(Received for publication, June 2, 1995; and in revised form, September 5, 1995)
From the
To elucidate the catalytic mechanism of monodehydroascorbate
(MDA) reductase from cucumber, its interaction with MDA radical was
investigated by the use of pulse radiolysis. When approximately
equimolar MDA radical to the fully reduced MDA reductase was generated,
the fully reduced enzyme reacted first with MDA radical to form the red
semiquinone, and the semiquinone further reacted with MDA radical to
form the oxidized enzyme. At a low ratio (<20) of MDA radical to
enzyme concentration, the fully reduced enzyme reacted quantitatively
with MDA radical to form the semiquinone with a second-order rate
constant of 2.6 10
M
s
at pH 7.4. At excess MDA radical to enzyme
concentration, a similar rate constant was obtained from the decay of
MDA radical. These results suggest that the reaction of the semiquinone
with MDA radical occurs at the same rate or rate-limiting step of the
oxidation of the fully reduced enzyme by MDA radical. The rate
constants decreased with an increase in NaCl concentration, suggesting
that the localization of cationic groups of amino acid residue near the
active site may provide electrostatic guidance to the anionic substrate
of MDA radical.
Ascorbate (AsA) (
)is critically
involved in cellular defense against oxidative injury, serving as a
reductant in scavenging reactive species of oxygen and radical
species(1, 2, 3, 4, 5) . In
these processes, MDA radical is produced by univalent oxidation of
AsA
(6, 7, 8, 9) .
Regeneration of AsA
from MDA radical is indispensable
to keep the defense activity of AsA
against oxidative
stress. Rapid removal of MDA radical is also necessary to protect cells
from the putative damaging effects of MDA radicals.
In mammalian
cells, enzymatic activity responsible for the NADH-dependent
regeneration of AsA from MDA radical has been
demonstrated(10, 11, 12, 13) . In
plants, NAD(P)H-dependent reducing activity for MDA radical has been
found not only in chloroplasts (14) but also in
non-photosynthetic tissues (15, 16) and in
algae(17, 18) . This activity regenerates
AsA
from MDA radical in chloroplasts and other
components for scavenging hydrogen peroxide by ascorbate peroxidase (14, 19) . A peculiar enzyme with this activity, named
MDA reductase, has been purified from cucumber fruit(20) ,
soybean root nodules(21) , and potato tubers(22) . The
enzyme from cucumber is a soluble monomeric enzyme, with a molecular
mass of 47 kDa, that contains 1 molecule of FAD per enzyme molecule.
Recently, cDNAs of the enzyme from cucumber seedlings (23) and
pea (24) have been cloned and the amino acid sequences deduced.
In addition, an overproduced system of MDA reductase of cucumber using
cDNA of the enzyme was established in Escherichia coli, and
the enzyme produced in E. coli was purified to a crystalline
state and partially characterized(25) .
Because of the
instability of its substrate, MDA radical, the molecular activity of
MDA reductase can be measured only by the rate of oxidation of NADH in
the presence of AsA-AsA
oxidase. On
the other hand, it is possible to measure the catalytic action of MDA
reductase directly by using pulse radiolysis to generate MDA radical (26, 27, 28, 29) . By the use of
this technique, we have directly observed the reaction of MDA radical
with hepatic NADH-cytochrome b
reductase(29) . The b
reductase has
been shown to be a good electron donor for MDA radical in mammalian
cells, and its reaction mechanism is supposed to be similar to that of
MDA reductase(30) . In this report, pulse radiolysis has been
used to study the reaction mechanism of MDA reductase. We have
confirmed that MDA radical reacts with the fully reduced form of MDA
reductase with a diffusion-controlled rate.
The recombinant MDA reductase was overproduced in E. coli transformed with pET-CMR, an expression plasmid possessing the
cDNA of cucumber (cytosolic) MDA reductase(23) . The enzyme was
purified to a homogeneous state, as reported previously(25) .
All other reagents were commercially obtained as the analytical grade.
The concentration of MDA reductase was determined on the basis of FAD
bound to the enzyme, using a molar extinction coefficient of 9.63
mM cm
at 450
nm(20) . The concentration of MDA radical generated by pulse
radiolysis was estimated from the absorbance at 360 nm, using a molar
extinction coefficient of 3300 M
cm
(28) .
Samples of MDA reductase for
pulse radiolysis were prepared as follows. Solutions containing 10
mM potassium phosphate buffer (pH 7.4) and 5 mM
AsA were bubbled with N
O gas for 5 min. A
concentrated solution of MDA reductase (420 µM) was added
to the solution. Subsequently, 1 equivalent NADH to the enzyme was
added to the solution to give a complex of reduced enzyme with
NAD
that exhibits distinctive long-wavelength
absorbance. A fresh enzyme solution was used for each pulse. Pulse
radiolysis experiments were performed with an electron linear
accelerator in the Institute of Scientific and Industrial Research
(Osaka University)(29, 31, 32) . The pulse
width and energy were 8 ns and 27 MeV, respectively. Absorption changes
were measured using a fast spectrophotometric system composed a Nikon
monochromator, an R-928 photomutiplier, and Unisoku data analyzing
system.
Hydrated electron (e),
OH
, and H
are produced by pulse
radiolysis of aqueous solutions. In the presence of 5 mM
AsA
, MDA radical is produced via the following
reactions by pulse radiolysis of N
O-saturated aqueous
solution.
The participation of H in this system can be
neglected, since the total yield of e
and OH
is considerably larger than that of
H
at neutral pH(27) . A transient spectrum of
MDA radical with an absorption maximum at 360 nm was observed at 100 ns
after pulse radiolysis. On pulse radiolysis of the fully NADH-reduced
form of MDA reductase in the presence of AsA
, the
absorption changes due to the oxidation of FADH
of
the enzyme were observed as shown in Fig. 1. Under the
experimental conditions, 30 µM MDA radical was generated
in 28 µM fully reduced form of the enzyme. The absorption
at 600 nm, characteristic of a charge transfer band between reduced
enzyme and NAD
, decreased, but that at 500 nm
increased. On the other hand, the absorption increase at 450 nm
consisted of a fast and a slow phase. At 525 nm, only a slow absorption
change was observed. This indicates that at least two distinct
reactions are involved in the oxidation of the NADH-reduced MDA
reductase by MDA radical.
Figure 1:
Absorption
changes after pulse radiolysis of the fully reduced form of MDA
reductase measured at 600, 525, 500, 450, and 360 nm in the presence of
AsA and N
O at pH 7.4. The reaction
mixture contained 28 µM MDA reductase, 30 µM NADH, 5 mM AsA
, and 10 mM potassium phosphate buffer at pH 7.4.
The kinetic difference spectra at 1, 100,
and 800 µs after the pulse are shown in Fig. 2A.
The spectrum at 1 µs after the pulse with an absorption maximum at
360 nm corresponds to the MDA radical. In this stage, little oxidation
of the enzyme-FADH-NAD
complex was
observed. The spectrum at 100 µs has twin absorption peaks at 455
and 500 nm, which is similar to that of the difference spectrum of the
semiquinone minus that of the E-FADH
-NAD
complex in Fig. 2B. In this time range, no
absorption change was observed at 525 nm, an isosbestic point between
the semiquinone and the fully reduced enzyme (Fig. 2). On the
other hand, the difference spectrum at 800 µs is not similar to
that of the semiquinone minus E-FADH
-NAD
but to that of the oxidized form minus
E-FADH
-NAD
. No absorption change was
observed at 500 nm, an isosbestic point between the oxidized and the
semiquinone forms, on this time scale. Therefore, the slower phase seen
in Fig. 1at 525 and 450 nm is attributable to the oxidation of
the semiquinone to the oxidized form of the enzyme. From these results,
under the conditions where equimolar MDA radical and NADH-reduced MDA
reductase were reacted, MDA radical reacted first with the fully
reduced enzyme to form the semiquinone enzyme. Subsequently, the
semiquinone thus formed further reacted with MDA radical to form the
oxidized form of the enzyme. From the absorbance changes of Fig. 2A, 13 µM of the red semiquinone was
formed initially in 28 µM of the fully reduced enzyme, and
subsequently 10 µM of the semiquinone was further oxidized
to the fully oxidized form of the enzyme.
Figure 2:
Kinetic difference spectra after pulse
radiolysis of MDA reductase at pH 7.4 (A) and the difference
spectra of the semiquinone form of MDA reductase (straight
line) and the oxidized enzyme (dashed line) minus the
fully reduced form of the enzyme in complex with NAD,
respectively (B). A, the spectra were taken at 1
µs (
), 100 µs (&cjs0571;), and 800 µs (
) after
pulse radiolysis. Experimental conditions are the same as in Fig. 1. B, enzyme concentration was 74 µM in 50 mM phosphate buffer (pH 7.4). The semiquinone was
obtained by illumination for 20 min with a 1-kW tungsten lamp. The
fully reduced form was obtained by the addition of 80 µM NADH.
When the similar experiment was performed in the presence of NADH (50-200 µM), further slow absorption change at 460 nm was observed with a half time of 2 ms (data not shown). This absorption change is attributable to the re-reduction of the oxidized enzyme with NADH, since a similar absorption change was not seen in the presence of 1 equivalent of NADH to the enzyme. The rate constant of this process depends on the concentration of NADH.
For determination of the rate
constant of the reduction of MDA radical with the fully reduced MDA
reductase, 1-2 µM MDA radical is generated in a
solution containing 28-50 µM MDA reductase. Typical
examples are shown in Fig. 3A. Under the present
conditions, MDA radical reacted quantitatively with the fully reduced
enzyme to form the semiquinone, and no further slow absorption change
was observed. The absorption changes at 460 and 600 nm obeyed
pseudo-first-order kinetics. Fig. 3B shows the
dependence of the apparent first-order rate constant on the
concentration of MDA reductase, giving a straight line. From the slope
of the line, the second-order rate constant of the reaction is
estimated to be 2.8 10
M
s
. The effect of NaCl
on the rate constant was also examined. As shown in Fig. 4, the
rate constants decreased nearly to 0.5
10
M
s
with an increase in
NaCl concentration.
Figure 3:
Absorption changes after pulse radiolysis
of the fully reduced form of MDA reductase measured at 600 and 460 nm
in the presence of AsA and N
O (A) and concentration dependence of apparent rate constants of
the oxidation of the fully reduced MDA reductase from the decrease of
absorption at 600 nm and the increase of absorption at 460 nm. The
reaction mixture contained 38 µM MDA reductase, 40
µM NADH, 5 mM AsA
, and 10
mM phosphate buffer pH 7.4 (A).
Figure 4:
NaCl concentration dependence of the rate
constant of the fully reduced form of MDA reductase with MDA radical at
pH 7.4. The reaction mixture contained 41.2 µM MDA
reductase, 60 µM NADH, 10 mM phosphate buffer (pH
7.4), and 5 mM AsA and NaCl at indicated
concentrations.
The rate constant for the reaction of MDA radical with the semiquinone form of the enzyme was not determined directly in the present experiment, since the semiquinone state for pulse radiolysis experiments could not be prepared as a stable form.
At excess MDA radical concentration (20-40 µM) to
the fully reduced MDA reductase (1-3 µM), the decay
of MDA radical was followed at 360 nm. Fig. 5A shows
the absorption changes at 360 nm in the absence and presence of the
E-FADH-NAD
complex. In the presence
of 2.2 µM E-FADH
-NAD
complex, an initial rapid decrease that obeyed pseudo-first-order
kinetics was observed, whereas in the absence of the enzyme a similar
initial decay was not seen on this time scale. Under the conditions, 4
µM MDA radical, which corresponds to approximately two
equivalents of the enzyme, decayed within 200 µs. This indicates
that the fully reduced MDA reductase is oxidized through two successive
one-electron transfers to MDA radical. The decay of MDA radical
remaining at the end of reaction with the enzyme occurs due to
spontaneous disproportionation in the time range of 100 ms (data not
shown). When the same experiments were performed in various doses to
generate MDA radical at the indicated concentration, the apparent
initial rate of the decay was linear to the concentration of MDA
radical (Fig. 5B). From the slope of the figures, the
second-order rate constant is calculated to be 2.6
10
M
s
.
Figure 5:
Absorption changes of MDA radical measured
at 360 nm in the absence and presence of the fully reduced form of MDA
reductase (A) and AsA concentration
dependence of the initial decay at 360 nm (B). The reaction
mixture contained 2.2 µM MDA reductase, 2.2 µM NADH, 10 mM phosphate buffer (pH 7.4), and 5 mM
AsA
.
The present experiments clearly show that MDA reductase is an effective scavenger of MDA radical. The reaction scheme for NADH-dependent MDA radical reduction by cucumber MDA reductase can be described as follows, which has been also supported by a ping-pong mechanism as evidenced by reaction kinetics(20) .
The enzyme FAD is reduced by NADH to form a charge-transfer
complex, E-FADH-NAD
in (20, 25) . Then, the reduced enzyme
donates the electron to MDA radical through two successive one-electron
transfers, and the FAD semiquinone, E-FAD-NAD
,
is an intermediate ( and ). We find that the
charge-transfer complex is oxidized by MDA radical to form the red
semiquinone of the enzyme. The anionic red semiquinone form of the
enzyme has also been shown by photochemical reduction of the enzyme in
the presence of 1 mol equivalent NAD
to the
enzyme(25) . By contrast, the semiquinone cannot be obtained by
the photochemical reduction in the absence of NAD
.
Furthermore, preliminary studies using pulse radiolysis showed that a
blue semiquinone is transiently observed by the reaction of MDA radical
with the fully dithionite-reduced enzyme in the absence of
NAD
. (
)From these results, the semiquinone
form of MDA reductase is the blue species, and it is converted into the
red species by the binding of NAD
.
The second-order
rate constant (k) between MDA radical and the
fully reduced MDA reductase in is 2.6
10
M
s
. On
the other hand, the good agreement of the rate constants determined
under both conditions MDA&cjs1138;
MDA reductase and MDA
reductase
MDA&cjs1138;suggests that k
k
and that formation of the semiquinone
of the enzyme is principally rate limiting. Based on this estimate, a
lower limit of the second-order rate constant of 2.6
10
M
s
is
obtained for the reaction of MDA radical with the semiquinone of the
enzyme (k
). A similar reaction sequence has been
proposed for NADH-cytochrome b
reductase. However,
the rate constants of k
and k
in MDA reductase are about 50 times larger than those in b
reductase (k
= 4.3
10
M
s
, k
= 3.7
10
M
s
)(29) . We have proposed that the electron
transfer from b
reductase to MDA radical simply
occurs at a flavin edge to the solvent through bimolecular
collision(30) . In contrast, the high rate constants of MDA
reductase and a high specificity of MDA radical to the enzyme (25) cannot be explained by a similar mechanism. It seems
likely that MDA reductase has an active site structure that accepts MDA
radical. This may be reflected in the homology in amino acid sequence
between MDA reductase and bovine b
reductase(23) . However, from the kinetic analysis of
both NADH oxidation in the steady state (25) and the reaction
of MDA radical with the reduced species of the enzyme in the present
study, we have no evidence of the complex formation of the reduced
species of MDA reductase with MDA radical. The MDA reductase does not
exhibit saturation kinetics in the range of 50 µM MDA
radical. One of possible factors for the facilitated reaction is that
the localization of cationic groups of amino acid residue near the
active site provides electrostatic guidance to the anionic substrate,
MDA radical, whose pK
is
-0.45(28) . This is supported by the result that the rate
constant of MDA reductase with MDA radical decreases with an increase
in NaCl concentration (Fig. 4). On the other hand, a
second-order rate constant for the reduction of MDA reductase by NADH (k
) was determined to be 1.25
10
M
s
by
stopped flow analysis(25) . This process has been shown to
proceed at the similar rate to those of and with electrostatic guidance of NADH(25) . This
suggests that the same residues of MDA reductase seem to facilitate
rapid interaction of both the electron donor and the acceptor with the
enzyme-FAD. A similar ionic strength effect was obtained in the
reaction of O
with copper-zinc superoxide
dismutase(33) . Although the high rate constant of the enzyme
reaction is governed by the specific geometry and redox potential of
the redox center, it is clear that modulation of electrostatic effects
near the active site affect the rate by as much as 1 order of
magnitude.
If the concentration of NADH is more than several
hundred-fold of that of MDA radical, the rate-limiting step of the
reaction catalyzed with MDA reductase is the oxidation of the reduced
enzyme with MDA radical rather than the reduction of the enzyme with
NADH. To keep the rapid enzymatic rate of regeneration of
AsA and avoid the injury with the radical, the ratio
of the concentration of NADH to MDA radical must be kept more than
several hundred in cells where MDA reductase functions. In cells where
concentrations of NADH are typically very high as compared with that of
MDA radical, the reaction of MDA reductase with MDA radical is rate
limiting for the overall process. In illuminated chloroplasts, MDA
radical generated by AsA
peroxidase is reduced in a
reaction mediated by photoreduced ferredoxin(34) , and
NADP
is also reduced to NADPH in photosystem I,
resulting in a high ratio of NADPH to MDA radical. Probably, in the
stroma, the ratio of NADPH to MDA radical would be kept high so that
the reaction catalyzed with chloroplastic MDA reductase is not limited
by the reductive half-reaction, which would have a higher affinity for
NADPH than cytosolic isozymes does.
In conclusion, MDA reductase is
an effective scavenger of MDA radical. The second-order rate constant
for MDA reductase is the greatest value measured for the reaction of
MDA radical with biological molecules (29) . In contrast to
plant cells, such efficient scavenge system of MDA radical has not been
yet realized in mammalian cells, though NADPH-dependent regenerating
activity of AsA from MDA radical has been found. The
presence or absence of the system is necessary to clarify.