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PubMed1983

Ligational effects on reduction of myoglobin and horseradish peroxidase by inorganic reagents.

Previous studies of the reduction of metmyoglobin and adducts by dithionite have been extended to horseradish peroxidase and its complexes. In addition, the reduction of metmyoglobin, horseradish peroxidase and adducts by a much bulkier reactant, cobalt(II) sepulchrate has been studied. Similar patterns of kinetic behavior were observed, namely, direct reduction of cyanide and imidazole adducts of the iron(III) proteins and indirect (via dissociation) reduction of the fluoride adduct. In the reduction of horseradish ferriperoxidase by cobalt(II) sepulchrate, three steps are observed and the spectral properties of the intermediate(s) and their kinetic behavior delineated. The final product is ferroperoxidase confirmed by spectral properties and its behavior on oxygenation. Reduction of cytochrome c(III) and Hipip by cobalt(II) sepulchrate appears to be a uniphasic reaction and second-order rate constants have been determined.

Animals↗
PubMed1988

The oxidation-reduction kinetics of cytochromes b, c1 and c in initially fully reduced mitochondrial membranes are in agreement with the Q-cycle hypothesis.

Stopped-flow experiments were performed to distinguish between two hypotheses, the Q-cycle and the SQ-cycle, each describing the pathway of electron transfer in the QH2:cytochrome c oxidoreductases. It was observed that, when mitochondrial membranes from the yeast Saccharomyces cerevisiae were poised at a low redox potential with appropriate amounts of sodium dithionite to completely reduce cytochrome b, the kinetics of oxidation of cytochrome b showed a lag period of maximally 100 ms. Under the same experimental conditions, the oxidation-reduction kinetics of cytochromes c + c1 showed transient behaviour. These results do not support the presence of a mobile species of semiquinone in the QH2:cytochrome c oxidoreductases, as envisaged in the SQ-cycle, but are consistent with a Q-cycle mechanism in which the two quinone-binding domains do not exchange electrons directly on the timescale of turnover of the enzyme.

Antimycin A↗
PubMed1990

Effects of oxidants and reductants on the efficiency of excitation transfer in green photosynthetic bacteria.

The efficiency of energy transfer in chlorosome antennas in the green sulfur bacteria Chlorobium vibrioforme and Chlorobium limicola was found to be highly sensitive to the redox potential of the suspension. Energy transfer efficiencies were measured by comparing the absorption spectrum of the bacteriochlorophyll c or d pigments in the chlorosome to the excitation spectrum for fluorescence arising from the chlorosome baseplate and membrane-bound antenna complexes. The efficiency of energy transfer approaches 100% at low redox potentials induced by addition of sodium dithionite or other strong reductants, and is lowered to 10-20% under aerobic conditions or after addition of a variety of membrane-permeable oxidizing agents. The redox effect on energy transfer is observed in whole cells, isolated membranes and purified chlorosomes, indicating that the modulation of energy transfer efficiency arises within the antenna complexes and is not directly mediated by the redox state of the reaction center. It is proposed that chlorosomes contain a component that acts as a highly quenching center in its oxidized state, but is an inefficient quencher when reduced by endogenous or exogenous reductants. This effect may be a control mechanism that prevents cellular damage resulting from reaction of oxygen with reduced low-potential electron acceptors found in the green sulfur bacteria. The redox modulation effect is not observed in the green gliding bacterium Chloroflexus aurantiacus, which contains chlorosomes but does not contain low-potential electron acceptors.

Bacteria↗
PubMed1990

Soluble cytochromes and a photoactive yellow protein isolated from the moderately halophilic purple phototrophic bacterium, Rhodospirillum salexigens.

Three soluble cytochromes were found in two strains of the halophilic non-sulfur purple bacterium Rhodospirillum salexigens. These are cytochromes C2, C and c-551. Cytochrome C2 was recognized by the presence of positive charge at the site of electron transfer (measured by laser flash photolysis), although the protein has an overall negative charge (pI = 4.7). Cytochrome C2 has a high redox potential (300 mV) and is monomeric (13 kDa). Cytochrome c was recognized from its characteristic absorption spectrum. It has a redox potential of 95 mV, an isoelectric point of 4.3, and is isolated as a dimer (33 kDa) of identical subunits (14 kDa), a property which is typical of this family of proteins. R. salexigens cytochrome c-551 has an absorption spectrum similar to the low redox potential Rb. sphaeroides cytochrome c-551.5. It also has a low redox potential (-170 mV), is very acidic (pI = 4.5), and is monomeric (9 kDa), apparently containing 1 heme per protein. The existence of abundant membrane-bound cytochromes c-558 and c-551 which are approximately half reduced by ascorbate and completely reduced by dithionite suggests the presence of a tetraheme reaction center cytochrome in R. salexigens, although reaction centers purified in a previous study (Wacker et al., Biochim. Biophys. Acta (1988) 933, 299-305) did not contain a cytochrome. The most interesting observation is that R. salexigens contains a photoactive yellow protein (PYP), previously observed only in the extremely halophilic purple sulfur bacterium Ectothiorhodospira halophila. The R. salexigens PYP appears to be slightly larger than that of Ec. halophila (16 kDa vs. 14 kDa). Otherwise, these two yellow proteins have similar absorption spectra, chromatographic properties and kinetics of photobleaching and recovery.

Ascorbic Acid↗
PubMed1993

Reductive inactivation of the mitochondrial three subunit NADH dehydrogenase.

The 3-subunit iron-sulfur flavoprotein (NADH-artificial electron acceptor oxidoreductase) derived from complex I (EC 1.6.5.3) is rapidly and irreversibly inactivated in the presence of NADH. The rate of inactivation increases with a decrease of the enzyme concentration. The activities with ferricyanide, menadione and cytochrome c were lost synchronously during preincubation of the enzyme in the presence of NADH or dithionite under either aerobic or anaerobic conditions. The titration of the inactivation rate with the NADH/NAD+ pair suggests that reduction of a component with Em' = -325 mV (n = 2) is a prerequisite for a loss of the enzyme activity. Among the compounds tested only FMN and NAD+ were able to protect the enzyme against the reductive inactivation. NADH-induced loss of the enzyme activity in diluted solutions is accompanied with the synchronous appearance of a fluorescence characteristic for free FMN. It is concluded that the reduction of flavin leads to a strong decrease of FMN affinity to its specific binding site, and possible implications of the redox-dependent affinity changes in operation of NADH-ubiquinone reductase are discussed.

Cytochrome c Group↗
PubMed1996

Rapid transmembrane movement of C6-NBD-labeled phospholipids across the inner membrane of Escherichia coli.

In this study we have investigated the transmembrane movement of short chain fluorescently labeled phospholipids across the inner membrane of Escherichia coli. Exogenously added C6-NBD-labeled phospholipids rapidly flip across the inner membrane of E. coli, as was shown by a dithionite reduction assay applied to inverted inner membrane vesicles (IIMV) isolated from wild type E. coli cells. The rate of transmembrane movement of the phospholipid probes incorporated into IIMV is temperature dependent, and shows no phospholipid head group specificity. C6-NBD-labeled phospholipids translocate across the membrane of IIMV incubated at 37 degrees C with a t1/2 of 7 min. After the incorporation into IIMV C6-NBD-PG is partially converted to CL by CL-synthase. If IIMV are pretreated with proteinase K the conversion of this fluorescent probe to C6-NBD-CL is not observed anymore, suggesting that the catalytic domain of CL-synthase is at the cytoplasmic site of the plasma membrane of E. coli. Newly synthesized C6-NBD-CL also flips across the inner membrane although at a slower rate than the other phospholipid probes. The transmembrane movement occurs in both directions and is not influenced by treatment of the IIMV with a sulfhydryl reagent or a proteinase, nor by the presence of ATP, or a deltapH across the membrane of the IIMV. However, the transmembrane movement of the C6-NBD-labeled phospholipid probes is not observed in LUVETs (large unilamellar vesicles made by extrusion technique) prepared of wild type E. coli lipids, indicating that the rapid transmembrane movement of phospholipids across the inner membrane of E. coli is a protein-mediated process.

4-Chloro-7-nitrobenzofurazan↗
PubMed1975

Nitrogenase. VII. Effect of component ratio, ATP and H2 on the distribution of electrons to alternative substrates.

Some kinetic properties of purified component I (Mo-Fe protein) and component II (Fe protein) of nitrogenase (EC 1.7.99.2) from Azotobacter vinelandii have been examined. The apparent Km values for reducible substrates (0.1 atm for N2, 0.01 atm for acetylene) and dithionite (0.5 mM) are similar for osmotically shocked cell lysates and purified components. However, the ATP dependence of acetylene and N2 reduction varies sigmoidally with ATP concentration and as a function of the relative and absolute concentration of components I and II in the assay. Acetylene is reduced in preference to N2 in competitive assays when component I is in relative excess. Acetylene reduction is not as dependent upon ATP concentration as is N2 reduction, so that acetylene is also a preferred substrate at lower ATP levels. Hydrogen specifically inhibits N2 reduction, diverting electrons to acetylene when both substrates are present in the assay. We propose a model of the enzyme activity, in which the substrates for reduction are bound to component I with electrons being activated by component II. ATP may be involved in activating electrons and in maintaining the appropriate conformation or reduction state of components to allow effective reduction of substrates. The relative rate of reduction of alternative substrates is dependent on the concentration of the particular state(s) capable of reacting with each substrate. The concentration of a particular state of component I is a function of components I, II and ATPL

Acetylene↗
PubMed1975

Effects of sulfhydryl agents on the activation of tryptophan-5-monooxygenase from bovine pineal glands.

Partially purified tryptophan-5-monooxygenase (L-tryptophan, tetrahydropteridine: oxygen oxidoreductase (5-hydroxylating) EC 1.14.16.4)from bovine pineal gland was activated by preincubation with sulfhydryl agents such as dithiothreitol, L-cysteine, cysteamine, L-cysteine ethylester, N-acetyl-L-cysteine, 2-mercaptoethanol and reduced glutathione, at alkaline pH (optimum pH equals 8.5). Dithiothreitol was the most effective of these, leading to approximately 50-fold activation of the enzyme after preincubation. Fe-2+ or other reducing agents such as borohydride, dithionite and ascorbate facilitated the velocity of the activation in the presence of sulfhydryl agents. In the absence of sulfhydryl agents, no activation was observed even in the presence of Fe-2+ or other reducing agents, suggesting an obligatory role or sulhydryl agents during the activation. The relative velocity and full extent of the activation were dependent on the concentrations of both the sulfhydryl agent and the enzyme in the activation mixture. The kinetic analysis of the activation indicated that the sulfhydryl agent reacts with more than 2 sites in the enzyme; one type of site is reduced by sulfhydryl agents, Fe-2+ or other reducing agents and the other specifically modified by a sulfhydryl agent. The activated enzyme did not require any exogenous Fe-2+ for its catalytic activity, but some roles of iron maybe exist in its catalytic reaction. The optimum pH for catalytic reaction of the activated enzyme was approximately 6.5. The apparent Km for L-tryptophan and pteridine cofactor, tetrahydro-pteridine (2-amino-4-hydroxy-6,7-dimethyl-5,6,7,8-tetrahydropterin), of the activated enzyme were 30 and 35 muM respectively.

2,2'-Dipyridyl↗
PubMed1975

Nitrate reductase from bacteroides of Rhizobium japonicum: enzyme characteristics and possible interaction with nitrogen fixation.

The soluble nitrate reductase of Rhizobium japonicum bacteroids has been purified and its properties compared to those of aerobically grown cells. The enzymes from both sources are similar with molecular weights of about 70 000 suggesting no close relationship with the molybdo-protein component of nitrogenase. Nitrite, the product of nitrate reductase, strongly inhibited the nitrogenase activity from bacteroids, at concentrations less than 100 muM. Thus, an interference in the rate of nitrogen fixation is possible as a result of nitrate reductase activity. A study of the distribution of nitrate reductase in bacteroids indicates that a proportion of the total activity is membrane-bound but that this activity is similar to that in the soluble fraction. Purified nitrate reductase required reduced viologen dyes for activity. Neither NADPH or NADH or FAD could substitute as electron donors. Dithionite is a strong inhibitor and inactivated nitrate reductase from all sources examined. This inactivation is prevented by methyl viologen. Purified nitrate reductase from bacteroids and bacteria Rhizobium japonicum is practically unaffected by exposure to oxygen.

Chromatography, Gel↗
PubMed1979

Purification and properties of the membrane-bound hydrogenase from Proteus mirabilis.

The cytoplasmic membrane-bound hydrogenase of the facultative anaerobe, Proteus mirabilis, has been solubilized and purified to homogeneity. The purified enzyme exhibited a maximal specific activity of about 780 mumol H2 oxidized/min per mg protein (benzyl viologen reduction). The hydrogenase has a molecular weight of 205 000 and is composed of two subunits with a molecular weight of 63 000 and two of 33 000. The absorption spectrum of the enzyme was characteristic of non-heme iron proteins. The millimolar extinction coefficients at 400 and 280 nm are 106 and 390, respectively. The hydrogenase has about 24 iron atoms and 24 acid-labile sulfide atoms/molecule. Amino acid analyses revealed the presence of 39 half-cystine residues/molecule and a preponderance of acidic amino acids. The hydrogenase in its oxidized form exhibits an EPR signal of the HiPIP-type with g values at 2.025 and 2.018. Upon reduction with either dithionite or H2 the signal disappears; no other signals were detectable.

Amino Acids↗
PubMed1981

Iron-sulphur clusters in fumarate reductase from Vibrio succinogenes.

(1) The fumarate reductase complex from Vibrio succinogenes contains one FAD molecule, one [4Fe-4S]3+(3+,2+) and one [2Fe-2S]2+(2+,1+) cluster per enzyme molecule. Both clusters can be partly reduced by succinate. In the presence of excess Na2S2O4 and fumarate, the [2Fe-2S] cluster is completely oxidized, whereas the other cluster is largely reduced. (2) The [2Fe-2S] cluster is localized in the Mr, 31,000 subunit. The EPR spectrum of the reduced cluster in the isolated subunit differs slightly in line width, but not in g-value, from the spectrum of reduced, intact enzyme complex. The demonstrates that the immediate environment of th cluster is little perturbed by dissociating this subunit from the FAD-containing Mr 79,000 subunit. The temperature dependence of the power-saturation behaviour has, however, greatly decreased in the isolated subunit, the saturation at 11 K of the paramagnetic cluster being much less than in the enzyme complex. Moreover, the temperature dependence of th power-saturation behaviour of this cluster in the enzyme is greater with succinate as reducing agent, than with dithionite. (3) The [4Fe-4S] cluster is located on the Mr 79,000 subunit. This cluster is unstable in air when the subunit has been dissociated from the enzyme complex.

Chemical Phenomena↗
PubMed1975

Hybrids of chemical derivatives of Escherichia coli alkaline phosphatase.

The activities of hybrid dimers of alkaline phosphatase containing two chemically modified subunits have been investigated. One hybrid species was prepared by dissociation and reconstitution of a mixture of two variants produced by chemical modification of the native enzyme with succinic anhydride and tetranitromethane, respectively. The succinyl-nitrotyrosyl hybrid was separated from the other members of the hybrid set by DEAE-Sephadex chromatography and then converted to a succinyl-aminotyrosyl hybrid by reduction of the modified tyrosine residues with sodium dithionite. A comparison of the activities of these two hybrids with the activities of the succinyl, nitrotyrosyl and aminotyrosyl derivatives has shown that either the subunits of alkaline phosphatase function independently or if the subunits turnover alternately in a reciprocating mechanism, then the intrinsic activity of each subunit must be strongly dependent on its partner subunit.

Alkaline Phosphatase↗
PubMed1975

Magnetic circular dichroism studies on microsomal aryl hydrocarbon hydroxylase: comparison with cytochrome b-5 and cytochrome P-450-cam.

Magnetic circular dichroism spectra are reported for the visible and near ultraviolet spectral regions of liver microsomes from dimethylbenzanthracene-treated rats. The sequential addition of NADH, dithionite, and carbon monoxide enables us to determine contributions to the magnetic circular dichroism by cytochromes b-5 and P-450, which dominate the spectra. The magnetic circular dichroism of the microsomal preparation is compared with that of purified oxidized and reduced cytochrome -b-5 from pig liver and with the camphor-complexed and camphor-free oxidized, reduced, and reduced carbonmonoxy cytochrome P-450-cam from Pseudomonas putida. The magnetic circular dichroism spectra of the membrane bound cytochrome -b-5 are similar to those of the purified protein, indicating that little or no alteration in the environment of the heme occurs during the isolation procedure. The soluble bacterial cytochrome P-450 also appears to be a suitable model for microsomal P-450, although differences in the magnetic circular dichroism intensity are observed for the two enzymes. No effect of dimethylbenzanthracene on the magnetic circular dichroism spectra of induced compared to control rat microsomes could be observed.

Animals↗
PubMed1977

Spectroscopic studies of the oxidation-reduction properties of three forms of ferredoxin from Desulphovibrio gigas.

Electron paramagnetic resonance spectra were recorded of three forms of Desulphovibrio gigas ferredoxin, FdI, FdI' and FdII. The g = 1.94 signal seen in dithionite-reduced samples is strong in FdI, weaker in FdI' and very small in FdII. The g = 2.02 signal in the oxidized proteins is weak in FdI and strongest in FdII. It is concluded that most of the 4Fe-4S centres in FdI change between states C- and C2-; FdI' contain both types of centre. There is no evidence that any particular centre can change reversibly between all three oxidation states. Circular dichroism spectra show differences between FdI and FdII even in the diamagnetic C2- state. The redox potentials of the iron-sulphur centres of the three oligomers (forms) are different. After formation of the apo-protein of FdII and reconstitution with iron and sulphide, the protein behaves more like FdI, showing a strong g = 1.94 signal in the reduced states.

Binding Sites↗
PubMed1986

Formation of an inactive cytochrome P-450Fe(II)-metabolite complex after administration of amiodarone in rats, mice and hamsters.

Administration of amiodarone hydrochloride (50-150 mg/kg i.p. daily) to rats, mice or hamsters resulted in the in vivo formation of a cytochrome P-450Fe(II)-amiodarone metabolite complex absorbing at 453 nm, unable to bind CO and biologically inactive. In rats, the amount of complex present in hepatic microsomes was small 24 hr after administration of a single dose of amiodarone (100 mg/kg i.p.) but was increased 2.5-times by pretreatment with phenobarbital and 8-times by pretreatment with dexamethasone phosphate. In addition, the complex increased linearly with time as the doses of amiodarone were repeated daily. When both enhancing factors were combined (treatment for 3 days with both dexamethasone and amiodarone), the amount of complex present in liver microsomes reached 0.78 nmol/mg protein or 40% of total cytochrome P-450 in rats. In these rats, in vitro disruption of the complex with potassium ferricyanide suppressed its Soret peak at 453 nm, increased by 70% the CO-binding spectrum of dithionite-reduced microsomes, and restored several monooxygenase activities. The 453 nm-absorbing complex was also formed in vitro upon incubation of amiodarone or N-desethylamiodarone with NADPH, EDTA and microsomes from dexamethasone-treated rats. The formation of the complex was smaller with microsomes from phenobarbital-treated rats and was not detected with microsomes from control rats. We conclude that amiodarone forms an inactive cytochrome P-450Fe(II)-metabolite complex in rats, mice and hamsters.

Amiodarone↗
PubMed1988

Role of hydrogenase 1 of Clostridium pasteurianum in the reduction of metronidazole.

Competition studies between the phosphoroclastic reaction and the metronidazole reduction reaction using dialyzed crude cell-free extracts of Clostridium pasteurianum which were essentially devoid of Hydrogenase 1 activity demonstrated that this enzyme plays an important role in the reduction of metronidazole. To determine further the exact function for Hydrogenase 1 in the reduction of the drug, this enzyme was highly purified from C. pasteurianum. Metronidazole reduction activity copurified with Hydrogenase 1 specific activity throughout the purification procedure. Drug reduction required the presence of an electron carrier and could not be accomplished by the enzyme alone. Ferredoxin, and also the low potential electron carrier dyes, methyl and benzyl viologen, and the flavin coenzymes, FAD and flavin mononucleotide (FMN), could couple the reduction of metronidazole. Hydrogenase 1 activity and its metronidazole reduction activity were inactivated irreversibly in the presence of oxygen. Metronidazole could be reduced only by an electron carrier-Hydrogenase 1 mechanism or directly by sodium dithionite.

Clostridium↗
PubMed1988

Formation of similar species to carbon monoxide during hepatic microsomal metabolism of cannabidiol on the basis of spectral interaction with cytochrome P-450.

Cannabidiol induced a carbon monoxide-like complex with mouse hepatic microsomal cytochrome P-450 during NADPH-dependent metabolism in vitro on a spectral basis. The reduction by dithionite was required for the maximal development of a spectrum. The complex showed a peak at 450 nm which shifted to 419 or 423 nm, respectively, by further addition of hemoglobin or myoglobin. Cannabidiol-induced complex formation required molecular oxygen, and was decreased by the addition of inhibitors of cytochrome P-450-dependent monoxygenase. Pretreatment of mice with phenobarbital (80 or 100 mg/kg, i.p. for 3 days) but not 3-methylcholanthrene (80 mg/kg, i.p.) increased the complex formation. In contrast, pretreatment with cobaltous chloride (40 mg/kg, i.p. for 3 days) decreased the complex formation. 8,9-Dihydro- and 1,2,8,9-tetrahydrocannabidiols also induced the same spectrum as that of above complex, whereas cannabidiol monomethyl- and dimethylethers reduced this ability. In addition, both cannabidivarin and cannabigerol induced the complex formation, although delta 9-tetrahydrocannabinol, cannabinol and cannabielsoin did not. Olivetol but not d-limonene induced the spectrum of the complex to some extent. These results indicate that cannabidiol induces a carbon monoxide-like complex with cytochrome P-450 during hepatic microsomal metabolism, and suggest that phenobarbital-inducible cytochrome P-450s mediate at least one of the metabolic steps of CBD to form the complex, as well as the importance of the resorcinol moiety of CBD for the complex formation.

Animals↗
PubMed1994

Potential antitumour mitosenes: relationship between in vitro DNA interstrand cross-link formation and DNA damage in Escherichia coli K-12 strains.

This investigation was aimed at determining the possible relationship between DNA interstrand cross-linking and the cytotoxic activity of potential antitumour mitosene compounds. Mitosenes, possessing two good leaving groups at C-1 and C-10, were found to be able to cross-link calf thymus DNA under hypoxic conditions following sodium dithionite (Na2S2O4) reduction at pH 7.0 and pH 5.5. DNA interstrand cross-linking was pH dependent for most of the mitosenes used, with a higher amount of cross-links formed at pH 5.5 compared to pH 7.0. Without reduction or under aerobic conditions no cross-link formation was detected. The importance of DNA damage for the toxic effect of these mitosenes was assayed by comparing the survival in a DNA repair deficient and a DNA repair proficient E. coli K-12 strain. A correlation between the number of cross-links formed in calf thymus DNA in vitro and the IC50 values in the DNA repair deficient E. coli strain was found. The effect of hypoxia on toxicity of mitosenes was studied in Chinese hamster V79 cells. In these cells, mitosenes appeared to be very active. Under severe hypoxic conditions toxicity of these mitosenes increased, most likely due to the increased lifetime of the activated mitosene species as compared to aerobic conditions. The results suggest that DNA cross-linking following reductive activation is important for the eventual activity of mitosenes in a bacterial system. Increased activity of mitosenes under hypoxic conditions in the V79 cells indicates that these mitosenes may be more active in hypoxic parts of tumours.

Animals↗