Role of Conserved Tyrosine 343 in Intramolecular Electron
Transfer in Human Sulfite Oxidase*
Changjian
Feng
,
Heather L.
Wilson§,
John K.
Hurley¶,
James T.
Hazzard¶,
Gordon
Tollin¶
,
K. V.
Rajagopalan§**, and
John H.
Enemark

From the
Department of Chemistry, University of
Arizona, Tucson, Arizona 85721, the § Department of
Biochemistry, Duke University Medical Center, Durham, North Carolina
27710, and the ¶ Department of Biochemistry and Molecular
Biophysics, University of Arizona, Tucson, Arizona 85721
Received for publication, October 9, 2002
 |
ABSTRACT |
Tyrosine 343 in human sulfite oxidase (SO) is
conserved in all SOs sequenced to date. Intramolecular electron
transfer (IET) rates between reduced heme (FeII) and
oxidized molybdenum (MoVI) in the recombinant wild-type and
Y343F human SO were measured for the first time by flash photolysis.
The IET rate in wild-type human SO at pH 7.4 is about 37% of that in
chicken SO with a similar decrease in kcat.
Steady-state kinetic analysis of the Y343F mutant showed an increase in
Kmsulfite and a decrease in
kcat resulting in a 23-fold attenuation in the
specificity constant
kcat/Kmsulfite
at the optimum pH value of 8.25. This indicates that Tyr-343 is
involved in the binding of the substrate and catalysis within the
molybdenum active site. Furthermore, the IET rate constant in
the mutant at pH 6.0 is only about one-tenth that of the wild-type enzyme, suggesting that the OH group of Tyr-343 is vital for efficient IET in SO. The pH dependences of IET rate constants in the wild-type and mutant SO are consistent with the previously proposed coupled electron-proton transfer mechanism.
 |
INTRODUCTION |
In vertebrates the molybdenum cofactor-containing enzyme sulfite
oxidase (SO,1 EC 1.8.3.1)
catalyzes the oxidation of sulfite to sulfate with the reduction of two
equivalents of ferricytochrome c (Equation 1) (1, 2),
according to the following equation.
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(Eq. 1)
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This is the terminal reaction in the oxidative degradation of
sulfur-containing compounds and is physiologically essential. The
absence of SO activity in humans is characterized by dislocation of
ocular lenses, mental retardation, and in severe cases, attenuated growth of the brain and early death. These severe neurological symptoms
result from the inability to properly produce the molybdenum cofactor
and also from point mutations in the SO protein itself (3).
The x-ray crystal structure of the dimeric chicken liver SO has
recently been reported (4). Each subunit consists of an N-terminal
b5-type cytochrome domain and two larger
segments that constitute the central molybdenum binding and C-terminal
interface domains. The molybdenum center in SO has square pyramidal
coordination geometry. Solvent access to the deeply buried active site
is via a channel that opens onto the equatorial Mo
O group. As
expected for binding an anionic substrate, the sulfite-binding pocket
is very positively charged and consists of three arginines (Arg-138, Arg-190, and Arg-450), Trp-204, and Tyr-322. Tyr-322 is also in close
proximity to the molybdenum coordination sphere (Fig.
1A).

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Fig. 1.
A, the substrate binding site in chicken
SO (4). Water molecules are depicted as solid black circles.
Dotted lines indicate selected hydrogen bonds. B,
a possible CEPT mechanism in which Tyr-322 in chicken SO (the
equivalent of Tyr-343 in human SO) acts as an intermediary proton
shuttle (adapted from Ref. 10).
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In the generally accepted catalytic cycle (Fig.
2, Refs. 5
9), the MoVI
center of the fully oxidized SO reacts with sulfite to produce sulfate.
The two-electron-reduced SO, which is in the
MoIV/FeIII state, undergoes intramolecular
electron transfer (IET) to generate the
MoV/FeII state that can be detected by electron
paramagnetic resonance (EPR) spectroscopy. One-electron transfer to
exogenous cyt cox, leaving the enzyme in the
one-electron-reduced state MoV/FeIII,
accomplishes re-oxidation of the FeII center. A second Mo
Fe IET step (giving MoVIFeII), followed by
reduction of a second equivalent of cyt cox,
returns the enzyme to the fully oxidized resting state. We have
previously shown that exogenous flavin radicals generated in
situ with a laser pulse will rapidly reduce the heme center of SO
by one electron (Fig. 2) and have used this unique technique to
investigate IET between the molybdenum and iron centers as a function
of pH values (10
12). Note that this process corresponds to the second
IET step noted above. Combining this kinetic information with the crystal structure (4) and the pulsed EPR results (13
17) for the
MoV form of the two-electron-reduced state of SO has led to
a plausible proposal for a coupled electron-proton transfer (CEPT)
reaction at the molybdenum center (18) as shown in Scheme
1.

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Fig. 2.
Postulated oxidation state changes occurring
at the molybdenum and iron centers of SO during the catalytic oxidation
of sulfite and the concomitant reduction of cyt c. The
one-electron reduction indicated by a dashed arrow
connecting MoVIFeIII and
MoVIFeII can be initiated with a laser pulse in
a solution containing dRF and a sacrificial electron donor
(semicarbazide in this study).
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In the crystal structure of chicken SO (4), Tyr-322 is within
hydrogen-bonding distance of the equatorial Mo
O group, is also
accessible to water molecules (Fig. 1A), and was proposed to
serve as a proton shuttle between Mo
O and OH
or
H2O (10). Fig. 1B gives a detailed schematic
view of part of a possible CEPT mechanism in which Tyr-322 acts as an
intermediary proton shuttle. A water molecule bound to the molybdenum
active site pocket is poised to donate a proton to the equatorial
MoVI = O group during reduction to MoV; for
the reverse process an OH
anion in the pocket accepts a
proton from MoV
OH. Competing hydrogen-bonding
interactions of the Mo
OH moiety with Tyr-322 and with the anion
occupying the active site may also be responsible for the well known
equilibrium between two EPR-distinct forms of SO observed for the
sulfite-reduced enzyme (15). This tyrosine residue is conserved in all
SOs from animals (6), plants (19), and bacteria (20). The mutation of
this conserved tyrosine should provide direct evidence for
its role in IET, and this is the goal of the present study.
The most extensively studied examples of SO are from rat, human, and
chicken livers, and all three enzymes show a very high degree of
sequence similarity with 68% sequence identity between human and
chicken SO (21
23). The close similarity of the pulsed EPR spectra for
native chicken and human SO indicates that the structures of their
molybdenum centers are essentially identical (13, 14). Native SO has
been purified from bovine, chicken, rat, and human livers. Rat (22, 24)
and human (25
27) SO have been successfully cloned and expressed in
active forms. Due to the intricacies of assembling and inserting the
molybdenum center into the overexpressed apoenzyme, it is
extraordinarily difficult to develop suitable expression systems for
recombinant SO having high levels of enzyme activity. Expression of
these proteins in Escherichia coli makes it possible to use
site-directed mutagenesis to incisively probe the roles of specific
residues in catalysis (25, 26, 28). This is particularly true for human
SO where, in addition to structural studies, site-directed mutagenesis
can be expected to critically evaluate the involvement of specific amino acid residues in pathological human SO deficiency. In the present
experiments we explore the role of the conserved Tyr-343 in human SO
(the equivalent of Tyr-322 in chicken SO) by comparative steady-state
kinetic and flash-induced IET studies of the wild-type and the Y343F mutant.
 |
EXPERIMENTAL PROCEDURES |
Site-directed Mutagenesis--
The Y343F mutation was introduced
into pTG918 (24) using the Transformer Site-directed Mutagenesis kit
(Clontech) with the mutagenic primer Y343F
(GGCGGGATTTCAAAGGCTTCTC). Mutations were verified by sequence analysis
performed at the Duke University DNA Analysis facility.
Expression and Purification of Wild-type and Y343F Human
SO--
Both recombinant wild-type and Y343F human SO were expressed
and purified as previously described (24, 25) with the following modifications. After the phenyl-Sepharose column, fractions exhibiting an A414/A280 ratio
greater than 0.89 were pooled and further purified using the gel
filtration column Zorbax GF-250 (Agilent Technologies). Fractions
exhibiting an A414/A280
ratio of 0.96 or greater were then pooled and used in the experiments
described in this study. The molybdenum content of purified SO proteins
was determined using a Perkin-Elmer Zeeman/3030 atomic absorption
spectrometer as previously described (25, 29). Enzyme concentrations
were determined by using molar extinction coefficients of 99,900 and 113,000 M
1 cm
1 at 413 nm for
the oxidized chicken and human SO, respectively.
Laser Flash Photolysis--
Laser flash photolysis experiments
were performed anaerobically on 0.50-ml solutions containing ~90
µM 5-deazariboflavin (dRF) and 0.5 mM freshly
prepared semicarbazide as a sacrificial reductant. The methodology used
has been described previously (10, 30). The published method (30) was
used for studying the effect of solution viscosity on IET in human SO.
The laser apparatus and associated visible absorbance detection system
have been extensively described (31) as has the basic photochemical
process by which 5-deazariboflavin semiquinone (dRFH·) is
generated by reaction between triplet state dRF and the sacrificial reductant and used to reduce redox-active proteins (32
34). Further details concerning the photochemical process, which are of particular relevance to the SO system, are presented below. Non-linear
least-squares fitting of experimental data at 513 nm was generally
performed using an implementation of the Levenberg-Marquart algorithm
(10), provided as part of the Microcal Origin (version 7.0;
Northampton, MA) software package for data processing and display.
Transient absorbance changes at 555 nm were analyzed using the computer fitting procedure SIFIT, obtained from OLIS Inc. (Jefferson, GA).
Steady-state Kinetics--
Steady-state enzyme kinetic
studies were performed aerobically in a Varian Cary-300
spectrophotometer. Initial velocities were determined by following the
reduction of a freshly prepared oxidized cytochrome c (type
VI, Sigma) solution at 550 nm, using an extinction coefficient
change of 19,630 M
1 cm
1 (35).
SO was routinely assayed at 25 °C in 100 mM Tris buffer and adjusted to pH 8.0 with acetic acid. The steady-state kinetic pH
profile study was conducted using a saturating concentration of
cytochrome c, 15 µM (7-fold greater than
Km), and varying the concentration of sulfite
between 2 and 496 µM. The buffers used over the pH range
6.0-8.7 were 100 mM Bis-Tris (pH 6.0
6.5), Bis-Tris
propane (pH 6.7
7.5), and Tris (pH 7.5
8.7) adjusted to the
appropriate pH with acetic acid to minimize the possibility of anion
inhibition (36). The buffer used over the pH range 8.7
9.5 was 100 mM glycine adjusted to the appropriate pH with NaOH. The
total concentrations of the wild-type and Y343F human SO are 2.6 × 10
10 M and 4.3 × 10
10
1.1 × 10
9 M, respectively.
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RESULTS |
Photochemical Reduction of Human SO by Deazariboflavin
Semiquinone--
The photochemical reduction of the FeIII
heme moiety of human SO was monitored by laser flash photolysis-induced
transient absorbance changes. The flash-induced difference spectrum is
shown in Fig. 3. The peak wavelengths
(555 and 523 nm) and isosbestic points (513, 533, 545, and 565 nm)
observed in the flash-induced difference spectra are identical to those
observed in the steady-state reduced minus oxidized difference
spectrum for heme reduction (inset of Fig. 3). These spectra
confirm that the transient absorbance changes observed at 555 nm are
directly related to reduction and re-oxidation of the b-type
heme prosthetic group (11). No detectable spectral contribution from
the molybdenum cofactor was observed. It is important to note that
human SO (wild-type and Y343F mutant) has photochemical reduction
properties that are similar to the native chicken SO.

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Fig. 3.
Flash-induced difference spectrum of
wild-type human SO obtained 20 ms after the laser flash. The
dotted lines with peaks at 555 and 523 nm were obtained by a
Gaussian fit to the data. Anaerobic solutions contained 10 µM SO, 0.5 mM semicarbazide hydrochloride and
~90 µM dRF. Within the resolution of the experiment,
the flash-induced difference spectrum is identical to that obtained by
steady-state reduction (inset). Difference spectra obtained
at longer times were similar in appearance indicating that no further
reduction occurred.
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Fig. 4, A and B
show typical transient kinetic traces of absorbance changes at 555 nm
upon laser flash photoexcitation of a solution containing oxidized
wild-type human SO (Fig. 4A) or native chicken SO (Fig.
4B), dRF, and semicarbazide. The kinetic behavior can be
fully described in terms of the minimal set of reactions shown in
Equations 2-5. dRFH· is generated by the laser pulse in the
presence of the sacrificial electron donor semicarbazide
(AH2) (Equation 3). The initial positive deflection of
absorbance from zero in Fig. 4 is due to net reduction of the SO heme
center to the FeII form (Equation 4), which has an
absorbance maximum at 555 nm (Fig. 3). The slow decrease in absorbance
that follows the initial rapid increase is due to the net IET from
FeII to MoVI, which establishes an equilibrium
between the MoVIFeII and
MoVFeIII forms of SO (Equation 5). The kinetics
of this latter process is independent of the concentrations of human
and chicken SO, indicating that it is due to a first-order IET process,
which is the reaction that is of particular interest to us in this
study.
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(Eq. 2)
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(Eq. 3)
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(Eq. 4)
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(Eq. 5)
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For a case such as that shown in Fig. 4, in which the
photochemically induced reduction of SO occurs much faster than
subsequent IET, excellent values for the overall IET rate constant
ket (= kf + kr) and parameters a and b
can be obtained by fitting the heme re-oxidation phase with the simple
exponential function given in Equation 6 (the geometrical meanings of
the parameters a and b are shown in Fig.
4A).
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(Eq. 6)
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Based on Equation 5, the parameters a and b
in Equation 6 should have the meanings expressed in Equations 7 and 8
(where A0 is simply the absorbance extrapolated
to t = 0, assuming that the photochemically induced
reduction of SO is instantaneous).
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(Eq. 7)
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(Eq. 8)
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(Eq. 9)
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Thus the individual IET rate constants kf
and kr can be calculated from
ket and Keq (=
b/a, Equation 9). The IET rate constants and
Keq values for the wild-type human SO (Fig.
4A) and the native chicken SO (Fig. 4B) under the
same conditions are shown in Table I.

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Fig. 4.
Transient kinetic trace obtained at 555 nm
upon photo-excitation of a solution containing (A)
10.8 µM wild-type human SO or
(B) 14.9 µM
chicken SO, ~90
µM dRF, and 0.5 mM
semicarbazide in 10 mM Tris buffer (pH
7.4). pH was adjusted with HCl. The solid line
indicates a single-exponential fit to the IET phase.
Keq = b/a.
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Table I
Flash photolysis kinetic parameters for human and chicken SO
Solutions for flash photolysis experiments contained 0.5 mM
semicarbazide, 10 mM Tris. The pH value was adjusted to 7.4 with HCl.
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The IET rate constant in wild-type human SO exhibits a linear
dependence on the negative 0.6th power of the viscosity (Fig. 5), which is similar to that for chicken
SO (
0.7th power, Ref. 30). Control experiments using either sucrose
or polyethylene glycol 400 as viscosogen indicate that it is viscosity
itself that is responsible for the dependence of IET rates on the
solution composition. The viscosity effect suggests that the IET in
human SO may also involve significant conformational change, which has been suggested to involve domain rearrangement in the chicken enzyme
(30, 37).

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Fig. 5.
Fit of viscosity dependence of
ket for wild-type human SO in sucrose
solution using modified Kramer's theory (30).
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The observed rate constant for the initial heme reduction is protein
concentration-dependent as expected for a bimolecular process (Equation 4). Electron transfer from dRFH· to the heme
of SO can best be observed spectrophotometrically as a decrease in
absorbance at 513 nm, which is close to the absorbance maximum for
dRFH· and is an isosbestic point for oxidized and reduced heme
cofactor. The calculated second order rate constants
(k1) for the reduction of wild-type human SO and
chicken SO are listed in Table I. As is evident, these are the same
within experimental error.
Steady-state Kinetics of Human SO--
The steady-state oxidation
of sulfite to sulfate as catalyzed by human SO using cytochrome
c as the electron acceptor yields plots of initial velocity
versus substrate concentration that display typical
saturation kinetics. The kcat values of both
wild-type and Y343F were found to vary significantly over the pH range
7.0-9.5 (Fig. 6), and both exhibit
bell-shaped pH dependences. Optimum pH values for activities of
wild-type and Y343F are ~8.1 and ~8.3, respectively, which are not
significantly different. The values of kcat,
Kmsulfite and
kcat/Kmsulfite
around the optimum pH value are shown in Table
II.

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Fig. 6.
The pH dependence of
kcat values of wild-type and Y343F
human SO. Experiments were performed in 0.1 M buffers
(see "Experimental Procedures") between pH 7.0 and 9.5 at 25 °C,
with varying concentrations of sulfite and 15 µM
cytochrome c.
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The Dependence of IET Rates on pH Values--
Fig.
7A shows the dependence of the
kf and kr magnitudes of
wild-type human SO on [OH
] between pH 7.3 and 8.4, in
solutions containing low anion concentrations ([Cl
]
~6.5 mM). Note that both kf and
kr decrease significantly as [OH
] increases. The situation is quite different in
solutions containing 20 mM Na2SO4
as shown in Fig. 7B in which kf
decreases slightly with increasing [OH
], while
kr is effectively unchanged. The previously
reported inhibition of chicken SO by
SO
anions (11) is also observed in
the human enzyme as both kf and
kr are substantially smaller in Fig.
7B compared with Fig. 7A.

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Fig. 7.
Dependence of the rate constants
kf and kr of
wild-type human SO on hydroxide ion concentration. A, 0.5 mM semicarbazide hydrochloride, 6 mM HCl;
B, 20 mM Na2SO4, 0.5 mM semicarbazide hydrochloride, 6 mM HCl. For
all experiments, the Tris base concentration was chosen so as to
achieve the appropriate pH value while keeping [Cl ]
between 5 and 6 mM.
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We attempted to observe IET in the Y343F mutant in the same
pH range as that for the wild-type enzyme. However, no decay subsequent to heme reduction due to IET was observed at 555 nm within this pH
range. Even at as low a pH as 6.4, the FeII re-oxidation
phase was still not observable and only began to be detectable at pH
6.2. The influence of pH on the IET rate constants of the Y343F mutant
were determined between pH 5.8 and 6.2. The SO activities at these low
pH values were measured to make certain that the enzyme was still
active. It was observed that the mutant remains functional even at pH
5.6 (kcat 1.25 s
1). In addition,
the magnitudes of the signal change at 555 nm in flash photolysis
experiments were comparable to those observed with the wild-type
protein. Fig. 8 shows the effects of pH
on Keq for IET in the wild-type and Y343F human
SO. Note the shift in the pH range for the mutant compared with the
wild-type enzyme. Fig. 9 compares the
kinetic traces of the absorbance changes at 555 nm upon laser flash
photoexcitation of solutions of Y343F or wild-type human SO at pH 6.0. Note the significant difference in the time scale for these two
proteins. Fig. 10 shows the pH dependence of IET rates in the Y343F mutant at low anion concentrations ([Ac
] ~12 mM, [Cl
] ~0.5
mM) between pH 5.8 and 6.2. Due to the slow IET, acetic acid was used to adjust the pH value of the buffer, and experiments on
Y343F at high anion concentrations were not conducted at various pH
values. The pH profiles of the IET rate constants for the mutant show
significant differences from those for wild-type protein (compare Fig.
10 with Fig. 7A). Note that kr for
the mutant increases slightly with increasing [OH
], but
kf is actually unchanged.

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Fig. 8.
Effects of pH values on
Keq for IET in wild-type and Y343F human
SO. The conditions for flash photolysis experiments are the same
as shown in the captions of Figs. 7 and 10, respectively.
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Fig. 9.
Transient kinetic trace obtained at 555 nm
upon photo-excitation of a solution containing 12.4 µM Y343F (A) or 9.0 µM wild-type human SO (B), and
~90 µM dRF and
0.5 mM semicarbazide in 20 mM
Bis-Tris. pH value was adjusted to 6.0 with HAc. The
solid line indicates a single exponential fit to the IET
phase.
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Fig. 10.
Dependence of the rate constants
kf and kr of
the Y343F mutant on hydroxide ion concentration. Solutions
contained 0.5 mM semicarbazide hydrochloride and 20 mM Bis-Tris. Acetic acid was used to adjust the pH value.
For all experiments, the free [Ac ] in the solution was
16 18 mM.
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 |
DISCUSSION |
IET Rates of Wild-type Human and Chicken SO--
The observed IET
rate constant for the wild-type human SO at pH 7.4 is 491 s
1, which is ~37% of that obtained for chicken SO
(1318 s
1) under the same conditions (Fig. 4, Table I).
The Keq values for IET in wild-type human SO and
chicken SO are 0.73 and 1.63, respectively, which must contribute to
the difference in IET rates of these two proteins. Note that the
kcat value of wild-type human SO (35 s
1) is also about 50% of that for chicken SO (73 s
1) under standard assay conditions. Taken together, the
steady-state kinetic and flash photolysis results are consistent with a
recent comprehensive kinetic study of chicken SO (36), which suggests that at pH 7.4 the IET step contributes to the overall kinetic barrier
to catalysis. The similarity in the heme reduction rates (k1) of human and chicken SO is consistent with
the very high degree (68%) of amino acid sequence homology in human
and chicken SO and suggests that the exposure of the heme cofactors in
the two proteins is similar.
The pH Dependence of IET Rates in Wild-type Human SO--
In
wild-type human SO, at low anion concentrations, both
kf and kr decrease
significantly with increasing [OH
] (Fig.
7A), which is quite different from the pattern observed previously for chicken SO (10) for which kf
decreased but kr remained constant. The reason
for this difference is not clear yet. A possible explanation is that
the interdomain docking for human SO during the IET reaction may be
disfavored upon increasing pH values, making both forward and reverse
electron transfer slower.
In the presence of high anion concentrations for wild-type human SO
only kf decreases with increasing
[OH
], while kr does not change
as much (Fig. 7B). However, under comparable conditions,
kr for chicken SO increases with increasing [OH
], while kf is effectively
unchanged (10), which is remarkably different from wild-type human SO.
These differences can be explained by the previously proposed mechanism
(10) involving CEPT processes (Scheme 1).
Electron transfer processes in biological systems are often coupled to
protonation/deprotonation events, and these effects may influence the
kinetics of electron transfer (38). In the case of molybdoenzymes
Stiefel has proposed that one-electron transfers are coupled to proton
transfers (39). Although this hypothesis is widely accepted and several
model systems designed to investigate such processes have been reported
(40, 41), only a few studies on CEPT in molybdenum enzymes have been
carried out (10, 42).
Scheme 1 can be algebraically expressed by Equations 10 and 11 (derived
by a similar procedure to that shown in the supplemental materials for
Ref. 10) as follows.
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(Eq. 10)
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(Eq. 11)
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(Eq. 12)
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In chicken SO, at high anion concentrations, if the parameter
d
[OH
], the apparent
kf is independent of [OH
]
(Equation 10); when the parameter e is comparable to
[OH
], the apparent kr increases
with increasing [OH
] (Equation 11, Ref. 10). Since the
parameters d and e are proportional to the
dissociation constants KB1 and
KB2 (see Scheme 1 for definitions), respectively, it is entirely possible that at sufficiently low values
for these two constants, the apparent kf could
decrease with increasing [OH
] (when d is
comparable to [OH
]), whereas the apparent
kr could be independent of [OH
]
(when e
[OH
]) over the entire pH range
investigated as is observed in Fig. 7B. Furthermore, an
increase in the association constants KX1 and
KX2 may play a similar role according to
Equations 10 and 11. Thus, the remarkable difference in the pH
dependence of the apparent kf and
kr values of human and chicken SO at high anion
concentrations may be a consequence of a much stronger binding of
anions (X
, OH
) to the active site of
human SO than to that of chicken SO, which could be related to
differences in the charge distribution around the molybdenum active
site in these two proteins.
The pH Dependence of Steady-state Kinetics in Y343F
Mutant--
The pH profiles for kcat (Fig. 6)
indicate that the mutation in Tyr-343 does not shift the optimum pH
value for SO activity, whereas the values of the kinetic constants
Kmsulfite and
kcat do show an appreciable departure from the
equivalent parameters associated with wild-type enzyme. The elevation
in Km for sulfite in the Y343F mutant is in accord
with a role for Tyr-343 in partially stabilizing the binding of the negatively charged sulfite molecule to the catalytic site (Fig. 1A). The increase in Km combined with the
decrease in kcat results in a decrease of nearly
23-fold in the apparent second order rate constant
(kcat/Kmsulfite)
at pH 8.25 (Table II). Previous detailed kinetic study has shown that
the human SO R160Q mutant has a greatly increased Km for sulfite and a marked decrease in kcat
resulting in an extreme decrease (1000-fold) in the specificity
constant
kcat/Kmsulfite
(26). The considerable difference in the magnitude of the changes of
kcat/Kmsulfite
for the Y343F and R160Q mutants can be explained by the different positions of Tyr-343 and Arg-160 in the active site of human SO. In the
crystal structure of chicken SO, Tyr-322 (the equivalent of Tyr-343 in
human SO) is only weakly hydrogen-bonded to the sulfate anion found
in the active site pocket (Tyr
O ···
OSO
= 2.93 Å), whereas the
positively charged Arg-138 (the equivalent of Arg-160 in human SO) is
directly involved in the attraction of the negatively charged sulfate
ion (Arg
O ··· OSO
= 2.63 Å), which is in full accord with the findings reported here.
The pH Dependence of IET Rates in the Y343F Mutant--
The
observed significant shift in Keq for the Y343F
mutant (Fig. 8) indicates that the mutation in Tyr-343 disfavors the
forward IET process (Equation 5), which may be due to the removal of
hydrogen-bonding between the equatorial MoV
OH group and
the hydroxyl group of Tyr-343 (Fig. 1B). Hydrogen bonding to
MoV
OH can stabilize the +5 oxidation-state, thereby
making the reduction of MoVI to MoV more
favorable. The absence of this hydrogen bonding in the Y343F mutant is
expected to decrease Keq substantially (see
below). Modulation of reduction potentials by hydrogen bonding is a
general phenomenon in biological systems (43, 44). The effect of
hydrogen bonding on the reduction potential in molybdenum complexes has been extensively studied (45
48). The NH ··· S hydrogen
bond makes a significant contribution to the positive shift of the
reduction potential of MoV/MoIV in
monooxomolybdenum complexes with
o-(acylamino)benzenethiolate ligands (48).
At pH 6.0, the ket value of Y343F is 46 s
1 (compared with 411 s
1 for the wild-type
under the same conditions, see Fig. 9). For the mutant,
Keq does decrease from 0.36 to 0.24 (Table
III). However, such a small shift in
Keq (i.e. a thermodynamic factor)
cannot account for such a large change in the IET rate constants
(~10-fold). The most plausible explanation is that the hydrophobic
phenylalanine in the Y343F mutant may hinder direct access
of water or H+ to the equatorial Mo=O group (Fig.
1B), thus retarding efficient CEPT (i.e. a
kinetic factor). This is clearly shown by the shift in the dependence
of Keq for IET to lower pH values in the Y343F mutant (Fig. 8).
View this table:
[in this window]
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|
Table III
Flash photolysis kinetic parameters for wild-type and Y343F human SO at
pH 6.0
Solutions contained 0.5 mM semicarbazide hydrochloride, 20 mM Bis-Tris. The pH value was adjusted to 6.0 with HAc.
|
|
For the Y343F mutant at low anion concentrations,
kr increases with increasing
[OH
], while kf is effectively
unchanged (Fig. 10), which is quite different from the wild-type
behavior (Fig. 7A). These data can also be explained by the
mechanism discussed above for the wild-type enzyme (Scheme 1; Equations
10 and 11). For the sake of brevity, only the analysis for
kf app will be presented. For Y343F at the
sufficiently high dissociation constant KB1 and low association constant KX1 (making
d
[OH
]), the apparent value for
kf could become pH-independent over the entire
pH range investigated, which suggests that the binding of
[X
] to the molybdenum active site may not be as
effective in the absence of the OH group of Tyr-343. It is reasonable
to expect hydrogen-bonding of the hydroxyl of Tyr-343 to anions because of its close proximity to the anion-binding site (Fig. 1A).
Thus it is possible that Tyr-343 plays a role in anchoring the anion close to the molybdenum center.
Role of Tyr-343 in the Inhibitory Effect of
Sulfate--
At pH 6.0, the ket values of the
Y343F mutant without and with 50 mM sulfate are 46 and 2.4 s
1, respectively, while for the wild-type under the same
conditions the corresponding ket values are 434 and 7.7 s
1, respectively. The inhibition by
SO
anions in the wild-type SO is
still apparent in the Y343F mutant, which demonstrates that the OH
group of Tyr-343 is not required for sulfate inhibition. Furthermore,
the data presented in this paper clearly demonstrate that the OH group
of Tyr-343 in human SO plays an important role in IET whether or not
anions are bound to the enzyme active site, in contrast to an earlier
suggestion that this is only true when sulfate is bound (10).
In conclusion, this study has provided direct evidence for
the important role of the conserved active site tyrosine (Tyr-343 in
human SO) in transferring protons during the CEPT process and the
binding of substrate to the active site. In addition, these results
provide experimental rationale for the close proximity of the
equivalent tyrosine residue (Tyr-322) to the coordinated ligands of the
molybdenum center observed in the crystal structure of chicken SO
(4).
 |
ACKNOWLEDGEMENTS |
We thank Dr. Andrew Pacheco and Professor
Fraser Armstrong for helpful discussions. We acknowledge the technical
assistance of Mr. Ralph Wiley and Ms. Sandra Jaramillo.
 |
FOOTNOTES |
*
This work was supported by Grants GM 37773 (to J. H. E.),
DK 15057 (to G. T.), and GM 44283 (to K. V. R.) from the National Institutes of Health.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. Fax:
520-621-9288; E-mail: gtollin@u.arizona.edu.
**
To whom correspondence should be addressed. Fax: 919-684-8919;
E-mail: raj@biochem.duke.edu.

To whom correspondence should be addressed. Fax: 520-626-8065;
E-mail: jenemark@u.arizona.edu.
Published, JBC Papers in Press, November 11, 2002, DOI 10.1074/jbc.M210374200
 |
ABBREVIATIONS |
The abbreviations used are:
SO, sulfite oxidase;
IET, intramolecular electron transfer;
EPR, electron paramagnetic
resonance;
dRF, 5-deazariboflavin;
dRFH·, 5-deazariboflavin
semiquinone;
CEPT, coupled electron-proton transfer;
ket, observed IET rate constant.
 |
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