Pulsed EPR study of amino acid and tetrahydropterin binding in a tyrosine hydroxylase nitric oxide complex: Evidence for substrate rearrangements in the formation of the oxygen-reactive complex

Article abstract of DOI:10.1021/bi4010914

Tyrosine hydroxylase is a nonheme iron enzyme found in the nervous system that catalyzes the hydroxylation of tyrosine to form l-3,4- dihydroxyphenylalanine, the rate-limiting step in the biosynthesis of the catecholamine neurotransmitters. Catalysis requires the binding of three substrates: tyrosine, tetrahydrobiopterin, and molecular oxygen. We have used nitric oxide as an O₂ surrogate to poise Fe(II) at the catalytic site in an S = ³/₂, {FeNO}⁷ form amenable to EPR spectroscopy. ²H-electron spin echo envelope modulation was then used to measure the distance and orientation of specifically deuterated substrate tyrosine and cofactor 6-methyltetrahydropterin with respect to the magnetic axes of the {FeNO}⁷ paramagnetic center. Our results show that the addition of tyrosine triggers a conformational change in the enzyme that reduces the distance from the {FeNO}⁷ center to the closest deuteron on 6,7-²H-6-methyltetrahydropterin from >5.9 A to 4.4 ± 0.2 A. Conversely, the addition of 6-methyltetrahydropterin to enzyme samples treated with 3,5-²H-tyrosine resulted in reorientation of the magnetic axes of the S = ³/₂, {FeNO}⁷ center with respect to the deuterated substrate. Taken together, these results show that the coordination of both substrate and cofactor direct the coordination of NO to Fe(II) at the active site. Parallel studies of a quaternary complex of an uncoupled tyrosine hydroxylase variant, E332A, show no change in the hyperfine coupling to substrate tyrosine and cofactor 6-methyltetrahydropterin. Our results are discussed in the context of previous spectroscopic and X-ray crystallographic studies done on tyrosine hydroxylase and phenylalanine hydroxylase.

Full text of DOI:10.1021/bi4010914

Article
pubs.acs.org/biochemistry
Pulsed EPR Study of Amino Acid and Tetrahydropterin Binding in a
Tyrosine Hydroxylase Nitric Oxide Complex: Evidence for Substrate
Rearrangements in the Formation of the Oxygen-Reactive Complex
Matthew D. Krzyaniak,†,§ Bekir E. Eser,‡ Holly R. Ellis,‡,∥ Paul F. Fitzpatrick,⊥ and John McCracken*,†
†Department of Chemistry, Michigan State University, East Lansing, Michigan 48824, United States
‡Department of Chemistry, Texas A&M University, College Station, Texas 77843, United States
⊥Department of Biochemistry, University of Texas Health Science Center, San Antonio, Texas 78229, United States
S
* Supporting Information
ABSTRACT: Tyrosine hydroxylase is a nonheme iron
enzyme found in the nervous system that catalyzes the
hydroxylation of tyrosine to form L-3,4-dihydroxyphenylala-
nine, the rate-limiting step in the biosynthesis of the
catecholamine neurotransmitters. Catalysis requires the bind-
ing of three substrates: tyrosine, tetrahydrobiopterin, and
molecular oxygen. We have used nitric oxide as an O2
3
surrogate to poise Fe(II) at the catalytic site in an S = /2,
2
{FeNO}7 form amenable to EPR spectroscopy. H-electron spin echo envelope modulation was then used to measure the
distance and orientation of specifically deuterated substrate tyrosine and cofactor 6-methyltetrahydropterin with respect to the
magnetic axes of the {FeNO}7 paramagnetic center. Our results show that the addition of tyrosine triggers a conformational
change in the enzyme that reduces the distance from the {FeNO}7 center to the closest deuteron on 6,7-2H-6-
methyltetrahydropterin from >5.9 Å to 4.4 0.2 Å. Conversely, the addition of 6-methyltetrahydropterin to enzyme samples
3
treated with 3,5-2H-tyrosine resulted in reorientation of the magnetic axes of the S = /2, {FeNO}7 center with respect to the
deuterated substrate. Taken together, these results show that the coordination of both substrate and cofactor direct the
coordination of NO to Fe(II) at the active site. Parallel studies of a quaternary complex of an uncoupled tyrosine hydroxylase
variant, E332A, show no change in the hyperfine coupling to substrate tyrosine and cofactor 6-methyltetrahydropterin. Our
results are discussed in the context of previous spectroscopic and X-ray crystallographic studies done on tyrosine hydroxylase and
phenylalanine hydroxylase.
Mutations in TyrH have been associated with L-DOPA
responsive forms of Segawa’s syndrome and Parkinson’s
disease,2,3 and they have been implicated in bipolar affective
disorder.4
yrosine hydroxylase (TyrH) is a nonheme Fe enzyme
Tfound in the brain and adrenal gland of humans that
catalyzes the hydroxylation of the amino acid L-tyrosine to form
L-3,4-dihydroxyphenylalanine (L-DOPA).1 This reaction is the
rate-limiting step in the biosynthesis of the catecholamine
neurotransmitters dopamine, epinephrine, and norepinephrin
(Scheme 1), making it vital to nervous system function.
The chemical mechanism of tyrosine hydroxylation requires
the binding of tyrosine (tyr), a tetrahydropterin, with
tetrahydrobiopterin (BH4), the physiological substrate, and
O2 to a catalytic site that houses an Fe(II) facially coordinated
by the side chains of two histidines and a glutamate.5 X-ray
crystallographic studies of TyrH are of Fe(III) forms of the
enzyme and show that the metal ion is either 5- or 6-coordinate
with the coordination sphere completed by water ligands.6,7
Detailed X-ray absorption and variable-temperature variable-
field MCD spectroscopic studies have shown that tyrosine and
BH4 do not bind directly to the Fe(II), but that their binding
leads to structural changes that result in the metal center
transitioning from 6- to 5-coordinate.8 These changes at the
catalytic site result in a ≥100-fold enhancement of O2 reactivity
Scheme 1. Hydroxylation Reactions Catalyzed by Tyrosine
Hydroxylase
Received: August 10, 2013
Revised: October 5, 2013
Published: October 29, 2013
© 2013 American Chemical Society
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with the Fe(II) and thus trigger the start of a two-step catalytic
mechanism.5,8 The first step involves reaction of the Fe(II)-
bound O2 with BH4 and leads to the hydroxylation of the C4a
carbon to yield 4a-hydroxy-biopterin (Scheme 1). In this
reaction, BH4 supplies two electrons for the heterolytic cleavage
of the O−O bond, leading to the formation of the hydroxy-
pterin product and a Fe(IV)-oxo intermediate; the Fe(IV)-oxo
species has been trapped for TyrH and characterized by
blocks O2 binding to the active site, allowing us to prepare
samples with chemically active substrate and cofactor.
Electronic structure calculations show that the g = 2.0 axis of
the {FeNO}7 paramagnetic center lies within 5° of the Fe−NO
bond25 and that the Fe−NO bond is highly covalent with
considerable double-bond character. Taken together, these
findings provide a structural reference point for interpreting our
hyperfine couplings.26,27 Hoffman and co-workers combined
this approach with 35 GHz 2H-ENDOR spectroscopy to
characterize substrate binding to the active sites of naphthalene
1,2 dioxygenase and ACC oxidase.28,29 Following their lead, we
Mossbauer spectroscopy.9 The second step of the catalytic
̈
mechanism involves attack of the Fe(IV)-oxo species on the
phenol side chain of tyrosine to produce L-DOPA by
electrophilic aromatic substitution.10,11
2
used 9 GHz H-ESEEM spectroscopy to study the structural
relationship between the {FeNO}7 center and substrate taurine
in an α-ketoglutarate-dependent hydroxylase, taurine dioxyge-
nase.30 An encouraging result from both the ENDOR and
ESEEM studies was that the effective dipole−dipole distances
Our understanding of the changes in protein structure that
accompany the increased reactivity with oxygen once both
substrates are bound is incomplete. In the case of TyrH, only
structures of the inactive ferric enzyme are available, with and
without bound dihydrobiopterin; these do not show any change
in structure upon binding of this cofactor analogue.6,7 In
contrast, fluorescence anisotropy analyses of TyrH have shown
that the conformation of a mobile loop important for the
coupling of BH4 oxidation to the hydroxylation of tyrosine12 is
altered significantly upon binding of 6-methyl-5-deazatetrahy-
dropterin, with a further smaller change when an amino acid is
also bound.13 Structural data for the other two pterin-
dependent, nonheme Fe aromatic amino acid hydroxylases,
phenylalanine hydroxylase (PheH) and tryptophan hydroxylase
(TrpH), provide some additional insight because all three of
these enzymes are thought to utilize the same catalytic
mechanism.14,15 Comparison of the X-ray structures of the
Fe(II) form of PheH without ligands to a binary complex with
BH4 showed no significant changes in protein structure upon
pterin binding.15 Subsequent studies of a ternary complex of
PheH treated with BH4 and the slow amino acid substrates
thienylalanine and norleucine showed that the addition of the
amino acid induced a conformational change in the protein that
tightened the structure about the Fe(II) site and reduced the
distance between the metal ion and C4a of BH4 from 6.1 to 4.5
Å.16 The structure of the Fe(III) form of chicken TrpH
similarly shows that two active-site loops close in response to
the binding of tryptophan and imidazole.17 However, there is
no structure available of any aromatic amino acid hydroxylase
with just the amino acid substrate bound, although hydrogen/
deuterium exchange studies of PheH show that the phenyl-
alanine does bind in the absence of a pterin.18
2
derived from the anisotropy of the H hyperfine coupling
agreed well with distances between Fe and the appropriate
ligand protons, as predicted from X-ray crystal structures.
Furthermore, the measured orientations of the dipolar coupling
vector with respect to the Fe−NO bond axis were in general
agreement with chemical models for the catalytic mechanism.
MATERIALS AND METHODS
■
6-Methyltetrahydropterin (6-MPH4) and 6-methylpterin were
purchased from Schircks Laboratories (Jona, Switzerland). 6-
(2-Hydroxy-1-methyl-2-nitrosohydrazino)-N-methyl-1-hexan-
amine (MAHMA NONOate), ethylenediaminetetraacetic acid
(EDTA), L-tyrosine, and glycerol were from Sigma-Aldrich (St.
Louis, MO). L-3,5-2H2-tyrosine and deuterium gas were from
Cambridge Isotopes (Andover, MA). Potassium chloride and
ferrous ammonium sulfate were from Fisher (Pittsburgh, PA).
All other chemicals were of the highest purity commercially
available. Deuterated 6-MPH4 was synthesized by reduction of
6-methylpterin to the level of tetrahydropterin using deuterium
gas, as previously described.31
Wild-type TyrH was expressed in Escherichia coli and purified
as previously described.19 To remove iron from the protein, the
ammonium sulfate pellet at the end of the purification was
resuspended in 5 mM EDTA, 200 mM Hepes (pH 7.5), 10%
glycerol, and 0.1 M KCl and incubated on ice for 1 h. The
enzyme solution was then dialyzed against the same buffer
without EDTA and concentrated using Amicon Ultra-15 and
Ultra-4 centrifugal filters (Millipore Corp., Bedford, MA). The
enzyme samples for ESEEM were brought to a final glycerol
concentration of 30% (v/v) during the concentration stage.
The iron content of the apoenzyme was measured using a
PerkinElmer AAnalyst600 atomic absorption instrument.32
Typical iron content of an apoenzyme preparation was ∼0.1
equiv.
Stock solutions of MAHMA NONOate were prepared in
0.01 M NaOH just before the experiment and were kept on ice.
Concentrations of the MAHMA NONOate solutions were
determined in 0.01 M KOH using an ε250 of 7.3 mM−1 cm−1.33
Highly concentrated (∼50 mM) stock solutions of tyrosine and
3,5-2H2-tyrosine were prepared at pH 10. Stock solutions of the
protiated and deuterated 6-MPH4 were prepared in 2 mM HCl
to prevent autoxidation until the sample was made anaerobic.
Ferrous ammonium sulfate solutions were prepared fresh in 2
mM HCl. ESEEM samples were prepared inside an anaerobic
cuvette at 25 °C. Apo-TyrH (0.9−1.2 mM) and tyrosine (1−
1.7 equiv for complexes containing tyrosine) were placed at the
bottom of the cuvette. A ferrous ammonium sulfate solution
In this article, we present the results of an EPR spectroscopic
study, using the pulsed EPR method of electron spin echo
envelope modulation (ESEEM), to determine the structural
relationships between the paramagnetic Fe center and the
substrates tyrosine and 6-methyltetrahydropterin (6-MPH4) in
enzyme−substrate complexes. A comparative study of a TyrH
variant, E332A, is also presented; previous biochemical and
spectroscopic studies have shown that this residue plays a role
in the binding of the pterin cofactor and the successful coupling
of tetrahydropterin oxidation to amino acid hydroxylation.8,19
We have used the approach developed by Salerno20 and
broadly applied by Lipscomb and co-workers for EPR studies of
nonheme Fe enzymes.21−23 Specifically, nitric oxide (NO) has
been used as an O2 surrogate to allow EPR detection of the
24
3
Fe(II) site by creating an S = /2, {FeNO}7 species. For
TyrH, the resulting EPR signals under the conditions of our
measurements stem from the ms = 1/2 Kramer’s doublet and
show nearly axial spectra with effective g values near g = 4 and
2. In addition to enabling EPR detection, the binding of NO
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(0.9 equiv in ∼10 μL) was placed on the lower neck of the
cuvette. 6-MPH4 (∼1.5 equiv) and MAHMA NONOate
solutions were either placed in the side arms or on the upper
neck of the cuvette for large and small volumes, respectively.
Buffer conditions were 100 mM Mops (pH 7.0), 0.3 M KCl,
and 30% glycerol. The contents of the cuvette (total volume of
∼250 μL) were made anaerobic by the application of argon-
vacuum exchange for at least 20 min. The anaerobic enzyme
solution was then mixed with ferrous ammonium sulfate and
incubated for 10 min. This was followed by mixing with 6-
MPH4 (if the complex was to contain 6-MPH4). After a few
minutes of incubation, MAHMA NONOate (∼0.6 equiv of the
enzyme) at the upper neck of the cuvette was introduced to the
enzyme−substrate mixture. After ∼3 min, ∼200 μL of the
reaction mixture was quickly transferred to the quartz EPR
tubes (4 mm o.d., 707-SQ-250M, Wilmad, Buena, NJ) using a
glass pipet and immediately frozen in liquid nitrogen. (The
half-life of MAHMA NONOate is ∼35 s under these buffer and
temperature conditions (data not shown).) UV−vis spectra
collected at increasing concentrations of MAHMA NONOate
showed that NO was saturating under the concentrations used.
EPR measurements were made on a Bruker E-680X
spectrometer operating at X-band and equipped with a model
ER 4118X-MD-X5-W1 probe that employed a 5 mm dielectric
resonator. The temperature was maintained at 4.0 K using an
Oxford Instruments liquid helium flow system equipped with a
CF-935 cryostat and an ITC-503 temperature controller.
ESEEM data were collected using a three-pulse (stimulated
echo) sequence: 90°−τ−90°−T−90°, with 90° microwave
pulse widths of 16 ns (fwhm). The tau values were chosen to
suppress the hydrogen matrix contribution; because of short
phase memory times, they were restricted to less than 200 ns.
An integration window of 24 ns was used to acquire the spin
echo amplitude, and data set lengths were 512 points. A four-
step phase-cycling scheme was used to eliminate two-pulse spin
echoes and the dc offset voltage of the detection system from
the data.
nearly axial spectra with features near g = 4 and 2. These
spectra were modeled using a standard approach for S = /2
transition ions with a spin Hamiltonian that consisted of a zero-
field splitting term and an isotropic, or spin-only, electronic
Zeeman term with go = 2.00 (eq 1).38
3
⎡
⎤
2
z
5
4
E
D
2
x
2
y
̂
̂
̂
̂ ̂
(S − S )
H = g β S ·H + D S −
+
⎢
⎥
⎦
e
o
⎣
∼
(1)
∼
For our simulations, we took D = 10 cm−1 on the basis of
findings from EPR, magnetic susceptibility, and Mossbauer
̈
studies of other nonheme Fe dioxygenases and {FeNO}7 model
complexes.21,23,26,39 TyrH spectra were treated as composites of
two paramagnetic species and were fit by varying the rhombic
terms in the ZFS interaction, E/D, the strains in E, and the
speciation of the paramagnetic centers. Fitting was restricted to
the g = 4 region of the EPR spectrum and accomplished using
the function fminsearch available in MATLAB (The Math-
works, Natick, MA).
2
Orientation selected H-ESEEM spectra were fit across the
EPR spectrum using the saffron routine from EasySpin 4.040 for
spectral simulation together with a combination of the
MATLAB global optimization routines patternsearch and
fminsearch for nonlinear least-squares optimization of the
spin Hamiltonian parameters that describe the hyperfine
3
interaction. These calculations were done for the S = /2
electron spin system using the principal axis system (PAS) of
the ZFS tensor as the reference frame and the values for D, E,
and go that were obtained from fitting the corresponding cw-
EPR spectra. The deuterium hyperfine interaction was
described by a spin Hamiltonian that included nuclear Zeeman,
electron−nuclear hyperfine and nuclear quadrupole interac-
tions
gnβN
h
̂
̂
̂
̂
̂
Hhf = −
I ·H + S ·A·∼I + I ·Q·∼I
∼ ∼
∼
(2)
∼
where gn = 0.8574 for 2H, βN is the nuclear magneton,
S
and ∼
I
are the electron and nuclear spin angular momentum o∼perators,
The deuterium contributions to the ESEEM spectra were
elucidated using the ratio method and were performed using
the analysis software of the Bruker Xepr program.34 Three-
pulse ESEEM data were collected for samples containing
deuterated substrate or cofactor and matching reference
samples containing the corresponding protiated substrate
and/or cofactor. Both data sets were phase-corrected, and the
real portion of each was normalized to 1. The two normalized
data sets were then divided, tapered with a Hamming window
function, and Fourier transformed. ESEEM spectra were
obtained by taking the absolute value of the real part of the
transforms. Because the three-pulse ESEEM function is the sum
of product terms for the two coherence transfer pathways that
lead to formation of the stimulated echo,35 this procedure is
approximate and can give rise to cross-terms that appear as
peaks in the FFT if the modulations are deep.36 For the studies
presented here, we found that the quotient deuterium spectra
in the g = 2 region were free from such distortions but that
spectra in the g = 4 region, where deeper overlapping
respectively, and
H
is the applied magnetic field. Because the
deuterated tyrosi∼ne substrate and pterin cofactor are not
2
coordinated to Fe, the measured H-hyperfine couplings were
modeled with a through-space dipolar interaction described by
g g ββ
e n
hre3ff
e n
T =
(3)
The resulting axial deuterium hyperfine tensor, with principal
elements of (−T, −T, 2T), was rotated into the ZFS reference
frame using a standard “zyz” Euler rotation scheme to obtain
the 3 × 3 hyperfine coupling matrix, A, as denoted in eq 2. The
deuterium nuclear quadrupole interaction (nqi) was also
constrained to have axial symmetry because the electric field
gradient at the nucleus is dominated by the electrons of its σ
bond to carbon. The principal values of the nqi coupling tensor
are then (e2qQ)/(4h)(−1, −1, 2), and subsequent trans-
formation to the ZFS axis system by an Euler rotation scheme
yields the nqi coupling tensor, Q (eq 2). Because both the
hyperfine and the nuclear quadrupole interactions were
constrained to axial symmetry, only the second and third
Euler angles of the zy′z′′ rotation scheme will serve to reorient
1
modulations from coupled 14N and H were observed, often
2
contained spurious peaks. The resulting uncertainty in H-
ESEEM amplitudes was considered in our analysis and will be
discussed below.
Continuous wave EPR spectra were analyzed using the
pepper module of EasySpin 4.0.37 The cw-EPR spectra
observed for the various {FeNO}7 derivatives of TyrH showed
2
the tensors in the ZFS reference frame. As a result, our H-
ESEEM simulations considered six adjustable parameters: the
dipolar coupling strength, T, two Euler angles to orient the
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hyperfine tensor, βhf and γhf, the quadrupole coupling constant,
e2qQ/h, and two Euler angles, βnqi and γnqi, to transform the nqi
coupling tensor.
RESULTS
■
The cw-EPR spectra for all of the TyrH complexes studied
showed nearly axial spectra with line shape features near g = 4
and 2. As the g = 4 feature is most sensitive to changes in the
symmetry of the zero-field splitting interaction, it is the focus of
Figure 1, where the experimental spectra are portrayed with
The ESEEM analysis described in the Results section was
based on a comparison of simulated and experimentally
determined 2H spectra. To facilitate this comparison, the
time domain output from the saffron (EasySpin) calculation
was normalized to 1 and then treated with the same analysis
procedure as described for our experimental data. Using this
approach, it was possible to fit 2H-ESEEM peak amplitudes and
line shapes. The fitting procedure was based on minimizing χ2
for each sample, where
calc
exp 2
N
f max
(Y − Y
)
ij
ij
χ2 =
∑ ∑
σ2
i=1 j=f min
(4)
The sums of eq 4 are taken over discrete ESEEM spectra, i,
collected at N different magnetic field positions and across the
2H-ESEEM line shape at each field position for frequency bins
indexed from fmin to fmax. Typically, our analysis considered 5
to 6 ESEEM spectra collected at different field positions across
the EPR spectrum. The indices for line shape comparisons were
determined from the individual spectra by selecting a span of
2
8−12 points that encompassed the H-ESEEM response. A
constant value for the experimental standard deviation, σ = 0.1,
was estimated from the noise typically observed over the 5−10
MHz range of our ESEEM spectra and was used for all of the
fits. The overall quality of a fit for each sample was judged by
normalizing eq 4
Figure 1. g = 4 region of cw-EPR spectra collected for {FeNO}7
derivatives of TyrH treated with (a) 6-MPH4, (b) tyr, and (c) 6-MPH4
plus tyr as well as (d) the E332A variant of TyrH treated with 6-MPH4
plus tyr. The solid lines are the experimental spectra, and the
simulations performed using the parameters in Table 1 are shown with
dashed lines. The experimental data were collected under the following
conditions: microwave frequency, 9.68 GHz; microwave power, 0.0063
mW; field modulation amplitude, 0.8 mT; 10 kHz modulation
frequency; time constant, 40 ms; and sample temperature, 4.0 K.
χ2
χν2 =
npts − L
(5)
where npts is the total number of data points considered in the
analysis and L is the number of parameters varied in the fitting
procedure. For the calculations described in this article, npts
ranged from 55 to 65 and L = 6.
The errors in the spin Hamiltonian parameters that were
derived from the fitting procedure described above are reported
as 1 SD. This corresponds to an increase in the best fit value,
or minimum value, of χ2ν by 1. These standard deviations were
estimated in two ways, and the largest values obtained are
reported along with the overall fitting results in Table 2. The
first approach to estimating parameter errors was based on
numerical calculation of the covariance matrix, C, using the
Hamiltonian parameters that gave the best fit value of χ2.41,42
The standard deviations were then given by the square roots of
the diagonal elements of C. This approach assumes that the χ2
function is quadratic near the minimum found for each
parameter. In practice, we found that the minima in plots of χ2
versus T, βhf, and e2qQ/h were quadratic or cusplike and that
the above procedure worked well for estimating the standard
deviation in their values. The remaining three parameters, γhf,
βnqi, and γnqi, showed individual χ2 functions that were either
edgelike or were characterized by shallow wells that were less
than χ2ν + 1 in depth. For these parameters, errors were also
estimated directly from plots of χ2 versus individual parameter
values and compared with those obtained from the covariance
matrix. For these cases, the largest value determined for the
standard deviation is reported in Table 2.
solid lines. These spectra were analyzed using EasySpin as
discussed above, and the results of the simulations are provided
as dashed lines in Figure 1 and summarized by the spin
Hamiltonian parameters given in Table 1. Corresponding full
EPR spectra, showing both the g = 4 and g = 2 regions, are
provided in Figure S1 of the Supporting Information. Figure 1a
shows the cw-EPR line shape in the g = 4 region for TyrH
treated with 6-MPH4 and NO. This spectrum is similar to that
obtained for TyrH without added tyrosine or 6-MPH4 (data
Table 1. Cw-EPR Parameters of Tyrosine Hydroxylase NO
Complexes
sample
percent contribution
|E|/D
Estrain (MHz)
TyrH{6-MPH4}
95
5
0
7800
3700
6400
1900
1900
3700
3200
4000
0.025
0.017
0.043
0.020
0.050
0.020
0.049
TyrH{tyr}
91
9
TyrH{tyr, 6-MPH4}
E332A{tyr, 6-MPH4}
63
37
58
42
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not shown) in that a single broad feature centered at g = 4.00
characterized by |E|/D = 0 dominates the spectrum. For TyrH
treated with tyrosine and NO (Figure 1b), the data were
analyzed as a composite of two species: a majority species
(91%) with |E|/D = 0.02 and a minority species characterized
by |E|/D = 0.04. The cw-EPR lineshapes in the g = 4 region for
the two quaternary complexes, wild-type TyrH and the variant
E332A treated with tyr, 6-MPH4, and NO, are shown in Figure
1 panels c and d, respectively. These spectra are nearly identical
and show that two species are also present at 4.0 K: a majority
species that represents about 60% of the {FeNO}7 sites with an
|E|/D = 0.02 and a species that accounts for the remaining 40%
of the EPR active sites characterized by an |E|/D = 0.05. Table 1
also shows that the strain, or spread, in E values decreases
modestly as substrates are added. This observation is in
agreement with findings from the X-ray studies of PheH and
TrpH where the protein structures were observed to tighten
around the Fe(II) site as a result of substrate and cofactor
binding.16,17
The results tabulated in Table 1 show that all of the
paramagnetic centers being considered in this work are
mixtures characterized by modest differences in |E|/D of 0.02
to 0.03. Previous EPR studies of coordinatively unsaturated
{FeNO}7 model complexes have shown that changes in the
rhombicity, |E|/D, of the ZFS interaction of 0.04 to 0.05 can
arise from modest changes of 18° in Fe−NO bond angle
brought about by steric crowding.43 It is also possible that
differences in the orientation of the bound NO ligand with
respect to rotation about the Fe−NO bond could lead to the
observed speciation. A recent DFT study of the {FeNO}7
adduct of taurine−α-ketoglutarate dioxygenase reported two
minima for the potential energy surface describing rotation
about the Fe−NO bond, with projections of the NO favoring
alignment with the carboxylate oxygens of the glutamate ligand
and bound α-ketoglutarate.27 Therefore, we chose to treat this
speciation explicitly in our orientation-selective analysis of the
ESEEM data, assuming that NO was coordinated to a unique
position on Fe for each sample and that the speciation observed
in the cw-EPR spectra arose from two conformations or
rotamers present in frozen solution. In principle, treating two
rotamers with a single set of Hamiltonian parameters for the
purpose of defining the hyperfine interaction (eq 2) is
problematic because the values of γhf and γnqi for each rotamer
should be distinct. In practice, it was found that the near-axial
nature of the paramagnetic centers, with a majority having |E|/
D = 0.02, made our measurements less sensitive to γhf and
allowed us to approximate the spin Hamiltonian with single
values for γhf and γnqi.
Figure 2a shows normalized three-pulse ESEEM data sets for
the ternary complex of TyrH/NO/tyr (solid line) and the
corresponding sample made with 3,5-2H-tyrosine (dashed line)
collected at 300 mT (see Scheme 1 for the substrate numbering
key). The time domain data sets were both normalized to 1 and
are nearly identical. The quotient modulation function is shown
in Figure 2b and reveals 2H ESEEM with a modulation depth of
about 6% of the overall signal amplitude. The corresponding
2H-ESEEM frequency spectrum of these data is represented by
the open and filled circles (with error bars) centered about the
deuterium Larmor frequency of 2.0 MHz in Figure 3e. Figure 3
also shows the results of repeating the measurements described
above at five other magnetic field positions spanning the EPR
spectrum from g = 4 to g = 2. In the g = 4 region, Figure 3a,b,
broader line shapes that are nearly flat at their maximum
Figure 2. (a) Three-pulse ESEEM data collected for TyrH/NO/
tyrosine (solid line) and TyrH/NO/3,5-2H-tyrosine (dashed line)
under the following conditions: field strength, 300 mT; microwave
frequency, 9.684 GHz; tau, 156 ns; and sample temperature, 4.0 K. (b)
Ratio of the two time domain data sets shown in panel a.
amplitude, were observed. Spectra obtained in the g = 2 region,
Figure 3e,f, show larger amplitudes but narrower line shapes.
For all six field positions, the data points displayed as filled
circles with error bars were used for analysis. These data points
are centered about the deuterium Larmor frequency and were
chosen because they exceeded an amplitude threshold of 0.4−
0.6 and are most likely free of spurious peaks that can result
from data division. Evidence for these spurious peaks can be
seen in all six of the ESEEM quotient spectra shown in Figure 3
by tracing through the points represented by the open circles
2
on the low- and high-frequency sides of the H-ESEEM peaks.
2
The solid lines shown in Figure 3 are simulated H-ESEEM
spectra that represent the best fit of the spin Hamiltonian
model provided in eq 2 for a single, coupled deuteron to the
data. The simulations took into account the complex nature of
the cw-EPR spectrum of the TyrH/NO/tyr sample, as
summarized in Table 1, by including contributions from both
paramagnetic centers, |E|/D = 0.02 and 0.04, weighted by the
scaling factors, 0.91 and 0.09, respectively. The results yield a
2H-dipolar coupling, T = 0.18 MHz, which corresponds to an
effective dipole−dipole distance of 4.1 Å, as obtained from eq 3.
The orientation of the principal axis system (PAS) of the
hyperfine coupling tensor with respect to the PAS of the ZFS
tensor was described by the Euler angles βhf = 26° and γhf = 0°.
These parameters and those obtained for the 2H-nuclear
quadrupole interaction are provided in Table 2. The overall
quality of the nonlinear least-squares fit to the data was
expressed as χ2ν = 1.2 in Table 2 and was computed using eq 5.
The errors reported for the best fit spin Hamiltonian
parameters determined for the coupled tyrosine deuteron were
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standard deviations, Table S3, and a full covariance matrix,
Table S4, in the Supporting Information. The standard
deviations are the square roots of the diagonal elements of
the covariance matrix and are all smaller than the values derived
from the single-parameter χ2 plots. However, the off-diagonal
elements of the covariance matrix show a substantial degree of
coupling between γhf and γnqi and lesser interactions between
the parameters that describe the deuterium nuclear quadrupole
interaction, e2qQ/h, βnqi, and γnqi. The effects of this covariance,
or coupling, between γnqi and γhf were explored further by
repeating the fitting procedure with the values for the hyperfine
coupling parameters, T and βhf, fixed to their best fit values of
0.18 MHz and 26°, respectively. For each optimization, γhf was
set to a fixed value between 0 and 90° and the three parameters
that describe the nqi, e2qQ/h, βnqi, and γnqi, were varied to find a
minimum in χ2. Although the best fits to the data were realized
for values of γhf in the range from 0 to 20°, optimizations that
gave χ2 values below the 1σ threshold defined by χ2ν + 1 could
be found for all values of γhf over the 0−90° range. Each of
these calculations yielded values for e2qQ/h and βnqi that varied
little from those found for the best fit solution, 0.27 MHz and
62°, respectively (Table 2), and fell within the standard
deviations measured from the single-parameter χ2 plots (Figure
S2). In contrast, the optimized values of γnqi tracked the fixed
value assigned to γhf so that |γnqi − γhf| = 38 7°. The modest
dependence of the spectral fitting on γhf is not surprising given
that the cw-EPR spectrum is nearly axial. The sensitivity of
these simulations to βnqi and |γnqi − γhf| is expected because
these angles serve to define the relative orientation of the nqi
with respect to the electron−nuclear hyperfine interaction.
2
Figure 3. H-ESEEM spectra (open and filled circles) obtained by
Fourier transformation of ESEEM data from the ternary complexes of
TyrH/NO/3,5-2H-tyrosine divided by TyrH/NO/tyr. Data were
collected at 9.684 GHz using the following field positions and tau
values: (a) 190 mT, 124 ns; (b) 200 mT, 116 ns; (c) 225 mT, 104 ns;
(d) 275 mT, 84 ns; (e) 300 mT, 156 ns; and (f) 320 mT, 148 ns. The
solid lines in each frame are best fit ESEEM simulations to the data
points represented by the filled circles with error bars using the
following spin Hamiltonian parameters: gn = 0.8574, T = 0.18 MHz,
βhf = 26°, γhf = 0°, e2qQ/h = 0.27 MHz, βnqi = 62°, and γnqi = 38°.
2
Figure 4 shows a composite drawing of the H-ESEEM
spectra collected for the quaternary complex of TyrH/NO/
3,5-2H-tyr/6-MPH4 collected at six different field positions
across EPR spectrum. Overall, these data show smaller 2H-peak
amplitudes than those observed for the ternary complex with
just 3,5-2H-tyr and NO (Figure 3). In the g = 4 region at 190
(Figure 4a) and 210 mT (Figure 4b), amplitudes were reduced
by about 20%, whereas in the g = 2 region, amplitude
reductions ranged from 20 to 50%. Unfortunately, as shown by
the open circles in the spectra, these data were also plagued by
distortions or spurious peaks that stem from the division
process used to reveal the deuterium ESEEM spectra. As a
result, the line shape data were less useful in guiding the
analysis. The results of spectral simulations are shown with
solid lines in Figure 4 and yield a dipolar coupling of T = 0.12
MHz, or an effective dipole−dipole distance of 4.7 Å, for a
determined from plots of χ2 as a function of changing individual
parameter values while holding the other spin Hamiltonian
parameters at their best fit values. For the calculation at hand
on the TyrH/NO/3,5-2H-tyr sample, these single-parameter χ2
plots are provided as Figure S2 of the Supporting Information.
The standard deviations given in Table 2 are derived from
measuring the width of each parameter distribution at χ2ν + 1, χ2
= 130 for this case (marked with an arrow in Figure S2). For
this sample, the single-parameter χ2 plots for the dipolar
coupling, T, βhf, and e2qQ/h were narrow and nearly parabolic
in shape, whereas the plots obtained for γhf, βnqi, and γnqi were
broader, reflecting greater uncertainty.
2
model that considered a single, coupled H. The Euler angles
for orienting the hyperfine coupling tensor within the ZFS axis
system were βhf = 94 10° and γhf = 20°. The value of χ2ν = 2.2
obtained for these fits was substantially worse than for the
ternary complex TyrH/NO/3,5-2H-tyr. Single-parameter χ2
plots and the covariance matrix calculated from the best fit
A covariance analysis was also done using the best fit
parameter set from Figure 3, and the results are tabulated as
2
Table 2. H-ESEEM Analysis Results
sample
T (MHz)
reff (Å)
βhf (o)
e2qQ/h (MHz)
βnqi (o)
|γnqi − γhf|
χν2
TyrH {2H-tyr}
0.18 0.01
0.12 0.01
0.12 0.01
0.14 0.01
0.14 0.01
<0.06
4.1 0.1
4.7 0.2
4.7 0.2
4.4 0.2
4.4 0.2
>5.9
26
5
0.27 0.05
0.34 0.06
0.29 0.05
0.22 0.06
0.20 0.06
62 12
69 10
44 16
70 57
66 60
38 20
20 80
14 43
26 90
18 90
1.2
2.2
1.2
1.2
1.0
TyrH {2H-tyr, 6-MPH4}
E332A {2H-tyr, 6-MPH4}
94 10
89
66
65
9
5
5
2
TyrH {tyr, H-6-MPH4}
2
E332A {tyr, H-6-MPH4}
TyrH {2H-6-MPH4}
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2
Figure 5. Three-pulse H-ESEEM spectra obtained for the ternary
complex TyrH/NO/6,7-2H-6-MPH4 (dashed line) and the quaternary
complex TyrH/NO/tyr/6,7-2H-6-MPH4 (solid line) at (a) 178 mT, τ
= 132 ns and (b) 320 mT, τ = 148 ns.
2
Figure 4. H-ESEEM spectra (open and filled circles) obtained by
Fourier transformation of ESEEM data from the quaternary complexes
of TyrH/NO/3,5-2H-tyr/6-MPH4 divided by TyrH/NO/tyr/6-MPH4.
Data were collected at 9.68 GHz using the following field positions
and tau values: (a) 190 mT, 124 ns; (b) 210 mT, 112 ns; (c) 225 mT,
104 ns; (d) 250 mT, 92 ns; (e) 300 mT, 156 ns; and (f) 320 mT, 148
ns. The solid lines in each frame are best fit ESEEM simulations to the
data points represented by the filled circles with error bars using the
following spin Hamiltonian parameters: gn = 0.8574, T = 0.12 MHz,
βhf = 94°, γhf = 20°, e2qQ/h = 0.34 MHz, βnqi = 71°, and γnqi = 40°.
2
illustrates a complication that we avoided in our H-ESEEM
analysis by not considering data collected at field positions
below 190 mT (g = 3.6).
2
Figure 6 shows a composite drawing of H-ESEEM spectra
observed for the quaternary complex of TyrH/NO/tyr/6,7-2H-
6-MPH4 at five magnetic-field positions across the EPR
spectrum. Overall, the 2H-ESEEM spectral amplitudes are
greater for this sample, and the lineshapes are narrowed and
more symmetrical about the 2H-Larmor frequency as compared
parameters are shown in Figure S5 and Tables S6 and S7,
respectively. Although this quaternary complex shows a higher
percentage of more rhombic {FeNO}7 centers, 40% of the sites
have |E|/D = 0.05; our spectral fits were also insensitive to γhf.
As γhf was varied from 0 to 90° in the fitting procedure,
optimized values of χ2ν varied from a low of 2.2 at γhf values of
20−40° to a maximum of χ2ν = 2.6 at 90°. The best fit values of
e2qQ/h = 0.34 MHz and βnqi = 69° varied by just 0.02 MHz
and 2° over this full range of γhf, whereas γnqi tracked the γhf
values such that |γnqi − γhf| = 20 8°.
2
to those observed for the H-tyr samples shown above. The
solid lines in Figure 6 are the best fit simulations to the data
points showed as filled circles and yield a dipolar coupling of T
= 0.14 MHz, or an effective dipolar distance of 4.4 Å. The
orientation of the hyperfine coupling tensor with respect to the
ZFS PAS was given by βhf = 66 5°, and a γhf value of 0° was
used for the simulations shown. Single-parameter χ2 plots and
the covariance matrix calculated from the best fit parameters are
shown in Figure S8 and Tables S9 and S10, respectively. As
with the previous analyses, fits of these data were not sensitive
to γhf, and optimizations done as a function of γhf gave
parameters for the nqi of e2qQ/h = 0.22 MHz, βnqi = 70°, and
|γnqi − γhf| = 26°. The error estimates for these parameters
(Table 2) come from the single-parameter χ2 plots of Figure S8.
The errors for the Euler angles describing the orientation of the
nqi principal axes with respect to the ZFS PAS are quite large
for this sample, as reflected in the unique edgelike shape of the
single-parameter χ2 plot for βnqi and the shallow nature of the
global minima found for γnqi.
Figure 5 shows 2H-ESEEM spectra collected for two different
samples treated with 6,7-2H-6-MPH4 at 178 (Figure 5a) and
320 mT (Figure 5b) (the pterin numbering scheme is provided
in Scheme 1). The spectra represented with the solid lines are
for a quaternary complex of TyrH/NO/tyr/6,7-2H-6-MPH4
and show peaks centered at the deuterium Larmor frequency of
1.2 MHz at 178 mT and 2.1 MHz at 320 mT. The spectra
represented by the dashed lines are from a ternary complex of
TyrH/NO/6,7-2H-6-MPH4 and are void of any H-ESEEM
response. Figure 5a also shows a peak at 1.6 MHz that is
present for both samples. This peak is typical of the distortions
that we observed in the g = 4 region from data division and
2
Composite figures of the 2H-ESEEM spectra collected for the
quaternary complexes of the E332A variant of TyrH with
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2
2
Figure 7. H-ESEEM spectra (open and filled circles) obtained by
Fourier transformation of ESEEM data from the ternary complexes of
E332A/NO/tyrosine/6,7-2H-6-MPH4 divided by E332A/NO/tyr/6-
MPH4. Data were collected at 9.68 GHz using the following field
positions and tau values: (a) 190 mT, 124 ns; (b) 210 mT, 112 ns; (c)
250 mT, 92 ns; (d) 300 mT, 156 ns; and (e) 320 mT, 148 ns. The
solid lines in each frame are best fit ESEEM simulations to the data
points represented by the filled circles with error bars using the
following spin Hamiltonian parameters: gn = 0.8574, T = 0.14 MHz,
βhf = 65°, γhf = 0°, e2qQ/h = 0.20 MHz, βnqi = 66°, and γnqi = 18°.
Figure 6. H-ESEEM spectra (open and filled circles) obtained by
Fourier transformation of ESEEM data from the ternary complexes of
TyrH/NO/tyrosine/6,7-2H-6-MPH4 divided by TyrH/NO/tyr/6-
MPH4. Data were collected at 9.68 GHz using the following field
positions and tau values: (a) 190 mT, 124 ns; (b) 210 mT, 112 ns; (c)
250 mT, 92 ns; (d) 300 mT, 156 ns; and (e) 320 mT, 148 ns. The
solid lines in each frame are best fit ESEEM simulations to the data
points represented by the filled circles with error bars using the
following spin Hamiltonian parameters: gn = 0.8574, T = 0.14 MHz,
βhf = 66°, γhf = 0°, e2qQ/h = 0.22 MHz, βnqi = 70°, and γnqi = 26°.
6,7-2H-6-MPH4 and 3,5-2H-tyr are shown in Figures 7 and 8,
respectively. A comparison of the spectra for E332A/NO/tyr/
6,7-2H-6-MPH4 (Figure 7) and wild-type TyrH/NO/tyr/
6,7-2H-6-MPH4 (Figure 6) show only subtle differences in
amplitude and line shape across the EPR spectrum, with the
most notable difference being a difference in line shape at 250
mT (compare Figures 6c and 7c). The solid lines in Figure 7
are the best fit simulations to the spectral data and provide
nearly identical values to those obtained above for the wild-type
enzyme (Table 2). A comparison of the field-dependent
ESEEM spectra obtained for the quaternary complexes of
E332A/NO/3,5-2H-tyr/6-MPH4 (Figure 8) and wild-type
TyrH/NO/3,5-2H-tyr/6-MPH4 (Figure 4) appear to differ
little except for the poorer signal-to-noise ratio observed for the
wild-type protein samples. Analyses of these data led to
simulations summarized by the solid lines drawn in Figure 8
and provide a dipolar coupling of T = 0.12 MHz and a
hyperfine tensor orientation described by βhf = 89 9°. The χν2
value for the fit of these data was 1.2, supplying more
confidence for the analysis of the wild-type TyrH/NO/3,5-2H-
tyr/6-MPH4 data presented above. Single-parameter χ2 plots to
support the data analyses presented for the quaternary
complexes of E332A/NO/tyr/6,7-2H-6-MPH4 and E332A/
NO/3,5-2H-tyr/6-MPH4 are provided in Figures S11 and S12
in the Supporting Information.
DISCUSSION
■
2
The H-ESEEM spectra described here establish that substrate
amino acid and tetrahydropterin cofactor bind to TyrH
differently in the quaternary complex, TyrH/NO/tyr/6-
MPH4, as compared to their positions in the respective ternary
complexes, TyrH/NO/tyr and TyrH/NO/6-MPH4. For the
TyrH/NO/6,7-2H-6-MPH4 ternary complex, no H-ESEEM
2
could be detected above the noise floor of the experiment. The
noise level can be estimated from these spectra as 0.3 to 0.4 at g
= 4 and 0.1 to 0.2 at g = 2 (Figure 5). Spectral simulations done
using the Euler angles determined for the TyrH/NO/tyr/
6,7-2H-6-MPH4 quaternary complex showed that a reff of 5.9 Å
2
would provide H signal amplitudes just above the noise level.
Therefore, for the ternary complex of TyrH/NO/6,7-2H-6-
MPH4, we conclude that the closest deuteron is greater than 5.9
Å from the {FeNO}7 center. H-ESEEM spectra obtained at
2
multiple field positions from the cofactor 6,7-2H-6-MPH4 when
tyrosine is bound to the enzyme showed a dipolar coupling of T
= 0.14 0.01 MHz, which translates into an effective dipole−
dipole distance of 4.4 0.2 Å (eq 3). These findings provide a
structural explanation for the observation that the initial
reaction between Fe(II)-bound O2 and BH4 in TyrH requires
the binding of tyrosine.44 Specifically, the binding of substrate
tyrosine results in structural changes at the active site that bring
the 6-MPH4 cofactor into position for the first step of the
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determined only for the ternary complex of TyrH/NO/3,5-2H-
tyr.
Figure 9a summarizes the deuterium coupling data for the
ternary complex of TyrH/NO/3,5-2H-tyr. The near-axial
2
Figure 8. H-ESEEM spectra (open and filled circles) obtained by
Fourier transformation of ESEEM data from the ternary complexes of
E332A/NO/3,5-2H-tyr/6-MPH4 divided by E332A/NO/tyr/6-MPH4.
Data were collected at 9.68 GHz using the following field positions
and tau values: (a) 190 mT, 124 ns; (b) 210 mT, 112 ns; (c) 225 mT,
104 ns; (d) 250 mT, 92 ns; (e) 300 mT, 156 ns; and (f) 320 mT, 148
ns. The solid lines in each frame are best fit ESEEM simulations to the
data points represented by the filled circles with error bars using the
following spin Hamiltonian parameters: gn = 0.8574, T = 0.12 MHz,
βhf = 89°, γhf = 20°, e2qQ/h = 0.29 MHz, βnqi = 44°, and γnqi = 34°.
2
Figure 9. Cone diagrams for schematic representation of the H spin
Hamiltonian parameters given in Table 2 and obtained for (a) the
ternary complex of TyrH/NO/3,5-2H-tyr and (b) the quaternary
complexes of TyrH/NO/3,5-2H-tyr/6-MPH4 (red) and TyrH/NO/
tyr/6,7-2H-6-MPH4 (green). (c) Stick diagram of the catalytic site of
PheH crystallized with thienylalanine and BH4 (taken from PDB file
1MMK). The vectors show possible orientations for the Fe−NO bond
axis.
catalytic mechanism. The effective cofactor distances deter-
mined in this study are similar to those determined for PheH
by X-ray crystallography. Those studies showed that the
addition of the substrate analog thienylalanine to PheH loaded
with BH4 resulted in the movement of the C6 carbon of bound
BH4 from a distance of 7.3 Å from the Fe to 5.0 Å.15,16
symmetry of the ZFS and the associated lack of sensitivity to
γhf, as summarized above, create a model where βhf and reff
describe a cone about the g = 2.00 axis, an axis that is nearly
coincident with the Fe−NO bond,25 where the coupled
deuteron is located. Our results show a single deuteron at an
effective distance of 4.1 Å from the paramagnetic center that
lies on the base of a cone that makes a 26° angle with the Fe−
NO bond axis. For this particular adduct, a second cone
representing the information held in the nqi parameters can be
constructed with its symmetry axis parallel to the Fe−NO bond
direction and its apex centered on the coupled nucleus. This
second cone makes an angle of βnqi = 62° with its symmetry
axis. A vector (Figure 9a) that is colinear with the C−2H bond
associated with this deuteron is then confined to the surface of
this cone and positioned |γnqi − γhf| = 38° from where the
cone surface intersects the plane defined by the Fe−NO bond
In addition to the structural information derived from the
effective dipolar distance, the 2H-ESEEM analysis also provides
Euler angles, βhf and γhf, that define the position of the dipolar
coupling vector in the ZFS axis system as well as a set of nqi
angles that define the orientation of the C−2H bond of the
coupled deuteron within that same axis system. Overall, we
found that our analyses were not sensitive to γhf. Although
specific values or a range of values for this parameter were
needed to obtain the best fit values of χ2ν, it was possible to fit
the data with modest adjustments of the nqi parameters so that
any value of γhf could be used to obtain a fit that fell beneath
the threshold defined by the best fit value of χ2ν + 1. Although
there was some dependence of the best fit values of e2qQ/h,
βnqi, and |γnqi − γhf| on the value of γhf chosen for an
optimization, their variation easily fell within the uncertainty
derived from single-parameter χ2 plots. The errors provided for
the analysis results in Table 2 show that the dipolar coupling
strength, T, and orientation, βhf, can be determined with
reasonable certainty for all of the enzyme forms studied. The
angles describing the orientation of the nqi were accurately
2
and the coupled H.
When 6-MPH4 is added to the protein to form the
quaternary complex of TyrH/NO/3,5-2H-tyr/6-MPH4, the
effective dipolar distance for the coupled tyr deuteron lengthens
to 4.7 Å, and the angle that reff makes with the Fe−NO bond
axis is increased to 94° (Figure 9b). Although substantial
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structural changes in response to pterin binding to TyrH have
been observed in fluorescence anisotropy studies of a flexible
polypeptide loop associated with the metal binding site,45 X-ray
crystallographic studies of TyrH have shown that no structural
changes are associated with the binding of 7,8-dihydrobiopterin
to the ferric enzyme.7 Considering these previous findings, it is
most likely that the large change in βhf reflects a change in the
coordination of NO with respect to bound tyrosine when 6-
MPH4 is bound. It is instructive to view these changes in terms
of the X-ray crystal structure for the catalytic domain of PheH
crystallized in the presence of BH4 and soaked anaerobically
with a slow-substrate, thienylalanine, PDB designation 1MMK.
A stick drawing of the Fe site of this structure created with
MacPyMol is shown in Figure 9c. The structure shows a four-
coordinate Fe(II) with the side chain of the glutamate ligand
coordinated to Fe(II) in a bidentate fashion. The two histidine
ligands that make up the balance of the facial triad are bound so
that one is nearly planar with the glutamate oxygens forming an
equatorial plane and the other is bound axially, essentially trans
to the closest proton of the substrate analogue. The protons
shown in this structure were added by the viewing program
with the positions occupied by the closest substrate deuteron
and the 6,7 deuterons of 6-MPH4 colored in purple. This
structure shows that the closest proton on thienylalanine is 4.1
Å from the Fe(II) center, whereas the distances to the
deuterons on the 6 and 7 carbons of BH4 are 5.6 and 7.1 Å
study indicates that the cofactor is bound closer to the Fe in the
TyrH/NO/tyr/6-MPH4 complex than what is predicted from
the corresponding PheH crystal structure.
The possibility that NO remains bound to the position where
it is in-line with tyr, along the vector labelled 1 in Figure 9c, for
the quaternary complex containing 6-MPH4 was examined by
2
constraining fits of the H-ESEEM data from the TyrH/NO/
3,5-2H-tyr/6-MPH4 (Figure 4) and E332A/NO/3,5-2H-tyr/6-
MPH4 (Figure 8) samples so that βhf was <45°. For both
enzyme samples, a minimum in χ2 was found for βhf = 17°, with
the normalized value, χ2ν, being 3.1 for wild-type TyrH and 2.1
for the E332A variant. These values are 0.9 higher than the χν2
values reported for the βhf ≅ 90° solutions discussed above
(Table 2). Our reasons for favoring the βhf ≅ 90° solutions
were that they gave the best values of χ2ν and that this
orientation for the principle axis of the ZFS tensor provided a
ready explanation for the combined lengthening of the effective
dipolar distance observed for 3,5-2H-tyr in response to pterin
binding and the shorter dipolar distance determined for
coupled 6,7-2H-6-MPH4 in the TyrH quaternary complex as
discussed above.
Recently, Olsson and co-workers have used the results of
DFT calculations and NMR relaxation measurements to
propose a structure for the catalytic site of PheH in the
presence of BH4 and bound O2. Their structure, shown in
Figure 6a of their paper, is based on the 1MMK crystallographic
structure for the catalytic domain of PheH in the presence of
bound BH4 and thienylalanine. This structure brings the C4-
carbonyl oxygen of BH4 to within 1.9 Å of the Fe(II), close
enough to bind to the metal ion, but it leaves the distance
between the C6−2H on the pterin and Fe(II) at 5.8 Å, which is
1.4 Å longer than the distance measured in our study.46
Unfortunately, their structure shows the pterin binding to the
metal at essentially the same position where we propose the
NO to bind when both substrate tyr and 6-MPH4 are present.
At this point, the best fits to our data preclude pterin binding to
Fe(II), but more measurements will be needed to evaluate this
intriguing proposal.
2
from the Fe(II), respectively. Our H-ESEEM results for the
ternary complex of TyrH/NO/3,5-2H-tyr fit well with this
structure if the NO is coordinated at a position that is opposite
the axial histidine ligand, or in-line with the substrate,
consistent with the vector labelled 1 in Figure 9c.
For the quaternary complexes of TyrH/NO/3,5-2H-tyr/6-
MPH4 and Tyr/NO/tyr/6,7-2H-6-MPH4, the orientations of
the 2H-dipolar coupling vectors can be explained if NO
coordinates to Fe in an equatorial, or off-line, fashion like that
of the vector labelled 2 in Figure 9c. However, the distance
from the {FeNO}7 center to the closest coupled deuteron of
the pterin as predicted from the dipolar coupling of 0.14 MHz
is 4.4 Å, which is 1.2 Å shorter than the distance between Fe(II)
and the C6−proton of cofactor BH4 in the PheH structure of
Figure 9c. Likewise, the dipolar coupling of 0.12 MHz
measured for the closest deuteron of substrate tyrosine
translates into a dipole−dipole distance of 4.7 Å, a 0.6 Å
increase from the 4.1 Å distance measured for the TyrH/NO/
3,5-2H-tyr ternary complex by ESEEM and estimated from the
X-ray crystal structure of PheH reproduced in Figure 9c. These
discrepancies are consistent with a modest distribution of
unpaired electron spin density onto the NO ligand with the
coupling between the {FeNO}7 paramagnetic center and the
closest deuteron of tyr providing a measure of the size of the
effect. Specifically, if one assumes that both substrate tyr and Fe
remain at the same physical distance from one another and that
the presence of bound 6-MPH4 serves only to redirect the
coordination of NO, then the reorientation of the ligand results
in a 0.06 MHz reduction in the dipolar coupling. A reduction of
this size in the dipolar coupling measured for the C6−2H of 6-
MPH4, T = 0.14 MHz, would increase the dipolar distance
estimated from the point dipole−dipole approximation from
4.4 to 5.3 Å. Although this estimate is in much better
agreement with the pterin locale reported in the PheH crystal
structure, it is a crude approximation that begs further
theoretical and experimental study. It is possible that the
short effective dipolar distance measured for the C6−2H in this
2
Table 2 summarizes our H-ESEEM findings for quaternary
complexes of the variant E332A. A comparison of these findings
with those of the wild-type enzyme shows that the structural
relationship between the {FeNO}7 paramagnetic center and
coupled deuterons of both substrate tyrosine and cofactor 6-
MPH4 are identical given experimental error. This is consistent
with previous spectroscopic studies of the E332A TyrH/tyr/6-
MPH4 complex that showed that the mutant still exhibits the
change from 6- to 5-coordinate upon binding both substrates
and that the electronic properties of the mutant complex are
very similar to that of the wild-type enzyme.8 Both results are
somewhat surprising because the side chain of this glutamate
residue forms two hydrogen bonds with the pterin cofactor and
the E332A enzyme oxidizes tetrahydropterins without hydrox-
ylating tyrosine.19 (Although the mutant enzyme also exhibits a
10-fold increase in the Km value for 6-MPH4, the concen-
trations of substrates used here were sufficiently high to
saturate the mutant protein.) One explanation for the
uncoupling of the mutant enzyme despite formation of an
apparently normal reactive complex, previously proposed by
Chow et al., is that a critical role of Glu332 is to transfer a
proton to the proper oxygen atom in the proposed peroxo
intermediate, facilitating the heterolytic cleavage of the
oxygen−oxygen bond required for formation of the Fe(IV)-
oxo hydroxylating intermediate.8 Alternatively, the lost hydro-
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Biochemistry
Article
gen bonds because of the mutation may destabilize the peroxo
intermediate sufficiently that it breaks down unproductively
before the Fe(IV)-oxo is formed; spectroscopic analyses carried
out at liquid-helium temperatures would be unlikely to detect
such kinetic effects. Because the present study has used only a
single dipolar coupling to characterize the positions of the
cofactor and substrate, it is not possible to form conclusions
about their conformations with respect to the coordination
sphere of Fe. An intriguing issue that may pertain to the
coupling of the two hydroxylation reactions that comprise the
TyrH catalytic mechanism involves the coordination of NO to
Fe(II) and how well the behavior observed in our studies
represents the actual chemistry of O2. Previous biochemical
studies have shown that the addition of both substrate and
cofactor enhance the binding constant for O2 by a factor of 100.
The work presented here shows that when both cofactor 6-
MPH4 and substrate tyrosine are bound to TyrH the
coordination of NO to Fe(II) is directed to an equatorial
position, which most likely poises the system for the first
hydroxylation reaction with pterin but also positions the Fe−
NO bond so that it is perpendicular to the substrate protons.
Presumably, this bond will have to reorient prior to
commencement of substrate hydroxylation. The E332 mutation
to alanine may lead to a structural change that impedes this
reorientation of the Fe(IV)O species.
∥Department of Chemistry and Biochemistry, Auburn Uni-
versity, Auburn, Alabama, United States.
Funding
This work was supported by NIH grants GM-47291 (P.F.F.)
and RR-15880 (J.M.) and by grants from The Welch
Foundation (AQ-1245) to P.F.F. and the Michigan Economic
Development Corporation to J.M.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
A special thanks is due to Professor Stefan Stoll for his
assistance in getting us started with the EasySpin software.
■
ABBREVIATIONS USED
■
TyrH, tyrosine hydroxylase; PheH, phenylalanine hydroxylase;
BH4, tetrahydrobiopterin; 6-MPH4, 6-methyltetrahydropterin;
DOPA, dihydroxyphenylalanine; tyr, tyrosine; EPR, electron
paramagnetic resonance; ESEEM, electron spin echo envelope
modulation; hf, hyperfine; nqi, nuclear quadrupole interaction;
PAS, principal axis system; MAHMA NONOATE, 6-(2-
hydroxy-1-methyl-2-nitrosohydrazino)-N-methyl-1-hexan-
amine; EDTA, ethylenediamine tetraacetic acid; TACN, 1,4,7-
triazacyclononane
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■
SUMMARY
■
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ASSOCIATED CONTENT
■
S
* Supporting Information
Cw-EPR spectra collected for {FeNO}7 derivatives of TyrH
treated with 6-MPH4, tyr, 6-MPH4 plus tyr, and the E332A
variant of TyrH treated with 6-MPH4 plus tyr; single parameter
χ2 plots for the TyrH[3,5-2H-tyr], TyrH[3,5-2H-tyr, 6-MPH4],
and TyrH[tyr, 6,7-2H-6-MPH4] samples as well as for the
E332A variant of TyrH, E332A[tyr, 6,7-2H-6- MPH4] and
TyrH, E332A[3,5-2H-tyr, 6- MPH4]; and covariance matrices
calculated using the best fit results for the TyrH[3,5-2H-tyr],
TyrH[3,5-2H-tyr, 6-MPH4], and TyrH[tyr, 6,7-2H-6-MPH4]
2H-ESEEM data. This material is available free of charge via the
Internet at http://pubs.acs.org.
AUTHOR INFORMATION
■
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Corresponding Author
*Tel.: (517)355-9715. Fax: (517)353-1793. E-mail: mccracke@
msu.edu.
Present Addresses
§Department of Chemistry, University of Alabama, Tuscaloosa,
Alabama, United States.
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