Flavin Peptide Research Articles

Identification of the covalently bound flavin of L-gulono-γ-lactone oxidase

It has been previously reported ( Nakagawa et al. (1975) J. Biochem. (Tokyo) 77 , 221–232 ) that L-gulono-γ-lactone oxidase contains a flavin moiety covalently linked to the protein. Properties of a partially purified flavin peptide, liberated by pronase digestion, indicate the presence of histidyl flavin, but not 8α-[N(3)-histidyl]-riboflavin. In the present study, a purified trypticchymotryptic flavin peptide was acid-hydrolyzed to the amino acyl flavin (a mixture of its riboflavin level as well as its 2′, 5′ -anhydro form). High voltage electrophoresis, fluorescence excitation spectrum, and pK a of fluorescence quenching, in comparison with synthetic compounds all show that 8α-[N(1)-histidyl]-riboflavin is the structure of the covalently bound flavin of this enzyme.

Ascorbate-synthesizing System in Rat Liver Microsomes<subtitle>II. A Peptide-bound Flavin as the Prosthetic Group of L-Gulono-γ-lactone Oxidase</subtitle>

L-Gulono-gamma-lactone oxidase [EC 1.1.3.8] was purified 80-fold from rat liver microsomes. In confirmation of our previous finding with a cruder preparation, the purified enzyme was shown to contain an L-gulono-gamma-lactone-reducible pigment as a prosthetic group. This pigment was not liberated from the protein by acid ammonium sulfate, 10% trichloroacetic acid or 2 M area, but was effectively released by proteolytic digestion. The pigment thus released showed a reduced-minus-oxidized difference spectrum characteristic of a flavin compound. The pigment was liberated from a trichloroacetic acid-treated preparation of the enzyme by pronase digestion and purified by Florisil column chromatography and paper chromatography. The absorption spectrum as well as the fluorescence emission and excitation spectra of the purified pigment indicated that it was actually a flavin peptide. It was, however, different not only from FMN but also from flavin peptides isolated from other sources such as succinate dehydrogenase [EC 1.3.99.1] and monoamine oxidase [EC 1.4.3.4] as regards the pH dependence of fluorescence intensity and the Rf value on thin-layer chromatography. A preliminary analysis showed that the purified flavin compound contained several amino acid residues. Alkaline photolysis of the purified flavin peptide suggested that the isoalloxazine ring of the flavin is involved in its binding to the peptide. The hypsochromic shift of the absorption peak in the near-ultraviolet region suggested further that the linkage between the flavin and the peptide may be mediated by the 8-methyl group of the isoalloxazine nucleus. It can be concluded that the prosthetic group of gulonolactone oxidase is a flavin which is covalently bound to the enzyme protein.

The covalently bound flavin of Chromatium cytochrome c552. 2. Sequence of flavin peptides and flavin-tyrosine interaction.

The previous paper in this series described the isolation of tryptic-chymotryptic and peptic flavin peptides from Chromatium cytochrome c552 in homogeneous form and presented evidence that the FAD component is covalently linked to a cysteinyl residue via a hemiacetal linkage to the 8α position of the isoalloxazine ring. The amino acid sequence of the peptic and tryptic-chymotryptic flavin peptides were found to be Tyr-Thr-Cys(FAD)-Tyr and Thr-Cys(FAD)-Tyr, respectively. However, the presence of the NH2-tyrosyl residue in the former, imparts markedly greater stability to the cysteinyl flavin linkage, and even results in essentially quenched fluorescence of the flavin after performic acid oxidation of the sulfur moiety. Oxidation at 40 °C, which destroys tyrosine, results in the same level of fluorescence as found when the tryptic-chymotryptic peptide is oxidized at 0 °C. Removal of this tyrosyl residue from the peptic peptide, oxidized at 0 °C, by aminopeptidase M also leads to an increase in fluorescence. Evidence for a tyrosyl-flavin interaction has been further obtained from CD spectra. The peptic peptide has a broad, positive Cotton effect with a maximum at 484 to 490 nm, which is enhanced after treatment with pyrophosphatase and phosphatase and negative bands at 305 and 375 nm. The tryptic-chymotryptic peptide, on the other hand, has a positive band at 340 nm, as in FAD, but only a small negative band at 380 nm. Thus the tyrosyl-flavin interaction appears to be responsible for the stabilization of the thiohemiacetal bond in the peptide and perhaps also in the protein.

The covalently bound flavin prosthetic group of β-cyclopiazonate oxidocyclase

It has been reported that /3-cyclopiazonate oxidocyclase from Penicillium cyclopium, an enzyme which catalyzes the dehydrogenation and cyclization of P-cyclopiazonic to a-cyclopiazonic acid, contains covalently bound flavin [I] . A flavin peptide, rele: sed by digestion with pronase has been partially purified and preliminary results suggested that the flavin is at the dinucleotide level. The present paper is the first report of a collaborative study between the laboratories in South Africa and in San Francisco, aimed at defining the structure of the flavin site of this enzyme. It has been found that the flavin is attached to the protein by way of the 8 (Y group, as in other enzymes containing covalently bound flavin [2] and that the amino acid substituent is histidine, as in succinate dehydrogenase [3,4] but the nature of the linkage of the flavin to the imidazole is different from that in succinate dehydrogenase. The unusual properties of the flavin peptide from

A novel form of covalently bound flavin from thiamine dehydrogenase

Thiamine dehydrogenase contains 1 mole of FAD per mole of enzyme covalently linked to the protein. A pure flavin peptide has been obtained from tryptic-chymotryptic digests of the enzyme. The hypsochromic shift of the second absorption maximum shows that the peptide is substituted at the 8α position of the flavin. The pH-fluorescence curve of the flavin peptide is similar to that of flavin peptides containing 8α-[N(3)-histidyl]-flavin but differs from the latter in having a pKa of fluorescence quenching over 1 pH unit higher. The amino acyl riboflavin derived from this peptide by acid hydrolysis is readily distinguished from both the N(3) and N(1) isomers of 8α-histidyl riboflavin by its pKa of fluorescence quenching and mobility in high voltage electrophoresis. Drastic acid hydrolysis, however, liberated 1 mole of histidine per mole of flavin, showing that this is the amino acid substituted at the 8α position. Attachment of the flavin appears to be to the imidazole ring but not via either of the ring nitrogens. These results indicate that a novel type of covalently bound flavin exists in thiamine dehydrogenase.

Structure of the flavin site of [formula omitted] flavocytochrome [formula omitted

Two flavin peptides have been isolated from Chromatium cytochrome c 552 by digestion with pepsin and with trypsin-chymotrypsin, respectively. Acid hydrolysis and aminopeptidase digestion of the peptic peptide shows the presence of 1 mole each of threonine and cysteine and 2 of tyrosine, per mole of FAD. Edman degradation gives the sequence: tyr-thr-cys (flavin)-tyr. Tryptic-chymotryptic digestion yields a flavin tripeptide of the structure: thr-cys (flavin)-tyr. The N-terminal tyrosine in the peptic tetrapeptide shows a strong interaction with the flavin, as judged by CD spectra, and this may account for the resistance of the bridge sulfur to oxidation by cold performic acid. Both peptides show abnormally low fluorescence in the pure state (1 to 5% relative to riboflavin) and positive iodoplatinic test; the peptic peptide yields a positive, the trypticchymotryptic peptide negative ninhydrin reaction. The electrophoretic mobility of the major product of aminopeptidase digestion (presumably the thiazolidine form of cysteinyl-8α-FAD thiohemiacetal) and other properties of the two peptides are in accord with previous suggestions that the linkage of the flavin to the peptide is a thiohemiacetal.

Covalently bound flavin in D-6-hydroxynicotine oxidase from Arthrobacter oxidans.

D-6-hydroxynicotine oxidase contains 1 mole of FAD covalently bound to one mole of enzyme. To identify the covalent linkage between FAD and protein, an amino acid derivative of riboflavin (HNO-flavin) was isolated and purified. It was obtained from flavin peptides by hydrolysis with 6 N HCl 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 8α-(N-3-histidyl)-riboflavin. Hydrolysis of HNO-flavin in 6 N HCl at 125°C liberated 1 mole of histidine per mole of 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 8a- (N-3-histidyl) -flavin-adenine-dinucleotide in the active center.

Covalently Bound Flavin in d‐6‐Hydroxynicotine Oxidase from Arthrobacter oxidans

A substituted riboflavin (HNO‐flavin) was isolated from d‐6‐hydroxynicotine oxidase. It was obtained from flavin peptides by hydrolysis with 6 N HCl 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 8α‐(N‐3‐histidyl)‐riboflavin. Hydrolysis of HNO‐flavin in 6N HCl at 125°C 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.

Studies on succinate dehydrogenase. 8 -Histidyl-FAD as the active center of succinate dehydrogenase.

1 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. 2 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. 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.

The covalently-bound flavin of hepatic monoamine oxidase. 2. Identification and properties of cysteinyl riboflavin.

In the previous paper in this series it was shown that a pure flavin pentapeptide isolated from hepatic monoamine oxidase contains 1 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.

The covalently-bound flavin of hepatic monoamine oxidase. 1. Isolation and sequence of a flavin peptide and evidence for binding at the 8alpha position.

A method has been elaborated for the large scale isolation of outer membranes of beef liver mitochondria. From these monoamine oxidase was partially purified and digested with trypsin and chymotrypsin to yield peptides containing riboflavin covalently linked to the peptide chain. A pure pentapeptide of riboflavin‐5′‐phosphate has been obtained by a series of chromatographic procedures. Edman degradation, followed by dansylation, revealed the amino acid sequence: Ser‐Gly‐Gly‐X‐Tyr, X being the amino acid to which the flavin is attached via the 8α‐carbon of the riboflavin. This site of attachment is concluded on the basis of analysis of the optical spectra in the neutral and cationic forms of the flavoquinone and of the electron spin resonance spectrum of the free radical cation.

Sequence and structure of a cysteinyl flavin peptide from monoamine oxidase

Previous studies in this laboratory have shown that the active center of hepatic monoamine oxidase contains flavin dinucleotide covalently linked to the peptide chain via the 8α position of the flavin but that, unlike in succinate dehydrogenase, the linkage is not to histidine but to another amino acid. A pure flavin pentapeptide has now been isolated from monoamine oxidase which yields on acid hydrolysis or digestion with aminopeptidase M 1 mole each of serine and tyrosine and 2 of glycine and gives a positive test for sulfur. Oxidation of the peptide with performic acid yields, in addition to the amino acids mentioned, cysteic acid. The physical and chemical properties of the peptide are in accord with the conclusion that the amino acid substituted on the 8α group of the flavin is cysteine in thioether linkage. Edman degradation followed by dansylation revealed the sequence: ▪

Structure of the covalently bound flavin of monoamine oxidase

Flavin peptides derived from monoamine oxidase, free from succinate dehydrogenase flavin, were obtained by digestion of outer membranes of beef liver mitochondria with trypsin and chymotrypsin and purification by various chromatographic methods. The flavin peptides show the same hypsochromic shift of the optical absorption spectrum as flavin peptides from succinate dehydrogenase: the 370 mμ band of the neutral oxidized flavin is shifted to 340 mμ, whereas the cation shows a peak at 370 mμ. The ESR spectrum of the monoamine oxidase flavin cation radical also resembles that of succinate dehydrogenase flavin in that the total width is reduced from 49 G (in riboflavin) to at least 45 G, and the line width from 3.8 G (in riboflavin) to 2.3 G. The covalently bound flavins of monoamine oxidase and succinate dehydrogenase differ, however, in that the former shows the same fluorescence intensity between pH 3.4 and 8, while the latter is quenched with a pK of 4.5 ± 0.1. These observations indicate that the FAD of monoamine oxidase is covalently linked to the peptide chain through the 8α-CH 3 group of riboflavin but histidine is not the immediate substituent, as in succinate dehydrogenase. Hydrolysis of flavin peptides from monoamine oxidase in 6 N HCl at 95° yields a derivative chromatographically distinct from free flavins which is ninhydrin-positive and thus contains an amino acid bound to the 8α-position.

Identification of the Covalently Bound Flavin of Succinate Dehydrogenase as 8α-(Histidyl) Flavin Adenine Dinucleotide

Abstract Acid hydrolysis of flavin peptides from the active center of mammalian succinate dehydrogenase yields a substituted riboflavin which was isolated in pure form. It contains a substituent attached to position 8α of riboflavin. Drastic acid hydrolysis of this compound and catalytic hydrogenation yield nearly 1 mole of free histidine. Histidine is also liberated on neutral photolysis. The presence of histidine is confirmed by behavior on high voltage electrophoresis at various pH values and by acid titration curves. Linkage of the 8α-CH3 group of riboflavin is to one of the imidazole ring nitrogens since neither the flavin peptide nor its acid derivative give a Pauly reaction. This assignment is in full accord with the characteristic pH-fluorescence curve of covalently bound flavin. The pK of the fluorescence quenching agrees with that expected for the imidazole nitrogen in histidyl flavin.

Interaction of purine with the prosthetic group of succinic dehydrogenase.

No data are to be found in the literature comparing on a molar basis the fluorescence, at dinucleotide and mono-nucleotide level, of the flavin peptide released from succinic dehydrogenase (SD) by tryptic digestion, neither has it been ascertained whether an internal quenching due to the purine moiety of the molecule exists in the peptide bound flavin-dinucleotide, as it does in flavin-adenine dinucleotide.