19 results on '"*FLAVINS"'
Search Results
2. Redox properties of wild-type, Cys69Ala, and Cys69Ser <em>Azotobacter vinelandii</em> flavodoxin II as measured by cyclic voltammetry and EPR spectroscopy.
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Steensma, Elles, Heering, Hendrik A., Hagen, Wilfred R., and Van Mierlo, Carlo P.M.
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PROTEINS , *FLAVINS , *NUCLEOTIDES , *BIOELECTROCHEMISTRY , *ELECTRON paramagnetic resonance , *NEOMYCIN - Abstract
This study deals with the detailed electrochemistry and complete EPR-monitored titrations of flavodoxin II of Azotobacter vinelandii (ATCC 478). Since wild-type flavodoxin dimerises via intermolecular disulphide bond formation between Cys69 residues. Cys69 has been replaced by both an alanine and a serine residue. Redox properties of she C69A and C69S flavodoxin mutants were compared to those of wild-type flavodoxin. In the presence of the promotor neomycin, C69A and C69S flavodoxin showed a reversible response of the semiquinone/hydroquinone couple at the glassy carbon electrode. However, the addition of dithiothreitol proved to be necessary for the stabilisation of the wild-type flavodoxin response. EPR-monitored redox titrations of wild-type and C69A flavodoxin at high and low pH confirmed the redox potentials measured using cyclic voltammetry. The pH dependence of the semiquinone/hydroquinone redox potentials cannot be described using a model assuming one redox-linked pK. Instead, the presence of at least two redox-linked protonation sites is suggested: pKred,1 = 5.39 ± 0.08. pKox = 7.29 ± 0.14, and pKred,2 = 7.84 ± 0.14 with Em,7 = -459 ± 4 mV, and a constant redox potential at high pH of -485 ± mV. The dependence of the semiquinone/hydroquionone redox potential on temperature is -0.5 ± 0.1 mV · K-1, yielding ΔH° = 28.6 ± 1.5 kJ · mol-1 and ΔS° = -50.0 ± 6.2 J · mol -1 · K-1. No significant differences in redox properties of wild-type. C69A, and C69S flavodoxin were observed. The electrochemical data suggest that replacement of Cys69 in the vicinity of the FMN by either an alanine or a serine residue does not alter the dielectric properties and structure of A. vinelandii flavodoxin 11. [ABSTRACT FROM AUTHOR]
- Published
- 1996
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3. Fluorescence Properties of Reduced Flavins and Flavoproteins.
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Visser, Antonie J. W. G., Ghisla, Sandro, Massey, Vincent, Muller, Franz, and Veeger, Cees
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FLAVINS , *FLAVOPROTEINS , *FLUORESCENCE , *RADIOACTIVITY , *PROTEINS , *PROTEOMICS - Abstract
Fluorescence lifetimes and polarized emission properties of reduced flavin were measured using several model compounds and flavoproteins. Depending on the conditions of solvent and temperature or reduction method the lifetimes vary between 1 and 15 ns. The longer lifetime values are found in several forms of reduced lactate oxidase, in which a good correlation exists between fluorescence intensity and lifetime. In practically all flavoproteins the fluorescence is heterogeneous. Several mechanisms are proposed to explain the observed heterogeneity in lifetimes. The reduced models in glycerol at subzero temperature exhibit high degrees of polarization of the fluorescence, whereas distinct depolarization is encountered in several reduced flavoproteins suggesting a certain mobility of the flavin chromophor. [ABSTRACT FROM AUTHOR]
- Published
- 1979
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4. On the Structure of Flavin-Oxygen Intermediates Involved in Enzymatic Reactions.
- Author
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Ghisla, Sandro, Entsch, Barrie, Massey, Vincent, and Husein, Mazhar
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FLAVOPROTEINS , *FLAVINS , *PEROXIDES , *COENZYMES , *PROTEINS - Abstract
During the catalytic reactions of flavoprotein hydroxylases and bacterial luciferase, flavin peroxides are formed as intermediates [see Massey, V. and Hemmerich, P. (1976) in The Enzymes, 3rd edn (P. Boyer, ed.) pp. 421-505, Academic Press, New York]. These intermediates have been postulated to be C(4a) derivatives of the flavin coenzyme. To test this hypothesis, modified flavin coenzymes carrying an oxygen substituent at position C (4a) of the isoalloxazine ring were synthesized. They are tightly bound by the apoenzymes of D-amino acid oxidase, p-hydroxybenzoate hydroxylase and lactate oxidase; the resulting complexes show spectral properties closely similar to those of the transient oxygen adducts of the hydroxylases. The optical spectra of the lumiflavin model compounds were found to be highly dependent on the solvent environment and nature of the substituents. Under appropriate conditions they simulate satisfactorily the spectra of the transient enzymatic oxygen adducts. The results support the proposal that the primary oxygen adducts formed with these flavo-proteins on reaction of the reduced enzymes with oxygen are flavin C(4a) peroxides. [ABSTRACT FROM AUTHOR]
- Published
- 1977
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5. The Dissociation of Flavin Coenzymes from Trypsin-Solubilized NADPH/Cytochrome <em>c</em>(P-450) Reductase of Pig-Liver Microsomes.
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Trout, Gordon E.
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TRYPSIN , *PANCREATIC secretions , *CYTOCHROME c , *ENZYMES , *PROTEINS , *FLAVINS - Abstract
The change in fluorescence emission at 520 nm after excitation at 365 nm was used to investigate the effect of pH and ionic strength on the dissociation of flavin cofactors from microsomal NADPH/cytochrome c (P-450) reductase. In the unmodified enzyme both the FAD and FMN moieties appeared to dissociate at a similar rate and followed first-order kinetics. The rate constant for the dissociation was increased by low pH and high ionic strength, particularly in the range pH 4.4-3.8 (0.02 M acetate buffer) where the rate constants increased 80-fold. Modification of the enzyme by treatment with p-chloromercuribenzoate enhanced the rate of flavin dissociation and, in the region of pH 4, resulted in a biphasic increase in fluorescence consistent with two simultaneous parallel first-order dissociations. It was concluded that p-chloromercuribenzoate treatment modified the protein so that the two flavin cofactor dissociated at different rates. Using the measured rate constants for the dissociations, and the known variation in fluorescence of flavin nucleotides with pH, and analogue computer simulation of the dissociation as well as manual curve-fitting procedure showed that the biphasic response could be explained as a simultaneous rapid dissociation of FAD and a slower loss of FMN from the protein. [ABSTRACT FROM AUTHOR]
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- 1976
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6. Identification and Properties of 8-Hydroxyflavin--Adenine Dinucleotide in Election-Transferring Flavoprotein from <em>Peptostreptococcus elsdenii</em>.
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Ghisla, Sandro and Mayhew, Stephen G.
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FLAVOPROTEINS , *BIOCHEMISTRY , *AMINO acids , *FLAVINS , *HYDROGEN-ion concentration , *PROTEINS - Abstract
A new flavin prosthetic group has been isolated in pure form from the electron-transferring flavoprotein of Peptostreptococcus elsdenii. Its structure has been established as the FAD derivative of 7-methyl-8-hydroxyisoalloxazine:[This symbol cannot be presented in ASCII format]Proof of this structure has been obtained by chemical synthesis of 7-methyl-8-hydroxyisoalloxazine models, and by stepwise degradation of the native compound to 7-methyl-8-hydroxyalloxazine. The orange chromophore is characterized by a strong absorption band with a maximum at 472 nm (ε = 41 000 M&Sup-1; cm⊃-1;) and a pK at 4.8 due to the ionisation of the C(8)-OH group. The properties of a series of functionally substituted derivatives of 8-hydroxy flavins and lumichromes have been investigated to provide a basis for interpreting the effects of pH on the spectroscopic properties of the 8-hydroxy derivatives of FAD and FMN. The 8-hydroxy derivative of FAD is bound by apo-D-amino acid oxidase; the complex shows no catalytic activity. The 8-hydroxy derivative of FMN is bound by apoflavodoxin to give a complex which has catalytic activity similar to that of native flavodoxin. The complex is reversibly reduced by dithionite, first to a relatively stable semiquinone and further to the dihydroflavin form. [ABSTRACT FROM AUTHOR]
- Published
- 1976
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7. Purification and Properties of Cyclopentanone Oxygenase of <em>Pseudomonas</em> NCIB 9872.
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Griffith, Martin and Trudgill, Peter W.
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OXYGENASES , *FLAVINS , *PSEUDOMONAS , *POLYACRYLAMIDE , *ELECTROPHORESIS , *PROTEINS , *BIOCHEMISTRY - Abstract
Cyclopentanone oxygenase from Pseudomonas NCIB 9872 has been purified some 40-fold. It gives a single peak in the ultracentrifuge and a single major protein band on polyacrylamide gels contaminated with about 5% of a slower migrating impurity. Flavin dissociates from the protein during electrophoresis. The enzyme has a molecular weight of about 200000 and is a hornopolymeric assemblage of either three or four subunits of molecular weight 54000–58000. The prosthetic group is FAD and values of about 2.5 are typically obtained for the number of moles bound to each mole of holoenzyme. Some FAD probably dissociates during purification and it seems likely that each subunit binds one FAD in the undamaged protein. The unitary stoichiometry of cyclopentanone, oxygen and NADPH is typical of mixed-function oxygenases with external electron donors. The oxygenated product has been identified as 1-oxa-2-oxocyclohexane (5-valerolactone) and enzyme is therefore systematically named as cyclopentanone, NADPH: oxygen oxidoreductase (1,2-lactouizing) (EC 1.14.13.—). Catalytically functional sulfhydryl groups are probably present but the biphasic nature of the inhibition curve when enzyme is titrated with p-hydroxymercuribenzoate reveals the presence of six titratable groups that are not all equivalent. The enzyme has a pH optimum of 7.7. The Km, for cyclopentanone is below 1 μM but not experimentally determinable. The response to variation of NADPH concentration is sigmoidal, indicative of homotrophic binding, a Hill plot gave an interaction number of 3, perhaps indicative of cooperativity between three subunits. Anaerobic reduction of enzyme-bound FAD by addition of NADPH required only slightly in excess of two electron equivalents. No evidence for the formation of stable semiquinones was obtained in this or in photoreduction experiments. Though the enzyme displays sensitivity towards transition metal chelating agents the small amounts of Fe and Cu present make a role in electron transport unlikely. The possibility that they may be involved in maintenance of quaternary structure has not been investigated. [ABSTRACT FROM AUTHOR]
- Published
- 1976
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8. Properties of Immobilized Flavodoxin from Peptostreptococcus elsdenii.
- Author
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Mayhew, Stephen G. and Strating, Marijke J. J.
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PROTEINS , *SEPHAROSE , *HYDROGEN , *FLUORIMETRY , *CHROMATOGRAPHIC analysis , *FLAVINS - Abstract
The small flavoprotein, flavodoxin, isolated from Peptostreptococcus elsdenii, has been covalently coupled to CNBr-activated Sepharose 4B. The immobilized protein replaces ferredoxin as an electron carrier in hydrogen production from dithionite or pyruvate in the presence of ferredoxin-free extracts of P. elsdenii; compared with soluble flavodoxin, its activities in these systems are 13% and 3.5% respectively. Acid treatment reversibly dissociates FMN from the immobilized protein. The dissociation constant of the complex with FMN, determined by fluorimetric titration, is 1.5 (± 0.4) nM, and is therefore very little different from that of soluble flavodoxin. Like soluble apoflavodoxin, the immobilized apoprotein is highly specific for flavins with an N-10 side-chain of 5 carbon atoms and a C-5' phosphate group. Approximately half of the flavin impurity in commercial preparations of FMN (12-15% of the total flavin), and similar impurity in synthetic analogues of FMN, is not separated by conventional purification procedures, but it is readily and conveniently removed by affinity chromatography with apoflavodoxin as the immobilized ligand. The immobilized protein is stable for long periods; its capacity for FMN decreases by only 20% after 15 cycles of flavin dissociation and reassociation during several months. [ABSTRACT FROM AUTHOR]
- Published
- 1975
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9. Flavin dynamics in oxidized Clostridium beijerinckii flavodoxin as assessed by time-resolved polarized fluorescence.
- Author
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Leenders, Rik, Van Hoek, Arie, Van Iersel, Martijn, Veeger, Cees, and Visser, Antonie J. W. G.
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FLAVINS , *FLUORESCENCE , *BOTULINUM toxin , *CLOSTRIDIUM botulinum , *CLOSTRIDIUM , *IODIDES , *PHOSPHATES , *PROTEINS - Abstract
The time-resolved fluorescence characteristics of flavin in oxidized flavodoxin isolated from the anaerobic bacterium Clostridium beijerinckii have been examined. The fluorescence intensity decays were analyzed using the maximum-entropy method. It is demonstrated that there exist large differences in fluorescence behaviour between free and protein-bound FMN. Three fluorescence lifetime components are found in oxidized flavodoxin, two of which are not present in the fluorescence-intensity decay of free FMN. The main component is distributed at 30 ps. with relative contribution of 90%. Another minor component (4% contribution) is distributed at 0.5 ns. The third component is distributed at 4.8 ns (6%), coinciding with the main distribution present in the fluorescence decay of free FMN. The results allowed us to determine the dissociation constant. Kd = 2.61 × 10 10M (at 20°C). Collisional fluorescence-quenching experiments revealed that the flavin moiety responsible for the longest fluorescence lifetime is, at least partially, exposed to the solvent. The shortest lifetime is not affected significantly, indicating that it possibly originates from an active-site conformation in which the flavin is more or less buried in the protein and not accessible to iodide. The fluorescence anisotropy behaviour of free and protein-bound FMN was examined and analyzed with the maximum-entropy method. It was found that an excess of apoflavodoxin is required to detect differences between free and protein-bound FMN. In free FMN one single distribution of rotational correlation times is detected, whereas in flavodoxin the anisotropy decay is composed of more than one distribution. Associative analysis of fluorescence anisotropy decays shows that part of the 4.8 ns fluorescence lifetime present in the flavodoxin fluorescence decay, is coupled to a rotational correlation time similar to that of free FMN in solution, while another part of this lifetime is coupled to a longer correlation time of about, 1 ns. This finding is in accordance with earlier studies [Barman, B. G. & Tollin, G. (1972) Biochemistry II. 4746–4754] in which it was proposed that the first binding step of the flavin to the protein involves the phosphate group rather than another part of the FMN. The two shortest fluorescence lifetimes, which do not carry information on the long-term rotational behaviour of the protein, seem nonetheless to be associated with a longer rotational correlation time which is comparable to overall protein tumbling. These lifetime components probably originate from a complex in which the flayin-ring system is more or less immobilized within the protein matrix. [ABSTRACT FROM AUTHOR]
- Published
- 1993
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10. Structural and redox relationships between <em>Paracoccus denitrificans</em>, porcine and human electron-transferring flavoproteins.
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Watmough, Nicholas J., Kiss, Julie, and Frerman, Frank E.
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PROTEINS , *PEPTIDES , *HELIX (Mollusks) , *BIOMOLECULES , *TRYPTOPHAN , *AMINO acids , *QUINONE , *EQUILIBRIUM , *BACTERIAL proteins , *SPECTRUM analysis , *FLAVOPROTEINS , *FLAVINS - Abstract
Electron-transferring flavoprotein (ETF) was purified from the bacterium Paracoccus denitrificans and the structural and redox relationships to the porcine and human ETFs were investigated. The three proteins have essentially identical subunit masses and the a-helix content of the bacterial and porcine ETFs are very similar, indicating global structural similarity. An anti-(porcine ETF) polyclonal antibody that crossreacts with the human large and small subunits also crossreacts strongly with the large subunit of Paracoccus ETF. However, crossreactivity with the small subunit is very weak. Nonetheless, an amino-terminal peptide and four internal peptides of the small bacterial subunit show extensive sequence identity with the human small subunit. Local similarities in environment are also indicated by the intrinsic tryptophan fluorescence emission spectra of porcine and Paracoccus ETFs. Although the visible spectra of porcine and Paracoccus ETFs are virtually identical, flavin fluorescence in the bacterial protein is only 15% that of the mammalian protein. Further, the circular dichroic spectrum of the flavin in the bacterial protein is significantly more intense, suggesting that the microenvironment of the isoalloxazine ring is different in the two proteins. Enzymatic or photochemical reduction of Paracoccus ETF rapidly yields an anionic semiquinone; formation of the fully reduced flavin in the bacterial ETF is very slow. The spacing of the oxidation-reduction potentials of the flavin couples in the bacterial ETF is essentially identical to that in porcine ETF as judged from the disproportionation equilibrium of the bacterial ETF flavin semiquinone. Together, the enzymatic reduction and disproportionation equilibria suggest that the flavin potentials of the two ETFs must be very close. The data indicate that the structural properties of the bacterial and mammalian proteins and the thermodynamic properties of the flavin prosthetic group of the proteins are very similar. [ABSTRACT FROM AUTHOR]
- Published
- 1992
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11. Transient kinetics of flavin-photosensitized oxidation of reduced redox proteins.
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Navarro, José A., De la Rosa, Miguel A., and Tollin, Gordon
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FLAVINS , *PIGMENTS , *OXIDATION-reduction reaction , *PROTEINS , *DYNAMICS , *BIOMOLECULES - Abstract
We have used laser flash photolysis to investigate the kinetics of oxidation of reduced plastocyanins obtained from spinach and the green alga Monoraphidium braunii by the triplet states of lumiflavin, riboflavin and FMN. We have compared the results of these experiments with the kinetics of reduction of the oxidized forms of these proteins by the corresponding flavin semiquinones, as well as with the kinetics of flavin oxidation and reduction of cytochrome c552 (Class I c-type cytochrome, generic name c553) from Monoraphidium. in all cases, the rate constants for oxidation were one or two orders of magnitude larger than for reduction, consistent with the greater thermodynamic driving force for the oxidation reaction. Similar steric and electrostatic effects were observed for both reactions with all proteins, suggesting that the same (or closely adjacent) sites were being utilized for electron removal and entry. The two algal proteins were quite similar to one another in their redox properties, consistent with their physiological role of being able to substitute for one another in photosynthetic electron transport. In contrast, the algal plastocyanin was more reactive than the spinach protein in both oxidation and reduction, suggesting differences in their steric properties at the site of electron transfer. [ABSTRACT FROM AUTHOR]
- Published
- 1991
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12. Flavin-photosensitized oxidation of reduced <em>c</em>-type cytochromes. Reaction mechanism and comparison with photoreduction of oxidized cytochromes by flavin semiquinones.
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Roncel, Mercedes, Hervás, Manuel, Navarro, José A., De la Rosa, Miguel A., and Tollin, Gordon
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CYTOCHROMES , *OXIDATION , *CHEMICAL reduction , *FLAVINS , *QUINONE , *PROTEINS , *HORSES - Abstract
In order to compare the oxidation and reduction reactions of c-type cytochromes (cytochromes c552 from the green alga Monoraphidium braunii and horse heart cytochrome c) by different flavins (lumiflavins, riboflavin and FMN), laser flash photolysis studies have been carried out using either reduced or oxidized protein in the presence of triplet or semiquinone flavin, respectively. The reaction kinetics clearly demonstrate the cytochrome oxidation is mediated by the flavin triplet state. The rate constants for reduction are 20-100 times smaller than those for oxidation, indicating that the triplet state is a more effective reactant than is the semiquinone. This is attributed to its excited state nature and correspondingly high free energy content. The rate constants for both the reduction and oxidation of cytochrome c552 by riboflavin are significantly smaller than those obtained with lumiflavin, suggesting a steric interference of the ribityl side chain in the flavin &mdah; cytochrome interaction. The comparison between oxidation and reduction indicates that the former process is less affected by steric hindrance than the latter. Both reduction and oxidation of cytochrome c552 by FMN show an ionic strength dependence with the same sign, consistent with a negatively charged reaction site on the cytochrome. The magnitude of the electrostatic effect is slightly smaller for reduction than it is for oxidation. A pattern quite similar to that observed with cytochrome c552 was obtained when parallel experiments were carried out with horse cytochrome c, although differences were observed in the steric and electrostatic properties of the electron transfer site(s) in these two cytochromes. These results suggest that the same or closely adjacent sites on the proteins are involved in the oxidation and reduction reactions. The biochemical implications of this are discussed. [ABSTRACT FROM AUTHOR]
- Published
- 1990
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13. Purification and properties of <em>N</em>5,<em>N</em>10-methylenetetrahydromethanopterin reductase from <em>Methanobacterium thermoautotrophicum</em> (strain Marbugh).
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Ma, Kesen and Thauer, Rudolf K.
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METHANOTHERMOBACTER thermautotrophicus , *COENZYMES , *POLYACRYLAMIDE , *GEL electrophoresis , *PROTEINS , *FLAVINS - Abstract
The reduction of N5,N10-methylenetetrahydromethanopterin (CH2 = H4MPT) to N5-methyltetrahydromethanopterin (CH3-H4MPT) is an intermediate step in methanogenesis from CO2 and H2. The reaction is catalyzed by CH2 = H4MPT reductase. The enzyme from Methanobacterium thermoautotrophicum (strain Marburg) was found to be specific for reduced coenzyme F420 as electron donor: neither NADH or NADPH nor reduced viotogen dyes could substitute for the reduced 5-deazaflavin. The reductase was purified over 100-fold to apparent homogeneity. Sodium dodecyl sulfate/polyacrylamide gel electrophoresis revealed only one protein band at the 36-kDa position. The apparent molecular mass of the native enzyme was determined by gel filtration to be in the order of 150 kDa. The purified enzyme was colourless. It did not contain flavin or iron. The ultraviolet/ visible spectrum was almost identical to that of albumin, suggesting the absence of a chromophoric prosthetic group. Reciprocal plots of the enzyme activity versus the substrate concentration at different constant concentrations of the second substrate yielded straight lines intersecting at one point on the abscissa to the left of the vertical axis. This intersecting pattern is characteristic of a ternary complex catalytic mechanism. The Km for CH2 = H4MPT and for the reduced coenzyme F420 were determined to be 0.3 mM and 3 µM. respectively. Vmax was 6000 µmol · min-1 · mg protein-1 (kcat = 3600 s-1). The CH2 = H4MPT reductase was stable in the presence of air; at 4°C less than 10% activity was lost within 24 h. [ABSTRACT FROM AUTHOR]
- Published
- 1990
- Full Text
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14. A two-dimensional 1H NMR study on <em>Megasphaera elsdenii</em> flavodoxin in the reduced state.
- Author
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van Mierlo, Carlo P. M., Vervoort, Jacques, Müller, Franz, and Bacher, Adelbert
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AMINO acids , *PROTEINS , *FLAVINS , *NUCLEAR magnetic resonance , *BIOCHEMISTRY , *ORGANIC chemistry - Abstract
Assignments for the 137 amino acid residues of Megasphaera elsdenii flavodoxin in the reduced state have been made using the sequential resonance assignment procedure. Several hydroxyl and sulfhydryl protons were observed at 41 °C at pH 8.3. Spin systems were sequentially assigned using phase-sensitive two-dimensional-correlated spectroscopy and phase-sensitive nuclear Overhauser enhancement spectroscopy. Spectra of the protein in H2O and of protein preparations either completely or partly exchanged against ²H2O were obtained. Use of the fast electron shuttle between the paramagnetic semiquinone and the diamagnetic hydroquinone state greatly simplified the NMR spectra, making it possible to assign easily the ¹H resonances of amino acid residues located in the immediate neighbourhood of the isoalloxazine ring. The majority of the nuclear Overhauser effect contacts between the flavin and the apoprotein correspond to the crystal structure of the flavin domain of Clostridium MP flavodoxin, but differences are also observed. The assignments provide the basis for the structure determination of M. elsdenii flavodoxin in the reduced state as well as for assigning the resonances of the oxidized flavodoxin. [ABSTRACT FROM AUTHOR]
- Published
- 1990
- Full Text
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15. Properties of the complexes of riboflavin 3′,5′-bisphosphate and the apoflavodoxins from <em>Megasphaera elsdenii</em> and <em>Desulfovibrio vulgaris</em>.
- Author
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Vervoort, Jacques, van Berkel, Willem J. H., Mayhew, Stephen G., Müller, Franz, Bacher, Adelbert, Nielsen, Peter, and LeGall, Jean
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VITAMIN B2 , *PROTEINS , *FLAVINS , *DISSOCIATION (Chemistry) , *SPECTRUM analysis , *BIOMOLECULES , *HYDROQUINONE - Abstract
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]
- Published
- 1986
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16. Studies with general acyl-CoA dehydrogenase from pig kidney. Inactivation by a novel type of 'suicide' inhibitor, 3,4-pentadienoyl-CoA.
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Wenz, Alexandra, Ghisla, Sandro, and Thorpe, Colin
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FLAVOPROTEINS , *DEHYDROGENASES , *ENZYMES , *FLAVINS , *PROTEINS , *HYDROGEN , *PROTONS - Abstract
3,4-Pentadienoyl-CoA, an allenic substrate analog, is a potent inhibitor of the flavoprotein pig-kidney general acyl-CoA dehydrogenase. The analog reacts very rapidly (k = 2.4 × 103 min-1) with the native oxidized enzyme to form a covalent flavin adduct probably involving tile isoalloxazine position N-5. This species is inactive, but activity may be regained by two pathways. The allenic thioester can be displaced (k = 0.3 min-1) by a large excess of octanoyl-CoA substrate upon reversal of covalent adduct formation. Alternatively, tile enzyme inactivator adduct slowly decomposes (t½ = 75 min) to form the strongly thermodynamically favoured 2,4-diene and catalytically active, oxidized enzyme. During this latter process 15–20% of the activity is irreversibly lost probably due to covalent modification of the protein. These data suggest that 3,4-pentadicnoyl-CoA should be considered a suicide substrate of the acyl-CoA dehydrogenase. The mechanism of the reactions, and in particular the 3,4→2,4 tautomerization, are consistent with a catalytic sequence initiated by abstraction of an α-hydrogen as a proton. [ABSTRACT FROM AUTHOR]
- Published
- 1985
17. A proton-nuclear-magnetic-resonance study at 500 MHz on <em>Megasphaera elsdenii</em> flavodoxin. A study on the stability, proton exchange and the assignment of some resonance lines.
- Author
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Moonen, Chrit T.W. and Müller, Franz
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NUCLEAR magnetic resonance spectroscopy , *PROTONS , *PROTEINS , *OXIDATION-reduction reaction , *FLAVINS , *OVERHAUSER effect (Nuclear physics) , *HYDROGEN-ion concentration - Abstract
¹H NMR studies were performed on the three redox states of Megasphaera elsdenii flavodoxin. The results show that the protein is remarkably stable, as concluded from amide proton exchange studies. Some amide protons are still present in the ¹H NMR spectrum even after one month in ²H2O at 33°C (pH 8.3). The reactivity of the exchangeable protons can be grouped into three categories, i.e. tj ... 5 min., 10s ... tj ... 5 min, and tj ... 10s. The amide proton exchange reactions are hardly dependent on the redox state. Optimal resolution of ¹H NMR spectra is obtained at 33 ° C, independent of the redox state. No conformational change of the protein is observed in the pH range between 6 and 8.5. Assignments of resonances to protons of flavin and of some amino acid residues are established in both the oxidized and the hydroquinone state using chemically and isotopically substituted flavins and the driven nuclear Overhauser technique. Preliminary two-dimensional ¹H-¹H correlated spectra show that the protein is amenable to two-dimensional NMR techniques. Previous assignments are confirmed by this technique. [ABSTRACT FROM AUTHOR]
- Published
- 1984
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18. Succinate Dehydrogenase Mutants of <em>Bacillus subtilis</em> Lacking Covalently Bound Flavin in the Flavoprotein Subunit.
- Author
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Hederstedt, Lars
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SUCCINATE dehydrogenase , *DEHYDROGENASES , *FLAVINS , *PROTEINS , *FLAVOPROTEINS , *BACILLUS (Bacteria) - Abstract
Succinate dehydrogenase consists of two unequal subunits: Fp and lp. An FAD group is covalently linked to a histidyl residue in the Fp subunit. The mechanism by which flavin is attached to protein is not known Covalently bound flavin was studied in wild-type and succinate-dehydrogenase-negative Bacillus subtilis. The ft subunit of succinate dehydrogenase was found to be the only (major) flavinylated protein in the cell. Mutants lacking covalently bound flavin and still containing the Fp polypeptide arc described. It is shown that the flavin is not essential for assembly and membrane binding of succinate dehydrogenase in B. sibtilis. [ABSTRACT FROM AUTHOR]
- Published
- 1983
- Full Text
- View/download PDF
19. Comparative Electron-Nuclear Double Resonance Study of Two Flavoproteins.
- Author
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Eriksson, L.E. Göran, Ehrenberg, Anders, and Hyde, James D.
- Subjects
- *
FLAVOPROTEINS , *PROTEINS , *FLAVINS , *PIGMENTS , *COENZYMES , *DEHYDROGENASES - Abstract
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]
- Published
- 1970
- Full Text
- View/download PDF
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