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PubMed2000

Radiolytic studies of trimethylamine dehydrogenase. Spectral deconvolution of the neutral and anionic flavin semiquinone, and determination of rate constants for electron transfer in the one-electron reduced enzyme.

Trimethylamine dehydrogenase from the pseudomonad Methylophilus methylotrophus has been examined using the technique of pulse radiolysis to rapidly introduce a single reducing equivalent into the enzyme. Using enzyme that has had its iron-sulfur center rendered redox-inert by prior reaction with ferricenium hexafluorophosphate, we determined the spectral change associated with formation of both the anionic and neutral forms that were generated at high and low pH, respectively, of the unique 6-cysteinyl-FMN of the enzyme. With native enzyme, electron transfer was observed within the radiolytically generated one-electron reduced enzyme but only at low pH (6.0). The kinetics and thermodynamics of this electron transfer in one-electron reduced enzyme may be compared with that studied previously in the two-electron reduced enzyme. In contrast to previous studies with two-electron reduced enzyme in which a pK(a) of approximately 8 was determined for the flavin semiquinone, in the one-electron reduced enzyme the semiquinone was not substantially protonated even at pH 6. 0. These results indicate that reduction of the iron-sulfur center of the enzyme significantly decreases the pK(a) of the flavin semiquinone of the active site. This provides further evidence, in conjunction with the strong magnetic interaction known to exist between the centers in the two-electron reduced enzyme, that the two redox-active centers in trimethylamine dehydrogenase are in intimate contact with one another in the active site of the enzyme.

Catalysis↗
PubMed2000

The C termini of constitutive nitric-oxide synthases control electron flow through the flavin and heme domains and affect modulation by calmodulin.

The sequences of nitric-oxide synthase flavin domains closely resemble that of NADPH-cytochrome P450 reductase (CPR). However, all nitric-oxide synthase (NOS) isoforms are 20-40 residues longer in the C terminus, forming a "tail" that is absent in CPR. To investigate its function, we removed the 33 and 42 residue C termini from neuronal NOS (nNOS) and endothelial NOS (eNOS), respectively. Both truncated enzymes exhibited cytochrome c reductase activities without calmodulin that were 7-21-fold higher than the nontruncated forms. With calmodulin, the truncated and wild-type enzymes reduced cytochrome c at approximately equal rates. Therefore, calmodulin functioned as a nonessential activator of the wild-type enzymes and a partial noncompetitive inhibitor of the truncated mutants. Truncated nNOS and eNOS plus calmodulin catalyzed NO formation at rates that were 45 and 33%, respectively, those of their intact forms. Without calmodulin, truncated nNOS and eNOS synthesized NO at rates 14 and 20%, respectively, those with calmodulin. By using stopped-flow spectrophotometry, we demonstrated that electron transfer into and between the two flavins is faster in the absence of the C terminus. Although both CPR and intact NOS can exist in a stable, one-electron-reduced semiquinone form, neither of the truncated enzymes do so. We propose negative modulation of FAD-FMN interaction by the C termini of both constitutive NOSs.

Amino Acid Sequence↗
PubMed2003

Blue light perception in plants. Detection and characterization of a light-induced neutral flavin radical in a C450A mutant of phototropin.

The LOV2 domain of Avena sativa phototropin and its C450A mutant were expressed as recombinant fusion proteins and were examined by optical spectroscopy, electron paramagnetic resonance, and electron-nuclear double resonance. Upon irradiation (420-480 nm), the LOV2 C450A mutant protein gave an optical absorption spectrum characteristic of a flavin radical even in the absence of exogenous electron donors, thus demonstrating that the flavin mononucleotide (FMN) cofactor in its photogenerated triplet state is a potent oxidant for redox-active amino acid residues within the LOV2 domain. The FMN radical in the LOV2 C450A mutant is N(5)-protonated, suggesting that the local pH close to the FMN is acidic enough so that the cysteine residue in the wild-type protein is likely to be also protonated. An electron paramagnetic resonance analysis of the photogenerated FMN radical gave information on the geometrical and electronic structure and the environment of the FMN cofactor. The experimentally determined hyperfine couplings of the FMN radical point to a highly restricted delocalization of the unpaired electron spin in the isoalloxazine moiety. In the light of these results a possible radical-pair mechanism for the formation of the FMN-C(4a)-cysteinyl adduct in LOV domains is discussed.

Avena↗
PubMed1996

Binding of Net-Fla, a netropsin-flavin hybrid molecule, to DNA: molecular mechanics and dynamics studies in vacuo and in water solution.

We have studied the binding of the hybrid netropsin-flavin (Net-Fla) molecule onto four sequences containing four A. T base pairs. Molecular mechanics minimizations in vacuo show numerous minimal conformations separated by one base pair. 400 ps molecular dynamics simulations in vacuo have been performed using the lowest minima as the starting conformations. During these simulations, the flavin moiety of the drug makes two hydrogen bonds with an amino group of a neighboring guanine. A 200 ps molecular dynamics simulation in explicit water solution suggests that the binding of Net-Fla upon the DNA substrate is enhanced by water bridges. A water molecule bridging the amidinium of Net-Fla to the N3 atom of an adenine seems to be stuck in the drug-DNA complex during the whole simulation. The fluctuations of the DNA helical parameters and of the torsion angles of the sugar-phosphate backbone are very similar in the simulations in vacuo and in water. The time auto-correlation functions for the DNA helical parameters decrease rapidly in the picosecond range in vacuo. The same functions computed from the water solution molecular dynamics simulations seem to have two modes: the rapid mode is similar to the behavior in vacuo, and is followed by a slower mode in the 10 ps range.

Binding Sites↗
PubMed2001

Advances in flavin and flavoprotein optical spectroscopy.

Flavins and flavoproteins are versatile redox cofactors that can perform both one- and two-electron transfer. Because they are highly colored in all three oxidation states, optical spectroscopy has been exploited for decades to study these redox changes. This review summarizes the application of optical spectroscopies to flavins and flavoproteins since 1990. Special emphasis is placed on new techniques, such as Stark spectroscopy, as well as significant refinements in more well known techniques, such as resonance Raman spectroscopy and ultrafast spectroscopy.

DNA Repair↗
PubMedDate not supplied

Multidomain flavin-dependent sulfhydryl oxidases.

Eukaryotic flavin-dependent sulfhydryl oxidases catalyze oxidative protein folding with the generation of disulfides and the reduction of oxygen to hydrogen peroxide. This review deals principally with the Quiescinsulfhydryl oxidases (QSOX) that are found in multiple forms in multicellular organisms and singly in a number of protozoan parasites. QSOX is an ancient fusion of thioredoxin domains and an FAD-binding module, ERV1/ALR. Interdomain disulfide exchanges transmit reducing equivalents from substrates to the flavin cofactor and thence to molecular oxygen. The in vitro substrate specificity of avian QSOX1 and the likely substrates of QSOXs in vivo are discussed. The location of QSOX immunoreactivity and mRNA expression levels in human cells and tissues is reviewed. Generally, there is a marked association of QSOX1 expression with cell types that have a high secretory load of disulfide-containing peptides and proteins. The abundance of sulfhydryl oxidases in the islets of Langerhans suggests that oxidative protein folding may directly contribute to the oxidative stress believed to be a factor in the progression to type II diabetes. Finally, the structure and mechanism of QSOX proteins is compared to their smaller stand-alone cousins: yeast ERV1p and ERV2p, the mammalian augmenter of liver regeneration (ALR), and the viral ALR homologs.

Amino Acid Motifs↗
PubMed2003

Oxidative folding of interleukin-2 is impaired in flavin-deficient jurkat cells, causing intracellular accumulation of interleukin-2 and increased expression of stress response genes.

Secretory proteins such as interleukin (IL)-2 undergo oxidative folding (disulfide formation) in the endoplasmic reticulum (ER) before secretion. Studies in yeast have suggested that oxidative folding depends on the flavoprotein Ero1p; unfolded proteins accumulate in the ER, triggering cellular stress response. Here, human lymphoid cells (Jurkat cells) were used to model effects of cellular flavin supply on secretion of IL-2 (containing one disulfide bond) and cellular stress response. Cells were cultured in media containing 0.85, 3.1, 12.6 or 300.6 nmol/L riboflavin for 5 wk, representing severely deficient, moderately deficient, physiologic and pharmacologic plasma concentrations in humans, respectively. Transport rates of riboflavin were increased in severely and moderately deficient cells compared with cells cultured in physiologic medium; this increase was not sufficient to prevent intracellular depletion of riboflavin, as judged by glutathione reductase activity and intracellular concentrations of glutathione. Intracellular accumulation of IL-2 was greater in severely deficient cells than in other groups. Nevertheless, severely deficient cells secreted normal amounts of IL-2 into the extracellular space, mediated by increased transcriptional activity of the IL-2 gene. Riboflavin-deficient cells responded to intracellular accumulation of IL-2 with increased expression of genes encoding ubiquitin-activating enzyme E1 and X box-binding protein, consistent with cellular stress. These findings are consistent with the hypothesis that flavin deficiency may cause cellular stress by accumulation of unfolded proteins in human cells.

Biological Transport↗
PubMed2001

Effects of flavin-binding motif amino acid mutations in the NADH-cytochrome b5 reductase catalytic domain on protein stability and catalysis.

Porcine NADH-cytochrome b5 reductase catalytic domain (Pb5R) has the RXY(T/S)+(T/S) flavin-binding motif that is highly conserved among the structurally related family of flavoprotein reductases. Mutations were introduced that alter the Arg(63), Tyr(65), and Ser(99) residues within this motif. The mutation of Tyr(65) to either alanine or phenylalanine destabilized the protein, produced an accelerated release of FAD in the presence of 1.5 M guanidine hydrochloride, and decreased the k(cat) values of the enzyme. These results indicate that Tyr(65) contributes to the stability of the protein and is important in the electron transfer from NADH to FAD. The mutation of Ser(99) to either alanine or valine, and of Arg(63) to either alanine or glutamine increased both the K(m) values for NADH (K(m)(NADH)) and the dissociation constant for NAD(+) (K(d)(NAD+)). However, the mutation of Ser(99) to threonine and of Arg(63) to lysine had very little effect on the K(m)(NADH) and K(d)(NAD+) values, and resulted in small changes in the absorption and circular dichroism spectra. These results suggest that the hydroxyl group of Ser(99) and the positive charge of Arg(63) contribute to the maintenance of the properties of FAD and to the effective binding of Pb5R to both NADH and NAD(+). In addition, the mutation of Arg(63) to either alanine or glutamine increased the apparent K(m) values for porcine cytochrome b5 (Pb5), while changing Arg(63) to lysine did not. The positive charge of Arg(63) may regulate the electron transfer through the electrostatic interaction with Pb5. These results substantiate the important role of the flavin-binding motif in Pb5R.

Amino Acid Motifs↗
PubMed1986

Studies in a model system on the effect of hydrogen bonding at hetero atoms of oxidized flavin on its electron acceptability.

The effect of hydrogen bonding at hetero atoms of oxidized flavin on its electron acceptability was studied by the ab initio molecular orbital method. The calculations were carried out for all possible lumiflavin-H2O complexes and for some lumiflavin-formamide complexes. Calculated data showed that the magnitudes of hydrogen bonding energy at the hetero atoms are in the order of N(3)H greater than N(5) greater than O(12) greater than N(1) greater than O(14). It was found that the atomic orbital coefficient of the lowest unoccupied molecular orbital is the largest at N(5) and that hydrogen bonding at N(1), N(5), O(12), and O(14) increases the electron acceptability of the oxidized flavin at N(5), while hydrogen bonding at N(3)H decreases it.

Chemical Phenomena↗
PubMed1989

Effects of pH and ionic strength on the binding of egg white riboflavin binding protein with flavins.

The effects of pH and ionic strength on the equilibrium constants and rate constants (binding and dissociation rate constants) between riboflavin binding protein (RBP) and flavins (riboflavin, 3-carboxymethylriboflavin [CMRF], and FMN) were studied by fluorometry. The equilibrium constant and the binding rate constant between RBP and riboflavin were pH-independent between pH 6 and 9, and both constants were also independent of the ionic strength, while the constants between RBP and CMRF or FMN were dependent on both pH and ionic strength. The dissociation rate constants between RBP and the flavins used here were not so dependent on pH and ionic strength in the pH region 6 to 9, and the patterns of pH profiles as a whole were similar to each other, although the constants for FMN were about 30-60 times larger than those for CMRF or riboflavin. RBP had lower affinity for FMN than for riboflavin in the neutral pH region, which is based on the small binding rate constant and the large dissociation rate constant for FMN. The former is due to an electrostatic repulsion force between negative net charges of RBP and the phosphate group of FMN, and the latter is due to steric interference by the phosphate group of FMN.

Animals↗
PubMed1991

On the ligand-protein and ligand-flavin interactions in NADPH-adrenodoxin reductase as studied by 31P- and 13C-NMR. Use of 13C-enriched FAD as a probe.

The interaction between 2',5'-ADP and NADPH-adrenodoxin reductase from bovine adrenocortical mitochondria was examined by titrating the enzyme with 2',5'-ADP, while the 31P-signals of 2',5'-ADP were being monitored by 31P-NMR. From the titration profile, the dissociation constant for the complex of the enzyme with 2',5'-ADP was estimated to be 0.22 +/- 0.05 mM. Adrenodoxin reductase was reconstituted with 13C-enriched FADs. The 13C-enriched FADs used were [2-13C]-, [4,10 alpha-13C2]-, and [4 alpha-13C]FAD. The 13C-NMR spectra of these reconstituted enzyme preparations showed 13C-resonance peaks corresponding to the enriched carbon atoms at 160.6 , 165.1, 136.6, and 152.4 ppm (2-, 4-, 4 alpha-, and 10 alpha-13C atoms, respectively). When 2',5'-ADP was bound to the reconstituted enzyme, these 13C-resonance peaks did not shift appreciably from those of the unbound enzyme, whereas in the complex of the reconstituted enzyme with NADP+, the signals for 4- and 10 alpha-13C shifted to higher fields by 2.1 and 0.7 ppm, respectively and the 4 alpha-13C signal shifted to a lower field by 1.4 ppm. These results suggest that in the complex of the enzyme with NADP+ the pyridine moiety is located in the vicinity of C(4 alpha)-C(4) region and that the pi-electron density of the 4 alpha-position of flavin is decreased in the enzyme-NADP+ complex. This argues in favor of the electron transfer from the dihydropyridine moiety of NADPH to the electron-deficient N(5) = C(4 alpha) region of flavin.

Adenosine Diphosphate↗
PubMed2002

FQR1, a novel primary auxin-response gene, encodes a flavin mononucleotide-binding quinone reductase.

FQR1 is a novel primary auxin-response gene that codes for a flavin mononucleotide-binding flavodoxin-like quinone reductase. Accumulation of FQR1 mRNA begins within 10 min of indole-3-acetic acid application and reaches a maximum of approximately 10-fold induction 30 min after treatment. This increase in FQR1 mRNA abundance is not diminished by the protein synthesis inhibitor cycloheximide, demonstrating that FQR1 is a primary auxin-response gene. Sequence analysis reveals that FQR1 belongs to a family of flavin mononucleotide-binding quinone reductases. Partially purified His-tagged FQR1 isolated from Escherichia coli catalyzes the transfer of electrons from NADH and NADPH to several substrates and exhibits in vitro quinone reductase activity. Overexpression of FQR1 in plants leads to increased levels of FQR1 protein and quinone reductase activity, indicating that FQR1 functions as a quinone reductase in vivo. In mammalian systems, glutathione S-transferases and quinone reductases are classified as phase II detoxification enzymes. We hypothesize that the auxin-inducible glutathione S-transferases and quinone reductases found in plants also act as detoxification enzymes, possibly to protect against auxin-induced oxidative stress.

Adaptation, Physiological↗
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↗
PubMed2001

Exploring the conformational equilibrium of E. coli thioredoxin reductase: characterization of two catalytically important states by ultrafast flavin fluorescence spectroscopy.

The conformational dynamics of wild-type Escherichia coli thioredoxin reductase (TrxR) and the mutant enzyme C138S were studied by ultrafast time-resolved fluorescence of the flavin cofactor in combination with circular dichroism (both in the flavin fingerprint and far-UV regions) and steady-state fluorescence and absorption spectroscopy. The spectroscopic data show two conformational states of the enzyme (named FO and FR), of which the physical characteristics differ considerably. Ultrafast fluorescence lifetime measurements make it possible to distinguish between the two different populations: Dominant picosecond lifetimes of approximately 1 ps (contribution 75%) and 7 ps (8%) are associated with the FO species in TrxR C138S. Long-lived fluorescence with two time constants in the range of 0.2-1 ns (total contribution 17%) originates from enzyme molecules in the FR conformation. The near absence of fast lifetime components in oxidized wild-type TrxR supports the idea of this enzyme being predominantly in the FR conformation. The emission spectrum of the FO conformation is blue-shifted with respect to that of the FR conformation. Because of the large difference in fluorescence characteristics, fluorescence measurements on time scales longer than 100 ps are fully determined by the fraction of enzyme molecules in the FR conformation. Binding of the thiol reagent phenyl mercuric acetate to wild-type enzyme and TrxR C138S stabilizes the enzymes in the FR conformation. Specific binding of the NADPH-analog, AADP(+), to the FR conformation resulted in dynamic fluorescence quenching in support of the multiple quenching sites model. Raising the temperature from 277K-323K resulted in a moderate shift to the FR conformation for TrxR C138S. High concentrations of the cosolvent glycerol triggered the domain rotation from the FO to the FR conformation.

Escherichia coli↗
PubMed1979

Fluorescence properties of reduced flavins and flavoproteins.

Fluorescence lifetimes and polarized emission properties of reduced flavin were measured using several model compounds and flavoproteins. Depending on the conditions of solvent and temperature or reduction method the lifetimes vary between 1 and 15 ns. The longer lifetime values are found in several forms of reduced lactate oxidase, in which a good correlation exists between fluorescence intensity and lifetime. In practically all flavoproteins the fluorescence is heterogeneous. Several mechanisms are proposed to explain the observed heterogeneity in lifetimes. The reduced models in glycerol at subzero temperature exhibit high degrees of polarization of the fluorescence, whereas distinct depolarization is encountered in several reduced flavoproteins suggesting a certain mobility of the flavin chromophor.

Flavins↗
PubMed1985

On the mechanism of flavin modification during inactivation of flavocytochrome b2 from baker's yeast by acetylenic substrates.

The reaction of 2-hydroxy-3-butynoate, a suicide substrate, with flavocytochrome b2 [F. Lederer (1974) Eur. J. Biochem. 88, 393-399] has been reinvestigated. It is shown that no inactivation occurs under anaerobic conditions. In the presence of ferricyanide, the partition ratio between oxidation and inactivation is 3200. Ketobutynoate has no effect on oxidized flavocytochrome b2. But it inactivates the reduced enzyme, while undergoing catalytic reduction to hydroxybutynoate. The partition ratio between reduction and inactivation is 5. Inactivation followed by borohydride reduction was carried out in parallel with lactate oxidase and flavocytochrome b2. The decomposition products of the initial adduct formed between flavin and inactivator were isolated and characterized. One of them (compound II) was obtained from both enzymes and is identical to the one previously isolated from hydroxybutynoate-inactivated lactate oxidase [Schonbrunn et al. (1976) Biochemistry 15, 1798-1807]. Its decarboxylated derivative (compound I) was also formed. Another major adduct, compound III, was isolated only from flavocytochrome b2. Its structure and the conditions in which it appears suggest it is formed from the same primary adduct as compounds I and II, but by a different decomposition mode, on the enzyme itself. Altogether these results strengthen the idea that inactivation is caused by reaction between oxidized flavin and an allenic carbanion, the isomerisation product of a normal reaction intermediate. It is proposed that differences in the rate-determining step of the redox reaction explain the differences in the inactivation process which are observed between flavocytochrome b2, lactate oxidase and hydroxyacid oxidase.

Biotransformation↗
PubMed1985

Nuclear magnetic resonance studies of the old yellow enzyme. 2. 13C NMR of the enzyme recombined with 13C-labeled flavin mononucleotides.

The apoenzyme of NADPH oxidoreductase, 'old yellow enzyme', was reconstituted with selectively 13C-enriched flavin mononucleotides and investigated by 13C NMR spectroscopy. The 13C NMR results confirm the results obtained by 15N NMR spectroscopy and yield additional information about the coenzyme-apoenzyme interaction. A strong deshielding of the C(2) and C(4) atoms of enzyme-bound FMN both in the oxidized and reduced state is observed, which is supposed to be induced by hydrogen-bond formation between the protein and the two carbonyl groups at C(2) and C(4) of the isoalloxazine ring system. The chemical shifts of all 13C resonances of the flavin in the two-electron-reduced state indicate that the N(5) atom is sp3-hybridized. From 31P NMR measurements it is concluded that the FMN phosphate group is not accessible to bulk solvent. The unusual 31P chemical shift of FMN in old yellow enzyme seems to indicate a different binding mode of the FMN phosphate group in this enzyme as compared to the flavodoxins. The 13C and 15N NMR data on the old-yellow-enzyme--phenolate complexes show that the atoms of the phenolate are more deshielded whereas the atoms of the enzyme-bound isoalloxazine ring are more shielded upon complexation. A non-linear correlation exists between the chemical shifts of the N(5) and the N(10) atoms and the pKa value of the phenolate derivative bound to the protein. Since the chemical shifts of N(5), N(10) and C(4a) are influenced most on complexation it is suggested that the phenolate is bound near the pyrazine ring of the isoalloxazine system. 15N NMR studies on the complex between FMN and 2-aminobenzoic acid indicate that the structure of this complex differs from that of the old-yellow-enzyme--phenolate complexes.

Apoenzymes↗
PubMed1991

Transient kinetics of flavin-photosensitized oxidation of reduced redox proteins. Comparison of c-type cytochromes and plastocyanins.

We have used laser flash photolysis to investigate the kinetics of oxidation of reduced plastocyanins obtained from spinach and the green alga Monoraphidium braunii by the triplet states of lumiflavin, riboflavin and FMN. We have compared the results of these experiments with the kinetics of reduction of the oxidized forms of these proteins by the corresponding flavin semiquinones, as well as with the kinetics of flavin oxidation and reduction of cytochrome c552 (Class I c-type cytochrome, generic name c553) from Monoraphidium. In all cases, the rate constants for oxidation were one or two orders of magnitude larger than for reduction, consistent with the greater thermodynamic driving force for the oxidation reaction. Similar steric and electrostatic effects were observed for both reactions with all proteins, suggesting that the same (or closely adjacent) sites were being utilized for electron removal and entry. The two algal proteins were quite similar to one another in their redox properties, consistent with their physiological role of being able to substitute for one another in photosynthetic electron transport. In contrast, the algal plastocyanin was more reactive than the spinach protein in both oxidation and reduction, suggesting differences in their steric properties at the site of electron transfer.

Chlorophyta↗