Inhibition of mitochondrial complex III by NO involves ubisemiquinone formation

IGLESIAS DE; BOMBICINO S; VALDEZ LB; BOVERIS A

Sierra de la Ventana

Congreso; XLIII Reunión Anual de la Sociedad Argentina de Biofísica; 2014

Sociedad Argentina de Biofísica

The effect of NO on mitochondrial complex III was studied using bovine heart-submitochondrial particles (SMP). GSNO (25-500 µM) and SPER-NO (2.5-30 µM) inhibited complex II-III activity (222 ± 4 nmol/min.mg protein) in a concentration dependent manner, and produced a hyperbolic increase in O2- production rate (up to 1.3 ± 0.1 nmol/min.mg protein). Considering that complex III produces O2- by autoxidation of ubisemiquinone (UQH?), the aim of this work was to evaluate through EPR the formation of UQH? as a result of NO interaction with complex III. In the presence of succinate, SMP showed an EPR signal at g~1.99 that could be attributed to UQH? implicated in the Q cycle. This signal was 42% increased by antimycin addition. The incubation of SMP with antimycin plus myxothiazol avoided the UQH? formation. The UQH? signal was completely absent if the SMP were previously denatured. In the presence of 500 µM GSNO (~1.3 µM NO) or 20 µM SPER-NO (~1.0 µM NO), the EPR signal was increased by 33% and 34%, respectively. When GSNO plus mixothiazol were simultaneously added to the reaction medium, the signal was not observed, similarly to the effect observed by the addition of antimycin plus myxothiazol. The EPR spectra obtained under N2 atmosphere were similar to the ones obtained in air saturated conditions, suggesting that UQH? signal is not caused by NOx species on complex III area. In conclusion, NO inhibits the ubiquinone-cytochrome b area leading to an UQH? steady-state concentration enhancement which, in turn, increases O2- production rate