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• W2152337502 abstract "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. 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. 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 (1Walsh C. Fisher J. Spencer R. Graham D.W. Ashton W.T. Brown J.E. Brown R.D. Rogers E.F. Biochemistry. 1978; 17: 1942-1951Crossref PubMed Scopus (128) Google Scholar, 2Walsh C. Acc. Chem. Res. 1980; 13: 148-155Crossref Scopus (396) Google Scholar, 3Light D.R. Walsh C. J. Biol. Chem. 1980; 255: 4264-4277Abstract Full Text PDF PubMed Google Scholar, 4Ghisla S. Massey V. Biochem. J. 1986; 239: 1-12Crossref PubMed Scopus (167) Google Scholar). 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 (5Lambooy J.P. Methods Enzymol. 1971; 18B: 437-444Crossref Scopus (12) Google Scholar), fluorine (6Kasai S. Sugimoto K. Miura R. Yamano T. Matsui K. J. Biochem. (Tokyo). 1983; 93: 397-402Crossref PubMed Scopus (13) Google Scholar), methylsulfonyl (7Moore E.G. Ghisla S. Massey V. J. Biol. Chem. 1979; 254: 8173-8178Abstract Full Text PDF PubMed Google Scholar, 8Raibekas A.A. Ramsey A.J. Jorns M.S. Biochemistry. 1993; 32: 4420-4429Crossref PubMed Scopus (12) Google Scholar) 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 (9Schopfer L.M. Massey V. Claiborne A. J. Biol. Chem. 1981; 256: 7329-7337Abstract Full Text PDF PubMed Google Scholar) and also for the covalent labeling of active site amino acid residues (10Moore E.G. Cardemil E. Massey V. J. Biol. Chem. 1978; 253: 6413-6422Abstract Full Text PDF PubMed Google Scholar, 11Raibekas A.A. Jorns M.S. Biochemistry. 1994; 33: 12649-12655Crossref PubMed Scopus (10) Google Scholar, 12Raibekas A.A. Jorns M.S. Biochemistry. 1994; 33: 12656-12664Crossref PubMed Scopus (8) Google Scholar). Elegant model studies by Bruice et al. (13Bruice T. Chan T.W. Taulane J.P. Yokoe I. Elliott D.L. Williams R.F. Novak M. J. Am. Chem. Soc. 1977; 99: 6713-6720Crossref PubMed Scopus (36) Google Scholar) 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. 1Y. V. S. N. Murthy and V. Massey, unpublished results. 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 (14Spencer R. Fisher J. Walsh C. Biochemistry. 1976; 15: 1043-1053Crossref PubMed Scopus (125) Google Scholar). 8-NH2-riboflavin, which was synthesized as described previously (15Berezovskii V.M. Tulchinskaya L.S. Polyakova N.A. Zh. Obshch. Khim. 1965; 35: 673-677Google Scholar), 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 (16Ghisla S. Mayhew S.G. Eur. J. Biochem. 1976; 63: 373-390Crossref PubMed Scopus (47) Google Scholar). The holoproteins and apoproteins were prepared as described previously: riboflavin binding protein from hen egg white (17Becvar J. Palmer G. J. Biol. Chem. 1982; 257: 5607-5617Abstract Full Text PDF PubMed Google Scholar), flavodoxin from Megasphera elsdenii (18Mayhew S.G. Massey V. J. Biol. Chem. 1969; 244: 794-802Abstract Full Text PDF PubMed Google Scholar, 19Mayhew S.G. Biochim. Biophys. Acta. 1971; 235: 289-302Crossref PubMed Scopus (103) Google Scholar), and OYE I, 2The abbreviations used are: OYE, Old Yellow Enzyme; HPLC, high performance liquid chromatography. overexpressed inEscherichia coli containing the plasmid pET-3b (20Saito K. Thiele D.J. Davio M. Lockridge O. Massey V. J. Biol. Chem. 1991; 266: 20720-20724Abstract Full Text PDF PubMed Google Scholar). The apoOYE I was prepared from the recombinant enzyme by using the procedure reported for the enzyme from brewers' bottom yeast (21Abramowitz A.S. Massey V. J. Biol. Chem. 1976; 251: 5321-5326Abstract Full Text PDF PubMed Google Scholar, 22Abramowitz A.S. Massey V. J. Biol. Chem. 1976; 251: 5327-5336Abstract Full Text PDF PubMed Google Scholar). The apoDAAO from pig kidney was obtained from Calzyme (San Luis Obisco, CA). 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 (23Massey V. Curti B. Ganther H. J. Biol. Chem. 1966; 241: 2347-2357Abstract Full Text PDF PubMed Google Scholar). 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). 10 mg of 8-amino-riboflavin was suspended in 3 ml of water in a test tube. To this suspension, 6n 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. 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. (14Spencer R. Fisher J. Walsh C. Biochemistry. 1976; 15: 1043-1053Crossref PubMed Scopus (125) Google Scholar). 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). 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 NaNO2were 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% 0.01m 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. 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. 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 (24Hodgson H.H. Heyworth F. J. Chem. Soc. 1949; : 1131-113?Crossref Scopus (10) Google Scholar, 25Suzuki N. Azuma T. Kaneko Y. Izawa Y. Tomioka H.J. J. Chem. Soc. Perkin Trans. I. 1987; 645 (and references therein)Google Scholar). 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 (26Yonezawa N. Hino T. Namie T. Katakai R. Synth. Commun. 1996; 26: 1575-1578Crossref Scopus (10) Google Scholar). 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 (SchemeFS1). 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. The λmax 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. 1 A). 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. 1 B) (molecular weight of the 8-CN-riboflavin is 387) and also by the 1H NMR spectrum (Fig. 1 C). 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. 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. 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. (13Bruice T. Chan T.W. Taulane J.P. Yokoe I. Elliott D.L. Williams R.F. Novak M. J. Am. Chem. Soc. 1977; 99: 6713-6720Crossref PubMed Scopus (36) Google Scholar). 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. 1 A). The pKa value of the reduced cyanoflavin was determined to be at pH 5.6 from the change in absorption spectrum with pH (Fig. 1 A, inset). Hence the electronegative 8-CN-group decreases the pKa of the reduced flavin from the usual value (27Bruice T.C. Prog. Bioorg. Chem. 1976; 4: 1-84Google Scholar) 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. 1 A. The reduction-oxidation potential for 8-CN-FAD was measured by using the xanthine/xanthine oxidase system and indigo tetrasulfonate as the reference dye (28Massey V. Curti B. Ronchi S. Zanetti G. Flavins and Flavoproteins 1990. Walter de Gruyter, Berlin1991: 59-66Crossref Google Scholar). 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. 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) (29Müller F. Massey V. J. Biol. Chem. 1969; 244: 4007-4016Abstract Full Text PDF PubMed Google Scholar). 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 (29Müller F. Massey V. J. Biol. Chem. 1969; 244: 4007-4016Abstract Full Text PDF PubMed Google Scholar). 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 (29Müller F. Massey V. J. Biol. Chem. 1969; 244: 4007-4016Abstract Full Text PDF PubMed Google Scholar), the oxidation-reduction potential at pH 7 would be predicted to be −50 mV, in perfect agreement with direct measurement. 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 Bruiceet al. (13Bruice T. Chan T.W. Taulane J.P. Yokoe I. Elliott D.L. Williams R.F. Novak M. J. Am. Chem. Soc. 1977; 99: 6713-6720Crossref PubMed Scopus (36) Google Scholar) and compatible with formation of a spirohydantoin (30Dudley K.H. Hemmerich P. J. Org. Chem. 1967; 32: 3049-3054Crossref PubMed Scopus (35) Google Scholar). 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. It is well known that flavins are photoreactive and their spectroscopic and photochemical properties have been the subject of intense studies (31Heelis P.F. Müller F. Chemistry and Biochemistry of Flavoenzymes. I. CRC Press, Inc., Boca Raton, FL1992: 171-194Google Scholar). 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 (32Weber G. Biochem. J. 1950; 47: 114-121Crossref PubMed Scopus (234) Google Scholar). The increased stability of FAD toward photolysis is attributed to this intramolecular stacking (33Miles D.W. Urry D.W. Biochemistry. 1968; 7: 2791-2799Crossref PubMed Scopus (80) Google Scholar). 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. 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 nmand shows considerable spectral shifts and resolution on binding (17Becvar J. Palmer G. J. Biol. Chem. 1982; 257: 5607-5617Abstract Full Text PDF PubMed Google Scholar). 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 10,400m−1 cm−1. The 5-deazaflavin-catalyzed photoreduction (34Massey V. Hemmerich P. J. Biol. Chem. 1977; 252: 5612-5614Abstract Full Text PDF PubMed Google Scholar) 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 λmax 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 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 (ε460 = 9.4 mm−1 cm−1) for the bound flavin. The extinction coefficient for 8-CN-FMN was determined as 11,000m−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 (35Massey V. Palmer G. Biochemistry. 1966; 5: 3181-3189Crossref PubMed Scopus (346) Google Scholar). 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 (36Mayhew S.G. Biochim. Biophys. Acta. 1971; 235: 276-288Crossref PubMed Scopus (56) Google Scholar). 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 (37Massey V. Müller F. Feldberg R. Schuman M. Sullivan P.A. Howell G.L. Mayhew S.G. Matthews R.G. Foust G.P. J. Biol. Chem. 1969; 244: 3999-4006Abstract Full Text PDF PubMed Google Scholar). 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 (38Schopfer L.M. Massey V. Kuby S.A. A Study of Enzymes. CRC Press, Inc., Boca Raton, FL1990: 247-283Google Scholar,39Stott K. Saito K. Thiele D.J. Massey V. J. Biol. Chem. 1993; 268: 6097-6106Abstract Full Text PDF PubMed Google Scholar). 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. (20Saito K. Thiele D.J. Davio M. Lockridge O. Massey V. J. Biol. Chem. 1991; 266: 20720-20724Abstract Full Text PDF PubMed Google Scholar) cloned a gene encoding an isoform of OYE (OYE I) from Saccharomyces carlsbergenesis, and its crystal structure has been determined (40Fox K.M. Karplus P.A. Structure. 1994; 2: 1089-1105Abstract Full Text Full Text PDF PubMed Scopus (229) Google Scholar). 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 ∼ 10−9m) (41Theorell H. Nygaard A.P. Acta. Chem. Scand. 1954; 8: 877-888Crossref Google Scholar). 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 λmax for the free flavin to protein-bound flavin shifted from 340 and 454 nm to 348 and 470 nm, with the ε470 value of 10,200m−1 cm−1. The oxidized form of OYE binds to a variety of ligands forming spectroscopically distinct complexes (38Schopfer L.M. Massey V. Kuby S.A. A Study of Enzymes. CRC Press, Inc., Boca Raton, FL1990: 247-283Google Scholar). 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 (21Abramowitz A.S. Massey V. J. Biol. Chem. 1976; 251: 5321-5326Abstract Full Text PDF PubMed Google Scholar, 22Abramowitz A.S. Massey V. J. Biol. Chem. 1976; 251: 5327-5336Abstract Full Text PDF PubMed Google Scholar). 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 (22Abramowitz A.S. Massey V. J. Biol. Chem. 1976; 251: 5327-5336Abstract Full Text PDF PubMed Google Scholar). 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 (22Abramowitz A.S. Massey V. J. Biol. Chem. 1976; 251: 5327-5336Abstract Full Text PDF PubMed Google Scholar). Native enzyme bindsp-chlorophenol with a Kd of 1 μm and has a maximum of 645 nm for the long wavelength cha" @default.
• W2152337502 created "2016-06-24" @default.
• W2152337502 creator A5020618593 @default.
• W2152337502 creator A5079221179 @default.
• W2152337502 date "1998-04-01" @default.
• W2152337502 modified "2023-09-27" @default.
• W2152337502 title "Synthesis and Properties of 8-CN-flavin Nucleotide Analogs and Studies with Flavoproteins" @default.
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