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PubMed1980

Stopped flow studies on the nonenzymatic reduction of methemoglobin by reduced flavin mononucleotide.

The nonenzymatic reduction of methemoglobin by the reduced form of flavin mononucleotide was studied under various conditions by following the reaction with a stopped flow apparatus. The reaction was very fast, compared with the reduction of the flavin by NADPH-flavin reductase of human erythrocytes, and followed a second order rate law: the rate constant (K) for the reduction of methemoglobin by reduced flavin mononucleotide was determined to be 5.5 X 10(6) M-1 S-1 in 50 mM phosphate buffer (pH 7.0) at 25 degrees C. The reaction was not influenced by changing phosphate buffer concentration from 10 to 100 mM. The rate of reduction at the physiological pH, 7.0, was about 95% of the maximal value that observed at around pH 6.4. Formation of deoxyhemoglobin and oxidized form of flavin mononucleotide by the reaction proceeded stoichiometrically in a ratio of unity. These results apparently indicate that the limiting step for the reduction of methemoglobin by the NADPH-flavin reductase system in human erythrocytes is the enzymatic reduction of flavin.

Flavin Mononucleotide↗
PubMed2001

Modeling of the bacterial luciferase-flavin mononucleotide complex combining flexible docking with structure-activity data.

Although the crystal structure of Vibrio harveyi luciferase has been elucidated, the binding sites for the flavin mononucleotide and fatty aldehyde substrates are still unknown. The determined location of the phosphate-binding site close to Arg 107 on the alpha subunit of luciferase is supported here by point mutagenesis. This information, together with previous structure-activity data for the length of the linker connecting the phosphate group to the isoalloxazine ring represent important characteristics of the luciferase-bound conformation of the flavin mononucleotide. A model of the luciferase-flavin complex is developed here using flexible docking supplemented by these structural constraints. The location of the phosphate moiety was used as the anchor in a flexible docking procedure performed by conformation search by using the Monte Carlo minimization approach. The resulting databases of energy-ranked feasible conformations of the luciferase complexes with flavin mononucleotide, omega-phosphopentylflavin, omega-phosphobutylflavin, and omega-phosphopropylflavin were filtered according to the structure-activity profile of these analogs. A unique model was sought not only on energetic criteria but also on the geometric requirement that the isoalloxazine ring of the active flavin analogs must assume a common orientation in the luciferase-binding site, an orientation that is also inaccessible to the inactive flavin analog. The resulting model of the bacterial luciferase-flavin mononucleotide complex is consistent with the experimental data available in the literature. Specifically, the isoalloxazine ring of the flavin mononucleotide interacts with the Ala 74-Ala 75 cis-peptide bond as well as with the Cys 106 side chain in the alpha subunit of luciferase. The model of the binary complex reveals a distinct cavity suitable for aldehyde binding adjacent to the isoalloxazine ring and flanked by other key residues (His 44 and Trp 250) implicated in the active site.

Binding Sites↗
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↗
PubMed1999

The effects of pH and semiquinone formation on the oxidation-reduction potentials of flavin mononucleotide. A reappraisal.

Calculation shows that there is poor agreement between frequently cited values for the midpoint redox potentials of the two one-electron steps in the reduction of flavin mononucleotide and equations for the lines that relate these potentials to pH and that use the published pKa values for the three redox states of the flavin [Draper, R. & Ingraham, L.L. (1969) Arch. Biochem. Biophys. 125, 802-808]. Equilibrium data for the first step in the reduction obtained by pulse radiolysis [Anderson, R.F. (1983) Biochim. Biophys. Acta 722, 158-162] show much closer agreement with theory and lead to values for the semiquinone formation constant of flavin mononucleotide that are close to those derived from measurements of the radical concentration using ESR spectroscopy. It is concluded that the data from the second method are more reliable. The redox potentials for flavin mononucleotide at pH 7.0 and 20 degrees C are calculated to be -0.207 V for the overall two-electron reduction (Em), -0.313 V for reduction of the oxidized flavin to the semiquinone (E2) and -0.101 V for the reduction of the semiquinone to the hydroquinone (E1). Information is provided to allow calculation of the three redox potentials at other pH values in the physiological range.

Benzoquinones↗
PubMed1984

A conformational approach to study the mode of binding of flavin mononucleotide to flavodoxin.

Energetically preferred conformers of Flavin mononucleotide (FMN) were determined using empirical potential energy functions. The minimum energy conformers were used to study the mode of its binding to apoflavodoxin. This study indicates that the conformers of FMN that initiate the binding process undergo significant changes in the position of the phosphate group to reach the final bound conformation. In the bound conformation the phosphate group leads to the formation of a network of hydrogen bonds with the apoflavodoxin and contributes significantly to the binding energy. This extra energy is required for FMN to overcome the repulsion from Met 56 and Glu 59 and to bind tightly to apoflavodoxin.

Binding Sites↗
PubMed1966

The modification of the cytotoxic effect of leucocidin by N-ethylmaleimide, flavine mononucleotide and mendione.

1. The movement of the cytoplasmic granules in the leucocidin-treated leucocyte is prevented in the presence of N-ethylmaleimide or menadione. This effect follows a change of state in the cytoplasm. It may not be due to reaction with SH groups. When granule movement is prevented in this way the subsequent addition of Ca(2+) and ATP does not induce the secretion of the proteins of the granules. 2. Menadione or iodoacetate stimulates some effects of suboptimum amounts of leucocidin. This effect probably follows a reaction with SH groups. 3. Flavine mononucleotide inhibits some effects of suboptimum amounts of leucocidin. 4. Leucocidin decreases the stimulation of glucose oxidation due to menadione but increases that due to flavine mononucleotide. Leucocidin decreases the adsorption of menadione by leucocytes but increases that of flavine mononucleotide. 5. The redox state of the nicotinamide-adenine nucleotide coenzymes is not altered during leucocidin action and flavine mononucleotide and menadione do not undergo significant continuous oxidation and reduction when added to the leucocyte.

Cytoplasmic Granules↗
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↗
PubMed1988

Flavodoxin from Anabaena 7120: uniform nitrogen-15 enrichment and hydrogen-1, nitrogen-15, and phosphorus-31 NMR investigations of the flavin mononucleotide binding site in the reduced and oxidized states.

Interactions between flavin mononucleotide (FMN) and apoprotein have been investigated in the reduced and oxidized states of the flavodoxin isolated from Anabaena 7120 (Mr approximately 21,000). 1H, 15N, and 31P NMR have been used to characterize the FMN-protein interactions in both redox states. These are compared with those seen in other flavodoxins. Uniformly enriched [15N]flavodoxin (greater than 95% isotopic purity) was isolated from Anabaena 7120 grown on K15NO3 as the sole nitrogen source. 15N insensitive nucleus enhanced by polarization transfer (INEPT) and nuclear Overhauser effect (NOE) studies of this sample provided information regarding protein structure and dynamics. A 1H-detected 15N experiment allowed the correlation of nitrogen resonances to those of their attached protons. Over 90% of the expected N-H cross peaks could be resolved in this experiment.

Binding Sites↗
PubMed1997

Cloning, sequencing, and analysis of a gene cluster from Chelatobacter heintzii ATCC 29600 encoding nitrilotriacetate monooxygenase and NADH:flavin mononucleotide oxidoreductase.

Nitrilotriacetate (NTA) is an important chelating agent in detergents and has also been used extensively in processing radionuclides. In Chelatobacter heintzii ATCC 29600, biodegradation of NTA is initiated by NTA monooxygenase that oxidizes NTA to iminodiacetate and glyoxylate. The NTA monooxygenase activity requires two component proteins, component A and component B, but the function of each component is unclear. We have cloned and sequenced a gene cluster encoding components A and B (nmoA and nmoB) and two additional open reading frames, nmoR and nmoT, downstream of nmoA. Based on sequence similarities, nmoR and nmoT probably encode a regulatory protein and a transposase, respectively. The NmoA sequence was similar to a monooxygenase that uses reduced flavin mononucleotide (FMNH2) as reductant; NmoB was similar to an NADH:flavin mononucleotide (FMN) oxidoreductase. On the basis of this information, we tested the function of each component. Purified component B was shown to be an NADH:FMN oxidoreductase, and its activity could be separated from that of component A. When the Photobacterium fischeri NADH:FMN oxidoreductase was substituted for component B in the complete reaction, NTA was oxidized, showing that the substrate specificity of the reaction resides in component A. Component A is therefore an NTA monooxygenase that uses FMNH2 and O2 to oxidize NTA, and component B is an NADH:FMN oxidoreductase that provides FMNH2 for NTA oxidation.

Amino Acid Sequence↗
PubMed1996

Evaluation of the electrostatic effect of the 5'-phosphate of the flavin mononucleotide cofactor on the oxidation--reduction potentials of the flavodoxin from desulfovibrio vulgaris (Hildenborough).

Two mutants of the Desulfovibrio vulgaris flavodoxin, T12H and N14H, were generated which, for the first time, place a basic residue within the normally neutral 5'-phosphate binding loop of the flavin mononucleotide cofactor binding site found in all flavodoxins. These histidine residues were designed to form an ion pair with the dianionic 5'-phosphate, either altering its ionization state or offsetting its negative charge to allow evaluation of the magnitude of its electrostatic effect on the redox properties of the cofactor. The midpoint potential for the oxidized/semiquinone couple was not significantly altered in either mutant. However, the midpoint potentials for the semiquinone/hydroquinone couple (Esq/hq) were less negative than that of the wild type, increasing by 28 and 15 mV relative to that of the wild type for the T12H and N14H mutants, respectively, at pH 6. 31P NMR spectroscopy suggests that, just as for wild type, the phosphate group in each mutant does not change its ionization state between pH 6 and 8. Therefore, the small increases in midpoint potential must be linked to the protonation of the histidine residues, either through favorable interactions with the anionic hydroquinone or by the partial compensation of the charge on the 5'-phosphate. Values for the pKa of His12 and His14 in the oxidized flavodoxin were determined by 1H NMR spectroscopy to be 6.71 and 6.93, respectively, which are only modestly elevated relative to the average value for histidines in proteins. This suggests that the histidines do not form strong ion-pairing interactions with the phosphate and/or that the effective charge on the 5'-phosphate may be substantially less than the reported formal dianionic charge. Either way, the data provide evidence for the rather weak electrostatic interaction between a charged group at this site and the anionic flavin hydroquinone. In contrast, Esq/hq reported for the apoflavodoxin-riboflavin complex, which lacks the 5'-phosphate group, is 180 mV less negative than that of the native flavodoxin. The re-evaluation of the redox and cofactor binding properties of the riboflavin complex generated values for the dissociation constants for the riboflavin complex in the oxidized, semiquinone, and hydroquinone oxidation states that are 2100-, 63000-, and 54-fold higher, respectively, than that for the naturally occurring flavin mononucleotide complex. The large redox potential shifts observed for both redox couples in the riboflavin complex are primarily the consequence of a decreased stabilization of the semiquinone rather than the result of the absence of the negative charge of the 5'-phosphate. It is concluded from this study that the negative charge on the phosphate group of the cofactor does not play a disproportionate role in decreasing Esq/hq, at most contributing equivalently with the acidic amino acid residues clustered around the flavin to an unfavorable electrostatic environment for the formation of the flavin hydroquinone anion.

Apoproteins↗
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↗
PubMed1999

Role of glutamate-59 hydrogen bonded to N(3)H of the flavin mononucleotide cofactor in the modulation of the redox potentials of the Clostridium beijerinckii flavodoxin. Glutamate-59 is not responsible for the pH dependency but contributes to the stabilization of the flavin semiquinone.

The midpoint potentials for both redox couples of the noncovalently bound flavin mononucleotide (FMN) cofactor in the flavodoxin are known to be pH dependent. While the pH dependency for the oxidized-semiquinone (ox/sq) couple is consistent with the formation of the blue neutral form of the flavin semiquinone, that of the semiquinone-hydroquinone (sq/hq) couple is more enigmatic. The apparent pK(a) of 6.7 for this couple in the flavodoxin from Clostridium beijerinckii has been attributed to the ionization of the FMN(HQ); however, nuclear magnetic resonance data strongly suggest the FMN(HQ) remains anionic over the entire pH range testable. As an alternative explanation, a specific glutamate residue (Glu59 in this flavodoxin), which is hydrogen-bonded to N(3)H of the FMN, has been postulated to be the primary redox-linked proton acceptor responsible for the pH effect in some flavodoxins. This model was directly tested in this study by permanently neutralizing Glu59 by its replacement with glutamine. This conservative substitution resulted in an increase of 86 mV (at pH 7) in midpoint potential of the sq/hq couple; however, the pH dependency of this couple was not altered. Thus, the redox-linked protonation of Glu59 clearly cannot be responsible for this effect as proposed. The pH dependency of the ox/sq couple was also similar to wild type, but the midpoint potential has decreased by 65 mV (pH 7). The K(d) values for the oxidized, semiquinone, and hydroquinone complexes increased by 43-, 590-, and 20-fold, respectively, relative to the wild type. Thus, the Glu59 to glutamine substitution substantially effects the stability of the semiquinone but, on a relative basis, slightly favors the formation of the hydroquinone. On the basis of (1)H-(15)N HSQC nuclear magnetic resonance spectroscopic studies, the increased temperature coefficients for the protons on N(3) and N(5) of the reduced FMN in E59Q suggest that the hydrogen-bonding interactions at these positions are significantly weakened in this mutant. The increase for N(5)H correlates with the reduced stability of the FMN(SQ) and the more negative midpoint potential for the ox/sq couple. On the basis of the X-ray structure, an "anchoring" role is proposed for the side chain carboxylate of Glu59 that stabilizes the structure of the 50's loop in such a way so as to promote the crucial hydrogen-bonding interaction that stabilizes the flavin semiquinone, contributing to the low potential of this flavodoxin.

Circular Dichroism↗
PubMed2005

Crystal structures of the tryptophan repressor binding protein WrbA and complexes with flavin mononucleotide.

The tryptophan repressor binding protein WrbA binds to the tryptophan repressor protein TrpR. Although the biological role of WrbA remains unclear, it has been proposed to function in enhancing the stability of TrpR-DNA complexes. Sequence database analysis has identified WrbA as a founding member of a flavodoxin-like family of proteins. Here we present crystal structures of WrbA from Deinococcus radiodurans and Pseudomonas aeruginosa and their complexes with flavin mononucleotide. The protomer structure is similar to that of previously determined long-chain flavodoxins; however, each contains a conserved inserted region unique to the WrbA family. Interestingly, each WrbA protein forms a homotetramer with 222 symmetry, unique among flavodoxin-like proteins, in which each protomer binds one flavin mononucleotide cofactor molecule.

Amino Acid Sequence↗
PubMed1994

A continuous, anaerobic spectrophotometric assay for chorismate synthase activity that utilizes photoreduced flavin mononucleotide.

A sensitive, continuous, spectrophotometric assay for chorismate synthase has been developed utilizing photoreduced flavin mononucleotide (FMNH2) as a cofactor under anaerobic conditions. The assay monitors directly the formation of chorismate from 5-enolpyruvylshikimate-3-phosphate (EPSP) at 275 nm with a precision of +/- 2 microM product. The assay conditions have been optimized with respect to FMNH2 (cofactor), EPSP (substrate) and enzyme concentrations, buffer type, and pH. The potential of the assay for detailed steady-state kinetic studies to elucidate the mechanism of action of this commercially important enzyme is also demonstrated.

Anaerobiosis↗
PubMed2002

Mechanisms of reactions of flavin mononucleotide triplet with aromatic amino acids.

Chemical reactions between the photoexcited triplet state of flavin mononucleotide and the aromatic amino acids, N-acetyl tryptophan (TrpH), N-acetyl tyrosine (TyrOH), and N-acetyl histidine (HisH) in aqueous solution have been studied in the pH range 2-12. Across the whole pH range, the principal mechanism of reaction of both TrpH and TyrOH is shown to be electron transfer. For HisH, the mechanism and rate of the reaction depend on the protonation state of the reactants. In acidic conditions (pH < 4), reaction does not occur. At 4 < pH < 11, the reaction proceeds via hydrogen atom abstraction with a rate constant varying from 3.0 x 10(6) to 2.5 x 10(8) M(-1) s(-1). In extremely basic solution (pH > 12) the mechanism switches to electron transfer.

Amino Acids, Aromatic↗