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PubMed1987

Purification and some properties of sulfur:ferric ion oxidoreductase from Thiobacillus ferrooxidans.

A sulfur:ferric ion oxidoreductase that utilizes ferric ion (Fe3+) as an electron acceptor of elemental sulfur was purified from iron-grown Thiobacillus ferrooxidans to an electrophoretically homogeneous state. Under anaerobic conditions in the presence of Fe3+, the enzyme reduced 4 mol of Fe3+ with 1 mol of elemental sulfur to give 4 mol of Fe2+ and 1 mol of sulfite, indicating that it corresponds to a ferric ion-reducing system (T. Sugio, C. Domatsu, O. Munakata, T. Tano, and K. Imai, Appl. Environ. Microbiol. 49:1401-1406, 1985). Under aerobic conditions, sulfite, but not Fe2+, was produced during the oxidation of elemental sulfur by this enzyme because the Fe2+ produced was rapidly reoxidized chemically by molecular oxygen. The possibility that Fe3+ serves as an electron acceptor under aerobic conditions was ascertained by adding o-phenanthroline, which chelates Fe2+, to the reaction mixture. Sulfur:ferric ion oxidoreductase had an apparent molecular weight of 46,000, and it is composed of two identical subunits (Mr = 23,000) as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Sulfur oxidation by this enzyme was absolutely dependent on the presence of reduced glutathione. The enzyme had an isoelectric point and a pH optimum at pH 4.6 and 6.5, respectively. Almost all the activity of sulfur:ferric ion oxidoreductase was observed in the osmotic shock fluid of the cells, suggesting that it was localized in the periplasmic space of the cells.

Glutathione↗
PubMed1987

Klebsiella pneumoniae 1,3-propanediol:NAD+ oxidoreductase.

Fermentative utilization of glycerol, a more reduced carbohydrate than aldoses and ketoses, requires the disposal of the two extra hydrogen atoms. This is accomplished by sacrificing an equal quantity of glycerol via an auxiliary pathway initiated by glycerol dehydratase. The product, 3-hydroxypropionaldehyde, is then reduced by 1,3-propanediol NAD+:oxidoreductase (1,3-propanediol dehydrogenase; EC 1.1.1.202), resulting in the regeneration of NAD+ from NADH. The pathway for the assimilation of glycerol is initiated by an NAD-linked dehydrogenase. In Klebsiella pneumoniae the two pathways are encoded by the dha regulon which is inducible only anaerobically. In this study 1,3-propanediol:NAD+ oxidoreductase was purified from cells grown anaerobically on glycerol. The enzyme was immunochemically distinct from the NAD-linked glycerol dehydrogenase and was an octamer or hexamer of a polypeptide of 45,000 +/- 3,000 daltons. When tested as a dehydrogenase, only 1,3-propanediol served as a substrate; no activity was detected with ethanol, 1-propanol, 1,2-propanediol, glycerol, or 1,4-butanediol. The enzyme was inhibited by chelators of divalent cations. An enzyme preparation inhibited by alpha,alpha'-dipyridyl was reactivated by the addition of Fe2+ or Mn2+ after removal of the chelator by gel filtration. As for glycerol dehydrogenase, 1,3-propanediol oxidoreductase is apparently inactivated by oxidation during aerobic metabolism, under which condition the enzyme becomes superfluous.

Alcohol Dehydrogenase↗
PubMed1992

Purification and some properties of sulfite:ferric ion oxidoreductase from Thiobacillus ferrooxidans.

Sulfite:ferric ion oxidoreductase in the plasma membrane of Thiobacillus ferrooxidans AP19-3 was purified to an electrophoretically homogeneous state. The enzyme had an apparent molecular weight of 650,000 and was composed of two subunits (M(rs), 61,000 and 59,000) as estimated by sodium sulfate-polyacrylamide gel electrophoresis. The Michaelis constants of sulfite:ferric ion oxidoreductase for Fe3+ and sulfite ions were 1.0 and 0.071 mM, respectively. Sulfite:ferric ion oxidoreductase suffered from end product inhibition by 1 mM Fe2+.

Electrophoresis↗
PubMed1993

Characterization of the baiH gene encoding a bile acid-inducible NADH:flavin oxidoreductase from Eubacterium sp. strain VPI 12708.

A cholate-inducible, NADH-dependent flavin oxidoreductase from the intestinal bacterium Eubacterium sp. strain VPI 12708 was purified 372-fold to apparent electrophoretic homogeneity. The subunit and native molecular weights were estimated to be 72,000 and 210,000, respectively, suggesting a homotrimeric organization. Three peaks of NADH:flavin oxidoreductase activity (forms I, II, and III) eluted from a DEAE-high-performance liquid chromatography column. Absorption spectra revealed that purified form III, but not form I, contained bound flavin, which dissociated during purification to generate form I. Enzyme activity was inhibited by sulfhydryl-reactive compounds, acriflavine, o-phenanthroline, and EDTA. Activity assays and Western blot (immunoblot) analysis confirmed that expression of the enzyme was cholate inducible. The first 25 N-terminal amino acid residues of purified NADH:flavin oxidoreductase were determined, and a corresponding oligonucleotide probe was synthesized for use in cloning of the associated gene, baiH. Restriction mapping, sequence data, and RNA blot analysis suggested that the baiH gene was located on a previously described, cholate-inducible operon > or = 10 kb long. The baiH gene encoded a 72,006-Da polypeptide containing 661 amino acids. The deduced amino acid sequence of the baiH gene was homologous to that of NADH oxidase from Thermoanaerobium brockii, trimethylamine dehydrogenase from methylotrophic bacterium W3A1, Old Yellow Enzyme from Saccharomyces carlsbergensis, and the product of the baiC gene of Eubacterium sp. strain VPI 12708, located upstream from the baiH gene in the cholate-inducible operon. Alignment of these five sequences revealed potential ligands for an iron-sulfur cluster, a putative flavin adenine dinucleotide-binding domain, and two other well-conserved domains of unknown function.

Amino Acid Sequence↗
PubMed1993

Electron microscopic analysis and structural characterization of novel NADP(H)-containing methanol: N,N'-dimethyl-4-nitrosoaniline oxidoreductases from the gram-positive methylotrophic bacteria Amycolatopsis methanolica and Mycobacterium gastri MB19.

The quaternary protein structure of two methanol:N,N'-dimethyl-4-nitrosoaniline (NDMA) oxidoreductases purified from Amycolatopsis methanolica and Mycobacterium gastri MB19 was analyzed by electron microscopy and image processing. The enzymes are decameric proteins (displaying fivefold symmetry) with estimated molecular masses of 490 to 500 kDa based on their subunit molecular masses of 49 to 50 kDa. Both methanol:NDMA oxidoreductases possess a tightly but noncovalently bound NADP(H) cofactor at an NADPH-to-subunit molar ratio of 0.7. These cofactors are redox active toward alcohol and aldehyde substrates. Both enzymes contain significant amounts of Zn2+ and Mg2+ ions. The primary amino acid sequences of the A. methanolica and M. gastri MB19 methanol:NDMA oxidoreductases share a high degree of identity, as indicated by N-terminal sequence analysis (63% identity among the first 27 N-terminal amino acids), internal peptide sequence analysis, and overall amino acid composition. The amino acid sequence analysis also revealed significant similarity to a decameric methanol dehydrogenase of Bacillus methanolicus C1.

Actinobacteria↗
PubMed1996

Succinate:quinone oxidoreductase (complex II) containing a single heme b in facultative alkaliphilic Bacillus sp. strain YN-2000.

Succinate:quinone oxidoreductase (EC 1.3.5.1) was first purified from the facultative alkaliphilic Bacillus sp. strain YN-2000 in the presence of Triton X-100. The isolated enzyme showed high succinate-ubiquinone oxidoreductase activity at pH 8.5. The Km for ubiquinone 1 and the Vmax of the enzyme were determined to be about 5 microM and 48 micromol of ubiquinone 1 per min per mg, respectively. The catalytic activity of the enzyme was 50% inhibited by 9 microM 2-thenoyltrifluoroacetone or 0.8 microM 2-n-heptyl-4-hydroxyquinoline- N-oxide. The enzyme consisted of three kinds of subunits with molecular masses of 66, 26, and 15 kDa, respectively, and contained 1.28 mol of covalently bound flavin adenine dinucleotide, 0.9 mol of heme b, 1.35 mol of menaquinone, 8.3 mol of nonheme iron, and 7.5 mol of inorganic sulfide per mol of enzyme. The enzyme showed symmetrical alpha absorption peaks at 556.5 and 554 nm in the reduced state at room temperature and 77 K, respectively. The potentiometric analysis of the enzyme yielded an Em,7 of heme b of about -64 mV (n = 1). Furthermore, the content of the enzyme was increased up to fivefold when the bacterium was grown at pH 10 compared with pH 7. These results indicate that the succinate:quinone oxidoreductase with a single heme b is involved in the respiratory chain of the alkaliphile at a very alkaline pH.

Bacillus↗
PubMed1996

Characterization of a fourth type of 2-keto acid-oxidizing enzyme from a hyperthermophilic archaeon: 2-ketoglutarate ferredoxin oxidoreductase from Thermococcus litoralis.

Thermococcus litoralis is a strictly anaerobic archaeon (archaebacterium) that grows at temperatures up to 98 degrees C by fermenting peptides. It is known to contain three distinct ferredoxin-dependent, 2-keto acid oxidoreductases, which use pyruvate, aromatic 2-keto acids such as indolepyruvate, or branched-chain 2-keto acids such as 2-ketoisovalerate, as their primary substrates. We show here that T. litoralis also contains a fourth member of this family of enzymes, 2-ketoglutarate ferredoxin oxidoreductase (KGOR). In the presence of coenzyme A, KGOR catalyzes the oxidative decarboxylation of 2-ketoglutarate to succinyl coenzyme A and CO2 and reduces T. litoralis ferredoxin. The enzyme was oxygen sensitive (half-life of approximately 5 min) and was purified under anaerobic conditions. It had an M(r) of approximately 210,000 and appeared to be an octomeric enzyme (alpha2beta2gamma2delta2) with four different subunits with M(r)s of 43,000 (alpha), 29,000 (beta), 23,000 (gamma), and 10,000 (delta). The enzyme contained 0.9 mol of thiamine PPi and at least four [4Fe-4S] clusters per mol of holoenzyme as determined by metal analyses and electron paramagnetic resonance spectroscopy. Significant amounts of other metals (Cu, Zn, Mo, W, and Ni) were not present (<0.1 mol/mol of holoenzyme). Pure KGOR did not utilize other 2-keto acids, such as pyruvate, indolepyruvate, or 2-ketoisovalerate, as substrates, and the apparent Km values for 2-ketoglutarate, coenzyme A, T. litoralis ferredoxin, and thiamine PPi were approximately 250, 40, 8, and 9 microM, respectively. The enzyme was virtually inactive at 25 degrees C and exhibited optimal activity above 90 degrees C (at pH 8.0) and at pH 8.0 (at 80 degrees C). KGOR was quite thermostable, with a half-life at 80 degrees C (under anaerobic conditions) of about 2 days. An enzyme analogous to KGOR has been previously purified from a mesophilic archaeon, but the molecular properties of T. litoralis KGOR more closely resemble those of the other oxidoreductases from hyperthermophiles. In contrast to these enzymes, however, KGOR appears to have a biosynthetic function rather than a role in energy conservation.

Acyl Coenzyme A↗
PubMed1999

Purification and molecular characterization of the tungsten-containing formaldehyde ferredoxin oxidoreductase from the hyperthermophilic archaeon Pyrococcus furiosus: the third of a putative five-member tungstoenzyme family.

Pyrococcus furiosus is a hyperthermophilic archaeon which grows optimally near 100 degreesC by fermenting peptides and sugars to produce organic acids, CO2, and H2. Its growth requires tungsten, and two different tungsten-containing enzymes, aldehyde ferredoxin oxidoreductase (AOR) and glyceraldehyde-3-phosphate ferredoxin oxidoreductase (GAPOR), have been previously purified from P. furiosus. These two enzymes are thought to function in the metabolism of peptides and carbohydrates, respectively. A third type of tungsten-containing enzyme, formaldehyde ferredoxin oxidoreductase (FOR), has now been characterized. FOR is a homotetramer with a mass of 280 kDa and contains approximately 1 W atom, 4 Fe atoms, and 1 Ca atom per subunit, together with a pterin cofactor. The low recovery of FOR activity during purification was attributed to loss of sulfide, since the purified enzyme was activated up to fivefold by treatment with sulfide (HS-) under reducing conditions. FOR uses P. furiosus ferredoxin as an electron acceptor (Km = 100 microM) and oxidizes a range of aldehydes. Formaldehyde (Km = 15 mM for the sulfide-activated enzyme) was used in routine assays, but the physiological substrate is thought to be an aliphatic C5 semi- or dialdehyde, e.g., glutaric dialdehyde (Km = 1 mM). Based on its amino-terminal sequence, the gene encoding FOR (for) was identified in the genomic database, together with those encoding AOR and GAPOR. The amino acid sequence of FOR corresponded to a mass of 68.7 kDa and is highly similar to those of the subunits of AOR (61% similarity and 40% identity) and GAPOR (50% similarity and 23% identity). The three genes are not linked on the P. furiosus chromosome. Two additional (and nonlinked) genes (termed wor4 and wor5) that encode putative tungstoenzymes with 57% (WOR4) and 56% (WOR5) sequence similarity to FOR were also identified. Based on sequence motif similarities with FOR, both WOR4 and WOR5 are also proposed to contain a tungstobispterin site and one [4Fe-4S] cluster per subunit.

Aldehyde Oxidoreductases↗
PubMed2005

WOR5, a novel tungsten-containing aldehyde oxidoreductase from Pyrococcus furiosus with a broad substrate Specificity.

WOR5 is the fifth and last member of the family of tungsten-containing oxidoreductases purified from the hyperthermophilic archaeon Pyrococcus furiosus. It is a homodimeric protein (subunit, 65 kDa) that contains one [4Fe-4S] cluster and one tungstobispterin cofactor per subunit. It has a broad substrate specificity with a high affinity for several substituted and nonsubstituted aliphatic and aromatic aldehydes with various chain lengths. The highest catalytic efficiency of WOR5 is found for the oxidation of hexanal (V(max) = 15.6 U/mg, K(m) = 0.18 mM at 60 degrees C). Hexanal-incubated enzyme exhibits S = 1/2 electron paramagnetic resonance signals from [4Fe-4S]1+ (g values of 2.08, 1.93, and 1.87) and W5+ (g values of 1.977, 1.906, and 1.855). Cyclic voltammetry of ferredoxin and WOR5 on an activated glassy carbon electrode shows a catalytic wave upon addition of hexanal, suggesting that ferredoxin can be a physiological redox partner. The combination of WOR5, formaldehyde oxidoreductase, and aldehyde oxidoreductase forms an efficient catalyst for the oxidation of a broad range of aldehydes in P. furiosus.

Aldehyde Oxidoreductases↗
PubMed1995

Potential role of NADH oxidoreductase-derived reactive O2 species in calf pulmonary arterial PO2-elicited responses.

Our laboratory has previously reported evidence that tone responses of isolated endothelium-removed calf pulmonary arteries elicited by changes in PO2 appear to be mediated via changes in H2O2 and guanosine 3',5'-cyclic monophosphate, and that the PO2 sensor mechanism is hypothesized to involve changes in superoxide anion (O2-.) production by a microsomal NADH-oxidoreductase, which is the major source of O2-. detected by lucigenin-elicited chemiluminescence (CL) in this tissue. In this study we examined if the flavoprotein-directed inhibitor of O2-. producing NAD(P)H oxidoreductases, diphenyliodonium (DPI), could be employed as an inhibitor of O2-. production by NADH oxidoreductase, which functions as a selective probe for PO2-elicited tone responses in calf pulmonary arterial smooth muscle. It was found that 1 microM DPI inhibited NADH-dependent production of CL in the arterial smooth muscle homogenate by 49% (n = 10). DPI reduced basal CL from endothelium-removed pulmonary arteries by 41% (n = 15). In endothelium-removed pulmonary arteries precontracted with U-46619, the hypoxic contraction of 2.3 +/- 0.5 g was reduced to 0.1 +/- 0.4 g (n = 7) by DPI, and the reoxygenation relaxation of 32.7 +/- 7.5% was decreased to 4.4 +/- 1.4% (n = 7). DPI did not have any significant effect on U-46619- or K(+)-elicited tone generation. DPI also did not alter the relaxation to H2O2 (1 microM-0.1 mM, n = 6), nitric oxide (0.42 nM-420 nM, n = 12), or isoproterenol (1 nM-1 microM, n = 6).(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1986

Three forms of xanthine: acceptor oxidoreductase in rat heart.

The enzyme xanthine: acceptor oxidoreductase found in rat heart equilibrates between three forms differing in electron acceptor specificity. Form D transfers electrons exclusively to NAD+ and accounts for 85% of total oxidoreductase activity. Form O transfers electrons to molecular oxygen and accounts for 8%. The D/O form prefers NAD+, but without NAD+ transfers electrons to oxygen. Interconversion from D to O and O to D forms is catalyzed by sulfhydryl group-modifying reagents: Cd2+, Cu2+, disulfiram, and heating with dithiothreitol. This suggests that sulfhydryl groups participate in the first stage of enzyme conversion. The NADH/NAD+ concentration ratio may regulate the dehydrogenase activity of xanthine:acceptor oxidoreductase (NAD+-dependent activity of D and D/O forms). Accumulating NADH inhibits hypoxanthine hydroxylation. The amount of form O increases during cardiac ischemia, facilitating superoxide radical-ion generation. Also, NADH/NAD+ does not regulate form O, promoting adenylate nucleotide pool depletion, especially in the heart which has low de novo purine nucleotide synthesis.

Animals↗
PubMed2005

High-level expression of the 1,3-propanediol oxidoreductase from Klebsiella pneumoniae in Escherichia coli.

As one of four key enzymes in glycerol dismutation process, 1,3-propanediol oxidoreductase (EC.1.1.1.202) is important in converting glycerol to 1,3-propanediol in Klebsiella pneumoniae. The dhaT gene encoding 1,3-propanediol oxidoreductase was amplified by polymerase chain reaction (PCR) using the genome DNA of K. pneumoniae as template, and then cloned into cloning vector pMD18-T. After DNA sequence was determined, the dhaT gene was subcloned into Escherichia coli expression vector pET-22b (+) and pET-28a (+). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis revealed that both the recombinant E. coli BL21 (DE3) (pET-22b (+)-dhaT) and E. coli BL21(DE3)(pET-28a (+)-dhaT) expressed predicted 42-kDa 1,3-propanediol oxidoreductase after induced by isopropyl-beta-D-thiogalac-topyranoside (IPTG), and the recombinant enzyme of E. coli BL21 (DE3) (pET-28a (+)-dhaT) was mostly in soluble form, and exhibited high activity (96.8 U/mL culture). The recombinant enzyme was purified and biochemically characterized. The apparent Km values of the enzyme for 1,3-propanediol and NAD+ were 8.5 and 0.21 mM, respectively. The enzyme had maximum activity at pH 9.5 and 30 degrees C.

Alcohol Dehydrogenase↗
PubMed1977

[Study of the NADH and NADPH-ferredoxin oxidoreductase activities in Clostridium acetobutylicum].

The NADH and NADPH-ferredoxin oxidoreductase have been studied in Clostridium acetobutylicum. Acetyl-CoA is an obligatory activator of NADH-ferredoxin reductase activity and NADH a competitive inhibitor of ferredoxin-NAD+ reductase activity. These regulations are the same when C. acetoburylicum moves from 'butylic-type metabolism' to 'butyric-type metabolism'; this demonstrates that NADH-ferredoxin oxidoreductase cna, through its reversible action, meet the very different cell needs imposed by these two types of culture. The physiological function of the clostridial NADPH-ferredoxin oxidoreductase was anabolic as it has been with other clostridia.

Acetyl Coenzyme A↗
PubMed1991

Molecular structure of rat hepatic 3 alpha-hydroxysteroid dehydrogenase. A member of the oxidoreductase gene family.

3-alpha-Hydroxysteroid dehydrogenase (3 alpha-HSD) (EC 1.1.1.50) is an important multifunctional oxidoreductase capable of metabolizing steroid hormones, polycyclic aromatic hydrocarbons, and prostaglandins. 3 alpha-HSD is also required for bile acid synthesis and has been suggested to play an important role in net bile acid transport across the hepatocyte (Stolz, A., Takikawa, H., Ookhtens, M., and Kaplowitz, N. (1989) Annu. Rev. Physiol. 51, 166-177). In order to characterize molecular forms and begin to determine its regulation, we now report the nucleotide sequence, tissue distribution, and homology to other members of the oxidoreductase superfamily. Rat hepatic 3 alpha-HSD cDNA encodes for a 322-amino acid protein with a predicted molecular weight of 37,022 expressed in a 2.4-kilobase (kb) message size. Northern blot analysis of total RNA revealed equivalent steady-state levels in liver and intestine in male rats with lower levels of expression in the colon and minimal expression in stomach, lung, and testis. Female liver contained approximately 2-3-fold greater steady-state levels of mRNA as compared to the male liver with equivalent intestinal expression. Two hybridizing bands, 2.4 and 1.4 kb, were identified in total RNA from the ovary. 3 alpha-HSD exhibits 75% amino acid sequence homology with bovine lung prostaglandin F synthetase and 50% homology with human aldose reductases. Amino acid sequence analysis with short chain alcohol dehydrogenases identified a possible NADP(H) cofactor-binding site at the amino terminus. The significant homology of 3 alpha-HSD with both prostaglandin F synthetase and aldose reductases suggest a subdivision of monomeric, NADPH reductases within the larger oxidoreductases superfamily.

3-Hydroxysteroid Dehydrogenases↗
PubMed1990

Detection and isolation of the NADPH-binding protein of the NADPH:O2 oxidoreductase complex of human neutrophils.

Neutrophils assayed with nitro blue tetrazolium (NBT) exhibit intracellular rather than extracellular superoxide-generating activity when stimulated with phorbol myristate acetate. Enzyme activity is stimulated by anionic detergents, reversibly inhibited by 2',3'-NADPH dialdehyde, and present in equal levels in membrane fractions obtained from phorbol myristate acetate-stimulated and resting cell suspensions. Solubilized membrane shows enzyme activity co-eluting on molecular sieving columns with the cytochrome b redox component of the oxidoreductase complex. Enzyme activity was resolved free of the cytochrome b component following passage of solubilized membrane extracts through QAE-Sephadex anion exchange columns. Enzyme activity measured by the NBT assay appears to be that associated with the NADPH binding protein of the oxidoreductase complex. When exposed to NBT and NADPH this component of the oxidoreductase generates superoxide independent of cytochrome b.

Carrier Proteins↗
PubMed1989

[Xanthine oxidoreductase activity in rat brain tissue: the changes after decapitation].

Since only little xanthine oxidase (XO) activity in mammalian brain was detected in earlier reports, the major end product of AMP degradation in the brain has been believed to be hypoxanthine. Our recent experimental study however, has indicated the presence of uric acid in the rat brain subjected to focal ischemia or cold injury. Allopurinol, a xanthine oxidoreductase inhibitor, has been found to markedly suppress the uric acid production in the same experimental settings. These results suggested that uric acid is generated from hypoxanthine by enzymatic reaction in injured brain tissue. The aim of this experiment is to prove the existence of xanthine oxidoreductase activity in brain tissue. Xanthine oxidoreductase activity in rat cerebral tissue was measured immediately or at 24-hour after decapitation. Under pentobarbital anesthesia, twenty Sprague-Dawley rats were killed by decapitation following washout of the blood by trans-cardiac perfusion with cold physiological saline. Immediately or after 24 hours of decapitation ischemia, the forebrain was removed and homogenized in 6 ml ice cold 0.05 M potassium phosphate buffer (pH 7.8) containing 1 mM phenylmethylsulfonyl fluoride, 0.3 mM EGTA, and 10 mM dithiothreitol. The homogenate was centrifuged at 100,000 g for 60 min and then the supernatant was dialyzed overnight against 0.05 M potassium phosphate buffer (pH 7.8). Aliquot of each dialyzed supernatant (sample) and standard xanthine solution with NAD was reacted at 37 degrees C for 15 min to measure the combined activity of xanthine dehydrogenase (XDH) and XO. For the measurement of XO, standard xanthine solution without NAD was used.(ABSTRACT TRUNCATED AT 250 WORDS)

Allopurinol↗
PubMed1988

The transition metal-mediated formation of the hydroxyl free radical during the reduction of molecular oxygen by ferredoxin-ferredoxin:NADP+ oxidoreductase.

The NADPH-supported enzymatic reduction of molecular oxygen by ferredoxin-ferredoxin:NADP+ oxidoreductase was investigated. The ESR spin trapping technique was employed to identify the free radical metabolites of oxygen. The spin trap 5,5-dimethyl-1-pyrroline N-oxide (DMPO) was used to trap and identify the oxygen-derived free radicals. [17O]Oxygen was employed to demonstrate that the oxygen-centered radicals arose from molecular oxygen. From the data, the following scheme is proposed: (Formula:see text). The formation of the free hydroxyl radical during the reduction of oxygen was demonstrated with quantitative competition experiments. The hydroxyl radical abstracted hydrogen from ethanol or formate, and the resulting scavenger-derived free radical was trapped with known rate constants. If H2O2 was added to the enzymatic reaction, a stimulation of the production of the hydroxyl radical was obtained. This stimulation was manifested in both the concentration and the rate of formation of the DMPO/hydroxyl radical adduct. Catalase was shown to inhibit formation of the hydroxyl radical adduct, further supporting the formation of hydrogen peroxide as an intermediate during the reduction of oxygen. All three components, ferredoxin, ferredoxin:NADP+ oxidoreductase, and NADPH, were required for reduction. Ferredoxin:NADP+ oxidoreductase reduces ferredoxin, which in turn is responsible for the reduction of oxygen to hydrogen peroxide and ultimately the hydroxyl radical. The effect of transition metal chelators on the DMPO/hydroxyl radical adduct concentration suggests that the reduction of chelated iron by ferredoxin is responsible for the reduction of hydrogen peroxide to the hydroxyl radical via Fenton-type chemistry.

Algorithms↗
PubMedDate not supplied

[NADH-vanadate-oxidoreductase in emotional-pain stress and its possible role in the regulation of Na+-K+-ATPase activity].

It is established that NADH-vanadate-oxidoreductase in brain and heart changes its activity depending on the period of the emotional pain stress (EPS) development. In early acute period of EPS (to 48 hours) NADH-vanadate-oxidoreductase is activated in the brain, while in the heart its activity lowers in comparison with normal animals. In the later period after EPS the stimulation of NADH-vanadate-oxidoreductase activity in the both organs in observed. This dynamics creates possibilities for different accumulation of such Na+,K+-ATPase inhibitor as vanadate (VO3-) in the heart and brain. An increase of pO2 of the blood, probably, influences the enhancement of the VO3- concentration in the blood and organs.

Animals↗