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(Received for publication, May 17,
1994; and in revised form, October 4, 1994) From the
NAD(P)H:quinone oxidoreductase (DT-diaphorase) appears to be a
2-electron transfer flavoprotein, which catalyzes the conversion of
quinones into hydroquinones. Upon photoreduction in the presence of
dimethylformamide, the enzyme forms a red semiquinone. In the absence
of dimethylformamide, only 10% of the radical form is thermodynamically
stabilized. This indicates a redox potential of the enzyme-bound
semiquinone/reduced flavin couple that is higher than the midpoint
potential for the oxidized flavin/semiquinone couple. The 2-electron
redox potential was determined to be -159 ± 3 mV at 25
°C, pH 7.0. In the presence of benzoquinone or 3-aminopyridine
adenine dinucleotide phosphate, as NADPH analogue, there is no change
in the redox properties of the enzyme flavin. A significant decrease is
observed in the presence of the competitive inhibitor dicumarol (E NAD(P)H:quinone oxidoreductase (EC 1.6.99.2), also known as
DT-diaphorase, is a flavoprotein widely distributed in animal tissues (1) . It is a dimeric enzyme with two molecules of FAD, one for
each subunit(2) . The name DT-diaphorase arose because of its
ability to use either NADH or NADPH as cofactors in its reduction of
quinone substrates. The enzyme is also remarkable in its high
sensitivity to inhibition by dicumarol. However, little is known about
the mechanism by which dicumarol inhibits the electron transfer
functions of this protein, except to note that the sequence of addition
of NAD(P)H and dicumarol appears important(3) . Another unique
property of the enzyme is the result of the initial observation by
Iyanagi and Yamazaki (4) that DT-diaphorase serves to transfer
2 electrons to a quinone, resulting in the formation of a hydroquinone
product, without the accumulation of a dissociated semiquinone. This
property has served as the starting point for a large number of studies
to examine the possible relationship of DT-diaphorase to oxygen
toxicity. By 1-electron reduction of quinones, semiquinones are
generated that react spontaneously with molecular oxygen to give the
parent quinone and superoxide. They can also react with nucleophiles,
such as reduced glutathione, depleting the reduced thiol and
nicotinamide nucleotide pools. Furthermore, quinone metabolites
contribute significantly to the toxic and carcinogenic effects of
aromatic hydrocarbons. By reducing quinones directly to hydroquinones,
DT-diaphorase is proposed to be a cellular device against their
toxicity(5) . Moreover, the enzyme can participate in the
metabolism of vitamin K and in the activation of anticancer drugs like
AZQ, ( DT-diaphorase overexpressed in Escherichia coli was
purified by Paulis Deng according to a previously published procedure (10) . NADPH, NADH, APADH, AADP
where
Figure 1:
Anaerobic photoreduction of
DT-diaphorase in the presence of DMF. DT-diaphorase in 50 mM phosphate buffer, pH 7.0, 15 mM EDTA, 1 µM 5-deazaflavin-3-sulfonate, 5 µM benzylviologen, and
75 mM DMF was irradiated under anaerobiosis at 25 °C.
Selected spectra are only shown at different times of irradiation: top solid line, oxidized enzyme after 170, 230, 300,
and 510 s; bottom solid line, after 750 s of irradiation. Inset, plot of absorbance at 474 nm versus absorbance
at 380 nm at various times of irradiation.
Figure 2:
Anaerobic determination of DT-diaphorase
redox potential by equilibration with a dye of lower redox potential.
DT-diaphorase 11.8 µM in 0.1 M phosphate buffer,
pH 7.0, 25 °C, containing 6 mM EDTA, 0.5 mM xanthine, 5 µM benzylviologen, and 25 µM 1-hydroxyphenazine was incubated under anaerobic conditions with
2.38
Moreover, from the calculated value of (E
Figure 3:
A, steady-state data for NADPH-PQQ
reductase activity of DT-diaphorase. DT-diaphorase activity was
determined spectrophotometrically by following the decrease in
absorbance at 350 nm at the concentrations of NAD(P)H shown using
different PQQ concentrations:
However, with DT-diaphorase, with all electron donors and
acceptors that we have studied, results such as those shown in Fig. 3have been obtained. With none of the substrates is there
evidence for the existence of a limiting velocity, and in all cases the
kinetic results are described within experimental error by the second
order rate constants k
where AH The second-order rate constants for
NADPH or its analogues, k
which sufficiently describes the experimental results.
Figure 4:
Reaction of the reduced flavoprotein with
PQQ. The reduced flavoprotein (17.3 µM before mixing) was
mixed with different concentrations of PQQ in 0.05 M phosphate
buffer, pH 7.0, at 25 °C. The graph shows changes at 450 nm (PQQ
concentrations of 52, 56.5, 135.5, 294, and 362 µM, traces from right to left). Inset,
direct plot of the observed rate as a function of PQQ
concentration.
Figure 5:
Reaction of the reduced flavoprotein with
AZQ. The reduced flavoprotein (28.3 µM before mixing) was
mixed with different concentrations of AZQ in 50 mM phosphate
buffer, pH 7.0, at 25 °C. The graph shows changes at 450 nm (AZQ
concentrations of 20, 40, 60, and 80 µM, traces from right to left). Inset, direct plot
of the observed rate as a function of AZQ
concentration.
where k
where k
In free solution the flavin radical exists in very rapid
equilibrium with its oxidized and fully reduced form with only a few
percent of the total flavin being in the radical form. It is well known
that when bound to a protein the stability of the flavin radical is
generally enhanced. In particular, Massey et al.(25) discussed some correlative properties of flavoproteins and
suggested that with few exceptions the semiquinoid form of the oxidases
is the red (or anionic) form, whereas the dehydrogenases/oxygenases are
remarkable in displaying little stabilization of any radical form.
Finally, the enzymes involved in obligatory 1-electron transfer
reactions almost uniformly appear to give thermodynamic stabilization
of the blue neutral flavin radical, and there is real evidence for the
catalytic functioning of the radical state. DT-diaphorase stabilizes
only 8-10% of the red semiquinone upon photoreduction or sodium
dithionite titration, corresponding to a negative difference in the
redox potentials of the couple EFAD/EFADH A ratio of binding constants for
oxidized and reduced flavin to the apoenzyme can be calculated from the
shift in redox potential of FAD when bound to DT-diaphorase. The
midpoint potential for the enzyme-bound FAD of -159 mV at pH 7.0
is not very different from that of free flavin (-207 mV at pH
7.0) and corresponds to a ratio of binding constants of 43 with the
reduced flavin bound more tightly to the apoenzyme than oxidized FAD.
This results in thermodynamically more favorable reduction of the
coenzyme. The presence of reduced benzoquinone and the reductive
pyridine nucleotide analogue AADP Regarding the steady-state and
stopped-flow data, a few points require comment. In our studies, four
electron donors and three different electron acceptors were used as
substrates. DT-diaphorase is remarkable in its ability to reduce a
variety of oxidants including quinones(28) ,
azo-compounds(34) , chromium VI compounds(35) ,
benzotriazine di-N-oxides(36) , and
nitro-compounds(37) . Many of these substrates, however, are
not suitable for kinetic studies because of their solubility or
spectral properties and because they are reduced too fast by
DT-diaphorase. In this study, using K
For
K
In none of these cases are there definable values of k
Volume 270,
Number 3,
Issue of January 20, 1995 pp. 1198-1204
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
REDOX POTENTIAL, STEADY-STATE, AND RAPID REACTION STUDIES (*)
= -234 ± 2 mV at pH 7.0).
The reaction mechanism of the flavoprotein has been investigated by
steady-state and stopped-flow kinetic methods using NADPH, NADH,
deamino-NADPH, and 3-acetylpyridine adenine dinucleotide reduced form
(APADH) as electron donors and K
Fe(CN)
,
4,5-dihydro-4,5-dioxo-1H-pyrrolo-[2,3-f]quinoline-2,7,9-tricarboxylic
acid (PQQ), and
2,5-diaziridinyl-3,6-bis(carboethoxyamino)-1,4-benzoquinone (AZQ) as
electron acceptors in 50 mM phosphate buffer, pH 7.0, 25
°C. No evidence could be obtained to indicate that semiquinoid
intermediates play a part in the catalytic mechanism of DT-diaphorase
with quinones as acceptors. The rates of the reduction by NADPH, NADH,
deamino-NADPH, and APADH (1.3 
10
, 8.8
10
, 8.3 10 and 9.8
10M min
,
respectively) as well as the rates of the reoxidation by PQQ and AZQ (9
10
and 2.8 10M min
,
respectively) are directly proportional to substrate concentration, and
there is no evidence of the formation of enzyme-substrate complexes. If
such complexes do indeed exist, the affinity of the enzyme for
substrate must be extremely low. Using K
Fe(CN) as electron acceptor, the rate of oxidation of fully reduced
enzyme is 4.6 10
M min
and it is accurately proportional to
ferricyanide concentration. This rate represents that of flavin
semiquinone formation, with the subsequent oxidation of the semiquinone
being much faster, since no spectral evidence for semiquinone formation
could be obtained. Studies were also conducted attempting to use
apo-DT-diaphorase reconstituted with PQQ as coenzyme. The lack of
activity toward AZQ, K
Fe(CN), and menadione
suggests that DT-diaphorase can use PQQ only as electron acceptor and
not as redox cofactor.
)mitomycin C, dinitrophenylaziridine (CB 1954), and
benzotriazine-di-N-oxide SR 4233 (6) by 2-electron
reduction processes. However, in spite of the many studies of the
biological role of DT-diaphorase and its possible use in cancer
chemotherapy, the redox properties of the flavoprotein were undefined
and the presumed obligatory 2-electron reduction of quinones rested
largely with the initial observation of Iyanagi and
Yamazaki(4) . In this study we determined the midpoint
potential for the couple FAD/FADH
in DT-diaphorase and the
properties of the radical form. Moreover, mechanistic studies are
reported using different electron donors (NADPH, NADH, deamino-NADPH,
and APADH) and three electron acceptors:
KFe(CN), PQQ, and AZQ.
KFe(CN) was chosen as the only obligatory
1-electron acceptor that is reported to be a good substrate for
DT-diaphorase(7) , PQQ as a coenzyme with quinoid
properties(8) , and AZQ as very important anticancer
nitrocompound that seems to be activated in vivo by
DT-diaphorase reduction. A possible explanation for the inhibitory
effect of dicumarol (9) on the NAD(P)H dehydrogenase activity
is also proposed.
,
deamino-NADPH, and PQQ were purchased from Sigma. All other chemicals
used were of analytical reagent grade. AZQ was a generous gift from Dr.
David Ross, School of Pharmacy, University of Colorado.
Enzyme Assays
DT-diaphorase activity was determined
spectrophotometrically by following the decrease in absorbance of the
electron donor at 340 nm, 350 nm (isosbestic point for the reduction of
PQQ), and 333 nm (isosbestic point for the reduction of AZQ) using
KFe(CN), PQQ, and AZQ as electron acceptors.
All determinations were made at 25 °C in 50 mM phosphate
buffer, pH 7.0, in the presence of excess FAD. Using PQQ as a
substrate, all the values were corrected for the contribution of the
free PQQ nonenzymatically reacting with NADPH or its
analogues(8) .Photochemical Reduction
Photoreduction of
DT-diaphorase was achieved by irradiating the protein under anaerobic
conditions in the presence of 15 mM EDTA, 1 µM 5-deazaflavin-3-sulfonate as catalyst, and 5 µM benzylviologen, at 25 °C in 50 mM phosphate buffer,
pH 7.0(11) . The concentrations of semiquinone and reduced
enzyme were calculated at 450 and 400 nm for each point in the
photoreduction using the following
equation:
A (ox - x) is the decrease in
absorbance in comparison to the spectrum of the oxidized enzyme,
,
, and
are the
extinction coefficients for oxidized, reduced, and radical enzyme
forms, respectively, and [red] and [sq] are the
molar concentration of reduced DT-diaphorase and its red semiquinone.
The extinction coefficients of the semiquinone at different wavelengths
were obtained by photoreduction of the enzyme in the presence of 75
mM DMF. When experiments were carried out in the presence of
AZQ or benzoquinone, they were photochemically reduced as described
above. After complete reduction the enzyme was added from the side arm
of the anaerobic cuvette in order to be itself photochemically reduced.
Dithionite Titration of
DT-diaphorase
DT-diaphorase (1.0 ml) was made anaerobic and
titrated with dithionite in 50 mM phosphate buffer, pH 7.0, at
25 °C by adding aliquots of an anaerobic dithionite solution from a
gas-tight syringe attached to the anaerobic cuvette by ground glass
joints. Benzylviologen (5 µM) was present during the
titration to ensure redox equilibration.Determination of Redox Potential
The redox
potential for the flavoprotein was determined at 25 °C, pH 7.0, 50
mM phosphate buffer by a spectrophotometric method employing a
reducing system of xanthine and xanthine oxidase and a suitable
mediator such as benzylviologen(12) .Stopped-flow Absorbance Spectrophotometry
The
stopped-flow apparatus has been described
previously(13, 14) . Anaerobiosis of the flow system
was achieved by equilibration overnight with an anaerobic solution of
protocatechuate-3,4-dioxygenase (a generous gift of Dr. David P.
Ballou, University of Michigan). Rate constants were obtained by
exponential fits using the software ``Program A'' (developed
by Chung-Jen Chiu, Rong Chung, Joel Dinverno, and Dr. David P. Ballou,
University of Michigan), which permits the analysis of experimental
data by exponential fits based on the Marquardt algorithm(15) .
This program permits the fixing of as many as five rate constants and
two amplitudes and subsequent curve fitting to evaluate the absorbance
of any intermediate species. It also permits the simulation of model
kinetic pathways for comparison with the experimental data.Anaerobic Reduction of Enzyme
To 10 ml of about 15
µM enzyme in the main compartment of a tonometer, 250
µM xanthine and 1.5 µM benzylviologen (the
redox mediator) were added. After anaerobiosis, 50 µl of xanthine
oxidase (A = 0.35) was added from the
side arm and the reduction was carried out at room
temperature(12) . When PQQ was used as electron acceptor, the
enzyme was reduced by
glucose-6-phosphate/glucose-6-phosphate-dehydrogenase using 50 mM glucose-6-phosphate, 0.1 µM NAD
, and
a catalytic amount of glucose-6-phosphate dehydrogenase.
Apoprotein Preparation
DT-diaphorase apoprotein
was prepared by dialyzing the holoenzyme at 4 °C against 2 M KBr as described elsewhere. ()Reconstitution with PQQ
was performed at ice temperature in 50 mM phosphate buffer, pH
7.0, 20% glycerol, and activity assays were carried out as described in
a previous section, in the presence of an excess of free PQQ.Equipment
Visible spectra were taken using a
Hewlett Packard 8452A diode array spectrophotometer. Photoreduction
experiments were carried out by irradiating the solution with a Sun Gun
(Smith Victoria Corp., Griffith, IN).
Photochemical Reduction
DT-diaphorase yields the
characteristic red semiquinone spectrum upon
photoreduction(11) . As shown in Fig. 1, a quantitative
production of red semiquinone was obtained in the presence of 75 mM DMF. From the plot of absorbance at 474 nm versus 380 nm
(inset of Fig. 1) the extinction of fully formed radical could
be calculated. In the presence of benzylviologen a slow
disproportionation occurs to yield 80% of the red semiquinone in
thermodynamically stabilized form. On the other hand, in the absence of
DMF, only 8% of semiquinone was obtained upon photoreduction. The
result suggests that the organic solvent changes the protein
environment or directly interacts with the
NC=O position of the
flavin(11) . The spectral changes after each irradiation were
complete within 10-15 min, and no further changes were detected
during periods up to 30 min in the dark. More than 30 min of
irradiation was required for complete reduction. On mixing with air, no
detectable radicals were observed; the reoxidation followed a
pseudo-first order reaction with a rate constant of 0.0462
min at ice temperature. Photoreduction in the
presence of 30 µM AADP
, as
NADP
/NADPH analogue, showed the same pattern. The
reoxidation rate at ice temperature was practically identical with that
previously determined in the absence of AADP
,
indicating that the autoxidation of the flavin is unaffected by the
presence of pyridine nucleotide. The same results were obtained in the
presence of 50 µM photoreduced AZQ or 100 µM photoreduced benzoquinone, suggesting that the presence of the
product does not increase the thermodynamic stabilization of the red
semiquinone. According to Clark (16) 8% of radical produced at
equilibrium corresponds to a negative separation of the two redox
potentials for FAD/FADH
(E
) and
FADH
/FADH
(E) in the
enzyme. From the Nernst equation (E
- E
) was calculated to be -0.083 V at 25
°C, pH 7.0.
Dithionite Titration of DT-diaphorase
Addition of
1 mol of dithionite/FAD, or 2 electron equivalents, brings complete
reduction of DT-diaphorase (results not shown). The concentration of
semiquinone was quantitated from the change in absorbance at 400 and
450 nm. The maximum percent of red semiquinone was calculated to be
10%. From the Nernst equation, (E - E
) was calculated to be -0.072 V, at 25
°C and pH 7.0, in quite good agreement with the value from the
photoreduction experiment.
Determination of Redox Potentials
To measure the
potential of the FAD/FADH couple in DT-diaphorase, a
reductive titration was done in the presence of 1-hydroxyphenazine (E at pH 7.0 = -0.172 V; (17) ) according to the method of Massey(12) . A
5-µl sample of 5 mM 1-hydroxyphenazine was added to 1 ml
of DT-diaphorase in 0.1 M phosphate buffer containing 0.5
mM xanthine, 6 mM EDTA, and 5 µM benzylviologen at pH 7.0 and 25 °C, with 2.38
10M milk xanthine oxidase added under
anaerobic conditions to start the reaction. The amounts of oxidized and
reduced indicator dye as well as the amounts of oxidized and reduced
DT-diaphorase were quantitated at 370 and 480 nm, respectively (Fig. 2). The log(ox/red) for the 1-hydroxyphenazine was plotted versus the log(ox/red) for the enzyme-bound-FAD (Fig. 2, inset) according to the method of
Minneart(18) . This plot gives a 1-unit slope and a value of
-159 ± 3 mV for the FAD/FADH
potential (Table 1). A similar titration was done in the presence of indigo
disulfonate (E = -125 mV at pH 7.0; (19) ), which undergoes a loss of absorbance at 610 nm during
reduction and shows an isosbestic point at 444 nm. The potential for
the FAD/FADH couple was found to be -159 ± 3
mV (Table 1) in excellent agreement with the value from the
1-hydroxyphenazine titration. From the Minnaert plot (cf.Fig. 2, inset), a 1-unit slope was calculated, as
expected for equilibrium between a 2-electron acceptor dye and a
2-electron donor without semiquinone formation. When the redox
titration in the presence of 1-hydroxyphenazine was repeated in the
presence of 10 µM photoreduced benzoquinone or 100
µM AADP the potential for the
FAD/FADH
couple was calculated to be -159 ± 3
mV and -165 ± 3 mV, respectively (Table 1),
suggesting that both the products of the reductive and the oxidative
half-reaction do not change the redox properties of the enzyme flavin.
However, a large decrease in the E for the
FAD/FADH couple was observed when the reduction of
DT-diaphorase was carried out in the presence of 7 µM phenosafranine (E = - 252 mV at
pH 7.0; (17) ) and 22.5 µM dicumarol. The amount
of reduced dye was quantitated by measuring the percent decrease at 530
nm compared with the maximum observed decrease at this wavelength
before the reduction of benzylviologen. The amount of reduced FAD was
quantitated by measuring the percent decrease at 456 nm corrected for
the contribution of the dye at this wavelength. The Minnaert plot gives
an E of -234 ± 2 mV for the
FAD/FADH
couple without semiquinone formation (Table 1). The lowering of E with dicumarol
implies that dicumarol binds preferentially to the oxidized form of the
enzyme. From the shift in midpoint potential of FAD (-207 mV, ref
12) upon binding to the enzyme (-159 mV) and the known
dissociation constant of FAD from the oxidized enzyme (K
= 1.8
10M)
a value of 4.2 10M for K
can be calculated using
the following
equation(16) .
10M milk xanthine oxidase.
The spectra shown from top to bottom were recorded at 0, 1.5, 3, 4.5,
6, 8, and 11 min. Inset, data obtained while monitoring
reduction of 1-hydroxyphenazine at 370 nm corrected for the
contribution of the FAD and the reduction of FAD at 480 nm corrected
for the contribution of the dye.
- E
), the redox potential for the
FAD/FADH
and FADH
/FADH
couples can be predicted to be approximately -0.2 and
-0.118 V, respectively.Steady-state Kinetics
Steady-state assays were
performed following the decrease in absorbance of NADPH or its
analogues at 25 °C, pH 7.0. Data such as those presented in Fig. 3were obtained using PQQ, AZQ, or KFe(CN) as electron acceptors. As far as we know, this is the first time
that PQQ, which is a well known cofactor for quinoproteins, has been
reported to be a substrate for DT-diaphorase. Using this coenzyme as
electron acceptor, the parallel pattern of the double-reciprocal plot
confirms that the reaction proceeds with a ping-pong mechanism, as
described previously for DT-diaphorase reacting with AZQ(20) .
The same result was obtained when KFe(CN) was
used as acceptor. The steady-state data may be described by a general
equation of the form shown
below(21) .
, 28 µM;
, 47
µM;
, 66 µM; 
, 84
µM; 
, 103 µM. B, replots
obtained from plot A. The graph was obtained by plotting the
intercepts of graph Aversus 1/PQQ
concentration.
and k for any set of substrate concentrations. Thus the reaction may be
formulated as shown in Reactions 1 and
2
= reduced pyridine nucleotide and B
= electron acceptor., cannot be determined
with accuracy from steady-state assays as it is impracticable to work
with electron donor concentrations sufficiently low to influence
significantly the rate of the reaction. Values of k for different electron acceptors, obtained from slope of
intercept plots such as that of Fig. 3B, are reported
in Table 2.
Reductive Half-reaction Experiments
The only rapid
reaction studies so far reported with DT-diaphorase are those of Hosoda et al.(22) . They reported an extremely fast second
order reduction of the enzyme by NADPH, with an estimated value of 3.1
10M s
at pH 7.0, 15 °C. Similar results were found in this study,
but because of the rapidity of the reaction, our data were obtained at
4 °C. The reductive half-reaction has been studied by stopped-flow
spectroscopy, monitoring the decrease in absorbance at 450 nm after
mixing 7.5 µM oxidized enzyme with electron donor
(10-50 µM; concentrations after mixing)
anaerobically. The observed changes in absorbance versus time
were monophasic, and the total change was always equal to the expected
change in absorbance for the complete reduction of the enzyme. Data
obtained at longer wavelengths showed no evidence of transient
charge-transfer complexes. With each of the 4 reduced pyridine
nucleotides studied, the observed rate of reduction was directly
proportional to the reduced pyridine nucleotide concentration, with the
second order rate constant shown in Table 3. Thus, from the rapid
reaction studies, there is no evidence for the formation of a complex
between oxidized enzyme and pyridine nucleotide. If such a complex does
exist, then it must be of low affinity, with K
values > 0.5 mM. The reaction of oxidized enzyme with
NADPH, NADH, deamino-NADPH, and APADH can be reduced
to
Oxidative Half-reaction Experiments
Hosoda et
al.(22) reported the very rapid reoxidation of reduced
enzyme by menadione, too fast to be measured by stopped-flow
spectrophotometry. We were able to confirm this finding; even at 4
°C the reoxidation of reduced enzyme by menadione, at
concentrations barely in excess of that of the enzyme, was complete
within the 3-ms dead time of our stopped-flow apparatus. For this
reason, in order to study the oxidative half-reaction, reduced
DT-diaphorase was prepared and reacted under anaerobic conditions at 25
°C with slower substrates: AZQ and PQQ. With both the substrates,
there were no detectable intermediates formed in the dead time of the
stopped-flow experiments ( Fig. 4and Fig. 5) or spectral
indications of intermediates at any wavelength. In particular only two
spectrophotometrically distinguishable forms of both the flavin and the
acceptor (oxidized and reduced) were detected in the 600-700 and
420-500 nm regions, where the PQQ semiquinone is reported to have
maximum absorbance(8) , as well as at 375 and 480 nm, where
both AZQ (23) and flavin radical can be easily observed. The
reactions followed second order kinetics, and again there was no
evidence for the formation of an enzyme-substrate complex. The
oxidative half-reaction can then be written
as
values are reported in Table 2, and found to be in good agreement with those obtained
from steady-state kinetics.Oxidative Half-reaction Using K
Since KFe(CN) as Electron AcceptorFe(CN) is an obligatory 1-electron acceptor, a flavin semiquinone
intermediate must occur in the oxidation of reduced enzyme.
Experimentally no flavin radical could be detected as an intermediate
and it was possible to monitor only the complete oxidation of the
enzyme. The rate traces were single exponential at all wavelengths and
a second order rate constant was calculated as shown in Table 2.
The mechanism to account for a stepwise 1-electron oxidation of fully
reduced flavin is the case of two irreversible consecutive
reactions
is faster than k. In order to confirm this observation, the
oxidation by KFe(CN) of
semiquinone-DT-diaphorase was carried out by stopped-flow measurements.
The red semiquinone was obtained by photoreduction in the presence of
DMF and was oxidized using KFe(CN) under
anaerobic conditions. The oxidized enzyme was completely formed in the
dead time of the stopped-flow instrument (3 ms), even at ferricyanide
concentrations barely in excess of the enzyme concentration, giving a
lower limit estimate of k as 3.7 10M min
, at least 80
times greater than the value of k
.Attempted Reconstitution of Deflavoenzyme with
PQQ
PQQ appears to be a water-soluble, organic, 1-electron
transfer redox cofactor, and quinoprotein dehydrogenases are reported
to operate via two single electron processes(24) .
DT-diaphorase, on the contrary, appears to react with PQQ as a
2-electron acceptor. It was considered interesting to check if the
enzyme can also use PQQ as coenzyme. Apo-DT-diaphorase was prepared as
described elsewhere and incubated with an excess of PQQ.
The enzyme showed no activity versus AZQ,
KFe(CN), or menadione, which is a typical
substrate for FAD-DT-diaphorase. The FAD-reconstituted protein, in
control experiments, was fully active when tested under the same
conditions.
and
EFADH
/EFADH
. These data suggest that
DT-diaphorase does not operate with a typical semiquinone intermediate
form of finite lifetime and thermodynamic considerations favor a
simultaneous 2-electron transfer mechanism. The results are in
agreement with earlier studies on DT-diaphorase(4) . This
property is peculiar to the protein and is presumably important for the
physiological role of the enzyme in its function in the cellular
detoxification of hydroquinones without semiquinone formation. Iyanagi
and Yamazaki (4) reported electron spin resonance studies as
direct experimental evidence of DT-diaphorase behaving as a 2-electron
donor. However, the possibility was not ruled out that an unstable
radical intermediate was present during the reoxidation of the
flavin(22) , and the redox properties of DT-diaphorase and the
reaction mechanism were not studied in detail. The present
determination of redox potentials and the photoreduction experiments
give further insight into this behavior. The results obtained with the
obligatory 1-electron acceptor, KFe(CN), are
particularly instructive. Although the intermediate formation of the
enzyme flavin radical is a clear requirement in this reaction, no
flavoprotein semiquinone could be detected, simply because the second
electron transfer was much faster than the first 1-electron transfer.
This possibility clearly exists with all of the acceptors studied.
Since DT-diaphorase uses FAD as coenzyme, it could participate in two
1-electron transfer reactions as well as in one 2-electron transfer
process. In the first case, the lifetime of the flavin radical could be
very short and the coenzyme would be fully oxidized before the release
of the product, as previously suggested in the reaction versus menadione(22, 26) . Of course, these arguments do
not rule out the possibility of a simple 2-electron transfer mechanism,
as commonly assumed. From the data reported in this work, the redox
potential for the EFAD/EFADH couple can be predicted
to be
-0.200 V. Many molecules like
quinone-epoxides(27) , 2-hydroxy-1,4-naphthoquinone (28) , benzoquinones(28) , and
anthraquinones(29) , which have redox potentials for the
quinone/quinone radical couple much lower than -0.200 V, are very
good acceptors of DT-diaphorase, arguing against single electron
transfer. While the possibility exists that binding of the substrate
can change the redox properties of the flavin and circumvent the
thermodynamic barrier against 1-electron transfer between FADH
and these molecules, it is important to note that with none of
the compounds tested was any evidence found for the formation of
radical intermediates. The result is clearly not surprising since in
studies with FAD analogues, we observed that the enzyme is
catalytically active when reconstituted with 5-deaza-FAD, which can be
oxidized only through a single 2-electron step(30) .
Furthermore, the enzyme cannot use PQQ as redox coenzyme but only as
electron acceptor. Remarkably, this coenzyme is involved in 1-electron
transfer catalysis in bacterial quinoprotein dehydrogenases (24) and seems to be present in mammalian tissues as a new
vitamin whose potential biochemical role is correlated with the
nonenzymatic oxidation of NAD(P)H(31) . The molecule
efficiently scavenges superoxide by oxidation and in pharmacologic
doses protects against glutathione depletion(32) . Indeed it
will be interesting to investigate further its function as a
DT-diaphorase substrate in order to clarify the role of both the enzyme
and PQQ in NAD(P)H metabolism. has no effect on the
midpoint potential of the enzyme-bound FAD, as well as the semiquinone
stability, suggesting that the thermodynamic electron transfer to and
from the enzyme is not controlled significantly by the substrate or the
product. This result is in accord with the steady-state kinetics, which
indicate lack of binding or at best very weak binding of substrates to
the enzyme. On the other hand, the presence of dicumarol results in a
change in the redox potential from -0.159 to -0.234 V,
making the reduction thermodynamically more difficult. While it is
tempting to speculate that this decrease might explain why dicumarol is
reported to be a competitive inhibitor of pyridine nucleotide and
uncompetitive inhibitor of the electron acceptor in spite of the fact
that its binding site seems to be different from the binding site of
NAD(P)H and its analogues(22) , its dramatic inhibition of
reduction rate by NAD(P)H (2) makes this explanation unlikely.
These results argue for a conformational change upon binding of
dicumarol, consistent with the observations reported with FAD
analogues
and proteolytic digestion
experiments(33) .Fe(CN),
PQQ, and AZQ as electron acceptor, the reaction was sufficiently slow
to be followed accurately by stopped-flow spectrophotometry. In
addition, both PQQ and AZQ can form semiquinone species sufficiently
stable to be detected by the stopped-flow technique. With all the
oxidative and the reductive substrates, there was no evidence for the
formation of an enzyme-substrate complex and two simple irreversible
half-reactions put together provide a sufficient scheme to account for
all the kinetic data. The schemes to which the rates apply are the
following. For AZQ and PQQ as electron
acceptors,
Fe(CN),
and K
for either electron
donor or acceptor, with catalytic velocities determined directly by the
concentration of the donor or the acceptor. It appears from the
experimental results that if a complex is formed at all, the affinity
of the enzyme for the substrate must be strikingly low and the true
rate-limiting step is the reoxidation of the flavin. This result is in
accordance with the lack of specificity shown by DT-diaphorase with
respect to the electron accepting substrate as well as the electron
donor, suggesting that the active site of this enzyme can accommodate
molecules of varying size and structure.
), 3-aminopyridine
adenine dinucleotide phosphate; PQQ,
4,5-dihydro-4,5-dioxo-1H-pyrrolo-[2,3-f]quinoline-2,7,9-tricarboxylic
acid.
)
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
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