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1.
The reduction of ferric leghemoglobin (Lb3+) from soybean (Glycine max (L.) Merr.) nodules by riboflavin, FMN and FAD in the presence of NAD(P)H was studied in vitro. The system NAD(P)H + flavin reduced Lb3+ to oxyferrous (Lb2+ · O2) or deoxyferrous (Lb2+) leghemoglobin in aerobic or anaerobic conditions, respectively. In the absence of O2 the reaction was faster and more effective (i.e. less NAD(P)H oxidized per mole Lb3+ reduced) than in the presence of O2; this phenomenon was probably because O2 competes with Lb3+ for reductant, thus generating activated O2 species. The flavin-mediated reduction of Lb3+ did not entail production of superoxide or peroxide, indicating that NAD(P)H-reduced flavins were able to reduce Lb3+ directly. The NAD(P)H + flavin system also reduced the complexes Lb3+ · nicotinate and Lb3+ · acetate to Lb2+ · O2, Lb2+ or Lb2+ · nicotinate, depending on the concentrations of ligands and of O2. In the presence of 200 M nitrite most Lb remained as Lb3+ in aerobic conditions but the nitrosyl complex (Lb2+ · NO) was generated in anaerobic conditions. The above-mentioned characteristics of the NAD(P)H + flavin system, coupled with its effectiveness in reducing Lb3+ at physiological levels of NAD(P)H and flavins in soybean nodules, indicate that this mechanism may be especially important for reducing Lb3+ in vivo.Abbreviations and Terminology FLbR ferric leghemoglobin reductase - Hb2+ /Hb3+ hemoglobin containing Fe2+ /Fe2+ - Lb2+ /Lb3+ leghemoglobin containing Fe2+ /Fe3+ - Lb3+ · nicotinate/acetate Lb in which nicotinate or acetate are complexed to Lb3+ - Lb2+ · O2/CO/NO/nicotinate Lb in which O2, CO, NO or nicotinate are complexed to Lb2+ - Rfl riboflavin - SOD superoxide dismutase (EC 1.15.1.1) Published as Paper No. 9237, Journal Series, Nebraska Agricultural Research DivisionWe thank M.B. Crusellas for his skillful drawings. M. Becana thanks the Spanish Ministry of Education and Science/Fulbright Commission for financial support.  相似文献   

2.
Ferric leghemoglobin reductase (FLbR) from soybean (Glycine max [L.] Merr) nodules catalyzed oxidation of NADH, reduction of ferric leghemoglobin (Lb+3), and reduction of dichloroindophenol (diaphorase activity). None of these reactions was detectable when O2 was removed from the reaction system, but all were restored upon readdition of O2. In the absence of exogenous electron carriers and in the presence of O2 and excess NADH, FLbR catalyzed NADH oxidation with the generation of H2O2 functioning as an NADH oxidase. The possible involvement of peroxide-like intermediates in the FLbR-catalyzed reactions was analyzed by measuring the effects of peroxidase and catalase on FLbR activities; both enzymes at low concentrations (about 2 μg/mL) stimulated the FLbR-catalyzed NADH oxidation and Lb+3 reduction. The formation of H2O2 during the FLbR-catalyzed NADH oxidation was confirmed using a sensitive assay based on the fluorescence emitted by dichlorofluorescin upon reaction with H2O2. The stoichiometry ratios between the FLbR-catalyzed NADH oxidation and Lb+3 reduction were not constant but changed with time and with concentrations of NADH and O2 in the reaction solution, indicating that the reactions were not directly coupled and electrons from NADH oxidation were transferred to Lb+3 by reaction intermediates. A study of the affinity of FLbR for O2 showed that the enzyme required at least micromolar levels of dissolved O2 for optimal activities. A mechanism for the FLbR-catalyzed reactions is proposed by analogy with related oxidoreductase systems.  相似文献   

3.
A. Puppo  L. Dimitrijevic  J. Rigaud 《Planta》1982,156(4):374-379
Superoxide anion is able to oxidize oxyleghemoglobin prepared from soybean nodules. Furthermore, ferrileghemoglobin is oxidized to leghemoglobin (IV) by hydrogen peroxide and this irreversible reaction leads to a complete inactivation of the hemoprotein. In scavenging O 2 - and H2O2, superoxide dismutase (EC 1.15.1.1) and catalase (EC 1.11.1.6) are able to limit these oxidations. The occurrence of these enzymes within soybean nodules and their main characteristics are reported here. A general scheme taking into account their roles in leghemoglobin protection in vivo is proposed.Abbreviations Lb leghemoglobin - SOD superoxide dismutase  相似文献   

4.
In cells, mitochondria, endoplasmic reticulum, and peroxisomes are the major sources of reactive oxygen species (ROS) under physiological and pathophysiological conditions. Cytochrome c (cyt c) is known to participate in mitochondrial electron transport and has antioxidant and peroxidase activities. Under oxidative or nitrative stress, the peroxidase activity of Fe3+cyt c is increased. The level of NADH is also increased under pathophysiological conditions such as ischemia and diabetes and a concurrent increase in hydrogen peroxide (H2O2) production occurs. Studies were performed to understand the related mechanisms of radical generation and NADH oxidation by Fe3+cyt c in the presence of H2O2. Electron paramagnetic resonance (EPR) spin trapping studies using 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) were performed with NADH, Fe3+cyt c, and H2O2 in the presence of methyl-β-cyclodextrin. An EPR spectrum corresponding to the superoxide radical adduct of DMPO encapsulated in methyl-β-cyclodextrin was obtained. This EPR signal was quenched by the addition of the superoxide scavenging enzyme Cu,Zn-superoxide dismutase (SOD1). The amount of superoxide radical adduct formed from the oxidation of NADH by the peroxidase activity of Fe3+cyt c increased with NADH and H2O2 concentration. From these results, we propose a mechanism in which the peroxidase activity of Fe3+cyt c oxidizes NADH to NAD, which in turn donates an electron to O2, resulting in superoxide radical formation. A UV-visible spectroscopic study shows that Fe3+cyt c is reduced in the presence of both NADH and H2O2. Our results suggest that Fe3+cyt c could have a novel role in the deleterious effects of ischemia/reperfusion and diabetes due to increased production of superoxide radical. In addition, Fe3+cyt c may play a key role in the mitochondrial “ROS-induced ROS-release” signaling and in mitochondrial and cellular injury/death. The increased oxidation of NADH and generation of superoxide radical by this mechanism may have implications for the regulation of apoptotic cell death, endothelial dysfunction, and neurological diseases. We also propose an alternative electron transfer pathway, which may protect mitochondria and mitochondrial proteins from oxidative damage.  相似文献   

5.
The accumulation of nitrosylleghemoglobin (LbNO) in nodulesand the properties of LbNO in vitro were investigated in connectionwith the inhibition of nitrogen fixation in soybean nodulesby nitrate. The leghemoglobin extracted under argon gas from nodules ofplants supplied with nitrate consisted mainly of LbNO, as judgedfrom the spectrum which corresponded to that of LbNO formedin vitro by the reaction of leghemoglobin with nitrite in thepresence of dithionite or by the combination of ferrous leghemoglobin(Lb2+) with nitric oxide. Further, LbNO formed in vivo was easilydissociated by visible light, as was LbNO formed in vitro. Thus,authentic LbNO does actually accumulate in nodules. Most of the leghemoglobin was of the ferrous type in nodulesof plants supplied with nitrate. Some LbNO appeared to be derivedfrom LbO2 which was deoxygenated by nitrite. The increase inlevels of LbNO in nodules paralleled the decrease in acetylenereducing activity. These results indicate that the decrease in nitrogenase activityin nodules of soybean plants supplemented with nitrate is causedby the decrease in levels of LbO2 that carries oxygen into bacteroids,which results from the formation of LbNO (Received August 22, 1989; Accepted December 4, 1989)  相似文献   

6.
The effect in vivo of hexavalent chromium (Cr6+) on the respiratory electron transport activity and production of superoxide (O2) radicals, was studied in submitochondrial particles (SMPs) prepared from mitochondria isolated from roots of 15‐day‐old pea (Pisum sativum L. cv. Azad) plants exposed to environmentally relevant (20 µm ) and acute (200 µm ) concentrations of chromium for 7 d. A concentration ‐dependent inactivation of electron transport activity from both NADH to O2 (NADH oxidase) and succinate to O2 (succinate oxidase) was observed. The electron transport activity was more sensitive to Cr6+ with NADH as the substrate than with succinate as the substrate. Although NADH dehydrogenase and succinate dehydrogenase were less affected, NADH: cytochrome c oxidoreductase and succinate: cytochrome c oxidoreductase activities were prominently affected by Cr6+. Cytochrome oxidase was the most susceptible complex of mitochondrial membranes to Cr6+, exhibiting maximal inactivation of activity both at 20 and 200 µm chromium concentrations. Cr6+ increased the generation of O2 radicals. This effect was more evident at 200 than at 20 µm . A significant increase in lipid peroxidation of mitochondrial membranes at 200 µm Cr6+ was the physiological impact of the metal‐induced enhanced generation of O2 radicals. An increase in superoxide dismutase (SOD) activity at 20 µm Cr6+ towards enhanced production of O2 radicals appeared to be a defence response in pea root mitochondria that, however, could not be sustained at 200 µm Cr6+. The results obtained concerning inactivation of mitochondrial electron transport and subsequent enhancement in the generation of O2 radicals suggest that root mitochondria are an important target of Cr6+‐induced oxidative stress in pea.  相似文献   

7.
《Free radical research》2013,47(4):219-227
The addition of 25μM hydrogen peroxide to 20μM metmyoglobin produces ferryl (FeIV = O) myoglobin. Optical spectroscopy shows that the ferryl species reaches a maximum concentration (60-70% of total haem) after 10 minutes and decays slowly (hours). Low temperature EPR spectroscopy of the high spin metmyoglobin (g = 6) signal is consistent with these findings. At this low peroxide concentration there is no evidence for iron release from the haem. At least two free radicals are detectable by EPR immediately after H2O2 addition, but decay completely after ten minutes. However, a longer-lived radical is observed at lower concentrations that is still present after 90 minutes. The monohydroxamate N-methylbutyro-hydroxamic acid (NMBH) increases the rate of decay of the fenyl species. In the presence of NMBH, none of the protein-bound free radicals are detectable; instead nitroxide radicals produced by oxidation of the hydroxamate group are observed. Similar results are observed with the trihydroxamate, desferoxamine. “Ferryl myoglobin” is still able to initiate lipid peroxidation even after the short-lived protein free radicals are no longer detectable (E.S.R. Newman, C.A. Rice-Evans and M.J. Davies (1991) Biochemical and Biophysical Research Communications 179, 1414-1419). It is suggested that the longer-lived protein radicals described here may be partly responsible for this effect. The mechanism of inhibition of initiation of lipid peroxidation by hydroxamate drugs, such as NMBH, may therefore be due to reduction of the protein-derived radicals, rather than reduction of ferryl haem.  相似文献   

8.
Lactate is a common substrate for major groups of strictly anaerobic bacteria, but the biochemistry and bioenergetics of lactate oxidation is obscure. The high redox potential of the pyruvate/lactate pair of E0′ = ?190 mV excludes direct NAD+ reduction (E0′ = ?320 mV). To identify the hitherto unknown electron acceptor, we have purified the lactate dehydrogenase (LDH) from the strictly anaerobic, acetogenic bacterium Acetobacterium woodii. The LDH forms a stable complex with an electron‐transferring flavoprotein (Etf) that exhibited NAD+ reduction only when reduced ferredoxin (Fd2?) was present. Biochemical analyses revealed that the LDH/Etf complex of A. woodii uses flavin‐based electron confurcation to drive endergonic lactate oxidation with NAD+ as oxidant at the expense of simultaneous exergonic electron flow from reduced ferredoxin (E0′ ≈ –500 mV) to NAD+ according to: lactate + Fd2? + 2 NAD+ → pyruvate + Fd + 2 NADH. The reduced Fd2? is regenerated from NADH by a sequence of events that involves conversion of chemical (ATP) to electrochemical and finally redox energy (Fd2? from NADH) via reversed electron transport catalysed by the Rnf complex. Inspection of genomes revealed that this metabolic scenario for lactate oxidation may also apply to many other anaerobes.  相似文献   

9.
In peroxisomes isolated from pea leaves (Pisum sativum L.) the production of superoxide free radicals (O2) by xanthine and NADH was investigated. In peroxisomal membranes, 100 micromolar NADH induced the production of O2 radicals. In the soluble fractions of peroxisomes, no generation of O2 radicals was observed by incubation with either NADH or xanthine, although xanthine oxidase was found located predominantly in the matrix of peroxisomes. The failure of xanthine to induce superoxide generation was probably due to the inability to fully suppress the endogenous Mn-superoxide dismutase activity by inhibitors which were inactive against xanthine oxidase. The generation of superoxide radicals in leaf peroxisomes together with the recently described production of these oxygen radicals in glyoxysomes (LM Sandalio, VM Fernández, FL Rupérez, LA del Río [1988] Plant Physiol 87: 1-4) suggests that O2 generation could be a common metabolic property of peroxisomes and further supports the existence of active oxygen-related rôles for peroxisomes in cellular metabolism.  相似文献   

10.
Several flavin-dependent enzymes of the mitochondrial matrix utilize NAD+ or NADH at about the same operating redox potential as the NADH/NAD+ pool and comprise the NADH/NAD+ isopotential enzyme group. Complex I (specifically the flavin, site IF) is often regarded as the major source of matrix superoxide/H2O2 production at this redox potential. However, the 2-oxoglutarate dehydrogenase (OGDH), branched-chain 2-oxoacid dehydrogenase (BCKDH), and pyruvate dehydrogenase (PDH) complexes are also capable of considerable superoxide/H2O2 production. To differentiate the superoxide/H2O2-producing capacities of these different mitochondrial sites in situ, we compared the observed rates of H2O2 production over a range of different NAD(P)H reduction levels in isolated skeletal muscle mitochondria under conditions that favored superoxide/H2O2 production from complex I, the OGDH complex, the BCKDH complex, or the PDH complex. The rates from all four complexes increased at higher NAD(P)H/NAD(P)+ ratios, although the 2-oxoacid dehydrogenase complexes produced superoxide/H2O2 at high rates only when oxidizing their specific 2-oxoacid substrates and not in the reverse reaction from NADH. At optimal conditions for each system, superoxide/H2O2 was produced by the OGDH complex at about twice the rate from the PDH complex, four times the rate from the BCKDH complex, and eight times the rate from site IF of complex I. Depending on the substrates present, the dominant sites of superoxide/H2O2 production at the level of NADH may be the OGDH and PDH complexes, but these activities may often be misattributed to complex I.  相似文献   

11.
Vanadate in the polymeric form of decavanadate, but not other forms, stimulated oxidation of NADH to NAD+ NADPH was also oxidized with comparable rates. This oxidation of NADH was accompanied by uptake of oxygen and generated hydrogen peroxide with the following stoichiometry: NADH + H+ + O2 → NAD+ + H2O2. The reaction followed second-order kinetics. The rate was dependent on the concentration of both NADH and vanadate and increased with decreasing pH. The reaction had an obligatory requirement for phosphate ions. Esr studies in the presence of the spin trap dimethyl pyrroline N oxide indicated the involvement of Superoxide anion as an intermediate. The reaction was sensitive to Superoxide dismutase and other scavengers of superoxide anions.  相似文献   

12.
锰超氧化物歧化酶(MnSOD)催化两分子超氧自由基歧化为分子氧和过氧化氢。超氧自由基被Mn3+SOD氧化成分子氧的反应以扩散的方式进行。超氧自由基被Mn2+SOD还原为过氧化氢的反应以快循环和慢循环两条途径平行进行。在慢循环途径中,Mn2+SOD与超氧自由基形成产物抑制复合物,然后该复合物被质子化而缓慢释放出过氧化氢。在快循环途径中,超氧自由基直接被Mn2+SOD转化为产物过氧化氢,快速循环有利于酶的复活与周转。本文提出温度是调节锰超氧化物歧化酶进入慢速或者快速循环催化途径的关键因素。随着在生理温度范围内的温度升高,慢速循环成为整个催化反应的主流,因而生理范围内的温度升高反而抑制该酶的活性。锰超氧化物歧化酶的双相酶促动力学特性可以用该酶保守活性中心的温度依赖性配位模型进行合理化解释。当温度降低时,1个水分子(或者OH-)接近Mn、甚至与Mn形成配位键,从而干扰超氧自由基与Mn形成配位键而避免形成产物抑制。因此在低温下该酶促反应主要在快循环通路中进行。最后阐述了几种化学修饰模式对...  相似文献   

13.
The photosensitizer flavin mononucleotide (FMN), in conjunction with the reducing agents diethylenetria-minepentaacetic acid (DTPA), hydrazine and hydroxylamines derived from nitroxides, generates superoxide radicals in a strictly light-dependent reaction in aerobic solution. Addition of superoxide dismutase (SOD) converts this system to a hydrogen peroxide generator. In the presence of horseradish peroxidase the latter system becomes a phenoxyl radical generator with appropriate phenolic substrates. Under anaerobic conditions FMN, hydrogen peroxide and an iron chelate generate ferryl and when this system is combined with dimethylsulfoxide, methyl radicals are produced. All the radicals can be generated with little contamination from other radicals, in high yields and the reaction can be terminated immediately upon cessation of illumination. Useful applications of this photochemical system include ESR studies of transient free radical species.  相似文献   

14.
Barry Halliwell 《Planta》1978,140(1):81-88
The enzyme horseradish peroxidase (EC 1.11.1.7) catalyses oxidation of NADH. NADH oxidation is prevented by addition of the enzyme superoxide dismutase (EC 1.15.1.1) to the reaction mixture before adding peroxidase but addition of dismutase after peroxidase has little inhibitory effect. Catalase (EC 1.11.1.6) inhibits peroxidase-catalysed NADH oxidation when added at any time during the reaction. Apparently the peroxidase uses hydrogen peroxide (H2O2) generated by non-enzymic breakdown of NADH to catalyse oxidation of NADH to a free-radical, NAD., which reduces oxygen to the superoxide free-radical ion, O2 .-. Some of the O2 .- reacts with peroxidase to give peroxidase compound III, which is catalytically inactive in NADH oxidation. The remaining O2 .- undergoes dismutation to O2 and H2O2. O2 .- does not react with NADH at significant rates. Mn2+ or lactate dehydrogenase stimulate NADH oxidation by peroxidase because they mediate a reaction between O2 .- and NADH. 2,4-Dichlorophenol, p-cresol and 4-hydroxycinnamic acid stimulate NADH oxidation by peroxidase, probably by breaking down compound III and so increasing the amount of active peroxidase in the reaction mixture. Oxidation in the presence of these phenols is greatly increased by adding H2O2. The rate of NADH oxidation by peroxidase is greatest in the presence of both Mn2+ and those phenols which interact with compound III. Both O2 .- and H2O2 are involved in this oxidation, which plays an important role in lignin synthesis.  相似文献   

15.
16.
Vanadyl (V(IV)) salts autoxidize in neutral aqueous solution yielding O2 plus vanadate (V(V)) and these, in turn, cause the oxidation of NADH, by a free radical chain reaction. This oxidation of NADH was inhibited by superoxide dismutase, but not by a scavenger of HO.. When H2O2 was present V(IV)) caused rapid oxidation of NADH by a process which was unaffected by superoxide dismutase but was inhibited by a scavenger of HO.. This appeared to be dependent upon reduction of H2O2 to OH plus HO., by V(IV)), followed by oxidation of NADH by HO.. Since there are reductants, within cells, capable of reducing V(V)) to V(IV), these reactions are likely to contribute to the toxicity of vanadate.  相似文献   

17.
In addition to well-known cell wall peroxidases, there is now evidence for the presence of this enzyme at the plasma membrane of the plant cells (surface peroxidase). Both are able to catalyze, through a chain of reactions involving the superoxide anion, the oxidation of NADH to generate hydrogen peroxide. The latter is oxidized by other wall-bound peroxidases to convert cinnamoyl alcohols into radical forms, which, then polymerize to generate lignin. However, there are other enzymes at the surface of plasma membranes capable of generating hydrogen peroxide (cell wall polyamine oxidase), superoxide anion (plasma membrane Turbo reductase), or both (plasma membrane flavoprotein?). These enzymes utilize NAD(P)H as a substrate. The Turbo reductase and the flavoprotein catalyze the univalent reduction of Fe3+ and then of O2 to produce Fe2+ and \(O_2^{\bar \cdot } \) , respectively. The superoxide anion, in the acidic environment of the cell wall, may then dismutate to H2O2. These superoxide anion- and hydrogen peroxide-generating systems are discussed in relation to their possible involvement in physiological and pathological processes in the apoplast of plant cells.  相似文献   

18.
Conversion of 2-methyl-3-hydroxypyridine-5-carboxylic acid (Cpd I) to α-(N-acetylaminomethylene)succinic acid (Cpd A) is catalyzed by an FAD protein, Cpd I oxygenase (Sparrow, et al., J. Biol. Chem. [1969] 244, 2590–2600) according to the equation: Cpd I + O2 + NADH + H+ → Cpd A + NAD+. When free FAD, FMN or riboflavin is added to reaction mixtures, oxidation of NADH remains dependent on presence of oxygenase and Cpd I, but is partially uncoupled from the oxygenation of Cpd I. Under these conditions, free reduced flavins appear in solution, as shown by their ability to reduce cytochrome c. These effects are not due to an increased rate of NADH oxidation. Such uncoupling may lead to appearance of artifactual species of activated oxygen or flavin that play no intermediate role in the oxygenase reaction.  相似文献   

19.
The photosensitized reduction of resorufin (RSF), the fluorescent product of Amplex Red, was investigated using electron spin resonance (ESR), optical absorption/fluorescence, and oxygen consumption measurements. Anaerobic reaction of RSF in the presence of the electron donor reduced nicotinamide adenine dinucleotide (NADH) demonstrated that during visible light irradiation (λ > 300 nm), RSF underwent one-electron reduction to produce a semiquinoneimine-type anion radical (RSF ‾) as demonstrated by direct ESR. Spin-trapping studies of incubations containing RSF, 5,5-dimethyl-1-pyrroline N-oxide (DMPO) and NADH demonstrated, under irradiation with visible light, the production of the superoxide dismutase (SOD)-sensitive DMPO/OOH adduct. Both absorption and fluorescence spectra of RSF in the presence of NADH demonstrated that the RSF ‾ was further reduced during irradiation with formation of its colorless dihydroquinoneimine form, dihydroresorufin (RSFH2). Both RSF ‾ and RSFH2, when formed in an aerobic system, were immediately oxidized by oxygen, which regenerated the dye and formed superoxide. Oxygen consumption measurements with a Clark-type oxygen electrode showed that molecular oxygen was consumed in a light-dependent process. The suppression of oxygen consumption by addition of SOD or catalase further confirmed the production of superoxide and hydrogen peroxide.  相似文献   

20.
The oxidation of Mn2+-pyrophosphate to Mn3+ by superoxide (O2?) was quantitative as evidenced from the formation of Mn3+-pyrophosphate and hydrogen peroxide and from the inhibition by superoxide dismutase. Using the competitive relation between Mn2+-pyrophosphate and superoxide dismutase for the O2?, the rate constant of Mn2+ oxidation was estimated to be about 6 × 106m?1 s?1. The oxidation of Mn2+-pyrophosphate by illuminated chloroplasts was also indicated to be stoichiometrically induced by O2?. In the presence of saturating amounts of the Mn2+, a double enhancement of hydrogen peroxide production and triple uptake of oxygen were found, as expected from the oxidation of Mn2+-pyrophosphate by O2?. Anaerobiosis or superoxide dismutase annuled these increments. We propose that the O2? generated as the sole initial step of the Mehler reaction oxidized Mn2+-pyrophosphate, and we discuss the role of free manganese in chloroplasts.  相似文献   

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