Flavin Peptide Research Articles

Facile access to foldable redox-active flavin-peptide conjugates.

A convenient approach for the synthesis of foldable redox-active flavin peptide conjugates was established. A model β-hairpin oligopeptide motif was utilized to demonstrate that azidolysine side-chains are readily functionalised with an alkyne-bearing flavine derivative. The folding equilibrium of the peptide backbone as well as the redox behaviour of the flavin moieties remains intact after the conjugation.

Directed Electron Transfer in Flavin Peptides with Oligoproline-Type Helical Conformation as Models for Flavin-Functional Proteins.

To mimic the charge separation in functional proteins we studied flavin‐modified peptides as models. They were synthesized as oligoprolines that typically form a polyproline type‐II helix, because this secondary structure supports the electron transfer properties. We placed the flavin as photoexcitable chromophore and electron acceptor at the N‐terminus. Tryptophans were placed as electron donors to direct the electron transfer over 0–3 intervening prolines. Spectroscopic studies revealed competitive photophysical pathways. The reference peptide without tryptophan shows dominant non‐specific ET dynamics, leading to an ion pair formation, whereas peptides with tryptophans have weak non‐specific ET and intensified directed electron transfer. By different excitation wavelengths, we can conclude that the corresponding ion pair state of flavin within the peptide environment has to be energetically located between the S1 and S4 states, whereas the directed electron transfer to tryptophan occurs directly from the S1 state. These photochemical results have fundamental significance for proteins with flavin as redoxactive cofactor.

Expression of zebrafish (Danio rerio) monoamine oxidase (MAO) in Pichia pastoris: Purification and comparison with human MAO A and MAO B

The expression, purification and characterization of zebrafish monoamine oxidase (zMAO) using the methylotropic yeast Pichia pastoris expression system is described. A 1L fermentation culture of Pichia pastoris containing the gene encoding zMAO under control of the methanol oxidase promotor expresses approximately 200mg of zMAO exhibiting 300 U of total activity. The enzyme is found in the mitochondrial fraction of the expression host and is purified in a 30% yield as a homogenous species with a M(r) of approximately 60,000 on SDS-PAGE and a mass of 58,525+/-40 Da from MALDI-TOF measurements. The zMAO preparation contains one mole of covalent flavin cofactor per mole of enzyme and exhibits >80% functionality. The covalent flavin exhibits fluorescence and EPR spectral properties consistent with known properties of 8 alpha-S-cysteinyl FAD. Chemical degradation of the flavin peptide results in the liberation of FAD. zMAO exhibits no immuno-chemical cross-reactivity with polyclonal anti-sera raised against human MAO A. The enzyme preparation exhibits reasonable thermostability up to a temperature of 30 degrees C. Benzylamine is oxidized with a k(cat) value of 4.7+/-0.1 min(-1) (K(m)=82+/-9 microM) and the enzyme oxidizes phenylethylamine with a k(cat) value of 204 min(-1) (K(m)=86+/-13 microM). The K(m) (O(2)) values determined for zMAO using either benzylamine or phenylethylamine as substrates ranges from 108(+/-5) to 140(+/-21)microM. The functional behavior of this teleost MAO relative to human MAO A and MAO B is discussed.

Cofactors in sarcosine oxidase from Corynebacterium sp. U-96.

Sarcosine oxidase from Corynebacterium sp. U-96 is a heterotetrameric enzyme that was reported to contain 1 mol of covalently bound FAD and 1 mol of non-covalently-bound FAD. This work describes the result of reinvestigation of the cofactors in this enzyme. The enzyme was found to contain 1 mol of non-covalently-bound NAD+, 1 mol of non-covalently-bound FAD, and 1 mol of covalent FMN. The covalent FMN was identified by the mass and amino acid sequence analyses of the flavin peptide.

High-level expression of human liver monoamine oxidase B in Pichia pastoris.

The high-level heterologous expression, purification, and characterization of the mitochondrial outer membrane enzyme human liver monoamine oxidase B (MAO B) using the methylotrophic yeast Pichia pastoris expression system are described. A 2-L culture of P. pastoris expresses approximately 1700 U of MAO B activity, with the recombinant enzyme associated tightly with the membrane fraction of the cell lysate. By a modification of the published procedure for purification of bovine liver MAO B [Salach, J. I. (1979) Arch. Biochem. Biophys. 192, 128-137], recombinant human liver MAO B is purified in a 34% yield ( approximately 200 mg from 2 L of cell culture). The isolated enzyme exhibits an M(r) of approximately 60, 000 on SDS-PAGE and 59,474 from electrospray mass spectrometry measurements, which is in good agreement with the mass predicted from the gene sequence and inclusion of the covalent FAD. One mole of covalent FAD per mole of MAO B is present in the purified enzyme and is bound by an 8alpha-S-cysteinyl(397) linkage, as identified by electrospray mass spectrometry of the isolated tryptic/chymotryptic flavin peptide. Recombinant human liver MAO B and bovine liver MAO B are shown to be acetylated at the seryl residues at their respective amino termini. The benzylamine oxidase activity of recombinant MAO B ranges from 3.0 to 3.4 U/mg and steady-state kinetic parameters for this enzyme preparation compare well with those published for the bovine liver enzyme: k(cat) = 600 min(-1), K(m)(benzylamine) = 0.50 mM, and K(m)(O(2)) = 0.33 mM. Kinetic isotope effect parameters using [alpha,alpha-(2)H(2)]benzylamine are also similar to those found for the bovine enzyme. Recombinant MAO B exhibits a (D)k(cat) = 4.7, a (D)[k(cat)/K(m)(benzylamine)] = 4.5, and a (D)[k(cat)/K(m)(O(2))] = 1.0. In contrast to bovine liver MAO B, no evidence was found for the presence of any anionic flavin radical either by UV-vis or by EPR spectroscopy in the resting form of the enzyme. These data demonstrate the successful heterologous expression of a functional, membrane-bound MAO B, which will permit a number of mutagenesis studies as structural and mechanistic probes not previously possible.

Influence of Flavin Analogue Structure on the Catalytic Activities and Flavinylation Reactions of Recombinant Human Liver Monoamine Oxidases A and B

Two riboflavin-deficient (rib5) Saccharomyces cerevisiae expression systems have been developed to investigate the influence of riboflavin structural alterations on the covalent flavinylation reaction and activity of recombinant human liver monoamine oxidases A and B (MAO A and B). Nineteen different riboflavin analogues were tested with MAO A and nine with MAO B. MAO expression and flavinylation were determined immunochemically with antisera to MAO and an anti-flavin antisera. Expression levels of both MAO A and B are invariant with the presence or absence of riboflavin or riboflavin analogues in the growth medium. Flavin analogues with a variety of seven and eight substitutions are found to be covalently incorporated and to confer catalytic activity. The selectivities of MAO A and MAO B for flavin analogue incorporation are found to be similar, although 8alpha-methylation of the flavin resulted in a higher level of catalytic activity for MAO B than for MAO A. N(3)-Methylriboflavin and 8-nor-8-aminoriboflavin are not covalently bound as they are not converted to their respective FAD forms by yeast. 5-Carba-5-deazaflavin and 7,8-nor-7-chlororiboflavin are not covalently incorporated into MAO A and do not support catalytic activity. A flavin peptide was isolated from MAO A containing 7-nor-7-bromo-FAD and was demonstrated to be covalently attached to Cys-406 by an 8alpha-S-thioether linkage by sequence analysis and by matrix-assisted laser desorption ionization time of flight mass spectroscopy. MAO A partially purified from yeast grown on 8-nor-8-chlororiboflavin exhibited an absorption spectrum indicating the covalent flavin is an 8-nor-8-S-thioflavin, suggesting a nucleophilic displacement mechanism that supports the quinone-methide mechanism previously suggested as a general mechanism for covalent flavin attachment.

The use of a water-soluble carbodiimide to study the interaction between Chromatium vinosum flavocytochrome c-552 and cytochrome c

The interaction between horse heart cytochrome c and Chromatium vinosum flavocytochrome c-552 was studied using the water-soluble reagent 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC). Treatment of flavocytochrome c-552 with EDC was found to inhibit the sulfide: cytochrome c reductase activity of the enzyme. SDS gel electrophoresis studies revealed that EDC treatment led to modification of carboxyl groups in both the M r 21000 heme peptide and the M r 46000 flavin peptide, and also to the formation of a cross-linked heme peptide dimer with an M r value of 42000. Both the inhibition of sulfide: cytochrome c reductase activity and the formation of the heme peptide dimer were decreased when the EDC modification was carried out in the presence of cytochrome c. In addition, two new cross-linked species with M r values of 34000 and 59000 were formed. These were identified as cross-linked cytochrome c-heme peptide and cytochrome c-flavin peptide species, respectively. Neither of these species were formed in the presence of a cytochrome c derivative in which all of the lysine amino groups had been dimethylated, demonstrating that EDC had cross-linked lysine amino groups on native cytochrome c to carboxyl groups on the heme and flavin peptides. A complex between cytochrome c and flavocytochrome c-552 was required for cross-linking to occur, since ionic strengths above 100 mM inhibited cross-linking.

Identification of the covalently bound flavins of D-gluconate dehydrogenases from Pseudomonas aeruginosa and Pseudomonas fluorescens and of 2-keto-D-gluconate dehydrogenase from Gluconobacter melanogenus.

An improved method is presented for the purification of 8 alpha-(N1-histidyl)riboflavin, 8 alpha-(N3-histidyl)riboflavin and their 2',5'-anhydro forms, which permits the isolation of sizeable quantities of each of these compounds from a synthetic mixture in pure form. Flavin peptides were isolated from the D-gluconate dehydrogenases of Pseudomonas aeruginosa and Pseudomonas fluorescens and from the 2-keto-D-gluconate dehydrogenase of Gluconobacter melanogenus. After conversion into the aminoacyl-riboflavin, the flavin in all three enzymes was identified as 8 alpha-(N3-histidyl)riboflavin. By sequential treatment with nucleotide pyrophosphatase and alkaline phosphatase, the flavin in each enzyme was shown to be in the dinucleotide form.

Identification of the covalently bound flavin of dimethylglycine dehydrogenase and sarcosine dehydrogenase from rat liver mitochondria.

Dimethylglycine dehydrogenase (EC 1.5.99.2) and sarcosine dehydrogenase (EC 1.5.99.1) are the folate binding proteins of rat liver mitochondria. These two enzymes contain covalently bound flavin and catalyze similar oxidative demethylation reactions (Wittwer, A. J., and Wagner, C. (1981) J. Biol. Chem. 256, 4102-4108). Flavin-peptides have been purified from these two enzymes after proteolytic digestion by trypsin and chymotrypsin. The spectral and chromatographic properties of these flavin peptides changed after treatment with nucleotide pyrophosphatase in a manner consistent with the conversion of an FAD-peptide to an FMN-peptide. The pKa for pH-dependent fluorescence quenching of the purified flavin-peptides was not affected by borohydride reduction which, in conjunction with the pKa values, indicated that the flavin was covalently linked via the 8 alpha position of the isoalloxazine ring to an imidazole N(3) of a histidine residue. Peptides from both enzymes showed histidylflavin at the N terminus. Amino acid composition and sequence analysis showed that the flavin-peptide from dimethylglycine dehydrogenase was His(flavin)-Ala-Ala-Gly-Leu. Amino acid composition and N-terminal analysis suggested the sequence of the flavin-peptide of sarcosine dehydrogenase was His(flavin)-(Ala, Gly,Thr)-Leu.

37] Covalent attachment of flavin to flavoproteins: Occurrence, assay, and synthesis

This chapter describes occurrence, assay, and synthesis of covalent attachment of flavin to flavoproteins. The first covalently bound flavin to be discovered was the flavin component of mammalian succinate dehydrogenase. Five different structures have been identified in this group of enzymes and chemically synthesized; four are adducts formed with the 8 α -methyl group of the flavin ring system, and the fifth is a substituent at C(6) of the aromatic ring of the flavin. It is essential to purify the flavin peptide first to remove materials which interfere in subsequent tests, such as hemes originating from cytochromes. To eliminate losses by having the flavin present in several phosphorylated forms, as a result of breakdown during manipulation, it is desirable to convert the crude peptide to the dephosphorylated form, by sequential treatment with nucleotide pyrophosphatase and alkaline phosphatase. Advantage may be taken of the different R f values of flavin peptides in these systems for the monophosphorylated and diphosphorylated forms, so that the procedure may be carried out both before and after digestion with the phosphatases. A more sensitive procedure for histidylflavins is based on the difference in fluorescence of the aminoacyl flavin between pH 3.2 and 7.

Identity of the active site flavin-peptide fragments from the human “A”-form and the bovine “B”-form of monoamine oxidase

The monoamine oxidase present in the mitochondria of human placenta was verified to be the clorygyline sensitive or A form. This property was not abolished following extensive phospholipase treatment and Triton X-100 extraction. Proteolytic digestion of the partially purified enzyme using trypsin and chymotrypsin and isolation of a peptide containing the covalently linked flavin coenzyme permitted determination of the amino acid composition and sequence of the flavin region of the catalytic site of the enzyme. The structure of the flavin peptide was found to be identical to that of the bovine liver enzyme which is clorgyline insensitive, hence, the B form. The flavin peptide segment of mitochondrial monoamine oxidase is thus conserved between the two forms and among mammalian species.

Identification and Properties of the Prosthetic Group of Choline Oxidase from <italic>Alcaligenes</italic> sp.

Choline oxidase from Alcaligenes sp. catalyzed the oxidation of choline and betaine aldehyde to betaine with concomitant consumption of oxygen and production of hydrogen peroxide. The values of Km for choline and betaine aldehyde were 0.87 and 6.2 mM, respectively. The molecular weight of the enzyme was estimated to be 66,000 by SDS-gel electrophoresis and 72,000 by gel-filtration using a high performance liquid chromatograph. The prosthetic group of the enzyme was identified as 8 alpha-[N(3)-histidyl]-FAD from the electrophoretic mobility at pH 6.25 of the hydrolysate of the methylated histidylflavin. The visible absorption spectrum of the enzyme showed peaks at 358 and 453 nm and a shoulder at about 480 nm. The covalently bound FAD was reduced on addition of either choline or betaine aldehyde under anaerobic conditions and was reoxidized by aeration. The enzyme was found to contain 1 mol of FAD per mol enzyme. Amino acid analysis of a purified flavin peptide gave the following molar ratios of amino acids to flavin: pro(1), Asp + Asn(3), Ser(1), His(1), and Arg(1). Aspartic acid was the N-terminal amino acid. The partial sequence of amino acids in the flavin peptide was as follows: Formula (See Text).

Thiols liberate covalently bonded flavin from monoamine oxidase

Although it was recommended that 2-mercaptoethanol should be added to all buffers used in monoamine oxidase purification, purification of the enzyme in the absence of thiols has yielded double the amount of enzyme. In fact the presence of 0.1 M 2-mercaptoethanol or dithioerythritol-SDS 1 was found to liberate about 50% of the covalently bonded FAD. This observation is surprising since the covalently bonded flavin has been reported to be 8-α-cysteinyl-FAD. There is a thioether linkage between the flavin and the protein and mild reducing agents such as mercaptoethanol would not normally cleave the thioether linkage. The importance of the present finding is that the thiols may be used possibly to liberate suicide substrate-flavin adducts from the enzyme for structural studies without isolating pure flavin peptides. The flavin can be removed simply by treatment of the enzyme with 0.1 M thiol solution containing 1% sodium dodecylsulfate followed by chromatography on Sephadex G-25.

Fumarate reductase of Escherichia coli. Elucidation of the covalent-flavin component.

Fumarate reductase is a membrane-bound terminal oxidase which is induced when Escherichia coli is grown anaerobically. The purified enzyme is composed of two polypeptide chains of 69,000 and 24,000 daltons and contains 1 mol of covalently bound flavin adenine dinucleotide per mol of enzyme. Fluorescence scanning of SDS-polyacrylamide gels of the protein shows that the flavin is attached to the large subunit. The hypsochromic shift of the 372 nm band of riboflavin to 350 nm in both native fumarate reductase and a flavin peptide released by proteolytic digestion indicates that the flavin is attached via position 8 alpha of riboflavin. Based on the spectral properties and pH-fluorescence dependence we have identified the linkage as 8 alpha-[N(3)-histidyl]FAD.

Identification of the covalently bound flavin prosthetic group of cholesterol oxidase.

Highly purified preparations of cholesterol oxidase from Schizophyllum commune contain a covalently bound flavin component. A flavin peptide has been obtained by digestion with trypsin-chymotrypsin and purification on a column of phosphocellulose. Digestion with nucleotide pyrophosphatase results in increased fluorescence at pH 3.4 and release of 5'-adenylate, showing that the flavin is in the dinucleotide form. The absorption spectrum of the flavin peptide shows the hypsochromic shift of the second absorption band characteristic of 8 alpha-substituted flavins. The fluorescence at pH 7 is extensively quenched even in the mononucleotide form, with a pKa at pH 5.8 in the flavin peptide and at 5.05 following acid hydrolysis to the aminoacyl flavin level. This suggests that histidine is the amino acid substituted at the 8 alpha position of the flavin and that N(1) of the imidazole ring is the site of attachment. These data, the reduction of the flavin by borohydride, and comparison of the mobilities in high voltage electrophoresis at two pH values with N(1)- and N(3)-histidyl riboflavin and their 2',5'-anhydro forms shows that the prosthetic group of cholesterol oxidase is 8 alpha-[N(1)-histidyl]-FAD.

Purification and Some Properties of Cholesterol Oxidase from <italic>Schizophyllum commune</italic> with Covalently Bound Flavin<xref ref-type="fn" rid="fn1"><sup>1</sup></xref>

Cholesterol oxidase [EC 1.1.3.6] from Schizophyllum commune was purified by an affinity chromatography using 3-O-succinylcholesterol-ethylenediamine (3-cholesteryl-3-[2-aminoethylamido]propionate) Sepharose gels. The resulting preparation was homogeneous as judged by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis. The molecular weight of the enzyme was estimated to be 53,000 by SDS-gel electrophoresis and 46,000 by sedimentation equilibrium. The enzyme contained 483 amino acid residues as calculated on the basis of the molecular weight of 53,000. The enzyme consumed 60 mumol of O2/min per mg of protein with 1.3 mM cholesterol at 37 degrees C. The enzyme showed the highest activity with cholesterol; 3 beta-hydroxysteroids, such as dehydroepiandrosterone, pregnenolone, and lanosterol, were also oxidized at slower rates. Ergosterol was not oxidized by the enzyme. The Km for cholesterol was 0.33 mM and the optimal pH was 5.0. The enzyme is a flavoprotein which shows a visible absorption spectrum having peaks at 353 nm and 455 nm in 0.1 M acetate buffer, pH 4.0. The spectrum was characterized by the hypsochromic shift of the second absorption peak of the bound flavin. The bound flavin was reduced on anaerobic addition of a model substrate, dehydroepiandrosterone. Neither acid not heat treatment released the flavin coenzyme from the enzyme protein. The flavin of the enzyme could be easily released from the enzyme protein in acid-soluble form as flavin peptides when the enzyme protein was digested with trypsin plus chymotrypsin. The mobilities of the aminoacyl flavin after hydrolysis of the flavin peptides on thin layer chromatography and high voltage electrophoresis differed from those of free FAD, FMN, and riboflavin. A pKa value of 5.1 was obtained from pH-dependent fluorescence quenching process of the aminoacyl flavin. AMP was detected by hydrolysis of the flavin peptides with nucleotide pyrophosphatase. The results indicate strongly that cholesterol oxidase from Schizophyllum commune contains FAD as the prothetic group, which is covalently linked to the enzyme protein. The properties of the bound FAD were comparable to those of N (1)-histidyl FAD.

46] Synthesis and isolation of 8α-substituted flavins and flavin peptides

Publisher Summary This chapter provides information on the synthesis and isolation of 8α-substituted flavins and flavin peptides. The presence of covalently bound flavin in succinate dehydrogenase was demonstrated in 1955 but a complete proof that its structure is 8α-[N (3)-histidyl]-FAD was published after some 15 years. Determination of flavin fluorescence has manifold use in the field of 8α-substituted flavins. First, it may provide a convenient means of estimating the quantity of flavin peptide during the early stages of purification in the presence of contaminating materials, such as heme peptides, which interfere with spectrophotometric determination. Second, it is often possible to predict whether a histidyl or cysteinyl residue is involved in the linkage. Despite these advances, the determination of the structure of a new type of covalently bound flavin remains a difficult task, particularly in enzymes containing a cysteinyl flavin, because these tend to be very unstable, particularly after hydrolysis to the aminoacyl flavin level.

Structure of the covalently bound flavin of Chlorobium cytochrome [formula omitted

Cytochrome c 553 ( Chlorobium thiosulfatophilum), is known to contain both a cytochrome moiety and a flavin component, which is not extracted by denaturation with trichloroacetic acid. In the present study a peptic flavin peptide was isolated and characterized. This confirms the hypothesis that the flavin is covalently linked to the polypeptide chain. Several lines of evidence suggest that the flavin is attached via its 8α position to the sulfur of a cysteinyl residue. These include a hypsochromic shift of the near ultraviolet maximum relative to riboflavin, an increase in fluorescence on oxidation with performic acid (from <6% to 70% that given by an equimolar concentration of riboflavin), with a further hypsochromic shift of the near ultraviolet maximum, and the presence of cysteine after acid hydrolyis but not after aminopeptidase M digestion. All of these properties have previously been observed in 8α-S- cysteinylriboflavin and its peptides. The cysteinyl flavin bond survives mild acid hydrolysis if the sulfur moiety is first oxidized to the sulfone prior to acid treatment. It is thus possible to obtain an amino acyl derivative by this method and show its identity to 8α-S- cysteinylsulfone-2′-5′-anhydroriboflavin obtained via synthetic methods. The flavin occurs as a dinucleotide, presumably FAD, and from the many properties in common with cysteinylriboflavin and its peptides it is concluded that the flavin structure of Chlorobium cytochrome c 553 is 8α-S- cysteinyl-FAD thioether. The amino acid sequence around the flavin site was found to be: ▪

Identification and properties of the covalently bound flavin of beta-cyclopiazonate oxidocyclase.

Beta-Cyclopiazonate oxidocyclase from Penicillium cyclopium has been previously shown to contain flavin dinucleotide in covalent linkage to the protein. In the present study, a pure flavin mononucleotide peptide was isolated from the enzyme by tryptic-chymotryptic digestion, chromatography on Florisil and on diethylaminoethylcellulose, and hydrolysis with nucleotide pyrophosphatase. The flavin peptide contains 9 amino acids, including histidine in linkage to the flavin, and Asx as the N-terminal residue. The fluorescence of the flavin in the FMN peptide is profoundly quenched even at pH 3.2, where protonation of the imidazole prevents queching of the flavin fluorescence by histidine. This quenching appears to be due to interaction of the flavin with a tryptophan residue, as the quenching is abolished by oxidation of the tryptophan with performic acid. Similarly, the fluorescence of the tryptophan in the peptide is quenched, presumably by the flavin. The flavin of beta-cyclopiazonate oxidocylcase is attached, by the way of the 8alpha-methylene group, to the imidazole ring of a histidine. The aminoacylflavin isolated from the enzyme is identical in the pKa of its imidazole group, in reduction by NaBH4, and in other properties with synthetic 8alpha-(N1-histidyl)riboflavin. The pKa of the histidylriboflavin component of the oxidocyclase is 5.2 before and 5.0 after acid modification of the ribityl chain, as is found in the synthetic derivative. It is concluded that the enzyme contains the N1 isomer of histidylriboflavin and that acid hydrolysis of flavin peptides isolated from the oxidocyclase, while liberating histidylriboflavin, also causes acid modification of the ribityl chain of the flavin moiety.

Identification of the covalently bound flavin of thiamin dehydrogenase.

Thiamin dehydrogenase, a flavoprotein isolated from an unidentified soil bacterium, contains 1 mol of covalently bound FAD/mol of enzyme. A flavin peptide, isolated from tryptic-chymotryptic digests of the enzyme and hydrolyzed to the FMN level, shows a pH-dependent fluorescence yield being maximal at pH 3.5 to 4.0 and decreasing over 90% at pH 7.5 with a pKa of 5.8. Acid hydrolysis of the peptide results in an aminoacylflavin which shows a pKa of fluorescence quenching of 5.2. Absorption and electron paramagnetic resonance spectral data show the covalent substituent to be at the 8alpha position of the flavin as is the case with all known enzymes containing covalently bound flavin. The aminoacylflavin gives a negative Pauly reaction but yields 1 mol of histidine on drastic acid hydrolysis thus showing an imidazole ring nitrogen as the 8alpha substituent of the flavin. The aminoacylflavin differs from synthetic 8alpha-[N(3)-histidyl]riboflavin or its acid-modified form in pKa of fluorescence quenching, in electrophoretic mobility, in being reduced by borohydride, and in being labile to storage, yielding 8-formylriboflavin. In all of these properties, however, the 8alpha-histidylriboflavin isolated from thiamin dehydrogenase is indistinguishable from 8alpha-[N(1)-histidyl]riboflavin. It is therefore concluded that the FAD moiety of thiamin dehydrogenase is covalently linked via the 8alpha-methylene group to the N(1) position of the imidazole ring of histidine.