Flavin Analogues Research Articles

Oxidative burst and electrolyte leakage induced by sulfhydryl blockers and by membrane permeabilizing reagents in different organs ofEgeria densa

Sulfhydryl blockers, such as N-ethylmaleimide, iodoacetate and heavy metals induce a transitory stimulation of O2 consumption and H2O2 production (oxidative burst) and a rapid release of electrolytes in leaves of various aquatic plants. The correlation between these two responses to N-ethylmaleimide or to Ag+ in separate organs and stages of leaf development was investigated inEgeria densa. Only adult leaves were able to respond to the sulfhydryl blockers with an oxidative burst, whereas this response was absent in immature growing leaves and in stem and root segments. In N-ethyl-maleimide- as well as in Ag+-treated adult leaves the oxidative burst was constantly associated with a relevant electrolyte leakage. These data are consistent with a model in which the SH reagent would first interact with a plasmalemma protein, leading to an increase in passive permeability to ions and to the activation of an oxidative enzyme of the type of the superoxide synthase described for granulocytes. In its turn, active-oxygen species produced by the activated oxidase might further damage the plasma membrane, increasing its passive permeability. Digitonin and nystatin, two reagents known to cause a permeabilization of lipid membranes, induced in adultE. densa leaves a transient increase in the rate of O2 consumption and H2O2 production and an electrolyte leakage very similar to those induced by sulfhydryl blockers. These effects, however, were not influenced by the flavin analogues diphenylene iodonium and quinacrine, and were partially inhibited by the presence of CN− and salicylhydroxamic acid, thus suggesting the involvement of a different oxidase in the oxidative burst elicited by these reagents.

Identification of a Reactive Cysteine in the Flavin-Binding Domain ofRhodotorula gracilisd-Amino Acid Oxidase

The holoenzyme form ofRhodotorula gracilisd-amino acid oxidase, an 80-kDa homodimer, reacted only to a limited extent with general thiol reagents (2,2′-dithiodipyridine, 5,5′-dithiobis(2-nitrobenzoic acid), andN-[7-(dimethylamino)-4-methylcoumarinyl]maleimide) (60% residual activity), whereas the monomeric apoprotein was completely inactivated and denatured by these reagents. To investigate the presence of thiol residue(s) in the active site of the enzyme, the apoprotein was reconstituted with the 8-(methylsulfonyl)-FAD chemical-affinity probe. Competitive inhibition between this analogue and FAD for apoprotein binding was observed. The covalent attachment of the flavin analogue to the apoprotein was complete after ≈20 h of incubation and the flavinylated enzyme, containing 8-(cysteinyl)-FAD, was monomeric and inactive. After HPLC isolation of the flavin-labeled tryptic peptides, Cys208 was identified as the only cysteine to react with the FAD analogue. These results show that a single cysteine ofR. gracilisd-amino acid oxidase reacts with the flavin analogue and that this is located near or at the FAD-binding domain.

Method for Preparing New Flavin Derivatives: Synthesis of Flavin−Thymine Nucleotides and Flavin−Oligonucleotide Adducts

In order to link to the 5‘-end of oligonucleotides the flavin analogs 9a,b possessing only one terminal hydroxy group on the side chain, the phosphoramidite and the H-phosphonate coupling methods w...

41] Syntheses and applications of flavin analogs as active site probes for flavoproteins

This chapter discusses the synthesis of a range of new flavins and their potentials as active-site probes for flavoproteins. A few new flavins with substitutions such as trifluoromethyl, chloro, azido, carbonyl, and mercapto at the N-5 position are successfully synthesized. The selective introduction of fluorine into biologically interesting molecules is fast emerging as an effective tool because of the unique properties of this halogen. Because fluorine is smallest next to hydrogen, its substitution often results in minimal steric constraints. Also, because fluorine can act as hydrogen bond acceptor, the replacement of hydroxyl with fluorine allows the molecule to retain its properties. The N-10 ribityl side chain of flavins is generally considered as only a binding anchor in flavoproteins, with no positive role in catalysis. However, the X-ray crystal structures of various flavoproteins such as Old Yellow Enzyme, acyl-CoA dehydrogenase, glutathione reductase, and lipoamide dehydrogenase show involvement of ribitylhydroxylgroups in hydrogen bonds that can regulate catalytically important amino acid residues.

Double Mediatory System Involving a Flavin Analog and a p-Benzoquinone Derivative for Photoinduced Electrochemical Oxidation of Benzyl Alcohol to Benzaldehyde in Acetonitrile

Abstract The photoinduced electrochemical oxidation of benzyl alcohol to benzaldehyde in acidic acetonitrile proceeded with practically 100% product selectivity and ca. 100% current efficiency under visible-light irradiation in the presence of riboflavin-2′,3′,4′,5′-tetraacetate (RF) as a mediator. A double mediatory system involving duroquinone as well as RF resulted in remarkable acceleration of the benzaldehyde formation with maintenance of both the high product selectivity and high current efficiency.

Studies with Flavin Analogs Provide Evidence That a Protonated Reduced FMN Is the Substrate-induced Transient Intermediate in the Reaction of Escherichia coli Chorismate Synthase

Chorismate synthase catalyzes the 1,4-elimination of phosphate and the C-(6-pro-R) hydrogen from 5-enolpyruvylshikimate 3-phosphate (EPSP) to generate chorismate. Although this reaction does not involve an overall change in redox state, the enzyme requires reduced FMN. To investigate the role of the flavin in catalysis we have employed chemically modified flavins: 1- and 5-deaza-, 2- and 4-thio-, 6-hydroxy-, 8-nor-6-methyl-, 8-methyl-sulfonyl-, 8-chloro-, 8-fluoro-, 8-nor-methyl-, 8-S-methyl-, 8-methoxy, 8-mercapto- and 8-amino-FMN. Photoreduction of 4-thio-FMN in the presence of chorismate synthase at pH 7.5 produced a reduced flavin species with an absorbance maximum at lambda = 410 nm indicative of monoanionic, reduced 4-thio-FMN. Binding of 8-mercapto- and 6-hydroxy-FMN to chorismate synthase in the presence of EPSP or (6R)-6-fluoro-EPSP resulted in an increase of the flavin analogs' pKa values by 4 and 1 pH units, respectively. On the basis of these findings it is concluded that chorismate synthase preferentially binds neutral flavin species, including the protonated reduced form, rather than anionic flavin species in the presence of EPSP or the 6-fluoro-substrate analog. Further support for this conclusion was obtained using 5-deaza- and 4-thio-FMN. Addition of EPSP to enzyme-bound, reduced 5-deaza-FMN produced spectral changes consistent with protonation of the flavin. Photoreduction of 4-thio-FMN in the presence of enzyme and the (6R)-6-fluoro-EPSP generated a reduced flavin species with absorbance properties of a neutral, reduced 4-thio-flavin. These results and their implications for the nature and kinetic properties of an observed flavin intermediate are discussed in the context of a possible role of reduced flavin as an electron donor to bound EPSP.

Is the NAD(P)H:Flavin Oxidoreductase from Escherichia coli a Member of the Ferredoxin-NADP+ Reductase Family?: EVIDENCE FOR THE CATALYTIC ROLE OF SERINE 49 RESIDUE

The NAD(P)H:flavin oxidoreductase from Escherichia coli, Fre, is a monomer of 26.1 kDa which catalyzes the reduction of free flavins by NADPH or NADH. The flavin reductase Fre is the prototype of a new class of flavin reductases able to transfer electrons with no prosthetic group. It has been suggested that the flavin reductase could belong to the ferredoxin-NADP+ reductase (FNR) family, on the basis of limited sequence homologies. A sequence, conserved within the ferredoxin-NADP+ reductase family and present in the flavin reductase, is important for recognition of the isoalloxazine ring. Within this sequence, we have mutated serine 49 of the flavin reductase into alanine or threonine. kcat value of the S49A mutant was 35-fold lower than kcat of the wild-type enzyme. Determination of real Kd values for NADPH and lumichrome, a flavin analog, showed that recognition of the flavin is strongly affected by the S49A mutation, whereas affinity for the nicotinamide cofactor is only weakly modified. This suggests that serine 49 is involved in the binding of the isoalloxazine ring. Moreover, the Kd value for 5-deazariboflavin, in which the N-5 position of the isoalloxazine ring has been changed to a carbon atom, is not affected by the serine 49 to alanine mutation. This is consistent with the concept that the N-5 position is the main site for serine 49-flavin interaction. In the ferredoxin-NADP+ reductase family, the equivalent serine residue, which has been shown to be essential for activity, is hydrogen-bonded to the N-5 of the FAD cofactor. Taken together, these data provide the first experimental support to the hypothesis that the flavin reductase Fre may belong to the ferredoxin-NADP+ reductase family.

The Mechanism and Substrate Specificity of the NADPH:Flavin Oxidoreductase from Escherichia coli

The NAD(P)H:flavin oxidoreductase from Escherichia coli, Fre, is a monomer of 26.2 kDa that catalyzes the reduction of free flavins by NADPh or NADH. Overexpression in E. coli now allows the preparation of large amounts of pure protein. Structural requirements for recognition of flavins as substrates and not as cofactors were studied by steady-state kinetics with a variety of flavin analogs. The entire isoalloxazine ring was found to be the essential part of the flavin molecule for interaction with the polypeptide chain. Methyl groups at C-7 and C-8 of the isoalloxazine ring and the N-3 of riboflavin also play an important role in that interaction, whereas the ribityl chain of the riboflavin is not required for binding to the protein. On the other hand, the presence of the 2'-OH of the ribityl chain stimulates the NADPH-dependent reaction significantly. Moreover, a study of competitive inhibitors for both substrates demonstrated that Fre follows a sequential ordered mechanism in which NADPH binds first followed by riboflavin. Lumichrome, a very good inhibitor of Fre, may be used to inhibit flavin reductase in E. coli growing cells. As a consequence, it can enhance the antiproliferative effect of hydroxyurea, a cell-specific ribonucleotide reductase inactivator.

2'-fluoro-2'-deoxy-D-arabinoflavin: characterization of a novel flavin and its effects on the formation and stability of two-electron-reduced mercuric ion reductase.

With the goal of generating a novel fluorine-containing flavin analogue with a reduction potential the same as normal flavin, 2'-fluoro-2'-deoxy-D-arabinoflavin has been synthesized. In its riboflavin and FAD forms, UV-visible spectral properties are similar to those of normal flavins, and tight binding to riboflavin binding protein and mercuric ion reductase occurs with very similar spectral changes. The reduction potential of the 2'-FaFAD analogue is determined to be -207 mV compared with -206 mV for FAD, indicating that the intervening 1'-methylene group insulates the redox-active isoalloxazine from the 2'-fluorine. With the intent of using the analogue as a fluorine NMR probe of the active site environments of two-electron-reduced mercuric ion reductase, apoenzyme was reconstituted and its behavior under reducing conditions examined. Whereas with normal enzyme, addition of two electrons gives rapid formation of a charge-transfer species where FAD remains oxidized and a disulfide is reduced to a thiol/thiolate pair, with the 2'-FaFAD enzyme, addition of two electrons gives rapid reduction of the flavin followed by slow transfer of electrons to the disulfide with very little development of the typical charge-transfer absorption. Analysis of crystal structure data suggests that having the fluorine in the alternate arabino stereochemistry places it much nearer the flavin-proximal cysteine/cystine sulfur, where it may inhibit both electron transfer from reduced flavin and the charge-transfer interaction between reduced thiolate and FAD.

Quinoline 2-oxidoreductase and 2-oxo-1,2-dihydroquinoline 5,6-dioxygenase from Comamonas testosteroni 63. The First Two Enzymes in Quinoline and 3-methylquinoline Degradation

The enzymes catalysing the first two steps of quinoline and 3-methylquinoline degradation by Comamonas testosteroni 63 were investigated. Quinoline 2-oxidoreductase, which catalyses the hydroxylation of (3-methyl-)quinoline to (3-methyl-)2-oxo-1,2-dihydroquinoline, was purified to apparent homogeneity. The native enzyme, with a molecular mass of 360 kDa, is composed of three non-identical subunits (87, 32, and 22 kDa), occurring in a ratio of 1.16:1:0.83. Containing FAD, molybdenum, iron, and acid-labile sulfur in the stoichiometric ratio of 2:2:8:8, the enzyme belongs to the molybdo-iron/sulfur flavoproteins. Molybdopterin cytosine dinucleotide is the organic part of the pterin molybdenum cofactor. Comparison of N-terminal amino acid sequences revealed similarities to a number of procaryotic molybdenum-containing hydroxylases. Especially the N-termini of the β-subunits of the quinoline 2-oxidoreductases from Comamonas testosteroni 63, Pseudomonas putida 86, and Rhodococcus spec. B1, and of quinoline-4-carboxylic acid 2-oxidoreductase from Agrobacterium spec. IB showed striking similarities. Further degradation of (3-methyl-)2-oxo-1,2-dihydroquinoline proceeds via dioxygenation at the benzene ring, i.e. at 5,6-position [Schach, S., Schwarz, G., Fetzner, S. & Lingens, F. (1993) Biol. Chem. Hoppe-Seyler 374, 175–181]. 2-Oxo-1,2-dihydroquinoline 5,6-dioxygenase was partially purified; NADH and oxygen are required for the reaction, and the enzymic activity is enhanced 1.5-fold by addition of Fe2+ ions. Unexpectedly, this aromatic ring dioxygenase did not separate into distinct protein components, but is apparently a single-component enzyme. The molecular mass was estimated to be about 260 kDa. 2-Oxo-1,2-dihydroquinoline 5,6-dioxygenase is very thermolabile. However, dithioerythritol and low concentrations of substrate had a moderately stabilizing effect. 2-Oxo-1,2-dihydroquinoline 5,6-dioxygenase is inhibited by sulfhydryl-blocking agents, by metal-chelating agents, and by the flavin analogues quinacrine and acriflavin.

Modeling an Elementary Step of the Enzyme Pyruvate Oxidase: Oxidation of a Thiamin Diphosphate-Bound Enamine Intermediate by a Flavin Analog

ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTModeling an Elementary Step of the Enzyme Pyruvate Oxidase: Oxidation of a Thiamin Diphosphate-Bound Enamine Intermediate by a Flavin AnalogChingfan C. Chiu, Ke Pan, and Frank JordanCite this: J. Am. Chem. Soc. 1995, 117, 26, 7027–7028Publication Date (Print):July 1, 1995Publication History Published online1 May 2002Published inissue 1 July 1995https://doi.org/10.1021/ja00131a036RIGHTS & PERMISSIONSArticle Views217Altmetric-Citations25LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InReddit PDF (260 KB) Get e-Alerts Get e-Alerts

Affinity probing of flavin binding sites. 1. Covalent attachment of 8-(methylsulfonyl)FAD to pig heart lipoamide dehydrogenase.

8-(Methylsulfonyl)FAD reacts with a single cysteine residue (Cys449) in pig apolipoamide dehydrogenase to generate a flavinylated enzyme containing covalently bound 8-(cysteinyl)FAD. Competitive behavior is observed in reconstitution reactions containing both FAD and 8-(methylsulfonyl)FAD. Covalently bound 8-(cysteinyl)FAD is shielded from solvent, as judged by spectral comparison with model 8-(alkylthio)-flavins in various solvents. Flavinylated lipoamide dehydrogenase is monomeric and catalytically inactive. Cys449 is located in the interface domain, near the active site histidine (His452). As shown previously, Cys449 is oxidized when native enzyme is treated with cupric ions. Cys449 is close to the isoalloxazine ring of FAD in native enzyme, as judged by alignment of the pig sequence with the structure of the homologous enzyme from Azotobacter vinelandii. The residue corresponding to Cys449 in A. vinlandii lipoamide dehydrogenase (Val447) is about 9 A from the carbonyl oxygen at C(2) in the pyrimidine ring of FAD. Approximation of a substituent at position 8 in FAD with Cys449 requires a 180 degrees flip of the isoalloxazine ring as compared with its orientation in the native structure. The different flavin orientation can explain the absence of dimerization and catalytic activity. Using the same method of apoenzyme preparation, noncovalent binding was observed with 8-chloroFAD, a less reactive flavin analogue. Relatively nonspecific covalent incorporation was observed with 8-chloroFAD when apoenzyme was prepared by an older method used in previous studies with this derivative [Moore, E.G., Cardemil, E., & Massey, V. (1978) J. Biol. Chem. 253, 6413-6422].

Use of laser flash photolysis time-resolved spectrophotometry to investigate interprotein and intraprotein electron transfer mechanisms

A description is given of the methodology developed in our laboratory for the application of laser flash photolysis to the elucidation of the kinetics and mechanism of electron transfer processes which occur intermolecularly between two protein molecules within a collisional complex, or intramolecularly between two redox centers within a single multisubunit or multidomain protein. This involves the use of flavin analogs, excited to their lowest triplet state by a laser flash, to initiate electron transfer, either by oxidation of a sacrificial donor followed by redox protein reduction via the flavin semiquinone, or by direct oxidation of a reduced redox protein by the flavin triplet. Time-resolved spectrophotometry is used to follow the course of the sequence of electron transfer events initiated by the laser flash. The application of this methodology to the following systems is described: cytochrome c/cytochrome c peroxidase; ferredoxin/ferredoxin NADP + reductase; cytochrome c/plastocyanin; flavocytochrome b 2; and sulfite oxidase.

Autoinhibition of murine macrophage-mediated oxidation of low-density lipoprotein by nitric oxide synthesis

Murine peritoneal macrophages treated with γ-interferon and lipopolysaccharide (activated cells) oxidized low-density lipoprotein (LDL) less readily than unstimulated cells. Activated cells expressed the enzyme nitric oxide synthase, whose activity was measured by the accumulation of nitrite in the culture supernatant. Treatment of activated macrophages with the arginine analogue N G -monomethyl-arginine (NMMA) inhibited nitric oxide synthesis and restored the ability of the cells to oxidize LDL. This treatment had no effect on the ability of unstimulated cells to oxidize LDL. Similarly, LDL oxidation by activated macrophages in arginine-free Ham's F-10 medium was identical to that of unstimulated cells, whereas restoration of arginine to the medium was associated with nitrite secretion and a decline in LDL oxidation by activated cells only. An inverse relationship between nitric oxide synthesis and LDL oxidation was also demonstrated in the presence of diphenylene iodonium, a flavin analogue which is a potent inhibitor of nitric oxide synthase. Thus nitric oxide synthesis appears to mediate the suppression of LDL oxidation which is associated with the activation of mouse macrophages by γ-interferon and lipopolysaccharide.

VECTORIAL ELECTRON TRANSPORT ACROSS LIPID VESICLE MEMBRANES DRIVE BY TWO PHOTOREACTIONS. II. PHOTOCHEMICAL REGENERATION OF REDUCED CYTOCHROME c BY FLAVIN TRIPLET STATE AND ETHYLENEDIAMINETETRAACETIC ACID IN THE INNER COMPARTMENT AND REDUCTION OF FERREDOXIN BY CHLOROPHYLL TRIPLET STATE IN THE OUTER COMPARTMENT

AbstractWe have attempted to mimic natural photosynthesis with regard to the photogeneration of a powerful reductant, using a negatively charged lipid bilayer vesicle system incorporating two photoreactions sensitized by a flavin analog (flavin mononucleotide [FMN]) and chlorophyll (chl) in their respective triplet states. Ethylenediamine‐tetraacetic acid (EDTA) in the inner aqueous compartment was used as a sacrificial electron donor to the FMN triplet, and ferredoxin in the outer aqueous compartment served as the final electron acceptor (mediated via triplet electron transfer chain in this multicomponent system to be elucidated. By itself, EDTA does not function as an effective donor to membran‐bound oxidized chl (chl+.), which is formed by electron transfer from triplet chl to the viologen follwed by transbilayer electron migration. This is a consequence of electrostatic repulsive interactions with the negatively charged membrane. This limitation is avoided when FMN is used as a photomediator between EDTA and chl+.. The overall reaction is dramatically increased in rate by enclosing cytochrom c together with EDTA and FMN in the inner compartment. The rate constant of the key step in the reaction, i.e. elctron transfer from reduced cytochrome c, generated via photoreduction by the FMN/EDTA system, to chl+. is increased 20‐fold over that obtained with cytochrome c alone as the elctron donor. One of the important constraints that limited the net electron transfer across the bilayer to 50% of the added cytochrome, i.e. inhibition by oxidized cytochrome c formed in the inner compartment, is avoided by the inclusion of the second photoreaction in this system, thus allowing photoreduction of all of the added ferredoxin to be achieved. This system provides a model for a photochemical energy storage process that utilizes two photorections operating in series resulting in electron flow across a lipid bilayer membrane.

Riboflavin 5′-pyrophosphate: A contaminant of commercial FAD, a coenzyme for FAD-dependent oxidases, and an inhibitor of FAD synthetase

Commercially available preparations of flavin adenine dinucleotide (FAD) have been found to be 94% pure, the remaining 6% being composed of four or five minor contaminants which can be separated from FAD by reverse-phase high-performance liquid chromatography. FAD purified in this manner has been shown to be 100% pure. One of the contaminants has been identified as riboflavin 5′-pyrophosphate (RPP) by spectroscopic and chemical methods of analysis. This compound has been shown to exhibit biological activity as a weak cofactor for two FAD-requiring enzymes. With the apo-protein of porcine d-amino-acid oxidase, values determined for RPP were 8.4 μ m for K m and 0.10 for V max compared to 0.47 μ m and 0.28 (36 U/mg), respectively, for FAD. With fungal glucose apooxidase, values determined for RPP were 474 n m for K m and 0.02 for V max and 45 n m and 0.09 (105 U/mg), respectively, for FAD. RPP can also inhibit FAD biosynthesis. For bovine liver FAD synthetase, a K i value for RPP against FMN was determined to be 9 μ m where K m for FMN was 5.5 μ m. These studies illustrate the value of riboflavin 5′-pyrophosphate as a flavin analog for use in the study of structure/function relationships within certain flavin-dependent enzymes.

Conformational changes in Chondrus crispus flavodoxin on dissociation of FMN and reconstitution with flavin analogues

The apoflavodoxin produced by precipitation of Chondrus crispus flavodoxin with trichloroacetic acid migrates as a single molecular species on non-denaturing PAGE, but at a much lower Rm than the flavoprotein. Values of s and D were significantly lower than for the flavodoxin, but their substitution in the Svedberg equation indicated the molecular mass was closely similar to that of the flavodoxin. This was confirmed by meniscus-depletion sedimentation-equilibrium studies. The Stokes radius of the apoflavodoxin was 3.65 nm, compared with 2.33 nm for the flavodoxin, and estimates of frictional coefficient ratio suggested the apoprotein was in extended conformation compared with the roughly globular shape of the flavodoxin. The Ka for FMN binding was 2.8 x 10(8)M, and the electrophoretic and physicochemical properties of the reconstituted flavoprotein were closely similar to those of the native flavodoxin. FAD, iso-FMN and thio-FMN were also bound effectively, but methyl-FMN and riboflavin were bound only weakly, if at all. The reconstituted flavoproteins were active to various extents in mediating electron transfer from NADPH to cytochrome c catalysed by flavodoxin-NADP+ oxidoreductase, the highest activity being with the thio-FMN flavodoxin.

Evidence for direct interaction between cysteine 138 and the flavin in thioredoxin reductase. A study using flavin analogs.

The flavoenzyme thioredoxin reductase from Escherichia coli contains an oxidation-reduction active disulfide made up of Cys135 and Cys138. Mutations changing each Cys residue to a Ser residue have been effected (Prongay, A. J., engelke, D. R., and Williams, C. H., Jr. (1989) J. Biol. Chem. 264, 2656-2664). The FAD prosthetic group of each altered thioredoxin reductase has been replaced with 1-deaza-FAD (a flavin analog with carbon substituted for nitrogen at position 1), 4-thio-FAD (a flavin analog with sulfur substituted for oxygen at position 4), and 6-thiocyanato-FAD. 1-Deaza-FAD-TRR(Cys135,Ser138) has absorbance and fluorescence spectral properties similar to the oxidized form of wild type apothioredoxin reductase reconstituted with 1-deaza-FAD. The absorbance spectrum of 1-deaza-FAD-TRR(Ser135,Cys138) is similar to the spectrum of the two-electron reduced form of wild type apothioredoxin reductase reconstituted with 1-deaza-FAD, indicating that it is a mixture of two species (O'Donnell, M. E., and Williams, C. H., Jr. (1984) J. Biol. Chem. 259, 2243-2251). The spectrum of one of these species of 1-deaza-FAD-TRR(Ser135,Cys138) resembles the spectrum of oxidized 1-deaza-FAD bound to wild type apothioredoxin reductase. The other species has an absorbance spectrum with a single peak at 400 nm (epsilon 400 = 11,100 M-1 cm-1) and resembles the spectrum of a thiolate adduct at the C4a position of the 1-deaza-FAD. The equilibrium between these species is pH-dependent, with a maximum of 50% C4a-adduct formation at low pH, and is linked to pK alpha values at 8.2 and 9.3. The absorbance spectrum of 4-thio-FAD-TRR(Cys135,Ser138) resembles the spectrum of the unbound 4-thio-FAD, whereas 4-thio-FAD-TRR(Ser135,Cys138) has a spectrum indicative of a mixture of 4-thio-FAD and FAD, suggesting a reaction between the 4-position of the flavin and Cys138. The binding of 6-thiocyanato-FAD to the apoprotein of the mutated enzymes showed no evidence for a reaction between the thiols and the group at the 6-position of the flavin.

Reconstitution of Escherichia coli photolyase with flavins and flavin analogs

Escherichia coli DNA photolyase contains two chromophore cofactors, 1,5-dihydroflavin adenine dinucleotide (FADH2) and (5,10-methenyltetrahydrofolyl)polyglutamate (5,10-MTHF). A procedure was developed for reversible resolution of apophotolyase and its chromophores. To investigate the structures important for the binding of FAD to apophotolyase and of photolyase to DNA, reconstitution experiments with FAD, FMN, riboflavin, 1-deazaFAD, 5-deazaFAD, and F420 were attempted. Only FAD and 5-deazaFAD showed high-affinity binding to apophotolyase. The apoenzyme had no affinity to DNA but did regain its specific binding to thymine dimer containing DNA upon binding stoichiometrically to FAD or 5-deazaFAD. Successful reduction of enzyme-bound FAD with dithionite resulted in complete recovery of photocatalytic activity.

Antimalarial action of flavin analogues seems not to be due to inhibition of glutathione reductase of host erythrocytes

A series of 10-(4′-chlorophenyl)-3-substituted flavins ( 1a-f) were examined with respect to their antimalarial properties. They were tested against Plasmodium falciparum in vitro and Plasmodium vinckei vinckei in uivo. The propostion that they might act through glutathione reductase (GR) (EC 1.6.4.2) inhibition has been studied. Inhibition of P. falciparum in vitro by these compounds shows only slight variation between analogues; in contrast, inhibition of human erythrocyte GR by members of the same series is highly variable, indicating that this is probably not their primary mode of antimalarial action. Results of the P. vinckei vinckei screen showed that 10-(4′-chlorophenyl)-3-methyl, 3-ethyl and 3-propyl substituted flavins are active in vivo over the dose range screened (10–70 mg/kg).