From the Department of Biochemistry and the Medical Research
Council Group in the Molecular Biology of Membranes, University of
Alberta, Edmonton, Alberta T6G 2H7, Canada
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INTRODUCTION |
Escherichia coli is capable of growing anaerobically by
inducing specialized respiratory chains that consist of a group of primary dehydrogenases, an intermediate electron carrier (menaquinone or ubiquinone), and a series of terminal reductases. Dimethyl sulfoxide
reductase (DmsABC)1 is one of
the terminal reductases (1), and it catalyzes reduction of dimethyl
sulfoxide and a variety of S- and N-oxides (2). This membrane-associated heterotrimer contains a catalytic subunit with
a molybdenum cofactor (DmsA, 87.4 kDa), an electron-transfer subunit
with four [4Fe-4S] clusters (DmsB, 23.1 kDa), and a
membrane-intrinsic anchor subunit (DmsC, 30.8 kDa) (3).
DmsC is a hydrophobic polypeptide that traverses eight times and
anchors the DmsAB dimer to the membrane (4). It is involved in
menaquinol (MQH2) binding and oxidation, and in electron
transport. DmsABC is able to draw electrons from the menaquinol pool to
DmsC and transfer them through DmsB to the catalytic subunit, DmsA. In
a recent electron paramagnetic resonance (EPR) study (5), it was shown
that DmsCH65 is involved in MQH2 binding and
oxidation. It was suggested that a mutation of the DmsC subunit,
DmsCH65R, may block binding of the MQH2 analog
HOQNO to the protein. However, there was no direct observation
available about this possible blockage of HOQNO binding.
For another terminal reductase, fumarate reductase, it has been
reported that there are two separate MQH2 binding sites
located in subunits C and D of the enzyme, respectively (6-8). The
MQH2 binding affinity of the site in subunit D is higher
than that of the site in subunit C. For DmsABC, however, the number and the location of MQH2 binding site(s) have not been
determined.
HOQNO is a structural analog of MQH2, and it is known as a
classical inhibitor of the mitochondrial cytochrome c
reductase. An earlier study showed that the fluorescence of HOQNO was
completely quenched by binding to submitochondrial particles of beef
heart (9). It was concluded that the inhibition of electron transfer by
HOQNO was caused by binding to the specific binding site. Fluorescence quenching was also observed when an HOQNO analog, NQNO, bound to the
mitochondrial cytochrome c reductase (10). NQNO is a specific inhibitor of the inner facing quinone-reaction center (Qi) of the mitochondrial cytochrome c
reductase. The fluorescence of HOQNO has provided a convenient means
for studying HOQNO binding and inhibition reactions. It is expected,
therefore, that a fluorescence spectroscopic study of the interaction
of HOQNO with DmsABC could provide useful information for determining
the number and the location of MQH2 binding site(s) in
DmsABC.
In this paper, we examine the binding stoichiometry and kinetics for
the interaction of HOQNO with DmsABC using fluorescence titration and
stopped-flow methods. We show that HOQNO binds to DmsABC with
approximately 1:1 stoichiometry and the interaction can be described by
a two-step equilibrium model. We also provide evidence that a mutation
of the DmsC subunit, DmsABCH65R, blocks the binding of the
MQH2 analog HOQNO to the protein as suggested recently in
an EPR study (5).
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EXPERIMENTAL PROCEDURES |
Bacterial Strains and Plasmids--
E. coli HB101
[supE44 hsdS20 2(rB
mB
) recA13 ara-14 proA2 lacY1 galK2
rpsL20 xyl-5 mtl-1] (11) was used for expression of wild-type and
mutant DmsABC. For overexpression of DmsABC and DmsABCH65R,
bacteria were transformed with pDMS160 and pDMS160-H65R, respectively (5, 12, 13). All manipulations of strains were carried out as described
by Sambrook et al. (14).
Growth of Cells and Preparation of Membrane Vesicles--
Cells
were grown anaerobically in 20-liter batches at 37 °C on a
glycerol-fumarate minimal medium for 48 h as described previously (15). Cells were harvested using a Pellicon membrane system (16),
washed, lysed by passage through a French pressure cell, and subjected
to differential centrifugation. The isolated membranes were resuspended
in 100 mM MOPS and 5 mM EDTA (pH 7.0), frozen in liquid nitrogen, and then stored at
70 °C until use (12). Protein concentrations were determined by a modified Lowry assay in the
presence of 1% SDS using a Bio-Rad serum albumin protein standard
(17). The concentration of DmsABC was calculated from the total
concentration of [4Fe-4S] clusters measured by the EPR spin
quantitation and by assuming that there are four [4Fe-4S] clusters
per DmsABC molecule (18, 19).
Quenching of HOQNO Fluorescence by Binding to DmsABC--
HOQNO
was obtained from Sigma. The concentration of HOQNO was determined
spectrophotometrically after diluting the ethanolic stock solution in 1 mM sodium hydroxide using an extinction coefficient of 9450 M
1 cm
1 at 346 nm (20).
Fluorescence measurements were carried out using a Perkin-Elmer LS-50B
luminescence spectrometer. The fluorescence emission spectrum of HOQNO
in 100 mM MOPS and 5 mM EDTA (pH 7.0) exhibits
a maximum at 479 nm with the excitation at 341 nm (data not shown). In
fluorescence titration experiments, aliquots of a 50 µM
HOQNO stock solution were added to the cuvette containing the protein
in 100 mM MOPS and 5 mM EDTA (pH 7.0), and
fluorescence emissions were measured at 479 nm (with excitation at 341 nm). The background fluorescence of the protein sample in the absence of HOQNO was subtracted from the fluorescence of the sample in the
presence of HOQNO.
Stopped-flow Experiment and Data Analysis--
The stopped-flow
experiments were performed using a Sequential Bio SX-17MV stopped-flow
spectrofluorimeter (Applied Photophysics Ltd., Leatherhead, UK). In a
typical experiment, DmsABC or DmsABCH65R (1 or 2 µM) in 100 mM MOPS and 5 mM EDTA
(pH 7.0) was mixed with an equal volume of various concentrations of
HOQNO (1-14 µM) in the same buffer. Temperature was
maintained at 25 °C. The fluorescence of HOQNO was excited at 341 nm, and the emissions above 400 nm were recorded through a cut off
filter SGG-400-1.00 (CVI Laser Co., Albuquerque, NM). For each
concentration of HOQNO, at least three runs were performed and 1000 or
2000 data points were collected. After averaging, data were fitted to
an appropriate equation using the software supplied by Applied
Photophysics. Under our experimental conditions, quenching of HOQNO
fluorescence by binding to DmsABC was biphasic, and the observed
fluorescence, F, was best fitted to a double exponential
equation,
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(Eq. 1)
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where Af and As are the
amplitudes of the fast and slow phase, and kf and
ks are the observed rates for the fast and slow
phase, respectively, t is time and b is an off
set value of the stopped-flow instrument.
The interaction of HOQNO with DmsABC can be described by the following
model (21-23) (see "Discussion"),
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(Eq. 2)
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where E represents DmsABC; L, the ligand
(HOQNO); EL, the initial complex of DmsABC with HOQNO before
the isomerization takes place; EL*, the final complex
product; k1 and k3 are
the rate constants for the forward reactions in
M
1 s
1 and s
1,
respectively, and k2 and
k4 are the rate constants for the reverse reactions in s
1. In the case where the bimolecular
process is much faster than the unimolecular process and the initial
concentration of L is much higher than the concentration of
E, the observed first-order rates for the fast phase
(kf) and the slow phase (ks) are
given by Equations 3 and 4, respectively,
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(Eq. 3)
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(Eq. 4)
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where Kd1 = k2/k1 is the dissociation
constant of the first equilibrium in Equation 2.
Under our experimental conditions, the plot of HOQNO fluorescence
against the concentration of HOQNO was linear up to 7 µM of HOQNO (after mixing) (data not shown). This was, therefore, the
highest concentration of HOQNO used in this work. Toward the low end of
the HOQNO concentrations used herein, the condition that initial
concentration of L is much higher than the concentration of
E (0.5 µM after mixing) does not hold.
However, the observed first-order rates for the fast phase,
kf, can still be analyzed using Equation 3 as
demonstrated by Halford (21).
From the fit of the observed kf data to Equation 3,
k1 and k2 can be
obtained. Under our experimental conditions, deviations of the observed
ks were too large to be fitted to Equation 4 to
yield reliable k3 and k4.
To evaluate the values of k3 and
k4, another approach using the Glint program
(Applied Photophysics) was employed. This program enables one to
globally analyze a complete data set measured at all wavelengths
according to a proposed reaction scheme and to obtain the reaction
parameters from the best fit of the calculated data to the experimental
data. Kinetic data irrelevant of wavelength (the case of this work) or
measured at a single wavelength can also be analyzed using this
program. In this work, using the reaction scheme (Equation 2), and the
k1 and k2 determined from
Equation 3, kinetic traces measured at various HOQNO concentrations
were analyzed using the Glint program, and the values of
k3 and k4 were evaluated
from the good fits of the calculated kinetic traces to the observed traces.
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RESULTS |
Stoichiometry of HOQNO Binding to DmsABC--
The binding of HOQNO
to DmsABC was examined by measuring the HOQNO fluorescence emission (at
479 nm with excitation at 341 nm) in the absence and in the presence of
DmsABC. In the absence of DmsABC, the plot of fluorescence measured by
titrating the buffer (100 mM MOPS and 5 mM
EDTA, pH 7.0) against the concentration of HOQNO gave a straight line
with a positive slope as shown in Fig.
1A. This fluorescence was
attributed to free HOQNO molecules (not bound to other compounds). In
the presence of DmsABC, however, the initial portion of each titration
curve was flat (Fig. 1A), indicating that there was no
increase of fluorescence intensity upon the addition of a certain
concentration of HOQNO to the protein. In other words, there was no
free HOQNO present in the system to emit fluorescence. It was clear
that the fluorescence of HOQNO was quenched in the presence of DmsABC
and this quenching was because of HOQNO binding to DmsABC. Further
addition of HOQNO to the protein caused a sudden increase of
fluorescence intensity, suggesting that free HOQNO became available
when the amount of HOQNO added to the protein was higher than a certain
concentration at a given concentration of DmsABC.

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Fig. 1.
Determination of the stoichiometry
of HOQNO binding to DmsABC. Panel A, titrations
of the buffer of 100 mM MOPS and 5 mM EDTA (pH
7.0) ( ), and 0.25 ( ), 0.5 (×), 0.75 ( ), and 1.0 ( )
µM DmsABC in the same buffer with HOQNO. Fluorescence
emission data at 479 nm (with excitation at 341 nm) were obtained by
subtracting the background fluorescence of the protein.
Panel B, plot of the concentration of HOQNO bound
to DmsABC against the concentration of the protein. The concentrations
of HOQNO bound to DmsABC were determined by extrapolating the linear
portions of the titration curves in panel A to
zero fluorescence.
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It was not possible to construct a Scatchard plot from the titration
data in Fig. 1A because of the sharp transition of the titration curves. To obtain the stoichiometry of HOQNO binding to
DmsABC, four concentrations of DmsABC (0.25, 0.5, 0.75, and 1.0 µM) were titrated with HOQNO in 100 mM MOPS
and 5 mM EDTA at pH 7.0 (Fig. 1A). The
extrapolations of the linear portions of the titration curves to zero
fluorescence yield four intercepts on the x-axis of the
plot. These intercepts represent the concentrations of HOQNO bound to
DmsABC at the given concentrations of DmsABC. Therefore, from the plot
of the intercepts against the concentration of DmsABC, Fig.
1B, the stoichiometry of HOQNO binding to DmsABC can be
determined to be approximately 1:1.
Effect of DmsABCH65R Mutation on HOQNO Binding to the
Protein--
It has been suggested in a recent EPR study (5) that
residue His-65 in the DmsC subunit may be involved in MQH2
binding and a His to Arg mutation, DmsABCH65R, may block
binding the MQH2 analog HOQNO to the protein. To verify this, titrations of 0.4 µM wild-type DmsABC and mutant
DmsABCH65R with HOQNO in 100 mM MOPS and 5 mM EDTA (pH 7.0) were carried out and the data obtained
were compared as shown in Fig. 2. For the
wild-type protein, the initial portion of the titration curve was flat
indicating quenching of HOQNO fluorescence because of HOQNO binding to
the protein. Further increase of HOQNO concentration caused a sharp
increase of fluorescence intensity. For the mutant DmsABCH65R, however, the titration data gave a
straight line with a positive slope, indicating that there was no
quenching of HOQNO fluorescence. In other words, in the case of
titration of DmsABCH65R, the HOQNO added to the protein was
in the free form and not bound to the protein. Fig. 2, thus,
demonstrates that the mutation of DmsABCH65R blocks HOQNO
binding to the protein.

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Fig. 2.
Comparison of fluorescence titration data for
wild-type DmsABC and the mutant DmsABCH65R.
0.4 µM wild-type DmsABC ( ) and mutant
DmsABCH65R ( ) were titrated with HOQNO in 100 mM MOPS and 5 mM EDTA (pH 7.0). Fluorescence
emission data at 479 nm (with excitation at 341 nm) were obtained by
subtracting the background fluorescence of the protein solution.
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Stopped-flow Studies of Interaction of HOQNO with DmsABC--
To
investigate the kinetics and mechanism of the interaction of HOQNO with
DmsABC, the stopped-flow fast kinetic technique was employed. Fig.
3 shows a plot of the observed
fluorescence intensities against the concentration of HOQNO obtained by
mixing various concentrations of HOQNO in buffer of 100 mM
MOPS and 5 mM EDTA (pH 7.0) with the same buffer or with 2 µM wild-type DmsABC (in the buffer). After mixing the
protein with HOQNO, a decrease of fluorescence intensity with time was
observed as shown in the inset of Fig. 3 indicating quenching of HOQNO
fluorescence. The magnitude of the initial fluorescence intensity
measured immediately after mixing HOQNO with DmsABC was very similar to
the magnitude of the fluorescence intensity measured in the absence of
the protein, indicating that no detectable reaction occurred during the
dead time of the stopped-flow instrument. The same results were also obtained by mixing 1 µM of DmsABC with various
concentrations of HOQNO in 100 mM MOPS and 5 mM
EDTA (pH 7.0) under the conditions in Figs. 5B and 6 (data
not shown). On the other hand, for the final fluorescence intensity
measured 50 s after mixing, the initial portion of the curve was
flat and the intercept on the x-axis (about 1 µM) obtained by extrapolating the linear portion of the curve to zero fluorescence was in good agreement with the 1:1 binding
stoichiometry obtained under the steady-state conditions (Figs. 1 and
2). Fig. 3 demonstrates that the fluorescence quenching observed after
mixing HOQNO with DmsABC is because of HOQNO binding to the
protein.

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Fig. 3.
Fluorescence quenching observed by mixing
HOQNO with wild-type DmsABC using the stopped-flow method. A
variety of concentrations of HOQNO in the buffer of 100 mM
MOPS and 5 mM EDTA at pH 7.0 were rapidly mixed with the
same buffer ( ) or mixed with 2 µM DmsABC in the same
buffer, ( ) and ( ). At a given concentration of HOQNO,
fluorescence quenching was observed from the decrease of the initial
fluorescence intensity ( ) (measured immediately after mixing) to the
final fluorescence intensity ( ) (measured 50 s after mixing).
The inset shows the decrease of fluorescence intensity with
time observed after mixing 2 µM DmsABC with 3 µM HOQNO.
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The DmsABCH65R mutant was used as a non-HOQNO binding
control for this work. Fig. 4 shows the
plot of the fluorescence intensities against the concentration of HOQNO
obtained by mixing 2 µM mutant DmsABCH65R
with various concentrations of HOQNO in 100 mM MOPS and 5 mM EDTA (pH 7.0). In contrast to Fig. 3, the plot of the
initial fluorescence intensity (measured immediately after mixing) and the final fluorescence intensity (measured 50 s after mixing) against the concentration of HOQNO gave two straight lines that not
only had the same slope but also overlapped each other within experimental error. Clearly, there was no significant quenching of
HOQNO fluorescence in this case. Fig. 4 indicates that the mutation of
DmsABCH65R blocks HOQNO binding to the protein, which
agrees with the steady-state titration data (Fig. 2). Furthermore, this
result serves as a good control for the stopped-flow experiment,
confirming that the nonlinear feature of the curve for the final
fluorescence intensity in Fig. 3 is not because of an artifact.

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Fig. 4.
Fluorescence observed by mixing
DmsABCH65R with HOQNO using the stopped-flow
method. 2 µM of
DmsABCH65R was rapidly mixed with various concentrations of
HOQNO in 100 mM MOPS and 5 mM EDTA at pH 7.0. , the initial fluorescence intensity measured immediately after
mixing; , the final fluorescence intensity measured 50 s after
mixing.
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As shown in Fig. 3, binding HOQNO to DmsABC causes a quenching of HOQNO
fluorescence, and this quenching process can be followed using the
stopped-flow method. To further investigate the kinetics of HOQNO
binding to DmsABC, 1 µM DmsABC was rapidly mixed with various concentrations of HOQNO using the stopped-flow method. Fig.
5A shows a typical trace of
fluorescence quenching observed after mixing 1 µM of
DmsABC with 2 µM HOQNO in 100 mM MOPS and 5 mM EDTA at pH 7.0 (25 °C) and the residuals for fitting
these data to Equation 1. The fluorescence quenching process was
completed about 50 s after mixing. The observed quenching trace
had two phases, a fast phase followed by a slow phase, and it was best fitted with the double exponential equation, Equation 1. This was also
true at other HOQNO concentrations used in this work (Fig.
5B). From the fit, the observed first-order rates and the amplitudes for the fast and slow phase at the given concentration of
HOQNO, kf, ks,
Af, and As, can be determined.
The first-order rate for the fast phase (kf) observed by mixing 1 µM DmsABC with various
concentrations of HOQNO in 100 mM MOPS and 5 mM
EDTA at pH 7.0 (25 °C) was plotted against the concentration of
HOQNO and the data obtained were fitted to Equation 3 as shown in Fig.
5B, from which the rate constants for the forward and
reverse reactions of the first equilibrium in Equation 2 were
determined to be k1 = (3.9 ± 0.3) × 105 M
1 s
1 and
k2 = 0.10 ± 0.01 s
1
respectively. Thus the dissociation constant for the first equilibrium, Kd1 = k2/k1, was calculated to
be about 260 nM.

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Fig. 5.
Kinetic analysis of fluorescence quenching
because of the interaction of HOQNO with DmsABC. A,
quenching of HOQNO fluorescence observed by mixing 1 µM
of DmsABC with 2 µM HOQNO in 100 mM MOPS and
5 mM EDTA at pH 7.0 (25 °C) and the fit of these data to
Equation 1. From the fit, the observed first-order rates and the
amplitudes for the fast and slow phase were determined to be
kf = 0.52 ± 0.06 s 1,
ks = 0.07 ± 0.008 s 1,
Af = 0.041 ± 0.003, and As = 0.035 ± 0.003, respectively. The lower part of the
figure shows the residuals of the fit. B, effect of HOQNO
concentration on kf. The kf data
were determined in the same way as described in panel A,
except varying HOQNO concentrations. The solid line
represents the fit of the kf data to Equation 3,
from which the rate constants for the first equilibrium in Equation 2
were determined to be k1 = (3.9 ± 0.3) × 105 M 1 s 1 and
k2 = 0.10 ± 0.01 s 1,
respectively.
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The rate constants for the forward and reverse reactions of the second
equilibrium in Equation 2, k3 and
k4, were evaluated with the Glint program
(Applied Photophysics). Using the reaction scheme described in Equation 2, and the k1 (=3.9 × 105
M
1 s
1) and
k2 (= 0.10 s
1) determined from the
fit as shown in Fig. 5B, k3 = 0.40 ± 0.04 s
1 and k4
0.01 s
1 were obtained from the good fits of the
calculated kinetic traces to the observed traces (data not shown).
Therefore, the association constant of the second equilibrium,
Ka2 = k3/k4
40, was
obtained. The upper limit of the overall dissociation constant for
Equation 2 was thus estimated to be about 6 nM,
i.e. Kdoverall = Kd1/(1 + Ka2)
6 nM.
To investigate the effect of the ligand (HOQNO) concentration on the
kinetics of HOQNO binding to DmsABC, the amplitudes for the fast phase
(Af) and the slow phase (As) were
plotted against the concentration of HOQNO (Fig.
6). The Af and
As were obtained by mixing 1 µM DmsABC with various concentrations of HOQNO in 100 mM MOPS and 5 mM EDTA at pH 7.0 (25 °C) and by fitting the observed
traces with Equation 1. The amplitude for the fast phase increased with
the increase of HOQNO concentration up to about 4 µM then
became saturated, whereas the amplitude for the slow phase increased
initially with HOQNO concentration up to 1 µM and then it
started to decrease with the increase of HOQNO concentration.

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Fig. 6.
Dependence of
Af and
As on HOQNO
concentration. The amplitudes for the fast phase
Af ( ) and slow phase As ( )
were obtained by mixing 1 µM of DmsABC with various
concentrations of HOQNO in 100 mM MOPS and 5 mM
EDTA at pH 7.0 (25 °C) and by fitting the observed traces to
Equation 1.
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DISCUSSION |
In bacterial anaerobic electron transport menaquinol shuttles
reducing equivalents from the primary dehydrogenases to the terminal
reductases within the cytoplasmic membrane. However, relatively little
is known about the interaction of menaquinol with these enzymes. In
this study, we have utilized direct steady state and rapid reaction
methodologies to investigate the binding of a fluorescent analog of
menaquinol to DmsABC. We have shown that HOQNO interacts with very high
affinity and 1:1 stoichiometry at a site within the DmsC subunit.
Mutation of His-65 to Arg in DmsC blocks quinol activity and eliminates
HOQNO binding. Our finding of only one exchangeable binding site, by
direct measurement, differs from studies with fumarate reductase (6-8)
and nitrate reductase (24), where two quinol binding sites have been
proposed by indirect methods.
In the stopped-flow studies, the biphasic nature of the observed
quenching process and the nonlinear increase of the observed slow rate
(ks) with HOQNO concentration reaching a plateau at
higher concentrations of HOQNO (data not shown) suggest that the
two-step mechanism (Equation 2) (21-23), a fast bimolecular process
followed by a slow unimolecular process, can be applied to describe the
quenching process resulting from binding HOQNO to DmsABC. In this
mechanism, it is assumed that a loosely bound complex of the HOQNO
(L) with DmsABC (E), EL, is formed
rapidly by a bimolecular association and then it is converted to a more tightly and specifically bound complex, EL*, by a slow
unimolecular process (isomerization). The isomerization may take place
through several possible pathways, such as rearrangement of
EL without any conformational change in E or
L, a conformational change in either E or
L, or conformational changes in both E and
L (22).
It is expected that stopped-flow kinetic studies can provide some
useful information about the bimolecular association of HOQNO with
DmsABC and the subsequent isomerization process. The effect of HOQNO
concentration on the amplitudes of the fast and slow phase may be
interpreted as follows. When the concentration of HOQNO is lower than
or comparable with the dissociation constant of the first equilibrium
in Equation 2, Kd1, but higher than the
overall dissociation constant, only a small amount of protein is
associated with the ligand (HOQNO) to form the complex EL by
the fast bimolecular process, which is observed as a fast phase of
quenching. The subsequent slow isomerization process of converting
EL to EL* would shift the first equilibrium in
Equation 2 to the right-hand side slowly. This slow shift of the first equilibrium is observed as a subsequent slow phase of quenching. Alternatively, when the concentration of HOQNO is increased to higher
than Kd1, more and more DmsABC molecules
are associated with HOQNO to give EL through the fast
bimolecular process, thus, less and less free DmsABC molecules are
available for the subsequent shift of the first equilibrium to the
right-hand side caused by the slow isomerization process. Therefore,
the amplitude of the observed fast phase of quenching increases with
increase of HOQNO concentration until becoming saturated, whereas the
observed slow phase of quenching decreases with increase of HOQNO
concentration, and this would finally result in that the amplitude of
the slow phase would be negligible compared with the amplitude of the
fast phase. These results suggest that the quenching of HOQNO
fluorescence occurs in the bimolecular step of the association of
E with L rather than in the unimolecular step of
forming EL* (see Equation 2).
Primary dehydrogenases and terminal reductases have been the subject of
a large number of steady-state kinetic investigations (see Refs. 24-31
as examples), but rapid reaction methodologies have been limited
because of the lack of available probes. We have now shown that
fluorescent HOQNO can be used to monitor the quinol binding reaction.
Although the steady-state methodology is useful for stoichiometry, the
affinity is so tight that Scatchard plots are not useful. The
stopped-flow method allows determination of rates and dissociation
constants. Although the three-dimensional structures of several
quinone-binding proteins have been determined (32, 33), it is not yet
possible to define a quinone binding motif. This technique will prove
useful in examining site-directed mutations in the quinol binding
region or in conformationally coupled changes that alter quinone
binding.
This work and a recent study on the interaction of HOQNO with fumarate
reductase using EPR and steady-state fluorescence spectroscopic methods
(34) demonstrate the utility of the characteristics of HOQNO
fluorescence and its structural analogy to MQH2 for
studying interactions of MQH2 with terminal reductases in
respiratory chains. Studies on the interactions of HOQNO with E. coli. nitrate reductase2
and with fumarate reductase3
are in progress.
We thank Dr. R. A. Rothery for helpful
discussions, assisting in protein preparations, and critically reading
the manuscript, and Dr. H. B. Dunford in the Department of
Chemistry, University of Alberta for providing the stopped-flow
facility.