To unveil what controls mitochondrial ROS detoxification, the
NADPH supply and GSH/
GSSG recycling for oxidative stress management were analyzed in
cancer and non-
cancer mitochondria. Therefore, proteomic and kinetomic analyses were carried out of the mitochondrial (i)
NADPH producing and (ii) GSH/
GSSG recycling
enzymes associated to oxidative stress management. The
protein contents of the eight
enzymes analyzed were similar or even higher in AS-30D rat
hepatoma mitochondria (HepM) than in rat liver (RLM) and rat heart (RHM) mitochondria, suggesting that the
NADPH/GSH/ROS pathway was fully functional in
cancer mitochondria. The Vmax values of IDH-2 were much greater than those of GDH, TH and ME, suggesting that IDH-2 is the predominant
NADPH producer in the three mitochondrial types; in fact, the GDH reverse reaction was favored. The Vmax values of GR and GPx were lower in HepM than in RLM, suggesting that the oxidative stress management is compromised in
cancer mitochondria. The Km values of IDH-2, GR and GPx were all similar among the different mitochondrial types. Kinetic modeling revealed that the oxidative stress management was mainly controlled by GR, GPx and IDH. Modeling and experimentation also revealed that, due to their higher IDH-2 activity and lower GPx activity presumably by acetylation, HepM (i) showed higher steady-state
NADPH levels; (ii) required greater
peroxide concentrations to achieve reliable steady-state fluxes and metabolite concentration; and (iii) endured higher
peroxide concentrations without collapsing their GSH/
GSSG ratios. Then, to specifically prompt lower GSH/
GSSG ratios under oxidative stress thus compromising
cancer mitochondria functioning, GPx should be re-activated.