Iron (III) reduction: A novel activity of the human NAD(P)H:oxidoreductase

NAD(P)H:quinone oxidoreductase (NQO1; EC 1.6.99.2) catalyzes a two-electron transfer involved in the protection of cells from reactive oxygen species. These reactive oxygen species are often generated by the one-electron reduction of quinones or quinone analogs. We report here on the previously unreported Fe(III) reduction activity of human NQO1. Under steady state conditions with Fe(III) citrate, the apparent Michaelis-Menten constant (Km(app)) was approximately 0.3 nM and the apparent maximum velocity (Vmax(app)) was 16 U mg(-1). Substrate inhibition was observed above 5 nM. NADH was the electron donor, Km(app)= 340 microM and Vmax(app) = 46 Umg(-1). FAD was also a cofactor with a Km(app) of 3.1 microM and Vmax(app) of 89 U mg(-1). The turnover number for NADH oxidation was 25 s(-1). Possible physiological roles of the Fe(III) reduction by this enzyme are discussed.     

A novel hydrogen peroxide sensor based on the direct electron transfer of horseradish peroxidase immobilized on silica-hydroxyapatite hybrid film

The direct electron transfer of immobilized horseradish peroxidase (HRP) on silica-hydroxyapatite (HAp) hybrid film-modified glassy carbon electrode (GCE) and its application as H(2)O(2) biosensors were investigated. On silica/HRP-HAp/GCE, HRP displayed a fast electron transfer process accompanied with one proton participate in. This sensor exhibited an excellent electrocatalytic response to the reduction of H(2)O(2) without the aid of an electron mediator. The proposed biosensor showed good reproducibility and high sensitivity to H(2)O(2) with the detection limit of 0.35 microM. In the range of 1.0-100 microM, the catalytic reduction current of H(2)O(2) was proportional to H(2)O(2) concentration. The apparent Michaelis-Menten constant (k(m)(app)) of the biosensor was calculated to be 21.8 microM, exhibiting a high enzymatic activity and affinity for H(2)O(2).     

Purification and properties of heterodisulfide reductase from Methanobacterium thermoautotrophicum (strain Marburg)

The reduction of the heterodisulfide of coenzyme M (H-S-CoM) and 7-mercaptoheptanoyl-L-threonine phosphate (H-S-HTP) is a key reaction in the metabolism of methanogenic bacteria. The heterodisulfide reductase catalyzing this step was purified 80-fold to apparent homogeneity from Methanobacterium thermoautotrophicum. The native enzyme showed an apparent molecular mass of 550 kDa. Sodium dodecyl sulfate/polyacrylamide gel electrophoresis revealed the presence of three different subunits of apparent molecular masses 80 kDa, 36 kDa, and 21 kDa. The enzyme, which was brownish yellow, contained per mg protein 7 +/- 1 nmol FAD, 130 +/- 10 nmol non-heme iron and 130 +/- 10 nmol acid-labile sulfur, corresponding to 4 mol FAD and 72 mol FeS/mol native enzyme. The purified heterodisulfide reductase catalyzed the reduction of CoM-S-S-HTP (app. Km = 0.1 mM) with reduced benzylviologen at a specific rate of 30 mumol.min-1.mg protein-1 (kcat = 68 s-1) and the reduction of methylene blue with H-S-CoM (app. Km = 0.2 mM) plus H-S-HTP (app. Km less than 0.05 mM) at a specific rate of 15 mumol.min-1.mg-1. The enzyme was highly specific for CoM-S-S-HTP and H-S-CoM plus H-S-HTP. The physiological electron donor/acceptor remains to be identified.     

Mechanism of elementary catalytic steps of pyruvate oxidase from Lactobacillus plantarum

Single steps in the catalytic cycle of pyruvate oxidase from Lactobacillus plantarum have been characterized kinetically and mechanistically by stopped-flow in combination with kinetic solvent isotope effect studies. Reversible substrate binding of pyruvate occurs with an on-rate of 6.5 x 10(4) M(-1) s(-1) and an off-rate of pyruvate of 20 s(-1). Decarboxylation of the intermediate lactyl-ThDP and the reduction of FAD which consists of two consecutive single electron-transfer steps from HEThDP to FAD occur with rates of about k(dec) = 112 s(-1) and k(red) = 422 s(-1). Flavin radical intermediates are not observed during reduction, and kinetic solvent isotope effects are absent, indicating that electron transfer and protonation processes are not rate limiting in the overall reduction process. Reoxidation of FADH(2) by O(2) to yield H(2)O(2) takes place at a pseudo-first-order rate of about 35 s(-1) in air-saturated buffer. A comparable value of about 35 s(-1) was estimated for the phosphorolysis of the acetyl-ThDP intermediate at phosphate saturation. In competition with phosphorolysis, enzyme-bound acetyl-ThDP is hydrolyzed with a rate k = 0.03 s(-1). This is the first report in which the reaction of enzyme-bound acetyl-ThDP with phosphate and OH(-) is monitored directly by FAD absorbance changes using the sequential stopped-flow technique.     

A new approach to the construction of potentiometric immunosensors.

An electrochemical immunosensor based on a new detection principle was developed. Laccase, which is able to catalyse the electroreduction of oxygen via the direct (mediatorless) mechanism was used as an enzyme label. The new detection method does not require the presence of an electrochemically active mediator, and the reaction substrates are atmospheric oxygen and electrons, the latter being taken by the active site of the enzyme label directly from the electrode. The formation of the complex between laccase-labelled antibody and antigen on the electrode surface resulted in a considerable (more than 300 mV) shift of the electrode potential. The rate of the increase of the electrode potential was inversely proportional to the concentration of the free antigen in the sample. The non-specific adsorption of conjugate and other proteins on the electrode could be eliminated by using a polyethylenimine-based polymer on the electrode surface. Insulin was used as a model analyte. The sensitivity limit for this antigen was approximately 3 micrograms ml-1.     

Unusual redox properties of electron-transfer flavoprotein from Methylophilus methylotrophus

The most positive redox potential ever recorded for a flavin adenine dinucleotide (FAD) containing protein has been measured for an electron-transfer flavoprotein (ETF) synthesized by Methylophilus methylotrophus. This potential value, 0.196 V versus the standard hydrogen electrode (vs SHE), was measured at pH 7.0 for the one-electron reduction of fully oxidized ETF (ETFox) to the red anionic semiquinone form of ETF (ETF.-). Quantitative formation of ETF.- was observed. The first successful reduction of ETF from M. methylotrophus to its two-electron fully reduced form was also achieved. Although addition of the second electron to ETF.- was extremely slow, the potential value measured for this reduction was -0.197 V vs SHE, suggesting a kinetic rather than thermodynamic barrier to two-electron reduction. These data are believed to be consistent with the postulated catalytic function of ETF to accept one electron from the iron-sulfur cluster of trimethylamine dehydrogenase (TMADH). The second electron reduction appears to have no catalytic function. The very positive potential measured for this ETF and the wide separation of potentials for the two electron reduction steps show that this ETF is a unique and interesting flavoprotein. In addition, this work highlights that while ETFs exhibit similar structural and spectral properties, they display wide variations in redox properties.     

Application of electrochemical impedance spectroscopy for monitoring allergen-antibody reactions using gold nanoparticle-based biomolecular immobilization method

Gold nanoparticles were used to enhance the immobilization amount and retain the immunoactivity of recombinant dust mite allergen Der f2 immobilized on a glassy carbon electrode (GCE). The interaction between allergen and antibody was studied by electrochemical impedance spectroscopy (EIS). Self-assembled Au colloid layer (?=16nm) deposited on (3-mercaptopropyl)trimethoxysilane (MPTS)-modified GCE offered a basis to control the immobilization of allergen Der f2. The impedance measurements were based on the charge transfer kinetics of the [Fe(CN)(6)](3-/4-) redox pair, compared with bare GCE, the immobilization of allergen Der f2 and the allergen-antibody interaction that occurred on the electrode surface altered the interfacial electron transfer resistance and thereby slowed down the charge transfer kinetics by reducing the active area of the electrode or by preventing the redox species in electrolyte solution from approaching the electrode. The interactions of allergen with various concentrations of monoclonal antibody were also monitored through the change of impedance response. The results showed that the electron transfer resistance increased with increasing concentrations of monoclonal antibody.     

Enhanced electron transfer for hemoglobin entrapped in a cationic gemini surfactant films on electrode and the fabrication of nitric oxide biosensor

The direct electrical communication between hemoglobin (Hb) and GCE surface was achieved based on the immobilization of Hb in a cationic gemini surfactant film and characterized by electrochemical techniques. The cyclic voltammograms showed that direct electron transfer between Hb and electrode surface was obviously promoted and then a novel unmediated nitric oxide (NO) biosensor was constructed in view of this protein-based electrode. This modified electrode showed an enzyme-like activity towards the reduction of NO and its amperometric response to NO was well-behaved with a rapid response time and displaying Michaelis-Menten kinetics with a calculated Km(app) value of 84.37 micromol L(-1). The detection limit was estimated to be 2.00 x 10(-8)mol L(-1). This biosensor was behaving as expected that it had a good stability and reproducibility, a higher sensitivity and selectivity and should has a potential application in monitoring NO released from biologic samples.     

Electrochemiluminescence determination of codeine or morphine with an organically modified silicate film immobilizing Ru(bpy)3(2+).

An ECL approach was developed for the determination of codeine or morphine based on tris(2,2'-bipyridine)ruthenium(II) (Ru(bpy)(3)(2+)) immobilized in organically modified silicates (ORMOSILs). Tetramethoxysilane (TMOS) and dimethyldimethoxysilane (DiMe-DiMOS) were selected as co-precursors for ORMOSILs, which were then immobilized on a surface of glassy carbon electrode (GCE) by a dip-coating process. Ru(bpy)(3)(2+) was immobilized in the ORMOSIL film via ion-association with poly(p-styrenesulphonate). The ORMOSIL-modified GCE presented good electrochemical and photochemical activities. In a flow system, the eluted codeine or morphine was oxidized on the modified GCE and reacted with immobilized Ru(bpy)(3)(2+) at a potential of +1.20 V (vs. Ag/AgCl). The modified electrode was used for the ECL determination of codeine or morphine and showed high sensitivity. The calibration curves were linear in the range 2 x 10(-8)-5 x 10(-5) mol/L for codeine and 1 x 10(-7)-3 x 10(-4) mol/L for morphine. The detection limit was 5 x 10(-9) mol/L for codeine and 3 x 10(-8) mol/L for morphine, at signal:noise ratio (S:N)=3. Both codeine and morphine showed reproducibility with RSD values <2.5% at 1.0 x 10(-6) mol/L. Furthermore, the modified electrode immobilized Ru(bpy)(3)(2+) was applied to the ECL determination of codeine or morphine in incitant samples.     

Electrochemical and kinetic analysis of electron-transfer reactions of Chlorella nitrate reductase.

Assimilatory nitrate reductase (NR) from Chlorella is homotetrameric, each subunit containing FAD, heme, and Mo-pterin in a 1:1:1 stoichiometry. Measurements of NR activity and steady-state reduction of the heme component under conditions of NADH limitation or competitive inhibition by nitrite suggested intramolecular electron transfer between heme and Mo-pterin was a rate-limiting step and provided evidence that heme is an obligate intermediate in the transfer of electrons between FAD and Mo-pterin. In addition to the physiological substrates NADH and nitrate, various redox mediators undergo reactions with one or more of the prosthetic groups. These reactions are coupled by NR to NADH oxidation or nitrate reduction. To test whether intramolecular redox reactions of NR were rate-determining, rate constants for redox reactions between NR and several chemically diverse mediators were measured by cyclic voltammetry in the presence of NADH or nitrate. Reduction of ferrocenecarboxylic acid, dichlorophenolindophenol, and cytochrome c by NADH-reduced NR was coupled to reoxidation at a glassy carbon electrode (ferrocene and dichlorophenolindophenol) or at a bis(4-pyridyl) disulfide modified gold electrode (cytochrome c), yielding rate constants of 10.5 x 10(6), 1.7 x 10(6), and 2.7 x 10(6) M-1 s-1, respectively, at pH 7. Kinetics were consistent with a second-order reaction, implying that intramolecular heme reduction by NADH and endogenous FAD was not limiting. In contrast, reduction of methyl viologen and diquat at a glassy carbon electrode, coupled to oxidation by NR and nitrate, yielded similar kinetics for the two dyes. In both cases, second-order kinetics were not obeyed, and reoxidation of dye-reduced Mo-pterin of NR by nitrate became limiting at low scan rates.(ABSTRACT TRUNCATED AT 250 WORDS)     

Electron transfer in human methionine synthase reductase studied by stopped-flow spectrophotometry

Human methionine synthase reductase (MSR) is a key enzyme in folate and methionine metabolism as it reactivates the catalytically inert cob(II)alamin form of methionine synthase (MS). Electron transfer from MSR to the cob(II)alamin cofactor coupled with methyl transfer from S-adenosyl methionine returns MS to the active methylcob(III)alamin state. MSR contains stoichiometric amounts of FAD and FMN, which shuttle NADPH-derived electrons to the MS cob(II)alamin cofactor. Herein, we have investigated the pre-steady state kinetic behavior of the reductive half-reaction of MSR by anaerobic stopped-flow absorbance and fluorescence spectroscopy. Photodiode array and single-wavelength spectroscopy performed on both full-length MSR and the isolated FAD domain enabled assignment of observed kinetic phases to mechanistic steps in reduction of the flavins. Under single turnover conditions, reduction of the isolated FAD domain by NADPH occurs in two kinetically resolved steps: a rapid (120 s(-1)) phase, characterized by the formation of a charge-transfer complex between oxidized FAD and NADPH, is followed by a slower (20 s(-1)) phase involving flavin reduction. These two kinetic phases are also observed for reduction of full-length MSR by NADPH, and are followed by two slower and additional kinetic phases (0.2 and 0.016 s(-1)) involving electron transfer between FAD and FMN (thus yielding the disemiquinoid form of MSR) and further reduction of MSR by a second molecule of NADPH. The observed rate constants associated with flavin reduction are dependent hyperbolically on NADPH and [4(R)-2H]NADPH concentration, and the observed primary kinetic isotope effect on this step is 2.2 and 1.7 for the isolated FAD domain and full-length MSR, respectively. Both full-length MSR and the separated FAD domain that have been reduced with dithionite catalyze the reduction of NADP+. The observed rate constant of reverse hydride transfer increases hyperbolically with NADP+ concentration with the FAD domain. The stopped-flow kinetic data, in conjunction with the reported redox potentials of the flavin cofactors for MSR [Wolthers, K. R., Basran, J., Munro, A. W., and Scrutton, N. S. (2003) Biochemistry, 42, 3911-3920], are used to define the mechanism of electron transfer for the reductive half-reaction of MSR. Comparisons are made with similar stopped-flow kinetic studies of the structurally related enzymes cytochrome P450 reductase and nitric oxide synthase.     

Determination of the redox potentials and electron transfer properties of the FAD- and FMN-binding domains of the human oxidoreductase NR1.

Human novel reductase 1 (NR1) is an NADPH dependent diflavin oxidoreductase related to cytochrome P450 reductase (CPR). The FAD/NADPH- and FMN-binding domains of NR1 have been expressed and purified and their redox properties studied by stopped-flow and steady-state kinetic methods, and by potentiometry. The midpoint reduction potentials of the oxidized/semiquinone (-315 +/- 5 mV) and semiquinone/dihydroquinone (-365 +/- 15 mV) couples of the FAD/NADPH domain are similar to those for the FAD/NADPH domain of human CPR, but the rate of hydride transfer from NADPH to the FAD/NADPH domain of NR1 is approximately 200-fold slower. Hydride transfer is rate-limiting in steady-state reactions of the FAD/NADPH domain with artificial redox acceptors. Stopped-flow studies indicate that hydride transfer from the FAD/NADPH domain of NR1 to NADP+ is faster than hydride transfer in the physiological direction (NADPH to FAD), consistent with the measured reduction potentials of the FAD couples [midpoint potential for FAD redox couples is -340 mV, cf-320 mV for NAD(P)H]. The midpoint reduction potentials for the flavin couples in the FMN domain are -146 +/- 5 mV (oxidized/semiquinone) and -305 +/- 5 mV (semiquinone/dihydroquinone). The FMN oxidized/semiquinone couple indicates stabilization of the FMN semiquinone, consistent with (a) a need to transfer electrons from the FAD/NADPH domain to the FMN domain, and (b) the thermodynamic properties of the FMN domain in CPR and nitric oxide synthase. Despite overall structural resemblance of NR1 and CPR, our studies reveal thermodynamic similarities but major kinetic differences in the electron transfer reactions catalysed by the flavin-binding domains.     

Over-expression in Escherichia coli and characterization of two recombinant isoforms of human FAD synthetase

FAD synthetase (FADS) (EC 2.7.7.2) is a key enzyme in the metabolic pathway that converts riboflavin into the redox cofactor FAD. Two hypothetical human FADSs, which are the products of FLAD1 gene, were over-expressed in Escherichia coli and identified by ESI-MS/MS. Isoform 1 was over-expressed as a T7-tagged protein which had a molecular mass of 63kDa on SDS-PAGE. Isoform 2 was over-expressed as a 6-His-tagged fusion protein, carrying an extra 84 amino acids at the N-terminal with an apparent molecular mass of 60kDa on SDS-PAGE. It was purified near to homogeneity from the soluble cell fraction by one-step affinity chromatography. Both isoforms possessed FADS activity and had a strict requirement for MgCl(2), as demonstrated using both spectrophotometric and chromatographic methods. The purified recombinant isoform 2 showed a specific activity of 6.8+/-1.3nmol of FAD synthesized/min/mg protein and exhibited a K(M) value for FMN of 1.5+/-0.3microM. This is the first report on characterization of human FADS, and the first cloning and over-expression of FADS from an organism higher than yeast.     

Mechanism of reduction of Corynebacterium sarcosine oxidase by dithiothreitol

The mechanism of the reduction of Corynebacterium sarcosine oxidase [EC 1.5.3.1] by dithiothreitol (DTT) was investigated. The reduction followed biphasic kinetics with second-order rate constants of 54 M-1 X S-1 and 5.4 M-1 X S-1 for the respective phases. When the oxidized enzyme was titrated with sarcosine under anaerobic conditions, no intermediate, such as a semiquinone or a charge-transfer complex, appeared during the reduction of the enzyme. On the other hand, on DTT titration, an intermediate with a semiquinoid character appeared, and its formation was maximum when half of the total FAD was reduced. An oxidized semiapoenzyme, which had lost 45% of the noncovalently-bound FAD present in the native enzyme, also showed biphasic kinetics in the reduction with DTT. The second-order rate constant was found to be 38 M-1 X S-1 for the fast phase. An intermediate was also formed and its concentration, estimated by electron spin resonance (ESR) measurement, was found to agree with that of the noncovalently-bound FAD. In addition, the oxidized semiapoenzyme, which had lost 95% of the noncovalently-bound FAD present in the native enzyme, was reduced with DTT much more slowly than the native enzyme. In this case, the second-order rate constant was found to be 0.4 M-1 X S-1, and no intermediate was observed during the titration with DTT. On the basis of these data, it is suggested that the noncovalently-bound FAD accepts electrons directly from DTT in the fast phase through the semiquinoid form, while the covalently-bound FAD accepts electrons from the reduced noncovalently-bound FAD in the slow phase without forming an intermediate.     

Interfacial electron transfer of glucose oxidase on poly(glutamic acid)-modified glassy carbon electrode and glucose sensing

The interfacial electron transfer of glucose oxidase (GOx) on a poly(glutamic acid)-modified glassy carbon electrode (PGA/GCE) was investigated. The redox peaks measured for GOx and flavin adenine dinucleotide (FAD) are similar, and the anodic peak of GOx does not increase in the presence of glucose in a mediator-free solution. These indicate that the electroactivity of GOx is not the direct electron transfer (DET) between GOx and PGA/GCE and that the observed electroactivity of GOx is ascribed to free FAD that is released from GOx. However, efficient electron transfer occurred if an appropriate mediator was placed in solution, suggesting that GOx is active. The PGA/GCE-based biosensor showed wide linear response in the range of 0.5–5.5 mM with a low detection limit of 0.12 mM and high sensitivity and selectivity for measuring glucose.     

Simultaneous voltammetric detection of dopamine and uric acid at their physiological level in the presence of ascorbic acid using poly(acrylic acid)-multiwalled carbon-nanotube composite-covered glassy-carbon electrode

The use of poly(acrylic acid) (PAA)-multiwalled carbon-nanotubes (MWNTs) composite-coated glassy-carbon disk electrode (GCE) (PAA-MWNTs/GCE) for the simultaneous determination of physiological level dopamine (DA) and uric acid (UA) in the presence of an excess of ascorbic acid (AA) in a pH 7.4 phosphate-buffered solution was proposed. PAA-MWNTs composite was prepared by mixing of MWNTs powder into 1 mg/ml PAA aqueous solution under sonication. GCE surface was modified with PAA-MWNTs film by casting. AA demonstrates no voltammetric peak at PAA-MWNTs/GCE. The PAA-MWNTs composite is of a high surface area and of affinity for DA and UA adsorption. DA exhibits greatly improved electron-transfer rate and is electro-catalyzed at PAA-MWNTs/GCE. Moreover, the electro-catalytic oxidation of UA at PAA-MWNTs/GCE is observed, which makes it possible to detect lower level UA. Therefore, the enhanced electrocatalytic currents for DA and UA were observed. The anodic peak currents at approximately 0.18 V and 0.35 V increase with the increasing concentrations of DA and UA, respectively, which correspond to the voltammetric peaks of DA and UA, respectively. The linear ranges are 40 nM to 3 microM DA and 0.3 microM to 10 microM UA in the presence of 0.3 mM AA. The lowest detection limits (S/N=3) were 20 nM DA and 110 nM UA.     

Redox properties of flavocytochrome c3 from Shewanella frigidimarina NCIMB400

The thermodynamic and catalytic properties of flavocytochrome c3 from Shewanella frigidimarina have been studied using a combination of protein film voltammetry and solution methods. As measured by solution kinetics, maximum catalytic efficiencies for fumarate reduction (kcat/Km = 2.1 x 10(7) M-1 s-1 at pH 7.2) and succinate oxidation (kcat/Km = 933 M-1 s-1 at pH 8.5) confirm that flavocytochrome c3 is a unidirectional fumarate reductase. Very similar catalytic properties are observed for the enzyme adsorbed to monolayer coverage at a pyrolytic graphite "edge" electrode, thus confirming the validity of the electrochemical method for providing complementary information. In the absence of fumarate, the adsorbed enzyme displays a complex envelope of reversible redox signals which can be deconvoluted to yield the contributions from each active site. Importantly, the envelope is dominated by the two-electron signal due to FAD [E degrees ' = -152 mV vs the standard hydrogen electrode (SHE) at pH 7.0 and 24 degrees C] which enables quantitative examination of this center, the visible spectrum of which is otherwise masked by the intense absorption bands due to the hemes. The FAD behaves as a cooperative two-electron center with a pH-dependent reduction potential that is modulated (pKox at 6.5) by ionization of a nearby residue. In conjunction with the kinetic pKa values determined for the forward and reverse reactions (7.4 and 8.6, respectively), a mechanism for fumarate reduction, incorporating His365 and an anionic form of reduced FAD, is proposed. The reduction potentials of the four heme groups, estimated by analysis of the underlying envelope, are -102, -146, -196, and -238 mV versus the SHE at pH 7.0 and 24 degrees C and are comparable to those determined by redox potentiometry.     

Nucleophilic participation of reduced flavin coenzyme in mechanism of UDP-galactopyranose mutase

UDP-galactopyranose mutase (UGM) requires reduced FAD (FAD(red)) to catalyze the reversible interconversion of UDP-galactopyranose (UDP-Galp) and UDP-galactofuranose (UDP-Galf). Recent structural and mechanistic studies of UGM have provided evidence for the existence of an FAD-Galf/p adduct as an intermediate in the catalytic cycle. These findings are consistent with Lewis acid/base chemistry involving nucleophilic attack by N5 of FAD(red) at C1 of UDP-Galf/p. In this study, we employed a variety of FAD analogues to characterize the role of FAD(red) in the UGM catalytic cycle using positional isotope exchange (PIX) and linear free energy relationship studies. PIX studies indicated that UGM reconstituted with 5-deaza-FAD(red) is unable to catalyze PIX of the bridging C1-OP(β) oxygen of UDP-Galp, suggesting a direct role for the FAD(red) N5 atom in this process. In addition, analysis of kinetic linear free energy relationships of k(cat) versus the nucleophilicity of N5 of FAD(red) gave a slope of ρ = -2.4 ± 0.4. Together, these findings are most consistent with a chemical mechanism for UGM involving an S(N)2-type displacement of UDP from UDP-Galf/p by N5 of FAD(red).     

Electron transfer in acetohydroxy acid synthase as a side reaction of catalysis. Implications for the reactivity and partitioning of the carbanion/enamine form of (alpha-hydroxyethyl)thiamin diphosphate in a "nonredox" flavoenzyme

Acetohydroxy acid synthases (AHAS) are thiamin diphosphate- (ThDP-) and FAD-dependent enzymes that catalyze the first common step of branched-chain amino acid biosynthesis in plants, bacteria, and fungi. Although the flavin cofactor is not chemically involved in the physiological reaction of AHAS, it has been shown to be essential for the structural integrity and activity of the enzyme. Here, we report that the enzyme-bound FAD in AHAS is reduced in the course of catalysis in a side reaction. The reduction of the enzyme-bound flavin during turnover of different substrates under aerobic and anaerobic conditions was characterized by stopped-flow kinetics using the intrinsic FAD absorbance. Reduction of enzyme-bound FAD proceeds with a net rate constant of k' = 0.2 s(-1) in the presence of oxygen and approximately 1 s(-1) under anaerobic conditions. No transient flavin radicals are detectable during the reduction process while time-resolved absorbance spectra are recorded. Reconstitution of the binary enzyme-FAD complex with the chemically synthesized intermediate 2-(hydroxyethyl)-ThDP also results in a reduction of the flavin. These data provide evidence for the first time that the key catalytic intermediate 2-(hydroxyethyl)-ThDP in the carbanionic/enamine form is not only subject to covalent addition of 2-keto acids and an oxygenase side reaction but also transfers electrons to the adjacent FAD in an intramolecular redox reaction yielding 2-acetyl-ThDP and reduced FAD. The detection of the electron transfer supports the idea of a common ancestor of acetohydroxy acid synthase and pyruvate oxidase, a homologous ThDP- and FAD-dependent enzyme that, in contrast to AHASs, catalyzes a reaction that relies on intercofactor electron transfer.     

The electrochemical preparation of FAD/ZnO with hemoglobin film-modified electrodes and their electroanalytical properties

Flavin adenine dinucleotide (FAD)-modified zinc oxide self-assembly films were prepared using repeated cyclic voltammetry. The electrochemical reaction of the hemoglobin with the FAD/ZnO self-assembly film-modified electrodes and their electrocatalytic properties were investigated. This paper describes the successful loading of the electrochemically active molecules of hemoglobin and FAD along with ZnO by electrochemical method. In addition to the cyclic voltammetry, an electrochemical quartz crystal microbalance was used to study the growth mechanism and the properties of the films. The FAD/zinc oxide films exhibited a single redox couple, which corresponded to the FAD redox couple. The electrocatalytic properties of the O2, H2O2, trichloroacetic acid and SO(3)2- were studied by the FAD/zinc oxide films in the absence or in the presence of hemoglobin. The electrocatalytic reduction current had been developed from the cathodic peak of the FAD/zinc oxide redox couple. The electrocatalytic process involved an interaction of hemoglobin and FAD/GC film-modified electrode to increase the electrocatalytic reduction current. The electrocatalytic reduction of O2 using the FAD/zinc oxide films was investigated by cyclic voltammetry and rotating ring-disk electrode methods.