Electrolytic regeneration of the reduced from the oxidized form of immobilized NAD.

A covalently bound adduct of nicotinamide adenine dinucleotide (NAD) with alginic acid has been found to be enzymatically active and to undergo electrochemical oxidation or reduction without significant loss of its enzymatic activity. The preparation of the adduct itself (from NAD+, alginic acid, and 1-cyclohexyl-3-(2-morpholinoethyl)-carbodiimide metho-p-toluenesulfonate) is also accomplished with substantially complete retention of enzymatic activity. This adduct has been converted from the oxidized to the reduced form by controlled potential electrolysis using mercury and stainless-steel electrodes. This electrolytically produced NADH complex could be oxidized again to the enzymatically active NAD+ complex by enzymatic reaction with the proton acceptor, 2,6-dichlorophenol indophenol, as catalyzed by diaphorase. Using this electrolytic method with immobilized NAD, it is now possible to carry out redox reactions in which NADH is enzymatically oxidized to NAD+, with the simultaneous electrolytic regeneration of the reduced form, NADH, from the oxidized form, NAD+, produced in the enzymatic reaction.     

On the oxidation of reduced nicotinamide dinucleotide phosphate by submitochondrial particles from beef heart

The oxidation of NADPH catalyzed by submitochondrial particles from beef heart in the absence and presence of NAD⁺ has been investigated. The data confirm earlier findings in this laboratory concerning the occurrence of an NADPH dehydrogenase with 2,6-dichlorophenolindophenol as the electron acceptor. This reaction is highly sensitive to palmityl-CoA, a feature further substantiating its possible relationship to nicotinamide nucleotide transhydrogenase. The particles also catalyzed a very low NADPH oxidase activity which probably proceeds via NADH dehydrogenase and is unrelated to transhydrogenase.     

Effects of pH, NADH, succinate and malate on the oxidation of glycine in spinach leaf mitochondria

The effect of external pH on several reactions catalyzed by glycine decarboxylase in spinach leaf mitochondria was investigated. Glycine-dependent oxygen consumption showed a pH optimum at 7.6, whereas the release of CO₂ and NH3 from glycine in the presence of oxaloacetate both showed pH maxima at 8.1. Glycine-dependent reduction of 2,6-dichlorophenolindophenol. on the other hand showed a pH optimum at 8.4. It is concluded that these three reactions have different rate-limiting steps. The rate of the glycine-bicarbonate exchange reaction catalyzed by glycine decarboxylase showed no optimum in the pH range investigated, pH 7–9, but increased with decreasing pH. This suggests that CO₂ may be the true substrate in this reaction.
The oxidation of glycine inhibited the oxidation of both malate, succinate and external NADH since the addition of malate, succinate or NADH to mitochondria oxidizing glycine in state 3 resulted in a rate of oxygen consumption which was lower than the sum of the rates when the substrates were oxidized individually. The addition of malate, succinate or NADH did not, however, decrease the rate of CO₂ or NH, release from glycine. It is suggested that the preferred oxidation of glycine by-spinach leaf mitochondria may constitute an important regulatory mechanism for the function of leaf mitochondria during photosynthesis.     

Photooxidase system of Rhodospirillum rubrum. I. Photooxidations catalyzed by chromatophores isolated from a mutant deficient in photooxidase activity

The aerobic photooxidations of reduced 2,6-dichlorophenolindophenol and of reaction-center bacteriochlorophyll (P-870) have been investigated in membrane vesicles (chromatophores) isolated from a non-phototrophic Rhodospirillum rubrum strain. In aerobic suspensions of wild-type chromatophores, continuous light elicits an increase of the levels of 2,6-dichlorophenolindophenol and of oxidized P-870, which reach steady-state values shortly after the onset of illumination. In contrast, light induces in mutant suspensions a transient increase of the levels of 2,6-dichlorophenolindophenol and of oxidized P-870, which fall to low steady-state values within a few seconds. These observations suggest that the mutation has altered a redox constituent located on the low-potential side of the photochemical reaction center, between a pool of acceptors and oxygen.Since endogenous cyclic photophosphorylation is catalyzed by mutant chromatophores at normal rates, it appears that the constituent altered by the mutation does not belong to the cyclic electron-transfer chain responsible for photophosphorylation. However, the system which mediates the aerobic photooxidations and the cyclic system are not completely independent: endogenous photophosphorylation is inhibited by oxygen in wild-type chromatophores but not in mutant chromatophores; in addition, the inhibitor of cyclic electron flow, 2-heptyl-4-hydroxyquinoline-N-oxide, enhances the aerobic photooxidation of reduced 2,6-dichlorophenolindophenol by chromatophores from both strains.These results support a tentative branched model for light-driven electron transfer. In that model, the constituent altered in the mutant strain is located in a side electron-transfer chain which connects the cyclic acceptors to oxygen.     

A modification of isocitrate and malate dehydrogenase assays for use in crude cell free extracts

A modification of the assays for isocitrate and malate dehydrogenase, using phenazine methosulphate and 2,6-dichlorophenolindophenol, permits measurements on cell-free extracts. Phenazine methosulfate at concentrations higher than 30 nmoles/3 ml prevents the accumulation of NADPH or NADH and thus reduces errors due to endogenous oxidation of these compounds. The use of 2,6-dichlorophenolindophenol rather than a tetrazolium salt as the terminal electron acceptor allows continuous spectrophotometric measurement of enzyme activities.Assay for NADP-specific isocitrate dehydrogenase can be performed in aerobic or anaerobic conditions. Assays for malate dehydrogenase should be run under anaerobic conditions because of the interference by oxygen on the phenazine methosulfate mediated reduction of 2,6-dichlorophenolindophenol by NADH. Under anaerobic conditions, where NADH oxidase is inoperative, the phenazine methosulfate/dichlorophenolindophenol assay is more sensitive than the assay using direct measurement of NADH at 340 nm.     

Myeloperoxidase catalysed cooxidative metabolism of methimazole: oxidation of glutathione and NADH by free radical intermediates

The myeloperoxidase catalysed oxidation of methimazole in the presence of NADH or GSH resulted in oxygen uptake suggesting that metabolism proceeded via a one electron mechanism. The GSH was oxidised to GSSG and the thiyl radical could be trapped with DMPO while NADH was oxidized to NAD+. Metabolism proceeded without the inactivation of the enzyme myeloperoxidase. Myeloperoxidase catalyzed oxidation of other substrates which proceed via one electron intermediates; 2,6-dimethylphenol, N,N,N',N'-tetramethyl-phenylenediamine and luminol, were all stimulated by methimazole providing further evidence for a methimazole free radical. The presence of iodide stimulated the oxidation of methimazole but inhibited the oxygen uptake in the presence of GSH or NADH suggesting that metabolism in this case proceeded by a two electron mechanism. In contrast, another S-thioureylene drug, thiourea; did not cause oxygen uptake when oxidised in the presence of GSH or NADH indicating that the myeloperoxidase oxidation of thiourea proceeded primarily by a two electron mechanism. The horseradish peroxidase catalysed one electron oxidation of p'p'-biphenol, and 3,3',5,5'-tetramethylbenzidine was reversibly inhibited by methimazole and thiourea by preventing the accumulation of oxidation products via reductive mechanisms whereas the reversible inhibition of guaiacol and luminol oxidation was the result of competitive inhibition. With p,p'-biphenol, and 3,3',5,5'-tetramethylbenzidine unstable adduct formation could be demonstrated.     

The non-enzymic oxidation of NADH by nitrosobenzene

Nitrosobenzene caused a biphasic oxidation of NADH or NADPH. The initial rapid phase of this oxidation, which is a stoichiometric reaction, was not associated with oxygen consumption and was unaffected by EDTA. In contrast, the slower phase of the NADH oxidation was inhibited by EDTA. It was associated with oxygen uptake and was catalytic, in the sense that many moles of NADH could be oxidized per mole of nitrosobenzene. Copper catalyzed this reaction. A mechanism which explains these observations is proposed.     

Characterization of a dihydrolipoyl dehydrogenase having diaphorase activity of Clostridium kluyveri

The Clostridium kluyveri bfmBC gene encoding a putative dihydrolipoyl dehydrogenase (DLD; EC 1.8.1.4) was expressed in Escherichia coli, and the recombinant enzyme rBfmBC was characterized. UV-visible absorption spectrum and thin layer chromatography analysis of rBfmBC indicated that the enzyme contained a noncovalently but tightly attached FAD molecule. rBfmBC catalyzed the oxidation of dihydrolipoamide (DLA) with NAD(+) as a specific electron acceptor, and the apparent K(m) values for DLA and NAD(+) were 0.3 and 0.5 mM respectively. In the reverse reaction, the apparent K(m) values for lipoamide and NADH were 0.42 and 0.038 mM respectively. Like other DLDs, this enzyme showed NADH dehydrogenase (diaphorase) activity with some synthetic dyes, such as 2,6-dichlorophenolindophenol and nitro blue tetrazolium. rBfmBC was optimally active at 40 degrees C at pH 7.0, and the enzyme maintained some activity after a 30-min incubation at 60 degrees C.     

Oxidation in H2O and D2O of 6-ethyl-5H-dibenz(c,e)azepine and 1-methylnicotinamide by aldehyde oxidase from rabbit liver

We report the solvent hydrogen isotope effects associated with the oxidation of 6-ethyl-5H-dibenz(c,e)azepine (6-ED) and 1-methylnicotinamide (1-MN) catalyzed by aldehyde oxidase from rabbit liver using two assay methods. The first uses 2,6-dichlorophenolindophenol (DCI) as electron acceptor in an indirect assay in which the bleaching of DCI is measured as the substrate is oxidized. The second uses molecular oxygen as electron acceptor in a direct assay in which the oxidation of 1-MN to its pyridones is accompanied by an increase in absorbance at 300 nm and the oxidation of 6-ED to its lactam product is accompanied by a decrease in absorbance at 335 nm. We have found a solvent hydrogen isotope effect close to unity in the turnover number for each substrate and for each assay method. The solvent hydrogen isotope effects on kcat/Km ranged from 0.4 to 1.1. We conclude that changes in bonding of hydrogen in solvent water, including hydrolysis of or general base attack on an enzyme-intermediate complex, do not play a rate-contributing role in the maximal velocity of oxidation of 1-MN and 6-ED catalyzed by aldehyde oxidase from rabbit liver.     

Ferrisiderophore reductase activity associated with an aromatic biosynthetic enzyme complex in Bacillus subtilis

The cytoplasmic fractions obtained from Bacillus subtilis strains W168 and WB2802 catalyzed reductive release of iron from the ferric chelate of 2,3-dihydroxybenzoic acid (ferri-DHB), the ferrisiderophore produced by B. subtilis. Ferrisiderophore reductase activity may insert iron into metabolism. This activity required a reductant (reduced nicotinamide adenine dinucleotide phosphate was preferred), was oxygen sensitive, and was stimulated by flavin mononucleotide plus certain divalent cations. The cytoplasmic fractions also reduced 2,6-dichlorophenolindophenol; this reaction was stimulated by flavin mononucleotide plus a divalent cation. Ferri-DHB and 2,6-dichlorophenolindophenol reductase activities were copurified by phosphocellulose and diethylaminoethyl-cellulose chromatography. Nondenaturing polyacrylamide gel electrophoresis of the purified material revealed that both ferri-DHB and 2,6-dichlorophenolindophenol reductase activities were located in a protein band at Rf 0.75. The chromatographic procedures purified a reductase known to be associated with two aromatic biosynthetic enzymes, chorismate synthase and dehydroquinate synthase. Therefore, a portion of the ferrisiderophore reductase activity in B. subtilis may be catalyzed by a reductase that also is essential for aromatic biosynthesis.     

Oxidation of 1,5-anhydro-D-glucitol to 1,5-anhydro-D-fructose catalyzed by an enzyme from bacterial membranes

Bacteria which grow on 1,5-anhydro-D-glucitol (AG) were isolated from soil. One such strain showing the highest AG-assimilating activity was further characterized and identified as a new strain of the Pseudomonas family (named Pseudomonas sp. NK-85001). A subcellular membranous fraction obtained from this strain catalyzed the oxidation of AG to 1,5-anhydro-D-fructose. This oxidation reaction consumed molecular oxygen as the terminal electron acceptor. The AG-oxidizing activity was further purified after solubilization. The AG oxidation catalyzed by this solubilized enzyme utilized molecular oxygen only in the presence of an electron mediator such as 2,6-dichlorophenolindophenol or phenazine methosulfate. Thus, the enzyme was suggested to be a dehydrogenase rather than an oxidase. The solubilized enzyme preparation also showed a strict substrate specificity. The observed specificity indicated that application of the enzyme for AG assay in clinical samples might be possible.     

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.     

Novel Dehalogenase Mechanism for 2,3-Dichloro-1-Propanol Utilization in Pseudomonas putida Strain MC4

A Pseudomonas putida strain (MC4) that can utilize 2,3-dichloro-1-propanol (DCP) and several aliphatic haloacids and haloalcohols as sole carbon and energy source for growth was isolated from contaminated soil. Degradation of DCP was found to start with oxidation and concomitant dehalogenation catalyzed by a 72-kDa monomeric protein (DppA) that was isolated from cell lysate. The dppA gene was cloned from a cosmid library and appeared to encode a protein equipped with a signal peptide and that possessed high similarity to quinohemoprotein alcohol dehydrogenases (ADHs), particularly ADH IIB and ADH IIG from Pseudomonas putida HK. This novel dehalogenating dehydrogenase has a broad substrate range, encompassing a number of nonhalogenated alcohols and haloalcohols. With DCP, DppA exhibited a k(cat) of 17 s(-1). (1)H nuclear magnetic resonance experiments indicated that DCP oxidation by DppA in the presence of 2,6-dichlorophenolindophenol (DCPIP) and potassium ferricyanide [K(3)Fe(CN)(6)] yielded 2-chloroacrolein, which was oxidized to 2-chloroacrylic acid.     

Importance of hydroxyl radical in the vanadium-stimulated oxidation of NADH

Vanadium compounds are known to stimulate the oxidation of NAD(P)H, but the mechanism remains unclear. This reaction was studied spectrophotometrically and by electron spin resonance spectroscopy (ESR) using vanadium in the reduced state (+4, vanadyl) and the oxidized state (+5, vanadate). In 25 mM sodium phosphate buffer at pH 7.4, vanadyl was slightly more effective in stimulating NADH oxidation than was vanadate. Addition of a superoxide generating system, xanthine/xanthine oxidase, resulted in a marked increase in NADH oxidation by vanadyl, and to a lesser extent, by vanadate. Decreasing the pH with superoxide present increased NADH oxidation for both vanadate and vanadyl. Addition of hydrogen peroxide to the reaction mixture did not change the NADH oxidation by vanadate, regardless of concentration or pH. With vanadyl however, addition of hydrogen peroxide greatly enhanced NADH oxidation which further increased with lower pH. Use of the spin trap DMPO in reaction mixtures containing vanadyl and hydrogen peroxide or a superoxide generating system resulted in the detection by ESR of hydroxyl. In each case, the hydroxyl radical signal intensity increased with vanadium concentration. Catalase was able to inhibit the formation of the DMPO--OH adduct formed by vanadate plus superoxide. These results show that the ability of vanadium to act in a Fenton-type reaction is an important process in the vanadium-stimulated oxidation of NADH.     

Characterization of one- and two-electron oxidations of glutathione coupled with lactoperoxidase and thyroid peroxidase reactions

Glutathione (GSH) was oxidized to GSSG in the presence of H2O2, tyrosine, and peroxidase. During the GSH oxidation catalyzed by lactoperoxidase, O2 was consumed and the formation of glutathione free radical was confirmed by ESR of its 5,5'-dimethyl-1-pyrroline-N-oxide adduct. When lactoperoxidase was replaced by thyroid peroxidase in the reaction system, the consumption of O2 and the formation of the free radical became negligibly small. These results led us to conclude that, in the presence of H2O2 and tyrosine, lactoperoxidase and thyroid peroxidase caused the one-electron and two-electron oxidations of GSH, respectively. It was assumed that GSH is oxidized by primary oxidation products of tyrosine, which are phenoxyl free radicals in lactoperoxidase reactions and phenoxyl cations in thyroid peroxidase reactions. When tyrosine was replaced by diiodotyrosine or 2,6-dichlorophenol, the difference in the mechanism between lactoperoxidase and thyroid peroxidase disappeared and both caused the one-electron oxidation of GSH. Iodides also served as an effective mediator of GSH oxidation coupled with the peroxidase reactions. In this case the two peroxidases both caused the two-electron oxidation of GSH.     

Oxidase Reaction of the Hybrid Mn-Peroxidase of the Fungus <Emphasis Type="Italic">Panus tigrinus</Emphasis> 8/18

The hybrid Mn-peroxidase of the fungus Panus tigrinus 8/18 oxidized NADH in the absence of hydrogen peroxide, this being accompanied by the consumption of oxygen. The reaction of NADH oxidation started after a period of induction and completely depended on the presence of Mn(II). The reaction was inhibited in the presence of catalase and super-oxide dismutase. Oxidation of NADH by the enzyme or by manganese(III)acetate was accompanied by the production of hydrogen peroxide and superoxide radicals. In the presence of NADH, the enzyme was transformed into a catalytically inactive oxidized form (compound III), and the latter was inactivated with bleaching of the heme. The substrate of the hybrid Mn-peroxidase (Mn(II)) reduced compound III to yield the native form of the enzyme and prevented its inactivation. It is assumed that the hybrid Mn-peroxidase used the formed hydrogen peroxide in the usual peroxidase reaction to produce Mn(III), which was involved in the formation of hydrogen peroxide and thus accelerated the peroxidase reaction. The reaction of NADH oxidation is a peroxidase reaction and the consumption of oxygen is due to its interaction with the products of NADH oxidation. The role of Mn(II) in the oxidation of NADH consisted in the production of hydrogen peroxide and the protection of the enzyme from inactivation.__________Translated from Biokhimiya, Vol. 70, No. 4, 2005, pp. 568–574.Original Russian Text Copyright © 2005 by Lisov, Leontievsky, Golovleva.     

Enzymatic reduction of complex redox dyes using NADH-dependent reductase from Bacillus subtilis coupled with cofactor regeneration

Conventional vat dyeing involves chemical reduction of dyes into their water-soluble leuco form generating considerable amounts of toxic chemicals in effluents. In the present study, a new β-nicotinamide adenine dinucleotide disodium salt (NADH)-dependent reductase isolated from Bacillus subtilis was used to reduce the redox dyes CI Acid Blue 74, CI Natural Orange 6, and CI Vat Blue 1 into their water-soluble leuco form. Enzymatic reduction was optimized in relation to pH and temperature conditions. The reductase was able to reduce Acid Blue 74 and Natural Orange 6 in the presence of the stoichiometrically consumed cofactor NADH; meanwhile, Vat Blue 1 required the presence of mediator 1,8-dihydroxyanthraquinone. Oxygen from air was used to reoxidize the dyes into their initial forms. The enzymatic reduction of the dyes was studied and the kinetic constants determined, and these were compared to the chemically-reduced leuco form. The enzyme responsible for the reduction showed homology to a NADH-dependent reductase from B. subtilis based on results from the MS/MS peptide mass mapping of the tryptically digested protein. Additionally, the reduction of Acid Blue 74 to its leuco form by reductase from B. subtilis was confirmed using NADH regenerated by the oxidation of formic acid with formate dehydrogenase from Candida boidinii in the same solution.     

Deiodination of l-thyroxine and its activity on the oxidation in vitro of reduced nicotinamide–adenine dinucleotide by peroxidase plus hydrogen peroxide

When l-thyroxine activates the oxidation of NADH by peroxidase+H(2)O(2), little removal of phenolic-ring iodine atoms becomes apparent until most of the NADH has been oxidized, after which it increases markedly. This extensive deiodination is accompanied by loss of the ability of thyroxine to catalyse the oxidation of NADH by peroxidase+H(2)O(2). The slight deiodination observed before the appearance of extensive deiodination is somewhat higher when the effect of thyroxine on NADH oxidation is greater, and lower when thyroxine has exerted a slighter effect. ICN (but not I(2) or thyronine) catalyses NADH oxidation, in both the presence and the absence of peroxidase+H(2)O(2): thyroxine+peroxidase+H(2)O(2) are thus comparable with ICN alone in their effects on NADH oxidation. The obvious conclusion from the above observation, namely that the active moiety is the halogen liberated from thyroxine (or ICN) is, however, not directly supported by some of the results obtained by measuring the degree of deiodination of thyroxine in the system. In an attempt to reconcile some apparently contradictory conclusions, it is suggested that, when thyroxine activates oxidation of NADH by peroxidase+H(2)O(2), the diphenyl ether structure is undergoing cyclic deiodination and iodination. This would be accompanied by the maintenance in the reaction medium of an oxidized form of iodine, similar to that liberated by ICN, which would be the actual active moiety, until the NADH concentration becomes so low that the diphenyl ether structure is ruptured oxidatively. An alternative explanation is that thyroxine is oxidized to a form that either oxidizes NADH or loses iodine in competing reactions.     

NADH oxidase activity of indoleamine 2,3-dioxygenase

The heme enzyme indoleamine 2,3-dioxygenase (IDO) was found to oxidize NADH under aerobic conditions in the absence of other enzymes or reactants. This reaction led to the formation of the dioxygen adduct of IDO and supported the oxidation of Trp to N-formylkynurenine. Formation of the dioxygen adduct and oxidation of Trp were accelerated by the addition of small amounts of hydrogen peroxide, and both processes were inhibited in the presence of either superoxide dismutase or catalase. Anaerobic reaction of IDO with NADH proceeded only in the presence of a mediator (e.g. methylene blue) and resulted in formation of the ferrous form of the enzyme. We propose that trace amounts of peroxide previously proposed to occur in NADH solutions as well as solid NADH activate IDO and lead to aerobic formation of superoxide and the reactive dioxygen adduct of the enzyme.     

The mechanism of oxidation of reduced nicotinamide dinucleotide phosphate by submitochondrial particles from beef heart.

1. Oxidation of NADPH by various acceptors catalyzed by submitochondrial particles and a partially purified NADH dehydrogenase from beef heart was investigated. Submitochondrial particles devoid of nicotinamide nucleotide transhydrogenase activity catalyze an oxidation of NADPH by oxygen. The partially purified NADH dehydrogenase prepared from these particles catalyzes an oxidation of NADPH by acetylpyridine-NAD. In both cases the rates of oxidation are about two orders of magnitude lower than those obtained with NADH as electron donor. 2. The kinetic characteristics of the NADPH oxidase reaction and reduction of acetylpyridine-NAD by NADPH are similar with regard to pH dependences and affinities for NADPH, indicating that both reactions involve the same binding site for NADPH. The binding of NADPH to this site appears to be rate limiting for the overall reactions. 3. At redox equilibrium NADPH and NADH reduce FMN and iron-sulphur center 1 of NADH dehydrogenase to the same extents. The rate of reduction of FMN by NADPH is at least two orders of magnitude lower than with NADH. 4. It is concluded that NADPH is a substrate of NADH dehydrogenase and that the nicotinamide nucleotide is oxidized by submitochondrial particles via the NADH--binding site of the enzyme.