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PubMed1991

A new reducing agent of flavins and its application to the assay of bacterial luciferase.

Copper(I) plus ethylenediaminetetraacetic acid, Cu(I)-EDTA, reduces flavins. Using Cu(I)-EDTA reduction of riboflavin 5'-phosphate, a new assay method for bacterial luciferase is established. In previous assay methods, flavin and aldehyde substrates for luciferase have been routinely added to luciferase at different times; the new assay permits aldehyde and Cu(I)-reduced flavin to be injected simultaneously into air-saturated buffer containing luciferase. In the new assay method using Cu(I), bovine serum albumin is not required, the initial emission intensity is more than twice and the overall yield is about twice of that in the so-called standard assay for luciferase.

Edetic Acid↗
PubMed1992

Flavin-sensitized photochemically induced dynamic nuclear polarization detection of pyrimidine dimer radicals.

A photochemically induced dynamic nuclear polarization (photo-CIDNP) study of carboxymethyllumiflavin-sensitized splitting of pyrimidine dimers has been carried out. In aqueous solution at high pH, an emission signal (delta 3.9 ppm) was observed from the dimer C(6)- and C(6')-protons of an N(1), N(1')-trimethylene-bridged thymine dimer (1). The dimer photo-CIDNP signal was seen only above pD 11.6 and was most intense at pD 12.9. Also observed were weak enhanced absorption signals from the product of splitting, trimethylenebis(thymine) (delta 1.7 and 7.2 ppm). In contrast, cis, syn-thymine dimer (3) gave no photo-CIDNP signals from the dimer. An enhanced absorption at 1.8 ppm, however, due to the product of splitting (thymine) was observed. It was found that dimer 1 and, to a lesser extent, dimer 3 quenched flavin fluorescence. An N(3),N(3')-dimethylated derivative of 1, however, failed to quench flavin fluorescence. Comparison of the pD profile of the dimer photo-CIDNP signal to the pKa values for thymidine dimer suggested that principally the dideprotonated dimer undergoes electron abstraction by the excited flavin.

Flavins↗
PubMed1999

Photosensitization of singlet oxygen formation by pterins and flavins. Time-resolved studies of oxygen phosphorescence under laser excitation.

To elucidate the biochemical roles of singlet molecular oxygen (1(O2)) in the light-dependent reactions photosensitized by biological blue-light photoreceptors, time-resolved measurements of photosensitized 1O2 phosphorescence (1270 nm) were performed in air-saturated aqueous ((D2)O) solutions of pterins (2-amino-4-hydroxy-6,7-dimethylpteridine (DMP) and 2-amino-4-hydroxy-6-tetrahydroxybutyl-(D-arabo)pteridine (TOP)) and flavins (riboflavin and flavin mononucleotide (FMN)) under excitation with nitrogen laser (337.1 nm) pulses. The 1(O2) quantum yields were found to be 0.16, 0.20, 0.50, and 0.50 for DMP, TOP, riboflavin, and FMN, respectively. The data indicate that pterins and flavins are rather efficient photosensitizers of 1(O2) production that might be important for their photobiological functions.

Flavins↗
PubMedDate not supplied

[Increased flavin synthesis in yeasts utilizing hydrocarbons].

Comparative studies of the process of possible overproduction of flavins by cultures with different flavinogenous activity grown on media with hydrocarbons and glucose have been carried out. The strains with a high flavinogenous activity, Candida guilliermondii and Torulopsis famata O-3, produced more flavins on media containing hydrocarbons than the cultures with a low flavinogenous activity. At a high content of iron in the medium, which is unfavourable for overproduction of riboflavin the rate of flavinogenesis is higher on hydrocarbons than on sugars, especially on alkanes with a longer chain in the strain O-3. Under the conditions of iron deficiency, the activity of flavinogenesis is higher on glucose in the case of both cultures. Iron deficiency in media containing hydrocarbons and their oxygenated derivative (cetyl alcohol, palmitic and acetic acids) has no such effect on the production of flavin by T. famata O-3 as in the glucose containing media. On media with ethanol, overproduction of the vitamin by the strain O-3 obeys the same relationships as on media with glucose. Possible factors that may have effect on the elevated synthesis are discussed.

Alkanes↗
PubMed1975

Interaction of flavins with electron-rich metals.

A complex of the electron-rich ion Cu(I) with the flavoquinone analogue 10-methylisoalloxazine has been synthesized and characterized by x-ray methods. The complex is unstable to oxygen. It is black-green in color, in contrast with the bright yellow, orange, or orange-brown crystalline complexes of 10-methylisoalloxazine or riboflavin with Cu(II), Ag(I), and Pb(II). These results are indicative of strong perturbation of the flavin electronic structure by the Cu(I) ion and suggest that this complex is a reasonable model for incipient transfer of an electron from a reduced metal to flavoquinone. the crystal structure is orthorhombic, Pna2-1, with unit cell constants a = 31.24(1) (figures in parentheses are estimated standard deviations), b = 12.862(4), c = 6.239(2) A, Pobs = 1.76 g per cm-3 and Pcalc = 1.77 g per cm-3 for Z = 4 and asymmetric formula CuClO4-2(C11H8N4O2). HCOOH. The final R factor based on 1250 counter-measured data is 8.8%. The 2 independent 10-methylisoalloxazine molecules, A and B, bind strongly to the cuprous ion throug N(5) of each flavin. The copper is approximately linearly coordinated with an N-Cu-N angle of 153(1) degrees, and Cu-N(5) distances of 1.94(2) A and 1.92(2) A. The next nearest atoms to Cu are the O(4) oxygens of each flavin, forming weak bonds with distances Cu-O(4) = 2.27(2) A and 2.21(2) A for molecules A and B. The dihedral angle between the 2 10-methylisoalloxazine molecules is 65.4 degrees.

Binding Sites↗
PubMed1992

Active-site structural comparison of streptococcal NADH peroxidase and NADH oxidase. Reconstitution with artificial flavins.

The apoproteins of the streptococcal NADH peroxidase (H2O2----2H2O) and NADH oxidase (O2----2H2O) stabilize the neutral forms of 6-hydroxy- and 6-mercapto-FAD, respectively. The redox behavior of the 6-hydroxy-FAD peroxidase closely mimics that of the native enzyme with both dithionite and NADH. Both oxidase and peroxidase preferentially stabilize the N(1)-protonated p-quinonoid species of 8-mercapto-FAD, and the 8-position of the bound flavin is accessible to solvent in both proteins. The 8-mercapto-FAD peroxidase yields an EH2 spectrum on reduction virtually identical to that seen with 8-mercapto-FAD glutathione reductase, but no distinct EH2.NADH form appears. The dramatic decreases in reactivity at the flavin 2- and 4-positions for both the peroxidase and the oxidase, assessed with the reconstituted 2- and 4-thio-FAD enzymes, suggest that these positions are buried by elements of both protein structures. Furthermore, reconstitution of the peroxidase with the higher potential 2- and 4-thioflavins yields enzyme forms which are fully reducible with 1.4 eq of NADH/FAD, giving rise to stable thio-FADH2.NAD+ complexes. This behavior closely mimics that of the native NADH oxidase and provides further evidence supporting the hypothesis that a major functional distinction between the two structurally related proteins is determined by the redox potential and/or NADH reactivity of the bound flavin coenzyme.

Binding Sites↗
PubMed1990

[Purification of flavin-containing liver polyamine oxidase and luminescent properties of the ferment].

A flavin-containing polyamine oxidase was isolated in an electrophoretically homogeneous state from cattle liver cytosol. The molecular mass, subunit composition and flavin content of the enzyme were determined; flavin, was shown to be covalently bound to the protein fragment of the polyamine oxidase molecule. Some optical and luminescent properties of the native and denatured enzyme were investigated. Denaturation and quenching were found to affect the luminescent properties of polyamine oxidase.

Animals↗
PubMedDate not supplied

[Supersynthesis of flavins in microorganisms and its molecular mechanism (review of the literature)].

The present review discusses the significance of fundamental research into regulation of flavin biosynthesis for development of the knowledge about mechanisms of overproduction of these compounds and their manufacturing. The pathways of riboflavin, FMN and FAD biosyntheses and their regulation in some bacteria, yeasts and fungi are considered, as well as the recent advances in flavin biotechnology. The modern trends in microbial and enzymatic production of flavins are discussed.

Bacteria↗
PubMed1988

Phagocyte NADPH-oxidase. Studies with flavin analogues as active site probes in triton X-100-solubilized preparations.

NADPH-oxidase of stimulated human neutrophil membranes was solubilized in Triton X-100 and activity reconstituted with FAD, 8-F-FAD, 8-phenyl-S-FAD, and 8-S-FAD. The enzyme had similar affinities for all the flavins with Km values in the 60-80 nM range. Vmax was found to increase 4-fold with increasing redox midpoint potential of the flavin. 8-F-FAD reconstituted with the enzyme was reactive toward thiophenol, suggesting exposure of the 8-position to solvent, a finding supported by unsuccessful attempts to label the enzyme with the photoaffinity probe 8-N3-[32P]FAD. Solubilized oxidase stabilized the red thiolate form of 8-S-FAD, a characteristic of flavoproteins of the dehydrogenase/electron transferase classes which stabilize the blue neutral form of the flavin semiquinone radical.

Cell Membrane↗
PubMed1983

Isolation and characterization of the protease-activated form of pyruvate oxidase. Evidence for a conformational change in the environment of the flavin prosthetic group.

Pyruvate oxidase is a tetrameric enzyme consisting of four identical subunits. The specific activity of the enzyme may be increased more than 20-fold by limited proteolytic digestion by alpha-chymotrypsin in the presence of pyruvate and thiamin pyrophosphate. This "activation" phenomenon is due to the specific cleavage of an Mr = approximately 2000 peptide from each subunit. The Mr = 2000 "activation peptide" (alpha) may be readily separated from the activated enzyme by high performance liquid chromatography under nondenaturing conditions. The alpha peptide is not required to maintain the modified tetramer in the activated state. Cleavage of the alpha peptide from each monomer is directly correlated with a substantial change in the visible spectrum of the flavin, characteristic of a shift from a hydrophobic to a more hydrophilic environment. Proteolytic cleavage by alpha-chymotrypsin in the absence of thiamin pyrophosphate irreversibly inactivates the enzyme by cleavage at a different site, producing an Mr = approximately 9000 "inactivation peptide" (beta). The beta peptide remains noncovalently associated with the inactivated tetramer. Cleavage of the beta peptide does not alter the spectrum of the flavin, even though the beta peptide contains the alpha peptide sequence. These results suggest that cleavage and release of the alpha peptide opens up the flavin active site and may be directly responsible for the observed stimulation of enzymatic activity.

Chromatography, High Pressure Liquid↗
PubMedDate not supplied

[Biosynthesis of flavins and its regulation in the yeast Pichia guilliermondii].

The nature of riboflavin precursors was studied in the yeast Pichia guilliermondii. By means of mutants with blocked GMP-synthetase the purine precursors of riboflavin were shown to belong to guanylic compounds. Accumulation of 2,4,5-triamino-6-oxypyrimidine, 2,5-diamino-6-oxy-4-ribitylaminopyrimidine, 2,6-dioxy-5-amino-4-ribitylaminopyrimidine (DOARAP) and 6,7-dimethyl-8-ribityllumasine occurs in the riboflavin-deficient mutants divided into five biochemical groups. This fact evidences for identity of riboflavin precursors in the yeast P. guilliermondii and Saccharomyces cerevisiae. Synthesis of DOARAP by the washed off cells of the mutants with the blocked lumasine synthetase is strongly inhibited by riboflavin; cycloheximide in the absence of riboflavin has no effect on this process. Consequently, flavinogenesis in P. guilliermondii is regulated according to the type of negative feedback by means of retroinhibition mechanism. A change in the content of flavins in the cells has no effect on synthesis of riboflavin synthetase; at the same time iron deficiency in the cells evokes derepression of this enzyme. Incubation of the cells rich in iron with o-phenantroline or alpha, alpha'-dipyridyl also causes derepression of riboflavin synthetase which is inhibited by cycloheximide. A deficiency of hem in the mutants which need epsilon-aminolevulinic acid does not affect the riboflavinsynthetase activity of the cells. Evidently, in P. guilliermondii a certain form of nonheminic iron might take part in regulating synthesis of riboflavin synthetase and other enzymes participating in riboflavin biosynthesis. Riboflavin overproduction is established to require formation of purines de novo. With the absence of flavinogenesis enzymes derepression a genetic disturbance in regulation of purinic nucleotides biosynthesis results in stimulation of flavinogenesis. The properties were studied for 680 time purified riboflavinkinase from cells of P. guilliermondii as well as for three phosphatases possessing the optimum of the activity at pH 3.5, 5.5 and 8.6, which ARE ABLE OF HYDROLYSING FMN. A change in the content of flavins and iron in the cells has no effect on the activity of riboflavinkinase in this species. Evidently, the mechanisms of riboflavin and flavin nucleotides biosynthesis regulation would be different in P. guilliermondii.

2,2'-Dipyridyl↗
PubMed1993

Effect of flavins on the rate of proteolytic digestion of muscle glycogen phosphorylase b.

The kinetics of tryptic proteolysis of rabbit skeletal muscle phosphorylase b has been registered by the diminishing of protein fluorescence intensity at lambda = 335 nm (excitation at 290 nm) or by the disappearance of the enzyme activity (0.02 M Hepes buffer, pH 6.8, 37 degrees C). The first procedure showed that flavins (riboflavin, FMN, FAD) protected the enzyme against tryptic digestion. Microscopic dissociation constants for the complexes of phosphorylase b with riboflavin, FMN and FAD were calculated from dependences of the initial digestion rate on the flavin concentration. They where equal to 30 +/- 1, 15.8 +/- 0.2 and 36 +/- 1 microM, respectively. No influence of FMN on the rate of the tryptic hydrolysis of phosphorylase b was observed when using the second procedure (enzyme activity test). FMN completely prevents the formation of 69-, 81- and 85-kDa fragments during 20 min incubation of phosphorylase b with trypsin.

Animals↗
PubMed2000

The FMN-binding domain of cytochrome P450BM-3: resolution, reconstitution, and flavin analogue substitution.

Cytochrome P450BM-3 is a self-sufficient bacterial protein containing three naturally fused domains which bind either heme, FMN, or FAD. Resolution of protein and FMN from the isolated FMN-containing domain of cytochrome P450Betamicro-3 was accomplished using trichloroacetic acid. The apoprotein thus prepared was shown to rebind FMN to regenerate the original holoprotein as indicated by both spectroscopy and activity measurements. To better understand how the protein/flavin interaction might contribute to reactivity, the association process was studied in detail. Fluorescence quenching was used to measure a dissociation constant of the flavin-protein complex of 31 nM, comparable to FMN-containing proteins of similar reactivity and higher than that of flavodoxins. Stopped-flow kinetics were performed, and a multistep binding process was indicated, with an initial k(on) value of 1.72 x 10(5) M(-)(1) s(-)(1). Preparation of the apoprotein allowed substitution of flavin analogues for the native FMN cofactor using 8-chloro-FMN and 8-amino-FMN. Both were found to bind efficiently to the protein with only minor variations in affinity. Reductive titrations established that, as in the native FMN-containing FMN-binding domain, the 8-amino-FMN-substituted domain does not produce a stable one-electron-reduced species during titration with sodium dithionite. The 8-chloro-FMN-substituted domain, however, had sufficiently altered redox properties to form a stable red anionic semiquinone. The 8-chloro-FMN-substituted FMN-binding domain was shown in reconstituted systems to retain most of the cytochrome c reductase activity of the native domain but only a very small amount of palmitic acid hydroxylase activity. The 8-amino-FMN-substituted FMN-binding domain showed no palmitic acid hydroxylase activity and only 30% of the native cytochrome c reductase activity, demonstrating the importance of thermodynamics to the mechanism of this protein.

Anaerobiosis↗
PubMed2000

Functional interactions in cytochrome P450BM3. Evidence that NADP(H) binding controls redox potentials of the flavin cofactors.

NADP(H) binding is essential for fast electron transfer through the flavoprotein domain of the fusion protein P450BM3. Here we characterize the interaction of NADP(H) with the oxidized and partially reduced enzyme and the effect of this interaction on the redox properties of flavin cofactors and electron transfer. Measurements by three different approaches demonstrated a relatively low affinity of oxidized P450BM3 for NADP(+), with a K(d) of about 10 microM. NADPH binding is also relatively weak (K(d) approximately 10 microM), but the affinity increases manyfold upon hydride ion transfer so that the active 2-electron reduced enzyme binds NADP(+) with a K(d) in the submicromolar range. NADP(H) binding induces conformational changes of the protein as demonstrated by tryptophan fluorescence quenching. Fluorescence quenching indicated preferential binding of NADPH by oxidized P450BM3, while no catalytically competent binding with reduced P450BM3 could be detected. The hydride ion transfer step, as well as the interflavin electron transfer steps, is readily reversible, as demonstrated by a hydride ion exchange (transhydrogenase) reaction between NADPH and NADP(+) or their analogues. Experiments with FMN-free mutants demonstrated that FAD is the only flavin cofactor required for the transhydrogenase activity. The equilibrium constants of each electron transfer step of the flavoprotein domain during catalytic turnover have been calculated. The values obtained differ from those calculated from equilibrium redox potentials by as much as 2 orders of magnitude. The differences result from the enzyme's interaction with NADP(H).

Animals↗
PubMed1986

Absolute stereochemistry of flavins in enzyme-catalyzed reactions.

The 8-demethyl-8-hydroxy-5-deaza-5-carba analogues of FMN and FAD have been synthesized. Several apoproteins of flavoenzymes were successfully reconstituted with these analogues. This and further tests established that these analogues could serve as general probes for flavin stereospecificity in enzyme-catalyzed reactions. The method used by us involved stereoselective introduction of label on one enzyme combined with transfer to and analysis on a second enzyme. Using as a reference glutathione reductase from human erythrocytes for which the absolute stereochemistry of catalysis is known from X-ray studies [Pai, E. F., & Schulz, G. E. (1983) J. Biol. Chem. 258, 1752-1758], we were able to determine the absolute stereospecificities of other flavoenzymes. We found that glutathione reductase (NADPH), general acyl-CoA dehydrogenase (acyl-CoA), mercuric reductase (NADPH), thioredoxin reductase (NADPH), p-hydroxybenzoate hydroxylase (NADPH), melilotate hydroxylase (NADH), anthranilate hydroxylase (NADPH), and glucose oxidase (glucose) all use the re face of the flavin ring when interacting with the substrates given in parentheses.

Acyl-CoA Dehydrogenases↗
PubMed1995

Specificity of inhibition of muscle glycogen phosphorylase b by flavins.

The inhibition of glycogen phosphorylase b from rabbit skeletal muscles by the derivatives of riboflavin, FMN, FAD, and 2', 3', 4', 5'-tetraacetylriboflavin substituted in positions 6 and 8 of the isoalloxazine part of the flavin molecule is found to be cooperative (the Hill coefficient, h, exceeds 1.0). The modification of the flavin molecule slightly changes the value of the Hill coefficient, but results in the increase of the "half-saturation" concentration [I]0.5.

Animals↗
PubMed1994

Ferric reductases or flavin reductases?

Assimilation of iron by microorganisms requires the presence of ferric reductases which participate in the mobilization of iron from ferrisiderophores. The common structural and catalytic properties of these enzymes are described and shown to be identical to those of flavin reductases. This strongly suggests that, in general, the reduction of iron depends on reduced flavins provided by flavin reductases.

Bacterial Proteins↗
PubMed1992

Spectrophotometric measurement of flavin-containing monooxygenase activity in freshly isolated rat hepatocytes and their cultures.

The determination of the mixed function flavin-containing monooxygenase activity in rat liver and in hepatocytes and their cultures by spectrophotometric measurement of the oxygenation of methimazole is complicated by an inhibition caused by some of the reagents used during this method. Optimal conditions were determined for measuring this enzyme activity in microsomal preparations of rat liver and its hepatocytes. Optimal flavin-containing monooxygenase activities were obtained for measurements performed in a 0.25 M N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine-EDTA buffer at pH 8.7 and at a methimazole concentration of 2 mM. Data are also presented which show that no interferences caused by either cytochrome P450-dependent enzymes or by the reduction of methimazole disulfide by glutathione have to be taken into account when determining methimazole oxygenation. Finally, the above assay was also used to study flavin-containing monooxygenase activity in primary monolayer cultures of hepatocytes for 6 days.

Animals↗