(Received for publication, May 17, 1994; and in revised form, December 1, 1994)
From the
NAD(P)H:quinone oxidoreductase (EC 1.6.99.2) (DT-diaphorase) is
an FAD-containing enzyme that catalyzes the 2-electron reduction of
quinones to hydroquinones using either NADH or NADPH as the electron
donor. In this study, FAD was removed by dialyzing the holoprotein
against 2 M KBr, and synthetic analogs of FAD were substituted
in the flavin binding site as structural probes. Spectral analysis
indicates that the benzoquinoid forms of 8-mercapto-FAD and
6-mercapto-FAD are stabilized on binding to the enzyme. This is
consistent with the fact that the native flavoprotein forms the anion
flavin radical upon photoreduction and suggests the presence of a
positive charge near the N(1)C(2)O position of the isoalloxazine ring.
Reactivity studies using 8-chloro- and 8-mercapto-flavins suggest that
the 8 position of the FAD is accessible to the solvent. However, the
rates of the reactions were dramatically decreased in the presence of
the competitive inhibitor, dicumarol. 6-Mercapto-, 6-thiocyanato-,
6-azido-, and 6-amino-flavins were also used as structural probes. The
results indicate that the 6 position is accessible to solvent.
Dicumarol binding increases the pK of the
enzyme-bound 6-mercapto-flavin from below pH 5.0 to higher than pH 9.0.
The results suggest that DT-diaphorase shows the same properties as the
C-C transhydrogenases, and the binding of dicumarol elicits a
conformational change or an adjustment in the polarity of the FAD
pocket. The enzyme reconstituted with oxidized 5-deaza-FAD has
significant catalytic activity, confirming that DT-diaphorase is an
obligatory 2-electron transfer enzyme and plays a role in the
detoxification of quinones and quinoid compounds by reducing them to
the relatively stable hydroquinones.
NAD(P)H:quinone oxidoreductase (EC 1.6.99.2) (DT-diaphorase) is
a cytosolic FAD-containing protein that catalyzes the reduction of
quinones to hydroquinones using either NADH or NADPH as the electron
donor(1) . Besides quinones, DT-diaphorase reduces nitro
compounds(2, 3) , azo-dyes(4) , and hexavalent
chromium compounds (5) as well as ferricyanide. The rat liver
enzyme expressed in Escherichia coli is a dimeric molecule
containing one non-covalently bound FAD per protein monomer of 30
kDa(6) . According to Iyanagi and Yamazaki (7) the
oxidative half-reaction is a 2-electron transfer process without
formation of the reactive semiquinone intermediate. Therefore,
DT-diaphorase is thought to function in the cellular detoxification of
quinones by shunting these compounds away from the free radical state.
This is presumed to result in a decrease of oxygen free radicals, which
are generated by redox cycling of semiquinones in the presence of
oxygen(8) . In addition, the enzyme seems to be involved in the
reductive activation of the cytotoxic anti-tumor quinones (important
drugs for cancer chemotherapy(9) ), and it can function as one
of the vitamin K reductases in the blood coagulation
process(10) . While the physiological roles of the enzyme have
been extensively studied, at present the structure-function
relationships are not clearly understood. Analysis of the primary
structure revealed that the enzyme does not share significant sequence
homology with other flavoproteins (11, 12) . Chemical
modification and site-directed mutagenesis studies suggest that
DT-diaphorase possesses different binding sites for NAD(P)H, quinones,
and the FAD prosthetic
group(6, 13, 14, 15, 16, 17) .
In particular, Ma et al.(18) proposed that the
isoalloxazine moiety of the flavin is located at a site close to the
NAD(P)H domain, and a -turn-
structure is involved in FAD
binding. Some information is available regarding the binding pocket of
nicotinamide and the competitive inhibitor dicumarol, but the
topography of the FAD domain is not well characterized. In this study,
the flavin cofactor was removed, and synthetic analogs of FAD were
substituted as structural probes, providing new insights into the
environment of the flavin binding site and into the interaction between
dicumarol and the enzyme.
DT-diaphorase overexpressed in E. coli was purified
by Paulis Deng according to a previously reported
procedure(6) . NADH, NADPH, NADP, and
dicumarol were from Sigma. 8-Mercapto-FAD was prepared from
8-chloro-FAD by reaction with sodium sulfide(19) .
6-Mercapto-FAD was prepared from 6-thiocyanato-FAD by reaction with
1,4-dithiothreitol(20) . The apoenzyme was prepared by
dialyzing the holoenzyme for 3 days at 4 °C against 200 mM potassium phosphate, pH 6.0, containing 0.3 mM EDTA, 20%
glycerol, 2 M KBr, and activated charcoal (about 1 g added to
200 ml of dialyzing fluid) to adsorb FAD as soon as it passed through
the dialysis membrane. When the yellow color of the enzyme was no
longer visible, the dialysis medium was changed to 200 mM potassium phosphate, pH 7.0, containing 0.3 mM EDTA and
20% glycerol. Reconstitution with oxidized flavin analogs was performed
in the same buffer at ice temperature with variable amounts of
apoprotein and excess of flavin. Unbound flavin was removed by repeated
ultrafiltration on a CM30 microconcentrator (Amicon). The spectrum of
the filtrate was recorded and subtracted from that of the mixture
before ultrafiltration to yield the spectrum of the reconstituted
enzyme. Dissociation constants were then calculated based on the
concentrations of the starting apoenzyme and the concentration of the
flavin in the filtrate.
DT-diaphorase assays were carried out at 25
°C in the reconstitution buffer by following the decrease in
absorbance at 340 nm in the presence of an excess of FAD or FAD
analogue. NADPH was used as electron donor, and
2-hydroxy-1,4-naphthoquinone was used as electron acceptor. The
activity of the apoenzyme due to residual FAD (1-5% initial) was
always measured and subtracted from the measured activity of
reconstituted enzyme. In all cases the double-reciprocal plots present
a set of parallel lines indicating a ping-pong mechanism as reported
for the native enzyme (21) but without definable values of V and K
for
electron donor or acceptor, as described for other
acceptors(22) .
Anaerobic experiments were performed in all glass apparatus (23) by sequential evacuation and re-equilibration with oxygen-free argon.
Photochemical reduction experiments were performed by irradiating the solution with a Sun Gun (Smith Victoria Corp., Griffith, IN). Visible spectra were taken using a Hewlett Packard 8452 A diode array spectrophotometer or a Varian Cary 3 UV-visible spectrophotometer. If not otherwise indicated, all the reactions were carried out at 25 °C in 0.2 M potassium phosphate, pH 7.0, containing 0.3 mM EDTA and 20% glycerol.
All the analogs are enzymatically active and can be reduced
by NAD(P)H under anaerobic conditions. Using
2-hydroxy-1,4-naphthoquinone as electron acceptor, there are no
definable values of k and K
, as already described for the native
enzyme(22) . The second order rate constants for the reaction
of the reduced flavoprotein with the quinone (k
)
are collected in Table 1along with the E
values of the flavin analogs.
Similar to results with the native enzyme, with all the FAD analogs the
reduction of the flavin appears to be much faster than the reoxidation,
since the observed steady-state rates were very little influenced by
the concentration of NADPH(22) .
Figure 1: Reaction of 8-chloro-FAD-DT-diaphorase with thiophenol. The reconstituted enzyme before (1) and after (2) 50 µM thiophenol at pH 6.0 and 25 °C is shown. Inset, plot of apparent rates versus thiophenol concentration. The rate of the reaction was calculated by following the change in absorbance at 490 nm.
Accessibility of the flavin 8 position was explored by reacting the
reconstituted enzyme with sulfur nucleophiles. Before the reaction, the
reconstituted enzyme was passed through a Sephadex G-25 column
equilibrated in 0.2 M phosphate buffer, pH 7.0, 0.3 mM EDTA, and 20% glycerol to remove excess of free flavin. Fig. 1shows the spectral changes encountered on the addition of
50 µM thiophenol to 8-chloro-FAD-DT-diaphorase. The
typical two-banded spectrum is replaced by an intense single maximum at
480 nm with a shoulder at 460 nm. This spectrum is clearly different
from that of free 8-phenylmercapto-FAD(29) , suggesting that
the protein can accommodate this somewhat bulky substituent without
release of the flavin. Solvent accessibility has also been examined
under the same conditions as in Fig. 1but with
1,4-dithiothreitol, sodium sulfide, and -mercaptoethanol (data not
shown) used as nucleophiles. As shown in Table 2, it is clear
that the 8-chloro group of 8-chloro-DT-diaphorase reacts faster than
does the 8-chloro group of the free flavin, suggesting that this
position is solvent-exposed and that the protein can stabilize the
tetrahedral intermediates of the reactions(32) . Monophasic
kinetics were observed with all thiol reagents, and simple second order
rate constants were calculated in each case. These results confirm that
the reconstituted enzyme is a homogeneous species and that no free
flavins are present.
In particular, sodium sulfide at pH 5 and pH 8
effects the displacement of chloride, giving a spectrum typical of the
benzoquinoid form of 8-mercapto-FAD(19) . A titration of free
8-mercapto-FAD with apoenzyme results in a non-resolved spectrum with
an extinction coefficient of 30,200 M cm
at 590 nm (Fig. 2). In this form,
the negative charge is carried over the N(1)C(2)O locus rather than on
the benzene subnucleus of the flavin, and its stabilization by the
enzyme suggests the presence of a positive charge in the FAD binding
site of DT-diaphorase. As expected, the decreased nucleophilicity of
this form results in much lower reactivity of the
enzyme-bound-8-mercapto-FAD toward thiol reagents. The second order
rate constants for iodoacetamide and methyl methanethiolsulfonate
reactions were 1.07 M
min
and 342 M
min
,
respectively (data not shown), much lower than the reaction with free
8-mercapto-FAD under the same conditions(32) .
Figure 2:
Titration of 8-mercapto-FAD with
apo-DT-diaphorase. 8-Mercapto-FAD (3.4 µM), curve
1, was titrated with apoenzyme 0.77 (2), 1.73 (3), and 3.4 µM (4) at pH 8.0. Inset, change in extinction coefficient at 520 () and
590 (
) nm on addition of apoenzyme.
By reacting
8-mercapto-FAD-DT-diaphorase with hydrogen peroxide, we further
confirmed the accessibility of the 8 position to solvent. The oxidation
occurs in two distinct steps. The extinction at 590 nm dropped fairly
rapidly to 16,000 M cm
,
and the maximum was shifted to 620 nm, suggesting the presence of a
deprotonated thiol oxide, as previously observed for 6-mercapto-FAD
reacted with hydrogen peroxide (33) and for 8-mercapto-FAD-NADH
peroxidase after peroxide addition(34) . The second order rate
constant for the initial oxidation of the flavin thiol was 37.5 M
min
(Fig. 3A). In the second step of the reaction,
the flavin-8-sulfenic acid undergoes further oxidation to either the
deprotonated sulfinic or sulfonic acid (without further detectable
spectral change between the latter) and with a second order rate
constant of 3.5 M
min
(Fig. 3B). Reactivity of the flavin thiol with m-chloroperbenzoic acid is also high and produces spectral
changes similar to those found on addition of hydrogen peroxide (data
not shown).
Figure 3:
Spectral course of the reaction of
8-mercapto-FAD-DT-diaphorase with 3.45 mM hydrogen peroxide. 1, starting spectrum; 2 and 3, spectra of
S-oxide derivative; 4, spectrum of sulfinate/sulfonate
derivative. Spectra in panelA were recorded before (curve1) and 2, 5, and 9 min after addition of
HO
. Spectra in panelB were
recorded at 12, 18, 25, 32, 42, and 89 min after
H
O
. The rates of the reaction were calculated
from the decrease in absorbance at 600 nm.
Addition of an excess of 1,4-dithiothreitol immediately
upon completion of the first reaction step or after the final oxidation
results in a displacement of S-oxide or
SO=/SO
=groups and the
formation of a 1,4-dithiothreitol derivative (spectrum similar to that
shown in Fig. 1), suggesting that the protein can stabilize the
tetrahedral intermediate of this reaction. This observation for
8-substituted flavin establishes a major difference from 6-SO-flavins
that can be reduced by 1,4-dithiothreitol and
6-SO
=/SO
= flavins that do not
react with 1,4-dithiothreitol(33) . The stability of the flavin
8-S-oxide derivative was also tested under anaerobic conditions in the
presence of catalase to avoid further oxidation. As expected, no
further spectral changes were detected after the addition of catalase,
indicating the absence of any nucleophilic groups in the vicinity of
the flavin 8 position that can displace the S-oxide group.
The same
conclusions can be reached using 8-OH-FAD as spectral probe at
different pH values (pH 4, 5.5, and 10). DT-diaphorase at pH 5.5
stabilizes the anionic form of 8-hydroxy-FAD with maximum at 480 nm
(data not shown). This result was taken as a further evidence for the
existence of a positive charge near the N(1)C(2)O position and the
absence of a negative charge near the 8 position of the flavin. The
binding with apoprotein, in fact, does not result in an increase in the
pK of the 8-OH-FAD enzyme (pK
= 4.8 for the free flavin) as observed, for example, in
Old Yellow Enzyme (35) .
Several of the properties of flavoprotein oxidases as a class have been attributed to the presence of such a positively charged group in the FAD binding site(36) . Since among these are the ability to form an N(5) adduct with sulfite, the reactivity of native enzyme and 8-mercapto-FAD with this reagent was also examined. No spectral perturbations were observed by reacting normal FAD-DT-diaphorase with 10 mM sodium sulfite, indicating no adduct formation at the flavin N(5) position.
As expected, the reaction of 8-mercapto-FAD-DT-diaphorase
(wavelength maximum 590 nm, = 30,200 M
cm
) with sulfite
yields 8-sulfonyl-FAD enzyme (wavelength maximum 460 nm,
=
9200 M
cm
), and no
further reaction occurs at the N(5) position (37) (data not
shown). A negatively charged protein residue near the N(5) position,
repelling approach to the sulfite ion, could be responsible for such
failure, as well as the presence of a hydrogen bond acceptor near the
N(1) locus of the flavin instead of a hydrogen bond donor. There is
generally a close correlation among the properties of the
8-mercapto-flavoprotein and the properties of the semiquinoid form of
the native enzyme(19) . In particular, the 8-mercapto enzyme is
easily reduced by substrate for those flavoproteins, which in the
native state give little or no thermodynamic stabilization of a
semiquinoid species. In the case of DT-diaphorase, the 8-mercapto-FAD
enzyme can be reduced by 70 µM NADH under anaerobic
conditions and can be slowly reoxidized by oxygen without any evidence
for a semiquinone intermediate. The same result was obtained for the
native FAD enzyme. This is consistent with the fact that the native
flavoprotein thermodynamically stabilizes only 8-10% of the red
anionic semiquinone, with maxima at 382 and 452 nm, upon photoreduction
in the presence of EDTA, 5-deazaflavin-3-sulfonate, and benzylviologen
as redox mediator(22) .
6-Azido-FAD-DT-diaphorase is also
converted to the 6-amino-FAD enzyme by aerobic turnover with NADPH.
Within seconds of the addition of a substoichiometric concentration of
NADP and an NADPH-generating system
(glucose-6-phosphate and glucose-6-phosphate dehydrogenase) to an
anaerobic solution of 6-azido-FAD enzyme, there was substantial
bleaching of the 429-nm absorption, followed by the appearance of the
6-amino-FAD spectrum after opening to air. This product can be also
reduced by NADPH under anaerobic conditions.
6-Thiocyanato-FAD binds
to DT-diaphorase with an absorption maximum at 441 nm. The resulting
holoprotein reacts very fast with 1,4-dithiothreitol, suggesting also
that the 6 position is accessible to solvent. A second order rate
constant of 1750 M min
was calculated for the reconstituted enzyme in comparison with
the value of 1000 M
min
obtained for the free flavin under the same conditions. As
expected for the presence of a positive charge near the N(1)C(2)O
position of the flavin, also in this case the anionic form of
6-mercapto-flavin was highly favored, as shown in Fig. 4by the
presence of the long wavelength absorbance.
Figure 4:
Conversion of
6-thiocyanato-FAD-DT-diaphorase to 6-mercapto-FAD-DT-diaphorase.
6-Thiocyanato-FAD enzyme (1) was reacted with 196 µM 1,4-dithiothreitol at 25 °C, and the time course of the
reaction was followed at 441 nm. 2, spectrum of the resulting
6-mercapto-FAD enzyme in the anionic form. Inset, plot of
apparent rates versus 1,4-dithiothreitol concentration in the
absence () and in the presence (
) of 30 µM dicumarol.
In keeping with the
benzene ring of the bound 6-mercapto-flavin being readily accessible to
solvent, reaction with methyl methanethiolsulfonate is very fast, and
the 6-mercapto-FAD spectrum can be regenerated by the addition of
1,4-dithiothreitol (data not shown). 6-Mercapto-FAD enzyme reacts also
with iodoacetamide with a second order rate constant of 85 M min
(150 M
min
for the free
flavin under the same conditions) and with m-chloroperbenzoic
acid or hydrogen peroxide to produce the flavin-6-S-oxide (180 M
min
for hydrogen
peroxide). As observed with lactate monooxygenase(33) , this
species is very resistant to further oxidation, and extended incubation
with excess of peracid is required for sulfinate/sulfonate formation.
It is probably the anion form of the S-oxide that is stabilized because
of a positively charged protein residue in the vicinity of the flavin
N(1) position, which stabilizes anionic flavin species, including the
6-mercapto flavin(33) .
Taken together, the preceding results describe a flavin binding pocket that is accessible to solvent along one entire edge of the isoalloxazine ring system, from position 8 to position 6. This feature seems to be common to all the pyridine nucleotide-dependent flavoproteins so far studied(36) . Furthermore, as indicated by the non-resolved spectrum, the general flavin environment is hydrophilic, and positively charged protein residue(s) exist near the N(1)C(2) locus. The enzyme does not react with sodium sulfite and forms a red semiquinone that is not thermodynamically stabilized. This description, although it constitutes a minimal picture of the flavin-protein interactions, suggests that DT-diaphorase shows the same properties as the C-C transhydrogenases according to the general classification proposed by Massey and Hemmerich (38) for flavoenzymes.
1-Deaza-FAD-DT-diaphorase was also reduced by NADPH and reoxidized by oxygen or diazaquinone as electron acceptor without any evidence for semiquinone formation.
An interesting observation was
made using 6-substituted FAD analogs. In this case, dicumarol binding
had no effect on the rates of the reactions described in a previous
section but increased the pK of the enzyme-bound
6-mercapto-FAD from below pH 5.0 to greater than pH 9.0. In Fig. 5, the spectrum of the anionic form of 6-mercapto-FAD
enzyme is shown and the neutral form obtained after adding dicumarol at
pH 7.0. At this pH, the free flavin is largely ionized
(pK
= 5.9(20) ) with the thiol
group bearing the negative charge. The negative charge is shifted
mainly to the N(1) locus after binding to the protein, as indicated by
the absorbance at long wavelengths (Fig. 5, inset). The
same effects of dicumarol were obtained from pH 5.5 to 8.5. Since the
pK
for dicumarol free or bound to the protein is
7.5(39) , the increase in the pK
of the
enzyme-bound 6-mercapto-FAD is not related to the ionization state of
the dicumarol. Possibly, binding of the competitive inhibitor elicits a
conformational change or an adjustment in the polarity of the FAD
binding site favoring such phenomena. Whatever the mechanism, the
ineffectiveness of dicumarol on the spectral properties of
8-mercapto-FAD at pH 5-8 suggests that the positive charge near
the N(1)C(2)O position of the flavin is not affected, although the
environment of the 8 position is changed since the rates of
nucleophilic substitution reactions are very much reduced.
Figure 5: Dicumarol effect on 6-mercapto-FAD-DT-diaphorase. Spectra of 6-mercapto-FAD-DT-diaphorase in the absence (1) and in the presence (2) of 37 µM dicumarol at pH 7.0. The same result was obtained in 5 mM sodium pyrophosphate buffer, pH 8.5, and 0.1 M sodium acetate, pH 5.0. Inset, titration of 6-mercapto-FAD with apo-DT-diaphorase. 9.3 µM 6-mercapto-FAD (1) was titrated with 1.7 (2) and 9.3 µM (3) apoenzyme at pH 8.5. Spectra are not corrected for dilution.
In
distinction to the results with dicumarol, the pK of 6-mercapto-FAD enzyme and the reactivity of the 8 position do
not change in the presence of 40-50 µM NADP
or quinones like benzoquinone and menadione,
which are known to be very good substrates for DT-diaphorase. These
observations could be interpreted as being due to different binding
sites for dicumarol and NADPH, despite dicumarol being a competitive
inhibitor against NADPH in steady-state assays(4) . However, no
kinetic evidence could be obtained for binding of either pyridine
nucleotides or quinones, suggesting that the binding affinity of the
enzyme for these compounds is at best very weak(22) . For this
reason, lack of effect of pyridine nucleotides and quinones on various
properties of the enzyme are difficult to interpret.
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