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We have used mixed monolayer protected gold clusters (MMPCs) to provide flavoenzyme model systems with a high affinity and ability to modulate cofactor reduction potential.
A new type of glycolate-oxidizing enzyme was found in the prasinophycean alga Mesostigma viride. This enzyme was characterized as the glycolate oxidase that strongly requires FMN, thus distinguishing it from most other Prasinophytes that possess glycolate dehydrogenase instead.
Phototropism, the bending response of plant organs to or away from a directional light source, is one of the best studied blue light responses in plants. Although phototropism has been studied for more than a century, recent advances have improved our understanding of the underlying signaling mechanisms involved. The NPH1 gene of Arabidopsis thaliana encodes a blue light-dependent autophosphorylating protein kinase with the properties of a photoreceptor for phototropism. NPH1 apoprotein noncovalently binds FMN to form the holoprotein nph1. The N-terminal region of the protein contains two LOV (light, oxygen, or voltage) domains that share homology with sensor proteins from a diverse group of organisms. These include the bacterial proteins NIFL and AER, both of which bind FAD, and the phy3 photoreceptor from Adiantium capillus-veneris. The LOV domain has therefore been proposed to reflect a flavin-binding site, regulating nph1 kinase activity in response to blue light-induced redox changes. Herein we demonstrate that the LOV domains of two nph1 proteins and phy3 bind stoichiometric amounts of FMN when expressed in Escherichia coli. The spectral properties of the chromopeptides are similar to the action spectrum for phototropism, implying that the LOV domain binds FMN to function as a light sensor. Thus, our findings support the earlier model that nph1 is a dual-chromophoric flavoprotein photoreceptor regulating phototropic responses in higher plants. We therefore propose the name phototropin to designate the nph1 holoprotein.
The polarized single-crystal absorption spectra of the oxidized and semiquinone forms of flavodoxin from Clostridium MP have been measured with a double beam recording microspectrophotometer. The spectra establish that the radical species in the crystal is the neutral (blue) falvine semiquinone. Combination of the spectra reported here with polarization data from previous fluorescence and stretched-film studies provides transition moment directions for the first two phi-phi transitions of the oxidized form. Predictions of molecular orbital theory are in good agreement with these experimental directions. The crystal spectra of the semiquinone indicate that the two lowest frequency transitions have the same detailed orbital origin as the corresponding transitions of the oxidized form; in the semiquinone these transitions appear at lower frequency, are closer together, and, as predicted from detailed considerations of transition probabilities, exhibit approximately half the absorption intensity. Our hypothesis of a common orbital origin suggests that semiquinone formation takes place by the addition of an electron to the lowest empty phi orbital of the oxidized form without any gross electronic rearrangement.
Microsomal NADPH-cytochrome P-450 reductase is the only mammalian flavoprotein known to contain both FAD and FMN as prosthetic groups. The discovery of the air-stable semiquinone [Masters, B. S. S., Kamin, H., Gibson, Q. H., & Williams, C. H., Jr. (1965) J. Biol. Chem. 240, 921-931] and its identification as a one-electron-reduced state [Iyanagi, T., & Mason, H. S. (1973) Biochemistry 12, 2297-2308] have engendered a number of studies to elucidate its unique catalytic mechanism. In this paper, 31P NMR spectroscopy is utilized to probe the localization of the free radical in this air-stable semiquinone form and to ascertain the environments of the FAD and FMN prosthetic groups as affected by the paramagnetic ion Mn(II). Consistent with conclusions drawn from studies utilizing FMN-free reductase [Vermilion, J. L., & Coon, M. J. (1978) J. Biol. Chem. 253, 8812-8819], the free radical was shown to reside on the FMN moiety by the broadening of its characteristic resonance in the 31P NMR spectrum. In addition, the effect of the paramagnetic ion Mn(II) was determined on the four resonances attributable to FAD and FMN and the additional ones contributed by NADP+ resulting from the oxidation of the physiological reductant NADPH. The addition of Mn(II) had little effect on the line widths of the FMN and FAD signals but resulted in an increase in their intensities due to a decrease in T1 relaxation times.(ABSTRACT TRUNCATED AT 250 WORDS)
Previous experiments in our laboratory with Saccharomyces cervisiae flavocytochrom b2 indicated that both fragments alpha and beta of the enzyme after cleavage by yeast proteases are required to form the flavin site. More detailed experiments have not been carried out on the nicked Hansenula anomala enzyme obtained by tryptic cleavage. A method has been devised that gives a quantitative separation in 4 M urea of beta, and alpha with its heme still bound. The characteristics of the various species: isolated alpha and beta and mixed alpha + beta were studied in 4 M urea and after elimination of this reagent by dialysis in the presence of FMN and 2-mercaptoethanol. Several methods, including heme spectroscopy, tryptophan fluorescence, sedimentation studies, and titration of bound flavin, were used. The results indicate that isolated alpha and beta have a folded globular structure after renaturation. The flavin binding to the alpha + beta mixture was important (50-100%) with recovery of the flavodehydrogenase activity. In contrast, binding was not detectable (< 0.5%, Kf > 10 mM) for isolated alpha and beta. As far as mononucleotide binding is concerned, such a cooperative requirement for two folding domains has never been reported in other enzymes. The present results are discussed together with others obtained in our laboratory which demonstrate that, as deduced from their sensitivity to trypsin, the structure of S. cerevisiae and H. anomala flavocytochrome b2 protomers is triglobular 'n-x-beta' (n and x combined within alpha). The tetramer assembly, which remains intact as a nicked enzyme (alpha beta)4 after the first trypsin cleavage, is broken down following a second cleavage of the chain into four cytochrome cores (n) and a functional T-flavodehydrogenase entity, a tetramer of the type (x beta)4.
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Bovine heart mitochondrial NADH-ubiquinone oxidoreductase (complex I) catalyzed NADH- and ubiquinone-1-dependent oxygen (O2) turnover to hydrogen peroxide that was stimulated by piericidin A and superoxide dismutase (SOD), but was insensitive to antimycin A, myxothiazol, and potassium cyanide. The extent of O2 consumption as a function of ubiquinone-1 did not correlate with piericidin A-sensitive rates of ubiquinone reduction. Decylubiquinone did not stimulate O2 consumption, but did initiate an SOD-sensitive cytochrome c reduction when complex I was isolated away from ubiquinol-cytochrome c oxidoreductase. Rates and extent of O2 turnover (ROS production) and ubiquinone reduction were higher than previously reported for submitochondrial particles (SMP) and isolated complex I. This ROS production was shown to co-isolate with complex I flavin.
Three different NAD(P)H-FMN reductases were extracted from Beneckea harveyi MB-20 cells and separated by DEAE-Sephadex A50 column chromatography. Further purification was achieved by affinity chromatography. In determinations of Km values for NADH, NADPH, and FMN, these three reductases exhibited different specificities and kinetic parameters. One reductase utilizes NADH, whereas a second one utilizes NADPH as the preferred substrate. The third, a newly described reductase species, exhibits about the same reaction rates with NADH and NADPH. The reaction mechanisms of the three enzyme forms have been deduced by steady state kinetic analysis. The highly pure (based on gel electrophoresis) NADPH-FMN reductase still exhibited a low (approximately 2%) activity for NADH, which activity was increased upon storage at 4 degrees but suppressed completely by the replacement of the phosphate buffer with sodium citrate buffer. This high specificity of NADPH-FMN reductase for NADPH under these conditions is useful for the assay of NADPH, notably in systems coupled to bacterial luciferase.
Interaction between cytochrome c and flavocytochrome b2 has been studied in presence of 2-p-toluidinylnaphthalene-6-sulfonate (TNS). Affinity of the probe to flavocytochrome b2 increases when the complex between the two proteins is obtained. Binding of TNS increases the fluorescence of flavocytochrome b2 FMN. When the stoichiometry of the complex between the two proteins is reached, TNS looses its affinity and stops binding on the flavocytochrome b2; consequently, FMN fluorescence increase is no more observed. The dissociation constant of the complex was found equal to 0.1 microM. A similar result was obtained for the interaction between cytochrome c and flavodehydrogenase domain. The latter was obtained by proteolysis of flavocytochrome b2.
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The inhibition of rabbit skeletal muscle glycogen phosphorylase B by FMN and its analogues with substituents in the positions 6 and 8 has been studied. Inhibiting action of FMN is manifested in reducing the limiting rate of enzymic reaction and in increasing the half-saturation concentration of AMP. The inhibitor half-saturation values (in microM) increase in the following order: FMN (13,5), 6-bromo-FMN (27), 8 alpha-hydroxy-FMN (30), 8-dimethylamino(nor)-FMN (33), 6-(N-acetyl-L-cysteine-S-yl)-FMN (44), 6-amino-FMN (96), 8-hydroxy(nor)-FMN (109), 6-nitro-FMN (170), 8 alpha-(N-acetyl-L-cysteine-S-yl)-FMN (260). The existence of the glycogen phosphorylase B complexes with FMN or its analogues has been proved by spectrophotometry and sedimentation in analytical ultracentrifuge. FMN has been shown to hinder AMP-induced transition of dimeric form of the enzyme to tetrameric one. AMP at high concentrations has been found to inhibit glycogen phosphorylase B.
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OBJECTIVES: To investigate plasma total homocysteine levels and its relation to B-vitamins and smoking in Graves' disease before and after antithyroid therapy. DESIGN: A longitudinal study taking place at four hospitals in Norway. METHODS AND SUBJECTS: Plasma total homocysteine, serum folate, serum cobalamin and riboflavin, flavin mononucleotide and flavin adenine dinucleotide in plasma were investigated in 182 patients with hyperthyroidism before treatment. The same parameters were reinvestigated in 112 of these patients after attaining euthyroid state. RESULTS: In hyperthyroidism, plasma total homocysteine was low, and inversely related to folate, cobalamin and riboflavin, and positively related to serum creatinine and age. Following antithyroid therapy, total homocysteine increased and the concentration of folate, cobalamin, riboflavin, flavin mononucleotide and flavin adenine dinucleotide decreased significantly. The most pronounced reduction (35%) was observed for flavin mononucleotide. In the hyperthyroid state, smokers had lower levels of folate and flavin mononucleotide than non-smokers. After restoration of euthyroidism, both folate and riboflavin were significantly lower in smokers than non-smokers. Plasma total homocysteine increased according to decreasing quartiles of B-vitamins. For riboflavin, this relation was confined to smokers. CONCLUSION: Plasma total homocysteine changes according to thyroid status. These changes may be partly attributable to altered folate, cobalamin but also riboflavin status, particularly in smokers.