flavin-adenine-dinucleotide and lumiflavin

The flavin adenine dinucleotide cofactor has an unusual bent configuration in photolyase and cryptochrome, and such a folded structure may have a functional role in initial photochemistry. Using femtosecond spectroscopy, we report here our systematic characterization of cyclic intramolecular electron transfer (ET) dynamics between the flavin and adenine moieties of flavin adenine dinucleotide in four redox forms of the oxidized, neutral, and anionic semiquinone, and anionic hydroquinone states. By comparing wild-type and mutant enzymes, we have determined that the excited neutral oxidized and semiquinone states absorb an electron from the adenine moiety in 19 and 135 ps, whereas the excited anionic semiquinone and hydroquinone states donate an electron to the adenine moiety in 12 ps and 2 ns, respectively. All back ET dynamics occur ultrafast within 100 ps. These four ET dynamics dictate that only the anionic hydroquinone flavin can be the functional state in photolyase due to the slower ET dynamics (2 ns) with the adenine moiety and a faster ET dynamics (250 ps) with the substrate, whereas the intervening adenine moiety mediates electron tunneling for repair of damaged DNA. Assuming ET as the universal mechanism for photolyase and cryptochrome, these results imply anionic flavin as the more attractive form of the cofactor in the active state in cryptochrome to induce charge relocation to cause an electrostatic variation in the active site and then lead to a local conformation change to initiate signaling.

Topics: Adenine; Benzoquinones; Cryptochromes; Deoxyribodipyrimidine Photo-Lyase; Electron Transport; Energy Transfer; Escherichia coli; Escherichia coli Proteins; Flavin-Adenine Dinucleotide; Flavins; Hydroquinones; Kinetics; Models, Chemical; Models, Molecular; Molecular Conformation; Molecular Structure; Mutation; Oxidation-Reduction; Photochemical Processes; Spectrophotometry; Substrate Specificity; Time Factors; Tryptophan

Riboflavin is an important water soluble vitamin (B2) required for metabolic reactions, normal cellular growth, differentiation and function. Mammalian brain cells cannot synthesize riboflavin and must import from systemic circulation. However, the uptake mechanism, cellular translocation and intracellular trafficking of riboflavin in brain capillary endothelial cells are poorly understood. The primary objective of this study is to investigate the existence of a riboflavin-specific transport system and delineate the uptake and intracellular regulation of riboflavin in immortalized rat brain capillary endothelial cells (RBE4). The uptake of [3H]-riboflavin is sodium, temperature and energy dependent but pH independent. [3H]-Riboflavin uptake is saturable with K(m) and V(max) values of 19 ± 3 μM and 0.235 ± 0.012 pmol/min/mg protein, respectively. The uptake process is inhibited by unlabelled structural analogs (lumiflavin, lumichrome) but not by structurally unrelated vitamins. Ca(++)/calmodulin and protein kinase A (PKA) pathways are found to play an important role in the intracellular regulation of [3H]-riboflavin. Apical and baso-lateral uptake of [3H]-riboflavin clearly indicates that a riboflavin specific transport system is predominantly localized on the apical side of RBE4 cells. A 628 bp band corresponding to a riboflavin transporter is revealed in RT-PCR analysis. These findings, for the first time report the existence of a specialized and high affinity transport system for riboflavin in RBE4 cells. The blood-brain barrier (BBB) is a major obstacle limiting drug transport inside the brain as it regulates drug permeation from systemic circulation. This transporter can be utilized for targeted delivery in enhancing brain permeation of highly potent drugs on systemic administration.

Topics: Animals; Biological Transport; Brain; Cells, Cultured; Dinitrophenols; Dose-Response Relationship, Drug; Endothelial Cells; Flavin Mononucleotide; Flavin-Adenine Dinucleotide; Flavins; Hydrogen-Ion Concentration; Ouabain; Rats; Riboflavin; Signal Transduction; Sodium; Sodium Azide; Substrate Specificity; Temperature; Time Factors; Tritium; Vitamin B Complex

Acyl-CoA dehydrogenase forms a complex with a substrate analog, 3-thiaacyl-CoA, exhibiting a charge-transfer (CT) band. The structure of a complex model of oxidized lumiflavin with deprotonated 3-thiabutanoate ethylthioester designed for the above CT complex was fully optimized by means of density functional theory (DFT), the spatial arrangement being similar to the corresponding X-ray structure reported previously. The electrostatic interaction between flavin and an anionic ligand, therefore, plays a major role in determination of the arrangement of the CT complex. When the excitation energies and oscillator strengths for the optimized structures of complex models including oxidized 8-substituted lumiflavins were calculated, the obtained wavelengths correlated well with observed values reported. Subsequently, we carried out DFT calculations for new complex models redesigned for complexes of oxidized 8-substituted FADs with an anionic ligand by introducing hydrogen bonds at the carbonyl group of the ligand with the 2'-hydroxyl group of the N10-ribityl of FAD and with the main-chain amide group of Glu376. The CT absorbing wavelengths of the new complex models exhibited better correlation with those observed previously. Consequently, comparison of substituent effects on the DFT calculations for the complex models will lead to a deeper understanding of the CT interaction and the effect of the hydrogen-bonding interaction on the CT framework.

Topics: Acyl-CoA Dehydrogenase; Electron-Transferring Flavoproteins; Flavin-Adenine Dinucleotide; Flavins; Hydrogen Bonding; Hydrogen-Ion Concentration; Models, Chemical; Models, Molecular; Molecular Conformation; Protein Structure, Secondary; Structure-Activity Relationship; Substrate Specificity

A pyrimidine-pyrimidone (6-4) photoproduct and a cyclobutane pyrimidine dimer (CPD) are major DNA lesions induced by ultraviolet irradiation, and (6-4) photolyase, an enzyme with flavin adenine dinucleotide (FAD) as a cofactor, repairs the former specifically by light illumination. We investigated resonance Raman spectra of (6-4) photolyase from Arabidopsis thaliana having neutral semiquinoid and oxidized forms of FAD, which were selectively intensity enhanced by excitations at 568.2 and 488.0 nm, respectively. DFT calculations were carried out for the first time on the neutral semiquinone. The marker band of a neutral semiquinone at 1606 cm(-1) in H(2)O, whose frequency is the lowest among various flavoenzymes, apparently splits into two comparable bands at 1594 and 1608 cm(-1) in D(2)O, and similarly, that at 1522 cm(-1) in H(2)O does into three bands at 1456, 1508, and 1536 cm(-1) in D(2)O. This D(2)O effect was recognized only after being oxidized once and photoreduced to form a semiquinone again, but not by simple H/D exchange of solvent. Some Raman bands of the oxidized form were observed at significantly low frequencies (1621, 1576 cm(-1)) and with band splittings (1508/1493, 1346/1320 cm(-1)). These Raman spectral characteristics indicate strong H-bonding interactions (at N5-H, N1), a fairly hydrophobic environment, and an electron-lacking feature in benzene ring of the FAD cofactor, which seems to specifically control the reactivity of (6-4) photolyase.

Topics: Amino Acid Sequence; Benzene; Coenzymes; Deoxyribodipyrimidine Photo-Lyase; Flavin-Adenine Dinucleotide; Flavins; Hydrogen Bonding; Molecular Conformation; Molecular Sequence Data; Pyrimidine Dimers; Quantum Theory; Sequence Alignment; Spectrum Analysis, Raman

We have studied the functional steps by which Saccharomyces cerevisiae mitochondria can synthesize FAD from cytosolic riboflavin (Rf). Riboflavin uptake into mitochondria took place via a mechanism that is consistent with the existence of (at least two) carrier systems. FAD was synthesized inside mitochondria by a mitochondrial FAD synthetase (EC 2.7.7.2), and it was exported into the cytosol via an export system that was inhibited by lumiflavin, and which was different from the riboflavin uptake system. To understand the role of the putative mitochondrial FAD carrier, Flx1p, in this pathway, an flx1Delta mutant strain was constructed. Coupled mitochondria isolated from flx1Delta mutant cells were compared with wild-type mitochondria with respect to the capability to take up Rf, to synthesize FAD from it, and to export FAD into the extramitochondrial phase. Mitochondria isolated from flx1Delta mutant cells specifically lost the ability to export FAD, but did not lose the ability to take up Rf, FAD, or FMN and to synthesize FAD from Rf. Hence, Flx1p is proposed to be the mitochondrial FAD export carrier. Moreover, deletion of the FLX1 gene resulted in a specific reduction of the activities of mitochondrial lipoamide dehydrogenase and succinate dehydrogenase, which are FAD-binding enzymes. For the flavoprotein subunit of succinate dehydrogenase we could demonstrate that this was not due to a changed level of mitochondrial FAD or to a change in the degree of flavinylation of the protein. Instead, the amount of the flavoprotein subunit of succinate dehydrogenase was strongly reduced, indicating an additional regulatory role for Flx1p in protein synthesis or degradation.

Topics: Base Sequence; Biological Transport; Cytosol; DNA Primers; Flavin-Adenine Dinucleotide; Flavins; Kinetics; Membrane Transport Proteins; Mitochondria; Molecular Sequence Data; Mutagenesis, Site-Directed; Riboflavin; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins

The influence of the amino acid residues surrounding the flavin ring in the flavodoxin of the cyanobacterium Anabaena PCC 7119 on the electron spin density distribution of the flavin semiquinone was examined in mutants of the key residues Trp(57) and Tyr(94) at the FMN binding site. Neutral semiquinone radicals of the proteins were obtained by photoreduction and examined by electron-nuclear double resonance (ENDOR) and hyperfine sublevel correlation (HYSCORE) spectroscopies. Significant differences in electron density distribution were observed in the flavodoxin mutants Trp(57) --> Ala and Tyr(94) --> Ala. The results indicate that the presence of a bulky residue (either aromatic or aliphatic) at position 57, as compared with an alanine, decreases the electron spin density in the nuclei of the benzene flavin ring, whereas an aromatic residue at position 94 increases the electron spin density at positions N(5) and C(6) of the flavin ring. The influence of the FMN ribityl and phosphate on the flavin semiquinone was determined by reconstituting apoflavodoxin samples with riboflavin and with lumiflavin. The coupling parameters of the different nuclei of the isoalloxazine group, as detected by ENDOR and HYSCORE, were very similar to those of the native flavodoxin. This indicates that the protein conformation around the flavin ring and the electron density distribution in the semiquinone form are not influenced by the phosphate and the ribityl of FMN.

Topics: Amino Acid Substitution; Anabaena; Electron Spin Resonance Spectroscopy; Flavin-Adenine Dinucleotide; Flavins; Flavodoxin; Hydrogen; Point Mutation; Riboflavin

X-ray crystallographic studies of several complexes involving FAD bound to p-hydroxybenzoate hydroxylase (PHBH) have revealed that the isoalloxazine ring system of FAD is capable of adopting in two positions on the protein. In one, the "in" form, the ring is surrounded by protein groups and has little contact with solvent; in the second, "out" form, the ring is largely solvent exposed. Using Raman difference spectroscopy, it has been possible to obtain Raman spectra for the flavin ring in both conformational states for different complexes in solution. The spectra consist of a rich assortment of isoalloxazine ring modes whose normal mode origin can be assigned by using density functional theory and ab initio calculations. Further insight into the sensitivity of these modes to changes in environment is provided by the Raman spectra of lumiflavin in the solid state, in DMSO and in aqueous solution. For the protein complexes, the Raman difference spectra of flavin bound to wt PHBH and wt PHBH plus substrate, p-hydroxybenzoate, provided examples of the "in" conformation. These data are compared to those for flavin bound to wt PHBH plus 2,4-dihydroxybenzoate, where X-ray analysis show that the flavin is "out". There are several spectral regions where characteristic differences exist for flavin in the "in" or "out" conformation, these occur near 1700, 1500, 1410, 1350, 1235, and 1145 cm(-)(1). These spectral features can be used as empirical marker bands to determine the populations of "in" and "out" for any complex of PHBH and to monitor changes in those populations with perturbations to the system, e.g., by changing temperature or pH. Thus, it will now be possible to determine the conformational state of the flavin in PHBH for those complexes that have resisted X-ray crystallographic analysis. Raman difference data are also presented for the Tyr222Phe mutant. The Raman data show that the isoalloxazine ring is predominantly "out" for Tyr222Phe. However, in the presence of the substrate p-hydroxybenzoate there is clear evidence from the Raman marker bands that a mixed population of "in" and "out" exists with the majority being in the "out" state. This is consistent with the conclusions drawn from crystallographic studies on this complex (Gatti, D. L., Palfey, B. A., Lah, M. S., Entsch, B., Massey, V., Ballou, D. P., and Ludwig, M. L. (1994) Science, 266, 110-114).

Topics: 4-Hydroxybenzoate-3-Monooxygenase; Amino Acid Substitution; Binding Sites; Flavin-Adenine Dinucleotide; Flavins; Mutagenesis, Site-Directed; Phenylalanine; Protein Conformation; Pseudomonas aeruginosa; Solvents; Spectrum Analysis, Raman; Tyrosine

1. Kinetic parameters of the oxidative part of glucose-oxidase reaction have been measured with 16 different electron-acceptors and glucose as a substrate. 2. In each case, the rate-limiting portion of the oxidative part of reaction was the formation of the E-FADH2.Acceptor-complex; this rate was pH-independent around the pH-optimum of the enzyme. 3. In each case, E-FADH2 acceptor-complex was undetectable in the steady-state kinetics, with the exception of cytochrome-c. 4. The rates of redox reactions between various forms of reduced 5-ethyl-lumiflavin and five different electron-acceptors have been examined with a conventional spectrophotometry. In each case, it was found that the reactions proceeded at high rates whenever thermodynamically feasible, and were totally prevented in the opposite case. 5. Molecular oxygen was able to oxidize only the neutral form of 5-ethyl-1,5-dihydrolumiflavin to its radical form, at a moderate rate; all other forms of reduced 5-ethyl-lumiflavin were not oxidized by O2. 6. By the comparison of enzymatic and model redox reactions, it was possible to establish the minimal mechanism of the oxidative part of the glucose-oxidase catalytic cycle.

Topics: Aspergillus niger; Flavin-Adenine Dinucleotide; Flavins; Glucose Oxidase; Kinetics; Models, Chemical; Oxidation-Reduction; Spectrophotometry

Flavins and reduced flavins were reacted with a variety of free radicals produced in dilute aqueous solution at pH 7 +/- 0.1 by radiation chemical methods. The radical .CH2C(CH3)2OH and the aliphatic beta, gamma, and delta radicals of ethanol, 2-propanol, and 1-butanol added to the radical form of flavin adenine dinucleotide (FAD) (FH.) to yield products that could not be reoxidized to flavin by oxygen. The first radical also added to FAD but with a much lower efficiency. In contrast, the alpha-carbon radicals .CH(OH)CH2OH, CH3(-3) CHOH, and (CH3)2COH appeared to undergo two reactions: FH. + RR'COH leads to FH- + RR'C==O FH. + RR'COH leads to FH- + RR'C==O + H+ The formate radical anion .CO2(-) reacted similarly, producing stoichiometric two-electron reduction of riboflavin and lumiflavin as well as FAD. While eaq- also seemed capable of this, it was found to react irreversibly with FADH2, which makes it a poor reagent for producing the dihydroflavin. The dihydro form of FAD was reoxidized to FAD by the species RS. and .BR2(-). In contrast to FAD, the alloxazine lumichrome underwent only one-electron reduction and oxidation by .CO2(-) and .Br2(-), respectively.

Topics: Alcohols; Chemical Phenomena; Chemistry; Flavin-Adenine Dinucleotide; Flavins; Formates; Free Radicals; Oxidation-Reduction; Riboflavin; Spectrometry, Gamma