New insights into the regulation of plant succinate dehydrogenase. On the role of the protonmotive force.

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(1/2) approximately 3 microm) 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(1/2) approximately 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.

Maesaquinone: a novel inhibitor of plant mitochondrial respiratory enzymes that react with ubiquinone.

The effect of maesaquinone, 2-(14-nonadecenyl)-3,6-dihydroxy-5-methyl-1,4-benzoquinone, on plant mitochondrial respiration has been investigated. In mitochondria isolated from thermogenic Arum maculatum spadices, this compound inhibits both cytochrome and alternative pathway activities. Kinetic analyses reveal that this inhibition is the result of potent effects of maesaquinone on the alternative oxidase (ID50 < 0.3 microM) and complex III (ID50 < 5 microM). Succinate dehydrogenase and external NADH dehydrogenase are also inhibited, albeit to a lesser extent (approximately 30% at 1 microM). These data suggest that maesaquinone specifically affects the interaction of the respective enzymes with ubiquinone.

Kinetic analysis of the mitochondrial quinol-oxidizing enzymes during development of thermogenesis in Arum maculatum L.

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.

Role of nonohmicity in the regulation of electron transport in plant mitochondria.

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 mitochondria in vivo.