Succinate-driven reverse electron transport in the respiratory chain of plant mitochondria. The effects of rotenone and adenylates in relation to malate and oxaloacetate metabolism

Abstract

The effects of rotenone on the succinate-driven reduction of matrix nicotinamide nucleotides were investigated in Percoll-purified mitochondria from potato (Solanum tuberosum) tubers. Depending on the presence of ADP or ATP, rotenone caused an increase or a decrease in the level of reduction of the matrix nicotinamide nucleotides. The increase in the reduction induced by rotenone in the presence of ADP was linked to the oxidation of the malate resulting from the oxidation of succinate. Depending on the experimental conditions, malic enzyme (at pH 6.6 or in the presence of added CoA) or malate dehydrogenase (at pH 7.9) were involved in this oxidation. At pH 7.9, the oxaloacetate produced progressively inhibited the succinate dehydrogenase. In the presence of ATP the production of oxaloacetate was stopped, and succinate dehydrogenase was protected from inhibition by oxaloacetate. However, previously accumulated oxaloacetate transitorily decreased the level of the reduction of the NAD+ driven by succinate, by causing the reversal of the malate dehydrogenase reaction. Under these conditions (i.e. presence of ATP), rotenone strongly inhibited the reduction of NAD+ by succinate-driven reverse electron flow. No evidence for an active reverse electron transport through a rotenone-insensitive path could be obtained. The inhibitory effect of rotenone was masked if malate had previously accumulated, owing to the malate-oxidizing enzymes which reduced part or all of the matrix NAD+.

Fluxes of Nicotinamide Adenine Dinucleotides through Mitochondrial Membranes in Human Cultured Cells

“…The kinetics of inhibition has been shown to be tightly dependent on the NAD ϩ content of the matrix which controls the activity of the malate dehydrogenase and therefore the production of oxaloacetate from the malate arising from the fumarate produced during succinate oxidation (1,12). We first observed that the oxidation of a low concentration of succinate was more rapidly inhibited in 1dBLCL as compared with 6dBLCL, supporting the hypothesis of a lower NAD ϩ content of these latter cells (Fig.…”

“…duced nitro blue tetrazolium was spectrophotometrically measured in a 1-ml cell containing 0.1 M Bicine buffer (pH 8.0), 500 mM ethanol, 1.7 mM ethosulfate, 30 M nitro blue tetrazolium, and 20 l of extract. NAD ϩ reduction was fluorimetrically monitored using a PerkinElmer fluorimeter (LS 50 B) at 37°C in a thermostated quartz cuvette (1 ϫ 1 cm), magnetically stirred, containing 2 ml of medium A (12).…”

“…We next estimated in situ the NAD content of mitochondria in dt-BLCL, still avoiding the isolation of the mitochondria. This was achieved by measuring the reduction of matrix NAD ϩ by reverse electron flow from succinate, which is known to reduce more than 90% of the matrix NAD ϩ pool, which then acts passively as an electron sink (4,12). BLCL were initially incubated 5 min under aerobic conditions at room temperature in the presence of digitonin (15 g/mg protein) in order to deprive mitochondria from any respiratory substrate.…”

Inhibitors of Succinate: Quinone Reductase/Complex II Regulate Production of Mitochondrial Reactive Oxygen Species and Protect Normal Cells from Ischemic Damage but Induce Specific Cancer Cell Death

“…Thus, the rotenone effect was attributed to maintenance of the NADH pool at its highest level during state 3 respiration, impeding the forward reaction of malate dehydrogenase to prevent production of the SDH inhibitory OAA, glutamate by transamination maintains OAA at a low level, while ATP was also shown to inhibit malate dehydrogenase (77) and was able to stimulate SDH activity to help decrease OAA levels (89)(90)(91)(92). For its own part, H 2 O 2 -induced inhibition of Complex II is probably mediated by maintaining high OAA levels through aconitase blockade (93,94).…”

Loss of the Mitochondrial Fatty Acid β-Oxidation Protein Medium-Chain Acyl-Coenzyme A Dehydrogenase Disrupts Oxidative Phosphorylation Protein Complex Stability and Function

“…However, treatment with the OXPHOS complex III inhibitor antimycin A resulted in elevated mitochondrial ROS generation in MCAD KO cells compared to controls. Why this is the case is not clear, but it may be due to succinate-driven reverse electron transfer through the respiratory chain 28 . In control cells, blocking complex III will cause reverse electron flow, with ubiquinol oxidized and NAD + reduced by complex I.…”