flavin-adenine-dinucleotide and 5-deazariboflavin

The cofactor flavin adenine dinucleotide (FAD) is required for the catalytic activity of a large class of enzymes known as flavoenzymes. Because flavin cofactors participate in catalysis via a number of different mechanisms, isoalloxazine analogues are valuable for mechanistic studies. We report improved chemical syntheses for the preparation of the two key analogues, 5-deazariboflavin and 1-deazariboflavin.

Topics: Catalysis; Flavin-Adenine Dinucleotide; Indicators and Reagents; Molecular Structure; Oxygenases; Riboflavin

N-Methyltryptophan oxidase (MTOX) is a flavoenzyme that catalyzes the oxidative demethylation of N-methyl-L-tryptophan and other N-methyl amino acids, including sarcosine, which is a poor substrate. The Escherichia coli gene encoding MTOX (solA) was isolated on the basis of its sequence homology with monomeric sarcosine oxidase, a sarcosine-inducible enzyme found in many bacteria. These studies show that MTOX is expressed as a constitutive enzyme in a wild-type E. coli K-12 strain, providing the first evidence that solA is a functional gene. MTOX expression is enhanced 3-fold by growth on minimal media but not induced by N-methyl-L-tryptophan, L-tryptophan, or 3-indoleacrylate. MTOX forms an anionic flavin semiquinone and a reversible, covalent flavin-sulfite complex (K(d) = 1.7 mM), properties characteristic of flavoprotein oxidases. Rates of formation (k(on) = 5.4 x 10(-3) M(-1) s(-1)) and dissociation (k(off) = 1.3 x 10(-5) s(-1)) of the MTOX-sulfite complex are orders of magnitude slower than observed with most other flavoprotein oxidases. The pK(a) for ionization of oxidized FAD at N(3)H in MTOX (8.36) is two pH units lower than that observed for free FAD. The MTOX active site was probed by characterization of various substrate analogues that act as competitive inhibitors with respect to N-methyl-L-tryptophan. Qualitatively similar perturbations of the MTOX visible absorption spectrum are observed for complexes formed with various aromatic carboxylates, including benzoate, 3-indole-(CH(2))(n)-CO(2)(-) and 2-indole-CO(2)(-). The most stable complex with 3-indole-(CH(2))(n)-CO(2)(-) is formed with 3-indolepropionate (K(d) = 0.79 mM), a derivative with the same side chain length as N-methyl-L-tryptophan. Benzoate binding is enhanced upon protonation of a group in the enzyme-benzoate complex (pK(EL) = 6.87) but blocked by ionization of a group in the free enzyme (pK(E) = 8.41), which is attributed to N(3)H of FAD. Difference spectra observed for the aromatic carboxylate complexes are virtually mirror images of those observed with sarcosine analogues (N,N'-dimethylglycine, N-benzylglycine). Charge-transfer complexes are formed with 3-indoleacrylate, pyrrole-2-carboxylate, and CH(3)XCH(2)CO(2)(-) (X = S, Se, Te).

Topics: Anaerobiosis; Benzoates; Binding Sites; Enzyme Stability; Escherichia coli; Escherichia coli Proteins; Flavin-Adenine Dinucleotide; Hydrogen-Ion Concentration; Indazoles; Ligands; Oxidation-Reduction; Oxidoreductases, N-Demethylating; Photochemistry; Recombinant Proteins; Riboflavin; Sarcosine Oxidase; Spectrophotometry; Substrate Specificity; Tryptophan

Transient kinetics of reduction and interprotein electron transfer in the human cytochrome b5 reductase-cytochrome b5 (b5R-b5) system was studied by laser flash photolysis in the presence of 5-deazariboflavin and EDTA at pH 7.0. Flash-induced reduction of the FAD cofactor of b5R by deazariboflavin semiquinone (in the absence of b5) occurred in a rapid second-order reaction (k2 = 3.1 x 10(8) M-1 s-1) and resulted in a neutral (blue) FAD semiquinone. The heme of cytochrome b5 (in the absence of b5R) was also rapidly reduced in this system with k2 = 3.1 x 10(8) M-1 s-1. When the two proteins were mixed at low ionic strength, a strong complex was formed. Although the heme of complexed b5 could be directly reduced by deazariboflavin semiquinone, the second-order rate constant was nearly an order of magnitude smaller than that of free b5 (k2 = 3.4 x 10(7) M-1 s-1). In contrast, access to the FAD of b5R by the external reductant was decreased by considerably more than an order of magnitude (k2 < 1 x 10(7) M-1 s-1). When an excess of b5R was titrated with small increments of b5 and then subjected to laser flash photolysis in the presence of deazariboflavin/EDTA, interprotein electron transfer from the b5R FAD semiquinone to the heme of b5 could be observed. At low ionic strength (I = 16 mM), the reaction showed saturation behavior with respect to the b5 concentration, with a limiting first-order rate constant for interprotein electron transfer k1 = 375 s-1, and a dissociation constant for protein-protein transient complex formation of approximately 1 microM. The observed rate constants for interprotein electron transfer decreased 23-fold when the ionic strength was increased to 1 M, indicating a plus-minus electrostatic interaction between the two proteins. Saturation kinetics were also observed at I = 56, 96, and 120 mM, with limiting first-order rate constants of 195, 155, and 63 s-1, respectively. In the presence of NAD+, the transient protein-protein complex was stabilized by approximately a factor of two, and limiting first-order rate constants of 360 s-1 were obtained at both I = 56 mM and I = 96 mM and 235 s-1 at I = 120 mM. Thus, NAD+ appears to stabilize as well as to optimize the protein-protein complex with respect to electron transfer. Another effect of NAD+ is to appreciably slow autoxidation and disproportionation of the FAD semiquinone.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Cytochrome Reductases; Cytochrome-B(5) Reductase; Cytochromes b5; Edetic Acid; Electron Transport; Flavin-Adenine Dinucleotide; Heme; Humans; Hydrogen-Ion Concentration; Kinetics; Lasers; NAD; Osmolar Concentration; Oxidation-Reduction; Photolysis; Protein Binding; Riboflavin; Spectrophotometry

Steady-state and laser flash photolysis techniques have been used to examine the photoreduction of yeast glutathione reductase by the one-electron reduction products of 5-deazariboflavin and the viologen analogue 1,1'-propylene-2,2'-bipyridyl. Steady-state photoreduction of the enzyme with the viologen generates the two-electron-reduced form, whereas photoreduction with deazaflavin generates the anion semiquinone. Flash photolysis indicates that the product of viologen radical reduction is also a semiquinone, suggesting that this species is rapidly further reduced by viologen in the steady-state experiment to form the EH2 enzyme. This reduction is apparently inhibited when deazaflavin is the photoreductant, perhaps due to complexation of the anion semiquinone with deazaflavin. Steady-state experiments demonstrate that complexation of the anion semiquinone with NADP+ also inhibits further reduction. Both one-electron reduction reactions of oxidized glutathione reductase proceed at close to diffusion-controlled rates (second-order rate constants = 10(8)-10(9) M-1 s-1), despite the relatively buried nature of the FAD cofactor. Addition of NADP+ and oxidized glutathione produced no effects on the kinetics of the initial entry of the electron into the enzyme. No kinetic evidence of intramolecular electron transfer involving the FAD and the protein disulfide was obtained during or subsequent to the initial one-electron reduction process. Thus, if this reaction occurs in the semiquinone, it must be quite rapid (k greater than 8000 s-1).

Topics: Chemical Phenomena; Chemistry; Diquat; Electrons; Flavin-Adenine Dinucleotide; Free Radicals; Glutathione; Glutathione Reductase; Kinetics; Lasers; NADP; Oxidation-Reduction; Photochemistry; Riboflavin; Time Factors; Viologens; Yeasts

Topics: Animals; Brevibacterium; Flavin Mononucleotide; Flavin-Adenine Dinucleotide; Liver; Methods; Nucleotidyltransferases; Oxidation-Reduction; Oxidoreductases; Rats; Riboflavin; Spectrophotometry; Structure-Activity Relationship

In order to facilitate interpretation of the deazaisoalloxazine system as a valid mechanistic probe of flavoenzyme catalysis, we have examined some of the fundamental chemical properties of this system. The enzymatic synthesis, on a micromole scale, of the flavin coenzyme analogues 5-deazariboflavin 5'-phosphate (deazaFMN) and 5-deazariboflavin 5'-diphosphate, 5' leads to 5'adenosine ester (deazaFAD) has been achieved. This latter synthesis is accomplished with a partially purified FAD synthetase complex (from Brevibacterium ammoniagenes), containing both phosphorylating and adenylylating activities, allowing direct conversion of the riboflavin analogue to the flavin adenine dinucleotide level. The structure of the reduced deazaflavin resulting from enzymatic and chemical reduction is established as the 1,5-dihydrodeazaflavin by proton magnetic resonance. Similarly, the C-5 position of the deazaflavins is demonstrated to be the locus for hydrogen transfer in deazaflavin redox reactions. Preparation of 1,5-dihydrodeazaflavins by sodium borohydride reduction stabilized them to autoxidation (t 1/2 approximately 40 h, 22 degrees C) although dihydrodeazaflavins are rapidly oxidized by other electron acceptors, including riboflavin, phenazine methosulfate, methylene blue, and dichlorophenolindophenol. Mixtures of oxidized and reduced deazaflavins undergo a rapid two-electron disproportionation (k = 22 M-1 S-1 0 degrees C), and oxidized deazaflavins form transient covalent adducts with nitroalkane anions at pH less than 5. Generalized methods for the synthesis of isotopically labeled flavin and deazaflavin coenzymes and their purification by adsorptive chromatography are given.

Topics: Brevibacterium; Flavin Mononucleotide; Flavin-Adenine Dinucleotide; Hydrogen-Ion Concentration; Methods; Multienzyme Complexes; Nucleotidyltransferases; Oxidation-Reduction; Riboflavin; Spectrophotometry, Ultraviolet