UNIPROT:O14944 - FACTA Search

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Query: UNIPROT:O14944 (EPR)
13,097 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Anaerobic reduction of the flavoprotein adrenodoxin reductase with NADPH yields a spectrum with long wavelength absorbance, 750 nm and higher. No EPR signal is observed. This spectrum is produced by titration of oxidized adrenodoxin reductase with NADPH, or of dithionite-reduced adrenodoxin reductase with NADP+. Both titrations yield a sharp endpoint at 1 NADP(H) added per flavin. Reduction with other reductants, including dithionite, excess NADH, and catalytic NADP+ with an NADPH generating system, yields a typical fully reduced flavin spectrum, without long wavelength absorbance. The species formed on NADPH reduction appears to be a two-electron-containing complex, with a low dissociation constant, between reduced adrenodoxin reductase and NADP+, designated ARH2-NADP+. Titration of dithionite-reduced adrenodoxin reductase with NADPH also produces a distinctive spectrum, with a sharp endpoint at 1 NADPH added per reduced flavin, indicating formation of a four-electron-containing complex between reduced adrenodoxin reductase and NADPH. Titration of adrenodoxin reductase with NADH, instead of NADPH, provides a curved titration plot rather than the sharp break seen with NADPH, and permits calculation of a potential for the AR/ARH2 couple of -0.291 V, close to that of NAD(P)H (-0.316 V). Oxidized adrenodoxin reductase binds NADP+ much more weakly (Kdiss=1.4 X 10(-5) M) than does reduced adrenodoxin reductase, with a single binding site. The preferential binding of NADP+ to reduced enzyme permits prediction of a more positive oxidation-reduction potential of the flavoprotein in the presence of NADP+; a change of about + 0.1 V has been demonstrated by titration with safranine T. From this alteration in potential, a Kdiss of 1.0 X 10(-8) M for binding of NADP+ to reduced adrenodoxin reductase is calculated. It is concluded that the strong binding of NADP+ to reduced adrenodoxin reductase provides the thermodynamic driving force for formation of a fully reduced flavoprotein form under conditions wherein incomplete reduction would otherwise be expected. Stopped flow studies demonstrate that reduction of adrenodoxin reductase by equimolar NADPH to form the ARH2-NADP+ complex is first order (k=28 s-1). When a large excess of NADPH is used, a second apparently first order process is observed (k=4.25 s-1), which is interpreted as replacement of NADPH for NADP+ in the ARH2-NADP+ complex. Comparison of these rate constants to catalytic flavin turnover numbers for reduction of various oxidants by NADPH, suggests an ordered sequential mechanism in which reduction of oxidant is accomplished by the ARH2-NADP+ complex, followed by dissociation of NADP+. The absolute dependence of NADPH-cytochrome c reduction on both adrenodoxin reductase and adrenodoxin is confirmed...
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PMID:Adrenodoxin reductase. Properties of the complexes of reduced enzyme with NADP+ and NADPH. 0 75

The soluble hydrogenase (hydrogen:NAD+ oxidoreductase (EC 1.12.1.2) from Alcaligenes eutrophus has been purified to homogeneity by an improved procedure, which includes preparative electrophoresis as final step. The specific activity of 57 mumol H2 oxidized/min per mg protein was achieved and the yield of pure enzyme from 200 g cells (wet weight) was about 16 mg/purification. After removal of non-functional iron, analysis of iron and acid-labile sulphur yielded average values of 11.5 and 12.9 atoms/molecule of enzyme, respectively. p-Chloromercuribenzoate was a strong inhibitor of hydrogenase and apparently competed with NAD not with H2. Chelating agents, CO and O2 failed to inhibit enzyme activity. The oxidized hydrogenase showed an EPR spectrum with a small signal at g = 2.02. On reduction the appearance of a high temperature (50--77 K) signal at g = 2.04, 1.95 and a more complex low temperature (less than 30 K) spectrum at g = 2.04, 2.0, 1.95, 1.93, 1.86 was observed. The pronounced temperature dependence and characteristic lineshape of the signals obtained with hydrogenase in 80--85% dimethylsulphoxide demonstrated that iron-sulphur centres of both the [2Fe-2S] and [4Fe-4S] types are present in the enzyme. Quantitation of the EPR signals indicated the existence of two identical centres each of the [4Fe-4S] and of the [2Fe-2S] type. The midpoint redox potentials of the [4Fe-4S] and the [2Fe-2S] centres were determined to be -445 mV and -325 mV, respectively. Spin coupling between two centres, indicated by the split feature of the low temperature spectrum of the native hydrogenase around g = 1.95, 1.93, has been established by power saturation studies. On reduction of the [Fe-4S] centres, the electron spin relaxation rate of the [2Fe-2S] centres was considerably increased. Treatment of hydrogenase with CO caused no change in EPR spectra.
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PMID:The iron-sulphur centres of soluble hydrogenase from Alcaligenes eutrophus. 22 63

Nonenzymatic reduction of methemoglobin (met-Hb) is effectively catalyzed by NADH and riboflavin (RF). At low concentrations of met-Hb the EPR spectra of the ternary complex are characterized by an increased, whereas those at high met-Hb concentrations--by a decreased intensity of the RF semiquinone signal, which suggests that the met Hb reduction by a Leu-form of RF proceeds at a higher rate than that by the RF semiquinone radical. Riboflavin also accelerates the met-Hb reduction by a NADH-Fe2+ or NADH-Fe(2+)-EDTA mixture. It was found that the rate of met-Hb reduction by the organic radical increases in the following order: NAD. < RF H. < RF H2.
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PMID:[Nonenzymatic reduction of methemoglobin by free radical forms of NADH and riboflavin]. 134 94

The enzymology of the heterodimeric (NiFe) and (NiFeSe) hydrogenases, the monomeric nickel-containing hydrogenases plus the multimeric F420-(NiFe) and NAD(+)-(NiFe) hydrogenases are summarized and discussed in terms of subunit localization of the redox-active nickel and non-heme iron clusters. It is proposed that nickel is ligated solely by amino acid residues of the large subunit and that the non-heme iron clusters are ligated by other cysteine-rich polypeptides encoded in the hydrogenase operons which are not necessarily homologous in either structure or function. Comparison of the hydrogenase operons or putative operons and their hydrogenase genes indicate that the arrangement, number and types of genes in these operons are not conserved among the various types of hydrogenases except for the gene encoding the large subunit. Thus, the presence of the gene for the large subunit is the sole feature common to all known nickel-containing hydrogenases and unites these hydrogenases into a large but diverse gene family. Although the different genes for the large subunits may possess only nominal general derived amino acid homology, all large subunit genes sequenced to date have the sequence R-X-C-X-X-C fully conserved in the amino terminal region of the polypeptide chain and the sequence of D-P-C-X-X-C fully conserved in the carboxyl terminal region. It is proposed that these conserved motifs of amino acids provide the ligands required for the binding of the redox-active nickel. The existing EXAFS (Extended X-ray Absorption Fine Structure) information is summarized and discussed in terms of the numbers and types of ligands to the nickel and the various redox species of nickel defined by EPR spectroscopy. New information concerning the ligands to nickel is presented based on site-directed mutagenesis of the gene encoding the large subunit of the (NiFe) hydrogenase-1 of Escherichia coli. Based on considerations of the biochemical, molecular and biophysical information, ligand environments of the nickel in different redox states of the (NiFe) hydrogenase are proposed.
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PMID:Structure-function relationships among the nickel-containing hydrogenases. 155 64

The role of phospholipid in the binding of coenzyme, NAD(H), to 3-hydroxybutyrate dehydrogenase, a lipid-requiring membrane enzyme, has been studied with the ultrafiltration binding method, which we optimized to quantitate weak ligand binding (KD in the range 10-100 microM). 3-Hydroxybutyrate dehydrogenase has a specific requirement of phosphatidylcholine (PC) for optimal function and is a tetramer quantitated both for the apodehydrogenase, which is devoid of phospholipid, and for the enzyme reconstituted into phospholipid vesicles in either the presence or absence of PC. We find that (i) the stoichiometry for NADH and NAD binding is 0.5 mol/mol of enzyme monomer (2 mol/mol of tetramer); (ii) the dissociation constant for NADH binding is essentially the same for the enzyme reconstituted into the mixture of mitochondrial phospholipids (MPL) (KD = 15 +/- 3 microM) or into dioleoyl-PC (KD = 12 +/- 3 microM); (iii) the binding of NAD+ to the enzyme-MPL complex is more than an order of magnitude weaker than NADH binding (KD approximately 200 microM versus 15 microM) but can be enhanced by formation of a ternary complex with either 2-methylmalonate (apparent KD = 1.1 +/- 0.2 microM) or sulfite to form the NAD-SO3- adduct (KD = 0.5 +/- 0.1 microM); (iv) the binding stoichiometry for NADH is the same (0.5 mol/mol) for binary (NADH alone) and ternary complexes (NADH plus monomethyl malonate); (v) binding of NAD+ and NADH together totals 0.5 mol of NAD(H)/mol of enzyme monomer, i.e., two nucleotide binding sites per enzyme tetramer; and (vi) the binding of nucleotide to the enzyme reconstituted with phospholipid devoid of PC is weak, being detected only for the NAD+ plus 2-methylmalonate ternary complex (apparent KD approximately 50 microM or approximately 50-fold weaker binding than that for the same complex in the presence of PC). The binding of NADH by equilibrium dialysis or of spin-labeled analogues of NAD+ by EPR spectroscopy gave complementary results, indicating that the ultrafiltration studies approximated equilibrium conditions. In addition to specific binding of NAD(H) to 3-hydroxybutyrate dehydrogenase, we find significant binding of NAD(H) to phospholipid vesicles. An important new finding is that the nucleotide binding site is present in 3-hydroxybutyrate dehydrogenase in the absence of activating phospholipid since (a) NAD+, as the ternary complex with 2-methylmalonate, binds to the enzyme reconstituted with phospholipid devoid of PC and (b) the apodehydrogenase, devoid of phospholipid, binds NADH or NAD-SO3- weakly (half-maximal binding at approximately 75 microM NAD-SO3- and somewhat weaker binding for NADH).(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Coenzyme binding by 3-hydroxybutyrate dehydrogenase, a lipid-requiring enzyme: lecithin acts as an allosteric modulator to enhance the affinity for coenzyme. 255 53

An efficient method of one-electron reduction of the anticancer agent mitoxantrone is described. The method depends on illumination of a suitable photosensitizer absorbing blue light [acriflavine, anthrapyrazole, or Ru(bpy)2+(3)] in the presence of the drug and an electron donor, such as NAD(P)H, in deaerated solutions. An EPR spectrum, assigned to a semiquinone of mitoxantrone, is generated under these conditions and identified by spectral simulation. Decay of this species, attributed to a radical-radical reaction, gives a second order rate constant of 1.7 x 10(2) M-1 s-1 in organic media [dimethylsulfoxide (DMSO)/pH 8 buffer, 1:1 vol/vol] but is more rapid (approximately 10(4) M-1 s-1) in aqueous media under comparable conditions. The considerably decreased lifetime of the mitoxantrone radical at pH 5 is attributed to an additional electron transfer, promoted by protonation of the radical, and/or to an accelerated recombination of neutral radicals, leading to an EPR-silent species. Parallel spectrophotometric studies on the generation of the mitoxantrone reduced species by photosensitized reduction are described. The method offers convenient access to a key radical species involved in the metabolism and possible mode of action of this clinical anticancer agent.
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PMID:Photosensitization of anticancer agents--8. One-electron reduction of mitoxantrone: an EPR and spectrophotometric study. 255 Sep 74

An EPR spectrum of the semiquinone radical of daunorubicin (D) was recorded upon illumination (480 nm) of the drug and NAD(P)H in deaerated aqueous and DMF/aqueous solutions. In the latter solvent system, an EPR spectrum with hyperfine structure was recorded. The kinetics of the photoinduced generation and decrease of the EPR signal intensity in the dark were measured. Second order rate constants for the radical recombination were derived for the two solvent systems. Photosensitized production of the superoxide radical, upon illumination of daunorubicin and NAD(P)H, in aerated aqueous or DMF/aqueous solutions, is evidenced by employing a spin trap DMPO (5,5-dimethyl-1-pyrroline-N-oxide) and an SOD assay. 5-Iminodaunorubicin (5-ID), in contrast to the parent compound (D), does not possess photosensitizing properties.
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PMID:Photosensitization by antitumor agents. 5. Daunorubicin-photosensitized oxidation of NAD(P)H in aqueous and N,N-dimethylformamide/aqueous solutions--an electron paramagnetic resonance study. 285 19

D-beta-Hydroxybutyrate dehydrogenase (D-3-hydroxybutyrate:NAD+ oxidoreductase, EC 1.1.1.30) is a lipid-requiring enzyme which specifically requires phosphosphatidylcholine for enzymic activity. The phosphatidylcholine modifies the binding and orientation of the coenzyme, NAD(H), with respect to the enzyme. In the present study, two derivatives of NAD, spin-labeled either at N-6 or C-8 of the adenine ring, were found to be active as coenzyme. The binding affinity of NADH to the enzyme was opitimized by increasing the salt concentration and increasing the pH from 6 to 8, with the pK at 6.8. Monomethylmalonate, a substrate analogue, was found to enhance NADH binding (Kd is reduced from 4 to 1 microM). Sulfite strongly enhances the binding of NAD+ via the enzyme-catalyzed formation of an adduct of sulfite with the nucleotide; the Kd for binding of NAD-sulfite is in the micromolar range, whereas NAD+ binding is more than a magnitude weaker. The binding of spin-labeled NAD(H) was further characterized by EPR spectroscopy. Increased sensitivity and resolution were obtained with the use of NAD(H) analogues perdeuterated in the spin-label moiety. For these analogues bound to D-beta-hydroxybutyrate dehydrogenase in phospholipid vesicles, EPR studies showed the spin-label moiety to be constrained and revealed two distinct components. Increasing the viscosity of the medium by addition of glycerol affected the EPR spectral characteristics of only the component with the smaller resolved averaged hyperfine splitting. The stage is now set to study motional characteristics of the enzyme, using these spin-labeled probes which mimic the coenzyme.
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PMID:Complex formation between nucleotides and D-beta-hydroxybutyrate dehydrogenase studied by fluorescence and EPR spectroscopy. 609 15

Crude extracts of Clostridium kluyveri, Clostridium spec. La 1, Clostridium sporogenes and Clostridium pasteurianum catalyse the NADH-dependent reduction of the nitro group of p-nitrobenzoate. The former three Clostridia also use pyruvate as electron donor for this reduction. The NADH-dependent reductases have been partially purified and characterized from Clostridium kluyveri. Nitroalkyl compounds as well as nitrite, sulfite, sulfate and hydroxylamine are no substrates. Based on chromatographic behavior, separation pattern, yields, stability, pH optima, molecular masses and EPR studies the three NADH-dependent nitroaryl group reducing enzymes in Clostridium kluyveri (three activities in Clostridium spec. La 1 and two activities in Clostridium sporogenes) are different from alcohol dehydrogenase, aldehyde dehydrogenase, 3-hydroxy-butyryl-CoA dehydrogenase, butyryrl-CoA dehydrogenase, 2-enoate reductase, ferredoxin-NAD and ferredoxin-NADP reductase. The physiological roles of the nitroaryl reductases are not known. The reductase activities show losses of 80-90% during classical protein purification procedures. One of the three nitroaryl reductases exhibits a pH optimum of 10.5. The crude extract reveals a pH optimum at 11.5. The first step of the reduction reaction leads to the nitroradical anion (1 electron transfer). The electron transfer to p-nitrobenzoate is also catalysed by ferrodoxin-NAD reductase from NADH and by ferredoxin-NADP reductase from NADP. Partially purified 2-oxo-acid synthases from Clostridium sporogenes catalyse with low rates the reduction of p-nitrobenzoate as well as 2-nitroethanol in the presence and absence of ferredoxin using pyruvate or 2-oxo-4-methylpentanoate as electron donors, respectively. The NADH-dependent reduction of p-nitro-benzoate accounts for at least 70% and the 2-oxo acid-dependent reduction for about 5% of the total nitroaryl reductase activity in the Clostridia. It seems that the pyridine nucleotide-dependent nitroaryl reductases are enzymes so far unknown in Clostridia.
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PMID:On nitroaryl reductase activities in several Clostridia. 632 15

4-Dimethylaminophenol (DMAP), after intravenous injection, rapidly forms ferrihaemoglobin and has been successfully used in the treatment of cyanide poisoning. Since DMAP produces many equivalents of ferrihaemoglobin, it was of interest to obtain further insight into this catalytic process. DMAP autoxidizes readily at pH regions above neutrality, a process which is markedly accelerated by oxyhaemoglobin. The resulting red-coloured product was identified as the 4-(N,N-dimethylamino) phenoxyl radical by EPR spectroscopy. The same radical was also produced by pulse radiolysis and oxidation with ferricyanide. The 4-(N,N-dimethylamino)phenoxyl radical is quite unstable and decays in a pseudo-first order reaction (k = 0.4 sec-1 at pH 8.5, 22 degrees) with the formation of p-benzoquinone and dimethylamine. This observed decay rate is identical with the rate of hydrolysis of N,N-dimethylquinonimine. When a solution containing the phenoxyl radical was extracted with ether, half the stoichiometric amount of DMAP was recovered. Hence it is apparent that the phenoxyl radical decays by disproportionation yielding DMAP and N,N-dimethylquinonimine. The latter product then quickly hydrolyses. The equilibrium of this disproportionation reaction is far towards the radical side, and the pseudo-first order hydrolysis controls the radical decay rate. p-Benzoquinone rapidly reacts with DMAP (k2 = 2 X 10(4) M-1 sec-1) with the formation of the 4-(N,N-dimethylamino)phenoxyl and the semiquinone radicals. This reaction explains the autocatalytic phenoxyl radical formation during autoxidation of DMAP. DMAP is not oxidized by H2O2 or O-.2 but the 4-(N,N-dimethylamino)phenoxyl radical is very rapidly reduced by O-.2 (k2 = 2 X 10(8) M-1 sec-1). In addition, the phenoxyl radical is quickly reduced by NAD(P)H or GSH with the formation of NAD(P)+ or GSSG. Since DMAP is also able to reduce two equivalents of ferrihaemoglobin (provided that the ferrohaemoglobin produced is trapped by carbon monoxide), electrophilic addition reactions of the phenoxyl radical seem unimportant in contrast to N,N-dimethylquinonimine. Hence, during the catalytic ferrihaemoglobin formation, DMAP is oxidized by oxygen which is activated by haemoglobin, and the phenoxyl radical oxidizes ferrohaemoglobin. This catalytic process is terminated by covalent binding of N,N-dimethylquinonimine to SH groups of haemoglobin (and GSH in red cells).
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PMID:Radical formation during autoxidation of 4-dimethylaminophenol and some properties of the reaction products. 632 8

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