Flavin Adenine Dinucleotide Molecule Research Articles

Partially Folded Structure of Flavin Adenine Dinucleotide-depleted Ferredoxin-NADP+ Reductase with Residual NADP+ Binding Domain

Maize ferredoxin-NADP(+) reductase (FNR) consists of flavin adenine dinucleotide (FAD) and NADP(+) binding domains with a FAD molecule bound noncovalently in the cleft between these domains. The structural changes of FNR induced by dissociation of FAD have been characterized by a combination of optical and biochemical methods. The CD spectrum of the FAD-depleted FNR (apo-FNR) suggested that removal of FAD from holo-FNR produced an intermediate conformational state with partially disrupted secondary and tertiary structures. Small angle x-ray scattering indicated that apo-FNR assumes a conformation that is less globular in comparison with holo-FNR but is not completely chain-like. Interestingly, the replacement of tyrosine 95 responsible for FAD binding with alanine resulted in a molecular form similar to apo-protein of the wild-type enzyme. Both apo- and Y95A-FNR species bound to Cibacron Blue affinity resin, indicating the presence of a native-like conformation for the NADP(+) binding domain. On the other hand, no evidence was found for the existence of folded conformations in the FAD binding domains of these proteins. These results suggested that FAD-depleted FNR assumes a partially folded structure with a residual NADP(+) binding domain but a disordered FAD binding domain.

Coumarin 6, Hypericin, Resorufins, and Flavins: Suitable Chromophores for Fluorescence Correlation Spectroscopy of Biological Molecules

In this work we show that the dyes coumarin 6, hypericin, 7-O-ethylresorufin and resorufin are suitable for fluorescence correlation spectroscopy (FCS) and demonstrate the use of these dyes in physiologically relevant protein studies. Since coumarins are metabolised by cytochromes P450, the binding of coumarin 6 to cytochrome P450 3A4 was investigated by FCS. Coumarin 6 appears to be a very bright non-covalent cytochrome P450 label. When titrating cytochrome P450 3A4 with coumarin 6, the diffusion time of the coumarin 6/ cytochrome P450 3A4 complex increases roughly two-fold at protein concentrations higher than 1 μmol l-1, indicating the formation of cytochrome aggregates. FCS of the flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD) shows that both endogenous dyes undergo photobleaching. Moreover, FAD appears to be present to great extent, as a non-fluorescent intramolecular complex. Analysis of the FCS data of the flavoprotein NADPH-cytochrome P450 oxidoreductase (molecular weight 76 500) yielded two components. While the slow component corresponds to a globular protein with the molecular weight about 75 000, the fast component appears to be due to free diffusing FMN and FAD molecules. The amount of free FMN and FAD increases with increasing laser power. At high laser power a complete photodissociation of FMN and FAD occurs.

Conversion of 4-hydroxyacetophenone into 4-phenyl acetate by a flavin adenine dinucleotide-containing Baeyer-Villiger-type monooxygenase.

An arylketone monooxygenase was purified from Pseudomonas putida JD1 by ion exchange and affinity chromatography. It had the characteristics of a Baeyer-Villiger-type monooxygenase and converted its substrate, 4-hydroxyacetophenone, into 4-hydroxyphenyl acetate with the consumption of one molecule of oxygen and oxidation of one molecule of NADPH per molecule of substrate. The enzyme was a monomer with an M(r) of about 70,000 and contained one molecule of flavin adenine dinucleotide (FAD). The enzyme was specific for NADPH as the electron donor, and spectral studies showed rapid reduction of the FAD by NADPH but not by NADH. Other arylketones were substrates, including acetophenone and 4-hydroxypropiophenone, which were converted into phenyl acetate and 4-hydroxyphenyl propionate, respectively. The enzyme displayed Michaelis-Menten kinetics with apparent K(m) values of 47 microM for 4-hydroxyacetophenone, 384 microM for acetophenone, and 23 microM for 4-hydroxypropiophenone. The apparent K(m) value for NADPH with 4-hydroxyacetophenone as substrate was 17.5 microM. The N-terminal sequence did not show any similarity to other proteins, but an internal sequence was very similar to part of the proposed NADPH binding site in the Baeyer-Villiger monooxygenase cyclohexanone monooxygenase from an Acinetobacter sp.

Alkyl-dihydroxyacetonephosphate Synthase: PRESENCE AND ROLE OF FLAVIN ADENINE DINUCLEOTIDE

Alkyl-dihydroxyacetonephosphate synthase is a peroxisomal enzyme involved in ether lipid synthesis. It catalyzes the exchange of the acyl chain in acyl-dihydroxyacetonephosphate for a long chain fatty alcohol, yielding the first ether linked intermediate, i.e. alkyl-dihydroxyacetonephosphate, in the pathway of ether lipid biosynthesis. Although this reaction is not a net redox reaction, the amino acid sequence of the enzyme suggested the presence of a flavin adenine dinucleotide (FAD)-binding domain. In this study we show that alkyl-dihydroxyacetonephosphate synthase contains an essential FAD molecule as cofactor, which is evidenced by fluorescence properties, UV-visible absorption spectra and the observation that the enzyme activity is dependent on the presence of this cofactor in a coupled in vitro transcription/translation assay. Furthermore, we could demonstrate that the FAD cofactor directly participates in catalysis. Upon incubation of the enzyme with the substrate palmitoyl-dihydroxyacetonephosphate, the flavin moiety is reduced, indicating that in this initial step the substrate is oxidized. Stopped flow experiments show that the reduction of the flavin moiety is a monophasic process yielding a oxygen stable, reduced enzyme species. Upon addition of hexadecanol to the reduced enzyme species, the flavin moiety is efficiently reoxidized. A hypothetical reaction mechanism is proposed that is consistent with the data in this paper and with previous studies.

A hyperactive NAD(P)H:Rubredoxin oxidoreductase from the hyperthermophilic archaeon Pyrococcus furiosus.

NAD(P)H:rubredoxin oxidoreductase (NROR) has been purified from the hyperthermophilic archaeon Pyrococcus furiosus. The enzyme is exceedingly active in catalyzing the NADPH-dependent reduction of rubredoxin, a small (5.3-kDa) iron-containing redox protein that had previously been purified from this organism. The apparent Vmax at 80 degrees C is 20,000 micromol/min/mg, which corresponds to a kcat/Km value of 300,000 mM(-1) s(-1). The apparent Km values measured at 80 degrees C and pH 8.0 for rubredoxin, NADPH, and NADH were 50, 5, and 34 microM, respectively. The enzyme did not reduce P. furiosus ferredoxin. NROR is a monomer with a molecular mass of 45 kDa and contains one flavin adenine dinucleotide molecule per mole but lacks metals and inorganic sulfide. The possible physiological role of this hyperactive enzyme is discussed.

Three-dimensional structure of human electron transfer flavoprotein to 2.1-A resolution.

Mammalian electron transfer flavoproteins (ETF) are heterodimers containing a single equivalent of flavin adenine dinucleotide (FAD). They function as electron shuttles between primary flavoprotein dehydrogenases involved in mitochondrial fatty acid and amino acid catabolism and the membrane-bound electron transfer flavoprotein ubiquinone oxidoreductase. The structure of human ETF solved to 2.1-A resolution reveals that the ETF molecule is comprised of three distinct domains: two domains are contributed by the alpha subunit and the third domain is made up entirely by the beta subunit. The N-terminal portion of the alpha subunit and the majority of the beta subunit have identical polypeptide folds, in the absence of any sequence homology. FAD lies in a cleft between the two subunits, with most of the FAD molecule residing in the C-terminal portion of the alpha subunit. Alignment of all the known sequences for the ETF alpha subunits together with the putative FixB gene product shows that the residues directly involved in FAD binding are conserved. A hydrogen bond is formed between the N5 of the FAD isoalloxazine ring and the hydroxyl side chain of alpha T266, suggesting why the pathogenic mutation, alpha T266M, affects ETF activity in patients with glutaric acidemia type II. Hydrogen bonds between the 4'-hydroxyl of the ribityl chain of FAD and N1 of the isoalloxazine ring, and between alpha H286 and the C2-carbonyl oxygen of the isoalloxazine ring, may play a role in the stabilization of the anionic semiquinone. With the known structure of medium chain acyl-CoA dehydrogenase, we hypothesize a possible structure for docking the two proteins.

A comparative study on stm imaging and electrocatalytic activity of different surfaces modified with flavin adenine dinucleotide

Flavin adenine dinucleotide (FAD) was modified onto the highly oriented pyrolytic graphite ( hopg) and glassy carbon electrode ( gce) surfaces with three methods, respectively. Corresponding image analysis for FAD-modified hopg surfaces has been performed by scanning tunnelling microscope (STM) for the first time. The molecular resolution STM image of FAD adsorbed on the freshly-cleaved hopg was obtained, the quantitative size determination suggests that the FAD molecules adsorb side lying on the substrate surface. The anodization treatment of hopg surface yields many pits, which were clearly observed under STM. These pits provide active sites on the hopg surface for modification and the treated hopg can strongly adsorb FAD molecules, the latter exhibiting an irregular cluster structure on such a surface. When FAD was electrochemically deposited on the substrate surface, a chain structure was successfully observed. The adsorbed FAD on anodized glassy carbon electrode ( gce) surface can effectively catalyze the reduction of glucose oxidase, hemoglobin and myoglobin, with a large decrease in the overvoltage, whereas the deposited FAD film exhibits excellent electrocatalysis towards dioxygen reduction.

Assembly of alcohol oxidase in peroxisomes of the yeast Hansenula polymorpha requires the cofactor flavin adenine dinucleotide.

The peroxisomal flavoprotein alcohol oxidase (AO) is an octamer (600 kDa) consisting of eight identical subunits, each of which contains one flavin adenine dinucleotide molecule as a cofactor. Studies on a riboflavin (Rf) auxotrophic mutant of the yeast Hansenula polymorpha revealed that limitation of the cofactor led to drastic effects on AO import and assembly as well as peroxisome proliferation. Compared to wild-type control cells Rf-limitation led to 1) reduced levels of AO protein, 2) reduced levels of correctly assembled and activated AO inside peroxisomes, 3) a partial inhibition of peroxisomal protein import, leading to the accumulation of precursors of matrix proteins in the cytosol, and 4) a significant increase in peroxisome number. We argue that the inhibition of import may result from the saturation of a peroxisomal molecular chaperone under conditions that normal assembly of a major matrix protein inside the target organelle is prevented.

Purification and properties of pentachlorophenol hydroxylase, a flavoprotein from Flavobacterium sp. strain ATCC 39723

A pentachlorophenol (PCP) hydroxylase which catalyzed the conversion of PCP to 2,3,5,6-tetrachlorohydroquinone and released iodide from triiodophenol in the presence of NADPH and oxygen was identified. The enzyme was purified by protamine sulfate precipitation, ammonium sulfate precipitation, hydrophobic chromatography, anion-exchange chromatography, gel filtration chromatography, and crystallization. The enzyme was a monomer with a molecular weight of 63,000. Under certain conditions, dimer and multimer conformations were also observed. The pI of the enzyme was pH 4.3. The optimal conditions for activity were a pH of 7.5 to 8.5 and a temperature of 40 degrees C. Each enzyme molecule contained one flavin adenine dinucleotide molecule. The Km for PCP was 30 microM and the Vmax was 16 mumol/min/mg of protein. The enzymatic reaction required 2 mol of NADPH per mol of halogenated substrate. On the basis of the data we present, it is likely that PCP hydroxylase is a flavoprotein monooxygenase. The addition of flavins to the reaction mixture did not stimulate the enzymatic reaction; however, we identified the photodegradation of triiodophenol and tribromophenol, but not PCP, by flavin mononucleotide or riboflavin and light.

Composition of the coenzyme F420-dependent formate dehydrogenase from Methanobacterium formicicum.

The coenzyme F420-dependent formate dehydrogenase from Methanobacterium formicicum was purified to electrophoretic homogeneity by anoxic procedures which included the addition of azide, flavin adenine dinucleotide (FAD), glycerol, and 2-mercaptoethanol to all buffer solutions to stabilize activity. The enzyme contains, in approximate molar ratios, 1 FAD molecule and 1 molybdenum, 2 zinc, 21 to 24 iron, and 25 to 29 inorganic sulfur atoms. Denaturation of the enzyme released a molybdopterin cofactor. The enzyme has a molecular weight of 177,000 and consists of one each of two different subunits, giving the composition alpha 1 beta 1. The molecular weight of the alpha-subunit is 85,000, and that of the beta-subunit is 53,000. The UV-visible spectrum is typical of nonheme iron-sulfur flavoprotein. Reduction of the enzyme facilitated dissociation of FAD, and the FAD-depleted enzyme was unable to reduce coenzyme F420. Preincubation of the FAD-depleted enzyme with FAD restored coenzyme F420-dependent activity.

Electrochemical Oxidation and Reduction of Flavin–Adenine Dinucleotide Adsorbed on a Mercury Electrode Surface

Abstract The electrochemical redox reaction of flavin–adenine dinucleotide (FAD) adsorbed on a hanging mercury drop electrode was studied in a pH 6.9 phosphate buffer by means of cyclic d.c. and a.c. voltammetry. Both the oxidized and reduced forms of FAD were strongly adsorbed on the mercury electrode surface. At the surface concentrations of FAD, Γ, lower than 5.0×10−11 mol cm−2, the cyclic d.c. and a.c. voltammetric behavior of adsorbed FAD was explained by the theory for a two-step one-electron surface redox reaction. The formal redox potential, semiquinone formation constant, and charge transfer rate constant for the surface redox reaction of FAD and the interaction parameter of adsorbed FAD species were determined. Cyclic d.c. voltammetry and differential capacity measurement showed that, as Γ exceeded 5×10−11 mol cm−2, the reorientation of adsorbed FAD molecules occurred. At Γ>5.8×10−11 mol cm−2, the semiquinone formation constant was very small and the behavior was interpreted by the theory of a single-step two-electron surface redox reaction. The electrochemical surface redox properties of FAD adsorbed on a mercury electrode surface appear to depend on the orientation mode of adsorbed FAD molecules.

Camphor revisited: involvement of a unique monooxygenase in metabolism of 2-oxo-delta 3-4,5,5-trimethylcyclopentenylacetic acid by Pseudomonas putida.

Previously, Pseudomonas putida was shown to degrade (+)-camphor, and cleavage of the first ring of the bicyclic structure involved two monooxygenases (a hydroxylase and a ring oxygen-inserting enzyme), a dehydrogenase, and spontaneous cleavage of an unstable oxygenation product (lactone). Cleavage of the second ring was not demonstrated but was assumed also to occur by ring oxygen insertion, since the predicted oxygenation product was extracted from whole-cell incubation systems. Our investigation established that metabolism of the first ring cleavage intermediate, 2-oxo-delta 3-4,5,5-trimethylcyclopentenylacetic acid, occurred through the sequential action of two inducible enzymes, a coenzyme A ester synthetase and an oxygenase. The oxygenase was purified to homogeneity and had a molecular weight of 106,000. This enzyme carried a single molecule of flavin adenine dinucleotide and consisted of two identical subunits. Iron was not present at a significant level. The oxygenase was specific for NADPH as the electron donor and absolutely specific for the coenzyme A ester of 2-oxo-delta 3-4,5,5-trimethylcyclopentenylacetic acid as the substrate. The reaction stoichiometry was compatible with this enzyme being a monooxygenase, and a mass spectral analysis of the methyl ester of the product confirmed the insertion of a single oxygen atom. The enzyme appeared to be analogous to, although distinct from. 2,5-diketocamphane 1,2-monooxygenase in catalyzing a "biological Baeyer-Villiger" reaction with the formation of a lactone. Structural analogy suggested that this lactone, like the first, was also unstable and susceptible to spontaneous ring opening, although this was not experimentally established.

A rapid kinetic study of divalent metal interactions with flavin coenzymes

Research in these laboratories has focussed in recent years on the kinetics of divalent metal ion interactions with coenzymes. The principal kinetic tool has been temperature-jump relaxation spectroscopy. A large amount of kinetic information is now available for several nucleotides (e.g. AMP) and inorganic phosphates. The purpose of this paper is to report the first rapid kinetic study of the mechanism of divalent metal ion interactions with the coenzymes flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD). The two compounds are structurally related to each other as well as to other coenzymes and phosphates that we have previously studied. FAD, for example, is structurally a combination of riboflavin phosphate and adenosine monophosphate (AMP). Ni(II) was chosen as the metal ion for these studies because of the large body of kinetic information that is already available for that ion. It can serve as a useful representative of divalent transition metal ion interactions with these and related coenzymes. The NiFMN system. Two relaxation effects were observed in the kinetic experiments: one (τ 1) on the order of 0.2 msec, the other (τ 2) at about 2 msec. The detailed concentration and pH dependencies of τ 1 and τ 2 are quite similar to those for the relaxation times found in the Ni-ribose phosphate and NiAMP systems respectively. The mechanism consistent with these observations is a dual-pathway, back-bound complex mechanism, shown schematically as I; ▪ in which NiL is the phosphate-bound complex, NiL′ the phosphate + base bound complex. The NiFAD System. FAD presents a number of different binding sites in the ionized phosphate bridge and the base nitrogens on both the adenine and isoalloxazine rings. The flexibility of this molecule facilitates both individual and simultaneous ring interactions with the phosphate-bound metal. The NiFAD system is unique in that four distinct relaxation times, τ–τ 2, were found and characterized. The relaxation times ranged from 90 μsec to 20 msec and were found to be only slightly pH and concentration dependent. Based on the large body of prior data from our laboratory on simple nucleotide systems, we were able to associate specific relaxation times with reaction steps in scheme II and to determine the rate constants. ▪ The mechanism shown as II quantitatively accounts for the number and behavior of all the relaxations steps. In this scheme, F, P and A refer to the flavin, phosphate, and adenine moieties of the FAD molecule, respectively. The first step (1–2) involves bridging to the phosphate moiety only, followed by species involving interactions with the phosphate plus the flavin (5) or adenine moieties (3). The final complex (4) involves simultaneous interactions with all the components of the molecule.

Lipoamide dehydrogenase from Malbranchea pulchella: isolation and characterization.

Lipoamide dehydrogenase (EC 1.6.4.3) has been isolated from a total homogenate of frozen mycelium of the thermophilic fungus Malbranchea pulchella var. sulfurea by a three-step procedure involving ammonium sulfate fractionation, Procion Brilliant Blue M-R--Sepharose 4B chromatography, and hydroxylapatite chromatography. The second step is the key purification step with the Procion Brilliant Blue M-R dye acting as an affinity ligand for the enzyme. The purified enzyme gave a single protein band on polyacrylamide gel electrophoresis in the presence and absence of sodium dodecyl sulfate. The enzyme is a dimer of molecular weight 102 000, and each monomer of 51 000 molecular weight binds one molecule of flavin adenine dinucleotide. Other properties determined include a pH optimum of 8.2, a strong specificity for the substrates dihydrolipoamide and nicotinamide adenine dinucleotide, the apparent lack of multiple enzymic forms, the presence of diaphorase activity, and resistance to temperature denaturation up to 60 degrees C. The amino acid composition and absorption spectrum of the enzyme were also determined. The properties of lipoamide dehydrogenase from this source are very similar to those reported for the enzyme from serveral other sources.

38] Fatty acid ω-hydroxylase (alkane hydroxylase) from Pseudomonas oleovorans

Publisher Summary This chapter describes the purification procedure of fatty acid ω-hydroxylase (alkane hydroxylase) from Pseudomonas oleovorans. The hydroxylation system induced in Pseudomonas oleovorans by growth on alkanes contains three proteins: NADH-rubredoxin reductase—a flavoprotein containing one molecule of flavin adenine dinucleotide (FAD) per polypeptide chain, rubredoxin—a red, nonheme iron protein, and the ω-hydroxylase, which is an unusual example of a mono-oxygenase containing nonheme iron as the prosthetic group. Electron transfer in this system is as follows: NADH reductase → rubredoxin → ω-hydroxylase → O 2 . Ferredoxin-NADP reductase may be substituted for the bacterial reductase, provided that NADPH is used in place of NADH as the primary electron donor. The activity of the hydroxylase is determined as the octane-dependent rate of NADPH oxidation in a reaction mixture containing an excess of reductase and rubredoxin. In the chapter, the various steps are summarized in a table and are carried out at 4°.