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1.
Cornelis Verduyn Marco L. F. Giuseppin W. Alexander Scheffers Johannes P. van Dijken 《Applied microbiology》1988,54(8):2086-2090
A catalase-negative mutant of the yeast Hansenula polymorpha consumed methanol in the presence of glucose when the organism was grown in carbon-limited chemostat cultures. The organism was apparently able to decompose the H2O2 generated in the oxidation of methanol by alcohol oxidase. Not only H2O2 generated intracellularly but also H2O2 added extracellularly was effectively destroyed by the catalase-negative mutant. From the rate of H2O2 consumption during growth in chemostat cultures on mixtures of glucose and H2O2, it appeared that the mutant was capable of decomposing H2O2 at a rate as high as 8 mmol · g of cells−1 · h−1. Glutathione peroxidase (EC 1.11.1.9) was absent under all growth conditions. However, cytochrome c peroxidase (CCP; EC 1.11.1.5) increased to very high levels in cells which decomposed H2O2. When wild-type H. polymorpha was grown on mixtures of glucose and methanol, the CCP level was independent of the rate of methanol utilization, whereas the level of catalase increased with increasing amounts of methanol in the substrate feed. Also, the wild type decomposed H2O2 at a high rate when cells were grown on mixtures of glucose and H2O2. In this case, an increase of both CCP and catalase was observed. When Saccharomyces cerevisiae was grown on mixtures of glucose and H2O2, the level of catalase remained low, but CCP increased with increasing rates of H2O2 utilization. From these observations and an analysis of cell yields under the various conditions, two conclusions can be drawn. (i) CCP is a key enzyme of H2O2 detoxification in yeasts. (ii) Catalase can effectively compete with mitochondrial CCP for hydrogen peroxide only if hydrogen peroxide is generated at the site where catalase is located, namely in the peroxisomes. 相似文献
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Nicoletta Guaragnella Antonella Bobba Ersilia Marra Sergio Giannattasio 《FEBS letters》2010,584(1):224-228
To investigate the role of cytochrome c (cyt c) release in yeast acetic acid-induced programmed cell death (AA-PCD), wild type (wt) and cells lacking metacaspase (Δyca1), cytochrome c (Δcyc1,7) and both (Δcyc1,7Δyca1) were compared for AA-PCD occurrence, hydrogen peroxide (H2O2) production and caspase activity. AA-PCD occurs in Δcyc1,7 and Δcyc1,7Δyca1 cells slower than in wt, but similar to that in Δyca1 cells, in which no cytochrome c release occurs. Both H2O2 production and caspase activation occur in these cells with early and extra-activation in Δcyc1,7 cells. We conclude that alternative death pathways can be activated in yeast AA-PCD, one dependent on cyt c release, which requires YCA1, and the other(s) independent on it. 相似文献
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Yeast cytochrome c peroxidase (CCP) efficiently catalyzes the reduction of H2O2 to H2O by ferrocytochrome c in vitro. The physiological function of CCP, a heme peroxidase that is targeted to the mitochondrial intermembrane space of Saccharomyces cerevisiae, is not known. CCP1-null-mutant cells in the W303-1B genetic background (ccp1Δ) grew as well as wild-type cells with glucose, ethanol, glycerol or lactate as carbon sources but with a shorter initial doubling time. Monitoring growth over 10 days demonstrated that CCP1 does not enhance mitochondrial function in unstressed cells. No role for CCP1 was apparent in cells exposed to heat stress under aerobic or anaerobic conditions. However, the detoxification function of CCP protected respiring mitochondria when cells were challenged with H2O2. Transformation of ccp1Δ with ccp1W191F, which encodes the CCPW191F mutant enzyme lacking CCP activity, significantly increased the sensitivity to H2O2 of exponential-phase fermenting cells. In contrast, stationary-phase (7-day) ccp1Δ-ccp1W191F exhibited wild-type tolerance to H2O2, which exceeded that of ccp1Δ. Challenge with H2O2 caused increased CCP, superoxide dismutase and catalase antioxidant enzyme activities (but not glutathione reductase activity) in exponentially growing cells and decreased antioxidant activities in stationary-phase cells. Although unstressed stationary-phase ccp1Δ exhibited the highest catalase and glutathione reductase activities, a greater loss of these antioxidant activities was observed on H2O2 exposure in ccp1Δ than in ccp1Δ-ccp1W191F and wild-type cells. The phenotypic differences reported here between the ccp1Δ and ccp1Δ-ccp1W191F strains lacking CCP activity provide strong evidence that CCP has separate antioxidant and signaling functions in yeast. 相似文献
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H2O2 production and cytochrome c peroxidase activity in mitochondria isolated from the trypanosomatid hemoflagellate Crithidia fasciculata 总被引:2,自引:0,他引:2
H2O2 production by coupled mitochondrial fractions from the protozoan, Crithidia fasciculata, has been measured spectrophotometrically by the formation of the stable enzyme-substrate complex with yeast cytochrome c peroxidase. H2O2 formation was observed with succinate, l-α-glycerophosphate, l-proline, α-ketoglutarate, and with endogenous substrate. The maximum rate of H2O2 generation obtained with each substrate in the presence of antimycin A was about 10% of the state 4 rate of O2 respiration, and only 1–2% of the carbonylcyanide m-fluorophenylhydrazone-uncoupled respiratory rate. Therefore, excess O2 uptake due to the formation of H2O2 cannot satisfactorily account for the low ADP:O ratios previously reported.Cytochrome c peroxidase activity was measured in mitochondrial preparations by recording the decrease in absorbance at 550 nm during the oxidation of horse heart ferrocytochrome c which was observed after addition of H2O2. The distribution of activity after sonic disruption of mitochondrial preparations was that expected for a soluble enzyme. The activity was proportional to the amount of enzyme protein added, and was abolished by heating at 100 °C for 3 min. Total cytochrome c peroxidase activity in mitochondrial fractions isolated from C. fasciculata was calculated to be 0.3% that of isolated yeast mitochondria, but it is suggested that the in vivo activity may be considerably higher than this estimate. 相似文献
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《BBA》2023,1864(4):149000
Cytochrome c oxidase (CcO) is a transmembrane heme‑copper metalloenzyme that catalyzes the reduction of O2 to H2O at the reducing end of the respiratory electron transport chain. To understand this reaction, we followed the conversion of CcO from Rhodobacter sphaeroides between several active-ready and carbon monoxide-inhibited states via attenuated total reflection Fourier-transform infrared (ATR FTIR) difference spectroscopy. Utilizing a novel gas titration setup, we prepared the mixed-valence, CO-inhibited R2CO state as well as the fully-reduced R4 and R4CO states and induced the “active ready” oxidized state OH. These experiments are performed in the dark yielding FTIR difference spectra exclusively triggered by exposure to O2, the natural substrate of CcO. Our data demonstrate that the presence of CO at heme a3 does not impair the catalytic oxidation of CcO when the cycle starts from the fully-reduced states. Interestingly, when starting from the R2CO state, the release of the CO ligand upon purging with inert gas yield a product that is indistinguishable from photolysis-induced states. The observed changes at heme a3 in the catalytic binuclear center (BNC) result from the loss of CO and are unrelated to electronic excitation upon illumination. Based on our experiments, we re-evaluate the assignment of marker bands that appear in time-resolved photolysis and perfusion-induced experiments on CcO. 相似文献
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Ruiz OH Gonzalez A Almeida AJ Tamayo D Garcia AM Restrepo A McEwen JG 《PLoS neglected tropical diseases》2011,5(10):e1353
Background
Paracoccidioides brasiliensis is a human thermal dimorphic pathogenic fungus. Survival of P. brasiliensis inside the host depends on the adaptation of this fungal pathogen to different conditions, namely oxidative stress imposed by immune cells.Aims and Methodology
In this study, we evaluated the role of alternative oxidase (AOX), an enzyme involved in the intracellular redox balancing, during host-P. brasiliensis interaction. We generated a mitotically stable P. brasiliensis AOX (PbAOX) antisense RNA (aRNA) strain with a 70% reduction in gene expression. We evaluated the relevance of PbAOX during interaction of conidia and yeast cells with IFN-γ activated alveolar macrophages and in a mouse model of infection. Additionally, we determined the fungal cell''s viability and PbAOX in the presence of H2O2.Results
Interaction with IFN-γ activated alveolar macrophages induced higher levels of PbAOX gene expression in PbWt conidia than PbWt yeast cells. PbAOX-aRNA conidia and yeast cells had decreased viability after interaction with macrophages. Moreover, in a mouse model of infection, we showed that absence of wild-type levels of PbAOX in P. brasiliensis results in a reduced fungal burden in lungs at weeks 8 and 24 post-challenge and an increased survival rate. In the presence of H2O2, we observed that PbWt yeast cells increased PbAOX expression and presented a higher viability in comparison with PbAOX-aRNA yeast cells.Conclusions
These data further support the hypothesis that PbAOX is important in the fungal defense against oxidative stress imposed by immune cells and is relevant in the virulence of P. brasiliensis. 相似文献8.
To gain further insight into yeast acetic acid-induced programmed cell death (AA-PCD) we analyzed the effects of the antioxidant N-acetyl-l-cysteine (NAC) on cell viability, hydrogen peroxide (H2O2) production, DNA fragmentation, cytochrome c (cyt c) release and caspase-like activation in wild type (wt) and metacaspase and/or cyt c-lacking cells. We found that NAC prevents AA-PCD in wt cells, by scavenging H2O2 and by inhibiting both cyt c release and caspase-like activation. This shows the occurrence of a reactive oxygen species (ROS)-dependent AA-PCD. Contrarily no NAC dependent change in AA-PCD of mutant cells was detectable, showing that a ROS-independent AA-PCD can also occur. 相似文献
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Adriana G. Prat Caroline Bolter Ulises Chavez Charles Taylor William Chefurka Julio F. Turrens 《Free radical biology & medicine》1991,11(6)
One of the most precise methods of determining hydrogen peroxide (H2O2) formation by biological systems is based on measuring the rate of enzyme-substrate complex formation between H2O2 and cytochrome c peroxidase (CCP). The main problem with this method is that CCP is not commercially available and has to be prepared in the laboratory. We have modified some currently available methods for purifying a highly active preparation of CCP in about 4 d. It includes a batch extraction of protein using DEAE-sepharose followed by concentration either by lyophilization or by passing the extract through a small DEAE-sepharose column instead of by ultrafiltration. The concentrated preparation is passed through a Sephadex G-75 column and the final CCP crystallized against water. The final preparations had a purity index (PI, ratio of absorbance at 408 nm/280 nm, equivalent to heme/protein ratio) above 1.2. These changes make the overall procedure very simple, preserving enzyme activity and spectral properties. In addition, we point out that special care has to be taken to eliminate cytochrome c from crude CCP extracts. Cytochrome c not only introduces an artifact when determining PI, but is also may act as a hydrogen donor for CCP when monitoring H2O2 formation, thus decreasing the sensitivity of this method. 相似文献
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《Inorganica chimica acta》1988,145(2):289-298
Manganese(II) complexes of long chain phosphines, MnX2(phosphine)(THF) XCl, Br, I; phosphine=P(C12H25)3, P(C14H29)3, P(C16H33)3, PPh(C12H25)2, PPh(C14H29)2, PPh(C16H33)2; THF=tetrahydrofuran have been prepared and characterised. These complexes react reversibly with molecular oxygen both in the solid state and in THF and toluene solution forming 1:1 Mn:O2 adducts. These adducts are monomeric in toluene and THF and molecular weight measurements confirm that the THF ligand remains coordinated in toluene solution leading to the formation of MnX2(phosphine)(THF)(O2) species. All the O2-adducts are highly coloured and binding curves have been constructed and Ko2 values calculated. Based on these Ko2 values the affinity for dioxygen is in the order XCl>Br>I in toluene solution, with Hill coefficient, n, indicating cooperativity (1-1.5). In THF dioxygen binding does not appear to be cooperative. 相似文献
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Hongyan Zhang Wenxia WangHeng Yin Xiaoming Zhao Yuguang Du 《Carbohydrate polymers》2012,87(3):2270-2278
Oligochitosan has been proved to trigger plant cell death. To gain some insights into the mechanisms of oligochitosan-induced cell death, the nature of oligochitosan-induced cell death and the role of calcium (Ca2+), nitric oxide (NO) and hydrogen peroxide (H2O2) were studied in tobacco suspension cells. Oligochitosan-induced cell death occurred in cytoplasmic shrinkage, phosphatidylserine externalization, chromatin condensation, TUNEL-positive nuclei, cytochrome c release and induction of programmed cell death (PCD)-related gene hsr203J, suggesting the activation of PCD pathway. Pretreatment cells with cyclosporin A, resulted in reducing oligochitosan-induced cytochrome c release and cell death, indicating oligochitosan-induced PCD was mediated by cytochrome c. In the early stage, cells undergoing PCD showed an immediate burst in free cytosolic Ca2+ ([Ca2+]cyt) elevation, NO and H2O2 production. Further study showed that these three signals were involved in oligochitosan-induced PCD, while Ca2+ and NO played a negative role in this process by modulating cytochrome c release. 相似文献
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N-Acetyltransferase Mpr1 of Saccharomyces cerevisiae can reduce intracellular oxidation levels and protect yeast cells under oxidative stress, including H2O2, heat-shock, or freeze-thaw treatment. Unlike many antioxidant enzyme genes induced in response to oxidative stress, the
MPR1 gene seems to be constitutively expressed in yeast cells. Based on a recent report that ethanol toxicity is correlated with
the production of reactive oxygen species (ROS), we examined here the role of Mpr1 under ethanol stress conditions. The null
mutant of the MPR1 and MPR2 genes showed hypersensitivity to ethanol stress, and the expression of the MPR1 gene conferred stress tolerance. We also found that yeast cells exhibited increased ROS levels during exposure to ethanol
stress, and that Mpr1 protects yeast cells from ethanol stress by reducing intracellular ROS levels. When the MPR1 gene was overexpressed in antioxidant enzyme-deficient mutants, increased resistance to H2O2 or heat shock was observed in cells lacking the CTA1, CTT1, or GPX1 gene encoding catalase A, catalase T, or glutathione peroxidase, respectively. These results suggest that Mpr1 might compensate
the function of enzymes that detoxify H2O2. Hence, Mpr1 has promising potential for the breeding of novel ethanol-tolerant yeast strains. 相似文献
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Cytochrome c oxidases (CcO) reduce O2 to H2O in the respiratory chain of mitochondria and many aerobic bacteria. In addition, some species of CcO can also reduce NO to N2O and water while others cannot. Here, the mechanism for NO-reduction in CcO is investigated using quantum mechanical calculations. Comparison is made to the corresponding reaction in a “true” cytochrome c-dependent NO reductase (cNOR). The calculations show that in cNOR, where the reduction potentials are low, the toxic NO molecules are rapidly reduced, while the higher reduction potentials in CcO lead to a slower or even impossible reaction, consistent with experimental observations. In both enzymes the reaction is initiated by addition of two NO molecules to the reduced active site, forming a hyponitrite intermediate. In cNOR, N2O can then be formed using only the active-site electrons. In contrast, in CcO, one proton-coupled reduction step most likely has to occur before N2O can be formed, and furthermore, proton transfer is most likely rate-limiting. This can explain why different CcO species with the same heme a3-Cu active site differ with respect to NO reduction efficiency, since they have a varying number and/or properties of proton channels. Finally, the calculations also indicate that a conserved active site valine plays a role in reducing the rate of NO reduction in CcO. 相似文献
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Hydrogen peroxide permeability of plasma membrane aquaporins of <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> 总被引:1,自引:0,他引:1
Hooijmaijers C Rhee JY Kwak KJ Chung GC Horie T Katsuhara M Kang H 《Journal of plant research》2012,125(1):147-153
Although aquaporins have been known to transport hydrogen peroxide (H2O2) across cell membranes, the H2O2-regulated expression patterns and the permeability of every family member of the plasma membrane intrinsic protein (PIP)
toward H2O2 have not been determined. This study investigates the H2O2-regulated expression levels of all plasma membrane aquaporins of Arabidopsis thaliana (AtPIPs), and determines the permeability of every AtPIP for H2O2 in yeast. Hydrogen peroxide treatment of Arabidopsis down-regulated the expression of AtPIP2 subfamily in roots but not in leaves, whereas the expression of AtPIP1 subfamily was not affected by H2O2 treatment. The growth and survival of yeast cells that expressed AtPIP2;2, AtPIP2;4, AtPIP2;5, or AtPIP2;7 was reduced in
the presence of H2O2, while the growth of yeast cells expressing any other AtPIP family member was not affected by H2O2. These results show that only certain isoforms of AtPIPs whose expression is regulated by H2O2 treatment are permeable for H2O2 in yeast cells, and suggest that the integrated regulation of aquaporin expression by H2O2 and the capacity of individual aquaporin to transport H2O2 are important for plant response to H2O2. 相似文献
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Zhi Li Hanxiao Sun Xuemei Mo Xiuying Li Bo Xu Peng Tian 《Applied microbiology and biotechnology》2013,97(11):4927-4936
The oleaginous yeast Rhodotorula glutinis has been known to be a potential feedstock for lipid production. In the present study, we investigated the enhancement of expression of malic enzyme (ME; NADP+ dependent; EC 1.1.1.40) from Mucor circinelloides as a strategy to improve lipid content inside the yeast cells. The 26S rDNA and 5.8S rDNA gene fragments isolated from Rhodotorula glutinis were used for homologous integration of ME gene into R. glutinis chromosome under the control of the constitutively highly expressed gene phosphoglycerate kinase 1 to achieve stable expression. We demonstrated that by increasing the expression of the foreign ME gene in R. glutinis, we successfully improved the lipid content by more than twofold. At the end of lipid accumulation phrase (96 h) in the transformants, activity of ME was increased by twofold and lipid content of the yeast cells was increased from 18.74 % of the biomass to 39.35 %. Simultaneously, there were no significant differences in fatty acid profiles between the wild-type strain and the recombinant strain. Over 94 % of total fatty acids were C16:0, C18:0, C16:1, C18:1, and C18:2. Our results indicated that heterologous expression of NADP+-dependent ME involved in fatty acid biosynthesis indeed increased the lipid accumulation in the oleaginous yeast R. glutinis. 相似文献
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Peroxiredoxins (Prxs) are ubiquitous antioxidant enzymes reducing toxic peroxides. Two distinct 2-Cys Prxs, Prx1 and Prx2, were identified in Vibrio vulnificus, a facultative aerobic pathogen. Both Prxs have two conserved catalytic cysteines, CP and CR, but Prx2 is more homologous in amino acid sequences to eukaryotic Prx than to Prx1. Prx2 utilized thioredoxin A as a reductant, whereas Prx1 required AhpF. Prx2 contained GGIG and FL motifs similar to the motifs conserved in sensitive Prxs and exhibited sensitivity to overoxidation. MS analysis and CP-SO3H specific immunoblotting demonstrated overoxidation of CP to CP-SO2H (or CP-SO3H) in vitro and in vivo, respectively. In contrast, Prx1 was robust and CP was not overoxidized. Discrete expression of the Prxs implied that Prx2 is induced by trace amounts of H2O2 and thereby residential in cells grown aerobically. In contrast, Prx1 was occasionally expressed only in cells exposed to high levels of H2O2. A mutagenesis study indicated that lack of Prx2 accumulated sufficient H2O2 to induce Prx1. Kinetic properties indicated that Prx2 effectively scavenges low levels of peroxides because of its high affinity to H2O2, whereas Prx1 quickly degrades higher levels of peroxides because of its high turnover rate and more efficient reactivation. This study revealed that the two Prxs are differentially optimized for detoxifying distinct ranges of H2O2, and proposed that Prx2 is a residential scavenger of peroxides endogenously generated, whereas Prx1 is an occasional scavenger of peroxides exogenously encountered. Furthermore, genome sequence database search predicted widespread coexistence of the two Prxs among bacteria. 相似文献