Stress-Induced Changes in Ubiquinone Concentration and Alternative Oxidase in Plant Mitochondria

We have investigated the influence of stress conditions such as incubation at 4°C and incubation in hyperoxygen atmosphere, on plant tissues. The ubiquinone (Q) content and respiratory activity of purified mitochondria was studied. The rate of respiration of mitochondria isolated from cold-treated green bell peppers (Capsicum annuum L) exceeds that of controls, but this is not so for mitochondria isolated from cold-treated cauliflower (Brassica oleracea L). Treatment with high oxygen does not alter respiration rates of cauliflower mitochondria. Analysis of kinetic data relating oxygen uptake with Q reduction in mitochondria isolated from tissue incubated at 4°C (bell peppers and cauliflowers) and at high oxygen levels (cauliflowers) reveals an increase in the total amount of Q and in the percentage of inoxidizable QH₂. The effects are not invariably accompanied by an induction of the alternative oxidase (AOX). In those mitochondria where the AOX is induced (cold-treated bell pepper and cauliflower treated with high oxygen) superoxide production is lower than in the control. The role of reduced Q accumulation and AOX induction in the defense against oxidative damage is discussed.     

Interactions Between the Cytochrome Pathway and the Alternative Oxidase in Isolated Acanthamoeba castellanii Mitochondria

The steady-state activity of the two quinol-oxidizing pathways of Acanthamoeba castellanii mitochondria, the phosphorylating cytochrome pathway (i.e. the benzohydroxamate(BHAM)-resistant respiration in state 3) and the alternative oxidase (i.e. the KCN-resistant respiration), is shown to be fixed by ubiquinone (Q) pool redox state independently of the reducing substrate (succinate or exogenous reduced nicotinamide adenine dinucleotide (NADH)), indicating that the active Q pool is homogenous. For both pathways, activity increases with the Q reduction level (up to 80%). However, the cytochrome pathway respiration partially inhibited (about 50%) by myxothiazol decreases when the Q reduction level increases above 80%. The decrease can be explained by the Q cycle mechanism of complex III. It is also shown that BHAM has an influence on the relationship between the rate of ADP phosphorylation and the Q reduction level when alternative oxidase is active, and that KCN has an influence on the relationship between the alternative oxidase activity and the Q reduction level. These unexpected effects of BHAM and KCN observed at a given Q reduction level are likely due to functional connections between the two pathways activities or to protein–protein interaction.     

The Alternative Oxidase of Yarrowia lipolytica Mitochondria Is Unable To Compete with the Cytochrome Pathway for Electrons

The activity of the cyanide-resistant alternative oxidase (pathway) of Yarrowia lipolytica mitochondria was studied as a function of the activity of the major, cyanide-sensitive, cytochrome pathway. The contribution of the alternative oxidase to the total respiration of mitochondria was evaluated by measuring the rate of oxygen consumption in the presence of cyanide (an inhibitor of the cytochrome pathway). The potential activity of the cytochrome pathway was evaluated spectrophotometrically, by measuring the oxidation rate of cytochrome c by ferricyanide, which accepts electrons from complex III (cytochrome c) of this pathway. The oxidation of succinate by mitochondria in the presence of ferricyanide and cyanide was accompanied by oxygen consumption due to the transfer of electrons through the alternative pathway. The subsequent addition of ADP or FCCP (an uncoupler of oxidative phosphorylation in the cytochrome pathway) completely inhibited the consumption of oxygen by the mitochondria. Under these conditions, the inhibition of the alternative pathway by benzohydroxamic acid failed to affect the transfer of electrons from cytochrome c to ferricyanide. Benzohydroxamic acid did not influence the rate of ferricyanide reduction by the cytochrome pathway occurring in controlled state 4, nor could it change the phosphorylation quotient ATP/O upon the oxidation of various substrates. These findings indicate that the alternative pathway is unable to compete with the cytochrome respiratory chain for electrons. The alternative pathway transfers only electrons that are superfluous for the cytochrome chain.     

The Content of Alternative Oxidase of Euglena Mitochondria is Increased by Growth in the Presence of Cyanide and is not Cytochrome o.

ABSTRACT A study of the effect of respiratory inhibitors on O₂ uptake of Euglena gracilis mitochondria, isolated from cells grown in the presence of cyanide or with ethanol as carbon source, was undertaken. The contents of cytochrome c oxidase and alternative oxidase were also determined. Inhibition of respiration by antimycin and cyanide was only partial and it was dependent on the oxidizable substrate used. Succinate oxidation was the most sensitive to cyanide whereas lactate oxidation was the most resistant. Cell growth in the presence of cyanide or with ethanol as carbon source brought about an enhanced content of alternative oxidase without a concomitant increase in cytochrome aa₃ content. However, a correlation between cyanide-resistant respiration and alternative oxidase content was not found. Analysis of heme types in mitochondrial membranes revealed the absence of heme O. The data suggest the presence of an inducible alternative oxidase in Euglena mitochondria which has high resistance to cyanide and contains heme B. A close relationship between Euglena alternative oxidase and bacterial quinol oxidases containing B-type heme is proposed.     

The Alternative Oxidase: in vivo Regulation and Function

Abstract: This review focuses on the biochemical regulation and function of the alternative oxidase in vivo. About 10 years ago, two activation mechanisms were discovered in isolated mitochondria, namely activation by reducing sulfur bonds in the protein and activation by an allosteric effect of pyruvate. It was proposed that plants would have a regulatory mechanism to modify alternative oxidase activity in vivo. However, more recent studies have shown that these two activation mechanisms may not play such an important role in regulation of alternative oxidase activity in vivo after all. Pyruvate and reduction of the sulfide bonds in the protein are definitely required for alternative oxidase activity, but they do not appear to be regulating the activity in vivo.
Despite the energy wasting nature of the alternative oxidase, there was no obvious physiological function for the pathway for many years. It is now more clear that the alternative oxidase can prevent the production of excess reactive oxygen species radicals by stabilizing the redox state of the mitochondrial ubiquinone pool, while allowing continued activity of the citric acid cycle. This may be important under conditions when the NADH supply is relatively high (reductant overflow), or when the cytochrome pathway is restrained. The cytochrome pathway might be inhibited by naturally occurring cyanide, nitric oxide, sulfide, high concentrations of CO₂, low temperatures, or by limited phosphate supply.     

Alternative Oxidase and Uncoupling Protein: Thermogenesis Versus Cell Energy Balance

The physiological role of an alternative oxidase and an uncoupling protein in plant and protists is discussed in terms of thermogenesis and energy metabolism balance in the cell. It is concluded that thermogenesis is restricted not only by a lower-limit size but also by a kinetically-limited stimulation of the mitochondrial respiratory chain.     

Thermolability of the Alternative Electron Transport Pathway in Higher Plant Mitochondria

Mitochondria from four plant species showing normal (Arum maculatum L., Arum italicum Mill., Sauromatum guttatum Schott) or induced (Solanum tuberosum L.) resistance to cyanide were submitted to temperature treatments up to 90 min at 45°C. The activity of the alternative, cyanide-resistant electron transport pathway was specifically and deeply altered by temperature treatments. Hydrogen sulfide was released in direct proportion to the reduction of activity of the alternative pathway. Only a small fraction (? 20%) of the total labile sulfide content of the mitochondria was associated with the operation of this pathway. In cyanide-resistant mitochondria, the cytochrome pathway was much more resistant to thermal inactivation than the alternative pathway. On the contrary, in cyanide-sensitive mitochondria (with no alternative pathway) the cytochrome pathway was highly sensitive to temperature treatments. These results indicate that the presence of a cyanide-resistant alternative pathway is correlated with a higher degree of resistance to thermal denaturation of the cytochrome pathway. They also strongly suggest that iron-sulfur proteins are regular components of the alternative pathway.     

Structure and Function of the Plant Alternative Oxidase: Its Putative Role in the Oxygen Defence Mechanism

Current understanding of the structure and function of the plant alternative oxidase is reviewed. In particular, the role of the oxidase in the protection of tissues against oxidative stress is developed.     

NaCl胁迫下交替呼吸途径对叶绿素含量及其荧光特性的影响

以菜豆幼苗作为试验材料,分析了NaCl胁迫下交替呼吸对叶绿素含量以及叶绿素荧光特性变化特征的影响,以探讨交替呼吸途径在逆境下的生理学作用以及植物在盐胁迫下光系统Ⅱ(PSⅡ)的调节作用机制。结果表明:(1)随着NaCl胁迫浓度(0、100、200、300mmol/L)的增高,菜豆幼苗叶片叶绿素含量显著下降,叶片光系统Ⅱ(PSⅡ)潜在最大光化学量子效率(Fv/Fm)、光适应下最大光化学效率(Fv′/Fm′)、PSⅡ光适应下实际光化学效率[Y(Ⅱ)]和光化学荧光猝灭(qP)与对照相比均显著性下降,而非光化学猝灭(NPQ)较对照组显著增加,同时交替呼吸容量在NaCl胁迫下也显著上升。(2)与单独NaCl胁迫相比,在NaCl胁迫下施加交替呼吸的抑制剂水杨基氧肟酸(SHAM)会导致菜豆幼苗叶片叶绿素含量、Fv/Fm、Fv′/Fm′、Y(Ⅱ)和qP进一步显著下降、NPQ进一步显著增加。研究认为,NaCl胁迫导致菜豆叶片光系统Ⅱ光化学效率下降和光能耗散增加,交替呼吸途径可有效缓解NaCl胁迫下菜豆叶绿素含量的减少以及光系统Ⅱ光化学反应效率的下降。     

Respiratory pathways and oxygen toxicity in Phanerochaete chrysosporium

Phanerochaete chrysosporium maintained on glucose as the carbon source contained severely impaired mitochondria that were characterised by the loss of both succinate dehydrogenase and cytochrome oxidase activities. These cells maintained a constant value for energy charge using anaerobic metabolism. Cells with these properties express lignin peroxidase when supplied with a pure oxygen atmosphere, which may reflect a response to accumulating reactive oxygen species. Cells maintained on cellulose retained fully functional mitochondria, but expressed lignin peroxidase without being exposed to a pure oxygen atmosphere. In the cells maintained on cellulose, mitochondrial function may be limited by the supply of glucose, leading to the accumulation of reactive oxygen species.     

Physiological Characteristics and Alternative Respiratory Pathway under Salt Stress in Two Wheat Cultivars Differing in Salt Tolerance

Changes in respiratory pathway, dry weight, contents of proline, ATP, Na⁺and K⁺were investigated under five salinity treatments in the leaves of plants of spring wheat (Triticum aestivumL.). Two cultivars differing in salt resistance, namely, 89122 (salt-tolerant) and Longchun13 (salt-sensitive), were used. The decrease in dry weight and K⁺content was observed with the increasing NaCl concentrations, but more in cv. 89122 plants than in Longchun13 plants. Contents of proline and Na⁺in both cvs increased greatly, but the former increased more in 89122 while the latter more in Longchun13 plants. In all salinity treatments tested, a salt-induced increase in the activity of the alternative pathway was found, although cytochrome pathway (CP) still remained the main electron transport pathway. ATP production changed in parallel with CP operation. Cv. 89122 plants could produce more ATP than cv. Longchun13 plants exposed to each salinity treatment and their ATP generation could even be stimulated in contrast to its rapidly decline in Longchun13 plants with increased salinity stress. The possible relationship between respiration metabolism and above mentioned physiological changes is discussed.     

Mitochondria Revisited. Alternative Functions of Mitochondria

This review explores the alternative functions of mitochondria inside the cell. In a general picture of mitochondrial functioning, the importance and uniqueness of these intrinsic functions make them irreplaceable by other intracellular compartments. Among these are, participation in apoptosis and cellular proliferation, regulation of the cellular redox state and level of second messengers, heme and steroid syntheses, production and transmission of a transmembrane potential, detoxication and heat production. In most of the listed functions, reactive oxygen species modulate a number of non-destructive cellular activities. Some of the mitochondrial functions are reviewed in detail.     

NADPH Oxidase System as a Superoxide-generating Cyanide-Resistant Pathway in the Respiratory Chain of Corynebacterium glutamicum

The respiratory chain of Corynebacterium glutamicum was investigated, especially with respect to a cyanide-resistant respiratory chain bypass oxidase. The membranes of C. glutamicum had NADH, succinate, lactate, and NADPH oxidase activities, and menaquinone, and cytochromes a ₅₉₈, b _562(558), and c ₅₅₀ as respiratory components. The NADH, succinate, lactate, and NADPH oxidase systems, all of which were more cyanide-resistant than N,N,N′,N′-tetramethyl-p-phenylene diamine oxidase activity (cytochrome aa ₃ terminal oxidase), had different sensitivities to cyanide; the cyanide sensitivity of these oxidase systems increased in the order, NADPH, lactate, NADH, and succinate. Taken together with the analysis of redox kinetics in the cytochromes and the effects of respiratory inhibitors, the results suggested that there is a cyanide-resistant bypass oxidase branching at the menaquinone site, besides cyanide-sensitive cytochrome oxidase in the respiratory chain. H⁺/O measurements with resting cells suggested that the cyanide-sensitive respiratory chain has two or three coupling sites, of which one is in NADH dehydrogenase and the others between menaquinone and cytochrome oxidase, but the cyanide-resistant bypass oxidase may not have any proton coupling site. NADPH and lactate oxidase systems were more resistant to UV irradiation than other systems and the UV insensitivity was highest in the NADPH oxidase system, suggesting that a specific quinone resistant to UV or no such a quinone works in at least NADPH oxidase system while the UV-sensitive menaquinone pool does in other oxidase systems. Furthermore, superoxide was generated in well-washed membranes, most strongly in the NADPH oxidase system. Thus, it was suggested that the cyanide-resistant bypass oxidase system of C. glutamicum is related to the NADPH oxidase system, which may be involved in generation of superoxide anions and probably functions together with superoxide dismutase and catalase.     

Specific Inhibition of the Cyanide-insensitive Respiratory Pathway in Plant Mitochondria by Hydroxamic Acids

Hydroxamic acids, R-CONHOH, are inhibitors specific to the respiratory pathway through the alternate, cyanide-insensitive terminal oxidase of plant mitochondria. The nature of the R group in these compounds affects the concentration at which the hydroxamic acids are effective, but it appears that all hydroxamic acids inhibit if high enough concentrations are used. The benzhydroxamic acids are effective at relatively low concentrations; of these, the most effective are m-chlorobenzhydroxamic acid and m-iodobenzhydroxamic acid. The concentrations required for half-maximal inhibition of the alternate oxidase pathway in mung bean (Phaseolus aureus) mitochondria are 0.03 mm for m-chlorobenzhydroxamic acid and 0.02 mm for m-iodobenzhydroxamic acid. With skunk cabbage (Symplocarpus foetidus) mitochondria, the required concentrations are 0.16 for m-chlorobenzhydroxamic acid and 0.05 for m-iodobenzhydroxamic acid. At concentrations which inhibit completely the alternate oxidase pathway, these two compounds have no discernible effect on either the respiratory pathway through cytochrome oxidase, or on the energy coupling reactions of these mitochondria. These inhibitors make it possible to isolate the two respiratory pathways and study their mode of action separately. These inhibitors also enhance an electron paramagnetic resonance signal near g = 2 in anaerobic, submitochondrial particles from skunk cabbage, which appears to be specific to the alternate oxidase and thus provides a means for its assay.     

The Effect of Growth and Measurement Temperature on the Activity of the Alternative Respiratory Pathway

A postulated role of the CN-resistant alternative respiratory pathway in plants is the maintenance of mitochondrial electron transport at low temperatures that would otherwise inhibit the main phosphorylating pathway and prevent the formation of toxic reactive oxygen species. This role is supported by the observation that alternative oxidase protein levels often increase when plants are subjected to growth at low temperatures. We used oxygen isotope fractionation to measure the distribution of electrons between the main and alternative pathways in mung bean (Vigna radiata) and soybean (Glycine max) following growth at low temperature. The amount of alternative oxidase protein in mung bean grown at 19°C increased over 2-fold in both hypocotyls and leaves compared with plants grown at 28°C but was unchanged in soybean cotyledons grown at 14°C compared with plants grown at 28°C. When the short-term response of tissue respiration was measured over the temperature range of 35°C to 9°C, decreases in the activities of both main and alternative pathway respiration were observed regardless of the growth temperature, and the relative partitioning of electrons to the alternative pathway generally decreased as the temperature was lowered. However, cold-grown mung bean plants that up-regulated the level of alternative oxidase protein maintained a greater electron partitioning to the alternative oxidase when measured at temperatures below 19°C supporting a role for the alternative pathway in response to low temperatures in mung bean. This response was not observed in soybean cotyledons, in which high levels of alternative pathway activity were seen at both high and low temperatures.     

线粒体呼吸功能与精子活力、核DNA损伤的相关性分析

为探讨线粒体呼吸功能与精子活力、核DNA损伤程度之间的相关性,按WHO标准收集34例不同活力的精液标本,采用蔗糖差速离心法或密度梯度离心法提取精子线粒体,通过铂氧电极-溶氧仪测定线粒体呼吸耗氧率并计算状态III呼吸、状态IV呼吸、呼吸控制率（RCR）、磷氧比（P／0）及氧化磷酸化效率（0PR）;应用精子染色质扩散（sperm chromatin dispersion,SCD）实验检测精子DNA损伤情况。结果表明：不同活力精子线粒体状态Ⅲ呼吸耗氧量之间具有显著差异俨〈0．01）;弱精子症组RcR和OPR与正常对照组比较,分别降低了17．03％（P〈0．05）和40．74％（P〈0。01）;精子DNA损伤程度与精子活力、状态III呼吸及OPR均呈极显著负相关（r值分别是-0．812、-0．788和-0．696）。以上结果提示：精子线粒体呼吸耗氧和氧化磷酸化功能与精子活力之间存在着密切的联系;精子DNA（包括mtDNA）损伤可能影响精子的正常功能。     

线粒体锚定对凋亡诱导因子抗氧化应激功能的影响

目的:构建带线粒体锚定信号肽的凋亡诱导因子(AIF)融合表达载体,研究在A549细胞中AIF线粒体锚定与其抗氧化应激功能的关系。方法:利用PCR将AIF原有线粒体定位信号(1-120 aa)替换成具有锚定功能的细胞色素c氧化酶Ⅳ亚型(COXⅣ)线粒体定位信号,并将COX-AIF克隆至pEGFP-N1和pDsRed1-N1载体,构建COX-AIF-GFP和COX-AIF-RFP融合表达载体;利用免疫印迹和激光共聚焦技术检测COX-AIF-GFP和COX-AIF-RFP的表达及其与线粒体的共定位;利用DCF染色和流式细胞技术检测A549细胞内过氧化物的水平。结果:表达了COX-AIF-GFP和COX-AIF-RFP融合蛋白,COX-AIF-GFP/RFP及AIF-RFP/RFP均定位于线粒体;与野生型AIF-RFP相比,COX-AIF-RFP可显著提高A549细胞的抗氧化应激能力。结论:AIF抗氧化应激能力依赖其在线粒体内膜的锚定。     

逆境胁迫对菜豆黄化苗转绿过程中交替呼吸途径与叶绿素荧光特征的影响

以菜豆黄化幼苗作为试验材料,探讨了铅(Pb)或PEG(聚乙二醇)胁迫下交替呼吸途径在植物转绿过程中对叶绿素含量以及叶绿素荧光特性的影响,以阐明逆境胁迫下植物交替呼吸途径的生理学作用。结果显示：(1)与菜豆黄化幼苗正常转绿过程(对照)相比,Pb或PEG胁迫导致菜豆黄化幼苗的叶绿素含量积累延迟,使叶片PSⅡ潜在最大光化学量子效率(F_v/F_m)、光适应下最大光化学效率(F_v′/F_m′)、PSⅡ光适应下实际光化学效率(Y(Ⅱ))和光化学荧光猝灭系数(qP)显著下降,而非光化学猝灭系数(NPQ)则显著增加。(2)在菜豆黄化幼苗转绿过程中,Pb或PEG胁迫导致其交替呼吸途径容量较对照均显著上升。(3)Pb或PEG胁迫下,交替呼吸途径抑制剂［水杨基氧肟酸(SHAM,1 mmol/L)］使菜豆黄化幼苗转绿过程中叶绿素含量、F_v/F_m、F_v′/F_m′、Y(Ⅱ)和qP进一步下降, NPQ却进一步增加,说明抑制交替呼吸途径会加剧Pb或PEG胁迫对PSⅡ反应中心活性的进一步抑制,使还原力积累加剧,造成热耗散进一步增加。研究表明,Pb或PEG胁迫均显著降低了菜豆黄化幼苗PSⅡ对光能的利用率,进而阻碍了菜豆黄化幼苗转绿进程;交替呼吸途径有助于在胁迫条件下缓解PSⅡ的过度还原,可能在一定程度上缓解了Pb或PEG胁迫对其转绿进程的阻碍作用。     

Nrf2-ARE信号通路在神经退行性疾病中的作用研究进展

神经退行性疾病(Neurodegenerative disease)是一类以神经元退行性病变为基础的慢性、进行性、不可逆的神经系统疾病的总称,主要包括阿尔茨海默病(Alzheimer's disease,AD)、帕金森病(Parkinson's disease,PD)、亨廷顿舞蹈病(Huntington disease,HD)、肌萎缩侧索硬化症(amyotrophic lateral sclerosis,ALS)、脊髓肌萎缩症(spinal muscular atrophy,SMA)、不同类型脊髓小脑共济失调(spinal cerebella ataxias,SCA)等。其病因和发病机制十分复杂,其中,氧化应激学说近年来受到了人们的广泛关注和普遍认可。而研究表明,Nrf2-ARE信号通路是体内抗氧化应答机制中最重要的通路之一,其能对氧化应激导致的神经细胞损伤产生保护作用,即阻止神经细胞的病变和凋亡,进而延缓神经退行性疾病的发生发展,因而有望成为神经退行性疾病的有效治疗靶标。本文就Nrf2-ARE信号通路结构特点及Nrf2-ARE信号通路在神经退行性疾病中的作用研究进展作一综述。     

Alternative oxidase from Arum and soybean: Its stabilization during purification

Complete purification of the alternative oxidase from plant mitochondria has not been achieved successfully, because of its instability on solubilization. We report here that the addition of pyruvate to the isolation medium stabilizes the activity of the solubilized enzyme. A procedure is described for the rapid isolation and partial purification of the cyanide-insensitive alternative oxidase from both Arum maculatum and soybean cotyledon ( Glycine max ) mitochondria. The degree of purification was 16- and 74-fold for Arum and soybean enzyme, respectively. The specific activities increased from 1 300 to 20 300 nmol oxygen consumed mg⁻¹ protein min⁻¹ (using duroquinol as substrate) after purification for the Arum erizyme and from 6 to 445 nmol oxygen consumed mg⁻¹ protein min⁻¹ for the soybean enzyme. A turnover for the partially purified Arum enzyme was estimated to be 47 electrons s⁻¹.
The partially purified enzyme from both Arum and soybean cotyledon mitochondria was sensitive to alternative oxidase inhibitors such as salicylhydroxamic acid, n -propyl gallate and octyl gallate, but not to myxottriazol, KCN or antimycin A. The activity of the enzyme could be stimulated by pyruvate, but not by malate and suceinate. The stability of the purified enzyme was also dependent on the continued presence of pyruvate. In the absence of pyruvace, the enzyme activity was lost in a time-dependent manner and the ability of pyruvate to recover the activity was also irreversibly lost.