Reactive Oxygen Species Production by Potato Tuber Mitochondria Is Modulated by Mitochondrially Bound Hexokinase Activity

Potato tuber (Solanum tuberosum) mitochondria (PTM) have a mitochondrially bound hexokinase (HK) activity that exhibits a pronounced sensitivity to ADP inhibition. Here we investigated the role of mitochondrial HK activity in PTM reactive oxygen species generation. Mitochondrial HK has a 10-fold higher affinity for glucose (Glc) than for fructose (K_MGlc = 140 μm versus K_MFrc = 1,375 μm). Activation of PTM respiration by succinate led to an increase in hydrogen peroxide (H₂O₂) release that was abrogated by mitochondrial HK activation. Mitochondrial HK activity caused a decrease in the mitochondrial membrane potential and an increase in oxygen consumption by PTM. Inhibition of Glc phosphorylation by mannoheptulose or GlcNAc induced a rapid increase in H₂O₂ release. The blockage of H₂O₂ release sustained by Glc was reverted by oligomycin and atractyloside, indicating that ADP recycles through the adenine nucleotide translocator and F₀F₁ATP synthase is operative during the mitochondrial HK reaction. Inhibition of mitochondrial HK activity by 60% to 70% caused an increase of 50% in the maximal rate of H₂O₂ release. Inhibition in H₂O₂ release by mitochondrial HK activity was comparable to, or even more potent, than that observed for StUCP (S. tuberosum uncoupling protein) activity. The inhibition of H₂O₂ release in PTM was two orders of magnitude more selective for the ADP produced from the mitochondrial HK reaction than for that derived from soluble yeast (Saccharomyces cerevisiae) HK. Modulation of H₂O₂ release and oxygen consumption by Glc and mitochondrial HK inhibitors in potato tuber slices shows that hexoses and mitochondrial HK may act as a potent preventive antioxidant mechanism in potato tubers.Production of reactive oxygen species (ROS) is an unavoidable consequence of aerobic respiration (Chance et al., 1979). The mitochondrial electron transport system (ETS) is the major site of ROS production in mammalian and nonphotosynthesizing plant cells (Puntarulo et al., 1991; Halliwell and Gutteridge, 2007). Depending on the mitochondrial respiratory states, a small portion of the consumable oxygen is partially reduced to generate ROS (Skulachev, 1996; Liu, 1997; Turrens, 1997; Møller, 2001; Considine et al., 2003; Smith et al., 2004). In plants, the monoelectronic reduction of oxygen by ETS leads to the production of superoxide radicals (O₂^·−) that can be dismutated by SOD, producing hydrogen peroxide (H₂O₂), and further decomposed by catalase and/or ascorbate-glutathione peroxidase cycles (Møller, 2001). An imbalance between the ROS production and antioxidant defenses can lead to an oxidative stress condition. Increased levels of ROS may be a consequence of the action of plant hormones, environmental stress, pathogens, or high levels of sugars and fatty acids (Bolwell et al., 2002; Couée et al., 2006; Gechev et al., 2006; Liu et al., 2007; Rhoads and Subbaiah, 2007). These conditions may lead to storage deterioration or impairment of seedling growth decreasing on crop yield. To avoid the harmful accumulation of ROS or to fine tune the steady-state levels of ROS, various enzymatic systems control the rate of ROS production in mitochondria (Schreck and Baeuerle, 1991; Møller, 2001).Mitochondrial ROS production is highly dependent on the membrane potential (ΔΨ_m) generated by the proton gradient formed across the inner mitochondrial membrane. High ΔΨ_m was shown to stimulate ROS production when the ETS is predominantly in a reduced state (i.e. when NADH, FADH₂, and O₂ are present in abundance but ADP or Pi levels are low). This condition is reached in resting metabolic states after a full oxidation of Glc or fatty acids. Stimulating electron flow by decreasing ΔΨ_m, either by the use of uncouplers or by coupling respiration to ATP synthesis, slows the ROS generation rate (Boveris and Chance, 1973; Korshunov et al., 1997). It has been observed that in isolated potato tuber (Solanum tuberosum) mitochondria (PTM) the uncoupling protein (referred to as PUMP in plants, or UCP in animals) causes a small decrease in ΔΨ_m when this proton carrier protein is activated by the presence of anionic fatty acids, a condition that blocks ROS generation (Vercesi et al., 1995, 2006). Nucleotides, such as ATP, antagonize this effect (Considine et al., 2003; Vercesi et al., 2006). On the other hand, fluctuations in free hexose levels due to environmental or developmental conditions (Morrell and ap Rees, 1986; Geigenberger and Stitt, 1993; Renz and Stitt, 1993) lead to variations in the oxygen consumption rate in heterotrophic tissues of plant (Brouquisse et al., 1991; Dieuaide et al., 1992). As a result, ROS-producing pathways may be either stimulated or repressed (Couée et al., 2006). Unlike PUMP activity, which is activated by an excess of free fatty acids, a specific mechanism for mitochondrial ROS production caused by an excess of hexose remains elusive.The metabolism of free hexoses begins by their phosphorylation in a reaction catalyzed by the hexokinase (HK):HK is a ubiquitous enzyme found in many organisms. In plants, the binding mechanism of HK to the outer mitochondrial membrane is not fully established, but some reports indicate that it may differ considerably from those properties described for mammal cells (Dry et al., 1983; Miernyk and Dennis, 1983; Rezende et al., 2006). It has been shown that in several mature and developing plant tissues, multiple HK isoforms are expressed with different kinetic properties and subcellular localizations. The HKs are found in cytosol, bound to the mitochondrial membrane, or in stroma of plastids in plant cells (Miernyk and Dennis, 1983; Galina et al., 1995; Damari-Weissler et al., 2007). Beyond its obvious role in glycolysis regulation, HK activity may also function as a sugar sensor, triggering a signal transduction pathway in plants (Rolland et al., 2006).In mammals, HK types I and II are associated with the mitochondrial outer membrane through the voltage-dependent anion channel (VDAC) and adenine nucleotide transporter (ANT). These associations were found in tissues with a high energy demand, such as heart, brain, and tumor cells (Arora and Pedersen, 1988; BeltrandelRio and Wilson, 1992; Wilson, 2003). In addition, recent evidence in mammalian cells has shown that binding of HK to VDAC located at the outer mitochondrial membrane is somehow involved in the protection against proapoptotic stimuli (Nakashima et al., 1986; Gottlob et al., 2001; Vander Heiden et al., 2001; Pastorino et al., 2002; Cesar and Wilson, 2004). Similar observations were reported for tobacco (Nicotiana tabacum) plant mitochondrial HK (mt-HK; Kim et al., 2006). However, it has been shown that drugs such as the fungicide clotrimazole and the anesthetic thiopental, which promptly disrupt the association between mt-HK and VDAC in mammalian mitochondria, are unable to promote this effect in maize (Zea mays) root mitochondria (Rezende et al., 2006). These observations suggest a different type of association of mt-HK with plant mitochondria. The binding of mt-HK with mitochondria in many plants involves a common N-terminal hydrophobic membrane anchor domain of about 24 amino acids that is related to the membrane targeting, but the exact mechanism of association is unknown (Damari-Weissler et al., 2007).Recently, our group demonstrated that mt-HK activity plays a key preventive antioxidant role by reducing mitochondrial ROS generation through a steady-state ADP recycling mechanism in rat brain neurons. The mitochondrial ADP recycling leads to a decrease in the ΔΨ_m coupled to the synthesis of ATP by oxidative phosphorylation (da-Silva et al., 2004; Meyer et al., 2006).Although plant HK is recognized to fulfill a catalytic function, the role of mt-HK activity in the regulation of both mitochondrial respiration and ROS production in plants is unknown. Recently, an authentic HK activity was detected in PTM (Graham et al., 2007) and its involvement in potato tuber glycolysis suggested, but its involvement in PTM ROS generation was not explored. We then raise the hypothesis that HK bound to PTM would contribute to produce a steady-state ADP recycling that regulates ROS formation. However, whether this association is capable of controlling the rate of ROS generation in plant mitochondria is unknown. Here, we aim to investigate the role of mt-HK activity in PTM physiology. The data indicate that mt-HK activity plays a key role as a regulator of ROS levels in respiring plant tissues exposed to high hexose levels.