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PubMed2005

Biosynthesis of covalently bound flavin: isolation and in vitro flavinylation of the monomeric sarcosine oxidase apoprotein.

The covalently bound FAD in native monomeric sarcosine oxidase (MSOX) is attached to the protein by a thioether bond between the 8alpha-methyl group of the flavin and Cys315. Large amounts of soluble apoenzyme are produced by controlled expression in a riboflavin-dependent Escherichia coli strain. A time-dependent increase in catalytic activity is observed upon incubation of apoMSOX with FAD, accompanied by the covalent incorporation of FAD to approximately 80% of the level observed with the native enzyme. The spectral and catalytic properties of the reconstituted enzyme are otherwise indistinguishable from those of native MSOX. The reconstitution reaction exhibits apparent second-order kinetics (k = 139 M(-)(1) min(-)(1) at 23 degrees C) and is accompanied by the formation of a stoichiometric amount of hydrogen peroxide. A time-dependent reduction of FAD is observed when the reconstitution reaction is conducted under anaerobic conditions. The results provide definitive evidence for autoflavinylation in a reaction that proceeds via a reduced flavin intermediate and requires only apoMSOX and FAD. Flavinylation of apoMSOX is not observed with 5-deazaFAD or 1-deazaFAD, an outcome attributed to a decrease in the acidity of the 8alpha-methyl group protons. Covalent flavin attachment is observed with 8-nor-8-chloroFAD in an aromatic nucleophilic displacement reaction that proceeds via a quininoid intermediate but not a reduced flavin intermediate. The reconstituted enzyme contains a modified cysteine-flavin linkage (8-nor-8-S-cysteinyl) as compared with native MSOX (8alpha-S-cysteinyl), a difference that may account for its approximately 10-fold lower catalytic activity.

Apoenzymes↗
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↗
PubMed1998

Electron transfer from flavin to iron in the Pseudomonas oleovorans rubredoxin reductase-rubredoxin electron transfer complex.

Rubredoxin reductase (RR) and rubredoxin form a soluble and physiological eT complex. The complex provides reducing equivalents for a membrane-bound omega-hydroxylase, required for the hydroxylation of alkanes and related compounds. The gene (alkT) encoding RR has been overexpressed and the enzyme purified in amounts suitable for studies of eT by stopped-flow spectroscopy. The eT reactions from NADH to the flavin of RR and from reduced RR to the 1Fe and 2Fe forms of rubredoxin have been characterized by transient kinetic and thermodynamic analysis. The reductive half-reaction proceeds in a one-step reaction involving oxidized enzyme and a two-electron-reduced enzyme-NAD+ charge-transfer complex. Flavin reduction is observed at 450 nm and charge-transfer formation at 750 nm; both steps are hyperbolically dependent on NADH concentration. The limiting flavin reduction rate (180 +/- 4 s-1) is comparable to the limiting rate for charge-transfer formation (189 +/- 7 s-1) and analysis at 450 and 750 nm yielded enzyme-NADH dissociation constants of 36 +/- 2 and 43 +/- 5 microM, respectively. Thermodynamic analysis of the reductive half-reaction yielded values for changes in entropy (DeltaS = -65.8 +/- 2.2 J mol-1 K-1), enthalpy (DeltaH = 37.8 +/- 0.6 kJ mol-1) and Gibbs free energy (DeltaG = 57.5 +/- 0.7 kJ mol-1 at 298 K) during hydride ion transfer to the flavin N5 atom. Spectral analysis of mixtures of 1Fe or 2Fe rubredoxin and RR suggest that conformational changes accompany eT complex assembly. Both the 1Fe (nonphysiological) and 2Fe (physiological) forms of rubredoxin were found to oxidize two electron-reduced rubredoxin reductase with approximately equal facility. Rates for the reduction of rubredoxin are hyperbolically dependent on rubredoxin concentration and the limiting rates are 72. 7 +/- 0.6 and 55.2 +/- 0.3 s-1 for the 1Fe and 2Fe forms, respectively. Analysis of the temperature dependence of eT to rubredoxin using eT theory revealed that the reaction is not adequately described as a nonadiabatic eT reaction (HAB >> 80 cm-1). eT to both the 1Fe and 2Fe forms of rubredoxin is therefore gated by an adiabatic process that precedes the eT reaction from flavin to iron. Possible origins of this adiabatic event are discussed.

Electron Transport↗
PubMed2004

The photoinduced triplet of flavins and its protonation states.

The photogenerated triplet states of riboflavin and flavin mononucleotide (FMN) have been examined by time-resolved electron paramagnetic resonance (EPR) spectroscopy at low temperature (T = 80 K). Because of the high time resolution of the utilized EPR instrumentation, the triplets are for the first time observed in the nonequilibrated electron-spin polarized state and not in their equilibrated forms with the population of the triplet sublevels governed by Boltzmann distribution. The electron-spin polarization pattern directly reflects the anisotropy of the intersystem crossing from the excited singlet-state precursor. Spectral analysis of the resulting enhanced absorptive and emissive EPR signals yields the zero-field splitting parameters, |D| and |E|, and the zero-field populations of the triplet at high accuracy. These parameters are sensitive probes for the protonation state of the flavin's isoalloxazine ring, as becomes evident by a comparison of the spectra recorded at different pH values of the solvent. The three protonation states of the flavins can furthermore be distinguished by the kinetics of the transient EPR signals, which are dominated by spin-lattice relaxation. The fastest decays are observed for the protonated FMN and riboflavin triplets, followed by the deprotonated flavin triplets. Slow decays are measured for the triplet states of neutral FMN and riboflavin. Because proton transfer is found to be slow on the time scale of spin-polarized triplet detection by transient EPR, the pH-dependent spin-relaxation and zero-field splitting parameters offer a novel approach to probe the protonation state of flavins in their singlet ground state through the characterization of their triplet-state properties.

Electron Spin Resonance Spectroscopy↗
PubMed2006

15N solid-state NMR provides a sensitive probe of oxidized flavin reactive sites.

Flavins are central to the reactivity of a wide variety of enzymes and electron transport proteins. There is great interest in understanding the basis for the different reactivities displayed by flavins in different protein contexts. We propose solid-state nuclear magnetic resonance (SS-NMR) as a tool for directly observing reactive positions of the flavin ring and thereby obtaining information on their frontier orbitals. We now report the SS-NMR signals of the redox-active nitrogens N1 and N5, as well as that of N3. The chemical shift tensor of N5 is over 720 ppm wide, in accordance with the predictions of theory and our calculations. The signal of N3 can be distinguished on the basis of coupling to 1H absent for N1 and N5, as well as the shift tensor span of only 170 ppm, consistent with N3's lower aromaticity and lack of a nonbonding lone pair. The isotropic shifts and spans of N5 and N1 reflect two opposite extremes of the chemical shift range for "pyridine-type" N's, consistent with their electrophilic and nucleophilic chemical reactivities, respectively. Upon flavin reduction, N5's chemical shift tensor contracts dramatically to a span of less than 110 ppm, and the isotropic chemical shift changes by approximately 300 ppm. Both are consistent with loss of N5's nonbonding lone pair and decreased aromaticity, and illustrate the responsiveness of the 15N chemical shift principal values to electronic structure. Thus. 15N chemical shift principal values promise to be valuable tools for understanding electronic differences that underlie variations in flavin reactivity, as well as the reactivities of other heterocyclic cofactors.

Binding Sites↗
PubMed1999

Chromatographic identification of a new flavin derivative in plain yogurt.

The presence of flavin derivatives in plain yogurt was assessed by high-performance liquid chromatography (HPLC) and thin-layer chromatography (TLC). The total amount of flavins in yogurts produced by different companies was variable and oscillated between 150.0 and 218.8 microg/100 g. Riboflavin (RF) was the predominant flavin. Besides RF, flavin adenine dinucleotide (FAD), flavin mononucleotide (FMN), 7alpha-hydroxyriboflavin (7alpha-HRF), 4'- or 5'-D-riboflavin-beta-D-galactoside (RFgal), and traces of 10-formylmethylflavin (10-FMF) and 10-hydroxyethylflavin (10-HEF) have been found. It is known that RFgal may be obtained using enzymes or cultures of different microorganisms, but its presence in foodstuffs has not been demonstrated, yet.

Chromatography, High Pressure Liquid↗
PubMed1976

Myeloma with xanthoderma due to an IgG lambdamonoclonal anti-flavin antibody.

When yellow skin and yellow hair developed in an elderly patient with multiple myeloma, we ruled out the usual causes of such pigmentation but identified a monoclonal IgGlambda (lgGGar) with anti-flavin antibody activity. Purified IgGGar was bright yellow, and the acid-dissociated chromophore was identified as riboflavin by chromatography and absorption spectroscopy. Native IgGGar contained 1.45 moles of flavin per mole of IgG, and increased to 2 moles with addition of riboflavin to saturation. The flavin was localized to the Fab fragment and was bound to IgGGar with high affinity. IgGGar showed strongest affinities for riboflavin, flavin mononucleotide and flavin adenine dinucleotide, and lower affinities for dinitrophenyl derivatives and naphthoquinone. The demonstration of hapten bound to the circulating monoclonal immunoglobulin in this case suggests the possibility of bound but colorless haptens on other myeloma proteins as well as on normal immunoglobulins.

Aged↗
PubMed2000

Design and properties of human D-amino acid oxidase with covalently attached flavin.

An "artificial flavinylation" approach was developed to replace a native noncovalent flavin prosthetic group with a covalently attached flavin analogue in recombinant human d-amino acid oxidase. The protein residue Gly-281 was replaced with Cys by site-directed mutagenesis, followed by reaction between mutated apoenzyme and the thiol-reactive flavin analogue, 8-methylsulfonyl FAD. The stoichiometric process of flavin attachment was accompanied by gain in enzymatic activity, reaching up to 26% activity of the recombinant native enzyme. The steady-state kinetic data together with the results of limited proteolysis and benzoate-binding studies suggest that, although mutation perturbs protein structural and catalytic properties, the flavinylation alone does not have any negative impact. We conclude that, despite the implemented restraints on its mobility, the covalently attached flavin is properly positioned within the protein active site and acts efficiently during d-amino acid oxidase catalytic turnover.

Base Sequence↗
PubMed2002

Reduced flavins promote oxidative DNA damage in non-respiring Escherichia coli by delivering electrons to intracellular free iron.

When cells are exposed to external H(2)O(2), the H(2)O(2) rapidly diffuses inside and oxidizes ferrous iron, thereby forming hydroxyl radicals that damage DNA. Thus the process of oxidative DNA damage requires only H(2)O(2), free iron, and an as-yet unidentified electron donor that reduces ferric iron to the ferrous state. Previous work showed that H(2)O(2) kills Escherichia coli especially rapidly when respiration is inhibited either by cyanide or by genetic defects in respiratory enzymes. In this study we established that these respiratory blocks accelerate the rate of DNA damage. The respiratory blocks did not substantially affect the amounts of intracellular free iron or H(2)O(2), indicating that that they accelerated damage because they increased the availability of the electron donor. The goal of this work was to identify that donor. As expected, the respiratory inhibitors caused a large increase in the amount of intracellular NADH. However, NADH itself was a poor reductant of free iron in vitro. This suggests that in non-respiring cells electrons are transferred from NADH to another carrier that directly reduces the iron. Genetic manipulations of the amounts of intracellular glutathione, NADPH, alpha-ketoacids, ferredoxin, and thioredoxin indicated that none of these was the direct electron donor. However, cells were protected from cyanide-stimulated DNA damage if they lacked flavin reductase, an enzyme that transfers electrons from NADH to free FAD. The K(m) value of this enzyme for NADH is much higher than the usual intracellular NADH concentration, which explains why its flux increased when NADH levels rose during respiratory inhibition. Flavins that were reduced by purified flavin reductase rapidly transferred electrons to free iron and drove a DNA-damaging Fenton system in vitro. Thus the rate of oxidative DNA damage can be limited by the rate at which electron donors reduce free iron, and reduced flavins become the predominant donors in E. coli when respiration is blocked. It remains unclear whether flavins or other reductants drive Fenton chemistry in respiring cells.

Cyanides↗
PubMed1993

Flavin dynamics in oxidized Clostridium beijerinckii flavodoxin as assessed by time-resolved polarized fluorescence.

The time-resolved fluorescence characteristics of flavin in oxidized flavodoxin isolated from the anaerobic bacterium Clostridium beijerinckii have been examined. The fluorescence intensity decays were analyzed using the maximum-entropy method. It is demonstrated that there exist large differences in fluorescence behaviour between free and protein-bound FMN. Three fluorescence lifetime components are found in oxidized flavodoxin, two of which are not present in the fluorescence-intensity decay of free FMN. The main component is distributed at 30 ps, with relative contribution of 90%. Another minor component (4% contribution) is distributed at 0.5 ns. The third component is distributed at 4.8 ns (6%), coinciding with the main distribution present in the fluorescence decay of free FMN. The results allowed us to determine the dissociation constant, Kd = 2.61 x 10(-10) M (at 20 degrees C). Collisional fluorescence-quenching experiments revealed that the flavin moiety responsible for the longest fluorescence lifetime is, at least partially, exposed to the solvent. The shortest lifetime is not affected significantly, indicating that it possibly originates from an active-site conformation in which the flavin is more or less buried in the protein and not accessible to iodide. The fluorescence anisotropy behaviour of free and protein-bound FMN was examined and analyzed with the maximum-entropy method. It was found that an excess of apoflavodoxin is required to detect differences between free and protein-bound FMN. In free FMN one single distribution of rotational correlation times is detected, whereas in flavodoxin the anisotropy decay is composed of more than one distribution. Associative analysis of fluorescence anisotropy decays shows that part of the 4.8 ns fluorescence lifetime present in the flavodoxin fluorescence decay, is coupled to a rotational correlation time similar to that of free FMN in solution, while another part of this lifetime is coupled to a longer correlation time of about 1 ns. This finding is in accordance with earlier studies [Barman, B. G. & Tollin, G. (1972) Biochemistry 11, 4746-4754] in which it was proposed that the first binding step of the flavin to the protein involves the phosphate group rather than another part of the FMN. The two shortest fluorescence lifetimes, which do not carry information on the long-term rotational behaviour of the protein, seem nonetheless to be associated with a longer rotational correlation time which is comparable to overall protein tumbling. These lifetime components probably originate from a complex in which the flavin-ring system is more or less immobilized within the protein matrix.

Clostridium↗
PubMed1966

Flavin-linked dehydrogenation of ether glycols by cell-free extracts of a soil bacterium.

Payne, W. J. (University of Georgia, Athens), and R. L. Todd. Flavin-linked dehydrogenation of ether glycols by cell-free extracts of a soil bacterium. J. Bacteriol. 91:1533-1536. 1966.-Cell-free extracts of bacterium TEG-5 grown on tetraethylene glycol dehydrogenated a variety of ether glycols and nonylphenoxy and secondary alcohol ethoxy derivatives. Nicotinamide nucleotides did not serve as electron acceptors, but ferricyanide was effective. Dialysis of crude extract depressed activity with tetraethylene glycol, which was restored then by flavine adenine dinucleotide (FAD) or boiled extract supernatant fluid (BES) but not by other flavins. Precipitatation of extract protein at pH 4.0 at 80% ammonium sulfate saturation dissociated FAD and yielded an inactive fraction. Activity was restorable by FAD and BES but not by other flavins. Ethylene glycol was not dehydrogenated by the acid ammonium sulfate fraction with FAD. Atabrine inhibited tetraethylene glycol oxidation, and the inhibition was relieved by FAD but not by other flavins. Tergitols which have sulfated ethoxy side chains on secondary alcohols were not dehydrogenated, but those with free ethoxy side chains on identical alcohols were.

Alcohols↗
PubMed1976

Site of action of nonheme iron in the malate (flavine adenine dinucleotide) pathway of Mycobacterium phlei.

Irradiation with ultraviolet light (360 nm) of cell-free extracts, electron-transport particles, and soluble components from Mycobacterium phlei resulted in the loss of malate oxidation by the flavine adenine dinucleotide pathway both in cell-free extracts and reconstituted systems. Addition of vitamin K1 restored the loss to the extent of 14% and 11% in cell-free extracts and reconstituted systems respectively. Electron-transport particles from M. phlei upon reduction with malate exhibited electron-paramagnetic resonance signals at g = 2.002 and 1.94, characteristic of napthosemiquinone and nonheme iron protein, respectively. Upon irradiating the particles with ultraviolet light (360 nm) these signals were not observed. Particulate flavine-adenine-dinucleotide-dependent malate dehydrogenase (EC 1.1.1.37) of M. phlei assayed by the 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyl tetrazolium bromide and phenazine methosulfate - 2,6-dichlorophenolindophenol systems, which trap electrons at cytochrome c and at the flavine level respectively, was inhibited by o-phenanthroline. These observations suggest that nonheme iron protein is sensitive to ultraviolet light (360 nm) and participates before or in combination with flavine in the malate (flavine adenine dinucleotide) pathway of M. phlei.

Alcohol Oxidoreductases↗
PubMed2004

Thermostable flavin reductase that couples with dibenzothiophene monooxygenase, from thermophilic Bacillus sp. DSM411: purification, characterization, and gene cloning.

Flavin reductase is essential for the oxygenases involved in microbial dibenzothiophene (DBT) desulfurization. An enzyme of the thermophilic strain, Bacillus sp. DSM411, was selected to couple with DBT monooxygenase (DszC) from Rhodococcus erythropolis D-1. The flavin reductase was purified to homogeneity from Bacillus sp. DSM411, and the native enzyme was a monomer of M(r) 16 kDa. Although the best substrates were flavin mononucleotide and NADH, the enzyme also used other flavin compounds and acted slightly on nitroaromatic compounds and NADPH. The purified enzyme coupled with DszC and had a ferric reductase activity. Among the flavin reductases so far characterized, the present enzyme is the most thermophilic and thermostable. The gene coded for a protein of 155 amino acids with a calculated mass of 17,325 Da. The enzyme was overproduced in Escherichia coli, and the specific activity in the crude extracts was about 440-fold higher than that of the wild-type strain, Bacillus sp. DSM411.

Bacillus↗
PubMed1998

Excited-state dynamics of fully reduced flavins and flavoenzymes studied at subpicosecond time resolution.

The photophysics of the fully reduced states of a number of flavins (flavin mononucleotide, flavin adenine dinucleotide and 3-N-methyllumiflavin) and flavoenzymes (glucose oxidase from Aspergillus niger and the flavodehydrogenase component isolated from flavocytochrome b2) was studied using subpicosecond laser excitation at lambda = 312 nm. The prompt transient absorption spectra (measured from 400 to 850 nm) were all closely similar in the case of the free flavins in aqueous solution. The decay of the transient absorbance obeyed biexponential kinetics with a fast component of lifetime ranging from 4 to 130 ps and a slower phase with a lifetime above 1 ns. The spectral structure changed appreciably during the rapid decay phase. In contrast, in the case of the enzymes only a very slight decay was apparent over the probed time interval (1 ns) and the shape of the spectrum remained unchanged. It is proposed that the two transient spectra appearing in the free flavins correspond to two conformations differing by their degree of nonplanarity, whereas in the flavoenzymes only one conformation is possible.

Aspergillus niger↗
PubMed2000

Initial-rate kinetics of the flavin reductase reaction catalysed by human biliverdin-IXbeta reductase (BVR-B).

The initial-rate kinetics of the flavin reductase reaction catalysed by biliverdin-IXbeta reductase at pH 7.5 are consistent with a rapid-equilibrium ordered mechanism, with the pyridine nucleotide binding first. NADPH binding to the free enzyme was characterized using stopped-flow fluorescence quenching, and a K(d) of 15.8 microM was calculated. Equilibrium fluorescence quenching experiments indicated a K(d) of 0.55 microM, suggesting that an enzyme-NADPH encounter complex (K(d) 15.8 microM) isomerizes to a more stable 'nucleotide-induced' conformation. The enzyme was shown to catalyse the reduction of FMN, FAD and riboflavin, with K(m) values of 52 microM, 125 microM and 53 microM, respectively. Lumichrome was shown to be a competitive inhibitor against FMN, with a K(i) of 76 microM, indicating that interactions with the isoalloxazine ring are probably sufficient for binding. During initial experiments it was observed that both the flavin reductase and biliverdin reductase activities of the enzyme exhibit a sharp optimum at pH 5 in citrate buffer. An initial-rate study indicated that the enzyme obeys a steady-state ordered mechanism in this buffer. The initial-rate kinetics in sodium acetate at pH 5 are consistent with a rapid-equilibrium ordered mechanism, indicating that citrate may directly affect the enzyme's behaviour at pH 5. Mesobiliverdin XIIIalpha, a synthetic biliverdin which binds to flavin reductase but does not act as a substrate for the enzyme, exhibits competitive kinetics with FMN (K(i) 0.59 microM) and mixed-inhibition kinetics with NADPH. This is consistent with a single pyridine nucleotide site and competition by FMN and biliverdin for a second site. Interestingly, flavin reductase/biliverdin-IXbeta reductase has also been shown to exhibit ferric reductase activity, with an apparent K(m) of 2.5 microM for the ferric iron. The ferric reductase reaction requires NAD(P)H and FMN. This activity is intriguing, as haem cleavage in the foetus produces non-alpha isomers of biliverdin and ferric iron, both of which are substrates for flavin reductase/biliverdin-IXbeta reductase.

Aerobiosis↗
PubMed1986

Interactions of pyridine nucleotides with redox forms of the flavin-containing NADH peroxidase from Streptococcus faecalis.

The flavin-containing NADH peroxidase of Streptococcus faecalis 10C1, which catalyzes the reaction: NADH + H+ + H2O2----NAD+ + 2H2O, has been purified to homogeneity in our laboratory for analyses of both its structure and redox behavior. Our findings indicate that the enzyme is a tetramer of four apparently identical subunits (Mr = 46,000/subunit), each containing one FAD coenzyme and a second non-flavin, nonmetal redox center. There is no evidence of nonequivalence among the flavins. Dithionite reduction of the enzyme occurs in two steps, with end points of 0.96 and 2.05 eq/FAD. The first step generates a two-electron reduced form of the enzyme (EH2) which is spectrally identical with that generated by aerobic addition of NADH. Our studies suggest that the long-wavelength absorbance band (lambda max approximately 540 nm) exhibited by this form results from charge-transfer interaction between the reduced non-flavin redox center and the oxidized flavin. A second type of long-wavelength charge-transfer absorbance band (lambda max approximately 770 nm) is generated on anaerobic addition of 1 eq of NADH to EH2 and results from interaction between oxidized FAD and the reduced pyridine nucleotide. Either the EH2 X NAD+ or the EH2 X NAD+ X NADH forms may be involved in the catalytic mechanism of the enzyme, as both are reactive with hydrogen peroxide.

Amino Acids↗
PubMed1985

The amino acid sequences of the flavin-peptides of dimethylglycine dehydrogenase and sarcosine dehydrogenase from rat liver mitochondria.

The flavoenzymes dimethylglycine dehydrogenase (EC 1.5.99.2) and sarcosine dehydrogenase (EC 1.5.99.1) contain covalently bound FAD linked via the 8 alpha-position of the isoalloxazine ring to the imidazole N(3) of a histidine residue (Cook, R. J., Misono, K. S., and Wagner, C. (1984) J. Biol. Chem. 259, 12475-12480). The flavin-peptides from tryptic digests of these two enzymes have been isolated and sequenced. Automated sequence analysis showed that the flavin-peptide from dimethylglycine dehydrogenase contained 25 amino acid residues in the following sequence: Ser-Glu-Leu-Thr-Ala-Gly-Ser- Thr-Trp-His(flavin)-Ala-Ala-Gly-Leu-Thr-Thr-Tyr-Phe-His-Pro-Gly-Ile-A sn-Leu-Lys. The sequence determined for the flavin-peptide from sarcosine dehydrogenase contained 14 amino acid residues Leu-Thr-Ser-Gly-Thr-Thr-Trp-His(flavin)-Thr-Ala-Gly-Leu-Gly-Arg.

Amino Acid Sequence↗
PubMedDate not supplied

Role of cytochromes P-450 and flavin-containing monooxygenase in the biotransformation of 4-fluoro-N-methylaniline.

In vivo and in vitro biotransformation of secondary aromatic amines was investigated using 4-fluoro-N-methylaniline as the model compound. Attention was focused on the role of cytochromes P-450 and the flavin-containing monooxygenase in formation of the various metabolic products. In vitro studies using microsomal preparations, purified reconstituted cytochromes P-450 IA1 and IIB1 and purified flavin-containing monooxygenase, demonstrated that N-demethylation, N-hydroxylation, and 4-hydroxylation accompanied by dehalogenation were all catalyzed by both the cytochrome P-450 system and the flavin-containing monooxygenase. The turnover rates of the two monooxygenase systems for the various metabolite formations and the reaction pathways involved, were shown to vary significantly. This study provides direct experimental support for the conclusion that the aromatic ring hydroxylation of secondary N-methylamines can be a consequence of flavin-containing monooxygenase catalyzed N-hydroxylation rather than of direct aromatic ring hydroxylation. The results obtained in vitro were compared with data from urine analysis of rats exposed to 4-fluoro-N-methylaniline. In this way it could be demonstrated that the major phase I biotransformation reactions for formation of urine excretable metabolites are (i) the cytochrome P-450-catalyzed N-demethylation followed by aromatic ring hydroxylation of the 4-fluoroaniline formed, and (ii) flavin-containing monooxygenase and cytochrome P-450-dependent formation of defluorinated 4-hydroxy-N-methylaniline.

Aniline Compounds↗