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A new flavin prosthetic group has been isolated in pure form from the electron-transferring flavoprotein of Peptostreptococcus elsdenii. Its structure has been established as the FAD derivative of 7-methyl-8-hydroxyisoalloxazine:[This symbol cannot be presented in ASCII format]Proof of this structure has been obtained by chemical synthesis of 7-methyl-8-hydroxyisoalloxazine models, and by stepwise degradation of the native compound to 7-methyl-8-hydroxyalloxazine. The orange chromophore is characterized by a strong absorption band with a maximum at 472 nm (ε = 41 000 M&Sup-1; cm⊃-1;) and a pK at 4.8 due to the ionisation of the C(8)-OH group. The properties of a series of functionally substituted derivatives of 8-hydroxy flavins and lumichromes have been investigated to provide a basis for interpreting the effects of pH on the spectroscopic properties of the 8-hydroxy derivatives of FAD and FMN. The 8-hydroxy derivative of FAD is bound by apo-D-amino acid oxidase; the complex shows no catalytic activity. The 8-hydroxy derivative of FMN is bound by apoflavodoxin to give a complex which has catalytic activity similar to that of native flavodoxin. The complex is reversibly reduced by dithionite, first to a relatively stable semiquinone and further to the dihydroflavin form. [ABSTRACT FROM AUTHOR]

The time dependence of the fluorescence of tryptophanyl and flavin residues in lipoamide dehydrogenase has been investigated with single-photon decay spectroscopy. When the two FAD molecules in the enzyme were directly excited the decay could only be analyzed in a sum of two exponentials with equal amplitudes. The phenomenon was observed at 4°C (τ1 = 0.8 ns, τ2 = 4.7 ns) and at 20°C (τ1 = 0.8 ns, `τ2 = 3.4 ns) irrespective of the emission and excitation wavelengths. This result reveals a difference in the nature of the two FAD centers. By excitation at 290 nm the fluorescence decay curves of tryptophan and FAD were obtained. The decays are analyzed in terms of energy transfer from tryptophanyl to flavin residues. The results, which are in good agreement with those obtained previously with static fluorescence methods, show that one of the two tryptophanyl residues within the subunit transfers its excitation energy to the flavin located at a distance of 1.5 nm. [ABSTRACT FROM AUTHOR]

The behaviour of bakers' yeast L-lactate dehydrogenase toward [2-²H]lactate, 3-chlorolactate and 2-hydroxy-3-butynoate is reported. By comparison with known facts concerning D-amino acid and lactic oxidases, the following conclusions are drawn. 1. Hydrogen removal at C-2 is rate-limiting in the overall reaction. This distinguishes L-lactate dehydrogenase from the oxidases where it is product dissociation which limits the rate. 2. The lack of dehydrohalogenation of 3-chlorolactate by L-lactate dehydrogenase, which differentiates the enzyme from the oxidases, can be explained if the reduction rate of ravin is much faster than for the oxidases. 3. Although only indirect evidence is presented, it seems most probable that the lactate C-2 hydrogen is removed as a proton, as in the case of D-amino acid oxidase and lactic oxidase. 4. The first steps of lactate oxidation are qualitatively similar for the two known enzymatically active forms of L-lactate dehydrogenase. [ABSTRACT FROM AUTHOR]

1. Initial velocity studies on β-cyclopiazonate oxidocyclase in the presence of the competitive inhibitor, 3-acetyl-5-scatyl-tetramic acid, indicates that this enzyme catalyzes the oxidocyclization of β-cyclopiazonic acid by a Ping-Pong-Bi-Bi mechanism. 2. It was possible to reduce the flavin coenzyme to the 3,4-dihydroflavin by consecutive cycles of reduction with BH4- and reoxidation by atmospheric oxygen. 8. The BH4-reduced enzyme shows a similar affinity for indole derivatives as does the native flavoquinone enzyme, but is inactive. β-Cyclopiazonic acid binds to the enzyme in a simple equilibrium, and the reduction of the 3,4-dihydroflavin to the 1,3,4,5.tetrahydroflavin or a conversion to 1,5-dihydroflavin does not proceed. 4. Partial reactivation of the enzyme by the photocatalyzed oxidation of the 3,4-dihydroflavin to the flavoquinone form was observed [ABSTRACT FROM AUTHOR]

Electron-transferring flavoprotein (ETF) was purified from the bacterium Paracoccus denitrificans and the structural and redox relationships to the porcine and human ETFs were investigated. The three proteins have essentially identical subunit masses and the a-helix content of the bacterial and porcine ETFs are very similar, indicating global structural similarity. An anti-(porcine ETF) polyclonal antibody that crossreacts with the human large and small subunits also crossreacts strongly with the large subunit of Paracoccus ETF. However, crossreactivity with the small subunit is very weak. Nonetheless, an amino-terminal peptide and four internal peptides of the small bacterial subunit show extensive sequence identity with the human small subunit. Local similarities in environment are also indicated by the intrinsic tryptophan fluorescence emission spectra of porcine and Paracoccus ETFs. Although the visible spectra of porcine and Paracoccus ETFs are virtually identical, flavin fluorescence in the bacterial protein is only 15% that of the mammalian protein. Further, the circular dichroic spectrum of the flavin in the bacterial protein is significantly more intense, suggesting that the microenvironment of the isoalloxazine ring is different in the two proteins. Enzymatic or photochemical reduction of Paracoccus ETF rapidly yields an anionic semiquinone; formation of the fully reduced flavin in the bacterial ETF is very slow. The spacing of the oxidation-reduction potentials of the flavin couples in the bacterial ETF is essentially identical to that in porcine ETF as judged from the disproportionation equilibrium of the bacterial ETF flavin semiquinone. Together, the enzymatic reduction and disproportionation equilibria suggest that the flavin potentials of the two ETFs must be very close. The data indicate that the structural properties of the bacterial and mammalian proteins and the thermodynamic properties of the flavin prosthetic group of the proteins are very similar. [ABSTRACT FROM AUTHOR]

Assignments for the 137 amino acid residues of Megasphaera elsdenii flavodoxin in the reduced state have been made using the sequential resonance assignment procedure. Several hydroxyl and sulfhydryl protons were observed at 41 °C at pH 8.3. Spin systems were sequentially assigned using phase-sensitive two-dimensional-correlated spectroscopy and phase-sensitive nuclear Overhauser enhancement spectroscopy. Spectra of the protein in H2O and of protein preparations either completely or partly exchanged against ²H2O were obtained. Use of the fast electron shuttle between the paramagnetic semiquinone and the diamagnetic hydroquinone state greatly simplified the NMR spectra, making it possible to assign easily the ¹H resonances of amino acid residues located in the immediate neighbourhood of the isoalloxazine ring. The majority of the nuclear Overhauser effect contacts between the flavin and the apoprotein correspond to the crystal structure of the flavin domain of Clostridium MP flavodoxin, but differences are also observed. The assignments provide the basis for the structure determination of M. elsdenii flavodoxin in the reduced state as well as for assigning the resonances of the oxidized flavodoxin. [ABSTRACT FROM AUTHOR]

The three-dimensional structure of flavocytochrome b2 (L-lactate dehydrogenase) from bakers' yeast (Saccharomyces cerevisiae) has recently been solved at 0.24-nm resolution [Mathews & Xia (1987) in Flavins and flavoproteins, Walter de Gruyter, Berlin, pp. 123-131]. We have used this structural information to investigate the roles of particular amino acid residues likely to be involved in the oxidation of L-lactate by kinetic analysis of mutant enzymes generated by site-directed mutagenesis of the isolated gene. The hydroxyl group of Tyr254 was expected to be important for the abstraction of the hydroxyl proton of L-lactate in the oxidation to pyruvate. Replacement of this tyrosine by phenylalanine reduced kcat from 190 ± 3 s-1 (25°C, pH 7.5) to 4.3 ± 0.1 s-1. This substitution had, however, no discernable effect on Km for lactate (0.54 ± 0.03 mM for the mutant compared with 0.49 ± 0.03 mM for the wild-type enzyme). Arg376 was expected to be essential for productive binding and orientation of L-lactate. Replacing Arg376 with lysine abolished all detectable activity. A total loss of enzymic activity was also observed when Lys349, thought likely to stabilize the anionic form of the flavin hydroquinone, was replaced by arginine. An amino acid residue replacement at a distance from the active site, Ala306 to serine, had a minor but significant effect on kcat (reduced from 190 s-1 to 160 s-1) and Km (increased from 0.49 mM to 0.83 mM) presumably arising from small conformational effects. The implications of these results are discussed in relation to the mechanism of L-lactate oxidation. [ABSTRACT FROM AUTHOR]

The reduction of flavins by BH4- is slow, but strongly catalyzed by light. The main product of the reaction is 1 ,5-dihydroflavin, i. e. normal leucoflavin, which is rapidly reoxidized upon admission of air. Under continuous aeration, however, a second reaction, irreversible by oxygen in the dark, becomes dominant yielding a tetrahydroflavin which, upon exhaustive reaction of BH4-, is autoxidized to give a new dihydroflavin of structure I. This compound is stable toward further oxidation in the crystalline state and in solution with organic solvents. Compound I has to be considered as a formal 3,4-dihydroflavin. It is photooxidizable to give starting flavin, reacts very slowly with S2O4- compared to flavoquinone (II), but is easily reduced by EDTA in the presence of light. The compound was also synthesized by an unequivocal route and found identical with the product obtained by BH4--reduction of lumiflavin. The light absorption spectrum of I shows absorption maxima at about 405, 320, 270, and 220 nm and is practically identical with that of the BH4---reduction product of the flavoproteins D- and L-amino acid oxidase. The BH4--reduction products of the amino acid oxidases are catalytically active. From this it follows that position 4 of the flavocoenzyme is not involved in enzymatic dehydrogenation. The first photo-excited triplet state of I has been investigated by optical and electron paramagnetic resonance spectroscopy. The structure of I was elucidated by means of infrared and nuclear magnetic resonance spectroscopy. [ABSTRACT FROM AUTHOR]

A substituted riboflavin (HNO-flavin) was isolated from D-6-hydroxynicotine oxidase. It was obtained from flavin peptides by hydrolysis with 6 N HCI at 95 °C or with aminopeptidase M. The riboflavin derivative had the spectral characteristics of 8α-substituted flavins. It showed a pH dependence of fluorescence with a pK of 4.65 and 86% quenching at pH 7. In thin-layer chromatography it was identical with 8aα-(N-3-histidy1)-riboflavin. Hydrolysis of HNO-flavin in 6N HCI at 125 ° liberated 1 mol histidine per mol flavin as shown by amino acid analysis Since FAD is the coenzyme of D-6-hydroxynicotine oxidase, these results are taken as evidence that this enzyme contains 8α-(N-3-histidyl)-flavin-adenine dinucleotide in the active center. [ABSTRACT FROM AUTHOR]

Improved methods have been devised for the isolation in μmole quantities of a pure flavin pentapeptide and its acid-hydrolysis product (SD-flavin) from inner-membrane preparations of heart mitochondria and from soluble, purified succinate dehydrogenase. SD-flavin differs from riboflavin in still having an amino acid covalently linked to the isoalloxazine ring system. SD-flavin may be compared with riboflavin and with various 8α-substituted synthetic flavins by optical spectrophotometry in the neutral and cationic states and by ESR and ENDOR spectrometry in the cationic radical state. On the basis of these experiments is was concluded that the FAD prosthetic group of mitochondrial succinate dehydrogenase is covalently linked through the 8α-position to the peptide backbone of the protein. This conclusion is in accord with the acid stability of the natural product and its tendency to yield riboflavin under reductive conditions. The unusual ph-fluorescence spectrum of the flavin strongly suggests that the 8α-methylene group is linked to an amino acid through a tertiary nitrogen group. [ABSTRACT FROM AUTHOR]

In the previous paper in this series it was shown that a pure flavin pentapeptide isolated from hepatic monoamine oxidase contains I mole each of serine and tyrosine and 2 moles of glycine, as well as an additional amino acid which is covalently linked to the 8α carbon of riboflavin. This amino acid has been identified as cysteine and the linkage to the flavin as a thioether on the basis of positive chloroplatinic and negative iodine-azide tests, performic acid oxidation or reduction with zinc, followed by acid hydrolysis, which yield cysteic acid or cysteine, respectively. The fluorescence properties of the flavin from monoamine oxidase agree with those expected for a thioether and its oxidation products. In regard to optical and electron spin resonance spectra, chemical stability, and fluorescence characteristics the flavin peptide isolated from the enzyme agrees excellently with the properties of synthetic cysteinyl 8α-riboflavin. [ABSTRACT FROM AUTHOR]