Monodehydroascorbate reductase from cucumber is a flavin adenine dinucleotide enzyme

Monodehydroascorbate reductase (EC 1.6.5.4) was purified from cucumber fruit to a homogeneous state as judged by polyacrylamide gel electrophoresis. The cucumber monodehydroascorbate reductase was a monomer with a molecular weight of 47,000. It contained 1 mol of FAD/mol of enzyme which was reduced by NAD(P)H and reoxidized by monodehydroascorbate. The enzyme had an exposed thiol group whose blockage with thiol reagents inhibited the electron transfer from NAD(P)H to the enzyme FAD. Both NADH and NADPH served as electron donors with Km values of 4.6 and 23 microM, respectively, and Vmax of 200 mol of NADH and 150 mol of NADPH oxidized mol of enzyme-1 s-1. The Km for monodehydroascorbate was 1.4 microM. The amino acid composition of the enzyme is presented. In addition to monodehydroascorbate, the enzyme catalyzed the reduction of ferricyanide and 2,6-dichloroindophenol but showed little reactivity with calf liver cytochrome b5 and horse heart cytochrome c. The kinetic data suggested a ping-pong mechanism for the monodehydroascorbate reductase-catalyzed reaction. Cucumber monodehydroascorbate reductase occurs in soluble form and can be distinguished from NADPH dehydrogenase, NADH dehydrogenase, DT diaphorase, microsome-bound NADH-cytochrome b5 reductase, and NADPH-cytochrome c reductase by its molecular weight, amino acid composition, and specificity of electron acceptors and donors.     

The reduced nicotinamide adenine dinucleotide "oxidase" of Acholeplasma laidlawii membranes.

An NADH dehydrogenase possessing a specific activity 3-5 times that of membrane-bound enzyme was obtained by extraction of Acholeplasma laidlawii membranes with 9.0% ethanol at 43 degrees C. This dehydrogenase contained only trace amounts of iron (suggesting an uncoupled respiration), a flavin ratio of 1:2 FAD to FMN and 30-40% lipid. Its resistance to sedimentation is probably due to the high flotation density of the lipids. It efficiently utilized ferricyanide, menadione and dichlorophenol indophenol as electron acceptors, but not O2, ubiquinone Q10 or cytochrome c. Lineweaver-Burk plots of the dehydrogenase were altered to linear functions upon extraction with 9.0% ethanol. A secondary site of ferricyanide reduction could not be explained by the presence of cytochromes, which these membranes lack. In comparison to other respiratory chain-linked NADH dehydrogenases in cytochrome-containing respiratory chains, this dehydrogenase was characterized by similar Km's with ferricyanide, dichlorophenol indophenol, menadione as electron acceptors, but considerably smaller V's with ferricyanide, dichlorophenol indophenol, menadione as electron acceptors, and smaller specific activities. It was not stimulated or reactivated by the addition of FAD, FMN, Mg2+, cysteine or membrane lipids, and was less sensitive to respiratory inhibitors than unextracted enzyme. The ineffectiveness of ADP stimulation on O2 uptake, the insensitivity to oligomycin and the very low iron content of A. laidlawii membranes were considered in relation to conservation of energy by these cells. Some kinetic properties of the dehydrogenation, the uniquely high glycolipid content and apparently uncoupled respiration at Site I were noteworthy characteristics of this NADH dehydrogenase from the truncated respiratory chain of A. laidlawii.     

The purification and properties of the respiratory-chain reduced nicotinamide–adenine dinucleotide dehydrogenase of Torulopsis utilis

1. An NADH-ferricyanide reductase activity has been isolated from the respiratory chain of Torulopsis utilis by using detergents. The isolated enzyme contains non-haem iron, acid-labile sulphide and FMN in the molar proportions 27.5:28.4:1. The preparation is free of FAD and largely free of cytochrome. 2. The enzyme catalyses ferricyanide reduction by NADPH at about 1% of the rate with NADH, and reacts poorly with acceptors other than ferricyanide. The rates of reduction of some acceptors are, as percentages of the rate with ferricyanide: menadione, 0.35%; lipoate, 0.01%; cytochrome c, 0.065%; dichlorophenolindophenol, 0.35%; ubiquinone-1, 0.08%. 3. Several properties of submitochondrial particles of T. utilis (non-haem iron, acid-labile sulphide, FMN and an NADH-reducible electron-paramagnetic-resonance signal) were found to co-purify with the NADH-ferricyanide reductase activity. Thus about 70% of the FMN and, within the limits of accuracy of the experiments, 100% of the non-haem iron and acid-labile sulphide of submitochondrial particles derived from T. utilis cells grown under conditions of glycerol limitation (but relatively low iron availability) can be attributed to the NADH-ferricyanide reductase. 4. It was also shown that the component of submitochondrial particles specifically bleached at 460nm by NADH [species 1 of Ragan & Garland (1971)] co-purifies with the NADH-ferricyanide reductase. 5. This successful purification of an NADH dehydrogenase from T. utilis forms a starting point for investigating the molecular properties of phenotypically modified mitochondrial NADH oxidation pathways that lack energy conservation between NADH and the cytochromes.     

Chaotropic resolution of high molecular weight (type I) NADH dehydrogenase, and reassociation of flavin-rich (type II) and flavin-poor subunits.

1. Type-I NADH dehydrogenase (Complex I) was solubilized and dissociated into subunits by NaClO4. NADH slows the dissociation. On subsequent stepwise addition of (NH4)2SO4 the dissociation is partly reversed, as is to be expected from the opposing effects of ClO-4 and SO-24, which are on the salting-in and salting-out sides, respectively, of the lyotropic series. 2. In consequence, the aggregates of subunits that are separated by (NH4)2-SO4 fractionation consist of randomly associated subunits as well as fragments of Type I enzyme. The fraction precipitating at 27% satd. (NH4)2SO4 is flavin-poor, that remaining soluble at 55% satd. (NH4)2SO4 flavin-rich and those separating between 27 and 55% satd. (NH4)2SO4 intermediate in composition. 3. The fraction remaining soluble at 55% satd. (NH4)2SO4 contains the purified low-molecular-weight iron-sulphur flavoprotein (Type-II dehydrogenase). It is a dimer consisting of one molecule of FMN, one 28-kilodalton and one 56-kilodalton subunit per protomer. Work of others indicates that it contains 4 Fe and 4 acid-labile S atoms per molecule of FMN. Sometimes the fraction remaining soluble at 55% satd. (NH4)2SO4 contained an additional small subunit (12 kilodaltons) and four additional Fe and acid labile S atoms per protomer. The sedimentation coefficients (s020,w) of the two preparations were 5.3 and 6.6 S, respectively, with calculated frictional ratios of 1.5 and 1.24, respectively. 4. The intermediate fractions are mixtures of the various subunits present in Complex I. Specifically a fraction separating at 55% satd. (NH4)2SO4 was found to be a mixture of two fragments, the pure iron-sulphur flavoprotein and a 26-S fragment that contained per protomer four subunits of 12 kilodaltons, one each of 28, 32, 56 and 77 kilodaltons, one molecule of FMN and 20 Fe and acid-labile S atoms. It was probably tetrameric or even larger. 5. The oxidoreductase activity of the intermediate fractions is dependent on the protein concentration, the activity with ferricyanide increasing and that with ferricytochrome c decreasing with increasing protein concentration. This is interpreted as an increased association of subunits present in the intermediate fractions. Similar results are obtained when flavin-rich and flavin-poor fractions are mixed. The association is cooperative. NADH favours the association of the subunits. 6. Association of the subunits is accompanied by a 10-fold increase in k2 (rate constant for intramolecular electron flow), a 10-fold decrease of the accessibility of ferricyanide to the reduced enzyme and a 10(4)-fold decrease of the accessibility of ferricytochrome c. The Ks (NADH) is also decreased. Although the changes are in the direction to be expected from a conversion of Type II enzyme to Type I, the value of k2 is still much less than in the latter enzyme.     

Formate dehydrogenase from Pseudomonas oxalaticus

Formate dehydrogenase (EC 1.2.1.2) from Pseudomonas oxalaticus has been isolated and characterized. The enzyme (molecular weight 315000) is a complex flavoprotein containing 2 FMN, 18--25 non-heme iron atoms and 15--20 acid-labile sulphides. In the last step of the purification, a sucrose gradient centrifugation, a second catalytically active species has been found apparently originating from a dissociation of the enzyme into two equal subunits. The enzyme is specific toward its natural substrate formate. It transfers electrons to NAD+, oxygen, ferricyanide, and a lot of nonphysiological acceptors (dyes). In addition electrons are transferred from NADH to these acceptors. The (reversible) removal of FMN requires a reduction step. Reincorporation has been followed by the reappearance of the reactivity against formate and by fluorescence titration. The deflavo enzyme also binds FAD and riboflavin. The resulting enzyme species show characteristic catalytic abilities. Activity against formate is peculiar to the FMN species.     

The NADH dehydrogenase of the respiratory chain of Escherichia coli. II. Kinetics of the purified enzyme and the effects of antibodies elicited against it on membrane-bound and free enzyme.

The purified respiratory chain NADH dehydrogenase of Escherichia coli oxidizes NADH with either dichlorophenolindophenol (DCIP). ferricyanide, or menadione as electron acceptors, with values for NADH are similar with the three electron acceptors (approximately 50 muM). The purified enzyme contains no flavin and has an absolute requirement for FAD, with Km values around 4 muM. The pH optimum of the enzyme appears to be between 6.5 and 7; the optimum is difficult to establish because of nonenzymatic reduction of DCIP at the lower pH values. Potassium cyanide stimulates the DCIP reductase activity about 2-fold, but has no effect on ferricyanide reductase. The enzyme exhibits hyperbolic kinetics with respect to NADH concentration in both the ferricyanide and DCIP reductase assays, but cooperatively is seen in the menadione reductase reaction. NAD+ is an effective competitive inhibitor of the reaction (Ki congruent to 20 muM); in the presence of NAD+, the NADH saturation curve becomes cooperative, even in the DCIP reductase assay. Many adenine containing nucleotides are competitive inhibitors of the enzyme. The apparent Ki values for these nucleotides as inhibitors of the purified enzyme, the membrane-bound NADH dehydrogenase, and the NADH oxidase are equivalent. An examination of inhibitory effects of a series of adenine nucleotides suggests that the inhibitors act as analogues of NAD+, which is the true physiological inhibitor. The results suggest that the enzyme in situ is always partially inhibited by the levels of NAD- in the E coli cell, and thus behaves in a cooperative fashion to changes in the NAD+/NADH ratio. An antibody has been elicited against the purified NADH dehydrogenase. Immunodiffusion and crossed immunoelectrophoresis show that the antibody is directed principally against the NADH dehydrogenase, with some activity against minor contaminants in the purified preparation. The antibody inhibits NADH dehydrogenase activity 50% at saturating levels. When this antibody preparation is used to examine solubilized membrane preparations, two major immunoprecipitates are found. A parallel inhibition of the membrane-bound NADH dehydrogenase and NADH oxidase activities is seen, supporting the hypothesis that the purified enzyme is indeed a component of the respiratory chain-dependent NADH oxidase pathway.     

A soluble NADH dehydrogenase (NADH: ferricyanide oxidoreductase) from Thermus aquaticus strain T351.

A soluble NADH dehydrogenase (NADH:ferricyanide oxidoreductase) has been obtained by simple disruption of cells of Thermus aquaticus strain T351, and purified. The enzyme is of low molecular mass, 50 000 Da, and displays many of the properties of the membrane-bound enzyme, including inhibition by both NADH and ferricyanide, and the same Km for ferricyanide. The enzyme contains 0.05 mol of FMN, 0.16 mol of labile sulphur and 2.2 mol of iron per mol of protein. The enzyme is inhibited by NAD and cupferron competitively with ferricyanide, and by ATP (but not ADP) competitively with NADH. The enzyme is particularly thermostable, having a half-life at 95 degrees C of 35 min. The effect of temperature on the molar absorption coefficient and the stability of NADH was determined.     

Reduction of 2,4,6-trinitrobenzenesulfonate by glutathione reductase and the effect of NADP+ on the electron transfer

Glutathione reductase has been found to catalyze an NAD(P)H-dependent electron transfer to 2,4,6-trinitrobenzenesulfonate (TNBS). In the presence of oxygen TNBS is not consumed in the reaction, but is rapidly reoxidized with concomitant production of hydrogen peroxide. Cytochrome c can replace oxygen as the final electron acceptor, indicating that a one-electron transfer takes place. The rate is slightly higher in the absence than in the presence of oxygen, ruling out superoxide anion as an obligatory intermediate in cytochrome c reduction. In the absence of oxygen (or cytochrome c), TNBS limits the reaction and accepts a total of four electrons. The TNBS-dependent NADPH (or NADH) oxidation is markedly stimulated by NADP+, and to a smaller extent also by NAD+. The TNBS-dependent reactions are inhibited by excess of NADPH but not by NADH. The kinetics of these reactions are consistent with a branching reaction mechanism in which a pathway including a ternary complex between the two-electron reduced enzyme and NADP+ has the highest turnover. NADPH-dependent reductions of ferricyanide or 2,6-dichloroindophenol catalyzed by glutathione reductase are also markedly influenced by NADP+. Evidently NADP+ facilitates a shift of the catalyzed reaction from the normal two-electron reduction of glutathione disulfide to a more unspecific one-electron reduction of other acceptors. Spectral as well as kinetic data suggest that the rate of radical formation limits the reactions with the artificial electron acceptors and that NADP+ promotes this rate-limiting step.     

Studies of the control of luminescence in Beneckea harveyi: properties of the NADH and NADPH:FMN oxidoreductases.

Highly purified NADH and NADPH:FMN oxidoreductases from Beneckea harveyi have been characterized with regard to kinetic parameters, association with luciferase, activity with artificial electron acceptors, and the effects of inhibitors. The NADH:FMN oxidoreductase exhibits single displacement kinetics while the NADPH:FMN oxidoreductase exhibits double displacement or ping-pong kinetics. This is consistent with the formation of a reduced enzyme as an intermediate in the reaction of catalyzed by the NADPH:FMN oxidoreductase. Coupling of either of the oxidoreductases to the luciferase reaction decreases the apparent Kms for NADH, NADPH, and FMN, supporting the suggestion of a complex between the oxidoreductases and luciferase. The soluble oxidoreductases are more efficient in producing light with luciferase than is a NADH dehydrogenase preparation obtained from the membranes of these bacteria. The soluble enzymes use either FMN or FAD as substrates for the oxidation of reduced pyridine nucleotides while the membrane NADH dehydrogenase is much more active with artificial electron acceptors such as ferricyanide and methylene blue. FMN and FAD are very poor acceptors. The evidence indicates that neither of the soluble oxidoreductases is derived from the membranes. Both enzymes are constitutive and do not depend on the synthesis of luciferase.     

Effect of calcium ions and inhibitors on internal NAD(P)H dehydrogenases in plant mitochondria.

Both the external oxidation of NADH and NADPH in intact potato (Solanum tuberosum L. cv. Bintje) tuber mitochondria and the rotenone-insensitive internal oxidation of NADPH by inside-out submitochondrial particles were dependent on Ca2+. The stimulation was not due to increased permeability of the inner mitochondrial membrane. Neither the membrane potential nor the latencies of NAD(+)-dependent and NADP(+)-dependent malate dehydrogenases were affected by the addition of Ca2+. The pH dependence and kinetics of Ca(2+)-dependent NADPH oxidation by inside-out submitochondrial particles were studied using three different electron acceptors: O2, duroquinone and ferricyanide. Ca2+ increased the activity with all acceptors with a maximum at neutral pH and an additional minor peak at pH 5.8 with O2 and duroquinone. Without Ca2+, the activity was maximal around pH 6. The Km for NADPH was decreased fourfold with ferricyanide and duroquinone, and twofold with O2 as acceptor, upon addition of Ca2+. The Vmax was not changed with ferricyanide as acceptor, but increased twofold with both duroquinone and O2. Half-maximal stimulation of the NADPH oxidation was found at 3 microM free Ca2+ with both O2 and duroquinone as acceptors. This is the first report of a membrane-bound enzyme inside the inner mitochondrial membrane which is directly dependent on micromolar concentrations of Ca2+. Mersalyl and dicumarol, two potent inhibitors of the external NADH dehydrogenase in plant mitochondria, were found to inhibit internal rotenone-insensitive NAD(P)H oxidation, at the same concentrations and in manners very similar to their effects on the external NAD(P)H oxidation.     

Diaphorases from Aerobacter aerogenes

Bernofsky, Carl (The University of Kansas, Kansas City), and Russell C. Mills. Diaphorases from Aerobacter aerogenes. J. Bacteriol. 92:1404-1414. 1966.-Five enzymes which catalyze the reduction of 2,6-dichlorophenol-indophenol by reduced nicotinamide adenine dinucleotide (NADH(2)) have been separated from sonic extracts of Aerobacter aerogenes B199 by diethylaminoethyl (DEAE) cellulose chromatography. Three major chromatographic fractions (enzymes I, II, and III) account for most of the activity in the extract. Of the two minor fractions, one is associated with cytochrome b(1). The other is extremely labile, and was not studied further. The chromatographed diaphorases appear to have a specific requirement for flavin mononucleotide. They are also readily inactivated by dilution; however, this can be prevented by a combination of phosphate buffer, bovine serum albumin, and flavin mononucleotide. The different enzymes are clearly distinguishable by their activities with NADH(2) and reduced nicotinamide adenine dinucleotide phosphate (NADPH(2)) in the presence of various electron acceptors (2,6-dichlorophenol-indophenol, ferricyanide, menadione, and cytochrome c), and by their responses to inhibitors (amobarbital, antimycin A, Atabrine, p-chloromercuribenzenesulfonate, dicumarol, and 2,4-dinitrophenol). With 2,6-dichlorophenol-indophenol as acceptor, enzymes I, II, and III have comparable activities with either NADH(2) or NADPH(2). With menadione and ferricyanide as acceptors, enzymes II and III exhibit very high, NADH(2)-specific activities. When cytochrome c is the acceptor, however, enzyme III shows greater activity with NADPH(2) as the electron donor. Ferricyanide is the most active acceptor for the cytochrome b(1)-containing fraction. Coenzyme Q(6) does not appear to serve as an acceptor. All the diaphorases, with the exception of that in the cytochrome b(1)-containing fraction, are inhibited by p-chloromercuribenzenesulfonate. Amobarbital is relatively ineffective and inhibits only the indophenol reductase activity of enzyme I. The menadione reductase activity of enzymes I, and II, and the diaphorases in the cytochrome b(1)-containing fraction are strongly inhibited by antimycin A, 2,4-dinitrophenol, dicumarol, and Atabrine. However, the menadione reductase activity of enzyme III is affected only by the last three of these inhibitors. The diaphorases in sonic-treated extracts do not appear to be associated with a particulate fraction.     

NADH dehydrogenase from bovine neutrophil membranes. Purification and properties

A membrane-associated NADH dehydrogenase from beef neutrophils was purified to homogeneity, using detergent (cholate plus Triton X-100) extraction and chromatography on DEAE-Sepharose CL-6B, agarose-hexane-NAD, and hydroxylapatite. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed an apparent subunit molecular weight of 17,500, but the enzyme was highly aggregated (Mr greater than 450,000) in nondenaturing gels containing 0.1% Triton X-100. The protein band in nondenaturing gels was also stained for activity using NADH and nitro blue tetrazolium. The enzyme showed greatest electron acceptor activity with ferricyanide (100%), followed by cytochrome c (3.5%), dichloroindophenol (2.7%), and cytochrome b5 (0.34%). No activity was seen with oxygen. The Km values for NADH and ferricyanide were 18 and 9.5 microM, respectively, and NAD+ was a weak competitive inhibitor (Ki = 118 microM). No activity was seen with NADPH. No effects were seen with mitochondrial respiratory inhibitors such as azide, cyanide, or rotenone, but p-chloromercuribenzoate was strongly inhibitory and N-ethylmaleimide was weakly inhibitory. No free flavin was detectable in enzyme preparations. Based upon kinetic, physical, and inhibition properties, this NADH dehydrogenase differs from those previously described in microsomes and erythrocyte plasma membrane.     

Purification and some properties of NAD(P)H dehydrogenase from Saccharomyces cerevisiae: Contribution to glycolytic methylglyoxal pathway

NAD(P)H dehydrogenase was purified approximately 480-fold from Saccharomyces cerevisiae with 6.5% activity yield. The enzyme was homogeneous on polyacrylamide gel electrophoresis. The molecular weight of the enzyme was estimated to be 40,000–44,000 by gel filtration on Sephadex G-150 column chromatography and SDS-polyacrylamide gel electrophoresis. The K_m values for NADPH and NADH were 7.3 μM and 0.1 mM, respectively. The activity of the enzyme increased approximately 4-fold with Cu²⁺. FAD, FMN and cytochrome c were not effective as electron acceptors, although Fe(CN)₆³⁻ was slightly effective. NADH generated by the reaction of lactaldehyde dehydrogenase in the glycolytic methylglyoxal pathway will be reoxidized by NAD(P)H dehydrogenase. NAD(P)H dehydrogenase thus may contribute to the reduction/oxidation system in the glycolytic methylglyoxal pathway to maintain the flux of methylglyoxal to lactic acid via lactaldehyde.     

Inhibition of NADH-dehydrogenase by low concentrations of NAD+]

Low concentrations of NAD+ inhibit the NADH: acceptor reductase reactions catalyzed by soluble NADH dehydrogenase from bovine heart mitochondria. The degree of incomplete inhibition of the enzyme depends on the nature and concentration of artificial electron acceptors and is manifested only at low concentrations of the latter. Marked inhibition was demonstrated for the 2.6-dichlorophenolindophenol-, ferricyanide- and O2-reductase reactions, being weakly pronounced during the measurement of the NADH: cytochrome c reductase activity. The inhibition of the above reactions by oxidized NAD+ isn't competitive towards NADH. A kinetic scheme is proposed, which postulates NADH: acceptor reductase reactions occurrence via two mechanisms, namely, a ping-pong mechanism and oxidation of the product-enzyme complex by the acceptor. It was shown that low concentrations of NAD+ also inhibit the NADH oxidase reaction catalyzed by complex I.     

Properties and synthesis regulation of NAD(P)H dehydrogenases from Rhodobacter capsulatus

Three NAD(P)H dehydrogenases were found and purified from a soluble fraction of cells of the purple non-sulfur bacterium Rhodobacter capsulatus, strain B10. Molecular mass of NAD(P)H, NADPH and NADH dehydrogenases are 67 000 (4 · 18 000), 35 000 and 39 000, and the isoelectric points are 4.6, 4.3 and 4.5, respectively. NAD(P)H dehydrogenase is characterized by a higher sensitivity to quinacrine, NADPH dehydrogenase by its sensitivity to p-chloromercuribenzoate and NADH dehydrogenase by its sensitivity to sodium arsenite. In contrast to the other two enzymes, NAD(P)H dehydrogenase is capable of oxidizing NADPH as well as NADH, but the ratio of their oxidation rates depends on the pH. All NAD(P)H dehydrogenases reacted with ferricyanide, 2,6-dichlorophenolindophenol, benzoquinone and naphthoquinone, but did not exhibit transhydrogenase, reductase or oxidase activity. Moreover, NADH dehydrogenase was also capable of reducing FAD and FMN. NAD(P)H and NADH dehydrogenases possessed cytochrome-c reductase activity, which was stimulated by menadione and ubiquinone Q₁. The activity of NAD(P)H and NADH dehydrogenases depended on culture-growth conditions. The activity of NAD(P)H dehydrogenase from cells grown under chemoheterotrophic aerobic conditions was the lowest and it increased notably under photoheterotrophic anaerobic conditions upon lactate or malate growth limitation. The activity of NADH dehydrogenase was higher from the cells grown under photoheterotrophic anaerobic conditions upon nitrate growth limitation and under chemoheterotrophic aerobic conditions. NADPH dehydrogenase synthesis dependence on R. capsulatus growth conditions was insignificant.     

Studies on NADH(NADPH)-cytochrome c reductase (FMN-containing) from yeast: steady-state kinetic properties of the flavoenzyme from top-fermenting ale yeast

A study of the steady-state kinetics of NADH(NADPH)-cytochrome c reductase (FMN-containing) from ale yeast (M. S. Johnson and S. A. Kuby (1985) J. Biol. Chem. 260, 12341-12350) has led to a postulated three-substrate random-ordered hybrid mechanism, where NAD(P)H and FMN add randomly and very likely in a steady-state fashion, followed by an ordered addition of cytochrome c. Kinetic parameters have been derived from this mechanism. Arrhenius plots showed large differences between NADH and NADPH, as the substrate-reductant. Menadione accelerated cytochrome c reduction and also O2 uptake, but vitamin K1 and coenzyme Q10 were ineffective as electron mediators, possibly as a result of their insolubility. With NADPH as the substrate-reductant, the order of the rate of reduction of electron acceptors was ferricyanide greater than DCIP greater than cytochrome c greater than oxygen; with menadione, the specificity sequence was cytochrome c greater than ferricyanide greater than DCIP greater than oxygen. With NADH, the order was ferricyanide greater than cytochrome c greater than oxygen greater than DCIP, which changed to cytochrome c greater than ferricyanide greater than oxygen greater than DCIP on addition of menadione. Cytochrome b5 was also reduced in the absence of oxygen. No transhydrogenase activity was observed, but the reduced thionicotinamide analogs of NADH and NADPH acted as substrates. Superoxide dismutase inhibited cytochrome c reduction in air by 50%, but O2-. was not necessary for cytochrome c reduction, as evidenced by the increase in rate in the absence of O2. The product of the reaction with oxygen appeared to be H2O2.     

NADH-specific dehydrogenase from onion root plasma membrane: purification and characterization

Summary Plasma membranes isolated from onion roots by twophase partition contain at least two different NAD(P)H-dehydrogenases. A 27 kDa electron transport protein oxidises both NADH and NADPH and exhibits maximal activity with quinones as electron acceptors. A distinct 31 kDa dehydrogenase is specific for NADH as donor and shows maximal activity with ferricyanide. This novel enzyme is responsible for most NADH-ferricyanide oxidoreductase activity of solubilized onion root plasma membranes and exhibits properties different to other purified NAD(P)H-dehydrogenases.Abbreviations DES diethylstilbestrol - FeCN potassium ferricyanide - NBT nitroblue tetrazolium - PHMB p-hydroxymercuribenzoate - PMSF phenylmethylsulfonylfluoride - PTA phosphotungstic acid - SHAM salicylhydroxamic acid - TTFA thenoyltrifluoroacetone     

Role of Thr(66) in porcine NADH-cytochrome b5 reductase in catalysis and control of the rate-limiting step in electron transfer

Site-directed mutagenesis of Thr(66) in porcine liver NADH-cytochrome b(5) reductase demonstrated that this residue modulates the semiquinone form of FAD and the rate-limiting step in the catalytic sequence of electron transfer. The absorption spectrum of the T66V mutant showed a typical neutral blue semiquinone intermediate during turnover in the electron transfer from NADH to ferricyanide but showed an anionic red semiquinone form during anaerobic photoreduction. The apparent k(cat) values of this mutant were approximately 10% of that of the wild type enzyme (WT). These data suggest that the T66V mutation stabilizes the neutral blue semiquinone and that the conversion of the neutral blue to the anionic red semiquinone form is the rate-limiting step. In the WT, the value of the rate constant of FAD reduction (k(red)) was consistent with the k(cat) values, and the oxidized enzyme-NADH complex was observed during the turnover with ferricyanide. This indicates that the reduction of FAD by NADH in the WT-NADH complex is the rate-limiting step. In the T66A mutant, the k(red) value was larger than the k(cat) values, but the k(red) value in the presence of NAD(+) was consistent with the k(cat) values. The spectral shape of this mutant observed during turnover was similar to that during the reduction with NADH in the presence of NAD(+). These data suggest that the oxidized T66A-NADH-NAD(+) ternary complex is a major intermediate in the turnover and that the release of NAD(+) from this complex is the rate-limiting step. These results substantiate the important role of Thr(66) in the one-electron transfer reaction catalyzed by this enzyme. On the basis of these data, we present a new kinetic scheme to explain the mechanism of electron transfer from NADH to one-electron acceptors including cytochrome b(5).     

Isolation and characterization of a NADH-dehydrogenase from rat liver mitochondria

Mitochondrial NADH dehydrogenase has been purified from rat liver mitochondria by protamine sulfate fractionation and DEAE-Sephadex chromatography. The enzyme is water-soluble and its molecular weight has been estimated at 400 +/- 50 kilodaltons. NADH-ferricyanide reductase and NADH cytochrome c reductase activities have been studied and the kinetic parameters have been determined. Both substrates, NADH and the electron acceptor (ferricyanide or cytochrome c) have an inhibitor effect on the reductase activities and the kinetic mechanism of the enzyme is ping-pong bi-bi.