Flavin Derivatives Research Articles

Molecular Photodiode Consisting of Flavin-Viologen-Tcnq LB Multilayers

A moleculear photodiode was fabricated with the hetero-Langmuir-Blodgett(LB) film consisting of an electron acceptor(A), an electron relay(R) and a sensitizer(S). TCNQ, viologen and flavin derivatives were used as A, R and S units, respectively. By aligning hetero-LB film of A/R/S units on ITO glass with an aluminum thin film, a molecular photodiode with the structure of Metal/Insulator/Metal(MIM) was constructed. Due to excitation by irradiation with a 460 nm monochromatic light source, the photo-induced unidirectional flow of electrons in the MIM device could be achieved and was detected as photocurrents. The direction of energy flow was in accordance with the energy level profile across the LB films. The photoswitching function was achieved and the rectifying characteristic was observed in the molecular device.

Covalent Attachment of FAD Derivatives to a Fusion Protein Consisting of 6-Hydroxy-D-nicotine Oxidase and a Mitochondrial Presequence: FOLDING, ENZYME ACTIVITY, AND IMPORT OF THE MODIFIED PROTEIN INTO YEAST MITOCHONDRIA

Autoflavinylation of 6-hydroxy-D-nicotine oxidase (6-HDNO) was successfully employed to modify the protein covalently with FAD derivatives. The model compounds N6-(2-aminoethyl)-FAD and N6-(6-carboxyhexyl)-FAD were spontaneously bound to a fusion protein consisting of the mitochondrial targeting sequence of Neurospora crassa F0-ATPase subunit 9 (Su9) attached to 6-HDNO. When translated in the rabbit reticulocyte lysate, Su9-6-HDNO was in the trypsin-sensitive apoenzyme form; when translated in the presence of flavins it adopted a trypsin-resistant conformation characteristic of the 6-HDNO holoenzyme. With flavin derivatives, Su9-6-HDNO exhibited approximately 50% of the 6-HDNO activity observed with FAD. The covalently modified Su9-6-HDNO was imported into Saccharomyces cerevisiae mitochondria with an efficiency equal to that of the apoenzyme. Apparently the increase in size and charge of the FAD moiety did not hamper translocation across the mitochondrial membranes. Yeast mutant ssc1-2 mitochondria deficient in mtHsp70 unfoldase activity imported the flavinylated Su9-6-HDNO protein. In mutant ssc1-3 mitochondria deficient in both mtHsp70 unfoldase and translocase activity Su9-6-HDNO was trapped as translocation intermediate; the Su9 presequence was passed to the matrix where it was proteolytically cleaved by the mitochondrial processing peptidase; (MPP); the translocation-arrested 6-HDNO moiety adopted a trypsin-sensitive conformation. Our results indicate that unfolding of the FAD-stabilized flavin-binding domain of 6-HDNO in passage through the mitochondrial general insertion pore does not require the activity of mtHsp70.

Theoretical and Experimental Studies of a Charge-Transfer Mechanism for Biomimetic Oxygenations of Phenol and Indol Derivatives

Abstract The structures and stabilities of organodioxide anions, charge-transfer (CT) complexes between carbanions and molecular oxygen, and related solvated clusters were investigated using ab initio and semiempirical MO plus intermolecular CT theories. The computational results, together with experimental evidence, have revealed the characteristics of potential curves, indicating that the nonradical mechanism via CT complexes is operative for base-catalyzed dioxygenation reactions of phenol and indole derivatives. The following conclusions were drawn from the present calculations: (1) The reactions of carbanion with triplet molecular oxygen proceed through a one-electron transfer (ET) mechanism from carbanion to 3O2. (2) The base-promoted phenol monoanion reacts with molecular oxygen via two different mechanism: one is the CT mechanism, where the triplet CT complex is formed and a spin inversion from a triplet CT complex to a singlet CT one occurs in aprotic solvents; the other involves the one ET mechanism assisted by hydrogen bonding in a protic solvent. (3) The one ET does not occur for the free monoanions of the indole derivatives; rather these species are readily oxygenated by organo-cobalt catalysts. (4) For the flavin derivatives, the CT mechanism is applicable in an aprotic solution, while both the CT and one ET mechanisms are conceivable in aqueous solution. The implications of these results to biological dioxygenations by metallo-dioxygenases are discussed in relation to biomimetic oxygenations of phenol and indole derivatives.

The design of an alternative, covalently flavinylated 6-hydroxy- d-nicotine oxidase by replacing the FAD-binding histidine by cysteine and reconstitution of the holoenzyme with 8-(methylsulfonyl)FAD

The cofactor of several flavoenzymes is autocatalytically bound to the polypeptide via a histidyl(N 3)-(8α)-FAD linkage which makes the generation of apoenzyme difficult. We introduced an alternative covalent protein-FAD bond at the active site of 6-hydroxy- n-nicotine oxidase (6HDNO) by replacing the FAD-binding histidine with cysteine. The resulting mutant enzyme was expressed with noncovalently attached cofactor. Incubation with 8-(methylsulfonyl)FAD, and less efficiently with 8-chloro-FAD, resulted in the spontaneous replacement of the noncovalently bound FAD by the flavin derivative and the formation of an 8-( N-acetylcysteinyl)FAD linkage. The flavinylated 6HDNO.cys exhibited close to wild-type activity levels. This strategy may be generally applicable to the attachment of artificially designed flavin derivatives to the active site of covalently flavinylated enzymes.

Model systems for flavoenzyme activity. The effects of specific hydrogen bonds on the 13C and 1H NMR of flavins.

We have developed a family of receptors designed to bind flavin derivatives using specific hydrogen bond interactions. These synthetic host molecules provide a model for specific flavoenzyme-cofactor interactions, allowing isolation and observation of the effects of hydrogen bonding on flavin NMR. We describe here the use of one of these receptors to study the effects of hydrogen bonding to O(2), N(3), and O(4) on flavin 1H and 13C NMR.

Chemical Modification of the N-10 Ribityl Side Chain of Flavins EFFECTS ON PROPERTIES OF FLAVOPROTEIN DISULFIDE OXIDOREDUCTASES

Three flavin derivatives modified at the 2'-position of the flavin N-10 ribityl side chain were synthesized: arabinoflavin, 2'-F-2'-deoxyarabinoflavin, and 2'-deoxyriboflavin. These were converted to the FAD level with FAD synthetase. Apoproteins of lipoamide dehydrogenase, glutathione reductase, and mercuric reductase, a family of flavoprotein oxidoreductases, were reconstituted with these flavins. Significant reduction of the catalytic activities was observed with the modified enzymes. During anaerobic reduction of the modified enzymes with substrate or dithiothreitol, decreased thermodynamic stability of the two-electron reduced enzyme forms (EH2) and the accumulation of the four-electron reduced forms (EH4) noted. This effect was more pronounced in case of arabino-FAD-reconstituted enzymes than with the other two. It was found that NAD+ binding influences the interaction between the flavin and the reduced disulfide in the 2'-F-arabino-FAD-lipoamide dehydrogenase, presumably by altering the relative oxidation-reduction potentials. 19F NMR data were obtained for different forms of the 2'-F-arabino-FAD-lipoamide dehydrogenase, which suggest marked conformational changes from one form to the other. The 19F NMR data for the oxidized forms of all three 2'-F-arabino-FAD proteins suggest that the fluorine experiences very similar chemical environments at the active sites.

Monoclonal antibodies against two electron reduced riboflavin and a quantification of affinity constants for this oxygen-sensitive molecule.

In order to create a protein environment that binds preferentially to the two-electron reduced form of flavin, monoclonal antibodies have been raised against a reduced flavin derivative. Due to the low fluorescence quantum yield and visible light absorption and to the instability of reduced flavin in an aerobic environment, it is not possible to determine the affinities of these antibodies for two-electron-reduced flavin using standard techniques. Because of its sensitivity, time-resolved fluorescence can be used to overcome this problem. This technique has been applied to study the binding of two antibodies, an IgG1 and an IgM, to reduced riboflavin (1,5-dihydroriboflavin) and oxidized riboflavin (riboflavin). The affinity of the IgG1 is more than 80 times larger for 1,5-dihydroriboflavin than for riboflavin. From analysis of the dynamical parameters of the system it is apparent that the internal motion of 1,5-dihydroriboflavin bound to IgG1 is much more restricted than that of riboflavin. In contrast, the affinity of the IgM is only slightly higher for 1,5-dihydroriboflavin than for riboflavin and the flexibility of binding of both flavin redox states in the antigen binding site is almost similar.

Flavinylation of monoamine oxidase B.

Monoamine oxidase B (MAO B) catalyzes the oxidative deamination of biogenic and xenobiotic amines. The oxidative step is coupled to the reduction of an obligatory cofactor, FAD, which is covalently linked to the enzyme at Cys397. In this study, we developed a novel riboflavin-depleted (Rib-) COS-7 cell line to study the flavinylation of MAO B. ApoMAO B can be obtained by expressing MAO B cDNA in these cells. We found that MAO B is expressed equally in the presence or absence of FAD and that apoMAO B can be inserted into the outer mitochondrial membrane. Flavinylation of MAO B was achieved by introducing MAO B cDNA and different flavin derivatives simultaneously into Rib- COS-7 cells via electroporation. Since the addition of riboflavin, FMN, or FAD resulted in equal levels of MAO B activity, we conclude that the flavin which initially binds to apoMAO B is FAD. In our previous work, we used site-directed mutagenesis to show that Glu34 in the dinucleotide-binding motif of MAO B is essential for MAO B activity, and we postulated that this residue is involved in FAD binding. In this study, we tested the role of residue 34 in flavin binding by expressing wild-type or mutant MAO B cDNA in Rib- COS-7 cells with the addition of [14C]FAD. We found that Glu34 is essential for both FAD binding and catalytic activity. Thus, FAD binds to MAO B in a dual manner at Glu34 noncovalently and Cys397 covalently. We conclude that Glu34 is critical for the initial non-covalent binding of FAD and is instrumental in delivering FAD to the covalent attachment site at Cys397.

Photooxidation of substituted benzyl alcohol by riboflavin

The oxidation of substituted benzyl alcohols using flavin derivatives as photochemical catalysts has been studied. The interpretation of the experimental results of these reactions using molecular orbital calculations is presented.

Surface Modification of Glassy Carbon by Riboflavin

Flavin derivatives catalyze the oxidation-reduction reactions in biological systems. All the known flavins form thermodynamically reversible systems. Hence flavins are good candidates for electron transfer mediators. In the present investigation riboflavin (vitamin B 2 ) is attached to the surface of glassy carbon by covalent linkage. The electrode thus modified is characterized by cyclic voltammetry and impedance measurements. From the voltammetric data the surface coverage is calculated to be 7.7×10 -9 mol/cm 2 . From the data of the impedance measurements, k o for the electron transfer across the modified electrode is calculated to be 2.5 s -1 and the value is comparable to that obtained from voltammetric data. The modified electrode is tested for its catalytic properties by investigating the reduction of oxygen and the oxidation of ascorbic acid

Structural characterization of oxidized lumiflavin hydrochloride hydrate: Comparison with the hydrogen bonding at the cofactor of flavoproteins

In order to examine the possibility of hydrogen bonding around the flavin ring, an X-ray diffraction study of the title compound was undertaken. Crystals of lumiflavin hydrochloride hydrate [2(C13H12N4O2) 4 HCl 7 H2O] are triclinic, space group\(P\bar 1\),Z=2,a=11.064(1),b=17.903(1),c=9.891(1)A, α=111.9(6), β-96.4(8), γ=91.6(7)°. We present the crystal structure of this compound and compare it with other flavin derivatives and with the structure of the active site of some flavoproteins.

Inhibitory Effect of Flavin Derivatives on the Lipid Peroxidation Attended to the In Vitro Metabolism of Trichloroacetic Acid.

The possibility of hepatic toxicity of trichloroethylene (TCE) related to the increase of lipid peroxide in the liver was investigated. Trichloroacetic acid (TCA) and dichloroacetic acid (DCA) were dominant metabolites of TCE in the liver, so that TCA was metabolized in vitro by microsomes and lipid peroxidation was measured as an oxygen uptake by the oxygen electrode method. The increase of lipid peroxidation with the metabolism of TCA and the TCA-induced lipid peroxidation was inhibited by mannitol, uric acid and flavin derivatives, but scarcely inhibited by superoxide dismutase and catalase. Moreover, non-enzymic DCA radical production induced by 2, 2'-azobis (2-amidinopropane) dihydrochloride was inhibited by radical scavengers and flavin derivatives. These results suggest that the active oxygen scarcely contributes to the lipid peroxidation with the metabolism of TCA and that the inhibitory effect of flavin derivatives depends on the affinity for DCA radical and lipid peroxide radical.

Bioluminescence mechanism on new systems

Abstract

The kinetic behavior of xanthine oxidase containing chemically modified flavins.

The steady-state and rapid kinetic properties of xanthine oxidase containing a series of FAD analogs of varying reduction potential have been investigated. From steady-state analysis, Vmax is found to exhibit a sigmoidal dependence on the flavin midpoint potential in the homologous series. This dependence is accurately described by a model in which the rate of catalysis is attenuated by the amount of partially reduced enzyme generated during turnover possessing an unfavorable distribution of reducing equivalents among the several redox-active centers of the protein. The model assumes that reducing equivalents equilibrate among these centers rapidly compared to the limiting rates for the reductive and oxidative half-reactions. This assumption is borne out by a quantitative analysis of the reductive and oxidative half-reactions of the several enzyme forms investigated in detail. It is demonstrated in these studies that xanthine oxidase containing low potential flavin derivatives such as 1-deaza, 6-hydroxy, or 8-hydroxy FAD exhibits low turnover not because of inherently slow rates of reduction by xanthine or oxidation by molecular oxygen, but because in partially reduced enzyme generated in the course of turnover reducing equivalents are distributed within the enzyme in such a way that the enzyme can participate in neither the reductive nor oxidative half-reactions. These results provide confirmation of the operation of a thermodynamic control mechanism in a simple electron-transferring system.

Cellobiose dehydrogenases of Sporotrichum (Chrysosporium) thermophile.

Both cellobiose dehydrogenases of Sporotrichum (Chrysosporium) thermophile, ATCC 42464, obtained after fractionation with DEAE-Trisacryl chromatography and named cellobiose dehydrogenase I and II have been purified to homogeneity by different chromatographic techniques. Both enzymes are slightly glycosylated flavocytochrome-b proteins with similar catalytic properties but with distinct molecular masses (91 kDa and 192 kDa for enzymes I and II, respectively) and isoelectric point (4.1 versus 3.45). Examination by SDS/PAGE clearly showed that the larger enzyme II is a homodimer, whose subunit is close to, but different from dehydrogenase I which is homogeneous by this technique. After limited digestion of both enzymes with papain, two main fractions with residual activity are formed, one carrying the heme, the other being the flavin component; each fraction is characterized by its particular chromatographic behaviour. The flavin carrying component shows an atypical (for flavoprotein) three-banded spectrum indicative of the presence of a flavin derivative. Both enzymes react very slowly with oxygen clearly forming some superoxide radicals and possibly hydrogen peroxide. Cellobiose and other cellodextrins are oxidized at their reducing glycosyl moiety to the corresponding aldonic acid. With the use of the autooxidable phenazinemethosulphate, cellulose (either in a hydrated form or crystalline) is also oxidized at free reducing ends so that appreciable amounts of cellobionic acid are released upon enzymatic hydrolysis.

Qualitative and Quantitative Assessment of Flavins in Cow's Milk

Although milk is one of the primary sources of riboflavin in the North American diet, the amounts of riboflavin and other flavin derivatives in milk have not been accurately quantified. In this study, a comprehensive assessment of milk was performed to identify and quantify greater than 95% of the flavins in this food. Riboflavin and flavin adenine dinucleotide (FAD) were the predominant flavins in all milk samples; 10-(2'-hydroxyethyl)flavin was next most common. The latter is potentially an antivitamin that may exert its effect at either the absorption or utilization level. Therefore, the presence of this analog should be considered when evaluating the riboflavin efficiency of milk. The large quantity of FAD indicates that precautions against underestimating FAD are warranted when determining total flavin content by fluorescence. This is particularly important because the fluorescence of FAD at pH 7.0 is only 10-20% of that for riboflavin. The presence of 7 alpha-hydroxyriboflavin (7-hydroxymethylriboflavin) in milk was ascertained by extensive testing. Traces of 8 alpha-hydroxyriboflavin (8-hydroxymethylriboflavin) were also detected. These metabolites may serve as indicators of cellular biochemical activity.

Chromophore function and interaction in Escherichia coli DNA photolyase: reconstitution of the apoenzyme with pterin and/or flavin derivatives

Native DNA photolyase, as isolated from Escherichia coli, contains a neutral flavin radical (FADH.) plus a pterin chromophore (5,10-methenyltetrahydropteroylpolyglutamate) and can be converted to its physiologically significant form by reduction of FADH. to fully reduced flavin (FADH2) with dithionite or by photoreduction. Either FADH2 or the pterin chromophore in dithionite-reduced native enzyme can function as a sensitizer in catalysis. Various enzyme forms (EFADox, EFADH., EFADH2, EPteFADox, EPteFADH., EPteFADH2, EPte) containing stoichiometric amounts of FAD in either of its three oxidation states and/or 5,10-methenyltetrahydrofolate (Pte) have been prepared in reconstitution experiments. Studies with EFADox and EPte showed that these preparations retained the ability to bind the missing chromophore. The results suggest that there could be considerable flexibility in the biological assembly of holoenzyme since the order of binding of the enzyme's chromophores is apparently unimportant, the binding of FAD is unaffected by its redox state, and enzyme preparations containing only one chromophore are reasonably stable. The same catalytic properties are observed with dithionite-reduced native enzyme or EFADH2. These preparations do not exhibit a lag in catalytic assays whereas lags are observed with preparations containing FADox or FADH. in the presence or absence of pterin. Photochemical studies show that these lags can be attributed to enzyme activation under assay conditions in a reaction involving photoreduction of enzyme-bound FADox or FADH. to FADH2. EPte is catalytically inactive, but catalytic activity is restored upon reconstitution of EPte with FADox. The results show that pterin is not required for dimer repair when FADH2 acts as the sensitizer but that FADH2 is required when dimer repair is initiated by excitation of the pterin chromophore. The relative intensity of pterin fluorescence in EPte, EPteFADH., EPteFADox, or EPteFADH2 has been used to estimate the efficiency of pterin singlet quenching by FADH. (93%), FADox (90%), or FADH2 (58%). Energy transfer from the excited pterin to flavin is energetically feasible and may account for the observed quenching of pterin fluorescence and also explain why photoreduction of FADox or FADH. is accelerated by the pterin chromophore. An irreversible photobleaching of the pterin chromophore is accelerated by FADH2 in a reaction that is accompanied by a transient oxidation of FADH2 to FADH.. Both pterin bleaching and FADH2 oxidation are inhibited by substrate.

A chemical model of cytochrome P-450 with electron-transfer activity

In this communication, we describe a new system of reductive dioxygen activation at thiolato-metalloporphyrin complexes by NaBH 4 as a reducing agent in the presence of flavin derivative as an electron-transfer agent in benzene as a solvent

ChemInform Abstract: Facile Synthesis of N‐10 Flavin Disulfides.

AbstractStarting with o‐dinitrobenzene (I), the flavin derivative (V) is obtained by base‐catalyzed coupling of (I) with the amine (II) followed by catalytic hydrogenation and cyclocondensation with alloxan (IV).

Further Investigations about the Flavin in the L-Amino Acid Oxidase and a Possible Flavin in Photosystem II Complexes from the Cyanobacterium Anacystis nidulans

The absorption spectrum of the previously purified ʟ-amino acid oxidase from the cyanobacterium Anacystis nidulans has shown considerable variation with each preparation and the spectrum in several preparations was quite different from the absorption spectrum of other simple flavoproteins (E. K. Pistorius and A. E. Gau, Biochim. Biophys. Acta 849, 203, 1986). Here we show that the spectral complexity and variability of the ʟ-amino acid oxidase can be largely explained by the presence of a modified flavin derivative of yet unknown structure besides oxidized FAD and FAD semiquinone. After removal from the enzyme this modified chromophore has absorption maxima at 260, 396 and in the 600 nm region. This derivative of FAD seems to be formed in variable amounts during the purification of the enzyme. On the other hand, extraction of Anacystis photosystem II complexes which contain the flavoprotein, almost exclusively yields modified flavin derivatives and practically no authentic oxidized FAD. The spectrum of the chromophores which have been extracted from photosystem II complexes at different purification stages, is either similar (although not identical) to the spectrum of the chromophore extracted from the isolated ʟ-amino acid oxidase or similar to the spectrum of reduced flavin. All extracted chromophores show a fluorescence emission in the 420 to 560 nm region when excited with light of 390 nm. These results indicate that the flavin present in the ʟ-amino acid oxidase protein as well as in photosystem II complexes from A. nidulans rapidly undergoes modification reactions of yet unknown nature to yield several closely related FAD derivatives. This might possibly be the reason why so far no flavin has been detected in photosystem II. The presence of such modified flavin derivatives in photosystem II complexes of A. nidulans as shown here is an additional support of our hypothesis that an unusual flavin is functional on the donor side of photosystem II.