2.4.7  An alternative oxidase

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In both plants and animals, cytochrome oxidase is sensitive to a number of inhibitors, the best known of which are carbon monoxide and cyanide. Plants, however, show resistance to both carbon monoxide and cyanide because they are equipped with an alternative oxidase. This enzyme does not translocate H+ and therefore is not linked to ATP formation. The enzyme is a quinol oxidase and appears to consist of one to three polypeptides of about 25–35 kDa, depending on the plant species. The proteins are encoded in nuclear genes which show tissue-specific expression. Cyanide-insensitive O2 uptake is inhibited by hydroxamic acids (SHAM — salicyl hydroxamic acid — is the most commonly used) and n-propylgallate.

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Figure 2.27 Regulation of alternative oxidase. (a) The alternative oxidase exists as a dimer linked together by disulphide bonds. Reduction of these bonds is required to allow activation of the oxidase; this activation occurs by a direct interaction between pyruvate and the oxidase. Oxidation inactivates the oxidase even when pyruvate is present. (b) Putative feed-forward regulation of the alternative oxidase to prevent fermentation from accumulated pyruvate and formation of reactive oxygen intermediates (ROI) frona over-reduced ubiquinone (Q). In vivo, reduction of the oxidase is apparently achieved via NAD(P)H which will accumulate when carbon flux through the TCA cycle is high and/ or the cytochrome chain is inhibited. Small solid arrows indicate activation pathways; dashed arrows indicate potentially deleterious side reactions. (Original drawing courtesy David Day)

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Figure 2.28 Experimental evidence for schemes in Figure 2.27. (a) and (b) represent oxygen electrode recordings obtained with a suspension of mitochondria isolated from tobacco leaves. NADH is added as the electron donor, and ADP ensures that electron transport is not restricted by the rate of oxidative phosphorylation. Addition of KCN inhibits cytochrome oxidase and thus allows alternative oxidase activity to be measured. At times indicated by (1)-(4), samples of mitochondria were taken from the reaction vessel, their proteins separated by SDS-PAGE in the absence of reductant, and alternative oxidase protein (AOX) bands visualised by immunoblotting (shown schematically as inserts). At (1), pyruvate has been added to the mitochondria but the rate of cyanide-sensitive respiration remains low because AOX protein is largely oxidised (the covalently linked dimer is evident at 70 kD in the blot); however, when isocitrate is added at (2) to generate NADPH in the mitochondrial matrix, AOX becomes reduced (major band at 35 kD is seen in the blot) and cyanide—insensitive O2 uptake becomes rapid as AOX is activated. At (3), isocitrate has been added and AOX is reduced, but respiration remains slow because pyruvate is not present to activate the enzyme fully. At (4), the redox status,of AOX does not change, but AOX activity increases dramatically upon pyruvate addition. Thus both a reductant such as NADPH and an activator such as pyruvate must be present before the alternative oxidase is fully engaged. In this case NADPH was generated from isocitrate via NADP-linked isocitrate dehydrogenase in the mitochondrial matrix. (Based on Vanlerberghe et al. 1995)

Activity of the alternative oxidase is regulated by a complicated feed-forward mechanism (Figure 2.27, with experimental evidence in Figure 2.28). The oxidase exists in mitochondria as a dimer which can be inactivated by covalent linkage via disulphide bonds. Activation of the enzyme involves reduction of that bond, probably via matrix NADPH in a thioredoxin-mediated reaction. The reduced (but not the oxidised) enzyme is stimulated allosterically by pyruvate and some other 2-oxo acids (such as glyoxylate), which interact directly with the oxidase.

This activation by pyruvate was discovered almost by accident during collaborative research involving Harvey Millar and David Day (Australian National University) and Joe Wiskich (University of Adelaide). It began when Jim Siedow (Duke University, USA) emailed the ANU group to say that he could not repeat their published results showing that soybean root mitochondria have alternative oxidase activity. As became apparent, this difference was due to substrate. Harvey Millar (a fourth year honours student at the time) had been using a mixture of malate plus pyruvate as TCA cycle substrates for his isolated mitochondria, whereas Jim Siedow was using succinate. Harvey and David had gone to Adelaide to use the so-called ‘Q’ electrode of Joe Wiskich to investigate apparent differences between substrates in more detail. However, they could not repeat their results from Canberra. Much to their consternation, soybean root mitochondria in Adelaide were completely sensitive to cyanide. However, the Wiskich group routinely used a mixture of malate plus glutamate to drive the TCA cycle, and when pyruvate was added to the reaction vessel, mitochondrial preparations showed a dramatic stimulation of respiration despite the presence of cyanide. Significantly, the level of ubiquinol remained unchanged, implying that this effect was not simply due to oxidation of added pyruvate. A few frantic weeks of experimentation followed, resulting in a publication by Millar et al. (1993) on organic acid activation of the alternative oxidase of plant mitochondria, and illustrating the value of global communication between research groups.

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Figure 2.29 Electron flux via either cytochrome oxidase (Cyt ox) or the alternative oxidase (Alt ox) varies according to the oxidation/reduction status of the quinone pool (Q). At low levels of reduced Q (i.e. a QH2/Q ratio of 0.25 or thereabouts) electron flow via Cyt ox is near capacity, whereas Alt ox contributes little to the overall flux of electrons to molecular oxygen, and thus production of H20. The Alt ox pathway becomes progressively engaged at higher values of the QH2/ Q ratio, and the extent of that engagement is greatly enhanced by pyruvate due to direct activation ofthe Alt ox pathway
(Original drawing courtesy Harvey Millar; derived from data in Hoefhagel et al. 1995 and fitted to the Q pool model of Van den Bergen et al. 1994).

Ubiquinol (Figure 2.23) is substrate for the alternative oxidase whose activity is also governed partly by the degree of reduction of the quinone pool. Where pyruvate is absent, the ratio of Qreduced/Qtotal (QH2/Q ratio) must be very high to activate the oxidase; when pyruvate is present, the alternative oxidase becomes active at a much lower QH2/Q ratio (Figure 2.29) and can compete with the cytochrome chain for electrons, and efficiency of ATP generation diminishes. In Figure 2.29, ubiquinone reduction is presented as the ratio of fully reduced ubiquinol to total ubiquinone (oxidised or reduced). Kinetics of the cytochrome pathway are effectively linear with respect to ubiquinone reduction. Kinetics of the alternative pathway are sigmoidal with significant activity only apparent once the ubiquinone pool is half reduced. When pyruvate is present, alternative pathway kinetics shift to the left, allowing greater electron flux at lower ubiquinone pool reduction levels. Under those circumstances, the alternative pathway will compete with the cytochrome pathway for reduced ubiquinone. Saturation of electron flux via either pathway is not observed in plant mitochondria as the mitochondrial dehydrogenases that provide the driving force for ubiquinone reduction usually become limiting long before either oxidase capacity is saturated. Consequently, the rate of electron flux at the steady-state level of ubiquinone reduction (ordinate in Figure 2.29) will be at the point of intersection between dehydrogenase and oxidase pathway kinetics (open circles in Figure 2.29).

These different controlling factors seem to form part of a regulatory mechanism that ensures that the alternative oxidase is ‘turned on’ when carbon flux through the cell is high or when the cytochrome chain is inhibited (e.g. by high cytosolic ATP/ADP). Under these conditions, pyruvate and reduced pyridine nucleotide levels will be relatively high, ensuring reduction and activation of the oxidase. This mechanism may point to a protective role for the alternative oxidase, preventing accumulation of pyruvate (which may lead to fermentation) and over-reduction of respiratory chain components (which may cause generation of damaging reactive oxygen species such as superoxide ions). Exposure of plants to low temperatures may cause disruption of the cytochrome path (probably via lipid phase changes) and the alternative oxidase may play a role here, since cold treatment stimulates its synthesis. Alternative oxidase synthesis is also induced by other conditions of stress including pathogen attack and ethylene-triggered processes such as fruit ripening, as well as by cytochrome chain inhibitors, all suggesting a protective role.

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