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PubMedDate not supplied

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.

Binding Sites↗
PubMed1994

alpha/beta barrel evolution and the modular assembly of enzymes: emerging trends in the flavin oxidase/dehydrogenase family.

Alpha/beta barrels have an ill-defined origin. Evidence exists which favours their divergent evolution from a common ancestral barrel and convergent evolution to a stable fold. However, recent sequence and structural information for the flavin oxidase/dehydrogenase family of barrel enzymes indicate that sub-families of alpha/beta barrels have evolved divergently. The modular fusion of barrel domains with core structures from other gene families has also contributed to the evolution of related but catalytically distinct enzyme molecules within each sub-family of the flavin oxidases/dehydrogenases. An analysis of the structures and sequences of the flavin oxidases/dehydrogenases has now enabled studies focusing on the evolutionary origins and modular assembly of this important family of proteins to be initiated.

Amino Acid Sequence↗
PubMed2005

Exploring the mechanisms of molecular recognition by flavins.

The design and development of chemical models for enzymes depends on the fundamental principles observed in biological systems. Molecular recognition of flavins by various receptors has attracted attention due to their applications as chemical models for flavoenzymes. The area of molecular recognition is being investigated through research at the interface of chemistry and biology. In this review, the literature has been surveyed to provide comprehensive coverage of the synthetic methodology of different flavins and receptors and their molecular-recognition studies. Various applications of flavin-receptor complexes have also been highlighted.

Flavins↗
PubMed2004

Preparation and redox properties of N,N,N-1,3,5-trialkylated flavin derivatives and their activity as redox catalysts.

Eight different flavin derivatives have been synthesized and the electronic effects of substituents in various positions on the flavin redox chemistry were investigated. The redox potentials of the flavins, determined by cyclic voltammetry, correlated with their efficiency as catalysts in the H2O2 oxidation of methyl p-tolyl sulfide. Introduction of electron-withdrawing groups increased the stability of the reduced catalyst precursor.

Alkylation↗
PubMed1992

Granule-associated flavin adenine dinucleotide (FAD) is responsible for eosinophil autofluorescence.

Unstained human eosinophils exhibit marked autofluorescence in comparison to other leukocytes due to a granule-associated fluorescent substance. Fluorescence spectroscopy of granule extracts reveals excitation maxima at approximately 380 and approximately 450 nm with a single emission at approximately 520, characteristic of flavins. The fluorescent material from eosinophil granule extracts was characterized by fluorescence, high-performance liquid chromatographic, and enzymatic analyses. First, acidification to pH 2.6 resulted in increased fluorescence, indicative of flavin adenine dinucleotide (FAD). Second, because flavin mononucleotide (FMN) and riboflavin cannot be distinguished by acidification, high-performance liquid chromatography was performed and revealed a predominance of FAD and smaller amounts (< 15%) of both FMN and riboflavin. Third, the presence of FAD was clearly demonstrated by reconstitution of the activity of D-amino acid oxidase, a FAD-dependent enzyme, when granule extracts were added to the apoenzyme. Thus, we have identified FAD as the predominant fluorophore in eosinophil granules. The small amounts of FMN and riboflavin detected may result from the hydrolysis of FAD under the acidic conditions of granule extraction. Because fluorescent material is deposited onto target cells by eosinophils, it is possible that granule-associated flavoproteins may act as a source of hydrogen peroxide and/or superoxide, which, in conjunction with eosinophil peroxidase, could yield potent cytotoxic agents.

Chromatography, High Pressure Liquid↗
PubMed1978

Flavin-catalyzed photooxidation of an acridan drug: a reinvestigation.

Anaerobic irradiation of flavin derivatives (IIalpha and IIbeta) in the presence of 2-chloro-9-(3-dimethylaminopropyl)acridan phosphate (Ialpha) resulted in the reduction of the flavins to IValpha and IVbeta and oxidation of an equimolar amount of Ialpha to IIIalpha. This photoreduction occurred from the first excited triplet state of IIalpha and IIbeta and proceeded via a covalent intermediate (V) between flavin and the acridan derivative. If the N-10 of the acridan was blocked by a methyl group, e.g., Ic, V could be observed spectrophotometrically at -40 degrees, decomposing homolytically on warming. With N-10 unsubstituted acridan derivatives, e.g., Ialpha and Ibeta, V could not be observed because of fast heterolytic decomposition, yielding IValpha, IVbeta, and the acridine compounds IIIalpha and IIIbeta. All photoreactions showed a kinetic isotope effect between 1.50 and 2.20 when position 9 of the acridan compounds was substituted by deuterium instead of hydrogen.

Acridines↗
PubMed1997

Identification of a reactive cysteine in the flavin-binding domain of Rhodotorula gracilis D-amino acid oxidase.

The holoenzyme form of Rhodotorula gracilis D-amino acid oxidase, an 80-kDa homodimer, reacted only to a limited extent with general thiol reagents (2,2'-dithiodipyridine, 5,5'-dithiobis(2-nitrobenzoic acid), and N-[7-(dimethylamino)-4-methylcoumarinyl]maleimide) (60% residual activity), whereas the monomeric apoprotein was completely inactivated and denatured by these reagents. To investigate the presence of thiol residue(s) in the active site of the enzyme, the apoprotein was reconstituted with the 8-(methylsulfonyl)-FAD chemical-affinity probe. Competitive inhibition between this analogue and FAD for apoprotein binding was observed. The covalent attachment of the flavin analogue to the apoprotein was complete after approximately 20 h of incubation and the flavinylated enzyme, containing 8-(cysteinyl)-FAD, was monomeric and inactive. After HPLC isolation of the flavin-labeled tryptic peptides, Cys208 was identified as the only cysteine to react with the FAD analogue. These results show that a single cysteine of R. gracilis D-amino acid oxidase reacts with the flavin analogue and that this is located near or at the FAD-binding domain.

Affinity Labels↗
PubMed1993

Paradoxical stabilization of the neutral flavin semiquinone of xanthine dehydrogenase at high pH.

The pH dependence of the behavior of chicken liver xanthine dehydrogenase in the course of reductive titrations with sodium dithionite has been examined. Below pH 8.5, the behavior of xanthine dehydrogenase is similar to that of the much better understood milk xanthine oxidase, with the amount of neutral semiquinone accumulating transiently in the course of the titration increasing somewhat as the pH decreases. At pH 10, however, an anomalously large accumulation of the neutral semiquinone is observed by both UV/visible and EPR spectroscopy. Treatment of xanthine dehydrogenase with the thiol reagent iodoacetamide significantly diminishes the ability of the enzyme to stabilize the neutral flavin semiquinone at high pH. These data are consistent with the presence of a protein thiol in the immediate vicinity of the flavin, whose ionization above pH 8.5 results in thermodynamic stabilization of the neutral flavin semiquinone over the anionic form.

Animals↗
PubMed2000

A mutant sarcosine oxidase in which activity depends on flavin adenine dinucleotide.

The covalent flavin attachment site in the Arthrobacter sarcosine oxidase (cysteine at position 318) was replaced with serine, and the mutational effect of C318S was analyzed. Wild type and C318S with a C-terminal 6-histidine tag were constructed and homogeneously purified by the single step. The covalently binding to flavin was not essential to the enzyme activity because the C318S mutant exhibited extremely weak activity. Moreover, the activity of the mutant was recovered in the presence of flavin adenine dinucleotide (FAD), and significantly increased as the concentration of FAD increased. This dependence of the mutant on FAD indicates that the noncovalent binding of free FAD to the mutant enzyme is reversible.

Amino Acid Sequence↗
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

Fluorescence polarization in a planar array of pigment molecules: theoretical treatment and application to flavins incorporated into artificial membranes.

A quantitative fluorescence polarization theory of molecules bound to two-dimensional plane layers has been developed when the electronic transition moments of absorption and emission are parallel within the fluorescent molecules. The transition moments are assumed to be in preferred orientation with respect to the normal to the plane and to be randomly oriented within the plane (rotational symmetry with the normal as axis of symmetry). Three basic model distributions of transition moments are investigated quantitatively. These model distributions represent a simplification but in most cases may be expected to describe reality with sufficient accuracy. For all distributions, two cases of different mobility of molecules are treated: (a) the lifetime of fluorescence is small compared with the characteristic relaxation time of the distribution, and (b) the lifetime of fluorescence is long, so that a complete reorientation of transition moments during the excited state can take place. From the quantitative calculations four characteristic quantities are derived, which are appropriate for the analysis of experimental data. Experiments are carried out with phosphatidylcholine bilayer membranes which contain three differently substituted amphiphilic flavins. All three flavins yield similar data. Their analyses predict free and fast mobility of the flavin chromophore.

Flavins↗
PubMed1995

Use of flavin photochemistry to probe intraprotein and interprotein electron transfer mechanisms.

Photoexcitation of flavin analogs generates the lowest triplet state (via intersystem crossing from the fist excited singlet state) in the nanosecond time domain and with high quantum efficiency. The triplet, being a strong oxidant, can abstract a hydrogen atom (or an electron) from a reduced donor in a diffusion-controlled reaction. If the donor is a redox protein, the oxidation process can be used to initiate an electron transfer sequence involving either intramolecular or intermolecular reactions. If the donor is an organic compound such as EDTA, the neutral flavin semiquinone will be produced by H atom abstraction; this is a strong reductant and can subsequently transfer a hydrogen atom (or an electron) to an oxidized redox protein, thereby again initiating a sequence of intramolecular or intermolecular processes. If flavin photoexcitation is accomplished using a pulsed light source, the initiation of these protein electron transfer reactions can be made to occur in the nanosecond to microsecond time domain, and the sequence of events can be followed by time-resolved spectrophotometry to obtain rate constants and thus mechanistic information. The present paper describes this technology, and selected examples of its use in the investigation of redox protein mechanisms are given.

Cytochromes↗
PubMed1976

Magnetic studies of Chromatium flavocytochrome C552. A mechanism for heme-flavin interaction.

Electron paramagnetic resonance and magnetic susceptibility studies of Chromatium flavocytochrome C552 and its diheme flavin-free subunit at temperatures below 45 degrees K are reported. The results show that in the intact protein and the subunit the two low-spin (S = 1/2) heme irons are distinguishable, giving rise to separate EPR signals. In the intact protein only, one of the heme irons exists in two different low spin environments in the pH range 5.5 to 10.5, while the other remains in a constant environment. Factors influencing the variable heme iron environment also influence flavin reactivity, indicating the existence of a mechanism for heme-flavin interaction.

Binding Sites↗
PubMed1996

Phage antibodies against an unstable hapten: oxygen sensitive reduced flavin.

It is difficult to raise antibodies against haptens and antigens that are unstable under the physiological conditions of the serum. Here we have used a phage antibody library to isolate antibody fragments against oxygen sensitive reduced flavin, by selection of the phage under anaerobic and reducing conditions at pH 5 and a pre-elution step with the oxidized flavin. The binding of the reduced hapten to one of the antibody fragments was characterised by time-resolved polarised fluorescence, and shown to be highly specific for the reduced flavin.

Amino Acid Sequence↗
PubMed1994

Purification and semienzymic synthesis of flavin adenine dinucleotide-3'-phosphate.

Nonradioactive immunoassays incorporating an element of amplification in their detection system require the use of components that are highly purified. Flavin adenine dinucleotide-3'-phosphate (FADP) is the primary substrate used in such an amplification assay. For incorporation into a simple, single-pot assay system, the concentration of contaminating flavin adenine dinucleotide (a prosthetic group for the enzyme D-aminoacid oxidase used in the amplification cascade assay) in this primary substrate must be minimized to achieve maximum sensitivity. Production of the substrate to a high degree of purity has been achieved using apo-glucose oxidase to specifically remove contaminating flavin adenine dinucleotide from solution and hydrolysis of a cyclic intermediate as a final production protocol by ribonuclease T2 to give the product in high yield. The use of continuous ultrafiltration reactors at each stage is described and compared to a final production step utilizing immobilized ribonuclease T2. These reactors allow large volumes of material to be handled and assist in the scale-up of these processes. The suitability of each protocol is assessed for the commercial production of FADP.

Apoproteins↗
PubMed1979

The pH dependence of the reactions of flavin triplet states with amino acids. A laser flash photolysis study.

The pH dependence of the rate constants of reaction of several amino acids with the triplet states of flavin mononucleotide in aqueous solution has been determined. In addition, the relative contributions of hydrogen atom transfer, electron transfer and physical deactivation to the overall process of triplet quenching by amino acids have been estimated. Analogous experiments to those with amino acids were carried out with EDTA as the substrate. The results indicate that the flavin triplet state abstracts an electron from EDTA but does not form an excited state flavin-EDTA complex as suggested in a previous study.

Amino Acids↗
PubMed1988

Characterization of a new flavin metabolite from human urine.

A new flavin metabolite comprising approximately 5% of the total flavin of human urine was isolated and characterized using absorption and fluorescence spectra, oxidation-reduction and hydrolysis data, and ninhydrin reactions. The flavin is a derivative associated with a peptide residue in ester linkage from an amino acid carboxyl to the ribityl chain of riboflavin, probably at the 5'-terminus.

Adult↗
PubMed2004

Human neuronal nitric oxide synthase can catalyze one-electron reduction of adriamycin: role of flavin domain.

We have analyzed the mechanism of one-electron reduction of adriamycin (Adr) using recombinant full-length human neuronal nitric-oxide synthase and its flavin domains. Both enzymes catalyzed aerobic NADPH oxidation in the presence of Adr. Calcium/calmodulin (Ca(2+)/CaM) stimulated the NADPH oxidation of Adr. In the presence or absence of Ca(2+)/CaM, the flavin semiquinone radical species were major intermediates observed during the oxidation of the reduced enzyme by Adr. The FAD-NADPH binding domain did not significantly catalyze the reduction of Adr. Neither the FAD semiquinone (FADH*) nor the air-stable semiquinone (FAD-FMNH*) reacted rapidly with Adr. These data indicate that the fully reduced species of FMN (FMNH(2)) donates one electron to Adr, and that the rate of Adr reduction is stimulated by a rapid electron exchange between the two flavins in the presence of Ca(2+)/CaM. Based on these findings, we propose a role for the FAD-FMN pair in the one-electron reduction of Adr.

Calcium↗