The Effect of Substrate, Dihydrobiopterin, and Dopamine
on the EPR Spectroscopic Properties and the Midpoint Potential of the
Catalytic Iron in Recombinant Human Phenylalanine Hydroxylase*
Peter-L.
Hagedoorn
,
Peter P.
Schmidt§,
K. Kristoffer
Andersson§,
Wilfred R.
Hagen
,
Torgeir
Flatmark¶, and
Aurora
Martínez¶
From the
Kluyver Department of Biotechnology, Delft
University of Technology, Julianalaan 67, 2628 BC Delft, The
Netherlands, the § Department of Biochemistry, University of
Oslo, N-0316 Oslo, Norway, and the ¶ Department of Biochemistry
and Molecular Biology, University of Bergen, Årstadveien 19, N-5009
Bergen, Norway
Received for publication, October 17, 2000, and in revised form, April 9, 2001
 |
ABSTRACT |
Phenylalanine hydroxylase (PAH) is a
tetrahydrobiopterin (BH4) and non-heme
iron-dependent enzyme that hydroxylates L-Phe to L-Tyr. The paramagnetic ferric iron at the active site
of recombinant human PAH (hPAH) and its midpoint potential at pH 7.25 (Em(Fe(III)/Fe(II))) were studied by EPR
spectroscopy. Similar EPR spectra were obtained for the tetrameric
wild-type (wt-hPAH) and the dimeric truncated hPAH(Gly103-Gln428) corresponding to the
"catalytic domain." A rhombic high spin Fe(III) signal with a g
value of 4.3 dominates the EPR spectra at 3.6 K of both enzyme
forms. An Em = +207 ± 10 mV was measured for
the iron in wt-hPAH, which seems to be adequate for a thermodynamically
feasible electron transfer from BH4 (Em (quinonoid-BH2/BH4) = +174
mV). The broad EPR features from g = 9.7-4.3 in the
spectra of the ligand-free enzyme decreased in intensity upon the
addition of L-Phe, whereas more axial type signals were
observed upon binding of 7,8-dihydrobiopterin (BH2), the
stable oxidized form of BH4, and of dopamine. All three
ligands induced a decrease in the Em value of the
iron to +123 ± 4 mV (L-Phe), +110 ± 20 mV
(BH2), and
8 ± 9 mV (dopamine). On the basis of
these data we have calculated that the binding affinities of
L-Phe, BH2, and dopamine decrease by 28-, 47-, and 5040-fold, respectively, for the reduced ferrous form of the
enzyme, with respect to the ferric form. Interestingly, an
Em value comparable with that of the ligand-free,
resting form of wt-hPAH, i.e. +191 ± 11 mV, was
measured upon the simultaneous binding of both L-Phe and
BH2, representing an inactive model for the iron
environment under turnover conditions. Our findings provide new
information on the redox properties of the active site iron relevant
for the understanding of the reductive activation of the enzyme and the
catalytic mechanism.
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INTRODUCTION |
Phenylalanine hydroxylase
(PAH,1 phenylalanine
4-monooxygenase, EC 1.14.16.1) is a tetrahydrobiopterin
(BH4) and non-heme iron-dependent enzyme that
hydroxylates L-Phe to L-Tyr using dioxygen. PAH
is found mainly in the liver, and mutations in human PAH (hPAH) result
in a dysfunction associated with the autosomal recessive disorder
phenylketonuria, which is the most prevalent inborn error of amino acid
metabolism. In recent years important progress has been made in the
elucidation of the crystal structure of the ligand-free PAH from human
(1, 2) and rat (3) and of the homologous enzyme tyrosine hydroxylase
(TH) (4). PAH and TH are structurally and functionally closely related
enzymes containing a 2-His-1-carboxylate facial triad motif (5, 6)
anchoring the mononuclear non-heme iron atom. The crystal structures of
the complexes of rat TH and hPAH with the oxidized cofactor analogue
dihydrobiopterin (BH2) have also been determined (7, 8).
Moreover, the structure of the ternary complex
hPAH·BH2·L-Phe has recently been studied by
NMR spectroscopy and molecular docking (9). These structural studies
support the proposal that an iron-peroxo-tetrahydropterin complex forms
during the catalytic cycle (10), and may either act as the
hydroxylating intermediate itself or be the precursor of a ferryl oxo
intermediate capable of aromatic hydroxylation (11, 12). Thus,
L-Phe seems to bind at the second coordination sphere of
the iron with a distance between the hydroxylation sites (C4a in the
pterin and C4 in L-Phe) of 6.3 Å, which is adequate for
the intercalation of iron-coordinated molecular oxygen (9). Moreover,
the crystal structure of hPAH complexed with diverse catecholamines has
revealed that the inhibitors bind to the iron by bidentate coordination
through the catechol hydroxyl groups (13), as observed earlier by
resonance Raman spectroscopy of PAH and TH (14, 15).
Although these recent structural studies have provided further insight
into the function of the iron and the pterin in the catalytic reaction
of the aromatic amino acid hydroxylases, little is yet known about the
details of electron transfer reactions and the catalytic mechanism. It
seems clear that no product or intermediate is released prior to the
binding of all substrates, and the first observable product of the
pterin is a 4a-hydroxytetrahydropterin, which is dehydrated to
quinonoid-dihydrobiopterin (q-BH2)
either spontaneously or in a reaction catalyzed by pterin
4a-carbinolamine dehydratase (16, 17). PAH isolated from rat liver and
recombinant rat PAH contain the catalytic iron in the ferric high spin
(S = 5/2) state (18-21). In the catalytic reaction
Fe(III) is reduced to Fe(II) by BH4, termed "reductive
activation" of the enzyme (22), and in vitro this
reduction is an obligate step that occurs in the pre-steady state
period (18). Some experimental evidence has been presented in favor of
Fe(II) during subsequent turnovers (16), but this has not been proven,
and a redox cycling from the Fe(II) to Fe(III) and even Fe(IV) has
alternatively been proposed (16, 23). The reductive activation produces
q-BH2 directly (22) and, although some
controversy exists in the early literature about the number of electron
equivalents consumed in this reduction and the requirement for
dioxygen, 1.2 ± 0.1 pterin-derived electrons seem to be consumed
per Fe(III) site, i.e. about 1 reduced tetrahydropterin/2 Fe(III)-PAH subunits, under either aerobic or anaerobic conditions (16,
24).
Both BH4 and the substrate L-Phe also have
important regulatory functions, which seem to be of physiological
significance (25); inhibitory catecholamines regulate the activity of
TH in an interplay with phosphorylation (26-28). The conformational changes induced by the substrate, the natural pterin cofactor (and its
inactive analogue), and the catecholamine inhibitors have been studied
at the level of the tertiary and quaternary structure of both PAH and
TH (25, 29, 30). However, it is not known to what extent the binding of
substrate, cofactor, and catecholamines at the active site affects the
coordination environment of the catalytic iron and its reactivity.
Earlier EPR spectroscopic studies on rat PAH have revealed that the
coordination geometry of the ferric iron depends on the buffer ions and
the presence of ligands coordinating at the first and the second
coordination sphere (20, 21). Although the enzyme isolated from rat
liver seems to contain a stoichiometric amount of iron per enzyme
subunit, not all of the iron has been found to be catalytically active (18-20). Moreover, the same proportion of iron that coordinates to
catecholamines is reduced by the tetrahydropterin cofactor and
participates in catalysis (20).
In the present study we have further characterized the X-band EPR
spectroscopic properties of both the tetrameric wild-type and a dimeric
truncated form of human PAH corresponding to the catalytic domain, as
well as the effect of the substrate L-Phe, reduced and
oxidized pterin cofactor, and the inhibitor dopamine. We also report
for the first time the midpoint potential of the iron in the wild-type
human PAH as isolated and its modulation upon the binding of substrate,
oxidized pterin cofactor (BH2), and dopamine.
 |
EXPERIMENTAL PROCEDURES |
Expression and Purification of the Wild-type and Truncated Form
of hPAH--
Expression in Escherichia coli (TB1 cells) of
human wild-type PAH (wt-hPAH) and the truncated
hPAH(Gly103-Gln428), i.e.
N102/
C24-hPAH as fusion proteins with maltose-binding protein, purification of the fusion proteins by affinity
chromatography on amylose resin, and their cleavage by the restriction
protease factor Xa (New England Biolabs) were performed as described
(31, 32). The tetrameric form of wt-hPAH and the dimeric
hPAH(Gly103-Gln428), corresponding to the
"catalytic domain," were separated from aggregated/higher
oligomeric forms and from maltose-binding protein and factor Xa by size
exclusion chromatography on HiLoad Superdex 200 HR prepacked column
(1.6 × 60 cm) from Amersham Pharmacia Biotech (32). Protein
concentration was estimated spectrophotometrically using the absorption
coefficients A280 nm (1 mg
ml
1 cm
1) = 1.0 for wt-hPAH and 0.9 for hPAH(Gly103-Gln428);
the assay of PAH activity was performed as described (32).
Preparation of Samples for EPR Spectroscopy--
Enzyme samples
initially were prepared in 20 mM NaHepes, 0.2 M
NaCl, pH 7.0. The iron content of the enzyme samples, measured by
atomic absorption spectroscopy, was as previously reported (1.8-2.0
atoms Fe/tetramer for wt-hPAH and 0.8-0.9 atoms Fe/dimer for
hPAH(Gly103-Gln428)) (32). In the experiments
for the initial characterization of the iron center and the effect of
ligands on the X-band EPR spectrum of hPAH, enzyme samples of either
wt-hPAH or hPAH(Gly103-Gln428) were prepared in
20 mM NaHepes, 0.2 M NaCl, pH 7.0. Additions and incubations (5 min, pH 7.0, 25 °C) of either of the following compounds: L-Phe, dopamine,
L-erythro-7,8-dihydrobiopterin (BH2, Dr. B. Schircks' Laboratories) and 6-methyl-5,6,7,8-tetrahydropterin (6-MPH4, Dr. B. Schircks Laboratories) in the presence of
dithiothreitol (DTT), were performed in the EPR tubes prior to freezing
the samples. We have previously shown that DTT alone does not reduce
the Fe(III) in rat PAH in the presence of dioxygen (20).
For the EPR-monitored redox titrations the samples were prepared in 50 mM Mops buffer, 0.2 M KCl, pH 7.25, and the
final enzyme (wt-hPAH) concentration was 100-120 µM
subunit. Nonspecifically bound Cu(II), giving rise to characteristic
EPR signals around g = 2.0-2.3, was removed by incubation
of the enzyme with 5 mM EDTA followed by three cycles of
dilution and concentration in EDTA-free Mops buffer using Centricon 30 microconcentrators (Amicon). This treatment did not result in any
significant change in the shape or intensity of the Fe(III) signal
around g = 4.3.
EPR Spectroscopy and Redox Titrations--
A first series of EPR
analysis was performed on the recombinant hPAH in the absence and
presence of ligands at 9.64 GHz microwave frequency on a Bruker ESP300E
equipped with an Oxford Instruments cryostat 900 at 3.6 K. Other EPR
parameters are given in the figure legends. The EPR spectra were
smoothed with the polynomial filter (n = 15) provided
in the WINEPR software (Bruker) and were base-line corrected.
Quantification of the Fe(III) EPR was performed by comparing the double
integral of the spectra of hPAH with the double integral of a 500 µM transferrin standard at several temperatures (which
excludes an error that might be introduced by very different D values).
In the cases in which quantification was performed using simulated
spectra, similar results were obtained.
Room temperature potentiometric titrations for subsequent EPR
monitoring were performed in a 2-ml anaerobic cell under purified argon. The bulk potential of the stirred solution was measured using a
platinum wire electrode with respect to the potential of a Radiometer
REF201 Ag/AgCl reference electrode. Reported potentials were all
expressed relative to the normal hydrogen electrode. 100 µM subunit hPAH in 50 mM Mops, 0.2 M KCl, pH 7.25 was poised at various potentials in the
presence of 100 µM each of the following mediators:
N,N,N',N'-tetramethyl-p-phenylenediamine,2,6-dichlorophenolindophenol, phenazine ethosulfate, methylene blue, resorufin,
indigosulfonate, phenosafranin, safranin O, neutral red, benzyl
viologen, and methyl viologen. Sodium dithionite and
K3Fe(CN)6 were used as reductant and oxidant,
respectively. Redox equilibrium was obtained as judged by the
attainment of a stable solution potential within a few minutes after
the addition of the titrant to the reaction mixture. The samples were
transferred anaerobically to EPR tubes and directly frozen in liquid
nitrogen. Midpoint potentials (Em) were obtained
from the experimental data points with a least squares fit to the
Nernst equation (n = 1). In addition to the titration of the ligand free enzyme, titrations in the presence of either L-Phe (5 mM), BH2 (5 mM), both L-Phe (5 mM) and
BH2 (5 mM), or dopamine (1 mM) were
also performed. The EPR-monitored titrations of hPAH samples were
performed at a microwave frequency of 9.41 GHz and a microwave power of
8 mW, with a 10-Gauss (1.0 mT) modulation amplitude and a
modulation frequency of 100 kHz at 29 K. These temperature values were
used to optimize the signal-to-noise ratio under nonsaturating
conditions. Simulation of the spectra was performed as described
(33).
 |
RESULTS |
EPR Spectra of Recombinant Human Phenylalanine
Hydroxylase--
Tris has been found to be an inhibitor of the enzyme,
competitive to the pterin cofactor (22), and our previous EPR
spectroscopic studies on rat PAH demonstrated that Tris, in its base
form, induces changes in the active site iron (20). Consequently, we
have used Hepes and Mops buffers in the present EPR studies.
Recombinant wt-hPAH revealed a low temperature (3.6 K) EPR spectrum
typical for high spin (S = 5/2) Fe(III) (Fig.
1, spectrum A). The spectrum is dominated by a resonance centered around g = 4.3, with an
accompanying broad signal with g value of ~9.7, which is
characteristic of ferric iron in a rhombic environment, with an E/D
value of ~1/3. Some minor iron species showing weak signals with g
values spread from 9.7 to 7.0 and 5.3 to 4.3 with various E/D values
between 0.05 and 0.33 were also present, indicating some
microheterogeneity in the coordination geometry of the enzyme-bound
iron. Nevertheless, the iron environment appears to be more homogenous
in the recombinant hPAH than previously observed for the hepatic rat
and bovine enzymes, which have been isolated through procedures
including preincubation of the crude extracts with ferrous ions and DTT
(34). Those PAH preparations showed a split of the major resonance at
g = 4.3 and a higher proportion of iron in a less rhombic
environment (gmax values in the range of 7 to 6 and E/D < 0.05) (20).

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Fig. 1.
EPR spectra of wt-hPAH. A,
wt-hPAH (100 µM subunit) as isolated in 20 mM
NaHepes, 0.2 M NaCl, pH 7.0; B, same as
spectrum A with 1 mM L-Phe;
C, same as spectrum A with 0.5 mM
6-MPH4 and 5 mM DTT; D, same as
spectrum B with 0.5 mM 6-MPH4 and 5 mM DTT (turnover conditions). All incubations of the enzyme
samples were performed at 25 °C for 5 min prior to freezing the
samples in the EPR tubes. EPR parameters were 9.64 GHz microwave
frequency, 0.1 mW microwave power, 1 mT modulation amplitude, and a
modulation frequency of 100 kHz at 3.6 K.
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Reduction of High Spin (S = 5/2) Fe(III) by
Tetrahydrobiopterin and the Effect of
L-Phe--
Quantification of the Fe(III) EPR in wild-type
hPAH gave numbers that were in reasonable agreement with the total iron
content measured by atomic absorption spectroscopy, i.e.
0.45-0.52 atoms iron/subunit for recombinant wt-hPAH (32). This value
is also similar to the catalytically active iron measured in rat PAH
(19, 21, 35). Thus, although the isolated rat and bovine liver enzyme
preparations contain about 1 atom iron/subunit, only a fraction of it
has been found to be reduced by the cofactor and thus to participate in
catalysis (19, 20).
The effect of preincubation of the samples with either
L-Phe or 6-MPH4 as well as with both compounds
simultaneously (turnover conditions) on the EPR spectrum of wt-hPAH is
shown in Fig. 1. For a better estimation of the amount of iron that is
not in the completely rhombic environment (E/D = 1/3, g = 4.3, resonance at 160 mT), the corresponding zero line for each
spectrum is shown. The area enclosed by the recorded spectra between 70 and 100 mT and this zero line correlates with iron in a less rhombic
environment. A comparison of these areas in spectra 1A and 1B shows
that preincubation of wt-hPAH (spectrum A) with the substrate
L-Phe (spectrum B) results in a decrease of these signals.
Moreover, a concomitant increase of the intensity of the major signal
at g = 4.3 is observed. Quantification, performed by double
integration of the spectra shown in 1A and 1B between 60 and 240 mT
reveals that the total intensity of both spectra is identical within
the error range of the method (10%). By contrast, when the reducing
cofactor analogue 6-MPH4 is added to the resting form of
the enzyme, the amount of ferric iron decreases to 35%, as judged by
the decrease of the double integral of the major signal at g = 4.3 (Fig. 1, spectrum C). This reduction is slightly enhanced (only 28% of
the iron is in the ferric state) if 6-MPH4 is added to the
enzyme preincubated with L-Phe, i.e. at turnover
conditions (Fig. 1, spectrum D).
Effect of Truncation of the Enzyme and the Addition of Oxidized
Cofactor Analogue (BH2) on the EPR Spectrum--
The
deletion of the N-terminal regulatory and C-terminal tetramerization
domains of hPAH results in an activated (about 3-fold) dimeric
hPAH(Gly103-Gln428) form that does not show any
significant further activation by prior incubation with
L-Phe, and contrary to the full-length wt-hPAH, it does not
bind L-Phe with positive cooperativity (32). As seen by the
crystal structures of this truncated form (1) and of rat PAH containing
the catalytic and regulatory domains (3), the negative effect exerted
by the regulatory domain on the catalytic activity is not accompanied
by significant structural changes around the 6-coordinating iron site.
Accordingly, no significant differences were observed in the main
features of the X-band EPR spectra of the full-length and truncated
hPAH forms either in the absence or presence of L-Phe (Fig.
1, spectra A and B and Fig.
2, spectra A and
B). However, there seem to be differences in the
coordination environment between the iron in the full-length and the
isolated catalytic domain as indicated by the decreased P1/2 values for the microwave power saturation
behavior of the truncated PAH (Table I),
which may indicate a more homogenous iron coordination environment for
this form.

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Fig. 2.
EPR spectra of the double truncated form of
hPAH. A, hPAH(Gly103-Gln428)
(or N102/ C24) (533 µM subunit) in 20 mM NaHepes, 0.2 M NaCl, pH 7.0. B, hPAH(Gly103-Gln428) (480 µM subunit) with 2 mM L-Phe.
C, hPAH(Gly103-Gln428) (600 µM subunit) with 600 µM BH2.
EPR parameters were 9.64 GHz microwave frequency, 0.1 mW microwave
power, 1 mT modulation amplitude, and a modulation frequency of 100 kHz
at 3.6 K.
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Table I
Microwave power saturation (P1/2) of the iron signal at g = 4.3 of different forms of hPAH at different temperatures
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The oxidized cofactor analogue BH2 inhibits both wt-hPAH
and hPAH(Gly103-Gln428) by a competitive type
of inhibition versus the natural pterin cofactor
BH4, with a Ki of 120 µM
for wt-hPAH and 100 µM for the truncated form (9). The
structure of the complex between
hPAH(Gly103-Gln428) and BH2 has
recently been solved both by NMR spectroscopy and docking in the
ligand-free crystal structure of the enzyme (9) and by x-ray
crystallography (8). We have examined here the effect of
BH2 on the EPR spectrum of this double truncated form. The
addition of BH2 at concentrations of 500 µM
results in the appearance of a less rhombic type of high spin ferric
species with g values of 7.4, 4.3, and (1.8) from the lowest and 5.8, (1.7), and (1.5) from the middle Kramer's doublet with E/D = 0.07 (Fig. 2, spectrum C). The g values below 2 (in parentheses)
can be estimated only from rhombograms, as any g or D strain has
severe broadening effects in these g value regions. It seems that
spectrum 2C consists of two major species, i.e. one with
E/D = 0.07 and one with E/D = 0.33, as well as some minor
species with intermediate E/D values that distort the signal at g
value = 5.8.
Determination of the Midpoint Potential (Em) of the
Catalytic Iron in hPAH and the Effects of Ligand Binding at the Active
Site--
Studies intended to determine the Em
(Fe(III)/Fe(II)) for the active site iron in hPAH by direct
electrochemistry using activated glassy carbon as the working electrode
were unsuccessful with all the promoters used. Thus, EPR-monitored
redox titrations of the iron in wt-hPAH (100-120 µM
enzyme subunit in Mops buffer at pH 7.25) were performed in the absence
of ligands and in the presence of L-Phe (Fig.
3, spectrum A), BH2 (Fig. 3,
spectrum B), and L-Phe and BH2 simultaneously
bound (Fig. 3, spectrum C) at 29 K. For the samples with
BH2 the ratio of the intensities of the signals with g
values of 5.8 and 7.4 is higher in the EPR spectra taken at 29 K (Fig.
3, spectrum B) than in those taken at 3.6 K (Fig. 2,
spectrum C), substantiating the assignment of the signal at
g value = 7.4 to the lowest and at g value = 5.8 to the
middle Kramer's doublet.

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Fig. 3.
EPR spectra of the samples used in the redox
titrations. The samples contained wt-hPAH (100 µM
subunit) in 50 mM Mops buffer, 0.2 M KCl, pH
7.25, with 5 mM L-Phe (A), 5 mM BH2 (B), 5 mM
L-Phe and 5 mM BH2 (C),
or 1 mM dopamine (D). The spectra were taken at
a microwave frequency of 9.41 GHz and a microwave power of 8 mW, with a
1 mT modulation amplitude and a modulation frequency of 100 kHz at 29 K.
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We have also studied the effect of dopamine on both the EPR spectrum of
wt-hPAH and the Em. The crystal structure of the
complexes between the catalytic domain of hPAH and several catecholamines shows that these inhibitors bind to the iron by bidentate coordination through the catechol group (13) in agreement with earlier resonance Raman spectroscopic studies (14, 15, 36). The
resulting elimination of two coordinating water ligands and the changes
in coordination geometry of the active site iron upon addition of
dopamine are accompanied by an increase of the less rhombic type EPR
signals with g values at 7.0-7.2, 4.7-4.9, and
(1.90-1.92) from the lowest and at 5.8 from the middle Kramer's doublet, corresponding to an E/D value of 0.045-0.055 and a decrease of rhombic EPR signal at g = 4.3 (Fig. 3, spectrum
D).
To determine the Em, the intensity of
the signals at g = 4.3 (Fig. 3, spectra A-C)
and at g = 7.09 (spectrum D) was followed during the
titrations. As shown in Fig. 4, the
Fe(III) in all the samples was reduced in the range of
200 to + 300 mV. The values of the estimated midpoint potential
Em at pH 7.25 in the absence and presence of ligands
binding at the active site are summarized in Table
II. Em = + 207 ± 10 mV for the resting form of the enzyme in the absence of ligands was
found to be decreased by about 84, 100, and 210 mV upon the binding of
L-Phe, BH2, and dopamine, respectively.
Moreover, when L-Phe and BH2 were bound
simultaneously, forming an inactive analogue (PAH-Fe(III)·L-Phe·BH2) of the turnover
active complex (PAH-Fe(II)·L-Phe·BH4), the
estimated Em value was 191 ± 11 mV.

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Fig. 4.
EPR monitored redox titrations. The
samples shown are: wt-hPAH (100 µM subunit) in 50 mM Mops buffer, 0.2 M KCl, pH 7.25, in the
absence of ligands ( ), with 5 mM L-Phe
( ), 5 mM BH2 ( ), 5 mM
L-Phe and 5 mM BH2 ( ), and 1 mM dopamine ( ). See also Table II and the legend to Fig.
3.
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Table II
Midpoint redox potential Em (Fe(III)/Fe(II)) at pH 7.25 in
samples of wt-hPAH in the absence and presence of L-Phe (5 mM), BH2 (5 mM), and dopamine (1 mM), and apparent Kd values for ligand binding to
the enzyme
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On the basis of the Em values in the absence
(Em(free)) and the presence of the ligands added
separately (Em(bound)), and by using the following
equation (37),
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(Eq. 1)
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where n is the number of electrons, F is the
Faraday constant (96485.3 C/mol), R is the Gass constant
(8.31451 J/kmol), and T is the temperature, we calculated
the apparent dissociation constants for the binding of
L-Phe, BH2, and dopamine to the reduced ferrous
form of PAH (Kd,red), using the
reported values for the binding of the ligands to the oxidized form of
the enzyme (Kd,ox) (38-40). As seen in
Table II, it is predicted that the binding affinities of
L-Phe, BH2, and dopamine decrease by 28-, 47-, and 5040-fold, respectively, for the reduced ferrous form of the enzyme
with respect to the ferric form.
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DISCUSSION |
The low temperature (4 K) X-band EPR spectra obtained for
recombinant hPAH, both the wt-hPAH and its N- and C-terminal truncated form (catalytic domain), are very similar to those obtained
previously for the hepatic rat and bovine PAH when the enzyme samples
are prepared in buffers that do not interact with the active site non-heme iron, i.e. potassium phosphate, NaHepes, and Mops
(20, 21). However, an important difference was observed between the isolated hepatic and the recombinant enzyme forms. Whereas isolated rat
liver PAH seems to contain more than 50% of the iron that does not
participate in catalysis (18-20), in hPAH the major EPR signal at g
= 4.3, characteristic of high spin (S = 5/2)
ferric iron in a rhombic coordination geometry, is largely reduced (to almost a quarter of the intensity in the enzyme as isolated) in the
presence of both L-Phe and tetrahydropterin (turnover
conditions) (Fig. 1D). The larger homogeneity of the iron
population in the recombinant hPAH makes it possible to determine the
midpoint potential (Em) of the active site iron, an
important novel finding in the present study.
The apparent Em at pH 7.25 for the catalytic iron
was measured both in the absence and presence of substrate, pterin cofactor analogue, and catecholamine inhibitor, all compounds binding
at the active site. Previously, the Em value of the
natural cofactor, i.e.
(q-BH2)/BH4 couple), has been
determined to be +174 mV at pH 7 (40), and the literature values vary
from +140 to +184 mV for cofactor analogues with various substituents at the 6-position (40-42). Thus, the Em value of
+207 ± 10 mV for the catalytic iron in the resting enzyme (Table
II) is in agreement with a thermodynamically feasible electron transfer from the reduced cofactor to the iron site, explaining the observed reductive activation of the enzyme by the tetrahydropterin
cofactors (22).
As seen from the EPR spectrum of the resting form of wt-hPAH, with a
major broad peak around g = 4.3 and minor features with g
values stretching from 9.7 to 4.5, the coordination geometry of the
active site iron seems to be rather flexible. This conclusion is
further supported by the crystal structure showing three water molecules in the first coordination sphere of the iron (1, 3). A
sharpening of the g = 4.3 signal and an increase of the P1/2 value for the microwave power saturation
behavior was observed in the presence of L-Phe, indicating a change in coordination environment of the active site iron in the
substrate-activated enzyme form. This effect is in good agreement with
our previous finding that the binding of L-Phe at the
active site is accompanied by a dissociation of one of the ferric
iron-coordinating water molecules (43, 44). The recent structural study
on the ternary PAH-Fe(III)·L-Phe·BH2
complex (9) supports the conclusion that the binding of
L-Phe may result in a 5-coordinated iron in the ferric form
of the enzyme. Even larger changes in the EPR spectra and the midpoint
potential were encountered on addition of the tetrahydrobiopterin
cofactor analogue BH2 and the inhibitor dopamine. Thus, a
less rhombic type of signal is observed in the presence of these
inhibitors (gmax values of 7.4 and 7.0 for
BH2 and dopamine, respectively) (Figs. 2C and 3)
indicating a decrease of the E/D value (from 0.33 to ~0.07 and 0.05, respectively) and a change in the coordination geometry of the iron.
These spectroscopic changes were also accompanied by a decrease of the
Em value of the iron to numbers, which are
significantly lower than those of the pterin cofactor redox couple
(Table II). Notably for the hPAH-dopamine complex it is clear that
reduction of the Fe(III) by the cofactor is not possible to achieve
because of a ~200 mV negative shift of the Em
value, and this effect could be of physiological significance in the
case of TH, which is regulated by feedback inhibition by catecholamines
and phosphorylation (12, 26, 27). The inhibition of PAH by
catecholamines appears to be competitive with respect to the
tetrahydropterin cofactors (45). Paramagnetic relaxation NMR
experiments have shown that catecholamines do not compete for the
cofactor binding site in hPAH and that both noradrenaline and dopamine
can be bound simultaneously with the cofactor analogue BH2
(9). Thus, the apparent competitive type of inhibition by
catecholamines versus the pterin cofactor may rather be due
to changes in the ligand field geometry of the active site iron as a
result of formation of the tight bidentate catecholate-Fe(III) complex
(13), lowering the Em value and thus stabilizing the
ferric state. The ~200-mV negative shift of the midpoint potential on
dopamine binding also explains the experimental finding that
recombinant human TH reconstituted with Fe(II) is rapidly oxidized upon
the addition of catecholamines forming a ferric blue-green complex
(46). Moreover, based on the large decrease of Em
upon dopamine binding, we estimated that the Kd
value for the binding of this inhibitor to the reduced enzyme was
increased by about 5040-fold (Table II). This large decrease in
affinity is in agreement with the calculated Kd
values for catecholamine binding to both the ferric and ferrous forms
of TH (47).
The changes in the midpoint potential of the active site iron induced
by the binding of L-Phe and BH2, both when
bound independently (reduction in Em) and when bound
simultaneously (comparable Em with the enzyme as
isolated) may be explained using as a frame the modeled structure of
the ternary complex of hPAH(Gly103-Gln428) with
L-Phe and BH2 (9) (Fig.
5) and the crystal structure of the
binary hPAH(Gly103-Gln428)·BH2
complex (8). Thus, the amino group of L-Phe seems to interact with the enzyme through a hydrogen bond with
Ser349, and this residue is hydrogen bonded with the
N
1 of the iron-coordinating
His285 in the resting unbound form of the enzyme (1, 9).
Hence, the binding of the substrate may disrupt the initial
hydrogen-bonding network of His285, resulting in a
modulation of the coordination geometry of the iron and a decrease of
the Em value as observed in this work. The
disruption of the hydrogen bond between Ser395 and
His331 in TH has also been related to a change in the
reactivity of Fe(II) with oxygen (48). The oxidized, inactive cofactor
analogue BH2, which inhibits the rat and recombinant human
PAH competitive to the cofactor BH4, appears to bind at the
same or overlapping sites to BH4 (9, 40, 49, 50). In the
structure of the binary complex of
hPAH(Gly103-Gln428) and BH2
determined either by NMR/molecular docking (9) (Fig. 5) or x-ray
crystallography (8), BH2 binds at the bottom of the wide
active site crevice structure. The pterin ring
-stacks with
Phe254 and interacts with Glu286,
Gly247, His264, and Leu249, whereas
the dihydroxypropyl side-chain at C-6 establishes interactions with Ala322. Although a similar binding site for
BH2 was found in the crystal structure and the NMR study,
some important differences in the detailed interactions were
encountered. Thus, in the NMR/docking structure, the N-3 and the amine
group at the C-2 hydrogen bond with the carboxyl group of
Glu286 and the distance between the O-4 atom of
BH2 and the iron (2.6 ± 0.3 Å) is compatible with
coordination (9). In the crystal structure, however, the N-3 and the
O-4 of BH2 interact with Glu286 and the iron,
respectively, through bridging water molecules, resulting in the
location of the pterin ring at distances not compatible for
coordination to the iron. The reasons for these discrepancies
are not clear, although alternative explanations have been proposed
(8). The less rhombic type of signals, which appear in the EPR spectra
of both the truncated form hPAH(Gly103-Gln428)
and the wt-hPAH in the presence of BH2, are compatible with a coordination of BH2 to the iron as indicated by the
solution structure (9). Nevertheless, the interaction of the pterin ring with an iron-coordinated water molecule might well result in a loss of flexibility and a change of the water-iron bonding distance and thereby in a less rhombic coordination geometry. Regardless of the detailed interactions between the pterin ring and the
iron, it seems clear that BH2 changes the coordination geometry of the metal and decreases its midpoint potential (Table II).
However, when bound simultaneously at opposite sides in the coordination environment of the iron in the ternary
PAH-Fe(III)·L-Phe·BH2 complex (9) (Fig. 5),
L-Phe and BH2 may exert compensatory changes in
the electronic environment of the iron, in agreement with the finding
that the midpoint potential in the ternary complex is more similar to
that in the resting unbound enzyme than in the binary complexes (Table
II). The L-Phe induced conformational change at the protein
level is well documented on the basis of intrinsic tryptophan
fluorescence spectroscopy, limited proteolysis, and dynamic light
scattering (reviewed in Refs. 16 and 17). Furthermore, it has been
shown that in the double truncated form of hPAH, BH2 binds
by an induced fit mechanism, involving a conformational change in the
protein at the active site (8) as well as in the dihydroxypropyl
side-chain of the cofactor (9). All of the structural and kinetic data
obtained so far are compatible with the proposed four-state
conformational model (51); i.e. a resting state, a
L-Phe activated state, a BH4/BH2
inhibited state, and a state of catalytic turnover. In this model the
resting state and the turnover state were found to be very similar by different indirect conformational probes.

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|
Fig. 5.
The structure of the active site of
hPAH (PDB accession number 1PAH (1)) with L-Phe and
BH2 bound according to the structure of the ternary complex
as resolved by NMR and molecular docking (9).
|
|
The inactive ternary PAH-Fe(III)·L-Phe·BH2
complex seems to provide a good model for the coordination environment
of the iron under turnover conditions. Although the midpoint potential
for the BH4/q-BH2 couple may change
when the cofactor binds to the enzyme, the +81 mV increase in
Em obtained when both ligands are added
simultaneously with respect to that obtained in the presence of
BH2 alone is in agreement with a thermodynamically favorable electron transfer from the cofactor to the iron at turnover conditions. As seen in Fig. 1, a larger reduction is obtained with
6-MPH4 when wt-hPAH is also complexed with
L-Phe (Fig. 1, spectrum D; turnover
conditions) than in the absence of the substrate (spectrum
C). Accordingly, although the affinity for tetrahydropterin cofactors is decreased for the L-Phe-activated enzyme (50), it has been reported previously that the reduction seems to be facilitated by the presence of L-Phe (24). This modulation
of the Em value upon ligand binding also agrees with
a type of mechanism for PAH involving the formation of a complex of all the reactants prior to catalysis. Thus, only when both the substrate and the tetrahydropterin cofactor are simultaneously bound to ferrous
PAH, dioxygen may bind and be activated at the open coordination position, as indicated by magnetic circular dichroism spectroscopic studies with the cofactor analogue 5-deaza-6-methyltetrahydropterin (52). Moreover, the Km values obtained for
L-Phe and BH4 with the ferrous form of PAH (25)
resemble the Kd,ox (Table II),
indicating that the decrease in affinity estimated for the independent
binding of substrate and cofactor analogue with respect to the binding
to the oxidized form of the enzyme is reversed when the ligands bind
forming the ternary complex. The midpoint reduction potential of the
heme iron in the BH4-dependent enzyme
nitric-oxide synthase has also been shown to be modulated similarly by
substrate and active site inhibitors (37). However, the absolute values
for the midpoint potentials determined for this enzyme are about
400-500 mV lower than those determined in this work for hPAH,
reflecting the different roles of BH4 in the reactions. The
Em values measured for nitric-oxide synthase have
been found adequate for a thermodynamically feasible reduction of the
heme by the flavin cofactors (53), and neither
4-hydroxytetrahydropterin nor q-BH2 has been
detected in the reaction of this enzyme. Recently, a protonated
trihydrobiopterin radical (54) has been shown to be formed in the redox
cycling of BH4 in nitric-oxide synthase (55, 56). Formation
of such a radical has been proposed but not detected for any of the
aromatic amino acid hydroxylases (12). Although the details of the
electron transfer reactions in the catalytic process of the aromatic
amino acid hydroxylases are not yet clear (16), our present findings
represent new information on the redox properties of the active site
iron that is relevant for our understanding of their catalytic mechanism.
 |
ACKNOWLEDGEMENTS |
We are very grateful to Dr. Per M. Knappskog
for the preparation of bacterial strains expressing human phenylalanine
hydroxylase and to Ali. J. S. Muñoz and Randi M. Svebak for
the expression, purification, and kinetic characterization of the
recombinant human phenylalanine hydroxylase.
 |
FOOTNOTES |
*
This work was supported by the Research Council of Norway,
L. Meltzers Høyskolefond, the Norwegian Cancer Society, the Norwegian Council on Cardiovascular Diseases, Rebergs legat, the Novo Nordisc Foundation, Marie-Curie Grant ERBFMBICT 961892, and the Training and
Mobility of Researchers and Biotechnology Programs of the European Union (ERBMRFXCT 980207 and BIO4-98-0385).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.
47-55-58-64-27; Fax: 47-55-58-64-00; E-mail:
aurora.martinez@ibmb.uib.no.
Published, JBC Papers in Press, April 11, 2001, DOI 10.1074/jbc.M009458200
 |
ABBREVIATIONS |
The abbreviations used are:
PAH, phenylalanine
hydroxylase;
BH2, L-erythro-7,8-dihydrobiopterin;
BH4, (6R)-L-erythro-5,6,7,8-tetrahydrobiopterin;
DTT, dithiothreitol;
Em, midpoint
oxidation-reduction potential;
Mops, 4-morpholinepropanesulfonic acid;
6-MPH4, 6-methyl-5,6,7,8-tetrahydropterin;
mT, millitesla;
P1/2, microwave power at 50% saturation
of the EPR signal;
hPAH(Gly103-Gln428), an N-
and C-terminal truncated form of human phenylalanine hydroxylase,
i.e.
N102/
C24-hPAH;
TH, tyrosine hydroxylase;
wt-hPAH, recombinant human wild-type phenylalanine hydroxylase.
 |
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