Flavin Mononucleotide Binding Research Articles

Flavodoxin from Anabaena 7120: uniform nitrogen-15 enrichment and hydrogen-1, nitrogen-15, and phosphorus-31 NMR investigations of the flavin mononucleotide binding site in the reduced and oxidized states

Interactions between flavin mononucleotide (FMN) and apoprotein have been investigated in the reduced and oxidized states of the flavodoxin isolated from Anabaena 7120 (Mr approximately 21,000). 1H, 15N, and 31P NMR have been used to characterize the FMN-protein interactions in both redox states. These are compared with those seen in other flavodoxins. Uniformly enriched [15N]flavodoxin (greater than 95% isotopic purity) was isolated from Anabaena 7120 grown on K15NO3 as the sole nitrogen source. 15N insensitive nucleus enhanced by polarization transfer (INEPT) and nuclear Overhauser effect (NOE) studies of this sample provided information regarding protein structure and dynamics. A 1H-detected 15N experiment allowed the correlation of nitrogen resonances to those of their attached protons. Over 90% of the expected N-H cross peaks could be resolved in this experiment.

A conformational approach to study the mode of binding of flavin mononucleotide to flavodoxin.

Energetically preferred conformers of Flavin mononucleotide (FMN) were determined using empirical potential energy functions. The minimum energy conformers were used to study the mode of its binding to apoflavodoxin. This study indicates that the conformers of FMN that initiate the binding process undergo significant changes in the position of the phosphate group to reach the final bound conformation. In the bound conformation the phosphate group leads to the formation of a network of hydrogen bonds with the apoflavodoxin and contributes significantly to the binding energy. This extra energy is required for FMN to overcome the repulsion from Met 56 and Glu 59 and to bind tightly to apoflavodoxin.

Purification and properties of dihydroorotate oxidase from Crithidia fasciculata and Trypanosoma brucei

Dihydroorotate oxidases have been highly purified from the parasitic protozoa Crithidia fasciculata and Trypanosoma brucei. The Crithidia enzyme was purified 4200-fold from a crude soluble protein extract in four steps. The protein is a dimer as judged from the native (Mr 60 000) and subunit (Mr 32 700) molecular weights. The purified enzyme exhibits a characteristic flavin electronic spectrum, and each mole of native dimer contains 1.0 mol of tightly bound flavin mononucleotide. Under anaerobic conditions, the flavin chromophore is reduced upon addition of L-dihydroorotate. In air-saturated buffer, the enzyme catalyzes the conversion of L-dihydroorotate to orotate with concomitant reduction of equimolar amounts of molecular oxygen to hydrogen peroxide. A variety of low molecular weight oxidants (e.g., quinones or ferricyanide) may replace oxygen as the electron acceptor during catalysis. The dihydroorotate oxidase of T. brucei was purified 1400-fold to apparent homogeneity by a highly similar isolation procedure. The estimated native (Mr 62 000) and subunit (Mr 30 500) molecular weights indicated a dimeric protein comparable in size to the enzyme from Crithidia. These results suggest that dihydroorotate oxidation is mediated by flavoprotein oxidases in these parasitic protozoa rather than by pterin-linked hydroxylases as recently proposed [Kidder, G. W., & Nolan, L.L. (1973) Biochem. Biophys. Res. Commun. 53, 929-936; Gutteridge, W. E., Dave, D., & Richards, W. H. G. (1979) Biochim. Biophys. Acta 582, 390-401].

Participation of the iron-sulphur cluster and of the covalently bound coenzyme of trimethylamine dehydrogenase in catalysis

Bacterial trimethylamine dehydrogenase contains a novel type of covalently bound flavin mononucleotide and a tetrameric iron-sulphur centre. The dehydrogenase takes up 1.5mol of dithionite/mol of enzyme and is thereby converted into the flavin quinol-reduced (4Fe-4S) form, with the expected bleaching of the visible absorption band of the flavin and the emergence of signals of typical reduced ferredoxin in the electronparamagnetic-resonance spectrum. On reduction with a slight excess of substrate, however, unusual absorption and electron-paramagnetic-resonance spectra appear quite rapidly. The latter is attributed to extensive interaction between the reduced (4Fe-4S) centre and the flavin semiquinone. The species of enzyme arising during the catalytic cycle were studied by a combination of rapid-freeze e.p.r. and stopped-flow spectophotometry. The initial reduction of the flavin to the quinol form is far too rapid to be rate-limiting in catalysis, as is the reoxidation of the substrate-reduced enzyme by phenazine methosulphate. Formation of the spin-spin-interacting species from the dihydroflavin is considerably slower, however, and it may be the rate-limiting step in the catalytic cycle, since its rate of formation agrees reasonably well with the catalytic-centre activity determined in steady-state kinetic assays. In addition to the interacting form, a second form of the enzyme was noted during reduction by trimethylamine, differing in absorption spectrum, the structure of which remains to be determined.

Reversible binding of riboflavin and pteridines to melanin in vitro

1. 1. Binding of riboflavin, flavin mononucleotide, flavin-adenine dinucleotide, 6,7-dimethyl-8-ribityllumazine, isoxanthopterin and pterin to melanosomes or natural and synthetic melanin has been studied in vitro. 2. 2. Binding of riboflavin to melanosomes or melanin is greater than the binding of the other compounds. 3. 3. The known distribution patterns of the fluorescent substances in the pigmented integuments of cold-blooded vertebrates are explained by the selective binding of these compounds to melanosomes.

Preparation of the lactate oxidase apoenzyme and studies on the binding of flavin mononucleotide to the apoenzyme.

1. Lactate oxidase from Mycobacterium smegmatis is completely resolved into free flavin and apoenzyme by treatment with acid (NH4)2SO4. 2. Reconstitution involves rapid binding of FMN, but the recovery of enzyme activity was slower and appeared to be biphasic. 3. The preparation of the holoenzyme obtained differs from the native enzyme in specific activity, extinction coefficients and mobility on disc-gel electrophoresis. 4. Dialysis of this reconstituted enzyme in 0.1 M-sodium phosphate buffer, pH 7.0, at 0 degrees C for 1 week yields a preparation which closely resembles the native enzyme.

The Structure of the Oxidized Form of Clostridial Flavodoxin at 1.9-A Resolution: DESCRIPTION OF THE FLAVIN MONONUCLEOTIDE BINDING SITE

Abstract An electron density map of the oxidized form of flavodoxin from Clostridium strain MP has been calculated to a resolution of 1.9 A employing isomorphous and anomalous scattering data from two heavy atom derivatives, Smiii and Aui. In the region from 3.0 to 1.9 A, the largest structure amplitudes (85% of the total | F | s) have been included in the computation. The over-all figure of merit is 0.808. The molecular model resulting from interpretation of this map has a chain length of 138 residues, in accord with the sequence (Tanaka, M., Haniu, M., Yasunobu, K. T., and Mayhew, S. G. (1974) J. Biol. Chem. 249, 4393–4396). As reported earlier, the molecule possesses a high degree of secondary structure, including a central parallel sheet and four helices. More than 70% of the residues appear to participate in backbone hydrogen bonding. Atomic coordinates, incorporating the sequence data, have been optimized by use of Diamond's model-building program. The flavin mononucleotide prosthetic group adopts a somewhat extended conformation. As expected for the oxidized state, the isoalloxazine ring is planar. It is partly shielded from solvent by a tryptophan residue and interacts on its inner surface with methionine. Protein atoms are also in appropriate positions for interaction with atoms N-1, O-2, and N-3 of the flavin ring, but N-5 appears too far removed to form a hydrogen bond with the apoprotein. The ribityl phosphate moiety also makes multiple contacts with protein groups. Two of the three O' oxygens are in positions where they may participate in hydrogen bonding; the phosphate is closely surrounded by four hydroxyamino acids and four backbone NH groups. However, the prosthetic group is accessible on one side to solvent. Peaks likely to represent solvent can be observed near the dimethylbenzene end of the isoalloxazine ring and adjacent to the 3'-ribityl oxygen. While the mutual arrangement of protein and ribityl phosphate is nearly identical with that found by Watenpaugh et al. (Watenpaugh, K. D., Sieker, L. C., Jensen, L. H., LeGall, J., and Dubourdieu, M. (1972) Proc. Nat. Acad. Sci. U. S. A. 69, 3185–3188) in Desulfovibrio vulgaris flavodoxin, the orientation and interactions of the flavin ring differ strikingly in the two flavodoxins. The structure of the oxidized form of Clostridium MP flavodoxin is very similar to that deduced earlier for the semiquinone form at 3.25 A, despite large differences in the x-ray intensities (Ri = 0.66).

The binding and spectral alterations of oxidized flavin mononucleotide by bacterial luciferase

The binding of oxidized flavin mononucleotide (FMN) to bacterial luciferase was studied by equilibrium dialysis. A Scatchard plot of the data indicates a single FMN binding site per luciferase molecule, with a dissociation constant of 2.4 × 10 −4 M at 2° in 0.05 M Bis-Tris, 0.2 M NaCl, pH 7.0. The visible absorbance spectrum of luciferase-bound FMN is altered considerably relative to the spectrum of free FMN. The spectrum of the bound flavin shows an apparent splitting of the 443-nm peak yielding well-defined maxima at 458 nm and 434 nm.

The binding of riboflavin-5'-phosphate in a flavoprotein: flavodoxin at 2.0-Angstrom resolution.

The crystal structure of the oxidized form of flavodoxin from Desulfovibrio vulgaris has been studied at 2.0-A resolution, and a detailed description of the region around the flavin mononucleotide binding site is now available. The flavin is between a tyrosine group, roughly parallel to it on one side, and a tryptophan, about 45 degrees from being parallel, on the other side. The two carbonyl groups and two nitrogen atoms of the flavin are hydrogen bonded to the peptide chain of the protein, while the two methyl groups are exposed at the surface of the protein. The phosphate group of the flavin mononucleotide is inside the protein and extensively hydrogen bonded to it. The ribityl group is hydrogen bonded both to the protein and to water on the surface of the protein.