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PubMed1996

Pulmonary artery NADPH-oxidase is activated in hypoxic pulmonary vasoconstriction.

An NADPH-oxidase complex containing at least two protein components (gp91-phox and p22-phox) and a unique low redox potential (-245 mV) cytochrome b-245 is the source of superoxide generated for bacterial killing in neutrophils and has been suggested as the oxygen sensor in the carotid body. In pure cultures of smooth muscle cells from calf small pulmonary arteries (300 microns diameter) the presence of the 91 kD protein specific to this cytochrome was demonstrated by Western blot analysis with monoclonal antibody 48. Low-temperature-difference spectrophotometry of homogenates of these cells demonstrated the characteristic cytochrome b-245 spectrum when titrated between redox potentials of -152 and -345 mV, consistent with the low redox potential form. When these same cells were exposed to hypoxia (approximately 40 mmHg), superoxide production increased significantly from 1.4 +/- 0.2 to 73 +/- 12 nmoles.min-1 mg-1 protein. Hypoxic generation of superoxide was inhibited by the NADPH-oxidase inhibitor diphenyleneiodonium (DPI: 10 microM) but not by the mitochondrial inhibitor myxathiazole (10 microM). The hypoxic superoxide increase was significantly greater than that observed from smooth muscle cells from large pulmonary arteries or from large or small systemic arteries. Fluorescence immunocytochemistry revealed the presence of the NADPH-oxidase protein in the walls of pulmonary arteries in rat lung slices, and confocal microscopy showed the complex to be widely distributed in the vicinity of the arterial smooth muscle walls. In hypoxia or norepinephrine (NP)-induced vasoconstriction of pulmonary artery rings from cats, the sensitivity to inhibition by DPI was observed to be significantly greater for hypoxia (ED50 = 0.8 microM) than for NP-induced (ED50 = 13.4 microM) constriction. Together these observations demonstrate that the unique cytochrome b-245 containing NADPH-oxidase is present in pulmonary artery smooth muscle and that an NADPH-oxidase or NADH-oxidoreductase complex is activated to release superoxide by hypoxic conditions. It is concluded that a trans-membrane NADPH-oxidase is the most likely and that activation of this system may be involved in the initiation of hypoxic pulmonary vasoconstriction.

Animals↗
PubMed2004

Enhanced reduction of Cr(VI) by direct electric current in a contaminated clay.

The probable relation between diffuse double-layer processes and redox reactions that enhance degradation or conversion of contaminants under an applied electric field were examined in a clay medium. Kaolinite clay, precontaminated with hexavalent chromium, was the test soil medium. Analyte, containing ferrous iron, was transported through the kaolinite clay using direct electric current. The Cr(VI) reduction to Cr(III) was followed by measuring the soil redox potential and pH at discrete locations in the clay bed. The post-test distribution of Cr showed significantly more Cr(III) than Cr(VI) at low to slightly acidic pH distribution (2 < pH < 6) in clay. The stoichiometric analyses of measured chromium and iron species concentrations versus the measured redox potentials were compared to Nernst equation predictions of an equivalent aqueous system. An average of +0.37 V shift was measured from the linear Nernstian prediction of cell potential. The applied electric field appeared to provide additional "cathodic current" to drive forth the redox reactions. The redox potential shift was explained by possible overpotential development at the clay-water interfaces due to double-layer polarization under the applied field.

Aluminum Silicates↗
PubMed1995

Identification of potential redox-sensitive cysteines in cytosolic forms of fructosebisphosphatase and glyceraldehyde-3-phosphate dehydrogenase.

Tertiary-structure modeling suggests the occurrence of disulfide bonds in the cytosolic form of fructose-bisphosphatase (EC 3.1.3.11) in spinach (Spinacia oleracea L.), sugarbeet (Beta vulgaris L.) and potato (Solanum tuberosum L.). Redox modulation could then control the AMP sensitivity of fructosebisphosphatase in the cytosol, as suggested by the experiments of E. Khayat et al. (1993, Plant Physiol. 101, 57-64). Modeling also reveals two cysteine residues correctly positioned to form a disulfide bond and hence potentially redox-sensitive in the cytosolic glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.12) from the facultative crassulacean metabolism plant Mesembryanthemum crystallinum L.

Amino Acid Sequence↗
PubMed1999

Comparative redox and pKa calculations on cytochrome c3 from several Desulfovibrio species using continuum electrostatic methods.

A comparative study of the pH-dependent redox mechanisms of several members of the cytochrome c3 family has been carried out. In a previous work, the molecular determinants of this dependency (the so-called redox-Bohr effect) were investigated for one species using continuum electrostatic methods to find groups with a titrating range and strength of interaction compatible with a mediating role in the redox-Bohr effect. Here we clarify these aspects in the light of new and improved pKa calculations, our findings supporting the hypothesis of propionate D from heme I being the main effector in the pH-dependent modulation of the cytochrome c3 redox potentials in all the c3 molecules studied here. However, the weaker (but significant) role of other titrating groups cannot be excluded, their importance and identity changing with the particular molecule under study. We also calculate the relative redox potentials of the four heme centers among the selected members of the c3 family, using a continuum electrostatic method that takes into account both solvation and interaction effects. Comparison of the calculated values with available data for the microscopic redox potentials was undertaken, the quality of the agreement being dependent upon the choice of the dielectric constant for the protein interior. We find that high dielectric constants give best correlations, while low values result in better magnitudes for the calculated potentials. The possibility that the crystallographic calcium ion in c3 from Desulfovibrio gigas may be present in the solution structure was tested, and found to be likely.

Calcium↗
PubMed1998

Generating controlled reducing environments in aerobic recombinant Escherichia coli fermentations: effects on cell growth, oxygen uptake, heat shock protein expression, and in vivo CAT activity.

The independent control of culture redox potential (CRP) by the regulated addition of a reducing agent, dithiothreitol (DTT) was demonstrated in aerated recombinant Escherichia coli fermentations. Moderate levels of DTT addition resulted in minimal changes to specific oxygen uptake, growth rate, and dissolved oxygen. Excessive levels of DTT addition were toxic to the cells resulting in cessation of growth. Chloramphenicol acetyltransferase (CAT) activity (nmoles/microgram total protein min.) decreased in batch fermentation experiments with respect to increasing levels of DTT addition. To further investigate the mechanisms affecting CAT activity, experiments were performed to assay heat shock protein expression and specific CAT activity (nmoles/microgram CAT min.). Expression of such molecular chaperones as GroEL and DnaK were found to increase after addition of DTT. Additionally, sigma factor 32 (sigma32) and several proteases were seen to increase dramatically during addition of DTT. Specific CAT activity (nmoles/microgram CAT min. ) varied greatly as DTT was added, however, a minimum in activity was found at the highest level of DTT addition in E. coli strains RR1 [pBR329] and JM105 [pROEX-CAT]. In conjunction, cellular stress was found to reach a maximum at the same levels of DTT. Although DTT addition has the potential for directly affecting intracellular protein folding, the effects felt from the increased stress within the cell are likely the dominant effector. That the effects of DTT were measured within the cytoplasm of the cell suggests that the periplasmic redox potential was also altered. The changes in specific CAT activity, molecular chaperones, and other heat shock proteins, in the presence of minimal growth rate and oxygen uptake alterations, suggest that the ex vivo control of redox potential provides a new process for affecting the yield and conformation of heterologous proteins in aerated E. coli fermentations.

Aerobiosis↗
PubMed2006

Direct electrochemistry of human, bovine and porcine cytochrome P450c17.

The direct electrochemistry of human, bovine and porcine cytochrome P450c17 (CYP17) has been examined on an edge-oriented pyrolytic graphite electrode. The recombinant protein was immobilized on an electrode modified with a surfactant to simulate the environment of a biological membrane, and hence physiological electron-transfer conditions. The P450 enzymes all retained 'electron-transfer' activity while immobilized at the electrode surface as assessed by the presence of catalytic signals under aerobic conditions. The redox potentials for porcine P450c17 were more positive (anodic) than both the human and bovine forms, perhaps reflecting the differences in substrate specificity for these species. In addition, these enzymes were all influenced by pH, consistent with a single proton associated with the single electron-transfer event. Ionic strength of the buffer medium also shifted the redox potentials towards positive, suggesting that electrostatic forces contribute to the protein environment required for the electron-transfer process. The effect of substrate on the redox potential for each P450c17 was measured in the presence of pregnenolone, progesterone, 17alpha-hydroxypregnenolone and 17alpha-hydroxyprogesterone. However, no influence on the redox parameters was observed.

Animals↗
PubMed2003

Structural, spectroscopic, and redox consequences of a central ligand in the FeMoco of nitrogenase: a density functional theoretical study.

Broken symmetry density functional and electrostatics calculations have been used to shed light on which of three proposed atoms, C, N, or O, is most likely to be present in the center of the FeMoco, the active site of nitrogenase. At the Mo(4+)4Fe(2+)3Fe(3+) oxidation level, a central N(3-) anion results in (1) calculated Fe-N bond distances that are in very good agreement with the recent high-resolution X-ray data of Einsle et al.; (2) a calculated redox potential of 0.19 eV versus the standard hydrogen electrode (SHE) for FeMoco(oxidized) + e(-) --> FeMoco(resting), in good agreement with the measured value of -0.042 V in Azotobacter vinelandii; and (3) average Mössbauer isomer shift values (IS(av) = 0.48 mm s(-1)) compatible with experiment (IS(av) = 0.40 mm s(-1)). At the more reduced Mo(4+)6Fe(2+)1Fe(3+) level, the calculated geometry around a central N(3-) anion still correlates well with the X-ray data, but the average Mössbauer isomer shift value (IS(av) = 0.54 mm s(-1)) and the redox potential of -2.21 eV show a much poorer agreement with experiment. These calculated structural, spectroscopic, and redox data indicate the most likely iron oxidation state for the resting FeMoco of nitrogenase to be 4Fe(2+)3Fe(3+). At this favored oxidation state, oxygen or carbon coordination leads to (1) Fe-O distances in poor agreement and Fe-C distances in good agreement with experiment and (2) calculated redox potentials of +0.97 eV for O(2-) and -1.31 eV for C(4-). The calculated structural parameters and/or redox data suggest either O(2-) or C(4-) is unlikely as a central anion.

Molybdoferredoxin↗
PubMed2002

Molecular mechanism of the electron transfer reaction in cytochrome P450(cam)--putidaredoxin: roles of glutamine 360 at the heme proximal site.

We characterized electron transfer (ET) from putidaredoxin (Pdx) to the mutants of cytochrome P450(cam) (P450(cam)), in which one of the residues located on the putative binding site to Pdx, Gln360, was replaced with Glu, Lys, and Leu. The kinetic analysis of the ET reactions from reduced Pdx to ferric P450(cam) (the first ET) and to ferrous oxygenated P450(cam) (the second ET) showed the dissociation constants (K(m)) that were moderately perturbed for the Lys and Leu mutants and the distinctly increased for the Glu mutant. Although the alterations in K(m) indicate that Gln360 is located at the Pdx binding site, the effects of the Gln360 mutations (0.66-20-fold of that of wild type) are smaller than those of the Arg112 mutants (25-2500-fold of that of wild type) [Unno, M., et al. (1996) J. Biol. Chem. 271, 17869-17874], allowing us to conclude that Gln360 much less contributes to the complexation with Pdx than Arg112. The first ET rate (35 s(-1) for wild-type P450(cam)) was substantially reduced in the Glu mutant (5.4 s(-1)), while less perturbation was observed for the Lys (53 s(-1)) and Leu (23 s(-1)) mutants. In the second ET reaction, the retarded ET rate was detected only in the Glu mutant but not in the Lys and Leu mutants. These results showed the smaller mutational effects of Gln360 on the ET reactions than those of the Arg112 mutants. In contrast to the moderate perturbations in the kinetic parameters, the mutations at Gln360 significantly affected both the standard enthalpy and entropy of the redox reaction of P450(cam), which cause the negative shift of the redox potentials for the Fe(3+)/Fe(2+) couple by 20-70 mV. Since the amide group of Gln360 is located near the carbonyl oxygen of the amide group of the axial cysteine, it is plausible that the mutation at Gln360 perturbs the electronic interaction of the axial ligand with heme iron, resulting in the reduction of the redox potentials. We, therefore, conclude that Gln360 primarily regulates the ET reaction of P450(cam) by modulating the redox potential of the heme iron and not by the specific interaction with Pdx or the formation of the ET pathway that are proposed as the regulation mechanism of Arg112.

Amino Acid Sequence↗
PubMed1975

Delayed fluorescence from Rhodopseudomonas viridis following single flashes.

Delayed fluorescence from Rhodopseudomonas viridis membrane fragments has been studies using a phosphoroscope employing single, short actinic flashes, under conditions of controlled redox potential and temperature. The emission spectrum shows that delayed fluorescence is emitted by the bulk, antenna bacteriochlorophyll. The energy for delayed fluorescence, however, must be stored in a reaction-center complex including the photooxidized form (P+) of the primary electron-donor (P) and the photoreduced form (X MINUS) of the primary electron-acceptor. This is shown by the following observations: (1) Delayed luminescence is quenched (a) at low redox potentials which allow cytochromes to reduce P+ rapidly after the flash, (b) at higher redox potentials which, by oxidizing P chemically, prevent the photochemical formation of P+X minus, and (c) upon transfer of an electron from X minus to a secondary acceptor, Y. (2) Under conditions that prevent the reduction of P+ by cytochromes and the oxidation of X minus by Y, the decay kinetics of delayed fluorescence are identical with those of P+X minus, as measured from optical absorbance changes. The main decay route for P+X minus under these conditions has a rate-constant of approximately 10-3-s-minus 1. In contrase, a comparison of the intensities of delayed and prompt fluorescence indicates that the process in which P+X minus returns energy to the bulk bacteriochlorophyll has a rate-constant of 3.7 s-minus 1, at 295 degrees K and pH 7.8. The decay kinetics of P+X minus and delayed fluorescence change little with temperature, whereas the intensity of delayed fluorescence increases with increasing temperature, having an activation energy of 12.5 kcal mol-mol- minus 1. We conclude that the main decay route involves tunneling of an electron from X minus to P+, without the promotion of P to an excited state. Delayed fluorescence requires such a promotion, followed by transfer of energy to the bulk bacteriochlorophyll, and this combination of events is rare. The activation energy, taken with potentiometric data, indicates that the photochemical conversion of PX to P+X minus results in increases of both the energy and the entropy of the system, by 16.6 kcal-mol- minus 1 and 8.8 cal-mol- minus 1-deg- minus 1. The intensity of delayed fluorescence depends strongly on the pH; the origin of this effect remains unclear.

Calorimetry↗
PubMed2005

Crystal structure of nitrated human manganese superoxide dismutase: mechanism of inactivation.

A cellular consequence of the reaction of superoxide and nitric oxide is enhanced peroxynitrite levels. Reaction of peroxynitrite with manganese superoxide dismutase (MnSOD) causes nitration of the active-site residue Tyr34 and nearly complete inhibition of catalysis. We report the crystal structures at 2.4 A resolution of human MnSOD nitrated by peroxynitrite and the unmodified MnSOD. A comparison of these structures showed no significant conformational changes of active-site residues or solvent displacement. The side chain of 3-nitrotyrosine 34 had a single conformation that extended toward the manganese with O1 of the nitro group within hydrogen-bonding distance (3.1 A) of Nepsilon2 of the second-shell ligand Gln143. Also, nitration of Tyr34 caused a weakening, as evidenced by the lengthening, of a hydrogen bond between its phenolic OH and Gln143, part of an extensive hydrogen-bond network in the active site. Inhibition of catalysis can be attributed to a steric effect of 3-nitrotyrosine 34 that impedes substrate access and binding, and alteration of the hydrogen-bond network that supports proton transfer in catalysis. It is also possible that an electrostatic effect of the nitro group has altered the finely tuned redox potential necessary for efficient catalysis, although the redox potential of nitrated MnSOD has not been measured.

Binding Sites↗
PubMedDate not supplied

[Effect of some electron acceptors on metabolism of methanogenic biotic community].

The dynamics of redox potential, pH, CH4, H2, CO2, N2 formation under the community development under anaerobic conditions in presence of acceptors of electrons (potassium ferricyanide, molecular oxygen, sodium nitrate and sodium sulphate) has been investigated. It has been established that the optimal value at the redox potential under the development of methanogenic community was created at the expense of microorganisms vital activity. The decrease of methanogenesis under the electron acceptors introduction is connected with the increase of the culture medium redox potential.

Anaerobiosis↗
PubMed1999

NifL of Klebsiella pneumoniae: redox characterization in relation to the nitrogen source.

In Klebsiella pneumoniae, NifL modulates the activity of the transcriptional activator NifA in response to combined nitrogen or external molecular oxygen. We recently showed that K. pneumoniae NifL is a flavoprotein which apparently senses oxygen through a redox-sensitive, conformational change. In order to study whether the nitrogen signal might be transmitted to NifA through a stable modification of NifL we characterized the redox properties of NifL synthesized in Escherichia coli in the presence of different nitrogen sources. FAD analyses showed that purified NifL carried FAD as cofactor independent of nitrogen and oxygen availability. The redox potential of NifL synthesized in the presence of ammonium was -277+/-5 mV at pH 8.0 and 25 degrees C, as determined by reduction with dithionite or with enzymatic reduction by xanthine oxidase in the presence of methyl viologen as redox mediator. When synthesized under nitrogen-limiting conditions, NifL showed a redox potential of -274+/-6 mV at pH 8.0 and 25 degrees C. Fully reduced NifL fractions, synthesized under either condition listed above, reoxidized rapidly in the presence of molecular oxygen. These results indicate that for NifL synthesized in E. coli, the redox potential of the NifL-bound FAD is not influenced by the nitrogen source. The two NifL fractions differed, however, in that a non-flavin specific absorbance at 420 nm was found only in NifL synthesized in the presence of ammonium.

Ammonia↗
PubMed1998

Flavonoid deactivation of ferrylmyoglobin in relation to ease of oxidation as determined by cyclic voltammetry.

Fourteen flavonoid aglycones, and the flavonoid glycoside rutin, with redox potentials ranging from 0.20 (myricetin) to 0.83 V (chrysin) vs. NHE, as determined by cyclic voltammetry at 23 degrees C in aqueous 50 mM phosphate, ionic strength 0.16 (NaCl) with pH = 7.4 and compared with redox potentials determined for four cinnamic acid derivatives, were all found to reduce ferrylmyoglobin, MbFe(IV)=O, to metmyoglobin, MbFe(III). Reaction stoichiometry depends strongly on the number of hydroxyl groups in the flavonoid B-ring. All compounds with 3',4'-dihydroxy substitution reduce 2 equivalents of MbFe(IV)=O, whereas naringenin, hesperitin and kaempferol, with one hydroxyl group in the B-ring, reduce with a one-to-one stoichiometry. As studied spectrophotometrically under pseudo-first-order conditions with flavonoids in excess, rutin and apigenin react with MbFe(IV)=O with very similar and moderately high activation enthalpies of deltaH298++ = 69 +/- 1 kJ mol(-1) and deltaH298++ = 65 +/- 3 kJ mol(-1), respectively, and with positive activation entropies of deltaS298++ = 23 +/- 4 J mol(-1) K(-1) and deltaS298++ = 13 +/- 9 J mol(-1) K(-1), respectively, in agreement with outer-sphere electron transfer as rate determining. For the fifteen plant polyphenols only qualitative relations exist between redox potential and rate constants rather than a linear free energy relationship (r2 = 0.503), and especially the flavone apigenin was found more efficient as reducing agent. For the flavanones, a linear relation (r2 = 0.971) indicate that, in the absence of a 2,3 double bond, removal of the 4-carbonyl group or addition of a 3-hydroxy group only has minor effect on reactivity. The flavonols are the most efficient reducing agents, effectively reducing MbFe(IV)=O to MbFe(III) and establishing a steady state distribution between the flavonol and MbFe(III) and oxymyoglobin, MbFe(II)O2. Oxidised flavonols reduces MbFe(III) to MbFe(II)O2 very efficiently and much faster than the parent flavonol.

Animals↗
PubMed2004

EDTA and mixed-ligand complexes of tetravalent and trivalent plutonium.

EDTA forms stable complexes with plutonium that are integral to nuclear material processing, radionuclide decontamination, and the potentially enhanced transport of environmental contamination. To characterize the aqueous Pu(4+/3+)EDTA species formed under the wide range of conditions of these processes, potentiometry, spectrophotometry, and cyclic voltammetry were used to measure solution equilibria. The results reveal new EDTA and mixed-ligand complexes and provide more accurate stability constants for previously identified species. In acidic solution (pH < 4) and at 1:1 ligand to metal ratio, PuY (where Y4- is the tetra-anion of EDTA) is the predominant species, with an overall formation constant of log beta110 = 26.44. At higher pH, the hydrolysis species, PuY(OH)- and PuY(OH)(2)2-, form with the corresponding overall stability constants log beta(11 - 1) = 21.95 and log beta(11 - 2) = 15.29. The redox potential of the complex PuY at pH = 2.3 was determined to be E(1/2) = 342 mV. The correlation between redox potential, pH, and the protonation state of PuY- was derived to estimate the redox potential of the Pu(4+/3+)Y complex as a function of pH. Under conditions of neutral pH and excess EDTA relative to Pu4+, PuY(2)4- forms with an overall formation constant of log beta120 = 35.39. In the presence of ancillary ligands, mixed-ligand complexes form, as exemplified by the citrate and carbonate complexes PuY(citrate)3- (log beta1101 = 33.45) and PuY(carbonate)2- (log beta1101 = 35.51). Cyclic voltammetry shows irreversible electrochemical behavior for these coordinatively saturated Pu4+ complexes: The reduction wave is shifted approximately -400 mV from the reduction wave of the complex PuY, while the oxidation wave is invariant.

Journal Article↗
PubMed1998

Intramolecular electron transport in quinoprotein alcohol dehydrogenase of Acetobacter methanolicus: a redox-titration study

Quinohemoprotein-cytochrome c complex alcohol dehydrogenase (ADH) of acetic acid bacteria consists of three subunits, of which subunit I contains pyrroloquinoline quinone (PQQ) and heme c, and subunit II contains three heme c components. The PQQ and heme c components are believed to be involved in the intramolecular electron transfer from ethanol to ubiquinone. To study the intramolecular electron transfer in ADH of Acetobacter methanolicus, the redox potentials of heme c components were determined with ADH complex and the isolated subunits I and II of A. methanolicus, as well as hybrid ADH consisting of the subunit I/III complex of Gluconobacter suboxydans ADH and subunit II of A. methanolicus ADH. The redox potentials of hemes c in ADH complex were -130, 49, 188, and 188 mV at pH 7.0 and 24, 187, 190, and 255 mV at pH 4.5. In hybrid ADH, one of these heme c components was largely changed in the redox potential. Reduced ADH was fully oxidized with potassium ferricyanide, while ubiquinone oxidized the enzyme partially. The results indicate that electrons extracted from ethanol at PQQ site are transferred to ubiquinone via heme c in subunit I and two of the three hemes c in subunit II. Copyright 1998 Elsevier Science B.V.

Journal Article↗
PubMed1999

Complementation of DsbA deficiency with secreted thioredoxin variants reveals the crucial role of an efficient dithiol oxidant for catalyzed protein folding in the bacterial periplasm.

The thiol/disulfide oxidoreductase DsbA is the strongest oxidant of the thioredoxin superfamily and is required for efficient disulfide bond formation in the periplasm of Escherichia coli. To determine the importance of the redox potential of the final oxidant in periplasmic protein folding, we have investigated the ability of the most reducing thiol/disulfide oxidoreductase, E.coli thioredoxin, of complementing DsbA deficiency when secreted to the periplasm. In addition, we secreted thioredoxin variants with increased redox potentials as well as the catalytic a-domain of human protein disulfide isomerase (PDI) to the periplasm. While secreted wild-type thioredoxin and the most reducing thioredoxin variant could not replace DsbA, all more oxidizing thioredoxin variants as well as the PDI a-domain could complement DsbA deficiency in a DsbB-dependent manner. There is an excellent agreement between the activity of the secreted thioredoxin variants in vivo and their ability to oxidize polypeptides fast and quantitatively in vitro. We conclude that the redox potential of the direct oxidant of folding proteins and in particular its reactivity towards reduced polypeptides are crucial for efficient oxidative protein folding in the bacterial periplasm.

Bacterial Proteins↗
PubMed2000

Effect of substituents on microsomal reduction of benzo(c)fluorene N-oxides.

The potential benzo(c)fluorene antineoplastic agent benfluron (B) displays high activity against a broad spectrum of experimental tumours in vitro and in vivo. In order to suppress some of its undesirable properties, its structure has been modified. Benfluron N-oxide (B N-oxide) is one of benfluron derivatives tested. The main metabolic pathway of B N-oxide is its reduction to tertiary amine B. A key role of cytochrome P4502B and P4502E1 in B N-oxide reduction has been proposed in the rat. Surprisingly, B N-oxide is reduced also in the presence of oxygen although all other N-oxides undergo reduction only under anaerobic conditions. With the aim to determine the influence of the N-oxide chemical structure and its redox potential on reductase affinity, activity and oxygen sensitivity five relative benzo(c)fluorene N-oxides were prepared. A correlation between the redox potential measured and the non-enzymatic reduction ability of the substrate was found, but no effect of the redox potential on reductase activity was observed. Microsomal reductases display a high affinity to B N-oxide (apparent K(m) congruent with0. 2 mM). A modification of the side-chain or nitrogen substituents has led to only a little change in apparent K(m) values, but a methoxy group substitution on the benzo(c)fluorene moiety induced a significant K(m) increase (ten-fold). Based on kinetic study results, the scheme of mechanism of cytochrome P450 mediated benzo(c)fluorene N-oxides reduction have been proposed. All benzo(c)fluorene N-oxides under study were able to be reduced in the presence of oxygen. Changes in the B N-oxide structure caused an extent of anaerobic conditions preference. The relationship between the benzo(c)fluorene N-oxide structure and the profile of metabolites in microsomal incubation was studied and important differences in the formation of individual N-oxide metabolites were found.

Aerobiosis↗
PubMed1985

Response of a 'susceptible' Escherichia coli to metronidazole therapy: an investigation using experimental subcutaneous abscesses.

Earlier experiments under anaerobic conditions in vitro had indicated that Gram-negative facultative anaerobes were susceptible to therapeutically attainable concentrations of metronidazole. This prompted us to study the response of one such strain to metronidazole therapy. Mice with 3-day-old subcutaneous abscesses induced by 'susceptible' Escherichia coli (accompanied by potentiating agent and obligately anaerobic Bacteroides fragilis) were given metronidazole by mouth (250 mg/kg/dose 12 hourly) either as a two- or a four-dose course. This therapy reduced the viable numbers of E. coli/abscess by a mere 0.3 log10 and 0.7 log10, respectively. Assays showed that concentrations of metronidazole in abscess-contents compared favourably with those in the earlier in vitro experiments. But measurement of redox potentials in abscesses indicated that the conditions in these (-191 mV vs. Ag/AgCl) were not nearly as reduced as those in experiments in vitro (-650 mV). This, and the known dependence of metronidazole on low redox potentials for expression of its antimicrobial ability, appears to be the explanation for the poor anticoliform effect in the subcutaneous abscess model. Full assessment of the clinical implications of these observations must await the availability of data regarding redox potentials in clinical situations.

Abscess↗