolecular decay to the inactive enzyme-
product complex is associated to the spectroscopic change.
the 630 nm transition. This was only produced by tryptamine and se-
rotonin and was dependent on both indoleacetaldehyde and H
2 2
O
concentration. It was probably not observed by Medda et al. with
LSAO [29] because the products concentration, stoichiometric with
the protein, was too low in their experiments. The 630 nm band
was formed at slower rate than the radical band and was suppressed
by ADH or by catalase, without effect on the radical spectrum (Fig. 2).
This implies the absence of a dynamic equilibrium between the spe-
cies responsible for the two sets of bands. This is supported by the
pH-dependence of the radical spectrum at difference from the
630 nm band, which is pH insensitive (Fig. 5). An increase of the rad-
ical species was only observed in presence of KCN, which stabilizes
Cu(I) and bleaches the 630 nm band (Fig. 7). On the contrary, freezing
the solution stabilizes the Cu(II) state, as shown by the EPR spectrum
(Fig. 6) in the presence of tryptamine and all other substrates [28]. By
considering that the amount of Cu(I)-semiquinone was reported to
never exceeded 40% of the copper sites [28] and that it was not in
equilibrium with the 630 nm absorbing species, it seems possible to
suggest that each of the two species involves a single protein subunit.
This conclusion might apply to all substrates, since the intensity of the
radical vis–UV spectrum is independent of the substrate nature [24].
Medda et al. [29] proposed the formation of an irreversible enamine
derivative between reduced LSAO and indoleacetaldehyde because the
two aldehyde molecules, formed by the reductive half-reaction under
anaerobic conditions, were progressively lost upon incubation, in paral-
lel with protein inactivation. In the present case an additional effect is
4
. Discussion
The oxidation of tryptamine by pea seedling amine oxidase to 3-
indoleacetaldehyde and the subsequent enzyme inactivation by H O
2 2
was reported by Clarke and Mann [31]. The inactivation of LSAO by stoi-
chiometric tryptamine was reported by Medda et al. [29]. The latter re-
action was carried out under anaerobic conditions and assigned to the
slow formation of an irreversible covalent derivative of the reduced pro-
tein with the two initially formed moles indoleacetaldehyde per mole of
enzyme. The full inactivation of the enzyme and the loss of reactivity
with phenylhydrazine required more than 4 h. In the present work,
LCAO was rapidly reduced by an excess tryptamine over O in a closed
2
spectrophotometer cuvette, as shown by the appearance of the typical
Cu(I)-semiquinone spectrum with peaks at 464 nm, 434 mm, and
3
60 nm. A high concentration of indoleacetaldehyde, equivalent to
that of O consumed during turnover (0.27 mM) was produced togeth-
er with 0.27 mM H . Subsequently, substrate reduced LCAO was inac-
tivated by indoleacetaldehyde, more rapidly than in the conditions used
by Medda et al. [29]. H also contributed to the inactivation since both
ADH and catalase had a protective action. The inactivation of reduced
LCAO by H was reported in a previous work carried out with other
2
2 2
O
2 2
O
2 2
O
primary amines [24].
induced by H
signment to a back-reaction of the reduced protein which binds both
indoleacetaldehyde and H . It is doubtful whether the binding site
of indoleacetaldehyde is the aminoresorcinol or some other residue of
the active site, e.g. Lys [32]. The bleaching effect of copper binding inhib-
2 2
O of producing the 630 nm band and supporting the as-
Another important new result induced by the high products con-
centration was the formation of an intense band at 630 nm (Fig. 1).
A similar band was produced by serotonin at 650 nm. The intensity
of the Cu(I)-semiquinone spectrum was similar to that produced by
all other examined substrates [24], independent of the presence of
2 2
O
3
itors such as NaN on the 630 nm band together with the reported high

P. Pietrangeli et al. / Journal of Inorganic Biochemistry 109 (2012) 33–39
39
affinity of reduced TPQ-Cu(II) for H
transfer complex involving copper, H
possibly formed by the reduced cofactor. The implication of the indole
group is supported by the longer maximum wavelength, 650 nm, of
the 5-hydroxytryptamine (serotonin) derivative. It may be noted that
low energy transitions imputable to the indole group (420 and
2
O
2
[33] suggest that a charge-
, and indoleacetaldehyde is
CuAO
BSAO
HRP
LCAO
LSAO
LTQ
copper amine oxidase
bovine serum amine oxidase
horseradish peroxidase
Lathyrus cicera amine oxidase
lentil seedling amine oxidase
lysyl trihydroxyphenylalanine quinone
superoxide dismutase
2
O
2
6
50 nm) are found in the TTQ spectrum [34]. With substrates lacking
SOD
the indole group the back reaction with aldehyde and H
duce colorless adducts.
2
O
2
may pro-
TPQ
TTQ
VAP-1
2,4,5-trihydroxyphenylalanine quinone
tryptophyl tryptophanquinone
vascular adhesion protein-1
The above results were confirmed by stopped flow experiments.
They were aimed to determine two points in particular, namely (i)
the mechanism and rate of the reaction and (ii) which enzyme deriv-
ative was responsible for the formation of the enzyme-product com-
plex. As far as the first point is concerned, we can confidently state
that it involves at least one spectroscopically silent intermediate
since the reaction is first order at all reagents' concentrations ex-
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ADH
AO
aldehyde dehydrogenase
amine oxidase