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PubMed1979

Fumarate reductase of Escherichia coli. Elucidation of the covalent-flavin component.

Fumarate reductase is a membrane-bound terminal oxidase which is induced when Escherichia coli is grown anaerobically. The purified enzyme is composed of two polypeptide chains of 69,000 and 24,000 daltons and contains 1 mol of covalently bound flavin adenine dinucleotide per mol of enzyme. Fluorescence scanning of SDS-polyacrylamide gels of the protein shows that the flavin is attached to the large subunit. The hypsochromic shift of the 372 nm band of riboflavin to 350 nm in both native fumarate reductase and a flavin peptide released by proteolytic digestion indicates that the flavin is attached via position 8 alpha of riboflavin. Based on the spectral properties and pH-fluorescence dependence we have identified the linkage as 8 alpha-[N(3)-histidyl]FAD.

Escherichia coli↗
PubMedDate not supplied

[Flavin biogenesis in yeasts].

The biosynthesis of flavins and methods for its study in yeast are considered. The chemical structure of flavin precursors and enzymes catalyzing certain stages of GTP transformation into riboflavin and flavin nucleotides are characterized. Differences in formation of flavins in bacteria and yeast are shown as well as possible ways of further research in this field.

Chemical Phenomena↗
PubMed1980

Flavin analogs as mechanistic probes of adrenodoxin reductase-dependent electron transfer to the cholesterol side chain cleavage cytochrome P-450 of the adrenal cortex.

The intrinsic isotope effect on the reduction of the FAD-containing dehydrogenase electron transferase, adrenodoxin reductase, by (4S)-[2H]NADPH has been determined to be 7.1 to 7.7. The replacement of FAD by a series of FAD analogs at the active site of adrenodoxin reductase with oxidation-reduction potentials which vary over a range of 212 mV has made it possible to extrapolate to this limiting value from the variation in the observed isotope effect on Vmax with flavin midpoint potential. Stop-flow studies which allow the direct determination of the intrinsic isotope effect on the reductive half-reaction corroborate this result. During the steady state reduction of ferricyanide by the native enzyme under conditions of Vmax, this isotope effect is almost fully expressed (VH/VD = 6.7 to 6.8). In contrast, we observe a dramatic attenuation of the intrinsic isotope effect (due to hydride transfer to flavin) when the oxidative half-reaction is mediated by the natural acceptor protein, the 2Fe/2S ferredoxin, adrenodoxin. In a coupled three-protein system, the adrenodoxin-mediated reductions of both the artificial electron acceptor, cytochrome c, and the physiological electron acceptor, cytochrome P-450scc, by adrenodoxin reductase occur at similar rates and with similar kinetic isotope effects (1.9 to 2.0) when (4S)-[2H]NADPH is the reductant. We infer similar mechanisms for the reduction of both cytochromes. These results are in agreement with previous studies (Lambeth, J.D., and Kamin, H. (1979) J. Biol. Chem. 254, 2766-2774) which show that the reductive half-reaction is not solely rate-determining in adrenodoxin-mediated processes. The observation of a linear free energy relationship between Vmax and the flavin midpoint potential during steady state reduction of ferricyanide confirms that the reductive half-reaction is rate-determining in this assay. The relationship between Vmax and flavin midpoint potential in reactions which require adrenodoxin suggests that the midpoint potential of native adrenodoxin reductase has been optimized. Thus, the apoenzyme of adrenodoxin reductase tailors the midpoint potential of bound FAD in order to balance the activation energies of the reductive and oxidative half-reactions.

Adrenal Cortex↗
PubMed1981

Thermodynamic and electron paramagnetic resonance characterization of flavin in succinate dehydrogenase.

Thermodynamic parameters of succinate dehydrogenase flavin were determined potentiometrically from the analysis of free radical signal levels as a function of the oxidation-reduction potential. Midpoint redox potentials of consecutive 1-electron transfer steps are -127 and -31 mV at pH 7.0. This corresponds to a stability constant of intermediate stability, 2.5 x 10(-2), which suggests flavin itself may be a converter from n = 2 to n = 1 electron transfer steps. The pK values of the free radical (FlH . in equilibrium Fl . -) and the fully reduced form (FlH2 in equilibrium FlH-) were estimated as 8.0 +/- 0.2 and 7.7 +/- 0.2, respectively. Succinate dehydrogenase flavosemiquinone elicits an EPR spectrum at g = 2.00 with a peak to peak width of 1.2 mT even in the protonated form, suggesting the delocalization in the unpaired electron density. A close proximity of succinate dehydrogenase flavin and iron-sulfur cluster S-1 was demonstrated based on the enhancement of flavin spin relaxation by Center S-1.

Anaerobiosis↗
PubMed1983

Amino acid sequence around the covalently-bound flavin prosthetic group of Corynebacterium sarcosine oxidase.

A flavin peptide from Corynebacterium sarcosine oxidase was obtained by proteolytic digestion with trypsin and chymotrypsin, and purified by the method of Kenney et al. (1). Amino acid analyses of the flavin peptide gave the following results: Asx(1), Ala(1), Val(1), and His(1) per flavin group. By carboxypeptidase A digestion and partial acid hydrolysis, the structure of the flavin peptide was determined as (Formula: see text).

Amino Acid Sequence↗
PubMed1984

Apoenzyme of Pseudomonas cepacia salicylate hydroxylase. Preparation, fluorescence property, and nature of flavin binding.

The apoenzyme of Pseudomonas cepacia salicylate hydroxylase was prepared by a dialysis method. The apoprotein retains a dimeric structure and binds one FAD per monomer. Flavin binding results in both 81 and 60% of quenching and 15- and 5-nm blue shifts of FAD and protein fluorescence, respectively. A hydrophobic environment for the flavin site and a conformational difference between apoprotein and holoenzyme are thus indicated. Prior binding of NADH markedly retards the holoenzyme activity development upon a subsequent FAD addition. Flavin 1,N6-ethenoadenine dinucleotide binds to the apoenzyme much more weakly than FAD but this reconstituted holoenzyme and the FAD X enzyme both exhibit similar activities. The adenine moiety appears to be important to binding. The formation of holoenzyme from apoprotein and FAD involves minimally a two-step reversible process, an initial flavin-binding step followed by a conformational transition. At both 6 and 23 degrees C, the rates of hydroxylase activity recovery can be correlated with the rates of FAD binding, indicating that the initial FAD X apoenzyme complex is fully active and the subsequent slow conformational change has no significant effect on the catalytic efficiency. Overall dissociation constants calculated based on kinetic data are essentially identical with those determined by equilibrium measurements.

Apoenzymes↗
PubMed1982

Studies of the flavin adenine dinucleotide binding region in Escherichia coli pyruvate oxidase.

Experiments have been performed to probe the flavin adenine dinucleotide (FAD) binding region in Escherichia coli pyruvate oxidase. This enzyme functions as a membrane-associated flavoprotein coupled to the aerobic E. coli respiratory chain. The FAD moiety is noncovalently bound to pyruvate oxidase and can be removed reversibly to form apopyruvate oxidase. The addition of free FAD to apoenzyme results in the stoichiometric re-formation of the active flavoprotein. Using this technique, synthetic analogs of FAD were substituted in the flavin binding site and used as structural probes. Spectral analysis indicates that the benzoquinoid forms of 8-mercapto-FAD and 6-hydroxy-FAD are stabilized in the enzyme-binding site. This is consistent with the fact that the native flavoprotein forms a red (anion) radical upon photoreduction. These data suggest that the isoalloxazine ring may be poised for reduction via position N-5 by a carbanionic intermediate. The alpha-carbanion of hydroxyethylthiamin pyrophosphate, formed following the decarboxylation of pyruvate, is a likely candidate. The highly resolved visible spectrum of the native flavoprotein suggests that the flavin is buried in a hydrophobic environment. Reactivity studies using 8-chloro-FAD-pyruvate oxidase and 2-thio-FAD-pyruvate oxidase suggest that the C-8 position and C-2 position of the isoalloxazine ring may not be accessible to the solvent. Spectral perturbations observed with 6-hydroxy-FAD-pyruvate oxidase indicate, however, that the isoalloxazine C-6 position may be located near the binding site for the cofactor thiamin pyrophosphate. Restrictions to the accessibility of the active site of the enzyme are suggested by the fact that sulfite does not form an adduct with the flavin in the native enzyme.

Binding Sites↗
PubMedDate not supplied

[Oxidation of NADH by singlet oxygen generated by triplet flavin].

Mechanisms of NADH oxidation in the presence of flavines in the light under aerobic and anaerobic conditions has been studied by spectrophotometry method and by registering the kinetics of changes in pH, luminescence and oxygen content in solution. It is shown that singlet oxygen O2* generated by triplet flavine may be an agent that immediately oxidizes NADH under aerobic conditions. The rate constant of the reaction of O2* with NADH accounts for about 1.9 . 10(9) M-1 s-1 and maximum quantum yield of NADH photooxidation in the presence of flavine mononucleotide is 0.7. During aerobic oxidation of NADH the flavine molecules function as photosensitizers rather than as carriers of reduced equivalents.

Flavins↗
PubMed1994

Photochemical reaction sequences of blue light activated flavins: sensory transduction through free radical messengers.

Flavin photochemistry involves the production of transient flavin free radicals which in turn sensitize molecular oxygen to generate superoxide. The blue light induced photochemical chain reaction of flavins may be blocked at two different levels by photoresponse inhibitors. A protein-bound/lipid-bound flavin located on the biomembrane may take part in the blue-light mediated sensory transduction by 'releasing' a 'free radical messenger' equivalent into the cell interior.

Flavins↗
PubMed1975

Bacterial luciferase. Binding of oxidized flavin mononucleotide.

Bacterial luciferase catalyzes a bioluminescent oxidation of reduced flavin mononucleotide; the products include a photon and oxidized FMN. The experiments reported here show that luciferase binds oxidized flavin mononucleotide in a 1:1 molar ratio with an apparent dissociation constant of 1.2 times 10-4 M at 3 degrees in 0.05 M 2,2-bis(hydroxymethyl)-2,2'2"-nitriloethanol (bis-tris), pH 7.0. Analysis of the binding at temperatures between 3 and 30 degrees indicates an enthalpy of binding (delta H a) of minus 10.0 kcal per mol. The absorption spectrum of luciferase-bound FMN shows considerable alteration relative to that of free flavin. There is one major peak at 366 nm, and the 445-nm band is resolved into two distinct peaks at 434 and 458 nm; this spectrum is indicative of binding in a nonpolar environment. The circular dichroism spectrum of FMN bound to luciferase has structure which correlates well with the optical absorption spectrum of the bound flavin. The detail in the spectra of the bound FMN probably reflects the resolution of vibrational structure which is blurred in polar environments. The optical activity shown by the CD spectrum presumably results from binding in an electronically asymmetric fashion. Although FMN free in solution is highly fluorescent, FMN bound to luciferase is nonfluorescent, thus indicating that the emitting species is not an excited state of product FMN located in the same site in which luciferase binds oxidized FMN.

Calorimetry↗
PubMed1993

Kinetic destabilization of the hydroperoxy flavin intermediate by site-directed modification of the reactive thiol in bacterial luciferase.

Bacterial luciferase catalyzes the formation of visible light, FMN, and a carboxylic acid from FMNH2, O2, and the corresponding aldehyde. The reactive cysteinyl residue at position 106 of the alpha subunit has been replaced by serine, alanine, and valine by site-directed mutagenesis (Baldwin, T. O., Chen L. H., Chlumsky, L. J., Devine, J. H., and Ziegler, M. M. (1989) J. Biolumin. Chemilumin. 4, 40-48) and the kinetics of the reaction catalyzed by each mutant protein measured by stopped-flow spectrophotometry at pH 7 and 25 degrees C. The time courses for the formation and decay of the various intermediates for the three alpha C106 mutants have been followed by monitoring the absorbance at 380 and 445 nm and the emission of visible light using n-decanal as the aldehyde substrate. The time courses for these events have been incorporated into a comprehensive kinetic model; 16 individual rate constants have been obtained for this model by numeric simulations of the time courses for the wild-type enzyme and for the three alpha C106 mutants. The mutants catalyzed the production of visible light demonstrating that the reactive thiol is not involved in the bioluminescence reaction. All three mutants have been found to catalyze the formation of the C4a-hydroperoxy flavin intermediate with rate constants equal to that of the wild-type enzyme. These results are incompatible with those reported by Xi et al. who have suggested that the major pathway for the oxidation of alpha C106V-bound FMNH2 does not involve the C4a-hydroperoxy flavin as an intermediate (Xi, L., Cho, K.-W., Herndon, M.E., and Tu, S.-C. (1990) J. Biol. Chem. 265, 4200-4203). The rates of decay of the C4a-hydroperoxy flavin intermediate with the mutant enzymes were found to be two orders of magnitude faster than that of the wild-type enzyme. Luciferase has been shown to be inhibited at high levels of aldehyde substrate when the enzyme is assayed by injecting FMNH2 into an aerobic mixture of enzyme and aldehyde. This aldehyde inhibition has been shown to occur by the formation of a dead-end enzyme-aldehyde complex which blocks the binding of FMNH2 to the enzyme; loss of activity is due to the rapid nonenzymatic decomposition of the reduced flavin with molecular oxygen.

Escherichia coli↗
PubMed1976

Identification of the covalently bound flavin of thiamin dehydrogenase.

Thiamin dehydrogenase, a flavoprotein isolated from an unidentified soil bacterium, contains 1 mol of covalently bound FAD/mol of enzyme. A flavin peptide, isolated from tryptic-chymotryptic digests of the enzyme and hydrolyzed to the FMN level, shows a pH-dependent fluorescence yield being maximal at pH 3.5 to 4.0 and decreasing over 90% at pH 7.5 with a pKa of 5.8. Acid hydrolysis of the peptide results in an aminoacylflavin which shows a pKa of fluorescence quenching of 5.2. Absorption and electron paramagnetic resonance spectral data show the covalent substituent to be at the 8alpha position of the flavin as is the case with all known enzymes containing covalently bound flavin. The aminoacylflavin gives a negative Pauly reaction but yields 1 mol of histidine on drastic acid hydrolysis thus showing an imidazole ring nitrogen as the 8alpha substituent of the flavin. The aminoacylflavin differs from synthetic 8alpha-[N(3)-histidyl]riboflavin or its acid-modified form in pKa of fluorescence quenching, in electrophoretic mobility, in being reduced by borohydride, and in being labile to storage, yielding 8-formylriboflavin. In all of these properties, however, the 8alpha-histidylriboflavin isolated from thiamin dehydrogenase is indistinguishable from 8alpha-[N(1)-histidyl]riboflavin. It is therefore concluded that the FAD moiety of thiamin dehydrogenase is covalently linked via the 8alpha-methylene group to the N(1) position of the imidazole ring of histidine.

Alcohol Oxidoreductases↗
PubMed1982

Growth, erythrocyte glutathione reductase and liver flavin as indicators of riboflavin status in turkey poults.

1. Turkey poults were fed on an isolated soya protein diet supplemented with 0, 2, 4 or 8 mg riboflavin/kg between 7 and 28 d of age. Maximum body weight was attained with the 4 and 8 mg diets, and poults fed on the supplemented diet did not survive until 21 d of age. The erythrocyte glutathione reductase activation coefficient (EGRAC) of the poults was inversely related to dietary riboflavin at all ages. At 28 d of age the values for EGRAC of poults fed on the 2, 4 and 8 mg diets were 2.25, 1.59 and 1.25 respectively; at 31 d, following a single oral dose of 10 mg riboflavin administered on day 28, the same poults had EGRAC values of 1.36, 1.17 and 1.26. 2. In a second experiment, poults were fed on diets supplemented with 0, 2, 4, 8 or 12 mg riboflavin/kg. Maximum body weight was attained with the 4, 8 and 12 mg diets. At 28 d of age the EGRAC values for the 0, 2, 4, 8 and 12 mg treatments were 2.36, 2.34, 1.59, 1.28 and 1.18 respectively. The concentration of flavin in the liver was not related to riboflavin intake, whereas total flavin in the liver was positively related, being on average 94, 206, 400, 440 and 413 micrograms flavin/liver for the 0, 2, 4, 8 and 12 mg treatments respectively. 3. It is concluded that EGRAC is a sensitive indicator of riboflavin status in the turkey poult and that an EGRAC of 1.6 indicates a marginal riboflavin status. It is also concluded that liver total flavin reflects merely the effect of riboflavin on growth and liver size.

Animals↗
PubMed2001

Frontal gel chromatographic analysis of the interaction of a protein with self-associating ligands: aberrant saturation in the binding of flavins to bovine serum albumin.

Frontal gel chromatography is an accurate method to obtain the total free ligand concentration of a protein-ligand mixture in which ligands self-associate. The average number of bound ligands per protein molecule is obtained as a function of the total free ligand concentration. The method was applied to the interaction of bovine serum albumin with self-associating flavins. The binding curves for FMN and FAD leveled off at about 0.7 and 0.5, respectively. These data were simulated well by a binding model where flavins undergo isodesmic indefinite self-association and the monomer alone binds to a single binding site of albumin. The isodesmic association constants of FMN and FAD were (1.7 +/- 0.1) x 10(2) and (2.2 +/- 0.3) x 10(2) M(-1), respectively. The binding constants of the monomer of FMN and FAD were (7.6 +/- 0.2) x 10(2) and (3.5 +/- 0.2) x 10(2) M(-1), respectively. FMN competitively inhibited the binding of FAD to albumin. The affinity to flavins was in the following order at pH 5.8: lumiflavin, FMN, riboflavin, and FAD. The SH modification and the binding of palmitate did not affect the FMN binding to bovine albumin at pH 5.8. As pH increased from 5.8 to 9.0, the affinity to FMN of bovine albumin decreased 3-fold, whereas that of human albumin increased about 80-fold. The present study clearly showed how isodesmic self-association of a ligand can cause apparent saturation of the interaction of a protein with the ligand at levels lower than 1.

Animals↗
PubMed1963

FLAVIN SENSITIZED PHOTOREACTIONS: EFFECTS OF 3-(P-CHLOROPHENYL)- 1,1-DIMETHYLUREA.

Photooxidation reactions, which were inhibited by high concentrations of oxygen, were affected by the addition of 3-(p-chlorophenyl)-1,1-dimethylurea (CMU), but the response varied with the substrate being oxidized. With ascorbic acid and 2,3-diketogulonic acid, CMU reversed the inhibition caused by high concentrations of oxygen. With ethylenediaminetetraacetic acid (EDTA), tetraethylene tetramine, or Mn(++) as reductant, CMU itself inhibited the reactions. The photoreduction of flavins by EDTA and the bleaching of flavin mononucleotide under anaerobic conditions were also inhibited by CMU. Corresponding photoreactions sensitized by phthaleine dyes or methylene blue were completely insensitive to CMU. This compound therefore seems to change specifically the reactivity of excited flavin molecules.

Antimetabolites↗
PubMed2001

Mild and efficient flavin-catalyzed H2O2 oxidations.

Based on a previously discovered method for amine oxidations using flavins as catalysts and hydrogen peroxide as oxidant, a comparative kinetic study using NMR spectroscopy was undertaken with a series of flavins for amine and thioether oxidations. Included in this series is the newly prepared 7,8-difluoro-1,3-dimethyl-5-ethyl-5,10-dihydroalloxazine. This study shows that flavins, which bear electron-donating groups on the aromatic ring and/or the N-10 position, are less active and are deactivated during the course of the reaction. Moreover, flavins that are alkylated at the N-1 position instead of the N-10 position and having either no substituents or electron-withdrawing groups on the aromatic ring, remain the most active and stable.

Journal Article↗
PubMed1994

A nomenclature for the mammalian flavin-containing monooxygenase gene family based on amino acid sequence identities.

A nomenclature based on comparisons of amino acid sequences is proposed for the members of the mammalian flavin-containing monooxygenase (FMO) gene family. This nomenclature is based on evidence of a single gene family composed of five genes. The percentage identities of the amino acid sequences of the five known forms of mammalian FMO are between 52 and 57% in rabbit and between 50 and 58% across species lines. The identities of all orthologs are greater than 82%. There is no evidence for multiple, highly related forms of the enzyme or for more than one mammalian FMO gene family. In the proposed system, the mammalian flavin-containing monooxygenase gene family is designated as "FMO" and the individual genes are distinguished by an Arabic numeral. The FMOs known as the "liver" and "lung" enzymes become FMO1 and FMO2, and the more recently described forms of the enzymes become FMO3, FMO4, and FMO5. Human FMO gene designations, FMO1 and FMO3, remain unchanged, but the gene designated FMO2 becomes FMO4. Following convention, the genes and cDNA designations will be italicized and the mRNA and protein designations will be nonitalicized. The purpose of the proposed nomenclature is to provide for the unambiguous identification of orthologous forms of mammalian FMOs, regardless of the species or tissue in question. The proposed classification considers only members of the mammalian flavin-containing monooxygenase gene family and has no bearing on the generally accepted definition of a multisubstrate flavin-containing monooxygenase.

Amino Acid Sequence↗