INTRODUCTION

Top Abstract Introduction Materials & Methods Results & Discussion References

A wide variety of redox transformations in biological systems are catalyzed by flavoenzymes. The oxidation-reduction potential of the flavin and the transfer of electrons by the semiquinone or the fully reduced forms to the acceptor are among the most significant features of the chemistry of flavoprotein catalysis, which is in general controlled by the potential of the flavin. Hence it is possible to regulate and manipulate electron flow during catalysis by altering the redox potential of the flavin. Various structurally modified flavins with altered redox potentials have been synthesized previously and employed as mechanistic probes (1-4). While deazaflavin derivatives best represent the low potential probes, flavin analogs with various electron withdrawing groups at the 8-position constitute the high potential series. It is known that the introduction of electronegative groups like chlorine (5), fluorine (6), methylsulfonyl (7, 8) at the 8-position of isoalloxazines increases electrophilicity and shifts the potential to more positive values. However these functionalities are highly reactive and can undergo displacement reactions either with external nucleophiles or with nucleophilic amino acid residues like cysteine at the active site. Accordingly these properties have been exploited to advantage in determining the solvent accessibility of the 8-position of the flavin in various flavoproteins (9) and also for the covalent labeling of active site amino acid residues (10-12). Elegant model studies by Bruice et al. (13) with 8-CN-isoalloxazines have demonstrated that cyanylation at the 8-position affords an isoalloxazine with one of the highest known potentials along with very interesting chemical and spectral properties. Although the CN functionality is strongly electronegative, it is resistant to displacement reactions, since these would involve carbon-carbon bond breaking. Also the relatively small size of the CN substitution meets the steric requirements for a good active site probe. Hence various laboratories have attempted the synthesis of 8-CN-flavin nucleotide derivatives by displacement reactions on flavins having leaving groups at the 8-position (e.g. chlorine, fluorine, methylsulfonyl, etc.,) and also by the Sandmeyer reaction on 8-NH₂-riboflavin, but without success. Attempts to synthesize the aromatic building block with CN substitution for the construction of the flavin were also a failure. In view of the relevance of this analog as a high potential active site probe for flavoproteins, we decided to reinvestigate the above approaches. The aromatic building block, 3-nitro-4-chloro-6-CN-toluene, which was successfully synthesized, failed to react with ribitylamine.¹ However, we discovered that 8-NH₂-riboflavin can be converted to 8-CN-riboflavin under novel reaction conditions as described in this paper. The riboflavin derivative is readily converted to the FAD and FMN derivatives by the FAD synthetase of Brevibacterium ammoniagenes (14).

	MATERIALS AND METHODS

Top Abstract Introduction Materials & Methods Results & Discussion References

8-NH₂-riboflavin, which was synthesized as described previously (15), was a kind gift from Dr. S. Ghisla, University of Konstanz. Copper(II) cyanide and sodium cyanide were from Aldrich. 8-NH₂-FAD was prepared as reported previously (16).

Preparation of Flavoproteins and Their Corresponding Apoproteins-- The holoproteins and apoproteins were prepared as described previously: riboflavin binding protein from hen egg white (17), flavodoxin from Megasphera elsdenii (18, 19), and OYE I,² overexpressed in Escherichia coli containing the plasmid pET-3b (20). The apoOYE I was prepared from the recombinant enzyme by using the procedure reported for the enzyme from brewers' bottom yeast (21, 22). The apoDAAO from pig kidney was obtained from Calzyme (San Luis Obisco, CA).

Reconstitution of Apoproteins-- ApoRBP and apoflavodoxin concentrations were determined by titration with pure riboflavin and FMN, respectively. ApoDAAO concentration was determined by titrating pure FAD with apoprotein in the presence of benzoate (23). Reconstitution of the apoproteins with 8-CN-flavins was accomplished by mixing 1.5-fold excess of the flavin with apoprotein and incubating on ice for 3 h. Excess flavin was removed by a Centricon-30 microconcentrator (Amicon).

Synthesis of 8-CN-riboflavin-- 10 mg of 8-amino-riboflavin was suspended in 3 ml of water in a test tube. To this suspension, 6 N HCl was added until a clear solution was obtained. This solution was cooled to 0 °C on ice, and three aliquots each of 40 µl of saturated sodium nitrite solution were added with continuous shaking of the test tube. After 5 min, 300 µl of saturated urea solution was added to destroy the excess sodium nitrite. The cold diazo salt solution was then added with a glass transfer pipette to a 10-ml saturated solution of NaCN + CuCN (70:30) in a 50-ml glass beaker with vigorous stirring at room temperature. After 20 min, the reaction mixture was loaded on a 20-cc C-18 Sep-Pak (Millipore, Milford, CT) cartridge. The cartridge was prewashed thoroughly with excess water, methanol, and again with water before loading the reaction mixture. The Sep-Pak cartridge was eluted with water followed by 5% acetonitrile to remove salts and a red band of unknown structure. Elution with 15% acetonitrile gave the 8-CN-riboflavin and with 20% acetonitrile gave 8-chlororiboflavin. Evaporation of the flavin solutions with a Speed Vac concentrator gave 6 mg of the 8-CN-riboflavin as a yellow powder.

Conversion of 8-CN-riboflavin to FAD and FMN-- 8-CN-riboflavin was converted to the FAD level with partially purified FAD synthetase from B. ammoniagenes by incubating the flavin in 0.002 M potassium P_i, pH 7.5 at 25 °C, following the procedure of Spencer et al. (14). After 14 h, HPLC analysis of the incubation mixture showed 100% conversion to the FAD form. The reaction mixture was loaded on to the prewashed (as described in the case of 8-CN-riboflavin) 20-cc C-18 Sep-Pak cartridge and eluted with 100 ml of water to wash off salts and breakdown products from ATP. Elution with 5% acetonitrile in water gave pure flavin, which was concentrated on a Speed Vac concentrator to obtain 8-CN-FAD as a yellow powder. 8-CN-FMN was obtained by hydrolysis of the FAD in 0.05 M potassium P_i, pH 7, with snake venom phosphodiesterase (Naja naja venom).

Cyanylation of 8-NH₂-FAD-- To 500 µl of 8-NH₂-FAD, 100 µl of 6 N HCl was added at ice temperature. Then two 20-µl aliquots of saturated NaNO₂ were added with mixing. After 2 min, 100 µl of saturated urea solution was added. When effervescence stopped, 500 µl of a saturated solution of 70:30 NaCN + CuCN solution was added into the diazo-FAD solution at room temperature. After 3-5 min, the solution was passed through a prewashed small Sep-Pak cartridge, and the salts were washed off with water. The flavin was then eluted with 5% acetonitrile. HPLC analysis of the 5% acetonitrile fraction showed both 8-CN-FAD (~80%) and 8-CN-FMN (~20%) when eluted with 80% 0.01 M potassium P_i, pH 6, and 20% methanol on a reverse phase C-18 column. Interestingly, no formation of 8-chloro-FAD or 8-chloro-FMN was observed.

Enzyme Assays-- Old Yellow Enzyme activities were measured in 0.1 M phosphate, pH 7.0 at 25°, in a stopped flow spectrophotometer (Kinetic Instruments, Ann Arbor, MI) either under anaerobic conditions (when cyclohexenone was employed as electron acceptor) or under controlled oxygen concentrations when oxygen was electron acceptor, monitoring both the consumption of NADPH at 340 nm and in the same experiments the level of flavin oxidation/reduction at 470 nm. The concentrations of both NADPH and the acceptor (cyclohexenone or O₂) were varied systematically to determine true k_cat and K_m values.

	RESULTS AND DISCUSSION

Top Abstract Introduction Materials & Methods Results & Discussion References

Attempts to convert the 8-diazo salts of flavins to 8-CN-flavins by the Sandmeyer reaction were unsuccessful. The choice of the cyanylating reagent has a critical effect on the course of the reaction (24, 25). For the 8-diazo-flavins, in our hands, either sodium cyanide or cuprous cyanide alone were ineffective. Lately several copper-cyano complexes of the kind Na₃(Cu(CN)₄), K₃(Cu(CN)₄), K₂(Cu(CN)₄·NH₃) were shown to have large advantages in cyanylation reactions affording high yields of nitriles (26). Before trying these complexes for the present cyanylation, we tried treating the diazoflavin with a saturated solution of an approximately 3:1 mixture NaCN + CuCN and found 8-CN-riboflavin in about 60% yield (Scheme 1). The 8-chlororiboflavin was obtained as a side product in about 20% yield, presumably because of the CuCl formed from CuCN and HCl. It was found that the ratio between NaCN and CuCN is crucial for both the reaction to occur as well as to obtain isolatable yields of the cyanoflavin. When the CuCN ratio was increased, an insoluble solid was obtained with no isolation of any flavin.

View larger version (8K): [in this window] [in a new window]   Scheme 1.

The _max of the UV-visible spectrum for 8-CN-riboflavin is slightly shifted from 445 nm for the normal flavin spectrum to 456 nm. The near-UV peak is shifted from 375 nm for the normal flavin to 338 nm for 8-CN-riboflavin (Fig. 1A). The cyanoflavin is fluorescent with an emission maximum at 530 nm and excitation maxima at 338 and 456 nm, identical with the absorption spectrum. Further characterization of the new flavin was made by recording the positive ion fast atom bombardment mass spectrum which showed M⁺¹ at 388 (Fig. 1B) (molecular weight of the 8-CN-riboflavin is 387) and also by the ¹H NMR spectrum (Fig. 1C). In the proton NMR spectrum, the electron-deficient nature of the flavin is well evident from the fact that the C6 and C9 protons are down-shifted and well separated due to the CN substitution in the benzene ring. The aromatic protons are at 8.0 and 8.4 ppm compared with 7.9 and 8.0 ppm in the normal flavin. The lone methyl group on the C7 is seen as a singlet at 2.6 ppm, down-shifted from 2.3 ppm in normal flavin. The rest of protons from the ribityl side chain are recorded between 3.6 and 4.8 ppm.

View larger version (23K): [in this window] [in a new window]   Fig. 1.   A, absorption spectra for the oxidized and reduced forms of 8-CN-riboflavin. The solid line indicates oxidized flavin; the dotted line indicates anionic reduced flavin, pH 7; the broken line indicates neutral reduced flavin, pH 3. Inset, plot of the absorbance difference between 338 and 400 nm of reduced 8-CN-riboflavin as a function of pH. B, positive ion fast atom bombardment mass spectrum of 8-CN-riboflavin; C, 1H NMR of 8-CN-riboflavin, recorded with a General Electric GN500 MHz NMR instrument. Chemical shift values are reported in parts/million relative to tetramethylsilane.

The 8-CN-riboflavin was converted to the FAD level by reacting with FAD synthetase from B. ammoniagenes in 100% yield without any complications. This again reaffirms the fact that the small cyano substitution meets the steric requirements of the 8-position of the flavin. The 8-CN-FMN was obtained by hydrolysis of the HPLC-pure 8-CN-FAD with the phosphodiesterase from snake venom. This reaction is accompanied by an increase in extinction for the FMN at 456 nm of 8.8% and with a 10.25-fold increase in fluorescence. The cyanylation reaction also worked with the 8-NH₂-FAD, except that about 20% of the FAD got hydrolyzed to 8-CN-FMN because of the drastic acidic conditions of the diazotization reaction.

Reduction of 8-CN-riboflavin

It was found that the reduction of 8-CN-riboflavin to its dihydro form can be accomplished with several reducing agents such as dithiothreitol, dithionite, NaBH₄, photochemically with EDTA as photodonor, NADPH, and NADH. The reduction is fully reversible with oxygen. However the spectrum for reduced 8-CN-riboflavin is different from that of normal flavin, as reported previously for 8-CN-7-nor-3-methyl-lumiflavin by Bruice et al. (13). Reduced 8-CN-riboflavin has two well defined peaks above the 300 nm region with maxima at 312 and 372 nm at pH 7 and at 316 and 362 nm under acidic conditions(Fig. 1A).

The pK_a of Reduced Cyanoriboflavin

The pK_a value of the reduced cyanoflavin was determined to be at pH 5.6 from the change in absorption spectrum with pH (Fig. 1A, inset). Hence the electronegative 8-CN-group decreases the pK_a of the reduced flavin from the usual value (27) of ~6.7 to 5.6 in accord with the electron-deficient nature of the flavin. The spectra of the neutral and anionic reduced forms are shown in Fig. 1A.

Determination of Reduction-Oxidation Potential

The reduction-oxidation potential for 8-CN-FAD was measured by using the xanthine/xanthine oxidase system and indigo tetrasulfonate as the reference dye (28). Reduction of the 8-CN-FAD with the xanthine/xanthine oxidase system proceeded with isosbestic points at 352 and 406 nm and the reduction of the dye with isosbestic points at 338 and 502 nm. These wavelengths were used to monitor the reduction of the components in a mixture of the two. A plot of log(ox/red) of the dye against log(ox/red) of the flavin gave a midpoint potential for 8-CN-FAD of 50 mV, which is 158 mV more positive than that of normal flavin. This is in accord with the strongly electron withdrawing nature of the CN substituent, making it a more electron deficient system, shifting the potential to more positive values.

Reaction with Sulfite

Native flavin is known to form an N-5 adduct with sulfite, but the reaction is only half completed at saturating sulfite concentrations because of the high dissociation constant (~2.5 M) (29). The absorption spectrum of the flavin adduct is similar to that of reduced flavin with a new peak maximal at 320 nm. With a series of artificial flavins a good correlation was found to exist between the dissociation constant of the flavin-sulfite adduct and the two-electron redox potential of the flavin (29). The straight line obtained by plotting the logarithm of the dissociation constants of the complexes and the redox potentials of the flavins shows that the more positive the potential, the tighter the sulfite binding. It is reasonable that flavins with more positive potentials are the most electron deficient and prone for the addition of two electrons through adduct formation. As anticipated, the cyanoflavin formed a tight complex with sulfite, with a K_d of 0.73 mM (Fig. 2, inset). The absorption spectrum of the adduct has a peak in the near-UV region at 310 nm (Fig. 2), similar to that with normal flavin and distinctively different from that of reduced 8-CN-flavin. From the previously observed correlation of the redox potential and K_d for the complex (29), the oxidation-reduction potential at pH 7 would be predicted to be 50 mV, in perfect agreement with direct measurement.

View larger version (35K): [in this window] [in a new window]   Fig. 2.   Titration of 49 µM 8-CN-riboflavin with sulfite in 0.1 M potassium Pi, pH 7 at 25 °C. Spectra were recorded (in the descending order of absorbance at 450 nm) with 0, 0.3, 0.7, 1.2, 2.2, 4.2 mM and a large excess (~50 mM) of sodium sulfite. Care was taken to ensure that the equilibrium adduct formation was complete after each addition. Inset, double-reciprocal plot of the degree of saturation () versus the concentration of sulfite, yielding a Kd of 0.73 mM.

Hydrolysis of 8-CN-riboflavin

The hydrolysis of 8-CN-riboflavin in 0.5 M carbonate, pH 10.25, was studied. The spectral changes that were observed are similar to those of the cyanoisoalloxazine observed by Bruice et al. (13) and compatible with formation of a spirohydantoin (30). As is the case with the cyanoisoalloxazine derivative, repetitive spectral scans during the hydrolysis failed to provide any evidence for the 10a- or 4a-hydroxyl adducts, which were considered as possible intermediate species during hydrolysis.

Photostability of 8-CN-flavin Derivatives

It is well known that flavins are photoreactive and their spectroscopic and photochemical properties have been the subject of intense studies (31). It was found that 8-CN-riboflavin is highly photoreactive. When a sample of the flavin was left exposed to room light, it was transformed over a period of a few days into a stable product with absorbance maxima at 320 and 388 nm. A similar reaction was also observed with the 8-CN-FMN derivative. However when 8-CN-FAD was exposed under the same conditions, it was found to be extremely stable toward photoreaction. Only about 5% of the reaction was observed in the same time scale where 8-CN-riboflavin and 8-CN-FMN reacted completely. It is known that FAD forms an intramolecular complex between the adenine moiety and the isoalloxazine ring (32). The increased stability of FAD toward photolysis is attributed to this intramolecular stacking (33). The photoreaction involves the ribityl side chain, since 8-CN-lumiflavin is completely stable under the same conditions. The nature of the product and the chemistry of the photoreaction is under investigation and will be reported separately.

Riboflavin-binding Protein

Although this protein has no known catalytic activity, binding experiments of artificial flavins with this protein are useful in interpreting the structural characteristics of the flavin. Riboflavin binds to apoRBP with a dissociation constant of 1.3 nM and shows considerable spectral shifts and resolution on binding (17). 8-CN-riboflavin showed similar spectral changes to those of normal flavin upon binding to apoRBP. The flavin absorption peaks are red shifted from 340 and 454 nm to 344 and 470 nm with a decrease in the extinction for both peaks. The binding of 8-CN-riboflavin to apoRBP resulted in the complete quenching of the fluorescence. The dissociation constant was calculated to be ~0.27 µM and by standardization of apoprotein with native riboflavin, the extinction coefficient of the 8-CN-riboflavin was determined as 10,400 M¹ cm¹. The 5-deazaflavin-catalyzed photoreduction (34) of 8-CN-riboflavin-RBP proceeded to the fully reduced form without stabilizing any semiquinone. The spectrum for the reduced protein-bound flavin is almost identical to that of the free reduced flavin except that the _max is slightly red shifted by 4 nm to 376 nm. The initial oxidized flavin spectrum is regained rapidly on admission of air.

8-CN-FMN-Flavodoxin

8-CN-FMN binds to apoflavodoxin with quenching of the fluorescence and with a dissociation constant of ~0.45 µM. The binding of the flavin to the apoprotein results in 14% decrease in the extinction and the maxima of the absorption spectrum shift from 340 and 452 nm for the free flavin to 342 and 460 nm (₄₆₀ = 9.4 mM¹ cm¹) for the bound flavin. The extinction coefficient for 8-CN-FMN was determined as 11,000 M¹ cm¹ by standardizing the apoprotein with pure native FMN. Reduction of native flavodoxin by EDTA/light or with the xanthine/xanthine oxidase system proceeds to the fully reduced protein through the formation of neutral semiquinone (35). The same reduction process occurs with all flavodoxins substituted with artificial flavins, including 8-CN-FMN flavodoxin (Fig. 3). The blue neutral semiquinone shows maxima at 598 and 644 nm. Previous studies with native flavodoxin showed essentially quantitative formation of the neutral semiquinone at the midpoint of dithionite reduction (36). However, in case of the 8-CN-FMN-protein the plot of A₄₅₈ versus A₆₄₄ (see the inset in Fig. 3) shows that only ~75% of the radical species was formed. This suggests that the potentials of the EFl_ox/EFlH^· and EFlH^·/EFl_red (where Fl is flavin) couples are closer than those of the native flavoprotein. The reduced flavoprotein showed two well resolved peaks in the absorption spectrum similar to the unbound reduced flavin. However the peak in the near-UV region shifted further to the lower wavelength region and the band at 370 nm in the free flavin showed a bathochromic shift to 388 nm. Interestingly, 25% of the absorption at the 460 nm region is retained with a shifted absorption band at 485 nm (Fig. 3). There is no adduct formation with sulfite in accordance with studies of the native protein (37).

View larger version (25K): [in this window] [in a new window]   Fig. 3.   Reduction of 31.9 µM 8-CN-FMN-flavodoxin with xanthine/xanthine oxidase system. The reduction was carried out in 100 mM potassium Pi, pH 7 at 25 °C, in the presence of 5 µM benzylviologen to ensure rapid equilibration of reducing equivalents. The solid line indicates the oxidized form of 8-CN-FMN-flavodoxin; the broken line indicates neutral semiquinone; the dotted line indicates reduced 8-CN-FMN-flavodoxin. Inset, plot of absorption at 458 nm versus absorption at 644 nm during the reduction.

Old Yellow Enzyme

Old Yellow Enzyme is the first discovered flavoprotein and was isolated from brewers' bottom yeast. It is a mixture of homodimers and a heterodimer with one FMN per subunit, products of separate genes (38, 39). As the physiological role of this protein is yet to be determined, structural and chemical reactivity studies in the direction of elucidating its function are the subject of extensive studies in this laboratory. Saito et al. (20) cloned a gene encoding an isoform of OYE (OYE I) from Saccharomyces carlsbergenesis, and its crystal structure has been determined (40). For the present investigations, this recombinant enzyme (OYE I) was used. The binding of apoOYE with 8-CN-FMN was followed by measuring the flavin fluorescence as well as changes in absorption spectrum. At the end point, the fluorescence was quenched almost completely with a residual intensity of ~6% of that of the free flavin. The titration plots suggested a binding affinity of the 8-CN-FMN to apoOYE similar to that with FMN (K_d ~ 10⁹ M) (41). The extinction coefficient of the cyano-FMN was determined as 11,000 M¹ cm¹ by standardizing the apoprotein with pure native FMN. The binding was accompanied by the usual resolution and the characteristic spectral shifts of the flavin spectrum found with the native FMN. The _max for the free flavin to protein-bound flavin shifted from 340 and 454 nm to 348 and 470 nm, with the ₄₇₀ value of 10,200 M¹ cm¹.

Binding of Phenols-- The oxidized form of OYE binds to a variety of ligands forming spectroscopically distinct complexes (38). Phenols are the most striking and well studied as they result in long wavelength charge transfer bands in the region 500-800 nm accompanied by strong perturbation in the flavin-visible absorption bands (21, 22). It was shown by a positive correlation between the energy of the charge transfer transition and the Hammett para constant that the phenol is the charge transfer donor and the oxidized flavin of the enzyme is the acceptor (22). A good correlation was also shown to exist between the redox potential of the flavin and the maximum of the long wavelength transition, from studies where the native flavin was replaced by a variety of synthetic flavin analogs of different oxidation-reduction potential (22). Native enzyme binds p-chlorophenol with a K_d of 1 µM and has a maximum of 645 nm for the long wavelength charge transfer band. The 8-CN-FMN enzyme binds p-chlorophenol with a K_d of 6.5 µM and the charge transfer maximum is shifted to 763 nm, but with less than the usual perturbations of the spectrum in the 450 nm region (Fig. 4). As the oxidation-reduction potential of the flavin becomes more positive, the wavelength maximum should shift to longer wavelengths, since it is expected to require less energy to form the charge transfer transition. As will be discussed later, the 115 nm red shift observed in case of 8-CN-FMN enzyme falls in line with the more positive potential compared with that of the native flavin.

View larger version (24K): [in this window] [in a new window]   Fig. 4.   Titration of 15.8 µM 8-CN-FMN-OYE with p-chlorophenol. Spectra were recorded on addition of 0, 3, 6, 12, 21, 51, 1051 µM of p-chlorophenol (in the increasing order of absorbance at 744 nm) in 100 mM potassium Pi, pH 7 at 25 °C.

Reduction of 8-CN-FMN-OYE-- The reduction of 8-CN-FMN enzyme with the xanthine/xanthine oxidase system with benzylviologen as mediator was carried out at 25 °C in 100 mM potassium P_i, pH 7 (Fig. 5). It was found to proceed through the formation of the anionic semiquinone to the two-electron reduced form. The plot of A₄₇₀ versus A₄₁₆ suggest that ~ 98% of the semiquinone radical species was formed (Fig. 5, inset). The reduced 8-CN-FMN enzyme showed a peak at 360 nm, a blue shift of 10 nm from that of the free cyanoflavin. Photoreduction of the 8-CN-FMN enzyme in the presence of EDTA resulted in ~100% anionic semiquinone without any further reduction to the fully reduced form.

View larger version (26K): [in this window] [in a new window]   Fig. 5.   Reduction of 27 µM 8-CN-FMN-OYE with xanthine/xanthine oxidase system. The reduction was carried out in 100 mM potassium Pi, pH 7 at 25 °C, with benzylviologen as mediator. The broken line indicates the oxidized 8-CN-FMN-OYE; the solid line indicates the anionic semiquinone; the dotted line indicates the reduced enzyme. Inset, plot of A471 versus A414 during the reduction.

Stabilization of Semiquinone-- With normal flavin semiquinone can be obtained either by one-electron reduction of the oxidized form or by one-electron oxidation of the reduced flavin. But in solution only about 2% of the total flavin is stabilized as semiquinone at pH 7 (42). This is because of the fact that in the following equation, the equilibrium always favors the disproportionation (right to left) of the semiquinone.

(Eq. 1)

However, several apoproteins upon binding the flavin stabilize larger amounts of the semiquinone form through electrostatic interactions between the flavin and the protein (43). When native OYE I was reduced with ~0.5 equivalents of NADPH, a mixture of reduced and oxidized forms of the enzyme was obtained. This mixture underwent a slow comproportionation reaction over a period of 4 h to form ~60-65% stable anionic semiquinone, establishing the thermodynamic stabilization of the semiquinone by the protein. When this reaction was carried out with 8-CN-FMN enzyme under identical conditions, it resulted in ~100% formation of stable anionic semiquinone. The reaction was found to be very fast when compared with the native enzyme, being complete within a few minutes, with a rate constant of 3 × 10⁴ M¹ s¹ for this comproportionation reaction. These results show that the potential for the EFl_ox/EFl_SQ couple is much higher than that of the EFl_SQ/EFl_red couple. The potential of the EFl_ox//EFl_SQ couple for 8CN-FMN OYE I was determined by equilibration versus toluidine blue using the xanthine/xanthine oxidase reduction method (28) and found to be 125 ± 3 mV. The potential of the second electron reduction, EFl_SQ/EFl_red, was determined similarly by equilibration versus phenosafranin to be 205 ± 13 mV. These values define the two electron potential of the 8-CN-FMN enzyme to be 40 ± 18 mV, similar to that of the free flavin. A linear relationship is expected for the energy of the long wavelength transition of the charge transfer complex between oxidized flavin and phenolate anion and the first 1-electron reduction potential of the flavin (43). As can be seen in Fig. 6, the 115 nm red shift of the transition with p-chlorophenol fits well with the previously determined values with other artificial flavins and that with the 8-CN-FMN enzyme.

View larger version (15K): [in this window] [in a new window]   Fig. 6.   Correlation of the energy of the long wavelength transition of various flavin forms of OYE and p-chlorophenol with the redox potential of the oxidized/semiquinone couple of the bound flavin. 8-CN-FMN enzyme, this work; remaining values from Ref. 43.

Steady State Turnover of 8-CN-FMN-OYE with -NADPH and Oxygen-- Enzyme-monitored turnover experiments were carried out by using a stopped flow spectrophotometer. A known concentration of enzyme (6.2 µM) in 100 mM potassium P_i, pH 7, was reacted with a range of concentrations of NADPH in the same buffer, equilibrated with different concentrations of oxygen at 25 °C. The concentrations of oxygen used were 189, 256, 433, and 743 µM. The reaction was followed at both 470 nm, from approach to steady state until final reoxidation by excess oxygen, and also at 340 nm (Fig. 7).

View larger version (13K): [in this window] [in a new window]   Fig. 7.   Representative kinetic traces from turnover reactions of 8-CN-FMN-OYE with NADPH and oxygen. In this figure. 6.2 µM enzyme (concentration after mixing) in 0.1 M phosphate, pH 7.0, 25 °C was reacted with 55 µM NADPH and 560 µM oxygen (concentrations after mixing) in a stopped flow spectrophotometer. The reaction was monitored at 470 nm to follow changes in flavin oxidation-reduction state, and at 340 nm, to follow oxidation of NADPH. The absorbance changes at 470 nm are shown on two time scales, the overall reaction (broken line) over 70 s, and the approach to steady state (solid line) over a period of 70 ms. The absorbance trace at 340 nm (dotted line) is over a period of 70 s, and comparison with the 470 nm trace demonstrates clearly that the enzyme remains in the reduced state until all the NADPH is consumed. Reaction rate constants for reduction were obtained by single exponential fits to the traces of approach to steady state and kobs values for the reoxidation by similar single exponential fits to the final reoxidation phase at 470 nm. Turnover numbers were calculated from changes in absorbance at 340 nm.

1

1

1

1

1

1

Steady State Turnover of 8-CN-FMN-OYE with -NADPH and Cyclohex-2-en-1-one-- It has been reported recently that cyclohexenone is an efficient electron acceptor wherein the carbon-carbon double bond is reduced to form cyclohexanone (39). Turnover experiments were carried out by reacting enzyme with mixtures of NADPH and cyclohexenone under anaerobic conditions in the stopped flow spectrophotometer. Oxidation of NADPH by the 8-CN-FMN reconstituted enzyme was monitored at 340 nm. It was found that the turnover numbers were independent of cyclohexenone concentration over the range of 50 µM to 1 mM. For 8-CN-FMN enzyme a turnover number of 7.5 min¹ was obtained with a K_m for NADPH of 0.2 µM, whereas the native OYE I has a turnover number³ of ~300 min¹. These results show that cyclohexenone is a poor electron acceptor for the 8-CN-FMN enzyme and the reoxidation of the reduced enzyme-bound cyanoflavin by cyclohexenone, unlike that of the native enzyme, is very slow, again a reflection of the high oxidation-reduction potential of the flavin. Attempts to measure directly the rate constant for oxidation of the reduced enzyme by cyclohexenone were unsuccessful, since the slowly produced oxidized enzyme reacted with remaining reduced enzyme to form anionic semiquinone (see earlier section). However, if the 8-CN-FMN enzyme functions like the normal enzyme, where it has been shown that the NADPH-cyclohexenone reductase reaction functions by a ping-pong kinetic mechanism, where k_cat = k_red·k_ox/(k_red + k_ox), it may be concluded that k_ox for cyclohexenone with the 8-CN-FMN enzyme = k_cat, i.e. 0.125 s¹, an 870-fold decrease from the value of 109 s¹ found for native enzyme.³ This large decrease in reaction rate constant compared with that with native enyzme is again consistent with the electron-withdrawing nature of the cyano substituent, which by stabilization of the anionic reduced flavin would make reduction of the olefinic bond of cyclohexenone thermodynamically less favorable. The magnitude of the decreased reactivity (~900-fold) should be compared with the 40-fold increase in the reduction reaction rate constant with NADPH and implies a late transition state in the reaction.

D-Amino Acid Oxidase

The binding of 8-CN-FAD to apoDAAO is accompanied by around 80% quenching of the fluorescence but by only small spectral shifts from free flavin to the bound flavin. The maxima of the free flavin shifted from 340 and 456 nm to 344 and 458 nm on binding to the apoprotein (Fig. 8). As in the case of the binding of native FAD, no vibronic resolution of peaks is observed on the binding of 8-CN-FAD. When the apoDAAO was titrated with FAD in presence of benzoate, the titration plots suggest tight binding similar to that found with native FAD (23).

View larger version (19K): [in this window] [in a new window]   Fig. 8.   Absorption spectra for the different forms of 8-CN-FAD-DAAO. The solid line indicates the oxidized 8-CN-FAD-DAAO; the dotted line indicates the anionic semiquinone obtained by 3 min light irradiation of the oxidized form in 0.1 M sodium pyrophosphate buffer, pH 8.5; the broken line indicates the reduced enzyme obtained by the anaerobic reaction of the oxidized enzyme with 50 M equivalents of D-alanine.

Photoreduction of 8-CN-FAD-DAAO-- When the 8-CN-FAD-DAAO was irradiated with light in presence of EDTA, it formed ~100% of anionic semiquinone (Fig. 8) with no further reduction to the two-electron reduced form on prolonged light irradiation. These results are similar to those found with native enzyme, where the anionic flavin semiquinone is also stabilized thermodynamically (35). The semiquinone has a sharp absorption maximum at 418 nm with an extinction coefficient of ~20,000 M¹ cm¹, somewhat lower than that found with SCN FMN OYE. However, when the photoreduction was carried out with the 8-CN-FAD enzyme-benzoate complex, the reaction resulted in essentially two-electron reduced enzyme with only ~5% intermediate semiquinone. These results are consistent with the fact that the binding of benzoate modulates the oxidation-reduction potential of the protein-bound flavin in such a way that two-electron reduction is facilitated and making the formation of the anionic radical more difficult. It was shown that in case of the native protein, the redox potentials for the EFl_ox/EFl_SQ and EFl_SQ/EFl_red couples are 98 and 204 mV for the holoenzyme and 260 and 140 mV for the benzoate complex (46). Although we have not attempted to determine the redox potentials of 8-CN-FAD DAAO, it is clear that a similar situation exists as with the native enzyme.

Reduction of 8-CN-FAD-DAAO with D-Alanine-- When 8-CN-FAD-DAAO was reacted anaerobically with 50-fold excess D-alanine, the spectrum for the two-electron reduced enzyme was formed rapidly (Fig. 8). The reduced enzyme has a peak at 370 nm. On opening to air, the oxidized enzyme spectrum was regained quickly once turnover was complete.

Benzoate Binding-- Pronounced spectral changes were observed in the region of 480 and 520 nm upon binding of benzoate to the oxidized form of D-amino acid oxidase (47, 48). The binding of sodium benzoate to 8-CN-FAD-D-amino acid oxidase resulted in similar spectral changes with marked resolution of the peaks as in the case of the benzoate-FAD-enzyme. The K_d for benzoate with the native enzyme is 3 × 10⁶ M at pH 8.5 with 1:1 stoichiometry (23). Titration plots with the 8-CN-FAD enzyme give a K_d for benzoate of ~120 µM, showing that benzoate binds with less affinity to the 8-CN-FAD enzyme. It is interesting to recall that DAAO from the yeast Rhodotorula gracilis also binds benzoate less tightly, with a K_d of 245 µM (49).

Sulfite Addition-- As discussed earlier, sulfite adds to the electrophilic N-5 of the oxidized form of flavins to afford adducts with similar but not identical spectral properties to those of the reduced flavins. However with the 8-CN-flavin, the adduct spectrum is completely different from its dihydroflavin spectrum and resembles more the reduced normal flavin spectrum. Native D-amino acid oxidase forms the adduct slowly (37) with a dissociation constant of 3.5 × 10³ M. The 8-CN-FAD enzyme forms the adduct with complete bleaching of both the 450 and 340 nm peaks, with the formation of a new peak at 324 nm as in the case of the unbound 8-CN-flavin. The addition was also found to be slow, as in the case of native enzyme, with a K_d of 9 µM. The tighter binding of sulfite to 8-CN-FAD enzyme than to normal enzyme is again consistent with the higher redox potential of the 8-CN-FAD enzyme.