Reduction of organic nitrates catalysed by xanthine oxidoreductase under anaerobic conditions.

Xanthine oxidoreductase catalyses the anaerobic reduction of glyceryl trinitrate (GTN), isosorbide dinitrate and isosorbide mononitrate to inorganic nitrite using xanthine or NADH as reducing substrates. Reduction rates are much faster with xanthine as reducing substrate than with NADH. In the presence of xanthine, urate is produced in essentially 1:1 stoichiometric ratio with inorganic nitrite, further reduction of which is relatively slow. Organic nitrates were shown to interact with the FAD site of the enzyme. In the course of reduction of GTN, xanthine oxidoreductase was progressively inactivated by conversion to its desulpho form. It is proposed that xanthine oxidoreductase is one of several flavoenzymes that catalyse the conversion of organic nitrate to inorganic nitrite in vivo. Evidence for its further involvement in reduction of the resulting nitrite to nitric oxide is discussed.     

Turkey liver xanthine dehydrogenase. Relation between nicotinamide-adenine dinucleotide oxidoreductase activity and the content of functional enzyme.

The turkey liver xanthine dehydrogenase-catalysed oxidation of NADH by Methylene Blue, by ferricyanide or by O2 is not dependent on the integrity of the active-centre persulphide groups. By contrast, the NADH-dichlorophenol-indophenol oxidoreductase and NADH-trinitrobenzenesulphonate oxidoreductase activities are directly proportional to the content of functional enzyme. These findings help to identify the sites of egress of electrons to oxidizing substrates.     

Evidence that a type-2 NADH:quinone oxidoreductase mediates electron transfer to particulate methane monooxygenase in methylococcus capsulatus.

NADH readily provides reducing equivalents to membrane-bound methane monooxygenase (pMMO) from Methylococcus capsulatus (Bath) in isolated membrane fractions, but detergent solubilization disrupts this electron-transfer process. Addition of exogenous quinones (especially decyl-plastoquinone and duroquinone) restores the NADH-dependent pMMO activity. Results of inhibitor and substrate dependence of this activity indicate the presence of only a type-2 NADH:quinone oxidoreductase (NDH-2). A 100-fold purification of the NDH-2 was achieved using lauryl-maltoside solubilization followed by ion exchange, hydrophobic-interaction, and gel-filtration chromatography. The purified NDH-2 has a subunit molecular weight of 36 kDa and exists as a monomer in solution. UV-visible and fluorescence spectroscopy identified flavin adenine dinucleotide (FAD) as a cofactor present in stoichiometric amounts. NADH served as the source of electrons, whereas NADPH could not. The purified NDH-2 enzyme reduced coenzyme Q(0), duroquinone, and menaquinone at high rates, whereas the decyl analogs of ubiquinone and plastoquinone were reduced at approximately 100-fold lower rates. Rotenone and flavone did not inhibit the NDH-2, whereas amytal caused partial inhibition but only at high concentrations.     

Participation of cytochrome b5 in CMP-N-acetylneuraminic acid hydroxylation in mouse liver cytosol

The activity of CMP-N-acetylneuraminic acid hydroxylase, that converts CMP-N-acetylneuraminic acid (CMP-NeuAc) to CPM-N-glycolylneuraminic acid (CMP-NeuGc), in mouse liver was determined by a newly developed HPLC method using non-radioactive CMP-NeuAc as a substrate. The activity was detected in the cytosol fraction but not in the microsomal fraction. Either NADH or NADPH was used as an electron donor by the cytosol enzyme, but NADH was much more efficiently used than NADPH. An antibody against cytochrome b5 markedly reduced the CMP-NeuAc hydroxylase activity when added to incubation mixture containing either NADH or NADPH as an electron donor. These data led us to postulate the following electron transport system, which is involved in the CMP-NeuAc hydroxylation in mouse liver cytosol: (formula; see text) where X, Y, and Z are components supposedly involved.     

Effect of NADH on hypoxanthine hydroxylation by native NAD+-dependent xanthine oxidoreductase of rat liver, and the possible biological role of this effect.

The course of the reaction sequence hypoxanthine leads to xanthine leads to uric acid, catalysed by the NAD+-dependent activity of xanthine oxidoreductase, was investigated under conditions either of immediate oxidation of the NADH formed or of NADH accumulation. The enzymic preparation was obtained from rat liver, and purified 75-fold (as compared with the 25000 g supernatant) on a 5'-AMP-Sepharose 4B column; in this preparation the NAD+-dependent activity accounted for 100% of total xanthine oxidoreductase activity. A spectrophotometric method was developed for continuous measurements of changes in the concentrations of the three purines involved. The time course as well as the effects of the concentrations of enzyme and of hypoxanthine were examined. NADH produced by the enzyme lowered its activity by 50%, resulting in xanthine accumulation and in decreases of uric acid formation and of hypoxanthine utilization. The inhibition of the Xanthine oxidoreductase NAD+-dependent activity by NADH is discussed as a possible factor in the regulation of IMP biosynthesis by the 'de novo' pathway or (from unchanged hypoxanthine) by ther salvage pathway.     

NADH oxidase activity of rat and human liver xanthine oxidoreductase: potential role in superoxide production

To characterise the NADH oxidase activity of both xanthine dehydrogenase (XD) and xanthine oxidase (XO) forms of rat liver xanthine oxidoreductase (XOR) and to evaluate the potential role of this mammalian enzyme as an O₂ ^•− source, kinetics and electron paramagnetic resonance (EPR) spectroscopic studies were performed. A steady-state kinetics study of XD showed that it catalyses NADH oxidation, leading to the formation of one O₂ ^•− molecule and half a H₂O₂ molecule per NADH molecule, at rates 3 times those observed for XO (29.2 ± 1.6 and 9.38 ± 0.31 min⁻¹, respectively). EPR spectra of NADH-reduced XD and XO were qualitatively similar, but they were quantitatively quite different. While NADH efficiently reduced XD, only a great excess of NADH reduced XO. In agreement with reductive titration data, the XD specificity constant for NADH (8.73 ± 1.36 μM⁻¹ min⁻¹) was found to be higher than that of the XO specificity constant (1.07 ± 0.09 μM⁻¹ min⁻¹). It was confirmed that, for the reducing substrate xanthine, rat liver XD is also a better O₂ ^•− source than XO. These data show that the dehydrogenase form of liver XOR is, thus, intrinsically more efficient at generating O₂ ^•− than the oxidase form, independently of the reducing substrate. Most importantly, for comparative purposes, human liver XO activity towards NADH oxidation was also studied, and the kinetics parameters obtained were found to be very similar to those of the XO form of rat liver XOR, foreseeing potential applications of rat liver XOR as a model of the human liver enzyme.     

Isolation and biochemical characterization of two soluble iron(III) reductases from Paracoccus denitrificans.

Two soluble enzymes (FerA and FerB) catalyzing the reduction of a number of iron(III) complexes by NADH, were purified to near homogeneity from the aerobically grown iron-limited culture of Paracoccus denitrificans using a combination of anion-exchange chromatography (Sepharose Q), chromatofocusing (Mono P), and gel permeation chromatography (Superose 12). FerA is a monomer with a molecular mass of 19 kDa, whereas FerB exhibited a molecular mass of about 55 kDa and consists of probably two identical subunits. FerA can be classified as an NADH:flavin oxidoreductase with a sequential reaction mechanism. It requires the addition of FMN or riboflavin for activity on Fe(III) substrates. In these reactions, the apparent substrate specificity of FerA seems to stem exclusively from different chemical reactivities of Fe(III) compounds with the free reduced flavin produced by the enzyme. Observations on reducibility of Fe(III) chelated by vicinal dihydroxy ligands support the view that FerA takes part in releasing iron from the catechol type siderophores synthesized by P. denitrificans. Contrary to FerA, the purified FerB contains a noncovalently bound redox-active FAD coenzyme, can utilize NADPH in place of NADH, does not reduce free FMN at an appreciable rate, and gives a ping-pong type kinetic pattern with NADH and Fe(III)-nitrilotriacetate as substrates. FerB is able to reduce chromate, in agreement with the fact that its N-terminus bears a homology to the previously described chromate reductase from Pseudomonas putida. Besides this, it also readily reduces quinones like ubiquinone-0 (Q0) or unsubstituted p-benzoquinone.     

Studies of the ferroxidase activity of native and chemically modified xanthine oxidoreductase.

The O2-utilizing (type O, oxidase) form of xanthine oxidoreductase is primarily responsible for its ferroxidase activity. This form of xanthine oxidoreductase has 1000 times the ferroxidase activity of the serum ferroxidase caeruloplasmin. It has the ability to catalyse the oxidative incorporation of iron into transferrin at very low Fe2+ and O2 concentrations. Furthermore, the pH optimum of the ferroxidase activity of the enzyme is compatible with the conditions of pH that normally exist in the intestinal mucosa, where it has been proposed that xanthine oxidoreductase may facilitate the absorption of ionic iron. Modification of the molybdenum (Mb) centres of the enzyme in vitro by treatment with cyanide, methanol or allopurinol completely abolishes its ferroxidase activity. The feeding of dietary tungsten to rats, which prevents the incorporation of molybdenum into newly synthesized intestinal xanthine oxidoreductase, results in the progressive loss of the ferroxidase activity of intestinal-mucosa homogenates. Removal of the flavin centres from the enzyme also results in the complete loss of ferroxidase activity; however, the ferroxidase activity of the flavin-free form of the enzyme can be restored with artificial electron acceptors that interact with the molybdenum or non-haem iron centres. The presence of superoxide dismutase or catalase in the assay system results in little inhibition of the ferroxidase activity of xanthine oxidoreductase.     

The NAD(P)H:flavin oxidoreductase from Escherichia coli. Evidence for a new mode of binding for reduced pyridine nucleotides.

The NAD(P)H:flavin oxidoreductase from Escherichia coli, named Fre, is a monomer of 26.2 kDa that catalyzes the reduction of free flavins using NADPH or NADH as electron donor. The enzyme does not contain any prosthetic group but accommodates both the reduced pyridine nucleotide and the flavin in a ternary complex prior to oxidoreduction. The specificity of the flavin reductase for the pyridine nucleotide was studied by steady-state kinetics using a variety of NADP analogs. Both the nicotinamide ring and the adenosine part of the substrate molecule have been found to be important for binding to the polypeptide chain. However, in the case of NADPH, the 2'-phosphate group destabilized almost completely the interaction with the adenosine moiety. Moreover, NADPH and NMNH are very good substrates for the flavin reductase, and we have shown that both these molecules bind to the enzyme almost exclusively by the nicotinamide ring. This provides evidence that the flavin reductase exhibits a unique mode for recognition of the reduced pyridine nucleotide. In addition, we have shown that the flavin reductase selectively transfers the pro-R hydrogen from the C-4 position of the nicotinamide ring and is therefore classified as an A-side-specific enzyme.     

The NADPH:O2 oxidoreductase of human neutrophils. Stoichiometry of univalent and divalent reduction of O2

The ratio of superoxide production to oxidation of NADPH affected by the NADPH:O2 oxidoreductase of human neutrophils is strongly influenced by pH, NADPH substrate concentration, aging of the enzyme, or its exposure to excess deoxycholate. Freshly prepared enzyme exhibited a Km for NADPH of 52 microM as determined by assaying NADPH oxidase activity, or approximately 33 microM by measurement of superoxide formation. In the range of 100-150 microM NADPH at pH 7.6 and in the presence of 0.06% deoxycholate, the univalent flux of electron equivalents given up by NADPH to O2 was 99%. Following storage of the oxidoreductase overnight on ice, its Km for NADPH rose to 125 microM as determined by monitoring oxidation of NADPH but was unaltered when measured in terms of superoxide production. Concomitantly, its capacity to affect univalent reduction of O2 fell approximately 20-30% under the same assay conditions. Univalent flux rates of less than 40% were observed with exposure of the enzyme to concentrations of deoxycholate in excess of 0.1% or to pH values below 6.0 or above 8.0. The capacity of the enzyme to carry out univalent reduction fell with increasing NADPH concentrations in a manner resembling that previously reported with increasing concentrations of xanthine in the case of xanthine oxidase (Fridovich, I. (1970) J. Biol. Chem. 245, 4053-4057). The reduced form of the neutrophil oxidoreductase, like xanthine oxidase, thus appears to be capable of conducting both 1- and 2-electron transfer steps in reducing O2. Its capacity to affect univalent reduction of O2 depends upon the concentration of electron donor (NADPH) supplied as well as conditions of storage and assay of the native enzyme.     

Purification and properties of a NAD(P)H:flavin oxidoreductase from the luminous bacterium, Beneckea harveyi.

A NAD(P)H:flavin oxidoreductase, which produces FMNH2, one of the substrates for the luciferase reaction in bioluminescent bacteria, has been purified with the aid of affinity chromatography on epsilon-aminohexanoyl-FMN-Sepharose. The purified enzyme, isolated from Beneckea harveyi, had a specific activity of 89 mumol of NADH oxidized/min/mg of protein at 23 degrees in the presence of saturating FMN and NADH and appeared homogeneous by several criteria on polyacrylamide gel electrophoresis. A molecular weight of 24,000 was estimated both by gel filtration and and sodium dodecyl sulfate gel electrophoresis indicating that the enzyme is composed of a single polypeptide chain. Kinetic studies showed that the higher specificity of the enzyme for NADH than NADPH and for riboflavin and FMN than FAD was primarily due to variations in the Michaelis constants for the different substrates. Initial velocity studies with all pairs of substrates gave intersecting patterns supporting a sequential mechanism for the NAD(P)H:flavin oxidoreductase.     

Nuclear 3alpha-hydroxysteroid dehydrogenase (3alphaOHD) activity for 5alpha-dihydrotestosterone in the rat prostate.

The in vitro examination of adult male rat prostatic 3alpha-hydroxysteroid dehydrogenase (3alphaOHD) activity using 5alpha-dihydrotestosterone4 as substrate indicates that significant levels of enzyme activity are associated with purified nuclei as well as with the cytosol fractions. Both the purified nuclear and the cytosol fractions exhibited higher levels of 3alphaOHD activity with NADH than with NADPH. The pH activity curves for the NADH and NADPH catalyzed reactions were different for both the nuclear and cytosol fractions. The results suggest the presence of a number of 3alphaOHD enzymes in rat prostate.     

Effect of triamcinolone administration on content of flavins in rabbit liver.

Triamcinoline acetonide (10 mg per kg of body weight a day) was administered to rabbit fed on a laboratory chow diet. The content of flavins in liver but not in kidney, muscle and brain started to decrease 24 h after a single dose. The activities of enzymes in the liver were determined: the activities of pyruvate dehydrogenase complex, lipoamide dehydrogenase (NADH:lipoamide oxidoreductase EC 1.6.4.3), NADH dehydrogenase (NADH : (acceptor) oxidoreductase EC 1.6.99.3) and D-amino acid oxidase (D-amino acid: oxygen oxidoreductase (deaminating) EC 1.4.3.3) were decreased but those of succinate dehydrogenase (succinate : (acceptor) oxidoreductase EC 1.3.99.1) and xanthine oxidase (xanthine : oxygen oxidoreductase EC 1.2.3.2) remained unchanged. The activities of enzymes in the kidney, however, remained unchanged except the decrease in the activity of pyruvate dehydrogenase complex.     

The ferrireductase paraferritin contains divalent metal transporter as well as mobilferrin

Inorganic iron can be transported into cells in the absence of transferrin. Ferric iron enters cells utilizing an integrin-mobilferrin-paraferritin pathway, whereas ferrous iron uptake is facilitated by divalent metal transporter-1 (DMT-1). Immunoprecipitation studies using antimobilferrin antibody precipitated the previously described large-molecular-weight protein complex named paraferritin. It was previously shown that paraferritin functions as an intracellular ferrireductase, reducing ferric iron to ferrous iron utilizing NADPH as the energy source. It functions in the pathway for the cellular uptake of ferric iron. This multipeptide protein contains a number of active peptides, including the ferric iron binding protein mobilferrin and a flavin monooxygenase. The immunoprecipitates and purified preparations of paraferritin also contained DMT-1. This identifies DMT-1 as one of the peptides constituting the paraferritin complex. Since paraferritin functions to reduce newly transported ferric iron to ferrous iron and DMT-1 can transport ferrous iron, these findings suggest a role for DMT-1 in conveyance of iron from paraferritin to ferrochelatase, the enzyme utilizing ferrous iron for the synthesis of heme in the mitochondrion.     

Redox potential and equilibria in the reductive half-reaction of Vibrio harveyi NADPH-FMN oxidoreductase

Vibrio harveyi NADPH:FMN oxidoreductase P (FRP(Vh)) is a homodimeric enzyme having a bound FMN per enzyme monomer. The bound FMN functions as a cofactor of FRP(Vh) in transferring reducing equivalents from NADPH to a flavin substrate in the absence of V. harveyi luciferase but as a substrate for FRP(Vh) in the luciferase-coupled bioluminescent reaction. As part of an integral plan to elucidate the regulation of functional coupling between FRP(Vh) and luciferase, this study was carried out to characterize the equilibrium bindings, reductive potential, and the reversibility of the reduction of the bound FMN in the reductive half-reaction of FRP(Vh). Results indicate that, in addition to NADPH binding, NADP(+) also bound to FRP(Vh) in either the oxidized (K(d) 180 microM) or reduced (K(d) 230 microM) form. By titrations with NADP(+) and NADPH and by an isotope exchange experiment, the reduction of the bound FMN by NADPH was found to be readily reversible (K(eq) = 0.8). Hence, the reduction of FRP(Vh)-bound FMN is not the committed step in coupling the NADPH oxidation to bioluminescence. To our knowledge, such an aspect of flavin reductase catalysis has only been clearly established for FRP(Vh). Although the reductive potentials and some other properties of a R203A variant of FRP(Vh) and an NADH/NADPH-utilizing flavin reductase from Vibrio fischeri are quite similar to that of the wild-type FRP(Vh), the reversal of the reduction of bound FMN was not detected for either of these two enzymes.     

NAD(P)H:flavin oxidoreductase of Escherichia coli. A ferric iron reductase participating in the generation of the free radical of ribonucleotide reductase

The active form of one subunit of Escherichia coli ribonucleotide reductase (protein B2) contains an organic free radical localized to tyrosine 122 of its polypeptide chain. When this radical is scavenged, e.g. by treatment with hydroxyurea, the enzyme is inactivated (protein B2/HU). E. coli contains an enzyme system consisting of at least three proteins that in the presence of NADPH, FMN, dithiothreitol, and oxygen introduce the tyrosyl radical into B2/HU (Eliasson, R., J?rnvall, H., and Reichard, P. (1986) Proc. Natl. Acad. Sci. U. S. A. 83, 2373-2377). One of the three proteins was identified as superoxide dismutase. We now identify a second protein, previously provisionally named Fraction c, as an NAD(P)H:flavin oxidoreductase (flavin reductase). After 4,000-fold purification the protein moved as a single band on sodium dodecyl sulfate gel electrophoresis with a molecular weight of 28,000-29,000. The enzyme contained no flavin but reduced riboflavin, FMN, and FAD by NADH, or riboflavin and FMN by NADPH. It is a powerful ferric iron reductase. We propose that its complementing activity during radical generation involves participation in the reduction of the ferric iron center of protein B2/HU. Radical formation is then linked to the reoxidation of iron by oxygen. The flavin reductase may also participate in other aspects of iron metabolism of E. coli.     

Vanadate-stimulated NADH oxidation by xanthine oxidase: an intrinsic property

Vanadate-dependent oxidation of NADH by xanthine oxidase does not require the presence of xanthine and therefore is not due to cooxidation. Addition of NADH or xanthine had no effect on the oxidation of the other substrate. Oxidation of NADH was high at acid pH and oxidation of xanthine was high at alkaline pH. The specific activity was relatively very high with NADH. Concentration-dependent oxidation of NADH Concentration-dependent oxidation of NADH was obtained in the presence of the polymeric form of vanadate, but not orthovanadate or metavanadate. Both NADH and NADPH were oxidized, as in the nonenzymatic system. Oxidation of NADH, but not xanthine, was inhibited by KCN, ascorbate, MnCl2, cytochrome c, mannitol, Tris, epinephrine, norepinephrine, and triiodothyronine. Oxidation of NADH was accompanied by uptake of oxygen and generation of H2O2 with a stoichiometry of 1:1:1 for NADH:O2:H2O2. A 240-nm-absorbing species was formed during the reaction which was different from H2O2 or superoxide. A mechanism of NADH oxidation is suggested wherein Vv and O2 receive one electron each successively from NADH followed by VIV giving the second electron to superoxide and reducing it to H2O2.     

Engineering the respiratory complex I to energy-converting NADPH:ubiquinone oxidoreductase

The respiratory complex I couples the electron transfer from NADH to ubiquinone with a translocation of protons across the membrane. Its nucleotide-binding site is made up of a unique Rossmann fold to accommodate the binding of the substrate NADH and of the primary electron acceptor flavin mononucleotide. Binding of NADH includes interactions of the hydroxyl groups of the adenosine ribose with a conserved glutamic acid residue. Structural analysis revealed that due to steric hindrance and electrostatic repulsion, this residue most likely prevents the binding of NADPH, which is a poor substrate of the complex. We produced several variants with mutations at this position exhibiting up to 200-fold enhanced catalytic efficiency with NADPH. The reaction of the variants with NAD(P)H is coupled with proton translocation in an inhibitor-sensitive manner. Thus, we have created an energy-converting NADPH:ubiquinone oxidoreductase, an activity so far not found in nature. Remarkably, the oxidation of NAD(P)H by the variants leads to an enhanced production of reactive oxygen species.     

Studies on chicken liver xanthine dehydrogenase with reference to the problem of non-equivalence of FAD moieties.

1. Reduction of chicken liver xanthine dehydrogenase (xanthine: NAD+ oxidoreductase, EC 1.2.1.37) by xanthine under anaerobic condition proceeded in two phases. This biphasicity may be due to functional and non-functional enzymes in the enzyme preparation. 2. Cyanolysis of a persulfide group of chicken liver enzyme resulted in an inactivation of the enzyme. The non-functional enzyme in the standard enzyme preparation was found to lack persulfide groups at the active sites. 3. The remaining NADH-Methylene Blue oxidoreductase activity, after KI treatment of the xanthine-reduced enzyme of a high flavin activity ratio, is not at the level of 50% of the initial activity, differing from the report suggesting non-equivalence of FAD chromophores. 4. The findings in the present report indicate that FAD chromophores of chicken liver enzyme are essentially equivalent.     

Enzyme-catalyzed redox reactions with the flavin analogues 5-deazariboflavin, 5-deazariboflavin 5'-phosphte, and 5-deazariboflavin 5'-diphosphate, 5' leads to 5'-adenosine ester.

The ability of 5-deazaisoalloxazines to substitute for the isoalloxazine (flavin) coenzyme has been examined with several flavoenzymes. Without exception, the deazaflavin is recognized at the active site and undergoes a redox change in the presence of the specific enzyme substrate. Thus, deazariboflavin is reduced catalytically by NADH in the presence of the Beneckea harveyi NAD(P)H:(flavin) oxidoreductase, the reaction proceeding to an equilibrium with an equilibrium constant near unity. This implies an E0 of -0.310 V for the deazariboflavindihydrodeazariboflavin couple, much lower than that for isoalloxazines. With this enzyme, both riboflavin and deazariboflavin show the same stereospecificity with respect to the pyridine nucleotide, and despite a large difference in Vmax for the two, both have the same rate-determining step (hydrogen transfer). Direct transfer of the hydrogen is seen between the nicotinamide and deazariboflavin in both reaction directions. DeazaFMN reconstituted yeast NADPH: (acceptor) oxidoreductase (Old Yellow Enzyme), and deazaFAD reconstituted D-amino acid:O2 oxidoreductase and Aspergillus niger D-glucose O2 oxidoreductase are all reduced by substrate at approximately 10(-5) the rate of holoenzyme; none are reoxidized by oxygen or any of the tested artificial electron acceptors, though deazaFADH-bound to D-amino acid:O2 oxidoreductase is rapidly oxidized by the imino acid product. Direct hydrogen transfer from substrate to deazaflavin has been demonstrated for both deazaFAD-reconstituted oxidases. These data implicate deazaflavins as a unique probe of flavin catalysis, in that any mechanism for the flavin catalysis must account for the deazaflavin reactivity as well.