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1.
Cu,Zn SOD is known to be inactivated by HO 2− and to be protected against that inactivation by a number of small molecules including formate, imidazole, and urate. This inactivation has been shown to be due to oxidation of a ligand field histidine residue by a bound oxidant formed by reaction of the active site Cu(II) with HO 2−. We now report that protective actions of both formate and NADH increase as the pH was raised in the range 8.0–9.5. This is taken to indicate increased accessibility of the Cu site with rising pH and/or increased reactivity of the bound oxidant toward exogeneous substrates at high pH. Formate appears to act as a sacrificial substrate that protects by competing with the endogenous histidine residue for reaction with the bound oxidant, or that repairs the damage by reducing the histidyl radical intermediate. The same is likely also true of NADH. 相似文献
2.
1. 1. The superoxide anion radical (O2−) reacts with ferricytochrome c to form ferrocytochrome c. No intermediate complexes are observable. No reaction could be detected between O2− and ferrocytochrome c. 2. 2. At 20 °C the rate constant for the reaction at pH 4.7 to 6.7 is 1.4 · 106 M−1 · s−1 and as the pH increases above 6.7 the rate constant steadily decreases. The dependence on pH is the same for tuna heart and horse heart cytochrome c. No reaction could be demonstrated between O2− and the form of cytochrome c which exists above pH ≈ 9.2. The dependence of the rate constant on pH can be explained if cytochrome c has pKs of 7.45 and 9.2, and O2− reacts with the form present below pH 7.45 with k = 1.4 · 106 M−1 · s−1, the form above pH 7.45 with k = 3.0 · 105 M−1 · s−1, and the form present above pH 9.2 with k = 0. 3. 3. The reaction has an activation energy of 20 kJ mol−1 and an enthalpy of activation at 25 °C of 18 kJ mol−1 both above and below pH 7.45. It is suggested that O2− may reduce cytochrome c through a track composed of aromatic amino acids, and that little protein rearrangement is required for the formation of the activated complex. 4. 4. No reduction of ferricytochrome c by HO2 radicals could be demonstrated at pH 1.2–6.2 but at pH 5.3, HO2 radicals oxidize ferrocytochrome c with a rate constant of about 5 · 105–5 · 106 M−1 · s−1
. 相似文献
3.
The reduction of ferricytochrome c by O 2− and CO 2− was studied in the pH range 6.6–9.2 and Arrhenius as well as Eyring parameters were derived from the rate constants and their temperature dependence. Ionic effects on the rate indicate that the redox process proceeds through a multiply-positively charged interaction site on cytochrome c. It is shown that the reaction with O 2− and correspondingly with O 2 of ferrocytochrome c) is by a factor of approx. 10 3 slower than warranted by factors such as redox potential. Evidence is adduced to support the view that this slowness is connected with the role of water in the interaction between O 2−/O 2 and ferri-ferrocytochrome c in the positively charged interaction site on cytochrome c in which water molecules are specifically involved in maintaining the local structure of cytochrome c and participate in the process of electron equivalent transfer. 相似文献
4.
1. Difference spectra, at room and liquid N 2 temperatures, of S 2O 42−-, and NO 2−-reduced intact cells and cell-free preparations of Nitrobacter agilis demonstrated the presence of cytochromes of the c- and a-types. Reduction of cytochromes by succinate, and to a limited extent, by NADPH also occurred, provided KCN (0.1 mM) was also present. 2. A particulate, heat-labile nitrite oxidase having an absolute requirement for O2 was prepared from N. agilis cells using sonic oscillation and differential centrifugation. The particles also possessed NADH oxidase, succinoxidase, formate oxidase and traces of NADPH oxidase activity. The stoichiometry of the nitrite oxidase reaction approached the theoretical value of 2 moles of NO2− consumed per mole of O2 consumed. The pH optimum of the nitrite oxidase system shifted to progressively more alkaline values as the NO2− concentration was increased, changing from a pH value of 6.8 at 0.6 mM KNO2 to pH 8.0 at 0.01 M KNO2 with apparent Km's of 0.2 and 1.2 mM NO2−, respectively. Computations of the HNO2 concentrations present under the above conditions showed an approx. 500-fold greater affinity for HNO2 which was independent of pH, suggesting the involvement of HNO2 as both a substrate and an inhibitor (at higher concentrations) of the nitrite oxidase system. The marked inhibition by NaN3, NaCN and Na2S, as well the light-reversible inhibition by CO, indicated the presence of cytochrome oxidase which was subsequently characterized. NO2− proved to be a competitive inhibitor of the nitrite oxidase system. 3. The particulate preparation also possessed a heat-labile nitrite-cytochrome c reductase activity which was energy independent and routinely measured under anaerobic conditions. As in the case of nitrite oxidase, the affinity of the enzyme for NO3− increased as the pH was lowered, but the pH optimum remained unaffected. In terms of calculated HNO2 concentration an approximately constant Km of about 0.2 μM was estimated at the several pH's examined. The inhibition by NO3− was shown to be competitive. The marked sensitivity of the reductase to several metal-binding agents implicated a metal component in the electron transport chain at the site prior to cytochrome c. 4. The membrane-like composition of the nitrite oxidase system is indicated. 相似文献
5.
Radioactive labelling techniques show that isolated broken chloroplasts can take up HCO 3− in the dark. There are two pools of binding sites for this ion on, or within, the thylakoid membranes. A smaller, high affinity pool exists at a concentration of one HCO 3− bound per 380–400 chlorophyll molecules. Removal of HCO 3− bound in this pool requires special conditions and results in greater than 90% inhibition of oxygen evolution. The inhibition is fully reversed when HCO 3− is added back. HCO 3− bound in the small pool does not necessarily exchange with free HCO 3− in the dark or in light. Evidence presented suggests that this site is very near the site of action of 3-(3,4-dichlorophenyl)-1,1-dimethyl urea. A second, much larger, pool of HCO 3− binding sites also exists in a concentration approaching that of the bulk chlorophyll. These sites have a much lower affinity for HCO 3−, and their function has not yet been determined. 相似文献
6.
N-acetylcysteine has been widely used as an antioxidant in vivo and in vitro. Its reaction with four oxidant species has therefore been examined. N-acetylcysteine is a powerful scavenger of hypochlorous acid (H---OCl); low concentrations are able to protect 1-antiproteinase against inactivation by HOCl. N-acetylcysteine also reacts with hydroxyl radical with a rate constant of 1.36 × 10 10 M −1s −1, as determined by pulse radiolysis. It also reacts slowly with H 2O 2, but no reaction of N-acetylcysteine with superoxide (O 2−) could be detected within the limits of our assay procedures. 相似文献
7.
Nitrogen dioxide (NO 2•) is a key biological oxidant. It can be derived from peroxynitrite via the interaction of nitric oxide with superoxide, from nitrite with peroxidases, or from autoxidation of nitric oxide. In this study, submicromolar concentrations of NO 2• were generated in < 1 μs using pulse radiolysis, and the kinetics of scavenging NO 2• by glutathione, cysteine, or uric acid were monitored by spectrophotometry. The formation of the urate radical was observed directly, while the production of the oxidizing radical obtained on reaction of NO 2• with the thiols (the thiyl radical) was monitored via oxidation of 2,2′-azino-bis-(3-ethylthiazoline-6-sulfonic acid). At pH 7.4, rate constants for reaction of NO 2• with glutathione, cysteine, and urate were estimated as 2 × 10 7, 5 × 10 7, and 2 × 10 7 M −1 s −1, respectively. The variation of these rate constants with pH indicated that thiolate reacted much faster than undissociated thiol. The dissociation of urate also accelerated reaction with NO 2• at pH > 8. The thiyl radical from GSH reacted with urate with a rate constant of 3 × 10 7 M −1 s −1. The implications of these values are: (i) the lifetime of NO 2• in cytosol is < 10 μs; (ii) thiols are the dominant ‘sink’ for NO 2• in cells/tissue, whereas urate is also a major scavenger in plasma; (iii) the diffusion distance of NO 2• is 0.2 μm in the cytoplasm and < 0.8 μm in plasma; (iv) urate protects GSH against depletion on oxidative challenge from NO 2•; and (v) reactions between NO 2• and thiols/urate severely limit the likelihood of reaction of NO 2• with NO• to form N 2O 3 in the cytoplasm. 相似文献
8.
The mechanism of the vanadate (V( v))-dependent oxidation of NADH was different in phosphate buffers and in phosphate-free media. In phosphate-free media (aqueous medium or HEPES buffer) the vanadyl (V( v)) generated by the direct V( v)-dependent oxidation of NADH formed a complex with V( v). In phosphate buffers V( v) autoxidized instead of forming a complex with V( v). The generated superoxide radical (O 2−) initiated, in turn, a high-rate free radical chain oxidation of NADH. Phosphate did not stimulate the V( v)-dependent NADH oxidation catalyzed by O 2−-generating systems. Monovanadate proved to be a stronger catalyzer of NADH oxidation as compared to polyvanadate. 相似文献
9.
A procedure is described for preparing particles from cells of Micrococcus denitrificans which were broken osmotically after treatment with lysozyme. 1. 1. The preparations catalysed ATP synthesis coupled to O2 uptake or NO3− reduction. With NADH or succinate as the electron donors the P:O ratios were about 1.5 and 0.5, respectively; and the P:NO3− ratios were about 0.9 and 0.06, respectively. 2. 2. Addition of ADP or Pi to the reaction mixture increased the rates of NADH-dependent O2 uptake and NO3− reduction. Addition of 1 mM 2,4-dinitrophenol, which inhibited phosphorylation by 50–60%, increased the basal rates of electron transport. 3. 3. Evidence derived from spectrophotometry and from the differential inhibition by antimycin A of O2 and NO3− reduction leads to the conclusion that the nitrate reductase interacted with the respiratory chain in the region of the b-type cytochrome, and that the c-type cytochrome present was not involved in the reduction of NO3− to NO2−.
Abbreviations: TMPD; tetramethyl-p-phenylenediamine 相似文献
10.
The thermoluminescence band observed in chloroplasts after flash excitation at ambient temperatures has recently been identified as being due to recombination of the electron on the semiquinone form of the secondary plastoquinone acceptor, Q B, with positive charges on the oxygen-evolving enzyme, S 2 and S 3 (Rutherford, A.W., Crofts, A.R. and Inoue, Y. (1982) Biochim. Biophys. Acta 682, 457–465). Further investigation of this thermoluminescence confirms this assignment and provides information on the function of PS II. The following data are reported: (1) Washing of chloroplasts with ferricyanide lowers the concentration of Q B− in the dark and predictable changes in the extent of the thermoluminescence band are observed. (2) The thermoluminescence intensity arising from S 2Q B− is approximately one half of that arising from S 3Q B−. (3) Preflash treatment followed by dark adaptation results in changes in the intensity of the thermoluminescence band recorded after a series of flashes. These changes can be explained according to the above assignments for the origin of the thermoluminescence and if Q B− provides an important source of deactivating electrons for the S states. Computer simulations of the preflash data are reported using the above assumptions. Previously unexplained data already in the literature (Läufer, A. and Inoue, Y. (1980) Photobiochem. Photobiophys. 1, 339–346) can be satisfactorily explained and are simulated using the above assumptions. (4) Lowering the pH to pH 5.5 results in a shift of the S 2Q B− thermoluminescence band to higher temperatures while that arising from S 3Q B− does not shift. This effect is interpreted as indicating that Q B− is protonated and the S 2 to S 3 reaction involves deprotonation while the S 1 to S 2 reaction does not. 相似文献
11.
Previously, we showed that oxidation of tryptophan-32 (Trp-32) residue was crucial for H 2O 2/bicarbonate (HCO 3−)-dependent covalent aggregation of human Cu,Zn SOD1 (hSOD1). The carbonate anion radical (CO 3−)-induced oxidation of Trp-32 to kynurenine-type oxidation products was proposed to cause the aggregation of hSOD1. Here we used the matrix-assisted laser desorption ionization–time of flight mass spectroscopy, high-performance liquid chromatography–electrospray ionization mass spectroscopy, and liquid chromatography mass spectroscopy methods to characterize products. Results show that a peptide region (31–36) of hSOD1 containing the Trp-32 residue (VWGSIK) is oxidatively modified to the N-formylkynurenine (NFK)- and kynurenine (Kyn)-containing peptides (V(NFK)GSIK) and (V(Kyn)GSIK) during HCO −-dependent peroxidase activity of hSOD1. Also, UV photolysis of a cobalt complex that generates authentic CO 3− radical induced a similar product profile from hSOD1. Similar products were obtained using a synthetic peptide with the same amino acid sequence ( i.e., VWGSIK). We propose a mechanism involving a tryptophanyl radical for CO 3−-induced oxidation of Trp-32 residue (VWGSIK) in hSOD1 to V(NFK)GSIK and V(Kyn)GSIK. 相似文献
12.
1. The nitrite oxidase particles obtained by sonic oscillation of Nitrobacter agilis cells also possessed appreciable formate oxidase activity, ranging from about 25 to 50% of the nitrite oxidase activity depending upon the N. agilis strain. Both activities distributed themselves in the same pattern and proportions during differential centrifugation, and resided solely in the pellet resulting from high-speed centrifugation. 2. Difference spectra of formate-reduced particles or intact cells demonstrated the presence of cytochromes of the c- and a-types like those of the NO2−-reduced material. Under anaerobic conditions NO3− or fumarate acted as an alternate electron acceptor in place of O2 in formate oxidation. Under aerobic conditions increasing NO3− concentrations resulted in (a) an increased role of NO3− as a terminal electron acceptor compared to O2, (b) a greater total enzymatic transfer of electrons from formate than if O2 were the sole electron acceptor, and (c) a partial inhibition of O2 uptake suggestive of a competition for electrons by the two acceptors. The formate oxidase system failed to catalyze consistently the transfer of electrons to either added mammalian cytochrome c or Fe(CN)63−. The marked sensitivity of the system to certain inhibitors implicated cytochrome oxidase as an integral part of the formate oxidase. The system was also inhibited significantly by a variety of chelating agents, indicating a metal component in the formate dehydrogenase or early portion of the electron transfer sequence. 3. The stoichiometry of the formate oxidase system was shown to approach the theoretical value of 2 moles of CO2 evolved per mole of O2 or per 2 moles of formate consumed. 4. To a limited extent, phosphorylation occurred concomittantly with the oxidation of formate in the presence of the cell-free particulate system. 相似文献
13.
The perchlorate (ClO 4−)-respiring organism, strain perc1ace, can grow using nitrate (NO 3−) as a terminal electron acceptor. In resting cell suspensions, NO 3− grown cells reduced ClO 4−, and ClO 4− grown cells reduced NO 3−. Activity assays showed that nitrate reductase (NR) activity was 1.31 μmol min −1 (mg protein) −1 in ClO 4− grown cells, and perchlorate reductase (PR) activity was 4.24 μmol min −1 (mg protein) −1 in NO 3− grown cells. PR activity was detected within the periplasmic space, with activities as high as 14 μmol min −1 (mg protein) −1. The NR had a pH optimum of 9.0 while the PR had an optimum of 8.0. This study suggests that separate terminal reductases are present in strain perclace to reduce NO 3− and ClO 4−. 相似文献
14.
Bovine mitochondrial malate dehydrogenase (EC 1.1.1.37) was inactivated by the specific modifications of a single histidine residue upon reaction with iodoacetamide. NADH protected against this loss of activity and reaction with the histidine residue, suggesting that the histidine is at the NADH binding site. N-Ethylmaleimide also modified the enzyme by reacting with 1 sulfhydryl residue. The reaction rate with N-ethylmaleimide was increased by decreasing the pH from neutrality or by the addition of urea. NADH protected against the modification of the sulfhydryl group under all the conditions tested, again suggesting active site specificity for this inactivation. This enzyme has a subunit weight of 33,000 and is a dimer. The native malate dehydrogenase will bind only 1 mol of NADH and it is thus assumed that there is only a single active site per dimer. 相似文献
15.
The embryo of oviparous species is confronted by a highly oxidative stress generating as it grows and must rely on effective antioxidant system for protection. Proteins of avian egg albumin have been suggested to play the major redox-modulatory role during embryo development. Recently, we found that ovotransferrin (OTf) undergoes distinct thiol-linked self-cleavage in a redox-dependent process. In this study, we explore that OTf is SOD mimic protein with a potent superoxide anion (O 2−) scavenging activity. The O 2− scavenging activity was investigated using the natural xanthine/xanthine oxidase (X/XOD) coupling system. OTf exhibited O 2− scavenging activity in a dose-dependent manner and showed remarkably higher scavenging activity than the known antioxidants, ascorbate or serum albumin. The isolated half-molecules of OTf exhibited higher scavenging activity than the intact molecule, whereas the N-lobe showed much greater activity. OTf dramatically quenched the O 2− flux but had no effect on the urate production in the X/XOD system, indicating its unique specificity to scavenge O 2− but not oxidase inhibition. Strikingly, metal-bound OTf exhibited greater O 2− dismutation capacity than the apo-protein, ranging from moderate (Zn 2+-OTf and Fe 2+-OTf) to high (Mn 2+-OTf and Cu 2+-OTf) activity with the Cu 2+-OTf being the most potent scavenger. In a highly sensitive fluorogenic assay, the metal-bound OTf exhibited significant increase in the rate of H 2O 2 production in the X/XOD reaction than the apo-OTf, providing evidence that Zn 2+-, Mn 2+- and Cu 2+-OTf possess SOD mimic activity. This finding is the first to describe that OTf is an O 2− scavenging molecule, providing insight into its novel SOD-like biological function, and heralding a fascinating opportunity for its potential candidacy as antioxidant drug. 相似文献
16.
Resonance Raman measurements have been performed with solutions of iodine-complexed synthetic amyloses ( DP 25–200), malto-oligomers ( DP 3–18, and -cylodextrin. Interest was focused on the minimum chain length for helical complex formation and a possible preferred length for the polyiodine chain. Four fundamental vibrations are observed at 164, 112, 52 and 24 cm −1. The 112 cm −1 Raman line was shown to arise both from free I 3− (enhanced at 363.8 nm excitation) and from bound iodine (relatively most intense at 457.9 nm excitation). The main signal of complexed iodine at 164 cm −1is enhanced at an excitation wavelength close to the long wavelength absorption maximum. This signal is observed firt with malto-octaose and -cyclodextrin. The less intense signals at 52 and 24 −1 are only detected at DP 15 and higher. Raman spectra give no evidence for a preferred length of the polyiodine chain. Significantly identical Raman spectra are obtained when using different molar ratios of I 2/KI solution or I 2 solution initially free of I − ions. The results are discussed in view of previous assignments of the Raman lines to I 2−, I 3−/I 2, and I 5− subunits. Our findings are incompatible with I 3− units as the only bound species. They are compatible with both I 3−/I 2 and I 3− subunits under certain conditions. In the case of I 2 solution used for complexation we favour the polyiodine chain model proposed previously by Cramer 35,36. The I 3− ions formed could function mainly as chain initiators, as has been suggested by Cesàro and Brant 30. 相似文献
17.
Hydrogen peroxide, produced by inflammatory and vascular cells, induces oxidative stress that may contribute to endothelial dysfunction. In smooth muscle cells, H 2O 2 induces production of O 2− by activating NADPH oxidase. However, the mechanisms whereby H 2O 2 induces oxidative stress in endothelial cells are poorly understood. We examined the effects of H 2O 2 on O 2− levels on porcine aortic endothelial cells (PAEC). Treatment with 60 μmol/L H 2O 2 markedly increased intracellular O 2− levels (determined by conversion of dihydroethidium to hydroxyethidium) and produced cytotoxicity (determined by propidium iodide staining) in PAEC. Overexpression of human manganese superoxide dismutase in PAEC reduced O 2− levels and attenuated cytotoxicity resulting from treatment with H 2O 2. L-NAME, an inhibitor of nitric oxide synthase (NOS), and apocynin, an inhibitor of NADPH oxidase, reduced O 2− levels in PAEC treated with H 2O 2, suggesting that both NOS and NADPH oxidase contribute to H 2O 2-induced O 2− in PAEC. Inhibition of NADPH oxidase using apocynin and NOS rescue with L-sepiapterin together reduced O 2− levels in PAEC treated with H 2O 2 to control levels. This suggests interaction-distinct NOS and NADPH oxidase pathways to superoxide. We conclude that H 2O 2 produces oxidative stress in endothelial cells by increasing intracellular O 2− levels through NOS and NADPH oxidase. These findings suggest a complex interaction between H 2O 2 and oxidant-generating enzymes that may contribute to endothelial dysfunction. 相似文献
18.
Crystals of calcium oxalate monohydrate (COM) in the renal tubule form the basis of most kidney stones. Tubular dysfunction resulting from COM-cell interactions occurs by mechanism(s) that are incompletely understood. We examined the production of reactive oxygen intermediates (ROI) by proximal (LLC-PK1) and distal (MDCK) tubular epithelial cells after treatment with COM (25–250 μg/ml) to determine whether ROI, specifically superoxide (O 2•−), production was activated, and whether it was sufficient to induce oxidative stress. Employing inhibitors of cytosolic and mitochondrial systems, the source of ROI production was investigated. In addition, intracellular glutathione (total and oxidized), energy status (ATP), and NADH were measured. COM treatment for 1–24 h increased O 2•− production 3–6-fold as measured by both lucigenin chemiluminescence in permeabilized cells and dihydrorhodamine fluorescence in intact cells. Using selective inhibitors we found no evidence of cytosolic production. The use of mitochondrial probes, substrates, and inhibitors indicated that increased O 2•− production originated from mitochondria. Treatment with COM decreased glutathione (total and redox state), indicating a sustained oxidative insult. An increase in NADH in COM-treated cells suggested this cofactor could be responsible for elevating O 2•− generation. In conclusion, COM increased mitochondrial O 2•− production by epithelial cells, with a subsequent depletion of antioxidant status. These changes may contribute to the reported cellular transformations during the development of renal calculi. 相似文献
19.
1. Rate constants for reduction of paraquat ion (1,1′-dimethyl-4,4′-bipyridy-lium, PQ 2+) to paraquat radical (PQ +·) by e−aq and CO 2−· have been measured by pulse radiolysis. Reduction by e−aq is diffusion controlled ( k = 8.4·10 10 M −1·s −1) and reduction by CO 2−· is also very fast k = 1.5·10 10 M −1·s −1). 2. The reaction of paraquat radical with oxygen has been analysed to give rate constants of 7.7·108 M−1·s−1 and 6.5·108 M−1·s−1 for the reactions of paraquat radical with O2 and O2−·, respectively. The similarity in these rate constants is in marked contrast to the difference in redox potentials of O2 and O2−· (− 0.59 V and + 1.12 V, respectively). 3. These rate constants, together with that for the self-reaction of O2−·, have been used to calculate the steady-state concentration of O2−· under conditions thought to apply at the site of reduction of paraquat in the plant cell. On the basis of these calculations the decay of O2−· appears to be governed almost entirely by its self-reaction, and the concentration 5 μm away from the thylakoid is still 90% of that at the thylakoid itself. Thus, O2−· persists long enough to diffuse as far as the chloroplast envelope and tonoplast, which are the first structures to be damaged by paraquat treatment. O2−· is therefore sufficiently long-lived to be a candidate for the phytotoxic product formed by paraquat in plants. 相似文献
20.
The binuclear copper active site of Carcinus hemocyanin has been reconstituted by incubating apohemocyanin with Cu(I) in the presence of Br - ions. At constant Cu(I) concentration the kinetics of reconstitution depends on both pH and Br - concentration. The process is faster at pH 6.0 than at pH 7.0 and in both cases the reaction is accelerated by increasing Br - concentration from 0.1 M to 0.4 M. At pH 6.0 a time-dependent inactivation of the O 2-binding properties of reconstituted hemocyanin is observed. This effect is attributed to a perturbation in the active site microenvironment caused by unspecifically bound copper. Br - ions show a protective effect probably by chelating excess metal. 相似文献
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