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PubMed1998

Reaction of the NAD(P)H:flavin oxidoreductase from Escherichia coli with NADPH and riboflavin: identification of intermediates.

Flavin reductase catalyzes the reduction of free flavins by NAD(P)H. As isolated, Escherichia coli flavin reductase does not contain any flavin prosthetic group but accommodates both the reduced pyridine nucleotide and the flavin substrate in a ternary complex prior to oxidoreduction. The reduction of riboflavin by NADPH catalyzed by flavin reductase has been studied by static and rapid kinetics absorption spectroscopies. Static absorption spectroscopy experiments revealed that, in the presence of riboflavin and reduced pyridine nucleotide, flavin reductase stabilizes, although to a small extent, a charge-transfer complex of NADP+ and reduced riboflavin. In addition, reduction of riboflavin was found to be essentially irreversible. Rapid kinetics absorption spectroscopy studies demonstrated the occurrence of two intermediates with long-wavelength absorption during the catalytic cycle. Such intermediate species exhibit spectroscopic properties similar to those of charge-transfer complexes of oxidized flavin and NAD(P)H, and reduced flavin and NAD(P)+, respectively, which have been identified as intermediates during the reaction of flavoenzymes of the ferredoxin-NADP+ reductase family. Thus, a minimal kinetic scheme for the reaction of flavin reductase with NADPH and riboflavin can be proposed. After formation of the Michaelis complex of flavin reductase with NADPH and riboflavin, a first intermediate, identified as a charge-transfer complex of NADPH and riboflavin, is formed. It is followed by a second charge-transfer intermediate of enzyme-bound NADP+ and reduced riboflavin. The latter decays, yielding the Michaelis complex of flavin reductase with NADP+ and reduced riboflavin, which then dissociates to complete the reaction. These results support the initial hypothesis of a structural similarity between flavin reductase and the enzymes of the ferredoxin-NADP+ reductase family and extend it at a functional level.

Catalysis↗

PubMed1999

Structure-function correlations of the reaction of reduced nicotinamide analogues with p-hydroxybenzoate hydroxylase substituted with a series of 8-substituted flavins.

Structural and kinetic studies have revealed two flavin conformations in p-hydroxybenzoate hydroxylase (PHBH), the in-position and the out-position. Conversion between these two conformations is believed to be essential during catalysis. Although substrate hydroxylation occurs while the flavin in PHBH is in the in-conformation, the position of the flavin during reduction by NADPH is uncertain. To investigate the catalytic importance of the out-conformation of the flavin and to clarify the mechanism of flavin reduction in PHBH, we report quantitative structure-reactivity relationships (QSAR) using PHBH substituted separately with nine derivatives of FAD modified in the 8-position and four dihydronicotinamide analogues as reducing agents. The 8-position of the FAD isoalloxazine ring was chosen for modification because in PHBH it has minimal interactions with the protein and is accessible to solvent. The chemical sequence of events during catalysis by PHBH was not altered when using any of the modified flavins, and normal products were obtained. Although the rate of reduction of PHBH reconstituted with flavin derivatives is expected to be dependent on the redox potential of the flavin, no strict correlation was observed. Instead, the rate of reduction correlated with the kappa-substituent constant, which is based on size and hydrophobicity of the 8-substituent on the FAD. Substituents that sterically hinder attainment of the out-conformation decreased the rate of flavin reduction much more than expected on the basis of the redox potential of the flavin. The results of this QSAR analysis are consistent with the hypothesis that the flavin in PHBH must move to the out-conformation for proper formation of the charge-transfer complex between NADPH and FAD that is necessary for rapid flavin reduction.

4-Hydroxybenzoate-3-Monooxygenase↗

PubMed1990

Electrochemical superoxidation of flavins: generation of active precursors in luminescent model systems.

Using 3-methyllumiflavin and tetraacetyliriboflavin as examples, we have shown that the socalled "fully oxidized" flavins can be "superoxidized" at an anodic potential of 1.8 to 1.9 V giving flavin radical cation transients which are rapidly transformed in subsequent chemical reactions. An attack by H2O subsequent to the superoxidation of 3-methyllumiflavin provides a route for the formation of 4a-hydroxy-3-methyllumiflavin radical cation, as evident from the subsequent decomposition to the protonated form of the starting flavin. When 3-methyllumiflavin is superoxidized in the presence of a base, a recycling process occurs, allowing superoxidized flavin to be trapped in a slower, competitive conversion. The relatively more stable trapped product is active in reacting with H2O2 to emit chemiluminescence. Electrochemical oxidation of H2O2 in acetonitrile at 1.30 V in the presence of an oxidized flavin results in a direct protonation of the flavin by H+ generated from the electrolysis of H2O2. Minor reactions presumably provide alternative formations of the 4a-hydroperoxy- and 4a-hydroxy-flavin radical cation transients by the direct addition of HOO. and HO. radicals, which also arise in the oxidation of H2O2, to protonated flavin. Under such conditions the superoxidized flavin radical cation is apparently also formed, either directly or by process(es) such as decomposition of the flavin 4a-adduct radical cations. Subsequent reductions of either the superoxidized flavin or the flavin 4a-adduct radical cations lead to an almost steady level of luminescence.(ABSTRACT TRUNCATED AT 250 WORDS)

Flavins↗

PubMed1998

Chromatographic determination of flavin derivatives in baker's yeast.

The presence of flavin derivatives in baker's yeast was tested by high-performance liquid chromatography and thin-layer chromatography. In yeast samples, besides flavin adenine dinucleotide and flavin mononucleotide, small amounts of riboflavin and traces of 10-formylmethylflavin have been found. Total amount of flavins was calculated to be 17.9 +/- 2.9 micrograms/g of fresh yeast. The distribution of flavin adenine dinucleotide, flavin mononucleotide, riboflavin and 10-formylmethylflavin in total flavin content were estimated to be 71.5%, 25.8%, 1.7% and below 0.05%, respectively. In some samples we have additionally detected small amounts (0.8% of total flavins) of new flavin derivative which has been identified as 4',5'-riboflavin cyclic phosphate by means of its chromatographic and chemical behaviour. This compound seems to be a product of flavin adenine dinucleotide degradation and probably has been earlier mistaken for flavin mononucleotide. Its formation is dependent on pH conditions.

Chromatography, High Pressure Liquid↗

PubMed1993

Properties of a high-potential flavin analogue and its use as an active site probe with clostridial flavodoxin.

The reduction potential of flavin bearing a methylsulfonyl moiety (MeSO2) in place of a methyl group at position 8 is increased by more than 150 mV as compared with normal flavin. This substitution is accompanied by a substantial increase in reactivity with various reductants, including NADH, and greatly (10(3)-fold) enhanced susceptibility toward nucleophilic attack by sulfite at N(5). 1,5-Dihydro-8-(methylsulfonyl)riboflavin exhibits two intense, well-resolved absorption bands (lambda max = 310, 362 nm) in a region where most other reduced flavins exhibit weak, characterless absorption. This unusual spectrum is attributable to a shift of pi-electron density from the N(5) atom into the benzene ring. It is observed only with reduced flavins bearing a strongly electronegative substituent (MeSO2, CN) at the 8-position. The effect is abolished by replacing the hydrogen at N(5) with a bulky group, like sulfite, which interferes with sp2 hybridization at N(5). Reaction of 8-MeSO2-substituted flavins with thiols results in nucleophilic displacement of MeSO2- in a reaction that is about 10(3)-fold faster than an analogous nucleophilic displacement reaction observed with 8-halo-substituted flavins. The flavin ring acts as a redox switch in controlling electrophilicity at the 8-position, as judged by the fact that the displacement reactions are observed only with the oxidized flavins. Initial studies to evaluate 8-MeSO2-substituted flavins as active site probes were conducted with flavodoxin from Clostridium beijerinckii MP. 8-MeSO2FMN is rapidly bound to apoflavodoxin, accompanied by absorbance and fluorescence changes similar to those observed for FMN binding. 1,5-Dihydro-8-MeSO2FMN flavodoxin exhibits spectral properties (lambda max = 323, 382 nm) similar to those of the corresponding free flavin, except for a bathochromic shift due to a change in the polarity of the flavin environment. As judged by peak resolution and intensity, the spectral properties of 1,5-dihydro-FMN flavodoxin (lambda max = 311, 362 nm) appear to lie about midway between those observed for the free 1,5-dihydro forms of FMN versus 8-MeSO2FMN. This suggests that the protein environment may favor enhanced resonance delocalization of pi-electron density into the benzene ring of bound 1,5-dihydro-FMN, as compared with the free flavin. This hypothesis is consistent with previous NMR studies and with a proposal that electron transfer from reduced flavodoxin to other redox proteins occurs through this region of the ring. 8-MeSO2FMN bound to flavodoxin reacts readily with exogenous thiols but does not react with sulfite.(ABSTRACT TRUNCATED AT 400 WORDS)

Binding Sites↗

PubMed1974

The Flavin Content of Clovers Relative to Symbiosis with a Riboflavin-requiring Mutant of Rhizobium trifoli.

A riboflavin-requiring auxotroph of Rhizobium trifolii (T1/D-his(r)-15) formed ineffective root nodules on red clover and on two cultivars of subterranean clover, but produced almost fully effective nodules on several other cultivars of subterranean clover. Fluorescence and bioassay measurements of the flavin content of the roots and shoots of these cultivars revealed no differences between cultivars which could be correlated with the differences in symbiotic response. The concentration of flavin in nodules formed by the auxotroph (in the absence of riboflavin), by the effective parent strain (T1), or by a partly effective mutant (penicillin-resistant) of T1 was roughly proportional to the effectiveness of the nodules. Effective nodules contained 20 times as much flavin, and ineffective nodules 3 to 4 times as much flavin as non-nodulated root tissue. Approximately 20 to 30% of the flavins in both root and nodule tissue was flavin adenine dinucleotide and 70 to 80% was riboflavin + flavin mononucleotide. Most of the flavin adenine dinucleotide in macerated nodules was associated with host cell fragments, and none was detected in a cell-free fraction. Bacteroids accounted for approximately 20% of flavins in effective nodules and also contained more riboflavin + flavin mononucleotide than cultured rhizobial cells. The total flavin content of noninoculated roots increased from about 1.2 nmoles to 1.7 nmoles flavin/g of tissue after 3 days' exposure to 80 mum riboflavin. Exposure of only the upper or lower portion of preinoculated roots indicated negligible translocation, as effective nodulation occurred only on parts of the root in direct contact with riboflavin. Plants grown in a medium containing combined nitrogen (100 or 300 mum nitrogen added as (NH(4))(2)SO(4)), but no added riboflavin showed an increased root flavin content (about 2.1 nmoles flavin/g tissue) and a partly effective response when inoculated with the mutant. Nitrogen also promoted some upward translocation of exogenous riboflavin in the roots.

Journal Article↗

PubMed1985

Properties and catalytic function of the two nonequivalent flavins in sarcosine oxidase.

Sarcosine oxidase from Corynebacterium sp. U-96 contains 1 mol of noncovalently bound flavin and 1 mol of covalently bound flavin per mole of enzyme. Anaerobic titrations of the enzyme with either sarcosine or dithionite show that both flavins are reducible and that two electrons per flavin are required for complete reduction. Absorption increases in the 510-650-nm region, attributed to the formation of a blue neutral flavin radical, are observed during titration of the enzyme with dithionite or substrate, during photochemical reduction of the enzyme, and during reoxidation of substrate-reduced enzyme. Fifty percent of the enzyme flavin forms a reversible, covalent complex with sulfite (Kd = 1.1 X 10(-4) M), accompanied by a complete loss of catalytic activity. Sulfite does not prevent reduction of the sulfite-unreactive flavin by sarcosine but does interfere with the reoxidation of reduced enzyme by oxygen. The stability of the sulfite complex is unaffected by excess acetate (an inhibitor competitive with sarcosine) or by removal of the noncovalent flavin to form a semiapoprotein preparation where 75% of the flavin reacts with sulfite (Kd = 9.4 X 10(-5) M) while only 3% remains reducible with sarcosine. The results indicate that oxygen and sulfite react with the covalently bound flavin and suggest that sarcosine is oxidized by the noncovalently bound flavin.

Aerobiosis↗

PubMed1976

Identification and properties of the covalently bound flavin of beta-cyclopiazonate oxidocyclase.

Beta-Cyclopiazonate oxidocyclase from Penicillium cyclopium has been previously shown to contain flavin dinucleotide in covalent linkage to the protein. In the present study, a pure flavin mononucleotide peptide was isolated from the enzyme by tryptic-chymotryptic digestion, chromatography on Florisil and on diethylaminoethylcellulose, and hydrolysis with nucleotide pyrophosphatase. The flavin peptide contains 9 amino acids, including histidine in linkage to the flavin, and Asx as the N-terminal residue. The fluorescence of the flavin in the FMN peptide is profoundly quenched even at pH 3.2, where protonation of the imidazole prevents queching of the flavin fluorescence by histidine. This quenching appears to be due to interaction of the flavin with a tryptophan residue, as the quenching is abolished by oxidation of the tryptophan with performic acid. Similarly, the fluorescence of the tryptophan in the peptide is quenched, presumably by the flavin. The flavin of beta-cyclopiazonate oxidocylcase is attached, by the way of the 8alpha-methylene group, to the imidazole ring of a histidine. The aminoacylflavin isolated from the enzyme is identical in the pKa of its imidazole group, in reduction by NaBH4, and in other properties with synthetic 8alpha-(N1-histidyl)riboflavin. The pKa of the histidylriboflavin component of the oxidocyclase is 5.2 before and 5.0 after acid modification of the ribityl chain, as is found in the synthetic derivative. It is concluded that the enzyme contains the N1 isomer of histidylriboflavin and that acid hydrolysis of flavin peptides isolated from the oxidocyclase, while liberating histidylriboflavin, also causes acid modification of the ribityl chain of the flavin moiety.

Amino Acids↗

PubMed1996

Is the NAD(P)H:flavin oxidoreductase from Escherichia coli a member of the ferredoxin-NADP+ reductase family?. Evidence for the catalytic role of serine 49 residue.

The NAD(P)H:flavin oxidoreductase from Escherichia coli, Fre, is a monomer of 26.1 kDa which catalyzes the reduction of free flavins by NADPH or NADH. The flavin reductase Fre is the prototype of a new class of flavin reductases able to transfer electrons with no prosthetic group. It has been suggested that the flavin reductase could belong to the ferredoxin-NADP+ reductase (FNR) family, on the basis of limited sequence homologies. A sequence, conserved within the ferredoxin-NADP+ reductase family and present in the flavin reductase, is important for recognition of the isoalloxazine ring. Within this sequence, we have mutated serine 49 of the flavin reductase into alanine or threonine. kcat value of the S49A mutant was 35-fold lower than kcat of the wild-type enzyme. Determination of real Kd values for NADPH and lumichrome, a flavin analog, showed that recognition of the flavin is strongly affected by the S49A mutation, whereas affinity for the nicotinamide cofactor is only weakly modified. This suggests that serine 49 is involved in the binding of the isoalloxazine ring. Moreover, the Kd value for 5-deazariboflavin, in which the N-5 position of the isoalloxazine ring has been changed to a carbon atom, is not affected by the serine 49 to alanine mutation. This is consistent with the concept that the N-5 position is the main site for serine 49-flavin interaction. In the ferredoxin-NADP+ reductase family, the equivalent serine residue, which has been shown to be essential for activity, is hydrogen-bonded to the N-5 of the FAD cofactor. Taken together, these data provide the first experimental support to the hypothesis that the flavin reductase Fre may belong to the ferredoxin-NADP+ reductase family.

Alanine↗

PubMed1996

Studies with flavin analogs provide evidence that a protonated reduced FMN is the substrate-induced transient intermediate in the reaction of Escherichia coli chorismate synthase.

Chorismate synthase catalyzes the 1,4-elimination of phosphate and the C-(6-pro-R) hydrogen from 5-enolpyruvylshikimate 3-phosphate (EPSP) to generate chorismate. Although this reaction does not involve an overall change in redox state, the enzyme requires reduced FMN. To investigate the role of the flavin in catalysis we have employed chemically modified flavins: 1- and 5-deaza-, 2- and 4-thio-, 6-hydroxy-, 8-nor-6-methyl-, 8-methyl-sulfonyl-, 8-chloro-, 8-fluoro-, 8-nor-methyl-, 8-S-methyl-, 8-methoxy, 8-mercapto- and 8-amino-FMN. Photoreduction of 4-thio-FMN in the presence of chorismate synthase at pH 7.5 produced a reduced flavin species with an absorbance maximum at lambda = 410 nm indicative of monoanionic, reduced 4-thio-FMN. Binding of 8-mercapto- and 6-hydroxy-FMN to chorismate synthase in the presence of EPSP or (6R)-6-fluoro-EPSP resulted in an increase of the flavin analogs' pKa values by 4 and 1 pH units, respectively. On the basis of these findings it is concluded that chorismate synthase preferentially binds neutral flavin species, including the protonated reduced form, rather than anionic flavin species in the presence of EPSP or the 6-fluoro-substrate analog. Further support for this conclusion was obtained using 5-deaza- and 4-thio-FMN. Addition of EPSP to enzyme-bound, reduced 5-deaza-FMN produced spectral changes consistent with protonation of the flavin. Photoreduction of 4-thio-FMN in the presence of enzyme and the (6R)-6-fluoro-EPSP generated a reduced flavin species with absorbance properties of a neutral, reduced 4-thio-flavin. These results and their implications for the nature and kinetic properties of an observed flavin intermediate are discussed in the context of a possible role of reduced flavin as an electron donor to bound EPSP.

Enzyme Inhibitors↗

PubMed1984

Nuclear-magnetic-resonance investigation of 15N-labeled flavins, free and bound to Megasphaera elsdenii apoflavodoxin.

Flavin derivatives, enriched with 15N (approximately equal to 95%) at the four nitrogen atoms of the isoalloxazine ring, have been investigated in the oxidized and the two-electron reduced state by the 15N nuclear magnetic resonance technique. The measurements were conducted with aqueous and chloroform solutions of flavin. A comparison of the chemical shifts of the N(1) and N(5) atoms of oxidized flavin in the two solvents revealed that these atoms are sensitive indicators for possible hydrogen-bridge formation to these atoms. The N(5) atom of oxidized flavin resonates at low field and shifts about 300 ppm upfield upon reduction. A pKa of 6.8 was determined from pH-dependent 15N NMR measurements of the two-electron reduced flavin molecule. In addition it is also shown that reduced flavin in aqueous solution possesses a more coplanar structure than in chloroform solution. The 15N chemical shifts of flavin bound to Megasphaera elsdenii apoflavodoxin indicate that various hydrogen bridges are formed between the prosthetic group and the apoprotein. Especially the N(1) atom of the prosthetic group in the oxidized state seems to form a strong hydrogen bond with the apoprotein. In the reduced state the prosthetic group is bound in the anionic form and possesses an almost coplanar structure. These results are in agreement with published crystallographic data on the related flavodoxin from Clostridium MP. Where possible 15N-1H, 15N-15N and 13C-15N coupling constants were determined. Some of the coupling constants are useful parameters for the elucidation of the planarity of free and protein-bound flavin and for the evaluation of the interaction between flavin and apoprotein. Spin-lattice relaxation measurements show that the relaxation of the 15N(3)H group of flavin is predominantly determined by dipole-dipole interaction. The calculated rotational correlation times of flavin in two different solvents were determined and are in good agreement with published results.

Apoproteins↗

PubMed1994

Vibrio harveyi NADPH-flavin oxidoreductase: cloning, sequencing and overexpression of the gene and purification and characterization of the cloned enzyme.

NAD(P)H-flavin oxidoreductases (flavin reductases) from luminous bacteria catalyze the reduction of flavin by NAD(P)H and are believed to provide the reduced form of flavin mononucleotide (FMN) for luciferase in the bioluminescence reaction. By using an oligonucleotide probe based on the partial N-terminal amino acid sequence of the Vibrio harveyi NADPH-FMN oxidoreductase (flavin reductase P), a recombinant plasmid, pFRP1, was obtained which contained the frp gene encoding this enzyme. The DNA sequence of the frp gene was determined; the deduced amino acid sequence for flavin reductase P consists of 240 amino acid residues with a molecular weight of 26,312. The frp gene was overexpressed, apparently through induction, in Escherichia coli JM109 cells harboring pFRP1. The cloned flavin reductase P was purified to homogeneity by following a new and simple procedure involving FMN-agarose chromatography as a key step. The same chromatography material was also highly effective in concentrating diluted flavin reductase P. The purified enzyme is a monomer and is unusual in having a tightly bound FMN cofactor. Distinct from the free FMN, the bound FMN cofactor showed a diminished A375 peak and a slightly increased 8-nm red-shifted A453 peak and was completely or nearly nonfluorescent. The Kms for FMN and NADPH and the turnover number of this flavin reductase were determined. In comparison with other flavin reductases and homologous proteins, this flavin reductase P shows a number of distinct features with respect to primary sequence, redox center, and/or kinetic mechanism.

Amino Acid Sequence↗

PubMed1989

Differences in protein structure of xanthine dehydrogenase and xanthine oxidase revealed by reconstitution with flavin active site probes.

The native flavin, FAD, was removed from chicken liver xanthine dehydrogenase and milk xanthine oxidase by incubation with CaCl2. The deflavoenzymes, still retaining their molybdopterin and iron-sulfur prosthetic groups, were reconstituted with a series of FAD derivatives containing chemically reactive or environmentally sensitive substituents in the isoalloxazine ring system. The reconstituted enzymes containing these artificial flavins were all catalytically active. With both the chicken liver dehydrogenase and the milk oxidase, the flavin 8-position was found to be freely accessible to solvent. The flavin 6-position was also freely accessible to solvent in milk xanthine oxidase, but was significantly less exposed to solvent in the chicken liver dehydrogenase. Pronounced differences in protein structure surrounding the bound flavin were indicated by the spectral properties of the two enzymes reconstituted with flavins containing ionizable -OH or -SH substituents at the flavin 6- or 8-positions. Milk xanthine oxidase either displayed no preference for binding of the neutral or anionic flavin (8-OH-FAD) or a slight preference for the anionic form of the flavin (6-hydroxy-FAD, 6-mercapto-FAD, and possibly 8-mercapto-FAD). On the other hand, the chicken liver dehydrogenase had a dramatic preference for binding the neutral (protonated) forms of all four flavins, perturbing the pK of the ionizable substituent greater than or equal to 4 pH units. These results imply the existence of a strong negative charge in the flavin binding site of the dehydrogenase, which is absent in the oxidase.

Animals↗

PubMed1985

Potentiometric studies of native and flavin-substituted Old Yellow Enzyme.

We have measured the redox potentials for the flavin cofactor of native Old Yellow Enzyme and for a series of chemically modified flavin derivatives bound to the apoprotein. These flavin derivatives have midpoint potentials ranging from -120 to -300 mV in free solution. For the native enzyme, the midpoint potential of the first one-electron couple EFMNox + e- in equilibrium EFMN-. is E1 = -245 +/- 5 mV and that for the second one-electron couple EFMN-. + e- in equilibrium EFMN red is E2 = -215 +/- 5 mV in 0.1 M phosphate buffer at pH 7.0 and 25 degrees C. Thus, the apoprotein lowers the two-electron midpoint potential of FMN below its value in free solution (Emid,bound = -230 mV, Emid,free = -210 mV). A similar effect on the two-electron midpoint potentials of most of the chemically modified flavins is observed upon binding to the apoprotein. Therefore, the relative order of the respective midpoint potentials of this series of flavins is maintained upon binding to the apoenzyme. However, the effect of the apoprotein on the separation between E1 and E2 varies considerably over this series of flavin derivatives, resulting in quite different levels of thermodynamic stability for the one-electron-reduced (semiquinone) forms of the flavin-substituted enzymes. The optical absorption spectra of these flavin-substituted enzymes were determined in the presence of several phenolic compounds which are known to bind to the native enzyme and to give characteristic long wavelength transitions. We have found that the positions of the wavelength maxima of these transitions are shifted to longer wavelengths as the measured redox potentials of the enzyme-bound flavins are increased. The relationship between the energy of these long wavelength transitions (expressed as vCT = 1/lambda max) and the redox potential of the enzyme-bound flavin is interpreted as further evidence that these transitions arise from charge-transfer complexes between the phenolic compounds and the flavin.

Dithionite↗

PubMed1977

Studies on the flavins in rat liver mitochondrial outer membranes.

The incorporation of radioactivity derived from [2-14C] riboflavin into the flavins of rat liver mitochondrial outer membranes was studied. These membranes were found to contain about 0.6 nmol of non-covalently bound flavins per mg protein; the majority is in the form of FAD (73%) and FMN (24%). The membranes also contain about 1.5 nmol per mg of covalently bound flavins. After labeling, radioactive flavins appeared in the non-covalently bound flavins for about 4 h. Most of this radioactivity was in FAC (77%). Neither the rate nor extent of this labeling was affected by cycloheximide (1 mg/kg) administered 30 min prior to the radioactive riboflavin. With the covalently bound flavins, radioactivity was incorporated into the coenzymes for at least 18 h, but the rate of incorporation was much slower. After cycloheximide, radioactive flavins continued to appear in covalently bound flavins for about 2 h, but then stopped. Labeling of both types of flavins after [14C] riboflavin was considerably slower than the incorporation of [3H] leucine into outer membrane proteins. These results suggest that with flavoproteins from the mitochondrial outer membranes, the incorporation of flavins occurs after synthesis of the various apoenzymes is complete.

Animals↗

PubMed2000

Probing the mechanisms of the biological intermolecular transfer of reduced flavin.

NAD(P)H-flavin oxidoreductases [flavin reductases (FR)] are a class of enzymes capable of producing reduced flavin for bacterial bioluminescence and other biological processes. Bacterial luciferase utilizes oxygen, reduced FMN (FMNH2) and a long-chain aliphatic aldehyde as substrates for light emission. The Vibrio harveyi luciferase and FRP (for which we have cloned the gene and determined the crystal structure) is a model for the elucidation of the reduced flavin transfer mechanism using both a flavin reductase single-enzyme assay monitoring the NADPH oxidation and a flavin reductase-luciferase coupled assay measuring bioluminescence intensity or quantum output. The FRP exhibits a ping-pong kinetic pattern in the single-enzyme assay but changes to a sequential pattern in the coupled assay. Furthermore, FMN at >2x10(-6) mol/L reduced both the light intensity and quantum yield of the coupled reaction by noncompetitively inhibiting NADPH and competitively inhibiting luciferase. These results support a scheme in which the luciferase forms specific complex(es) with FRP. Indeed, such complexes were shown by fluorescence anisotropy to exist between luciferase and monomeric FRP either in the holo- or apoenzyme form. Furthermore, the reduced flavin cofactor of FRP is transferred directly to luciferase for bioluminescence, whereas the reduced flavin product of FRP is inefficient in supporting the luminescence reaction. The mechanism of reduced flavin transfer is apparently flavin and flavin reductase specific.

Bacteria↗

PubMed1994

Identification of the genes encoding NAD(P)H-flavin oxidoreductases that are similar in sequence to Escherichia coli Fre in four species of luminous bacteria: Photorhabdus luminescens, Vibrio fischeri, Vibrio harveyi, and Vibrio orientalis.

Genes encoding NAD(P)H-flavin oxidoreductases (flavin reductases) similar in both size and sequence to Fre, the most abundant flavin reductase in Escherichia coli, were identified in four species of luminous bacteria, Photorhabdus luminescens (ATCC 29999), Vibrio fischeri (ATCC 7744), Vibrio harveyi (ATCC 33843), and Vibrio orientalis (ATCC 33934). Nucleotide sequence analysis showed Fre-like flavin reductases in P. luminescens and V. fischeri to consist of 233 and 236 amino acids, respectively. As in E. coli Fre, Fre-like enzymes in luminous bacteria preferably used riboflavin as an electron acceptor when NADPH was used as an electron donor. These enzymes also were good suppliers of reduced flavin mononucleotide (FMNH2) to the bioluminescence reaction. In V. fischeri, the Fre-like enzyme is a minor flavin reductase representing < 10% of the total FMN reductase. That the V. fischeri Fre-like enzyme has no appreciable homology in amino acid sequence to the major flavin reductase in V. fischeri, FRase I, indicates that at least two different types of flavin reductases supply FMNH2 to the luminescence system in V. fischeri. Although Fre-like flavin reductases are highly similar in sequence to luxG gene products (LuxGs), Fre-like flavin reductases and LuxGs appear to constitute two separate groups of flavin-associated proteins.

Amino Acid Sequence↗

PubMed1999

Crystal structure of NAD(P)H:flavin oxidoreductase from Escherichia coli.

Flavin reductases use flavins as substrates and are distinct from flavoenzymes which have tightly bound flavins. The reduced flavin can serve to reduce ferric complexes and iron proteins. In Escherichia coli, reactivation of ribonucleotide reductase is achieved by reduced flavins produced by flavin reductase. The crystal structure of E. coli flavin reductase reveals that the enzyme structure is similar to the structures of the ferredoxin reductase family of flavoproteins despite very low sequence similarities. The main difference between flavin reductase and structurally related flavoproteins is that there is no binding site for the AMP moiety of FAD. The direction of the helix in the flavin binding domain, corresponding to the phosphate binding helix in the flavoproteins, is also slightly different and less suitable for phosphate binding. Interactions for flavin substrates are instead provided by a hydrophobic isoalloxazine binding site that also contains a serine and a threonine, which form hydrogen bonds to the isoalloxazine of bound riboflavin in a substrate complex.

Adenosine Monophosphate↗