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PubMed2005

H2O2-induced changes in mitochondrial activity in isolated mouse pancreatic acinar cells.

This study employed confocal laser scanning microscopy to monitor the effect of H2O2 on cytosolic as well as mitochondrial calcium (Ca2+) concentrations, mitochondrial inner membrane potential (psi m) and flavine adenine dinucleotide (FAD) oxidation state in isolated mouse pancreatic acinar cells. The results show that incubation of pancreatic acinar cells with H2O2, in the absence of extracellular Ca2+ ([Ca2+],) led to an increase either in cytosolic and in mitochondrial Ca2+ concentration. Additionally, H2O2 induced a depolarization of mitochondria and increased oxidized FAD level. Pretreatment of cells with the mitochondrial inhibitors rotenone or cyanide inhibited the response induced by H2O2 on mitochondrial inner membrane potential but failed to block oxidation of FAD in the presence of H2O2. However, the H2O2-evoked effect on FAD state was blocked by pretreatment of cells with the mitochondrial uncoupler, carbonyl cyanide p-trifluoromethoxy-phenylhydrazone (FCCP). On the other hand, perfusion of cells with thapsigargin (Tps), an inhibitor of the SERCA pump, led to an increase in mitochondrial Ca2+ concentration and in oxidized FAD level, and depolarized mitochondria. Pretreatment of cells with thapsigargin inhibited H2O2-evoked changes in mitochondrial Ca2+ concentration but not those in membrane potential and FAD state. The present results have indicated that H2O2 can evoke marked changes in mitochondrial activity that might be due to the oxidant nature of H2O2. This in turn could represent the mechanism of action of ROS to induce cellular damage leading to cell dysfunction and generation of pathologies in the pancreas.

Animals↗
PubMed1989

Mechanism of inhibition of erythrocyte glutathione reductase by mitomycin-C.

Mitomycin-C (MT-C) inhibits human erythrocyte glutathione reductase (EGR). We found that Km values of EGR for GSSG, NADPH, and FAD were 1.3 X 10(-4) mol/l, 2.3 X 10(-5) mol/l, and 3.9 X 10(-7) mol/l respectively, and that EGR was inhibited by MT-C non-competitively with respect to both the substrate GSSG (Ki = 4.8 X 10(-5) mol/l) and the cofactor NADPH (Ki = 2.2 X 10(-4) mol/l), and competitively with respect to FAD (Ki = 4.2 X 10(-5) mol/l). On the other hand, we demonstrated that FAD makes a complex with MT-C, the dissociation constant (K = 6.5 X 10(-5) mol/l) of which was obtained from fluorescence quenching of FAD with MT-C. It also became clear that spectra wf FAD at around 375 nm changes with increasing MT-C concentration. So, the data strongly suggest that the mechanism of inhibition of EGR by MT-C is mainly due to complex formation of the co-enzyme FAD with MT-C.

Erythrocytes↗
PubMed1992

Selective chemical modification of amino acid residues in the flavin adenine dinucleotide binding site of NADPH-ferredoxin reductase.

1. An apo-NADPH-ferredoxin reductase was prepared from holo-NADPH-ferredoxin reductase (EC 1.18.1.2) from bovine adrenocortical mitochondria. 2. Amino acid residues of the apo-reductase were modified selectively, to identify the FAD-binding site of the reductase, with chemical reagents such as diethylpyrocarbonate, 5,5'-dithiobis(2-nitrobenzoate), tetranitromethane, pyridoxal 5'-phosphate, p-nitrophenylglyoxal, diisopropylfluorophosphate and N-bromosuccinimide. The binding of FAD to the apo-reductase was measured as quenching of the fluorescence of FAD caused by the binding between apo-reductase and FAD. The quenching was blocked when the apo-reductase was modified with diethylpyrocarbonate and restored on the addition of hydroxylamine. 3. The blocking of the quenching occurred in a competitive manner as to FAD in the presence of diethylpyrocarbonate. However, when the apo-reductase was modified with 5,5'-dithiobis(2-nitrobenzoate), the blocking of the quenching occurred in a non-competitive manner. 4. These results suggested that a histidyl residue of the apo-reductase is essential for the binding of FAD to the reductase. This was confirmed by amino acid sequencing of the modified apo-reductase.

Amino Acid Sequence↗
PubMed1992

Preparation and characterization of monoclonal antibodies against 4-aminobenzoate hydroxylase from Agaricus bisporus.

A monoclonal antibody (mAb, A) recognizing the FAD-binding domain of 4-aminobenzoate hydroxylase (4-aminobenzoate, NAD(P)H:oxygen oxidoreductase (1-hydroxylating, decarboxylating), EC 1.14.13.27) from Agaricus bisporus, a common edible mushroom, had been produced (Tsuji, H., Ogawa, T., Bando, N., Kimoto, M. and Sasaoka, K. (1990) J. Biol. Chem. 265, 16064-16067). In the present study, three other mAbs (B1, B2 and B3) against the enzyme have been further prepared in order to facilitate the structural characterization of the enzyme. The three new mAbs immunoblotted the enzyme. The four mAbs, including A, were specific for different epitopes on the enzyme. B1 and B2 immunoprecipitated the apoenzyme and the immunoprecipitation was inhibited in the presence of FAD, whereas B3 failed to immunoprecipitate the apoenzyme in the absence or presence of FAD. B1 and B2 competed with FAD for the binding to the apoenzyme. These findings show that B1 and B2 recognize the FAD-binding domain of the enzyme in analogy with A. The immunoblotting analyses of the peptides obtained from the enzyme by digestion with lysyl endopeptidase (EC 3.4.21.50) provided useful knowledge as to the location of the epitopes to the mAbs on the enzyme, suggesting that the FAD-binding domain of the enzyme can be located and characterized by detailed investigations on the location of the epitopes.

Agaricus↗
PubMed2006

Reversible resolution of flavin and pterin cofactors of His-tagged Escherichia coli DNA photolyase.

Escherichia coli photolyase catalyzes the repair of cyclobutane pyrimidine dimers (CPD) in DNA under near UV/blue-light irradiation. The enzyme contains flavin adenine dinucleotide (FAD) and methenyltetrahydrofolate (MTHF) as noncovalently bound light sensing cofactors. To study the apoprotein-chromophore interactions we developed a new procedure to prepare apo-photolyase. MTHF-free photolyase was obtained by binding the C-terminal His-tagged holoenzyme to a metal-affinity column at neutral pH and washing the column with deionized water. Under these conditions the flavin remains bound and the defolated enzyme can be released from the column with 0.5 M imidazole pH 7.2. The MTHF-free protein was still capable of DNA repair, showing 70% activity of native enzyme. Fluorescence polarization experiments confirmed that MTHF binding is weakened at low ionic strength. Apo-photolyase was obtained by treating the His-tagged holoenzyme with 0.5 M imidazole pH 10.0. The apo-photolyase thus obtained was highly reconstitutable and bound nearly stoichiometric amounts of FAD(ox). Photolyase reconstituted with FAD(ox) had about 34% activity of native enzyme, which increased to 83% when FAD(ox) was reduced to FADH(-). Reconstitution kinetics performed at 20 degrees C showed that apo-photolyase associates with FADH(-) much faster (k(obs) approximately 3,000 M(-1) s(-1)) than with FAD(ox) (k(obs)=16 [corrected] M(-1) s(-1)). The dissociation constant of the photolyase-FAD(ox) complex is about 2.3 microM and that of E-FADH(-) is not higher than 20 nM (pH 7.2).

Apoenzymes↗
PubMed2006

Heterotetrameric sarcosine oxidase: structure of a diflavin metalloenzyme at 1.85 A resolution.

The crystal structure of heterotetrameric sarcosine oxidase (TSOX) from Pseudomonas maltophilia has been determined at 1.85 A resolution. TSOX contains three coenzymes (FAD, FMN and NAD+), four different subunits (alpha, 103 kDa; beta, 44 kDa; gamma, 21 kDa; delta, 11 kDa) and catalyzes the oxidation of sarcosine (N-methylglycine) to yield hydrogen peroxide, glycine and formaldehyde. In the presence of tetrahydrofolate, the oxidation of sarcosine is coupled to the formation of 5,10-methylenetetrahydrofolate. The NAD+ and putative folate binding sites are located in the alpha-subunit. The FAD binding site is in the beta-subunit. FMN is bound at the interface of the alpha and beta-subunits. The FAD and FMN rings are separated by a short segment of the beta-subunit with the closest atoms located 7.4 A apart. Sulfite, an inhibitor of oxygen reduction, is bound at the FMN site. 2-Furoate, a competitive inhibitor with respect to sarcosine, is bound at the FAD site. The sarcosine dehydrogenase and 5,10-methylenetetrahydrofolate synthase sites are 35 A apart but connected by a large internal cavity (approximately 10,000 A3). An unexpected zinc ion, coordinated by three cysteine and one histidine side-chains, is bound to the delta-subunit. The N-terminal half of the alpha subunit of TSOX (alphaA) is closely similar to the FAD-binding domain of glutathione reductase but with NAD+ replacing FAD. The C-terminal half of the alpha subunit of TSOX (alphaB) is similar to the C-terminal half of dimethylglycine oxidase and the T-protein of the glycine cleavage system, proteins that bind tetrahydrofolate. The beta-subunit of TSOX is very similar to monomeric sarcosine oxidase. The gamma-subunit is similar to the C-terminal sub-domain of alpha-TSOX. The delta-subunit shows little similarity with any PDB entry. The alphaA domain/beta-subunit sub-structure of TSOX closely resembles the alphabeta dimer of L-proline dehydrogenase, a heteroctameric protein (alphabeta)4 that shows highest overall similarity to TSOX.

Binding Sites↗
PubMed2004

Neuronal RNA oxidation is a prominent feature of familial Alzheimer's disease.

An in situ approach was used to identify the oxidized RNA nucleoside 8-hydroxyguanosine (8OHG) in the frontal cortex of familial Alzheimer's disease (FAD) with a mutation in presenilin-1 (PS-1) or amyloid beta protein precursor (AbetaPP) gene (n = 13, age 47-81 years). Neurons with marked 8OHG immunoreaction in the cytoplasm were widely distributed in the superior/middle frontal gyrus of FAD. Relative intensity measurements of neuronal 8OHG immunoreactivity showed that there was a significant increase in FAD compared with controls (n = 15, age 59-81 years), while there was no difference in relative 8OHG between the PS-1 and the AbetaPP FAD. Interestingly, a presymptomatic case carrying a PS-1 mutation showed a considerable level of relative 8OHG, and the increased levels of neuronal 8OHG in FAD were more prominent in cases with a lower percentage area of Abeta42 burden. These results suggest that oxidative stress is an early event involved in the pathological cascade of FAD.

Aged↗
PubMed1998

Familial Alzheimer's disease genes in Japanese.

More than 40 missense mutations and a splice-site mutation in the presenilin 1 (PS-1) gene, two missense mutations of presenilin 2 (PS-2), and more than three missense mutations of amyloid precursor protein (APP) cosegregate with early onset familial Alzheimer's disease (FAD). In order to determine the incidence of mutations of these genes in Japanese patients, we screened 25 early onset FAD families, one late-onset FAD case, 33 early onset AD cases and five late-onset AD cases for mutations in the coding regions of the genes using SSCP analysis. Four different missense mutations of the PS-1 gene, including a novel mutation, Glu273Ala, were identified in five early onset FAD families and one missense mutation of PS-1 in one isolated AD patient. While no missense mutations of PS-2 were detected, four silent nucleotide substitutions were observed. Our data indicate that PS-1 mutations account for 20.0% of early onset FAD cases in Japan. Since mutations in PS-2 and APP genes were not found in the remaining cases, which could be explained only partially by apolipoprotein E epsilon4, important FAD genes or risk-factor genes remain to be identified.

Adult↗
PubMedDate not supplied

Genetic risk factors in Japanese Alzheimer's disease patients: alpha1-ACT, VLDLR, and ApoE.

We studied the polymorphism of alpha1-antichymotrypsin (ACT), very low density lipoprotein receptor (VLDLR) and apolipoprotein E (ApoE) genes in 200 control subjects and 65 patients with Alzheimer's disease (AD) in Japanese. The subjects consisted of 30 patients with early onset familial Alzheimer's disease (FAD), a patient with late onset FAD, 29 patients with an early onset isolated form of AD, and 5 patients with late onset AD. ApoE genotypes were significantly different between controls and FAD (p < 0.0005) or AD (p < 0.05), and patients carrying at least one ApoE epsilon4 allele were found in 44% of FAD and 34.3% of AD; both were significantly different (p < 0.001) from the controls (12.5%). ACT genotypes and allele frequencies were not different among these groups except for genotypes between ApoE epsilon4 FAD and ApoE epsilon4 controls (p = 0.019). There was a slight but significant increase of the 5 repeat allele of VLDLR in AD (p = 0.014), but the difference was rather diminished in the presence of an ApoE epsilon4 allele. None of combinations of ACT and VLDLR genotypes in the presence or absence of an ApoE epsilon4 allele gave significant difference. Thus, we conclude that among the reported genetic risk factors, ApoE epsilon4 is the only definite risk factor for both FAD and AD, and the VLDLR polymorphism might be associated with AD cases in Japanese.

Alleles↗
PubMed1999

Antimutagenic effects and possible mechanisms of action of vitamins and related compounds against genotoxic heterocyclic amines from cooked food.

Possible antimutagenic activity of 26 vitamins and related compounds - ascorbic acid, beta-carotene, cyanocobalamin, folic acid, nicotinic acid, nicotinamide, pantothenic acid, pyridoxale, pyridoxamine, pyridoxine, retinal, retinol, retinoic acid, retinyl acetate, retinyl palmitate, riboflavin, riboflavin 5'-phosphate, flavin adenine dinucleotide (FAD), alpha-tocopherol, alpha-tocopherol acetate, vitamins K(1), K(3), K(4), 1, 4-naphthoquinone, and coenzyme Q(10) - was tested against six heterocyclic amine (HCA) mutagens, i.e., 2-amino-3-methyl-imidazo[4, 5-f]quinoline (IQ), 2-amino-3,4-dimethyl-imidazo[4,5-f]quinoline (MeIQ), 2-amino-3,8-dimethyl-imidazo[4,5-f]quinoxaline (MeIQx), 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), 2-amino-6-methyl-dipyrido[1,2-a:3',2'-d]imidazole (Glu-P-1) and 3-amino-1-methyl-5H-pyrido[4,3-b]indole (Trp-P-2) in the Salmonella/reversion assay using tester strains Salmonella typhimurium TA 98 and TA 100. Retinol, retinal, riboflavin, riboflavin 5'-phosphate, FAD, vitamins K(1), K(3), K(4), 1, 4-naphthoquinone, and coenzyme Q(10) caused a concentration-dependent decrease in the mutagenicity of all six mutagens in both tester strains. Quantification of antimutagenic potencies by calculating ID(50)1000; vitamin K(1): 401-740; vitamin K(3) (menadione): 85-590; vitamin K(4): 45-313; 1,4-naphthoquinone: 170-290; coenzyme Q(10): 490-860. In general, there were no major differences between HCAs tested except in part with Trp-P-2 nor between the two tester strains. In enzyme kinetic experiments with Salmonella, retinol, vitamins K(3), and K(4) behaved as competitive inhibitors of IQ induced mutagenesis. However, at the highest concentration of menadione (200 nmol/plate) and of riboflavin 5'-phosphate (2000 nmol/plate), non-competitive inhibition was observed. At other concentrations of riboflavin 5'-phosphate and at all concentrations of FAD, meaningful interpretation of enzyme kinetics were not possible. Reduction of the activity of 7-ethoxy- and 7-methoxyresorufin-O-dealkylases with IC(50) values of 2.03-30.8 microM indicated strong inhibition of 1A1 and 1A2 dependent monooxygenases by menadione and retinol. Riboflavin 5'-phosphate and FAD were less effective (IC(50): 110-803.7 microM). Nicotinamide-adenine-dinucleotidephosphate (NADPH) cytochrome P-450 reductase was not affected by retinoids but stimulated by naphthoquinones and both riboflavin derivatives up to about 50 and 80%, respectively. Again, the mutagenic activity of N-hydroxy-2-amino-3-methyl-imidazo[4,5-f]quinoline (N-OH-IQ) in Salmonella was not suppressed by K-vitamins but marginally reduced by retinol, retinal, and FAD but distinctly by riboflavin 5'-phosphate. In various experiments designed for modulation of the mutagenic response, inhibition of metabolic activation of IQ to N-OH-IQ was found to be the only relevant mechanism of antimutagenesis of menadione while a weak contribution of an other way seemed possible for retinol and FAD.

Animals↗
PubMed2001

Fluorescence correlation spectroscopy of flavins and flavoenzymes: photochemical and photophysical aspects.

Fluorescence Correlation Spectroscopy (FCS) was used to investigate the excited-state properties of flavins and flavoproteins in solution at the single molecule level. Flavin mononucleotide (FMN), flavin adenine dinucleotide (FAD) and lipoamide dehydrogenase served as model systems in which the flavin cofactor is either free in solution (FMN, FAD) or enclosed in a protein environment as prosthetic group (lipoamide dehydrogenase). Parameters such as excitation light intensity, detection time and chromophore concentration were varied in order to optimize the autocorrelation traces. Only in experiments with very low light intensity ( < 10 kW/cm2), FMN and FAD displayed fluorescence properties equivalent to those found with conventional fluorescence detection methods. Due to the high triplet quantum yield of FMN, the system very soon starts to build up a population of non-fluorescent molecules, which is reflected in an apparent particle number far too low for the concentration used. Intramolecular photoreduction and subsequent photobleaching may well explain these observations. The effect of photoreduction was clearly shown by titration of FMN with ascorbic acid. While titration of FMN with the quenching agent potassium iodide at higher concentrations ( > 50 mM of I-) resulted in quenched flavin fluorescence as expected, low concentrations of potassium iodide led to a net enhancement of the de-excitation rate from the triplet state, thereby improving the fluorescence signal. FCS experiments on FAD exhibited an improved photostability of FAD as compared to FMN: As a result of stacking of the adenine and flavin moieties, FAD has a considerably lower triplet quantum yield. Correlation curves of lipoamide dehydrogenase yielded correct values for the diffusion time and number of molecules at low excitation intensities. However, experiments at higher light intensities revealed a process which can be explained by photophysical relaxation or photochemical destruction of the enzyme. As the time constant of the process induced at higher light intensities resembles the diffusion time constant of free flavin, photodestruction with the concomitant release of the cofactor offers a reasonable explanation.

Ascorbic Acid↗
PubMed1999

Kinetics and thermodynamics of free flavins and the flavin-based redox active site within glucose oxidase dissolved in solution or sequestered within a sol-gel-derived glass.

We report on the steady-state and time-resolved fluorescence from the redox active site flavine adenine dinucleotides (FADs) that are bound to glucose oxidase (GOx) when this enzyme is dissolved in aqueous solution or sequestered within a sol-gel-derived glass. To the best of our knowledge, this represents the first report on the actual dynamics of an enzyme active site when the enzyme is part of a sol-gel-derived glass. The results from these experiments show that the "free" FAD intramolecular folding/unfolding kinetics are slowed 3-10-fold within the glass vs solution. The intramolecular exciplex formation event (i.e., excited-state FAD residue folding/unfolding) is completely arrested for the GOx-bound FAD if the enzyme is sequestered within a glass in the absence of glucose. This is significantly different from the behavior of GOx dissolved in solution. However, despite this difference in behavior, the GOx molecules that are sequestered within the glasses continue to function somewhat like GOx dissolved in aqueous solution if they are challenged with glucose. We also found that the GOx molecules do not leach from the glass and they exhibit rotational mobility that is only 2-fold less than GOx dissolved in aqueous solution at 20 degrees C. In aqueous solution or within these glasses, the enzyme pocket that hosts the FAD redox sites opens up by 25-30% when GOx is challenged with glucose. Finally, we present preliminary analytical results for film-based sol-gel-derived biosensors that contain GOx, L-amino acid oxidase or cholesterol oxidase wherein the intrinsic FAD fluorescence produces the analytical signal.

Binding Sites↗
PubMed1995

Characterization of lipoamide dehydrogenase from Escherichia coli lacking the redox active disulfide: C44S and C49S.

Lipoamide dehydrogenase from Escherichia coli, a dimeric flavoprotein in the pyridine nucleotide-disulfide oxidoreductase family of enzymes, catalyzes the reduction of NAD+ by dihydrolipoamide. The two electrons are transferred via a redox active disulfide and FAD. Cys44 and Cys49 comprise the redox active disulfide, Cys44 interchanging with dihydrolipoamide and Cys49 interacting with the flavin. Each of these residues has been mutated to serine (C44S, C49S). The altered enzymes showed minute amounts of activity, 0.003% for C44S and 0.012% for C49S using the physiological substrates dihydrolipoamide and NAD+. These very low activities were expected, since the disulfide was no longer present in C44S and C49S, making dithiol-disulfide interchange impossible. However, the enzymes were capable of catalyzing reactions using NADH as the electron donor and alternate electron acceptors: K3Fe(CN)6, thio-NAD+, DCIP, and O2. These activities with NADH indicated that interaction of C44S and C49S with pyridine nucleotides was not affected greatly by the mutation. The pH dependence of the charge-transfer absorbance of C44S gives pKa values of 2.7, associated with titration of Cys49, and 9.5, associated with titration of the acid-base catalyst, His444'. A pKa of 5.1 was estimated for Cys44 in C49S from the pH dependence of its reactivity with methyl methanethiosulfonate. The fluorescence of the FAD in oxidized wild type lipoamide dehydrogenase is markedly temperature dependent, while the remaining fluorescence of two-electron-reduced enzyme is independent of temperature. The fluorescence of the FAD in C44S and in C49S is likewise independent of temperature. The FAD of C44S and C49S is stoichiometrically titrated by 1 equiv of sodium dithionite. However, the FAD of C44S is markedly less completely reduced by 1 equiv of NADH than is the FAD of C49S. Ferricyanide stoichiometrically reoxidizes the FADH2 of both altered forms of the enzyme.

Cysteine↗
PubMed1995

Discovery of a third coenzyme in sarcosine oxidase.

Denaturation of recombinant sarcosine oxidase or the natural enzyme isolated from Corynebacterium sp. P-1 with guanidine hydrochloride releases noncovalently bound FAD and a second UV-absorbing component (peak 2) which comigrates with NAD+ during reversed-phase HPLC. Both FAD and peak 2 are also found in extracts prepared by incubating sarcosine oxidase at 37 degrees C for 30 min, a procedure which causes partial (approximately 50%) release of the enzyme's noncovalently bound FAD. Peak 2 in the 37 degrees C extract is heat labile and decomposes upon boiling for 5 min at pH 8.0. A similar instability was observed with NAD+. Reaction of the 37 degrees C extract from sarcosine oxidase with phosphodiesterase yields nicotinamide mononucleotide, AMP, and FMN, as expected for a mixture containing NAD+ and FAD. Peak 2 was converted to NADH upon reaction of the 37 degrees C extract with yeast alcohol dehydrogenase in the presence of ethanol. Guanidine hydrochloride extracts, prepared from recombinant or natural enzyme, contain 1 mol of NAD+/mol of FAD. Since sarcosine oxidase contains 1 mol of noncovalently bound FAD, the results show that the enzyme also contains 1 mol of NAD+. The NAD+ is tightly bound and is not lost during enzyme purification. It is not susceptible toward hydrolysis by NADase, reduction by alcohol dehydrogenase, or nucleophilic attack by cyanide. Unlike the flavins in sarcosine oxidase, NAD+ is not reduced by sarcosine and is not in redox equilibrium with the flavins.

Chromatography, High Pressure Liquid↗
PubMed1991

Electronic energy transfer and fluorescence quenching in the active sites of mercuric reductase.

The FAD-containing enzyme mercuric reductase has been studied by means of steady-state and time-resolved fluorescence spectroscopy. The fluorescence relaxation of the excited state of the isoalloxazine ring of FAD can be described by a sum of two exponential functions. The two lifetimes are not due to a different lifetime of each of the two FAD molecules of mercuric reductase. The FAD molecules are quenched dynamically by a quencher that is not sensitive to the solvent viscosity. In vitro activation induces a dynamic quenching of fluorescence, while upon binding of NADP+ the FAD molecules are both statically and dynamically quenched. Time-resolved fluorescence anisotropy experiments of mercuric reductase in water show that the isoalloxazine ring probably undergoes a rapid and restricted vibrational motion of small amplitude. Electronic energy transfer occurs between the two FAD molecules at a rate of about 3.4 x 10(7) s-1. The angle between the emission transition dipole of the donor and the absorption transition dipole of the acceptor is 137 +/- 2 degrees (or 43 +/- 2 degrees). From previous X-ray data of glutathione reductase we find that the corresponding angle is 160 degrees. This suggests that the isoalloxazine rings of mercuric reductase and glutathione reductase are mutually tilted in slightly different ways.

Binding Sites↗
PubMed1991

Kinetic comparison of reduction and intramolecular electron transfer in milk xanthine oxidase and chicken liver xanthine dehydrogenase by laser flash photolysis.

A comparative study using laser flash photolysis of the kinetics of reduction and intramolecular electron transfer among the redox centers of chicken liver xanthine dehydrogenase and of bovine milk xanthine oxidase is described. The photogenerated reductant, 5-deazariboflavin semiquinone, reacts with the dehydrogenase (presumably at the Mo center) in a second-order manner, with a rate constant (k = 6 x 10(7) M-1 s-1) similar to that observed with the oxidase [k = 3 x 10(7) M-1 s-1; Bhattacharyya et al. (1983) Biochemistry 22, 5270-5279]. In the case of the dehydrogenase, neutral FAD radical formation is found to occur by intramolecular electron transfer (kobs = 1600 s-1), presumably from the Mo center, whereas with the oxidase the flavin radical forms via a bimolecular process involving direct reduction by the deazaflavin semiquinone (k = 2 x 10(8) M-1 s-1). Biphasic rates of Fe/S center reduction are observed with both enzymes, which are due to intramolecular electron transfer (kobs approximately 100 s-1 and kobs = 8-11 s-1). Intramolecular oxidation of the FAD radical in each enzyme occurs with a rate constant comparable to that of the rapid phase of Fe/S center reduction. The methylviologen radical, generated by the reaction of the oxidized viologen with 5-deazariboflavin semiquinone, reacts with both the dehydrogenase and the oxidase in a second-order manner (k = 7 x 10(5) M-1 s-1 and 4 x 10(6) M-1 s-1, respectively). Alkylation of the FAD centers results in substantial alterations in the kinetics of the reaction of the viologen radical with the oxidase but not with the dehydrogenase. These results suggest that the viologen radical reacts directly with the FAD center in the oxidase but not in the dehydrogenase, as is the case with the deazaflavin radical. The data support the conclusion that the environments of the FAD centers differ in the two enzymes, which is in accord with other studies addressing this problem from a different perspective [Massey et al. (1989) J. Biol. Chem. 264, 10567-10573]. In contrast, the rate constants for intramolecular electron transfer among the Mo, FAD, and Fe/S centers in the two enzymes (where they can be determined) are quite similar.

Animals↗
PubMed1982

Reduction, oxidation, and addition reactions between free radicals and flavins.

Flavins and reduced flavins were reacted with a variety of free radicals produced in dilute aqueous solution at pH 7 +/- 0.1 by radiation chemical methods. The radical .CH2C(CH3)2OH and the aliphatic beta, gamma, and delta radicals of ethanol, 2-propanol, and 1-butanol added to the radical form of flavin adenine dinucleotide (FAD) (FH.) to yield products that could not be reoxidized to flavin by oxygen. The first radical also added to FAD but with a much lower efficiency. In contrast, the alpha-carbon radicals .CH(OH)CH2OH, CH3(-3) CHOH, and (CH3)2COH appeared to undergo two reactions: FH. + RR'COH leads to FH- + RR'C==O FH. + RR'COH leads to FH- + RR'C==O + H+ The formate radical anion .CO2(-) reacted similarly, producing stoichiometric two-electron reduction of riboflavin and lumiflavin as well as FAD. While eaq- also seemed capable of this, it was found to react irreversibly with FADH2, which makes it a poor reagent for producing the dihydroflavin. The dihydro form of FAD was reoxidized to FAD by the species RS. and .BR2(-). In contrast to FAD, the alloxazine lumichrome underwent only one-electron reduction and oxidation by .CO2(-) and .Br2(-), respectively.

Alcohols↗
PubMed1981

Studies on the microsomal mixed-function oxidase system: mechanism of action of hepatic NADPH-cytochrome P-450 reductase.

The mechanism of hepatic NADPH-cytochrome P-450 reductase has been investigated by using a stopped-flow technique. The reduction of the oxidized native enzyme (FAD-FMN) by NADPH proceeds by both one-electron equivalent and two-electron eqiuvalent mechanisms. The air-stable semiquinone form (FAD-FMNH.) of the native enzyme, which is characterized by an absorption shoulder at 635 nm, is also rapidly reduced to another semiquinone form (FADH-FMNH2) by NADPH with the disappearance of the shoulder at 635 nm, but the absorbance change at 585 nm is relatively constant. The FAD moiety in the FMN-depleted enzyme is rapidly reduced by NADPH, and reduced FAD is oxidized in successive one-electron steps by O2 or potassium ferricyanide. These results indicate the possibility of intra-molecular one-electron transfer between FAD and FMN. The rate of cytochrome P-450 reduction decreases in the presence of FMN-depleted enzyme but is nearly restored to the value of the original enzyme with FMN-reconstituted enzyme. These data suggest that FAD is the low-potential flavin, which serves as an electron acceptor from NADPH. On the other hand, FMN, which is the high-potential flavin, appears to participate as an electron carrier in the process of electron transfer from NADPH to cytochrome P-450 during the mixed-function catalytic cycle.

Animals↗