From the Department of Biological Chemistry, University of Michigan, Ann Arbor, Michigan 48109
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ABSTRACT |
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A high potential analog of riboflavin with a cyano function at the 8-position was synthesized by employing novel reaction conditions, starting from 8-amino-riboflavin. This was converted to the FAD level with FAD synthetase. The reduced 8-CN-riboflavin, unlike normal reduced flavin, has a distinctive absorption spectrum with two distinctive peaks in the near ultraviolet region. The oxidation-reduction potential of the new flavin was determined to be -50 mV, ~160 mV more positive than that of normal riboflavin. The 8-CN-riboflavin and 8-CN-FMN were found to be photoreactive and need to be protected from exposure to light. However such complications were not encountered with protein-bound flavins. The apoproteins of flavodoxin and Old Yellow Enzyme (OYE) were reconstituted with the 8-CN-FMN and apoDAAO was reconstituted with 8-CN-FAD. Spectral properties of the enzyme-bound neutral and anionic semiquinones were determined from these reconstituted proteins. In the case of 8-CN-FMN-OYE I, it was shown that the comproportionation reaction of a mixture of reduced and oxidized enzyme bound flavin is very rapid, compared with the same reaction with native protein, resulting in ~100% thermodynamically stable anionic semiquinone. In the case of 8-CN-OYE I, it was shown that the rate of reduction of the enzyme bound flavin by NADPH is ~40 times faster, and the rate of reoxidation of reduced enzyme bound flavin by oxygen is an order of magnitude slower than with the normal FMN enzyme. This is in accord with the high oxidation-reduction potential of the flavin, which thermodynamically stabilizes the reduced enzyme.
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INTRODUCTION |
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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).
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MATERIALS AND METHODS |
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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 Pi, 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 Pi, 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 Pi, 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 kcat and Km values.
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RESULTS AND DISCUSSION |
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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.
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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.
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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 pKa of Reduced Cyanoriboflavin
The pKa 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 pKa 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 Kd 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 Kd
for the complex (29), the oxidation-reduction potential at pH 7 would
be predicted to be 50 mV, in perfect agreement with direct
measurement.
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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 cm
1. 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 mM
1 cm
1) for the bound flavin.
The extinction coefficient for 8-CN-FMN was determined as 11,000 M
1 cm
1 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 EFlox/EFlH·
and EFlH·/EFlred (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).
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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 (Kd ~ 109 M)
(41). The extinction coefficient of the cyano-FMN was determined as
11,000 M
1 cm
1 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 M
1 cm
1.
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 Kd 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 Kd 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.
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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 Pi, 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.
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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.
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(Eq. 1) |
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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 Pi,
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).
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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 min
1 was obtained with a
Km for NADPH of 0.2 µM, whereas the
native OYE I has a turnover number3 of ~300
min
1. 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 kcat = kred·kox/(kred + kox), it may be concluded that
kox for cyclohexenone with the 8-CN-FMN
enzyme = kcat, i.e. 0.125 s
1, an 870-fold decrease from the value of 109 s
1 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).
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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 cm
1, 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 EFlox/EFlSQ and
EFlSQ/EFlred 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 Kd 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 Kd 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 Kd 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 Kd 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.
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ACKNOWLEDGEMENTS |
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We are very grateful to Dr. Sandro Ghisla, University of Konstanz, Germany for the samples of 8-aminoriboflavin and ribitylamine.
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FOOTNOTES |
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* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: Dept. of Biological
Chemistry, University of Michigan Medical School, Ann Arbor, MI
48109-0606. Tel. 313-764-7196; Fax: 313-763-4581; E-mail:
massey{at}umich.edu.
1 Y. V. S. N. Murthy and V. Massey, unpublished results.
2 The abbreviations used are: OYE, Old Yellow Enzyme; HPLC, high performance liquid chromatography.
3 B. J. Brown and V. Massey, unpublished results.
4
We were able to accomplish the reversal of OYE
redox chemistry by reconstituting the apoprotein with 8-CN-FMN. The
reductase activity of the native enzyme toward ,
-unsaturated
carbonyl compounds was changed so that the enzyme now acted
preferentially to dehydrogenate the corresponding saturated carbonyl
compounds, using molecular oxygen as acceptor (Y. V. S. N. Murthy and V. Massey, manuscript in preparation).
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REFERENCES |
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