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S Ghisla

Publications and source records attributed to S Ghisla.

At least 109 records · Page 6Linked to original sources
PubMed1984

4-Thioflavins as active site probes of flavoproteins. Reactions with sulfite.

4-Thioflavins react with sulfite under aerobic conditions to yield highly fluorescent products with absorption maxima around 410 nm. These products have been identified as 4-hydroxy-4-sulfonylflavins, and have been shown to arise from a series of reactions following the O2-dependent reoxidation of an intermediate with absorption maxima at 363 and 465 nm. Under anaerobic conditions, the same intermediate is formed, but decays to a 350 nm absorbing species, which is probably the N(5)-sulfite adduct of 4-thioflavin. A plausible mechanism is described for the formation of the derivatives, and several of their chemical and physical properties are described. Distinctly different results between different proteins are obtained when sulfite reacts with enzyme-bound 4-thioflavins. 4-Thio-FAD-D-amino acid oxidase and 4-thio-FMN-lactate oxidase react rapidly to yield the N(5)-sulfite adducts, as occurs with the native enzymes. 4-Thio-FAD-p-hydroxybenzoate hydroxylase reacts slowly in a manner paralleling the reaction with the free 4-thioflavins.

4-Hydroxybenzoate-3-Monooxygenase↗
PubMed1984

4-Thioflavins as active site probes of flavoproteins. General properties.

4-Thioflavins (oxygen at position 4 replaced by sulfur) have been studied as potential active site probes of flavoproteins. They react readily with thiol reagents, with large spectral changes, which should be useful for testing the accessibility of the flavin 4-position in flavoproteins. They have an oxidation-reduction potential at pH 7 of -0.055 V, approximately 0.15 V higher than that of native flavins. The spectral characteristics in the fully reduced state show two clear absorption bands, dependent on the ionization state (pK = 4.5). The lowest energy band of the neutral dihydroflavin has a maximum at approximately 485 nm while that of the anion is approximately 425 nm. This should be useful in defining the ionization state of the reduced flavin in flavoproteins. The spectral characteristics of the semiquinoid forms of 4-thioflavins have been determined bound to the apoproteins of flavodoxin and D-amino acid oxidase. The neutral radical has an absorption maximum at 730 nm, while the anion radical has an unusually sharp peak at 415 nm. The reduced forms of 4-thioflavins, free and enzyme bound, react with O2 to regenerate oxidized 4-thioflavin. Reduced 4-thio-FAD p-hydroxybenzoate hydroxylase, however, in its reaction with O2, undergoes a substantial conversion to the native FAD-enzyme. 4-Thioflavins are unusually susceptible to attack by nucleophiles such as hydroxylamine and amines to form the respective 4-hydroxyimino- and 4-aminoflavins, offering the possibility of forming stable covalent flavin-protein linkages with suitably positioned protein residues. Thiols also react with 4-thioflavins, promoting their conversion to the normal (4-oxo) flavin coenzymes. Such reactivity has been found with the apoenzymes of glucose oxidase and lactate oxidase, providing evidence for a thiol residue in the active site of these enzymes.

4-Hydroxybenzoate-3-Monooxygenase↗
PubMed1984

Mechanistic studies with general acyl-CoA dehydrogenase and butyryl-CoA dehydrogenase: evidence for the transfer of the beta-hydrogen to the flavin N(5)-position as a hydride.

Butyryl-CoA dehydrogenase from Megasphera elsdenii catalyzes the exchange of the alpha- and beta-hydrogens of substrate with solvent [Gomes, B., Fendrich, G., & Abeles, R. H. (1981) Biochemistry 20, 1481-1490]. The stoichiometry of this exchange was determined by using 3H2O label as 1.94 +/- 0.1 per substrate molecule. The rate of 3H label incorporation into substrate under anaerobic conditions is monophasic, indicating that both the alpha- and beta-hydrogens exchange at the same rate. The exchange in 2H2O leads to incorporation of one 2H each into the alpha- and the beta-positions of butyryl-CoA, as determined by companion 1H NMR experiments and confirmed by mass spectroscopic analysis. In contrast, with general acyl-CoA dehydrogenase from pig kidney, only exchange of the alpha-hydrogen was found. The beta-hydrogen is the one that is transferred (reversibly) to the flavin 5-position during substrate dehydrogenation. This was demonstrated by reacting 5-3H- and 5-2H-reduced 5-deaza-FAD-general acyl-CoA dehydrogenase with crotonyl-CoA. Only one face of the reduced flavin analogue is capable of transferring hydrogen to substrate. The rate of this reaction is 11.1 s-1 for 5-deaza-FAD-enzyme and 2.2 s-1 for [5-2H]deaza-FAD-enzyme, yielding an isotope effect of 5. These values compare with a rate of 2.6 s-1 for the reaction of native reduced enzyme with crotonyl-CoA. The two reduced enzymes (normal vs. 5-deaza-FAD-enzyme) thus react at similar rates, indicating a similar mechanism.(ABSTRACT TRUNCATED AT 250 WORDS)

Acyl Coenzyme A↗
PubMed1984

Structure of a novel flavin chromophore from Avena coleoptiles, the possible 'blue light' photoreceptor.

A yellow chromophore has been isolated from Avena coleoptiles grown in the dark. It had previously been shown by Zenk [Zenk, M. H. (1967) Z. Pflanzenphysiol. 56, 122 - 140] to be a flavin of still unidentified structure, and had been suggested to be the 'blue light' photoreceptor in this organism. The structure of this flavin (F1-X) has been identified as 5'-malonylriboflavin by a combination of physicochemical techniques, and by its identity with a sample obtained synthetically. The 5'-malonylester linkage is relatively labile towards hydrolysis and photolysis, it isomerizes to an equilibrium mixture containing probably the 4' isomer. The electronic spectra of 5'-malonylriboflavin (absorption and fluorescence) are practically identical to those of normal riboflavin (vitamin B2).

Carrier Proteins↗
PubMed1983

Oxidation of 2-thioflavins by peroxides. Formation of flavin 2-S-oxides.

The reaction of 2-thioriboflavin (sulfur replacing the oxygen substituent at position 2 alpha) with hydrogen peroxide at pH approximately 10 leads to a blue flavin (lambda max = 565 nm) which was purified in stable, homogeneous form. Titrations of 2-thioflavins with m-chloroperoxybenzoic acid also yield the same blue flavins with consumption of 1 eq of peracid. Anaerobic reduction of the blue flavin by sodium dithionite requires 4e- eq, and leads to formation of 1,5-dihydro-2-thioflavin. Oxidation of the latter with O2 restores the original 2-thioflavin. pH titration of the blue flavin shows two pKa values of 2.4 and 6.6, with no apparent ionization in the pH range 8-11. These results suggest that the blue flavin is a flavin 2-S-oxide. The visible absorption spectra of flavin 2-S-oxides show a pronounced dependence on solvent polarity. This property suggests that these flavin analogs may be useful hydrophilic/hydrophobic probes of flavoprotein active sites. Flavin 2-S-oxides can be oxidized further to the 2-sulfinate and 2-sulfonate analogs, some properties of which are described.

Chromatography, High Pressure Liquid↗
PubMed1983

Bioluminescence emission from the reaction of luciferase-flavin mononucleotide radical with O2-..

The blue neutral luciferase flavin radical has been shown not to be in a catalytically significant equilibrium with species leading to emission of light [Kurfürst, M., Ghisla, S., Presswood, R., & Hastings, J. W. (1982) Eur. J. Biochem. 123, 355-361]. It is shown here that this radical can nevertheless react with O2-. to form a species that is competent in light emission. From its properties, the species formed is deduced to be luciferase-FMNH 4a-hydroperoxide, a key intermediate in the normal luciferase reaction. Although it is concluded that this intermediate can undergo a reversible homolytic dissociation to yield free superoxide and the corresponding luciferase radical, the slowness of these steps precludes a catalytic significance for these pathways in the normal bioluminescent reaction.

Flavin Mononucleotide↗
PubMed1982

Structure and catalytic inactivity of the bacterial luciferase neutral flavin radical.

A luciferase-bound neutral flavin semiquinone radical can be formed upon the oxidation of the luciferase-FMNH2 complex by molecular oxygen. This species can also be formed anaerobically by comproportionation of FMN and FMNH2 in the presence of luciferase. The radical is kinetically stable (t1/2 approximately 20 h at 0 degree C in air; the Arrhenius delta H not equal to decay being about 170 kJ/mol) and can be prepared in pure form by Sephadex G-25 chromatography at 0-4 degrees C. The pure enzyme-bound radical is inactive for light emission either with or without aldehyde, and is not in (relevantly rapid) equilibrium with the luciferase 4a-peroxyflavin, the active intermediate in the bioluminescent reaction.

Benzoquinones↗
PubMed1981

Inactivation of general acyl-CoA dehydrogenase from pig kidney by a metabolite of hypoglycin A.

Pig kidney general acyl-CoA dehydrogenase is irreversibly inactivated by methylenecyclopropylacetyl-CoA, a metabolite of the hypoglycemic amino acid hypoglycin from Blighia sapida, to less that 2% of native activity. Octanoyl-CoA affords strong protection against this inhibition. During inactivation, about 80% of the enzyme FAD is covalently and irreversibly modified with the residual inhibition possibly resulting from modification of the protein. Denaturation of the inactivated enzyme yields several modified flavin derivatives in addition to about 20% unmodified FAD. From spectral comparison, the structure of one of these species is tentatively assigned to a derivative of 4a,5-dihydroflavin, while two further products resemble 6-, and 8-substituted flavins. These results suggest that methylenecyclopropylacetyl-CoA (and consequently the methylenecyclopropylmethano moiety of hypoglycin) be considered "suicide" substrates.

Acyl-CoA Dehydrogenases↗
PubMed1981

Molecular complex of lumiflavin and 2-aminobenzoic acid: crystal structure, crystal spectra, and solution properties.

The molecular complex lumiflavin-2-aminobenzoic acid monohydrate (C13H12N4O2.C7H7NO2.H2O) crystallizes from from aqueous solution as red triclinic prisms. The space group is P1 with cell dimensions a = 9.660 A, b = 14.866 A, c = 7.045 A, alpha = 95.44 degrees , beta = 95.86 degrees, and gamma = 105.66 degrees . The crystal structure was solved by direct methods and refined by block-diagonal least-squares procedures to an R value of 0.050 on the basis of 1338 observed reflections. The structure is composed of stacks of alternating lumiflavin adn un-ionized (neutral) 2-aminobenzoic acid molecules. Two different modes of stacking interaction are observed. In one, 2-aminobenzoic acid overlaps all three of the isoalloxazine rings, at a mean distance of 3.36 A; in the other, 2-aminobenzoic acid interacts distance of 3.36 A; in the other, 2-aminobenzoic acid interacts with the pyrazine and dimethylbenzene moieties, at a distance of 3.42 A. Perpendicular to the stacking direction, the molecules form a continuous sheet. Each flavin is hydrogen bonded via O(2) and NH(3) to two symmetrically related aminobenzoates; the water of crystallization forms three hydrogen bonds, bridging two flavins, via O(4) and N(5), and one aminobenzoic acid. The red color of the crystals results from a charge-transfer transition involving stacked flavin and 2-aminobenzoic acid. The red color of the crystals results from a charge-transfer transition involving stacked flavin and 2-aminobenzoic acid molecules. Measurements of the polarized optical absorption spectra of crystals show that the transition moment direction for the long wavelength absorbance (beyond 530 nm) contains an out-of-plane component which can only arise from a charge-transfer interaction. Since the amino N does not make exceptionally close interactions with isoalloxazine atoms in either stacking mode (minimum interatomic distance 3.52 A), the charge transfer is presumed to involve pi orbitals of the 2-aminobenzoic acid donor.

Chemical Phenomena↗
PubMed1980

Chemical synthesis and some properties of 6-substituted flavins.

A number of derivatives of riboflavin and of 3-methyllumiflavin substituted in the 6 position have been synthesized starting with 6-nitro flavins, reduction to the 6-amino flavin, and diazotization, followed by reaction with the appropriate nucleophile. The absorption spectra, oxidation-reduction potentials, and the electron spin resonance spectra of the radical cationic forms of several of these synthetic compounds have been determined, including 6-S-cysteinyl-3-methyllumiflavin and 6-S-cysteinylriboflavin. The latter has been shown to be identical with the dephosphorylated form of the aminoacyl flavin isolated from trimethylamine dehydrogenase [Steenkamp, D. J., Kenney, W. C. & Singer, T. P. (1978) J. Biol. Chem. 253, 2812-2817; Steenkamp, D. J., McIntire, W., & Kenney, W. C. (1978) J. Biol. Chem. 253, 2818-2824] in regard to absorption specturm, photochemical properties, and mobility in high-voltage electrophoresis and in thin-layer chromatography. An unusually pronounced interaction between the amino group and the isoalloxazine ring system was deduced from the absorption spectra of 6-amino-3-methyllumiflavin and 6-aminoriboflavin.

Electron Spin Resonance Spectroscopy↗
PubMed1980

Studies on the reaction mechanism of lactate oxidase. Formation of two covalent flavin-substrate adducts on reaction with glycollate.

L-Lactate oxidase from Mycobacterium smegmatis catalyzes the oxidative decarboxylation of glycollate, with formate, CO2, and H2O as the major products. In addition, some "uncoupling" of the normal reaction occurs, with glyoxylate and H2O adition, some "uncoupling" of the normal reaction occurs, with glyoxylate and H2O2 as products. Glyoxylate is also a substrate (presumably as its hydrate); in this case, the reaction products are oxalate and H2O2. Evidence is presented that the enzyme recognizes glycollate as a prochiral substrate, differentiating between the Re- and Si-faces of the alpha carbon atom. Two highly fluorescent species are formed concomitantly from the reaction with glycollate; they are proposed to be covalent alpha-glycollyl adducts to the reduced flavin position N(5). One of these adducts is labile and in rapid equilibrium with oxidized enzyme and glycollate, and with the complex of reduced enzyme and glyoxylate; this adduct is a catalytically competent intermediate. The other adduct is comparatively stable (t 1/2 for decay = 20 min at 25 degrees C) and does not react with O2. It is formed at a rate approximately 1% that of the catalytic adduct, but because of its lack of reaction with O2 and its stability, it gradually accumulates during catalytic turnover, resulting in catalytically incompetent enzyme. An isotope effect of approximately 4 is found in the reduction of oxidized enzyme flavin and in the formation of the labile fluorescent adduct, when alpha-2H2-glycollate or (R)-glycollate-2-d is used, but not with the (S)-glycollate-2-d enantiomer. It is concluded that the catalytic adduct is formed by hydrogen abstraction from the Re-face of glycollate.

Aerobiosis↗
PubMed1979

Covalent adducts of lactate oxidase. Photochemical formation and structure identification.

Lactate oxidase forms tight complexes with a variety of mono- and dicarboxylic acids. Most of these undergo facile photoreactions involving decarboxylation of the carboxylic acid and formation of covalent adducts at position N(5) of the flavin, characterized by absorption maxima from 325 to 365 nm and fluorescence emission in the range 440 to 490 nm. The properties of the adducts are strongly dependent on the nature of the substituent. Enzyme-bound N(5)-acyl adducts and N(5)-CH2-R derivatives are stable in the dark. Glycollyl- and alpha-lactyl adducts, however, decay to oxidized enzyme with half-lives in the order of minutes. Upon denaturation of the enzyme, the N(5)-alkyl adducts decay rapidly or are oxidized by oxygen. Reduced lactate oxidase is also photoalkylated in the presence of halogenated carboxylic acids. Bromoacetate yields an N(5)-carboxymethyl adduct; with beta-bromopropionate, a C(4a)-beta-propionyl derivate is formed. The N(5) adduct is identical with that from the photochemical reaction of oxidized enzyme and malonic acid. When the native coenzyme FMN is substituted by 2-S-FMN, qualitatively the same photoproducts are formed. The adducts obtained with the 2-S-FMN enzyme show the expected bathochromic shifts in absorption spectra. The results indicate that the photoreactivity of the enzyme is restricted to the positions C(4a) and N(5) of the flavin.

Carboxylic Acids↗
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↗
PubMed1979

Suicide inactivation of the flavoenzyme D-lactate dehydrogenase by alpha-hydroxybutynoate.

The acetylenic alpha-hydroxy acid 2-hydroxy-3-butynoate (alpha HB) is a substrate and an irreversible inactivator of the FAD-containing flavoenzyme D-lactate dehydrogenase from Megasphaera elsdenii. On the average, the enzyme undergoes five catalytic turnovers with alpha HB in air at pH 7.0 before being inactivated. Irreversible inactivation is due to the conversion of the flavin to a pink adduct with visible absorption peaks at 522, 382, and 330 nm and weak fluorescence with an emission maximum at 635 nm. The adduct is stable and can be released from the enzyme and purified. It retains a structure analogous to FAD since it binds to the FAD-specific apo-D-amino acid oxidase. It can be further converted to an FMN analogue with phosphodiesterase which binds to the FMN-specific apoflavodoxin. Experiments were conducted to test whether inactivation was initiated by an alpha HB allene carbanion or the dehydrogenation product of alpha HB. Kinetic studies proved inconclusive in that a rapid equilibrium between an oxidized enzyme--allene carbanion pair and reduced enzyme--keto acid pair would make these two species kinetically equivalent. The olefinic substrate 2-hydroxy-3-butenoate, however, produced no flavin adduct. Since the keto acid derived from the oxidation of this alpha-hydroxy acid is expected to be as reactive as 2-keto-3-butynoate, it is concluded that an allene carbanion produced by abstraction of the alpha-hydrogen of alpha HB is the reactive species which covalently adds to the flavin.

Alkynes↗