Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:1.6.99.3 (diaphorase)
5,903 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The respiratory chain NADH:ubiquinone oxidoreductase (NADH dehydrogenase or Complex I) of mitochondria comprises some 30 different subunits, and one FMN and 4 or 5 iron-sulfur clusters as internal redox groups. The bacterial glucose dehydrogenase, which oxidizes glucose to gluconolactone in the periplasmatic space and transfers the electrons to ubiquinone, is a single polypeptide chain with pyrolloquinoline quinone as the only redox group. We report here that the two different enzymes have the same ubiquinone binding domain motif and we discuss the predicted membrane folding of this domain with regard to its role in the proton translocating function of the two enzymes.
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PMID:The same domain motif for ubiquinone reduction in mitochondrial or chloroplast NADH dehydrogenase and bacterial glucose dehydrogenase. 214 3

Several NAD(P)H-dependent ferri-reductase activities were detected in sub-cellular extracts of the yeast Saccharomyces cerevisiae. Some were induced in cells grown under iron-deficient conditions. At least two cytosolic iron-reducing enzymes having different substrate specificities could contribute to iron assimilation in vivo. One enzyme was purified to homogeneity: it is a flavoprotein (FAD) of 40 kDa that uses NADPH as electron donor and Fe(III)-EDTA as artificial electron acceptor. Isolated mitochondria reduced a variety of ferric chelates, probably via an 'external' NADH dehydrogenase, but not the siderophore ferrioxamine B. A plasma membrane-bound ferri-reductase system functioning with NADPH as electron donor and FMN as prosthetic group was purified 100-fold from isolated plasma membranes. This system may be involved in the reductive uptake of iron in vivo.
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PMID:Iron-reductases in the yeast Saccharomyces cerevisiae. 218 97

A catalytic component of the bovine mitochondrial NADH:ubiquinone oxidoreductase complex (Complex I) is a soluble NADH dehydrogenase iron-sulfur flavoprotein (FP). FP is composed of three subunits of Mr 51,000, 24,000, and 9,000, and contains FMN and two iron-sulfur clusters. Previous studies by others with the use of various chemical probes had suggested that, except for an access for NADH to the 51-kDa subunit, the FP polypeptides are buried within Complex I and shielded from the medium. In the present study, monospecific antibodies were raised to each of the three FP subunits, and used in conjunction with Complex I, submitochondrial particles (SMP), mitoplasts, and intact mitochondria as sources of antigens. Results of enzyme-linked immunosorbent assays and 125I-protein A labeling experiments indicated that epitopes from the 51-, 24-, and 9-kDa subunits of FP are exposed to the medium in Complex I and SMP, but not in mitoplasts and mitochondria. Appropriate enzymatic assays showed that none of the antibodies inhibited the NADH dehydrogenase activity of isolated FP or the NADH oxidase activity of SMP. These results have been discussed in relation to the structure of Neurospora Complex I deduced from membrane crystals of the isolated enzyme complex by Leonard et al. [K. Leonard, H. Haiker, and H. Weiss (1987) J. Mol. Biol. 194, 277-286].
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PMID:Studies on the structure of NADH:ubiquinone oxidoreductase complex: topography of the subunits of the iron-sulfur flavoprotein component. 246 82

1. An NADH-dependent nitroreductase from the inner membrane of ox liver mitochondria copurified with Complex I of the respiratory redox chain (NADH:ubiquinone oxidoreductase, EC 1.6.5.3). 2. The corresponding nitroreductase from ox heart mitochondria co-purified with the NADH-cytochrome c reductase of Mahler, Sarkar & Vernon [(1952) J. Biol. Chem. 199, 585-597] [NADH: (acceptor) oxidoreductase, EC 1.6.99.3], a component of Complex I that contains the FMN. 3. The mitochondrial nitroreductase activity is attributed to the flavoprotein component of Complex I.
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PMID:Nitroreductase activity of NADH dehydrogenase of the respiratory redox chain. 249 90

The electrostatically stabilized complex between Anabaena variabilis ferredoxin--NADP+ reductase and Azotobacter vinelandii flavodoxin has been covalently cross-linked by treatment with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide. The covalent complex exhibits a molecular mass and FMN/FAD content consistent with that expected for a 1:1 stoichiometry of the two flavoproteins. Immunochemical cross-reactivity is exhibited by the covalent complex with rabbit antisera prepared separately against each protein. The complex retains NADPH-ferricyanide diaphorase activity although the Km for ferricyanide is increased twofold and the turnover number is decreased by a factor of two when compared to native reductase. NADPH-cytochrome-c reductase activity of the complex is observed at a level that is quite similar to that determined at saturating concentrations of flavodoxin, while it is only 1-2% of that exhibited by the reductase in the presence of ferredoxin. No stimulation of cytochrome-c reductase activity is observed on adding ferredoxin to the cross-linked complex. Stopped-flow data show that covalent cross-linking of the flavodoxin to the reductase reduces the rate of electron transfer from its semiquinone form to cytochrome c by a factor of 60. Anaerobic titrations of the reduced complex with NADP+ show the semiquinone/quinol couple of the flavodoxin is increased 100 mV relative to the free form and the quinone/quinol couple of complexed ferredoxin-NADP+ reductase is increased by only 25 mV, relative to the free protein. Addition of NADPH to the cross-linked complex reduces the FAD of the reductase as well as the FMN moiety of flavodoxin to a mixture of semiquinone and quinol forms.
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PMID:Preparation and properties of a cross-linked complex between ferredoxin--NADP+ reductase and flavodoxin. 250 11

The steady-state kinetics of oxidation of the mitochondrial NADH: ubiquinone oxidoreductase (complex I, EC 1.6.99.3) by artificial electron acceptors--p-quinones and inorganic complexes has been investigated. A limiting stage in the NADH: ferricyanide reductase reaction is a reductive half-reaction. Ferricyanide interacts with negative-charged protein groups taking part in the NADH binding. The rate constants of the quinone reduction by complex I vary from 1.10(6) to 4.10(3) M-1s-1. The NADH, NAD+ and ADP-ribose inhibition data indicate that oxidizers in the rotenono-insensitive reaction interact with the redox centre near the NAD+/NADH binding site, most probably with FMN.
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PMID:[Reaction of complex I of the mitochondrial electron transport chain with artificial oxidizers]. 251 53

NADPH-sulfite reductase flavoprotein (SiR-FP) was purified from a Salmonella typhimurium cysG strain that does not synthesize the hemoprotein component of the sulfite reductase holoenzyme. cysJ, which codes for SiR-FP, was cloned from S. typhimurium LT7 and Escherichia coli B, and both genes were sequenced. Physicochemical analyses and deduced amino acid sequences indicate that SiR-FP is an octamer of identical 66-kDa peptides and contains 4 FAD and 4 FMN per octamer. Potentiometric titrations of SiR holoenzyme, SiR-FP, and FMN-depleted SiR-FP yielded the following redox potentials for the prosthetic groups at pH 7.7: E'1 (FMNH./FMN) = -152 mV; E'2 (FMNH2/FMNH.) = -327 mV; E'3 (FADH./FAD) = -382 mV; E'4 (FADH2/FADH.) = -322 mV. Microcoulometric titration of SiR-FP at 25 degrees C yielded data which were in full agreement with these potentials. Spectroscopic and catalytic studies of native SiR-FP and of SiR-FP depleted of FMN support the following electron flow sequence: NADPH----FAD----FMN. FMN can then contribute electrons to the hemoprotein component of sulfite reductase, as well as to cytochrome c and various diaphorase acceptors. The FMN is postulated to cycle between the FMNH2 and FMNH. oxidation states during catalysis; in this sense SiR-FP shares a catalytic mechanism with NADPH-cytochrome P-450 oxidoreductase. SiR-FP domains involved in binding FMN, FAD, and NADPH are proposed from amino acid sequence homologies with Desulfovibrio vulgaris flavodoxin (Dubourdieu, M., and Fox, J.L. (1977) J. Biol. Chem. 252, 1453-1463) and spinach ferredoxin-NADP+ oxidoreductase (Karplus, P.A., Walsh, K.A., and Herriott, J. R. (1984) Biochemistry 23, 6576-6583). Comparison of the deduced amino acid sequences of SiR-FP and NADPH-cytochrome P-450 oxidoreductase (Porter, T. D., and Kasper, C.B. (1985) Proc. Natl. Acad. Sci. U. S.A. 82, 973-977) also showed identities that suggest these two proteins are descended from a common precursor, which contained binding regions for both FMN and FAD.
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PMID:Characterization of the flavoprotein moieties of NADPH-sulfite reductase from Salmonella typhimurium and Escherichia coli. Physicochemical and catalytic properties, amino acid sequence deduced from DNA sequence of cysJ, and comparison with NADPH-cytochrome P-450 reductase. 255 Apr 23

The protomeric chain of Hansenula anomala flavocytochrome b2 was previously shown to be built as the covalent association of two functional domains: an L-lactate dehydrogenase domain and a cytochrome c reductase domain, joined together by a proteolytically sensitive zone. This paper concerns the specific cleavage of this latter zone with a H. anomala proteinase(s) preparation and the purification of the resulting L-lactate dehydrogenase moiety of the molecule with at least 25% recovery, (i.e. one order of magnitude more than for the previously published method). A preliminary characterization of this dehydrogenase domain indicates that it is a tetramer (Mr = 4 x 39000) containing FMN as expected and not heme. It has high L-lactate:ferricyanide oxidoreductase activity (about 70% that of the whole flavocytochrome b2) and the same Km for L(+)-lactate as flavocytochrome b2, but it has no L-lactate:cytochrome c oxidoreductase activity. Its flavin semiquinone is stabilized in the presence of pyruvate as in flavocytochrome b2. The subcellular origin of the H. anomala proteinase in the preparation has not yet been elucidated.
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PMID:Isolation of the flavodehydrogenase domain of Hansenula anomala flavocytochrome b2 after mild proteolysis by an H. anomala proteinase. 265 53

Comparison of the amino acid sequence of rat liver NADPH-cytochrome P-450 oxidoreductase with that of flavoproteins of known three-dimensional structure suggested that residues Tyr-140 and Tyr-178 are involved in binding of FMN to the protein. To test this hypothesis, NADPH-cytochrome P-450 oxidoreductase was expressed in Escherichia coli using the expression-secretion vector pIN-III-ompA3, and site-directed mutagenesis was employed to selectively alter these residues and demonstrate that they are major determinants of the FMN-binding site. Bacterial expression produced a membrane-bound 80-kDa protein containing 1 mol each of FMN and FAD per mol of enzyme, which reduced cytochrome c at a rate of 51.5 mumol/min/mg of protein and had absorption spectra and kinetic properties very similar to those of the rat liver enzyme. Replacement of Tyr-178 with aspartate abolished FMN binding and cytochrome c reductase activity. Incubation with FMN increased catalytic activity to a maximum of 8.6 mumol/min/mg of protein. Replacement of Tyr-140 with aspartate did not eliminate FMN binding, but reduced cytochrome c reductase activity about 5-fold, suggesting that FMN may be bound in a conformation which does not permit efficient electron transfer. Substitution of phenylalanine at either position 140 or 178 had no effect on FMN content or catalytic activity. The FAD level in the Asp-178 mutant was also decreased, suggesting that FAD binding is dependent upon FMN; FAD incorporation may occur co-translationally and require prior formation of an intact FMN domain.
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PMID:Structural analysis of the FMN binding domain of NADPH-cytochrome P-450 oxidoreductase by site-directed mutagenesis. 270 80

Considerable evidence suggests that the release of iron from ferritin is a reductive process. A role in this process has been proposed for two hepatic enzymes, namely xanthine oxidoreductase and an NADH oxidoreductase. The abilities of xanthine and NADH to serve as a source of reducing power for the enzyme-mediated release of ferritin iron (ferrireductase activity) were compared with turkey liver and rat liver homogenates. The maximal velocity (Vmax.) for the reaction with NADH was 50 times greater than with xanthine; however, the substrate concentration required to achieve half-maximal velocity (Km) was 1000 times less with xanthine than with NADH. NADPH could be substituted for NADH with little loss in activity. Dicoumarol did not inhibit the reaction with NADH or NADPH, demonstrating that the ferrireductase activity with those substrates was not the result of the liver enzyme 'DT-diaphorase' [NAD(P)H dehydrogenase (quinone)]. A flavin nucleotide was required for ferrireductase activity with rat and turkey liver cytosol when xanthine, NADH or NADPH was used as the reducing substrate. FMN yielded twice the activity with NADH or NADPH, whereas FAD was twice as effective with xanthine as substrate. Kinetic comparisons, differences in lability and partial chromatographic resolution of the ferrireductase activities with the two types of reducing substrates strongly indicate that the ferrireductase activities with xanthine and NADH are catalysed by separate enzyme systems contained in liver cytosol. Complete inhibition by allopurinol of the ferrireductase activity endogenous to undialysed liver cytosol preparations and the ability of xanthine to restore equivalent activity to dialysed preparations indicate that the source of reducing power for the endogenous activity is xanthine. These studies suggest that xanthine, NADH or NADPH can serve as a source of reducing power for the enzyme-mediated reduction of ferritin iron, with a flavin nucleotide serving as the shuttle of electrons from the enzymes to the ferritin iron.
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PMID:The mobilization of ferritin iron by liver cytosol. A comparison of xanthine and NADH as reducing substrates. 277 99


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