From the Department of Biology, University of Padova,
Viale G. Colombo 3, 35121, Padova, Italy, the
§ Department of Molecular Physics, Huygens Laboratory,
Leiden University, P. O. Box 9504, 2300 RA Leiden, The
Netherlands, and the ¶ Leiden Institute of Chemistry, Gorlaeus
Laboratories, Leiden University, P. O. Box 9502, 2300 RA Leiden, The Netherlands
Received for publication, September 27, 2002, and in revised form, November 22, 2002
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ABSTRACT |
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The dinuclear copper enzyme tyrosinase (Ty) from
genetically engineered Streptomyces antibioticus has been
investigated in its paramagnetic half-met form [Cu(I)-Cu(II)]. The
cw EPR, pulsed EPR, and hyperfine
sublevel correlation spectroscopy
(HYSCORE) experiments on the half-met-Ty and on its complexes with
three different types of competitive inhibitor are reported. The first type includes p-nitrophenol, a very poor substrate for the
monooxygenase activity of Ty. The second type comprises
hydroxyquinones, such as kojic acid and L-mimosine,
and the third type of inhibitor is represented by toluic acid. The
electronic and structural differences of the half-met-Ty form induced
at the cupric site by the different inhibitors have been determined.
Probes of structural effects are the hyperfine coupling constants of
the non coordinating N The reaction mechanism of the binuclear copper enzyme tyrosinase
(Ty)1 (EC 1.14.18.1) is far
from completely understood. Like catechol oxidase and the
respiratory pigment hemocyanin (Hc), tyrosinase contains a so-called
"type-3 copper" active site (1). Despite the wide
distribution of Ty in nature, from bacteria to man, no crystal
structure of the enzyme is available to date. It is current practice to
use the crystal structures of other members of the type-3 copper
protein family as structural models for Ty. These are the deoxygenated
form of Panulirus interruptus Hc (2, 3), the deoxy and oxy
forms of subunit II from Limulus polyphemus Hc (4-6), the
oxy form of the odg domain of Octopus dofleini Hc (7) and,
recently, the deoxy, met, and inhibitor-bound forms of the
Ipomoea batatas catechol oxidase (8). The common structural theme of the type-3 active site in all crystallized proteins is the
presence of six conserved histidine ligands that, three by three,
co-ordinate the two copper ions through their N The proteins can be distinguished according to their activity. Hcs
reversibly bind oxygen under physiological conditions. They exhibit no
enzymatic activity except for a very small catecholase activity for Hcs
deriving from one of the two phyla in which Hcs have been detected,
i.e. the molluscs (10) (the other one being the arthropods).
Catechol oxidases oxidize catechols to o-quinones, but only
the Tys can catalyze the monooxygenation of monophenols to
o-diphenols (catechols) as well as the oxidation of
diphenols to o-quinones with molecular oxygen as a
co-substrate. Direct structural information on Tys would be helpful for
proposing a model for the molecular mechanism of its complex enzymatic
activity, but the structural investigation of Tys is hindered by two
issues, the very low level of expression of these enzymes in natural
systems and their limited accessibility to common spectroscopic
techniques. In the present study the first issue was solved by the use
of an overexpression system for bacterial Ty from S. antibioticus, which allowed the production of milligram amounts of
Ty. The second issue was addressed by the generation of a paramagnetic
active site through partial reduction of the met form. The partial
reduction of the EPR silent met-Ty to the half-met [Cu(I)-Cu(II)]
form has been previously described for Neurospora
crassa Ty (11), and on the basis of cw EPR spectroscopy of this
derivative and its inhibitor-bound complexes, a reaction mechanism was
proposed whereby the hydroxy group of the incoming phenol binds at an
axial coordination position of one copper of
[Cu(II)-O To shed more light on these questions, we have performed cw EPR, pulsed
EPR, and hyperfine sublevel
correlation spectroscopy (HYSCORE) experiments
on half-met-tyrosinase from S. antibioticus and on its
complexes with three types of competitive inhibitor. The first type is
represented by p-nitrophenol, a very poor substrate for the
monooxygenase activity of Ty. On the time scale of the sample
preparation it forms a stable complex with half-met-Ty. The second type
of inhibitor is formed by hydroxyquinones such as kojic acid and
L-mimosine. These molecules can be considered intermediate
state analogues since they exhibit a structural analogy to the quinone
reaction products as well as to the diphenolic substrates while
occurring in an oxidation state that is not suitable for reaction. The
third type of inhibitor is represented by toluic acid. This is a
competitive inhibitor and a substrate analogue in which the carboxylate
group, which is conjugated to the aromatic ring, is expected to bind to
the cupric center of half-met-Ty. The aim of the present work is to
characterize the half-met-Ty form and to explore the electronic and
structural differences brought about by binding of the inhibitors.
Probes for such structural effects are the hyperfine coupling constants
from the non-coordinating histidyl nitrogens of half-met-Ty. These have
been obtained from simulations of the electron spin echo envelope
modulation (ESEEM) spectra and HYSCORE spectra of half-met-Ty (15).
The host for the expression of tyrosinase, S. antibioticus (LMD 86.18), was obtained from the collection of the
Kluyver Laboratory of the Delft Technical University, The Netherlands.
The plasmid that contains the tyrosinase operon (melC1 and
melC2) and the thiostrepton resistance marker was kindly
provided by Prof. E. Katz (16). Tyrosinase was purified following the
procedure described previously (17). The purified protein was obtained
as a mixture of [Cu(II)-OH The selective reduction of one of the two coppers in the active site
was carried out by mixing 1 ml of 0.5 mM met-Ty in 20 mM phosphate buffer at pH 6.8 with NaNO2 and
ascorbic acid buffered solutions to a final concentration of 40 mM and 4 mM, respectively (modified from Refs.
11 and 12). The formation of the enzymatically inactive half-met-Ty was
followed by monitoring the specific activity of the protein solution
with respect to the conversion of DOPA (as described in Ref. 20). When
no further decrease of activity was detected, the protein solution,
which has a pale green color, was purified with a desalting column
(Sephadex G-25), which was previously equilibrated with 20 mM phosphate buffer at pH 6.8. The residual activity was
less than 4% of the starting activity indicating less than 4% of the
Ty being present in the red, oxy, and met form. The latter forms of Ty
are EPR-inactive, anyway. The half-met-Ty solution was then
concentrated in an Amicon® ultrafiltration system to a final
concentration of 0.7 mM.
The complexes of half-met-Ty with inhibitor were obtained by addition
of small aliquots of buffered inhibitor solution at a concentration of
20 mM to achieve a final concentration of 2 mM.
After addition of the inhibitor, the samples were frozen immediately by
immersion in liquid nitrogen.
The 95-GHz (W-band) ESE-detected EPR experiment for a frozen solution
of half-met-tyrosinase (T = 1.2 K) was performed
on a home-built spectrometer described in Ref. 21 except for the microwave bridge, which was replaced by a bridge from the Department of
Microwave Equipments for Millimeter Waveband ESR Spectroscopy in
Donetsk, Ukraine. Microwave pulses of 100 and 150 ns were used, and the
time between the microwave pulses was 300 ns. The repetition rate of
the pulse sequence was 66 Hz. The 9-GHz (X-band) continuous wave EPR
experiments were performed on a Bruker ESP380E EPR spectrometer. The
microwave frequency was 9.40 GHz for half-met-Ty, half-met-Ty with
L-mimosine, half-met-Ty with toluic acid, and half-met-Ty with kojic acid and 8.95 GHz for half-met-Ty with
p-nitrophenol. The modulation field was 0.5 millitesla, and
the modulation frequency 100 kHz. To verify that no copper was removed
from the active site of the protein by the excess of inhibitors, the cw
EPR spectra of a 2-mM CuSO4 solution plus
inhibitor at the same concentration as the Ty sample were collected.
These bear no resemblance to the spectra presented here for the Ty
inhibitor complexes.
The ESEEM experiments were performed with a 3-pulse sequence in which
the microwave pulses had a length of 16 ns. The microwave frequency was
9.74 GHz for half-met-Ty, 9.75 GHz for half-met-Ty with
L-mimosine, and 9.73 GHz for half-met-Ty with
p-nitrophenol. The time T between the second and
third microwave pulse was scanned, starting at 56 ns and incremented
with steps of 8 ns. In total, 2048 data points were acquired. Two
series of modulation patterns were recorded. In the first series, in
which the time The HYSCORE experiments were performed with a
Simulations of the cw EPR spectra have been carried out with the
software EPR (22). According to the procedure outlined and discussed in
Ref. 23, the ESEEM spectra were simulated as follows. The expectation
value of the electron spin operator histidyl nitrogens. By using the available
crystal structures of hemocyanin as a template in combination with the
spectroscopic results, a structural model for the active site of
half-met-Ty is obtained and a model for the binding modes of both mono-
and diphenols could be proposed.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
nitrogens. Despite
the lack of a three-dimensional structure this structural theme could
also be identified in Streptomyces antibioticus Ty in the
oxidized [Cu(II)-Cu(II)] state by paramagnetic 1H NMR
(9). In that study, the protein was found to exhibit a well resolved
paramagnetic spectrum despite its size (30 kDa), and the protein
ligands to the binuclear copper site were identified as six N
nitrogens. The other common structural feature of type-3 sites is the
binding mode of molecular oxygen to the deoxy form [Cu(I)-Cu(I)],
which in all cases results in a
µ-
2:
2 peroxide-bound dicupric
site. The spectroscopic properties typical for the binuclear cupric
µ-
2:
2 peroxide complex have been
described for the oxygenated forms of all proteins of this family
(1).
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-Cu(II)] met-Ty (~90%),
[Cu(II)-O
20 °C. The protein
concentrations of the solutions used in the EPR experiments were
determined by measurement of the optical absorption at 280 nm using a
molar extinction coefficient of 82.1 mM
1
cm
1 (19).
between the first and second microwave pulse was
fixed at 120 ns for half-met-Ty and 144 ns for half-met-Ty with
p-nitrophenol or L-mimosine, the modulation
patterns were measured at different magnetic field settings. In the
second series the modulation patterns were measured at different
values and the magnetic field was fixed to the position of maximum
absorption in the ESE-detected EPR spectrum (335 millitesla for
half-met-tyrosinase, 332.5 millitesla for half-met-tyrosinase with
p-nitrophenol, and 337.5 millitesla half-met-tyrosinase with
L-mimosine). The background of the modulation patterns was removed by fitting and subsequently subtracting a Gaussian function. No
window function was used. The data were zero-filled to 4096 data points
and subsequently Fourier-transformed to obtain magnitude ESEEM spectra.
/2-
-
/2-T2-
-T1-
/2 microwave pulse
sequence with
/2 pulses of 16 ns and a
pulse of 24 ns. In a
HYSCORE spectrum, cross-peaks from nitrogens are expected at
(±
Q, ±
DQ) and
(±
DQ, ±
Q), where the first coordinate represents the frequency related to T1 and the second
coordinate the frequency related to T2. The symbol
Q corresponds to one of the three quadrupole frequencies
of the cancelled MS manifold and the symbol
DQ to the so-called
MI = 2 or
double-quantum transition of the non-cancelled MS manifold.
For half-met-tyrosinase the magnetic field setting was 335 millitesla,
for half-met-Ty with nitrophenol it was 332.5 millitesla, and for
half-met-Ty with L-mimosine it was 337.5 millitesla. The
microwave frequency was the same as for the ESEEM experiments. The time
between the first and second microwave pulse was fixed at 192 ns.
In total 512 × 256 data points were acquired in the
T1 and T2 direction, respectively. Starting
times of T1 and T2 were 168 and 136 ns, respectively, and the time increments were 16 ns for both. The background of the resulting two-dimensional modulation patterns was
subtracted by a quadratic fit first to the slices at fixed T2 and then to the slices at fixed T1.
Apodization was performed with a Hamming window function. The data were
zero-filled to 1024 × 512 points and transformed into the
frequency domain by a two-dimensional Fourier transformation. The
spectra are represented as magnitude contour spectra.
where
(Eq. 1)
e is the Bohr magneton,
B0 the magnetic field,
In this equation gN is the g value of nitrogen,
(Eq. 2)
n the nuclear magneton,
and the
values of the electron spin using the appropriate
product formula (23). Finally, to simulate a spectrum of a frozen
solution, about 3100 directions of
B0 are considered. The
decay of the echo and the modulations and the dead time of the
spectrometer were taken into account as described in Ref. 23 with time
constants of 400 µs, 4 µs, and 298 ns, respectively.
The principal values of the g tensor are called gxx,
gyy, and gzz, and the principal axes are called
x, y, and z. For each nitrogen, the
quadrupole tensor is specified by principal values Qx'x', Qy'y', and
Qz'z' and principal axes x',
y', and z', and the hyperfine tensor is specified
by principal values Ax"x",
Ay"y", and Az"z" and
principal axes x", y", and z". The
principal values of the quadrupole tensor are related to the parameters e2qQ and that describe the electric field gradient at
the nitrogen according to Qx'x' = e2qQ(
1)/4, Qy'y' =
e2qQ(
+ 1)/4 and Qz'z' = e2qQ/2.
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RESULTS |
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cw and ESE-detected EPR--
The continuous wave EPR spectra at 9 GHz of frozen solutions of S. antibioticus
half-met-tyrosinase and half-met-Ty with the inhibitors
p-nitrophenol, L-mimosine, kojic acid, and
toluic acid are presented in Fig. 1. To
obtain accurate g values for half-met-Ty, an ESE-detected EPR spectrum
was recorded at 95 GHz (Fig. 2), which
provided for superior g value resolution. Simulations of the cw EPR
spectra are included in Fig. 1, and the g values (gxx, gyy, gzz) and copper hyperfine values
(Axx, Ayy, Azz) of the simulations are given in Table I.
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The spectra are characterized by large gzz and
Azz values, consistent with a singly occupied molecular
orbital in the ground state of
dx2y2 or dxy character
on copper (26). For half-met-Ty, the g values obtained from independent
simulation of the spectra at 9 and 95 GHz are consistent with those
reported previously for the half-met derivative of N. crassa
Ty (11, 12). The gzz value amounts to 2.298 and the
rhombicity ( gyy
gxx ) to 0.018. The
spectrum of half-met-Ty with p-nitrophenol is similar to
that of half-met-Ty. Only around g = 2.1 do the spectra differ.
When L-mimosine binds, the cw EPR spectrum is characterized
by larger gzz and Azz values. Superhyperfine
structure becomes visible in the gxx/gyy region and on the copper hyperfine band at g = 2.5. For half-met-Ty with kojic acid, the gzz and Azz values become even
larger. The complex of half-met-Ty with toluic acid is characterized by
the largest gzz and smallest Azz value. It is
clear that binding of the inhibitors investigated in this study results
in significant changes in EPR parameters. As we are dealing here with
competitive inhibitors they must bind at or close to the copper site.
However, the fact that nitrophenol can also act as a substrate, albeit
a very poor one (45), is diagnostic of binding to the copper. The only
inhibitor/enzyme complex of a type-3 copper protein of which a
structure has been reported in the literature is catechol oxidase (8),
where the thiourea inhibitor binds directly to the copper site. Other
(indirect) structural evidence on the binding mode of inhibitors
relates to the release of a water molecule or an OH
moiety from the first coordination shell of the paramagnetic copper in
half-met-Ty upon inhibitor binding (23), making it likely that the
leaving moiety is replaced by an oxygen of the inhibitor. For the
met-form of Ty there has also been found direct evidence for
coordination to the copper since the contact-shifted NMR signals of the
inhibitors have been observed in the 600-MHz NMR spectra of the
inhibitor/Ty complexes.2
Taking all this evidence into account it appears that the simplest way
to interpret the present data is to assume that the inhibitors directly
bind to the copper.
ESEEM--
In Fig. 3, three-pulse
ESEEM spectra for frozen solutions of half-met-Ty, half-met-Ty with
p-nitrophenol, and half-met-Ty with L-mimosine
are presented, recorded at the magnetic field of maximum absorption in
the ESE-detected EPR spectrum. The ESEEM spectrum of half-met-Ty (Fig.
3a) is characterized by three bands at about 0.5, 1.0, and
1.5 MHz and a broad and weak band around 4 MHz, typical for a remote
(amino) nitrogen of a histidine that ligates to copper (27). An
additional band is present at 1.7 MHz. For half-met-Ty with
p-nitrophenol (Fig. 3b), the three bands at 0.5, 1.0, and 1.5 MHz become more pronounced compared with those for
half-met-Ty. One band at 1.6 MHz dominates the ESEEM spectrum for
half-met-Ty with L-mimosine (see Fig. 3c).
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For each of these samples a series of ESEEM spectra were collected for
different magnetic field settings and different values of the time between the first and second microwave pulses. For half-met-Ty a number
of these are shown in Fig. 4. The
frequencies of the bands below 2 MHz are almost independent of the
value of the magnetic field, but their relative intensities vary. When the value of the time
is changed, the relative intensities are affected more. For example, at
= 120 ns or 144 ns, the band at
1.5 MHz dominates, and at
= 208 ns, this band is weaker than the one at 0.5 MHz. The intensities of the broad bands around 4 MHz
also vary.
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HYSCORE--
Fig. 5a
shows the HYSCORE spectrum of half-met-Ty, recorded at the field of
maximum absorption in the ESE-detected EPR spectrum. On the diagonal,
bands are visible at the same frequency as the bands in the
corresponding ESEEM spectrum. For example, the three sharp bands appear
at (1,
2) = (0.5, 0.5), (1.0, 1.0), and (1.5, 1.5) MHz. Two strong cross-peaks, labeled I and II are
visible at (1.7, 3.7) and (3.7, 1.7) MHz. Weak cross-peaks III-VI
correlate 0.5, 1.0, and 1.5 MHz to about 4.8 MHz. The frequencies
correlated by the cross-peaks are summarized in Table
II. A more resolved pattern is observed
in the HYSCORE spectrum of half-met-Ty with p-nitrophenol
(Fig. 5b). Similar to the case of half-met-Ty, the bands on
the diagonal correspond to the bands in the ESEEM spectrum. The
frequencies of the sharp bands (0.5, 1.0, 1.5 MHz) are connected to
about 4.8 MHz by the weak cross-peaks XI-XIII. The peak marked by XVI
occurs at the same position as the one marked by I in the spectrum for
half-met-Ty, but its intensity is smaller. The HYSCORE spectrum for
half-met-Ty with L-mimosine is shown in Fig. 5c. The spectrum is dominated by cross-peak XVII at (1.6, 4.2) MHz. A
cluster of six cross-peaks connects the frequencies 0.4, 1.2, and 1.6 MHz to 3.8 and 4.2 MHz. The frequencies 0.4 and 1.2 MHz are not
resolved in the ESEEM spectrum.
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DISCUSSION |
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cw EPR-- The controlled oxidation of only one copper in the binuclear type-3 active site provides a sensitive paramagnetic probe for the investigation of the interaction between the metal ion and substrates or transition state analogue inhibitors. In this study S. antibioticus tyrosinase has been first purified from an overexpression system and then converted to the half-met form following the protocol described for N. crassa Ty (11, 12). The cw EPR spectra of the half-met derivative of bacterial tyrosinase are similar to those of the other half-met derivatives of type-3 sites (cf. Table I) suggesting that at least for the half-met derivative a substantial structural similarity exists within this "type" of active sites.
When competitive inhibitors are added to a solution of bacterial
half-met-Ty, the gzz value and copper hyperfine component Azz are affected. As already mentioned in the Introduction,
the inhibitors used can be divided into three types: monophenols
(p-nitrophenol), hydroxyquinones (L-mimosine and
kojic acid), and carboxylated substrates (toluic acid). When the
paramagnetic properties are analyzed in terms of coordination number of
the cupric copper, all complexes have large gzz and
Azz values (cf. Table I). In Fig.
6, a Vänngård-Peisach-Blumberg
plot (28) is shown in which in addition to the properties of the
various half-met-Ty complexes also a number of four- and
five-coordinate Cu(II) model complexes and copper proteins are
included. Two different linear correlations between gzz and
Azz values can be identified in the plot (Fig. 6B). It is known that coordination shells with the same
number of ligands but of different composition may give rise to shifts in A and g values and so care must be exercised in drawing conclusions from the location of new points in such a diagram. We notice, however,
that the points on the four-coordinate correlation line all derive from
complexes with aromatic-like ligands and as such appear to lie on a
single line. Half-met-Ty and half-met-Ty with p-nitrophenol
are close to the line that corresponds to the four coordinate copper
centers (only 3N1O complexes have been included in the plot), while
half-met-Ty with L-mimosine or kojic acid is closer to the
line for the five-coordinated model complexes (3N2O). Still close to
the linear regression of five-coordinated model complexes but in a
different region of the plot are the values referring to the toluic
acid complex. It is therefore tentatively concluded that the cupric
site of half-met-Ty is four-coordinated. It remains four-coordinated
when a monophenol binds and becomes five-coordinated when a
hydroxyquinone or toluic acid binds.
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ESEEM and HYSCORE--
More information about the coupling of the
histidines to copper is obtained from the HYSCORE spectra and
simulations of the ESEEM spectra. At X-band, the hyperfine interaction
and the nuclear Zeeman interaction of the remote nitrogens of the
histidines are about equal in magnitude. In one of the MS
manifolds, these two interactions cancel and the frequency of the three
nuclear transitions is determined by the quadrupole interaction (29).
Three relatively sharp bands are expected from this manifold, usually
between 0 and 2 MHz. The other MS manifold gives rise to
one band around 4 MHz that corresponds to the MI = 2 transition. To retrieve information about the coupling of the
histidines to copper, we use the cross-peaks in the HYSCORE spectra to
identify the four frequencies that stem from one nitrogen. Once the
frequencies have been identified, simulation of a large number of ESEEM
spectra yields quantitative information about the hyperfine and
quadrupole interaction for each nitrogen.
In a HYSCORE spectrum, cross-peaks are expected at (±Q,
±
DQ) and (±
DQ, ±
Q),
where
DQ is the
MI = 2 transition and
Q one of the three bands below 2 MHz. The cross-peaks
III-VI in the HYSCORE spectrum of half-met-Ty (Fig. 5a)
correlate the frequencies 0.5, 1.0, and 1.5 MHz to about 4.8 MHz
(cf. Table II). These frequencies therefore stem from one
remote nitrogen of a histidine. Cross-peaks I, II, and VII correlate
1.6 MHz to about 3.8 MHz and indicate the contribution of a nitrogen
from a second histidine. Given the relatively large intensity of
cross-peaks I and II, both 1.6 and 3.8 MHz correspond to fundamental
frequencies of the second nitrogen. Sum and difference frequencies are
not observed. When p-nitrophenol binds (Fig. 5b),
the frequencies of the bands of the first nitrogen, correlated by
cross-peaks VIII-XIII, are virtually unchanged, as was the case for
the ESEEM spectra. The contribution of the second nitrogen has become
considerably less pronounced, as indicated by the relatively low
intensity of peak XVI compared with I. Cross-peaks XIV, XV and XVI are
tentatively assigned to the second nitrogen, whose frequencies thereby
become 0.5, 1.0, 1.6, and 3.7 MHz. For half-met-Ty with
L-mimosine (Fig. 5c), more drastic changes occur
as regards the intensity distribution. Cross-peaks XVII-XXIV correlate
0.5, 1.1, and 1.6 MHz to
MI = 2 frequencies at 3.8 and
4.2 MHz. It is unclear whether the frequencies 3.8 and 4.2 MHz belong
to one nitrogen.
The simulations of the ESEEM spectra of half-met-Ty and its inhibitor
complexes are included in Figs. 3 and 4 underneath the experimental
spectra. Based on the HYSCORE data, the simulations for half-met-Ty
include two nitrogens. For half-met-Ty with p-nitrophenol, only the dominant nitrogen is considered in the simulation as the
contribution of the second nitrogen is minor. For half-met-Ty with
L-mimosine no direct evidence for a second nitrogen is
present in the HYSCORE spectrum and one nitrogen is included in the
simulation. In the ESEEM simulations, the hyperfine and quadrupole
tensors for each nitrogen enter as parameters. In
principle, this would mean 11 free parameters for each nitrogen, six
for the hyperfine tensor and five for the (traceless) quadrupole
tensor. Fortunately, this number can be limited considerably. From
nuclear quadrupole resonance studies (32), it is known that the
principal axes of the quadrupole tensor of the remote histidine
nitrogens are (within 10 °) directed as follows: one axis along the
N-H bond (x'), the second perpendicular to the
imidazole plane (z'), and the third perpendicular to the
first two (y'). The two parameters e2qQ and that specify the principal values of the quadrupole tensor are read
from the frequencies of the three quadrupole transitions observed in
the ESEEM spectra (initial values of 1.5 and 0.6 MHz, respectively).
The directions of the principal axes of the hyperfine tensor are based
on those found for the remote histidine nitrogens in the blue-copper
site of P. aeruginosa azurin (30), one axis along the
Cu-N
direction (x"), one perpendicular to the imidazole plane and subsequently orthogonalised to the first (z"), and
the third orthogonal to the first two (y"). To perform the
simulations structural input is needed for the active site. Because at
present no crystallographic data exists for tyrosinase, we used the
data published for subunit II from L. polyphemus hemocyanin
(PDB ID 1LL1) (4, 5), the amino acid sequence and the spectroscopic properties of which are similar to those of Ty. Initial guesses for the
principal values of the hyperfine tensors are taken from an ESEEM study
of green half-met hemocyanin (31). The simulations are performed using
the principal axes system of the g tensor as a reference system.
Because of the dx2
y2 nature of the
singly occupied molecular orbital and the near-axial g tensor we chose
the z-axis of the g tensor perpendicular to the plane
spanned by the bonds connecting the copper with N
s of histidines 324 and 364 (amino acid numbering from L. polyphemus sequence).3
The simulations performed with the initial parameter set already
qualitatively reproduce the bands in the spectrum and their relative
intensity, provided that the remote nitrogen of histidine 364 is
assigned the largest hyperfine coupling from Ref. 31. The positions of
the bands, their relative intensities, and the lineshapes then were
optimized by slight variations of the principal parameters of the
tensors, i.e. Ax"x",
Ay"y", Az"z",
e2qQ and . For half-met-Ty with L-mimosine,
the x" and y" directions of the hyperfine tensor
had to be set parallel to the x' and y' directions to reduce the intensities of the bands at 0.5 and 1.0 MHz.
For all three complexes, the frequencies of the bands are nicely
reproduced for most values of the magnetic field and of the time .
The dominant nitrogen gives rise to bands at 0.5, 1.0, and 1.5 MHz and
a
MI = 2 feature at about 4 MHz. For half-met-Ty with
p-nitrophenol, the intensity of these bands is largest, and for half-met-Ty with L-mimosine, only the band at 1.5 MHz
has significant intensity. The relative intensities of the bands also generally agree with those in the experimental spectra. The simulations can in principle be improved by further optimizing the relative orientations of the hyperfine and the quadrupole tensor, but we chose
not to do so because it would lead to too many free parameters.
The optimized hyperfine and quadrupole tensors are given in
Table III for the three
complexes. Note that as a consequence of the nearly axial g tensor, the
x and y components of the hyperfine and
quadrupole principal axes are arbitrary and they are determined within
a rotation around the z-axis. These components are included nonetheless in the table (where we chose the x and
y directions about parallel to the Cu-N directions of
histidines 324 and 364, respectively) to specify the relative
orientation of the principal directions of the quadrupole and hyperfine
tensors. For half-met-Ty, the N
of histidine 364 dominates the ESEEM
spectrum and is characterized by the largest hyperfine coupling
(Aiso = 1.80 MHz). The second nitrogen, that of histidine
324 (Aiso = 1.35 MHz), makes a minor contribution to the
spectrum. When p-nitrophenol binds, the hyperfine coupling
of the N
of histidine 364 increases and dominates the ESEEM spectrum
to such an extent that the contribution of the N
of histidine 324 to
the ESEEM spectrum becomes undetectable. When L-mimosine
binds, the change in the ESEEM pattern is compatible with a decrease in
the hyperfine coupling of the nitrogen of histidine 364 (Aiso = 1.50 MHz). For all cases, the values of
e2qQ and
are virtually identical to those read from the
spectra.
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Structural Implication for the Binding of Inhibitors--
The
structural model for the half-met derivative of S. antibioticus Ty that emerges from the analysis of the
cw EPR data implies a four-coordinated cupric site, where three of the
ligands are likely to be the conserved histidines. The absence of well
resolved superhyperfine features from the directly coordinated
nitrogens, however, does not allow us to directly "count" the
copper bound nitrogens in the cw EPR spectrum. The contributions of at
least two distinct remote histidyl nitrogens have been identified in the nitrogen region of the HYSCORE spectra and accordingly the contribution of two non-equivalent nitrogens has been used to obtain
the best simulation to the field and -dependent ESEEM data. The failure to detect the contribution of the third histidine residue in the pulsed EPR experiment is probably related to the magnitude of the hyperfine interaction of its remote nitrogen that may
be too far from the so-called "exact cancellation condition." Support for the presence of three ligand histidine residues is provided
by the highly conserved sequences in the active site region of all
proteins with a type-3 site that always show a pattern of six conserved
metal ligand histidines. This pattern is present also in S. antibioticus Ty. More importantly, experimental evidence of
coordination by three His residues for each copper ion of S. antibioticus Ty comes from a paramagnetic NMR study (9), where the
signals from a total of six N
-coordinated histidines were assigned
in the NMR spectra of the met [Cu(II)-OH
-Cu(II)]
resting form. The N
coordination for the ligand histidine fits with
the parameters obtained for the hyperfine interaction of the remote
nitrogen from the simulation of the ESEEM data (32).
The nature of the fourth ligand previously has been unambiguously
proven by hyperfine sublevel correlation (HYSCORE) spectroscopy to be
an oxygen of a water or hydroxyl group (15), which provided a means for
the evaluation of the hyperfine interaction. Its magnitude appeared to
be diagnostic of an equatorial coordination of this exogenous ligand
(15). The data presented in this study do not provide an indication on
whether or not the second (cuprous) ion is involved in the coordination
of this water molecule. What is stated above can be summarized in a
structural model (Fig. 7A) where the paramagnetic center is on the CuB and the latter
is tetra coordinated. The choice of CuB and not
CuA as the paramagnetic center in the half-met-Ty is based
on the observation that only this assignment gave consistent results in
the simulation of the quadrupole data for the histidine N atoms. The
coordination geometry of the metal ion, however, can only tentatively
be considered as tetragonally distorted.
|
The definition of a structural model for the half-met derivative provides a tool to investigate the interaction with different types of competitive inhibitor. The first molecule investigated was p-nitrophenol. The presence of a nitro substituent in the para position results in a very poor substrate that in the native protein acts as a competitive inhibitor of L-Dopa oxidation with an inhibition constant of 700 µM.4 Binding to the cupric center is demonstrated by both the changes in the lineshape of the cw EPR spectrum and by the absence of the contribution from the bound water molecule in the HYSCORE spectrum of the half-met-Ty with p-nitrophenol (15). The virtually unchanged g|| and A|| values suggest that the Cu(II) remains four-coordinated and that the coordination geometry is not significantly modified upon p-nitrophenol binding. The binding may simply be an exchange of the bound hydroxyl moiety coordinated in the trans position to His-324 as indicated in Fig. 7B. It should be mentioned that this coordination mode for p-nitrophenol is more likely to be relevant for the inhibition mechanism of p-nitrophenol on met-Ty rather than for the binding mode of phenols in the monooxygenation reaction where the binding of the phenol substrate occurs to a Cu(II)-peroxo complex.
A more complex effect on the coordination of Cu(II) has to be assumed to rationalize the modification observed upon binding of a bidentate hydroxyquinone such as kojic acid or L-mimosine. These are among the strongest competitive inhibitors of bacterial tyrosinase with inhibition constants of 4 and 30 µM, respectively (17). In this case the disappearance of the signal of the bound water molecule in the HYSCORE spectrum upon inhibitor binding observed previously (15) is accompanied by a more significant change in the paramagnetic properties of the Cu(II) in the active site. The g|| and A|| values for both hydroxyquinone complexes are indicative of penta-coordination, and this can be achieved if both oxygen atoms of the inhibitors coordinate to the Cu(II) as shown in Fig. 7C. In agreement with the proposed binding mode for hydroxyquinones, an x-ray absorption edge study on the met-form of N. crassa Ty indicates a substantially distorted tetra-coordinated Cu(II) ion (33). Furthermore, the extended x-ray absorption fine structure data indicate an increase in the number of atoms in the first coordination shell of the metal ions in the active site upon binding of L-mimosine to met-Ty (33).
Indirect evidence that bidentate ligands bind with both oxygen atoms to Cu(II) is found in the analysis of the half-met-Ty complex with toluic acid. In hydroxyquinones the ligand oxygens conjugated to the aromatic ring are 2.7 Å apart, while in the carboxylic group of toluic acid the two oxygen ligands are 2.3 Å apart. The structural consequence of this difference, when assuming bidentate ligation, is a more distorted coordination geometry for the carboxylic complex than for the hydroxyquinone complex, as actually observed for the toluic acid half-met-Ty complex.
The proposed binding mode where both oxygen atoms of the hydroxyquinones bind to the Cu(II) has several important implications for the enzymatic reaction mechanism of tyrosinase. First, it does not assume a different coordination mode for mono- and bidentate molecules. This would mean that both substrates dock into the same region of the active site. The second aspect is that the proposed binding mode does not imply a structural rearrangement of the diphenol reaction intermediate when the monophenols are converted to diquinones. It is important to point out that it is not possible solely on the data presented here to exclude a role for the Cu(I) in the binding. Furthermore, no simple correlation can be made between the observed inhibition constant and the structure of the inhibitors, although in general bidentate hydroxyquinone ligands seem to bind stronger than monodentate inhibitors.
In conclusion, a transition state analogue as shown in Fig.
7C, which is based on the data obtained for inhibitors such
as kojic acid and L-mimosine, provides a model for the
binding of the diphenols that are generated as intermediates during
turnover of the enzyme, when monophenols are converted into quinones
(Scheme 3 in Ref. 15).
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FOOTNOTES |
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* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed. Tel.:
31-71-527-4256; Fax: 31-71-527-4349; E-mail:
canters@chem.leidenuniv.nl.
Published, JBC Papers in Press, December 6, 2002, DOI 10.1074/jbc.M206394200
2 L. Bubacco and G. W. Canters, unpublished results.
3 An analogous calculation was carried out using the crystal structure of the odg subunit of O. dofleinii Hc kindly provided by M. E. Cuff (7). This protein, the overall sequence of which is closer to that of bacterial Ty than that of L. polyphemus Hc, provided essentially the same answer.
4 L. Bubacco, M. Gastel, E. J. J. Groenen, E. Vijgenboom, and G. W. Canters, unpublished result.
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ABBREVIATIONS |
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The abbreviations used are: Ty(s), tyrosinase(s); Hc(s), hemocyanin(s); HYSCORE, hyperfine sublevel correlation spectroscopy; ESEEM, electron spin echo envelope modulation; cw, continuous wave.
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