CONICET | Buscador de Institutos y Recursos Humanos

The effects of NO on complex III were studied using bovine heart submitochondrial particles (SMP). Complex II-III activity (222 ± 4 nmol/min.mg protein) was inhibited by 50% in the presence of 1.25 μM NO, released from 500 µM GSNO or 30 uM SPER-NO. Neither GSNO nor SPER-NO were able to inhibit complex II (220 ± 9 nmol/min.mg protein) suggesting that NO affects complex III area. Complex II-III activity was also decreased (36%) when SMP were incubated with L-arginine and mtNOS cofactors, indicating that the inhibition was produced by endogenous NO. GSNO (500 uM) reduced cytochrome b562 by 71% in an [O2] independent manner. Hyperbolic increases in O2- (up to 1.3 ± 0.1 nmol/min.mg protein) and H2O2 (up to 0.64 ± 0.05 nmol/min.mg protein) productions were observed with a maximal effect at 500 μM GSNO. SMP incubated in the presence of succinate showed an EPR signal (g=1.99) compatible with a stable semiquinone (UQH.), which was increased (42%) by antimycin and abolished by antimycin plus myxothiazol supplementations. In the presence of 1.25 uM NO (500 μM GSNO or 30 μM SPER-NO) the UQH. signal was increased by 35%. When GSNO and myxothiazol were simultaneously added to the medium, the UQH. signal was not observed, similarly to the effect of antimycin plus myxothiazol. EPR spectra obtained under N2 atmosphere were comparable to the ones obtained in air saturated conditions, suggesting that UQH? signal is not caused by NOx species on complex III. To conclude, NO interacts with complex III in an [O2] independent manner producing antimycin-like effects. This behaviour comprises the inhibition of electron transfer, the interruption of the oxidation of cytochromes b, and the enhancement of [UQH.]ss which, in turn, leads to an increase in O2- and H2O2 production rates.