Xanthine dehydrogenase (EC 1.2, 1.37) was isolated from chicken livers and immobilized by adsorpt... more Xanthine dehydrogenase (EC 1.2, 1.37) was isolated from chicken livers and immobilized by adsorption to a Sepharose derivative, prepared by reaction of n‐octy‐lamine with CNBr‐activated Sepharose 4B. Using a crude preparation of enzyme for immobilization it was observed that relatively more activity was adsorbed that protein, but the yield of immobilized activity increased as a purer enzyme preparation was used. As more activity and protein were bound, relatively less immobilized activity was recovered. This effect was probably due to blocking of active xanthine dehydrogenase by protein impurities. The kinetics of free and immobilized xanthine dehydrogenase were studied in the pH range 7.5–9.1. The Km and V values estimated for free xanthine dehydrogenase increase as the pH increases; the K'm and V values for the immobilized enzyme go through a minimum at pH 8.1. By varying the amount of enzyme activity bound per unit volume of gel, it was shown that K'm is larger than Km as...
Scheme 1 Ionisations of fully reduced flavin. ).......5~ N e A) The importance of the N(l)-H ioni... more Scheme 1 Ionisations of fully reduced flavin. ).......5~ N e A) The importance of the N(l)-H ionisation has long been recognized. It strongly influences the reactivity of the reduced moleeule, in particular towards oxygen. A shift of this pK also reflects changes in the redox state of the isoalloxazine [1]. B) The ionisation of N(3)-H does not appear to have a major role in influencing the catalytic properties of the flavin. In some cases, e.g. with oxidized glycollate oxidase, the pKa of N(3)-H is lowered frorn -10 to -6.7 due to the interaction with the
The condensation of 3-hydroximino-2-butanone (1) with 5-amino-6-ribitylamino-2,4(1H,3H)- pyrimidi... more The condensation of 3-hydroximino-2-butanone (1) with 5-amino-6-ribitylamino-2,4(1H,3H)- pyrimidinedione (2) yields 6,7-dimethyl-8-ribityllumazine (3). At slightly alkaline pH, the carbonyl group of 1 reacts preferentially with the 5-amino group of 2 (regioselectivity, 4:1). Under acidic conditions, the reaction occurs with higher yield and marginal regioselectivity of opposite direction (1:1.4). Appropriately 13C-labeled samples of 1 afford 3 labeled at C-6α, C-6, C-7 or C-7α. [6α,7α-13C2]-3 was prepared by condensation of 2 with [1,4-13C2]diacetyl. The lumazines 3 were converted to riboflavin by the enzyme, riboflavin synthase, with almost quantitative yield. By this procedure, any C-atom of the carbocyclic moiety of riboflavin can be selectively labeled with 13C at high abundance. Phosphorylation yields the respectively 13C-labeled FMN samples.
The interaction of flavinium salts with triphenylphosphine is described. The complexes which can ... more The interaction of flavinium salts with triphenylphosphine is described. The complexes which can be isolated in high yields in the crystalline state contain one mole triphenylphosphine per mole flavin. Triphenylphosphine is covalently bound to the N (5)-atom of the isoalloxazine ring system. Dissociation constants and rate constants have been determined spectrophotometrically. Evidence for the structure of the complex was obtained from light absorption, infrared and nuclear magnetic resonance studies. The similarities between the flavin-triphenylphosphine adduct and the earlier described flavin-sulfite adduct are described and the possible biological relevance of these complexes is discussed.
Maleimide spin label was covalently bound to sulfhydryl residues of ᴅ-amino acid oxidase and lipo... more Maleimide spin label was covalently bound to sulfhydryl residues of ᴅ-amino acid oxidase and lipoamide dehydrogenase. Labeling of native ᴅ-amino acid oxidase resulted in a non-homogeneous EPR-spectrum, which consisted of 4 moles of spin label bound immobile to the enzyme (mol.-weight 90.000). A detailed analysis of the spectrum, the kinetics of the reaction of the spin label with the protein and the temperature dependence of the spectrum showed that the spectrum originates from two different pairs of sulfhydryl groups. The spin labeled lipoamide dehydrogenase yielded also a mixed EPR-spectrum of two different bound spin labels, i.e. an immobile and a semimobile species. The temperature dependence gave for both types of spectra a transition point at 10°C. Titration with urea gave only for the immobile species a transition at 1.5 M. Part of the semimobile species seems to be bound near the active site. ᴅ-amino acid oxidase was also specifically labeled, near the active site, with a su...
Quantitative light absorption data of the neutral free flavosemiquinone are reported. It is shown... more Quantitative light absorption data of the neutral free flavosemiquinone are reported. It is shown that the visible range of the spectrum of the radical is drastically dependent on the solvent. The spectra of the neutral blue flavosemiquinone are compared with those of the blue flavoprotein radicals and conclusions are drawn concerning the environment of the protein‐bound flavosemiquinone.The stabilization of the neutral blue flavosemiquinone through N(5)‐alkylation and/or π‐complex formation with aromatic compounds is described.The light absorption characteristics of a tautomeric neutral, but red‐coloured, flavosemiquinone are described. The results are compared with those obtained from the free anionic and protein‐bound red flavosemiquinones. The two types of radicals cannot be distinguished from each other by electron spin resonance spectrometry but may be recognized by means of light absorption spectrophotometry. The possible biological significance of the neutral red flavosemiqu...
Proceedings of the National Academy of Sciences, 2001
The PHOT1 ( NPH1 ) gene from Avena sativa specifies the blue light receptor for phototropism, pho... more The PHOT1 ( NPH1 ) gene from Avena sativa specifies the blue light receptor for phototropism, phototropin, which comprises two FMN-binding LOV domains and a serine/threonine protein kinase domain. Light exposure is conducive to autophosphorylation of the protein kinase domain. We have reconstituted a recombinant LOV2 domain of A. sativa phototropin with various 13 C/ 15 N-labeled isotopomers of the cofactor, FMN. The reconstituted protein samples were analyzed by NMR spectroscopy under dark and light conditions. Blue light irradiation is shown to result in the addition of a thiol group (cysteine 450) to the 4a position of the FMN chromophore. The adduct reverts spontaneously in the dark by elimination. The light-driven flavin adduct formation results in conformational modification, which was diagnosed by 1 H and 31 P NMR spectroscopy. This conformational change is proposed to initiate the transmission of the light signal via conformational modulation of the protein kinase domain con...
Proceedings of the National Academy of Sciences, 1981
Transient fluorescent species are observed in the bioluminescent reactions of three reduced flavi... more Transient fluorescent species are observed in the bioluminescent reactions of three reduced flavin mononucleotides with aliphatic aldehydes and oxygen, catalyzed by bacterial luciferase. In each case the fluorescence spectral distribution is similar to that of the bioluminescence but is readily distinguishable from it on the basis of a significantly greater signal strength. The corrected bioluminescence maxima using Beneckea harveyi luciferase are 479 nm (iso-FMNH 2 ), 490 nm (FMNH 2 ), and 560 nm (2-thio-FMNH 2 ). In an ethanol glass at 77 K, 2-thioriboflavin is fluorescent (ϕ F = 0.03, λ max = 562 nm). These results are interpreted by a sensitized chemiluminescence mechanism in which the flavins bound to luciferase act as acceptors of excitation energy. For 2-thio-FMNH 2 , this acceptor species appears to be the oxidized 2-thio-FMN on the basis of the spectral evidence, whereas for the other flavins, some form of reduced species is a more likely candidate.
Unequivocal syntheses of N(1 or 3)‐mono‐substituted 7, 8‐dimethylalloxazines are described. The b... more Unequivocal syntheses of N(1 or 3)‐mono‐substituted 7, 8‐dimethylalloxazines are described. The borohydride reduction of various alloxazines has been studied under aerobic and anaerobic conditions, in the absence of light. These reactions are discussed in relation to other work on 7, 8‐dimethylisoalloxazines (flavins) and on certain flavoproteins.
After more than one‐half century of investigations, the mechanism of bioluminescence from the FMN... more After more than one‐half century of investigations, the mechanism of bioluminescence from the FMNH2 assisted oxygen oxidation of an aliphatic aldehyde on bacterial luciferase continues to resist elucidation. There are many types of luciferase from species of bioluminescent bacteria originating from both marine and terrestrial habitats. The luciferases all have close sequence homology, and in vitro, a highly efficient light generation is obtained from these natural metabolites as substrates. Sufficient exothermicity equivalent to the energy of a blue photon is available in the chemical oxidation of the aldehyde to the corresponding carboxylic acid, and a luciferase‐bound FMNH‐OOH is a key player. A high energy species, the source of the exothermicity, is unknown except that it is not a luciferin cyclic peroxide, a dioxetanone, as identified in the pathway of the firefly and the marine bioluminescence systems. Besides these natural substrates, variable bioluminescence properties are f...
Xanthine dehydrogenase (EC 1.2, 1.37) was isolated from chicken livers and immobilized by adsorpt... more Xanthine dehydrogenase (EC 1.2, 1.37) was isolated from chicken livers and immobilized by adsorption to a Sepharose derivative, prepared by reaction of n‐octy‐lamine with CNBr‐activated Sepharose 4B. Using a crude preparation of enzyme for immobilization it was observed that relatively more activity was adsorbed that protein, but the yield of immobilized activity increased as a purer enzyme preparation was used. As more activity and protein were bound, relatively less immobilized activity was recovered. This effect was probably due to blocking of active xanthine dehydrogenase by protein impurities. The kinetics of free and immobilized xanthine dehydrogenase were studied in the pH range 7.5–9.1. The Km and V values estimated for free xanthine dehydrogenase increase as the pH increases; the K'm and V values for the immobilized enzyme go through a minimum at pH 8.1. By varying the amount of enzyme activity bound per unit volume of gel, it was shown that K'm is larger than Km as...
Scheme 1 Ionisations of fully reduced flavin. ).......5~ N e A) The importance of the N(l)-H ioni... more Scheme 1 Ionisations of fully reduced flavin. ).......5~ N e A) The importance of the N(l)-H ionisation has long been recognized. It strongly influences the reactivity of the reduced moleeule, in particular towards oxygen. A shift of this pK also reflects changes in the redox state of the isoalloxazine [1]. B) The ionisation of N(3)-H does not appear to have a major role in influencing the catalytic properties of the flavin. In some cases, e.g. with oxidized glycollate oxidase, the pKa of N(3)-H is lowered frorn -10 to -6.7 due to the interaction with the
The condensation of 3-hydroximino-2-butanone (1) with 5-amino-6-ribitylamino-2,4(1H,3H)- pyrimidi... more The condensation of 3-hydroximino-2-butanone (1) with 5-amino-6-ribitylamino-2,4(1H,3H)- pyrimidinedione (2) yields 6,7-dimethyl-8-ribityllumazine (3). At slightly alkaline pH, the carbonyl group of 1 reacts preferentially with the 5-amino group of 2 (regioselectivity, 4:1). Under acidic conditions, the reaction occurs with higher yield and marginal regioselectivity of opposite direction (1:1.4). Appropriately 13C-labeled samples of 1 afford 3 labeled at C-6α, C-6, C-7 or C-7α. [6α,7α-13C2]-3 was prepared by condensation of 2 with [1,4-13C2]diacetyl. The lumazines 3 were converted to riboflavin by the enzyme, riboflavin synthase, with almost quantitative yield. By this procedure, any C-atom of the carbocyclic moiety of riboflavin can be selectively labeled with 13C at high abundance. Phosphorylation yields the respectively 13C-labeled FMN samples.
The interaction of flavinium salts with triphenylphosphine is described. The complexes which can ... more The interaction of flavinium salts with triphenylphosphine is described. The complexes which can be isolated in high yields in the crystalline state contain one mole triphenylphosphine per mole flavin. Triphenylphosphine is covalently bound to the N (5)-atom of the isoalloxazine ring system. Dissociation constants and rate constants have been determined spectrophotometrically. Evidence for the structure of the complex was obtained from light absorption, infrared and nuclear magnetic resonance studies. The similarities between the flavin-triphenylphosphine adduct and the earlier described flavin-sulfite adduct are described and the possible biological relevance of these complexes is discussed.
Maleimide spin label was covalently bound to sulfhydryl residues of ᴅ-amino acid oxidase and lipo... more Maleimide spin label was covalently bound to sulfhydryl residues of ᴅ-amino acid oxidase and lipoamide dehydrogenase. Labeling of native ᴅ-amino acid oxidase resulted in a non-homogeneous EPR-spectrum, which consisted of 4 moles of spin label bound immobile to the enzyme (mol.-weight 90.000). A detailed analysis of the spectrum, the kinetics of the reaction of the spin label with the protein and the temperature dependence of the spectrum showed that the spectrum originates from two different pairs of sulfhydryl groups. The spin labeled lipoamide dehydrogenase yielded also a mixed EPR-spectrum of two different bound spin labels, i.e. an immobile and a semimobile species. The temperature dependence gave for both types of spectra a transition point at 10°C. Titration with urea gave only for the immobile species a transition at 1.5 M. Part of the semimobile species seems to be bound near the active site. ᴅ-amino acid oxidase was also specifically labeled, near the active site, with a su...
Quantitative light absorption data of the neutral free flavosemiquinone are reported. It is shown... more Quantitative light absorption data of the neutral free flavosemiquinone are reported. It is shown that the visible range of the spectrum of the radical is drastically dependent on the solvent. The spectra of the neutral blue flavosemiquinone are compared with those of the blue flavoprotein radicals and conclusions are drawn concerning the environment of the protein‐bound flavosemiquinone.The stabilization of the neutral blue flavosemiquinone through N(5)‐alkylation and/or π‐complex formation with aromatic compounds is described.The light absorption characteristics of a tautomeric neutral, but red‐coloured, flavosemiquinone are described. The results are compared with those obtained from the free anionic and protein‐bound red flavosemiquinones. The two types of radicals cannot be distinguished from each other by electron spin resonance spectrometry but may be recognized by means of light absorption spectrophotometry. The possible biological significance of the neutral red flavosemiqu...
Proceedings of the National Academy of Sciences, 2001
The PHOT1 ( NPH1 ) gene from Avena sativa specifies the blue light receptor for phototropism, pho... more The PHOT1 ( NPH1 ) gene from Avena sativa specifies the blue light receptor for phototropism, phototropin, which comprises two FMN-binding LOV domains and a serine/threonine protein kinase domain. Light exposure is conducive to autophosphorylation of the protein kinase domain. We have reconstituted a recombinant LOV2 domain of A. sativa phototropin with various 13 C/ 15 N-labeled isotopomers of the cofactor, FMN. The reconstituted protein samples were analyzed by NMR spectroscopy under dark and light conditions. Blue light irradiation is shown to result in the addition of a thiol group (cysteine 450) to the 4a position of the FMN chromophore. The adduct reverts spontaneously in the dark by elimination. The light-driven flavin adduct formation results in conformational modification, which was diagnosed by 1 H and 31 P NMR spectroscopy. This conformational change is proposed to initiate the transmission of the light signal via conformational modulation of the protein kinase domain con...
Proceedings of the National Academy of Sciences, 1981
Transient fluorescent species are observed in the bioluminescent reactions of three reduced flavi... more Transient fluorescent species are observed in the bioluminescent reactions of three reduced flavin mononucleotides with aliphatic aldehydes and oxygen, catalyzed by bacterial luciferase. In each case the fluorescence spectral distribution is similar to that of the bioluminescence but is readily distinguishable from it on the basis of a significantly greater signal strength. The corrected bioluminescence maxima using Beneckea harveyi luciferase are 479 nm (iso-FMNH 2 ), 490 nm (FMNH 2 ), and 560 nm (2-thio-FMNH 2 ). In an ethanol glass at 77 K, 2-thioriboflavin is fluorescent (ϕ F = 0.03, λ max = 562 nm). These results are interpreted by a sensitized chemiluminescence mechanism in which the flavins bound to luciferase act as acceptors of excitation energy. For 2-thio-FMNH 2 , this acceptor species appears to be the oxidized 2-thio-FMN on the basis of the spectral evidence, whereas for the other flavins, some form of reduced species is a more likely candidate.
Unequivocal syntheses of N(1 or 3)‐mono‐substituted 7, 8‐dimethylalloxazines are described. The b... more Unequivocal syntheses of N(1 or 3)‐mono‐substituted 7, 8‐dimethylalloxazines are described. The borohydride reduction of various alloxazines has been studied under aerobic and anaerobic conditions, in the absence of light. These reactions are discussed in relation to other work on 7, 8‐dimethylisoalloxazines (flavins) and on certain flavoproteins.
After more than one‐half century of investigations, the mechanism of bioluminescence from the FMN... more After more than one‐half century of investigations, the mechanism of bioluminescence from the FMNH2 assisted oxygen oxidation of an aliphatic aldehyde on bacterial luciferase continues to resist elucidation. There are many types of luciferase from species of bioluminescent bacteria originating from both marine and terrestrial habitats. The luciferases all have close sequence homology, and in vitro, a highly efficient light generation is obtained from these natural metabolites as substrates. Sufficient exothermicity equivalent to the energy of a blue photon is available in the chemical oxidation of the aldehyde to the corresponding carboxylic acid, and a luciferase‐bound FMNH‐OOH is a key player. A high energy species, the source of the exothermicity, is unknown except that it is not a luciferin cyclic peroxide, a dioxetanone, as identified in the pathway of the firefly and the marine bioluminescence systems. Besides these natural substrates, variable bioluminescence properties are f...
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