Gossypol Inhibits Electron Transport and Stimulates ROS Generation in Yarrowia lipolytica Mitochondria

Abstract

This work studied the effect of gossypol on the mitochondrial respiratory chain of Yarrowia lipolytica. The compound was shown to inhibit mitochondrial electron transfer and stimulate generation of reactive oxygen species. The inhibition kinetics in oxidation of various substrates (NADH, succinate, α-glycerophosphate and pyruvate + malate) by isolated mitochondria was investigated. Analysis of the kinetic parameters showed gossypol to inhibit two fragments of the mitochondrial electron transfer chain: a) between coenzyme Q and cytochrome b with K(IIIi) of 118.3 μM (inhibition by the noncompetitive type), and b) at the level of exogenous NADH dehydrogenase with of K(Ii) 17.2 μM (inhibition by the mixed type).

Keywords: Gossypol; Kinetics of inhibition; Mitochondria; ROS production; Yarrowia lipolytica..

Fig. (1)

Influence of gossypol concentrations on mitochondrial respiration in the presence of different exogenous substrates. 1, NADH; 2, α-glycerophosphate; 3, pyruvate + malate; 4, succinate.

Fig. (2)

Dixon plots of gossypol inhibition of oxygen consumption by mitochondria during oxidation of NADH (1), α-glycerophosphate (2), pyruvate + malate (3) and succinate (4). Experimental values are taken from Fig. (1).

Fig. (3)

Influence of gossypol on H₂O₂ generation by mitochondria. Mitochondria (0.8 mg protein/ml) were incubated in a reaction medium containing 0.6 M mannitol, 20 mM Tris-phosphate buffer (pH 7.2), 10 μM CCCP, 20 μM DCDHF-DA and 5 U horseradish peroxidase. Pyruvate and malate were added at a concentration of 5 mM each; succinate, 10 mM; antimycin A, 5 µM; gossypol, 250 µM.

Fig. (4)

Dependence of the initial reaction rates of oxygen uptake by mitochondria on NADH concentration in the absence (1) and presence (2) of gossypol (30 µM).