Regulation of succinate dehydrogenase was investigated using tightly coupled potato tuber mitochondria in a novel fashion by simultaneously measuring the oxygen uptake rate and the ubiquinone (Q) reduction level. We found that the activation level of the enzyme is unambiguously reflected by the kinetic dependence of the succinate oxidation rate upon the Q-redox poise. Kinetic results indicated that succinate dehydrogenase is activated by both ATP (K ½ ϳ 3 M) and ADP. The carboxyatractyloside insensitivity of these stimulatory effects indicated that they occur at the cytoplasmic side of the mitochondrial inner membrane. Importantly, our novel approach revealed that the enzyme is also activated by oligomycin (K ½ ϳ 16 nM). Time-resolved kinetic measurements of succinate dehydrogenase activation by succinate furthermore revealed that the activity of the enzyme is negatively affected by potassium. The succinate-induced activation (؎K ؉ ) is prevented by the presence of an uncoupler. Together these results demonstrate that in vitro activity of succinate dehydrogenase is modulated by the protonmotive force. We speculate that the widely recognized activation of the enzyme by adenine nucleotides in plants is mediated in this manner. A mechanism that could account for such regulation is suggested and ramifications for its in vivo relevance are discussed.Succinate dehydrogenase is a membrane-bound component of the respiratory chain of aerobic organisms (1). It couples the reduction of ubiquinone (Q) 1 to the oxidation of succinate and is, as such, a Krebs cycle as well as a respiratory chain enzyme. This duality suggests a potentially important role in the control of energy metabolism, a reason why the enzyme's regulation has been the subject of previous extensive investigations in both mammalian (2, 3) and plant (4, 5) systems.The isolation of succinate dehydrogenase from mammalian sources yields a predominantly deactivated enzyme due to the presence of tightly bound oxaloacetate (6, 7). The isolated enzyme, however, can be activated by substrates and substrate analogues (8) as well as by many anions, acid pH (9, 10), reducing treatments, (11) and QH 2 (2, 12). Additionally, succinate dehydrogenase is activated by ATP (3) via a mechanism that, even to date, is poorly understood. In the presence of ADP or an uncoupler, the mammalian enzyme is rapidly deactivated (3). Gutman et al. (3) attributed physiological relevance to these observations reasoning that deactivation of succinate dehydrogenase would benefit the cell when the ATP:ADP ratio is low, because it would allow faster NADH oxidation and hence more efficient ATP synthesis. The subsequently formed ATP would then activate the enzyme to allow turnover of the Krebs cycle to proceed in an unhindered fashion (3).In plants, succinate dehydrogenase is activated by substrates, QH 2 , ATP, anions, and acid pH in a manner similar to that observed in mammalian systems (4, 5). The effect of ADP on the plant enzyme, however, has not been established conclusively. Oestreicher...

The dependence of the rate of oxygen uptake upon the ubiquinone (Q)-pool reduction level in mitochondria isolated during the development of thermogenesis of Arum maculatum spadices has been investigated. At the alpha-stage of development, the respiratory rate was linearly dependent upon the reduction level of the Q-pool (Qr) both under state-3 and -4 conditions. Progression through the beta/gamma to the delta-stage resulted in a non-linear dependence of the state-4 rate on Qr. In the delta-stage of development, both state-3 and -4 respiratory rates were linearly dependent upon Qr due to a shift in the engagement of the alternative oxidase to lower levels of Qr. Western blot analysis revealed that increased alternative oxidase activity could be correlated with expression of a 35 kDa protein. Respiratory control was only observed with mitochondria in the alpha-stage of development. At the beta/gamma-stage of development, the addition of ADP resulted in a significant oxidation of the Q-pool which was accompanied by a decrease in the respiratory rate. This was due either to decreased contribution of the alternative pathway to the overall respiratory rate under state 3 or by deactivation of succinate dehydrogenase activity by ADP. Cold-storage of the spadices at the beta-stage of development led to increased activity of both the cytochrome pathway and succinate dehydrogenase, without any change in alternative oxidase activity. Results are discussed in terms of how changes in the activation level of the alternative oxidase and succinate dehydrogenase influence the activity and engagement of the quinol-oxidizing pathways during the development of thermogenesis in A. maculatum.

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The relationship between the respiratory rate and the membrane ionic current on the protonmotive force has been investigated in percoll purified potato mitochondria. The dependence of the membrane ionic current on the membrane potential was monitored using a methyltriphenylphosphonium-sensitive electrode and determining the maximal net rate of depolarization following the addition of a respiratory inhibitor. We have confirmed that a nonohmic relationship exists between the ionic conductance and membrane potential. Addition of ATPase inhibitors markedly increased the initial rate of dissipation suggesting that in their absence the dissipation rate induced by respiratory inhibitors is partially offset by H+-efflux due to the hydrolysis of endogenous ATP. This was corroborated by direct measurement of endogenous ATP levels which decreased significantly following dissipation of the membrane potential. Results are discussed in terms of the regulation of electron transport in plant mitochondna in vivo.It is generally accepted that the free energy which is liberated from the redox reactions catalyzed by the respiratory chain is conserved as a proton electrochemical gradient across the inner mitochondrial membrane (2). The magnitude of this force has been shown in a number of systems to be unrelated to the rate of electron flux since the rate of respiration can be substantially inhibited by malonate or antimycin resulting in only a minor depression of the protonmotive force (6,8,21,24,27). This has been variously attributed to the nonohmic behavior of the inner membrane (5,15,27), to heterogeneity ofthe coupling of the mitochondria (11), to localized protonic coupling (29), and to variations in the stoichiometry of the redox driven proton pumps (redox slippage) (31). Estimates of the passive conductance of the inner membrane to protons in rat liver mitochondria (5,15) of respiration under ADP limited conditions (9) which has been attributed either to the activity of an endogenous H+/ K+ antiporter (8, 13) and/or to the operation of nonphosphorylative pathways (22). It has been suggested that nonphosphorylative pathways such as the alternative and rotenone-insensitive pathways constitute a natural slippage (or energy overflow) pathway in plant mitochondria (10, 16) (since they bypass the proton-pumping sites) and, as such, modulation of proton conductance may not be as important as in other tissues.In contrast to this suggestion there is some evidence in the literature to suggest that proton conductance does regulate respiratory activity. For instance, in isolated mitochondria it has been found that the state 4 rate is generally much slower than the rate of respiration prior to the initial addition of ADP (7) and, furthermore, that the decreased respiratory rate is associated with an increased membrane potential (7,22). These differences have been attributed to the build-up of endogenous ATP and decreased H+ conductance through the ATPase (7). However, the mechanism whereby this results in a decreased respirator...

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