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.     

Regulation of alternative oxidase activity in higher plants

Plant mitochondria contain two terminal oxidases: cytochrome oxidase and the cyanideinsensitive alternative oxidase. Electron partioning between the two pathways is regulated by the redox poise of the ubiquinone pool and the activation state of the alternative oxidase. The alternative oxidase appears to exist as a dimer which is active in the reduced, noncovalently linked form and inactive when in the oxidized, covalently linked form. Reduction of the oxidase in isolated tobacco mitochondria occurs upon oxidation of isocitrate or malate and may be mediated by matrix NAD(P)H. The activity of the reduced oxidase is governed by certain other organic acids, notably pyruvate, which appear to interact directly with the enzyme. Pyruvate alters the interaction between the alternative oxidase and ubiquinol so that the oxidase becomes active at much lower levels of ubiquinol and competes with the cytochrome pathway for electrons. These requirements for activation of the alternative oxidase constitute a sophisticated feed-forward control mechanism which determines the extent to which electrons are directed away from the energy-conserving cytochrome pathway to the non-energy conserving alternative oxidase. Such a mechanism fits well with the proposed role of the alternative oxidase as a protective enzyme which prevents over-reduction of the cytochrome chain and fermentation of accumulated pyruvate.     

A critique of the use of inhibitors to estimate partitioning of electrons between mitochondrial respiratory pathways in plants

The contribution of individual plant mitochondrial respiratory pathways to total respiration is commonly assessed by titration with specific inhibitors of different components in the branched electron transport chain. A pathway's contribution is equal to the activity when the other branch is blocked by an inhibitor multiplied by the degree (0-1.0) to which this activity is engaged when both pathways are operating. According to Bahr and Bonner (1973. J. Biol. Chem. 218: 3441–3445) the plot of the activities of identical titrations, one performed in the absence and the other in the presence of a specific inhibitor of the other branch of the respiratory chain, yields a straight line whose slope indicates the engagement of the titrated pathway during uninhibited respiration. An initial slope of zero may occur if electron flux is diverted between pathways during titrations. However, beyond the breakpoint (representing the point of pathway saturation), a straight line is obtained with a slope representing engagement. This technique assumes that the kinetics of inhibiting a specific component of the respiratory chain are independent of the absolute rate of electron flux through the total pathway. To test this assumption, the activity of respiratory pathways in isolated soybean (Glycine max [L]. Merr. cv. Stevens) mitochondria was titrated with specific inhibitors of the cytochrome and alternative oxidases. Under these conditions, the electron flux through a given pathway was manipulated by poising the rate of succinate oxidation with the succinate dehydrogenase inhibitor malonate. Construction of activity plots in the presence versus absence of malonate failed to result in straight lines for either KCN (when titrating the cytochrome pathway) or salicylhydroxamic acid (when titrating the alternative pathway). Rather, the resultant plots were always curvilinear whenever the activity in the presence of malonate divided by the activity in the absence of malonate was less than 1.0. In no case could the real engagement of the pathway be precisely estimated from the titration data. Titrations of cytochrome pathway activity in isolated potato tuber (Solanum tuberosum L. cv. Sabago and Canabex) mitochondria (which lack the alternative oxidase) showed that as the inhibitor concentration was increased, so did the reduction status of the ubiquinone pool, to a new steady state. The dependence of inhibition kinetics on the rate of flux through the pathway, and the increase in ubiquinone pool reduction upon KCN addition, are explained in terms of the elasticity of component enzymes as outlined in the theory of metabolic control analysis. The implications of this finding for the use of titrations to estimate engagement of plant respiratory pathways are discussed.     

The alternative oxidase in roots of poa annua after transfer from high-light to low-light conditions

The activity of the alternative pathway can be affected by a number of factors, including the amount and reduction state of the alternative oxidase protein, and the reduction state of the ubiquinone pool. To investigate the importance of these factors in vivo, we manipulated the rate of root respiration by transferring the annual grass Poa annua L. from high-light to low-light conditions, and at the same time from long-day to short-day conditions for four days. As a result of the low-light treatment, the total respiration rate of the roots decreased by 45%, in vitro cytochrome c oxidase capacity decreased by 49%, sugar concentration decreased by 90% and the ubiquinone concentration increased by 31%, relative to control values. The absolute rate of oxygen uptake via the alternative pathway, as determined using the 18O-isotope fractionation technique, did not change. Conversely, the cytochrome pathway activity decreased during the low-light treatment; its activity increased upon addition of exogenous sugars to the roots. Interestingly, no change was observed in the concentration of the alternative oxidase protein or in the reduction state of the protein. Also, there was no change in the reduction state of the ubiquinone pool. In conclusion, the concentration and activity of the alternative oxidase were not changed, even under severe light deprivation.     

Phytochrome-driven changes in respiratory electron transport partitioning in soybean (Glycine max. L.) cotyledons

After it was observed that light induces changes in electron partitioning between the cytochrome and the alternative pathway, the focus interest was directed to assessing what type of photoreceptors are involved and the extent of such modifications. Studies on 5-day-old soybean (Glycine max L.) cotyledons using an oxygen isotope fractionation technique showed that phytochrome is involved in changes in electron partitioning between the cytochrome and the alternative respiratory pathway. A follow-up of a previous study, showing that 5 min of white light caused changes in mitochondrial electron partitioning, demonstrated that while blue light was not involved in any such changes, red light caused a significant shift of electrons toward the alternative pathway. The major shift, observed after 24 h of light, is mainly due to both a decrease in the activity of the cytochrome pathway and an increase in the activity of the alternative pathway. The involvement of a phytochrome receptor was confirmed by demonstration of reversibility by far-red light. The implications of the possible involvement of phytochrome in the regulation of mitochondrial electron transport are 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.     

In situ quantification of mitochondrial respiration in permeabilized fibers of a marine invertebrate with low aerobic capacity

The aim of this study was to design a protocol to allow the assessment of normal and alternative pathways for electron transport in mitochondria using an in situ approach (on permeabilized fibers) in high-resolution respirometry. We measured the oxygen consumption of permeabilized fibers from Nereis (Neanthes) virens with different substrates and the presence of ADP. To estimate the alternative oxidase (AOX) activity, antimycin A was introduced in order to inhibit complex III. Moreover, the apparent complex IV (COX) excess capacity was evaluated using different substrates to assess the implication of this complex in the partitioning of electrons during its progressive inhibition. Our in situ method enabled to quantify the activity of the normal COX pathway as well as the AOX pathway when different substrates were oxidized by either complex I, complex II or both. Using this approach, we confirmed that according to the substrates used, each pathway has a different role and consequently is otherwise involved in the partitioning of electrons through the electron transport system, and suggested that the AOX activity is triggered not only by the redox state of the cell but also by the type of substrates provided to mitochondria.     

Inhibition of electron transfer from ferrocytochrome b to ubiquinone, cytochrome c1 and duroquinone by antimycin.

The effect of antimycin on (i) the respiratory activity of the KCN-insensitive pathway of mitochondria of Neurospora grown on chloramphenicol (chloramphenicol-grown) with durohydroquinone and succinate or NADH as substrate, (ii) the electron transfer from the b-type cytochromes to ubiquinone with durohydroquinone as electron donor as well as (iii) the electron transfer from the b-type cytochromes to duroquinone with succinate as electron donor in chloramphenicol-grown Neurospora and beef heart submitochondrial particles was studied. All experiments were performed in the uncoupled state. 1. The respiratory chain of chloramphenicol-grown Neurospora mitochondria branches at ubiquinone into two pathways. Besides the cytochrome oxidase-dependent pathway, a KCN-insensitive branch equiped with a salicylhydroxamate-sensitive oxidase exists. Durohydroquinone, succinate or NADH are oxidized via both pathways. The durohydroquinone oxidation via the KCN-insensitive pathway is inhibited by antimycin, wheras the succinate or NADH oxidation is not. The titer for ful inhibition is one mol antimycin per mol cytochrome b-563 or cytochrome b-557. 2. The electron transfer from durohydroquinone to ubiquinone, which takes place in the KCN-inhibited state, does not occur in the antimycin-inhibited state. 3. The reduction of duroquinone by succinate in the presence of KCN is inhibited by antimycin. The titer for full inhibition is one mol antimycin per mol cytochrome b-566 or cytochrome b-562 for beef heart (or cytochrome b-563 or cytochrome b-557 for Neurospora). 4. When electron transfer from the b-type cytochromes to cytochrome C1, ubiquinone and duroquinone is inhibited by antimycin, the hemes of cytochrome b-566 and cytochrome b-562 (or cytochrome b-563 and cytochrome b-557) are in the reduced state. 5. The experimental results suggest that the two b-type cytochromes form a binary complex the electron transferring activity of which is inhibited by antimycin, the titer for full inhibition being one mol of antimycin per mol of complex. The electron transfer from the b-type cytochromes to ubiquinone is inhibited in a non-linear fashion.     

Measurements of the Engagement of Cyanide-Resistant Respiration in the Crassulacean Acid Metabolism Plant Kalanchoë daigremontiana with the Use of On-Line Oxygen Isotope Discrimination

Discrimination against ¹⁸O during dark respiration in tissues of Kalanchoë daigremontiana, Medicago sativa, and Glycine max was measured using an on-line system that enabled direct measurements of the oxygen fractionation of samples in a gas-phase leaf disk electrode unit. Discrimination factors for cytochrome pathway respiration were 18.6 to 19.8%_o for all tissues. However, discrimination in cyanide-resistant respiration was significantly higher in green tissues (30.4-31.2%_o) compared with nongreen tissues (25.3-25.9%_o). Using these discrimination factors, the partitioning of electron transport to these pathways was calculated from measurements of discrimination in the absence of inhibitors. Changes in flux through the alternative pathway were measured during the light and dark phases of Crassulacean acid metabolism in leaf disks of K. daigremontiana. The flux of electrons through the alternative pathway was higher during deacidification than during the other phases of Crassulacean acid metabolism. The increase in alternative pathway electron flux accounted for all of the increased respiration in the light phase. Despite this increase, simultaneous measurements of malate concentration and respiratory flux confirm that only a small proportion of the total malate decarboxylation occurs in the mitochondria.     

Characterization of the mitochondrial respiratory pathways in Candida albicans

Candida albicans is an opportunistic oral pathogen. The flexibility of this microorganism in response to environmental changes includes the expression of a cyanide-resistant alternative respiratory pathway. In the present study, we characterized both conventional and alternative respiratory pathways and determined their ADP/O ratios, inhibitor sensitivity profiles and the impact of the utilization of either pathway on susceptibility to commonly used antimycotics. Oxygen consumption by isolated mitochondria using NADH or malate/pyruvate as respiratory substrates indicated that C. albicans cells express both cytoplasmic and matrix NADH-ubiquinone oxidoreductase activities. The ADP/O ratio was higher for malate/pyruvate (2.2±0.1), which generate NADH in the matrix, than for externally added NADH (1.4±0.2). In addition, malate/pyruvate respiration was rotenone-sensitive, and an enzyme activity assay further confirmed that C. albicans cells express Complex I activity. Cells grown in the presence of antimycin A expressed the cyanide-insensitive respiratory pathway. Determination of the respiratory control ratio (RCR) and ADP/O ratios of mitochondria from these cells indicated that electron transport from ubiquinone to oxygen via the alternative respiratory pathway was not coupled to ATP production; however, an ADP/O ratio of 0.8 was found for substrates that donate electrons at Complex I. Comparison of antifungal susceptibility of C. albicans cells respiring via the conventional or alternative respiratory pathways showed that respiration via the alternative pathway does not reduce the susceptibility of cells to a series of clinically employed antimycotics (using Fungitest®), or to the naturally occurring human salivary antifungal peptide, histatin 5.     

The Role of the Alternative Oxidase in Stabilizing the in Vivo Reduction State of the Ubiquinone Pool and the Activation State of the Alternative Oxidase

A possible function for the alternative (nonphosphorylating) pathway is to stabilize the reduction state of the ubiquinone pool (Q_r/Q_t), thereby avoiding an increase in free radical production. If the Q_r/Q_t were stabilized by the alternative pathway, then Q_r/Q_t should be less stable when the alternative pathway is blocked. Q_r/Q_t increased when we exposed roots of Poa annua (L.) to increasing concentrations of KCN (an inhibitor of the cytochrome pathway). However, when salicylhydroxamic acid, an inhibitor of the alternative pathway, was added at the same time, Q_r/Q_t increased significantly more. Therefore, we conclude that the alternative pathway stabilizes Q_r/Q_t. Salicylhydroxamic acid increasingly inhibited respiration with increasing concentrations of KCN. In the experiments described here the alternative oxidase protein was invariably in its reduced (high-activity) state. Therefore, changes in the reduction state of the alternative oxidase cannot account for an increase in activity of the alternative pathway upon titration with KCN. The pyruvate concentration in intact roots increased only after the alternative pathway was blocked or the cytochrome pathway was severely inhibited. The significance of the pyruvate concentration and Q_r/Q_t on the activity of the alternative pathway in intact roots is discussed.     

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.     

The mechanism of mitochondrial superoxide production by the cytochrome bc1 complex

Production of reactive oxygen species (ROS) by the mitochondrial respiratory chain is considered to be one of the major causes of degenerative processes associated with oxidative stress. Mitochondrial ROS has also been shown to be involved in cellular signaling. It is generally assumed that ubisemiquinone formed at the ubiquinol oxidation center of the cytochrome bc(1) complex is one of two sources of electrons for superoxide formation in mitochondria. Here we show that superoxide formation at the ubiquinol oxidation center of the membrane-bound or purified cytochrome bc(1) complex is stimulated by the presence of oxidized ubiquinone indicating that in a reverse reaction the electron is transferred onto oxygen from reduced cytochrome b(L) via ubiquinone rather than during the forward ubiquinone cycle reaction. In fact, from mechanistic studies it seems unlikely that during normal catalysis the ubisemiquinone intermediate reaches significant occupancies at the ubiquinol oxidation site. We conclude that cytochrome bc(1) complex-linked ROS production is primarily promoted by a partially oxidized rather than by a fully reduced ubiquinone pool. The resulting mechanism of ROS production offers a straightforward explanation of how the redox state of the ubiquinone pool could play a central role in mitochondrial redox signaling.     

Substrate kinetics of the Acanthamoeba castellanii alternative oxidase and the effects of GMP

In Acanthamoeba castellanii mitochondria, the apparent affinity values of alternative oxidase for oxygen were much lower than those for cytochrome c oxidase. For unstimulated alternative oxidase, the K(Mox) values were around 4-5 microM both in mitochondria oxidizing 1 mM external NADH or 10 mM succinate. For alternative oxidase fully stimulated by 1 mM GMP, the KK(Mox) values were markedly different when compared to those in the absence of GMP and they varied when different respiratory substrates were oxidized (K(Mox) was around 1.2 microM for succinate and around 11 microM for NADH). Thus, with succinate as a reducing substrate, the activation of alternative oxidase (with GMP) resulted in the oxidation of the ubiquinone pool, and a corresponding decrease in K(Mox). However, when external NADH was oxidized, the ubiquinone pool was further reduced (albeit slightly) with alternative oxidase activation, and the K(Mox) increased dramatically. Thus, the apparent affinity of alternative oxidase for oxygen decreased when the ubiquinone reduction level increased either by changing the activator or the respiratory substrate availability.     

In situ quantification of mitochondrial respiration in permeabilized fibers of a marine invertebrate with low aerobic capacity

The aim of this study was to design a protocol to allow the assessment of normal and alternative pathways for electron transport in mitochondria using an in situ approach (on permeabilized fibers) in high-resolution respirometry. We measured the oxygen consumption of permeabilized fibers from Nereis (Neanthes) virens with different substrates and the presence of ADP. To estimate the alternative oxidase (AOX) activity, antimycin A was introduced in order to inhibit complex III. Moreover, the apparent complex IV (COX) excess capacity was evaluated using different substrates to assess the implication of this complex in the partitioning of electrons during its progressive inhibition. Our in situ method enabled to quantify the activity of the normal COX pathway as well as the AOX pathway when different substrates were oxidized by either complex I, complex II or both. Using this approach, we confirmed that according to the substrates used, each pathway has a different role and consequently is otherwise involved in the partitioning of electrons through the electron transport system, and suggested that the AOX activity is triggered not only by the redox state of the cell but also by the type of substrates provided to mitochondria.     

The electron partitioning between the cytochrome and alternative respiratory pathways during chilling recovery in two cultivars of maize differing in chilling sensitivity

Chilling effects on respiration during the recovery period were studied in two maize (Zea mays L.) cultivars differing in their tolerance to chilling: Penjalinan, a chilling-sensitive cultivar, and Z7, a chilling-tolerant cultivar. Both cultivars were exposed to 5 degrees C for 5 d, after which measurements were taken at 25 degrees C. Chlorophyll fluorescence analysis in dark-adapted leaves showed less damage in cv Z7 than in cv Penjalinan during recovery from the chilling treatment. Studies of the electron partitioning between the cytochrome and the alternative respiratory pathways during chilling recovery using the oxygen isotope fractionation technique showed that, although total leaf respiration was not affected by the chilling treatment in either of the two cultivars, electron partitioning to the alternative pathway was significantly increased in the more stressed chilling-sensitive cv Penjalinan, suggesting that increased activity of the alternative pathway is not related to the plant tolerance to chilling. These results suggest a possible role of the alternative pathway in plants under stress rather than specifically contributing to plant resistance to chilling.     

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.     

The effect of pH on the alternative oxidase activity in isolated Acanthamoeba castellanii mitochondria

Mitochondria of Acanthamoeba castellanii possess a cyanide-resistant GMP-stimulated ubiquinol alternative oxidase in addition to the cytochrome pathway. In a previous work it has been observed that an interaction between the two ubiquinol-oxidizing pathways exists in intact A. castellanii mitochondria and that this interaction may be due to a high sensitivity of the alternative oxidase to matrix pH. In this study we have shown that the alternative oxidase activity reveals a pH-dependence with a pH optimum at 6.8 whatever the reducing substrate may be. The GMP stimulation of alternative oxidase is also strongly dependent on pH implicating probably protonation/deprotonation processes at the level of ligand and protein with an optimum pH at 6.8. The ubiquinone redox state-dependence of alternative oxidase activity is modified by pH in such a way that the highest activity for a given ubiquinone redox state is observed at pH 6.8. Thus pH, binding of GMP, and redox state of ubiquinone collaborate to set the activity of the GMP-stimulated alternative oxidase in isolated A. castellanii mitochondria. The high pH sensitivity of the alternative oxidase could link inactivation of the cytochrome pathway proton pumps to activation of the alternative oxidase with acceleration of redox free energy dissipation as a consequence.     

The alternative respiration pathway in plants: Role and regulation

In the past few years, knowledge of the nature and regulation of the alternative oxidase in plant mitochondria has increased greatly. The protein has been characterized and mechanisms that regulate its activity have been described. The consequences of these regulatory mechanisms are that in vivo the cytochrome pathway and the alternative pathway may compete for electrons. The implications for the interpretation of the 'Bahr and Bonner' inhibitor titrations, formerly used to estimate the partitioning of electrons over the two pathways, are discussed.
It is proposed that activation and engagement of the alternative oxidase may keep Q reduction levels low in order to prevent harmful high levels of free radical production. A model is presented for the regulation of alternative oxidase protein induction, involving a signalling function of active oxygen species.