The Regulation of Electron Partitioning between the Cytochrome and Alternative Pathways in Soybean Cotyledon and Root Mitochondria

The regulation of electron partitioning between the cytochrome (Cyt) and alternative pathways in soybean (Glycine max L. cv Ransom) mitochondria in the absence of added inhibitors has been studied using the oxygen isotope fractionation technique. This regulation can depend on several factors, including the amount of alternative oxidase protein, the redox status of the alternative oxidase regulatory sulfhydryl-disulfide system, the degree of activation by [alpha]-keto acids, and the concentration and redox state of the ubiquinone pool. We studied electron partitioning onto the alternative pathway in mitochondria isolated from etiolated and light-grown cotyledons and roots to ascertain how these factors interact in different tissues. In light-grown cotyledon mitochondria there is some partitioning to the alternative pathway in state 4, which is increased dramatically by either pyruvate or dithiothreitol. In etiolated cotyledon mitochondria, the alternative pathway shows little ability to compete for electrons with the Cyt pathway under any circumstances. In root mitochondria, control of alternative pathway activity is exercised by both the ubiquinone pool and the regulatory sulfhydryl-disulfide system. In addition, oxygen isotope fractionation by the Cyt and alternative pathways in mitochondria were identical to the fractionation for the respective pathways seen in intact tissue, suggesting that residual respiration is not present in the absence of inhibitors.     

Partitioning of Electrons between the Cytochrome and Alternative Pathways in Intact Roots

To test the hypothesis that the cytochrome pathway is not invariably saturated when the alternative pathway is engaged, we titrated root respiration of several species with KCN (an inhibitor of the cytochrome pathway), both in the absence and presence of an inhibitor of the alternative pathway (salicylhydroxamic acid, SHAM). The slopes of the resultant KCN [rho] plots ([rho]cyt) were then used to determine whether the cytochrome pathway was saturated in each species. The species used were Festuca ovina ssp. ovina L., Phaseolus vulgaris L., and six Poa species (Poa pratensis L., Poa compressa L., Poa trivialis L., Poa alpina L., Poa costiniana Vick., and Poa fawcettiae Vick.). Although the cytochrome pathway was saturated in a number of species (i.e. [rho]cyt values were 1.0), several others exhibited [rho]cyt values of less than 0.5. Alternative pathway capacity correlated negatively with [rho]cyt, with [rho]cyt values of less than 1.0 occurring in tissues in which the alternative pathway capacity was greater than 25 to 30% of total respiration. The species that did not show full engagement of the cytochrome pathway rarely exhibited SHAM inhibition in the absence of KCN. We conclude that this lack of SHAM inhibition is not due to a lack of alternative pathway engagement but rather to the diversion of electrons from the alternative pathway to the unsaturated cytochrome path following the addition of SHAM.     

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.     

Pathways for Electron Tunneling in Cytochrome c Oxidase

Warburg showed in 1929 that the photochemical action spectrum for CO dissociation from cytochrome c oxidase is that of a heme protein. Keilin had shown that cytochrome a does not react with oxygen, so he did not accept Warburg's view until 1939, when he discovered cytochrome a ₃. The dinuclear cytochrome a ₃-Cu_B unit was found by EPR in 1967, whereas the dinuclear nature of the Cu_A site was not universally accepted until oxidase crystal structures were published in 1995. There are negative redox interactions between cytochrome a and the other redox sites in the oxidase, so that the reduction potential of a particular site depends on the redox states of the other sites. Calculated electron-tunneling pathways for internal electron transfer in the oxidase indicate that the coupling-limited rates are 9×10⁵ (Cu _A a) and 7×10⁶ s^–1 (a a ₃); these calculations are in reasonable agreement with experimental rates, after corrections are made for driving force and reorganization energy. The best Cu_A-a pathway starts from the ligand His204 and not from the bridging sulfur of Cys196, and an efficient a-a ₃ path involves the heme ligands His378 and His376 as well as the intervening Phe377 residue. All direct paths from Cu_A to a ₃ are poor, indicating that direct Cu_A a ₃ electron transfer is much slower than the Cu_A a reaction. The pathways model suggests a means for gating the electron flow in redox-linked proton pumps.     

Compartmentation of Alternative Oxidase in Plant Mitochondria

Capacity of alternative pathway mediated, CN-resistant respirationwas measured in mitochondria isolated from three plant species:Iris bulbs, potato tuber callus and Petunia cells, grown insuspension culture. Succinate, NADH or a combination of succinateand NADH were used as respiratory substrates. In all three plantspecies electrons from exogenous NADH appeared to have no accessto the alternative pathway connected with succinate mediatedrespiration and vice versa. Therefore, besides a (functional)compartmentation of Q-pools a compartmentation of alternativepathways in plant mitochondria is proposed as well. (Received October 19, 1985; Accepted January 29, 1986)     

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.     

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.     

Thioredoxin m4 Controls Photosynthetic Alternative Electron Pathways in Arabidopsis

In addition to the linear electron flow, a cyclic electron flow (CEF) around photosystem I occurs in chloroplasts. In CEF, electrons flow back from the donor site of photosystem I to the plastoquinone pool via two main routes: one that involves the Proton Gradient Regulation5 (PGR5)/PGRL1 complex (PGR) and one that is dependent of the NADH dehydrogenase-like complex. While the importance of CEF in photosynthesis and photoprotection has been clearly established, little is known about its regulation. We worked on the assumption of a redox regulation and surveyed the putative role of chloroplastic thioredoxins (TRX). Using Arabidopsis (Arabidopsis thaliana) mutants lacking different TRX isoforms, we demonstrated in vivo that TRXm4 specifically plays a role in the down-regulation of the NADH dehydrogenase-like complex-dependent plastoquinone reduction pathway. This result was confirmed in tobacco (Nicotiana tabacum) plants overexpressing the TRXm4 orthologous gene. In vitro assays performed with isolated chloroplasts and purified TRXm4 indicated that TRXm4 negatively controls the PGR pathway as well. The physiological significance of this regulation was investigated under steady-state photosynthesis and in the pgr5 mutant background. Lack of TRXm4 reversed the growth phenotype of the pgr5 mutant, but it did not compensate for the impaired photosynthesis and photoinhibition sensitivity. This suggests that the physiological role of TRXm4 occurs in vivo via a mechanism distinct from direct up-regulation of CEF.In plant thylakoids, photosynthesis involves a linear electron flow (LEF) from water to NADP⁺ via PSII, cytochrome b₆/f, PSI, and soluble carriers. LEF produces NADPH and generates a transthylakoidal electrochemical proton gradient that drives the synthesis of ATP. Besides LEF, cyclic electron flow (CEF) can also occur, involving only PSI (for review, see Johnson, 2011; Kramer and Evans, 2011). These additional reactions include two main distinct pathways involving either the Proton Gradient Regulation5 (PGR5)/PGRL1 complex (Munekage et al., 2002; DalCorso et al., 2008) or the NADH dehydrogenase-like complex (NDH; for review, see Battchikova et al., 2011; Ifuku et al., 2011). The functioning of either CEF pathway, which generates a pH gradient ΔpH without any accumulation of NADPH, is thought to achieve the appropriate ATP/NADPH balance required for the biochemical needs of the plant, especially under certain environmental conditions such as low CO₂ (Golding and Johnson, 2003), heat (Clarke and Johnson, 2001), cold (Clarke and Johnson, 2001), drought (Golding and Johnson, 2003; Kohzuma et al., 2009), high light (Munekage et al., 2004), or dark-to-light transitions (Joliot and Joliot, 2005; Fan et al., 2007). CEF-generated ΔpH is also involved in photoprotection owing to the down-regulation of PSII via nonphotochemical quenching (Munekage et al., 2004; Takahashi et al., 2009). Very recently, the role of the PGR5 protein as a regulator of LEF has been established. It has proved to be essential in the protection of PSI from photodamage (Suorsa et al., 2012).The two cyclic pathways are redundant (Munekage et al., 2004), sharing ferredoxin (Fd) as a common stromal electron donor (Yamamoto et al., 2011) and electron carriers from plastoquinone (PQ) to PSI with LEF. Thus, LEF and either of the CEF pathways may be in competition. The molecular events that allow CEF to challenge LEF remain enigmatic, particularly when considering that the conditions that require CEF are also those under which LEF is in excess. Efforts to understand the appropriate functioning of CEF have led to the proposition of several models segregating cyclic and linear pathways at a structural level (for review, see Eberhard et al., 2008; Cardol et al., 2011; Johnson, 2011; Rochaix, 2011). According to the restricted diffusion model, founded on the uneven distribution of the photosynthetic protein complexes in the thylakoids, there is little competition between CEF and LEF, as CEF occurs in stroma lamellae where PSI is concentrated while LEF takes place in the grana stacks. In line with the supercomplex model, whose relevance was demonstrated in the microalga Chlamydomonas reinhardtii, CEF happens within tightly bound supercomplexes containing PSI, with its own light-harvesting complex (LHCI), the PSII light-harvesting complex (LHCII), cytochrome b₆/f, Fd, Fd NADP reductase (FNR), and the integral membrane protein PGRL1 (Iwai et al., 2010). In higher plants, an association between NDH and PSI subunits suggests the formation of such supercomplexes (Peng et al., 2009). The availability of FNR, found either free in the stroma or bound to the thylakoids (Zhang et al., 2001), has also been proposed to modulate partitioning between LEF and CEF (Joliot and Joliot, 2006; Joliot and Johnson, 2011). In addition, more dynamic models that illustrate competitive processes involved in the distribution of electrons between the cyclic and linear flows have been proposed. The competition between cytochrome b₆/f and FNR for electrons from Fd could regulate the segregation between LEF and CEF (Breyton et al., 2006; Yamamoto et al., 2006; Hald et al., 2008). A few years ago, Joliot and Joliot (2006) suggested that the ATP/ADP ratio was one of the parameters that triggered on the transition between LEF and CEF. It was also established that the redox poise of chloroplast stroma contributed to the regulation of the photosynthetic pathway and played an important role in defining the extent of CEF. Breyton et al. (2006) scrutinized this redox regulation and established that the fraction of PSI complexes engaged in CEF could be modulated by changes in the stromal redox state. Overreduction of the NADPH pool was involved in the repartition between LEF and CEF (Joliot and Joliot, 2006). The NADPH/NADP⁺ ratio was proposed as a regulator of PGR-dependent CEF in vivo (Okegawa et al., 2008).All the published data supporting a role for the redox status in the regulation of CEF urged us to investigate a putative role of thioredoxins (TRX) in the regulation of CEF. TRX are ubiquitous disulfide reductases regulating the redox status of target proteins (for review, see Lemaire et al., 2007; Meyer et al., 2009). In chloroplast, TRX mediate the light regulation of numerous enzymes, among which some belong to the Calvin cycle (for review, see Schürmann and Buchanan, 2008; Montrichard et al., 2009; Lindahl et al., 2011). Global proteomic approaches have revealed that well-known photosynthetic complex subunits may be partners of TRX, such as PsbO in PSII, plastocyanin, Rieske Fe-S protein in cytochrome b₆/f, and PsaK and PsaN in PSI (for review, see Montrichard et al., 2009; Lindahl et al., 2011). Furthermore, regarding the regulation of photosynthesis, TRX have also been involved in state transitions (Rintamäki et al., 2000; Buchanan and Balmer, 2005), and their participation in the control of the redox poise of the electron transport chain has also been suggested (Johnson, 2003).In this work, we have investigated the possible role of TRX in the regulation of CEF. Using Arabidopsis (Arabidopsis thaliana) mutants with altered expression of genes encoding different plastid TRX, we have established in vivo the inhibitor activity of TRXm4 on the NDH-dependent pathway for plastoquinone reduction. This result was confirmed in transplastomic tobacco (Nicotiana tabacum) plants overexpressing the TRXm4 orthologous gene. Moreover, in vitro assays performed with isolated chloroplasts indicated that TRXm4 negatively controls the PGR-dependent electron flow as well.     

Specificity of the Organic Acid Activation of Alternative Oxidase in Plant Mitochondria

The claim that succinate and malate can directly stimulate the activity of the alternative oxidase in plant mitochondria (A.M. Wagner, C.W.M. van den Bergen, H. Wincencjusz [1995] Plant Physiol 108: 1035-1042) was reinvestigated using sweet potato (Ipomoea batatas L.) mitochondria. In whole mitochondria, succinate (in the presence of malonate) and both L- and D-malate stimulated respiration via alternative oxidase in a pH- (and NAD+)-dependent manner. Solubilized malic enzyme catalyzed the oxidation of both L- and D-malate, although the latter at only a low rate and only at acid pH. In submitochondrial particle preparations with negligible malic enzyme activity, neither L- nor D-malate stimulated alternative oxidase activity. However, even in the presence of high malonate concentrations, some succinate oxidation was observed via the alternative oxidase, giving the impression of stimulation of the oxidase. Neither L-malate nor succinate (in the presence of malonate) changed the dependence of alternative oxidase activity on ubiquinone reduction state in submitochondrial particles. In contrast, a large change in this dependence was observed upon addition of pyruvate. Half-maximal stimulation of alternative oxidase by pyruvate occurred at less than 5 [mu]M in submitochondrial particles, one-twentieth of that reported for whole mitochondria, suggesting that pyruvate acts on the inside of the mitochondrion. We suggest that malate and succinate do not directly stimulate alternative oxidase, and that reports to the contrary reflect intra-mitochondrial generation of pyruvate via malic enzyme.     

Regulation of Electron Transport in Plant Mitochondria under State 4 Conditions

The regulation of electron transport in pea (Pisum sativum L.) leaf mitochondria under state 4 conditions has been investigated by simultaneously monitoring oxygen uptake, the steady-state reduction level of ubiquinone, and membrane potential. Membrane potentials were measured using a methyltriphenylphosphonium electrode while a voltametric technique was used to monitor changes in the steady-state reduction levels of quinone. It was found that the addition of glycine to mitochondria oxidising malate in state 4 led to a marked increase in the rate of O₂ uptake and increased both the membrane potential and reduction level of the quinone pool. Increases in the state 4 respiratory rate were attributed to both an increase in driving flux, due to increased Q-pool reduction, and in membrane potential. Due to the nonohmic behavior of the inner membrane, under these conditions, an increase in potential would result in a considerable rise in proton conductance. Measurement of dual substrate oxidation, in the presence of n-propylgallate, revealed that the increase in respiratory activity was not mediated by the alternative oxidase. Similar increases in membrane potential and the level of Q-pool reduction were observed even in the presence of rotenone suggesting that the rotenone-insensitive pathway is a constitutive feature of plant mitochondria and may play a role in facilitating rapid state 4 rates even in the presence of a high energy charge.     

Malate Oxidation in Plant Mitochondria via Malic Enzyme and the Cyanide-insensitive Electron Transport Pathway

Malate oxidation in plant mitochondria proceeds through the activities of two enzymes: a malate dehydrogenase and a NAD⁺-dependent malic enzyme. In cauliflower, mitochondria malate oxidation via malate dehydrogenase is rotenone- and cyanide-sensitive. Addition of exogenous NAD⁺ stimulates the oxidation of malate via malic enzyme and generates an electron flux that is both rotenone- and cyanide-insensitive. The same effects of exogenous NAD⁺ are also observed with highly cyanide-sensitive mitochondria from white potato tubers or with mitochondria from spinach leaves. Both enzymes are located in the matrix, but some experimental data also suggest that part of malate dehydrogenase activity is also present outside the matrix compartment (adsorbed cytosolic malate dehydrogenase?). It is concluded that malic enzyme and a specific pool of NAD⁺/NADH are connected to the cyanide-insensitive alternative pathway by a specific rotenone-insensitive NADH dehydrogenase located on the inner face of the inner membrane. Similarly, malate dehydrogenase and another specific pool of NAD⁺/NADH are connected to the cyanide- (and antimycin-) sensitive pathway by a rotenone-sensitive NADH dehydrogenase located on the inner face of the inner membrane. A general scheme of electron transport in plant mitochondria for the oxidation of malate and NADH can be given, assuming that different pools of ubiquinone act as a branch point between various dehydrogenases, the cyanide-sensitive cytochrome pathway and the cyanide-insensitive alternative pathway.     

线粒体、细胞色素c与细胞凋亡

线粒体是细胞的一个独特而重要的细胞器,它为细胞各种生命活动提供能量。许多研究表明,线粒体的作用远比人们了解的复杂和多样。近年来研究发现,线粒体与细胞凋亡密切相关,表现在如下一些方面。１．细胞凋亡早期线粒体跨膜电位（ΔΨｍ）下降自１９９３年以来,Ｋｒｏ...     

交替氧化酶和解偶联蛋白在植物线粒体中的作用及其相互关系

植物线粒体不仅含有细胞色素C呼吸途径相关蛋白,也包括交替氧化酶（抗氰交替途径）和解偶联蛋白（解偶联途径）,这些途径中的蛋白都直接影响植物线粒体能量代谢。本文介绍了交替氧化酶和解偶联蛋白在植物线粒体能量代谢中的作用及其相互关系的研究进展。     

线粒体,细胞包素c与细胞凋亡

线粒体是细胞的一个独特而重要的细胞器,它为细胞各种生命活动提供能量.许多研究表明,线粒体的作用远比人们了解的复杂和多样.近年来研究发现,线粒体与细胞凋亡密切相关,表现在如下一些方面.     

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.     

Signals Regulating the Expression of the Nuclear Gene Encoding Alternative Oxidase of Plant Mitochondria

Suspension cells of tobacco (Nicotiana tabacum L. cv Bright Yellow) were used to investigate signals regulating the expression of the nuclear gene Aox1 encoding the mitochondrial alternative oxidase (AOX) protein responsible for cyanide-resistant respiration in plants. We found that an increase in the tricarboxylic acid cycle intermediate citrate (either after its exogenous supply to cells or after inhibition of aconitase by monofluoroacetate) caused a rapid and dramatic increase in the steady-state level of Aox1 mRNA and AOX protein. This led to a large increase in the capacity for AOX respiration, defined as the amount of salicylhydroxamic acid-sensitive O2 uptake by cells in the presence of potassium cyanide. The results indicate that citrate may be an important signal metabolite regulating Aox1 gene expression. A number of other treatments were also identified that rapidly induced the level of Aox1 mRNA and AOX capacity. These included short-term incubation of cells with 10 mM acetate, 2 [mu]M antimycin A, 5 mM H2O2, or 1 mM cysteine. For some of these treatments, induction of AOX occurred without an increase in cellular citrate level, indicating that other signals (possibly related to oxidative stress conditions) are also important in regulating Aox1 gene expression. The signals influencing Aox1 gene expression are discussed with regard to the potential function(s) of AOX to modulate tricarboxylic acid cycle metabolism and/or to prevent the generation of active oxygen species by the mitochondrial electron transport chain.