The interaction between mitochondrial NADH-ubiquinone oxidoreductase and ubiquinol-cytochrome c oxidoreductase. Evidence for stoicheiometric association.

1. The NADH-ubiquinone oxidoreductase complex (Complex I) and the ubiquinol-cytochrome c oxidoreductase complex (Complex III) combine in a 1:1 molar ratio to give NADH-cytochrome c oxidoreductase (Complex I-Complex III). 2. Experiments on the inhibition of the NADH-cytochrome c oxidoreductase activity of mixtures of Complexes I and III by rotenone and antimycin indicate that electron transfer between a unit of Complex I-Complex III and extra molecules of Complexes I or III does not contribute to the overall rate of cytochrome c reduction. 3. The reduction by NADH of the cytochrome b of mixtures of Complexes I and III is biphasic. The extents of the fast and slow phases of reduction are determined by the proportion of the total Complex III specifically associated with Complex I. 4. Activation-energy measurements suggest that the structural features of the Complex I-Complex III unit promote oxidoreduction of endogenous ubiquinone-10.     

Keto-acid oxidoreductases in the anaerobic protozoa.

In anaerobes, decarboxylation of pyruvate is executed by the enzyme pyruvate:ferredoxin oxidoreductase, which donates electrons to ferredoxin. The pyruvate:ferredoxin oxidoreductase and its homologues utilise many alternative substrates in bacterial anaerobes. The pyruvate:ferredoxin oxidoreductase from anaerobic protozoa, such as Giardia duodenalis, Trichomonas vaginalis, and Entamoeba histolytica have retained this diversity in usage of alternative keto acids for energy production utilising a wide variety of substrates. In addition to this flexibility, both T. vaginalis and G. duodenalis have alternative enzymes that are active in metronidazole-resistant parasites and that do not necessarily involve donation of electrons to characterized ferredoxins. Giardia duodenalis has two oxoacid oxidoreductases, including pyruvate:ferredoxin oxidoreductase and T. vaginalis has at least three. These alternative oxoacid oxidoreductases apparently do not share homology with the characterized pyruvate:ferredoxin oxidoreductase in either organism. Independently, both G. duodenalis and T. vaginalis have retained alternative oxoacid oxidoreductase activities that are clearly important for the survival of these parasitic protists.     

Metabolism of L-fucose and L-rhamnose in Escherichia coli: differences in induction of propanediol oxidoreductase.

Escherichia coli is capable of growing on L-fucose or L-rhamnose as a sole source of carbon and energy. When grown under anaerobic conditions on either sugar, a nicotinamide adenine dinucleotide-linked L-lactaldehyde:propanediol oxidoreductase activity is induced. The functioning of this enzyme results in the regeneration of oxidized nicotinamide adenine dinucleotide. Conditions of induction of the enzyme activity were studied and were found to display different characteristics on each sugar. In the rhamnose-grown cells, the increase in enzyme activity detected under inducing conditions was accompanied by the synthesis of propanediol oxidoreductase, as measured by the appearance in the extracts of a protein that reacts with propanediol oxidoreductase antibodies. In contrast, in fucose-grown cells, the level of propanediol oxidoreductase as measured by enzyme antibody-reacting material was high under noninducing and inducing conditions. Thus, the increase in enzyme activity detected in going from noninducing to inducing conditions in fucose-grown cells did not depend on the appearance of the specific protein but on the activation of the propanediol oxidoreductase already present in the cells in an inactive form. The propanediol oxidoreductase of both homologous systems should consequently be regulated by different control mechanisms.     

Microbial metabolism of quinoline and related compounds. XII. Isolation and characterization of the quinoline oxidoreductase from Rhodococcus spec. B1 compared with the quinoline oxidoreductase from Pseudomonas putida 86.

Quinoline oxidoreductase from Rhodococcus spec. B1 was purified 39-fold to apparent homogeneity in a 5-step procedure with a recovery of 26%. The Mr of the native enzyme as determined by gel chromatography was 300,000. SDS polyacrylamide gel electrophoresis of the enzyme revealed 3 protein bands corresponding to Mr 82,000, 32,000, and 18,000. The enzyme contains 1.3 atoms of molybdenum, 8 atoms of iron, 8 atoms of acid-labile sulphur, 2 molecules of FAD and 2 molecules of molybdopterin cytosine dinucleotide. Cyanide, 4-hydroxymercuribenzoate and methanol were effective as inhibitors. The amino-terminal protein sequences of the 3 subunits of quinoline oxidoreductase from Rhodococcus B1 compared to those of quinoline oxidoreductase from Pseudomonas putida 86 revealed no difference among 71 amino acids examined.     

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+.     

NN''-dicyclohexylcarbodi-imide-sensitivity of bovine heart mitochondrial NADH: ubiquinone oxidoreductase. Inhibition of activity and binding to subunits.

Dicyclohexylcarbodi-imide (DCCD) inhibition of NADH: ubiquinone oxidoreductase was studied in submitochondrial particles and in the isolated form, together with the binding of the reagent to the enzyme. DCCD inhibited the isolated enzyme in a time- and concentration-dependent manner. Over the concentration range studied, a maximum inhibition of 85% was attained within 60 min. The time course for the binding of DCCD to the enzyme was similar to that of activity inhibition. The NADH:ubiquinone oxidoreductase activity of the submitochondrial particles was also sensitive to DCCD, and the locus of binding of the inhibitor was studied by subsequent resolution of the enzyme into subunit polypeptides. Only two subunits (molecular masses 13.7 and 21.5 kDa) were labelled by [14C]DCCD, whereas, when the enzyme in its isolated form was treated with [14C]DCCD, six subunits (13.7, 16.1, 21.5, 39, 43 and 53 kDa) were labelled. Comparison with the subunit labelling of F1F0-ATPase and ubiquinol:cytochrome c oxidoreductase indicated that the labelling pattern of NADH:ubiquinone oxidoreductase, and enzyme complex with a multitude of subunits, is unique and not due to contamination by other inner-membrane proteins. The correlation between the electron- and proton-transport functions and the DCCD-binding components remains to be established.     

Propanediol oxidoreductases of Escherichia coli, Klebsiella pneumoniae and Salmonella typhimurium. Aspects of interspecies structural and regulatory differentiation.

The enzyme propanediol oxidoreductase, which converts the lactaldehyde formed in the metabolism of fucose and rhamnose into propane-1,2-diol under anaerobic conditions, was investigated in Escherichia coli, Klebsiella pneumoniae and Salmonella typhimurium. Structural analysis indicated that the enzymes of E. coli and K. pneumoniae have the same Mr and pI, whereas that of Salm. typhimurium also has the same Mr but a slightly different pI. One-dimensional peptide mapping showed identity between the E. coli and K. pneumoniae enzymes when digested with alpha-chymotrypsin, Staphylococcus aureus V8 proteinase or subtilisin. In the case of Salm. typhimurium, this held only for the subtilisin-digested enzymes, indicating that the hydrophobic regions were preserved to a considerable extent. Anaerobically, the three species induced an active propanediol oxidoreductase when grown on fucose or rhamnose. An inactive propanediol oxidoreductase was induced in Salm. typhimurium by either fucose or rhamnose under aerobic conditions, and this was activated once anaerobiosis was established. An inactive propanediol oxidoreductase was also induced in E. coli under aerobic conditions, but only by growth on fucose. The inactive enzyme was not induced by either of the sugars in K. pneumoniae.     

Purification of the solubilized NADPH:O2 oxidoreductase of human neutrophils. Isolation of its catalytically inactive cytochrome b and flavoprotein redox centers

The membrane-bound NADPH:O2 oxidoreductase of human neutrophils has been solubilized in approximately 70% yield and purified on concanavalin A-Sepharose and gel sieving columns of varying bed volumes and sieving ranges. The half-life of the solubilized oxidoreductase stored at 2-4 degrees C in the presence of 25% glycerol at pH 8.6 is approximately 30 h. The oxidoreductase contains a flavoprotein identifiable by its fluorescence spectrum for FAD which binds weakly to concanavalin A-Sepharose and elutes from gel sieving columns at a molecular weight range of approximately 51,000. This flavoprotein accounts for approximately 70% of the total FAD content found in granular membrane fractions recovered from activated neutrophils. Recovery of oxidoreductase activity from both concanavalin A-Sepharose affinity and gel sieving columns is affected by the resolution of the flavoprotein free of the cytochrome b component of the oxidoreductase. The resolved flavoprotein and cytochrome b appear unable to catalyze either NADH nor NADPH oxidase activities with O2, ferricyanide, or nitroblue tetrazolium salt serving as electron acceptors.     

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.     

Preparation of chick brain synaptosomes and synaptosomal membranes.

A method is described for the preparation of synaptosomes and synaptosomal membranes from chicken brain. Procedures for isolating rat synaptosomal membranes could not be used directly; several modifications of existing procedures are reported. Purity of the subcellular and subsynaptosomal fractions was monitored by electron microscopy and measurements of ferrocytochrome c: oxygen oxidoreductase (EC 1.9.3.)), monoamine: oxygen oxidoreductase (deaminating) EC 1.4.3.4), rotenone-insensitive NADH: cytochrome c oxidoreductase (EC 1.6.99.3), NADPH: cytochrome c oxidoreductase (EC 1.6.99.1), orthophosphoric monoester phosphohydrolase (EC 3.1.3.2), ATP phosphohydrolase (EC 3.6.1.4), and levels of RNA. Microsomes are the main contaminant of the synaptosomal membrane fraction. Mitochondrial and lysosomal enzymes occur in lesser amounts. No myelin contamination was observed. Marker enzymes for contaminants suggest that these synaptosomal membranes are as pure as membranes described by others, and the specific activity of a neuronal membrane marker, (Na+ -K+)-activated ATPase, is as high as other preparations. Levels of this enzyme in the membrane fraction are enriched 13-fold over homogenate ATPase levels.     

Immunodetection and photostability of NADPH-protochlorophyllide oxidoreductase in Pinus pinea L.

Antibody against the light-dependent NADPH-protochlorophyllide oxidoreductase of oat was used to detect a protein of the same molecular weight in cotyledons of 40-day-old dark-grown seedlings of Pinus pinea L. Exposure of the seedlings to light resulted in a rapid decrease in protochlorophyllide content without the concomitant decrease in 38 kDa protein which is observed on transfer of dark-grown angiosperm seedlings to light. The stability of the light-dependent NADPH-protochlorophyllide oxidoreductase in pine in the absence of accumulated substrate is consistent with either (1) a different mechanism of regulation of chlorophyll synthesis in gymnosperms or (2) a higher proportion of stable extra-plastidic protein reacting with the antibody to the light-dependent NADPH-protochlorophyllide oxidoreductase than is the case in angiosperms.Abbreviations Chl chlorophyll - Chlide chlorophyllide - NADPH-Pchlide oxidoreductase NADPH protochlorophyllide oxidoreductase - NC nitrocellulose - PBS phosphate buffered saline - Pchlide protochlorophyllide - SDS sodum dodecyl sulphate - SDS-PAGE sodium dodecyl sulphate polyacrylamide gel electrophoresis     

DNA strand scission and free radical production in menadione-treated cells. Correlation with cytotoxicity and role of NADPH quinone acceptor oxidoreductase.

Menadione (MD; 2-methyl-1,4-naphthoquinone), a redox cycling quinone was shown to induce single (ss)- and double (ds)-strand DNA breaks in human MCF-7 cells. This DNA damage was mediated via the hydroxyl radical as evidenced by electron spin resonance spectroscopy (ESR) studies utilizing the spin trap, 5,5-dimethyl-1-pyrroline-1-oxide. The free radical production and DNA damage were shown to play a role in MD cytotoxicity as revealed by the reversal of MD toxicity and inhibition of hydroxyl radical production by exogenously added catalase. The role of NADPH quinone acceptor oxidoreductase in the metabolism of MD was evaluated. Purified quinone acceptor oxidoreductase in combination with MD resulted in the production of significant levels of the hydroxyl radical as measured by ESR. Dicumarol, an inhibitor of quinone acceptor oxidoreductase, decreased the production of the hydroxyl radical and attenuated DNA strand breaks in MCF-7 cells treated with MD.     

The efficient export of NADP-containing glucose-fructose oxidoreductase to the periplasm of Zymomonas mobilis depends both on an intact twin-arginine motif in the signal peptide and on the generation of a structural export signal induced by cofactor binding.

The periplasmic, NADP-containing glucose-fructose oxidoreductase of the gram-negative bacterium Zymomonas mobilis belongs to a class of redox cofactor-dependent enzymes which are exported with the aid of a signal peptide containing a so-called twin-arginine motif. In this paper we show that the replacement of one or both arginine residues results in drastically reduced translocation of glucose-fructose oxidoreductase to the periplasm, showing that this motif is essential. Mutant proteins which, in contrast to wild-type glucose-fructose oxidoreductase, bind NADP in a looser and dissociable manner, were severely affected in the kinetics of plasma membrane translocation. These results strongly suggest that the translocation of glucose-fructose oxidoreductase into the periplasm uses a Sec-independent apparatus which recognizes, as an additional signal, a conformational change in the structure of the protein, most likely triggered by cofactor binding. Furthermore, these results suggest that glucose-fructose oxidoreductase is exported in a folded form. A glucose-fructose oxidoreductase:beta-galactosidase fusion protein is not lethal to Z. mobilis cells and leads to the accumulation of the cytosolic preform of wild-type glucose-fructose oxidoreductase expressed in trans but not of a typical Sec-substrate (OmpA), indicating that the glucose-fructose oxidoreductase translocation apparatus can be blocked without interfering with the export of essential proteins via the Sec pathway.     

Specific immobilization of in vivo biotinylated bacterial luciferase and FMN:NAD(P)H oxidoreductase.

Bacterial bioluminescence, catalyzed by FMN:NAD(P)H oxidoreductase and luciferase, has been used as an analytical tool for quantitating the substrates of NAD(P)H-dependent enzymes. The development of inexpensive and sensitive biosensors based on bacterial bioluminescence would benefit from a method to immobilize the oxidoreductase and luciferase with high specific activity. Toward this end, oxidoreductase and luciferase were fused with a segment of biotin carboxy carrier protein and produced in Escherichia coli. The in vivo biotinylated luciferase and oxidoreductase were immobilized on avidin-conjugated agarose beads with little loss of activity. Coimmobilized enzymes had eight times higher bioluminescence activity than the free enzymes at low enzyme concentration and high NADH concentration. In addition, the immobilized enzymes were more stable than the free enzymes. This immobilization method is also useful to control enzyme orientation, which could increase the efficiency of sequentially operating enzymes like the oxidoreductase-luciferase system.     

The recognition of a special ubiquinone functionally central in the ubiquinone-cytochrome b-c2 oxidoreductase.

Although the energy conserving membranes of the photosynthetic bacterium Rhodopseudomonas sphaeroides contain a 25 (+/- 3)-fold molar excess of ubiquinone over the photochemical reaction center, the activity of the ubiquinone-cytochrome b-c2 oxidoreductase is unaffected by quinone extraction until only 3, or at most 4, ubiquinones remain; only then does further extraction prevent the function of the oxidoreductase. Since 2 of these last ubiquinones are integral parts of the photochemical reaction center, we conclude that the ubiquinone-cytochrome b-c2 oxidoreductase requires only 1, or at most 2, molecules of ubiquinone-10 for its function. Earlier kinetic data identified a major electron donor to ferricytochrome c2 as a single molecule (known as Z) which requires 2 electrons and 2 protons for its equilibrium reduction. Hence, we identify a single molecule of quinone, probably ubiquinone-10 in a special environment, as a major electron donor to ferricytochrome c2 in the ubiquinone cytochrome b-c2 oxidoreductase.     

Purified NAD(P)H-quinone oxidoreductase enhances the mutagenicity of dinitropyrenes in vitro.

The effect of highly purified rat liver cytosolic NAD(P)H-quinone oxidoreductase [EC 1.6.99.2] on the mutagenicity of 1,3- 1,6- and 1,8-dinitropyrene (DNP) was studied in the Ames Salmonella typhimurium mutagenicity assay. NAD(P)H-quinone oxidoreductase over the range of 0.02-0.8 micrograms/plate (38-1500) units increased up to threefold the mutagenicity of all three DNPs in S. typhimurium TA 98. In TA98NR, a strain deficient in "classical" nitro-reductase, the mutagenicity of 1,6- and 1,8-DNP was essentially unchanged, whereas that of 1,3-DNP was markedly reduced. NAD(P)H-quinone oxidoreductase enhanced the mutagenicity of 1,6- and 1,8-DNP to approximately equivalent extents in TA98NR and TA98. The mutagenicity of 1,3-DNP in TA98NR was potently enhanced by the addition of NAD(P)H-quinone oxidoreductase in a dose-responsive manner. In the presence of 0.8 micrograms NAD(P)H-quinone oxidoreductase, 1,3-DNP displayed a mutagenic response in TA98NR that was comparable to that obtained in TA98. NAD(P)H-quinone oxidoreductase was found to increase the mutagenicity of 1,6- but not 1,3- or 1,8-DNP to mutagenic intermediates in TA98/1,8-DNP6, a strain deficient in O-acetyltransferase activity. The results suggest that NAD(P)H-quinone oxidoreductase not only catalyzes reduction of the parent DNP but also that of partially reduced metabolites generated from that DNP. Such reductive metabolism may lead to increased formation of the penultimate mutagenic species.     

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.     

Chemical and spectral properties of carbon monoxide: methylene blue oxidoreductase. The molybdenum-containing iron-sulfur flavoprotein from Pseudomonas carboxydovorans

Carbon monoxide:methylene blue oxidoreductase, the key enzyme of CO-oxidation in energy metabolism of the carboxydobacterium Pseudomonas carboxydovorans, has been isolated in good yield and purity and found to contain FAD, molybdenum, iron, and labile sulfide in the ratio of 1:1:4:4. The enzyme is, therefore, a new molybdenum-containing iron-sulfur flavoprotein, exhibiting chemical and spectral properties quite similar to those of xanthine oxidase. Analytical data on the spectral characteristics of the enzyme in the oxidized and various reduced states are presented. Carbon monoxide:methylene blue oxidoreductase turned out to be photoreducible in the presence of EDTA and urea and was subject to reoxidation by air oxygen; no flavoprotein semiquinone was formed. Unphysiological electron acceptors, e.g. methylene blue, were used as oxidizing substrates whereas NAD or NADP turned out to be ineffective. Methylene blue reduction with CO was not affected by the presence of allopurinol, and carbon monoxide:methylene blue oxidoreductase was not able to catalyze the reduction of methylene blue with xanthine, adenine, or aldehydes. CO was the only reducing substrate used by the enzyme. Carbon monoxide:methylene blue oxidoreductase formed no sulfite adduct, and the reactivity with ferricyanide or cytochrome c was significant but slow. As known for other molybdenum hydroxylases, carbon monoxide:methylene blue oxidoreductase was rapidly inactivated by methanol, but the enzyme exhibited no ability to catalyze the oxidation of NADH with methylene blue, and NAD was not able to overcome methanol inhibition.     

Dihydroxyacetone reductase from Mucor javanicus. 1. Isolation and properties.

An NADPH-dependent oxidoreductase has been extracted from the mycelium of the fungus Mucor Javanicus (Wehmer) and enriched 1000-fold with respect to the protein contained in the crude extract after centrifugation at 2600 X g. The molecular weight of the enzyme was estimated by gel filtration to be about 100 000; electrophoresis under dissociating conditions indicates four subunits of molecular weight about 28 000. Data on stability and activity of the enzyme as a function of pH and temperature are reported. From a kinetic study and product analysis of the reduction of the two enantiomeric trans-1-decalones and also from a kinetic study of the oxidation of the two diastereomeric pairs of trans-1-decalols it follows that the enzymes is an e-Si oxidoreductase (according to the nomenclature proposed by Dutler et al., Eur. J. Biochem. 22 [1971]203-212 and Prelog and Helmchen, Helv. Chim. Acta, 55 [1972] 2581-2598). This classification is amply confirmed by the kinetic behaviour of a large number of alicyclic substrates. Using (4-2HSi-labelled coenzyme to reduce (9S)-trans-1,4-decalindione, it was shown that the enzyme is HSi (= HS = HB)-stereospecific with respect to the coenzyme. It is demonstrated that the oxidoreductase from Mucor javanicus can be used for the preparation of optically pure chiral alcohols and ketones. In the following paper evidence is presented that the natural substrate of the enzyme is dihydroxyacetone.     

Inactivation of clostridial ferredoxin and pyruvate-ferredoxin oxidoreductase by sodium nitrite.

Clostridial ferredoxin and pyruvate-ferredoxin oxidoreductase activity was investigated after in vitro or in vivo treatment with sodium nitrite. In vitro treatment of commercially available Clostridium pasteurianum ferredoxin with sodium nitrite inhibited ferredoxin activity. Inhibition of ferredoxin activity increased with increasing levels of sodium nitrite. Ferredoxin was isolated from normal C. pasteurianum and Clostridium botulinum cultures and from cultures incubated with 1,000 micrograms of sodium nitrite per ml for 45 min. The activity of in vivo nitrite-treated ferredoxin was decreased compared with that of control ferredoxin. Pyruvate-ferredoxin oxidoreductase isolated from C. botulinum cultures incubated with 1,000 micrograms of sodium nitrite per ml showed less activity than did control oxidoreductase. It is concluded that the antibotulinal activity of nitrite is due at least in part to inactivation of ferredoxin and pyruvate-ferredoxin oxidoreductase.