Biochemical characterization of mitochondrial complex III inhibition by nitric oxide.

VALDEZ LB; IGLESIAS DE; BOMBICINO SS; BOVERIS A

Kyoto

Congreso; 17th Biennial Meeting of Society for Free Radical Research International - SFRRI 2014; 2014

Society for Free Radical Research International

The aim of this work was to study the effect of NO on electron transfer between cyt. b and cyt. c, using phosphorylating electron transfer particles (ETPH-Mg2+) and GSNO as NO donor. After 2 min of incubation, 500 uM GSNO released 240 nM NO to the reaction medium. Succinate-cytochrome c reductase activity (222+/-4 nmol/min.mg protein) was inhibited (51%) by 500 uM GSNO; this inhibition was independent on [O2]. Succinate-cytochrome c reductase was also reduced (36%) when ETPH-Mg2+ were incubated in the presence of L-arginine and mtNOS cofactors, suggesting that this inhibitory effect is caused by mtNOS-produced NO. In the presence of 500 uM GSNO cyt. b5622+/cyt. b5623+ ratio was 2.5. O2- and H2O2 production rates were hyperbolically increased with a maximal effect at 500 uM GSNO (1.3+/-0.1 nmol O2-/min.mg protein; 0.64+/-0.05 nmol H2O2/min.mg protein); the O2-/H2O2 ratio was 1.98. ETPH-Mg2+ incubated in the presence of succinate for 1 min showed an EPR signal at g~1.99, which was increased by GSNO or antimycin addition. When ETPH-Mg2+ were incubated in the presence of GSNO for 5 min, EPR spectra showed an additional signal at g~2.02, suggesting that two ubiquinol pools are partially oxidized by NO. NO leads to electron transfer inhibition between cyt. b and c, with a [UQH°]ss enhancement which, in turn, generates an increase in O2- and H2O2 production rates.