Localisation of the Sites of Action of Cadmium on Oxidative Phosphorylation in Potato Tuber Mitochondria Using Top‐Down Elasticity Analysis

Abstract

The aim of this study was to identify the significant sites of action of cadmium on oxidative phosphorylation in potato tuber mitocondria. We simplified the system to three convenient subsystems linked via the production or consumption of a common intermediate, namely protonmotive force. The three subsystems were substrate oxidation, which produces protonmotive force, and the proton leak reactions and the phosphorylation reactions, which consume protonmotive force. By measuring the effect of cadmium on the kinetic response of each subsystem to protonmotive force (top-down elasticity analysis), we found that cadmium stimulated proton leak reactions and strongly inhibited substrate oxidation, but had no measurable effect on the phosphorylation reactions. Cadmium therefore decreases the amount of ATP produced/oxygen consumed (the effective P/O ratio) not by inhibiting the phosphorylation reactions directly, but by inhibiting the production of protonmotive force and by diverting proton flux from phosphorylation reactions to the proton leak reactions.The toxicity of heavy metals on aquatic and terrestrial organisms has become a major topic of toxicological research over recent decades, in particular because heavy metal concentrations have considerably increased in the environment, predominantly due to accelerated industrial contamination and air pollution [l, 21. The various heavy metals show similar characteristics. They are easily taken up into living organisms, where they accumulate in several tissues [3-61, due to a long biological half-life [7]. Once incorporated, heavy metals cause a variety of cytotoxic reactions within living cells [3-71 and affect essential metabolic pathways in plant cells [l, 6, 81. In plants, the overall result of the heavy metal impact is a decrease in growth rate and finally death [9]. This wide range of toxicity is explained by the chemical characteristics of heavy metals ; the most characteristic feature of heavy metals like cadmium, mercury and lead is a unique high affinity for the free electron pairs of SH groups [3-61. SH groups are important in enzyme function, either oxygen consumption by the substrate oxidation reactions) ; J p , rate of oxygen consumption required to pump protons out at the rate equal to the rate of proton return through the phosphorylation system; JL, rate of oxygen consumption required to pump protons out at a rate equal to the rate of proton return through the proton leak; E+ overall elasticity coefficient of a block of reactions to the common intermediate dp; the subscripts and superscripts S, P and L refer to the following three blocks of reactions: the substrate oxidation, the phosphorylation system and the proton leak branch, respectively ; Ph,MeP+, methyltriphenylphosphonium cation.Enzyme. Hexokinase; type 111: from baker's yeast (EC 2.7.1.1.).by determining structure and conformation or by direct involvement in the catalysed metabolic reaction in the active centre of the enzyme. Therefore heavy metals, as SH group reagents, affect most metabolic pathw...

Top-down control analysis of the cadmium effects on molluscan mitochondria and the mechanisms of cadmium-induced mitochondrial dysfunction

“…In contrast, the phosphorylation subsystem was not affected by Cd exposure. Earlier research conducted in potato tuber mitochondria also yielded similar results, showing suppressed substrate oxidation and stimulated proton leak but no effects on phosphorylation subsystem (37,41,42). This similarity of mitochondrial responses to Cd between oysters and plants suggests that the sites of Cd action in mitochondria are evolutionarily conserved and that the high sensitivity of substrate oxidation and proton leak subsystems to Cd effects can be expected in other animals.…”

“…Thus, in the physiological range of membrane potentials (170 -180 mV), only about 13-16% of respiration of phosphorylating control mitochondria is due to the futile proton leak, whereas during exposure to 12.5 M Cd 2ϩ this proportion rises to 35-55%. A similar decrease in phosphorylation efficiency in response to Cd was observed in potato tuber mitochondria (41), suggesting that this may be another common mechanism of Cd effects on mitochondrial bioenergetics. Surprisingly, the phosphorylation subsystem was not affected by Cd in oyster mitochondria under the assay conditions used in this study.…”

“…Although the top-down control analysis gives a relatively low-resolution picture of mitochondrial function, it provides a quantitative insight into the overall control structure of oxidative phosphorylation, pinpoints critical functions of mitochondrial bioenergetics that are most sensitive to Cd and focuses future searches for molecular targets of Cd. The top-down control analysis has been previously used to identify mitochondrial sites of Cd action in plants (37,(41)(42)(43); however, to our knowledge, it has never been used to investigate Cd effects in animal mitochondria.…”

Effects of temperature and cadmium exposure on the mitochondria of oysters (Crassostrea virginica) exposed to hypoxia and subsequent reoxygenation

“…In the absence of Cd or temperature stress, the substrate oxidation subsystem conferred the greatest degree of control over the state 3 respiration. The same trend is also found in insects (Chamberlin, 2004a;, mammalian liver (Brown et al, 2007;Ciapaite et al, 2009) and plant mitochondria (Kesseler and Brand, 1994b;Kesseler et al, 1992). In marked contrast, control over state 3 respiration of mammalian muscle mitochondria is shared between the substrate oxidation and phosphorylation subsystems (34-44% and 51-54%, respectively) with a small contribution (7-8%) of the proton leak (Lombardi et al, 2000).…”

“…The phosphorylation subsystem (including F O , F 1 -ATPase, adenylate and inorganic phosphate transporters) uses ∆p to synthesize ATP, and the proton leak subsystem involves all futile cation cycles that dissipate ∆p without ATP production. Proton leak reactions reflect inherent inefficiency of mitochondria, but can also fulfill important physiological functions such as control of ROS production (Brand, 2000;Miwa and Brand, 2003;Rofle and Brand, 1997 (Chamberlin, 2004b;Dufour et al, 1996), Cd exposure (Kesseler and Brand, 1994a;Kesseler and Brand, 1994b;Kesseler and Brand, 1994c;Kesseler and Brand, 1995;Kurochkin et al, 2011) and developmentally induced apoptosis (Chamberlin, 2004a). However, this approach has not been used to explore how rapid changes in oxygen levels affect the mitochondrial function of animals that often experience transient environmental hypoxia.…”