flavin-adenine-dinucleotide and quinone

Electron bifurcation, or the coupling of exergonic and endergonic oxidation-reduction reactions, was discovered by Peter Mitchell and provides an elegant mechanism to rationalize and understand the logic that underpins the Q cycle of the respiratory chain. Thought to be a unique reaction of respiratory complex III for nearly 40 years, about a decade ago Wolfgang Buckel and Rudolf Thauer discovered that flavin-based electron bifurcation is also an important component of anaerobic microbial metabolism. Their discovery spawned a surge of research activity, providing a basis to understand flavin-based bifurcation, forging fundamental parallels with Mitchell's Q cycle and leading to the proposal of metal-based bifurcating enzymes. New insights into the mechanism of electron bifurcation provide a foundation to establish the unifying principles and essential elements of this fascinating biochemical phenomenon.

Topics: Benzoquinones; Electron Transport; Electron Transport Chain Complex Proteins; Flavin-Adenine Dinucleotide; Hydroquinones; Mitochondria; NAD; Oxidation-Reduction

This review considers quinone-dependent alcohol dehydrogenases and FAD-dependent alcohol oxidases, enzymes that are present in numerous methylotrophic eu- and prokaryotes and significantly differ in their primary and quaternary structure. The cofactors of the enzymes are bound to the protein polypeptide chain through ionic and hydrophobic interactions. Microorganisms containing these enzymes are described. Methods for purification of the enzymes, their physicochemical properties, and spatial structures are considered. The supposed mechanism of action and practical application of these enzymes as well as their producers are discussed.

Topics: Alcohol Dehydrogenase; Alcohol Oxidoreductases; Animals; Benzoquinones; Flavin-Adenine Dinucleotide; Humans; Models, Molecular; Molecular Structure

Redox reactions pervade living cells. They are central to both anabolic and catabolic metabolism. The ability to maintain redox balance is therefore vital to all organisms. Various regulatory sensors continually monitor the redox state of the internal and external environments and control the processes that work to maintain redox homeostasis. In response to redox imbalance, new metabolic pathways are initiated, the repair or bypassing of damaged cellular components is coordinated and systems that protect the cell from further damage are induced. Advances in biochemical analyses are revealing a range of elegant solutions that have evolved to allow bacteria to sense different redox signals.

Topics: Bacteria; Bacterial Proteins; Benzoquinones; Cysteine; Flavin Mononucleotide; Flavin-Adenine Dinucleotide; Hemeproteins; Iron-Sulfur Proteins; NAD; NADP; Oxidation-Reduction; Oxidative Stress; Signal Transduction

The family of flavoenzymes in which the flavin coenzyme redox cofactor is covalently attached to the protein through an amino acid side chain is covered in this review. Flavin-protein covalent linkages have been shown to exist through each of five known linkages: (a) 8alpha-N(3)-histidyl, (b) 8alpha-N(1)-histidyl, (c) 8alpha-S-cysteinyl, (d) 8alpha-O-tyrosyl, or (e) 6-S-cysteinyl with the flavin existing at either the flavin mononucleotide or flavin adenine dinucleotide (FAD) levels. This class of enzymes is widely distributed in diverse biological systems and catalyzes a variety of enzymatic reactions. Current knowledge on the mechanism of covalent flavin attachment is discussed based on studies on the 8alpha-S-cysteinylFAD of monoamine oxidases A and B, as well as studies on other flavoenzymes. The evidence supports an autocatalytic quinone-methide mechanism of protein flavinylation. Proposals to explain the structural and mechanistic advantages of a covalent flavin linkage in flavoenzymes are presented. It is concluded that multiple factors are involved and include: (a) stabilization of the apoenzyme structure, (b) steric alignment of the cofactor in the active site to facilitate catalysis, and (c) modulation of the redox potential of the covalent flavin through electronic effects of 8alpha-substitution.

Topics: Benzoquinones; Catalysis; Flavin-Adenine Dinucleotide; Flavins; Mass Spectrometry; Models, Chemical; Monoamine Oxidase; Oxidation-Reduction; Phosphorylation

Monotopic membrane-bound flavoproteins, sulfide:quinone oxidoreductases (SQRs), have a variety of physiological functions, including sulfide detoxification. SQR enzymes are classified into six groups. SQRs use the flavin adenine dinucleotide (FAD) cofactor to transfer electrons from sulfide to quinone. A type VI SQR of the photosynthetic purple sulfur bacterium, Thiocapsa roseopersicina (TrSqrF), has been previously characterized, and the mechanism of sulfide oxidation has been proposed. This paper reports the characterization of quinone binding site (QBS) of TrSqrF composed of conserved aromatic and apolar amino acids. Val331, Ile333, and Phe366 were identified near the benzoquinone ring of enzyme-bound decylubiquinone (dUQ) using the TrSqrF homology model. In silico analysis revealed that Val331 and Ile333 alternately connected with the quinone head group via hydrogen bonds, and Phe366 and Trp369 bound the quinones via hydrophobic interactions. TrSqrF variants containing alanine (V331A, I333A, F366A) and aromatic amino acid (V331F, I333F, F366Y), as well as a C-terminal α-helix deletion (CTD) mutant were generated. These amino acids are critical for quinone binding and, thus, catalysis. Spectroscopic analyses proved that all mutants contained FAD. I333F replacement resulted in the lack of the charge transfer complex. In summary, the interactions described above maintain the quinone molecule's head in an optimal position for direct electron transfer from FAD. Surprisingly, the CTD mutant retained a relatively high level of specific activity while remaining membrane-anchored. This is a unique study because it focuses on the QBS and the oxidative stage of a type VI sulfide-dependent quinone reduction. KEY POINTS: • V331, I333, F366, and W369 were shown to interact with decylubiquinone in T. roseopersicina SqrF • These amino acids are involved in proper positioning of quinones next to FAD • I333 is essential in formation of a charge transfer complex from FAD to quinone.

Topics: Amino Acids; Benzoquinones; Binding Sites; Flavin-Adenine Dinucleotide; Oxidation-Reduction; Quinone Reductases; Sulfides

Alzheimer's disease (AD) is one of the most prominent neurodegenerative diseases. Results from animal and cellular models suggest that FAD-deficient forms of NAD(P)H quinone oxidoreductase 1 (NQO1) may accelerate the aggregation of Alzheimer's amyloid-β peptide (Aβ1-42). Here, we examined in vitro whether NQO1 and its FAD-deficient P187S mutation (NQO1*2) directly interact with Aβ1-42 and modify its rate of aggregation. When monitored using the fluorescence of either noncovalent thioflavin T (ThT) or HiLyte Fluor 647 (HF647) dye covalently attached to the Aβ1-42 peptide, the aggregation kinetics of Aβ1-42 were markedly more rapid in the presence of NQO1*2 than the wild-type (WT) NQO1. Experiments using apo-NQO1 indicate that this increase is linked to the inability of NQO1*2 to bind to FAD. Furthermore, dicoumarol, an NQO1 inhibitor that binds near the FAD-binding site and stabilizes NQO1*2, markedly decreased the aggregation kinetics of Aβ1-42. Imaging flow cytometry confirmed in-vitro coaggregation of NQO1 isoforms and Aβ1-42. Aβ1-42 alone forms rod-shaped fibril structures while in the presence of NQO1 isoforms, Aβ1-42 is incorporated in the middle of larger globular protein aggregates surrounded by NQO1 molecules. Isothermal titration calorimetry (ITC) analysis indicates that Aβ1-42 interacts with NQO1 isoforms with a specific stoichiometry through a hydrophobic interaction with positive enthalpy and entropy changes. These data define the kinetics, mechanism, and shape of coaggregates of Aβ1-42 and NQO1 isoforms and the potential relevance of FAD-deficient forms of NQO1 for amyloid aggregation diseases.

Topics: Amyloid beta-Peptides; Animals; Benzoquinones; Flavin-Adenine Dinucleotide; Mutation; NAD; NAD(P)H Dehydrogenase (Quinone); NADH, NADPH Oxidoreductases

p-Nitrophenol 4-monooxygenase PnpA, the key enzyme in the hydroquinone pathway of p-nitrophenol (PNP) degradation, catalyzes the monooxygenase reaction of PNP to p-benzoquinone in the presence of FAD and NADH. Here, we determined the first crystal structure of PnpA from Pseudomonas putida DLL-E4 in its apo and FAD-complex forms to a resolution of 2.04 Å and 2.48 Å, respectively. The PnpA structure shares a common fold with hydroxybenzoate hydroxylases, despite a low amino sequence identity of 14-18%, confirming it to be a member of the Class A flavoprotein monooxygenases. However, substrate docking studies of PnpA indicated that the residues stabilizing the substrate in an orientation suitable for catalysis are not observed in other homologous hydroxybenzoate hydroxylases, suggesting PnpA employs a unique catalytic mechanism. This work expands our understanding on the reaction mode for this enzyme class.

Topics: Bacterial Proteins; Benzoquinones; Binding Sites; Biocatalysis; Crystallography, X-Ray; Flavin-Adenine Dinucleotide; Models, Molecular; Molecular Structure; Nitrophenols; Oxygenases; Protein Binding; Protein Conformation; Pseudomonas putida; Substrate Specificity

Acetohydroxyacid synthase (AHAS) catalyzes the first step of branched-chain amino acid (BCAA) biosynthesis, a pathway essential to the lifecycle of plants and microorganisms. This enzyme is of high interest because its inhibition is at the base of the exceptional potency of herbicides and potentially a target for the discovery of new antimicrobial drugs. The enzyme has conserved attributes from its predicted ancestor, pyruvate oxidase, such as a ubiquinone-binding site and the requirement for FAD as cofactor. Here, we show that these requirements are linked to the regulation of AHAS, in relationship to its anabolic function. Using various soluble quinone derivatives (

Topics: Acetolactate Synthase; Benzoquinones; Flavin-Adenine Dinucleotide; Humans; Models, Molecular; Mycobacterium tuberculosis; Oxidation-Reduction; Saccharomyces cerevisiae; Tuberculosis; Ubiquinone

The flavin adenine dinucleotide-dependent glucose dehydrogenase (FAD-GDH) from

Topics: Aspergillus; Benzoquinones; Flavin-Adenine Dinucleotide; Fungal Proteins; Glucose Dehydrogenases; Kinetics; Oxidation-Reduction; Phenothiazines

Recent studies implicate hydrogen sulfide (H

Topics: Bacterial Proteins; Benzoquinones; Biocatalysis; Disulfides; Flavin-Adenine Dinucleotide; Kinetics; Models, Chemical; Models, Molecular; Molecular Structure; Mutation; Protein Domains; Protein Multimerization; Protein Structure, Secondary; Quinone Reductases; Staphylococcus aureus; Substrate Specificity; Sulfides; Sulfites; Sulfur

The bioelectrochemical behavior of three triphenylmethane (TPM) dyes commonly used as pH indicators, and their application in mediated electron transfer systems for glucose oxidase bioanodes in biofuel cells was investigated. Bromophenol Blue, Bromothymol Blue, Bromocresol Green were compared bioelectrochemically against two widely used mediators, benzoquinone and ferrocene carboxy aldehyde. Biochemical studies were performed in terms of enzymatic oxidation, enzyme affinity, catalytic efficiency and co-factor regeneration. The different features of the TPM dyes as mediators are determined by the characteristics in the oxidation/reduction processes studied electrochemically. The reversibility of the oxidation/reduction processes was also established through the dependence of the voltammetric peaks with the sweep rates. All three dyes showed good performances compared to the FA and BQ when evaluated in a half enzymatic fuel cell. Potentiodynamic and power response experiments showed maxima power densities of 32.8 μW cm(-2) for ferrocene carboxy aldehyde followed by similar values obtained for TPM dyes around 30 μW cm(-2) using glucose and mediator concentrations of 10 mmol L(-1) and 1.0 mmol L(-1), respectively. Since no mediator consumption was observed during the bioelectrochemical process, and also good redox re-cycled processes were achieved, the use of triphenylmethane dyes is considered to be promising compared to other mediated systems used with glucose oxidase bioanodes and/or biofuel cells.

Topics: Aldehydes; Aspergillus niger; Benzoquinones; Biocatalysis; Bioelectric Energy Sources; Bromcresol Green; Bromphenol Blue; Bromthymol Blue; Electrochemical Techniques; Electrodes; Electron Transport; Enzymes, Immobilized; Ferrous Compounds; Flavin-Adenine Dinucleotide; Fungal Proteins; Glucose; Glucose Oxidase; Horseradish Peroxidase; Molecular Structure; Oxidation-Reduction; Spectrophotometry

Reactive oxygen species (ROS) production from mitochondrial complex II (succinate-quinone reductase, SQR) has become a focus of research recently since it is implicated in carcinogenesis. To date, the FAD site is proposed as the ROS producing site in complex II, based on studies done on Escherichia coli, whereas the quinone binding site is proposed as the site of ROS production based on studies in Saccharomyces cerevisiae. Using the submitochondrial particles from the adult worms and L(3) larvae of the parasitic nematode Ascaris suum, we found that ROS are produced from more than one site in the mitochondrial complex II. Moreover, the succinate-dependent ROS production from the complex II of the A. suum adult worm was significantly higher than that from the complex II of the L(3) larvae. Considering the conservation of amino acids crucial for the SQR activity and the high levels of ROS production from the mitochondrial complex II of the A. suum adult worm together with the absence of complexes III and IV activities in its respiratory chain, it is a good model to examine the reactive oxygen species production from the mitochondrial complex II.

Topics: Animals; Ascaris suum; Benzoquinones; Binding Sites; Electron Transport Complex II; Flavin-Adenine Dinucleotide; Larva; Mitochondria; Reactive Oxygen Species

The tcpRXABCYD operon of Cupriavidus necator JMP134 is involved in the degradation of 2,4,6-trichlorophenol (2,4,6-TCP), a toxic pollutant. TcpA is a reduced flavin adenine dinucleotide (FADH2)-dependent monooxygenase that converts 2,4,6-TCP to 6-chlorohydroxyquinone. It has been implied via genetic analysis that TcpX acts as an FAD reductase to supply TcpA with FADH2, whereas the function of TcpB in 2,4,6-TCP degradation is still unclear. In order to provide direct biochemical evidence for the functions of TcpX and TcpB, the two corresponding genes (tcpX and tcpB) were cloned, overexpressed, and purified in Escherichia coli. TcpX was purified as a C-terminal His tag fusion (TcpX(H)) and found to possess NADH:flavin oxidoreductase activity capable of reducing either FAD or flavin mononucleotide (FMN) with NADH as the reductant. TcpX(H) had no activity toward NADPH or riboflavin. Coupling of TcpX(H) and TcpA demonstrated that TcpX(H) provided FADH2 for TcpA catalysis. Among several substrates tested, TcpB showed the best activity for quinone reduction, with FMN or FAD as the cofactor and NADH as the reductant. TcpB could not replace TcpX(H) in a coupled assay with TcpA for 2,4,6-TCP metabolism, but TcpB could enhance TcpA activity. Further, we showed that TcpB was more effective in reducing 6-chlorohydroxyquinone than chemical reduction alone, using a thiol conjugation assay to probe transitory accumulation of the quinone. Thus, TcpB was acting as a quinone reductase for 6-chlorohydroxyquinone reduction during 2,4,6-TCP degradation.

Topics: Bacterial Proteins; Benzoquinones; Chlorophenols; Chromatography, Gel; Cloning, Molecular; Cupriavidus necator; Electrophoresis, Polyacrylamide Gel; Flavin Mononucleotide; Flavin-Adenine Dinucleotide; FMN Reductase; Hydrogen-Ion Concentration; Molecular Structure; NAD(P)H Dehydrogenase (Quinone); Operon; Temperature

To understand the biochemical basis for the function of the rotenone-insensitive internal NADH-quinone (Q) oxidoreductase (Ndi1), we have overexpressed mature Ndi1 in Escherichia coli membranes. The Ndi1 purified from the membranes contained one FAD and showed enzymatic activities comparable with the original Ndi1 isolated from Saccharomyces cerevisiae. When extracted with Triton X-100, the isolated Ndi1 did not contain Q. The Q-bound form was easily reconstituted by incubation of the Q-free Ndi1 enzyme with ubiquinone-6. We compared the properties of Q-bound Ndi1 enzyme with those of Q-free Ndi1 enzyme, with higher activity found in the Q-bound enzyme. Although both are inhibited by low concentrations of AC0-11 (IC(50) = 0.2 microm), the inhibitory mode of AC0-11 on Q-bound Ndi1 was distinct from that of Q-free Ndi1. The bound Q was slowly released from Ndi1 by treatment with NADH or dithionite under anaerobic conditions. This release of Q was prevented when Ndi1 was kept in the reduced state by NADH. When Ndi1 was incorporated into bovine heart submitochondrial particles, the Q-bound form, but not the Q-free form, established the NADH-linked respiratory activity, which was insensitive to piericidin A but inhibited by KCN. Furthermore, Ndi1 produces H(2)O(2) as isolated regardless of the presence of bound Q, and this H(2)O(2) was eliminated when the Q-bound Ndi1, but not the Q-free Ndi1, was incorporated into submitochondrial particles. The data suggest that Ndi1 bears at least two distinct Q sites: one for bound Q and the other for catalytic Q.

Topics: Animals; Benzoquinones; Binding Sites; Cattle; Electron Transport Complex I; Flavin-Adenine Dinucleotide; Hydrogen Peroxide; Mitochondria, Heart; NAD; NADH Dehydrogenase; Octoxynol; Oxidation-Reduction; Saccharomyces cerevisiae Proteins

A new approach for studying intramolecular electron transfer in multicenter enzymes is described. Two fumarate reductases, adsorbed on an electrode in a fully active state, have been studied using square-wave voltammetry as a kinetic method to probe the mechanism of the long-range electron transfer to and from the buried active site. Flavocytochrome c(3) (Fcc(3)), the globular fumarate reductase from Shewanella frigidimarina, and the soluble subcomplex of the membrane-bound fumarate reductase of Escherichia coli (FrdAB) each contain an active site FAD that is redox-connected to the surface by a chain of hemes or Fe-S clusters, respectively. Using square-wave voltammetry with large amplitudes, we have measured the electron-transfer kinetics of the FAD cofactor as a function of overpotential. The results were modeled in terms of the FAD group receiving or donating electrons either via a direct mechanism or one involving hopping via the redox chain. The FrdAB kinetics could be described by both models, while the Fcc(3) data could only be fit on the basis of a direct electron-transfer mechanism. This raises the likelihood that electron transfer can occur via a superexchange mechanism utilizing the heme groups to enhance electronic coupling. Finally, the FrdAB data show, in contrast to Fcc(3), that the maximum ET rate at high overpotential is related to the turnover number for FrdAB measured previously so that electron transfer is the limiting step during catalysis.

Topics: Benzoquinones; Binding Sites; Computer Simulation; Cytochrome c Group; Electrons; Escherichia coli; Flavin-Adenine Dinucleotide; Heme; Kinetics; Models, Chemical; Oxidation-Reduction; Protein Conformation; Shewanella; Succinate Dehydrogenase

Complex II from the thermoacidophilic archaeon Acidianus ambivalens, an archetype of an emerging class of succinate dehydrogenases (SDH), was extracted from intact membranes and purified to homogeneity. The complex contains one molecule of covalently bound FAD and 10 Fe atoms. EPR studies showed that the complex contains the canonical centres S1 ([2Fe-2S]2+/1+) and S2 ([4Fe-4S]+2/+1) but lacks centre S3 ([3Fe-4S]+1/0); these observations agree with the fact that the iron-sulfur subunit contains an extra cysteine that may allow the binding of a new centre, most probably a tetranuclear one. Succinate-driven oxygen consumption is observed in intact membranes indicating that in vivo, complex II operates as a succinate:quinone oxidoreductase, despite missing the typical anchor domain subunits. The pure complex was found to contain bound caldariella quinone, the enzyme physiological partner. An alternative membrane anchoring for this new type of SDHs, based on the amphipathic nature of the putative helices found in SdhC, is suggested.

Topics: Amino Acid Sequence; Benzoquinones; Cell Membrane; Cysteine; Electron Spin Resonance Spectroscopy; Electron Transport Complex II; Electrophoresis, Polyacrylamide Gel; Flavin-Adenine Dinucleotide; Iron; Models, Biological; Molecular Sequence Data; Multienzyme Complexes; Oxidation-Reduction; Oxidoreductases; Oxygen; Protein Structure, Tertiary; Sequence Homology, Amino Acid; Succinate Dehydrogenase; Sulfhydryl Compounds; Sulfolobaceae; Sulfur; Ultraviolet Rays

The amitochondriate eukaryote Giardia lamblia contains an NAD(P)H:menadione oxidoreductase (EC 1.6.99.2) (glQR) that catalyses the two-electron transfer oxidation of NAD(P)H with a quinone as acceptor. The gene encoding this protein in G. lamblia was expressed in Escherichia coli. The purified recombinant protein had an NAD(P)H oxidoreductase activity, with NADPH being a more efficient electron donor than NADH. Menadione, naphthoquinone and several artificial electron acceptors served as substrate for the enzyme. glQR shows high amino acid similarity to its homologues in vertebrates and also to a series of hypothetical proteins from bacteria. Although glQR is considerably smaller than the mammalian enzymes, three-dimensional modelling shows similar arrangement of the secondary structural elements. Most amino acid residues of the mammalian enzymes that participate in substrate binding or catalysis are conserved. Conservation of these features and the similarity in substrate specificity and in susceptibility to inhibitors establish glQR as an authentic member of this protein family.

Topics: Amino Acid Sequence; Animals; Benzoquinones; Binding Sites; Cloning, Molecular; Escherichia coli; Flavin-Adenine Dinucleotide; Giardia lamblia; Humans; Mice; Models, Molecular; Molecular Sequence Data; NAD(P)H Dehydrogenase (Quinone); NADP; Rats; Sequence Alignment; Sequence Analysis, DNA; Sequence Homology, Amino Acid; Vertebrates

Topics: Benzoquinones; Catechols; Chelating Agents; Cyanides; Enzyme Inhibitors; Flavin-Adenine Dinucleotide; Oxidoreductases; Quinacrine; Quinones; Research; Vegetables