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1.
Aerobic organisms are afforded with an antioxidant enzymatic apparatus that more recently has been recognized to include cytochrome
c, as it is able to prevent hydrogen peroxide generation by returning electrons from the superoxide ion back to the respiratory
chain. The present study investigated the glutathione peroxidase (GPx), superoxide dismutase (SOD) and cytochrome c-like antioxidant activities of para Mn(III)TMPyP in isolated rat liver mitochondria (RLM) and mitoplasts. In RLM, Mn IIITMPyP decreased the lipid-peroxide content associated with glutathione (GSH) depletion consistent with the use of GSH as a
reducing agent for high valence states of Mn IIITMPyP. SOD and cytochrome c antioxidant activities were also investigated. Mn IITMPyP was able to reduce ferric cytochrome c, indicating the potential to remove a superoxide ion by returning electrons back to the respiratory chain. In antimicyn A-poisoned
mitoplasts, Mn IIITMPyP efficiently decreased the EPR signal of DMPO-OH adduct concomitant with GSH depletion. The present results are consistent
with SOD and GPx activities for Mn IIITMPyP and do not exclude cytochrome c-like activity. However, considering that para Mn IIITMPyP more efficiently reduces, rather than oxidizes, superoxide ion; electron transfer from the Mn IITMPyP to the respiratory chain might not significantly contribute to the superoxide ion removal, since most of Mn IITMPyP is expected to be produced at the expense of NADPH/GSH oxidation. The present results suggest GPx-like activity to be
the principal antioxidant mechanism of Mn IIITMPyP, whose efficiency is dependent on the NADPH/GSH content in cells. 相似文献
2.
In the presence of Fe 3+ and complexing anions, the peroxidation of unsaturated liver microsomal lipid in both intact microsomes and in a model system containing extracted microsomal lipid can be promoted by either NADPH and NADPH : cytochrome c reductase or by xanthine and xanthine oxidase. Erythrocuprein effectively inhibits the activity promoted by xanthine and xanthine oxidase but produces much less inhibition of NADPH-dependent peroxidation. The singlet-oxygen trapping agent, 1,3-diphenylisobenzofuran, had no effect on NADPH-dependent peroxidation but strongly inhibited the peroxidation promoted by xanthine and xanthine oxidase. NADPH-dependent lipid peroxidation was also shown to be unaffected by hydroxyl radical scavengers.. The addition of catalase had no effect on NADPH-dependent lipid peroxidation, but it significantly increased the rate of malondialdehyde formation in the reaction promoted by xanthine and xanthine oxidase. These results demonstrate that NADPH-dependent lipid peroxidation is promoted by a reaction mechanism which does not involve either superoxide, singlet oxygen, HOOH, or the hydroxyl radical. It is concluded that NADPH-dependent lipid peroxidation is initiated by the reduction of Fe 3+ followed by the decomposition of hydroperoxides to generate alkoxyl radicals. The initiation reaction may involve some form of the perferryl ion or other metal ion species generated during oxidation of Fe 2+ by oxygen. 相似文献
3.
Class Ib ribonucleotide reductases (RNR) utilize a di-nuclear manganese or iron cofactor for reduction of superoxide or molecular oxygen, respectively. This generates a stable tyrosyl radical (Y·) in the R2 subunit (NrdF), which is further used for ribonucleotide reduction in the R1 subunit of RNR. Here, we report high-resolution crystal structures of Bacillus anthracis NrdF in the metal-free form (1.51 Å) and in complex with manganese (MnII/MnII, 1.30 Å). We also report three structures of the protein in complex with iron, either prepared anaerobically (FeII/FeII form, 1.32 Å), or prepared aerobically in the photo-reduced FeII/FeII form (1.63 Å) and with the partially oxidized metallo-cofactor (1.46 Å). The structures reveal significant conformational dynamics, likely to be associated with the generation, stabilization, and transfer of the radical to the R1 subunit. Based on observed redox-dependent structural changes, we propose that the passage for the superoxide, linking the FMN cofactor of NrdI and the metal site in NrdF, is closed upon metal oxidation, blocking access to the metal and radical sites. In addition, we describe the structural mechanics likely to be involved in this process. 相似文献
4.
The stability of (all- E)-β-carotene toward dietary iron was studied in a mildly acidic (pH 4) micellar solution as a simple model of the postprandial gastric conditions. The oxidation was initiated by free iron (Fe II, Fe III) or by heme iron (metmyoglobin, MbFe III). Fe II and metmyoglobin were much more efficient than Fe III at initiating β-carotene oxidation. Whatever the initiator, hydrogen peroxide did not accumulate. Moreover, β-carotene markedly inhibited the conversion of Fe II into Fe III. β-Carotene oxidation induced by Fe II or MbFe III was maximal with 5–10 eq Fe II or 0.05–0.1 eq MbFe III and was inhibited at higher iron concentrations, especially with Fe II. UPLC/DAD/MS and GC/MS analyses revealed a complex distribution of β-carotene-derived products including Z-isomers, epoxides, and cleavage products of various chain lengths. Finally, the mechanism of iron-induced β-carotene oxidation is discussed. Altogether, our results suggest that dietary iron, especially free (loosely bound) Fe II and heme iron, may efficiently induce β-carotene autoxidation within the upper digestive tract, thereby limiting its supply to tissues (bioavailability) and consequently its biological activity. 相似文献
5.
1. The NADPH-oxidizing activity of a 100 000 × g particulate fraction of the postnuclear supernatant obtained from guinea-pig phagocytosing polymorphonuclear leucocytes has been assayed by simultaneous determination of oxygen consumption, NADPH oxidation and O ?2 generation at pH 5.5 and 7.0 and with 0.15 mM and 1 mM NADPH.2. The measurements of oxygen consumption and NADPH oxidation gave comparable results. The stoichiometry between the oxygen consumed and the NADPH oxidized was 1 : 1.3. A markedly lower enzymatic activity was observed, under all the experimental conditions used, when the O ?2 generation assay was employed as compared to the assays of oxygen uptake and NADPH oxidation.4. The explanation of this difference came from the analysis of the effect of superoxide dismutase and of cytochrome c which removes O ?2 formed during the oxidation of NADPH.5. Both superoxide dismutase and cytochrome c inhibited the NADPH-oxidizing reaction at pH 5.5. The inhibition was higher with 1 mM NADPH than with 0.15 mM NADPH.6. Both superoxide dismutase and cytochrome c inhibited the NADPH-oxidizing reaction at pH 7.0 with 1 mM NADPH but less than at pH 5.5 with 1 mM NADPH.7. The effect of superoxide dismutase at pH 7.0 with 0.15 mM NADPH was negligible.8. In all instances the inhibitory effect of cytochrome c was greater than that of superoxide dismutase.9. It was concluded that the NADPH-oxidizing reaction studied here is made up of three components: an enzymatic univalent reduction of O 2; an enzymatic, apparently non-univalent, O 2 reduction and a non-enzymatic chain reaction.10. These three components are variably and independently affected by the experimental conditions used. For example, the chain reaction is freely operative at pH 5.5 with 1 mM NADPH but is almost absent at pH 7.0 with 0.15 mM NADPH, whereas the univalent reduction of O 2 is optimal at pH 7.0 with 1 mM NADPH. 相似文献
6.
Nicotianamine (NA) occurs in all plants and chelates metal cations, including Fe II, but reportedly not Fe III. However, a comparison of the Fe II and Zn II affinity constants of NA and various Fe III-chelating aminocarboxylates suggested that NA should chelate Fe III. High-voltage electrophoresis of the FeNA complex formed in the presence of Fe III showed that the complex had a net charge of 0, consistent with the hexadentate chelation of Fe III. Measurement of the affinity constant for Fe III yielded a value of 10 20.6, which is greater than that for the association of NA with Fe II (10 12.8). However, capillary electrophoresis showed that in the presence of Fe II and Fe III, NA preferentially chelates Fe II, indicating that the Fe IINA complex is kinetically stable under aerobic conditions. Furthermore, Fe complexes of NA are relatively poor Fenton reagents, as measured by their ability to mediate H 2O 2-dependent oxidation of deoxyribose. This suggests that NA will have an important role in scavenging Fe and protecting the cell from oxidative damage. The pH dependence of metal ion chelation by NA and a typical phytosiderophore, 2′-deoxymugineic acid, indicated that although both have the ability to chelate Fe, when both are present, 2′-deoxymugineic acid dominates the chelation process at acidic pH values, whereas NA dominates at alkaline pH values. The consequences for the role of NA in the long-distance transport of metals in the xylem and phloem are discussed. 相似文献
7.
An oxalate-bridged binuclear iron(III) complex, [(acac) 2Fe(μ-ox)Fe(acac) 2], (acac −=acetylacetonate anion and ox 2−=oxalate anion) was prepared. The complex crystallized as two types of crystals under different conditions: one had 1,2-dichloroethane as a solvent molecule of crystallization 2, the other did not 1. Both compounds have been characterized by X-ray crystallography, infrared spectroscopy, and thermogravimetric analysis. Compound 1 has also been characterized by UV-Vis and 1H NMR spectroscopies, mass spectrometry, and electrochemistry. In both crystals, each iron(III) is coordinated in an octahedral arrangement by the oxygen atoms of an oxalate-bridging ligand and four oxygen atoms belonging to peripheral acac ligands in an octahedral arrangement. The intermetallic distance of Fe?Fe is 5.4368(9) Å in 1 and 5.438(2) Å in 2. Two iron(III) ions in each crystal are bridged by the oxalate and both lie in the oxalate-plane. The results of thermal analyses imply that the thermal stability of 2 is lower than that of 1. Cyclic voltammograms of 1 in acetonitrile and dichloromethane at low temperature showed two consecutive, quasi-Nernstian, one-electron reduction steps corresponding to the reduction of Fe III-Fe III to Fe III-Fe II followed by the reduction of Fe III-Fe II to Fe II-Fe II. The electrochemical comproportionation constants ( Kc) of the equilibrium (Fe III-Fe III) + (Fe II-Fe II) ? 2(Fe III-Fe II) are 10 8.9 in acetonitrile medium and 10 8.5 in dichloromethane, respectively. The considerably large Kc values indicate that the main factor contributing to the stabilization of the Fe III-Fe II mixed-valence state is electronic delocalization through the oxalate-bridge. 相似文献
8.
In pregnant females, placenta is the most important source of lipid hydroperoxides and other reactive oxygen species (ROS).
The increased production of lipid peroxides is often linked to preeclampsia. In our study, we revealed that NADPH- and iron-dependent
lipid peroxidation in human placental microsomes (HPM) occurred. In the presence of Fe 2+ ion, HPM produced small amounts of thiobarbituric acid-reactive substances (TBARS) – a final product of lipid peroxidation.
NADPH caused a strong increase of iron stimulated TBARS formation. TBARS formation was inhibited by superoxide dismutase,
butylated hydroxytoluene and α-tocopherol but not by mannitol or catalase. TBARS and superoxide radical production was inhibited
in similar manner by cytochrome P450 inhibitors. The results obtained led us to the following conclusions: (1) microsomal
lipid peroxidation next to mitochondrial lipid peroxidation may by an important source of lipid hydroperoxides in blood during
pregnancy and (2) superoxide radical released by microsomal cytochrome P450 is an important factor in NADPH- and iron-dependent
lipid peroxidation in HPM. 相似文献
9.
Electrically permeabilized neutrophils were used to study the mechanism of activation of the respiratory burst by the chemotactic agent formyl-methionyl-leucyl-phenylalanine (fMLP). Permeabilization was assessed by flow cytometry, radioisotope trapping, and by the requirement for exogenous NADPH for oxygen consumption. A respiratory burst could be elicited by fMLP, phorbol ester, or diacylglycerol in permeabilized cells suspended in EGTA-buffered medium with 100 nM free Ca2+. The fMLP response persisted even in cells depleted of intracellular Ca2+ stores by pretreatment with ionomycin. Therefore, a change in cytosolic free Ca2+ ([Ca2+]i) is not required for receptor-mediated stimulation of the respiratory burst. The responses induced by phorbol ester and diacylglycerol were largely inhibited by H7, a protein kinase C antagonist. In contrast, the stimulation of oxygen consumption by fMLP was unaffected by H7. These results suggest that a third signaling pathway, distinct from changes in [Ca2+]i and activation of protein kinase C, is involved in the response of neutrophils to chemoattractants. 相似文献
10.
The relationship between the degradation reaction of cytochrome and lipid peroxidation was studied utilizing bovine adrenal cortex mitochondria. The two reactions were found to be closely correlated in terms of their response to storage of the mitochondrial preparation, stimulation by Fe 2+, inhibition by EDTA and their initiation by cumene hydroperoxide. Both reactions were also found not to be inhibited by catalase, superoxide dismutase, 1,4-diazabicyclo-(2,2,2)-octane and alcohols, indicating that H 2O 2, superoxide, singlet oxygen and hydroxyl radicals do not participate in these reactions. Yet, diphenylamine proved to be a powerful inhibitor for both reactions, suggesting the involvement of a radical species. Cumene hydroperoxide could induce these two reactions at below 0.1 mM concentrations in the presence of molecular oxygen. The chemiluminescence observed during the Fe 2+-mediated lipid peroxidation reaction which was not inhibited by either superoxide dismutase or 1,4-diazabicyclo-(2,2,2)-octane, was biphasic: one was a rapid burst; and the other was a slowly increasing emission. The latter portion of the emission of light coincided with the formation of malondialdehyde. These results indicate that in adrenal cortex mitochondria the degradation of cytochrome is closely related to lipid peroxidation. 相似文献
11.
The system, which contains NADPH, purified cytochrome P-450 reductase, and adriamycin, produces H 2O 2 and in appreciable amounts with oxygen consumption and NADPH oxidation under aerobic conditions. Such an adriamycin-induced NADPH oxidation system, however, does not cause the decomposition of unsaturated fatty acids in microsomal phospholipid micelles, suggesting no direct participation of the active oxygen species and semiquinone radicals of adriamycin in lipid peroxidation. Adriamycin produces a co-ordination complex with Fe 3+ and ADP, which, but no Fe 3+-ADP complex, could be reduced by NADPH-cytochrome P-450 reductase at the expence of NADPH. The decomposition of unsaturated fatty acids in phospholipid micelles is achieved by the Fe 3+-ADP-adriamycin complex and strikingly enhanced by enzymatically reduced iron-ADP-adriamycin complex. 相似文献
12.
Magnetite (Fe IIFe III2O 4) is often considered as a stable end product of the bioreduction of Fe III minerals (e.g., ferrihydrite, lepidocrocite, hematite) or of the biological oxidation of Fe II compounds (e.g., siderite), with green rust (GR) as a mixed Fe II‐Fe III hydroxide intermediate. Until now, the biotic transformation of magnetite to GR has not been evidenced. In this study, we investigated the capability of an iron‐reducing bacterium, Shewanella putrefaciens, to reduce magnetite at circumneutral pH in the presence of dihydrogen as sole inorganic electron donor. During incubation, GR and/or siderite (Fe IICO 3) formation occurred as secondary iron minerals, resulting from the precipitation of Fe II species produced via the bacterial reduction of Fe III species present in magnetite. Taking into account the exact nature of the secondary iron minerals and the electron donor source is necessary to understand the exergonic character of the biotic transformation of magnetite to GR, which had been considered to date as thermodynamically unfavorable at circumneutral pH. This finding reinforces the hypothesis that GR would be the cornerstone of the microbial transformations of iron‐bearing minerals in the anoxic biogeochemical cycle of iron and opens up new possibilities for the interpretation of the evolution of Earth's history and for the understanding of biocorrosion processes in the field of applied science. 相似文献
13.
Addition of nifurtimox (a nitrofuran derivative used for the treatment of Chagas' disease) to rat liver microsomes produced an increase of (a) electron flow from NADPH to molecular oxygen, (b) generation of both superoxide anion radical (O 2?) and hydrogen peroxide, and (c) lipid peroxidation. The nifurtimox-stimulated NADPH oxidation was greatly inhibited by NADP + and p-chloromercuribenzoate, and to a lesser extent by SKF-525-A and metyrapone. These inhibitions reveal the function of both the NADPH-cytochrome P-450 ( c) reductase and cytochrome P-450 in nifurtimox reduction. Superoxide dismutase, catalase (in the presence of superoxide dismutase), and hydroxyl radical scavengers (mannitol, 5,5-dimethyl-1-pyrroline-1-oxide) inhibited the nifurtimox-stimulated NADPH oxidation, in accordance with the additional operation of a reaction chain including the hydroxyl radical. Further evidence supporting the role of superoxide anion and hydroxyl radicals in the nifurtimox-induced NADPH oxidation resulted from the effect of specific inhibitors on NADPH oxidation by O 2? (generated by the xanthine oxidase reaction) and by OH. (generated by an iron chelate or the Fenton reaction). Production of O 2? by rat kidney, testes and brain microsomes was significantly stimulated by nifurtimox in the presence of NADPH. It is postulated that enhanced formation of free radicals is the basis for nifurtimox toxicity in mammals, in good agreement with the postulated mechanism of the trypanocide effect of nifurtimox on Trypanosoma cruzi. 相似文献
14.
The rates of electron exchange between ferricytochrome c (C III)3 and ferrocytochrome c (C II) were observed as a function of the concentrations of ferrihexacyanide (Fe III) and ferrohexacyanide (Fe II) by monitoring the line widths of several proton resonances of the protein. Addition of Fe II to C III homogeneously increased the line widths of the two downfield paramagnetically shifted heme methyl proton resonances to a maximal value. This was interpreted as indicating the formation of a stoichiometric complex, C III·Fe II, in the over-all reaction: Values for , respectively, were calculated from the maximal change in line width observed at pH 7.0 and 25 °C. Changes in the line width of C III in the presence of Fe II and either KCl or Fe III suggest that complexation is principally ionic, that Fe III and Fe II compete for a common site. Addition of saturating concentrations of Fe III to C III produced only minor changes in the nuclear magnetic resonance spectrum of C III suggesting that complexation occurs on the protein surface.Addition of Fe III to C II in the presence of excess Fe II (to retain most of the protein as C II) increased the line width of the methyl protons of ligated methionine 80. A value for k?2 ≈ 2.08 × 10 4 s ?1 was calculated from the dependence of linewidth on the concentration of Fe II at 24 °C. These rates are shown to be consistent with the over-all rates of reduction and oxidation previously determined by stopped flow measurements, indicating that k2 and k?2 were rate limiting. From the temperature dependence the enthalpies of activation are 7.9 and 15.2 kcal/mol for k2 and k?2, respectively. 相似文献
15.
Heterocyclic nitrogenous bases, such as pyridine and imidazole which bind to heme-iron in cytochromes, inhibited the respiratory burst in intact neutrophils and NADPH-dependent oxygen consumption in lysed cells. Inhibition was accompanied by a spectral change in reduced cytochrome b558 as judged by low-temperature spectroscopy at 77 K. The position and shape of the alpha-band of the cytochrome were significantly altered upon exposure to pyridine or some other bases. Both inhibition and spectral changes were reversible. The results are consistent with the view that cytochrome b558 is involved in the NADPH oxidase system in neutrophils. 相似文献
16.
Salts of Cu I, II, Ni II, Sn II, In III and a sub-fraction of the cupoprotein ceruloplasmin induced phagokinesis of cultured aortal endothelial cells. A variant aortal endothelial cell line was highly sensitive; cells travelled up to 1000 μm in 24 h in response to 2× 10 ?6 M SnCl 2. Other metal ions tested (Zn II, Co II, Mn II, Cr II, Fe III, Al III, Sb III and Mo II) were not active. The motility response of endothelial cells to Cu ions in vitro is proposed as a model system for studying early events in neovascularization and as a sensitive assay for detecting angiogenic activity in fractions from cells and tissues. 相似文献
17.
Four reductions of the R2 subunit of mouse ribonucleotide reductase have been studied and found to exhibit different behaviour
from that of Escherichia coli R2. An important difference is that there is no stable met-R2 (Fe 2
II I) form of mouse R2. With hydroxyurea, hydrazine and hydroxylamine uniphasic kinetics are observed for the combined reduction
of radical Tyr ˙ and Fe 2
II I components to tyrosine and Fe 2
II respectively. The rate constants, determined at 370 nm (emphasising Fe III decay) and 417 nm (emphasising Tyr ˙ decay), differ by factors of 2–3, allowing some mechanistic features to be defined. The studies with hydrazine are particularly
important. In the case of E. coli R2, a first phase corresponding to two-equivalent reduction of the met-R2 component has been observed [18]. It is likely
that the four times slower second phase reaction of active E. coli R2 also corresponds to the Fe 2
II I → Fe 2
II change and is followed by fast intramolecular Fe 2
II reduction of the higher potential Tyr ˙. The latter changes are believed to hold also for (active) mouse R2. The Fe IIFe III semi forms have been detected at low levels by EPR for mouse R2 (9%) and E. coli (∼5%) in previous studies. Further substrate reduction of Fe IIFe III occurs at a comparable rate to account for the transient behaviour of Fe IIFe III. For mouse R2 the combined Fe III decay processes (which we are unable to separate) give smaller uniphasic rate constants at 370 nm than at 417 nm. A fitted-base-line
(FBL) treatment of absorbance changes at 417 nm targets more closely the Tyr ˙ decay as a means of monitoring the rate-determining step. The FBL method gives rate constants k (M –1 s –1) at 25 °C and pH 7.5 for hydroxyurea (1.46), hydrazine (0.163) and hydroxylamine (4.4). Surprisingly, phenylhydrazine, with
a less strong reduction potential (0.25 V), gives a substantially faster reduction of the Tyr ˙ as the only redox step (rate constant 27 M –1 s –1). In this case a slower second phase at 370 nm is independent of reductant and corresponds to rate-controlling release of
Fe III. Overall the results indicate a more reactive redox centre for mouse R2 and help develop further an understanding of factors
affecting the reactivity of R2.
Received: 11 October 1996 / Accepted: 11 February 1997 相似文献
18.
The cytochrome (cyt) c′, cyt c556, and cyt c2 genes from Rhodopseudomonas palustris have been cloned; recombinant cyt c′ and cyt c556 have been expressed, purified, and characterized. Unlike mitochondrial cyt c, these two proteins are structurally similar to cyt b562, in which the heme is embedded in a four-helix bundle. The hemes in both recombinant proteins form covalent thioether links to two Cys residues. UV/vis spectra of the Fe II and Fe III states of the recombinant cyts are identical with those of the corresponding native proteins. Equilibrium unfolding measurements in guanidine hydrochloride solutions confirm that native Fe II-cyt c556 is more stable than the corresponding state of Fe III-cyt c556 (ΔΔ Gf°=22 kJ/mol). 相似文献
19.
Distinct chemical species of iron were investigated by Mössbauer spectroscopy during iron uptake into cucumber roots grown in unbuffered nutrient solution with or without 57Fe-citrate. Mössbauer spectra of iron deficient roots supplied with 10–500 μM 57Fe-citrate for 30–180 min and 24 h and iron-sufficient ones, were recorded. The roots were analysed for Fe concentration and Fe reductase activity. The Mössbauer parameters in the case of iron-sufficient roots revealed high-spin iron(III) components suggesting the presence of Fe III-carboxylate complexes, hydrous ferric oxides and sulfate–hydroxide containing species. No Fe II was detected in these roots. However, iron-deficient roots supplied with 0.5 mM 57Fe III-citrate for 30 min contained significant amount of Fe II in a hexaaqua complex form. This is a direct evidence for the Strategy I iron uptake mechanism. Correlation was found between the decrease in Fe reductase activity and the ratio of Fe II–Fe III components as the time of iron supply was increased. The data may refer to a higher iron reduction rate as compared to its uptake/reoxidation in the cytoplasm in accordance with the increased reduction rate in iron deficient Strategy I plants. 相似文献
20.
Capability to release superoxide anion in response to phorbol myristate acetate by intact cells has been compared with Kinetic properties of NADPH oxidase by lysates of human monocytes and monocyte-derived macrophages. Maturation of monocytes in vitro is accompanied by substantial decrease of the capability to release superoxide anion in response to phorbol myristate acetate. Exposure of mature macrophages to recombinant interferon gamma enhances respiratory burst activity up to 3-4 fold. Modifications of NADPH oxidase accompany changes in the ability to release superoxide anion. The affinity of the oxidase for its substrate is higher in monocytes and gamma interferon treated macrophages, while Vmax is not changed. 相似文献
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