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Megasphaera elsdenii and Desulfovibrio vulgaris apoflavodoxins have been reconstituted with riboflavin 3′,5′- bisphosphate. Several biochemical and biophysical properties of the complexes have boon investigated and the results are compared with the properties of the native proteins. The dissociation constant of the modified complex of M. elsdenii flavodoxin is increased by a factor of about 23 by comparison with that of the native protein. The rate constant for the formation of the complex of M. elsdenii flavodoxin is about 26 times lower than that for tile native protein. The redox potential of the transition between the oxidized and semiquinone state is similar to that of the native protein. On the other hand, the redox potential of the semiquinone-hydroquinone transition is about 20 mV more negative than that of the native protein. Absorbance and circular dichroic spectra of the protein-bound artificial prosthetic group and the protein- bound natural prosthetic group are very similar. In both the oxidized and m the tully reduced state only minor differences in interaction between the isoalloxazine ring and the apoprotein for the two flavin derivatives are found by 13C and 15N NMR. 31P-NMR studies show that the 5′-phosphate group of the two flavin derivatives is bound in the same way and that it is dianionic in the complex. In contrast, the 3′-phosphate group in riboflavin 3′,5′-bisphosphate is monoanionic or even neutral when bound to the protein. The 3′-phosphate group is also dose to or on the surface of the protein. Desulfovibrio vulgaris apoflavodoxin has an affinity for riboflavin 3′,5′-bisphosphate which is 10 times higher _ I as compared to Megasphaera eisdeaii apoflavodoxin (Ka = 108 M-1). Also the association rate constant of Desulfovibrio vulgaris apoprotein and riboflavin 3′,5′-bisphosphate is found to be 10 times faster than for the Megasphaera elsdenii flavodoxin reaction. The dissociation behaviour of native Desulfovibrio vulgaris flavodoxin measured under identical conditions as for the riboflavin 3′,5′-bisphosphate analog gives a value (Kd &assymp; 0.2 nM) which is considerably lower than reported earlier [Dubourdieu, M., MacKnight, M. L. & Tollin, G. (1974) Biochem. Biophys. Res. Commun. 60, 649–655]. The results are discussed in the fight of the existing crystallographic data of flavodoxins and the recently proposed theory on the regulation of the redox potential in flavoproteins [Moonen, C. T. W., Vervoort, J. & Müller, F. (1984) in Flavins andflavoproteins, pp. 493–496, Walter de Gruyter, Berlin]. [ABSTRACT FROM AUTHOR]

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 -122-140] to be a flavin of still unidentified structure, and had been suggested to be "blue light' photoreceptor in this organism. The structure of this flavin (F1-X) has been obtained synthetically. The 5' malonylester linkage is relatively labile towards hydrolysis and photolysis, it flavin (absorption and florescence) are practically identical to those of normal riboflavin (vitamin B2. [ABSTRACT FROM AUTHOR]

Homogenates of Propionibacterium freudenreichii transform riboflavin into 5,6-dimethyl-benzimidazole. This process is stimulated by nicotinamide. Homogenates of Propionibacterium shermanii form only small amounts of 5,6-dimethylbenzimidazole from riboflavin in the absence of nicotinamide, but also form appreciable amounts in the presence of nicotinamide. The stimulation of the 5,6-dimethylbenzimidazole-forming system by nicotinamide shows a lag phase which is abolished by preincubation of the homogenate with nicotinamide. Since no lag phase is observed when nicotinamide is replaced by nicotinate, nicotinate seems to be the true stimulating agent. These observations are in agreement with the fact that nicotinamide is rapidly split to nicotinate in homogenates of P. freudenreichii. The 5,6-dimethylbenzimidazole-forming homogenate system is only active at a high buffer concentration (0.3–0.5 M) and in the presence of oxygen. The system has a pronounced oxygen optimum. Flavin mononucleotide and flavin-adenine dinucleotide are better substrates for the 5,6-dimethylbenzimidazole-forming homogenate system than riboflavin. But with [1′-14C]riboflavin as substrate the specific radioactivity of 5,6-dimetbylbenzimidazole is higher than the specific radioactivity of flavin-adenine dinucleotide and lower than the specific radioactivity of flavin mononucleotide. Therefore flavin mononucleotide is the immediate substrate for the formation of 5,6-dimethylbenzimidazole. A tentative reaction sequence for the transformation of flavin mononucleotide into 5,6-dimethylbenzimidazole is discussed. [ABSTRACT FROM AUTHOR]

The photoreaction of flavoquinones (lumiflavin, riboflavin, FMN etc. and their 3-alkylated derivatives) with propargylamine-type acetylenic substrates, R4-Cα≡ Cβ-Cγ~HR3-NR2R1, yields a mixture of two adducts, which result from covalent Cα fixation of the Cγ-deprotonated substrate to either position C(4a) or N(5) in the flavin nucleus. The N(5) adduct is a dihydroflavin-5-trimethine-cyanine with very intense (ε > 20000 M-1 cm-1) absorption maxima in the region 380–450 nm depending on the R1, R2. This absorption allows recognition of minute amounts of this species of flavocyanine even in complex mixtures. Flavocyanines can be reconverted to starting flavin by base. Its spectral properties are identical with those obtained for the pargyline-flavin inhibitor complex from bovine kidney or pig liver monoamine oxidase. The C(4a) adduct is a 4a:5-propenylidene-dihydroflavin, the cyclisation probably arising through secondary attack of the Cγ carbon at N(5). It cannot be reconverted to starting flavin in relevant yields. Its chromophor (λmax ≈ 360 nm) is identical with that of lactate-oxidase inhibitor complex. If N(3) is alkylated, these cyclic adducts undergo cleavage of the 4—4a bond upon treatment with base, followed by aromatization of the substrate substituent to yield novel pyrroloquinoxal-4-ones. Upon treatment with LiAIH4, however, the adducts are hydrogenated at the N(1)=C(10a) double bond, which by concomitant base hydrolysis yields 1,2-dihydroquinoxaline derivatives. Their autoxidation yields a pyrrolo-quinoxalinium cation which exhibits spectral identity with the product isolated from the α-hydroxybutynoate-flavin inhibitor complex of Mycobacterium smegmatis lactate oxidase, after borohydride treatment. The scope and limitations of covalent adduct formation in flavin-dependent bioeatalysis will be discussed. [ABSTRACT FROM AUTHOR]

The presence of higher (>0.2 M) concentrations of divalent anions A2- (hydrogenphosphate, sulfate) is found to accelerate as well as to change entirely the course of riboflavin photolysis: instead of 10-dealkylation to yield lumichrome, intramolecular addition of the 2'-hydroxyl group is found to occur at the peri-position C(9). The reaction is analogous to the "photohydration" of the flavin nucleus in the cationic state as described by Schöllnhammer and Hemmerich [Eur. J. Biochem. (1974) 44, 561-577]. The final product of the new addition reaction arises from autoxidation of dihydroflavin intermediate and exhibits the structure ... It is thus representative for the new class of flavins ("cyclo-dehydrofalvins"). Earlier reports on "anomalous" flavin photodegradation products absorbing around 410 nm [Holmström (1964) Ark. Kem. 22, 281; Massey and Atherton (1962) J. Biol. Chem. 237, 2965] are readily explained. The reaction is found to depend strictly on the presence of a nucleophilic function in the N(10)-side chain, e.g. N(10)-CH2-C(OH)RR' or even N(10)-(CH2)2-SO3-. Quenching experiments suggest that the new reaction occurs via the singlet state ¹Flox*, while the normal photolysis is mediated by the triplit ³ Flox*. The new photoaddition is thought to occur via a Flavin-A2- complex which creates sterically favorable conditions for C(9)/O(2'α)-interaction. [ABSTRACT FROM AUTHOR]

Homogenates of Propionibacterium freudenreichii transform riboflavin into 5,6-dimethyl-benzimidazole. This process is stimulated by nicotinamide. Homogenates of Propionibacterium shermanii form only small amounts of 5,6-dimethylbenzimidazole from riboflavin in the absence of nicotinamide, but also form appreciable amounts in the presence of nicotinamide. The stimulation of the 5,6-dimethylbenzimidazole-forming system by nicotinamide shows a lag phase which is abolished by preincubation of the homogenate with nicotinamide. Since no lag phase is observed when nicotinamide is replaced by nicotinate, nicotinate seems to be the true stimulating agent. These observations are in agreement with the fact that nicotinamide is rapidly split to nicotinate in homogenates of P. freudenreichii. The 5,6-dimethylbenzimidazole-forming homogenate system is only active at a high buffer concentration (0.3–0.5 M) and in the presence of oxygen. The system has a pronounced oxygen optimum. Flavin mononucleotide and flavin-adenine dinucleotide are better substrates for the 5,6-dimethylbenzimidazole-forming homogenate system than riboflavin. But with [1′-14C]riboflavin as substrate the specific radioactivity of 5,6-dimetbylbenzimidazole is higher than the specific radioactivity of flavin-adenine dinucleotide and lower than the specific radioactivity of flavin mononucleotide. Therefore flavin mononucleotide is the immediate substrate for the formation of 5,6-dimethylbenzimidazole. A tentative reaction sequence for the transformation of flavin mononucleotide into 5,6-dimethylbenzimidazole is discussed. [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]

Succinate dehydrogenase flavocoenzyme (‘SD-flavin’), previously shown to be an 8α-substituted riboflavin derivative containing a tertiary nitrogen homoconjugated to the flavin nucleus, was subjected to further hydrolysis and to reduction under acid conditions. Both conditions resulted in the liberation of 1 mole of histidine per mole of flavin. This proves histidine to be the covalent link between flavin and peptide backbone in succinate dehydrogenase and imidazole to be the tertiary nitrogen function homoconjugated to the flavin. 8α-Histidyl-riboflavin has been synthesized starting from riboflavin chemically and shown to be completely identical with the natural product in optical, ESR and NMR spectra, pH-fluorescence curve and behavior on thin-layer and paper chromatography, as well as paper electrophoresis. [This equation cannot be represented into ASCII Text] 3. Both the natural compound isolated by acid hydrolysis of flavin peptide and the synthetic one contain two isomers, which may be separated by high voltage electrophoresis. The isomers appear to be the N(1)- and N(3)-imidazole substituted compounds. Digestion of the flavin peptide with aminopeptidase M yields only one isomer but on treatment with 6-N HCl this is gradually converted to a mixture of the two isomers. The absolute assignment of the natural isomer is suggested as 8α-[N(3)-histidyl]-riboflavin on the basis of imidazole quaternization with CH3I, reductive cleavage of the flavin-imidazole bond and identification of the methyl-histidine liberated as 1-methyl-histidine. [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]