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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. Osidatlon 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 m 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. [ABSTRACT FROM AUTHOR]

Reports in the literature indicate that the flavin prosthetic group of Chromatium cytochrome c552 is not acid-extractable but is dissociated from the protein by various treatments, including prolonged incubation with urea solution, which are not expected to break covalent bonds. In the present paper it is established by isolation of peptic and of tryptic-chymotryptic flavin peptides that the flavin component is covalently linked to the protein. The peptide chain is attached to the 8α group of the FAD, as in other enzymes containing covalently bound flavin, as evidenced by a hypochromic shift of the near ultraviolet absorption maximum, the characteristics of the hyperfine EPR spectrum, and the release of 8-carboxy-FMN from the cytochrome on oxidation with cold performic acid with concomitant hydrolysis of the pyrophosphate linkage. The fluorescence of the flavin peptides, at the FAD, FMN and riboflavin level, is extensively quenched, with less than 1% of the quantum yield of fluorescence, compared with riboflavin, in the peptic peptide. On oxidation of the tryptic-chymotryptic peptide with performic acid the fluorescence increases to 50% of that given by an equimolar concentration of riboflavin. This increase is accompanied by a further hypochromic shift of the near ultraviolet absorption maximum. This behavior, the tendency of the flavin peptide to undergo autooxidation, and the positive chloroplatinic acid test resemble the properties of cysteinylflavin thioether and its peptides and suggest that the flavin is bonded to a cysteine residue, as in monoamine oxidase. The presence of cysteine in both flavin peptides from cytochrome c552 has been verified by the liberation of cysteine on acid hydrolysis. Despite these similarities to peptides containing a cysteinyl flavin thioether. the peptic and tryptic-chymotryptic peptides from cytochrome c552 show several properties which preclude a thioether linkage. Evidence is summarized to indicate that the flavin is linked via a thiohemiacetal bond to a cysteinyl residue in the polypeptide chain. Thus, the flavin released from the peptides by acid hydrolysis is in every respect identical to 8-formylriboflavin. Further, two flavin components were detected on high voltage electrophoresis after aminopeptidase M digestion of the peptic peptide, which had mobilities expected for the aminoacylflavin and a thiazolidine derivative. A thiohemiacetal structure also ac-counts for the much greater lability of the flavin peptide linkage than that found for monoamine oxidase. Thus from the evidence presented, it is concluded that the structure of the covalently bound flavin in Chromatium cytochrome c552 is 8α-S-cysteinyl-8α-hydroxy-FAD. [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. New flavins have been isolated from purified prepartions of an electron-transferring flavo-protein (ETF) from Peptostreptococcus elsdenii and glycolate oxidase from pig liver. The structures of these new species have been established as FAD and FMN derivatives of 6-hydroxy-7,8-dimethyl-isoalloxazine, the chemical synthesis of which is described in tlhe accompanying paper by Schöllnhammer and Hemmerich. The chromophores are yellow at pH 5 and green at pH 9 due to an ionization at pK 7.1. 2. 6-OH-FAD is bound by apo-ETF and its pK is decreased. The complex is reduced by NADH and it couples the oxidation of NADH to the reduction of dichlorophenolindophenol. Unlike the complex of FAD and apo-ETF, 6-OH-FAD · ETF does not couple the oxidation of NADH to the reduction of butyryl-CoA dehydrogenase. 3. 6-OH-FMN is bound by the FMN-specific protein apoflavodoxin from P. elsdenii and the pK is increased to ≈9. This complex is reduced by sodium dithionite and an intermediate, presumed to be the semi-quinone, is formed at half rduction. [ABSTRACT FROM AUTHOR]

1. The method of "photohydroxylation" of azaheteroaromatics, known in the phenazine and a alloxazine series, has been applied to flavin (= isoalloxazine) derivatives FI[SUBox]H. The products habe been identified as 6- and 9-hydroxyflavins (Fl[SUBox]-6/9-OH). On this basis it was possible to assign a 6-hydroxyflavin structure to the new flavocoenzyme chromophores described by Mayhew et al. in the accompanying paper. 2. In agreement with the data obtained by Dekker et al. in a recent laser study on alloxazine, we find that, the reaction proceeds via, addition of water, alcohols or carboxylic acids by the lowest excited singlet state of flavin with synchronous intramolecular hydrogen shift from the site of RO-addition [= C(6) or C(9)] towards N(5) and subsequent oxidation of the 6/9-hydroxy1.5-dihydroflavin formed. 3. The photoaddition reaction occurs in competition with photodehydrogenation of available CH-bonds by the lowest excited flavin triplet, in which case the CH-substrate may be provided by the solvent ROH or, when R = H, by methyl groups of a second flavin molecule (= flavin autophotolysis). This photodehydrogenation may be suppressed by O[SUB2]-dependent triplet quenching and/or by applying a positive charge to the flavin (RFl[SUBox]H[SUP+], R = H or alkyl) in order to inhibit the reaction with itself. 4. Differentiation of 6- and 9-hydroxyflavin isomers was possible by double resonance evaluation of the proton coupling between the residual Ar-H and the adjacent methyl group, by evaluation of the electron paramagnetic resonance hyperfine coupling for the residual Ar-H in the radical cations 1,5-RHFl-6/9-OH, and by Formation of cupric chelates from 6-hydroxy-flavin as compared to the lack of metal affinity in the 9-isomer. [SUP1]H-Nuclear magnetic resonance, electron paramagnetic resonance and visible spectra and their dependence on the state of ionisation of hydroxyflavins are discussed. Hydroxyflavin anions as well as their N(1)-alkylated... [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]

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]

The circular dichroism spectra of bakers' yeast L-actate dehydrogenase and some of its derivatives (without flavin and/or heme) have been investigated in order to obtain some information about the interactions between the prosthetic chromophores or between these chromophores and the protein moiety. The following derivatives were studied: (a) the cytochrome b2 (flavohemoprotein), (b) the apocytochrome b2 (following FMN removal), (c) the noyou cytochromique b2 (low molecular weight core isolated following proteolytic degradation), (d) the apo-noyau cytochromique b2 (obtained by removal of the heme from the latter). It is shown that the circular dichroism spectra of the protein moieties in the ultraviolet region (200-250 nm) are not affected, either by the removal of the flavin from the flavocytochrome b2, or by the removal of the heine from the noyau cytochromique b2. The significance of the values calculated for the α-helical content is discussed. The oxido-reduction state of the ferric ion does not affect the circular dichroism spectra in this region. The circular dichroism bands associated with the heme chromophore are essentially the same in the apocytochrome b2 and in the noyau cytochromique b2 i. e. both without the flavin moiety, but are markedly changed in the flavocytochrome b2, in particular with an inversion of the strong γ band. Noticeable differences are observed with a homologous enzyme extracted from the yeast Hansenula anomala, especially in the γ band region. In conclusion there appears to be no prosthetic group-protein interaction detectable by circular dichroism measurements, i. e. no important modification of the peptide bond geometry occurs when the flavin is removed from the enzyme or when the oxido-reduction state of the heine is varied. However, a strong effect is exerted by the flavin on the symmetry of the heine environment, this effect being probably au indirect one, via the protein moiety, as discussed in this paper. [ABSTRACT FROM AUTHOR]

The dye proflavin (3,6 diaminoacridine) binds to a single site on the ficin molecule (EC 3.4.4.12). The value of the dissociation constant (Kp) for the enzyme-dye complex was 3.5±1.8 × 10-4 M measured spectrophotometrically and 1.55± 0.61 × 10-4 by equilibrium dialysis. Interaction with proflavin increased the catalytic constant (kcat) for the ficin-catalysed hydrolysis of benzoyl-L-arginine ethyl ester although the value of KM app was unaltered. Dye-binding did not significantly affect the value of kcat for the ficin-catalysed hydrolysis of p-nitrophenyl hippurate, a substrate for which acylation is not rate-limiting in catalysis. The dependence of initial velocity of benzoyl-L-arginine ethyl ester hydrolysis on dye concentration enabled the computation of a value of Kp=1.61 7times; 10-4 M assuming a kinetic model involving formation of a ternary dye-enzyme-substrate complex in which substrate is more rapidly hydrolyzed than in the binary enzyme-substrate complex. Dye-binding leads to an enhanced nucleophilicity of the essential thiol group of ficin. It is concluded that deacylation is not the rate-limiting step in the ficin-catalysed hydrolysis of benzoyl-L-arginine ethyl ester. Possible explanations of the proflavin-induced acceleration of benzoyl L-arginine ethyl ester hydrolysis by ficin are discussed, including consideration of an allosteric modification mechanism. [ABSTRACT FROM AUTHOR]

NADPH-diaphorase was extensively purified from Bacillus subtilis NY. FMN is most probably the prosthetic group of this enzyme. NAPH is oxidized by oxygen as the terminal acceptor only in the presence of catalytic amounts of free flavins (Km for FMN was 1.6 x 10-6 M at pH 8.8). NADPH was directly oxidized in the presence of various electron acceptors such as quinines, ferricyaide and tetrazolium salts. Cytochrome c was a very poor acceptor in this system. Km for NAPH was 2.0 x 10-4 M and K1 for NADPH+ as a competitive inhibitor was 1.1 x 10-3 M at pH 7.2 Mn++ or Mg++ at mM concentration showed some stimulation of enzyme activity. The enzyme was partially inhibited by various cheating agents and p-hydroxymercuribenzoate but not by several typical inhibitors of the respiratory chain. It is suggested that this flavoprotein can serve as a useful tool for NADP+ regeneration in coupled enzyme reactions. [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]

D-6-Hydroxynicotine oxidase from Arthrobacter oxidans grown on citrate and DL-nicotine was obtained as a homogeneous protein as judged by ultracentrifugation and disc electrophoresis. From the sedimentation and diffusion coefficients of 3.54 S and 6.69 F, respectively, and from sedimentation equilibrium measurements a molecular weight of 53000 was calculated. D-6-Hydroxynicotine oxidase consists of one polypeptide chain and contains one mol FAD per mol enzyme. The flavin was shown to be covalently bound to the protein. In the presence of oxygen, D-6-hydroxynicotine oxidase converts D-6-hydroxynicotine [6-hydroxypyridyl-(3)]-(4-N-methylaminopropyl)-ketone. Molecular oxygen could be replaced as electron acceptor by methylene blue and 2,6-dichlorophenolindophenol but not by one-electron acceptors Anaerobic reduction of the enzyme with substrate did not reveal a semiquinoid flavin intermediate. The spectral changes obtained by the photochemical reduction of D-6-hydroxynicotine oxidase in the presence of EDTA, however, indicated an anionic semiquinone form. D-6-Hydroxynicotine oxidase proved to he highly specific for the structural and absolutely specific for the stereochemical configuration of its substrate. Among others, L-6-hydroxynicotine and the reaction product were found to be competitive inhibitors. [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]

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 add 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. [ABSTRACT FROM AUTHOR]

The proton electron-nuclear double resonance (ENDOW) of flavin rascals in NADPH dehydrogenase and a flavoprotein from Azotobacter vinelandii have been studied at --160 and --120°. By combination of the apoprotein of NADPH dehydrogenase with [8-Me-2H3]FMN, it was demonstrated that the CH3(8) group of the coenzyme gives an ENDOR signal. The ENDOR signals from CH3(8) of the two proteins showed different hyperfine couplings which are consistent with the assignments of a radical anion, or a neutral rascal enolized at 0(4), in NADPH dehydrogenase and a neutral rascal protonated at N(5) in the Azotobacter flavoprotein. Analysis of the matrix-ENDOR signals in H2O and 2H2O yields information concerning the exchangeability of protons both in the constituent water and on the apoprotein in the vicinity of the rascal. [ABSTRACT FROM AUTHOR]

The neutral flavosemiquinone has been studied in detail by electron spin resonance spectroscopy. Isotopic (15N, 2H) and chemical substitutions have been carried out. A hyperfine coupling scheme of the neutral flavosemiquinone is described where N(5), N(10), CH3(10), CH3(8) and H(6) are involved. The highest spin density is located at N(5) as has also been found for the anionic and chelated form of the flavosemiquinone. The exchangeable proton in the neutral flavosemiquinone is attached to N(5) having a coupling constant of 7.6 G which is about the same magnitude as the coupling constant to N(5). The hyperfine couplings are interpreted in terms of spin densities and comparison is made with available quantum chemical calculations. Evidence is presented that two tautomeric forms of the neutral flavosemiquinone have to be considered which can be obtained in a pure form by suitable substitution of flavin derivatives. The two types of neutral flavosemiquinones are easily distinguishable by their electron spin resonance and light absorptions. [ABSTRACT FROM AUTHOR]

The complexes formed between Zn2+ or Cd2+ and half-reduced flavins (isoalloxazines) in neutral aqueous as well as non-aqueous media were studied by electron spin resonance. Formation of 1:1 radical chelates between anionic radicals and Zn2+ as well as Cd2+ were found to occur. The stability constants at 20° for the chelates were estimated. A number of flavin derivatives were investigated. The spin density distribution was mapped by means of isotopic substitutions (²H, 15N, 64Zn, 67Zn, 111Cd, and 114Cd). The spin distribution within the ligand is similar to that found earlier for the anionic flavin radical. Spin delocalization to the metals was observed and hyperfine coupling constants for magnetic isotopes determined by direct measurements and spectra simulations. The difference in spin delocalization between Zn and Cd is discussed. [ABSTRACT FROM AUTHOR]