©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
DT-diaphorase
REDOX POTENTIAL, STEADY-STATE, AND RAPID REACTION STUDIES (*)

(Received for publication, May 17, 1994; and in revised form, October 4, 1994)

Gabriella Tedeschi (1) Shiuan Chen (2) Vincent Massey (1)(§)

From the  (1)Department of Biological Chemistry, The University of Michigan Medical School, Ann Arbor, Michigan, 48109-0606 and the (2)Division of Immunology, Beckman Research Institute of the City of Hope, Duarte, California 91010

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

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(m) = -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(3)Fe(CN)(6), 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 times 10^9, 8.8 times 10^8, 8.3 times 10^8 and 9.8 times 10^6M min, respectively) as well as the rates of the reoxidation by PQQ and AZQ (9 times 10^4 and 2.8 times 10^6M 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(3)Fe(CN)(6) as electron acceptor, the rate of oxidation of fully reduced enzyme is 4.6 times 10^7M 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(3)Fe(CN)(6), and menadione suggests that DT-diaphorase can use PQQ only as electron acceptor and not as redox cofactor.


INTRODUCTION

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, (^1)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(2) 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: K(3)Fe(CN)(6), PQQ, and AZQ. K(3)Fe(CN)(6) 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.


MATERIALS AND METHODS

DT-diaphorase overexpressed in Escherichia coli was purified by Paulis Deng according to a previously published procedure (10) . NADPH, NADH, APADH, AADP, 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 K(3)Fe(CN)(6), 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:

where DeltaA (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. (^2)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).


RESULTS

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 N(1)C(2)=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(2) (E) in the enzyme. From the Nernst equation (E - E) was calculated to be -0.083 V at 25 °C, pH 7.0.


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.



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(2) couple in DT-diaphorase, a reductive titration was done in the presence of 1-hydroxyphenazine (E(m) 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 times 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(2) potential (Table 1). A similar titration was done in the presence of indigo disulfonate (E(m) = -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(2) 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(2) 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(m) for the FAD/FADH(2) couple was observed when the reduction of DT-diaphorase was carried out in the presence of 7 µM phenosafranine (E(m) = - 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(2) 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 times 10M)^2 a value of 4.2 times 10M for K can be calculated using the following equation(16) .


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 times 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.





Moreover, from the calculated value of (E - E), the redox potential for the FAD/FADH and FADH/FADH(2) 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 K(3)Fe(CN)(6) 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 K(3)Fe(CN)(6) was used as acceptor. The steady-state data may be described by a general equation of the form shown below(21) .


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: , 28 µM; , 47 µM; times, 66 µM; bullet, 84 µM; circle, 103 µM. B, replots obtained from plot A. The graph was obtained by plotting the intercepts of graph Aversus 1/PQQ concentration.



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(1) and k(2) for any set of substrate concentrations. Thus the reaction may be formulated as shown in Reactions 1 and 2

and

where AH(2) = reduced pyridine nucleotide and B = electron acceptor.

The second-order rate constants for NADPH or its analogues, k(1), 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(2) 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 times 10^8M 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(d) values > 0.5 mM. The reaction of oxidized enzyme with NADPH, NADH, deamino-NADPH, and APADH can be reduced to



which sufficiently describes the experimental results.

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


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(2) 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(3)Fe(CN)(6) as Electron Acceptor

Since K(3)Fe(CN)(6) 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

where k(3) is faster than k(2). In order to confirm this observation, the oxidation by K(3)Fe(CN)(6) 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 K(3)Fe(CN)(6) 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(3) as 3.7 times 10^9M min, at least 80 times greater than the value of k(2).

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^2 and incubated with an excess of PQQ. The enzyme showed no activity versus AZQ, K(3)Fe(CN)(6), 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.


DISCUSSION

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 and EFADH/EFADH(2). 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, K(3)Fe(CN)(6), 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(2) 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,^2 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.

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 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^2 and proteolytic digestion experiments(33) .

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(3)Fe(CN)(6), 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,

For K(3)Fe(CN)(6),

In none of these cases are there definable values of k and K(m) 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.


FOOTNOTES

*
This work was supported by Grant GM-11106 from the U. S. Public Health Service and grant 963 GI from the American Heart Association, Greater Los Angeles Affiliate (to S. C.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed.

(^1)
The abbreviations used are: AZQ, 2,5-diaziridinyl-3,6-bis(carboethoxyamino)-1,4-benzoquinone; DMF, dimethylformamide; deamino-NADPH, nicotinamide hypoxanthine dinucleotide phosphate reduced form; APADH, 3-acetylpyridine adenine dinucleotide reduced form; AADP, 3-aminopyridine adenine dinucleotide phosphate; PQQ, 4,5-dihydro-4,5-dioxo-1H-pyrrolo-[2,3-f]quinoline-2,7,9-tricarboxylic acid.

(^2)
Tedeschi, G., Chen, S., and Massey, V., J. Biol. Chem., in press.


REFERENCES

  1. Ernster, L. (1987) Chem. Scripta 27A, 1-13
  2. Huang, M.-T., Miwa, G., and Lu, A. Y. H. (1979) J. Biol. Chem. 254, 3930-3934 [Abstract]
  3. Huang, M.-T., Miwa, G., and Lu, A. Y. H. (1978) Biochem. Biophys. Res. Commun. 83, 1253-1259 [Medline] [Order article via Infotrieve]
  4. Iyanagi, T., and Yamazaki, I., (1970) Biochim. Biophys. Acta 216, 282-294 [Medline] [Order article via Infotrieve]
  5. Prochaska, H. J., and Talalay, P. (1991) in Oxidative Stress: Oxidants and Antioxidants (Sies, H., ed) pp. 195-211, Academic Press, Ltd., New York
  6. Riley, R. J., and Workman, P. (1992) Biochem. Pharmacol. 43, 1657-1669 [CrossRef][Medline] [Order article via Infotrieve]
  7. Ernster, L. (1967) Methods Enzymol. 10, 309-317
  8. Sugioka, K., Nakano, M., Naito, I., Tero-Kubota, S., and Ikegami, Y. (1988) Biochim. Biophys. Acta 964, 175-182 [Medline] [Order article via Infotrieve]
  9. Ordonez, I. D., and Cadenas, E. (1992) Biochem. J. 286, 481-490 [Medline] [Order article via Infotrieve]
  10. Chen, H., Ma, J. X., Forrest, G. L., Deng, P. S. K., Martino, P. A., Lee, T. D., and Chen, S. (1992) Biochem. J. 284, 855-860 [Medline] [Order article via Infotrieve]
  11. Massey, V., and Hemmerich, P. (1978) Biochemistry 17, 9-17 [Medline] [Order article via Infotrieve]
  12. Massey, V. (1990) in Flavins and Flavoproteins (Curti, B., Ronchi, S., and Zanetti, G., eds) pp. 59-66, Walter de Gruyter & Co., Berlin
  13. Beaty, N., and Ballou, D. P. (1981) J. Biol. Chem. 256, 4611-4618 [Abstract/Free Full Text]
  14. Brissette, P., Ballou, D. P., and Massey, V. (1989) Anal. Biochem. 181, 234-238 [Medline] [Order article via Infotrieve]
  15. Bevington (1969) in Data Reduction and Error Analysis for the Physical Sciences , pp. 235-242, McGraw Hill, Inc., New York
  16. Clark, W. M. (1960) Oxidation Reduction Potentials of Organic Systems , pp. 184-188, The Williams & Wilkins Co., Baltimore
  17. Muller, O. (1942) J. Biol. Chem. 145, 425-441
  18. Minnaert, K. (1965) Biochim. Biophys. Acta 110, 42-56 [Medline] [Order article via Infotrieve]
  19. Fultz, M. L., and Durst, R. A. (1982) Anal. Chim. Acta 140, 1-8 [CrossRef]
  20. Siegel, D., Gibson, N. W., Preusch, P. C., and Ross, D. (1990) Cancer Res. 50, 7293-7300 [Abstract]
  21. Dalziel, K. (1957) Acta Chem. Scand. 11, 1706-1723
  22. Hosoda, S., Nakamura, W., and Hayashi, K. (1974) J. Biol. Chem. 249, 6416-6423 [Abstract/Free Full Text]
  23. Butler, J., Hoey, B. M., and Lea, J. S. (1987) Biochim. Biophys. Acta 925, 144-149 [Medline] [Order article via Infotrieve]
  24. McWhirter, R. B., and Klapper, M. H. (1990) Biochemistry 29, 6919-6926 [Medline] [Order article via Infotrieve]
  25. Massey, V., Müller, F., Feldberg, R., Schuman, M., Sullivan, P. A., Howell, L., Mayhew, S. G., Matthews, R. G., and Foust, G. P. (1969) J. Biol. Chem. 244, 3999-4006 [Abstract/Free Full Text]
  26. Chen, S., and Liu, X. F. (1992) Mol. Pharmacol. 42, 545-548 [Abstract]
  27. Brunmark, A., Cadenas, E., Segura-Aguilar, J., Lind, C., and Ernster, L. (1988) Free Radical Biol. & Med. 5, 133-143
  28. Buffinton, G. D., Öllinger, K., Brunmark, A., and Cadenas, E. (1988) Biochem. J. 257, 561-571
  29. Dodd, N. J. F., and Mukherjee, T. (1984) Biochem. Pharmacol. 33, 379-385 [Medline] [Order article via Infotrieve]
  30. Blankenhorn, G. (1976) Eur. J. Biochem. 67, 67-80 [Abstract]
  31. Duine, J. A. (1990) Trends Biochem. Sci. 15, 96-97 [Medline] [Order article via Infotrieve]
  32. Nishigori, H., Yasunaga, M., Mizumara, M., Lee, J. W., Iwatsuru, M. (1989) Life Sci. 45, 593-598 [Medline] [Order article via Infotrieve]
  33. Chen, S., Deng, P. S. K., Bailey, J. M., and Swiderek, K. M. (1994) Protein Sci. 3, 51-57 [Abstract/Free Full Text]
  34. Huang, M-T., Miwa, G. T., and Lu, A. Y. H. (1979) J. Biol. Chem. 254, 3930-3934 [Abstract]
  35. De Flora, S., Morelli, A., Basso, C., Romano, M., Serra, D., and De Flora, A. (1985) Cancer Res. 45, 3188-3196 [Abstract]
  36. Riley, R. J., and Workman, P. (1992) Biochem. Pharmacol. 43, 167-174 [Medline] [Order article via Infotrieve]
  37. Sugimura, T., Okabe, K., and Nagao, M. (1966) Cancer Res. 26, 1717-1721 [Medline] [Order article via Infotrieve]

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