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Results for “Flavin Mononucleotide”

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At least 19 recordsLinked to original sources

PubMed1975

The thermochemical characterization of sodium dithionite, flavin mononucleotide, flavin-adenine dinucleotide and methyl and benzyl viologens as low-potential reductants for biological systems.

The heat of reaction (deltaH) of Fe(CN)63-, Methyl Viologen, FMN and FAD with S2O42- in aqueous buffer solutions was measured calorimetrically. In addition deltaH values for reduction of Fe(CN)63-, FMN and FAD by reduced Methyl Viologen were determined. The resulting calorimetric data and corresponding E0 values were combined to yield thermodynamic data for these simple reducing agents in a form useful for applications to biological reactions. Thermodynamic data for the reduction of spinach ferredoxin are also presented.

Calorimetry↗

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↗

PubMed1978

Metabolism of injected flavins studied by using double-labeled [14C]flavin adenine dinucleotide and [14C, 32P]flavin mononucleotide.

The metabolism of flavins in mouse was studied with [F-(2)-14C, A-(2,8)-14C]FAD and [F-(2)-14C, 32P]FMN. Ninety minutes after injection, radioactive isoalloxazine nucleus of double-labeled FAD was markedly incorporated into FAD, FMN and riboflavin in the liver, whereas a small amount of radioactive adenine nucleus of double-labeled FAD was found in FAD in the liver. In the case of FMN, radioactive isoalloxazine nucleus of double-labeled FMN was markedly incorporated into FAD, FMN and riboflavin in the liver, whereas only a minute amount of radioactive phosphorus was incorporated into FMN and FAD in the same organ. These results indicate that FMN and FAD injected are rapidly hydrolyzed and resynthesized in animal body.

Animals↗

PubMed1975

Flavin mononucleotide reductase of luminous bacteria.

NAD(P)H: FMN oxidoreductase (flavin reductase) couples in vitro to bacterial luciferase. This reductase, which is also postulated to supply reduced flavin mononucleotide in vivo as a substrate for the bioluminescent reaction, has been partially purified and characterized from two species of luminous bacterial. From Photobacterium fischeri the enzyme has a M. W. determined by Sephadex gel filtration, of 43,000 and may have a subunit structure. The turnover number at 20 degrees C, based on a purity estimate of 20 percent, is 1.7 times 10-4 moles of NADH oxidized per min per mole of reductase. The reductase isolated from Beneckea harveyi has an apparent molecular weight of 23,000; its purity was too low to permit estimation of specific activity. Using a spectrophotometric assay at 340 nm with the P. fischeri reductase, both NADH (Km, 8 times 10-5 M) and NADPH (Km, 4 times 10-4 M) were enzymatically oxidized, the Vmax with NADH being approximately twice that of NADPH. Of the flavins tested in this assay, only FMN (Km, 7.3 times 10-5 M) and FAD (Km, 1.4 times 10-4 M) were effective, FMN having a Vmax three times that of FAD. In the coupled assay, i.e., measuring the bioluminescence intensity of the reaction with added luciferase, the optimum FMN concentration was nearly 100 times less than in the spectrophotometric assay. The studies reported suggest the existence of a functional reductase-luciferase complex.

Dicumarol↗

PubMed1975

Inhibitory effect of flavin mononucleotide on the hemolysis of rabbit erythrocytes by staphylococcal alpha-toxin.

Flavin mononucleotide diminished the hemolytic action of staphylococcal alpha-toxin on rabbit erythrocytes by competitive inhibition, probably by its interaction with the alpha-toxin binding sites on the cell membrane.

Animals↗

PubMed1991

Interaction of flavin mononucleotide with dimeric and tetrameric forms of muscle phosphorylase beta.

Interaction of flavin mononucleotide (FMN) with dimeric and tetrameric forms of rabbit muscle glycogen phosphorylase beta has been studied under the conditions when allosteric activator binding sites are saturated by AMP (1 mM AMP; pH 6.8; 17 degrees C). Simultaneous use of schlieren optical system and photoelectric scanning absorption optical system of analytical ultracentrifuge Spinco, model E, makes it possible to register the oligomeric state of the enzyme and calculate the degree of saturation of individual oligomeric enzyme forms by FMN. The apparent association constant for the equilibrium dimer in equilibrium with tetramer decreased with increasing FMN concentration. The microscopic dissociation constants for the complexes of dimeric and tetrameric forms of glycogen phosphorylase beta with FMN have been found to be equal to 10 and 79 microM, respectively.

Adenosine Monophosphate↗

PubMed1990

Analysis of the reduction of nitroxides by flavin mononucleotide.

This article describes a simple method to prepare hydroxylamines from nitroxides by photo-activated flavin mononucleotide. The half-time of reduction varied from 2 to 38.4 s for a series of nitroxides. For most nitroxides short exposures to light (min) were sufficient to produce significant amounts of hydroxylamine; longer periods of exposure increased the yields of other products. Proxyl (2,2,5-trimethyl-5-alkylpyrrolidine-N-oxyl) nitroxides were unusually reactive with a much higher yield of products which could not be reoxidized by ferricyanide to the nitroxides. Optimum conditions for reversible reduction depend on the nitroxide and the amounts of other reducible substances such as oxygen and ferricyanide that may be present.

Cyclic N-Oxides↗

PubMed1992

Fatty acid-enhanced binding of flavin mononucleotide to bacterial luciferase measured by steady-state fluorescence.

Bacterial luciferase catalyzes the oxidation of reduced flavin mononucleotide and tetradecanal resulting in the emission of light. We have investigated the interactions of a recombinant luciferase from a terrestrial bacterium Xenorhabdus luminescens with the reaction products, FMN and myristic acid, using steady-state fluorescence spectroscopy. Quenching of the intrinsic fluorescence and FMN fluorescence on binding of FMN to luciferase was found to be greatly stimulated in the presence of myristic acid, corresponding to a reduction of more than 30-fold in the FMN dissociation constant of the enzyme. In addition, the FMN-luciferase complex exhibits distinct fatty acid-dependent fluorescence properties. These results indicate that luciferase forms a ternary complex with FMN and myristic acid with a significantly different conformation from that of the binary FMN-luciferase complex.

Enterobacteriaceae↗

PubMed1991

Spinach glycolate oxidase and yeast flavocytochrome b2 are structurally homologous and evolutionarily related enzymes with distinctly different function and flavin mononucleotide binding.

A comparison of the three-dimensional structures of the flavin mononucleotide (FMN)-dependent enzymes glycolate oxidase, flavocytochrome b2, and trimethylamine dehydrogenase is presented. Their flavin-binding domains all have the same structural motif, the 8-fold beta/alpha-barrel domain, which is also present in a large number of other enzymes. FMN is bound in a similar fashion in all three enzymes. The binding site is at the carboxyl-terminal end of the eight beta-strands of the barrel where the active site is invariably found in this type of domain structure. The similarity of the structures of glycolate oxidase and flavocytochrome b2 extends to the loop regions and even outside the beta/alpha-barrels with a root mean square deviation of 0.93 A for 311 superimposed C alpha-atoms and with a sequence identity of 37%. A detailed analysis of their active sites shows, however, that the orientation of FMN is significantly different in the two structures due to different conformations of residues in the end of strand one. Thus, in flavocytochrome b2 a hydrogen bond is formed between the FMN N-5 position and the main chain amide of Ala-198, while in glycolate oxidase, the ring system is tilted away from the strand, creating a pocket on the re-side of the FMN ring where a water molecule is bound. Model building shows that this site could accommodate the hydroperoxide moiety of a FMN-4a-hydroperoxide intermediate. Thus, in the course of evolution, a few mutations in, and close to, the active sites have fine tuned these enzymes to exert their specific functions as an oxidase or transferase, respectively.

Alcohol Oxidoreductases↗

PubMed1976

Effects of pressure upon the fluorescence of the riboflavin binding protein and its flavin mononucleotide complex.

The effect of pressure in the range of 10(-3)-10 kbars upon the ultraviolet fluorescence of the riboflavin binding protein and the fluorescences of its complex with flavin mononucleotide has been studied. The fluorescence spectrum of the isolated protein showed a reversible red shift of 12nm (1000 cm-1) at high pressure, indicating the reversible exposure of the tryptophan to solvent. From the pressure dependence of the visible fluorescence of the protein-flavin complex in the region of 1-4 kbars the volume change in dissociation of the protein-ligand complex was estimated to be +3.3ml/mol. A very sharp increase in fluorescence-up to 30-fold of the low-pressure value-takes place in the region 5-8 kbars. This increase is due to release of the flavin from the complex and is assigned to pressure denaturation of the protein. The midpoint, rho 1/2, of this transition was found at 6.5 kbars and the change in volume, delta, in the reaction (native-to-denatured) was calculated to be -74ml/mol. Addition of up to 30% methanol results in a progressive decrease both in delta and rho 1/2, in agreement with the concept that hydrophobic bonding stabilizes the native structure.

Bromosuccinimide↗

PubMed1992

Crystal structure of oxidized flavodoxin from a red alga Chondrus crispus refined at 1.8 A resolution. Description of the flavin mononucleotide binding site.

In order to describe the detailed conformation of the oxidized flavodoxin from a eukaryotic red alga, Chondrus crispus, the crystal structure has been refined by a restrained least-squares method. The crystallographic R factor is 0.168 for 13,899 reflections with F greater than 2 sigma F between 6.0 and 1.8 A resolution. The refined model includes 173 amino acid residues, flavin mononucleotide (FMN) and 110 water molecules. The root-mean-square deviation in bond lengths from ideal values is 0.015 A, and the mean co-ordinate error is estimated to be 0.2 A. The FMN is located at the periphery of the molecule. The orientation of the isoalloxazine ring is such that the C-7 and C-8 methyl groups are exposed to solvent and the pyrimidine moiety is buried in the protein. Three peptide segments, T8-T13, T55-T58 and D94-C103, are involved in FMN binding. The first segment of T8-T13 enfolds the phosphate group of the FMN. The three oxygen atoms in the phosphate group form extensive hydrogen bonds with amide groups of the main chain and the O gamma atoms of the side-chains in this segment. T55 O and W56 N epsilon 1 in the second segment form hydrogen bonds with O-2 in the ribityl moiety and one of the oxygen atoms in the phosphate group, respectively. The O gamma H of T58 forms a hydrogen bond with the N-5 atom in the isoalloxazine ring, which is expected to be protonated in the semiquinone form. The third segment is in contact with the isoalloxazine ring. It appears that the hydrogen bond acceptor of the NH of Asp94 in the third segment is O-2 rather than N-1 in the isoalloxazine ring. The isoalloxazine ring is flanked by the side-chains of Trp56 and Tyr98; it forms an angle of 38 degrees with the indole ring of Trp56 and is almost parallel to the benzene ring of Tyr98. The environment of the phosphate group is conserved as in other flavodoxins whereas that of the isoalloxazine ring differs. The relationship between the hydrogen bond to the N-5 in the ring and the redox potential for the oxidized/semiquinone couple is discussed.

Amino Acid Sequence↗

PubMed1975

Preparation of the lactate oxidase apoenzyme and studies on the binding of flavin mononucleotide to the apoenzyme.

1. Lactate oxidase from Mycobacterium smegmatis is completely resolved into free flavin and apoenzyme by treatment with acid (NH4)2SO4. 2. Reconstitution involves rapid binding of FMN, but the recovery of enzyme activity was slower and appeared to be biphasic. 3. The preparation of the holoenzyme obtained differs from the native enzyme in specific activity, extinction coefficients and mobility on disc-gel electrophoresis. 4. Dialysis of this reconstituted enzyme in 0.1 M-sodium phosphate buffer, pH 7.0, at 0 degrees C for 1 week yields a preparation which closely resembles the native enzyme.

Alcohol Oxidoreductases↗

PubMed1975